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Sample records for podocytes induces intracellular

  1. Valsartan inhibits amylin-induced podocyte damage.

    PubMed

    Huang, Fengjuan; Wang, Qingzhu; Ma, Xiaojun; Wu, Lina; Guo, Feng; Qin, Guijun

    2016-07-01

    Previous studies have described the deposition of amylin in the kidney of patients with type 2 diabetes mellitus (T2DM). These deposits play a critical role in the pathogenesis of diabetic nephropathy (DN), although the mechanism underlying this effect is unknown. Thus, this study was undertaken to investigate whether amylin aggregation stimulates the local angiotensin II type 1 receptor (AT1R) in podocytes, and to examine its role in podocyte apoptosis. Amylin-induced apoptosis was investigated in vitro in differentiated, conditionally immortalized mouse podocytes and in vivo in KM mice. Expression of genes including nephrin, podocin, AT1R and desmin was measured through quantitative real time PCR, western blot and immunohistochemistry. Apoptosis was determined by flow cytometry, while the cellular distribution of podocin and nephrin was investigated by immunofluorescence. The ultra-structure of glomeruli was examined by transmission electron microscopy (TEM). Amylin enhanced apoptosis in a dose-dependent manner in vitro. The peptide also suppressed podocin and nephrin expression, but enhanced that of AT1R and desmin. Both effects were significantly blocked by valsartan, which inhibits angiotensin II type 1 receptor. These findings suggest that amylin activates a local intracellular RAS in podocytes and induces damage and apoptosis. PMID:27102209

  2. Astragaloside IV prevents high glucose-induced podocyte apoptosis via downregulation of TRPC6.

    PubMed

    Yao, Xing-Mei; Liu, Yu-Jun; Wang, Yun-Man; Wang, Hao; Zhu, Bing-Bing; Liang, Yong-Ping; Yao, Wei-Guo; Yu, Hui; Wang, Nian-Song; Zhang, Xue-Mei; Peng, Wen

    2016-06-01

    Diabetic nephropathy (DN) is one of the most important causes of end‑stage renal disease. Astragaloside IV (AS-IV) is a saponin isolated from Astragalus membranaceus, which possesses various pharmacological activities. AS‑IV prevents podocyte apoptosis and ameliorates renal injury in DN; however, few studies have focused on its effects on ion channels. The transient receptor potential channel 6 (TRPC6) is an important Ca2+‑permeable ion channel in podocytes, which is involved in high glucose (HG)-induced podocyte apoptosis. The aim of the present study was to investigate whether AS‑IV prevented HG‑induced podocyte apoptosis via TRPC6. Cultured podocytes were pre‑treated with 10, 20 or 40 µM AS‑IV for 1 h prior to HG exposure for 24 h. Apoptosis, cell viability, expression of TRPC6, nuclear factor of activated T cells (NFAT2) and B‑cell lymphoma 2‑associated X protein (Bax), as well as the intracellular Ca2+ concentration were subsequently analyzed. The results indicated that HG induced podocyte apoptosis and upregulation of TRPC6, and increased intracellular Ca2+. Furthermore, enhanced NFAT2 and Bax expression was detected. Conversely, AS‑IV protected HG‑induced podocyte apoptosis, downregulated TRPC6 expression and suppressed intracellular Ca2+ in HG-stimulated podocytes. AS‑IV also suppressed NFAT2 and Bax expression. These results suggest that AS‑IV may prevent HG-induced podocyte apoptosis via downregulation of TRPC6, which is possibly mediated via the calcineurin/NFAT signaling pathway. PMID:27109610

  3. Podocytes

    PubMed Central

    Reiser, Jochen; Altintas, Mehmet M.

    2016-01-01

    Podocytes are highly specialized cells of the kidney glomerulus that wrap around capillaries and that neighbor cells of the Bowman’s capsule. When it comes to glomerular filtration, podocytes play an active role in preventing plasma proteins from entering the urinary ultrafiltrate by providing a barrier comprising filtration slits between foot processes, which in aggregate represent a dynamic network of cellular extensions. Foot processes interdigitate with foot processes from adjacent podocytes and form a network of narrow and rather uniform gaps. The fenestrated endothelial cells retain blood cells but permit passage of small solutes and an overlying basement membrane less permeable to macromolecules, in particular to albumin. The cytoskeletal dynamics and structural plasticity of podocytes as well as the signaling between each of these distinct layers are essential for an efficient glomerular filtration and thus for proper renal function. The genetic or acquired impairment of podocytes may lead to foot process effacement (podocyte fusion or retraction), a morphological hallmark of proteinuric renal diseases. Here, we briefly discuss aspects of a contemporary view of podocytes in glomerular filtration, the patterns of structural changes in podocytes associated with common glomerular diseases, and the current state of basic and clinical research. PMID:26918173

  4. Growth Hormone Induces Transforming Growth Factor-Beta-Induced Protein in Podocytes: Implications for Podocyte Depletion and Proteinuria.

    PubMed

    Chitra, P Swathi; Swathi, T; Sahay, Rakesh; Reddy, G Bhanuprakash; Menon, Ram K; Kumar, P Anil

    2015-09-01

    The glomerular podocytes form a major size selective barrier for the filtration of serum proteins and reduced podocyte number is a critical event in the pathogenesis of proteinuria during diabetic nephropathy (DN). An elevated level of growth hormone (GH) is implicated as a causative factor in the development of nephropathy in patients with type 1 diabetes mellitus. We have previously shown that podocytes express GH receptor and are a target for GH action. To elucidate the molecular basis for the effects of GH on podocyte depletion, we conducted PCR-array analyses for extracellular matrix and adhesion molecules in podocytes. Our studies reveal that GH increases expression of a gene that encodes transforming growth factor-beta-induced protein (TGFBIp) expression. Similarly, microarray data retrieved from the Nephromine database revealed elevation of TGFBIp in patients with DN. Treatment with GH results in increased secretion of extracellular TGFBIp by podocytes. Both GH and TGFBIp induced apoptosis and epithelial mesenchymal transition (EMT) of podocytes. Exposure of podocytes to GH and TGFBIp resulted in increased migration of cells and altered podocyte permeability to albumin across podocyte monolayer. Administration of GH to rats induced EMT and apoptosis in the glomerular fraction of the kidney. Therefore, we conclude that the GH-dependent increase in TGFBIp in the podocyte is one of the mechanisms responsible for podocyte depletion in DN. PMID:25740786

  5. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    SciTech Connect

    Li, Ruizhao; Zhang, Li; Shi, Wei; Zhang, Bin; Liang, Xinling; Liu, Shuangxin; Wang, Wenjian

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  6. Acute ethanol induces apoptosis by stimulating TRPC6 via elevation of superoxide in oxygenated podocytes.

    PubMed

    Lu, Xiao-Yu; Liu, Bing-Chen; Wang, Li-Hua; Yang, Li-Li; Bao, Qing; Zhai, Yu-Jia; Alli, Abdel A; Thai, Tiffany L; Eaton, Douglas C; Wang, Wei-Zhi; Ma, He-Ping

    2015-05-01

    Our recent studies indicate that hydrogen peroxide (H2O2) only at high concentrations can cause oxidative stress in renal epithelial cells and induce apoptosis of podocytes. Consistently, the present study shows that H2O2, even at 1 mM, failed to induce intracellular oxidative stress and apoptosis of the podocytes due to efficient activity of catalase, an enzyme which degrades H2O2 to produce water and oxygen (O2). However, H2O2 acted as a source of O2 to allow acute ethanol to induce superoxide production and cause apoptosis of the podocytes. In contrast, acute ethanol alone did not elevate intracellular superoxide, even though it stimulates expression and translocation of p47phox to the plasma membrane. Inhibition of catalase abolished not only O2 production from H2O2 degradation, but also NOX2-dependent superoxide production in the podocytes challenged by both H2O2 and acute ethanol. In parallel, acute ethanol in the presence of H2O2, but neither ethanol nor H2O2 alone, stimulated transient receptor potential canonical 6 (TRPC6) channels and caused TRPC6-dependent elevation of intracellular Ca2+. These data suggest that exogenous H2O2 does not induce oxidative stress due to rapid degradation to produce O2 in the podocytes, but the oxygenated podocytes become sensitive to acute ethanol challenge and undergo apoptosis via a TRPC6-dependent elevation of intracellular Ca2+. Since cultured podocytes are considered in hypoxic conditions, H2O2 may be used as a source of O2 to establish an ischemia-reperfusion model in some type of cultured cells in which H2O2 does not directly induce intracellular oxidative stress. PMID:25601712

  7. Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes

    PubMed Central

    Ozawa, Shota; Ueda, Shuko; Imamura, Hiromi; Mori, Kiyoshi; Asanuma, Katsuhiko; Yanagita, Motoko; Nakagawa, Takahiko

    2015-01-01

    Differentiated podocytes, a type of renal glomerular cells, require substantial levels of energy to maintain glomerular physiology. Mitochondria and glycolysis are two major producers of ATP, but the precise roles of each in podocytes remain unknown. This study evaluated the roles of mitochondria and glycolysis in differentiated and differentiating podocytes. Mitochondria in differentiated podocytes are located in the central part of cell body while blocking mitochondria had minor effects on cell shape and migratory ability. In contrast, blocking glycolysis significantly reduced the formation of lamellipodia, a cortical area of these cells, decreased the cell migratory ability and induced the apoptosis. Consistently, the local ATP production in lamellipodia was predominantly regulated by glycolysis. In turn, synaptopodin expression was ameliorated by blocking either mitochondrial respiration or glycolysis. Similar to differentiated podocytes, the differentiating podocytes utilized the glycolysis for regulating apoptosis and lamellipodia formation while synaptopodin expression was likely involved in both mitochondrial OXPHOS and glycolysis. Finally, adult mouse podocytes have most of mitochondria predominantly in the center of the cytosol whereas phosphofructokinase, a rate limiting enzyme for glycolysis, was expressed in foot processes. These data suggest that mitochondria and glycolysis play parallel but distinct roles in differentiated and differentiating podocytes. PMID:26677804

  8. A novel podocyte gene, semaphorin 3G, protects glomerular podocyte from lipopolysaccharide-induced inflammation

    PubMed Central

    Ishibashi, Ryoichi; Takemoto, Minoru; Akimoto, Yoshihiro; Ishikawa, Takahiro; He, Peng; Maezawa, Yoshiro; Sakamoto, Kenichi; Tsurutani, Yuya; Ide, Shintaro; Ide, Kana; Kawamura, Harukiyo; Kobayashi, Kazuki; Tokuyama, Hirotake; Tryggvason, Karl; Betsholtz, Christer; Yokote, Koutaro

    2016-01-01

    Kidney diseases including diabetic nephropathy have become huge medical problems, although its precise mechanisms are still far from understood. In order to increase our knowledge about the patho-physiology of kidney, we have previously identified >300 kidney glomerulus-enriched transcripts through large-scale sequencing and microarray profiling of the mouse glomerular transcriptome. One of the glomerulus-specific transcripts identified was semaphorin 3G (Sema3G) which belongs to the semaphorin family. The aim of this study was to analyze both the in vivo and in vitro functions of Sema3G in the kidney. Sema3G was expressed in glomerular podocytes. Although Sema3G knockout mice did not show obvious glomerular defects, ultrastructural analyses revealed partially aberrant podocyte foot processes structures. When these mice were injected with lipopolysaccharide to induce acute inflammation or streptozotocin to induce diabetes, the lack of Sema3G resulted in increased albuminuria. The lack of Sema3G in podocytes also enhanced the expression of inflammatory cytokines including chemokine ligand 2 and interleukin 6. On the other hand, the presence of Sema3G attenuated their expression through the inhibition of lipopolysaccharide-induced Toll like receptor 4 signaling. Taken together, our results surmise that the Sema3G protein is secreted by podocytes and protects podocytes from inflammatory kidney diseases and diabetic nephropathy. PMID:27180624

  9. A novel podocyte gene, semaphorin 3G, protects glomerular podocyte from lipopolysaccharide-induced inflammation.

    PubMed

    Ishibashi, Ryoichi; Takemoto, Minoru; Akimoto, Yoshihiro; Ishikawa, Takahiro; He, Peng; Maezawa, Yoshiro; Sakamoto, Kenichi; Tsurutani, Yuya; Ide, Shintaro; Ide, Kana; Kawamura, Harukiyo; Kobayashi, Kazuki; Tokuyama, Hirotake; Tryggvason, Karl; Betsholtz, Christer; Yokote, Koutaro

    2016-01-01

    Kidney diseases including diabetic nephropathy have become huge medical problems, although its precise mechanisms are still far from understood. In order to increase our knowledge about the patho-physiology of kidney, we have previously identified >300 kidney glomerulus-enriched transcripts through large-scale sequencing and microarray profiling of the mouse glomerular transcriptome. One of the glomerulus-specific transcripts identified was semaphorin 3G (Sema3G) which belongs to the semaphorin family. The aim of this study was to analyze both the in vivo and in vitro functions of Sema3G in the kidney. Sema3G was expressed in glomerular podocytes. Although Sema3G knockout mice did not show obvious glomerular defects, ultrastructural analyses revealed partially aberrant podocyte foot processes structures. When these mice were injected with lipopolysaccharide to induce acute inflammation or streptozotocin to induce diabetes, the lack of Sema3G resulted in increased albuminuria. The lack of Sema3G in podocytes also enhanced the expression of inflammatory cytokines including chemokine ligand 2 and interleukin 6. On the other hand, the presence of Sema3G attenuated their expression through the inhibition of lipopolysaccharide-induced Toll like receptor 4 signaling. Taken together, our results surmise that the Sema3G protein is secreted by podocytes and protects podocytes from inflammatory kidney diseases and diabetic nephropathy. PMID:27180624

  10. Csk regulates angiotensin II-induced podocyte apoptosis.

    PubMed

    Zhang, Lu; Ren, Zhilong; Yang, Qian; Ding, Guohua

    2016-07-01

    Increasing data have shown that angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. The mechanism underlying Ang II-induced podocyte apoptosis has not been established. C-terminal Src kinase (Csk) is a cytoplasmic kinase that interacts with scaffolding proteins involved in cell growth, adhesion, and polarization, and the role of Csk in regulating cellular apoptosis has gradually attracted attention. This study evaluates the role of Csk in Ang II-induced podocyte apoptosis. In vivo, Wistar rats were randomly subjected to a normal saline or Ang II infusion. In vitro, we exposed differentiated mouse podocytes to Ang II. Ang II increased Csk expression and induced podocyte apoptosis, stimulated Csk translocation and binding to Caveolin-1, and stimulated decreased Fyn pY416, increased Fyn pY529, and nephrin dephosphorylation. Csk knockdown prevented Ang II-induced podocyte apoptosis, reduced Fyn kinase inactivation, and increased the interaction between nephrin and the activated form of Fyn, accompanied by a reduced interaction between Csk and Caveolin-1. These findings indicate that Ang II induces podocyte injury via a Csk-dependent pathway. PMID:27225249

  11. Albumin-associated free fatty acids induce macropinocytosis in podocytes

    PubMed Central

    Chung, Jun-Jae; Huber, Tobias B.; Gödel, Markus; Jarad, George; Hartleben, Björn; Kwoh, Christopher; Keil, Alexander; Karpitskiy, Aleksey; Hu, Jiancheng; Huh, Christine J.; Cella, Marina; Gross, Richard W.; Miner, Jeffrey H.; Shaw, Andrey S.

    2015-01-01

    Podocytes are specialized epithelial cells in the kidney glomerulus that play important structural and functional roles in maintaining the filtration barrier. Nephrotic syndrome results from a breakdown of the kidney filtration barrier and is associated with proteinuria, hyperlipidemia, and edema. Additionally, podocytes undergo changes in morphology and internalize plasma proteins in response to this disorder. Here, we used fluid-phase tracers in murine models and determined that podocytes actively internalize fluid from the plasma and that the rate of internalization is increased when the filtration barrier is disrupted. In cultured podocytes, the presence of free fatty acids (FFAs) associated with serum albumin stimulated macropinocytosis through a pathway that involves FFA receptors, the Gβ/Gγ complex, and RAC1. Moreover, mice with elevated levels of plasma FFAs as the result of a high-fat diet were more susceptible to Adriamycin-induced proteinuria than were animals on standard chow. Together, these results support a model in which podocytes sense the disruption of the filtration barrier via FFAs bound to albumin and respond by enhancing fluid-phase uptake. The response to FFAs may function in the development of nephrotic syndrome by amplifying the effects of proteinuria. PMID:25915582

  12. Cell cycle re-entry sensitizes podocytes to injury induced death.

    PubMed

    Hagen, Manuel; Pfister, Eva; Kosel, Andrea; Shankland, Stuart; Pippin, Jeffrey; Amann, Kerstin; Daniel, Christoph

    2016-07-17

    Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy. PMID:27232327

  13. Toll-like Receptor 9 Can be Activated by Endogenous Mitochondrial DNA to Induce Podocyte Apoptosis

    PubMed Central

    Bao, Wenduona; Xia, Hong; Liang, Yaojun; Ye, Yuting; Lu, Yuqiu; Xu, Xiaodong; Duan, Aiping; He, Jing; Chen, Zhaohong; Wu, Yan; Wang, Xia; Zheng, Chunxia; Liu, Zhihong; Shi, Shaolin

    2016-01-01

    Toll-like receptor 9 (TLR9) senses bacterial DNA characteristic of unmethylated CpG motifs to induce innate immune response. TLR9 is de novo expressed in podocytes of some patients with glomerular diseases, but its role in podocyte injury remains undetermined. Since TLR9 activates p38 MAPK and NFkB that are known to mediate podocyte apoptosis, we hypothesized that TLR9 induces podocyte apoptosis in glomerular diseases. We treated immortalized podocytes with puromycin aminonucleosides (PAN) and observed podocyte apoptosis, accompanied by TLR9 upregulation. Prevention of TLR9 upregulation by siRNA significantly attenuated NFκB p65 or p38 activity and apoptosis, demonstrating that TLR9 mediates podocyte apoptosis. We next showed that endogenous mitochondrial DNA (mtDNA), whose CpG motifs are also unmethylated, is the ligand for TLR9, because PAN induced mtDNA accumulation in endolysosomes where TLR9 is localized, overexpression of endolysosomal DNase 2 attenuated PAN-induced p38 or p65 activity and podocyte apoptosis, and DNase 2 silencing was sufficient to activate p38 or p65 and induce apoptosis. In PAN-treated rats, TLR9 was upregulated in the podocytes, accompanied by increase of apoptosis markers. Thus, de novo expressed TLR9 may utilize endogenous mtDNA as the ligand to facilitate podocyte apoptosis, a novel mechanism underlying podocyte injury in glomerular diseases. PMID:26934958

  14. A vital role for myosin-9 in puromycin aminonucleoside-induced podocyte injury by affecting actin cytoskeleton.

    PubMed

    Yuan, Yanggang; Zhao, Chuanyan; An, Xiaofei; Wu, Lin; Wang, Hui; Zhao, Min; Bai, Mi; Duan, Suyan; Zhang, Bo; Zhang, Aihua; Xing, Changying

    2016-06-01

    Podocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored both in vitro and in vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies. PMID:26902808

  15. Phospholipase C Epsilon (PLCε) Induced TRPC6 Activation: A Common but Redundant Mechanism in Primary Podocytes

    PubMed Central

    Kalwa, Hermann; Storch, Ursula; Demleitner, Jana; Fiedler, Susanne; Mayer, Tim; Kannler, Martina; Fahlbusch, Meike; Barth, Holger; Smrcka, Alan; Hildebrandt, Friedhelm; Gudermann, Thomas; Dietrich, Alexander

    2016-01-01

    In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca2+ concentration [Ca2+]i. Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca2+ influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain-of-function mutants of TRPC6, while others carry loss-of-function mutants of PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCβ and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLCε in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor-operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLCε siRNA and by a PLCε loss-of-function mutant isolated from a patient with FSGS. PLCε-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho-GEF activation which induced Rho-mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTPγS-induced TRPC6 activation, pointing to a redundant role of PLCε-mediated TRPC6 activation at least in podocytes. PMID:25521631

  16. PGC-1α overexpression protects against aldosterone-induced podocyte depletion: role of mitochondria

    PubMed Central

    Zhao, Min; Yuan, Yanggang; Bai, Mi; Ding, Guixia; Jia, Zhanjun; Huang, Songming; Zhang, Aihua

    2016-01-01

    Growing evidence has shown that podocyte number is a critical determinant for the development of glomerulosclerosis and progressive renal failure. We previously reported that mitochondrial dysfunction (MtD) is an early event in podocyte injury. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is an important modulator of mitochondrial biogenesis. Here, we investigated the role of PGC-1α overexpression in podocyte depletion and the involvement of mitochondria in this process. Following chronic aldosterone (Aldo) infusion for 14 days, we observed a remarkable podocyte loss, podocyte phenotypic changes, and albuminuria in WT mice. However, all these abnormalities were significantly attenuated in PGC-1α transgenic mice. Next, we examined mitochondrial function in both genotypes with or without Aldo infusion. As expected, Aldo-induced MtD in glomeruli was markedly improved in PGC-1α transgenic mice. In vitro, Aldo induced podocyte detachment and phenotypic changes in line with MtD in dose- and time-dependent manners. Similarly, ethidium bromide, an inducer of MtD, mimicked Aldo effects on podocyte detachment and phenotypic alterations. Notably, overexpression of PGC-1α in podocytes entirely reversed Aldo-induced podocyte detachment, phenotypic changes, and MtD. Taken together, these findings demonstrate that PGC-1α protects against podocyte depletion and phenotypic changes possibly by maintaining normal mitochondrial function. PMID:26943584

  17. Cdc42 deficiency induces podocyte apoptosis by inhibiting the Nwasp/stress fibers/YAP pathway

    PubMed Central

    Huang, Z; Zhang, L; Chen, Y; Zhang, H; Zhang, Q; Li, R; Ma, J; Li, Z; Yu, C; Lai, Y; Lin, T; Zhao, X; Zhang, B; Ye, Z; Liu, S; Wang, W; Liang, X; Liao, R; Shi, W

    2016-01-01

    Podocyte apoptosis is a major mechanism that leads to proteinuria in many chronic kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. The Rho family of small GTPases has been shown to be required in maintaining podocyte structure and function. Recent studies have indicated that podocyte-specific deletion of Cdc42 in vivo, but not of RhoA or Rac1, leads to congenital nephrotic syndrome and glomerulosclerosis. However, the underlying cellular events in podocyte controlled by Cdc42 remain unclear. Here, we assessed the cellular mechanisms by which Cdc42 regulates podocyte apoptosis. We found that the expression of Cdc42 and its activity were significantly decreased in high glucose-, lipopolysaccharide- or adriamycin-injured podocytes. Reduced Cdc42 expression in vitro and in vivo by small interfering RNA and selective Cdc42 inhibitor ML-141, respectively, caused podocyte apoptosis and proteinuria. Our results further demonstrated that insufficient Cdc42 or Nwasp, its downstream effector, could decrease the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Moreover, our data indicated that the loss of stress fibers caused by Cdc42/Nwasp deficiency also decreased Yes-associated protein (YAP) mRNA and protein expression, and induced podocyte apoptosis. Podocyte apoptosis induced by Cdc42/Nwasp/stress fiber deficiency was significantly inhibited by overexpressing-active YAP. Thus, the Cdc42/Nwasp/stress fibers/YAP signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary Cdc42 would be one potent way to prevent proteinuria kidney diseases. PMID:26986510

  18. The Notch pathway mediates the angiotensin II-induced synthesis of extracellular matrix components in podocytes.

    PubMed

    Yao, Min; Wang, Xiaomei; Wang, Xiaomeng; Zhang, Tao; Chi, Yanqing; Gao, Feng

    2015-07-01

    The Notch pathway is known to contribute to the development of glomerular disease. Angiotensin II (Ang II), an important member of the renin-angiotensin system, stimulates the accumulation of extracellular matrix components in glomerular disease; however, the exact mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of the Notch pathway on the synthesis of extracellular matrix components in Ang II-stimulated podocytes. Mouse podocytes were stimulated with Ang II (10-6 mol/l). The activation of the Notch pathway was inhibited by a vector carrying short hairpin RNA (shRNA) targeting Notch1 (sh-Notch1) or by γ-secretase inhibitor (GSI). The protein levels of Notch1, Notch intracellular domain 1 (NICD1), hairy and enhancer of split-1 (Hes1), matrix metalloproteinase (MMP)-2, MMP-9, transforming growth factor-β1 (TGF-β1), type IV collagen and laminin were determined by western blot analysis. The Notch1, Hes1, MMP-2, MMP-9, TGF-β1, type IV collagen and laminin mRNA levels were detected by RT-PCR. The MMP-2 and MMP-9 activity was measured using a cell active fluorescence assay kit. The levels of TGF-β1, type IV collagen and laminin were determined in the culture medium of the podocytes by enzyme-linked immunosorbent assay (ELISA). Our results revealed that Ang II upregulated Notch1, NICD1, Hes1, TGF-β1, type IV collagen and laminin expression and downregulated MMP-2 and MMP-9 expression in the cultured podocytes. The inhibition of the Notch pathway by sh-Notch1 or GSI increased MMP-2 and MMP-9 expression, decreased the TGF-β1 level and suppressed type IV collagen and laminin expression. The inhibition of the Notch pathway by sh-Notch1 or GSI also increased MMP-2 and MMP-9 activity, and decreased TGF-β1 levels, type IV collagen levels and laminin secretion. These findings indicate that the Notch pathway potentially mediates the Ang II-induced synthesis of extracellular matrix components in podocytes through the

  19. MicroRNA-29a Promotion of Nephrin Acetylation Ameliorates Hyperglycemia-Induced Podocyte Dysfunction

    PubMed Central

    Lin, Chun-Liang; Lee, Pei-Hsien; Hsu, Yung-Chien; Lei, Chen-Chou; Ko, Jih-Yang; Chuang, Pei-Chin; Huang, Yu-Ting; Wang, Shao-Yu; Wu, Shin-Long; Chen, Yu-Shan; Chiang, Wen-Chih; Reiser, Jochen

    2014-01-01

    Podocyte dysfunction is a detrimental feature in diabetic nephropathy, with loss of nephrin integrity contributing to diabetic podocytopathy. MicroRNAs (miRs) reportedly modulate the hyperglycemia-induced perturbation of renal tissue homeostasis. This study investigated whether regulation of histone deacetylase (HDAC) actions and nephrin acetylation by miR-29 contributes to podocyte homeostasis and renal function in diabetic kidneys. Hyperglycemia accelerated podocyte injury and reduced nephrin, acetylated nephrin, and miR-29a levels in primary renal glomeruli from streptozotocin-induced diabetic mice. Diabetic miR-29a transgenic mice had better nephrin levels, podocyte viability, and renal function and less glomerular fibrosis and inflammation reaction compared with diabetic wild-type mice. Overexpression of miR-29a attenuated the promotion of HDAC4 signaling, nephrin ubiquitination, and urinary nephrin excretion associated with diabetes and restored nephrin acetylation. Knockdown of miR-29a by antisense oligonucleotides promoted HDAC4 action, nephrin loss, podocyte apoptosis, and proteinuria in nondiabetic mice. In vitro, interruption of HDAC4 signaling alleviated the high glucose–induced apoptosis and inhibition of nephrin acetylation in podocyte cultures. Furthermore, HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), the enrichment of H3K9Ac in miR-29a proximal promoter, and miR-29a transcription in high glucose–stressed podocytes. In conclusion, hyperglycemia impairs miR-29a signaling to intensify HDAC4 actions that contribute to podocyte protein deacetylation and degradation as well as renal dysfunction. HDAC4, via epigenetic H3K9 hypoacetylation, reduces miR-29a transcription. The renoprotective effects of miR-29a in diabetes-induced loss of podocyte integrity and renal homeostasis highlights the importance of post-translational acetylation reactions in podocyte microenvironments. Increasing miR-29a action may

  20. Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

    SciTech Connect

    Piwkowska, Agnieszka; Rogacka, Dorota; Angielski, Stefan; Jankowski, Maciej

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} activates the insulin signaling pathway and glucose uptake in podocytes. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} induces time-dependent changes in AMPK phosphorylation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} enhances insulin signaling pathways via AMPK activation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} stimulation of glucose uptake is AMPK-dependent. -- Abstract: Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H{sub 2}O{sub 2}) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H{sub 2}O{sub 2}-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H{sub 2}O{sub 2} (100 {mu}M) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5 min ({Delta} 183%, P < 0.05), 3 min ({Delta} 414%, P < 0.05), and 10 min ({Delta} 35%, P < 0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H{sub 2}O{sub 2}>. Furthermore, H{sub 2}O{sub 2} inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10 min; {Delta} -32%, P < 0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPK{alpha}; 78% at 3 min and 244% at 10 min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100 {mu}M, 2 h). Moreover, Compound C significantly reduced the effect of H{sub 2}O{sub 2} on IR phosphorylation by about 40% (from 2.07 {+-} 0.28 to 1.28 {+-} 0.12, P < 0.05). In addition, H{sub 2}O{sub 2} increased glucose uptake in podocytes

  1. PEDF inhibits AGE-induced podocyte apoptosis via PPAR-gamma activation.

    PubMed

    Ishibashi, Yuji; Matsui, Takanori; Ohta, Keisuke; Tanoue, Ryuichiro; Takeuchi, Masayoshi; Asanuma, Katsuhiko; Fukami, Kei; Okuda, Seiya; Nakamura, Kei-ichiro; Yamagishi, Sho-ichi

    2013-01-01

    Advanced glycation end products (AGEs) formed at an accelerated rate under diabetes, elicit oxidative and pro-apoptotic reactions in various types of cells, including podocytes, thus being involved in the development and progression of diabetic nephropathy. Recently, we, along with others, have found that pigment epithelium-derived factor (PEDF), a glycoprotein with potent neuronal differentiating activity, inhibits AGE-elicited mesangial and tubular cell damage through its anti-oxidative properties. However, the effects of PEDF on podocyte loss, one of the characteristic features of diabetic nephropathy remain unknown. In this study, we investigated whether and how PEDF could protect against AGE-elicited podocyte apoptosis in vitro. AGEs decreased PEDF mRNA level in podocytes, which was blocked by neutralizing antibody raised against receptor for AGEs (RAGE-Ab). PEDF or RAGE-Ab was found to inhibit the AGE-induced up-regulation of RAGE mRNA level, oxidative stress generation and resultant apoptosis in podocytes. All of the beneficial effects of PEDF on AGE-exposed podocytes were blocked by the treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPARγ). Further, although PEDF did not affect protein expression levels of PPARγ, it significantly restored the PPARγ transcriptional activity in AGE-exposed podocytes. The present results demonstrated for the first time that PEDF could block the AGE-induced apoptotic cell death of podocytes by suppressing RAGE expression and subsequent ROS generation partly via PPARγ activation. Our present study suggests that substitution of PEDF proteins may be a promising strategy for preventing the podocyte loss in diabetic nephropathy. PMID:23108227

  2. Human Induced Pluripotent Stem Cell-Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation.

    PubMed

    Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake; Nishinakamura, Ryuichi

    2016-06-01

    Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. PMID:26586691

  3. Overexpression of miR-34c inhibits high glucose-induced apoptosis in podocytes by targeting Notch signaling pathways

    PubMed Central

    Liu, Xiang-Dong; Zhang, Lian-Yun; Zhu, Tie-Chui; Zhang, Rui-Fang; Wang, Shu-Long; Bao, Yan

    2015-01-01

    Recent findings have shown that microRNAs play critical roles in the pathogenesis of diabetic nephropathy. miR-34c has been found to inhibit fibrosis and the epithelial-mesenchymal transition of kidney cells. However, the role of miR-34c in diabetic nephropathy has not been well studied. The current study was designed to investigate the role and potential underlying mechanism of miR-34c in regulating diabetic nephropathy. After treating podocytes with high glucose (HG) in vitro, we found that miR-34c was downregulated and that overexpression of miR-34c inhibited HG-induced podocyte apoptosis. The direct interaction between miR-34c and the 3’-untranslated region (UTR) of Notch1 and Jagged1 was validated by dual-luciferase reporter assay. Moreover, Notch1 and Jagged1 as putative targets of miR-34c were downregulated by miR-34c overexpression in HG-treated podocytes. Overexpression of miR-34c inhibited HG-induced Notch signaling pathway activation, as indicated by decreased expression of the Notch intracellular domain (NICD) and downstream genes including Hes1 and Hey1. Furthermore, miR-34c overexpression increased the expression of the anti-apoptotic gene Bcl-2, and decreased the expression of the pro-apoptotic protein Bax and cleaved Caspase-3. Additionally, the phosphorylation of p53 was also downregulated by miR-34c overexpression. Taken together, our findings suggest that miR-34c overexpression inhibits the Notch signaling pathway by targeting Notch1 and Jaggged1 in HG-treated podocytes, representing a novel and potential therapeutic target for the treatment of diabetic nephropathy. PMID:26191142

  4. Melatonin protects podocytes from angiotensin II-induced injury in an in vitro diabetic nephropathy model.

    PubMed

    Ji, Zhen-Zhong; Xu, Yan-Cheng

    2016-07-01

    Diabetic nephropathy (DN) is a severe renal disorder characterized by podocyte damage and accumulation of extracellular matrix leading to further glomerulosclerosis, possibly via the activation of inflammatory signaling and the generation of oxidative stress. Melatonin has been considered a robust anti‑oxidant, and is able to attenuate DN in several animal models. In the present study, cell viability was examined by CCK-8 assay. Cell apoptosis, intracellular reactive oxygen species and mitochondrial membrane potential (MMP) levels were measured by flow cytometry. Western blot analysis was performed to detect the expression of Nephrin, B cell lymphoma‑2 (Bcl‑2)‑associated X protein (Bax), Bcl-2, Caspase-3, Janus kinase (JAK)2 and Signal transducer and activator of transcription (STAT)3 in podocytes. Based on an in vitro podocyte injury model, the present study found that the application of melatonin significantly reduced AngII‑induced apoptosis and increased the proliferative rate of cells, as evidenced by decreased expression levels of apoptotic proteins, including Caspase‑3 and Bax, and a change in the ratio of Bax/Bcl‑2. Further investigation demonstrated that a reduction in oxidative stress and the recovery of mitochondrial function were involved in this protective effect. In addition, the Jak/STAT signaling pathway was inhibited, indicating that cytokine‑mediated inflammation was also targeted by melatonin. The present study demonstrated for the first time, to the best our knowledge, that melatonin exerted an anti-apoptotic effect in AngII-mediated podocyte injury, and indicates a possible mechanism to explain the protective effect of melatonin in DN. PMID:27220903

  5. Calpain-Mediated Cleavage of Calcineurin in Puromycin Aminonucleoside-Induced Podocyte Injury

    PubMed Central

    Ding, Fangrui; Li, Xuejuan; Li, Baihong; Guo, Jifan; Zhang, Yanqin; Ding, Jie

    2016-01-01

    The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus are widely used in the treatment of proteinuria diseases. As the direct target of these drugs, calcineurin has previously been demonstrated to play a role in proteinuria diseases. However, aside from its immune-related effects, the local status of calcineurin in renal inherent cells has not been fully explored in the settings of proteinuria disease and podocyte injury. In this study, calcineurin activity and protein expression in the well-known puromycin aminonucleoside (PAN)-induced podocyte injury model were examined. Interestingly, we found that calcineurin activity was abnormally increased in PAN-treated podocytes, whereas the expression of the full-length 60-kDa calcineurin protein was decreased. This result suggests that there may be another activated form of calcineurin that is independent of the full-length phosphatase. To investigate whether calpain is involved in regulating calcineurin, we exposed PAN-treated podocytes to both pharmacological inhibitors of calpain and specific siRNAs against calpain. Calpain blockade reduced the enhanced calcineurin activity and restored the down-regulated expression of 60-kDa calcineurin. In addition, purified calpain protein was incubated with podocyte extracts, and a 45-kDa fragment of calcineurin was identified; this finding was confirmed in PAN-induced podocyte injury and calpain inhibition experiments. We conclude that calcineurin activity is abnormally increased during PAN-induced podocyte injury, whereas the expression of the full-length 60-kDa calcineurin protein is down-regulated due to over-activated calpain that cleaves calcineurin to form a 45-kDa fragment. PMID:27171192

  6. Diet-Induced Podocyte Dysfunction in Drosophila and Mammals.

    PubMed

    Na, Jianbo; Sweetwyne, Mariya T; Park, Ae Seo Deok; Susztak, Katalin; Cagan, Ross L

    2015-07-28

    Diabetic nephropathy is a major cause of end-stage kidney disease. Characterized by progressive microvascular disease, most efforts have focused on injury to the glomerular endothelium. Recent work has suggested a role for the podocyte, a highly specialized component of the glomerular filtration barrier. Here, we demonstrate that the Drosophila nephrocyte, a cell analogous to the mammalian podocyte, displays defects that phenocopy aspects of diabetic nephropathy in animals fed chronic high dietary sucrose. Through functional studies, we identify an OGT-Polycomb-Knot-Sns pathway that links dietary sucrose to loss of the Nephrin ortholog Sns. Reducing OGT through genetic or drug means is sufficient to rescue loss of Sns, leading to overall extension of lifespan. We demonstrate upregulation of the Knot ortholog EBF2 in glomeruli of human diabetic nephropathy patients and a mouse ob/ob diabetes model. Furthermore, we demonstrate rescue of Nephrin expression and cell viability in ebf2(-/-) primary podocytes cultured in high glucose. PMID:26190114

  7. Redox signaling via lipid raft clustering in homocysteine-induced injury of podocytes

    PubMed Central

    Zhang, Chun; Hu, Jun-Jun; Xia, Min; Boini, Krishna M.; Brimson, Christopher; Li, Pin-Lan

    2010-01-01

    Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91phox) and cytosolic subunit (p47phox). Then, confocal microscopy showed that gp91phox and p47phox could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their co-localization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-β-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91phox or LR-p47phox transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91phox and p47phox were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys. PMID:20036696

  8. Constitutional Nephrin Deficiency in Conditionally Immortalized Human Podocytes Induced Epithelial-Mesenchymal Transition, Supported by β-Catenin/NF-kappa B Activation: A Consequence of Cell Junction Impairment?

    PubMed

    Ghiggeri, Gian Marco; Gigante, Maddalena; Di Donato, Armando

    2013-01-01

    The kidney glomerular podocytes are the cellular target of many chronic nephropathies both determined and acquired genetically. Mutations that affected the expression and/or the function of nephrin, a key component of the slit-diaphragm, are often causes of these pathologies. Recent findings showed that murine podocytes could undergo epithelial-mesenchymal transformation (EMT), suggesting new hypotheses about the pathogenesis of glomerular fibrosis. Here, we show that also human podocytes can undergo EMT, but more importantly nephrin ablation itself can trigger this phenotypic transformation. In fact, a model of human podocyte with engineered nephrin deficiency constitutionally expressed high levels of α-SMA, vimentin, fibronectin, and other hallmarks of EMT. Since it is known that cell contact abrogation is one of the triggers of EMT, we reasoned that nephrin loss could account for such cell junction disruption and cause the EMT. Therefore, we demonstrated that also normal podocytes could spontaneously undergo EMT if grown in Ca(2+)-free medium, which is known to impair cell contacts. The analysis of the main intracellular signal transduction pathways evidenced some major anomalies consequent with the nephrin abrogation. The most intriguing was the activation of β-catenin pathway, which plays a critical role in podocyte ontogenesis as well as in the nephrin expression and EMT regulation. Also other important signaling proteins, like NF-κB, p53, and retinoblastoma protein (RB), showed important activity modifications. Interestingly, most of the above indicated signaling pathway alterations were again reproducible by cell junction rupture, induced by Ca(2+) deprivation. Finally, immunofluorescence analysis on kidney sections of patients with NS of Finnish type confirmed the constitutive expression of α-SMA. PMID:24392227

  9. Constitutional Nephrin Deficiency in Conditionally Immortalized Human Podocytes Induced Epithelial-Mesenchymal Transition, Supported by β-Catenin/NF-kappa B Activation: A Consequence of Cell Junction Impairment?

    PubMed Central

    Ghiggeri, Gian Marco

    2013-01-01

    The kidney glomerular podocytes are the cellular target of many chronic nephropathies both determined and acquired genetically. Mutations that affected the expression and/or the function of nephrin, a key component of the slit-diaphragm, are often causes of these pathologies. Recent findings showed that murine podocytes could undergo epithelial-mesenchymal transformation (EMT), suggesting new hypotheses about the pathogenesis of glomerular fibrosis. Here, we show that also human podocytes can undergo EMT, but more importantly nephrin ablation itself can trigger this phenotypic transformation. In fact, a model of human podocyte with engineered nephrin deficiency constitutionally expressed high levels of α-SMA, vimentin, fibronectin, and other hallmarks of EMT. Since it is known that cell contact abrogation is one of the triggers of EMT, we reasoned that nephrin loss could account for such cell junction disruption and cause the EMT. Therefore, we demonstrated that also normal podocytes could spontaneously undergo EMT if grown in Ca2+-free medium, which is known to impair cell contacts. The analysis of the main intracellular signal transduction pathways evidenced some major anomalies consequent with the nephrin abrogation. The most intriguing was the activation of β-catenin pathway, which plays a critical role in podocyte ontogenesis as well as in the nephrin expression and EMT regulation. Also other important signaling proteins, like NF-κB, p53, and retinoblastoma protein (RB), showed important activity modifications. Interestingly, most of the above indicated signaling pathway alterations were again reproducible by cell junction rupture, induced by Ca2+ deprivation. Finally, immunofluorescence analysis on kidney sections of patients with NS of Finnish type confirmed the constitutive expression of α-SMA. PMID:24392227

  10. Calcineurin inhibitors cyclosporin A and tacrolimus protect against podocyte injury induced by puromycin aminonucleoside in rodent models.

    PubMed

    Shen, Xiujin; Jiang, Hong; Ying, Meike; Xie, Zhoutao; Li, Xiayu; Wang, Haibing; Zhao, Jie; Lin, Chuan; Wang, Yucheng; Feng, Shi; Shen, Jia; Weng, Chunhua; Lin, Weiqiang; Wang, Huiping; Zhou, Qin; Bi, Yan; Li, Meng; Wang, Lingyan; Zhu, Tongyu; Huang, Xiaoru; Lan, Hui-Yao; Zhou, Jing; Chen, Jianghua

    2016-01-01

    Podocyte injury and the appearance of proteinuria are features of minimal-change disease (MCD). Cyclosporin A (CsA) and tacrolimus (FK506) has been reported to reduce proteinuria in patients with nephrotic syndrome, but mechanisms remain unknown. We, therefore, investigated the protective mechanisms of CsA and FK506 on proteinuria in a rat model of MCD induced by puromycin aminonucleoside (PAN) and in vitro cultured mouse podocytes. Our results showed that CsA and FK506 treatment decreased proteinuria via a mechanism associated to a reduction in the foot-process fusion and desmin, and a recovery of synaptopodin and podocin. In PAN-treated mouse podocytes, pre-incubation with CsA and FK506 restored the distribution of the actin cytoskeleton, increased the expression of synaptopodin and podocin, improved podocyte viability, and reduced the migrating activities of podocytes. Treatment with CsA and FK506 also inhibited PAN-induced podocytes apoptosis, which was associated with the induction of Bcl-xL and inhibition of Bax, cleaved caspase 3, and cleaved PARP expression. Further studies revealed that CsA and FK506 inhibited PAN-induced p38 and JNK signaling, thereby protecting podocytes from PAN-induced injury. In conclusion, CsA and FK506 inhibit proteinuria by protecting against PAN-induced podocyte injury, which may be associated with inhibition of the MAPK signaling pathway. PMID:27580845

  11. Calcineurin inhibitors cyclosporin A and tacrolimus protect against podocyte injury induced by puromycin aminonucleoside in rodent models

    PubMed Central

    Shen, Xiujin; Jiang, Hong; Ying, Meike; Xie, Zhoutao; Li, Xiayu; Wang, Haibing; Zhao, Jie; Lin, Chuan; Wang, Yucheng; Feng, Shi; Shen, Jia; Weng, Chunhua; Lin, Weiqiang; Wang, Huiping; Zhou, Qin; Bi, Yan; Li, Meng; Wang, Lingyan; Zhu, Tongyu; Huang, Xiaoru; Lan, Hui-Yao; Zhou, Jing; Chen, Jianghua

    2016-01-01

    Podocyte injury and the appearance of proteinuria are features of minimal-change disease (MCD). Cyclosporin A (CsA) and tacrolimus (FK506) has been reported to reduce proteinuria in patients with nephrotic syndrome, but mechanisms remain unknown. We, therefore, investigated the protective mechanisms of CsA and FK506 on proteinuria in a rat model of MCD induced by puromycin aminonucleoside (PAN) and in vitro cultured mouse podocytes. Our results showed that CsA and FK506 treatment decreased proteinuria via a mechanism associated to a reduction in the foot-process fusion and desmin, and a recovery of synaptopodin and podocin. In PAN-treated mouse podocytes, pre-incubation with CsA and FK506 restored the distribution of the actin cytoskeleton, increased the expression of synaptopodin and podocin, improved podocyte viability, and reduced the migrating activities of podocytes. Treatment with CsA and FK506 also inhibited PAN-induced podocytes apoptosis, which was associated with the induction of Bcl-xL and inhibition of Bax, cleaved caspase 3, and cleaved PARP expression. Further studies revealed that CsA and FK506 inhibited PAN-induced p38 and JNK signaling, thereby protecting podocytes from PAN-induced injury. In conclusion, CsA and FK506 inhibit proteinuria by protecting against PAN-induced podocyte injury, which may be associated with inhibition of the MAPK signaling pathway. PMID:27580845

  12. sPLA2 IB induces human podocyte apoptosis via the M-type phospholipase A2 receptor

    PubMed Central

    Pan, Yangbin; Wan, Jianxin; Liu, Yipeng; Yang, Qian; Liang, Wei; Singhal, Pravin C.; Saleem, Moin A.; Ding, Guohua

    2014-01-01

    The M-type phospholipase A2 receptor (PLA2R) is expressed in podocytes in human glomeruli. Group IB secretory phospholipase A2 (sPLA2 IB), which is one of the ligands of the PLA2R, is more highly expressed in chronic renal failure patients than in controls. However, the roles of the PLA2R and sPLA2 IB in the pathogenesis of glomerular diseases are unknown. In the present study, we found that more podocyte apoptosis occurs in the kidneys of patients with higher PLA2R and serum sPLA2 IB levels. In vitro, we demonstrated that human podocyte cells expressed the PLA2R in the cell membrane. After binding with the PLA2R, sPLA2 IB induced podocyte apoptosis in a time- and concentration-dependent manner. sPLA2 IB-induced podocyte PLA2R upregulation was not only associated with increased ERK1/2 and cPLA2α phosphorylation but also displayed enhanced apoptosis. In contrast, PLA2R-silenced human podocytes displayed attenuated apoptosis. sPLA2 IB enhanced podocyte arachidonic acid (AA) content in a dose-dependent manner. These data indicate that sPLA2 IB has the potential to induce human podocyte apoptosis via binding to the PLA2R. The sPLA2 IB-PLA2R interaction stimulated podocyte apoptosis through activating ERK1/2 and cPLA2α and through increasing the podocyte AA content. PMID:25335547

  13. Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress.

    PubMed

    Xu, S; Nam, S M; Kim, J-H; Das, R; Choi, S-K; Nguyen, T T; Quan, X; Choi, S J; Chung, C H; Lee, E Y; Lee, I-K; Wiederkehr, A; Wollheim, C B; Cha, S-K; Park, K-S

    2015-01-01

    Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca(2+) increase due to depletion of luminal ER Ca(2+). Palmitate-induced ER Ca(2+) depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca(2+) pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca(2+) depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca(2+) loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca(2+) depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA

  14. Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress

    PubMed Central

    Xu, S; Nam, S M; Kim, J-H; Das, R; Choi, S-K; Nguyen, T T; Quan, X; Choi, S J; Chung, C H; Lee, E Y; Lee, I-K; Wiederkehr, A; Wollheim, C B; Cha, S-K; Park, K-S

    2015-01-01

    Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca2+ increase due to depletion of luminal ER Ca2+. Palmitate-induced ER Ca2+ depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca2+ pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca2+ depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca2+ loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca2+ depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to

  15. Sialic acid attenuates puromycin aminonucleoside-induced desialylation and oxidative stress in human podocytes.

    PubMed

    Pawluczyk, Izabella Z A; Ghaderi Najafabadi, Maryam; Patel, Samita; Desai, Priyanka; Vashi, Dipti; Saleem, Moin A; Topham, Peter S

    2014-01-15

    Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx-crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid. PAN treatment of podocytes resulted in a loss of sialic acid from podocyte proteins. This was accompanied by a reduction, in the expression of sialyltransferases and a decrease in the key enzyme of sialic acid biosynthesis N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). PAN treatment also attenuated expression of the antioxidant enzyme superoxide dismutase (mSOD) and concomitantly increased the generation of superoxide anions. Sialic acid supplementation rescued podocyte protein sialylation and partially restored expression of sialyltransferases. Sialic acid also restored mSOD mRNA expression and quenched the oxidative burst. These data suggest that PAN-induced aberrant sialylation occurs as a result of modulation of enzymes involved sialic acid metabolism some of which are affected by oxidative stress. These data suggest that sialic acid therapy not only reinstates functionally important negative charge but also acts a source of antioxidant activity. PMID:24200502

  16. Sequential signaling cascade of IL-6 and PGC-1α is involved in high glucose-induced podocyte loss and growth arrest

    SciTech Connect

    Kim, Dong Il; Park, Soo Hyun

    2013-06-14

    Highlights: •The pathophysiological role of IL-6 in high glucose-induced podocyte loss. •The novel role of PGC-1α in the development of diabetic nephropathy. •Signaling of IL-6 and PGC-1α in high glucose-induced dysfunction of podocyte. -- Abstract: Podocyte loss, which is mediated by podocyte apoptosis, is implicated in the onset of diabetic nephropathy. In this study, we investigated the involvement of interleukin (IL)-6 in high glucose-induced apoptosis of rat podocytes. We also examined the pathophysiological role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in this system. High glucose treatment induced not only podocyte apoptosis but also podocyte growth arrest. High glucose treatment also increased IL-6 secretion and activated IL-6 signaling. The high glucose-induced podocyte apoptosis was blocked by IL-6 neutralizing antibody. IL-6 treatment or overexpression induced podocyte apoptosis and growth arrest, and IL-6 siRNA transfection blocked high glucose-induced podocyte apoptosis and growth arrest. Furthermore, high glucose or IL-6 treatment increased PGC-1α expression, and PGC-1α overexpression also induced podocyte apoptosis and growth arrest. PGC-1α siRNA transfection blocked high glucose-induced podocyte apoptosis and growth arrest. Collectively, these findings showed that high glucose promoted apoptosis and cell growth arrest in podocytes via IL-6 signaling. In addition, PGC-1α is involved in podocyte apoptosis and cell growth arrest. Therefore, blocking IL-6 and its downstream mediators such as IL6Rα, gp130 and PGC-1α may attenuate the progression of diabetic nephropathy.

  17. Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis.

    PubMed

    Lenoir, Olivia; Jasiek, Magali; Hénique, Carole; Guyonnet, Léa; Hartleben, Björn; Bork, Tillmann; Chipont, Anna; Flosseau, Kathleen; Bensaada, Imane; Schmitt, Alain; Massé, Jean-Marc; Souyri, Michèle; Huber, Tobias B; Tharaux, Pierre-Louis

    2015-01-01

    The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN. PMID:26039325

  18. Smad3 deficiency protects mice from obesity-induced podocyte injury that precedes insulin resistance.

    PubMed

    Sun, Yu B Y; Qu, Xinli; Howard, Victor; Dai, Lie; Jiang, Xiaoyun; Ren, Yi; Fu, Ping; Puelles, Victor G; Nikolic-Paterson, David J; Caruana, Georgina; Bertram, John F; Sleeman, Mark W; Li, Jinhua

    2015-08-01

    Signaling by TGF-β/Smad3 plays a key role in renal fibrosis. As obesity is one of the major risk factors of chronic and end-stage renal disease, we studied the role of Smad3 signaling in the pathogenesis of obesity-related renal disease. After switching to a high fat diet, the onset of Smad3 C-terminal phosphorylation, increase in albuminuria, and the early stages of peripheral and renal insulin resistance occurred at 1 day, and 4 and 8 weeks, respectively, in C57BL/6 mice. The loss of synaptopodin, a functional marker of podocytes, and phosphorylation of the Smad3 linker region (T179 and S213) appeared after 4 weeks of the high fat diet. This suggests a temporal pattern of Smad3 signaling activation leading to kidney injury and subsequent insulin resistance in the development of obesity-related renal disease. In vivo, Smad3 knockout attenuated the high fat diet-induced proteinuria, renal fibrosis, overall podocyte injury, and mitochondrial dysfunction in podocytes. In vitro palmitate caused a rapid activation of Smad3 in 30 min, loss of synaptopodin in 2 days, and impaired insulin signaling in 3 days in isolated mouse podocytes. Blockade of either Smad3 phosphorylation by SIS3 (a Smad3 inhibitor) or T179 phosphorylation by flavopiridol (a CDK9 inhibitor) prevented the palmitate-induced loss of synaptopodin and mitochondrial function in podocytes. Thus, Smad3 signaling plays essential roles in obesity-related renal disease and may be a novel therapeutic target. PMID:25945408

  19. Podocyte-Specific VEGF-A Gain of Function Induces Nodular Glomerulosclerosis in eNOS Null Mice

    PubMed Central

    Veron, Delma; Aggarwal, Pardeep K.; Velazquez, Heino; Kashgarian, Michael; Moeckel, Gilbert

    2014-01-01

    VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF164 gain of function in eNOS−/− mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF164 gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS−/− mice with podocyte-specific VEGF164 gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF164 gain of function decreased glomerular S-nitrosylation of laminin in eNOS−/− mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilson–like nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin. PMID:24578128

  20. Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization.

    PubMed

    Forst, Anna-Lena; Olteanu, Vlad Sorin; Mollet, Géraldine; Wlodkowski, Tanja; Schaefer, Franz; Dietrich, Alexander; Reiser, Jochen; Gudermann, Thomas; Mederos y Schnitzler, Michael; Storch, Ursula

    2016-03-01

    Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X4) blocker 5-BDBD. Moreover, mechanical P2X4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X4 channels as mechanotransducers in podocytes. PMID:26160898

  1. Protection of podocytes from hyperhomocysteinemia-induced injury by deletion of gp91phox gene

    PubMed Central

    Zhang, Chun; Hu, Jun-Jun; Xia, Min; Boini, Krishna M.; Brimson, Christopher A.; Laperle, Laura A.; Li, Pin-Lan

    2010-01-01

    In the present study, mice lacking gp91phox gene were used to address the role of NADPH oxidase in hyperhomocysteinemia-induced podocyte injury. It was found that the folate-free diet increased plasma homocysteine levels, but failed to increase O2.− production in the glomeruli from gp91phox gene knockout (gp91−/−) mice, compared with wild-type (gp91+/+) mice. Proteinuria and glomerular damage index (GDI) were significantly lower, while the glomerular filtration rate (GFR) was higher in gp91−/− than gp91+/+ mice when they were on the folate-free diet (Urine albumin excretion: 21.23±1.88 vs. 32.86±4.03 μg/24 h; GDI: 1.17±0.18 vs. 2.59 ±0.49; and GFR: 53.01±4.69 vs. 40.98±1.44 μL/min). Hyperhomocysteinemia-induced decrease in nephrin expression and increase in desmin expression in gp91+/+ mice were not observed in gp91−/− mice. Morphologically, foot process effacement and podocyte loss due to hyperhomocysteinemia were significantly attenuated in gp91−/− mice. In in vitro studies of podocytes, homocysteine was found to increase gp91phox expression and O2.− generation, which was substantially inhibited by gp91phox siRNA. Functionally, homocysteine-induced decrease in vascular endothelial growth factor-A (VEGF-A) production was abolished by gp91phox siRNA or diphenyleneiodonium, a NADPH oxidase inhibitor. These results suggest that the functional integrity of NADPH oxidase is essential for hyperhomocysteinemia-induced podocyte injury and glomerulosclerosis. PMID:20116427

  2. Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis

    SciTech Connect

    Dong, Chenglong; Zheng, Haining; Huang, Shanshan; You, Na; Xu, Jiarong; Ye, Xiaolong; Zhu, Qun; Feng, Yamin; You, Qiang; Miao, Heng; Ding, Dafa; Lu, Yibing

    2015-10-01

    Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12–24 h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy. - Highlights: • High glucose leads to increased autophagy in podocytes at an early stage. • The early autophagic response protects against high glucose-induced apoptosis. • Heme oxygenase-1 enhances autophagy and decreases high glucose -mediated apoptosis. • Heme oxygenase-1 induces autophagy through the activation of AMPK.

  3. Pterostilbene and allopurinol reduce fructose-induced podocyte oxidative stress and inflammation via microRNA-377.

    PubMed

    Wang, Wei; Ding, Xiao-Qin; Gu, Ting-Ting; Song, Lin; Li, Jian-Mei; Xue, Qiao-Chu; Kong, Ling-Dong

    2015-06-01

    High dietary fructose is an important causative factor in the development of metabolic syndrome-associated glomerular podocyte oxidative stress and injury. Here, we identified microRNA-377 (miR-377) as a biomarker of oxidative stress in renal cortex of fructose-fed rats, which correlated with podocyte injury and albuminuria in metabolic syndrome. Fructose feeding increased miR-377 expression, decreased superoxide dismutase (SOD) expression and activity, and caused O2(-) and H2O2 overproduction in kidney cortex or glomeruli of rats. This reactive oxygen species induction increased p38 MAPK phosphorylation and thioredoxin-interacting protein (TXNIP) expression and activated the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome to produce interleukin-1β in kidney glomeruli of fructose-fed rats. These pathological processes were further evaluated in cultured differentiated podocytes exposed to 5mM fructose, or transfected with miR-377 mimic/inhibitor and TXNIP siRNA, or co-incubated with p38 MAPK inhibitor, demonstrating that miR-377 overexpression activates the O2(-)/p38 MAPK/TXNIP/NLRP3 inflammasome pathway to promote oxidative stress and inflammation in fructose-induced podocyte injury. Antioxidants pterostilbene and allopurinol were found to ameliorate fructose-induced hyperuricemia, podocyte injury, and albuminuria in rats. More importantly, pterostilbene and allopurinol inhibited podocyte miR-377 overexpression to increase SOD1 and SOD2 levels and suppress the O2(-)/p38 MAPK/TXNIP/NLRP3 inflammasome pathway activation in vivo and in vitro, consistent with the reduction of oxidative stress and inflammation. These findings suggest that miR-377 plays an important role in glomerular podocyte oxidative stress, inflammation, and injury driven by high fructose. Inhibition of miR-377 by antioxidants may be a promising therapeutic strategy for the prevention of metabolic syndrome-associated glomerular podocyte injury. PMID:25746774

  4. MicroRNA-218 promotes high glucose-induced apoptosis in podocytes by targeting heme oxygenase-1.

    PubMed

    Yang, Haibo; Wang, Qingjun; Li, Sutong

    2016-03-18

    Emerging evidence has demonstrated that microRNAs (miRNAs) play a mediatory role in the pathogenesis of diabetic nephropathy. In this study, we found that miR-218 was upregulated in high glucose (HG) treated podocytes, which are essential components of the glomerular filtration barrier and a major prognostic determinant in diabetic nephropathy. Additionally, up-regulation of miR-218 was accompanied by an increased rate of podocyte death and down-regulation in the level of nephrin, a key marker of podocytes. However, inhibition of miR-218 exerted the opposite effect. In addition, the dual-luciferase reporter assay showed that miR-218 directly targeted the 3'-untranslated region of heme oxygenase-1 (HO-1), and further study confirmed an increase of HO-1 in HG-treated podocytes transfected with anti-miR-218. Knockdown of HO-1 blocked the anti-apoptotic effect of anti-miR-218. Furthermore, inhibition of miR-218 was associated with decreased expression of the known pro-apoptotic molecule p38-mitogen-activated protein kinase (p38-MAPK) activation. Following preconditioning with SB203580, an inhibitor of p38-MAPK, the stimulatory effect of HG on podocyte apoptosis was strikingly ameliorated. These findings suggested that miR-218 accelerated HG-induced podocyte apoptosis through directly down-regulating HO-1 and facilitating p38-MAPK activation. PMID:26876575

  5. Melanocortin 1 receptor agonist protects podocytes through catalase and RhoA activation.

    PubMed

    Elvin, Johannes; Buvall, Lisa; Lindskog Jonsson, Annika; Granqvist, Anna; Lassén, Emelie; Bergwall, Lovisa; Nyström, Jenny; Haraldsson, Börje

    2016-05-01

    Drugs containing adrenocorticotropic hormone have been used as therapy for patients with nephrotic syndrome. We have previously shown that adrenocorticotropic hormone and a selective agonist for the melanocortin 1 receptor (MC1R) exert beneficial actions in experimental membranous nephropathy with reduced proteinuria, reduced oxidative stress, and improved glomerular morphology and function. Our hypothesis is that MC1R activation in podocytes elicits beneficial effects by promoting stress fibers and maintaining podocyte viability. To test the hypothesis, we cultured podocytes and used highly specific agonists for MC1R. Podocytes were subjected to the nephrotic-inducing agent puromycin aminonucleoside, and downstream effects of MC1R activation on podocyte survival, antioxidant defense, and cytoskeleton dynamics were studied. To increase the response and enhance intracellular signals, podocytes were transduced to overexpress MC1R. We showed that puromycin promotes MC1R expression in podocytes and that activation of MC1R promotes an increase of catalase activity and reduces oxidative stress, which results in the dephosphorylation of p190RhoGAP and formation of stress fibers through RhoA. In addition, MC1R agonists protect against apoptosis. Together, these mechanisms protect the podocyte against puromycin. Our findings strongly support the hypothesis that selective MC1R-activating agonists protect podocytes and may therefore be useful to treat patients with nephrotic syndromes commonly considered as podocytopathies. PMID:26887829

  6. Shear stress induces cell apoptosis via a c-Src-phospholipase D-mTOR signaling pathway in cultured podocytes

    SciTech Connect

    Huang, Chunfa; Bruggeman, Leslie A.; Hydo, Lindsey M.; Miller, R. Tyler

    2012-06-10

    The glomerular capillary wall, composed of endothelial cells, the glomerular basement membrane and the podocytes, is continually subjected to hemodynamic force arising from tractional stress due to blood pressure and shear stress due to blood flow. Exposure of glomeruli to abnormal hemodynamic force such as hyperfiltration is associated with glomerular injury and progressive renal disease, and the conversion of mechanical stimuli to chemical signals in the regulation of the process is poorly understood in podocytes. By examining DNA fragmentation, apoptotic nuclear changes and cytochrome c release, we found that shear stress induced cell apoptosis in cultured podocytes. Meanwhile, podocytes exposed to shear stress also stimulated c-Src phosphorylation, phospholipase D (PLD) activation and mammalian target of rapamycin (mTOR) signaling. Using the antibodies against c-Src, PLD{sub 1}, and PLD{sub 2} to perform reciprocal co-immunoprecipitations and in vitro PLD activity assay, our data indicated that c-Src interacted with and activated PLD{sub 1} but not PLD{sub 2}. The inhibition of shear stress-induced c-Src phosphorylation by PP{sub 2} (a specific inhibitor of c-Src kinase) resulted in reduced PLD activity. Phosphatidic acid, produced by shear stress-induced PLD activation, stimulated mTOR signaling, and caused podocyte hypertrophy and apoptosis.

  7. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro.

    PubMed

    Lv, Zhimei; Hu, Mengsi; Ren, Xiaoxu; Fan, Minghua; Zhen, Junhui; Chen, Liqun; Lin, Jiangong; Ding, Nannan; Wang, Qun; Wang, Rong

    2016-01-01

    Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation. PMID:26881253

  8. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro

    PubMed Central

    Lv, Zhimei; Hu, Mengsi; Ren, Xiaoxu; Fan, Minghua; Zhen, Junhui; Chen, Liqun; Lin, Jiangong; Ding, Nannan; Wang, Qun; Wang, Rong

    2016-01-01

    Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation. PMID:26881253

  9. Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS

    PubMed Central

    Sun, Li-na; Liu, Xiang-chun; Chen, Xiang-jun; Guan, Guang-ju; Liu, Gang

    2016-01-01

    Aim: Caveolin-1 (cav-1) is a major multifunctional scaffolding protein of caveolae. Cav-1 is primarily expressed in mesangial cells, renal proximal tubule cells and podocytes in kidneys. Recent evidence shows that the functional connections between cav-1 and ROS play a key role in many diseases. In this study we investigated whether regulating the functional connections between cav-1 and ROS in kidneys contributed to the beneficial effects of curcumin in treating diabetic nephropathy in vitro and in vivo. Methods: Cultured mouse podocytes (mpc5) were incubated in a high glucose (HG, 30 mmol/L) medium for 24, 48 or 72 h. Male rats were injected with STZ (60 mg/kg, ip) to induce diabetes. ROS generation, SOD activity, MDA content and caspase-3 activity in the cultured cells and kidney cortex homogenate were determined. Apoptotic proteins and cav-1 phosphorylation were analyzed using Western blot analyses. Results: Incubation in HG-containing medium time-dependently increased ROS production, oxidative stress, apoptosis, and cav-1 phosphorylation in podocytes. Pretreatment with curcumin (1, 5, and 10 μmol/L) dose-dependently attenuated these abnormalities in HG-treated podocytes. Furthermore, in HG-containing medium, the podocytes transfected with a recombinant plasmid GFP-cav-1 Y14F (mutation at a cav-1 phosphorylation site) exhibited significantly decreased ROS production and apoptosis compared with the cells transfected with empty vector. In diabetic rats, administration of curcumin (100 or 200 mg/kg body weight per day, ig, for 8 weeks) not only significantly improved the renal function, but also suppressed ROS levels, oxidative stress, apoptosis and cav-1 phosphorylation in the kidneys. Conclusion: Curcumin attenuates high glucose-induced podocyte apoptosis in vitro and diabetic nephropathy in vivo partly through regulating the functional connections between cav-1 phosphorylation and ROS. PMID:26838071

  10. Angiotensin II down-regulates nephrin–Akt signaling and induces podocyte injury: roleof c-Abl

    PubMed Central

    Yang, Qian; Ma, Yiqiong; Liu, Yipeng; Liang, Wei; Chen, Xinghua; Ren, Zhilong; Wang, Huiming; Singhal, Pravin C.; Ding, Guohua

    2016-01-01

    Recent studies have shown that nephrin plays a vital role in angiotensin II (Ang II)–induced podocyte injury and thus contributes to the onset of proteinuria and the progression of renal diseases, but its specific mechanism remains unclear. c-Abl is an SH2/SH3 domain–containing nonreceptor tyrosine kinase that is involved in cell survival and regulation of the cytoskeleton. Phosphorylated nephrin is able to interact with molecules containing SH2/SH3 domains, suggesting that c-Abl may be a downstream molecule of nephrin signaling. Here we report that Ang II–infused rats developed proteinuria and podocyte damage accompanied by nephrin dephosphorylation and minimal interaction between nephrin and c-Abl. In vitro, Ang II induced podocyte injury and nephrin and Akt dephosphorylation, which occurred in tandem with minimal interaction between nephrin and c-Abl. Moreover, Ang II promoted c-Abl phosphorylation and interaction between c-Abl and SH2 domain–containing 5′-inositol phosphatase 2 (SHIP2). c-Abl small interfering RNA (siRNA) and STI571 (c-Abl inhibitor) provided protection against Ang II–induced podocyte injury, suppressed the Ang II-induced c-Abl–SHIP2 interaction and SHIP2 phosphorylation, and maintained a stable level of nephrin phosphorylation. These results indicate that c-Abl is a molecular chaperone of nephrin signaling and the SHIP2-Akt pathway and that the released c-Abl contributes to Ang II–induced podocyte injury. PMID:26510503

  11. Mice with mutant Inf2 show impaired podocyte and slit diaphragm integrity in response to protamine-induced kidney injury.

    PubMed

    Subramanian, Balajikarthick; Sun, Hua; Yan, Paul; Charoonratana, Victoria T; Higgs, Henry N; Wang, Fang; Lai, Ka-Man V; Valenzuela, David M; Brown, Elizabeth J; Schlöndorff, Johannes S; Pollak, Martin R

    2016-08-01

    Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury. PMID:27350175

  12. Ursodeoxycholic acid and 4-phenylbutyrate prevent endoplasmic reticulum stress-induced podocyte apoptosis in diabetic nephropathy.

    PubMed

    Cao, Ai-Li; Wang, Li; Chen, Xia; Wang, Yun-Man; Guo, Heng-Jiang; Chu, Shuang; Liu, Cheng; Zhang, Xue-Mei; Peng, Wen

    2016-06-01

    Endoplasmic reticulum (ER) stress, resulting from the accumulation of misfolded and/or unfolded proteins in ER membranes, is involved in the pathogenesis of diabetic nephropathy (DN). The aim of this study was to investigate the role of ER stress inhibitors ursodeoxycholic acid (UDCA) and 4-phenylbutyrate (4-PBA) in the treatment of DN in db/db mice. Findings have revealed that diabetic db/db mice were more hyperglycemic than their non-diabetic controls, and exhibited a marked increase in body weight, water intake, urine volume, fasting plasma glucose, systolic blood pressure, glucose and insulin tolerance. UDCA (40 mg/kg/day) or 4-PBA (100 mg/kg/day) treatment for 12 weeks resulted in an improvement in these biochemical and physical parameters. Moreover, UDCA or 4-PBA intervention markedly decreased urinary albuminuria and attenuated mesangial expansion in diabetic db/db mice, compared with db/db mice treated with vehicle. These beneficial effects of UDCA or 4-PBA on DN were associated with the inhibition of ER stress, as evidenced by the decreased expression of BiP, phospho-IRE1α, phospho-eIF2α, CHOP, ATF-6 and spliced X-box binding protein-1 in vitro and in vivo. UDCA or 4-PBA prevented hyperglycemia-induced or high glucose (HG)-induced apoptosis in podocytes in vivo and in vitro via the inhibition of caspase-3 and caspase-12 activation. Autophagy deficiency was also seen in glomeruli in diabetic mice and HG-incubated podocytes, exhibiting decreased expression of LC3B and Beclin-1, which could be restored by UDCA or 4-PBA treatment. Taken together, our results have revealed an important role of ER stress in the development of DN, and UDCA or 4-PBA treatment may be a potential novel therapeutic approach for the treatment of DN. PMID:26999661

  13. Phosphodiesterase 5 inhibition ameliorates angiontensin II-induced podocyte dysmotility via the protein kinase G-mediated downregulation of TRPC6 activity.

    PubMed

    Hall, Gentzon; Rowell, Janelle; Farinelli, Federica; Gbadegesin, Rasheed A; Lavin, Peter; Wu, Guanghong; Homstad, Alison; Malone, Andrew; Lindsey, Thomas; Jiang, Ruiji; Spurney, Robert; Tomaselli, Gordon F; Kass, David A; Winn, Michelle P

    2014-06-15

    The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr(69)/Thr(70) (Thr(69) in mice and Thr(70) in humans) and Ser(321)/Ser(322) (Ser(321) in mice and Ser(322) in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr(69) in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-dl-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3',4'-(methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr(69) dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease. PMID:24740790

  14. Reversal by Growth Hormone of Homocysteine-induced Epithelial-to-Mesenchymal Transition through Membrane Raft-Redox Signaling in Podocytes

    PubMed Central

    Li, Cai-Xia; Xia, Min; Han, Wei-Qing; Li, Xiao-Xue; Zhang, Chun; Boini, Krishna M.; Liu, Xiao-Cheng; Li, Pin-Lan

    2011-01-01

    Epithelial-to-Mesenchymal Transition (EMT) is an important pathogenic mechanism mediating glomerular injury or sclerosis in a variety of renal and systemic diseases such as hyperhomocysteinemia (hHcys). The present study was designed to test whether Hcys-induced EMT in podocytes is reversed by growth hormone (GH), a hormone regulating cell differentiation and growth and to explore the cellular and molecular mechanism mediating its action. It was found that Hcys induced significant EMT in podocytes, as shown by marked decreases in slit diaphragm-associated protein P-cadherin and zonula occludens-1 as epithelial markers and by dramatic increases in the expression of mesenchymal markers, fibroblast specific protein-1 and α-smooth muscle actin, which were detected by all examinations via immunocytochemistry, real time RT-PCR and Western blot analysis. When podocytes were treated with GH at 25 ng/mL, however, Hcys failed to induce podocyte EMT. Using electromagnetic spin resonance spectrometry, Hcys-induced superoxide (O2.−) production via NADPH oxidase was found to be significantly inhibited by GH (66%). Functionally, GH was shown to substantially inhibit Hcys-induced increases in the permeability of podocyte monolayers and to block the decrease in podocin expression in these cells. In addition, NADPH oxidase subunit, gp91phox and GH receptors aggregated in membrane raft clusters, which produced O2.− in response to Hcys and could be blocked by GH, membrane raft disruptors filipin and MCD or NADPH oxidase inhibitor, apocynin. It is concluded that Hcys-induced podocyte EMT is associated with transmembrane membrane raft-redox signaling and that GH reverses this Hcys-induced EMT protecting podocytes from functional disturbance. PMID:21691087

  15. Extract of Rhizoma Polygonum cuspidatum reduces early renal podocyte injury in streptozotocin‑induced diabetic rats and its active compound emodin inhibits methylglyoxal‑mediated glycation of proteins.

    PubMed

    Sohn, Eunjin; Kim, Junghyun; Kim, Chan Sik; Jo, Kyuhyung; Kim, Jin Sook

    2015-10-01

    Podocyte injury contributes to renal damage and, eventually, to the occurrence of proteinuria in diabetic nephropathy. The aim of the present study was to investigate the effect of an ethanol extract from Rhizoma Polygonum cuspidatum (P. cuspidatum) on proteinuria and podocyte injury, and elucidate the underlying mechanism for streptozotocin (STZ)‑induced diabetic nephropathy. The protective effects of P. cuspidatum extract (PCE) on renal podocytes in STZ‑induced diabetic rats were also investigated. PCE (100 or 350 mg/kg/day) was administered to STZ‑induced diabetic rats for 16 weeks, and blood glucose levels, body weight and proteinuria were measured. A double labeling technique with the terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed and synaptopodin expression was observed. In addition, cleaved caspase‑3, methylglyoxal (MGO) and 8‑hydroxydeoxyguanosine (8‑OHdG) expression levels were measured. STZ‑induced diabetic rats developed hyperglycemia and proteinuria. Increased apoptosis of the podocytes and increased cleaved caspase‑3, MGO and 8‑OHdG expression levels, as well as decreased synaptopodin expression were detected in the glomeruli of STZ‑induced diabetic rats. However, treatment with PCE for 16 weeks restored protein levels to normal, and reduced podocyte loss and apoptosis. Levels of caspase‑3 and MGO expression, as well as oxidative stress were ameliorated by PCE treatment. In addition, emodin, a biologically active ingredient of PCE, exerted an MGO scavenging effect and inhibited MGO‑derived advanced glycation end‑product formation. These findings indicate that PCE may be administered to prevent proteinuria and podocyte loss in STZ‑induced diabetic rats partly by inhibiting podocyte apoptosis and cleaved caspase‑3 expression, and by restoring the balance of oxidative stress and MGO expression. PMID:26299942

  16. Extract of Rhizoma Polygonum cuspidatum reduces early renal podocyte injury in streptozotocin-induced diabetic rats and its active compound emodin inhibits methylglyoxal-mediated glycation of proteins

    PubMed Central

    SOHN, EUNJIN; KIM, JUNGHYUN; KIM, CHAN SIK; JO, KYUHYUNG; KIM, JIN SOOK

    2015-01-01

    Podocyte injury contributes to renal damage and, eventually, to the occurrence of proteinuria in diabetic nephropathy. The aim of the present study was to investigate the effect of an ethanol extract from Rhizoma Polygonum cuspidatum (P. cuspidatum) on proteinuria and podocyte injury, and elucidate the underlying mechanism for streptozotocin (STZ)-induced diabetic nephropathy. The protective effects of P. cuspidatum extract (PCE) on renal podocytes in STZ-induced diabetic rats were also investigated. PCE (100 or 350 mg/kg/day) was administered to STZ-induced diabetic rats for 16 weeks, and blood glucose levels, body weight and proteinuria were measured. A double labeling technique with the terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed and synaptopodin expression was observed. In addition, cleaved caspase-3, methylglyoxal (MGO) and 8-hydroxydeoxyguanosine (8-OHdG) expression levels were measured. STZ-induced diabetic rats developed hyperglycemia and proteinuria. Increased apoptosis of the podocytes and increased cleaved caspase-3, MGO and 8-OHdG expression levels, as well as decreased synaptopodin expression were detected in the glomeruli of STZ-induced diabetic rats. However, treatment with PCE for 16 weeks restored protein levels to normal, and reduced podocyte loss and apoptosis. Levels of caspase-3 and MGO expression, as well as oxidative stress were ameliorated by PCE treatment. In addition, emodin, a biologically active ingredient of PCE, exerted an MGO scavenging effect and inhibited MGO-derived advanced glycation end-product formation. These findings indicate that PCE may be administered to prevent proteinuria and podocyte loss in STZ-induced diabetic rats partly by inhibiting podocyte apoptosis and cleaved caspase-3 expression, and by restoring the balance of oxidative stress and MGO expression. PMID:26299942

  17. Lipopolysaccharide induces inducible nitric oxide synthase-dependent podocyte dysfunction via a hypoxia-inducible factor 1α and cell division control protein 42 and Ras-related C3 botulinum toxin substrate 1 pathway

    PubMed Central

    Mashmoushi, Ahmad K.; Oates, Jim C.

    2015-01-01

    Urine protein loss in immune complex-mediated diseases such as lupus nephritis is associated with podocyte foot process effacement (podocytopathy) but is not always dependent on glomerular immune complex deposition. Several murine and human studies have associated lupus nephritis with inducible nitric oxide synthase (iNOS) expression in what appear to be podocytes. This study was conducted to determine mechanisms of immune-complex-independent and iNOS-dependent podocyte dysfunction. Conditionally immortalized podocytes were cultured with lipopolysaccharide (LPS) and nitric oxide (NO), superoxide (SO), or peroxynitrite donors in the presence or absence of inhibitors of iNOS, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or monocyte chemotactic protein 1 (MCP-1), or with sepiapterin to increase coupling of iNOS homodimers. Podocyte NO, SO, and MCP-1 production and nitrotyrosine modifications were determined. The podocytopathy phenotype was determined by measuring cell motility and membrane permeability to albumin. This study determined that NO produced by iNOS is sufficient and necessary to induce podocytopathy. NO probably induces this phenotype via hypoxia-inducible factor 1α and cell division control protein 42 and Ras-related C3 botulinum toxin substrate 1 pathways. With LPS stimulation, neither SO nor peroxynitrite produced by uncoupled iNOS or NADPH oxidase nor MCP-1 was sufficient to induce the full phenotype. This study supports the notion that iNOS may induce autocrine podocyte dysfunction. Thus, targeting iNOS or the pathways of its induction may have therapeutic benefit. PMID:25765888

  18. Protective Action of Anandamide and Its COX-2 Metabolite against l-Homocysteine-Induced NLRP3 Inflammasome Activation and Injury in Podocytes.

    PubMed

    Li, Guangbi; Xia, Min; Abais, Justine M; Boini, Krishna; Li, Pin-Lan; Ritter, Joseph K

    2016-07-01

    Recent studies have demonstrated that l-homocysteine (Hcys)-induced podocyte injury leading to glomerular damage or sclerosis is attributable to the activation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome. Given the demonstrated anti-inflammatory effects of endocannabinoids, the present study was designed to test whether anandamide (AEA) or its metabolites diminish NLRP3 inflammasome activation and prevent podocyte injury and associated glomerular damage during hyperhomocysteinemia (hHcys). AEA (100 μM) inhibited Hcys-induced NLRP3 inflammasome activation in cultured podocytes, as indicated by elevated caspase-1 activity and interleukin-1β levels, and attenuated podocyte dysfunction, as shown by reduced vascular endothelial growth factor production. These effects of AEA were inhibited by the cyclooxygenase-2 (COX-2) inhibitor celecoxib (CEL). In mice in vivo, AEA treatment attenuated glomerular NLRP3 inflammasome activation induced by hHcys accompanying a folate-free diet, on the basis of inhibition of hHcys-induced colocalization of NLRP3 molecules and increased interleukin-1β levels in glomeruli. Correspondingly, AEA prevented hHcys-induced proteinuria, albuminuria, and glomerular damage observed microscopically. Hcys- and AEA-induced effects were absent in NLRP3-knockout mice. These beneficial effects of AEA against hHcys-induced NLRP3 inflammasome activation and glomerular injury were not observed in mice cotreated with CEL. We further demonstrated that prostaglandin E2-ethanolamide (PGE2-EA), a COX-2 product of AEA, at 10 μM had a similar inhibitory effect to that of 100 μM AEA on Hcys-induced NLRP3 inflammasome formation and activation in cultured podocytes. From these results, we conclude that AEA has anti-inflammatory properties, protecting podocytes from Hcys-induced injury by inhibition of NLRP3 inflammasome activation through its COX-2 metabolite, PGE2-EA. PMID:27189966

  19. Tadalafil Integrates Nitric Oxide-Hydrogen Sulfide Signaling to Inhibit High Glucose-induced Matrix Protein Synthesis in Podocytes*

    PubMed Central

    Lee, Hak Joo; Feliers, Denis; Mariappan, Meenalakshmi M.; Sataranatarajan, Kavithalakshmi; Choudhury, Goutam Ghosh; Gorin, Yves; Kasinath, Balakuntalam S.

    2015-01-01

    Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H2S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H2S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase β. Tadalafil rapidly increased the expression and activity of the H2S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H2S and nitric oxide (NO). Nω-Nitro-l-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H2S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1

  20. Vascular Smooth Muscle Cells Contribute to APOL1-induced Podocyte Injury in HIV milieu

    PubMed Central

    Lan, Xiqian; Wen, Hongxiu; Saleem, Moin A.; Mikulak, Joanna; Malhotra, Ashwani; Skorecki, Karl; Singhal, Pravin C

    2015-01-01

    Clinical reports have demonstrated that higher rates of non-diabetic glomerulosclerosis in African Americans can be attributed to two coding sequence variants (G1 and G2) in the APOL1 gene; however, the underlying mechanism is still unknown. Kidney biopsy data suggest enhanced expression of APOL1/APOL1 variants (Vs) in smooth muscle cells (SMCs) of renal vasculature. Since APOL1 is a secretory protein of relatively low molecular weight (41 kDa), SMCs may be a contributory endocrine/paracrine source of APOL1 wild type (WT)/APOL1Vs in the glomerular capillary perfusate percolating podocytes. In the present study, we tested the hypothesis that an HIV milieu stimulated secretion of APOL1 and its risk variants by arterial SMCs contributes to podocyte injury. Human umbilical artery smooth muscle cells (HSMCs)-treated with conditioned media (CM) of HIV-infected peripheral mononuclear cells (PBMC/HIV-CM), CM of HIV-infected U939 cells, or recombinant IFN-γ displayed enhanced expression of APOL1. Podocytes co-cultured in trans-wells with HSMCs-over expressing APOL1WT showed induction of injury; however, podocytes co-cultured with HSMC-over expressing either APOL1G1 or APOL1G2 showed several fold greater injury when compared to HSMC- over expressing APOL1WT. Conditioned media collected from HSMC-over-expressing APOL1G1/APOL1G2 (HSMC/APOL1G1-CM or HSMC/APOL1G2-CM) also displayed higher percentages of injured podocytes in the form of swollen cells, leaky lysosomes, loss of viability, and enhanced sensitivity to adverse host factors when compared to HSMC/APOL1WT-CM. Notably, HSMC/APOL1WT-CM promoted podocyte injury only at a significantly higher concentrations compared to HSMC/APOL1G1/G2-CM. We conclude that HSMCs could serve as an endocrine/paracrine source of APOL1Vs, which mediate accelerated podocyte injury in HIV milieu. PMID:25796344

  1. Inhibition of p66ShcA Longevity Gene Rescues Podocytes from HIV-1-induced Oxidative Stress and Apoptosis*

    PubMed Central

    Husain, Mohammad; Meggs, Leonard G.; Vashistha, Himanshu; Simoes, Sonia; Griffiths, Kevin O.; Kumar, Dileep; Mikulak, Joanna; Mathieson, Peter W.; Saleem, Moin A.; Del Valle, Luis; Pina-Oviedo, Sergio; Wang, Jin Ying; Seshan, Surya V.; Malhotra, Ashwani; Reiss, Krzysztof; Singhal, Pravin C.

    2009-01-01

    Glomerular visceral epithelial cells (podocytes) play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated nephropathy. A key question concerns the mechanism(s) by which the HIV-1 genome alters the phenotype of the highly specialized, terminally differentiated podocytes. Here, using an in vitro system of conditionally immortalized differentiated human podocytes (CIDHPs), we document a pivotal role for the p66ShcA protein in HIV-1-induced reactive oxygen species generation and CIDHP apoptosis. CIDHP transfected with truncated HIV-1 construct (NL4-3) exhibit increased reactive oxygen species metabolism, DNA strand breaks, and a 5-fold increase in apoptosis, whereas the opposite was true for NL4-3/CIDHP co-transfected with mu-36p66ShcA (mu-36) dominant negative expression vector or isoform-specific p66-small interfering RNA. Phosphorylation at Ser-36 of the wild type p66ShcA protein, required for p66ShcA redox function and inhibition of the potent stress response regulator Foxo3a, was unchanged in mu-36/NL4-3/CIDHP but increased in NL4-3/CIDHP. Acute knockdown of Foxo3a by small interfering RNA induced a 50% increase in mu-36/NL4-3/CIDHP apoptosis, indicating that Foxo3a-dependent responses promote the survival phenotype in mu-36 cells. We conclude that inhibition of p66ShcA redox activity prevents generation of HIV-1 stress signals and activation of the CIDHP apoptosis program. PMID:19383602

  2. Twisted Gastrulation, a BMP Antagonist, Exacerbates Podocyte Injury

    PubMed Central

    Yamada, Sachiko; Nakamura, Jin; Asada, Misako; Takase, Masayuki; Matsusaka, Taiji; Iguchi, Taku; Yamada, Ryo; Tanaka, Mari; Higashi, Atsuko Y.; Okuda, Tomohiko; Asada, Nariaki; Fukatsu, Atsushi; Kawachi, Hiroshi; Graf, Daniel; Muso, Eri; Kita, Toru; Kimura, Takeshi; Pastan, Ira; Economides, Aris N.; Yanagita, Motoko

    2014-01-01

    Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7) in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1), a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs) and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury. PMID:24586548

  3. The Expression Profile of Complement Components in Podocytes.

    PubMed

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  4. The Expression Profile of Complement Components in Podocytes

    PubMed Central

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  5. 3,4-DGE is cytotoxic and decreases HSP27/HSPB1 in podocytes.

    PubMed

    Sanchez-Niño, Maria Dolores; Poveda, Jonay; Sanz, Ana Belen; Carrasco, Susana; Ruiz-Ortega, Marta; Selgas, Rafael; Egido, Jesus; Ortiz, Alberto

    2014-03-01

    Hyperglycemia is the key driver of diabetic complications and increased concentrations of glucose degradation products. The study of peritoneal dialysis solution biocompatibility has highlighted the adverse biological effects of glucose degradation products. Recently, 3,4-dideoxyglucosone-3-ene (3,4-DGE) was identified as the most toxic glucose degradation product in peritoneal dialysis fluids. In addition, 3,4-DGE is present in high-fructose corn syrup, and its precursor 3-deoxyglucosone is increased in diabetes. The role of 3,4-DGE in glomerular injury had not been addressed. We studied the effects of 3,4-DGE on cultured human podocytes and in vivo in mice. 3,4-DGE induced apoptosis in podocytes in a dose- and time-dependent manner. 3,4-DGE promoted the release of cytochrome c from mitochondria and activation of caspase-3. While high glucose concentrations increased the levels of the podocyte intracellular antiapoptotic protein HSP27/HSPB1, 3,4-DGE decreased the expression of podocyte HSP27/HSPB1. Apoptosis induced by 3,4-DGE was caspase-dependent and could be prevented by the broad-spectrum caspase inhibitor zVAD-fmk. Antagonism of Bax by a Ku-70-derived peptide also prevented apoptosis. Intravenous administration of 3,4-DGE to healthy mice resulted in a decreased expression of HSP27/HSPB1 and caspase-3 activation in whole kidney and in podocytes in vivo. In conclusion, 3,4-DGE induces apoptotic cell death in cultured human podocytes, suggesting a potential role in glomerular injury resulting from metabolic disorders. PMID:24337777

  6. MicroRNA-30 family members regulate calcium/calcineurin signaling in podocytes

    PubMed Central

    Wu, Junnan; Zheng, Chunxia; Wang, Xiao; Yun, Shifeng; Zhao, Yue; Liu, Lin; Lu, Yuqiu; Ye, Yuting; Zhu, Xiaodong; Zhang, Changming; Shi, Shaolin; Liu, Zhihong

    2015-01-01

    Calcium/calcineurin signaling is critical for normal cellular physiology. Abnormalities in this pathway cause many diseases, including podocytopathy; therefore, understanding the mechanisms that underlie the regulation of calcium/calcineurin signaling is essential. Here, we showed that critical components of calcium/calcineurin signaling, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, are the targets of the microRNA-30 family (miR-30s). We found that these 5 genes are highly expressed as mRNA, but the level of the proteins is low in normal podocytes. Conversely, protein levels were markedly elevated in podocytes from rats treated with puromycin aminonucleoside (PAN) and from patients with focal segmental glomerulosclerosis (FSGS). In both FSGS patients and PAN-treated rats, miR-30s were downregulated in podocytes. In cultured podocytes, PAN or a miR-30 sponge increased TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 expression; calcium influx; intracellular Ca2+ concentration; and calcineurin activity. Moreover, NFATC3 nuclear translocation, synaptopodin degradation, integrin β3 (ITGB3) activation, and actin fiber loss, which are downstream of calcium/calcineurin signaling, were induced by miR-30 reduction but blocked by the calcineurin inhibitor FK506. Podocyte-specific expression of the miR-30 sponge in mice increased calcium/calcineurin pathway component protein expression and calcineurin activity. The mice developed podocyte foot process effacement and proteinuria, which were prevented by FK506. miR-30s also regulated calcium/calcineurin signaling in cardiomyocytes. Together, our results identify miR-30s as essential regulators of calcium/calcineurin signaling. PMID:26436650

  7. HIV-1 expression induces cyclin D1 expression and pRb phosphorylation in infected podocytes: cell-cycle mechanisms contributing to the proliferative phenotype in HIV-associated nephropathy

    PubMed Central

    Nelson, Peter J; Sunamoto, Masaaki; Husain, Mohammad; Gelman, Irwin H

    2002-01-01

    Background The aberrant cell-cycle progression of HIV-1-infected kidney cells plays a major role in the pathogenesis of HIV-associated nephropathy, however the mechanisms whereby HIV-1 induces infected glomerular podocytes or infected tubular epithelium to exit quiescence are largely unknown. Here, we ask whether the expression of HIV-1 genes in infected podocytes induces cyclin D1 and phospho-pRb (Ser780) expression, hallmarks of cyclin D1-mediated G1 → S phase progression. Results We assessed cyclin D1 and phospho-pRb (Ser780) expression in two well-characterized models of HIV-associated nephropathy pathogenesis: HIV-1 infection of cultured podocytes and HIV-1 transgenic mice (Tg26). Compared to controls, cultured podocytes expressing HIV-1 genes, and podocytes and tubular epithelium from hyperplastic nephrons in Tg26 kidneys, had increased levels of phospho-pRb (Ser780), a target of active cyclin D1/cyclin-dependent kinase-4/6 known to promote G1 → S phase progression. HIV-1-infected podocytes showed markedly elevated cyclin D1 mRNA and cyclin D1 protein, the latter of which did not down-regulate during cell-cell contact or differentiation, suggesting post-transcriptional stabilization of cyclin D1 protein levels by HIV-1. The selective suppression of HIV-1 transcription by the cyclin-dependent kinase inhibitor, flavopiridol, abrogated cyclin D1 expression, underlying the requirement for HIV-1 encoded products to induce cyclin D1. Indeed, HIV-1 virus deleted of nef failed to induce cyclin D1 mRNA to the level of other single gene mutant viruses. Conclusions HIV-1 expression induces cyclin D1 and phospho-pRb (Ser780) expression in infected podocytes, suggesting that HIV-1 activates cyclin D1-dependent cell-cycle mechanisms to promote proliferation of infected renal epithelium. PMID:12241561

  8. Cell Biology and Pathology of Podocytes

    PubMed Central

    Greka, Anna; Mundel, Peter

    2013-01-01

    As an integral member of the filtration barrier in the kidney glomerulus, the podocyte is in a unique geographical position: It is exposed to chemical signals from the urinary space (Bowman’s capsule), it receives and transmits chemical and mechanical signals to/from the glomerular basement membrane upon which it elaborates, and it receives chemical and mechanical signals from the vascular space with which it also communicates. As with every cell, the ability of the podocyte to receive signals from the surrounding environment and to translate them to the intracellular milieu is dependent largely on molecules residing on the cell membrane. These molecules are the first-line soldiers in the ongoing battle to sense the environment, to respond to friendly signals, and to defend against injurious foes. In this review, we take a membrane biologist’s view of the podocyte, examining the many membrane receptors, channels, and other signaling molecules that have been implicated in podocyte biology. Although we attempt to be comprehensive, our goal is not to capture every membrane-mediated pathway but rather to emphasize that this approach may be fruitful in understanding the podocyte and its unique properties. PMID:22054238

  9. GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway

    PubMed Central

    Chen, Z; Wan, X; Hou, Q; Shi, S; Wang, L; Chen, P; Zhu, X; Zeng, C; Qin, W; Zhou, W; Liu, Z

    2016-01-01

    GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases. PMID:26794661

  10. The two kidney to one kidney transition and transplant glomerulopathy: a podocyte perspective.

    PubMed

    Yang, Yan; Hodgin, Jeffrey B; Afshinnia, Farsad; Wang, Su Q; Wickman, Larysa; Chowdhury, Mahboob; Nishizono, Ryuzoh; Kikuchi, Masao; Huang, Yihung; Samaniego, Milagros; Wiggins, Roger C

    2015-06-01

    The attrition rate of functioning allografts beyond the first year has not improved despite improved immunosuppression, suggesting that nonimmune mechanisms could be involved. Notably, glomerulopathies may account for about 40% of failed kidney allografts beyond the first year of engraftment, and glomerulosclerosis and progression to ESRD are caused by podocyte depletion. Model systems demonstrate that nephrectomy can precipitate hypertrophic podocyte stress that triggers progressive podocyte depletion leading to ESRD, and that this process is accompanied by accelerated podocyte detachment that can be measured in urine. Here, we show that kidney transplantation "reverse nephrectomy" is also associated with podocyte hypertrophy and increased podocyte detachment. Patients with stable normal allograft function and no proteinuria had levels of podocyte detachment similar to levels in two-kidney controls as measured by urine podocyte assay. By contrast, patients who developed transplant glomerulopathy had 10- to 20-fold increased levels of podocyte detachment. Morphometric studies showed that a subset of these patients developed reduced glomerular podocyte density within 2 years of transplantation due to reduced podocyte number per glomerulus. A second subset developed glomerulopathy by an average of 10 years after transplantation due to reduced glomerular podocyte number and glomerular tuft enlargement. Reduced podocyte density was associated with reduced eGFR, glomerulosclerosis, and proteinuria. These data are compatible with the hypothesis that podocyte depletion contributes to allograft failure and reduced allograft half-life. Mechanisms may include immune-driven processes affecting the podocyte or other cells and/or hypertrophy-induced podocyte stress causing accelerated podocyte detachment, which would be amenable to nonimmune therapeutic targeting. PMID:25388223

  11. Podocyte Mitosis – A Catastrophe

    PubMed Central

    Lasagni, L; Lazzeri, E; Shankland, S.J; Anders, H.-J; Romagnani, P

    2013-01-01

    Podocyte loss plays a key role in the progression of glomerular disorders towards glomerulosclerosis and chronic kidney disease. Podocytes form unique cytoplasmic extensions, foot processes, which attach to the outer surface of the glomerular basement membrane and interdigitate with neighboring podocytes to form the slit diaphragm. Maintaining these sophisticated structural elements requires an intricate actin cytoskeleton. Genetic, mechanic, and immunologic or toxic forms of podocyte injury can cause podocyte loss, which causes glomerular filtration barrier dysfunction, leading to proteinuria. Cell migration and cell division are two processes that require a rearrangement of the actin cytoskeleton; this rearrangement would disrupt the podocyte foot processes, therefore, podocytes have a limited capacity to divide or migrate. Indeed, all cells need to rearrange their actin cytoskeleton to assemble a correct mitotic spindle and to complete mitosis. Podocytes, even when being forced to bypass cell cycle checkpoints to initiate DNA synthesis and chromosome segregation, cannot complete cytokinesis efficiently and thus usually generate aneuploid podocytes. Such aneuploid podocytes rapidly detach and die, a process referred to as mitotic catastrophe. Thus, detached or dead podocytes cannot be adequately replaced by the proliferation of adjacent podocytes. However, even glomerular disorders with severe podocyte injury can undergo regression and remission, suggesting alternative mechanisms to compensate for podocyte loss, such as podocyte hypertrophy or podocyte regeneration from resident renal progenitor cells. Together, mitosis of the terminally differentiated podocyte rather accelerates podocyte loss and therefore glomerulosclerosis. Finding ways to enhance podocyte regeneration from other sources remains a challenge goal to improve the treatment of chronic kidney disease in the future. PMID:23176147

  12. Characterization of prostanoid receptors in podocytes.

    PubMed

    Bek, M; Nüsing, R; Kowark, P; Henger, A; Mundel, P; Pavenstädt, H

    1999-10-01

    Prostaglandins participate in the regulation of important glomerular functions and are involved in the pathogenesis of glomerular diseases. This study investigates the influence of prostaglandins on membrane voltage, ion conductances, cAMP accumulation, and cytosolic calcium activity ([Ca2+]i) in differentiated podocytes. Prostaglandin E2 (PGE2) caused a concentration-dependent depolarization and an increase of the whole cell conductance in podocytes (EC50 approximately 50 nM). Compared with PGE2, the EP2/EP3/EP4 receptor agonist 11-deoxy-PGE1 caused an equipotent depolarization, whereas the DP receptor agonist BW 245 C, the EP1/EP3 receptor agonist sulprostone, and the IP receptor agonist iloprost were at least 100 to 1000 times less potent than PGE2. The EP2 receptor agonist butaprost did not change membrane voltage of podocytes. The depolarizing effect of PGE2 was increased in an extracellular solution with a reduced Cl- concentration (from 145 to 32 mM). PGE2 and the prostaglandin agonists, but not the IP receptor agonist iloprost and the EP2 receptor agonist butaprost, induced a time- and concentration-dependent cAMP accumulation in podocytes. In fura-2 fluorescence experiments, PGE2, sulprostone, PGF2alpha, fluprostenol (a potent FP agonist), and U-46619 (a selective thromboxane A2 agonist) induced a biphasic increase of [Ca2+]i in 60 to 80% of podocytes. In reverse transcription-PCR studies, podocyte mRNA for the EP1, EP4, FP, and TP receptor could be amplified. These data indicate that in podocytes, PGE2 regulates distinct cellular functions via the EP1 and EP4 receptor, thereby increasing [Ca2+]i and cAMP, respectively. Furthermore, PGF1alpha and U-46619 increase [Ca2+]i via their specific receptors. PMID:10505684

  13. Apoptotic cell death induced by intracellular proteolysis.

    PubMed

    Williams, M S; Henkart, P A

    1994-11-01

    To mimic the injection of granzymes into target cells by cytotoxic lymphocytes or the activation of endogenous proteases in programmed cell death, the proteases chymotrypsin, proteinase K, or trypsin were loaded into the cytoplasm of several different cell types using the osmotic lysis of pinosomes technique. Internalization of these proteases caused cell lysis within several hours, accompanied by extensive nuclear damage in most but not all combinations of target cells and proteases. This nuclear damage, quantitated by DNA release from nuclei, was associated with apoptotic features including DNA fragmentation into nucleosomal ladders, chromatin condensation, nuclear fragmentation, and membrane blebbing. Agents reported to block programmed cell death, including aurintricarboxylic acid, inhibitors of energy metabolism, and protein or RNA synthesis, failed to block this protease-induced death, although some inhibited nuclear damage. In separate experiments, introduction of staphylococcal nuclease into cells led to near complete (at least 75% of total) nucleosomal DNA fragmentation within 6 to 8 h. Condensation of chromatin did not accompany this fragmentation to the same extent, and there was approximately a 10-h lag between half-maximal DNA fragmentation and 50% loss of membrane integrity. The results suggest that activation of intracellular proteases during cell death by any molecular pathway could give rise to apoptotic morphology and DNA fragmentation. PMID:7930626

  14. Vesicular demyelination induced by raised intracellular calcium.

    PubMed

    Smith, K J; Hall, S M; Schauf, C L

    1985-11-01

    Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 microM, 10 micrograms/ml) or low (1-1.5 microM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15-20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca2+, and could be modulated in severity by varying this concentration. Other divalent cations (Ba2+, Co2+, Mg2+, Mn2+, Ni2+, Sr2+) could not substitute for Ca2+. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn2+ (greater than or equal to 1 microM), Ni2+ (greater than or equal to 1-10 microM), Co2+ (greater than or equal to 100 microM) or Mn2+ (greater than or equal to 100 microM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (greater than or equal to 1 microM), an ionophore with a greater selectivity for Ca2+ than A23187, also induced a prompt Ca2+-dependent

  15. Podocyte injury and its consequences.

    PubMed

    Nagata, Michio

    2016-06-01

    Podocytes maintain the glomerular filtration barrier, and the stability of this barrier depends on their highly differentiated postmitotic phenotype, which also defines the particular vulnerability of the glomerulus. Recent podocyte biology and gene disruption studies in vivo indicate a causal relationship between abnormalities of single podocyte molecules and proteinuria and glomerulosclerosis. Podocytes live under various stresses and pathological stimuli. They adapt to maintain homeostasis, but excessive stress leads to maladaptation with complex biological changes including loss of integrity and dysregulation of cellular metabolism. Podocyte injury causes proteinuria and detachment from the glomerular basement membrane. In addition to "sick" podocytes and their detachment, our understanding of glomerular responses following podocyte loss needs to address the pathways from podocyte injury to sclerosis. Studies have found a variety of glomerular responses to podocyte dysfunction in vivo, such as disruption of podocyte-endothelial cross talk and activation of podocyte-parietal cell interactions, all of which help us to understand the complex scenario of podocyte injury and its consequences. This review focuses on the cellular aspects of podocyte dysfunction and the adaptive or maladaptive glomerular responses to podocyte injury that lead to its major consequence, glomerulosclerosis. PMID:27165817

  16. Sesquiterpene Lactones Inhibit Advanced Oxidation Protein Product-Induced MCP-1 Expression in Podocytes via an IKK/NF-κB-Dependent Mechanism

    PubMed Central

    Zhao, Yan; Chen, Si-jia; Wang, Jian-cheng; Niu, Hong-xin; Jia, Qian-qian; Chen, Xiao-wen; Du, Xiao-yan; Lu, Lu; Huang, Bo; Zhang, Quan; Chen, Yue; Long, Hai-bo

    2015-01-01

    Inflammation is a relevant factor in the pathogenesis of diabetes nephropathy (DN). Sesquiterpene lactones (SLs), originally isolated from Tanacetum parthenium, have been reported to exhibit anti-inflammatory effects but few studies have examined their effects on DN. To determine whether advanced oxidation protein products (AOPPs) can induce the expression of chemokine monocyte chemoattractant protein- (MCP-) 1 in cultured mouse podocytes and to explore the mechanisms of the potential renoprotection of SLs, we treated podocytes with AOPPs and SLs (parthenolide and its derivatives micheliolide, compound 1, and compound 2). MCP-1 mRNA and protein expression were tested using quantitative real-time PCR and ELISA, respectively, and the protein levels of IKKβ, phospho-IKKβ, IκBα, NF-κB p65, phospho-NF-κB p65, and tubulin were analyzed by Western blotting. AOPPs activated the expression of MCP-1 mRNA and protein in a dose- and time-dependent manner, activated IKKβ and NF-κB p65, and promoted IκBα degradation. The IKK/NF-κB inhibitor parthenolide decreased AOPP-induced MCP-1 expression. Pretreatment with SLs inhibited MCP-1 mRNA and protein expression and suppressed IKKβ and NF-κB p65 phosphorylation and IκBα degradation. Taken together, these findings provide a novel explanation for the anti-inflammatory effects of SLs that will ultimately benefit DN and potentially other inflammatory and immune renal diseases. PMID:25664142

  17. Progenitor Cells and Podocyte Regeneration

    PubMed Central

    Shankland, Stuart J.; Pippin, Jeffrey W.; Duffield, Jeremy S.

    2014-01-01

    The very limited ability of adult podocytes to proliferate in vivo is clinically significant because: podocytes form a vascular barrier which is functionally critical to the nephron; podocyte hypoplasia is a characteristic of disease; and inadequate regeneration of podocytes is a major cause of persistent podocyte hypoplasia. Excessive podocyte loss or inadequate replacement leads to glomerulosclerosis in many progressive kidney diseases. Thus, restoration of podocyte cell density is almost certainly reliant on regeneration by podocyte progenitors. However such putative progenitors have remained elusive until recently. In this review we describe the developmental processes leading to podocyte and parietal epithelial cell (PEC) formation during glomerulogenesis. We compare evidence that in normal human kidneys PECs expressing ‘progenitor’ markers CD133 and CD24 can differentiate into podocytes in vitro and in vivo with evidence from animal models suggesting a more limited role of PEC-capacity to serve as podocyte progenitors in adults. We will highlight tantalizing new evidence that specialized vascular wall cells of afferent arterioles including those which produce renin in healthy kidney, provide a novel local progenitor source of new PECs and podocytes in response to podocyte hypoplasia in the adult, and draw comparisons with glomerulogenesis. PMID:25217270

  18. High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes.

    PubMed

    Thilo, Florian; Lee, Marlene; Xia, Shengqiang; Zakrzewicz, Andreas; Tepel, Martin

    2014-07-18

    Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxidative stress and syndecan-4 (SDC-4) in human podocytes. Human podocytes were exposed to control conditions (5.6 mmol/L D-glucose), high glucose (30 mmol/L D-glucose or L-glucose), 100 μmol/L peroxynitrite, or high glucose and the superoxide dismutase mimetic tempol (100 μmol/L). TRPC6 and SDC-4 transcripts and protein expression were measured using RT-PCR and in-cell Western assay. Intracellular reactive oxygen species (ROS) and cytosolic calcium were measured using fluorescent dye techniques. High D-glucose increased TRPC6 transcripts to 8.66±4.08 (p<0.05) and TRPC6 protein expression to 1.44±0.07 (p<0.05) without altering SDC-4 transcripts or protein expression. The D-glucose induced increase of TRPC6 expression was blocked by tempol. Increased oxidative stress using peroxynitrite significantly increased TRPC6 transcripts to 4.29±1.26 (p<0.05) and TRPC6 protein expression to 1.28±0.05 (p<0.05) without altering SDC-4 transcripts or protein expression. In human podocytes transfected with scrambled siRNA, high D-glucose increased ROS after 90 min to 3.55±0.08 arbitrary units while 5.6 mmol/L D-glucose increased ROS to 2.49±0.09 (p<0.001) only. The increase in ROS was inhibited by tempol and by SDC-4 knockdown. High glucose modifies TRPC6 channels and ROS production via SDC-4 in human podocytes. PMID:24942878

  19. Gene expression patterns in glucose-stimulated podocytes

    SciTech Connect

    Han, Seung Hyeok; Yang, Sanghwa; Jung, Dong Sub; Li, Jin Ji; Kim, Jin Ju; Kwak, Seung Jae; Kim, Dong Ki; Moon, Sung Jin; Lee, Jung Eun; Han, Dae-Suk; Kang, Shin-Wook

    2008-06-06

    To explore the mechanisms of podocyte injury under diabetic conditions, we performed an expression profile in glucose-stimulated podocytes. Differential gene expression profiles between conditionally immortalized mouse podocytes cultured in medium containing 5.6 and 30 mM glucose were measured with oligonucleotide microarrays. Of the genes identified, heme oxygenase-1, vascular endothelial growth factor-A, and thrombospondin-1 showed a consistently increased pattern, whereas angiotensin-converting enzyme-2 and peroxisomal proliferator activator receptor-{gamma} were down-regulated. These results were validated using real-time PCR and western blotting in podocytes, and with immunohistochemistry on renal tissues from streptozotocin-induced diabetic rats. Not only is this the first report of gene expression profiling of podocyte injury under diabetic conditions, but the identified genes are promising targets for future diabetes research.

  20. Cyclosporine A protects podocytes by regulating WAVE1 phosphorylation

    PubMed Central

    Li, Xuejuan; Ding, Fangrui; Wang, Suxia; Li, Baihong; Ding, Jie

    2015-01-01

    Accumulating evidence suggests that podocytes are direct targets of many classic antiproteinuric drugs. The immunosuppressive drug cyclosporine A (CsA), which is a calcineurin inhibitor, is used to treat proteinuric kidney diseases. One novel mechanism by which CsA reduces proteinuria is by directly stabilizing the podocyte cytoskeleton. Previous studies showed that calcineurin can directly regulate WAVE1 within mouse striatal slices. In this study, WAVE1 was expressed in podocytes and was localized in the podocyte cell bodies and foot processes (FPs). WAVE1 expression increased in both in vivo and in vitro models of puromycin aminonucleoside (PAN)-induced podocyte injury. CsA restored WAVE1 expression and also partially rescued the disordered F-actin arrangement after PAN injury. Co-immunoprecipitation assays showed that calcineurin directly interacted with WAVE1 and regulated WAVE1 phosphorylation in podocytes. Synaptopodin is a well-characterized target of CsA. WAVE1 overexpression and synaptopodin knockdown experiments directly demonstrated that WAVE1 expression is not dependent on synaptopodin expression, and vice versa. Overexpression of WAVE1 using a WAVE1 plasmid disrupted F-actin structure and promoted podocyte migration compared with the empty vector group. Therefore, WAVE1 may be a novel molecular target for the maintenance of podocyte FPs and for antiproteinuric treatment in the future. PMID:26634693

  1. Urinary exosomal Wilms' tumor-1 as a potential biomarker for podocyte injury

    PubMed Central

    Zhou, Hua; Kajiyama, Hiroshi; Tsuji, Takayuki; Hu, Xuzhen; Leelahavanichkul, Asada; Vento, Suzanne; Frank, Rachel; Kopp, Jeffrey B.; Trachtman, Howard; Star, Robert A.

    2013-01-01

    Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We aimed to determine if urinary exosomal WT-1 could serve as a noninvasive biomarker of podocyte injury. We examined WT-1 by Western blot in a human podocyte-like cell line, a mouse model of podocyte injury, and human subjects with podocyte disorders. WT-1 was detected in exosomal fraction of the conditioned media from podocytes and increased 48 h after hTGF-β1 stimulation. Cellular WT-1 decreased in podocytes following hTGF-β1 incubation. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinary exosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunteers or steroid-sensitive nephrotic syndrome (SSNS) patients. Urinary exosomal WT-1 was also significantly decreased in patients in remission for either FSGS or SSNS or following steroid treatment in six SSNS subjects. We conclude that urinary exosomal WT-1 is a promising noninvasive biomarker with apparent podocyte specificity that can detect early progression and treatment-induced regression of podocyte injury in FSGS or SSNS. These results warrant longitudinal, prospective studies in a large cohort with a range of podocyte diseases. PMID:23761678

  2. Urinary exosomal Wilms' tumor-1 as a potential biomarker for podocyte injury.

    PubMed

    Zhou, Hua; Kajiyama, Hiroshi; Tsuji, Takayuki; Hu, Xuzhen; Leelahavanichkul, Asada; Vento, Suzanne; Frank, Rachel; Kopp, Jeffrey B; Trachtman, Howard; Star, Robert A; Yuen, Peter S T

    2013-08-15

    Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We aimed to determine if urinary exosomal WT-1 could serve as a noninvasive biomarker of podocyte injury. We examined WT-1 by Western blot in a human podocyte-like cell line, a mouse model of podocyte injury, and human subjects with podocyte disorders. WT-1 was detected in exosomal fraction of the conditioned media from podocytes and increased 48 h after hTGF-β1 stimulation. Cellular WT-1 decreased in podocytes following hTGF-β1 incubation. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinary exosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunteers or steroid-sensitive nephrotic syndrome (SSNS) patients. Urinary exosomal WT-1 was also significantly decreased in patients in remission for either FSGS or SSNS or following steroid treatment in six SSNS subjects. We conclude that urinary exosomal WT-1 is a promising noninvasive biomarker with apparent podocyte specificity that can detect early progression and treatment-induced regression of podocyte injury in FSGS or SSNS. These results warrant longitudinal, prospective studies in a large cohort with a range of podocyte diseases. PMID:23761678

  3. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    EPA Science Inventory

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  4. Podocyte-Specific GLUT4-Deficient Mice Have Fewer and Larger Podocytes and Are Protected From Diabetic Nephropathy

    PubMed Central

    Guzman, Johanna; Jauregui, Alexandra N.; Merscher-Gomez, Sandra; Maiguel, Dony; Muresan, Cristina; Mitrofanova, Alla; Diez-Sampedro, Ana; Szust, Joel; Yoo, Tae-Hyun; Villarreal, Rodrigo; Pedigo, Christopher; Molano, R. Damaris; Johnson, Kevin; Kahn, Barbara; Hartleben, Bjoern; Huber, Tobias B.; Saha, Jharna; Burke, George W.; Abel, E. Dale; Brosius, Frank C.; Fornoni, Alessia

    2014-01-01

    Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria. PMID:24101677

  5. Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleoside nephrotoxicity

    PubMed Central

    Xia, Li; Zhou, Mingyan; Kalhorn, Thomas F.; Ho, Horace T. B.; Wang, Joanne

    2009-01-01

    Plasma membrane monoamine transporter (PMAT) is a novel polyspecific organic cation transporter that transports organic cations and the purine nucleoside, adenosine. PMAT is expressed in the kidney, but the specific localization and function of this transporter in renal cells are unclear. In this study, we developed a polyclonal antibody toward a 14-amino acid sequence in the last intracellular loop of PMAT and determined the precise cellular localization of PMAT in human and rat kidneys. Surprisingly, we found that the PMAT protein was predominantly expressed in the glomerulus with minimal expression in tubular cells. Within the glomerulus, dual-color immunofluorescence labeling showed that the PMAT protein was specifically localized to the visceral glomerular epithelial cells, i.e., podocytes. There was no significant PMAT immunoreactivity in mesangial or glomerular endothelial cells. We further showed that puromycin aminonucleoside (PAN), a classic podocyte toxin that induces massive proteinuria and severe glomerulopathy, is transported by PMAT. Expression of PMAT in Madin-Darby canine kidney cells significantly increased cell sensitivity to PAN. Decynium 22, a potent PMAT inhibitor, abolished PAN toxicity in PMAT-expressing cells. Together, our data suggest that PMAT is specifically expressed in podocytes and may play an important role in PAN-induced kidney injury. PMID:19357181

  6. cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

    PubMed

    Li, Xiaoying; Tao, Hua; Xie, Kewei; Ni, Zhaohui; Yan, Yucheng; Wei, Kai; Chuang, Peter Y; He, John Cijiang; Gu, Leyi

    2014-01-01

    Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP. PMID:24642777

  7. Downregulation of MicroRNA-30 Facilitates Podocyte Injury and Is Prevented by Glucocorticoids

    PubMed Central

    Wu, Junnan; Zheng, Chunxia; Fan, Yun; Zeng, Caihong; Chen, Zhaohong; Qin, Weisong; Zhang, Changming; Zhang, Wanfen; Wang, Xiao; Zhu, Xiaodong; Zhang, Mingchao; Zen, Ke

    2014-01-01

    MicroRNAs (miRNAs) are essential for podocyte homeostasis, and the miR-30 family may be responsible for this action. However, the exact roles and clinical relevance of miR-30s remain unknown. In this study, we examined the expression of the miR-30 family in the podocytes of patients with FSGS and found that all members are downregulated. Treating cultured human podocytes with TGF-β, LPS, or puromycin aminonucleoside (PAN) also downregulated the miR-30 family. Podocyte cytoskeletal damage and apoptosis caused by treatment with TGF-β or PAN were ameliorated by exogenous miR-30 expression and aggravated by miR-30 knockdown. Moreover, we found that miR-30s exert their protective roles by direct inhibition of Notch1 and p53, which mediate podocyte injury. In rats, treatment with PAN substantially downregulated podocyte miR-30s and induced proteinuria and podocyte injury; however, transfer of exogenous miR-30a to podocytes of PAN-treated rats ameliorated proteinuria and podocyte injury and reduced Notch1 activation. Finally, we demonstrated that glucocorticoid treatment maintains miR-30 expression in cultured podocytes treated with TGF-β, LPS, or PAN and in the podocytes of PAN-treated rats. Glucocorticoid-sustained miR-30 expression associated with reduced Notch1 activation and alleviated podocyte damage. Taken together, these findings demonstrate that miR-30s protect podocytes by targeting Notch1 and p53 and that the loss of miR-30s facilitates podocyte injury. In addition, sustained miR-30 expression may be a novel mechanism underlying the therapeutic effectiveness of glucocorticoids in treating podocytopathy. PMID:24029422

  8. Statin-sensitive endocytosis of albumin by glomerular podocytes.

    PubMed

    Eyre, Jeanette; Ioannou, Kyriakos; Grubb, Blair D; Saleem, Moin A; Mathieson, Peter W; Brunskill, Nigel J; Christensen, Erik I; Topham, Peter S

    2007-02-01

    Glomerular podocytes are critical regulators of glomerular permeability via the slit diaphragm and may play a role in cleaning the glomerular filter. Whether podocytes are able to endocytose proteins is uncertain. We studied protein endocytosis in conditionally immortalized mouse and human podocytes using FITC-albumin by direct quantitative assay and by fluorescence microscopy and electron microscopy in mouse podocytes. Furthermore, in vivo uptake was studied in human, rat, and mouse podocytes. Both mouse and human podocytes displayed specific one-site binding for FITC-albumin with K(d) of 0.91 or 0.44 mg/ml and B(max) of 3.15 or 0.81 microg/mg cell protein, respectively. In addition, they showed avid endocytosis of FITC-albumin with K(m) of 9.48 or 4.5 mg/ml and V(max) of 474.3 or 97.4 microg.mg cell protein(-1).h(-1), respectively. Immunoglobulin and transferrin were inefficient competitors of this process, indicating some specificity for albumin. Accumulation of endocytosed albumin could be demonstrated in intracellular vesicles by fluorescence confocal microscopy and electron microscopy. Endocytosis was sensitive to pretreatment with simvastatin. In vivo accumulation of albumin was found in all three species but was most pronounced in the rat. We conclude that podocytes are able to endocytose protein in a statin-sensitive manner. This function is likely to be highly significant in health and disease. In addition, protein endocytosis by podocytes may represent a useful, measurable phenotypic characteristic against which potentially injurious or beneficial interventions can be assessed. PMID:17032937

  9. [Treatment of nephrotic syndrome: immuno- or rather podocyte therapy?].

    PubMed

    Lewko, Barbara

    2016-01-01

    Nephrotic syndrome (NS) is a group of clinical symptoms resulting from massive proteinuria caused by impairment of the glomerular filtration barrier. The filtration barrier comprises glomerular basement membrane with endothelial cells lining its inner side and a podocyte monolayer covering its outer aspect. As well as forming part of the glomerular filter, podocytes also regulate synthesis of other components of the filtration barrier. Therefore, integrity of these cells is crucial for maintaining the normal ultrafiltration function. The pathogenesis of idiopathic nephrotic syndrome (INS) was proposed to be associated with autoimmunity and appearance in the circulation of a still unknown protein permeability factor (PF) inducing changes in the glomerular filtration barrier. Several candidate PFs have been identified to date, and current results indicate that podocytes are target cells for all of them. Changes in podocyte structure and functions induced by these factors are typical for changes observed in patients with nephrotic proteinuria. Most pharmacotherapeutic approaches in NS are based on various immunosuppressive agents and are targeted toward minimizing proteinuria. It appears, however, that these drugs not only target the cells of the immune system but also act directly on podocytes. Thus, it can be concluded that detailed studies on mechanisms regulating podocyte functions as well as designing drugs to protect these cells are required for effective therapy of NS. PMID:27180964

  10. Krüppel-like Factor 15 (KLF15) Is a Key Regulator of Podocyte Differentiation*

    PubMed Central

    Mallipattu, Sandeep K.; Liu, Ruijie; Zheng, Feng; Narla, Goutham; Ma'ayan, Avi; Dikman, Steven; Jain, Mukesh K.; Saleem, Moin; D'Agati, Vivette; Klotman, Paul; Chuang, Peter Y.; He, John C.

    2012-01-01

    Podocyte injury resulting from a loss of differentiation is the hallmark of many glomerular diseases. We previously showed that retinoic acid (RA) induces podocyte differentiation via stimulation of the cAMP pathway. However, many podocyte maturity markers lack binding sites for RA-response element or cAMP-response element (CREB) in their promoter regions. We hypothesized that transcription factors induced by RA and downstream of CREB mediate podocyte differentiation. We performed microarray gene expression studies in human podocytes treated with and without RA to identify differentially regulated genes. In comparison with known CREB target genes, we identified Krüppel-like factor 15 (KLF15), a kidney-enriched nuclear transcription factor, that has been previously shown to mediate cell differentiation. We confirmed that RA increased KLF15 expression in both murine and human podocytes. Overexpression of KLF15 stimulated expression of differentiation markers in both wild-type and HIV-1-infected podocytes. Also, KLF15 binding to the promoter regions of nephrin and podocin was increased in RA-treated podocytes. Although KLF15−/− mice at base line had minimal phenotype, lipopolysaccharide- or adriamycin-treated KLF15−/− mice had a significant increase in proteinuria and podocyte foot process effacement with a reduction in the expression of podocyte differentiation markers as compared with the wild-type treated mice. Finally, KLF15 expression was reduced in glomeruli isolated from HIV transgenic mice as well as in kidney biopsies from patients with HIV-associated nephropathy and idiopathic focal segmental glomerulosclerosis. These results indicate a critical role of KLF15 in mediating podocyte differentiation and in protecting podocytes against injury. PMID:22493483

  11. Myo1e Impairment Results in Actin Reorganization, Podocyte Dysfunction, and Proteinuria in Zebrafish and Cultured Podocytes

    PubMed Central

    Mao, Jianhua; Wang, Dayan; Mataleena, Parikka; He, Bing; Niu, Dadi; Katayama, Kan; Xu, Xiangjun; Ojala, Juha RM; Wang, Wenjing; Shu, Qiang; Du, Lizhong; Liu, Aimin; Pikkarainen, Timo; Patrakka, Jaakko; Tryggvason, Karl

    2013-01-01

    Background Podocytes serve as an important constituent of the glomerular filtration barrier. Recently, we and others identified Myo1e as a key molecular component of the podocyte cytoskeleton. Results Myo1e mRNA and protein was expressed in human and mouse kidney sections as determined by Northern blot and reverse transcriptase PCR, and its expression was more evident in podocytes by immunofluorescence. By specific knock-down of MYO1E in zebrafish, the injected larvae exhibited pericardial edema and pronephric cysts, consistent with the appearance of protein in condensed incubation supernate. Furthermore, specific inhibition of Myo1e expression in a conditionally immortalized podocyte cell line induced morphological changes, actin cytoskeleton rearrangement, and dysfunction in cell proliferation, migration, endocytosis, and adhesion with the glomerular basement membrane. Conclusions Our results revealed that Myo1e is a key component contributing to the functional integrity of podocytes. Its impairment may cause actin cytoskeleton re-organization, alteration of cell shape, and membrane transport, and podocyte drop-out from the glomerular basement membrane, which might eventually lead to an impaired glomerular filtration barrier and proteinuria. PMID:23977349

  12. Wnt/β-catenin signalling and podocyte dysfunction in proteinuric kidney disease

    PubMed Central

    Zhou, Lili; Liu, Youhua

    2016-01-01

    Podocytes are unique, highly specialized, terminally differentiated cells that are integral components of the kidney glomerular filtration barrier. Podocytes are vulnerable to a variety of injuries and in response they undergo a series of changes ranging from hypertrophy, autophagy, dedifferentiation, mesenchymal transition and detachment to apoptosis, depending on the nature and extent of the insult. Emerging evidence indicates that Wnt/β-catenin signalling has a central role in mediating podocyte dysfunction and proteinuria. Wnts are induced and β-catenin is activated in podocytes in various proteinuric kidney diseases. Genetic or pharmacologic activation of β-catenin is sufficient to impair podocyte integrity and causes proteinuria in healthy mice, whereas podocyte-specific ablation of β-catenin protects against proteinuria after kidney injury. Mechanistically, Wnt/β-catenin controls the expression of several key mediators implicated in podocytopathies, including Snail1, the renin–angiotensin system and matrix metalloproteinase 7. Wnt/β-catenin also negatively regulates Wilms tumour protein, a crucial transcription factor that safeguards podocyte integrity. Targeted inhibition of Wnt/β-catenin signalling preserves podocyte integrity and ameliorates proteinuria in animal models. This Review highlights advances in our understanding of the pathomechanisms of Wnt/β-catenin signalling in mediating podocyte injury, and describes the therapeutic potential of targeting this pathway for the treatment of proteinuric kidney disease. PMID:26055352

  13. APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability

    PubMed Central

    Lan, Xiqian; Jhaveri, Aakash; Cheng, Kang; Wen, Hongxiu; Saleem, Moin A.; Mathieson, Peter W.; Mikulak, Joanna; Aviram, Sharon; Malhotra, Ashwani; Skorecki, Karl

    2014-01-01

    Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent. PMID:24899058

  14. APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability.

    PubMed

    Lan, Xiqian; Jhaveri, Aakash; Cheng, Kang; Wen, Hongxiu; Saleem, Moin A; Mathieson, Peter W; Mikulak, Joanna; Aviram, Sharon; Malhotra, Ashwani; Skorecki, Karl; Singhal, Pravin C

    2014-08-01

    Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent. PMID:24899058

  15. Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis

    PubMed Central

    Fornoni, Alessia; Sageshima, Junichiro; Wei, Changli; Merscher-Gomez, Sandra; Robier, Aguillon-Prada; Jauregui, Alexandra N.; Li, Jing; Mattiazzi, Adela; Ciancio, Gaetano; Chen, Linda; Zilleruelo, Gaston; Abitbol, Carolyn; Chandar, Jayanthi; Seeherunvong, Wacheree; Ricordi, Camillo; Ikehata, Masami; Rastaldi, Maria Pia; Reiser, Jochen; Burke, George W.

    2013-01-01

    Focal segmental glomerulosclerosis (FSGS) is a prevalent glomerular disease characterized by proteinuria, progression to end stage renal disease and recurrence of proteinuria after kidney transplantation in approximately one third of patients. It has been suggested that rituximab might treat recurrent FSGS through an unknown mechanism. Rituximab recognizes CD20 on B-lymphocytes but might also bind sphingomyelin-phosphodiesterase-acid-like-3b (SMPDL-3b) and regulates acid-sphyngomyelinase (ASMase) activity. We hypothesized that rituximab prevents recurrent FSGS and preserves podocyte SMPDL-3b expression. We studied 41 patients at high risk for recurrent FSGS, 27 of whom were treated with rituximab at time of kidney transplant. Incidence of nephrotic-range proteinuria and change in estimated glomerular filtration rate (ΔeGFR) were analyzed. SMPDL-3b immunostaining was performed in post-reperfusion kidney biopsies. SMPDL-3b protein, ASMase activity, and cytoskeleton remodeling were studied in cultured normal human podocytes that had been exposed to patient sera with or without rituximab. Rituximab treatment was associated with lower incidence of post-transplant proteinuria and decreased ΔeGFR. The number of SMPDL-3b+ podocytes in post-reperfusion biopsies was reduced in patients who developed recurrent FSGS. Rituximab partially prevented SMPDL-3b and ASMase downregulation that was observed in podocytes treated with the sera of patients with recurrent FSGS. Either SMPDL-3b overexpression or treatment with rituximab prevented disruption of the actin cytoskeleton and podocyte apoptosis induced by patient sera. This effect was diminished in cultured podocytes where the gene encoding SMPDL-3b was silenced. Our study suggests that treatment of high-risk patients with rituximab at time of kidney transplant might prevent recurrent FSGS by modulating podocyte function in an SMPDL-3b–dependent manner. PMID:21632984

  16. Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis.

    PubMed

    Fornoni, Alessia; Sageshima, Junichiro; Wei, Changli; Merscher-Gomez, Sandra; Aguillon-Prada, Robier; Jauregui, Alexandra N; Li, Jing; Mattiazzi, Adela; Ciancio, Gaetano; Chen, Linda; Zilleruelo, Gaston; Abitbol, Carolyn; Chandar, Jayanthi; Seeherunvong, Wacheree; Ricordi, Camillo; Ikehata, Masami; Rastaldi, Maria Pia; Reiser, Jochen; Burke, George W

    2011-06-01

    Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized by proteinuria, progression to end-stage renal disease, and recurrence of proteinuria after kidney transplantation in about one-third of patients. It has been suggested that rituximab might treat recurrent FSGS through an unknown mechanism. Rituximab not only recognizes CD20 on B lymphocytes, but might also bind sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) protein and regulate acid sphingomyelinase (ASMase) activity. We hypothesized that rituximab prevents recurrent FSGS and preserves podocyte SMPDL-3b expression. We studied 41 patients at high risk for recurrent FSGS, 27 of whom were treated with rituximab at time of kidney transplant. SMPDL-3b protein, ASMase activity, and cytoskeleton remodeling were studied in cultured normal human podocytes that had been exposed to patient sera with or without rituximab. Rituximab treatment was associated with lower incidence of posttransplant proteinuria and stabilization of glomerular filtration rate. The number of SMPDL-3b(+) podocytes in postreperfusion biopsies was reduced in patients who developed recurrent FSGS. Rituximab partially prevented SMPDL-3b and ASMase down-regulation that was observed in podocytes treated with the sera of patients with recurrent FSGS. Overexpression of SMPDL-3b or treatment with rituximab was able to prevent disruption of the actin cytoskeleton and podocyte apoptosis induced by patient sera. This effect was diminished in cultured podocytes where SMPDL-3b was silenced. Our study suggests that treatment of high-risk patients with rituximab at time of kidney transplant might prevent recurrent FSGS by modulating podocyte function in an SMPDL-3b-dependent manner. PMID:21632984

  17. The β isoform of GSK3 mediates podocyte autonomous injury in proteinuric glomerulopathy.

    PubMed

    Li, Changbin; Ge, Yan; Dworkin, Lance; Peng, Ai; Gong, Rujun

    2016-05-01

    Converging evidence points to glycogen synthase kinase (GSK) 3 as a key player in the pathogenesis of podocytopathy and proteinuria. However, it remains unclear if GSK3 is involved in podocyte autonomous injury in glomerular disease. In normal kidneys, the β isoform of GSK3 was found to be the major GSK3 expressed in glomeruli and intensely stained in podocytes. GSK3β expression in podocytes was markedly elevated in experimental or human proteinuric glomerulopathy. Podocyte-specific somatic ablation of GSK3β in adult mice attenuated proteinuria and ameliorated podocyte injury and glomerular damage in experimental adriamycin (ADR) nephropathy. Mechanistically, actin cytoskeleton integrity in podocytes was largely preserved in GSK3β knockout mice following ADR insult, concomitant with a correction of podocyte hypermotility and lessened phosphorylation and activation of paxillin, a focal adhesion-associated adaptor protein. In addition, GSK3β knockout diminished ADR-induced NFκB RelA/p65 phosphorylation selectively at serine 467; suppressed de novo expression by podocytes of NFκB-dependent podocytopathic mediators, including B7-1, cathepsin L, and MCP-1; but barely affected the induction of NFκB target pro-survival factors, such as Bcl-xL. Moreover, the ADR-elicited podocytopenia and podocyte death were significantly attenuated in GSK3β knockout mice, associated with protection against podocyte mitochondrial damage and reduced phosphorylation and activation of cyclophilin F, a structural component of mitochondria permeability transition pores. Overall, our findings suggest that the β isoform of GSK3 mediates autonomous podocyte injury in glomerulopathy by integrating multiple podocytopathic signalling pathways. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26876299

  18. Estradiol induces functional inactivation of p53 by intracellular redistribution.

    PubMed

    Molinari, A M; Bontempo, P; Schiavone, E M; Tortora, V; Verdicchio, M A; Napolitano, M; Nola, E; Moncharmont, B; Medici, N; Nigro, V; Armetta, I; Abbondanza, C; Puca, G A

    2000-05-15

    Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm. PMID:10825127

  19. GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    PubMed Central

    Kim, Sang-Hoon; Kim, Hyun-Jung; Kim, Chan-Wha

    2016-01-01

    Podocyte foot processes are interdigitated to form the slit diaphragm and are crucial for the glomerular filtration barrier. Glucocorticoid-induced transcript 1 (GLCCI1) is transcriptionally regulated, but its signaling pathway in podocytes is unknown. The main objective of this study was to investigate the regulation of podocyte foot process proteins and to investigate the role of GLCCI1 in the phosphoinositide 3-kinase (PI3K) pathway using high glucose-induced podocytes and streptozotocin-induced diabetic rats. In podocytes and rat kidneys, GLCCI1 was found to be highly specific for the glomerulus and podocyte foot processes similar to other podocyte-specific proteins (nephrin, podocin, synatopodin and podocalyxin) based on reverse transcription-PCR, western blotting, immunofluorescence and immunoelectron microscopy analyses. In addition, the decrease in the GLCCI1 expression level under hyperglycemic conditions was restored by treatment with a PI3K inhibitor (wortmannin). Immunofluorescence analysis confirmed that GLCCI1 colocalized with nephrin and synaptopodin both in vivo and in vitro. Finally, immunoelectron microscopy data from streptozotocin-induced diabetic rats showed that GLCCI1 also localized in podocyte foot processes. Hence, GLCCI1 is a component of podocyte foot processes, and its expression appears to be regulated via the PI3K pathway. PMID:27174202

  20. Cyclin-dependent kinase 2 protects podocytes from apoptosis

    PubMed Central

    Saurus, Pauliina; Kuusela, Sara; Dumont, Vincent; Lehtonen, Eero; Fogarty, Christopher L.; Lassenius, Mariann I.; Forsblom, Carol; Lehto, Markku; Saleem, Moin A.; Groop, Per-Henrik; Lehtonen, Sanna

    2016-01-01

    Loss of podocytes is an early feature of diabetic nephropathy (DN) and predicts its progression. We found that treatment of podocytes with sera from normoalbuminuric type 1 diabetes patients with high lipopolysaccharide (LPS) activity, known to predict progression of DN, downregulated CDK2 (cyclin-dependent kinase 2). LPS-treatment of mice also reduced CDK2 expression. LPS-induced downregulation of CDK2 was prevented in vitro and in vivo by inhibiting the Toll-like receptor (TLR) pathway using immunomodulatory agent GIT27. We also observed that CDK2 is downregulated in the glomeruli of obese Zucker rats before the onset of proteinuria. Knockdown of CDK2, or inhibiting its activity with roscovitine in podocytes increased apoptosis. CDK2 knockdown also reduced expression of PDK1, an activator of the cell survival kinase Akt, and reduced Akt phosphorylation. This suggests that CDK2 regulates the activity of the cell survival pathway via PDK1. Furthermore, PDK1 knockdown reduced the expression of CDK2 suggesting a regulatory loop between CDK2 and PDK1. Collectively, our data show that CDK2 protects podocytes from apoptosis and that reduced expression of CDK2 associates with the development of DN. Preventing downregulation of CDK2 by blocking the TLR pathway with GIT27 may provide a means to prevent podocyte apoptosis and progression of DN. PMID:26876672

  1. Overexpression of Mafb in Podocytes Protects against Diabetic Nephropathy

    PubMed Central

    Yoh, Keigyou; Ojima, Masami; Okamura, Midori; Nakamura, Megumi; Hamada, Michito; Shimohata, Homare; Moriguchi, Takashi; Yamagata, Kunihiro; Takahashi, Satoru

    2014-01-01

    We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target. PMID:24722438

  2. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    PubMed Central

    Xin, Wei; Li, Zhaoping; Chen, Liyong

    2016-01-01

    It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN). Here we used shRNA transfection to knockdown the insulin receptor (IR) gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN. PMID:27077005

  3. Genetic causes of proteinuria and nephrotic syndrome: Impact on podocyte pathobiology

    PubMed Central

    Akchurin, Oleh; Reidy, Kimberly J.

    2014-01-01

    In the past 20 years, multiple genetic mutations have been identified in patients with congenital nephrotic syndrome (CNS) and both familial and sporadic focal segmental glomerulosclerosis (FSGS). Characterization of the genetic basis of CNS and FSGS has led to the recognition of the importance of podocyte injury to the development of glomerulosclerosis. Genetic mutations induce injury due to effects on the podocyte’s structure, actin cytoskeleton, calcium signaling, and lysosomal and mitochondrial function. Transgenic animal studies have contributed to our understanding of podocyte pathobiology. Podocyte endoplasmic reticulum stress response, cell polarity, and autophagy play a role in maintenance of podocyte health. Further investigations related to the effects of genetic mutations on podocytes may identify new pathways for targeting therapeutics for nephrotic syndrome. PMID:24584664

  4. Transient light-induced intracellular oxidation revealed by redox biosensor

    SciTech Connect

    Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  5. Shiga Toxin Promotes Podocyte Injury in Experimental Hemolytic Uremic Syndrome via Activation of the Alternative Pathway of Complement

    PubMed Central

    Locatelli, Monica; Buelli, Simona; Pezzotta, Anna; Corna, Daniela; Perico, Luca; Tomasoni, Susanna; Rottoli, Daniela; Rizzo, Paola; Conti, Debora; Thurman, Joshua M.; Remuzzi, Giuseppe; Zoja, Carlamaria

    2014-01-01

    Shiga toxin (Stx)–producing Escherichia coli is the offending agent of postdiarrhea-associated hemolytic uremic syndrome (HUS), a disorder of glomerular ischemic damage and widespread microvascular thrombosis. We previously documented that Stx induces glomerular complement activation, generating C3a responsible for microvascular thrombosis in experimental HUS. Here, we show that the presence of C3 deposits on podocytes is associated with podocyte damage and loss in HUS mice generated by the coinjection of Stx2 and LPS. Because podocyte adhesion to the glomerular basement membrane is mediated by integrins, the relevance of integrin-linked kinase (ILK) signals in podocyte dysfunction was evaluated. Podocyte expression of ILK increased after the injection of Stx2/LPS and preceded the upregulation of Snail and downregulation of nephrin and α-actinin-4. Factor B deficiency or pretreatment with an inhibitory antibody to factor B protected mice against Stx2/LPS-induced podocyte dysregulation. Similarly, pretreatment with a C3a receptor antagonist limited podocyte loss and changes in ILK, Snail, and α-actinin-4 expression. In cultured podocytes, treatment with C3a reduced α-actinin-4 expression and promoted ILK-dependent nuclear expression of Snail and cell motility. These results suggest that Stx-induced activation of the alternative pathway of complement and generation of C3a promotes ILK signaling, leading to podocyte dysfunction and loss in Stx-HUS. PMID:24578132

  6. β-Arrestin-1 Drives Endothelin-1–Mediated Podocyte Activation and Sustains Renal Injury

    PubMed Central

    Buelli, Simona; Rosanò, Laura; Gagliardini, Elena; Corna, Daniela; Longaretti, Lorena; Pezzotta, Anna; Perico, Luca; Conti, Sara; Rizzo, Paola; Novelli, Rubina; Morigi, Marina; Zoja, Carlamaria; Remuzzi, Giuseppe; Bagnato, Anna

    2014-01-01

    Activation of endothelin-A receptor (ETAR) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through β-arrestin signaling. Here, we investigated whether this pathogenetic pathway could affect podocyte phenotype in proliferative glomerular disorders. In cultured mouse podocytes, ET-1 caused loss of the podocyte differentiation marker synaptopodin and acquisition of the mesenchymal marker α-smooth muscle actin. ET-1 promoted podocyte migration via ETAR activation and increased β-arrestin-1 expression. Activated ETAR recruited β-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and β-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1–induced migration as confirmed by Snail knockdown experiments. Silencing of β-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ETAR-driven signaling. In vitro findings were confirmed in doxorubicin (Adriamycin)-induced nephropathy. Mice receiving Adriamycin developed renal injury with loss of podocytes and hyperplastic lesion formation; β-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ETAR antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular β-arrestin-1 and Snail. Increased β-arrestin-1 levels in podocytes retrieved from crescents of patients with proliferative glomerulopathies confirmed the translational relevance of these findings and suggest the therapeutic potential of ETAR antagonism for a group of diseases still needing a specific treatment. PMID:24371298

  7. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

    PubMed Central

    Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  8. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli.

    PubMed

    Ilatovskaya, Daria V; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca(2+) concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  9. Cannabidiol induces intracellular calcium elevation and cytotoxicity in oligodendrocytes.

    PubMed

    Mato, Susana; Victoria Sánchez-Gómez, María; Matute, Carlos

    2010-11-01

    Heavy marijuana use has been linked to white matter histological alterations. However, the impact of cannabis constituents on oligodendroglial pathophysiology remains poorly understood. Here, we investigated the in vitro effects of cannabidiol, the main nonpsychoactive marijuana component, on oligodendrocytes. Exposure to cannabidiol induced an intracellular Ca(2+) rise in optic nerve oligodendrocytes that was not primarily mediated by entry from the extracellular space, nor by interactions with ryanodine or IP(3) receptors. Application of the mitochondrial protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM) completely prevented subsequent cannabidiol-induced Ca(2+) responses. Conversely, the increase in cytosolic Ca(2+) levels elicited by FCCP was reduced after previous exposure to cannabidiol, further suggesting that the mitochondria acts as the source of cannabidiol-evoked Ca(2+) rise in oligodendrocytes. n addition, brief exposure to cannabidiol (100 nM-10 μM) led to a concentration-dependent decrease of oligodendroglial viability that was not prevented by antagonists of CB(1), CB(2), vanilloid, A(2A) or PPARγ receptors, but was instead reduced in the absence of extracellular Ca(2+). The oligodendrotoxic effect of cannabidiol was partially blocked by inhibitors of caspase-3, -8 and -9, PARP-1 and calpains, suggesting the activation of caspase-dependent and -independent death pathways. Cannabidiol also elicited a concentration-dependent alteration of mitochondrial membrane potential, and an increase in reactive oxygen species (ROS) that was reduced in the absence of extracellular Ca(2+). Finally, cannabidiol-induced cytotoxicity was partially prevented by the ROS scavenger trolox. Together, these results suggest that cannabidiol causes intracellular Ca(2+) dysregulation which can lead to oligodendrocytes demise. PMID:20645411

  10. Puromycin aminonucleoside increases podocyte permeability by modulating ZO-1 in an oxidative stress-dependent manner.

    PubMed

    Ha, Tae-Sun; Park, Hye-Young; Seong, Su-Bin; Ahn, Hee Yul

    2016-01-01

    Puromycin aminonucleoside (PAN)-induced nephrosis is a widely studied animal model of human idiopathic nephrotic syndrome because PAN injection into rats results in increased glomerular permeability with the characteristic ultrastructural changes in podocytes similar to human nephrosis. To investigate the role of zonula occludens (ZO)-1 and oxidative stress on PAN-induced podocyte phenotypical changes and hyperpermeability in vitro, we cultured rat and mouse podocytes and treated with various concentrations of PAN. PAN treatment increased oxidative stress level of podocytes significantly with the induction of Nox4. In addition, PAN changed the ultrastructure of podocytes, such as shortening and fusion of microvilli, and the separation of intercellular gaps, which were improved by anti-oxidative vitamin C and Nox4 siRNA. PAN also disrupted the intercellular linear ZO-1 staining and induced inner cytoplasmic re-localization of ZO-1 protein, resulting in increased podocyte intercellular permeability. PAN reduced ZO-1 protein amount and mRNA expression in a dose-dependent manner, which means that PAN could also modulate ZO-1 protein transcriptionally. However, the decreased ZO-1 protein of podocytes by PAN was improved by Nox4 siRNA transfection. Furthermore, vitamin C mitigated the quantitative and distributional disturbances of ZO-1 protein caused by PAN. Our results demonstrate that the phenotypical changes of intercellular ZO-1 by oxidative stress via Nox4 likely contribute to the glomerular hyperpermeability caused by PAN. PMID:26683996

  11. Spironolactone inhibits podocyte motility via decreasing integrin β1 and increasing integrin β3 in podocytes under high-glucose conditions.

    PubMed

    Li, Zhuo; Zhang, Li; Shi, Wei; Chen, Yuanhan; Zhang, Hong; Liu, Shuangxin; Liang, Xinling; Ling, Ting; Yu, Chunping; Huang, Zhongshun; Tan, Xiaofan; Zhao, Xinchen; Ye, Zhiming; Zhang, Bin; Wang, Wenjian; Li, Ruizhao; Ma, Jianchao

    2015-11-01

    Integrin β1 and β3 expression by podocytes is required to maintain glomerular structural integrity. Previous studies have shown that aldosterone (ALD) is involved in glomerular podocyte injury, and mineralocorticoid receptor (MR) blocker spironolactone effectively reduces proteinuria in patients with diabetic nephropathy. The present study was designed to observe the effects of spironolactone on β1 and β3 integrin expression and podocyte motility under in vitro diabetic conditions. Immortalized mouse podocytes were cultured in media containing normal glucose (NG) levels, high glucose (HG) or HG plus spironolacton. The expression of β1 and β3 integrin in podocytes was detected by reverse transcription quantitative polymerase chain reaction, immunofluorescence and western blot analyses. The effects of spironolacton on podocyte motility was further evaluated using a wound healing assay. HG stimulation markedly decreased mRNA and protein expression of integrin β1, and significantly increased mRNA and protein expression of integrin β3 in cultured podocytes. However, simultaneous treatment with spironolacton (10‑7 mol/l) significantly attenuated HG-mediated increases in integrin β3 and decreases in integrin β1 expression. Furthermore, the migration of podocytes induced by HG was abrogated by concomitant treatment with spironolacton. In conclusion, the present study suggested that HG decreased the expression of integrin β1 in cultured podocytes, accompanied with an increase of integrin β3. Spironolactone inhibited cell motility and stabilized podoctyes treated with HG, probably through partly normalizing the expression of integrin β1 and decreasing the expression of integrin β3. PMID:26352002

  12. Sirtuin1 Maintains Actin Cytoskeleton by Deacetylation of Cortactin in Injured Podocytes

    PubMed Central

    Motonishi, Shuta; Wada, Takehiko; Ishimoto, Yu; Ohse, Takamoto; Matsusaka, Taiji; Kubota, Naoto; Shimizu, Akira; Kadowaki, Takashi; Tobe, Kazuyuki

    2015-01-01

    Recent studies have highlighted the renoprotective effect of sirtuin1 (SIRT1), a deacetylase that contributes to cellular regulation. However, the pathophysiologic role of SIRT1 in podocytes remains unclear. Here, we investigated the function of SIRT1 in podocytes. We first established podocyte-specific Sirt1 knockout (SIRT1pod−/−) mice. We then induced glomerular disease by nephrotoxic serum injection. The increase in urinary albumin excretion and BUN and the severity of glomerular injury were all significantly greater in SIRT1pod−/− mice than in wild-type mice. Western blot analysis and immunofluorescence showed a significant decrease in podocyte-specific proteins in SIRT1pod−/− mice, and electron microscopy showed marked exacerbation of podocyte injury, including actin cytoskeleton derangement in SIRT1pod−/− mice compared with wild-type mice. Protamine sulfate-induced podocyte injury was also exacerbated by podocyte-specific SIRT1 deficiency. In vitro, actin cytoskeleton derangement in H2O2-treated podocytes became prominent when the cells were pretreated with SIRT1 inhibitors. Conversely, this H2O2-induced derangement was ameliorated by SIRT1 activation. Furthermore, SIRT1 activation deacetylated the actin-binding and -polymerizing protein cortactin in the nucleus and facilitated deacetylated cortactin localization in the cytoplasm. Cortactin knockdown or inhibition of the nuclear export of cortactin induced actin cytoskeleton derangement and dissociation of cortactin from F-actin, suggesting the necessity of cytoplasmic cortactin for maintenance of the actin cytoskeleton. Taken together, these findings indicate that SIRT1 protects podocytes and prevents glomerular injury by deacetylating cortactin and thereby, maintaining actin cytoskeleton integrity. PMID:25424328

  13. Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia

    PubMed Central

    Zhang, Chun; Xia, Min; Boini, Krishna M.; Li, Cai-Xia; Abais, Justine M.; Li, Xiao-Xue; Laperle, Laura A.; Li, Pin-Lan

    2012-01-01

    The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1) in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O2.−) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91phox (gp91−/−), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91+/+) mice, hHcys induced by a folate-free (FF) diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91−/− mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91+/+ mice with hHcys were all significantly attenuated in gp91−/− mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury. PMID:21647593

  14. Autophagy Induced by Intracellular Infection of Propionibacterium acnes

    PubMed Central

    Nakamura, Teruko; Furukawa, Asuka; Uchida, Keisuke; Ogawa, Tomohisa; Tamura, Tomoki; Sakonishi, Daisuke; Wada, Yuriko; Suzuki, Yoshimi; Ishige, Yuki; Minami, Junko; Akashi, Takumi

    2016-01-01

    Background Sarcoidosis is caused by Th1-type immune responses to unknown agents, and is linked to the infectious agent Propionibacterium acnes. Many strains of P. acnes isolated from sarcoid lesions cause intracellular infection and autophagy may contribute to the pathogenesis of sarcoidosis. We examined whether P. acnes induces autophagy. Methods Three cell lines from macrophages (Raw264.7), mesenchymal cells (MEF), and epithelial cells (HeLa) were infected by viable or heat-killed P. acnes (clinical isolate from sarcoid lymph node) at a multiplicity of infection (MOI) of 100 or 1000 for 1 h. Extracellular bacteria were killed by washing and culturing infected cells with antibiotics. Samples were examined by colony assay, electron-microscopy, and fluorescence-microscopy with anti-LC3 and anti-LAMP1 antibodies. Autophagy-deficient (Atg5-/-) MEF cells were also used. Results Small and large (≥5 μm in diameter) LC3-positive vacuoles containing few or many P. acnes cells (LC3-positive P. acnes) were frequently found in the three cell lines when infected by viable P. acnes at MOI 1000. LC3-positive large vacuoles were mostly LAMP1-positive. A few small LC3-positive/LAMP1-negative vacuoles were consistently observed in some infected cells for 24 h postinfection. The number of LC3-positive P. acnes was decreased at MOI 100 and completely abolished when heat-killed P. acnes was used. LC3-positive P. acnes was not found in autophagy-deficient Atg5-/- cells where the rate of infection was 25.3 and 17.6 times greater than that in wild-type Atg5+/+ cells at 48 h postinfection at MOI 100 and 1000, respectively. Electron-microscopic examination revealed bacterial cells surrounded mostly by a single-membrane including the large vacuoles and sometimes a double or multi-layered membrane, with occasional undigested bacterial cells in ruptured late endosomes or in the cytoplasm. Conclusion Autophagy was induced by intracellular P. acnes infection and contributed to intracellular

  15. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides:

    NASA Astrophysics Data System (ADS)

    Chen, Junping; Patil, Swanand; Seal, Sudipta; McGinnis, James F.

    2006-11-01

    Photoreceptor cells are incessantly bombarded with photons of light, which, along with the cells' high rate of oxygen metabolism, continuously exposes them to elevated levels of toxic reactive oxygen intermediates (ROIs). Vacancy-engineered mixed-valence-state cerium oxide nanoparticles (nanoceria particles) scavenge ROIs. Our data show that nanoceria particles prevent increases in the intracellular concentrations of ROIs in primary cell cultures of rat retina and, in vivo, prevent loss of vision due to light-induced degeneration of photoreceptor cells. These data indicate that the nanoceria particles may be effective in inhibiting the progression of ROI-induced cell death, which is thought to be involved in macular degeneration, retinitis pigmentosa and other blinding diseases, as well as the ROI-induced death of other cell types in diabetes, Alzheimer's disease, atherosclerosis, stroke and so on. The use of nanoceria particles as a direct therapy for multiple diseases represents a novel strategy and suggests that they may represent a unique platform technology.

  16. KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria.

    PubMed

    Hayashi, Kaori; Sasamura, Hiroyuki; Nakamura, Mari; Azegami, Tatsuhiko; Oguchi, Hideyo; Sakamaki, Yusuke; Itoh, Hiroshi

    2014-06-01

    The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria. PMID:24812666

  17. Podocyte directed therapy of nephrotic syndrome-can we bring the inside out?

    PubMed

    Müller-Deile, Janina; Schiffer, Mario

    2016-03-01

    Several of the drugs currently used for the treatment of glomerular diseases are prescribed for their immunotherapeutic or anti-inflammatory properties, based on the current understanding that glomerular diseases are mediated by immune responses. In recent years our understanding of podocytic signalling pathways and the crucial role of genetic predispositions in the pathology of glomerular diseases has broadened. Delineation of those signalling pathways supports the hypothesis that several of the medications and immunosuppressive agents used to treat glomerular diseases directly target glomerular podocytes. Several central downstream signalling pathways merge into regulatory pathways of the podocytic actin cytoskeleton and its connection to the slit diaphragm. The slit diaphragm and the cytoskeleton of the foot process represent a functional unit. A breakdown of the cytoskeletal backbone of the foot processes leads to internalization of slit diaphragm molecules, and internalization of slit diaphragm components in turn negatively affects cytoskeletal signalling pathways. Podocytes display a remarkable ability to recover from complete effacement and to re-form interdigitating foot processes and intact slit diaphragms after pharmacological intervention. This ability indicates an active inside-out signalling machinery which stabilizes integrin complex formations and triggers the recycling of slit diaphragm molecules from intracellular compartments to the cell surface. In this review we summarize current evidence from patient studies and model organisms on the direct impact of immunosuppressive and supportive drugs on podocyte signalling pathways. We highlight new therapeutic targets that may open novel opportunities to enhance and stabilize inside-out pathways in podocytes. PMID:25939817

  18. Renal Lipotoxicity-Associated Inflammation and Insulin Resistance Affects Actin Cytoskeleton Organization in Podocytes

    PubMed Central

    Vivas, Yurena; Velasco, Ismael; Yeo, Tet-Kin; Chen, Sheldon; Medina-Gomez, Gema

    2015-01-01

    In the last few decades a change in lifestyle has led to an alarming increase in the prevalence of obesity and obesity-associated complications. Obese patients are at increased risk of developing hypertension, heart disease, insulin resistance (IR), dyslipidemia, type 2 diabetes and renal disease. The excess calories are stored as triglycerides in adipose tissue, but also may accumulate ectopically in other organs, including the kidney, which contributes to the damage through a toxic process named lipotoxicity. Recently, the evidence suggests that renal lipid accumulation leads to glomerular damage and, more specifically, produces dysfunction in podocytes, key cells that compose and maintain the glomerular filtration barrier. Our aim was to analyze the early mechanisms underlying the development of renal disease associated with the process of lipotoxicity in podocytes. Our results show that treatment of podocytes with palmitic acid produced intracellular accumulation of lipid droplets and abnormal glucose and lipid metabolism. This was accompanied by the development of inflammation, oxidative stress and endoplasmic reticulum stress and insulin resistance. We found specific rearrangements of the actin cytoskeleton and slit diaphragm proteins (Nephrin, P-Cadherin, Vimentin) associated with this insulin resistance in palmitic-treated podocytes. We conclude that lipotoxicity accelerates glomerular disease through lipid accumulation and inflammation. Moreover, saturated fatty acids specifically promote insulin resistance by disturbing the cytoarchitecture of podocytes. These data suggest that renal lipid metabolism and cytoskeleton rearrangements may serve as a target for specific therapies aimed at slowing the progression of podocyte failure during metabolic syndrome. PMID:26545114

  19. Interconnected Network Motifs Control Podocyte Morphology and Kidney Function

    PubMed Central

    Azeloglu, Evren U.; Hardy, Simon V.; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y.; Fang, Wei; Xiong, Huabao; Neves, Susana R.; Jain, Mohit R.; Li, Hong; Ma’ayan, Avi; Gordon, Ronald E.; He, John Cijiang; Iyengar, Ravi

    2014-01-01

    Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3′,5′-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element–binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor–driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease. PMID:24497609

  20. Alteration of Forkhead Box O (Foxo4) Acetylation Mediates Apoptosis of Podocytes in Diabetes Mellitus

    PubMed Central

    Chuang, Peter Y.; Dai, Yan; Liu, Ruijie; He, Helen; Kretzler, Matthias; Jim, Belinda; Cohen, Clemens D.; He, John C.

    2011-01-01

    The number of kidney podocytes is reduced in diabetic nephropathy. Advanced glycation end products (AGEs) accumulate in patients with diabetes and promote the apoptosis of podocyte by activating the forkhead box O4 (Foxo4) transcription factor to increase the expression of a pro-apoptosis gene, Bcl2l11. Using chromatin immunoprecipitation we demonstrate that AGE-modified bovine serum albumin (AGE-BSA) enhances Foxo4 binding to a forkhead binding element in the promoter of Bcl2lll. AGE-BSA also increases the acetylation of Foxo4. Lysine acetylation of Foxo4 is required for Foxo4 binding and transcription of Bcl2l11 in podocytes treated with AGE-BSA. The expression of a protein deacetylase that targets Foxo4 for deacetylation, sirtuin (Sirt1), is down regulated in cultured podocytes by AGE-BSA treatment and in glomeruli of diabetic patients. SIRT1 over expression in cultured murine podocytes prevents AGE-induced apoptosis. Glomeruli isolated from diabetic db/db mice have increased acetylation of Foxo4, suppressed expression of Sirt1, and increased expression of Bcl2l11 compared to non-diabetic littermates. Together, our data provide evidence that alteration of Foxo4 acetylation and down regulation of Sirt1 expression in diabetes promote podocyte apoptosis. Strategies to preserve Sirt1 expression or reduce Foxo4 acetylation could be used to prevent podocyte loss in diabetes. PMID:21858169

  1. Wnt/β-Catenin Signaling Mediated-UCH-L1 Expression in Podocytes of Diabetic Nephropathy.

    PubMed

    Zhang, Hongxia; Luo, Weili; Sun, Yonghong; Qiao, Yanchun; Zhang, Liying; Zhao, Zhilian; Lv, Shijun

    2016-01-01

    Increasing studies identified podocyte injury as a key early risk factor resulting in diabetic nephropathy (DN). The ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) participates in podocyte differentiation and injury, which is elevated in the podocytes of a variety of nephritis. Whether UCH-L1 expression is positively related to podocyte injury of DN remains unclear. In this study, elevated expression of UCH-L1 and its intrinsic mechanism in high glucose (HG)-stimulated murine podocytes were investigated using western blot and real-time quantitative PCR. Kidney biopsies of DN patients and health individuals were stained by immunofluorescence (IF) method. The morphological and functional changes of podocytes were tested by F-actin staining and cell migration assay. Results demonstrated that HG induced upregulation of UCH-L1 and activation of the Wnt/β-catenin signaling pathway in podocytes. However, blocking of the Wnt pathway by dickkopf related protein 1 (DKK1) eliminated the above changes. Furthermore, IF staining confirmed that, compared with healthy individuals, the expression of UCH-L1 and β-catenin were obviously increased in kidney biopsy of DN patients. Overexpression of UCH-L1 remodeled its actin cytoskeleton, increased its cell migration and impacted its important proteins. All the findings manifested that Wnt/β-catenin/UCH-L1 may be a new potential therapy method in the treatment of DN in future. PMID:27571062

  2. Podocyte apoptosis is prevented by blocking the Toll-like receptor pathway

    PubMed Central

    Saurus, P; Kuusela, S; Lehtonen, E; Hyvönen, M E; Ristola, M; Fogarty, C L; Tienari, J; Lassenius, M I; Forsblom, C; Lehto, M; Saleem, M A; Groop, P-H; Holthöfer, H; Lehtonen, S

    2015-01-01

    High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN. PMID:25950482

  3. Non-canonical NFκB activation promotes chemokine expression in podocytes

    PubMed Central

    Valiño-Rivas, Lara; Gonzalez-Lafuente, Laura; Sanz, Ana B.; Ruiz-Ortega, Marta; Ortiz, Alberto; Sanchez-Niño, Maria D.

    2016-01-01

    TNF-like weak inducer of apoptosis (TWEAK) receptor Fn14 is expressed by podocytes and Fn14 deficiency protects from experimental proteinuric kidney disease. However, the downstream effectors of TWEAK/Fn14 in podocytes are poorly characterized. We have explored TWEAK activation of non-canonical NFκB signaling in cultured podocytes. In cultured podocytes, TWEAK increased the expression of the chemokines CCL21, CCL19 and RANTES in a time-dependent manner. The inhibitor of canonical NFκB activation parthenolide inhibited the CCL19 and the early RANTES responses, but not the CCL21 or late RANTES responses. In this regard, TWEAK induced non-canonical NFκB activation in podocytes, characterized by NFκB2/p100 processing to NFκB2/p52 and nuclear migration of RelB/p52. Silencing by a specific siRNA of NIK, the upstream kinase of the non-canonical NFκB pathway, prevented CCL21 upregulation but did not modulate CCL19 or RANTES expression in response to TWEAK, thus establishing CCL21 as a non-canonical NFκB target in podocytes. Increased kidney Fn14 and CCL21 expression was also observed in rat proteinuric kidney disease induced by puromycin, and was localized to podocytes. In conclusion, TWEAK activates the non-canonical NFκB pathway in podocytes, leading to upregulation of CCL21 expression. The non-canonical NFκB pathway should be explored as a potential therapeutic target in proteinuric kidney disease. PMID:27353019

  4. Drug Targets for Oxidative Podocyte Injury in Diabetic Nephropathy

    PubMed Central

    Usman, Muhammad

    2015-01-01

    Diabetic nephropathy (DN) is one the most prevalent chronic complications of diabetes mellitus that affects as much as one-third of diabetic patients irrespective of the type of diabetes. Hyperglycemia is the key trigger for DN that initiates a number of microscopic and ultramicroscopic changes in kidney architecture. Microscopic changes include thickening of the glomerular basement membrane (GBM), tubular basement membrane (TBM), mesangial proliferation, arteriosclerosis, and glomerulotubular junction abnormalities (GTJA). Among the ultramicroscopic changes, effacement of podocytes and decrease in their density seem to be the centerpiece of DN pathogenesis. These changes in kidney architecture then produce functional deficits, such as microalbuminuria and decreased glomerular filtration rate (GFR). Among several mechanisms involved in inflicting damage to podocytes, injuries sustained by increased oxidative stress turns out to be the most important mechanism. Different variables that are included in increased production of reactive oxygen species (ROS) include a hyperglycemia-induced reduction in glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation via hyperglycemia, advanced glycation end products (AGEs), protein kinase C (PKC), and renin-angiotensin-aldosterone system (RAAS). Unfortunately, control of podocyte injury hasn’t received much attention as a treatment approach for DN. Therefore, this review article is mainly concerned with the exploration of various treatment options that might help in decreasing the podocyte injury, mainly by reducing the level of NADPH oxidase-mediated generation of ROS. This article concludes with a view that certain NADPH oxidase inhibitors, RAAS inhibitors, statins, antidiabetic drugs, and antioxidant vitamins might be useful in decreasing podocyte injury and resultant structural and functional kidney impairments in DN. PMID:26798569

  5. Drug Targets for Oxidative Podocyte Injury in Diabetic Nephropathy.

    PubMed

    Bhatti, Adnan Bashir; Usman, Muhammad

    2015-01-01

    Diabetic nephropathy (DN) is one the most prevalent chronic complications of diabetes mellitus that affects as much as one-third of diabetic patients irrespective of the type of diabetes. Hyperglycemia is the key trigger for DN that initiates a number of microscopic and ultramicroscopic changes in kidney architecture. Microscopic changes include thickening of the glomerular basement membrane (GBM), tubular basement membrane (TBM), mesangial proliferation, arteriosclerosis, and glomerulotubular junction abnormalities (GTJA). Among the ultramicroscopic changes, effacement of podocytes and decrease in their density seem to be the centerpiece of DN pathogenesis. These changes in kidney architecture then produce functional deficits, such as microalbuminuria and decreased glomerular filtration rate (GFR). Among several mechanisms involved in inflicting damage to podocytes, injuries sustained by increased oxidative stress turns out to be the most important mechanism. Different variables that are included in increased production of reactive oxygen species (ROS) include a hyperglycemia-induced reduction in glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation via hyperglycemia, advanced glycation end products (AGEs), protein kinase C (PKC), and renin-angiotensin-aldosterone system (RAAS). Unfortunately, control of podocyte injury hasn't received much attention as a treatment approach for DN. Therefore, this review article is mainly concerned with the exploration of various treatment options that might help in decreasing the podocyte injury, mainly by reducing the level of NADPH oxidase-mediated generation of ROS. This article concludes with a view that certain NADPH oxidase inhibitors, RAAS inhibitors, statins, antidiabetic drugs, and antioxidant vitamins might be useful in decreasing podocyte injury and resultant structural and functional kidney impairments in DN. PMID:26798569

  6. Cells of Renin Lineage Are Progenitors of Podocytes and Parietal Epithelial Cells in Experimental Glomerular Disease

    PubMed Central

    Pippin, Jeffrey W.; Sparks, Matthew A.; Glenn, Sean T.; Buitrago, Sandra; Coffman, Thomas M.; Duffield, Jeremy S.; Gross, Kenneth W.; Shankland, Stuart J.

    2014-01-01

    Glomerular injury leads to podocyte loss, a process directly underlying progressive glomerular scarring and decline of kidney function. The inherent repair process is limited by the inability of podocytes to regenerate. Cells of renin lineage residing alongside glomerular capillaries are reported to have progenitor capacity. We investigated whether cells of renin lineage can repopulate the glomerulus after podocyte injury and serve as glomerular epithelial cell progenitors. Kidney cells expressing renin were genetically fate-mapped in adult Ren1cCreER×Rs-tdTomato-R, Ren1cCre×Rs-ZsGreen-R, and Ren1dCre×Z/EG reporter mice. Podocyte depletion was induced in all three cell-specific reporter mice by cytotoxic anti-podocyte antibodies. After a decrease in podocyte number, a significant increase in the number of labeled cells of renin lineage was observed in glomeruli in a focal distribution along Bowman's capsule, within the glomerular tuft, or in both locations. A subset of cells lining Bowman's capsule activated expression of the glomerular parietal epithelial cell markers paired box protein PAX2 and claudin-1. A subset of labeled cells within the glomerular tuft expressed the podocyte markers Wilms tumor protein 1, nephrin, podocin, and synaptopodin. Neither renin mRNA nor renin protein was detected de novo in diseased glomeruli. These findings provide initial evidence that cells of renin lineage may enhance glomerular regeneration by serving as progenitors for glomerular epithelial cells in glomerular disease characterized by podocyte depletion. PMID:23769837

  7. Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture.

    PubMed

    New, Laura A; Martin, Claire E; Scott, Rizaldy P; Platt, Mathew J; Keyvani Chahi, Ava; Stringer, Colin D; Lu, Peihua; Samborska, Bozena; Eremina, Vera; Takano, Tomoko; Simpson, Jeremy A; Quaggin, Susan E; Jones, Nina

    2016-08-01

    Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton. PMID:26802179

  8. LMX1B is Essential for the Maintenance of Differentiated Podocytes in Adult Kidneys

    PubMed Central

    Burghardt, Tillmann; Kastner, Jürgen; Suleiman, Hani; Rivera-Milla, Eric; Stepanova, Natalya; Lottaz, Claudio; Kubitza, Marion; Böger, Carsten A.; Schmidt, Sarah; Gorski, Mathias; de Vries, Uwe; Schmidt, Helga; Hertting, Irmgard; Kopp, Jeffrey; Rascle, Anne; Moser, Markus; Heid, Iris M.; Warth, Richard; Spang, Rainer; Wegener, Joachim; Mierke, Claudia T.; Englert, Christoph

    2013-01-01

    Mutations of the LMX1B gene cause nail–patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes. PMID:23990680

  9. Podocyte-associated mRNA profiles in kidney tissue and in urine of patients with active lupus nephritis

    PubMed Central

    dos Santos, Mariane; Bringhenti, Rafael N; Rodrigues, Patrícia G; do Nascimento, Jonathan F; Pereira, Sane V; Zancan, Rafael; Monticielo, Odirlei A; Gasparin, Andrese A; de Castro, Waldir P; Veronese, Francisco V

    2015-01-01

    Aim: Glomerular deposition of immune complexes and inflammation induce podocyte injury in lupus nephritis (LN). This study hypothesized that the severity of the histological lesions of LN affects podocyte-associated mRNAs profiles in kidney tissue and in urine. Methods: Thirty-three patients with LN were grouped according to the presence of mild mesangial (classes I and II) or moderate-to-severe immune complex deposition, proliferation and/or inflammation (classes III, IV and V) in kidney biopsy. Tissue and urine mRNA of nephrin, podocin, podocalyxin, α-actinin-4, transient receptor potential cation channel 6, and of growth factors VEGF-A and TGF-β1 and the transcription factor FOXP3 were measured using real time polymerase chain reaction. These mRNAs were correlated with histological severity of LN, extent of glomerular immune deposits, and tissue infiltrating cells. Results: Podocyte-associated mRNAs were inhibited in renal tissue of patients with LN irrespective of histological class when compared to controls. However, significantly higher expression of podocyte mRNAs in urine, including those of growth factors and FOXP3, were found in patients with moderate-to-severe nephritis, mostly in class III and IV proliferative forms. The number of invading CD8+ T cells, B cells and macrophages correlated positively with urine podocyte-associated mRNAs. Urine podocyte mRNAs also correlated with proteinuria. Conclusions: Inhibition of podocyte-associated mRNAs in kidney tissue suggests that podocyte injury occurs regardless of class severity of LN. Increased urinary excretion of podocyte mRNAs, mostly in patients with moderate-to-severe lesions, may reflect a greater burden of glomerular damage with detachment of podocytes into the urine. PMID:26191151

  10. MiRNA expression profile and miRNA-mRNA integrated analysis (MMIA) during podocyte differentiation.

    PubMed

    Li, Zhigui; Wang, Lifeng; Xu, Jing; Yang, Zhuo

    2015-06-01

    The podocyte is a prominent cell type, which encases the capillaries of glomerulus. Podocyte-selective deletion of Dicer or Drosha was reported to induce proteinuria and glomerulosclerosis, suggesting the essential role of microRNA (miRNA) in podocytes for renal function. However, no comprehensive miRNA expression or miRNA-mRNA integrated analysis (MMIA) can be found during podocyte differentiation. Herein, miRNA and mRNA microarrays are presented, which were carried out in differentiated and undifferentiated mouse podocyte cell lines (MPC5). A total of 50 abnormal miRNAs (26 down-regulated and 24 up-regulated) were identified in differentiated and undifferentiated podocytes. Using MMIA, 80 of the 743 mRNAs (>twofold change) were predicted for potential crosstalk with 30 miRNAs of the 50 abnormal miRNAs. In addition, the gene ontology of mRNAs and the pathway analysis of miRNAs revealed a new potential-regulated network during podocyte differentiation. The expressions of three remarkably changed miRNAs (miR-34c, miR-200a and miR-467e) and four mRNAs (Runx1t1, Atp2a2, Glrp1, and Mmp15), were randomly chosen for further validation by the quantitative real-time polymerase chain reaction, and their expression trends were consistent with the microarray data. Reference searching was also conducted to confirm our data and to find potential new molecules and miRNA-target pairs involved in the podocyte differentiation. The dual luciferase reporter assay for miR-200a/GLRX and let-7b/ARL4D confirmed the prediction of MMIA. The results of this study provide a detailed integration of mRNA and miRNA during podocyte differentiation. The molecular integration mode will open up new perspectives for a better understanding of the mechanism during podocyte differentiation. PMID:25433550

  11. Up-regulation of the homophilic adhesion molecule sidekick-1 in podocytes contributes to glomerulosclerosis.

    PubMed

    Kaufman, Lewis; Potla, Uma; Coleman, Sarah; Dikiy, Stanislav; Hata, Yutaka; Kurihara, Hidetake; He, John C; D'Agati, Vivette D; Klotman, Paul E

    2010-08-13

    Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide. Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, alpha-actinin-4, nephrin, JAM4, and beta-catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function. PMID:20562105

  12. N-WASP Is Required for Stabilization of Podocyte Foot Processes

    PubMed Central

    Schell, Christoph; Baumhakl, Lisa; Salou, Sarah; Conzelmann, Ann-Christin; Meyer, Charlotte; Helmstädter, Martin; Wrede, Christoph; Grahammer, Florian; Eimer, Stefan; Kerjaschki, Dontscho; Walz, Gerd; Snapper, Scott

    2013-01-01

    Alteration of cortical actin structures is the common final pathway leading to podocyte foot process effacement and proteinuria. The molecular mechanisms that safeguard podocyte foot process architecture and maintain the three-dimensional actin network remain elusive. Here, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin nucleation, is required to stabilize podocyte foot processes. Mice lacking N-WASP specifically in podocytes were born with normal kidney function but developed significant proteinuria 3 weeks after birth, suggesting an important role for N-WASP in maintaining foot processes. In addition, inducing deletion of N-WASP in adult mice resulted in severe proteinuria and kidney failure. Electron microscopy showed an accumulation of electron-dense patches of actin and strikingly altered morphology of podocyte foot processes. Although basic actin-based processes such as cell migration were not affected, primary cultures of N-WASP–deficient podocytes revealed significant impairment of dynamic actin reorganization events, including the formation of circular dorsal ruffles. Taken together, our findings suggest that N-WASP–mediated actin nucleation of branched microfilament networks is specifically required for the maintenance of foot processes, presumably sustaining the mechanical resistance of the filtration barrier. PMID:23471198

  13. High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

    SciTech Connect

    Zhang, Yue; Li, Hongbo; Hao, Jun; Zhou, Yi; Liu, Wei

    2014-08-15

    Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. - Highlights: • HG up-regulated the expression of Cdk5 and p35, and Cdk5 activity in podocytes. • HG activated TGF-β1 pathway and SB431542 inhibited Cdk5 expression and activity. • HG increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. • Inhibition of Egr-1

  14. Nuclear translocation of IQGAP1 protein upon exposure to puromycin aminonucleoside in cultured human podocytes: ERK pathway involvement.

    PubMed

    Rigothier, Claire; Saleem, Moin Ahson; Bourget, Chantal; Mathieson, Peter William; Combe, Christian; Welsh, Gavin Iain

    2016-10-01

    IQGAP1, a protein that links the actin cytoskeleton to slit diaphragm proteins, is involved in podocyte motility and permeability. Its regulation in glomerular disease is not known. We have exposed human podocytes to puromycin aminonucleoside (PAN), an inducer of nephrotic syndrome in rats, and studied the effects on IQGAP1 biology and function. In human podocytes exposed to PAN, a nuclear translocation of IQGAP1 was observed by immunocytolocalization and confirmed by Western blot after selective nuclear/cytoplasmic extraction. In contrast to IQGAP1, IQGAP2 expression remained cytoplasmic. IQGAP1 nuclear translocation was associated with a significant decrease in its interaction with nephrin and podocalyxin. Activation of the ERK pathway was observed in PAN treated podocytes with a preponderant nuclear localization of the phosphorylated form of ERK (P-ERK). The interaction between IQGAP1 and P-ERK increased upon podocyte exposure to PAN. Inhibitors of ERK pathway activation blocked IQGAP1 nuclear translocation (p<0.02). Chromatin interaction protein assays demonstrated an interaction of IQGAP1 with chromatin and with Histone H3, which increased in response to PAN. In summary, PAN induces the ERK dependent translocation of IQGAP1 into the nuclei in human podocytes which leads to the interaction of IQGAP1 with chromatin and Histone H3, and decreased interactions between IQGAP1 and slit-diaphragm proteins. Therefore, IQGAP1 may have a role in podocyte gene regulation in glomerular disease. PMID:27377965

  15. Identification of Chromosomal Shigella flexneri Genes Induced by the Eukaryotic Intracellular Environment

    PubMed Central

    Runyen-Janecky, L. J.; Payne, S. M.

    2002-01-01

    Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium Shigella flexneri is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the S. flexneri chromosome fused to a promoterless green fluorescent protein gene (gfp). Bacteria containing promoter fusions that had a higher level of gfp expression when S. flexneri was intracellular (in Henle cells) than when S. flexneri was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (uhpT, bioA, and lysA) were involved in metabolic processes. The uhpT gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (pstS and phoA) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (sufA, sitA, and fhuA) were identified. The sufA promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the S. flexneri uhpT and pstS genes by allelic exchange. The uhpT mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The pstS mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line. PMID:12117948

  16. Intracellular mediators of potassium-induced aldosterone secretion

    SciTech Connect

    Ganguly, A.; Chiou, S.; Davis, J.S. )

    1990-01-01

    We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) in {sup 3}H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium.

  17. Tacrolimus restores podocyte injury and stabilizes the expression of Cabin1 in 5/6 nephrectomized rats.

    PubMed

    Wen, Yueqiang; Liu, Lingling; Zhou, Peilan; Li, Huiyuan; Wang, Zebin; Zhang, Yajie; Liang, Jianbo

    2016-05-01

    Podocyte injury is a vital factor, which induces massive proteinuria. Studies have shown that tacrolimus (TAC) protected podocyte via stabilizing cytoskeleton. Our latest study indicates that calcineurin binding protein 1 (Cabin1) undergoes nuclear translocation during podocytes injury. Whether TAC targets on Cabin1 during podocyte injury is still not clear. This study establishes non-immunological proteinuric model. To observe the effect of the treatment of TAC on Cabin1 expression in 5/6 nephrectomized rats. Sprague-Dawley rats were injected with TAC (0.2 mg/kg/day) for 4-8 weeks after 5/6 nephrectomy. Then, rats were sacrificed in the eighth week after operation, renal tissues were processed for morphological studies under light and electrical microscope. Cabin1 expression and distribution were detected by western blot and indirect immunofluorescence staining. In 5/6 nephrectomized rats, urinary protein excretion reached 90.2 ± 30.1 mg/24 h, glomerular sclerosis index and tubulointerstitial fibrosis score were significantly increased, and widespread of podocyte foot processes fusion was found. Moreover, Cabin1 protein expression was markedly increased, and its distribution became much more obviously in podocytes nuclei. In TAC treated rats, urinary protein excretion significantly decreased (44.9 ± 22.5 mg/24 h), glomerular sclerosis and tubulointerstitial fibrosis were alleviated, and podocyte foot processes fusion was inhibited. Furthermore, TAC alleviated the increased protein expression and abnormal distribution of Cabin1. In conclusion, TAC restores podocyte injury and stabilizes the expression of Cabin1. Cabin1 may become a new target to demonstrate the mechanism of TAC in podocyte injury. PMID:26905810

  18. Assessment of urinary angiotensinogen as a marker of podocyte injury in proteinuric nephropathies.

    PubMed

    Eriguchi, Masahiro; Yotsueda, Ryusuke; Torisu, Kumiko; Kawai, Yasuhiro; Hasegawa, Shoko; Tanaka, Shigeru; Noguchi, Hideko; Masutani, Kosuke; Kitazono, Takanari; Tsuruya, Kazuhiko

    2016-02-15

    Urinary protein (UP) is widely used as a clinical marker for podocyte injury; however, not all proteinuric nephropathies fit this model. We previously described the elevation of urinary angiotensinogen (AGT) accompanied by AGT expression by injured podocytes in a nitric oxide inhibition rat model (Eriguchi M, Tsuruya K, Haruyama N, Yamada S, Tanaka S, Suehiro T, Noguchi H, Masutani K, Torisu K, Kitazono T. Kidney Int 87: 116-127, 2015). In this report, we performed the human and animal studies to examine the significance and origin of urinary AGT. In the human study, focal segmental glomerulosclerosis (FSGS) patients presented with higher levels of urinary AGT, corrected by UP, than minimal-change disease (MCD) patients. Furthermore, AGT was evident in podocin-negative glomerular segmental lesions. We also tested two different nephrotic models induced by puromycin aminonucleoside in Wistar rats. The urinary AGT/UP ratio and AGT protein and mRNA expression in sieved glomeruli from FSGS rats were significantly higher than in MCD rats. The presence of AGT at injured podocytes in FSGS rats was detected by immunohistochemistry and immunoelectron microscopy. Finally, we observed the renal tissue and urinary metabolism of exogenous injected human recombinant AGT (which is not cleaved by rodent renin) in FSGS and control rats. Significant amounts of human AGT were detected in the urine of FSGS rats, but not of control rats. Immunostaining for rat and human AGT identified that only rat AGT was detected in injured podocytes, and filtered human AGT was seen in superficial proximal tubules, but not in injured podocytes, suggesting AGT generation by injured podocytes. In conclusion, the urinary AGT/UP ratio represents a novel specific marker of podocyte injury. PMID:26632605

  19. pH sensing by intracellular Salmonella induces effector translocation.

    PubMed

    Yu, Xiu-Jun; McGourty, Kieran; Liu, Mei; Unsworth, Kate E; Holden, David W

    2010-05-21

    Salmonella enterica is an important intracellular bacterial pathogen of humans and animals. It replicates within host-cell vacuoles by delivering virulence (effector) proteins through a vacuolar membrane pore made by the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS). T3SS assembly follows vacuole acidification, but when bacteria are grown at low pH, effector secretion is negligible. We found that effector secretion was activated at low pH from mutant strains lacking a complex of SPI-2-encoded proteins SsaM, SpiC, and SsaL. Exposure of wild-type bacteria to pH 7.2 after growth at pH 5.0 caused dissociation and degradation of SsaM/SpiC/SsaL complexes and effector secretion. In infected cells, loss of the pH 7.2 signal through acidification of host-cell cytosol prevented complex degradation and effector translocation. Thus, intravacuolar Salmonella senses host cytosolic pH, resulting in the degradation of regulatory complex proteins and effector translocation. PMID:20395475

  20. Counting glomeruli and podocytes: rationale and methodologies

    PubMed Central

    Puelles, Victor G.; Bertram, John F.

    2015-01-01

    Purpose of review There is currently much interest in the numbers of both glomeruli and podocytes. This interest stems from greater understanding of the effects of suboptimal fetal events on nephron endowment, the associations between low nephron number and chronic cardiovascular and kidney disease in adults, and the emergence of the podocyte depletion hypothesis. Recent findings Obtaining accurate and precise estimates of glomerular and podocyte number has proven surprisingly difficult. When whole kidneys or large tissue samples are available, design-based stereological methods are considered gold-standard because they are based on principles that negate systematic bias. However, these methods are often tedious and time-consuming, and oftentimes inapplicable when dealing with small samples such as biopsies. Therefore, novel methods suitable for small tissue samples, and innovative approaches to facilitate high through put measurements, such as magnetic resonance imaging (MRI) to estimate glomerular number and flow cytometry to estimate podocyte number, have recently been described. Summary This review describes current gold-standard methods for estimating glomerular and podocyte number, as well as methods developed in the past 3 years. We are now better placed than ever before to accurately and precisely estimate glomerular and podocyte number, and to examine relationships between these measurements and kidney health and disease. PMID:25887899

  1. Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

    SciTech Connect

    Zhang, Shangrong; Wang, Yifan; Li, Shu Jie

    2014-06-13

    Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor.

  2. Insulin increases glomerular filtration barrier permeability through PKGIα-dependent mobilization of BKCa channels in cultured rat podocytes.

    PubMed

    Piwkowska, Agnieszka; Rogacka, Dorota; Audzeyenka, Irena; Kasztan, Małgorzata; Angielski, Stefan; Jankowski, Maciej

    2015-08-01

    Podocytes are highly specialized cells that wrap around glomerular capillaries and comprise a key component of the glomerular filtration barrier. They are uniquely sensitive to insulin; like skeletal muscle and fat cells, they exhibit insulin-stimulated glucose uptake and express glucose transporters. Podocyte insulin signaling is mediated by protein kinase G type I (PKGI), and it leads to changes in glomerular permeability to albumin. Here, we investigated whether large-conductance Ca²⁺-activated K⁺ channels (BKCa) were involved in insulin-mediated, PKGIα-dependent filtration barrier permeability. Insulin-induced glomerular permeability was measured in glomeruli isolated from Wistar rats. Transepithelial albumin flux was measured in cultured rat podocyte monolayers. Expression of BKCa subunits was detected by RT-PCR. BKCa, PKGIα, and upstream protein expression were examined in podocytes with Western blotting and immunofluorescence. The BKCa-PKGIα interaction was assessed with co-immunoprecipitation. RT-PCR showed that primary cultured rat podocytes expressed mRNAs that encoded the pore-forming α subunit and four accessory β subunits of BKCa. The BKCa inhibitor, iberiotoxin (ibTX), abolished insulin-dependent glomerular albumin permeability and PKGI-dependent transepithelial albumin flux. Insulin-evoked albumin permeability across podocyte monolayers was also blocked with BKCa siRNA. Moreover, ibTX blocked insulin-induced disruption of the actin cytoskeleton and changes in the phosphorylation of PKG target proteins, MYPT1 and RhoA. These results indicated that insulin increased filtration barrier permeability through mobilization of BKCa channels via PKGI in cultured rat podocytes. This molecular mechanism may explain podocyte injury and proteinuria in diabetes. PMID:25952906

  3. Podocytes degrade endocytosed albumin primarily in lysosomes.

    PubMed

    Carson, John M; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  4. Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes

    PubMed Central

    Carson, John M.; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B.; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  5. Plekhh2, a novel podocyte protein downregulated in human focal segmental glomerulosclerosis, is involved in matrix adhesion and actin dynamics.

    PubMed

    Perisic, Ljubica; Lal, Mark; Hulkko, Jenny; Hultenby, Kjell; Önfelt, Björn; Sun, Ying; Dunér, Fredrik; Patrakka, Jaakko; Betsholtz, Christer; Uhlen, Mathias; Brismar, Hjalmar; Tryggvason, Karl; Wernerson, Annika; Pikkarainen, Timo

    2012-11-01

    Pleckstrin homology domain-containing, family H (with MyTH4 domain), member 2 (Plekhh2) is a 1491-residue intracellular protein highly enriched in renal glomerular podocytes for which no function has been ascribed. Analysis of renal biopsies from patients with focal segmental glomerulosclerosis revealed a significant reduction in total podocyte Plekhh2 expression compared to controls. Sequence analysis indicated a putative α-helical coiled-coil segment as the only recognizable domain within the N-terminal half of the polypeptide, while the C-terminal half contains two PH, a MyTH4, and a FERM domain. We identified a phosphatidylinositol-3-phosphate consensus-binding site in the PH1 domain required for Plekhh2 localization to peripheral regions of cell lamellipodia. The N-terminal half of Plekkh2 is not necessary for lamellipodial targeting but mediates self-association. Yeast two-hybrid screening showed that Plekhh2 directly interacts through its FERM domain with the focal adhesion protein Hic-5 and actin. Plekhh2 and Hic-5 coprecipitated and colocalized at the soles of podocyte foot processes in situ and Hic-5 partially relocated from focal adhesions to lamellipodia in Plekhh2-expressing podocytes. In addition, Plekhh2 stabilizes the cortical actin cytoskeleton by attenuating actin depolymerization. Our findings suggest a structural and functional role for Plekhh2 in the podocyte foot processes. PMID:22832517

  6. Spironolactone promotes autophagy via inhibiting PI3K/AKT/mTOR signalling pathway and reduce adhesive capacity damage in podocytes under mechanical stress

    PubMed Central

    Li, Dong; Lu, Zhenyu; Xu, Zhongwei; Ji, Junya; Zheng, Zhenfeng; Lin, Shan; Yan, Tiekun

    2016-01-01

    Mechanical stress which would cause deleterious adhesive effects on podocytes is considered a major contributor to the early progress of diabetic nephropathy (DN). Our previous study has shown that spironolactone could ameliorate podocytic adhesive capacity in diabetic rats. Autophagy has been reported to have a protective role against renal injury. The present study investigated the underlying mechanisms by which spironolactone reduced adhesive capacity damage in podocytes under mechanical stress, focusing on the involvement of autophagy. Human conditional immortalized podocytes exposed to mechanical stress were treated with spironolactone, LY294002 or rapamycin for 48 h. The accumulation of LC3 puncta was detected by immunofluorescence staining. Podocyte expression of mineralocorticoid receptor (MR), integrin β1, LC3, Atg5, p85-PI3K, p-Akt, p-mTOR were detected by Western blotting. Podocyte adhesion to collagen type IV was also performed with spectrophotometry. Immunofluorescence staining showed that the normal level of autophagy was reduced in podocytes under mechanical stress. Decreased integrin β1, LC3, Atg5 and abnormal activation of the PI3K/Akt/mTOR pathway were also detected in podocytes under mechanical stress. Spironolactone up-regulated integrin β1, LC3, Atg5 expression, down-regulated p85-PI3K, p-Akt, p-mTOR expression and reduced podocytic adhesive capacity damage. Our data demonstrated that spironolactone inhibited mechanical-stress-induced podocytic adhesive capacity damage through blocking PI3K/Akt/mTOR pathway and restoring autophagy activity. PMID:27129295

  7. Spironolactone promotes autophagy via inhibiting PI3K/AKT/mTOR signalling pathway and reduce adhesive capacity damage in podocytes under mechanical stress.

    PubMed

    Li, Dong; Lu, Zhenyu; Xu, Zhongwei; Ji, Junya; Zheng, Zhenfeng; Lin, Shan; Yan, Tiekun

    2016-08-01

    Mechanical stress which would cause deleterious adhesive effects on podocytes is considered a major contributor to the early progress of diabetic nephropathy (DN). Our previous study has shown that spironolactone could ameliorate podocytic adhesive capacity in diabetic rats. Autophagy has been reported to have a protective role against renal injury. The present study investigated the underlying mechanisms by which spironolactone reduced adhesive capacity damage in podocytes under mechanical stress, focusing on the involvement of autophagy. Human conditional immortalized podocytes exposed to mechanical stress were treated with spironolactone, LY294002 or rapamycin for 48 h. The accumulation of LC3 puncta was detected by immunofluorescence staining. Podocyte expression of mineralocorticoid receptor (MR), integrin β1, LC3, Atg5, p85-PI3K, p-Akt, p-mTOR were detected by Western blotting. Podocyte adhesion to collagen type IV was also performed with spectrophotometry. Immunofluorescence staining showed that the normal level of autophagy was reduced in podocytes under mechanical stress. Decreased integrin β1, LC3, Atg5 and abnormal activation of the PI3K/Akt/mTOR pathway were also detected in podocytes under mechanical stress. Spironolactone up-regulated integrin β1, LC3, Atg5 expression, down-regulated p85-PI3K, p-Akt, p-mTOR expression and reduced podocytic adhesive capacity damage. Our data demonstrated that spironolactone inhibited mechanical-stress-induced podocytic adhesive capacity damage through blocking PI3K/Akt/mTOR pathway and restoring autophagy activity. PMID:27129295

  8. Deficiency of the Planar Cell Polarity Protein Vangl2 in Podocytes Affects Glomerular Morphogenesis and Increases Susceptibility to Injury

    PubMed Central

    Rocque, Brittany L.; Babayeva, Sima; Li, Jane; Leung, Vicki; Nezvitsky, Lisa; Cybulsky, Andrey V.; Gros, Philippe

    2015-01-01

    The planar cell polarity (PCP) signaling pathway is crucial for tissue morphogenesis. Van Gogh-like protein 2 (Vangl2) is central in the PCP pathway; in mice, Vangl2 loss is embryonically lethal because of neural tube defects, and mutations in Vangl2 are associated with human neural tube defects. In the kidney, PCP signaling may be important for tubular morphogenesis and organization of glomerular epithelial cells (podocytes) along the glomerular basement membrane. Podocyte cell protrusions (foot processes) are critical for glomerular permselectivity; loss of foot process architecture results in proteinuria and FSGS. Previously, we showed a profound effect of PCP signaling on podocyte shape, actin rearrangement, cell motility, and nephrin endocytosis. To test our hypothesis that the PCP pathway is involved in glomerular development and function and circumvent lethality of the ubiquitous Vangl2 mutation in the Looptail mouse, we generated a mouse model with a podocyte-specific ablation of the Vangl2 gene. We report here that podocyte-specific deletion of Vangl2 leads to glomerular maturation defects in fetal kidneys. In adult mice, we detected significantly smaller glomeruli, but it did not affect glomerular permselectivity in aging animals. However, in the context of glomerular injury induced by injection of antiglomerular basement membrane antibody, deletion of Vangl2 resulted in exacerbation of injury and accelerated progression to chronic segmental and global glomerular sclerosis. Our results indicate that Vangl2 function in podocytes is important for glomerular development and protects against glomerular injury in adult animals. PMID:25145929

  9. Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts

    PubMed Central

    Chacón-Díaz, Carlos; Altamirano-Silva, Pamela; González-Espinoza, Gabriela; Medina, María-Concepción; Alfaro-Alarcón, Alejandro; Bouza-Mora, Laura; Jiménez-Rojas, César; Wong, Melissa; Barquero-Calvo, Elías; Rojas, Norman; Guzmán-Verri, Caterina

    2015-01-01

    Canine brucellosis caused by Brucella canis is a disease of dogs and a zoonotic risk. B. canis harbors most of the virulence determinants defined for the genus, but its pathogenic strategy remains unclear since it has not been demonstrated that this natural rough bacterium is an intracellular pathogen. Studies of B. canis outbreaks in kennel facilities indicated that infected dogs displaying clinical signs did not present hematological alterations. A virulent B. canis strain isolated from those outbreaks readily replicated in different organs of mice for a protracted period. However, the levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-12 in serum were close to background levels. Furthermore, B. canis induced lower levels of gamma interferon, less inflammation of the spleen, and a reduced number of granulomas in the liver in mice than did B. abortus. When the interaction of B. canis with cells was studied ex vivo, two patterns were observed, a predominant scattered cell-associated pattern of nonviable bacteria and an infrequent intracellular replicative pattern of viable bacteria in a perinuclear location. The second pattern, responsible for the increase in intracellular multiplication, was dependent on the type IV secretion system VirB and was seen only if the inoculum used for cell infections was in early exponential phase. Intracellular replicative B. canis followed an intracellular trafficking route undistinguishable from that of B. abortus. Although B. canis induces a lower proinflammatory response and has a stealthier replication cycle, it still displays the pathogenic properties of the genus and the ability to persist in infected organs based on the ability to multiply intracellularly. PMID:26438796

  10. Brucella canis is an intracellular pathogen that induces a lower proinflammatory response than smooth zoonotic counterparts.

    PubMed

    Chacón-Díaz, Carlos; Altamirano-Silva, Pamela; González-Espinoza, Gabriela; Medina, María-Concepción; Alfaro-Alarcón, Alejandro; Bouza-Mora, Laura; Jiménez-Rojas, César; Wong, Melissa; Barquero-Calvo, Elías; Rojas, Norman; Guzmán-Verri, Caterina; Moreno, Edgardo; Chaves-Olarte, Esteban

    2015-12-01

    Canine brucellosis caused by Brucella canis is a disease of dogs and a zoonotic risk. B. canis harbors most of the virulence determinants defined for the genus, but its pathogenic strategy remains unclear since it has not been demonstrated that this natural rough bacterium is an intracellular pathogen. Studies of B. canis outbreaks in kennel facilities indicated that infected dogs displaying clinical signs did not present hematological alterations. A virulent B. canis strain isolated from those outbreaks readily replicated in different organs of mice for a protracted period. However, the levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-12 in serum were close to background levels. Furthermore, B. canis induced lower levels of gamma interferon, less inflammation of the spleen, and a reduced number of granulomas in the liver in mice than did B. abortus. When the interaction of B. canis with cells was studied ex vivo, two patterns were observed, a predominant scattered cell-associated pattern of nonviable bacteria and an infrequent intracellular replicative pattern of viable bacteria in a perinuclear location. The second pattern, responsible for the increase in intracellular multiplication, was dependent on the type IV secretion system VirB and was seen only if the inoculum used for cell infections was in early exponential phase. Intracellular replicative B. canis followed an intracellular trafficking route undistinguishable from that of B. abortus. Although B. canis induces a lower proinflammatory response and has a stealthier replication cycle, it still displays the pathogenic properties of the genus and the ability to persist in infected organs based on the ability to multiply intracellularly. PMID:26438796

  11. Glutamate-induced intracellular calcium oscillations in astrocytes with confocal microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zhu, Geng; Wu, Yuxiang; Luo, Qingming

    2006-02-01

    Changes in the intracellular Ca 2+ concentration ([Ca 2+]i) play a crucial role involved in the modulation of signal transduction, development, and plasticity in the CNS. Glial cells can respond to various stimuli with an increase in [Ca 2+]i. In this paper, we used confocal microscopy to study calcium transient induced by glutamate in cultured astrocytes. Firstly, 100 μM glutamate induced long-time intracellular calcium oscillations in astrocytes and only a single spike under calcium-free solution. When the concentration of glutamate decreased to 1 μM, only a single spike could be induced. It shows that intracellular calcium oscillations depend on agonist concentration and extracellular Ca 2+. Secondly, we investigated amplitude of responses under different stimulation. The amplitude of initial peak induced by 100 μM glutamate decreased in Ca 2+-free condition, whereas the duration of kinetics was prolonged. But both the amplitude and area of a single spike induced by 1 μM Glu decreased in Ca 2+-free condition. The results show that areaof peak is more accurate than amplitude to display transients of [Ca 2+]i. All results above suggest that astrocytes are not passive, they display diverse temporal and spatial increases in [Ca 2+]i in response to a variety of stimuli. These [Ca 2+]i increases provide a possible means for information coding.

  12. Podocytic cytoskeletal disaggregation and basement-membrane detachment in puromycin aminonucleoside nephrosis.

    PubMed Central

    Whiteside, C. I.; Cameron, R.; Munk, S.; Levy, J.

    1993-01-01

    Puromycin aminonucleoside--(PAN) treated rats develop acute nephrotic syndrome, mimicking human minimal lesion disease. In PAN nephrosis, podocyte detachment from the glomerular basement membrane (GBM) is the most likely cause of massive proteinuria in this model. To elucidate further the mechanisms of PAN-induced cellular dysfunction, new methods were employed to visualize podocyte cytoskeletal aggregation and to measure fibrillar attachment to the GBM. Adult Sprague-Dawley rats (n = 4/group) received a single tail-vein injection of PAN (75 mg/kg). On days 1, 2, 3, and 5 following injection, 24-hour urine collections were obtained for creatinine clearance, albuminuria, and total proteinuria. Then kidneys from each group were fixed by perfusion. Podocytic cytoskeleton was visualized by scanning electron microscopy. Subepithelial GBM staining and attachment fiber number, observed on digitized images of transmission electron micrographs, were quantitated with computer-based density analysis. A significant reduction in attachment fiber number in the GBM lamina rara externa occurred by day 5. On scanning electron micrographs, the secondary and tertiary podocytic processes were observed to be formed by highly aggregated cytoskeleton, which became partially disaggregated by day 3, was totally absent by day 5, and normalized by day 20. Immunogold staining revealed that actin and vinculin localized to the tertiary podocytic processes in the normal state were dispersed into the cell body following PAN. Podocyte cytoskeletal disaggregation precedes, and detachment from the GBM occurs simultaneously with, the onset of massive proteinuria in the PAN model. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:8494056

  13. Podocyte NF-κB is dispensable for the pathogenesis of renal ischemia-reperfusion injury.

    PubMed

    Yamashita, Maho; Yoshida, Tadashi; Hayashi, Matsuhiko

    2016-08-01

    Podocytes play a central role in the formation of the glomerular filtration barrier in the kidney, and their dysfunction has been shown to result in multiple proteinuric kidney diseases. In this study, we sought to determine whether NF-κB, a proinflammatory signaling, within podocytes was involved in renal ischemia-reperfusion (I/R) injury. Podocyte-specific IκBΔN transgenic (Pod-IκBΔN) mice, in which NF-κB was inhibited specifically in podocytes, were generated by the Cre-loxP technology, and their phenotype was compared with control mice after bilateral renal ischemia. The effect of systemic administration of a NF-κB inhibitor, pyrrolidinedithiocarbamate (PDTC), on renal I/R injury was also examined. Pod-IκBΔN mice were phenotypically normal before surgery. Following renal I/R injury, serum concentrations of urea nitrogen and creatinine were elevated in both Pod-IκBΔN and control mice to a similar extent, whereas PDTC treatment attenuated the elevation of these parameters. Renal histological damage in I/R-injured Pod-IκBΔN mice was also similar to I/R-injured control mice, although it was improved by PDTC treatment. Moreover, I/R induced accumulation of inflammatory cells, such as neutrophils and macrophages, was reduced by PDTC treatment, but not by podocyte-specific NF-κB inhibition. These results provide evidence that the NF-κB activity in podocytes does not contribute to the pathogenesis of renal I/R injury. PMID:27565904

  14. Association of Preeclampsia with Podocyte Turnover

    PubMed Central

    Bloemenkamp, Kitty W.M.; van der Zon, Tom; Zandbergen, Malu; Schutte, Joke M.; Bruijn, Jan A.; Bajema, Ingeborg M.; Baelde, Hans J.

    2014-01-01

    Background and objectives Preeclampsia is characterized by hypertension and proteinuria, and increased shedding of podocytes into the urine is a common finding. This finding raises the question of whether preeclamptic nephropathy involves podocyte damage. This study examined podocyte-related changes in a unique sample of renal tissues obtained from women who died of preeclampsia. Design, setting, participants, & measurements All patients with preeclampsia who died in The Netherlands since 1990 and had available autopsy tissue were identified using a nationwide database of the Dutch Pathology Registry (PALGA). This resulted in a cohort of 11 women who died from preeclampsia. Three control groups were also identified during the same time period, and consisted of normotensive women who died during pregnancy (n=25), and nonpregnant controls either with (n=14) or without (n=13) chronic hypertension. Glomerular lesions, including podocyte numbers, podocyte proliferation, and parietal cell activation, were measured. Results Patients with preeclampsia had prominent characteristic glomerular lesions. The results showed that the number of podocytes per glomerulus did not differ significantly between the patients with preeclampsia and the control groups. However, preeclampsia was associated with a significant increase in intraglomerular cell proliferation (7.3% [SD 9.4] of the glomeruli of patients with preeclampsia had Ki-67–positive cells versus 1.6% [SD 3.3] of the glomeruli of hypertensive controls and 1.1% [SD 1.3] of nonpregnant controls; P=0.004) and activated parietal epithelial cells on a podocyte location (34% [SD 13.1] of the glomeruli of patients with preeclampsia versus 18.0% [SD 15.3] of pregnant controls, 11.9% [SD 13.2] of hypertensive controls, and 10.8% [SD 13.4] of nonpregnant controls; P=0.01). Conclusions These findings suggest that the recently described mechanisms of podocyte replacement play a role in preeclampsia. These results provide key new

  15. Mitophagy induced by nanoparticle-peptide conjugates enabling an alternative intracellular trafficking route.

    PubMed

    Zhang, Zhaolei; Zhou, Lei; Zhou, Yanqing; Liu, Jinyin; Xing, Xiaoyun; Zhong, Jun; Xu, Guoqiang; Kang, Zhenhui; Liu, Jian

    2015-10-01

    The intracellular behaviors of nanoparticles are fundamentally important for the evaluation of their biosafety and the designs of nano carrier-assisted drug delivery with high therapeutic efficacy. It is still in a great need to discover how functionalized nanoparticles are transported inside the cells, for instance, in a complicated fashion of translocation between different types of cell organelles. Here we report a new understanding of the interactions between nanoparticles and cells by the development of polyoxometalates nanoparticle-peptide conjugates and investigation of their intracellular trafficking behaviors. The as-prepared nanoparticles are featured with a unique combination of fluorescence and high contrast for synchrotron X-ray-based imaging. Functional surface modification with peptides facilitates effective delivery of the nanoparticles onto the target organelle (mitochondria) and subsequent intracellular trafficking in a dynamic mode. Interestingly, our experimental results have revealed that autophagy of mitochondria (mitophagy) can be induced by NP-peptide as a cellular response for recycling the damaged organelles, through molecular mediation associated with the change of mitochondrial membrane potential. The biological effects induced by NP-peptide reciprocally affect the distribution patterns and fates of nanoparticles in the cell metabolism by providing an alternative route of intracellular trafficking. The new understanding of the mutual activities between nanoparticles and cells will enrich our approaches in the development of nanobiotechnology and nano-medicine for disease treatments. PMID:26142776

  16. Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels.

    PubMed

    Yang, Xinhui; Jiang, Jiangtao; Yang, Xinyan; Han, Jichun; Zheng, Qiusheng

    2016-07-01

    Licochalcone A (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER) stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamine B assay. Apoptosis was detected by flow cytometry with Annexin V/propidium iodide staining, and by fluorescent microscopy with Hoechst 33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B‑cell lymphoma (Bcl)‑extra large, Bcl‑2‑associated X protein, Bcl‑2‑interacting mediator of cell death, apoptotic protease activating factor‑1 (Apaf‑1), calpain 2, cysteinyl aspartate specific proteinase (caspase)‑3, caspase‑4 and caspase‑9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf‑1, caspase‑9 and caspase‑3. Exposure of T24 cells to LCA also triggered calpain 2 and caspase‑4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be

  17. Cadmium-induced cell killing in Sacharomyces cerevisiae involves increases in intracellular NO levels.

    PubMed

    Wu, Lihua; Chen, Yanfei; Gao, Huixian; Yin, Jingjing; Huang, Liqun

    2016-03-01

    Cadmium is a widespread environmental pollutant and poses some potential risks to human health. However, the signaling events controlling cadmium toxicity are not fully understood. In this study, we examined the effect of cadmium chloride on cell viability and the intracellular nitric oxide (NO) level in yeast cells. The results showed that exposure of yeast cells to cadmium (0-100 μM) could induce cell killing with significantly increased intracellular NO levels. Morphological analysis of the nuclei with 4('),6-diamidino-2-phenylindole staining and DNA strand breaks analysis showed that cadmium at 50 μM can induce cell apoptosis in yeast cells. Treatment of yeast cells with cadmium (50 μM) and the nitric oxide scavenger c-PTIO [2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-1-oxyl-3-oxide; 0.2 mM] showed that c-PTIO attenuated the cadmium-induced cell killing. Our findings indicated that cadmium-induced yeast cell killing is mediated by a directly increased intracellular NO level. PMID:26872495

  18. Podocyte Depletion in Thin GBM and Alport Syndrome

    PubMed Central

    Wang, Su Q.; Afshinnia, Farsad; Kershaw, David; Wiggins, Roger C.

    2016-01-01

    The proximate genetic cause of both Thin GBM and Alport Syndrome (AS) is abnormal α3, 4 and 5 collagen IV chains resulting in abnormal glomerular basement membrane (GBM) structure/function. We previously reported that podocyte detachment rate measured in urine is increased in AS, suggesting that podocyte depletion could play a role in causing progressive loss of kidney function. To test this hypothesis podometric parameters were measured in 26 kidney biopsies from 21 patients aged 2–17 years with a clinic-pathologic diagnosis including both classic Alport Syndrome with thin and thick GBM segments and lamellated lamina densa [n = 15] and Thin GBM cases [n = 6]. Protocol biopsies from deceased donor kidneys were used as age-matched controls. Podocyte depletion was present in AS biopsies prior to detectable histologic abnormalities. No abnormality was detected by light microscopy at <30% podocyte depletion, minor pathologic changes (mesangial expansion and adhesions to Bowman’s capsule) were present at 30–50% podocyte depletion, and FSGS was progressively present above 50% podocyte depletion. eGFR did not change measurably until >70% podocyte depletion. Low level proteinuria was an early event at about 25% podocyte depletion and increased in proportion to podocyte depletion. These quantitative data parallel those from model systems where podocyte depletion is the causative event. This result supports a hypothesis that in AS podocyte adherence to the GBM is defective resulting in accelerated podocyte detachment causing progressive podocyte depletion leading to FSGS-like pathologic changes and eventual End Stage Kidney Disease. Early intervention to reduce podocyte depletion is projected to prolong kidney survival in AS. PMID:27192434

  19. Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development, Function or Stress Response.

    PubMed

    Schell, Christoph; Kretz, Oliver; Bregenzer, Andreas; Rogg, Manuel; Helmstädter, Martin; Lisewski, Ulrike; Gotthardt, Michael; Tharaux, Pierre-Louis; Huber, Tobias B; Grahammer, Florian

    2015-01-01

    The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive Cxadr knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, Cxadr is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of Cxadr for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific Cxadr knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of Cxadr on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of Cxadr. PMID:26076477

  20. Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development, Function or Stress Response

    PubMed Central

    Schell, Christoph; Kretz, Oliver; Bregenzer, Andreas; Rogg, Manuel; Helmstädter, Martin; Lisewski, Ulrike; Gotthardt, Michael; Tharaux, Pierre-Louis; Huber, Tobias B.; Grahammer, Florian

    2015-01-01

    The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive Cxadr knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, Cxadr is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of Cxadr for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific Cxadr knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of Cxadr on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of Cxadr. PMID:26076477

  1. Intracellular monitoring of target protein production in Staphylococcus aureus by peptide tag‐induced reporter fluorescence

    PubMed Central

    Gauger, Tina; Weihs, Felix; Mayer, Sonja; Krismer, Bernhard; Liese, Jan; Kull, Melanie; Bertram, Ralph

    2012-01-01

    Summary An intracellular approach for monitoring protein production in Staphylococcus aureus is described. mCherry, fused to the dodecapeptide Tip, was capable of inducing tetracycline repressor (TetR). Time‐ and concentration‐dependent production of mCherry could be correlated to TetR‐controlled GFPmut2 activity. This approach can potentially be extended to native S. aureus proteins. PMID:21958360

  2. The inhibitory effect of angiotensin II on BKCa channels in podocytes via oxidative stress.

    PubMed

    Gao, Na; Wang, Hui; Zhang, Xiaochen; Yang, Zhuo

    2015-01-01

    Angiotensin II (Ang II) is an important active substance of the renin-angiotensin system (RAS). The present study has confirmed that abnormalities of Ang II may be related with cerebrovascular diseases, endocrine diseases, cardiovascular diseases, liver diseases, such as: cerebral hypoxia, diabetes, obesity, atrial fibrillation, and liver cirrhosis. However, understanding effects of Ang II on podocytes is not enough. This study was to investigate the effects of oxidative stress on the large conductance, Ca(2+)-activated K(+) channels (BKCa). Results from the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay showed that Ang II induced podocyte death in a concentration-dependent manner. The measurement of superoxide dismutase (SOD) generation demonstrated that Ang II decreased the total SOD of cellular levels. Meaningfully, pretreatment of a type of ROS scavenger formulations named N-(mercaptopropionyl)-glycine (N-MPG) could inhibit podocyte apoptosis induced by Ang II. Meanwhile, patch-clamp technique was used in this study to detect the effects of Ang II on currents of BKCa channel in podocytes. The results indicated that Ang II inhibited the current amplitude of BKCa channel and decreased the slope of I-V curve. Ang II also made the activation curves of BKCa channel shift to the left. These results may provide a theoretical basis for potential treatment of chronic glomerular disease in the future. PMID:25234195

  3. Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

    PubMed Central

    George, Britta; Verma, Rakesh; Soofi, Abdulsalam A.; Garg, Puneet; Zhang, Jidong; Park, Tae-Ju; Giardino, Laura; Ryzhova, Larisa; Johnstone, Duncan B.; Wong, Hetty; Nihalani, Deepak; Salant, David J.; Hanks, Steven K.; Curran, Tom; Rastaldi, Maria Pia; Holzman, Lawrence B.

    2012-01-01

    The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation — markers of focal adhesion complex–mediated Crk-dependent signaling — was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2–dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases. PMID:22251701

  4. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. PMID:26667172

  5. Susceptibility of Podocytes to Palmitic Acid Is Regulated by Stearoyl-CoA Desaturases 1 and 2

    PubMed Central

    Sieber, Jonas; Weins, Astrid; Kampe, Kapil; Gruber, Stefan; Lindenmeyer, Maja T.; Cohen, Clemens D.; Orellana, Jana M.; Mundel, Peter; Jehle, Andreas W.

    2014-01-01

    Type 2 diabetes mellitus is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are highly susceptible to saturated FFAs but not to protective, monounsaturated FFAs. We report that patients with diabetic nephropathy develop alterations in glomerular gene expression of enzymes involved in fatty acid metabolism, including induction of stearoyl-CoA desaturase (SCD)-1, which converts saturated to monounsaturated FFAs. By IHC of human renal biopsy specimens, glomerular SCD-1 induction was observed in podocytes of patients with diabetic nephropathy. Functionally, the liver X receptor agonists TO901317 and GW3965, two known inducers of SCD, increased Scd-1 and Scd-2 expression in cultured podocytes and reduced palmitic acid–induced cell death. Similarly, overexpression of Scd-1 attenuated palmitic acid–induced cell death. The protective effect of TO901317 was associated with a reduction of endoplasmic reticulum stress. It was lost after gene silencing of Scd-1/-2, thereby confirming that the protective effect of TO901317 is mediated by Scd-1/-2. TO901317 also shifted palmitic acid–derived FFAs into biologically inactive triglycerides. In summary, SCD-1 up-regulation in diabetic nephropathy may be part of a protective mechanism against saturated FFA-derived toxic metabolites that drive endoplasmic reticulum stress and podocyte death. PMID:23867797

  6. Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro

    PubMed Central

    Canfrán-Duque, Alberto; Pastor, Oscar; Reina, Manuel; Lerma, Milagros; Cruz-Jentoft, Alfonso J.

    2015-01-01

    Scope First- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation. Methods HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation. Results Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics. Conclusion Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials. PMID:26517556

  7. Asterosap-induced elevation in intracellular pH is indispensable for ARIS-induced sustained increase in intracellular Ca2+ and following acrosome reaction in starfish spermatozoa.

    PubMed

    Kawase, Osamu; Minakata, Hiroyuki; Hoshi, Motonori; Matsumoto, Midori

    2005-02-01

    In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6 +/- 0.1 to 7.7 +/- 0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction. PMID:15984164

  8. Ba2+ induces contraction in swine carotid artery by mobilizing intracellular Ca2+.

    PubMed

    Hai, C M; Murphy, R A

    1987-04-01

    Recent data suggest that Ba2+ activates the smooth muscle contractile apparatus. However, in vitro studies indicate that Ba2+ activates Ca2+ calmodulin-dependent enzymes only at very high concentrations. These observations might suggest that Ba2+ activates the contractile apparatus by a mechanism independent of myosin phosphorylation. We tested this hypothesis using intact and Triton X-100 skinned swine carotid medial strips. In intact tissues, Ba2+ stimulated dose-dependent contractions in the absence of extracellular Ca2+. However, in skinned tissues, Ba2+ induced much lower stress development at concentrations more than three orders of magnitude greater than needed for Ca2+-induced contraction. Ba2+ also failed to maintain stress in skinned tissues previously contracted with 4 microM Ca2+. The Ba2+-induced contraction in intact strips was associated with gradual increases in myosin phosphorylation from a basal level of approximately 5% to a sustained level of 30.0 +/- 0.8%. Partial intracellular calcium depletion with 100 microM histamine, 25 mM caffeine, and 5 mM ethyleneglycol-bis(beta-aminoethyl-ether)-N,N'-tetraacetic acid (EGTA) failed to abolish Ba2+-induced contractions. However, repeated contractions with Ba2+ in the absence of extracellular Ca2+ led to decreases in stress development. These observations suggest that Ba2+ induces stress development by mobilizing intracellular Ca2+ that was not released by histamine and caffeine. PMID:3565557

  9. Non-thermal Plasma Induces Apoptosis in Melanoma Cells via Production of Intracellular Reactive Oxygen Species

    PubMed Central

    Sensenig, Rachel; Kalghatgi, Sameer; Cerchar, Ekaterina; Fridman, Gregory; Shereshevsky, Alexey; Torabi, Behzad; Arjunan, Krishna Priya; Podolsky, Erica; Fridman, Alexander; Friedman, Gary; Azizkhan-Clifford, Jane; Brooks, Ari D.

    2012-01-01

    Non-thermal atmospheric pressure dielectric barrier discharge (DBD) plasma may provide a novel approach to treat malignancies via induction of apoptosis. The purpose of this study was to evaluate the potential of DBD plasma to induce apoptosis in melanoma cells. Melanoma cells were exposed to plasma at doses that did not induce necrosis, and cell viability and apoptotic activity were evaluated by Trypan blue exclusion test, Annexin-V/PI staining, caspase-3 cleavage, and TUNEL® analysis. Trypan blue staining revealed that non-thermal plasma treatment significantly decreased the viability of cells in a dose-dependent manner 3 and 24 h after plasma treatment. Annexin-V/PI staining revealed a significant increase in apoptosis in plasma-treated cells at 24, 48, and 72 h post-treatment (p<0.001). Caspase-3 cleavage was observed 48 h post-plasma treatment at a dose of 15 J/cm2. TUNEL® analysis of plasma-treated cells demonstrated an increase in apoptosis at 48 and 72 h post-treatment (p<0.001) at a dose of 15 J/cm2. Pre-treatment with N-acetyl-L-cysteine (NAC), an intracellular reactive oxygen species (ROS) scavenger, significantly decreased apoptosis in plasma-treated cells at 5 and 15 J/cm2. Plasma treatment induces apoptosis in melanoma cells through a pathway that appears to be dependent on production of intracellular ROS. DBD plasma production of intracellular ROS leads to dose-dependent DNA damage in melanoma cells, detected by γ-H2AX, which was completely abrogated by pre-treating cells with ROS scavenger, NAC. Plasma-induced DNA damage in turn may lead to the observed plasma-induced apoptosis. Since plasma is non-thermal, it may be used to selectively treat malignancies. PMID:21046465

  10. Intracellular NAD+ depletion enhances bortezomib-induced anti-myeloma activity

    PubMed Central

    Cagnetta, Antonia; Calimeri, Teresa; Acharya, Chirag; Fulciniti, Mariateresa; Tai, Yu-Tzu; Hideshima, Teru; Chauhan, Dharminder; Zhong, Mike Y.; Patrone, Franco; Nencioni, Alessio; Gobbi, Marco; Richardson, Paul; Munshi, Nikhil

    2013-01-01

    We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD+ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD+ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD+ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD+ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib. PMID:23823317

  11. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    PubMed

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. PMID:24136144

  12. EBP50 induces apoptosis in macrophages by upregulating nitric oxide production to eliminate intracellular Mycobacterium tuberculosis

    PubMed Central

    Guo, Yang; Deng, Yating; Huang, Zikun; Luo, Qing; Peng, Yiping; Chen, Jie; Jiang, Hong; Ye, Jianqing; Li, Junming

    2016-01-01

    Mycobacterium bovis BCG is known to have the capacity to inhibit the positioning of iNOS on BCG-containing phagosomes by interfering with EBP50, a scaffolding protein that controls the recruitment of inducible nitric oxide synthase (iNOS) at the vicinity of phagosomes in macrophages. However, knockdown of the expression of EBP50 still facilitates the intracellular survival of BCG, which suggested that EBP50 may have some other unknown antimycobacterial properties. In this study we show that overexpression of EBP50 by a recombinant lentivirus had no effect on the iNOS recruitment to M.tuberculosis-containing phagosomes, but significantly promoted the elimination of intracellular M.tuberculosis. We revealed in the present study that the enhancement of intracellular killing to M. tuberculosis upon EBP50 overexpression was due to the increased level of apoptosis in macrophages. We showed that EBP50 overexpression significantly increased the expression of iNOS and generation of nitric oxide (NO), and EBP50-induced apoptosis was NO-dependent and mediated by Bax and caspase-3. We found that M. tuberculosis decreases while Mycobacterium smegmatis increases the expression of EBP50 in RAW264.7 cells, which suggested that virulent mycobacteria are capable of modulating the antimycobacterial properties of macrophages by inhibiting the expression and interfering with the function of EBP50. PMID:26729618

  13. Intracellular NAD⁺ depletion enhances bortezomib-induced anti-myeloma activity.

    PubMed

    Cagnetta, Antonia; Cea, Michele; Calimeri, Teresa; Acharya, Chirag; Fulciniti, Mariateresa; Tai, Yu-Tzu; Hideshima, Teru; Chauhan, Dharminder; Zhong, Mike Y; Patrone, Franco; Nencioni, Alessio; Gobbi, Marco; Richardson, Paul; Munshi, Nikhil; Anderson, Kenneth C

    2013-08-15

    We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD⁺ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD⁺ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD⁺ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD⁺ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib. PMID:23823317

  14. Nickel Mobilizes Intracellular Zinc to Induce Metallothionein in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Leikauf, George D.; Pitt, Bruce R.; Wasserloos, Karla J.; Barchowsky, Aaron

    2009-01-01

    We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release. PMID:19097988

  15. β-Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

    PubMed Central

    Liu, J; Li, Q X; Wang, X J; Zhang, C; Duan, Y Q; Wang, Z Y; Zhang, Y; Yu, X; Li, N J; Sun, J P; Yi, F

    2016-01-01

    β-Arrestins are multifunctional proteins originally identified as negative adaptors of G protein-coupled receptors (GPCRs). Emerging evidence has also indicated that β-arrestins can activate signaling pathways independent of GPCR activation. This study was to elucidate the role of β-arrestins in diabetic nephropathy (DN) and hypothesized that β-arrestins contribute to diabetic renal injury by mediating podocyte autophagic process. We first found that both β-arrestin-1 and β-arrestin-2 were upregulated in the kidney from streptozotocin-induced diabetic mice, diabetic db/db mice and kidney biopsies from diabetic patients. We further revealed that either β-arrestin-1 or β-arrestin-2 deficiency (Arrb1−/− or Arrb2−/−) ameliorated renal injury in diabetic mice. In vitro, we observed that podocytes increased both β-arrestin-1 and β-arrestin-2 expression levels under hyperglycemia condition and further demonstrated that β-arrestin-1 and β-arrestin-2 shared common mechanisms to suppress podocyte autophagy by negative regulation of ATG12–ATG5 conjugation. Collectively, this study for the first time demonstrates that β-arrestin-1 and β-arrestin-2 mediate podocyte autophagic activity, indicating that β-arrestins are critical components of signal transduction pathways that link renal injury to reduce autophagy in DN. Modulation of these pathways may be an innovative therapeutic strategy for treating patients with DN. PMID:27054338

  16. Reactive oxygen species are involved in insulin-dependent regulation of autophagy in primary rat podocytes.

    PubMed

    Audzeyenka, Irena; Rogacka, Dorota; Piwkowska, Agnieszka; Rychlowski, Michal; Bierla, Joanna Beata; Czarnowska, Elżbieta; Angielski, Stefan; Jankowski, Maciej

    2016-06-01

    Autophagy is an intracellular defense mechanism responsible for the turnover of damaged or non-functional cellular constituents. This process provides cells with energy and essential compounds under unfavorable environmental conditions-such as oxidative stress and hyperglycemia, which are both observed in diabetes. The most common diabetes complication is diabetic nephropathy (DN), which can lead to renal failure. This condition often includes impaired podocyte function. Here we investigated autophagic activity in rat podocytes cultured with a high insulin concentration (300nM). Autophagy was activated after 60min of insulin stimulation. Moreover, this effect was abolished following pharmacological (apocynin) or genetic (siRNA) inhibition of NAD(P)H oxidase activity, indicating that insulin-dependent autophagy stimulation involved reactive oxygen species (ROS). We also observed a continuous and time-dependent increase of podocyte albumin permeability in response to insulin, and this process was slightly improved by autophagy inhibition following short-term insulin exposure. Our results suggest that insulin may be a factor affecting the development of diabetic nephropathy. PMID:27026581

  17. Human podocytes perform polarized, caveolae-dependent albumin endocytosis

    PubMed Central

    Dobrinskikh, Evgenia; Okamura, Kayo; Kopp, Jeffrey B.; Doctor, R. Brian

    2014-01-01

    The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes. PMID:24573386

  18. The Expression and Significance of Neuronal Iconic Proteins in Podocytes

    PubMed Central

    Sun, Yu; Zhang, Hongxia; Hu, Ruimin; Sun, Jianyong; Mao, Xing; Zhao, Zhonghua; Chen, Qi; Zhang, Zhigang

    2014-01-01

    Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes. PMID:24699703

  19. Intracellular accumulation of indium ions released from nanoparticles induces oxidative stress, proinflammatory response and DNA damage.

    PubMed

    Tabei, Yosuke; Sonoda, Akinari; Nakajima, Yoshihiro; Biju, Vasudevanpillai; Makita, Yoji; Yoshida, Yasukazu; Horie, Masanori

    2016-02-01

    Due to the widespread use of indium tin oxide (ITO), it is important to investigate its effect on human health. In this study, we evaluated the cellular effects of ITO nanoparticles (NPs), indium chloride (InCl3) and tin chloride (SnCl3) using human lung epithelial A549 cells. Transmission electron microscopy and inductively coupled plasma mass spectrometry were employed to study cellular ITO NP uptake. Interestingly, greater uptake of ITO NPs was observed, as compared with soluble salts. ITO NP species released could be divided into two types: 'indium release ITO' or 'tin release ITO'. We incubated A549 cells with indium release ITO, tin release ITO, InCl3 or SnCl2 and investigated oxidative stress, proinflammatory response, cytotoxicity and DNA damage. We found that intracellular reactive oxygen species were increased in cells incubated with indium release ITO, but not tin release ITO, InCl3 or SnCl2. Messenger RNA and protein levels of the inflammatory marker, interleukin-8, also increased following exposure to indium release ITO. Furthermore, the alkaline comet assay revealed that intracellular accumulation of indium ions induced DNA damage. Our results demonstrate that the accumulation of ionic indium, but not ionic tin, from ITO NPs in the intracellular matrix has extensive cellular effects. PMID:26378248

  20. A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death*

    PubMed Central

    de Araujo, Christiane B.; Russo, Lilian C.; Castro, Leandro M.; Forti, Fábio L.; do Monte, Elisabete R.; Rioli, Vanessa; Gozzo, Fabio C.; Colquhoun, Alison; Ferro, Emer S.

    2014-01-01

    Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides. PMID:24764300

  1. Application of confocal microscopy on glutamate-induced intracellular calcium transient in neurons

    NASA Astrophysics Data System (ADS)

    Zhu, Geng; Zhou, Wei; Zhang, Yuan; Liu, Xiuli; Wu, Yuxiang; Luo, Qingming

    2006-02-01

    Intracellular calcium, as an important second messenger, plays a significant role in cell signaling transduction and metabolism. Glutamate can induce the intracellular calcium transient through triggering diverse signaling pathways. To test the effect of glutamate to neurons, we loaded Fluo-3/Am in cultured rat hippocampal neurons, and then acquired two-dimensional fluorescent image by confocal microscopy and the analyzed fluorescent intensity. In cultured neurons, we observed two types of neurons that have different morphology: bipolar-type and pyramidal-type. Inducing [Ca 2+] i transient by glutamate, we found the amplitude and time constant of the response curves of bipolar neurons are larger than those of pyramidal neurons. Further, we induced [Ca 2+] ii transient under different concentrations of glutamate. Two different types of kinetic of the [Ca 2+] i transient have been found, corresponded to the two kinds of neuron. The amplitude of [Ca 2+] i transient increased when applying higher concentration of glutamate in pyramidal neurons; while it decreased in bipolar ones. Responses of neurons bathing in calcium-free extracellular solution to glutamate were different from those bathing in normal solution. [Ca 2+] i transient of pyramidal neurons caused by any concentration were totally blocked; while [Ca 2+] i transient in bipolar neurons caused by high concentration of glutamate (500μM) were partly inhibited. All of the phenomena suggest that different types of cultured hippocampal neurons may have different mechanism of the response to glutamate.

  2. Polymerization-Induced Self-Assembly of Galactose-Functionalized Biocompatible Diblock Copolymers for Intracellular Delivery

    PubMed Central

    2013-01-01

    Recent advances in polymer science are enabling substantial progress in nanobiotechnology, particularly in the design of new tools for enhanced understanding of cell biology and for smart drug delivery formulations. Herein, a range of novel galactosylated diblock copolymer nano-objects is prepared directly in concentrated aqueous solution via reversible addition–fragmentation chain transfer polymerization using polymerization-induced self-assembly. The resulting nanospheres, worm-like micelles, or vesicles interact in vitro with galectins as judged by a turbidity assay. In addition, galactosylated vesicles are highly biocompatible and allow intracellular delivery of an encapsulated molecular cargo. PMID:23941545

  3. Polymerization-induced self-assembly of galactose-functionalized biocompatible diblock copolymers for intracellular delivery.

    PubMed

    Ladmiral, Vincent; Semsarilar, Mona; Canton, Irene; Armes, Steven P

    2013-09-11

    Recent advances in polymer science are enabling substantial progress in nanobiotechnology, particularly in the design of new tools for enhanced understanding of cell biology and for smart drug delivery formulations. Herein, a range of novel galactosylated diblock copolymer nano-objects is prepared directly in concentrated aqueous solution via reversible addition-fragmentation chain transfer polymerization using polymerization-induced self-assembly. The resulting nanospheres, worm-like micelles, or vesicles interact in vitro with galectins as judged by a turbidity assay. In addition, galactosylated vesicles are highly biocompatible and allow intracellular delivery of an encapsulated molecular cargo. PMID:23941545

  4. Gravity-induced changes in intracellular potentials in elongating cortical cells of mung bean roots

    NASA Technical Reports Server (NTRS)

    Ishikawa, H.; Evans, M. L.

    1990-01-01

    Gravity-induced changes in intracellular potentials in primary roots of 2-day-old mung bean (Vigna mungo L. cv. black matpe) seedlings were investigated using glass microelectrodes held by 3-dimensional hydraulic micro-drives. The electrodes were inserted into outer cortical cells within the elongation zone. Intracellular potentials, angle of root orientation with respect to gravity, and position within the root of the impaled cortical cell were measured simultaneously. Gravistimulation caused intracellular potential changes in cortical cells of the elongation zone. When the roots were oriented vertically, the intracellular potentials of the outer cortical cells (2 mm behind the root apex) were approximately - 115 mV. When the roots were placed horizontally cortical cells on the upper side hyperpolarized to - 154 mV within 30 s while cortical cells on the lower side depolarized to about - 62 mV. This electrical asymmetry did not occur in cells of the maturation zone. Because attempts to insert the electrode into cells of the root cap were unsuccessful, these cells were not measured. The hyperpolarization of cortical cells on the upper side was greatly reduced upon application of N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of respiratory energy coupling. When stimulated roots were returned to the vertical, the degree of hyperpolarization of cortical cells on the previous upper side decreased within 30 s and approached that of cortical cells in non-stimulated roots. This cycle of hyperpolarization/loss of hyperpolarization was repeatable at least ten times by alternately turning the root from the vertical to the horizontal and back again. The very short (<30 s) lag period of these electrical changes indicates that they may result from stimulus-perception and transduction within the elongation zone rather than from transmission of a signal from the root cap.

  5. Gravity-induced changes in intracellular potentials in elongating cortical cells of mung bean roots.

    PubMed

    Ishikawa, H; Evans, M L

    1990-06-01

    Gravity-induced changes in intracellular potentials in primary roots of 2-day-old mung bean (Vigna mungo L. cv. black matpe) seedlings were investigated using glass microelectrodes held by 3-dimensional hydraulic micro-drives. The electrodes were inserted into outer cortical cells within the elongation zone. Intracellular potentials, angle of root orientation with respect to gravity, and position within the root of the impaled cortical cell were measured simultaneously. Gravistimulation caused intracellular potential changes in cortical cells of the elongation zone. When the roots were oriented vertically, the intracellular potentials of the outer cortical cells (2 mm behind the root apex) were approximately - 115 mV. When the roots were placed horizontally cortical cells on the upper side hyperpolarized to - 154 mV within 30 s while cortical cells on the lower side depolarized to about - 62 mV. This electrical asymmetry did not occur in cells of the maturation zone. Because attempts to insert the electrode into cells of the root cap were unsuccessful, these cells were not measured. The hyperpolarization of cortical cells on the upper side was greatly reduced upon application of N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of respiratory energy coupling. When stimulated roots were returned to the vertical, the degree of hyperpolarization of cortical cells on the previous upper side decreased within 30 s and approached that of cortical cells in non-stimulated roots. This cycle of hyperpolarization/loss of hyperpolarization was repeatable at least ten times by alternately turning the root from the vertical to the horizontal and back again. The very short (<30 s) lag period of these electrical changes indicates that they may result from stimulus-perception and transduction within the elongation zone rather than from transmission of a signal from the root cap. PMID:11537168

  6. Loss of the Podocyte-Expressed Transcription Factor Tcf21/Pod1 Results in Podocyte Differentiation Defects and FSGS

    PubMed Central

    Maezawa, Yoshiro; Onay, Tuncer; Scott, Rizaldy P.; Keir, Lindsay S.; Dimke, Henrik; Li, Chengjin; Eremina, Vera; Maezawa, Yuko; Jeansson, Marie; Shan, Jingdong; Binnie, Matthew; Lewin, Moshe; Ghosh, Asish; Miner, Jeffrey H.; Vainio, Seppo J.

    2014-01-01

    Podocytes are terminally differentiated cells with an elaborate cytoskeleton and are critical components of the glomerular barrier. We identified a bHLH transcription factor, Tcf21, that is highly expressed in developing and mature podocytes. Because conventional Tcf21 knockout mice die in the perinatal period with major cardiopulmonary defects, we generated a conditional Tcf21 knockout mouse to explore the role of this transcription factor in podocytes in vivo. Tcf21 was deleted from podocytes and podocyte progenitors using podocin-cre (podTcf21) and wnt4-cre (wnt4creTcf21) driver strains, respectively. Loss of Tcf21 from capillary-loop stage podocytes (podTcf21) results in simplified glomeruli with a decreased number of endothelial and mesangial cells. By 5 weeks of age, 40% of podTcf21 mice develop massive proteinuria and lesions similar to FSGS. Notably, the remaining 60% of mice do not develop proteinuria even when aged to 8 months. By contrast, earlier deletion of Tcf21 from podocyte precursors (wnt4creTcf21) results in a profound developmental arrest of podocyte differentiation and renal failure in 100% of mice during the perinatal period. Taken together, our results demonstrate a critical role for Tcf21 in the differentiation and maintenance of podocytes. Identification of direct targets of this transcription factor may provide new therapeutic avenues for proteinuric renal disease, including FSGS. PMID:24904088

  7. Loss of the podocyte-expressed transcription factor Tcf21/Pod1 results in podocyte differentiation defects and FSGS.

    PubMed

    Maezawa, Yoshiro; Onay, Tuncer; Scott, Rizaldy P; Keir, Lindsay S; Dimke, Henrik; Li, Chengjin; Eremina, Vera; Maezawa, Yuko; Jeansson, Marie; Shan, Jingdong; Binnie, Matthew; Lewin, Moshe; Ghosh, Asish; Miner, Jeffrey H; Vainio, Seppo J; Quaggin, Susan E

    2014-11-01

    Podocytes are terminally differentiated cells with an elaborate cytoskeleton and are critical components of the glomerular barrier. We identified a bHLH transcription factor, Tcf21, that is highly expressed in developing and mature podocytes. Because conventional Tcf21 knockout mice die in the perinatal period with major cardiopulmonary defects, we generated a conditional Tcf21 knockout mouse to explore the role of this transcription factor in podocytes in vivo. Tcf21 was deleted from podocytes and podocyte progenitors using podocin-cre (podTcf21) and wnt4-cre (wnt4creTcf21) driver strains, respectively. Loss of Tcf21 from capillary-loop stage podocytes (podTcf21) results in simplified glomeruli with a decreased number of endothelial and mesangial cells. By 5 weeks of age, 40% of podTcf21 mice develop massive proteinuria and lesions similar to FSGS. Notably, the remaining 60% of mice do not develop proteinuria even when aged to 8 months. By contrast, earlier deletion of Tcf21 from podocyte precursors (wnt4creTcf21) results in a profound developmental arrest of podocyte differentiation and renal failure in 100% of mice during the perinatal period. Taken together, our results demonstrate a critical role for Tcf21 in the differentiation and maintenance of podocytes. Identification of direct targets of this transcription factor may provide new therapeutic avenues for proteinuric renal disease, including FSGS. PMID:24904088

  8. Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells

    PubMed Central

    Bayles, Kenneth W.; Wesson, Carla A.; Liou, Linda E.; Fox, Lawrence K.; Bohach, Gregory A.; Trumble, W. R.

    1998-01-01

    We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes. PMID:9423876

  9. Protective role of intracellular glutathione against ethanol-induced damage in cultured rat gastric mucosal cells

    SciTech Connect

    Mutoh, H.; Hiraishi, H.; Ota, S.; Yoshida, H.; Ivey, K.J.; Terano, A.; Sugimoto, T. )

    1990-06-01

    This study investigated whether intracellular glutathione is cytoprotective against ethanol-induced injury to cultured rat gastric mucosal cells in vitro. Secondly, it investigated whether reduced glutathione or oxidized glutathione is responsible for this cytoprotection. Cytolysis was quantified by measuring 51Cr release from prelabeled cells. Concentrations of ethanol greater than 12% caused cell damage and increased 51Cr release in a dose-dependent and time-related fashion. When a substrate for glutathione synthesis, N-acetyl-L-cysteine, was provided to cultured cells for 4 h before challenge with ethanol, cytolysis was significantly decreased corresponding with an increase in cellular glutathione content. Pretreatment with diethyl maleate, which depletes reduced glutathione without forming oxidized glutathione, potentiated ethanol-induced cell damage in a dose-dependent manner with the decrease of cellular glutathione content. The administration of tert-butyl hydroperoxide (which is specifically reduced by glutathione peroxidase to generate oxidized glutathione from reduced glutathione) or diamide (which nonenzymatically oxidizes reduced glutathione to oxidized glutathione) enhanced ethanol injury. We conclude that in cultured gastric mucosal cells, (a) intracellular glutathione maintains integrity of gastric mucosal cells against ethanol in vitro; and (b) reduced glutathione rather than oxidized glutathione is responsible for this cytoprotection. We postulate that the presence of reduced glutathione is essential to allow glutathione peroxidase to catalyze the ethanol-generated toxic oxygen radical, hydrogen peroxide.

  10. Hepatitis C Virus Subgenomic Replicons Induce Endoplasmic Reticulum Stress Activating an Intracellular Signaling Pathway

    PubMed Central

    Tardif, Keith D.; Mori, Kazutoshi; Siddiqui, Aleem

    2002-01-01

    Hepatitis C virus (HCV) replicates from a ribonucleoprotein (RNP) complex that is associated with the endoplasmic reticulum (ER) membrane. The replication activities of the HCV subgenomic replicon are shown here to induce ER stress. In response to this stress, cells expressing HCV replicons induce the unfolded protein response (UPR), an ER-to-nucleus intracellular signaling pathway. The UPR is initiated by the proteolytic cleavage of a transmembrane protein, ATF6. The resulting cytoplasmic protein fragment of ATF6 functions as a transcription factor in the nucleus and activates selective genes required for an ER stress response. ATF6 activation leads to increased transcriptional levels of GRP78, an ER luminal chaperone protein. However, the overall level of GRP78 protein is decreased. While ER stress is also known to affect translational attenuation, cells expressing HCV replicons have lower levels of phosphorylation of the α subunit of eukaryotic initiation factor 2. Interestingly, cap-independent internal ribosome entry site-mediated translation directed by the 5′ noncoding region of HCV and GRP78 is activated in cells expressing HCV replicons. These studies provide insight into the effects of HCV replication on intracellular events and the mechanisms underlying liver pathogenesis. PMID:12097557

  11. Intracellular calcium affects prestin's voltage operating point indirectly via turgor-induced membrane tension

    NASA Astrophysics Data System (ADS)

    Song, Lei; Santos-Sacchi, Joseph

    2015-12-01

    Recent identification of a calmodulin binding site within prestin's C-terminus indicates that calcium can significantly alter prestin's operating voltage range as gauged by the Boltzmann parameter Vh (Keller et al., J. Neuroscience, 2014). We reasoned that those experiments may have identified the molecular substrate for the protein's tension sensitivity. In an effort to understand how this may happen, we evaluated the effects of turgor pressure on such shifts produced by calcium. We find that the shifts are induced by calcium's ability to reduce turgor pressure during whole cell voltage clamp recording. Clamping turgor pressure to 1kPa, the cell's normal intracellular pressure, completely counters the calcium effect. Furthermore, following unrestrained shifts, collapsing the cells abolishes induced shifts. We conclude that calcium does not work by direct action on prestin's conformational state. The possibility remains that calcium interaction with prestin alters water movements within the cell, possibly via its anion transport function.

  12. UVB radiation induces an increase in intracellular zinc in human epidermal keratinocytes.

    PubMed

    Stork, Christian J; Martorano, Lisa M; Li, Yang V

    2010-10-01

    Ultraviolet (UV) radiation is known to cause oxidative stress, inflammation, DNA damage and apoptotic cell death; however, many details of these malign mechanism have yet to be elucidated. In this study, the exposure of adult human epidermal keratinocytes (HEKa) with UVB (>100 mJ/cm(2)) resulted in the significant increase of intracellular zinc that was released from its storage and was detected by fluorescent zinc indicators. Toxicity testing revealed that UVB-induced zinc release in HEKa is associated with HEKa cell death. Cells that showed elevated intracellular zinc fluorescence upon UVB exposure were also stained by propidium iodide (PI), a traditional viability indicator whose fluorescent signal is as a result of its intercalating with DNA fragments and is unaffected by zinc concentration, showing significant colocalization [Pearson's correlation coefficients r=0.956 (n=6)]. The cytotoxicity of zinc was also determined by an MTT assay after applying the exogenous zinc (ZnCl2) along with its ionophore pyrithione (20 microM) into HEKa culture medium. A significant reduction in cell viability as a function of both zinc concentration and exposure time was observed. The treatments of 1, 10 and 100 microM ZnCl2 with pyrithione demonstrated 2.3, 60 and 84% cell deaths, respectively (control 0.5%) after 30 min. ZnCl2 (100 microM) was also found to induce complete HEKa death after 1 h. Thus, the present study demonstrates that UVB irradiation-induced increased zinc is detrimental to HEKa viability, and zinc may be a necessary step in UVB-induced cell death signaling pathways. PMID:20818483

  13. The protective effect of magnesium lithospermate B against glucose-induced intracellular oxidative damage

    SciTech Connect

    Qu, Jian; Ren, Xian; Hou, Rui-ying; Dai, Xing-ping; Zhao, Ying-chun; Xu, Xiao-jing; Zhang, Wei; Zhou, Gan; Zhou, Hong-hao; Liu, Zhao-qian

    2011-07-22

    Highlights: {yields} LAB reduced the ROS production in HEK293T cells cultured under oxidative stress. High dose of glucose enhanced the expression of HO-1 mRNA and HO-1 protein in a time-dependent manner. {yields} LAB enhanced the expression of HO-1 mRNA and HO-1 protein in a dose-dependent manner treated with high dose of glucose. {yields} LAB plays an important role against glucose-induced intracellular oxidative damage. {yields} The enhanced expression of HO-1 mRNA and HO-1 protein caused by LAB is regulated via Nrf2 signal pathway. -- Abstract: Objectives: To investigate the effects of magnesium lithospermate B (LAB) on intracellular reactive oxygen species (ROS) production induced by high dose of glucose or H{sub 2}O{sub 2}, we explored the influences of LAB on the expression of heme oxygenase-1 (HO-1) and nuclear factor E2-related factor-2 (Nrf2) in HEK293T cells after treatment with high dose of glucose. Materials and methods: The total nuclear proteins in HEK293T cells were extracted with Cytoplasmic Protein Extraction Kit. The ROS level was determined by flow cytometry. The mRNA and protein expression of HO-1 and Nrf2 were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Results: LAB reduced the ROS production in HEK293T cells cultured under oxidative stress. High dose of glucose enhanced the expression of HO-1 mRNA and HO-1 protein in a time-dependent manner. LAB enhanced the expression of HO-1 mRNA and HO-1 protein in a dose-dependent manner treated with high dose of glucose. The amount of Nrf2 translocation was enhanced after cells were pretreated with 50 {mu}mol/L or 100 {mu}mol/L LAB. Silencing of Nrf2 gene eliminated the enhanced expression of HO-1 protein induced by high dose of glucose plus LAB. Conclusions: LAB plays an important role against glucose-induced intracellular oxidative damage. The enhanced expression of HO-1 mRNA and HO-1 protein caused by LAB is regulated via Nrf2 signal pathway.

  14. Involvement of intracellular Na^+ accumulation in Hg^{+2} or Cd^{+2} induced cytotoxicity

    NASA Astrophysics Data System (ADS)

    Pourahmad, J.; O'Brien, P. J.

    2003-05-01

    Previously we showed that hepatocyte lysis induced by Hg^{+2} or Cd^{+2} could be partly attributed to mitochondrial toxicity [1, 2]. Similar changes in Na^+ homeostasis induced when Cd^{+2} or Hg^{+2} was incubated with hepatocytes. Cd^{+2} or Hg^{+2} induced cytotoxicity were prevented by Na^+ omission from the media or by the addition of the Na^+/H^+ exchange inhibitor 5-(N, N-dimethyl)-amiloride. Furthermore the omission of CI^- from the media or 2 addition of glycine, a CI^- channel blocker also prevented Cd^{+2} or Hg^{+2} induced hepatocyte toxicity. A hypotonic media also increased Cd^{+2} or Hg^{+2} induced hepatocyte cytotoxicity. This suggests that Cd^{+2} or Hg^{+2} cytotoxicity could be partly attributed to disruption of cell volume regulation mechanisms. The increased osmotic load caused by the uncontrolled accumulation of intracellular Na^+ in Cd^{+2} or Hg^{+2} treated hepatocytes likely resulted from the activation of Na^+/H^+ exchanger and the Na^+/HCO3^- cotransporter by the acidosis and ATP depletion caused by mitochondrial toxicity.

  15. Actin-associated Proteins in the Pathogenesis of Podocyte Injury

    PubMed Central

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies. PMID:24396279

  16. Methamphetamine induces a rapid increase of intracellular Ca(++) levels in neurons overexpressing GCaMP5.

    PubMed

    Yu, Seong-Jin; Wu, Kou-Jen; Bae, Eun K; Hsu, Man-Jung; Richie, Christopher T; Harvey, Brandon K; Wang, Yun

    2016-03-01

    In this study, methamphetamine (Meth)- and glutamate (Glu)-mediated intracellular Ca(++) (Ca(++) i) signals were examined in real time in primary cortical neurons overexpressing an intracellular Ca(++) probe, GCaMP5, by adeno-associated viral (AAV) serotype 1. Binding of Ca(++) to GCaMP increased green fluorescence intensity in cells. Both Meth and Glu induced a rapid increase in Ca(++) i, which was blocked by MK801, suggesting that Meth enhanced Ca(++) i through Glu receptor in neurons. The Meth-mediated Ca(++) signal was also blocked by Mg(++) , low Ca(++) or the L-type Ca(++) channel inhibitor nifedipine. The ryanodine receptor inhibitor dantrolene did not alter the initial Ca(++) influx but partially reduced the peak of Ca(++) i. These data suggest that Meth enhanced Ca(++) influx through membrane Ca(++) channels, which then triggered the release of Ca(++) from the endoplasmic reticulum in the cytosol. AAV-GCaMP5 was also injected to the parietal cortex of adult rats. Administration of Meth enhanced fluorescence in the ipsilateral cortex. Using immunohistochemistry, Meth-induced green fluorescence was found in the NeuN-containing cells in the cortex, suggesting that Meth increased Ca(++) in neurons in vivo. In conclusion, we have used in vitro and in vivo techniques to demonstrate a rapid increase of Ca(++) i by Meth in cortical neurons through overexpression of GCaMP5. As Meth induces behavioral responses and neurotoxicity through Ca(++) i, modulation of Ca(++) i may be useful to reduce Meth-related reactions. PMID:25377775

  17. Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes.

    PubMed

    Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D'Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C; Rastaldi, Maria Pia; Saleem, Moin A; Mavilio, Domenico; Mikulak, Joanna

    2015-01-01

    Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

  18. FGFR3 intracellular mutations induce tyrosine phosphorylation in the Golgi and defective glycosylation.

    PubMed

    Gibbs, Linda; Legeai-Mallet, Laurence

    2007-04-01

    Mutations of the Fibroblast Growth Factor Receptor 3 (FGFR3) gene have been implicated in a series of skeletal dysplasias including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The severity of these diseases ranges from mild dwarfism to severe dwarfism and to perinatal lethality, respectively. Although it is considered that the mutations give rise to constitutively active receptors, it remains unclear how the different mutations are functionally linked to the severity of the different pathologies. By examining various FGFR3 mutations in a HEK cell culture model, including the uncharacterized X807R mutation, it was found that only the mutations affecting the intracellular domain, induced premature receptor phosphorylation and inhibited receptor glycosylation, suggesting that premature receptor tyrosine phosphorylation of the native receptor inhibits its glycosylation. Moreover, these mutations appeared to be associated with elevated receptor signaling in the Golgi apparatus. In conclusion, although pathological severity could not be correlated with a single factor arising from FGFR3 mutations, these results suggest that intracellular domain mutations define a distinct means by which mutated FGFR3 could disrupt bone development. PMID:17320202

  19. Lead Poisoning Disturbs Oligodendrocytes Differentiation Involved in Decreased Expression of NCX3 Inducing Intracellular Calcium Overload

    PubMed Central

    Ma, Teng; Wu, Xiyan; Cai, Qiyan; Wang, Yun; Xiao, Lan; Tian, Yanping; Li, Hongli

    2015-01-01

    Lead (Pb) poisoning has always been a serious health concern, as it permanently damages the central nervous system. Chronic Pb accumulation in the human body disturbs oligodendrocytes (OLs) differentiation, resulting in dysmyelination, but the molecular mechanism remains unknown. In this study, Pb at 1 μM inhibits OLs precursor cells (OPCs) differentiation via decreasing the expression of Olig 2, CNPase proteins in vitro. Moreover, Pb treatment inhibits the sodium/calcium exchanger 3 (NCX3) mRNA expression, one of the major means of calcium (Ca2+) extrusion at the plasma membrane during OPCs differentiation. Also addition of KB-R7943, NCX3 inhibitor, to simulate Pb toxicity, resulted in decreased myelin basic protein (MBP) expression and cell branching. Ca2+ response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca2+ concentration reducing MBP expression. In contrast, over-expression of NCX3 in Pb exposed OPCs displayed significant increase MBP fluorescence signal in positive regions and CNPase expression, which recovered OPCs differentiation to counterbalance Pb toxicity. In conclusion, Pb exposure disturbs OLs differentiation via affecting the function of NCX3 by inducing intracellular calcium overload. PMID:26287169

  20. Palmitate induces insulin resistance without significant intracellular triglyceride accumulation in HepG2 cells.

    PubMed

    Lee, Jin-young; Cho, Hyang-Ki; Kwon, Young Hye

    2010-07-01

    Previous studies showed that increased release of free fatty acids from adipocytes leads to insulin resistance and triglyceride (TG) accumulation in the liver, which may progress into hepatic steatohepatitis. We and other investigators have previously reported that palmitate induces endoplasmic reticulum stress-mediated toxicity in several tissues. This work investigated whether palmitate could induce insulin resistance and steatosis in HepG2 cells. We treated cells with either saturated fatty acid (palmitate) or unsaturated fatty acid (oleate), and observed that palmitate significantly activated c-jun N-terminal kinase and inactivated protein kinase B. Both 4-phenylbutyric acid and glycerol significantly activated protein kinase B, confirming the involvement of endoplasmic reticulum stress in palmitate-mediated insulin resistance. Oleate, but not palmitate, significantly induced intracellular TG deposition and activated sterol regulatory element binding protein-1. Instead, diacylglycerol level and protein kinase C epsilon activity were significantly increased by palmitate, suggesting the possible role of diacylglycerol in palmitate-mediated lipotoxicity. Therefore, the present study clearly showed that palmitate impairs insulin resistance, but does not induce significant TG accumulation in HepG2 cells. PMID:20006364

  1. Intracellular insulin-like growth factor-1 induces Bcl-2 expression in airway epithelial cells.

    PubMed

    Chand, Hitendra S; Harris, Jennifer Foster; Mebratu, Yohannes; Chen, Yangde; Wright, Paul S; Randell, Scott H; Tesfaigzi, Yohannes

    2012-05-01

    Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and it can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and insulin-like growth factor-1 (IGF-1) coincided with induced Bcl-2 expression compared with controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using short hairpin RNA showed that intracellular IGF-1 (IC-IGF-1) was increasing Bcl-2 expression. Blocking epidermal growth factor receptor or IGF-1R activation also suppressed IC-IGF-1 and abolished the Bcl-2 induction. Induced expression and colocalization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and epidermal growth factor receptor pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases. PMID:22461702

  2. Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization

    NASA Astrophysics Data System (ADS)

    Chiappini, C.; De Rosa, E.; Martinez, J. O.; Liu, X.; Steele, J.; Stevens, M. M.; Tasciotti, E.

    2015-05-01

    The controlled delivery of nucleic acids to selected tissues remains an inefficient process mired by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and questionable safety as a result of toxicity issues arising from the typical materials and procedures employed. High efficiency and minimal toxicity in vitro has been shown for intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery has been difficult, as current interfacing strategies rely on complex equipment or active cell internalization through prolonged interfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etching of silicon can access the cytosol to co-deliver DNA and siRNA with an efficiency greater than 90%, and that in vivo the nanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in blood perfusion in a target region of the muscle.

  3. Depletion of Rab32 decreases intracellular lipid accumulation and induces lipolysis through enhancing ATGL expression in hepatocytes.

    PubMed

    Li, Qing; Wang, Jun; Wan, Ying; Chen, Dongfeng

    2016-03-18

    Nonalcoholic fatty liver disease (NAFLD) is a disease caused by the accumulation of lipids in hepatocytes. To date, however, the pathogenesis of NAFLD is still unclear. Recent studies have shown that Rab GTPases, a major protein family in vesicle trafficking, are associated with intracellular lipid accumulation. Here, we show that Rab32, the only Rab GTPase located in mitochondria, participates in hepatic steatosis. Ablation of Rab32 can decrease intracellular lipid accumulation in hepatocytes (HepG2, L02). Further studying the possible mechanism, we found that knockdown of Rab32 can enhance lipolysis instead of lipogenesis via inducing the expression of adipose triglyceride lipase (ATGL), a key enzyme on the surface of lipid droplets which has been proved to be significant in controlling intracellular lipid accumulation. Co-immunoprecipitation shows that Rab32 and ATGL are not directly associated. These findings suggest that knockdown of Rab32 indirectly affects lipolysis through increasing the expression of ATGL. Taken together, our study reveals that Rab32 can participate in regulating intracellular lipid accumulation and that knockdown of Rab32 can decrease intracellular lipid accumulation in hepatocytes. We also demonstrated that ablation of Rab32 can induce intracellular lipolysis by enhancing the expression of ATGL. PMID:26882978

  4. Erythropoietin ameliorates podocyte injury in advanced diabetic nephropathy in the db/db mouse.

    PubMed

    Loeffler, Ivonne; Rüster, Christiane; Franke, Sybille; Liebisch, Marita; Wolf, Gunter

    2013-09-15

    Podocyte damage and accumulation of advanced glycation end products (AGEs) are characteristics of diabetic nephropathy (DN). The pathophysiology of AGE-challenged podocytes, such as hypertrophy, apoptosis, and reduced cell migration, is closely related to the induction of the cell cycle inhibitor p27(Kip1) and to the inhibition of neuropilin 1 (NRP1). We have previously demonstrated that treatment with erythropoietin is associated with protective effects for podocytes in vitro. db/db mice with overt DN aged 15-16 wk were treated with either placebo, epoetin-β, or continuous erythropoietin receptor activator (CERA) for 2 wk. db/db mice compared with nondiabetic db/m control mice revealed the expected increases in body weight, blood glucose, albumin-to-creatinine ratio, and AGE accumulation. Whereas there were no differences in body weight, hyperglycemia and AGEs were observed among diabetic mice that received epoetin-β compared with CERA and placebo treatment, indicating that epoetin-β/CERA treatment does not interfere with the development of diabetes in this model. However, the albumin-to-creatinine ratio was significantly lower in db/db mice treated with epoetin-β or CERA. Furthermore, kidney weights in db/db mice were increased compared with db/m control mice, indicating renal hypertrophy, whereas the increase in renal weight in epoetin-β- or CERA-treated db/db mice was significantly lower than in placebo-treated control mice. Induction of p27(Kip1) and suppression of NRP1 were significantly reduced in the epoetin-β treatment group versus the CERA treatment group. Furthermore, erythropoietin treatment diminished the diabetes-induced podocyte loss. Together, independently from hematopoetic effects, epoetin-β or CERA treatment was associated with protective changes in DN, especially that NRP1 and p27(Kip1) expressions as well as numbers of podocytes returned to normal levels. Our data show, for the first time, that medication of overt DN with erythropoietin

  5. Metformin Ameliorates Podocyte Damage by Restoring Renal Tissue Podocalyxin Expression in Type 2 Diabetic Rats

    PubMed Central

    Zhai, Limin; Gu, Junfei; Yang, Di; Wang, Wei; Ye, Shandong

    2015-01-01

    Podocalyxin (PCX) is a signature molecule of the glomerular podocyte and of maintaining integrity of filtration function of glomerulus. The aim of this study was to observe the effect of different doses of metformin on renal tissue PCX expression in type 2 diabetic rats and clarify its protection on glomerular podocytes. Type 2 diabetic Sprague-Dawley (SD) rats in which diabetes was induced by high-fat diet/streptozotocin (HFD-STZ) were treated with different doses of metformin (150, 300, and 500 mg/kg per day, resp.) for 8 weeks. Various biochemical parameters, kidney histopathology, and renal tissue PCX expression levels were examined. In type 2 diabetic rats, severe hyperglycemia and hyperlipidemia were developed. Urinary albumin and PCX were markedly increased. Diabetes induced significant alterations in renal glomerular structure. In addition, protein and mRNA expression of renal tissue PCX were highly decreased. However, treatment of rats with different doses of metformin restored all these changes to a varying degree. These results suggested that metformin can ameliorate glomerular podocyte damage in type 2 diabetic rats, which may be partly associated with its role in restoring PCX expression and inhibiting urinary excretion of PCX with dose dependence. PMID:26075281

  6. Effects of overexpressing FoxO1 on apoptosis in glomeruli of diabetic mice and in podocytes cultured in high glucose medium.

    PubMed

    Li, Wen; Wang, Qingzhu; Du, Mengmeng; Ma, Xiaojun; Wu, Lina; Guo, Feng; Zhao, Shuiying; Huang, Fengjuan; Wang, Huanhuan; Qin, Guijun

    2016-09-16

    Podocyte apoptosis induced by high levels of glucose is a key event in the development and prognosis of diabetic nephropathy (DN). Forkhead transcription factor O1 (FoxO1) has been defined as a critical mediator of oxidative stress in animal models of diabetes and is involved in mitophagy. To test the role of FoxO1 in regulating podocyte apoptosis both in vivo and in vitro, we generated FoxO1 overexpression models. High-glucose (HG) induced podocyte apoptosis with decreased mitophagy. These changes were accompanied by mitochondrial dysfunction and more severe podocyte loss in mouse kidney. FoxO1 overexpression prevented the apoptosis induced by HG. Reduction of cell apoptosis and renal damage depended upon the expression of PTEN-induced putative kinase 1 (PINK1). These findings suggest that specific overexpression of renal FoxO1 decreases podocyte apoptosis, which may be explained in part by its regulation of PINK1, and that targeting FoxO1 may represent a novel therapeutic approach for DN. PMID:27475499

  7. Podocyte Dedifferentiation: A Specialized Process for a Specialized Cell

    PubMed Central

    May, Carl James; Saleem, Moin; Welsh, Gavin Iain

    2014-01-01

    The podocyte is one of the two cell types that contribute to the formation of the glomerular filtration barrier (GFB). It is a highly specialized cell with a unique structure. The key feature of the podocyte is its foot processes that regularly interdigitate. A structure known as the slit diaphragm can be found bridging the interdigitations. This molecular sieve comprises the final layer of the GFB. It is well accepted that the podocyte is the target cell in the pathogenesis of nephrotic syndrome. In nephrotic syndrome, the GFB no longer restricts the passage of macromolecules and protein is lost into the urine. A number of phenotypic and morphological changes are seen in the diseased podocyte and in the literature these have been described as an epithelial–mesenchymal transition (EMT). However, there is a growing appreciation that this term does not accurately describe the changes that are seen. Definitions of type-2 EMT are based on typical epithelial cells. While the podocyte is known as a visceral epithelial cell, it is not a typical epithelial cell. Moreover, podocytes have several features that are more consistent with mesenchymal cells. Therefore, we suggest that the term podocyte disease transformation is more appropriate. PMID:25324828

  8. Chylomicron remnant model emulsions induce intracellular cholesterol accumulation and cell death due to lysosomal destabilization.

    PubMed

    Wakita, Kyoko; Morita, Shin-ya; Okamoto, Naoko; Takata, Eriko; Handa, Tetsurou; Nakano, Minoru

    2015-05-01

    Chylomicron remnants, which carry dietary fats and cholesterol, play a role in promoting atherosclerosis. Chylomicron remnants are characterized by high cholesterol content at the surface, different from low-density lipoproteins (LDLs) containing high amounts of esterified cholesterol (CE) in the core. We prepared cholesterol-rich emulsions (TO-PC/cholesterol emulsions) as models for chylomicron remnants and compared their effects on J774 macrophages with acetylated-LDL (ac-LDL). Internalization of TO-PC/cholesterol emulsions into macrophages reduced cell viability, whereas ac-LDL did not. Surprisingly, there was no difference in intracellular free cholesterol content between cells incubated with TO-PC/cholesterol emulsions and with ac-LDL. Furthermore, cholesterol in TO-PC/cholesterol emulsions and ac-LDL both were internalized into J774 macrophages; however, incubation with TO-PC/cholesterol emulsions induced leakage of lysosomal protease, cathepsin-L, to cytosol, which was not observed for incubation with ac-LDL. Inhibition of the activity of cathepsin-L recovered the viability of macrophages that ingested TO-PC/cholesterol emulsions. We suggest an alternative fate of cholesterol-rich emulsions taken up by macrophages, which is different from other atherogenic lipoproteins rich in CE; internalization of TO-PC/cholesterol emulsions into macrophages induces rapid free cholesterol accumulation in lysosomes and cell death due to lysosomal destabilization. PMID:25661161

  9. Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-induced increase in intracellular and plasma Hsp72 in humans

    PubMed Central

    Magalhães, Flávio de Castro; Amorim, Fabiano Trigueiro; Passos, Renata L. Freitas; Fonseca, Michele Atalla; Oliveira, Kenya Paula Moreira; Lima, Milene Rodrigues Malheiros; Guimarães, Juliana Bohen; Ferreira-Júnior, João Batista; Martini, Angelo R. P.; Lima, Nilo R. V.; Soares, Danusa Dias; Oliveira, Edilamar Menezes

    2010-01-01

    In order to verify the effects of heat and exercise acclimation (HA) on resting and exercise-induced expression of plasma and leukocyte heat shock protein 72 (Hsp72) in humans, nine healthy young male volunteers (25.0 ± 0.7 years; 80.5 ± 2.0 kg; 180 ± 2 cm, mean ± SE) exercised for 60 min in a hot, dry environment (40 ± 0°C and 45 ± 0% relative humidity) for 11 days. The protocol consisted of running on a treadmill using a controlled hyperthermia technique in which the work rate was adjusted to elevate the rectal temperature by 1°C in 30 min and maintain it elevated for another 30 min. Before and after the HA, the volunteers performed a heat stress test (HST) at 50% of their individual maximal power output for 90 min in the same environment. Blood was drawn before (REST), immediately after (POST) and 1 h after (1 h POST) HST, and plasma and leukocytes were separated and stored. Subjects showed expected adaptations to HA: reduced exercise rectal and mean skin temperatures and heart rate, and augmented sweat rate and exercise tolerance. In HST1, plasma Hsp72 increased from REST to POST and then returned to resting values 1 h POST (REST: 1.11 ± 0.07, POST: 1.48 ± 0.10, 1 h POST: 1.22 ± 0.11 ng mL−1; p < 0.05). In HST2, there was no change in plasma Hsp72 (REST: 0.94 ± 0.08, POST: 1.20 ± 0.15, 1 h POST: 1.17 ± 0.16 ng mL−1; p > 0.05). HA increased resting levels of intracellular Hsp72 (HST1: 1 ± 0.02 and HST2: 4.2 ± 1.2 density units, p < 0.05). Exercise-induced increased intracellular Hsp72 expression was observed on HST1 (HST1: REST, 1 ± 0.02 vs. POST, 2.9 ± 0.9 density units, mean ± SE, p < 0.05) but was inhibited on HST2 (HST2: REST, 4.2 ± 1.2 vs. POST, 4.4 ± 1.1 density units, p > 0.05). Regression analysis showed that the lower the pre-exercise expression of intracellular Hsp72, the higher the exercise-induced increase (R = −0

  10. Activation of the IL-2 Receptor in Podocytes: A Potential Mechanism for Podocyte Injury in Idiopathic Nephrotic Syndrome?

    PubMed Central

    Zea, Arnold H.; Stewart, Tyrus; Ascani, Jeannine; Tate, David J.; Finkel-Jimenez, Beatriz; Wilk, Anna; Reiss, Krzysztof; Smoyer, William E.; Aviles, Diego H.

    2016-01-01

    The renal podocyte plays an important role in maintaining the structural integrity of the glomerular basement membrane. We have previously reported that patients with idiopathic nephrotic syndrome (INS) have increased IL-2 production. We hypothesized that podocytes express an IL-2 receptor (IL-2R) and signaling through this receptor can result in podocyte injury. To confirm the presence of the IL-2R, we tested a conditionally immortalized murine podocyte cell line by flow cytometry, qPCR, and Western blot. To test for the presence of the IL-2R in vivo, immunohistochemical staining was performed on human renal biopsies in children with FSGS and control. Podocytes were stimulated with IL-2 in vitro, to study signaling events via the JAK/STAT pathway. The results showed that stimulation with IL-2 resulted in increased mRNA and protein expression of STAT 5a, phosphorylated STAT 5, JAK 3, and phosphorylated JAK 3. We then investigated for signs of cellular injury and the data showed that pro-apoptotic markers Bax and cFLIP were significantly increased following IL-2 exposure, whereas LC3 II was decreased. Furthermore, mitochondrial depolarization and apoptosis were both significantly increased following activation of the IL-2R. We used a paracellular permeability assay to monitor the structural integrity of a podocyte monolayer following IL-2 exposure. The results showed that podocytes exposed to IL-2 have increased albumin leakage across the monolayer. We conclude that murine podocytes express the IL-2R, and that activation through the IL-2R results in podocyte injury. PMID:27389192

  11. The homophilic adhesion molecule sidekick-1 contributes to augmented podocyte aggregation in HIV-associated nephropathy.

    PubMed

    Kaufman, Lewis; Yang, Guozhe; Hayashi, Kayo; Ashby, James R; Huang, Li; Ross, Michael J; Klotman, Mary E; Klotman, Paul E

    2007-05-01

    The collapsing glomerulopathy of HIV-associated nephropathy (HIVAN) is characterized by podocyte dedifferentiation and proliferation. In affected glomeruli, proliferating podocytes adhere in aggregates to form glomerular pseudocrescents and fill an enlarged Bowman's space. Previously, we reported that sidekick-1 (sdk-1), an adhesion molecule of the immunoglobulin superfamily, was highly up-regulated in HIV-1 transgenic podocytes. In the current work, we explore how sdk-1 overexpression contributes to HIVAN pathogenesis. Murine podocytes infected with HIV-1 virus expressed significantly more sdk-1 than control-infected cells. Podocytes stably transfected with an sdk-1 expression construct grew in large aggregates with a simplified morphology characterized by a disorganized actin cytoskeleton, changes similar to podocytes in HIVAN. In contrast to controls, HIV-1 infected podocytes adhered to stably transfected sdk-1 podocyte aggregates in mixing studies. Furthermore, substrate-released cell sheets of wild-type podocytes were readily dissociated by mechanical stress, whereas HIV-1 podocytes remained in aggregates. The number of HIV-1 podocyte aggregates was significantly reduced in cells expressing a short hairpin RNA (shRNA) construct specific for sdk-1 compared with cells expressing control shRNA. Finally, in a HIVAN mouse model, sdk-1 protein was detected in podocytes in collapsed glomerular tufts and in glomerular pseudocrescents. These findings suggest that sdk-1 is an important mediator of cellular adhesion in HIV-infected podocytes and may contribute to podocyte clustering that is characteristic of pseudocrescent formation in HIVAN. PMID:17307840

  12. [Cadmium-induced cytotoxicity and intracellular Ca2+ alteration in hepatocytes and protection by selenium].

    PubMed

    Wang, Sha Sha; Chen, Long; Xia, Shu Kai; Jiang, Shan Xiang

    2006-08-01

    Cadmium (Cd)-induced cytotoxicity and intracellular Ca2+ alteration ([Ca2+]i) in hepatocytes and intervention with selenium (Se) were studied to discuss mechanism of Cd-induced hepatocyte injury and protective effect by Se. Freshly primary culture hepatocytes isolated from neonatal mice were randomly divided into a normal control group, four treatment groups with cadmium chloride (CdCl2,5,25,100,250 micromol/L, respectively), two treatment groups with sodium selenite (Na2SeO3,10,20 micromol/L, respectively), and eight treatment groups with CdCl2 (5,25,100, 250 micromol/L,respectively) of administered Na2SeO3 (10,20 micromol/L, respectively). Hepatocyte viability and its malondialdehyde (MDA) content as well as lactate dehydrogenase (LDH) activity in cultured medium were assayed, and the intracellular free Ca2+ level ([Ca2+]i) in hepatocytes was detected with laser scanning confocal microscope (LSCM) at 12 h after treatment. The results showed that hepatocyte viability significantly decreased in Cd-exposed groups with dosages, and had no significant differences in Se-treated groups compared with that of control group. Administration of Se increased or obviously raised the viability in Cd-exposed hepatocytes. We observed a dose-dependent increase of LDH activity and significantly higher values in cultured medium of 100 and 250 micromol/L CdC12 groups compared with that in control group,while Se-treated groups had no significant change. LDH activity of administered Se in 25,100,250 micromol/L CdCl2 groups decreased or significant decreased, respectively. Different dosages of Cd induced significant elevation of MDA concentration in hepatocytes, but administration of Se to hepatocytes is incapable of eliciting the same consequences as Cd. 10 and 20 micromol/L Na2SeO3 inhibited or significantly reduced MDA production in hepatocytes induced by 25,100 and 250 micromol/L CdCl2, respectively. [Ca2+]i fluorescence intensity was significantly higher in Cd-exposed hepatocytes

  13. Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes.

    PubMed

    Rathi, Satyajeet S; Saini, Harjot K; Xu, Yan-Jun; Dhalla, Naranjan S

    2004-08-01

    Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [Ca2+]i. PMID:15524176

  14. Oncolytic reovirus induces intracellular redistribution of Ras to promote apoptosis and progeny virus release.

    PubMed

    Garant, K A; Shmulevitz, M; Pan, L; Daigle, R M; Ahn, D-G; Gujar, S A; Lee, P W K

    2016-02-11

    Reovirus is a naturally oncolytic virus that preferentially replicates in Ras-transformed cells and is currently undergoing clinical trials as a cancer therapeutic. Ras transformation promotes reovirus oncolysis by enhancing virion disassembly during entry, viral progeny production, and virus release through apoptosis; however, the mechanism behind the latter is not well understood. Here, we show that reovirus alters the intracellular location of oncogenic Ras to induce apoptosis of H-RasV12-transformed fibroblasts. Reovirus infection decreases Ras palmitoylation levels and causes accumulation of Ras in the Golgi through Golgi fragmentation. With the Golgi being the site of Ras palmitoylation, treatment of target cells with the palmitoylation inhibitor, 2-bromopalmitate (2BP), prompts a greater accumulation of H-RasV12 in the Golgi, and a dose-dependent increase in progeny virus release and subsequent spread. Conversely, tethering H-RasV12 to the plasma membrane (thereby preventing its movement to the Golgi) allows for efficient virus production, but results in basal levels of reovirus-induced cell death. Analysis of Ras downstream signaling reveals that cells expressing cycling H-RasV12 have elevated levels of phosphorylated JNK (c-Jun N-terminal kinase), and that Ras retained at the Golgi body by 2BP increases activation of the MEKK1/MKK4/JNK signaling pathway to promote cell death. Collectively, our data suggest that reovirus induces Golgi fragmentation of target cells, and the subsequent accumulation of oncogenic Ras in the Golgi body initiates apoptotic signaling events required for virus release and spread. PMID:25961930

  15. Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein.

    PubMed

    St Croix, Claudette M; Wasserloos, K J; Dineley, K E; Reynolds, I J; Levitan, E S; Pitt, B R

    2002-02-01

    We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn(2+), plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn(2+) by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn(2+)-specific fluorophore Zinquin that were rapidly reversed by the Zn(2+) chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn(2+) in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn(2+) homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn(2+)-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn(2+) that were reversed by Zn(2+) supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn(2+) release from thiolate clusters. This effect was restored by Zn(2+) supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn(2+) and suggest that the ratio of MT to T can regulate Zn(2+) homeostasis in response to nitrosative stress. PMID:11792622

  16. Effect of simvastatin on the expression of nephrin, podocin, and vascular endothelial growth factor (VEGF) in podocytes of diabetic rat

    PubMed Central

    Chen, Shujun; Chen, Haiping; Liu, Qi; Ma, Qing

    2015-01-01

    This study aims to investigate the effects and mechanisms of simvastatin on podocyte injuries in diabetic rats. Streptozotocin was used to induce diabetes in a rat model. Three groups were tested: normal control (NC) group, diabetes mellitus control (DM) group, and simvastatin (SVT) group. The serum creatinine, cholesterol, and urinary albumin excretion rate (UAER) were measured 4 to 8 weeks after administering either saline or the drug. Renal pathological changes were observed, and immunohistochemistry was performed to determine the expression of nephrin, podocin, and vascular endothelial growth factor (VEGF). Real-time PCR was performed to detect the mRNA expression levels of nephrin, podocin, and VEGF. Serum creatinine levels and the UAER were higher in the DM group than in the NC group (P < 0.01). The protein and mRNA expression levels of nephrin and podocin were lower in the DM group than in the NC group (P < 0.01); whereas, the expression of VEGF protein and mRNA was higher in the DM group than in the NC group (P < 0.01). Simvastatin (SVT) could reduce serum creatinine levels and the UAER, maintain the expression of nephrin and podocin, reduce the expression of VEGF, and improve the pathological changes of podocytes, which were much more pronounced at 8 weeks (P < 0.01). Simvastatin could maintain the distribution of nephrin and podocin in podocytes, inhibit VEGF expression, and thus improve podocyte injuries and protect kidney functions in diabetic rats. PMID:26770424

  17. Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy

    PubMed Central

    Madhusudhan, Thati; Wang, Hongjie; Dong, Wei; Ghosh, Sanchita; Bock, Fabian; Thangapandi, Veera Raghavan; Ranjan, Satish; Wolter, Juliane; Kohli, Shrey; Shahzad, Khurrum; Heidel, Florian; Krueger, Martin; Schwenger, Vedat; Moeller, Marcus J.; Kalinski, Thomas; Reiser, Jochen; Chavakis, Triantafyllos; Isermann, Berend

    2015-01-01

    Endoplasmic reticulum (ER) stress is associated with diabetic nephropathy (DN), but its pathophysiological relevance and the mechanisms that compromise adaptive ER signalling in podocytes remain unknown. Here we show that nuclear translocation of the transcription factor spliced X-box binding protein-1 (sXBP1) is selectively impaired in DN, inducing activating transcription factor-6 (ATF6) and C/EBP homology protein (CHOP). Podocyte-specific genetic ablation of XBP1 or inducible expression of ATF6 in mice aggravates DN. sXBP1 lies downstream of insulin signalling and attenuating podocyte insulin signalling by genetic ablation of the insulin receptor or the regulatory subunits phosphatidylinositol 3-kinase (PI3K) p85α or p85β impairs sXBP1 nuclear translocation and exacerbates DN. Corroborating our findings from murine DN, the interaction of sXBP1 with p85α and p85β is markedly impaired in the glomerular compartment of human DN. Thus, signalling via the insulin receptor, p85, and XBP1 maintains podocyte homeostasis, while disruption of this pathway impairs podocyte function in DN. PMID:25754093

  18. Hyperoxia Induces Intracellular Acidification in Neonatal Mouse Lung Fibroblasts: Real-Time Investigation Using Plasmonically Enhanced Raman Spectroscopy.

    PubMed

    Panikkanvalappil, Sajanlal R; James, Masheika; Hira, Steven M; Mobley, James; Jilling, Tamas; Ambalavanan, Namasivayam; El-Sayed, Mostafa A

    2016-03-23

    It is important to understand the molecular mechanisms underlying oxygen toxicity, which contributes to multiple human disorders. The archetype model of oxygen toxicity is neonatal lung injury induced by hyperoxia exposure. Here, we utilized plasmonically enhanced Raman spectroscopy (PERS) in combination with fluorescence and proteomic analysis to provide comprehensive information on hyperoxia-induced biomolecular modifications in neonatal mouse lung fibroblasts (nMLFs). During this study, we made the novel observation that hyperoxia induces intracellular acidification in nMLF, which we probed in real-time using label-free PERS. We found that intracellular acidification induces conformational modifications in proteins followed by significant changes in Raman vibrations corresponding to aromatic amino acids such as phenylalanine and tryptophan as well as cysteine moieties. Hyperoxia-induced intracellular pH changes and subsequent modifications in protein expression and associated post-translational modifications within the cells were further validated by fluorescence and proteomic analysis. These new insights may help identifying unique oxidant stress-induced mechanisms in disease processes and may guide the development of more efficient therapeutic strategies. PMID:26938952

  19. Iberis amara Extract Induces Intracellular Formation of Reactive Oxygen Species and Inhibits Colon Cancer

    PubMed Central

    Plauth, Annabell; Wowro, Sylvia J.; Fischer, Cornelius; Abdel-Aziz, Heba; Sauer, Sascha

    2016-01-01

    Massively increasing global incidences of colorectal cancer require efficient treatment and prevention strategies. Here, we report unexpected anticancerogenic effects of hydroethanolic Iberis amara extract (IAE), which is known as a widely used phytomedical product for treating gastrointestinal complaints. IAE significantly inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC50) of 6 and 9 μg/ml, respectively, and further generated inhibitory effects in PC-3 prostate and MCF7 breast cancer cells. Inhibition of proliferation in HT-29 cells was associated with a G2/M phase cell cycle arrest including reduced expression of various regulatory marker proteins. Notably, in HT-29 cells IAE further induced apoptosis by intracellular formation of reactive oxygen species (ROS). Consistent with predictions derived from our in vitro experiments, bidaily oral gavage of 50 mg/kg of IAE over 4 weeks resulted in significant inhibition of tumor growth in a mouse HT-29 tumor xenograft model. Taken together, Iberis amara extracts could become useful alternatives for preventing and treating the progression of colon cancer. PMID:27050665

  20. APPL proteins promote TGFβ-induced nuclear transport of the TGFβ type I receptor intracellular domain

    PubMed Central

    Li, Chunyan; Bergh, Anders; Miaczynska, Marta; Heldin, Carl-Henrik; Landström, Marene

    2016-01-01

    The multifunctional cytokine transforming growth factor-β (TGFβ) is produced by several types of cancers, including prostate cancer, and promote tumour progression in autocrine and paracrine manners. In response to ligand binding, the TGFβ type I receptor (TβRI) activates Smad and non-Smad signalling pathways. The ubiquitin-ligase tumour necrosis factor receptor-associated factor 6 (TRAF6) was recently linked to regulate intramembrane proteolytic cleavage of the TβRI in cancer cells. Subsequently, the intracellular domain (ICD) of TβRI enters in an unknown manner into the nucleus, where it promotes the transcription of pro-invasive genes, such as MMP2 and MMP9. Here we show that the endocytic adaptor molecules APPL1 and APPL2 are required for TGFβ-induced nuclear translocation of TβRI-ICD and for cancer cell invasiveness of human prostate and breast cancer cell lines. Moreover, APPL proteins were found to be expressed at high levels in aggressive prostate cancer tissues, and to be associated with TβRI in a TRAF6-dependent manner. Our results suggest that the APPL–TβRI complex promotes prostate tumour progression, and may serve as a prognostic marker. PMID:26583432

  1. APPL proteins promote TGFβ-induced nuclear transport of the TGFβ type I receptor intracellular domain.

    PubMed

    Song, Jie; Mu, Yabing; Li, Chunyan; Bergh, Anders; Miaczynska, Marta; Heldin, Carl-Henrik; Landström, Marene

    2016-01-01

    The multifunctional cytokine transforming growth factor-β (TGFβ) is produced by several types of cancers, including prostate cancer, and promote tumour progression in autocrine and paracrine manners. In response to ligand binding, the TGFβ type I receptor (TβRI) activates Smad and non-Smad signalling pathways. The ubiquitin-ligase tumour necrosis factor receptor-associated factor 6 (TRAF6) was recently linked to regulate intramembrane proteolytic cleavage of the TβRI in cancer cells. Subsequently, the intracellular domain (ICD) of TβRI enters in an unknown manner into the nucleus, where it promotes the transcription of pro-invasive genes, such as MMP2 and MMP9. Here we show that the endocytic adaptor molecules APPL1 and APPL2 are required for TGFβ-induced nuclear translocation of TβRI-ICD and for cancer cell invasiveness of human prostate and breast cancer cell lines. Moreover, APPL proteins were found to be expressed at high levels in aggressive prostate cancer tissues, and to be associated with TβRI in a TRAF6-dependent manner. Our results suggest that the APPL-TβRI complex promotes prostate tumour progression, and may serve as a prognostic marker. PMID:26583432

  2. Hyperglycemia and mechanical stress: targeting the renal podocyte.

    PubMed

    Lewko, Barbara; Stepinski, Jan

    2009-11-01

    Hyperglycemia and deriving from glomerular hypertension mechanical stress are the key factors underlying pathogenesis of diabetic nephropathy (DN). Multiple direct and secondary effects of both these factors are mediated by complex signaling pathways with extensive interactions. The common signaling pathways stimulated by high glucose and mechanical insult may act in an additive manner, thereby accelerating the cell damage. Podocytes, the cells covering the outer aspect of glomerular basement membrane (GBM), are subjected not only to the load of filtered glucose but also to diverse mechanical forces. Bulging into the Bowman's space, they have no support from the apical side, which makes them particularly susceptible to the effects of mechanical strain. Both high glucose and mechanical stress may impair the protein systems anchoring the podocyte foot processes in GBM, therefore blunting resistance of these cells to mechanical forces. Modulation by these factors of expression and activity of numerous structural and functional proteins results in the (auto)inflammatory responses, dysfunction, apoptosis or necrosis of the podocytes. Loss of the podocytes is irreversible due to their inability to proliferate and to replenish damaged cells. Podocytes are injured early in the course of DN, which, most likely, underlies further glomerular and renal damage in diabetes. This review summarizes the effects of elevated glucose and mechanical stress that seem to be involved in podocyte impairment in diabetes, with particular focus on the possible interactions between these factors. PMID:19562677

  3. Plasma-activated medium-induced intracellular zinc liberation causes death of SH-SY5Y cells.

    PubMed

    Hara, Hirokazu; Taniguchi, Miko; Kobayashi, Mari; Kamiya, Tetsuro; Adachi, Tetsuo

    2015-10-15

    Plasma is an ionized gas consisting of ions, electrons, free radicals, neutral particles, and photons. Plasma-activated medium (PAM), which is prepared by the irradiation of cell-free medium with non-thermal atmospheric pressure plasma, induces cell death in various types of cancer cell. Since PAM contains reactive oxygen species (ROS), its anti-cancer effects are thought to be attributable to oxidative stress. Meanwhile, oxidative stress has been shown to induce the liberation of zinc (Zn(2+)) from intracellular Zn(2+) stores and to provoke Zn(2+)-dependent cell death. In this study, we thus examined whether Zn(2+) is involved in PAM-induced cell death using human neuroblastoma SH-SY5Y cells. Exposure to PAM triggered cell death in SH-SY5Y cells. The cell-permeable Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) protected against PAM-induced cell death. Zn(2+) imaging using the fluorescent Zn(2+) probe FluoZin-3 revealed that PAM elicited a rise of intracellular free Zn(2+). In addition, PAM stimulated PARP-1 activation, mitochondrial ROS generation, and the depletion of intracellular NAD(+) and ATP. These findings suggest that PAM-induced PARP-1 activation causes energy supply exhaustion. Moreover, TPEN suppressed all of these events elicited by PAM. Taken together, we demonstrated here that Zn(2+) released from intracellular Zn(2+) stores serves as a key mediator of PAM-induced cell death in SH-SY5Y cells. PMID:26319292

  4. Intracellular acidosis modulates the stretch-induced changes in E-C coupling of the rat atrium.

    PubMed

    Tavi, P; Han, C; Weckström, M

    1999-11-01

    By inducing a small reduction of the intracellular pH (0.18 units) with 20 mmol L-1 propionate we demonstrated that acidification changed the responses of isolated rat atria to stretch. Stretch (increase of the intra-atrial pressure) in normal pH increased the Ca2+ transients' amplitude (Indo-1 fluorescence) from 0.26 +/- 0.09 in 1 mmHg to 0.36 +/- 0.13 in 4 mmHg (P < 0.05, n=6), without affecting the diastolic [Ca2+]i level (n.s. n=6). The changes in Ca2+ balance during stretch were accompanied by a biphasic increase in the contraction force. Five minutes of continuous stretch increased the action potential duration (APD90%, P < 0.01, n=13) and decreased the APD15% (P < 0.001, n=13). During acidosis, the stretch-induced increase of the Ca2+ transient amplitude (0.4 +/- 0. 13 vs. 0.3 +/- 0.08, P < 0.05, n=6) was accompanied by the increase of the diastolic [Ca2+]i (1.16 +/- 0.07, P < 0.05, n=6) compared with non-acidotic control (1.06 +/- 0.06, n=6). Acidic intracellular pH also inhibited the stretch-induced changes in the action potentials (n=10) and slowed down the development of the contractile force during stretch. The results showed that acidosis modulates the mechanotransduction. It does this by interfering with the intracellular Ca2+ balance, inhibiting the Ca2+ extrusion mechanisms and reducing the Ca2+-buffering power of the cells. The physiological and pathological processes associated with stretch are therefore modulated by intracellular pH owing to its concerted effects on intracellular Ca2+ handling caused by a competitive inhibition of various Ca2+-binding molecules. PMID:10606822

  5. Dexamethasone-dependent modulation of cyclic GMP synthesis in podocytes.

    PubMed

    Lewko, Barbara; Waszkiewicz, Anna; Maryn, Anna; Gołos, Magdalena; Latawiec, Elżbieta; Daca, Agnieszka; Witkowski, Jacek M; Angielski, Stefan; Stępiński, Jan

    2015-11-01

    Podocytes may be direct target for glucocorticoid therapy in glomerular proteinuric disease. Permeability of podocytes largely depends on their capacity to migrate which involves the contractile apparatus in their foot processes. In this study, we examined the effect of synthetic glucocorticoid dexamethasone (DEX) on the ability of podocytes to produce cyclic guanosine monophosphate (cGMP) in the presence of vasoactive factors, atrial natriuretic peptide (ANP), nitric oxide (NO), and angiotensin II (Ang II). We investigated also the effects of cGMP and DEX on podocyte motility. Primary rat podocytes and immortalized mouse podocytes were pretreated with 1 µM DEX for 4 or 24 h. Glomerular hypertension was mimicked by subjecting the cells to mechanical stress. Total and subcellular cGMP levels were determined in podocytes incubated with 0.1 µM ANP, 1 µM S-nitroso-N-acetyl penicillamine (SNAP), and 1 µM Ang II. Cell motility was estimated by a wound-healing assay. The ANP-dependent production of cGMP increased after 4 h exposition to DEX, but was attenuated after 24 h. Adversely, a 24-h pretreatment with DEX augmented the NO-dependent cGMP synthesis. Ang II suppressed the ANP-dependent cGMP production and the effect was enhanced by DEX in mechanical stress conditions. Mechanical stress reduced total cGMP production in the presence of all stimulators, whereas extracellular to total cGMP ratio increased. 8-Br cGMP enhanced podocyte migration which was accompanied by F-actin disassembly. In the presence of DEX these effects were prevented. We conclude that DEX modulates the production of cGMP in podocytes stimulated with vasoactive factors such as Ang II, ANP, and NO, and the effect is time-dependent. cGMP increases podocyte motility, which is prevented by DEX. This mechanism may account for the antiproteinuric effect of glucocorticoids. PMID:26272337

  6. Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion

    PubMed Central

    Kirby, Brett S.; Hanna, Gabi; Hendargo, Hansford C.

    2014-01-01

    Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ∼42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., “PIPA”) restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ∼120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo. PMID:25305182

  7. Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells.

    PubMed

    Wu, Li-Hua; Li, Peng; Zhao, Qing-Li; Piao, Jin-Lan; Jiao, Yu-Fei; Kadowaki, Makoto; Kondo, Takashi

    2014-11-01

    Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-β-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway. PMID:25187044

  8. Detection of light-induced changes of intracellular ionized calcium concentration in Limulus ventral photoreceptors using arsenazo III

    PubMed Central

    Brown, J. E.; Brown, P. K.; Pinto, L. H.

    1977-01-01

    1. The metallochromic indicator dye, arsenazo III, was injected intracellularly into Limulus ventral photoreceptor cells to concentrations greater than 1 mM. 2. The absorption spectrum (450-750 nm) of the dye in single dark-adapted cells was measured by a scanning microspectrophotometer. When a cell was light-adapted, the absorption of the dye changed; the difference spectrum had two maxima at about 610 and 660 nm, a broad minimum at about 540 nm and an isosbestic point at about 585 nm. 3. When intracellular calcium concentration was raised in dark-adapted cells previously injected with arsenazo III, the difference spectum had two maxima at about 610 and 660 nm, a broad minimum at about 530 nm and an isosbestic point at about 585 nm. The injection of Mg2+ into dark-adapted cells previously injected with the dye induced a difference spectrum that had a single maximum at about 620 nm. Also, decreasing the intracellular pH of cells previously injected with the dye induced a difference spectrum that had a minimum at about 620 nm. The evidence suggests that there is a rise of intracellular ionized calcium when a Limulus ventral photoreceptor is light-adapted. 4. The intracellular calcium concentration, [Ca2+]1, in light-adapted photoreceptors was estimated to reach at least 10-4 M by compaing the light-induced difference spectra measured in ventral photoreceptors with a standard curve determined in microcuvettes containing 2mM arsenazo III in 400 mM-KCl, 1 mM-MgCl2 and 25 mM MOPS at pH 7·0. 5. In cells injected to less than 3 mM arsenazo III, light induced a transient decrease in optical transmission at 660 nm (T660). This decrease in T660 indicates that illumination of a ventral photoreceptor normally causes a transient increase of [Ca2+]1. 6. Arsenazo III was found to be sensitive, selective and rapid enough to measure light-induced changes of intracellular ionized calcium in Limulus ventral photoreceptor cells. PMID:17732

  9. Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys.

    PubMed

    Sugahara, Go; Kamiie, Junichi; Kobayashi, Ryosuke; Mineshige, Takayuki; Shirota, Kinji

    2016-06-01

    Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R. PMID:26854253

  10. Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys

    PubMed Central

    SUGAHARA, Go; KAMIIE, Junichi; KOBAYASHI, Ryosuke; MINESHIGE, Takayuki; SHIROTA, Kinji

    2016-01-01

    Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R. PMID:26854253

  11. Infrasound increases intracellular calcium concentration and induces apoptosis in hippocampi of adult rats.

    PubMed

    Liu, Zhaohui; Gong, Li; Li, Xiaofang; Ye, Lin; Wang, Bin; Liu, Jing; Qiu, Jianyong; Jiao, Huiduo; Zhang, Wendong; Chen, Jingzao; Wang, Jiuping

    2012-01-01

    In the present study, we determined the effect of infrasonic exposure on apoptosis and intracellular free Ca²⁺ ([Ca²⁺]i) levels in the hippocampus of adult rats. Adult rats were randomly divided into the control and infrasound exposure groups. For infrasound treatment, animals received infrasonic exposure at 90 (8 Hz) or 130 dB (8 Hz) for 2 h per day. Hippocampi were dissected, and isolated hippocampal neurons were cultured. The [Ca²⁺]i levels in hippocampal neurons from adult rat brains were determined by Fluo-3/AM staining with a confocal microscope system on days 1, 7, 14, 21 and 28 following infrasonic exposure. Apoptosis was evaluated by Annexin V-FITC and propidium iodide double staining. Positive cells were sorted and analyzed by flow cytometry. Elevated [Ca²⁺]i levels were observed on days 14 and 21 after rats received daily treatment with 90 or 130 dB sound pressure level (SPL) infrasonic exposure (p<0.01 vs. control). The highest levels of [Ca²⁺]i were detected in the 130 dB SPL infrasonic exposure group. Meanwhile, apoptosis in hippocampal neurons was found to increase on day 7 following 90 dB SPL infrasound exposure, and significantly increased on day 14. Upon 130 dB infrasound treatment, apoptosis was first observed on day 14, whereas the number of apoptotic cells gradually decreased thereafter. Additionally, a marked correlation between cell apoptosis and [Ca²⁺]i levels was found on day 14 and 21 following daily treatment with 90 and 130 dB SPL, respectively. These results demonstrate that a period of infrasonic exposure induced apoptosis and upregulated [Ca²⁺]i levels in hippocampal neurons, suggesting that infrasound may cause damage to the central nervous system (CNS) through the Ca²⁺‑mediated apoptotic pathway in hippocampal neurons. PMID:21946944

  12. Intracellular influx of calcium induced by quartz particles in alveolar macrophages.

    PubMed

    Tian, Feng; Zhu, Tong; Shang, Yu

    2010-01-15

    Historical studies report that cellular injury and silicosis are related to cytosolic free calcium (Ca2+). Moreover, reactive oxygen species (ROS) have been linked to cellular injury. However, the detail mechanism of the increase in [Ca2+]i and the relationship between [Ca2+]i and ROS production remains unknown. Quartz particle has been found to increase [Ca2+]i and activate the generation of ROS. Our hypothesis is that [Ca2+]i increase induced by quartz particle is from extracellular Ca2+ through the Ca2+ channel, and [Ca2+]i increase is believed to activate ROS production. In order to examine this hypothesis, we treated rat alveolar macrophages with quartz (SiO2) particles and used laser scanning confocal microscopy to measure [Ca2+]i and the fluorescence intensity of ROS. Time- and dose-dependent increases in [Ca2+]I and ROS in macrophages as well as cell viability were observed. Through chelating extracellular Ca2+ with ethylene glycol tetraacetic acid and releasing intracellular Ca2+ with thapsigargin, we found that 72.7% of the [Ca2+]i increase was due to the influx of Ca2+ from the extracellular environment, via Ca2+ channels in the plasma membrane. By adding mannitol to scavenge hydroxyl radicals (OH(.)), and removing surface iron from the quartz particles to reduce OH(.) generation, we observed a reduced level of ROS generation, whereas the increase in [Ca2+]i was unaffected. When using EGTA to reduce [Ca2+]i, we observed a decrease in ROS production. This study suggests that the [Ca2+]i influx was independent of OH(.) production, and the [Ca2+]i increase resulted in ROS production. These results further indicate that there is a strong relationship between cytosolic free Ca2+ content and cellular injury as well as silica exposure. PMID:19835900

  13. Thermally induced changes in intracellular pH and modulators of phosphofructokinase in trout white muscle

    PubMed

    Lehoux; Guderley

    1997-01-01

    The intracellular pH (pHi) and the concentrations of lactate and selected modulators of phosphofructokinase (PFK; EC 2.7.1.11) were measured in white epaxial muscle of 15 °C-acclimated rainbow trout (Oncorhynchus mykiss) maintained at 8, 15 or 22 °C for 48 h and sampled at rest and after 10 min of exhaustive exercise. The lactate accumulation resulting from exercise was 13 % smaller at 22 °C than at 8 and 15 °C. The estimated duration of burst performance was shorter at cold than at warm temperatures, whereas the average rate of lactate accumulation during burst performance was higher at 8 °C than at 15 and 22 °C. pHi rose when temperature decreased, but less than predicted by the imidazole alphastat hypothesis of Reeves. The effects of temperature on the pre-exercise concentrations of PFK modulators [adenylates, fructose 6-phosphate (F6P) and fructose 1,6-bisphosphate (FBP)] were generally negligible. In exhausted trout, adenylate concentrations were almost unaffected by temperature. In contrast, post-exercise FBP and F6P concentrations were significantly higher at low than at high temperatures. We interpret the response of F6P to temperature as an indication that the covariation of pHi and temperature is insufficient to prevent a cold-enhancement of PFK inhibition. Since F6P is a potent activator of PFK, we conclude that, in trout white muscle, thermally induced changes in F6P concentration probably help buffer the effects of temperature change on PFK activity. PMID:9318724

  14. A superoxide anion generator, pyrogallol induces apoptosis in As4.1 cells through the depletion of intracellular GSH content.

    PubMed

    Park, Woo Hyun; Han, Yong Whan; Kim, Suhn Hee; Kim, Sung Zoo

    2007-06-01

    We investigated the involvement of ROS such as H2O2 and O2*-, and GSH in As4.1 cell death induced by pyrogallol. The intracellular H2O2 levels were decreased or increased depending on the concentration and incubation time of pyrogallol. The levels of O2*- were significantly increased. Pyrogallol reduced the intracellular GSH content. And ROS scavengers, Tempol, Tiron, Trimetazidine and NAC could not significantly down-regulate the production of H2O2 and O2*-. However, these ROS scavengers slightly inhibited apoptosis. Interestingly, Tempol showing the recovery of GSH depletion induced by pyrogallol significantly decreased apoptosis without the significant reduction of intracellular O2*- levels. SOD and catalase did not change the level of H2O2 but decreased the level of O2*-. The inhibition of GSH depletion by these was accompanied with the decrease of apoptosis, as evidenced by sub-G1 DNA content, annexin V staining, mitochondria membrane potential (DeltaPsi(m)) and Western data. In addition, ROS scavengers and SOD did not alter a G2 phase accumulation of the cell cycle induced by pyrogallol. However, catalase changed the cell cycle distributions of pyrogallol-treated cells to those of pyrogallol-untreated cells. In summary, we have demonstrated that pyrogallol potently generates ROS, especially O2*-, in As4.1 JG cells, and Tempol, SOD and catalase could rescue to a lesser or greater extent cells from pyrogallol-induced apoptosis through the up-regulation of intracellular GSH content. PMID:17382355

  15. Effect of Interferon and Interferon Inducers on Infections with a Nonviral Intracellular Microorganism, Chlamydia trachomatis.

    PubMed

    Kazar, J; Gillmore, J D; Gordon, F B

    1971-06-01

    The effect of mouse interferon (IF) on the multiplication of Chlamydia trachomatis (strain MRC-1/G) in homologous (L-929) cell cultures and the effect of the IF inducers Newcastle disease virus (NDV) and polyriboinosinic acid-polyribocytidylic acid complex (poly I:C) on the experimental infection of mice with aerosolized C. trachomatis (strain MoPn) were investigated. Treatment of infected cell cultures with IF reduced the number of cells containing chlamydial inclusions and depressed the yield of chlamydiae as determined by titrations for infectivity. Growth of chlamydiae was reduced when cultures were exposed to IF 6 or 18 hr before infection, and slight reduction of the yield was also detectable in cell cultures treated with IF at early intervals (0 or 4 hr) after chlamydial infection. No effect of IF on penetration of chlamydiae into mouse cells was observed, whether phagocytic cells from peritoneal washings or L-929 cells were used, indicating that the inhibitory effect of IF occurs after chlamydiae enter the host cell. Additional evidence was obtained that a significant effect of IF occurs at an early stage in maturation of the intracellular chlamydiae. In mice exposed repeatedly to NDV aerosols and challenged with aerosolized MoPn 8 hr after the first exposure to NDV, mortality was delayed by 2 to 3 days and lung consolidation was slightly reduced at 3 days after infection. Yields of chlamydiae from lung pools of NDV-treated mice, taken at 3, 6, and 9 days after challenge, were not significantly different from those of controls. Similar results were obtained when mice were challenged with MoPn 8 hr after intranasal injection with 100 mug of poly I:C or 24 hr after intravenous injection with 200 mug of poly I:C. In contrast, administration of 0.2 ml of NDV (10(8.3) plaque-forming units) intravenously 10 hr before or 24 hr after challenge with MoPn accelerated mortality of mice by 2 to 3 days. In all experiments, detectable levels of IF in sera or 20% lung

  16. Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase 3 activation in HL-60 cells.

    PubMed

    Huang, R F; Huang, S M; Lin, B S; Wei, J S; Liu, T Z

    2001-05-11

    The cytotoxicity of homocysteine derivatives on chromosomal damage in somatic cells is not well established. The present study used reactive homocysteine derivative of homocysteine thiolactone (Hcy) to investigate its causal effect on apoptotic DNA injury in human promyeloid HL-60 cells. Our results demonstrated that Hcy induced cell death and features of apoptosis including increased phosphotidylserine exposure on the membrane surface, increased apoptotic cells with hypoploid DNA contents, and internucleosomal DNA fragmentation, all of which occurred in a time- and concentration-dependent manner. Hcy treatment also significantly increased intracellular reactive oxygen species H2O2, which coincided with the elimination of caspase 3 proenzyme levels and increased caspase 3 activity at the time of the appearance of apoptotic DNA fragmentation. Preincubation of Hcy-treated HL-60 cells with catalase completely scavenged intracellular H2O2, thus inhibiting caspase 3 activity and protecting cells from apoptotic DNA damage. In contrast, superoxide dismutase failed to inhibit Hcy-induced DNA damage. Taken together, these results demonstrate that Hcy exerted its genotoxic effects on HL-60 cells through an apoptotic pathway, which is mediated by the activation of caspase 3 activity induced by an increase in intracellular hydrogen peroxide. PMID:11432446

  17. The effect of albumin on podocytes: The role of the fatty acid moiety and the potential role of CD36 scavenger receptor

    SciTech Connect

    Pawluczyk, I.Z.A.; Pervez, A.; Ghaderi Najafabadi, M.; Saleem, M.A.; Topham, P.S.

    2014-08-15

    Evidence is emerging that podocytes are able to endocytose proteins such as albumin using kinetics consistent with a receptor-mediated process. To date the role of the fatty acid moiety on albumin uptake kinetics has not been delineated and the receptor responsible for uptake is yet to be identified. Albumin uptake studies were carried out on cultured human podocytes exposed to FITC-labelled human serum albumin either carrying fatty acids (HSA{sub +FA}) or depleted of them (HSA{sub −FA}). Receptor-mediated endocytosis of FITC-HSA{sub +FA} over 60 min was 5 times greater than that of FITC-HSA{sub −FA}. 24 h exposure of podocytes to albumin up-regulated nephrin expression and induced the activation of caspase-3. These effects were more pronounced in response to HSA{sub −FA.} Individually, anti-CD36 antibodies had no effect upon endocytosis of FITC-HSA. However, a cocktail of 2 antibodies reduced uptake by nearly 50%. Albumin endocytosis was enhanced in the presence of the CD36 specific inhibitor sulfo-N-succinimidyl oleate (SSO) while knock-down of CD36 using CD36siRNA had no effect on uptake. These data suggest that receptor-mediated endocytosis of albumin by podocytes is regulated by the fatty acid moiety, although, some of the detrimental effects are induced independently of it. CD36 does not play a direct role in the uptake of albumin. - Highlights: • The fatty acid moiety is essential for receptor mediated endocytosis of albumin. • Fatty acid depleted albumin is more pathogenic to podocytes. • CD36 is not directly involved in albumin uptake by podocytes.

  18. Structural responses of cells to intracellular magnetic force induced by superparamagnetic iron oxide nanoparticles

    PubMed Central

    Shen, Han; Tong, Sheng; Bao, Gang; Wang, Biao

    2014-01-01

    In this paper, we study the effects of intracellular force on human umbilical vein endothelial cells. We generated intracellular force on endothelial cells under different magnetic fields using the cell uptake of superparamagnetic iron oxide nanoparticles. Cell responses to intracellular force were observed using fluorescent microscopy. Our results indicated that nanoparticles were taken up by the cell by endocytosis and were deposited in lysosomes. Nanoparticles and lysosomes inside the cell could be relocated by the application of a magnetic force. The intracellular magnetic force could also be used to accelerate cell migration by adjusting the magnetic fields and giving the cell free culture space. No cytotoxicity of nanoparticles was found in our experiments. By comparing intracellular relocalization with migration of the whole cell, we obtained a better understanding of the self-defence mechanisms of cells based on their mechanical properties. Based on the promising mechanical properties and low cytotoxicity of our magnetic nanoparticles, their potential applications in cytomechanics and cell patterning are discussed. PMID:24336693

  19. Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

    PubMed Central

    Hsuan, S. L.; Kannan, M. S.; Jeyaseelan, S.; Prakash, Y. S.; Sieck, G. C.; Maheswaran, S. K.

    1998-01-01

    Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The

  20. Transgenic Strategies to Study Podocyte Loss and Regeneration

    PubMed Central

    Lombardi, Duccio

    2015-01-01

    Podocyte death and regeneration are major topics in kidney research but remain controversial. Data obtained in humans demonstrate the existence of cells sited along Bowman's capsule that behave as podocyte progenitors in vitro and in in vivo mouse models of podocyte injury xenotrasplanted with this human-derived population. However, this podocyte reservoir still remains elusive in murine models, where it could be more easily studied. Transgenic models can be a powerful tool to identify this population and to better understand its dynamics and hierarchies in both physiological and pathological conditions. Indeed, exploiting transgenic approaches allows detecting, at the single cell level, movements, cell death, and replacement. Moreover, through lineage tracing it is now possible to identify specific population increase and to point out clonal expansions during or after the regenerative processes. However, applying transgenic strategies to study glomerular regeneration requires the search of markers to unequivocally identify this progenitor population. Achieving this aim would lead to a deep comprehension of the biological processes that underlie glomerular regeneration and clarify how different cell pools interface during this phase. Here we discuss strategies that have been used and new approaches in transgenic models finalized to study podocyte loss and subsequent replacement. PMID:26089920

  1. Intracellular influx of calcium induced by quartz particles in alveolar macrophages

    SciTech Connect

    Feng Tian; Tong Zhu; Yu Shang

    2010-01-15

    Historical studies report that cellular injury and silicosis are related to cytosolic free calcium (Ca{sup 2+}). Moreover, reactive oxygen species (ROS) have been linked to cellular injury. However, the detail mechanism of the increase in [Ca{sup 2+}]{sub i} and the relationship between [Ca{sup 2+}]{sub i} and ROS production remains unknown. Quartz particle has been found to increase [Ca{sup 2+}]{sub i} and activate the generation of ROS. Our hypothesis is that [Ca{sup 2+}]{sub i} increase induced by quartz particle is from extracellular Ca{sup 2+} through the Ca{sup 2+} channel, and [Ca{sup 2+}]{sub i} increase is believed to activate ROS production. In order to examine this hypothesis, we treated rat alveolar macrophages with quartz (SiO{sub 2}) particles and used laser scanning confocal microscopy to measure [Ca{sup 2+}]{sub i} and the fluorescence intensity of ROS. Time- and dose-dependent increases in [Ca{sup 2+}]{sub I} and ROS in macrophages as well as cell viability were observed. Through chelating extracellular Ca{sup 2+} with ethylene glycol tetraacetic acid and releasing intracellular Ca{sup 2+} with thapsigargin, we found that 72.7% of the [Ca{sup 2+}]{sub i} increase was due to the influx of Ca{sup 2+} from the extracellular environment, via Ca{sup 2+} channels in the plasma membrane. By adding mannitol to scavenge hydroxyl radicals (OH.), and removing surface iron from the quartz particles to reduce OH. generation, we observed a reduced level of ROS generation, whereas the increase in [Ca{sup 2+}]{sub i} was unaffected. When using EGTA to reduce [Ca{sup 2+}]{sub i}, we observed a decrease in ROS production. This study suggests that the [Ca{sup 2+}]{sub i} influx was independent of OH. production, and the [Ca{sup 2+}]{sub i} increase resulted in ROS production. These results further indicate that there is a strong relationship between cytosolic free Ca{sup 2+} content and cellular injury as well as silica exposure.

  2. Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway

    PubMed Central

    Lee, Eunkyung; Choi, So-Young; Yang, Jae-Ho

    2016-01-01

    Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway. PMID:27382356

  3. Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway.

    PubMed

    Lee, Eunkyung; Choi, So-Young; Yang, Jae-Ho; Lee, Youn Ju

    2016-07-01

    Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway. PMID:27382356

  4. Bcl-2 overexpression inhibits generation of intracellular reactive oxygen species and blocks adriamycin-induced apoptosis in bladder cancer cells.

    PubMed

    Kong, Chui-Ze; Zhang, Zhe

    2013-01-01

    Resistance to induction of apoptosis is a major obstacle for bladder cancer treatment. Bcl-2 is thought to be involved in anti-apoptotic signaling. In this study, we investigated the effect of Bcl-2 overexpression on apoptotic resistance and intracellular reactive oxygen species (ROS) generation in bladder cancer cells. A stable Bcl-2 overexpression cell line, BIU87-Bcl-2, was constructed from human bladder cancer cell line BIU87 by transfecting recombinant Bcl-2 [pcDNA3.1(+)-Bcl-2]. The sensitivity of transfected cells to adriamycin (ADR) was assessed by MTT assay. Apoptosis was examined by flow cytometry and acridine orange fluorescence staining. Intracellular ROS was determined using flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were also investigated by the xanthinoxidase and visible radiation methods using SOD and CAT detection kits. The susceptibility of BIU87-Bcl-2 cells to ADR treatment was significantly decreased as compared with control BIU87 cells. Enhanced expression of Bcl-2 inhibited intracellular ROS generation following ADR treatment. Moreover, the suppression of SOD and CAT activity induced by ADR treatment was blocked in the BIU87-Bcl-2 case but not in their parental cells. The overexpression of Bcl-2 renders human bladder cancer cells resistant to ADR-induced apoptosis and ROS might act as an important secondary messenger in this process. PMID:23621258

  5. Probe Intracellular Trafficking of a Polymeric DNA Delivery Vehicle by Functionalization with an Aggregation-Induced Emissive Tetraphenylethene Derivative.

    PubMed

    Han, Xiongqi; Chen, Qixian; Lu, Hongguang; Ma, Jianbiao; Gao, Hui

    2015-12-30

    Characteristic aggregation-induced quenching of π-fluorophores imposed substantial hindrance to their utilization in nanomedicine for insight into microscopic intracellular trafficking of therapeutic payload. To address this obstacle, we attempted to introduce a novel aggregation-induced emission (AIE) fluorophore into the cationic polymer, which was further used for formulation of a gene delivery carrier. Note that the selective restriction of the intramolecular rotation of the AIE fluorophore through its covalent bond to the polymer conduced to immense AIE. Furthermore, DNA payload labeled with the appropriate fluorophore as the Förster resonance energy transfer (FRET) acceptor verified a facile strategy to trace intracellular DNA releasing activity relying on the distance limitation requested by FRET (AIE fluorophore as FRET donor). Moreover, the hydrophobic nature of the AIE fluorophore appeared to promote colloidal stability of the constructed formulation. Together with other chemistry functionalization strategies (including endosome escape), the ultimate formulation exerted dramatic gene transfection efficiency. Hence, this report manifested a first nanomedicine platform combining AIE and FRET for microscopic insight into DNA intracellular trafficking activity. PMID:26634294

  6. Triclocarban-induced change in intracellular Ca²⁺ level in rat thymocytes: cytometric analysis with Fluo-3 under Zn²⁺-free conditions.

    PubMed

    Miura, Yukari; Chen, Xiaohui; Yamada, Saki; Sugihara, Aya; Enkhjargal, Molomjamts; Sun, Yuanzhi; Kuroda, Keiko; Satoh, Masaya; Oyama, Yasuo

    2014-03-01

    Triclocarban (TCC) is an antimicrobial used in personal hygiene products. Recent health concerns arose after TCC was detected in the blood of human subjects who showered with soap containing TCC. In this study, the effect of TCC on intracellular Ca(2+) concentration in rat thymocytes was examined using Fluo-3, an indicator of intracellular Ca(2+). TCC at concentrations ranging from 0.1 μM to 3 μM increased intracellular Ca(2+) concentration biphasically: first by releasing Ca(2+) from intracellular Ca(2+) stores and then inducing Ca(2+) influx through store-operated Ca(2+) channels. The threshold TCC concentration to increase intracellular Ca(2+) concentration in this study was lower than the maximum TCC concentrations reported in human blood samples. Therefore, we anticipate that TCC at concentrations reported in human blood samples might disturb intracellular Ca(2+) signaling in human lymphocytes. PMID:24562054

  7. Podocyte Injury and Albuminuria in Mice with Podocyte-Specific Overexpression of the Ste20-Like Kinase, SLK

    PubMed Central

    Cybulsky, Andrey V.; Takano, Tomoko; Papillon, Joan; Guillemette, Julie; Herzenberg, Andrew M.; Kennedy, Chris R. J.

    2010-01-01

    SLK expression and activity are increased during kidney development and recovery from renal ischemia-reperfusion injury. In cultured cells, SLK promotes F-actin destabilization as well as apoptosis, partially via the p38 kinase pathway. To better understand the effects of SLK in vivo, a transgenic mouse model was developed where SLK was expressed in a podocyte-specific manner using the mouse nephrin promoter. Offspring of four founder mice carried the SLK transgene. Among male transgenic mice, 66% developed albuminuria at approximately 3 months of age, and the albuminuric mice originated from three of four founders. Overall, the male transgenic mice demonstrated about fivefold greater urinary albumin/creatinine compared with male non-transgenic mice. Transgenic and non-transgenic female mice did not develop albuminuria, suggesting that females were less susceptible to glomerular filtration barrier damage than their male counterparts. In transgenic mice, electron microscopy revealed striking podocyte injury, including poorly formed or effaced foot processes, and edematous and vacuolated cell bodies. By immunoblotting, nephrin expression was decreased in glomeruli of the albuminuric transgenic mice. Activation-specific phosphorylation of p38 was increased in transgenic mice compared with non-transgenic animals. Glomeruli of SLK transgenic mice showed around 30% fewer podocytes, and a reduction in F-actin compared with control glomeruli. Thus, podocyte SLK overexpression in vivo results in injury and podocyte loss, consistent with the effects of SLK in cultured cells. PMID:20889563

  8. Secondary Focal Segmental Glomerulosclerosis: From Podocyte Injury to Glomerulosclerosis

    PubMed Central

    Kim, Jae Seok; Han, Byoung Geun; Choi, Seung Ok; Cha, Seung-Kuy

    2016-01-01

    Focal segmental glomerulosclerosis (FSGS) is a common cause of proteinuria and nephrotic syndrome leading to end stage renal disease (ESRD). There are two types of FSGS, primary (idiopathic) and secondary forms. Secondary FSGS shows less severe clinical features compared to those of the primary one. However, secondary FSGS has an important clinical significance because a variety of renal diseases progress to ESRD thorough the form of secondary FSGS. The defining feature of FSGS is proteinuria. The key event of FSGS is podocyte injury which is caused by multiple factors. Unanswered questions about how these factors act on podocytes to cause secondary FSGS are various and ill-defined. In this review, we provide brief overview and new insights into FSGS, podocyte injury, and their potential linkage suggesting clues to answer for treatment of the disease. PMID:27088082

  9. The Importance of Podocyte Adhesion for a Healthy Glomerulus

    PubMed Central

    Lennon, Rachel; Randles, Michael J.; Humphries, Martin J.

    2014-01-01

    Podocytes are specialized epithelial cells that cover the outer surfaces of glomerular capillaries. Unique cell junctions, known as slit diaphragms, which feature nephrin and Neph family proteins in addition to components of adherens, tight, and gap junctions, connect adjacent podocyte foot processes. Single gene disorders affecting the slit diaphragm result in nephrotic syndrome in humans, characterized by massive loss of protein across the capillary wall. In addition to specialized cell junctions, interconnecting podocytes also adhere to the glomerular basement membrane (GBM) of the capillary wall. The GBM is a dense network of secreted, extracellular matrix (ECM) components and contains tissue-restricted isoforms of collagen IV and laminin in addition to other structural proteins and ECM regulators such as proteases and growth factors. The specialized niche of the GBM provides a scaffold for endothelial cells and podocytes to support their unique functions and human genetic mutations in GBM components lead to renal failure, thus highlighting the importance of cell–matrix interactions in the glomerulus. Cells adhere to ECM via adhesion receptors, including integrins, syndecans, and dystroglycan and in particular the integrin heterodimer α3β1 is required to maintain barrier integrity. Therefore, the sophisticated function of glomerular filtration relies on podocyte adhesion both at cell junctions and at the interface with the ECM. In health, the podocyte coordinates signals from cell junctions and cell–matrix interactions, in response to environmental cues in order to regulate filtration and as our understanding of mechanisms that control cell adhesion in the glomerulus develops, then insight into the effects of disease will improve. The ultimate goal will be to develop targeted therapies to prevent or repair defects in the filtration barrier and to restore glomerular function. PMID:25352829

  10. PARP-1 Hyperactivation and Reciprocal Elevations in Intracellular Ca2+ During ROS-Induced Nonapoptotic Cell Death

    PubMed Central

    Zhang, Fengjiao; Xie, Ruiye; Munoz, Frances M.; Lau, Serrine S.; Monks, Terrence J.

    2014-01-01

    The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia/reperfusion injury, and many other pathological conditions. DNA strand breaks caused by ROS lead to the activation of poly(ADP-ribose)polymerase-1 (PARP-1), the excessive activation of which can result in cell death. We have utilized a model in which 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes ROS-dependent cell death in human renal proximal tubule epithelial cells (HK-2), to further elucidate the role of PARP-1 in ROS-dependent cell death. TGHQ-induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca2+ concentrations, and subsequent nonapoptotic cell death in both a PARP- and Ca2+-dependent manner. Thus, inhibition of PARP-1 with PJ34 completely blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers and NAD consumption, and delayed HK-2 cell death. In contrast, chelation of intracellular Ca2+ with BAPTA completely abrogated TGHQ-induced cell death. Ca2+ chelation also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca2+ homeostasis. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Thus, pathways coupling PARP-1 hyperactivation to cell death are likely to be context-dependent, and therapeutic strategies designed to target PARP-1 need to recognize such variability. Our studies provide new insights into PARP-1-mediated nonapoptotic cell death, during which PARP-1 hyperactivation and elevations in intracellular Ca2+ are reciprocally coupled to amplify ROS-induced nonapoptotic cell death. PMID:24752504

  11. Ablation of developing podocytes disrupts cellular interactions and nephrogenesis both inside and outside the glomerulus

    PubMed Central

    Jia, Qunshan; McDill, Bradley W.; Sankarapandian, Bala; Wu, Sen; Liapis, Helen; Holzman, Lawrence B.; Capecchi, Mario R.; Miner, Jeffrey H.; Chen, Feng

    2008-01-01

    Podocyte loss in adults leads to glomerulosclerosis. However, the impact of podocyte loss on glomerulogenesis and the development of the kidney as a whole has not been directly studied. Here, we used a podocyte-specific Cre transgene to direct the production of diphtheria toxin (DTA) inside podocytes during nephrogenesis. Affected podocytes underwent translational arrest and apoptosis, leading to oliguria, proteinuria, hematuria, interstitial hemorrhage, and perinatal death. Glomerular cell-cell interactions were disrupted, even before overt podocyte apoptosis. VEGF production by podocytes was greatly decreased, and this was associated with reduced endothelial fenestration and altered glomerular vascular architecture. In addition to these glomerular anomalies, embryonic podocyte ablation also led to structural changes and increased apoptosis in proximal tubules. The collecting ducts, however, only showed molecular changes that are likely an indirect effect of the greatly reduced urine flow. Although podocyte loss significantly impacted the development and maintenance of the vasculature both inside and outside the glomerulus, our results suggest that there is a lack of long-range signaling from deep-seated, mature glomeruli to the differentiating cells in the outer nephrogenic zone. This study illustrates the tight integration of various cell types in the developing kidney and shows that the impact of podocyte loss during development is much greater than that in adults. This study also shows the specificity and effectiveness of a genetically controlled podocyte ablation system in mice where the additional readily available tools can further expand its applications. PMID:18842818

  12. A Potential Role for Mechanical Forces in the Detachment of Podocytes and the Progression of CKD

    PubMed Central

    Lemley, Kevin V.

    2015-01-01

    Loss of podocytes underlies progression of CKD. Detachment of podocytes from the glomerular basement membrane (GBM) rather than apoptosis or necrosis seems to be the major mechanism of podocyte loss. Such detachment of viable podocytes may be caused by increased mechanical distending and shear forces and/or impaired adhesion to the GBM. This review considers the mechanical challenges that may lead to podocyte loss by detachment from the GBM under physiologic and pathophysiologic conditions, including glomerular hypertension, hyperfiltration, hypertrophy, and outflow of filtrate from subpodocyte spaces. Furthermore, we detail the cellular mechanisms by which podocytes respond to these challenges, discuss the protective effects of angiotensin blockade, and note the questions that must be addressed to better understand the relationship between podocyte detachment and progression of CKD. PMID:25060060

  13. Podocyte-specific knockout of myosin 1e disrupts glomerular filtration.

    PubMed

    Chase, Sharon E; Encina, Christina V; Stolzenburg, Lindsay R; Tatum, Arthur H; Holzman, Lawrence B; Krendel, Mira

    2012-10-01

    Myosin 1e (myo1e) is an actin-dependent molecular motor that plays an important role in kidney functions. Complete knockout of myo1e in mice and Myo1E mutations in humans are associated with nephrotic syndrome and focal segmental glomerulosclerosis. In this paper, we tested the hypothesis that myo1e is necessary for normal functions of glomerular visceral epithelial cells (podocytes) using podocyte-targeted knockout of myo1e. Myo1e was selectively knocked out in podocytes using Cre-mediated recombination controlled by the podocin promoter. Myo1e loss from podocytes resulted in proteinuria, podocyte foot process effacement, and glomerular basement membrane disorganization. Our findings indicate that myo1e expression in podocytes is necessary for normal glomerular filtration and that podocyte defects are likely to represent the primary pathway leading to glomerular disease associated with Myo1E mutations. PMID:22811491

  14. Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression

    PubMed Central

    Jeruschke, Stefanie; Jeruschke, Kay; DiStasio, Andrew; Karaterzi, Sinem; Büscher, Anja K.; Nalbant, Perihan; Klein-Hitpass, Ludger; Hoyer, Peter F.; Weiss, Jürgen; Stottmann, Rolf W.; Weber, Stefanie

    2015-01-01

    Background Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte actin cytoskeleton. In contrast, a potential stabilization of microtubules by everolimus has not been studied so far. Methods To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury. Results Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involved in cytoskeletal reorganization, cell adhesion, migration and extracellular matrix composition to be affected. Everolimus was capable of protecting podocytes from injury, both on transcriptional and protein level. Rescued genes included tubulin beta 2B class IIb (TUBB2B) and doublecortin domain containing 2 (DCDC2), both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Validating gene expression data, Western-blot analysis in cultured podocytes demonstrated an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Interestingly, Tubb2bbrdp/brdp mice revealed a delay in glomerular podocyte development as showed by podocyte-specific markers Wilm’s tumour 1, Podocin, Nephrin and Synaptopodin. Conclusions Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton

  15. Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores.

    PubMed

    Kadiri, Lolahon R; Kwan, Alex C; Webb, Watt W; Harris-Warrick, Ronald M

    2011-09-01

    Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network. We analyzed the mechanisms by which DA evokes bursting in the AB neuron. Previous work showed that DA-evoked bursting is critically dependent on external calcium (Harris-Warrick RM, Flamm RE. J Neurosci 7: 2113-2128, 1987). Using two-photon microscopy and calcium imaging, we show that DA evokes the release of calcium from intracellular stores well before the emergence of voltage oscillations. When this release from intracellular stores is blocked by antagonists of ryanodine or inositol trisphosphate (IP(3)) receptor channels, DA fails to evoke AB bursting. We further demonstrate that DA enhances the calcium-activated inward current, I(CAN), despite the fact that it significantly reduces voltage-activated calcium currents. This suggests that DA-induced release of calcium from intracellular stores activates I(CAN), which provides a depolarizing ramp current that underlies endogenous bursting in the AB neuron. PMID:21676929

  16. Multifunctional basic motif in the glycine receptor intracellular domain induces subunit-specific sorting.

    PubMed

    Melzer, Nima; Villmann, Carmen; Becker, Kristina; Harvey, Kirsten; Harvey, Robert J; Vogel, Nico; Kluck, Christoph J; Kneussel, Matthias; Becker, Cord-Michael

    2010-02-01

    The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs. PMID:19959465

  17. Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission

    PubMed Central

    Jalini, Shirin; Ye, Hui; Tonkikh, Alexander A.; Charlton, Milton P.; Carlen, Peter L.

    2016-01-01

    Oxygen-glucose deprivation (OGD) leads to depression of evoked synaptic transmission, for which the mechanisms remain unclear. We hypothesized that increased presynaptic [Ca2+]i during transient OGD contributes to the depression of evoked field excitatory postsynaptic potentials (fEPSPs). Additionally, we hypothesized that increased buffering of intracellular calcium would shorten electrophysiological recovery after transient ischemia. Mouse hippocampal slices were exposed to 2 to 8 min of OGD. fEPSPs evoked by Schaffer collateral stimulation were recorded in the stratum radiatum, and whole cell current or voltage clamp recordings were performed in CA1 neurons. Transient ischemia led to increased presynaptic [Ca2+]i, (shown by calcium imaging), increased spontaneous miniature EPSP/Cs, and depressed evoked fEPSPs, partially mediated by adenosine. Buffering of intracellular Ca2+ during OGD by membrane-permeant chelators (BAPTA-AM or EGTA-AM) partially prevented fEPSP depression and promoted faster electrophysiological recovery when the OGD challenge was stopped. The blocker of BK channels, charybdotoxin (ChTX), also prevented fEPSP depression, but did not accelerate post-ischemic recovery. These results suggest that OGD leads to elevated presynaptic [Ca2+]i, which reduces evoked transmitter release; this effect can be reversed by increased intracellular Ca2+ buffering which also speeds recovery. PMID:26934214

  18. Overview on how oncogenic Kras promotes pancreatic carcinogenesis by inducing low intracellular ROS levels.

    PubMed

    Kong, Bo; Qia, Chengjia; Erkan, Mert; Kleeff, Jörg; Michalski, Christoph W

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease without clearly known disease causes. Recent epidemiological and animal studies suggest that the supplementation of dietary antioxidants (e.g., vitamins C and E) decreases cancer risk, implying that increased reactive oxygen species (ROS) may play a role in pancreatic carcinogenesis. However, oncogenic Kras mutations (e.g., Kras(G12D)), which are present in more than 90% of PDAC, have been proven to foster low intracellular ROS levels. Here, oncogenic Kras activates expression of a series of anti-oxidant genes via Nrf2 (nuclear factor, erythroid derived 2, like 2) and also mediates an unusual metabolic pathway of glutamine to generate NADPH. This can then be used as the reducing power for ROS detoxification, leading collectively to low ROS levels in pancreatic pre-neoplastic cells and in cancer cells. In adult stem cells and cancer stem cells, low ROS levels have been associated with the formation of a proliferation-permissive intracellular environment and with perseverance of self-renewal capacities. Therefore, it is conceivable that low intracellular ROS levels may contribute significantly to oncogenic Kras-mediated PDAC formation. PMID:24062691

  19. Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis.

    PubMed

    Navarro-Lérida, Inmaculada; Corvi, Maria Martha; Barrientos, Alberto Alvarez; Gavilanes, Francisco; Berthiaume, Luc Gérard; Rodríguez-Crespo, Ignacio

    2004-12-31

    A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and .NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker beta-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the .NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization. PMID:15485846

  20. Na+ /Ca2+ exchanger contributes to asterosap-induced elevation of intracellular Ca2+ concentration in starfish spermatozoa.

    PubMed

    Islam, M Sadiqul; Kawase, Osamu; Hase, Sumitaka; Minakata, Hiroyuki; Hoshi, Motonori; Matsumoto, Midori

    2006-05-01

    Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm. PMID:16719949

  1. Roles of intracellular Ca2+ and cyclic AMP in mast cell histamine release induced by radiographic contrast media.

    PubMed

    Saito, Mami; Itoh, Yoshinori; Yano, Takahisa; Sendo, Toshiaki; Goromaru, Takeshi; Sakai, Naoko; Oishi, Ryozo

    2003-04-01

    Mast cell histamine release is considered to be associated with the etiology of anaphylactoid reactions to iodinated radiographic contrast media (RCM). In the present study, the effects of various ionic and non-ionic RCM on histamine release from mast cells were compared, and the possible mechanisms of the histamine release were subsequently determined. Both ionic (ioxaglate and amidotrizoate) and non-ionic (iohexol, ioversol, iomeprol, iopamidol and iotrolan) RCM increased histamine release from the dissociated rat pulmonary cells, whereby ionic materials were more potent than non-ionic agents. There was no significant correlation between the extent of histamine release and the osmolarity of each RCM solution. In addition, hyperosmotic mannitol solution (1000 mOsm/kg) caused no marked histamine release. Thus, it is unlikely that the hyperosmolarity of RCM solutions contributes to the histamine release. RCM also stimulated, but to a lesser extent, the histamine release from rat peritoneal cells. The RCM-induced histamine release from both types of cells was inhibited by dibutyl cyclic AMP or combined treatment with forskolin and 3-isobutyl-1-methylxanthine. Corresponding to these results, RCM markedly reduced the cellular cyclic AMP content. On the other hand, the removal of intracellular but not the extracellular Ca2+ attenuated the RCM-induced mast cell histamine release. From these findings, it is suggested that the decrease in cellular cyclic AMP content and an increase in intracellular Ca2+ contribute at least in part to the RCM-induced mast cell histamine release. PMID:12690428

  2. Streptococcus pneumoniae Infection of Host Epithelial Cells via Polymeric Immunoglobulin Receptor Transiently Induces Calcium Release from Intracellular Stores*

    PubMed Central

    Asmat, Tauseef M.; Agarwal, Vaibhav; Räth, Susann; Hildebrandt, Jan-Peter; Hammerschmidt, Sven

    2011-01-01

    The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae (pneumococci) that interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. This interaction promotes bacterial colonization and bacterial internalization by initiating host signal transduction cascades. Here, we examined alterations of intracellular calcium ([Ca2+]i) levels in epithelial cells during host cell infections with pneumococci via the PspC-hpIgR mechanism. The release of [Ca2+]i from intracellular stores in host cells was significantly increased by wild-type pneumococci but not by PspC-deficient pneumococci. The increase in [Ca2+]i was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor U73122 abolished the increase in [Ca2+]i. In addition, we demonstrated the effect of [Ca2+]i on pneumococcal internalization by epithelial cells. Uptake of pneumococci was significantly increased after pretreatment of epithelial cells with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid-tetraacetoxymethyl ester or use of EGTA as an extracellular Ca2+-chelating agent. In contrast, thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ATPase, which increases [Ca2+]i in a sustained fashion, significantly reduced pIgR-mediated pneumococcal invasion. Importantly, pneumococcal adherence to pIgR-expressing cells was not altered in the presence of inhibitors as demonstrated by immunofluorescence microscopy. In conclusion, these results demonstrate that pneumococcal infections induce mobilization of [Ca2+]i from intracellular stores. This may constitute a defense response of host cells as the experimental reduction of intracellular calcium levels facilitates pneumococcal internalization by pIgR-expressing cells, whereas elevated calcium levels diminished bacterial internalization by host epithelial

  3. Genes and Podocytes – New Insights into Mechanisms of Podocytopathy

    PubMed Central

    Bierzynska, Agnieszka; Soderquest, Katrina; Koziell, Ania

    2014-01-01

    After decades of primarily morphological study, positional cloning of the NPHS1 gene was the landmark event that established aberrant podocyte genetics as a pivotal cause of malfunction of the glomerular filter. This ended any uncertainty whether genetic mutation plays a significant role in hereditary nephrotic syndromes (NS) and confirmed podocytes as critical players in regulating glomerular protein filtration. Although subsequent sequencing of candidate genes chosen on the basis of podocyte biology had less success, unbiased analysis of genetically informative kindreds and syndromic disease has led to further gene discovery. However, the 45 genes currently associated with human NS explain not more than 20–30% of hereditary and only 10–20% of sporadic cases. It is becoming increasingly clear both from genetic analysis and phenotypic data – including occasional response to immunosuppressive agents and post-transplant disease recurrence in Mendelian disease – that monogenic inheritance of abnormalities in podocyte-specific genes disrupting filter function is only part of the story. Recent advances in genetic screening technology combined with increasingly robust bioinformatics are set to allow identification and characterization of novel disease causing variants and more importantly, disease modifying genes. Emerging data also support a significant but incompletely characterized immunoregulatory component. PMID:25667580

  4. Protective Immunity to Vaccinia Virus Induced by Vaccination with Multiple Recombinant Outer Membrane Proteins of Intracellular and Extracellular Virions

    PubMed Central

    Fogg, Christiana; Lustig, Shlomo; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2004-01-01

    Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-μg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine. PMID:15367588

  5. Murine Double Minute-2 Prevents p53-Overactivation-Related Cell Death (Podoptosis) of Podocytes.

    PubMed

    Thomasova, Dana; Bruns, Hauke A; Kretschmer, Victoria; Ebrahim, Martrez; Romoli, Simone; Liapis, Helen; Kotb, Ahmed M; Endlich, Nicole; Anders, Hans-Joachim

    2015-07-01

    Murine double minute-2 (MDM2), an E3 ligase that regulates the cell cycle and inflammation, is highly expressed in podocytes. In podocyte injury, MDM2 drives podocyte loss by mitotic catastrophe, but the function of MDM2 in resting podocytes has not been explored. Here, we investigated the effects of podocyte MDM2 deletion in vitro and in vivo. In vitro, MDM2 knockdown by siRNA caused increased expression of p53 and podocyte death, which was completely rescued by coknockdown of p53. Apoptosis, pyroptosis, pyronecrosis, necroptosis, ferroptosis, and parthanatos were excluded as modes of occurrence for this p53-overactivation-related cell death (here referred to as podoptosis). Podoptosis was associated with cytoplasmic vacuolization, endoplasmic reticulum stress, and dysregulated autophagy (previously described as paraptosis). MDM2 knockdown caused podocyte loss and proteinuria in a zebrafish model, which was consistent with the phenotype of podocyte-specific MDM2-knockout mice that also showed the aforementioned ultrastructual podocyte abnormalities before and during progressive glomerulosclerosis. The phenotype of both animal models was entirely rescued by codeletion of p53. We conclude that MDM2 maintains homeostasis and long-term survival in podocytes by preventing podoptosis, a p53-regulated form of cell death with unspecific features previously classified as paraptosis. PMID:25349197

  6. Murine Double Minute-2 Prevents p53-Overactivation-Related Cell Death (Podoptosis) of Podocytes

    PubMed Central

    Bruns, Hauke A.; Kretschmer, Victoria; Ebrahim, Martrez; Romoli, Simone; Liapis, Helen; Kotb, Ahmed M.; Endlich, Nicole; Anders, Hans-Joachim

    2015-01-01

    Murine double minute-2 (MDM2), an E3 ligase that regulates the cell cycle and inflammation, is highly expressed in podocytes. In podocyte injury, MDM2 drives podocyte loss by mitotic catastrophe, but the function of MDM2 in resting podocytes has not been explored. Here, we investigated the effects of podocyte MDM2 deletion in vitro and in vivo. In vitro, MDM2 knockdown by siRNA caused increased expression of p53 and podocyte death, which was completely rescued by coknockdown of p53. Apoptosis, pyroptosis, pyronecrosis, necroptosis, ferroptosis, and parthanatos were excluded as modes of occurrence for this p53-overactivation-related cell death (here referred to as podoptosis). Podoptosis was associated with cytoplasmic vacuolization, endoplasmic reticulum stress, and dysregulated autophagy (previously described as paraptosis). MDM2 knockdown caused podocyte loss and proteinuria in a zebrafish model, which was consistent with the phenotype of podocyte-specific MDM2-knockout mice that also showed the aforementioned ultrastructual podocyte abnormalities before and during progressive glomerulosclerosis. The phenotype of both animal models was entirely rescued by codeletion of p53. We conclude that MDM2 maintains homeostasis and long-term survival in podocytes by preventing podoptosis, a p53-regulated form of cell death with unspecific features previously classified as paraptosis. PMID:25349197

  7. Discovery of new glomerular disease-relevant genes by translational profiling of podocytes in vivo

    PubMed Central

    Grgic, Ivica; Hofmeister, Andreas F.; Genovese, Giulio; Bernhardy, Andrea J.; Sun, Hua; Maarouf, Omar H.; Bijol, Vanesa; Pollak, Martin R.; Humphreys, Benjamin D.

    2014-01-01

    Identifying new biomarkers and therapeutic targets for podocytopathies such as focal segmental glomerulosclerosis (FSGS) requires a detailed analysis of transcriptional changes in podocytes over the course of disease. Here we used translating ribosome affinity purification (TRAP) to isolate and profile podocyte-specific mRNA in two different models of FSGS. Expressed eGFP-tagged ribosomal protein L10a in podocytes under the control of the Collagen-1α1 promoter enabled podocyte-specific mRNA isolation in a one-step process over the course of disease. This TRAP protocol robustly enriched known podocyte-specific mRNAs. We crossed col1α1-L10a mice with the actn4−/− and actn4+/K256E models of FSGS and analyzed podocyte transcriptional profiles at 2, 6 and 44 weeks of age. Two upregulated podocyte genes in murine FSGS (CXCL1 and DMPK) were found to be upregulated at the protein level in biopsies from patients with FSGS, validating this approach. There was no dilution of podocyte-specific transcripts during disease. These are the first podocyte-specific RNA expression datasets during aging and in two models of FSGS. This approach identified new podocyte proteins that are upregulated in FSGS and help define novel biomarkers and therapeutic targets for human glomerular disease. PMID:24940801

  8. Hyperoside reduces albuminuria in diabetic nephropathy at the early stage through ameliorating renal damage and podocyte injury.

    PubMed

    Zhang, Jisheng; Fu, Haiyan; Xu, Yan; Niu, Yunfei; An, Xiaofei

    2016-10-01

    Diabetic nephropathy (DN) is one of the major microvascular complications in diabetes. Podocyte injury such as slit diaphragm effacement is regarded as a determinant in the occurrence and development of albuminuria in DN. In this study, we examined the effect of hyperoside, an active flavonoid glycoside, on proteinuria and renal damage in a streptozotocin-induced DN mouse model at the early stage. The results showed that oral administration of hyperoside (30 mg/kg/day for 4 weeks could significantly decrease urinary microalbumin excretion and glomerular hyperfiltration in DN mice, but did not affect the glucose and lipid metabolism. Periodic acid-Schiff staining and transmission electron microscopy showed that glomerular mesangial matrix expansion and podocyte process effacement in DN mice were significantly improved by hyperoside. Further investigations via immunofluorescence staining, real-time reverse transcription polymerase chain reaction and Western blot analysis showed that the decreased slit diaphragm protein nephrin and podocin mRNA expression and protein levels in DN mice were restored by hyperoside treatment. Collectively, these findings demonstrated that hyperoside could decrease albuminuria at the early stage of DN by ameliorating renal damage and podocyte injury. PMID:27255369

  9. Pluronic-modified superoxide dismutase 1 attenuates angiotensin II-induced increase in intracellular superoxide in neurons.

    PubMed

    Yi, Xiang; Zimmerman, Matthew C; Yang, Ruifang; Tong, Jing; Vinogradov, Serguei; Kabanov, Alexander V

    2010-08-15

    Overexpressing superoxide dismutase 1 (SOD1; also called Cu/ZnSOD), an intracellular superoxide (O(2)(*-))-scavenging enzyme, in central neurons inhibits angiotensin II (AngII) intraneuronal signaling and normalizes cardiovascular dysfunction in diseases associated with enhanced AngII signaling in the brain, including hypertension and heart failure. However, the blood-brain barrier and neuronal cell membranes impose a tremendous impediment for the delivery of SOD1 to central neurons, which hinders the potential therapeutic impact of SOD1 treatment on these diseases. To address this, we developed conjugates of SOD1 with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer (Pluronic) (SOD1-P85 and SOD1-L81), which retained significant SOD1 enzymatic activity. The modified SOD1 effectively scavenged xanthine oxidase/hypoxanthine-derived O(2)(*-), as determined by HPLC and the measurement of 2-hydroxyethidium. Using catecholaminergic neurons, we observed an increase in neuronal uptake of SOD1-Pluronic after 1, 6, or 24h, compared to neurons treated with pure SOD1 or PEG-SOD1. Importantly, without inducing neuronal toxicity, SOD1-Pluronic conjugates significantly inhibited AngII-induced increases in intraneuronal O(2)(*-) levels. These data indicate that SOD1-Pluronic conjugates penetrate neuronal cell membranes, which results in elevated intracellular levels of functional SOD1. Pluronic conjugation may be a new delivery system for SOD1 into central neurons and therapeutically beneficial for AngII-related cardiovascular diseases. PMID:20493251

  10. ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells

    PubMed Central

    Wang, Caixia; Hu, Xiaoke; Gao, Yan; Ji, Yinglu

    2015-01-01

    Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30–40% corresponding to significant depletion (approximately 70–80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment. PMID:26339612

  11. Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction

    PubMed Central

    Gan, Ming; Moussaud, Simon; Jiang, Peizhou; McLean, Pamela J.

    2014-01-01

    The pathological hallmark of Parkinson’s disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation/aggregation. Using two neuronal cell models we found that extracellular ATP induced a significant increase in intracellular αsyn levels between 24 to 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells’ response to extracellular ATP. Although Ca2+ influx via P2X1 receptor is necessary for αsyn accumulation, Ca2+ influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD. PMID:25480524

  12. Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction.

    PubMed

    Gan, Ming; Moussaud, Simon; Jiang, Peizhou; McLean, Pamela J

    2015-02-01

    The pathologic hallmark of Parkinson's disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation and/or aggregation. Using 2 neuronal cell models, we found that extracellular adenosine triphosphate (ATP) induced a significant increase in intracellular αsyn levels between 24 and 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells' response to extracellular ATP. Although Ca(2+) influx via P2X1 receptor is necessary for αsyn accumulation, Ca(2+) influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD. PMID:25480524

  13. Copper-induced changes in intracellular thiols in two marine diatoms: Phaeodactylum tricornutum and Ceratoneis closterium.

    PubMed

    Smith, Cassandra L; Steele, Jessica E; Stauber, Jennifer L; Jolley, Dianne F

    2014-11-01

    Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted <10% of total glutathione after 48h. P. tricornutum also produced more phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper. PMID:25261820

  14. Occurrence of minimal change nephrotic syndrome in classical Hodgkin lymphoma is closely related to the induction of c-mip in Hodgkin-Reed Sternberg cells and podocytes

    PubMed Central

    Audard, Vincent; Zhang, Shao-Yu; Copie-Bergman, Christiane; Rucker-Martin, Catherine; Ory, Virginie; Candelier, Marina; Baia, Maryse; Lang, Philippe; Pawlak, André; Sahali, Djillali

    2010-01-01

    It is currently considered that idiopathic minimal change nephrotic syndrome (I-MCNS) is an immune-mediated glomerular disease. Its association with classical Hodgkin lymphoma (cHL-MCNS) suggests a molecular link between these two diseases, which remains to be elucidated. We analyzed the expression of c-mip (cmaf inducing protein) in lymphomatous tissues and kidney biopsies of patients with cHL-MCNS (n=8) and in lymphomatous tissues of isolated cHL (n=9). Because c-mip affects the regulatory loop involving Fyn, we investigated possible structural defects in this signaling pathway, using laser capture microdissection, RT-PCR and Western-blotting. We found that c-mip was selectively expressed in Hodgkin and Reed-Sternberg (HRS) cells and podocytes of patients with cHL-MCNS but is undetectable in patients with isolated cHL. We demonstrated that c-mip was specifically involved in the negative regulation of early proximal signaling through its interaction with PAG and Fyn. We showed that the upregulation of c-mip in cHL-MCNS was associated with a possible Fyn defect in HRS cells and podocytes, while Fyn was normally expressed in isolated cHL and normal podocytes. Moreover, we showed that c-mip was upregulated in Fyn-deficient podocytes. C-mip may be a useful marker of cHL-MCNS and its induction reflects the dysregulation of proximal signaling. PMID:20200355

  15. The Basic Domain of HIV-Tat Transactivating Protein Is Essential for Its Targeting to Lipid Rafts and Regulating Fibroblast Growth Factor-2 Signaling in Podocytes Isolated from Children with HIV-1–Associated Nephropathy

    PubMed Central

    Xie, Xuefang; Colberg-Poley, Anamaris M.; Das, Jharna R.; Li, Jinliang; Zhang, Aiping; Tang, Pingtao; Jerebtsova, Marina; Gutkind, J. Silvio

    2014-01-01

    Podocyte injury has a critical role in the pathogenesis of HIV-associated nephropathy (HIVAN). The HIV-1 transactivator of transcription (Tat), combined with fibroblast growth factor-2 (FGF-2), can induce the dedifferentiation and proliferation of cultured human podocytes. Cellular internalization of Tat requires interactions with heparan sulfate proteoglycans and cholesterol-enriched lipid rafts (LRs). However, the specific distribution of Tat in human podocytes and its ability to associate with LRs have not been documented. Here, we found that Tat is preferentially recruited to LRs in podocytes isolated from children with HIVAN. Furthermore, we identified arginines in the basic domain (RKKRRQRRR) of Tat as essential for (1) targeting Tat to LRs, (2) Tat-mediated increases in the expression of Rho-A and matrix metalloproteinase-9 in LRs, and (3) Tat-mediated enhancement of FGF-2 signaling in human podocytes and HIV-transgenic mouse kidneys and the exacerbation of renal lesions in these mice. Tat carrying alanine substitutions in the basic domain (AKKAAQAAA) remained localized in the cytosol and did not associate with LRs or enhance FGF-2 signaling in cultured podocytes. These results show the specific association of Tat with LRs in podocytes isolated from children with HIVAN, confirm Tat as a regulator of FGF-2 signaling in LRs, and identify the key domain of Tat responsible for promoting these effects and aggravating renal injury in HIV-transgenic mice. Moreover, these results provide a molecular framework for developing novel therapies to improve the clinical outcome of children with HIVAN. PMID:24578133

  16. Potentiation of Methylmercury-Induced Death in Rat Cerebellar Granular Neurons Occurs by Further Decrease of Total Intracellular GSH with BDNF via TrkB in Vitro.

    PubMed

    Sakaue, Motoharu; Maki, Takehiro; Kaneko, Takuya; Hemmi, Natsuko; Sekiguchi, Hitomi; Horio, Tomoyo; Kadowaki, Erina; Ozawa, Aisa; Yamamoto, Masako

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is a principal factor for neurogenesis, neurodevelopment and neural survival through a BDNF receptor, tropomyosin-related kinase (Trk) B, while BDNF can also cause a decrease in the intracellular glutathione (GSH) level. We investigated the exacerbation of methylmercury-induced death of rat cerebellar granular neurons (CGNs) by BDNF in vitro. Since methylmercury can decrease intracellular GSH levels, we hypothesized that a further decrease of the intracellular GSH level is involved in the process of the exacerbation of neuronal cell death. In the present study, we established that in CGN culture, a decrease of the intracellular GSH level was further potentiated with BDNF in the process of the methylmercury-induced neuronal death and also in GSH reducer-induced neuronal death. BDNF treatment promoted the decrease in GSH levels induced by methylmercury and also by L-buthionine sulfoximine (BSO) and diethyl maleate (DEM). The promoting effect of BDNF was observed in a TrkB-vector transformant of the rat neuroblastoma B35 cell line but not in the mock-vector transformant. These results indicate that the exacerbating effect of BDNF on methylmercury-induced neuronal death in cultures of CGNs includes a further decrease of intracellular GSH levels, for which TrkB is essential. PMID:27251509

  17. Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione.

    PubMed

    Zhu, Jingsong; Krom, Bastiaan P; Sanglard, Dominique; Intapa, Chaidan; Dawson, Clinton C; Peters, Brian M; Shirtliff, Mark E; Jabra-Rizk, Mary Ann

    2011-01-01

    Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent. PMID:22205973

  18. UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

    SciTech Connect

    Jee, Hye Jin; Kim, Hyun-Ju; Kim, Ae Jeong; Bae, Yoe-Sik; Bae, Sun Sik; Yun, Jeanho

    2009-06-05

    Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2{sup -/-} mouse embryonic fibroblasts (MEFs) while Akt1{sup -/-} MEFs show cell cycle arrest. Here, we find that Akt1{sup -/-} MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated {beta}-galactosidase (SA {beta}-gal) staining indicate that Akt1{sup -/-} MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1{sup -/-} MEFs suppressed SA {beta}-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1{sup -/-} MEFs, suggesting that UV light induces premature senescence in Akt1{sup -/-} MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

  19. Proteomic analysis of a podocyte vesicle-enriched fraction from human normal and pathological urine samples.

    PubMed

    Lescuyer, Pierre; Pernin, Agnès; Hainard, Alexandre; Bigeire, Caty; Burgess, Jennifer A; Zimmermann-Ivol, Catherine; Sanchez, Jean-Charles; Schifferli, Jürg A; Hochstrasser, Denis F; Moll, Solange

    2008-07-01

    Podocytes (glomerular visceral epithelial cells) release vesicles into urine. Podocyte vesicle-enriched fractions from normal and pathological human urine samples were prepared for proteomic analysis. An immunoadsorption method was applied and enrichment of podocyte vesicles was assessed. We identified 76 unique proteins. One protein, serum paraoxonase/arylesterase 1 (PON-1), was newly identified in normal human urine sample. We confirmed this result and showed PON-1 expression in normal human kidney. These results demonstrated the potential for using the urine samples enriched in podocyte vesicles as a starting material in studies aimed at discovery of biomarkers for diseases. PMID:21136901

  20. Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels.

    PubMed

    Skatchkov, Serguei N; Bukauskas, Feliksas F; Benedikt, Jan; Inyushin, Mikhail; Kucheryavykh, Yuriy V

    2015-06-17

    Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions. PMID:26011388

  1. Edwardsiella tarda-Induced Inhibition of Apoptosis: A Strategy for Intracellular Survival

    PubMed Central

    Zhou, Ze-jun; Sun, Li

    2016-01-01

    Edwardsiella tarda is a Gram-negative bacterial pathogen that can infect a wide range of freshwater and marine fish. One salient feature of E. tarda is the ability to survive and replicate in various host cells. In this study, we observed that E. tarda replicated robustly in the zebrafish cell line ZF4, and that E. tarda-infected cells exhibited no detectable signs of apoptosis. Global transcriptome analysis and quantitative real-time RT-PCR revealed that E. tarda infection generally significantly downregulated pro-apoptotic genes and upregulated anti-apoptotic genes. To investigate the role of apoptosis in E. tarda infection, two upregulated anti-apoptotic genes (Fech and Prx3) and two downregulated pro-apoptotic genes (Brms1a and Ivns1a) were overexpressed in zebrafish. Subsequent infection study showed that Fech and Prx3 overexpression significantly promoted E. tarda dissemination in and colonization of fish tissues, while Brms1a and Ivns1a overexpression significantly reduced E. tarda dissemination and colonization. Consistently, when Fech and Prx3 were knocked down in zebrafish, E. tarda infection was significantly inhibited, whereas Brms1a and Ivns1a knockdown significantly enhanced E. tarda infection. These results indicate for the first time that E. tarda prevents apoptosis in teleost as a strategy for intracellular survival, and that some putative apoptotic genes of teleost function in the apoptosis pathway probably in a manner similar to that in mammalian systems. PMID:27471679

  2. A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm.

    PubMed

    Romarowski, Ana; Sánchez-Cárdenas, Claudia; Ramírez-Gómez, Héctor V; Puga Molina, Lis del C; Treviño, Claudia L; Hernández-Cruz, Arturo; Darszon, Alberto; Buffone, Mariano G

    2016-03-01

    During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca(2+) ([Ca(2+)]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca(2+)]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca(2+)]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca(2+)]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca(2+)]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca(2+)]i originating in the sperm head promotes the initiation of AR. PMID:26819478

  3. Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels

    PubMed Central

    Skatchkov, Serguei N.; Bukauskas, Feliksas F.; Benedikt, Jan; Inyushin, Mikhail

    2015-01-01

    Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell–cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions. PMID:26011388

  4. Comparative proteomic analysis reveals intracellular targets for bacillomycin L to induce Rhizoctonia solani Kühn hyphal cell death.

    PubMed

    Zhang, Bao; Qin, Yuxuan; Han, Yuzhu; Dong, Chunjuan; Li, Pinglan; Shang, Qingmao

    2016-09-01

    Bacillomycin L, a natural iturinic lipopeptide produced by Bacillus amyloliquefaciens, is characterized by strong antifungal activity against a variety of agronomically important filamentous fungi including Rhizoctonia solani Kühn. To further understand its antifungal actions, proteomes were comparatively studied within R. solani hyphal cells treated with or without bacillomycin L. The results show that 39 proteins were alternatively expressed within cells in response to this lipopeptide, which are involved in stress response, carbohydrate, amino acid and nucleotide metabolism, cellular component organization, calcium homeostasis, protein degradation, RNA processing, gene transcription, and others, suggesting that, in addition to inducing cell membrane permeabilization, iturin exhibits antibiotic activities by targeting intracellular molecules. Based on these results, a model of action of bacillomycin L against R. solani hyphal cells was proposed. Our study provides new insight into the antibiotic mechanisms of iturins. PMID:27267622

  5. Intracellular delivery of poly(I:C) induces apoptosis of fibroblast-like synoviocytes via an unknown dsRNA sensor.

    PubMed

    Karpus, Olga N; Hsiao, Cheng-Chih; de Kort, Hanneke; Tak, Paul P; Hamann, Jörg

    2016-08-26

    Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS. PMID:27343555

  6. Three-dimensional podocyte-endothelial cell co-cultures: Assembly, validation, and application to drug testing and intercellular signaling studies.

    PubMed

    Li, Min; Corbelli, Alessandro; Watanabe, Shojiro; Armelloni, Silvia; Ikehata, Masami; Parazzi, Valentina; Pignatari, Chiara; Giardino, Laura; Mattinzoli, Deborah; Lazzari, Lorenza; Puliti, Aldamaria; Cellesi, Francesco; Zennaro, Cristina; Messa, Piergiorgio; Rastaldi, Maria Pia

    2016-04-30

    Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies. PMID:26924225

  7. Protective effect of nifedipine against cytotoxicity and intracellular calcium alterations induced by acetaminophen in rat hepatocyte cultures.

    PubMed

    Ellouk-Achard, S; Mawet, E; Thibault, N; Dutertre-Catella, H; Thevenin, M; Claude, J R

    1995-01-01

    Alteration of calcium homeostasis has been proposed to play a major role in cell necrosis induced by a variety of chemical agents such as acetaminophen (APAP). In this study, a potential protective effect of the dihydropyridine calcium channel blocking agent, nifedipine, was investigated in vitro on acetaminophen-induced hepatocyte damage. Rat hepatocytes were exposed during 20 hours to various concentrations of APAP (0.50 to 4.00 mM). The following metabolic and functional parameters were investigated: -lactate dehydrogenase (LDH) release as an indicator of plasma membrane integrity, -cell viability evaluated by the colorimetric MTT assay, and intracellular calcium concentration as evaluated by two fluorimetric methods: a scanning laser cytometer using indo-1-AM as fluorescent probe and a fluorescence plate reader using fluo-3-AM as calcium indicator. Incubation of hepatocytes with APAP alone in the range 0.50 to 4.00mM resulted in a dose-response relationship with regard to LDH release (243% to 750% of control) and to the loss of cell viability (0 to 67% of control). Moreover these results were correlated with a significant increase in cytosolic calcium content (189 to 406 nM). Nifedipine treatment prior to APAP exposure, partially prevented LDH release, the plasma membrane blebbing, and thereby the loss of viability. In addition, intracellular calcium level progressively returned within the limits of the control values with increasing concentrations of nifedipine. It can be concluded that, in vitro conditions, nifedipine pretreatment exhibits a preventive effect against acetaminophen hepatocyte injury. PMID:7497906

  8. A Case of Podocytic Infolding Glomerulopathy with Focal Segmental Glomerulosclerosis

    PubMed Central

    Iguchi, Akira; Sohma, Ayako; Yamazaki, Hajime; Ito, Tomoyuki; Saeki, Takako; Ito, Yumi; Imai, Naohumi; Osawa, Yutaka; Narita, Ichiei

    2013-01-01

    Podocytic infolding glomerulopathy (PIG) has been proposed as a new disease entity. A 14-year-old girl underwent renal biopsy at our institution because of a chance finding of proteinuria. Light microscopic findings revealed a minor glomerular abnormality, but under a higher magnification, after periodic acid methenamine silver staining, a bubbling appearance in the glomerular basement membrane (GBM) was observed. An electron microscopic examination revealed microspheres in the GBM, which were sparse but global. The patient was diagnosed as having PIG. After 3 years, her urinary protein had increased and a second biopsy was performed, showing focal segmental glomerulosclerosis in addition to a lesser degree of podocytic infolding than at the first biopsy. This is the first report of a case complicated by a different type of glomerulonephritis after being diagnosed as PIG. A few cases of PIG are complicated by focal segmental glomerulosclerosis, suggesting several mechanisms for the disorder. PMID:24027576

  9. Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

    SciTech Connect

    Banerjee, Chaitali; Goswami, Ramansu; Datta, Soma; Rajagopal, R.; Mazumder, Shibnath

    2011-10-01

    We had earlier shown that exposure to arsenic (0.50 {mu}M) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca{sup 2+}) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca{sup 2+} homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca{sup 2+} levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: > Altered Ca{sup 2+} homeostasis leads to arsenic-induced HKM apoptosis. > Calpain-2 plays a critical role in the process. > ERK is pro-apoptotic in arsenic-induced HKM apoptosis. > Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.

  10. Stress-induced inhibition of nonsense mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11

    PubMed Central

    Martin, Leenus; Gardner, Lawrence B.

    2014-01-01

    SLC7A11 encodes a subunit of the xCT cystine/glutamate amino acid transport system and plays a critical role in the generation of glutathione and the protection of cells from oxidative stress. Expression of SLC7A11 promotes tumorigenesis and chemotherapy resistance, but while SLC7A11 has been previously noted to be upregulated in hypoxic cells its regulation has not been fully delineated. We have recently shown that nonsense mediated RNA decay (NMD) is inhibited by cellular stresses generated by the tumor microenvironment, including hypoxia, and augments tumorigenesis. Here we demonstrate that the inhibition of NMD by various cellular stresses leads to the stabilization and upregulation of SLC7A11 mRNA and protein. The inhibition of NMD and upregulation of SLC7A11 augments intracellular cystine transport, and increases intracellular levels of cysteine and glutathione. Accordinglyy, the inhibition of NMD protects cells against oxidative stress via SLC7A11 upregulation. Together our studies identify a mechanism for the dynamic regulation of SLC7A11, through the stress-inhibited regulation of NMD, and add to the growing evidence that the inhibition of NMD is an adaptive response. PMID:25399695

  11. Intracellular potassium as a possible inducer of amino acid transport across hamster jejunal enterocytes.

    PubMed Central

    Cremaschi, D; James, P S; Meyer, G; Rossetti, C; Smith, M W

    1986-01-01

    Brush border membrane potentials (Vm), intracellular K+ activity (aiK) and alanine uptake were measured in different parts of villi in mid-jejunal tissue taken from hamsters fed different amounts of food at high and low environmental temperatures. Basal villus enterocytes (Y cells) were found to have lower values for Vm and aiK than upper villus enterocytes (O cells). Alanine uptake was confined to O cells. The position on the villus where values for Vm and aiK changed, and where alanine uptake could first be seen to take place, depended on the energy intake and environmental temperature at which hamsters were kept. Na+-dependent alanine uptake and Vm were both higher in O cells of hamsters fed 10 kcal day-1 at 30 degrees C (10 k/30 degrees C) compared with animals fed 30 kcal day-1 at an environmental temperature of 12 degrees C (30 k/12 degrees C hamsters). The rates at which enterocytes migrated along the crypt-villus axis, measured separately in thymidine-labelling experiments, were 6.9 and 16.1 micron h-1 for 10 k/30 degrees C and 30 k/12 degrees C hamsters respectively. Both Vm and aiK were estimated, from these measurements, to have increased significantly by the time enterocytes became 30 h old. Alanine uptake began 15 h later. Neither of these parameters were influenced by previous adaptation conditions. The close temporal and variable positional relationship found between changes in aiK and onset of transport suggests that early changes in electrolyte composition might initiate a second phase of development enabling the enterocyte to absorb nutrients. The possibility that other ions besides K+ might also be involved in this aspect of regulation is also discussed. PMID:3795055

  12. Sulfur mustard-induced increase in intracellular calcium: A mechanism of mustard toxicity

    SciTech Connect

    Ray, R.; Majerus, B.J.; Munavalli, G.S.; Petrali, J.P.

    1993-05-13

    The effect of sulfur mustard SM, bis-(2-chloroethyl) sulfide on intracellular free Ca2+ concentration (Ca2+)i was studied in vitro using the clonal mouse neuroblastoma-rat glioma hybrid NG108-15 and primary normal human epidermal keratinocyte (NHEK) cell culture models. SM depletes cellular glutathione (GSH) and thus may inhibit GSH-dependent Ca2+-ATPase (Ca2+ pump), leading to a high (Ca2+) and consequent cellular toxicity. Following 0.3 mM SM exposure, GSH levels decreased 20-34% between 1-6 hr in NG108-15 cells. SM increased (Ca2+)i, measured using the Ca2+-specific fluorescent probe Fluo-3 AM, in both NG108-15 cells (1030% between 2-6 hr) and NHEK (23-30% between 0.5-3 hr) . Depletion of cellular GSH by buthionine sulfoximine (1 mM), a specific GSH biosynthesis inhibitor, also increased Ca2+, (88% at 1 hr) in NHEK, suggesting that GSH depletion may lead to increased (Ca2+)i. Calcium, localized cytochemically with antimony, accumulated in increased amounts around mitochondria and endoplasmic reticula, in the cytosol, and in particular in the euchromatin regions of the nucleus beginning at 6 hr after 0.3 mM SM exposure of NG108-15 cells. Cell membrane integrity examined with the fluorescent membrane probe calcein AM was unaffected through 6 hr following 1 mM SM exposure; and cell viability (NG108-15 cells) measured by trypan blue exclusion was >80% of control through 9 hr following 0.3 mM SM exposure.

  13. Contribution of Endogenously Produced Reactive Oxygen Species to the Activation of Podocyte NLRP3 Inflammasomes in Hyperhomocysteinemia

    PubMed Central

    Abais, Justine M.; Xia, Min; Li, Guangbi; Gehr, Todd W. B.; Boini, Krishna M.; Li, Pin-Lan

    2013-01-01

    Hyperhomocysteinemia (hHcys) is an important pathogenic factor contributing to the progression of end-stage renal disease. Recent studies have demonstrated the implication of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated NLRP3 inflammasome activation in the development of podocyte injury and glomerular sclerosis during hHcys. However, it remains unknown which reactive oxygen species (ROS) are responsible for this activation of NLRP3 inflammasomes and how such action of ROS is controlled. The present study tested the contribution of common endogenous ROS including superoxide (O2•−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) to the activation of NLRP3 inflammasomes in mouse podocytes and glomeruli. In vitro, confocal microscopy and size exclusion chromatography demonstrated that dismutation of O2•− by 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) and decomposition of H2O2 by catalase prevented Hcys-induced aggregation of NLRP3 inflammasome proteins and inhibited Hcys-induced caspase-1 activation and IL-1β production in mouse podocytes. However, •OH scavenger tetramethylthiourea (TMTU) had no significant effect on either Hcys-induced NLRP3 inflammasome formation or activation. In vivo, scavenging of O2•− by TEMPOL and removal of H2O2 by catalase substantially inhibited NLRP3 inflammasome formation and activation in glomeruli of hHcys mice as shown by reduced colocalization of NLRP3 with ASC or caspase-1 and inhibition of caspase-1 activation and IL-1β production. Furthermore, TEMPOL and catalase significantly attenuated hHcys-induced glomerular injury. In conclusion, endogenously produced O2•− and H2O2 primarily contribute to NLRP3 inflammasome formation and activation in mouse glomeruli resulting in glomerular injury or consequent sclerosis during hHcys. PMID:24140862

  14. Opposite regulation of cocaine-induced intracellular signaling and gene expression by dopamine D1 and D3 receptors.

    PubMed

    Zhang, Jianhua; Xu, Ming

    2006-08-01

    Repeated exposure to cocaine induces persistent neuroadaptations that involve alterations in cellular signaling and gene expression mediated by dopamine (DA) receptors in the brain. Both dopamine D1 and D3 receptors mediate cocaine-induced behaviors and they are also coexpressed in the same neurons in the nucleus accumbens (NAc) and caudoputamen (CPu). We have investigated whether these two receptors coordinately regulate intracellular signaling and gene expression after acute and repeated cocaine administration. We found that extracellular signal-regulated kinase (ERK) activation and c-fos induction in the CPu following an acute cocaine administration is mediated by the D1 receptor and inhibited by the D3 receptor. ERK activation is necessary for acute cocaine-induced expression of fos family genes that include c-fos, fosB, and fra2. Furthermore, potential target genes of cAMP response element-binding (CREB) protein and/or AP-1 transcription complex, including dynorphin, neogenin, and synaptotagmin VII, are also oppositely regulated by D1 and D3 receptors after repeated cocaine injections. Lastly, such regulation requires proper ERK activation. These results suggest that D1 and D3 receptors oppositely regulate target gene expression by regulating ERK activation after cocaine administration. PMID:17105899

  15. Tau Protein Mediates APP Intracellular Domain (AICD)-Induced Alzheimer’s-Like Pathological Features in Mice

    PubMed Central

    Dawson, Hana N.; Pimplikar, Sanjay W.

    2016-01-01

    Amyloid precursor protein (APP) is cleaved by gamma-secretase to simultaneously generate amyloid beta (Aβ) and APP Intracellular Domain (AICD) peptides. Aβ plays a pivotal role in Alzheimer’s disease (AD) pathogenesis but recent studies suggest that amyloid-independent mechanisms also contribute to the disease. We previously showed that AICD transgenic mice (AICD-Tg) exhibit AD-like features such as tau pathology, aberrant neuronal activity, memory deficits and neurodegeneration in an age-dependent manner. Since AD is a tauopathy and tau has been shown to mediate Aβ–induced toxicity, we examined the role of tau in AICD-induced pathological features. We report that ablating endogenous tau protects AICD-Tg mice from deficits in adult neurogenesis, seizure severity, short-term memory deficits and neurodegeneration. Deletion of tau restored abnormal phosphorylation of NMDA receptors, which is likely to underlie hyperexcitability and associated excitotoxicity in AICD-Tg mice. Conversely, overexpression of wild-type human tau aggravated receptor phosphorylation, impaired adult neurogenesis, memory deficits and neurodegeneration. Our findings show that tau is essential for mediating the deleterious effects of AICD. Since tau also mediates Aβ-induced toxic effects, our findings suggest that tau is a common downstream factor in both amyloid-dependent and–independent pathogenic mechanisms and therefore could be a more effective drug target for therapeutic intervention in AD. PMID:27459671

  16. Effect of vanadate on ATP-induced increase in intracellular calcium ion levels in human umbilical vein endothelial cells.

    PubMed

    Nejime, Namie; Tada, Yukari; Kagota, Satomi; Kubota, Yoko; Shibuichi, Ikuo; Shinoda, Yuki; Yamamoto, Tomohiro; Watanabe, Yasuo; Shinozuka, Kazumasa

    2010-01-01

    We investigated the effect of ammonium vanadate (vanadate) on ATP-induced increases in intracellular calcium ion level ([Ca(2+)](i)) of human umbilical vein endothelial cells (HUVEC) by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe Calcium Green 1/AM. The ATP analogue 2-methylthio-ATP (2meS-ATP), at 10 microM, significantly increased the [Ca(2+)](i) of HUVEC, and this was abolished by 1 microM thapsigargin (a calcium pump inhibitor), whereas extracellular free calcium had no effect. Vanadate at 10 microM also significantly increased the [Ca(2+)](i) of HUVEC, which was abolished by 1 microM thapsigargin. However, vanadate at 1 microM did not exert such a significant effect. We thus examined the influence of < or =1 microM vanadate for 24 h on 2meS-ATP-induced increase in [Ca(2+)](i). Vanadate significantly reduced the action of 2meS-ATP at 1 microM but not at 0.1 microM. Endogenously released ATP is known to induce various actions on endothelial cells. The present results suggest that vanadate exerts a regulatory influence on the function of vascular endothelial cells. PMID:20522978

  17. Podocyte Pathology and Nephropathy – Sphingolipids in Glomerular Diseases

    PubMed Central

    Merscher, Sandra; Fornoni, Alessia

    2014-01-01

    Sphingolipids are components of the lipid rafts in plasma membranes, which are important for proper function of podocytes, a key element of the glomerular filtration barrier. Research revealed an essential role of sphingolipids and sphingolipid metabolites in glomerular disorders of genetic and non-genetic origin. The discovery that glucocerebrosides accumulate in Gaucher disease in glomerular cells and are associated with clinical proteinuria initiated intensive research into the function of other sphingolipids in glomerular disorders. The accumulation of sphingolipids in other genetic diseases including Tay–Sachs, Sandhoff, Fabry, hereditary inclusion body myopathy 2, Niemann–Pick, and nephrotic syndrome of the Finnish type and its implications with respect to glomerular pathology will be discussed. Similarly, sphingolipid accumulation occurs in glomerular diseases of non-genetic origin including diabetic kidney disease (DKD), HIV-associated nephropathy, focal segmental glomerulosclerosis (FSGS), and lupus nephritis. Sphingomyelin metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate have also gained tremendous interest. We recently described that sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is expressed in podocytes where it modulates acid sphingomyelinase activity and acts as a master modulator of danger signaling. Decreased SMPDL3b expression in post-reperfusion kidney biopsies from transplant recipients with idiopathic FSGS correlates with the recurrence of proteinuria in patients and in experimental models of xenotransplantation. Increased SMPDL3b expression is associated with DKD. The consequences of differential SMPDL3b expression in podocytes in these diseases with respect to their pathogenesis will be discussed. Finally, the role of sphingolipids in the formation of lipid rafts in podocytes and their contribution to the maintenance of a functional slit diaphragm in the glomerulus will be discussed. PMID:25126087

  18. Podocyte pathology and nephropathy - sphingolipids in glomerular diseases.

    PubMed

    Merscher, Sandra; Fornoni, Alessia

    2014-01-01

    Sphingolipids are components of the lipid rafts in plasma membranes, which are important for proper function of podocytes, a key element of the glomerular filtration barrier. Research revealed an essential role of sphingolipids and sphingolipid metabolites in glomerular disorders of genetic and non-genetic origin. The discovery that glucocerebrosides accumulate in Gaucher disease in glomerular cells and are associated with clinical proteinuria initiated intensive research into the function of other sphingolipids in glomerular disorders. The accumulation of sphingolipids in other genetic diseases including Tay-Sachs, Sandhoff, Fabry, hereditary inclusion body myopathy 2, Niemann-Pick, and nephrotic syndrome of the Finnish type and its implications with respect to glomerular pathology will be discussed. Similarly, sphingolipid accumulation occurs in glomerular diseases of non-genetic origin including diabetic kidney disease (DKD), HIV-associated nephropathy, focal segmental glomerulosclerosis (FSGS), and lupus nephritis. Sphingomyelin metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate have also gained tremendous interest. We recently described that sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is expressed in podocytes where it modulates acid sphingomyelinase activity and acts as a master modulator of danger signaling. Decreased SMPDL3b expression in post-reperfusion kidney biopsies from transplant recipients with idiopathic FSGS correlates with the recurrence of proteinuria in patients and in experimental models of xenotransplantation. Increased SMPDL3b expression is associated with DKD. The consequences of differential SMPDL3b expression in podocytes in these diseases with respect to their pathogenesis will be discussed. Finally, the role of sphingolipids in the formation of lipid rafts in podocytes and their contribution to the maintenance of a functional slit diaphragm in the glomerulus will be discussed. PMID:25126087

  19. Proteinuria in preeclampsia from a podocyte injury perspective.

    PubMed

    Henao, Daniel E; Saleem, Moin A

    2013-12-01

    Preeclampsia is a frequent cause of maternal and fetal morbidity and mortality worldwide. The underlying causes of this hypertensive complication have remained elusive. The placenta seems to be at the origin of the disease, as its removal appears to be the only effective treatment available. Many organs can potentially be affected. Nonetheless, kidney alterations are always present: proteinuria is one of the hallmarks for a preeclampsia diagnosis. VEGF is pivotal for maintaining glomerular filtration barrier function; hence, the elevated concentrations of placental-derived VEGF inhibitors, such as sFlt-1, may largely explain the renal alterations observed. Classically, glomerular endothelial injury was considered responsible for the renal impairment present in preeclampsia. Recent findings, however, have shown that podocytes are crucial in explaining the loss of filtration capacity of the preeclamptic kidney. The aims of this manuscript are to detail the main findings that associate podocyte injury with proteinuria in preeclampsia, and discuss the eventual applications of podocyte damage biomarkers in clinical practice. PMID:24214687

  20. Fullerenol Nanoparticles with Structural Activity Induce Variable Intracellular Actin Filament Morphologies.

    PubMed

    Jin, Junjiang; Dong, Ying; Wang, Ying; Xia, Lin; Gu, Weihong; Bai, Xue; Chang, Yanan; Zhang, Mingyi; Chen, Kui; Li, Juan; Zhao, Lina; Xing, Gengmei

    2016-06-01

    Fullerenol nanoparticles are promising for various biological applications; many studies have shown that they induce variable and diverse biological effects including side effects. Separation and purification of two fractions of fullerenols has demonstrated that they have varied chemical structures on the surfaces of their carbon cages. Actin is an important structural protein that is able to transform functional structures under varied physiological conditions. We assessed the abilities of the two fractions of fullerenols to attach to actin and induce variable morphological features in actin filament structures. Specifically the fullerenol fraction with a surface electric charge of -1.913 ± 0.008q (x10(-6) C) has percentages of C-OH and C=O on the carbon cage of 16.14 ± 0.60 and 17.55 ± 0.69. These features allow it to form intermolecular hydrogen bonds with actin at a stoichiometric ratio of four fullerenols per actin subunit. Molecular simulations revealed these specific binding sites and binding modes in atomic details in the interaction between the active fullerenol and actin filament. Conversely, these interactions were not possible for the other fraction of fullerenol with that percentages of C-OH and C=O on the carbon cage were 15.59 ± 0.01 and 1.94 ± 0.11. Neither sample induced appreciable cytotoxicity or acute cell death. After entering cells, active fullerenol binding to actin induces variable morphological features and may transform ATP-actin to ADP-actin. These changes facilitate the binding of ADF/cofilin, allowing cofilin to sever actin filaments to form cofilin/actin/fullerenol rods. Our findings suggest that fullerenol with structural activity binding disturbs actin filament structure, which may inhibit locomotion of cell or induce chronic side effects in to cells. PMID:27319217

  1. Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

    PubMed

    Shimizu, Shunichi; Yonezawa, Ryo; Negoro, Takaharu; Yamamoto, Shinichiro; Numata, Tomohiro; Ishii, Masakazu; Mori, Yasuo; Toda, Takahiro

    2015-11-01

    Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2

  2. Targets and Intracellular Signaling Mechanisms for Deoxynivalenol-Induced Ribosomal RNA Cleavage

    PubMed Central

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-01-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. PMID:22491426

  3. Targets and intracellular signaling mechanisms for deoxynivalenol-induced ribosomal RNA cleavage.

    PubMed

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-06-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. PMID:22491426

  4. Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells.

    PubMed

    Nascimento, Aline Rosa; Pimenta, Maristela Taliari; Lucas, Thais F G; Royer, Carine; Porto, Catarina Segreti; Lazari, Maria Fatima Magalhaes

    2012-09-15

    Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of G(i). AKT activation could be detected 5 min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was G(i)- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to G(i) and activation of both MEK/ERK1/2 and PI3K/AKT pathways. PMID:22819701

  5. Autophagy regulates hyperoxia-induced intracellular accumulation of surfactant protein C in alveolar type II cells.

    PubMed

    Zhang, Liang; Zhao, Shuang; Yuan, Li-Jie; Wu, Hong-Min; Jiang, Hong; Zhao, Shi-Meng; Luo, Gang; Xue, Xin-Dong

    2015-10-01

    Surfactant protein C (SP-C) deficiency is a risk factor for hyperoxia-induced bronchopulmonary dysplasia in newborn infants. However, the role of SP-C deficiency in the process is unclear. Here, using neonatal rat BPD model and MLE-12, mouse alveolar epithelial type II cell, we examined the changes of SP-C levels during hyperoxia. Immunohistochemistry, immunofluorescence, and ELISA analysis showed SP-C accumulation in alveolar epithelial type II cells. Electron microscopy further demonstrated the accumulation of lamellar bodies and the co-localization of lamellar bodies with autophagosomes in the cytoplasm of alveolar epithelial type II cells. The inhibition of autophagy with 3-Methyladenine and knockdown of Atg7 abolished hyperoxia-induced SP-C accumulation in the cytoplasm. Furthermore, inhibition of JNK signaling with SP600125 suppressed hyperoxia-induced Atg7 expression and SP-C accumulation. These findings suggest that hyperoxia triggers autophagy via JNK signaling-mediated Atg7 expression, which promotes the accumulation of SP-C within alveolar epithelial type II cells. Our data provide a potential approach for hyperoxic lung injury therapy by targeted pharmacological inhibition of autophagic pathway. PMID:26122393

  6. Enhanced intracellular calcium promotes metabolic and secretory disturbances in rat gastric mucosa during ethanol-induced gastritis.

    PubMed

    Hernández-Rincón, Ileana; Olguín-Martínez, Marisela; Hernández-Muñoz, Rolando

    2003-03-01

    Changes in the Ca(2+) homeostasis have been implicated in cell injury and death. However, Ca(2+) participation in ethanol-induced chronic gastric mucosal injury has not been elucidated. We have developed a model of ethanol-induced chronic gastric injury in rats, characterized by marked alterations in plasma membranes from gastric mucosa and a compensatory cell proliferation, which follows ethanol withdrawal. Therefore, the present study explored the possible role of intracellular Ca(2+) in the oxidative metabolism and in acid secretion in this experimental model. Glucose oxidation was greatly enhanced in the injured mucosa, as evaluated by CO(2) production by isolated mucosal preparations incubated with (14)C-radiolabeled glucose in different carbons. Oxygen consumption and acid secretion (aminopyrine accumulation) were also stimulated. A predominating secretory status was morphologically identified by electron microscopy in oxyntic cells of gastric mucosa from ethanol-treated rats. A coupling between secretory and metabolic effects induced by ethanol (demonstrated by an inhibitory effect of omeprazole in both parameters) was found. These ethanol-induced effects were also inhibited by addition of Ca(2+) chelators to isolated gastric mucosa samples. Lanthanum, a Ca(2+) channel blocker, inhibited ethanol-promoted increase of oxidative metabolism. In addition, a stimulated Ca(2+) uptake by mucosal minces and increased in vivo Ca(2+) levels in cytosolic and mitochondrial fractions, were also noticed. Enhanced glucose and oxygen consumptions were associated with higher ATP and NADP+ availability, whereas cytosolic NAD/NADH ratio (assessed by mucosal levels of lactate and pyruvate) was not significantly modified by the chronic ethanol administration. In conclusion, changes in Ca(2+) homeostasis, probably mainly due to increased extracellular Ca(2+) uptake, could mediate secretory and metabolic alterations found in the gastric mucosa from rats chronically treated with

  7. Diethyldithiocarbamate induces apoptosis in neuroblastoma cells by raising the intracellular copper level, triggering cytochrome c release and caspase activation.

    PubMed

    Matias, Andreza C; Manieri, Tânia M; Cipriano, Samantha S; Carioni, Vivian M O; Nomura, Cassiana S; Machado, Camila M L; Cerchiaro, Giselle

    2013-02-01

    Dithiocarbamates are nitrogen- and sulfur-containing compounds commonly used in pharmacology, medicine and agriculture. The molecular effects of dithiocarbamates on neuronal cell systems are not fully understood, especially in terms of their ability to accumulate copper ions inside the cell. In this work, the molecular effects of N,N-diethyldithiocarbamate (DEDTC) were studied in human SH-SY5Y neuroblastoma cells to determine the role of copper in the DEDTC toxicity and the pathway trigged in cell by the complex Cu-DEDTC. From concentration-dependent studies, we found that 5 μM of this compound induced a drastic decrease in viable cells with a concomitant accumulation in intracellular copper resulted from complexation with DEDTC, measured by atomic absorption spectroscopy. The mechanism of DEDTC-induced apoptosis in neuronal model cells is thought to occur through the death receptor signaling triggered by DEDTC-copper complex in low concentration that is associated with the activation of caspase 8. Our results indicated that the mechanism of cell death involves cytochrome c release forming the apoptosome together with Apaf-1 and caspase 9, converting the caspase 9 into its active form, allowing it to activate caspase 3 as observed by immunofluorescence. This pathway is induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions present in the culture medium and transports them into the cell, suggesting that the DEDTC by itself was not able to cause cell death and the major effect is from its copper-complex in neuroblastoma cells. The present study suggests a role for the influence of copper by low concentrations of DEDTC in the extracellular media, the absorption and accumulation of copper in the cell and apoptotic events, induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions. PMID:22951949

  8. Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP(+)/NADPH ratio.

    PubMed

    Itsumi, M; Inoue, S; Elia, A J; Murakami, K; Sasaki, M; Lind, E F; Brenner, D; Harris, I S; Chio, I I C; Afzal, S; Cairns, R A; Cescon, D W; Elford, A R; Ye, J; Lang, P A; Li, W Y; Wakeham, A; Duncan, G S; Haight, J; You-Ten, A; Snow, B; Yamamoto, K; Ohashi, P S; Mak, T W

    2015-11-01

    Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock. PMID:25882048

  9. BDNF-stimulated intracellular signalling mechanisms underlie exercise-induced improvement in spatial memory in the male Wistar rat.

    PubMed

    Bechara, Ranya G; Lyne, Ronan; Kelly, Áine M

    2014-12-15

    Exercise-induced improvements in learning are associated with neurotrophic and neurogenic changes in the dentate gyrus, but the intracellular signalling mechanisms that may mediate these improvements remain unknown. In the current study we investigate the effects of one week of forced exercise on spatial memory and analyse in parallel BDNF-stimulated signalling pathways in cells of the dentate gyrus. Additionally, we test whether a single intracerebroventricular (i.c.v.) injection of BDNF can mimic the observed cognitive and signalling changes. Male Wistar rats were assigned to exercised and sedentary groups and tested in a spatial task post-exercise. Tissue from the dentate gyrus was assessed for expression and release of BDNF, and for changes in expression and activation of TrkB, ERK and synapsin-1. In a separate set of experiments, male Wistar rats received a single i.c.v. injection of BDNF and were then tested in the same spatial learning task. Exercised and BDNF-treated (but not control) rats could successfully complete an object displacement task that tests spatial learning. Exercised rats and BDNF-treated rats displayed increases BDNF expression and ERK1 activation, while exercised rats showed increases in cell division, stimulated BDNF release, TrkB activation, and synapsin-1 expression in the dentate gyrus. We conclude that exercise-induced increases in BDNF in the dentate gyrus are sufficient to cause improvements in spatial memory by activating signalling cascades that enhance synaptic transmission in the hippocampus. PMID:24269499

  10. The intracellular sensor NOD2 induces microRNA-29 expression in human dendritic cells to limit IL-23 release.

    PubMed

    Brain, Oliver; Owens, Benjamin M J; Pichulik, Tica; Allan, Philip; Khatamzas, Elham; Leslie, Alasdair; Steevels, Tessa; Sharma, Sameer; Mayer, Alice; Catuneanu, Ana Maria; Morton, Victoria; Sun, Mei-Yi; Jewell, Derek; Coccia, Margherita; Harrison, Oliver; Maloy, Kevin; Schönefeldt, Susann; Bornschein, Simon; Liston, Adrian; Simmons, Alison

    2013-09-19

    NOD2 is an intracellular sensor that contributes to immune defense and inflammation. Here we investigated whether NOD2 mediates its effects through control of microRNAs (miRNAs). miR-29 expression was upregulated in human dendritic cells (DCs) in response to NOD2 signals, and miR-29 regulated the expression of multiple immune mediators. In particular, miR-29 downregulated interleukin-23 (IL-23) by targeting IL-12p40 directly and IL-23p19 indirectly, likely via reduction of ATF2. DSS-induced colitis was worse in miR-29-deficient mice and was associated with elevated IL-23 and T helper 17 signature cytokines in the intestinal mucosa. Crohn's disease (CD) patient DCs expressing NOD2 polymorphisms failed to induce miR-29 upon pattern recognition receptor stimulation and showed enhanced release of IL-12p40 on exposure to adherent invasive E. coli. Therefore, we suggest that loss of miR-29-mediated immunoregulation in CD DCs might contribute to elevated IL-23 in this disease. PMID:24054330

  11. Intracellular calcium disturbances induced by arsenic and its methylated derivatives in relation to genomic damage and apoptosis induction.

    PubMed

    Florea, Ana-Maria; Yamoah, Ebenezer N; Dopp, Elke

    2005-06-01

    Arsenic and its methylated derivatives are contaminants of air, water, and food and are known as toxicants and carcinogens. Arsenic compounds are also being used as cancer chemotherapeutic agents. In humans, inorganic arsenic is metabolically methylated to mono-, di-, and trimethylated forms. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In recent years, the correlation between arsenic exposure, cytotoxicity and genotoxicity, mutagenicity, and tumor promotion has been established, as well as the association of arsenic exposure with perturbation of physiologic processes, generation of reactive oxygen species, DNA damage, and apoptosis induction. Trivalent forms of arsenic have been found to induce apoptosis in several cellular systems with involvement of membrane-bound cell death receptors, activation of caspases, release of calcium stores, and changes of the intracellular glutathione level. It is well known that calcium ion deregulation plays a critical role in apoptotic cell death. A calcium increase in the nuclei might lead to toxic effects in the cell. In this review, we highlight the relationship between induced disturbances of calcium homeostasis, genomic damage, and apoptotic cell death caused by arsenic and its organic derivatives. PMID:15929885

  12. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells.

    PubMed

    Sen, Nilkantha; Das, Benu Brata; Ganguly, Agneyo; Banerjee, Bijoylaxhmi; Sen, Tanusree; Majumder, Hemanta K

    2006-11-01

    Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells. PMID:16707127

  13. Upregulation of α3β1-Integrin in Podocytes in Early-Stage Diabetic Nephropathy

    PubMed Central

    Sawada, Kaichiro; Kaneyama, Noriko; Shiraiwa, Sawako; Moriya, Hitomi; Miyatake, Han; Tanaka, Eitaro; Yamamoto, Naoyuki; Miyauchi, Masaaki; Kimura, Moritsugu; Wada, Takehiko; Fukagawa, Masafumi

    2016-01-01

    Background. Podocyte injury plays an important role in the onset and progression of diabetic nephropathy (DN). Downregulation of α3β1-integrin expression in podocytes is thought to be associated with podocyte detachment from the glomerular basement membrane, although the mechanisms remain obscure. To determine the mechanism of podocyte detachment, we analyzed the expression levels of α3β1-integrin in podocytes in early and advanced stages of DN. Methods. Surgical specimens from DN patients were examined by in situ hybridization, and the expression levels of α3- and β1-integrin subunits in glomeruli of early (n = 6) and advanced (n = 8) stages were compared with those of normal glomeruli (n = 5). Heat-sensitive mouse podocytes (HSMP) were cultured with TGF-β1 to reproduce the microenvironment of glomeruli of DN, and the expression levels of integrin subunits and the properties of migration and attachment were examined. Results. Podocytes of early-stage DN showed upregulation of α3- and β1-integrin expression while those of advanced stage showed downregulation. Real-time PCR indicated a tendency for upregulation of α3- and β1-integrin in HSMP cultured with TGF-β1. TGF-β1-stimulated HSMP also showed enhanced in vitro migration and attachment on collagen substrate. Conclusions. The results suggested that podocyte detachment during early stage of DN is mediated through upregulation of α3β1-integrin. PMID:27340677

  14. Cystinosin deficiency causes podocyte damage and loss associated with increased cell motility.

    PubMed

    Ivanova, Ekaterina A; Arcolino, Fanny O; Elmonem, Mohamed A; Rastaldi, Maria P; Giardino, Laura; Cornelissen, Elisabeth M; van den Heuvel, Lambertus P; Levtchenko, Elena N

    2016-05-01

    The involvement of the glomerulus in the pathogenesis of cystinosis, caused by loss-of-function mutations in cystinosin (CTNS, 17p13), is a matter of controversy. Although patients with cystinosis demonstrate glomerular lesions and high-molecular-weight proteinuria starting from an early age, a mouse model of cystinosis develops only signs of proximal tubular dysfunction. Here we studied podocyte damage in patients with cystinosis by analyzing urinary podocyte excretion and by in vitro studies of podocytes deficient in cystinosin. Urine from patients with cystinosis presented a significantly higher amount of podocytes compared with controls. In culture, cystinotic podocytes accumulated cystine compatible with cystinosin deficiency. The expression of podocyte specific genes CD2AP, podocalyxin, and synaptopodin and of the WT1 protein was evident in all cell lines. Conditionally immortalized podocyte lines of 2 patients with different CTNS mutations had altered cytoskeleton, impaired cell adhesion sites, and increased individual cell motility. Moreover, these cells showed enhanced phosphorylation of both Akt1 and Akt2 (isoforms of protein kinase B). Inhibition of Akt by a specific inhibitor (Akti inhibitor 1/2) resulted in normalization of the hypermotile phenotype. Thus, our study extends the list of genetic disorders causing podocyte damage and provides the evidence of altered cell signaling cascades resulting in impaired cell adhesion and enhanced cell motility in cystinosis. PMID:27083281

  15. Molecular and Cellular Events Mediating Glomerular Podocyte Dysfunction and Depletion in Diabetes Mellitus

    PubMed Central

    Anil Kumar, P.; Welsh, Gavin I.; Saleem, Moin A.; Menon, Ram K.

    2014-01-01

    The essential function of the kidney is to ensure formation of a relatively protein-free ultra-filtrate, urine. The rate of filtration and composition of the primary renal filtrate is determined by the transport of fluid and solutes across the glomerular filtration barrier consisting of endothelial cells, the glomerular basement membrane, and podocyte foot processes. In diabetes mellitus (DM), components of the kidney that enable renal filtration get structurally altered and functionally compromised resulting in proteinuria that often progresses to end-stage renal disease. Histological alterations in DM include early hypertrophy of glomerular and tubular components, subsequent thickening of basement membrane in glomeruli and tubules, progressive accumulation of extracellular matrix proteins in the glomerular mesangium and loss of podocytes, together constituting a clinical condition referred to as diabetic nephropathy (DN). The glomerulus has become the focus of research investigating the mechanism of proteinuria. In particular, the progressive dysfunction and/or loss of podocytes that is contemporaneous with proteinuria in DN have attracted intense scientific attention. The absolute number of podocytes predicts glomerular function and podocyte injury is a hallmark of various glomerular diseases. This review discusses the importance of podocytes in normal renal filtration and details the molecular and cellular events that lead to podocyte dysfunction and decreased podocyte count in DN. PMID:25309512

  16. Vps34 Deficiency Reveals the Importance of Endocytosis for Podocyte Homeostasis

    PubMed Central

    Bechtel, Wibke; Helmstädter, Martin; Balica, Jan; Hartleben, Björn; Kiefer, Betina; Hrnjic, Fatima; Schell, Christoph; Kretz, Oliver; Liu, Shuya; Geist, Felix; Kerjaschki, Dontscho; Walz, Gerd

    2013-01-01

    The molecular mechanisms that maintain podocytes and consequently, the integrity of the glomerular filtration barrier are incompletely understood. Here, we show that the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34) plays a central role in modulating endocytic pathways, maintaining podocyte homeostasis. In mice, podocyte-specific conditional knockout of Vps34 led to early proteinuria, glomerular scarring, and death within 3–9 weeks of age. Vps34-deficient podocytes exhibited substantial vacuolization and foot process effacement. Although the formation of autophagosomes and autophagic flux were impaired, comparisons between podocyte-specific Vps34-deficient mice, autophagy-deficient mice, and doubly deficient mice suggested that defective autophagy was not primarily responsible for the severe phenotype caused by the loss of Vps34. In fact, Rab5-positive endosomal compartments, endocytosis, and fluid-phase uptake were severely disrupted in Vps34-deficient podocytes. Vps34 deficiency in nephrocytes, the podocyte-like cells of Drosophila melanogaster, resulted in a block between Rab5- and Rab7-positive endosomal compartments. In summary, these data identify Vps34 as a major regulator of endolysosomal pathways in podocytes and underline the fundamental roles of endocytosis and fluid-phase uptake for the maintenance of the glomerular filtration barrier. PMID:23492732

  17. Identification of intracellular signaling pathways that induce myotonic dystrophy protein kinase expression during myogenesis.

    PubMed

    Carrasco, Marta; Canicio, Judith; Palacín, Manuel; Zorzano, Antonio; Kaliman, Perla

    2002-08-01

    Myotonic dystrophy (DM) is the most common inherited adult neuromuscular disorder. DM is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Decreased DMPK protein levels may contribute to the pathology of DM, as revealed by gene target studies. However, the postnatal regulation of DMPK expression and its pathophysiological role remain undefined. We studied the regulation of DMPK protein and mRNA expression during myogenesis in rat L6E9 myoblasts, mouse C2C12 myoblasts, and 10T1/2 fibroblasts stably expressing the myogenic transcription factor MyoD (10T1/2-MyoD). We detected DMPK as an 80-kDa protein mainly localized to the cytosolic fraction of skeletal muscle cells. DMPK expression and protein kinase activity were enhanced in IGF-II-differentiated cells. In L6E9 and C2C12 cells, DMPK expression was regulated through the same signaling pathways (i.e. phosphatidylinositol 3-kinase, nuclear factor-kappaB, nitric oxide synthase, and p38 mitogen-activated protein kinase) that had been described as being crucial for the myogenesis induced by either low serum or IGF-II. However, in 10T1/2-MyoD cells, p38 MAPK inhibition blocked cell fusion and caveolin-3 expression without affecting DMPK up-regulation. These results suggest that although DMPK is induced during myogenesis, its expression cannot be totally associated with the development of a fully differentiated phenotype. PMID:12130568

  18. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  19. Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes.

    PubMed

    Burghardt, Tillmann; Hochapfel, Florian; Salecker, Benjamin; Meese, Christine; Gröne, Hermann-Josef; Rachel, Reinhard; Wanner, Gerhard; Krahn, Michael P; Witzgall, Ralph

    2015-12-15

    Podocytes constitute the outer layer of the glomerular filtration barrier, where they form an intricate network of interdigitating foot processes which are connected by slit diaphragms. A hitherto unanswered puzzle concerns the question of whether slit diaphragms are established between foot processes of the same podocyte or between foot processes of different podocytes. By employing focused ion beam-scanning electron microscopy (FIB-SEM), we provide unequivocal evidence that slit diaphragms are formed between foot processes of different podocytes. We extended our investigations of the filtration slit by using dual-axis electron tomography of human and mouse podocytes as well as of Drosophila melanogaster nephrocytes. Using this technique, we not only find a single slit diaphragm which spans the filtration slit around the whole periphery of the foot processes but additional punctate filamentous contacts between adjacent foot processes. Future work will be necessary to determine the proteins constituting the two types of cell-cell contacts. PMID:26400546

  20. Osmolarity and glucose differentially regulate aldose reductase activity in cultured mouse podocytes.

    PubMed

    Lewko, Barbara; Latawiec, Elżbieta; Maryn, Anna; Barczyńska, Anna; Pikuła, Michał; Zieliński, Maciej; Rybczyńska, Apolonia

    2011-01-01

    Podocyte injury is associated with progression of many renal diseases, including diabetic nephropathy. In this study we examined whether aldose reductase (AR), the enzyme implicated in diabetic complications in different tissues, is modulated by high glucose and osmolarity in podocyte cells. AR mRNA, protein expression, and activity were measured in mouse podocytes cultured in both normal and high glucose and osmolarity for 6 hours to 5 days. Hyperosmolarity acutely stimulated AR expression and activity, with subsequent increase of AR expression but decrease of activity. High glucose also elevated AR protein level; however, this was not accompanied by respective enzyme activation. Furthermore, high glucose appeared to counteract the osmolarity-dependent activation of AR. In conclusion, in podocytes AR is modulated by high glucose and increased osmolarity in a different manner. Posttranslational events may affect AR activity independent of enzyme protein amount. Activation of AR in podocytes may be implicated in diabetic podocytopathy. PMID:22253613

  1. nTiO₂ induced changes in intracellular composition and nutrient stoichiometry in primary producer--cyanobacteria.

    PubMed

    Cherchi, Carla; Miljkovic, Milos; Diem, Max; Gu, April Z

    2015-04-15

    The widely and increasing use of nano-titanium dioxide (nTiO2) has led to its release in the environment and concerns of consequent impact on aquatic eco-relevant biota. Previous studies indicated possible physiological changes (i.e., nitrogen storage) induced by nano-titanium dioxide (nTiO2) exposure in algae, which will likely have ecological implications. This study investigated the short- (96 h) and long-term (21 days) ecotoxic impact of environmentally relevant nTiO2 concentrations on the cellular biochemical pools and nutrient stoichiometry in the nitrogen-fixing cyanobacteria Anabaena variabilis. Changes in nutrient element ratios and cellular composition were analyzed using both chemical elemental analysis and Fourier Transform Infrared (FT-IR) spectroscopy. Chemical elemental analysis showed that exposure to nTiO2 at varying dose concentrations and exposure duration led to statistically significant changes in intracellular C:N, C:P and N:P stoichiometries compared with those in the controls. In general, there seemed to be a decreasing trends of cellular C:N ratio and increase in the cellular C:P and N:P ratios with the increasing level of nTiO2 exposure. Further FT-IR analysis results revealed both temporal and dose-dependent change patterns of major macromolecules, including protein, lipids, nucleic acids and carbohydrates, in A. variabilis upon nTiO2 exposure. The relative ratio of amide II, lipids, nucleic acids and carbohydrates to the cellular protein content (quantified as amide I stretch) changed significantly within the initial 96 h of exposure and, both the magnitude of changes and levels of recovery seemed to be nTiO2 dose-dependent. This study, for the first time, demonstrated that the intracellular composition and nutrient stoichiometry changes could be induced by long-term and short-term exposures to nTiO2 to primary producers, which may have ecological implications for interspecies equilibriums and community dynamics in aquatic ecosystems

  2. Abnormalities in intracellular calcium regulation and contractile function in myocardium from dogs with pacing-induced heart failure.

    PubMed Central

    Perreault, C L; Shannon, R P; Komamura, K; Vatner, S F; Morgan, J P

    1992-01-01

    24 d of rapid ventricular pacing induced dilated cardiomyopathy with both systolic and diastolic dysfunction in conscious, chronically instrumented dogs. We studied mechanical properties and intracellular calcium (Ca2+i) transients of trabeculae carneae isolated from 15 control dogs (n = 32) and 11 dogs with pacing-induced cardiac failure (n = 26). Muscles were stretched to maximum length at 30 degrees C and stimulated at 0.33 Hz; a subset (n = 17 control, n = 17 myopathic) was loaded with the [Ca2+]i indicator aequorin. Peak tension was depressed in the myopathic muscles, even in the presence of maximally effective (i.e., 16 mM) [Ca2+] in the perfusate. However, peak [Ca2+]i was similar (0.80 +/- 0.13 vs. 0.71 +/- 0.05 microM; [Ca2+]o = 2.5 mM), suggesting that a decrease in Cai2+ availability was not responsible for the decreased contractility. The time for decline from the peak of the Cai2+ transient was prolonged in the myopathic group, which correlated with prolongation of isometric contraction and relaxation. However, similar end-diastolic [Ca2+]i was achieved in both groups (0.29 +/- 0.05 vs. 0.31 +/- 0.02 microM), indicating that Cai2+ homeostasis can be maintained in myopathic hearts. The inotropic response of the myopathic muscles to milrinone was depressed compared with the controls. However, when cAMP production was stimulated by pretreatment with forskolin, the response of the myopathic muscles to milrinone was improved. Our findings provide direct evidence that abnormal [Ca2+]i handling is an important cause of contractile dysfunction in dogs with pacing-induced heart failure and suggest that deficient production of cAMP may be an important cause of these changes in excitation-contraction coupling. PMID:1311723

  3. Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells.

    PubMed

    Simões-Wüst, A P; Grãos, M; Duarte, C B; Brenneisen, R; Hamburger, M; Mennet, M; Ramos, M H; Schnelle, M; Wächter, R; Worel, A M; von Mandach, U

    2010-10-01

    The use of preparations from Bryophyllum pinnatum in tocolysis is supported by both clinical (retrospective comparative studies) and experimental (using uterus strips) evidence. We studied here the effect of B. pinnatum juice on the response of cultured human myometrial cells to stimulation by oxytocin, a hormone known to be involved in the control of uterine contractions by increasing the intracellular free calcium concentration ([Ca2+]i). In this work, [Ca2+]i was measured online during stimulation of human myometrial cells (hTERT-C3 and M11) with oxytocin, which had been pre-incubated in the absence or in the presence of B. pinnatum juice. Since no functional voltage-gated Ca2+ channels could be detected in these myometrial cells, the effect of B. pinnatum juice was as well studied in SH-SY5Y neuroblastoma cells, which are known to have such channels and can be depolarised with KCl. B. pinnatum juice prevented the oxytocin-induced increase in [Ca2+]i in hTERT-C3 human myometrial cells in a dose-dependent manner, achieving a ca. 80% inhibition at a 2% concentration. Comparable results were obtained with M11 human primary myometrial cells. In hTERT-C3 cells, prevention of the oxytocin-induced increase in [Ca2+]i was independent of the extracellular Ca2+ concentration and of voltage-dependent Ca2+-channels. B. pinnatum juice delayed, but did not prevent the depolarization-induced increase in [Ca2+]i in SH-SY5Y cells. Taken together, the data suggest a specific and concentration-dependent effect of B. pinnatum juice on the oxytocin signalling pathway, which seems to corroborate its use in tocolysis. Such a specific mechanism would explain the rare and minor side-effects in tocolysis with B. pinnatum as well as its high therapeutic index. PMID:20381326

  4. Abnormalities in intracellular calcium regulation and contractile function in myocardium from dogs with pacing-induced heart failure

    NASA Technical Reports Server (NTRS)

    Perreault, C. L.; Shannon, R. P.; Komamura, K.; Vatner, S. F.; Morgan, J. P.

    1992-01-01

    24 d of rapid ventricular pacing induced dilated cardiomyopathy with both systolic and diastolic dysfunction in conscious, chronically instrumented dogs. We studied mechanical properties and intracellular calcium (Ca2+i) transients of trabeculae carneae isolated from 15 control dogs (n = 32) and 11 dogs with pacing-induced cardiac failure (n = 26). Muscles were stretched to maximum length at 30 degrees C and stimulated at 0.33 Hz; a subset (n = 17 control, n = 17 myopathic) was loaded with the [Ca2+]i indicator aequorin. Peak tension was depressed in the myopathic muscles, even in the presence of maximally effective (i.e., 16 mM) [Ca2+] in the perfusate. However, peak [Ca2+]i was similar (0.80 +/- 0.13 vs. 0.71 +/- 0.05 microM; [Ca2+]o = 2.5 mM), suggesting that a decrease in Cai2+ availability was not responsible for the decreased contractility. The time for decline from the peak of the Cai2+ transient was prolonged in the myopathic group, which correlated with prolongation of isometric contraction and relaxation. However, similar end-diastolic [Ca2+]i was achieved in both groups (0.29 +/- 0.05 vs. 0.31 +/- 0.02 microM), indicating that Cai2+ homeostasis can be maintained in myopathic hearts. The inotropic response of the myopathic muscles to milrinone was depressed compared with the controls. However, when cAMP production was stimulated by pretreatment with forskolin, the response of the myopathic muscles to milrinone was improved. Our findings provide direct evidence that abnormal [Ca2+]i handling is an important cause of contractile dysfunction in dogs with pacing-induced heart failure and suggest that deficient production of cAMP may be an important cause of these changes in excitation-contraction coupling.

  5. Intracellular Delivery by Shape Anisotropic Magnetic Particle-Induced Cell Membrane Cuts.

    PubMed

    Lin, Ming-Yu; Wu, Yi-Chien; Lee, Ji-Ann; Tung, Kuan-Wen; Zhou, Jessica; Teitell, Michael A; Yeh, J Andrew; Chiou, Pei Yu

    2016-08-01

    Introducing functional macromolecules into a variety of living cells is challenging but important for biology research and cell-based therapies. We report a novel cell delivery platform based on rotating shape anisotropic magnetic particles (SAMPs), which make very small cuts on cell membranes for macromolecule delivery with high efficiency and high survivability. SAMP delivery is performed by placing commercially available nickel powder onto cells grown in standard cell culture dishes. Application of a uniform magnetic field causes the magnetic particles to rotate because of mechanical torques induced by shape anisotropic magnetization. Cells touching these rotating particles are nicked, which generates transient membrane pores that enable the delivery of macromolecules into the cytosol of cells. Calcein dye, 3 and 40 kDa dextran polymers, a green fluorescence protein (GFP) plasmid, siRNA, and an enzyme (β-lactamase) were successfully delivered into HeLa cells, primary normal human dermal fibroblasts (NHDFs), and mouse cortical neurons that can be difficult to transfect. The SAMP approach offers several advantages, including easy implementation, low cost, high throughput, and efficient delivery of a broad range of macromolecules. Collectively, SAMP delivery has great potential for a broad range of academic and industrial applications. PMID:26882924

  6. Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death.

    PubMed

    Knight, Martin M; Roberts, Susan R; Lee, David A; Bader, Dan L

    2003-04-01

    Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2- transients and oscillations. Decreasing the power of the laser light decreased the percent-age of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was approximately 90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+ oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells. PMID:12661552

  7. Disruption of the intracellular Ca{sup 2+} homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocytes

    SciTech Connect

    Fu Min; Wu Meng; Wang Jifeng; Qiao Yanjiang; Wang Zhao . E-mail: zwang@tsinghua.edu.cn

    2007-03-23

    Aconitine is an effective ingredient in Aconite tuber, an important traditional Chinese medicine. Aconitine is also known to be a highly toxic diterpenoid alkaloid with arrhythmogenic effects. In the present study, we have characterized the properties of arrhythmic cytotoxicity and explored the possible mechanisms of aconitine-induced cardiomyocytes. Results show that aconitine induces significant abnormity in the spontaneous beating rate, amplitude of spontaneous oscillations and the relative intracellular Ca{sup 2+} concentration. Also, mRNA transcription levels and protein expressions of SR Ca{sup 2+} release channel RyR{sub 2} and sarcolemmal NCX were elevated in aconitine-induced cardiomyocytes. However, co-treatment with ruthenium red (RR), a RyR channel inhibitor, could reverse the aconitine-induced abnormity in intracellular Ca{sup 2+} signals. These results demonstrate that disruption of intracellular Ca{sup 2+} homeostasis in the cardiac excitation-contraction coupling (EC coupling) is a crucial mechanism of arrhythmic cytotoxicity in aconitine-induced cardiomyocytes. Moreover, certain inhibitors appear to play an important role in the detoxification of aconitine-induced Ca{sup 2+}-dependent arrhythmias.

  8. Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

    PubMed Central

    Hong, Jin Hee; Min, Cheol Hong; Jeong, Byeongha; Kojiya, Tomoyoshi; Morioka, Eri; Nagai, Takeharu; Ikeda, Masayuki; Lee, Kyoung J.

    2010-01-01

    Background Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca2+ spikes” (i.e., [Ca2+]c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms. Methodology/Principal Findings We addressed this issue based on a Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca2+ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca2+ spikes was increased to 13∼14%. Conclusions/Significance Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+]c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN. PMID:20224788

  9. Regulation by intracellular Ca sup 2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

    SciTech Connect

    Miyata, Yoshihiko Tokyo Metropolitan Inst. of Medical Science ); Nishida, Eisuke; Sakai, Hikoichi ); Koyasu, Shigeo; Yahara, Ichiro )

    1989-04-01

    Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes and stimulate the fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells. An increase in intracellular Ca{sup 2+} concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca{sup 2+} or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca{sup 2+} and cAMP. {sup 125}I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca{sup 2+}- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.

  10. Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella

    PubMed Central

    Trevijano-Contador, Nuria; Herrero-Fernández, Inés; García-Barbazán, Irene; Scorzoni, Liliana; Rueda, Cristina; Rossi, Suélen Andreia; García-Rodas, Rocío; Zaragoza, Oscar

    2015-01-01

    Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans. PMID:25531532

  11. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

    PubMed Central

    Li, Dan

    2014-01-01

    Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. PMID:24699332

  12. One Year of Enzyme Replacement Therapy Reduces Globotriaosylceramide Inclusions in Podocytes in Male Adult Patients with Fabry Disease

    PubMed Central

    Najafian, Behzad; Tøndel, Camilla; Svarstad, Einar; Sokolovkiy, Alexey; Smith, Kelly; Mauer, Michael

    2016-01-01

    Fabry nephropathy is associated with progressive accumulation of globotriaosylceramide (GL3) in podocytes. Reducing this GL3 burden may reduce podocyte injury. Sensitive methods to quantify podocyte GL3 content may determine whether a given strategy can benefit podocytes in Fabry disease. We developed an unbiased electron microscopic stereological method to estimate the average volume of podocytes and their GL3 inclusions in 6 paired pre- and post-enzyme replacement therapy (ERT) biopsies from 5 men with Fabry disease. Podocyte GL3 content was regularly reduced (average 73%) after 11–12 months of ERT. This was not detectable using a semi-quantitative approach. Parallel to GL3 reduction, podocytes became remarkably smaller (average 63%). These reductions in podocyte GL3 content or size were not significantly correlated with changes in foot process width (FPW). However, FPW after ERT was significantly correlated with the magnitude of the decrease in podocyte GL3 content from baseline to 11–12 months of ERT. Also podocytes exocytosed GL3 inclusions, a phenomenon correlated with their reduction in their GL3 content. Demonstrable after11–12 months, reduction in podocyte GL3 content allows for early assessment of treatment efficacy and shorter clinical trials in Fabry disease. PMID:27081853

  13. Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase C

    SciTech Connect

    Kang, Mi Sun; Jeong, Ju Yeon; Seo, Ji Heui; Jeon, Hyung Jun; Jung, Kwang Mook; Chin, Mi-Reyoung; Moon, Chang-Kiu; Bonventre, Joseph V.; Jung, Sung Yun; Kim, Dae Kyong . E-mail: proteinlab@hanmail.net

    2006-10-15

    Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca{sup 2+} levels ([Ca{sup 2+}]{sub i}). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A{sub 2} (cPLA{sub 2}) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca{sup 2+}]{sub i} and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca{sup 2+} in the culture media, D609 completely prevented cell death with parallel decrease in [Ca{sup 2+}]{sub i}. Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca{sup 2+}]{sub i} through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA{sub 2}, A-SMase, and PKC, or to the generation of ROS.

  14. IP{sub 3}-dependent intracellular Ca{sup 2+} release is required for cAMP-induced c-fos expression in hippocampal neurons

    SciTech Connect

    Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng; Johnson, Hong W.; Schell, Michael J.; Lord, Rebecca L.; Chawla, Sangeeta

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca{sup 2+} pool. Black-Right-Pointing-Pointer The submembraneous Ca{sup 2+} pool derives from intracellular ER stores. Black-Right-Pointing-Pointer Expression of IP{sub 3}-metabolizing enzymes inhibits cAMP-induced c-fos expression. Black-Right-Pointing-Pointer SRE-mediated and CRE-mediated gene expression is sensitive to IP{sub 3}-metabolizing enzymes. Black-Right-Pointing-Pointer Intracellular Ca{sup 2+} release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca{sup 2+} and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca{sup 2+} and cAMP signals, including some that are Ca{sup 2+}-responsive, some that are cAMP-responsive and some that detect coincident Ca{sup 2+} and cAMP signals. Because Ca{sup 2+} and cAMP can influence each other's amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca{sup 2+} are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca{sup 2+} buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP{sub 3} levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements - the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP{sub 3} metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by

  15. 4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells.

    PubMed

    Ji, Yun; Dai, Zhaolai; Wu, Guoyao; Wu, Zhenlong

    2016-01-01

    Excessive reactive oxygen species (ROS) induces oxidative damage to cellular constituents, ultimately leading to induction of apoptotic cell death and the pathogenesis of various diseases. The molecular mechanisms for the action of ROS in intestinal diseases remain poorly defined. Here, we reported that 4-hydroxy-2-nonenal (4-HNE) treatment led to capses-3-dependent apoptosis accompanied by increased intracellular ROS level and reduced glutathione concentration in intestinal epithelial cells. These effects of 4-HNE were markedly abolished by the antioxidant L-cysteine derivative N-acetylcysteine (NAC). Further studies demonstrated that the protective effect of NAC was associated with restoration of intracellular redox state by Nrf2-related regulation of expression of genes involved in intracellular glutathione (GSH) biosynthesis and inactivation of 4-HNE-induced phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). The 4-HNE-induced ERK1/2 activation was mediated by repressing mitogen-activated protein kinase phosphatase-1 (MKP-1), a negative regulator of ERK1/2, through a proteasome-dependent degradation mechanism. Importantly, either overexpression of MKP-1 or NAC treatment blocked 4-HNE-induced MKP-1 degradation, thereby protecting cell from apoptosis. These novel findings provide new insights into a functional role of MKP-1 in oxidative stress-induced cell death by regulating ERK1/2 MAP kinase in intestinal epithelial cells. PMID:27620528

  16. Exposure to electromagnetic field attenuates oxygen-glucose deprivation-induced microglial cell death by reducing intracellular Ca(2+) and ROS.

    PubMed

    Duong, Cao Nguyen; Kim, Jae Young

    2016-01-01

    Purpose The aim of this research was to demonstrate the protective effects of electromagnetic field (EMF) exposure on the human microglial cell line, HMO6, against ischemic cell death induced by in vitro oxygen-glucose deprivation (OGD). Materials and methods HMO6 cells were cultured for 4 h under OGD with or without exposure to EMF with different combinations of frequencies and intensities (10, 50, or 100 Hz/1 mT and 50 Hz/0.01, 0.1, or 1 mT). Cell survival, intracellular calcium and reactive oxygen species (ROS) levels were measured. Results OGD caused significant HMO6 cell death as well as elevation of intracellular Ca(2+) and ROS levels. Among different combinations of EMF frequencies and intensities, 50 Hz/1 mT EMF was the most potent to attenuate OGD-induced cell death and intracellular Ca(2+) and ROS levels. A significant but less potent protective effect was also found at 10 Hz/1 mT, whereas no protective effect was found at other combinations of EMF. A xanthine oxidase inhibitor reversed OGD-induced ROS production and cell death, while NADPH oxidase and mitochondrial respiration chain complex II inhibitors did not affect cell death. Conclusions 50 Hz/1 mT EMF protects human microglial cells from OGD-induced cell death by interfering with OGD-induced elevation of intracellular Ca(2+) and ROS levels, and xanthine oxidase is one of the main mediators involved in OGD-induced HMO6 cell death. Non-invasive treatment of EMF radiation may be clinically useful to attenuate hypoxic-ischemic brain injury. PMID:26882219

  17. miR-200 family promotes podocyte differentiation through repression of RSAD2

    PubMed Central

    Li, Zhigui; Yin, Hongqiang; Hao, Shuang; Wang, Lifeng; Gao, Jing; Tan, Xiaoyue; Yang, Zhuo

    2016-01-01

    Mature podocytes are highly differentiated cells with several characteristic phenotypic features that are involved in the glomerular filtration function. During kidney development, a series of changes of the morphological characteristics and cellular functions may happen in podocytes. The miR-200 family functions in various biological and pathological processes. But the underlying molecular mechanisms of miR-200 family that functions in podocyte differentiation remain poorly understood. Herein is shown that miR-200a, miR-200b and miR-429 are significantly upregulated during the differentiation of podocytes, with highest upregulation of miR-200a. In these cells, restraint of miR-200 family by RNA interference assay revealed a prominent inhibition of cell differentiation. More intriguingly, miR-200 family directly inhibited the radical S-adenosyl methionine domain-containing protein 2 (RASD2) expression. Moreover, further upregulation of RSAD2 combining with restraint of miR-200 family revealed a promotion of podocyte dedifferentiation and proliferation. In addition, the expression of RSAD2 is consistent with that of in vitro podocyte differentiation in prenatal and postnatal mouse kidney, and significantly down-regulated during the kidney development. Together, these findings indicate that miR-200 family may potentially promote podocyte differentiation through repression of RSAD2 expression. Our data also demonstrate a novel role of the antiviral protein RSAD2 as a regulator in cell differentiation. PMID:27251424

  18. Inhibiting Amadori-modified albumin formation improves biomarkers of podocyte damage in diabetic rats

    PubMed Central

    Cohen, Margo P; Shearman, Clyde W

    2013-01-01

    Recent studies have shown that urinary excretion of podocyte proteins is an indicator of podocyte injury, and that podocyte abnormalities and elevated concentrations of Amadori-modified glycated albumin (AGA) are linked to the development of diabetic nephropathy and to each other. We evaluated relationships between urinary markers of podocyte damage, increased AGA and filtration function in rats made diabetic by streptozotocin injection and treated for 8 weeks with a compound that inhibits the formation of AGA, with age-matched nondiabetic and diabetic rats serving as controls. Blood and urine were collected for measurement of glycated albumin, creatinine, albumin, nephrin, podocalyxin, and βig-h3 protein. The elevated circulating concentrations of glycated albumin and higher urinary levels of these podocyte markers as well as of albumin that were observed in diabetic rats compared with nondiabetic controls were significantly reduced in animals receiving test compound, and decrease in urinary biomarkers correlated with reduction in AGA. The results provide evidence that lowering the concentration of AGA, independent of filtration status and hyperglycemia, reduces urinary nephrin, podocalyxin, and βig-h3 protein, linking the increased glycated albumin associated with diabetes to podocyte abnormalities and shedding of podocyte proteins into the urine. PMID:24303153

  19. miR-200 family promotes podocyte differentiation through repression of RSAD2.

    PubMed

    Li, Zhigui; Yin, Hongqiang; Hao, Shuang; Wang, Lifeng; Gao, Jing; Tan, Xiaoyue; Yang, Zhuo

    2016-01-01

    Mature podocytes are highly differentiated cells with several characteristic phenotypic features that are involved in the glomerular filtration function. During kidney development, a series of changes of the morphological characteristics and cellular functions may happen in podocytes. The miR-200 family functions in various biological and pathological processes. But the underlying molecular mechanisms of miR-200 family that functions in podocyte differentiation remain poorly understood. Herein is shown that miR-200a, miR-200b and miR-429 are significantly upregulated during the differentiation of podocytes, with highest upregulation of miR-200a. In these cells, restraint of miR-200 family by RNA interference assay revealed a prominent inhibition of cell differentiation. More intriguingly, miR-200 family directly inhibited the radical S-adenosyl methionine domain-containing protein 2 (RASD2) expression. Moreover, further upregulation of RSAD2 combining with restraint of miR-200 family revealed a promotion of podocyte dedifferentiation and proliferation. In addition, the expression of RSAD2 is consistent with that of in vitro podocyte differentiation in prenatal and postnatal mouse kidney, and significantly down-regulated during the kidney development. Together, these findings indicate that miR-200 family may potentially promote podocyte differentiation through repression of RSAD2 expression. Our data also demonstrate a novel role of the antiviral protein RSAD2 as a regulator in cell differentiation. PMID:27251424

  20. The podocyte as a direct target for treatment of glomerular disease?

    PubMed

    Mallipattu, Sandeep K; He, John C

    2016-07-01

    The Centers for Disease Control and Prevention estimates more than 10% of adults in the United States, over 20 million Americans, have chronic kidney disease (CKD). A failure to maintain the glomerular filtration barrier directly contributes to the onset of CKD. The visceral epithelial cells, podocytes, are integral to the maintenance of this renal filtration barrier. Direct podocyte injury contributes to the onset and progression of glomerular diseases such as minimal change disease (MCD), focal segmental glomerular sclerosis (FSGS), diabetic nephropathy, and HIV-associated nephropathy (HIVAN). Since podocytes are terminally differentiated with minimal capacity to self-replicate, they are extremely sensitive to cellular injury. In the past two decades, our understanding of the mechanism(s) by which podocyte injury occurs has greatly expanded. With this newfound knowledge, therapeutic strategies have shifted to identifying targets directed specifically at the podocyte. Although the systemic effects of these agents are important, their direct effect on the podocyte proves to be essential in ameliorating glomerular disease. In this review, we highlight the mechanisms by which these agents directly target the podocyte independent of its systemic effects. PMID:27097894

  1. Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

    PubMed Central

    Yoo, Tae-Hyun; Pedigo, Christopher E.; Guzman, Johanna; Correa-Medina, Mayrin; Wei, Changli; Villarreal, Rodrigo; Mitrofanova, Alla; Leclercq, Farah; Faul, Christian; Li, Jing; Kretzler, Matthias; Nelson, Robert G.; Lehto, Markku; Forsblom, Carol; Groop, Per-Henrik; Reiser, Jochen; Burke, George William

    2015-01-01

    Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ3 integrin-dependent migration in vitro. Furthermore, αVβ3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target. PMID:24925721

  2. Sphingomyelinase-like phosphodiesterase 3b expression levels determine podocyte injury phenotypes in glomerular disease.

    PubMed

    Yoo, Tae-Hyun; Pedigo, Christopher E; Guzman, Johanna; Correa-Medina, Mayrin; Wei, Changli; Villarreal, Rodrigo; Mitrofanova, Alla; Leclercq, Farah; Faul, Christian; Li, Jing; Kretzler, Matthias; Nelson, Robert G; Lehto, Markku; Forsblom, Carol; Groop, Per-Henrik; Reiser, Jochen; Burke, George William; Fornoni, Alessia; Merscher, Sandra

    2015-01-01

    Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ3 integrin-dependent migration in vitro. Furthermore, αVβ3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target. PMID:24925721

  3. Defining the Molecular Character of the Developing and Adult Kidney Podocyte

    PubMed Central

    Brunskill, Eric W.; Georgas, Kylie; Rumballe, Bree; Little, Melissa H.; Potter, S. Steven

    2011-01-01

    Background The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease. Methodology/Principal Findings In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined. Conclusions/Significance The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells. PMID:21931791

  4. Cells of renin lineage take on a podocyte phenotype in aging nephropathy

    PubMed Central

    Pippin, Jeffrey W.; Glenn, Sean T.; Krofft, Ronald D.; Rusiniak, Michael E.; Alpers, Charles E.; Hudkins, Kelly; Duffield, Jeremy S.; Gross, Kenneth W.

    2014-01-01

    Aging nephropathy is characterized by podocyte depletion accompanied by progressive glomerulosclerosis. Replacement of terminally differentiated podocytes by local stem/progenitor cells is likely a critical mechanism for their regeneration. Recent studies have shown that cells of renin lineage (CoRL), normally restricted to the kidney's extraglomerular compartment, might serve this role after an abrupt depletion in podocyte number. To determine the effects of aging on the CoRL reserve and if CoRL moved from an extra- to the intraglomerular compartment during aging, genetic cell fate mapping was performed in aging Ren1cCre × Rs-ZsGreen reporter mice. Podocyte number decreased and glomerular scarring increased with advanced age. CoRL number decreased in the juxtaglomerular compartment with age. There was a paradoxical increase in CoRL in the intraglomerular compartment at 52 and 64 wk of age, where a subset coexpressed the podocyte proteins nephrin, podocin, and synaptopodin. Transmission electron microscopy studies showed that a subset of labeled CoRL in the glomerulus displayed foot processes, which attached to the glomerular basement membrane. No CoRL in the glomerular compartment stained for renin. These results suggest that, despite a decrease in the reserve, a subpopulation of CoRL moves to the glomerulus after chronic podocyte depletion in aging nephropathy, where they acquire a podocyte-like phenotype. This suggests that they might serve as adult podocyte stem/progenitor cells under these conditions, albeit in insufficient numbers to fully replace podocytes depleted with age. PMID:24647714

  5. Sensory neuron subpopulation-specific dysregulation of intracellular calcium in a rat model of chemotherapy-induced peripheral neuropathy.

    PubMed

    Yilmaz, E; Gold, M S

    2015-08-01

    The purpose of the present study was to test the prediction that the unique manifestation of chemotherapeutic-induced peripheral neuropathy (CIPN) would be reflected in a specific pattern of changes in the regulation of the intracellular Ca(2+) concentration ([Ca(2+)]i) in subpopulations of cutaneous neurons. To test this prediction, we characterized the pattern of changes in mechanical nociceptive threshold associated with paclitaxel administration (2mg/kg, iv, every other day for four days), as well as the impact of target of innervation and paclitaxel treatment on the regulation of [Ca(2+)]i in subpopulations of putative nociceptive and non-nociceptive neurons. Neurons innervating the glabrous and hairy skin of the hindpaw as well as the thigh were identified with retrograde tracers, and fura-2 was used to assess changes in [Ca(2+)]i. Paclitaxel was associated with a persistent decrease in mechanical nociceptive threshold in response to stimuli applied to the glabrous skin of the hindpaw, but not the hairy skin of the hindpaw or the thigh. However, in both putative nociceptive and non-nociceptive neurons, resting [Ca(2+)]i was significantly lower in neurons innervating the thigh after treatment. The magnitude of the depolarization-evoked Ca(2+) transient was also lower in putative non-nociceptive thigh neurons. More interestingly, while paclitaxel had no detectable influence on either resting or depolarization-evoked Ca(2+) transients in putative non-nociceptive neurons, in putative nociceptive neurons there was a subpopulation-specific decrease in the duration of the evoked Ca(2+) transient that was largely restricted to neurons innervating the glabrous skin. These results suggest that peripheral nerve length alone, does not account for the selective distribution of CIPN symptoms. Rather, they suggest the symptoms of CIPN reflect an interaction between the toxic actions of the therapeutic and unique properties of the neurons deleteriously impacted. PMID

  6. Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells.

    PubMed

    Robinson, Meghan; Chapani, Parv; Styan, Tara; Vaidyanathan, Ranjani; Willerth, Stephanie Michelle

    2016-08-01

    Pluripotent stem cells can become any cell type found in the body. Accordingly, one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells, which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next, we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture, hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells. PMID:27138845

  7. Podocyte Detachment Is Associated with Renal Prognosis in ANCA-Associated Glomerulonephritis

    PubMed Central

    Zou, Rong; Wang, Su-xia; Liu, Gang; Yu, Feng; Chen, Min; Zhao, Ming-Hui

    2016-01-01

    Abstract The prognosis of antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (ANCA-GN) is unfavorable despite immunosuppressive therapy. It has been suggested that the loss of podocytes is a hallmark of progressive kidney disease. However, it is unclear about podocyte injuries and their predictive values on the prognosis in ANCA-GN. Therefore, the current study aimed to investigate the podocyte injury in renal histopathology and its association with renal prognosis of patients with ANCA-GN. A total of 170 patients with ANCA-GN were recruited in this study. Morphometric investigation of podocytes by electron microscopy including foot process width (FPW), podocyte density per glomerulus (Nv), and glomerular basement membrane (GBM) width were measured and calculated in ANCA-GN patients. Cox regression analysis was used to analyze the association between podocyte injuries and prognosis of patients with ANCA-GN. Foot processes broadening, podocyte detachment, and GBM thickening could be observed in electron micrographs in the specimens of 158/170 (92.9%), 142/170 (83.5%), and 150/170 (88.2%) patients, respectively. Compared with normal controls, FPW and GBM width in ANCA-GN patients was significantly higher (1269.39 ± 680.19 vs 585.81 ± 77.16, P = 0.004; 668.23 ± 208.73 vs 354.23 ± 52.70, P = 0.000, respectively), while the podocyte density was significantly lower (55.90 ± 36.32 vs 255.23 ± 47.29, P = 0.000). The podocyte density was independently associated with the recovery of renal function in logistic regression analysis (OR, 1.083; 95% CI, 1.025–1.440; P = 0.005). Furthermore, multivariate analysis revealed that podocyte density was an independent predictor of end-stage renal disease (ESRD) (model A: HR, 0.950; 95% CI, 0.919–1.982; P = 0.002; model B: HR, 0.953; 95% CI, 0.922–0.985; P = 0.004). Podocyte structural damage and detachment occurred frequently in patients with ANCA

  8. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis

    PubMed Central

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D.; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  9. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis.

    PubMed

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  10. Involvement of Intracellular and Mitochondrial Aβ in the Ameliorative Effects of Huperzine A against Oligomeric Aβ42-Induced Injury in Primary Rat Neurons

    PubMed Central

    Lei, Yun; Yang, Ling; Ye, Chun Yan; Qin, Ming Yan; Yang, Huai Yu; Jiang, Hua Liang; Tang, Xi Can; Zhang, Hai Yan

    2015-01-01

    Considerable studies indicate huperzine A is a promising natural product to suppress neuronal damages induced by β-amyloid (Aβ), a key pathogenic event in the Alzheimer’s disease (AD). As an extension, the present study for the first time explored whether the beneficial profiles of huperzine A against oligomeric Aβ42 induced neurotoxicity are associated with the accumulation and detrimental function of intraneuronal/mitochondrial Aβ, on the basis of the emerging evidence that intracellular Aβ is more relevant to AD progression as compared with extracellular Aβ. Huperzine A treatment was shown to significantly attenuate the neurotoxicity of oligomeric Aβ42, as demonstrated by increased neuronal viability. Interestingly, our results proved that exogenous Aβ42 could accumulate intraneuronally in a dose- and time-dependent manner, while huperzine A treatment markedly reduced the level of intracellular Aβ42. Moreover, huperzine A treatment rescued mitochondrial dysfunction induced by oligomeric Aβ42, including adenosine triphosphate (ATP) reduction, reactive oxygen species (ROS) overproduction and membrane potential depolarization. Further study demonstrated that huperzine A also significantly reduced the level of Aβ42 in the mitochondria-enriched subcellular fractions, as well as the Aβ42 fluorescent signals colocalized with mitochondrial marker. This study indicates that interfering intracellular Aβ especially mitochondrial Aβ accumulation, together with ameliorating Aβ-associated mitochondrial dysfunction, may contribute to the protective effects of huperzine A against Aβ neurotoxicity. Above results may shed more light on the pharmacological mechanisms of huperzine A and provide important clues for discovering novel therapeutic strategies for AD. PMID:26024517

  11. Involvement of Intracellular and Mitochondrial Aβ in the Ameliorative Effects of Huperzine A against Oligomeric Aβ42-Induced Injury in Primary Rat Neurons.

    PubMed

    Lei, Yun; Yang, Ling; Ye, Chun Yan; Qin, Ming Yan; Yang, Huai Yu; Jiang, Hua Liang; Tang, Xi Can; Zhang, Hai Yan

    2015-01-01

    Considerable studies indicate huperzine A is a promising natural product to suppress neuronal damages induced by β-amyloid (Aβ), a key pathogenic event in the Alzheimer's disease (AD). As an extension, the present study for the first time explored whether the beneficial profiles of huperzine A against oligomeric Aβ(42) induced neurotoxicity are associated with the accumulation and detrimental function of intraneuronal/mitochondrial Aβ, on the basis of the emerging evidence that intracellular Aβ is more relevant to AD progression as compared with extracellular Aβ. Huperzine A treatment was shown to significantly attenuate the neurotoxicity of oligomeric Aβ(42), as demonstrated by increased neuronal viability. Interestingly, our results proved that exogenous Aβ(42) could accumulate intraneuronally in a dose- and time-dependent manner, while huperzine A treatment markedly reduced the level of intracellular Aβ(42). Moreover, huperzine A treatment rescued mitochondrial dysfunction induced by oligomeric Aβ(42), including adenosine triphosphate (ATP) reduction, reactive oxygen species (ROS) overproduction and membrane potential depolarization. Further study demonstrated that huperzine A also significantly reduced the level of Aβ(42) in the mitochondria-enriched subcellular fractions, as well as the Aβ(42) fluorescent signals colocalized with mitochondrial marker. This study indicates that interfering intracellular Aβ especially mitochondrial Aβ accumulation, together with ameliorating Aβ-associated mitochondrial dysfunction, may contribute to the protective effects of huperzine A against Aβ neurotoxicity. Above results may shed more light on the pharmacological mechanisms of huperzine A and provide important clues for discovering novel therapeutic strategies for AD. PMID:26024517

  12. Resveratrol Attenuates the Na+-Dependent Intracellular Ca2+ Overload by Inhibiting H2O2-Induced Increase in Late Sodium Current in Ventricular Myocytes

    PubMed Central

    Qian, Chunping; Ma, Jihua; Zhang, Peihua; Luo, Antao; Wang, Chao; Ren, Zhiqiang; Kong, Linghao; Zhang, Shuo; Wang, Xiaojing; Wu, Ying

    2012-01-01

    Background/Aims Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H2O2)-induced increase in late sodium current (INa.L) which augmented the reverse Na+-Ca2+ exchanger current (INCX), and the diastolic intracellular Ca2+ concentration in ventricular myocytes. Methods INa.L, INCX, L-type Ca2+ current (ICa.L) and intracellular Ca2+ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes. Results Resveratrol (10, 20, 40 and 80 µM) decreased INa.L in myocytes both in the absence and presence of H2O2 (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin (TTX, 4 µM), INa.L inhibitors, decreased INa.L in cardiomyocytes in the presence of 300 µM H2O2. H2O2 (300 µM) increased the reverse INCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H2O2 of the diastolic intracellular Ca2+ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca2+ transient rise and decay. As expected, 2 µM TTX had no effect on ICa.L. Conclusion Resveratrol protects the cardiomyocytes by inhibiting the H2O2-induced augmentation of INa.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias. PMID:23272101

  13. PKA-mediated responses in females' estrous cycle affect cocaine-induced responses in dopamine-mediated intracellular cascades.

    PubMed

    Weiner, J; Sun, W Lun; Zhou, L; Kreiter, C M; Jenab, S; Quiñones-Jenab, V

    2009-07-01

    An extensive body of literature provides evidence for both sexual dimorphism and menstrual cycle effects in drug abuse patterns and behavioral responses. However, the cellular mechanisms underlying sexually dimorphic responses to and hormonal effects on cocaine use remain unclear. We hypothesized that endogenous hormonal fluctuations during the estrous cycle of rats modulate cocaine's effects on dopamine- and PKA-mediated intracellular responses. To test this hypothesis, intact female rats at different stages of their cycle received a single injection of saline or cocaine (20 mg/kg) and were sacrificed after 15 or 60 min. The nucleus accumbens (NAc) and caudate putamen (CPu) were dissected and analyzed via Western blot for total and phosphorylated (p-thr34) dopamine- and 3'-5'-cyclic AMP-regulated phosphoprotein with molecular weight 32 kDa (DARPP-32), PP1, PP2B (CNA1 and CNB1 subunits), PKA, CREB, cFOS, and Delta-FosB. Our results show that saline-treated rats had estrous cycle-related differences in protein levels of pCREB, DARPP-32, p-thr34-DARPP-32, PP1, and CNA1. Saline-treated female rats in the estrus stage had higher levels of pCREB in the NAc, but cocaine-treatment lowered pCREB levels. The estrous cycle also significantly affected the magnitude of change for p-thr34-DARPP-32 protein levels in both the NAc and CPu. Sixty minutes of cocaine administration increased p-thr34-DARPP-32 levels in the NAc of rats during estrus and proestrus and in the CPu of rats in diestrus. Furthermore, cocaine-induced changes in PP1 protein levels in the NAc were also affected by the stage of the cycle; 60 min of cocaine administration increased PP1 levels in the NAc of rats during diestrus, whereas PP-1 levels decreased in rats during estrus. Taken together, these novel findings suggest that hormonal fluctuations during the estrous cycle may contribute to the previously reported sex differences in the PKA pathway and in behavioral responses to cocaine. PMID:19348873

  14. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    PubMed Central

    2013-01-01

    Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP) was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA). The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds) than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio) and B. megaterium (from Mobitec), we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus. PMID:23374160

  15. Allicin protects rat cardiomyoblasts (H9c2 cells) from hydrogen peroxide-induced oxidative injury through inhibiting the generation of intracellular reactive oxygen species.

    PubMed

    Chan, Jackie Yan-Yan; Tsui, Hei-Tung; Chung, Ivan Ying-Ming; Chan, Robbie Yat-Kan; Kwan, Yiu-Wa; Chan, Shun-Wan

    2014-11-01

    Oxidative stress is considered an important factor that promotes cell death in response to a variety of pathophysiological conditions. This study investigated the antioxidant properties of allicin, the principle ingredient of garlic, on preventing oxidative stress-induced injury. The antioxidant capacities of allicin were measured by using 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and hydrogen peroxide (H(2)O(2))-induced cell damage on H9c2 cardiomyoblasts. Allicin (0.3-10 μM) pre-incubation could concentration-dependently attenuate the intracellular reactive oxygen species (ROS) increase induced by H(2)O(2) on H9c2 cells. It could also protect H9c2 cells against H(2)O(2)-induced cell damage. However, the DPPH free radical scavenging activity of allicin was shown to be low. Therefore, it is believed that the protective effect of allicin on H9c2 cells could inhibit intracellular ROS production instead of scavenging extracellular H(2)O(2) or free radicals. For the observed protective effect on H9c2 cells, allicin might also be effective in reducing free radical-induced myocardial cell death in ischemic condition. PMID:24945597

  16. Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo.

    PubMed

    Kumamoto, Junichi; Tsutsumi, Moe; Goto, Makiko; Nagayama, Masaharu; Denda, Mitsuhiro

    2016-01-01

    Cry j1 is the major peptide allergen of Japanese cedar (Sugi), Cryptomeria japonica. Since some allergens disrupt epidermal permeability barrier homeostasis, we hypothesized that Cry j1 might have a similar effect. Intracellular calcium level in cultured human keratinocytes was measured with a ratiometric fluorescent probe, Fura-2 AM. Application of Cry j1 significantly increased the intracellular calcium level of keratinocytes, and this increase was inhibited by trypsin inhibitor or a protease-activated receptor 2 (PAR-2) antagonist. We found that Cry j1 itself did not show protease activity, but application of Cry j1 to cultured keratinocytes induced a rapid (within 30 s) and transient increase of protease activity in the medium. This transient increase was blocked by trypsin inhibitor or PAR-2 antagonist. The effect of Cry j1 on transepidermal water loss (TEWL) of cultured human skin was measured in the presence and absence of a trypsin inhibitor and PAR-2 antagonist. Cry j1 significantly impaired the barrier function of human skin ex vivo, and this action was blocked by co-application of trypsin inhibitor or PAR-2 antagonist. Our results suggested that interaction of Cry j1 with epidermal keratinocytes leads to the activation of PAR-2, which induces elevation of intracellular calcium and disruption of barrier function. Blocking the interaction of Cry j1 with epidermal keratinocytes might ameliorate allergic reaction and prevent disruption of epidermal permeability barrier homeostasis. PMID:26498292

  17. Human Immunodeficiency Virus Type 1 Subtype C Tat Fails To Induce Intracellular Calcium Flux and Induces Reduced Tumor Necrosis Factor Production from Monocytes▿

    PubMed Central

    Campbell, Grant R.; Watkins, Jennifer D.; Singh, Kumud K.; Loret, Erwann P.; Spector, Stephen A.

    2007-01-01

    Over 50% of all human immunodeficiency virus type 1 (HIV-1) infections worldwide are caused by subtype C strains, yet most research to date focuses on subtype B, the subtype most commonly found in North America and Europe. The HIV-1 trans-acting regulatory protein (Tat) is essential for regulating productive replication of HIV-1. Tat is secreted by HIV-infected cells and alters several functions of uninfected bystander cells. One such function is that, by acting at the cell membrane, subtype B Tat stimulates the production of tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) from human monocytes and can act as a chemoattractant. In this study, we show that the mutation of a cysteine to a serine at residue 31 of Tat commonly found in subtype C variants significantly inhibits the abilities of the protein to bind to chemokine (C-C motif) receptor 2 (CCR2), induce intracellular calcium flux, stimulate TNF and CCL2 production, and inhibit its chemoattractant properties. We also show that TNF is important in mediating some effects of extracellular Tat. This report therefore demonstrates the important functional differences between subtype C and subtype B Tat and highlights the need for further investigation into the different strains of HIV-1. PMID:17376903

  18. Nephrin phosphorylation regulates podocyte adhesion through the PINCH-1-ILK-α-parvin complex

    PubMed Central

    Zha, Dongqing; Chen, Cheng; Liang, Wei; Chen, Xinghua; Ma, Tean; Yang, Hongxia; van Goor, Harry; Ding, Guohua

    2013-01-01

    Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism. [BMB Reports 2013; 46(4): 230-235] PMID:23615266

  19. Lipid biology of the podocyte--new perspectives offer new opportunities.

    PubMed

    Fornoni, Alessia; Merscher, Sandra; Kopp, Jeffrey B

    2014-07-01

    In the past 15 years, major advances have been made in understanding the role of lipids in podocyte biology. First, susceptibility to focal segmental glomerulosclerosis (FSGS) and glomerular disease is associated with an APOL1 sequence variant, is expressed in podocytes and encodes apolipoprotein L1, an important component of HDL. Second, acid sphingomyelinase-like phosphodiesterase 3b encoded by SMPDL3b has a role in the conversion of sphingomyelin to ceramide and its levels are reduced in renal biopsy samples from patients with recurrent FSGS. Furthermore, decreased SMPDL3b expression is associated with increased susceptibility of podocytes to injury after exposure to sera from these patients. Third, in many individuals with membranous nephropathy, autoantibodies against the phospholipase A2 (PLA2) receptor, which is expressed in podocytes, have been identified. Whether these autoantibodies affect the activity of PLA2, which liberates arachidonic acid from glycerophospholipids and modulates podocyte function, is unknown. Fourth, clinical and experimental evidence support a role for ATP-binding cassette sub-family A member 1-dependent cholesterol efflux, free fatty acids and glycerophospolipids in the pathogenesis of diabetic kidney disease. An improved understanding of lipid biology in podocytes might provide insights to develop therapeutic targets for primary and secondary glomerulopathies. PMID:24861084

  20. Luciferase-based protein-denaturation assay for quantification of radiofrequency field-induced targeted hyperthermia: developing an intracellular thermometer

    PubMed Central

    Raoof, Mustafa; Zhu, Cihui; Kaluarachchi, Warna D.; Curley, Steven A.

    2013-01-01

    Background Several studies have reported targeted hyperthermia at the cellular level using remote activation of nanoparticles by radiofrequency waves. To date, methods to quantify intracellular thermal dose have not been reported. In this report we study the relationship between radio wave exposure and luciferase denaturation with and without intracellular nanoparticles. The findings are used to devise a strategy to quantify targeted thermal dose in a primary human liver cancer cell line. Methods Water-bath or non-invasive external RF generator (600W, 13.56 MHz) was used for hyperthermia exposures. Luciferase activity was measured using a bioluminescence assay and viability was assessed using Annexin V-FITC and Propidium iodide staining. Heat shock proteins were analyzed using western-blot analysis Results Duration-dependent luciferase denaturation was observed in SNU449 cells exposed to RF field that preceded measurable loss in viability. Loss of luciferase activity was higher in cetuximab-conjugated gold nanoparticle (C225-AuNP) treated cells. Using a standard curve from water-bath experiments, the intracellular thermal dose was calculated. Cells treated with C225-AuNP accumulated 6.07 times higher intracellular thermal dose than the untreated controls over initial 4 minutes of RF exposure. Conclusions Cancer cells when exposed to an external RF field exhibit dose-dependent protein denaturation. Luciferase denaturation assay can be used to quantify thermal dose delivered after RF exposures to cancer cells with and without nanoparticles. PMID:22515341

  1. [Commemorative lecture of receiving Imamura Memorial Prize. Characterization of immunosuppressive macrophages induced in mice infected with Mycobacterium intracellulare].

    PubMed

    Tomioka, H

    1993-12-01

    Functional changes in T lymphocytes and macrophages (M phi s) in host mice during the course of Mycobacterium intracellulare infection were studied. In both strains of mice, BALB/c or C57BL/6 (susceptible to M. avium complex) and CBA/JN or C3H/He (resistant to M. avium complex), the smooth, opaque and dome-shaped colonial (SmD) variants of M. intracellulare were easily eliminated from the sites after week 2 of infection. In contrast, the smooth, transparent and irregularly shaped colonial (SmT) variants showed steady growth in the former strains of mice and persisted for long time even in the latter strains of mice. No difference was found between persistence of the organisms in euthymic (+/+) and athymic (nu/nu) BALB/c mice during the first 4 weeks after infection. Thereafter, more rapid growth was seen in the spleens and lungs of nu/nu mice. Thus, matured T cells may be important for the prevention of the progression of M. intracellulare infection to the terminal state. Next, the profiles of generation and characteristics of splenic M phi s which suppress the Con A mitogenic response of splenic T cells in host CBA/JN or BALB/c mice during the course of M. intracellulare infection were investigated. In M. intracellulare--infected mice, reduction in some cellular functions of host splenic T cells, such as the Con A mitogenic response and mixed leucocyte reaction, were seen around 2 weeks after infection, and this was accompanied by appearance of immunosuppressive M phi s in spleen cells (SPCs).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8301920

  2. Endocannabinoid 2-AG and intracellular cannabinoid receptors modulate a low-threshold calcium spike-induced slow depolarizing afterpotential in rat thalamic paraventricular nucleus neurons.

    PubMed

    Zhang, L; Kolaj, M; Renaud, L P

    2016-05-13

    In rat paraventricular thalamic nucleus (PVT) neurons, activation of low-threshold calcium (Ca(2+)) channels triggers a low-threshold spike (LTS) which may be followed by slow afterpotentials that can dramatically influence action potential patterning. Using gluconate-based internal recording solutions, we investigated the properties of a LTS-induced slow afterdepolarization (sADP) observed in a subpopulation of PVT neurons recorded in brain slice preparations. This LTS-induced sADP required T-type Ca(2+) channel opening, exhibited variable magnitudes between neurons and a voltage dependency with a maximum near -50mV. The area under the sADP remained stable during control monitoring, but displayed gradual suppression in media where strontium replaced Ca(2+). The sADP was suppressed following bath application of 2-APB or ML204, suggesting engagement of transient receptor potential canonical (TRPC)-like channels. Further investigation revealed a reversible suppression during bath applications of membrane permeable cannabinoid receptor (CBR) blockers rimonabant, AM630 or SR144528 suggesting the presence of both CB1Rs and CB2Rs. Similar results were achieved by intracellular, but not bath application of the membrane impermeant CB1R blocker hemopressin, suggesting an intracellular localization of CB1Rs. Data from pharmacologic manipulation of endocannabinoid biosynthetic pathways suggested 2-arachidonlyglycerol (2-AG) as the endogenous cannabinoid ligand, derived via hydrolysis of diacylglycerol (DAG), with the latter formed from the pathway involving phosphatidylcholine-specific phospholipase D and phosphatic acid phosphohydrolase. The sADP suppression observed during recordings with pipettes containing LY294002, a PI3-kinase inhibitor, suggested a role for PI3kinase in the translocation of these TRPC-like channels to the plasma membrane. Drug-induced attenuation of the availability of 2-AG influences the number of action potentials that surmount the LTS evoked in PVT

  3. The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LβT2 cells by suppressing GnRH-induced intracellular calcium transients

    PubMed Central

    Dooley, Gregory P.; Tjalkens, Ronald B.; Hanneman, William H.

    2013-01-01

    The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH. PMID:24052811

  4. Huaiqihuang may protect from proteinuria by resisting MPC5 podocyte damage via targeting p-ERK/CHOP pathway

    PubMed Central

    Li, Tingxia; Mao, Jianhua; Huang, Lei; Fu, Haidong; Chen, Shuohui; Liu, Aimin; Liang, Yuqin

    2016-01-01

    The purpose of this study was to investigate the potential effects of Huaiqihuang (HQH) granule, a Chinese herbal medicine, in treating proteinuria and to reveal its possible mechanism. MPC5 podocytes were cultured in vitro at 37°C and induced with tunicamycin (TM). The TM-induced cells were treated with HQH at different concentrations. The cell proliferation was detected using the MTT assay. The optimal effective dose of HQH for MPC5 cells was determined by the MTT assay and LDH assay respectively. The influences of HQH on the proteinuria-related protein expression and the signaling pathway associated protein expression were also detected using quantitative reverse transcription PCR and Western blotting analysis. The results showed that the MPC5 cell model was successfully constructed in vitro. The HQH application could improve the harmful effects induced by TM on the MPC5 cells, including promoted cell proliferation and suppressed cell apoptosis. Furthermore, the protein expression, including podocin, nephrin, and synaptopodin was down-regulated by the TM treatment in the MPC5 cells. On contrary, the expression of these proteins was up-regulated after the HQH application. Also, the effect of TM on integrin α3 and integrin β1 expressions was also reversed by the HQH treatment. Moreover, the HQH application decreased the expression of p-ERK and DNA-damage-inducible transcript 3 (DDIT3 or CHOP) in the MPC5 cells, which was opposite to the effect observed in the cells treated with TM. Taken together, our study suggest that HQH application may protect podocytes from TM damage by suppressing the p-ERK/CHOP signaling pathway.

  5. Intracellular microlasers

    NASA Astrophysics Data System (ADS)

    Humar, Matjaž; Hyun Yun, Seok

    2015-09-01

    Optical microresonators, which confine light within a small cavity, are widely exploited for various applications ranging from the realization of lasers and nonlinear devices to biochemical and optomechanical sensing. Here we use microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explore two distinct types of microresonator—soft and hard—that support whispering-gallery modes. Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (˜500 pN μm-2) and its dynamic fluctuations at a sensitivity of 20 pN μm-2 (20 Pa). In a second form, whispering-gallery modes within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes.

  6. Cluster of Differentiation 38 (CD38) Mediates Bile Acid-induced Acinar Cell Injury and Pancreatitis through Cyclic ADP-ribose and Intracellular Calcium Release*

    PubMed Central

    Orabi, Abrahim I.; Muili, Kamaldeen A.; Javed, Tanveer A.; Jin, Shunqian; Jayaraman, Thottala; Lund, Frances E.; Husain, Sohail Z.

    2013-01-01

    Aberrant Ca2+ signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca2+ signals due to bile acid exposure is the intracellular Ca2+ channel ryanodine receptor. One putative activator of the ryanodine receptor is the nucleotide second messenger cyclic ADP-ribose (cADPR), which is generated by an ectoenzyme ADP-ribosyl cyclase, CD38. In this study, we examined the role of CD38 and cADPR in acinar cell Ca2+ signals and acinar injury due to bile acids using pharmacologic inhibitors of CD38 and cADPR as well as mice deficient in Cd38 (Cd38−/−). Cytosolic Ca2+ signals were imaged using live time-lapse confocal microscopy in freshly isolated mouse acinar cells during perifusion with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 μm). To focus on intracellular Ca2+ release and to specifically exclude Ca2+ influx, cells were perifused in Ca2+-free medium. Cell injury was assessed by lactate dehydrogenase leakage and propidium iodide uptake. Pretreatment with either nicotinamide (20 mm) or the cADPR antagonist 8-Br-cADPR (30 μm) abrogated TLCS-induced Ca2+ signals and cell injury. TLCS-induced Ca2+ release and cell injury were reduced by 30 and 95%, respectively, in Cd38-deficient acinar cells compared with wild-type cells (p < 0.05). Cd38-deficient mice were protected against a model of bile acid infusion pancreatitis. In summary, these data indicate that CD38-cADPR mediates bile acid-induced pancreatitis and acinar cell injury through aberrant intracellular Ca2+ signaling. PMID:23940051

  7. Hypoxia induces an increase in intracellular magnesium via transient receptor potential melastatin 7 (TRPM7) channels in rat hippocampal neurons in vitro.

    PubMed

    Zhang, Jing; Zhao, Fengbo; Zhao, Yin; Wang, Jing; Pei, Lei; Sun, Ning; Shi, Jing

    2011-06-10

    TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg(2+) fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg(2+)](i)) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg(2+)](o)). The results showed that the neuronal [Mg(2+)](i) was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg(2+)](i) reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg(2+)](o) decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg(2+)](i) peak immediately appeared in hippocampal neurons. This increase of [Mg(2+)](i) declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd(3+) or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg(2+)](i) increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg(2+)](i). By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg(2+)](i) induced by OGD or CI but also the basal levels of [Mg(2+)](i) were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg(2+)](i) after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg(2+)](i) via TRPM7 channels in rat hippocampal neurons. PMID:21487014

  8. Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca(2+) influx through transient receptor potential V1 (TRPV1) channels.

    PubMed

    Murakami, Satoshi; Sudo, Yuka; Miyano, Kanako; Nishimura, Hitomi; Matoba, Motohiro; Shiraishi, Seiji; Konno, Hiroki; Uezono, Yasuhito

    2016-02-01

    Non-selective transient receptor potential vanilloid (TRPV) cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM) is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1) and the Ca(2+)-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM) and pH 6.5 buffer elicited steep increases in the intracellular Ca(2+) concentration ([Ca(2+)]i), while treatment with THAM (pH 8.5) alone had no effect. However, treatment with THAM (pH 8.5) following capsaicin application elicited a profound, long-lasting increase in [Ca(2+)]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca(2+)]i increases, which could be a mechanism underlying pain induced by basic pH. PMID:26738986

  9. Evaluation of extra- and intracellular OH radical generation, cancer cell injury, and apoptosis induced by a non-thermal atmospheric-pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Ninomiya, Kazuaki; Ishijima, Tatsuo; Imamura, Masatoshi; Yamahara, Takayuki; Enomoto, Hiroshi; Takahashi, Kenji; Tanaka, Yasunori; Uesugi, Yoshihiko; Shimizu, Nobuaki

    2013-10-01

    In this study, we investigated the effects of a non-thermal atmospheric-pressure plasma jet inducing extracellular and intracellular OH radical generation as well as cell injury and apoptosis for the cultured human breast cancer cells. Increased OH radical generation in the extracellular culture medium (liquid phase) was observed with increased irradiation time, distance to the liquid surface, and voltage. From the voltage-response relationships for two breast cancer cell lines (invasive MDA-MB-231 cells and non-invasive MCF-7 cells) and normal breast cells (HMEC), the half-maximal effective peak-to-peak voltage (EV50) values were 16.7 ± 0.3 kV, 15.0 ± 0.4 kV and 11.2 ± 0.7 kV for MDA-MB-231, MCF-7 and HMEC cells, respectively. This indicated that there was almost no selective cancer cell injury induced by plasma jet irradiation under these conditions. Compared with control condition without a plasma jet, intracellular OH radical accumulation and apoptotic cells were observed with a plasma jet using conditions that induced injury to 50% of cells irrespective of the cancer cell line.

  10. The Chilean wild raspberry (Rubus geoides Sm.) increases intracellular GSH content and protects against H2O2 and methylglyoxal-induced damage in AGS cells.

    PubMed

    Jiménez-Aspee, Felipe; Theoduloz, Cristina; Ávila, Felipe; Thomas-Valdés, Samanta; Mardones, Claudia; von Baer, Dietrich; Schmeda-Hirschmann, Guillermo

    2016-03-01

    The Chilean raspberry Rubus geoides Sm. (Rosaceae) is a native species occurring in the Patagonia. Five R. geoides samples were assessed for phenolic content and composition, antioxidant activity, effect on total reduced glutathione (GSH) synthesis and protective effect against H2O2 and methylglyoxal (MGO)-induced stress in epithelial gastric AGS cells. The HPLC-DAD/ESI-MS profiles allowed the tentative identification of 39 phenolics including flavonol glycosides and tannins. R. geoides presented higher total phenolic and flavonoid content than Rubus idaeus. Two out of the five phenolic enriched R. geoides extracts (PEEs) exhibited better antioxidant activity than R. idaeus in the DPPH, FRAP and TEAC assays. A significant cytoprotective activity was observed when AGS cells were pre-incubated with extracts and subsequently challenged with H2O2 or MGO. Treatment with the PEEs increased the intracellular GSH content. R. geoides fruit extracts may induce the activation of intracellular protection mechanisms against oxidative and dicarbonyl-induced stress. PMID:26471634

  11. Cytoskeletal changes in podocytes associated with foot process effacement in Masugi nephritis.

    PubMed Central

    Shirato, I.; Sakai, T.; Kimura, K.; Tomino, Y.; Kriz, W.

    1996-01-01

    Foot process effacement represents the most characteristic change in podocyte phenotype under a great variety of experimental as well as human glomerulopathies. It consists in simplification up to a total disappearance of an interdigitating foot process pattern. Finally, podocytes affix to the glomerular basement membrane by outspread epithelial sheets. Structural and immunocytochemical techniques were applied to analyze the cytoskeletal changes associated with foot process effacement in Masugi nephritis. Three days after injection of the anti-glomerular-basement-membrane serum an interdigitating foot process pattern was almost fully lost; more than 90 percent of the outer glomerular capillary surface were covered by expanded sheets of podocyte epithelium that contain a highly organized cytoskeleton adhering to the basal cell membrane. Structurally, this cytoskeleton consists of an interwoven network of microfilaments with regularly distributed dense bodies, which obviously serve as cross-linkers within this network. Immunocytochemically, the expression of actin, alpha-actinin, and pp44 (a specific podocyte protein normally associated with the cytoskeleton of foot processes) were increased in this structure; alpha-actinin was especially prominent in the dense bodies. The results are consistent with the view that foot process effacement represents an adaptive change in cell shape including hypertrophy of the contractile apparatus, reinforcing the supportive role of podocytes. Several factors associated with increased distending forces to podocytes may underlie this phenotype change including loss of mesangial support, elevated glomerular pressures, and impairment of GBM substructure as well as of podocyte-GBM-contacts. Twenty-eight days after serum injection a remodeling of the foot process pattern was seen. It appears that this restitution depends on a preceding repair of mesangial support function to glomerular capillaries. Images Figure 1 Figure 2 Figure 3 Figure

  12. Enhanced Ca(2+)-induced Ca(2+) release from intracellular stores contributes to catecholamine hypersecretion in adrenal chromaffin cells from spontaneously hypertensive rats.

    PubMed

    Segura-Chama, Pedro; López-Bistrain, Patricia; Pérez-Armendáriz, Elia Martha; Jiménez-Pérez, Nicolás; Millán-Aldaco, Diana; Hernández-Cruz, Arturo

    2015-11-01

    Adrenal chromaffin cells (CCs) from spontaneously hypertensive rats (SHRs) secrete more catecholamine (CA) upon stimulation than CCs from normotensive Wistar Kyoto rats (WKY). Unitary CA exocytosis events, both spontaneous and stimulated, were amperometrically recorded from cultured WKY and SHR CCs. Both strains display spontaneous amperometric spikes but SHR CCs produce more spikes and of higher mean amplitude. After a brief stimulation with high K(+) or caffeine which produces voltage-gated Ca(2+) influx or intracellular Ca(2+) release, respectively, more spikes and of greater mean amplitude and unitary charge were recorded in SHR CCs. Consequently, peak cumulative charge was ~2-fold higher in SHR CCs. Ryanodine (10 μM), a specific blocker of the ryanodine receptors reduced depolarization-induced peak cumulative charge by ~10 % in WKY and ~77 % in SHR CCs, suggesting, a larger contribution of Ca(2+)-induced Ca(2+) release to CA exocytosis in SHR CCs. Accordingly, Ca(2+) imaging showed larger [Ca(2+)]i signals induced both by depolarization and caffeine in SHR CCs. Distribution amplitude histograms showed that small amperometric spikes (0-50 pA) are more frequent in WKY than in SHR CCs. Conversely, medium (50-190 pA) and large (190-290 pA) spikes are more numerous in SHR than in WKY CCs. This study reveals that the enhanced CA secretion in SHR CCs results from a combination of (1) larger depolarization-induced Ca(2+) transients, due to a greater Ca(2+)-induced intracellular Ca(2+) release, (2) more exocytosis events per time unit, and (3) a greater proportion of medium and large amperometric spikes probably due to a higher mean CA content per granule. Enhanced CA release by excessive amplification by Ca(2+) induced Ca(2+) release and larger granule catecholamine content contributes to the increased CA plasma levels and vasomotor tone in SHRs. PMID:25791627

  13. Lovastatin-induced decrease of intracellular cholesterol level attenuates fibroblast-to-myofibroblast transition in bronchial fibroblasts derived from asthmatic patients.

    PubMed

    Michalik, Marta; Soczek, Ewelina; Kosińska, Milena; Rak, Monika; Wójcik, Katarzyna Anna; Lasota, Sławomir; Pierzchalska, Małgorzata; Czyż, Jarosław; Madeja, Zbigniew

    2013-03-15

    Chronic inflammation of the airways and structural changes in the bronchial wall are basic hallmarks of asthma. Human bronchial fibroblasts derived from patients with diagnosed asthma display in vitro predestination towards TGF-β-induced fibroblast-to-myofibroblast transition (FMT), a key event in the bronchial wall remodelling. Statins inhibit 3-hydroxymethyl-3-glutaryl coenzyme A reductase, a key enzyme in the cholesterol synthesis pathway and are widely used as antilipidemic drugs. The pleiotropic anti-inflammatory effects of statins, independent of their cholesterol-lowering capacity, are also well established. Since commonly used anti-asthmatic drugs do not reverse the structural remodelling of the airways and statins have tentative anti-asthmatic activity, we have studied the effect of lovastatin on FMT in populations of human bronchial fibroblasts derived from asthmatic patients. We demonstrate that the intensity of FMT induced by TGF-β1 was strongly and dose-dependently attenuated by lovastatin. Furthermore, we show that neither the suppression of prenylation of signalling proteins nor the effect on reactive oxygen species formation are important for lovastatin-induced inhibition of myofibroblast differentiation. On the other hand, we show that a squalene synthase inhibitor, zaragozic acid A, reduced the TGF-β1-induced FMT to an extent comparable to lovastatin effect. Additionally we demonstrate that in bronchial fibroblast populations, both inhibitors (lovastatin and zaragozic acid A) attenuate the TGF-β1-induced Smad2 nuclear translocation in a manner dependent on intracellular cholesterol level. Our data suggest that statins can directly, by decrease of intracellular cholesterol level, affect basic cell signalling events crucial for asthmatic processes and potentially prevent perilous bronchial wall remodelling associated with intensive myofibroblast formation. PMID:23485731

  14. Inhibition of Thromboxane A2-Induced Arrhythmias and Intracellular Calcium Changes in Cardiac Myocytes by Blockade of the Inositol Trisphosphate Pathway

    PubMed Central

    Kosloski, L. M.; Gilbert, W. J. R.; Touchberry, C. D.; Moore, D. S.; Kelly, J. K.; Brotto, M.; Orr, J. A.

    2009-01-01

    We have recently reported that left atrial injections of the thromboxane A2 (TXA2) mimetic, (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2 -oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619), induced ventricular arrhythmias in the anesthetized rabbit. Data from this study led us to hypothesize that TXA2 may be inducing direct actions on the myocardium to induce these arrhythmias. The aim of this study was to further elucidate the mechanism responsible for these arrhythmias. We report that TXA2R is expressed at both the gene and protein levels in atrial and ventricular samples of adult rabbits. In addition, TXA2R mRNA was identified in single, isolated ventricular cardiac myocytes. Furthermore, treatment of isolated cardiac myocytes with U46619 increased intracellular calcium in a dose-dependent manner and these increases were blocked by the specific TXA2R antagonist, 7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ29548). Pretreatment of myocytes with an inhibitor of inositol trisphosphate (IP3) formation, gentamicin, or with an inhibitor of IP3 receptors, 2-aminoethoxydiphenylborate (2-APB), blocked the increase in intracellular calcium. In vivo pretreatment of anesthetized rabbits with either gentamicin or 2-APB subsequently inhibited the formation of ventricular arrhythmias elicited by U46619. These data support the hypothesis that TXA2 can induce arrhythmias via a direct action on cardiac myocytes. Furthermore, these arrhythmogenic actions were blocked by inhibitors of the IP3 pathway. In summary, this study provides novel evidence for direct TXA2-induced cardiac arrhythmias and provides a rationale for IP3 as a potential target for the treatment of TXA2-mediated arrhythmias. PMID:19741149

  15. Spatio-temporal activity patterns of odor-induced synchronized potentials revealed by voltage-sensitive dye imaging and intracellular recording in the antennal lobe of the cockroach.

    PubMed

    Watanabe, Hidehiro; Ai, Hiroyuki; Yokohari, Fumio

    2012-01-01

    In animals, odor qualities are represented as both spatial activity patterns of glomeruli and temporal patterns of synchronized oscillatory signals in the primary olfactory centers. By optical imaging of a voltage-sensitive dye (VSD) and intracellular recording from secondary olfactory interneurons, we examined possible neural correlates of the spatial and temporal odor representations in the primary olfactory center, the antennal lobe (AL), of the cockroach Periplaneta americana. Voltage-sensitive dye imaging revealed that all used odorants induced odor-specific temporal patterns of depolarizing potentials in specific combinations of anterior glomeruli of the AL. The depolarizing potentials evoked by different odorants were temporally synchronized across glomeruli and were termed "synchronized potentials." These observations suggest that odor qualities are represented by spatio-temporal activity patterns of the synchronized potentials across glomeruli. We also performed intracellular recordings and stainings from secondary olfactory interneurons, namely projection neurons and local interneurons. We analyzed the temporal structures of enanthic acid-induced action potentials of secondary olfactory interneurons using simultaneous paired intracellular recording from two given neurons. Our results indicated that the multiple local interneurons synchronously fired in response to the olfactory stimulus. In addition, all stained enanthic acid-responsive projection neurons exhibited dendritic arborizations within the glomeruli where the synchronized potentials were evoked. Since multiple local interneurons are known to synapse to a projection neuron in each glomerulus in the cockroach AL, converging inputs from local interneurons to the projection neurons appear to contribute the odorant specific spatio-temporal activity patterns of the synchronized potentials. PMID:22848191

  16. Intracellular Zn(2+) Increase in Cardiomyocytes Induces both Electrical and Mechanical Dysfunction in Heart via Endogenous Generation of Reactive Nitrogen Species.

    PubMed

    Tuncay, Erkan; Turan, Belma

    2016-02-01

    Oxidants increase intracellular free Zn(2+) concentration ([Zn(2+)]i) in ventricular myocytes, which contributes to oxidant-induced alterations in excitation-contraction coupling (ECC). However, it is not clear whether increased [Zn(2+)]i in cardiomyocytes via increased reactive nitrogen species (RNS) has a role on heart function under pathological conditions, such as hyperglycemia. In this study, first we aimed to investigate the role of increased [Zn(2+)]i under in vitro condition in the development of both electrical and mechanical dysfunction of isolated papillary muscle strips from rat heart via exposed samples to a Zn(2+)-ionophore (Zn-pyrithione; 1 μM) for 20 min. Under simultaneous measurement of intracellular action potential and contractile activity in these preparations, Zn-pyrithione exposure caused marked prolongation in action potential repolarization phase and slowdown in both contraction and relaxation rates of twitch activity. Second, in order to demonstrate an association between increased [Zn(2+)]i and increased RNS, we monitored intracellular [Zn(2+)]i under an acute exposure of nitric oxide (NO) donor sodium nitroprusside, SNP, in freshly isolated quiescent cardiomyocytes loaded with FluoZin-3. Resting level of free Zn(2+) is significantly higher in cardiomyocytes under hyperglycemic condition compared to those of the controls, which seems to be associated with increased level of RNS production in hyperglycemic cardiomyocytes. Western blot analysis showed that Zn-pyrithione exposure induced a marked decrease in the activity of protein phosphatase 1 and 2A, member of macromolecular protein complex of cardiac ryanodine receptors, RyR2, besides significant increase in the phosphorylation level of extracellular signal-regulated kinase1/2 as a concentration-dependent manner. Overall, the present data demonstrated that there is a cross-relationship between increased RNS production and increased [Zn(2+)]i level in cardiomyocytes under pathological

  17. Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels.

    PubMed

    Watanabe, Akihiko; Takayama-Watanabe, Eriko; Vines, Carol A; Cherr, Gary N

    2011-01-01

    Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca²(+)](i)). In quiescent non-motile sperm loaded with the Ca²(+) indicator Fluo-4, intracellular free Ca²(+) was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca²(+)](i) occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca²(+)](i) at motility-initiation was monitored using spectrofluorometry, large increases in [Ca²(+)](i) occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca²(+)](i) irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca²(+) had been chelated with BAPTA-AM. The relative levels of [Ca²(+)](i) were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca²(+)](i) in the resting sperm modulates the responsiveness to the SMIS. PMID:21261606

  18. Intracellular microlasers

    PubMed Central

    Humar, Matjaž; Yun, Seok Hyun

    2015-01-01

    Optical microresonators1 which confine light within a small cavity are widely exploited for various applications ranging from the realization of lasers2 and nonlinear devices3, 4, 5 to biochemical and optomechanical sensing6, 7, 8, 9, 10, 11. Here we employ microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explored two distinct types of microresonators: soft and hard, that support whispering-gallery modes (WGM). Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (~500 pN/μm2) and its dynamic fluctuations at a sensitivity of 20 pN/μm2 (20 Pa). In a second form, WGMs within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes. PMID:26417383

  19. Beta1 integrin expression by podocytes is required to maintain glomerular structural integrity.

    PubMed

    Pozzi, Ambra; Jarad, George; Moeckel, Gilbert W; Coffa, Sergio; Zhang, Xi; Gewin, Leslie; Eremina, Vera; Hudson, Billy G; Borza, Dorin-Bogdan; Harris, Raymond C; Holzman, Lawrence B; Phillips, Carrie L; Fassler, Reinhard; Quaggin, Susan E; Miner, Jeffrey H; Zent, Roy

    2008-04-15

    Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). beta1, the most abundantly expressed integrin subunit, binds at least 12 alpha subunits. beta1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted beta1 integrin in the podocyte by crossing beta1(flox/flox) mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;beta1(flox/flox) mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of beta1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus. PMID:18328474

  20. Renin-angiotensin blockade resets podocyte epigenome through Kruppel-like Factor 4 and attenuates proteinuria.

    PubMed

    Hayashi, Kaori; Sasamura, Hiroyuki; Nakamura, Mari; Sakamaki, Yusuke; Azegami, Tatsuhiko; Oguchi, Hideyo; Tokuyama, Hirobumi; Wakino, Shu; Hayashi, Koichi; Itoh, Hiroshi

    2015-10-01

    Proteinuria is a central component of chronic kidney disease and an independent risk factor for cardiovascular disease. Kidney podocytes have an essential role as a filtration barrier against proteinuria. Kruppel-like Factor 4 (KLF4) is expressed in podocytes and decreased in glomerular diseases leading to methylation of the nephrin promoter, decreased nephrin expression and proteinuria. Treatment with an angiotensin receptor blocker (ARB) reduced methylation of the nephrin promoter in murine glomeruli of an adriamycin nephropathy model with recovery of KLF4 expression and a decrease in albuminuria. In podocyte-specific KLF4 knockout mice, the effect of ARB on albuminuria and the nephrin promoter methylation was attenuated. In cultured human podocytes, angiotensin II reduced KLF4 expression and caused methylation of the nephrin promoter with decreased nephrin expression. In patients, nephrin promoter methylation was increased in proteinuric kidney diseases with decreased KLF4 and nephrin expression. KLF4 expression in ARB-treated patients was higher in patients with than without ARB treatment. Thus, angiotensin II can modulate epigenetic regulation in podocytes and ARB inhibits these actions in part via KLF4 in proteinuric kidney diseases. This study provides a new concept that renin-angiotensin system blockade can exert therapeutic effects through epigenetic modulation of the kidney gene expression. PMID:26108068

  1. Alteration of podocyte protein expression and localization in the early stage of various hemodynamic conditions.

    PubMed

    Li, Kai; Wang, Juan; Yin, Xiaohui; Zhai, Xiaoyue; Li, Zilong

    2013-01-01

    Given that podocalyxin (PCX) and nestin play important roles in podocyte morphogenesis and the maintenance of structural integrity, we examined whether the expression and localization of these two podocyte proteins were influenced in the early stage of various hemodynamic conditions. Mice kidney tissues were prepared by in vivo cryotechnique (IVCT). The distribution of glomeruli and podocyte proteins was visualized with DAB staining, confocal laser scanning microscopy and immunoelectron microscopy. The mRNA levels were examined by real-time quantitative PCR. The results showed the following: Under the normal condition, PCX stained intensely along glomerular epithelial cells, whereas nestin was clearly staining in the endothelial cells and appeared only weakly in the podocytes. Under the acute hypertensive and cardiac arrest conditions, PCX and nestin staining was not clear, with a disarranged distribution, but the colocalization of PCX and nestin was apparent under this condition. In addition, under the acute hypertensive and cardiac arrest conditions, the mRNA levels of PCX and nestin were significantly decreased. Collectively, the abnormal redistribution and decreased mRNA expressions of PCX and nestin are important molecular events at the early stage of podocyte injury during hemodynamic disorders. IVCT may have more advantages for morphological analysis when researching renal diseases. PMID:23502465

  2. Small Molecule Membrane Transporters in the Mammalian Podocyte: A Pathogenic and Therapeutic Target

    PubMed Central

    Zennaro, Cristina; Artero, Mary; Di Maso, Vittorio; Carraro, Michele

    2014-01-01

    The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development. PMID:25411800

  3. Role of altered insulin signaling pathways in the pathogenesis of podocyte malfunction and microalbuminuria

    PubMed Central

    Jauregui, Alexandra; Mintz, Daniel H; Mundel, Peter; Fornoni, Alessia

    2010-01-01

    Purpose of review In diabetic nephropathy (DN), insulin resistance and hyperinsulinemia correlate with the development of albuminuria. The possibility that altered insulin signaling in glomerular cells and particularly podocytes contributes to the development of DN will be discussed. Recent findings While normal podocytes uptake glucose in response to insulin, diabetic podocytes become insulin resistant in experimental DN prior to the development of significant albuminuria. Both clinical and experimental data suggest that insulin sensitizers may be renoprotective independently of their systemic effects on the metabolic control of diabetes. Summary We will review the clinical and experimental evidence that altered insulin signaling correlates with the development of DN in both type 1 and type 2 diabetes, and that insulin sensitizers may be superior to other hypoglycemic agents in the prevention of DN. We will then review potential mechanisms by which altered podocyte insulin signaling may contribute to the development of DN. Understanding the role of podocyte in glucose metabolism is important because it may lead to the discovery of novel pathogenetic mechanisms of DN, it may affect current strategies for prevention and treatment of DN, and it may allow for the identification of novel therapeutic targets. PMID:19724224

  4. Proteomic analysis of podocyte exosome-enriched fraction from normal human urine.

    PubMed

    Prunotto, Marco; Farina, Annarita; Lane, Lydie; Pernin, Agnès; Schifferli, Jürg; Hochstrasser, Denis F; Lescuyer, Pierre; Moll, Solange

    2013-04-26

    Urine results from a coordinated activity of glomerular and tubular compartments of the kidney. As a footprint of these cellular functional processes, urinary exosomes, and 40-80 nm membrane vesicles released after fusion with the plasma membrane into the extracellular environment by renal epithelial cells, are a source for identification of proteins and investigation of their role in the kidney. The aim of the present study was the identification of podocyte exosome proteins based on urine immunoabsorption using podocyte-specific CR1-immunocoated beads followed by proteomic analysis using LC MS/MS techniques. This methodology allowed the identification of 1195 proteins. By using a bioinformatic approach, 27 brain-expressed proteins were identified, in which 14 out of them were newly demonstrated to be expressed in the kidney at a mRNA level, and, one of them, the COMT protein, was demonstrated to be expressed in podocytes at a protein level. These results, attesting the reliability of the methodology to identify podocyte proteins, need now to be completed by further experiments to analyze more precisely their biological function(s) in the podocytes. PMID:23376485

  5. Genome-Wide Analysis of Wilms' Tumor 1-Controlled Gene Expression in Podocytes Reveals Key Regulatory Mechanisms.

    PubMed

    Kann, Martin; Ettou, Sandrine; Jung, Youngsook L; Lenz, Maximilian O; Taglienti, Mary E; Park, Peter J; Schermer, Bernhard; Benzing, Thomas; Kreidberg, Jordan A

    2015-09-01

    The transcription factor Wilms' tumor suppressor 1 (WT1) is key to podocyte development and viability; however, WT1 transcriptional networks in podocytes remain elusive. We provide a comprehensive analysis of the genome-wide WT1 transcriptional network in podocytes in vivo using chromatin immunoprecipitation followed by sequencing (ChIPseq) and RNA sequencing techniques. Our data show a specific role for WT1 in regulating the podocyte-specific transcriptome through binding to both promoters and enhancers of target genes. Furthermore, we inferred a podocyte transcription factor network consisting of WT1, LMX1B, TCF21, Fox-class and TEAD family transcription factors, and MAFB that uses tissue-specific enhancers to control podocyte gene expression. In addition to previously described WT1-dependent target genes, ChIPseq identified novel WT1-dependent signaling systems. These targets included components of the Hippo signaling system, underscoring the power of genome-wide transcriptional-network analyses. Together, our data elucidate a comprehensive gene regulatory network in podocytes suggesting that WT1 gene regulatory function and podocyte cell-type specification can best be understood in the context of transcription factor-regulatory element network interplay. PMID:25636411

  6. Zn2+ induces hyperpolarization by activation of a K+ channel and increases intracellular Ca2+ and pH in sea urchin spermatozoa

    PubMed Central

    Granados-González, Gisela; de De la Torre, Lucia García; Nishigaki, Takuya; Darszon, Alberto

    2014-01-01

    Zinc (Zn2+) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn2+ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pHi). In this study we found that submicromolar concentrations of free Zn2+ change membrane potential (Em) and increase the concentration of intracellular Ca2+ ([Ca2+]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn2+ response in sperm of this species mainly involves an Em hyperpolarization caused by K+ channel activation. The pharmacological profile of the Zn2+-induced hyperpolarization indicates that the cGMP-gated K+ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn2+. Considering that Zn2+ also induces [Ca2+]i fluctuations, our observations suggest that Zn2+ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn2+ in male gamete function. PMID:25092071

  7. Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii.

    PubMed

    Mahon, Cathal S; O'Donoghue, Anthony J; Goetz, David H; Murray, Patrick G; Craik, Charles S; Tuohy, Maria G

    2009-11-01

    monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus T. emersonii. PMID:19556294

  8. Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

    PubMed Central

    Mahon, Cathal S.; O'Donoghue, Anthony J.; Goetz, David H.; Murray, Patrick G.; Craik, Charles S.; Tuohy, Maria G.

    2009-01-01

    monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus T. emersonii. PMID:19556294

  9. Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1)-Harnessed SAINT-Based Lipid Carrier System

    PubMed Central

    Visweswaran, Ganesh Ram R.; Gholizadeh, Shima; Ruiters, Marcel H. J.; Molema, Grietje; Kok, Robbert J.; Kamps, Jan. A. A. M.

    2015-01-01

    Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes. PMID:26407295

  10. Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers

    PubMed Central

    Marino, Franca; Cosentino, Marco; Ferrari, Marco; Cattaneo, Simona; Frigo, Giuseppina; Fietta, Anna M; Lecchini, Sergio; Frigo, Gian Mario

    2004-01-01

    Background The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca++ entry through the plasma membrane of these cells. Results The PKC activator 12-O-tetradecanoylphorbol-13-acetate (PMA) concentration-dependently inhibited TTN-induced [Ca++]i rise, and this effect was reverted by the PKC inhibitors rottlerin (partially) and Ro 32-0432 (completely). PMA also inhibited TTN-induced IL-8 mRNA expression. In the absence of PMA, however, rottlerin (but not Ro 32-0432) per se partially inhibited TTN-induced [Ca++]i rise. The response of [Ca++]i to TTN was also sensitive to mibefradil and flunarizine (T-type Ca++-channel blockers), but not to nifedipine, verapamil (L-type) or ω-conotoxin GVIA (N-type). In agreement with this observation, PCR analysis showed the expression in human PMNs of the mRNA for all the α1 subunits of T-type Ca++ channels (namely, α1G, α1H, and α1I). Conclusions In human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels. PMID:15228623

  11. Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease

    PubMed Central

    Pullman, James M.; Nylk, Jonathan; Campbell, Elaine C.; Gunn-Moore, Frank J.; Prystowsky, Michael B.; Dholakia, Kishan

    2016-01-01

    A detailed microscopic analysis of renal podocyte substructure is essential to understand and diagnose nephrotic kidney disease. Currently only time consuming electron microscopy (EM) can resolve this substructure. We used structured illumination microscopy (SIM) to examine frozen sections of renal biopsies stained with an immunofluorescence marker for podocin, a protein localized to the perimeter of the podocyte foot processes and compared them with EM in both normal and nephrotic disease biopsies. SIM images of normal glomeruli revealed curvilinear patterns of podocin densely covering capillary walls similar to podocyte foot processes seen by EM. Podocin staining of all nephrotic disease biopsies were significantly different than normal, corresponding to and better visualizing effaced foot processes seen by EM. The findings support the first potential use of SIM in the diagnosis of nephrotic disease. PMID:26977341

  12. Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.

    PubMed

    Pullman, James M; Nylk, Jonathan; Campbell, Elaine C; Gunn-Moore, Frank J; Prystowsky, Michael B; Dholakia, Kishan

    2016-02-01

    A detailed microscopic analysis of renal podocyte substructure is essential to understand and diagnose nephrotic kidney disease. Currently only time consuming electron microscopy (EM) can resolve this substructure. We used structured illumination microscopy (SIM) to examine frozen sections of renal biopsies stained with an immunofluorescence marker for podocin, a protein localized to the perimeter of the podocyte foot processes and compared them with EM in both normal and nephrotic disease biopsies. SIM images of normal glomeruli revealed curvilinear patterns of podocin densely covering capillary walls similar to podocyte foot processes seen by EM. Podocin staining of all nephrotic disease biopsies were significantly different than normal, corresponding to and better visualizing effaced foot processes seen by EM. The findings support the first potential use of SIM in the diagnosis of nephrotic disease. PMID:26977341

  13. Lonidamine Induces Intracellular Tumor Acidification and ATP Depletion in Breast, Prostate and Ovarian Cancer Xenografts and Potentiates Response to Doxorubicin

    PubMed Central

    Nath, Kavindra; Nelson, David S.; Heitjan, Daniel F.; Leeper, Dennis B.; Zhou, Rong; Glickson, Jerry D.

    2015-01-01

    We demonstrate that the effects of lonidamine (LND, 100 mg/kg, i.p.) are similar for a number of xenograft models of human cancer including DB-1 melanoma and HCC1806 breast, BT-474 breast, LNCaP prostate and A2870 ovarian carcinomas. Following treatment with LND, each of these tumors exhibits a rapid decrease in intracellular pH, a small decrease in extracellular pH, a concomitant monotonic decrease in nucleoside triphosphate and increase in inorganic phosphate over a 2–3 hr period. We previously demonstrated that selective intracellular tumor acidification potentiates response of this melanoma model to melphalan (7.5 mg/kg, i.v.), producing an estimated 89% cell kill based on tumor growth delay analysis. We now show that in both DB-1 melanoma and HCC1806 breast carcinoma, LND potentiates response to doxorubicin producing 95% cell kill in DB-1 melanoma at 7.5 mg/kg, i.v. doxorubicin and 98% cell kill at 10.0 mg/kg doxorubicin, and in HCC1806 breast carcinoma producing a 95% cell kill at 12.0 mg/kg doxorubicin. Potentiation of doxorubicin can result from cation trapping of the weakly basic anthracycline. Recent experience with the clinical treatment of melanoma and other forms of human cancer suggests that these diseases will probably not be cured by a single therapeutic procedure other than surgery. A multimodality therapeutic approach will be required. As a potent modulator of tumor response to N-mustards and anthracyclines as well as tumor thermo- and radiosensitivity, LND promises to play an important clinical role in the management and possible complete local control of a number of prevalent forms of human cancer. PMID:25504852

  14. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae)

    PubMed Central

    Shane, Michael W.; Stigter, Kyla; Fedosejevs, Eric T.; Plaxton, William C.

    2014-01-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native ‘extremophile’ plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors’ knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  15. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae).

    PubMed

    Shane, Michael W; Stigter, Kyla; Fedosejevs, Eric T; Plaxton, William C

    2014-11-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native 'extremophile' plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors' knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  16. Gα12 Activation in Podocytes Leads to Cumulative Changes in Glomerular Collagen Expression, Proteinuria and Glomerulosclerosis

    PubMed Central

    Boucher, Ilene; Yu, Wanfeng; Beaudry, Sarah; Negoro, Hideyuki; Tran, Mei; Pollak, Martin; Henderson, Joel; Denker, Bradley M.

    2011-01-01

    Glomerulosclerosis is a common pathologic finding that often progresses to renal failure. The mechanisms of chronic kidney disease progression are not well-defined but may include activation of numerous vasoactive and inflammatory pathways. We hypothesized that podocytes are susceptible to filtered plasma components including hormones and growth factors that stimulate signaling pathways leading to glomerulosclerosis. Gα12 couples to numerous G-protein-coupled receptors (GPCR) and regulates multiple epithelial responses including proliferation, apoptosis, permeability and the actin cytoskeleton. Herein, we report that genetic activation of Gα12 in podocytes leads to time dependent increases in proteinuria and glomerulosclerosis. To mimic activation of Gα12-pathways, constitutively active Gα12(QL) was conditionally expressed in podocytes using Nphs2-Cre and LacZfloxed QLα12 transgenic mice. Some QLα12LacZ+/Cre+ mice developed proteinuria at 4-6m, and most were proteinuric by 12m. Proteinuria increased with age, and by 12-14m many demonstrated glomerulosclerosis with ultrastructural changes including foot process fusion and both mesangial and subendothelial deposits. QLα12LacZ+/Cre+ mice showed no changes in podocyte number, apoptosis, proliferation, or Rho/Src activation. Real-time PCR revealed no significant changes in Nphs1, Nphs2, Cd2ap, or Trpc6 expression, but Col4a2 message was increased in younger and older mice while Col4a5 was decreased in older mice. Confocal microscopy revealed disordered collagen IVα1/2 staining in older mice and loss of α5 without changes in other collagen IV subunits. Taken together, these studies suggest that Gα12 activation promotes glomerular injury without podocyte depletion through a novel mechanism regulating collagen (α)IV expression, and supports the notion that glomerular damage may accrue through persistent GPCR activation in podocytes. PMID:22249312

  17. Novel unbiased assay for circulating podocyte-toxic factors associated with recurrent focal segmental glomerulosclerosis.

    PubMed

    Kachurina, Nadezda; Chung, Chen-Fang; Benderoff, Erin; Babayeva, Sima; Bitzan, Martin; Goodyer, Paul; Kitzler, Thomas; Matar, Dany; Cybulsky, Andrey V; Alachkar, Nada; Torban, Elena

    2016-05-15

    Focal segmental glomerular sclerosis (FSGS) is an irreversible renal pathology characterized by podocyte detachment from the glomerular basement membrane, hyalinosis, and sclerosis. Clinically, it manifests with proteinuria and progressive loss of glomerular filtration. Primary idiopathic FSGS can occur in isolation and frequently progresses to end-stage renal disease, requiring dialysis or kidney transplantation. In 30-50% of these patients, proteinuria and FSGS recur in the renal allograft, suggesting the presence of a podocyte-toxic factor(s) in the recipient's serum. Currently, there is no reliable way to quantify the serum activity or predict the subset of FSGS patients at risk for recurrence after transplantation. We describe a novel in vitro method that measures the podocyte-toxic activity of sera from FSGS patients using cultured human podocytes; we compare this with the effect of compounds such as adriamycin. Using immunofluorescence microscopy followed by computerized image-processing analysis, we show that incubation of human podocytes with adriamycin leads to a dose-dependent disassembly of focal adhesion complexes (FACs). We then demonstrate that sera from patients with posttransplant recurrent or idiopathic FSGS cause a similar FAC disturbance. In contrast, sera from nonrecurrent FSGS patients do not affect FACs. In some FSGS patients, toxic effects of serum can be prevented by blockade of the tumor necrosis factor-α pathway. We propose that this method may be useful as a diagnostic tool to identify FSGS patients with serum podocyte-toxic activity that presumably places them at increased risk for recurrence in the renal allograft. PMID:26719363

  18. Effect of Cordyceps sinensis and Tripterygium wilfordii polyglycosidium on podocytes in rats with diabetic nephropathy.

    PubMed

    Hao, Li; Pan, Meng-Shu; Zheng, Yun; Wang, Rui-Feng

    2014-06-01

    The aim of the present study was to investigate the effects of Cordyceps sinensis (CS) and Tripterygium wilfordii polyglycosidium (TWP) on podocytes in rats with diabetic nephropathy (DN). DN rat models were established and divided randomly into normal control (group A), DN (group B), CS (group C), TWP (group D) and CS and TWP groups (group E). After 12 weeks, levels of 24-h urinary protein, blood urea nitrogen (BUN), serum creatinine (SCR), white blood cells, blood glucose (GLU), aspartate aminotransferase, alanine aminotransferase and kidney weight (KW)/body weight (BW) were determined. Renal pathological changes were evaluated using hematoxylin and eosin staining, whereas the structural changes in the podocytes were observed under a transmission electron microscope. The expression levels of nephrin and podocin were evaluated using immunofluorescence staining. Compared with group A, the SCR and BUN levels in group B were higher (P<0.05) and the GLU, KW/BW and the 24-h urine protein were markedly higher (P<0.01). Moreover, incidences of glomerular disorders, chronic tubulointerstitial damage and glomerular podocyte lesions in groups B, C, D and E were observed, compared with group A. The high cortical expression of nephrin and podocin protein decreased. Compared with group B, the KW/BW and 24-h urinary protein level in groups C, D and E were lower (P<0.01). The glomeruli, tubules and podocytes exhibited pathomorphological improvements and the nephrin and podocin protein expression levels were higher in the nephridial tissue. A decrease in KW/BW and the 24-h urinary protein level, as well as improvements in glomerular disorder, chronic tubulointerstitial damage and glomerular podocyte lesions, were observed in groups C, D and E. Therefore, the results demonstrated that CS and TWP exhibited a protective effect on the podocytes of rats with DN. Moreover, CS combined with TWP increased this protective effect. PMID:24926327

  19. Dedifferentiation of Immortalized Human Podocytes in Response to Transforming Growth Factor-β

    PubMed Central

    Herman-Edelstein, Michal; Thomas, Merlin C.; Thallas-Bonke, Vicki; Saleem, Moin; Cooper, Mark E.; Kantharidis, Phillip

    2011-01-01

    OBJECTIVE Diabetic nephropathy is associated with dedifferentiation of podocytes, losing the specialized features required for efficient glomerular function and acquiring a number of profibrotic, proinflammatory, and proliferative features. These result from tight junction and cytoskeletal rearrangement, augmented proliferation, and apoptosis. RESEARCH DESIGN AND METHODS Experiments were performed in conditionally immortalized human podocytes developed by transfection with the temperature-sensitive SV40-T gene. Cells were then cultured in the presence of transforming growth factor (TGF)-β1 or angiotensin II in the presence or absence of a selective inhibitor of the TGF-β type I receptor kinase, SB-431542. Gene and protein expression were then examined by real-time RT-PCR and immunofluorescence, and correlated with changes observed in vivo in experimental diabetes. RESULTS Treatment of cells with TGF-β1 resulted in dynamic changes in their morphology, starting with retraction and shortening of foot processes and finishing with the formation of broad and complex tight junctions between adjacent podocytes. This dedifferentiation was also associated with dose- and time-dependent reduction in the expression of glomerular epithelial markers (nephrin, p-cadherin, zonnula occludens-1) and increased expression of mesenchymal markers (α−smooth muscle actin, vimentin, nestin), matrix components (fibronectin, collagen I, and collagen IV α3), cellular proliferation, and apoptosis. The induction of diabetes in mice was also associated with similar changes in morphology, protein expression, and proliferation in glomerular podocytes. CONCLUSIONS In response to TGF-β and other TGF-dependent stimuli, mature podocytes undergo dedifferentiation that leads to effacement of foot processes, morphologic flattening, and increased formation of intercellular tight junctions. This simplification of their phenotype to a more embryonic form is also associated with reentry of mature

  20. DC-SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis.

    PubMed

    Cai, Minchao; Zhou, Tong; Wang, Xuan; Shang, Minghua; Zhang, Yueyue; Luo, Maocai; Xu, Chundi; Yuan, Weijie

    2016-03-01

    Podocytes, the main target of immune complex, participate actively in the development of glomerular injury as immune cells. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune molecular that has an immune recognition function, and is involved in mediation of cell adhesion and immunoregulation. Here we explored the expression of DC-SIGN on podocytes and its role in immune and inflammatory responses in lupus nephritis (LN). Expression of DC-SIGN and immunoglobulin (Ig)G1 was observed in glomeruli of LN patients. DC-SIGN was co-expressed with nephrin on podocytes. Accompanied by increased proteinuria of LN mice, DC-SIGN and IgG1 expressions were observed in the glomeruli from 20 weeks, and the renal function deteriorated up to 24 weeks. Mice with anti-DC-SIGN antibody showed reduced proteinuria and remission of renal function. After the podocytes were stimulated by serum of LN mice in vitro, the expression of DC-SIGN, major histocompatibility complex (MHC) class II and CD80 was up-regulated, stimulation of T cell proliferation was enhanced and the interferon (IFN)-γ/interleukin (IL)-4 ratio increased. However, anti-DC-SIGN antibody treatment reversed these events. These results suggested that podocytes in LN can exert DC-like function through their expression of DC-SIGN, which may be involved in immune and inflammatory responses of renal tissues. However, blockage of DC-SIGN can inhibit immune functions of podocytes, which may have preventive and therapeutic effects. PMID:26440060

  1. Thrombospondin 1 Deficiency Ameliorates the Development of Adriamycin-Induced Proteinuric Kidney Disease

    PubMed Central

    Maimaitiyiming, Hasiyeti; Zhou, Qi; Wang, Shuxia

    2016-01-01

    Accumulating evidence suggests that thrombospondin 1 (TSP1) is an important player in diabetic nephropathy. However, the role of TSP1 in podocyte injury and the development of non-diabetic proteinuric kidney disease is largely unknown. In the current study, by using a well-established podocyte injury model (adriamycin-induced nephropathy mouse model), we examined the contribution of TSP1 to the development of proteinuric kidney disease. We found that TSP1 was up-regulated in the glomeruli, notably in podocytes, in adriamycin injected mice before the onset of proteinuria. ADR treatment also stimulated TSP1 expression in cultured human podocytes in vitro. Moreover, increased TSP1 mediated ADR-induced podocyte apoptosis and actin cytoskeleton disorganization. This TSP1’s effect was through a CD36-dependent mechanism and involved in the stimulation of p38MAPK pathway. Importantly, in vivo data demonstrated that TSP1 deficiency protected mice from ADR induced podocyte loss and foot process effacement. ADR induced proteinuria, glomerulosclerosis, renal macrophage infiltration and inflammation was also attenuated in TSP1 deficient mice. Taken together, these studies provide new evidence that TSP1 contributes to the development of non-diabetic proteinuric kidney disease by stimulating podocyte injury and the progression of renal inflammation. PMID:27196103

  2. Macrophage activation for intracellular killing as induced by a Ca2+ ionophore. Dependence on L-arginine-derived nitrogen oxidation products.

    PubMed Central

    Buchmüller-Rouiller, Y; Corradin, S B; Mauël, J

    1992-01-01

    Mouse macrophages activated by interferon-gamma kill intracellular Leishmania by a process that depends on the generation of L-arginine-derived nitrogen oxidation products. Interferon-induced intracellular killing can be mimicked by exposure of macrophages to the Ca2+ ionophore A23187 in the presence of lipopolysaccharide. The mechanisms of this effect were therefore investigated. Destruction of the parasite was accompanied by accumulation of nitrite in the macrophage culture fluids. Leishmanicidal activity and nitrite production in cultures stimulated with ionophore A23187 and lipopolysaccharide were abrogated when cells were activated in medium containing arginase or the L-arginine analogues L-canavanine, guanidine or NG-monomethyl-L-arginine. L-Arginine was required during the lipopolysaccharide-induced triggering phase only. Indeed, macrophage priming with ionophore A23187 in L-arginine-depleted medium led to full microbicidal activity and nitrite generation provided that L-arginine was present during subsequent triggering by lipopolysaccharide. Addition of NG-monomethyl-L-arginine to ionophore-activated macrophages increased O2- production on phorbol myristate stimulation, while inhibiting glucose oxidation through the hexose monophosphate shunt pathway. Leishmanicidal activity and nitrite production were also inhibited when ionophore-treated cultures were incubated with excess iron, implying a role for iron as a defence mechanism against the toxicity of nitrogen derivatives. These results indicate that the ionophore-induced leishmanicidal activity occurs through a process similar to that evoked by interferon-gamma, i.e. the production of L-arginine-derived nitrogen oxidation products. PMID:1599422

  3. Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells.

    PubMed

    Lee, Sei-Jung; Lim, Kye-Taek

    2006-04-01

    This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays, respectively. SNL glycoprotein has an apparent cytotoxic and apoptotic effect at a concentration of 40 microg/ml after 4 h. To further verify the apoptotic effect, we investigated the changes in activity of the apoptotic-related proteins [Bid, cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)] triggered by SNL glycoprotein, using a western blot analysis. The results in this study indicated that SNL glycoprotein has a stimulatory effect on Bid activation, resulting in the release of cytochrome c, the stimulation of caspase-8, -9 and -3 activities, and the cleavage of PARP in HCT-116 cells. However, SNL glycoprotein did not significantly stimulate an increase in levels of intracellular reactive oxygen species (ROS). From the results in this experiment, it is suggested that SNL glycoprotein induces apoptosis through the mitochondrial apoptotic signal pathway in HCT-116 cells, rather than through intracellular ROS. PMID:16208518

  4. Production of intracellular reactive oxygen species and change of cell viability induced by atmospheric pressure plasma in normal and cancer cells

    NASA Astrophysics Data System (ADS)

    Ja Kim, Sun; Min Joh, Hea; Chung, T. H.

    2013-10-01

    The effects of atmospheric pressure plasma jet on cancer cells (human lung carcinoma cells) and normal cells (embryonic kidney cells and bronchial epithelial cells) were investigated. Using a detection dye, the production of intracellular reactive oxygen species (ROS) was found to be increased in plasma-treated cells compared to non-treated and gas flow-treated cells. A significant overproduction of ROS and a reduction in cell viability were induced by plasma exposure on cancer cells. Normal cells were observed to be less affected by the plasma-mediated ROS, and cell viability was less changed. The selective effect on cancer and normal cells provides a promising prospect of cold plasma as a cancer therapy.

  5. Protective effects of Alpha-lipoic acid on MeHg-induced oxidative damage and intracellular Ca(2+) dyshomeostasis in primary cultured neurons.

    PubMed

    Yang, Tianyao; Xu, Zhaofa; Liu, Wei; Xu, Bin; Deng, Yu

    2016-05-01

    Methylmercury (MeHg) is one of the ubiquitous environmental toxicant that leads to long-lasting neurological deficits in animals and humans. However, the mechanisms of MeHg-induced neuronal cell death are incompletely understood. Treatment of neuronal cells with MeHg (0-2 μM) for 0.5-12 h, or pretreated with LA (12.5-100 μM) for 0.5-6 h resulted in toxic effects of primary cultured neurons concentration- and time-dependently. For further experiments, 12.5, 25, and 50 μM of LA pretreatment for 3 h followed by 1 μM MeHg for 6 h were performed for the examination of the responses of neurons. Exposure of MeHg resulted in damages of neurons, which were shown by a loss of cell viability, and supported by high levels of lactate dehydrogenase (LDH) release, apoptosis, and morphological changes. In addition, neurons were sensitive to MeHg-mediated oxidative stress, a finding that is consistent with ROS over-production, leading to decrease Ca(2+)-ATPase activity and increase intracellular free calcium. Moreover, expressions of NMDA receptor subunits in neurons were down-regulated after MeHg exposure, and expression of NR2A mRNA and protein were much more sensitive to MeHg than those of NR1 and NR2B. On the contrary, pretreatment with LA presented a concentration-dependent prevention against MeHg-mediated cytotoxic effects of neurons. In conclusion, present results showed that oxidative stress and intracellular Ca(2+ )dyshomeostasis resulting from MeHg exposure contributed to neuronal injury. LA could attenuate MeHg-induced neuronal toxicity via its antioxidant properties in primary cultured neurons. PMID:26986620

  6. The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

    NASA Astrophysics Data System (ADS)

    Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

    2013-09-01

    We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

  7. Differential regulation of thrombin- or ATP-induced mobilization of intracellular Ca2+ by prostacyclin receptor in mouse mastocytoma cells.

    PubMed

    Negishi, M; Hashimoto, H; Ichikawa, A

    1991-04-15

    Thrombin induced an increase in [Ca2+]i in mouse mastocytoma P-815 cells. This increase was markedly reduced by prior exposure to pertussis toxin (PT) but not by removal of extracellular Ca2+, suggesting that thrombin stimulates phospholipase C via a PT-sensitive GTP-binding protein. ATP also induced an increase in [Ca2+]i. This increase was insensitive to PT but completely suppressed on removal of extracellular Ca2+, suggesting that ATP stimulates Ca2+ influx in a PT-insensitive manner. Iloprost, a stable prostacyclin analogue, increased the cellular cAMP level and dose-dependently inhibited the thrombin-induced increase in [Ca2+]i, whereas the ATP-induced increase in [Ca2+]i was markedly enhanced by iloprost. Cyclic AMP analogues, dibutyryl cAMP and 8-bromo cAMP, also inhibited the increase in [Ca2+]i induced by thrombin and promoted that by ATP, indicating that the inhibitory and stimulatory effects of iloprost are mediated by cAMP. These results suggest that the prostacyclin receptor differentially regulates two distinct Ca2+ mobilizing systems via cAMP in mastocytoma cells. PMID:1708239

  8. The NF-κB essential modulator (NEMO) controls podocyte cytoskeletal dynamics independently of NF-κB.

    PubMed

    Brähler, Sebastian; Ising, Christina; Barrera Aranda, Belén; Höhne, Martin; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul Thomas

    2015-10-01

    Maintenance of the glomerular filtration barrier with its fenestrated endothelium, the glomerular basement membrane, and the podocytes as the outer layer, is a major prerequisite for proper renal function. Tight regulation of the balance between plasticity and rigidity of the podocytes' architecture is required to prevent the onset of glomerular disease, mainly proteinuria. The underlying cellular signaling pathways that regulate the organization of the podocytes' cytoskeleton are still a matter of controversial debate. In this study, we investigated the role of the NF-κB signaling pathway in podocyte cytoskeletal dynamics. As previously published, genetic inhibition of the NF-κB essential modulator (NEMO) in podocytes does not affect glomerular function under physiological, nonstressed conditions nor does it alter the initial podocyte response in an experimental glomerulonephritis (NTN) model (Brähler S, Ising C, Hagmann H, Rasmus M, Hoehne M, Kurschat C, Kisner T, Goebel H, Shankland SJ, Addicks K, Thaiss F, Schermer B, Pasparakis M, Benzing T, Brinkkoetter PT. Am J Physiol Renal Physiol 303: F1473-F1475, 2012). Quite the contrary, podocyte-specific NEMO null mice recovered significantly faster and did not develop glomerulosclerosis and end-stage renal failure over time. Here, we show that cytoskeletal rearrangements and increased podocyte motility following stimulation with IL-1, TNF-α, or LPS depend on NEMO. NEMO also regulates the phosphorylation of the MAP kinase ERK1/2 and suppresses the activation of RhoA following stimulation with IL-1. The migratory response and altered ERK1/2 phosphorylation is independent of NF-κB signaling as demonstrated by expression of a mutant IκB resistant to phosphorylation and degradation. In conclusion, signaling through NEMO might not only be involved in the production of NF-κB proinflammatory chemokines but also regulates podocyte dynamics independently of NF-κB, most likely through small GTPases and MAP kinases. PMID

  9. Fluoxetine induces alkalinization of astroglial cytosol through stimulation of sodium-hydrogen exchanger 1: dissection of intracellular signaling pathways

    PubMed Central

    Ren, Jienan; Song, Dan; Bai, Qiufang; Verkhratsky, Alexei; Peng, Liang

    2015-01-01

    Clinical evidence suggest astrocytic abnormality in major depression (MD) while treatment with anti-psychotic drugs affects astroglial functions. Astroglial cells are involved in pH homeostasis of the brain by transporting protons (through sodium-proton transporter 1, NHE1, glutamate transporters EAAT1/2 and proton-lactate co-transporter MCT1) and bicarbonate (through the sodium-bicarbonate co-transporter NBC or the chloride-bicarbonate exchanger AE). Here we show that chronic treatment with fluoxetine increases astroglial pHi by stimulating NHE1-mediated proton extrusion. At a clinically relevant concentration of 1 μM, fluoxetine significantly increased astroglial pHi from 7.05 to 7.34 after 3 weeks and from 7.18 to 7.58 after 4 weeks of drug treatment. Stimulation of NHE1 is a result of transporter phosphorylation mediated by several intracellular signaling cascades that include MAPK/ERK1/2, PI3K/AKT and ribosomal S6 kinase (RSK). Fluoxetine stimulated phosphorylation of ERK1/2, AKT and RSK in a concentration dependent manner. Positive crosstalk exists between two signal pathways, MAPK/ERK1/2 and PI3K/AKT activated by fluoxetine since ERK1/2 phosphrylation could be abolished by inhibitors of PI3K, LY294002 and AKT, triciribine, and AKT phosphorylation by inhibitor of MAPK, U0126. As a result, RSK phosphorylation was not only inhibited by U0126 but also by inhibitor of LY294002. The NHE1 phoshorylation resulted in stimulation of NHE1 activity as revealed by the NH4Cl-prepulse technique; the increase of NHE1 activity was dependent on fluoxetine concentration, and could be inhibited by both U0126 and LY294002. Our findings suggest that regulation of astrocytic pHi and brain pH may be one of the mechanisms underlying fluoxetine action. PMID:25784857

  10. Sulfur mustard-induced increase in intracellular free calcium level and arachidonic acid release from cell membrane

    SciTech Connect

    Ray, R.; Legere, R.H.; Majerus, B.J.; Petrali, J.P.

    1995-12-31

    The mechanism of action of the alkylating agent bis-(2-chloroethyl)sulfide (sulfur mustard, SM) was studied using the in thai vitro mouse neuroblastoma-rat glioma hybrid NG 108-1 S clonal p cell line model. Following 0.3 mM SM exposure, cell viability remained high (>80% of untreated control) up to 9 hr and then declined steadily to about 40% of control after 20-24 hr. During the early period of SM exposure, when there was no significant cell viability loss, the following effects were observed. The cellular glutathione level decreased 20% after 1 hr and 34% after 6 hr. Between 2 and 6 hr, there was a time-dependent increase (about 10 to 30%) in intracellular free calcium (Ca2+), which was localized to the limiting membrane of swollen endoplasmic reticula and mitochondria, to euchromatin areas of the nucleus, and to areas of the cytosol and plasma membrane. Moreover,there was also a time-dependent increase in the release of isotopically labeled arachidonic acid ((3H)AA) from cellular membranes. Increase in (3H)AA release was 28% at 3 hr and about 60-80% between 6 and 9 hr. This increase in I3HIAA release was inhibited by quinacrine (20 uM), which is a phospholipase (PLA2) inhibitor. At 16 hr after SM exposure, there was a large increase (about 200% of control) in I3HIAA release, which was coincident with a 50% loss of cell viability. These results suggest a Ca2+-mediated toxic mechanism of SM via PLA2 activation and arachidonate release.

  11. Trajectories and single-particle tracking data of intracellular vesicles loaded with either SNAP-Crb3A or SNAP-Crb3B.

    PubMed

    Siebrasse, Jan Peter; Djuric, Ivona; Schulze, Ulf; Schlüter, Marc A; Pavenstädt, Hermann; Weide, Thomas; Kubitscheck, Ulrich

    2016-06-01

    Using a combined approach of pulse chase labeling and single-particle tracking of Crb3A or 3B loaded vesicles we collected trajectories of different vesicle population in living podocyte cells and evaluated statistically their different mobility patterns. Differences in their intracellular mobility and in their directed transport correspond well to the role of Crb3A and 3B in renal plasma membrane sorting (Djuric et al., 2016) [1]. PMID:27222870

  12. Quantitative podocyte parameters predict human native kidney and allograft half-lives

    PubMed Central

    Cibrik, Diane; Hodgin, Jeffrey B.; Wu, Fan; Zhang, Min; Kikuchi, Masao; Wickman, Larysa; Samaniego, Milagros; Bitzer, Markus; Wiggins, Jocelyn E.; Ojo, Akinlolu; Li, Yi; Wiggins, Roger C.

    2016-01-01

    BACKGROUND Kidney function decreases with age. A potential mechanistic explanation for kidney and allograft half-life has evolved through the realization that linear reduction in glomerular podocyte density could drive progressive glomerulosclerosis to impact both native kidney and allograft half-lives. METHODS Predictions from podometrics (quantitation of podocyte parameters) were tested using independent pathologic, functional, and outcome data for native kidneys and allografts derived from published reports and large registries. RESULTS With age, native kidneys exponentially develop glomerulosclerosis, reduced renal function, and end-stage kidney disease, projecting a finite average kidney life span. The slope of allograft failure rate versus age parallels that of reduction in podocyte density versus age. Quantitative modeling projects allograft half-life at any donor age, and rate of podocyte detachment parallels the observed allograft loss rate. CONCLUSION Native kidneys are designed to have a limited average life span of about 100–140 years. Allografts undergo an accelerated aging-like process that accounts for their unexpectedly short half-life (about 15 years), the observation that older donor age is associated with shorter allograft half-life, and the fact that long-term allograft survival has not substantially improved. Podometrics provides potential readouts for these processes, thereby offering new approaches for monitoring and intervention. FUNDING National Institutes of Health. PMID:27280173

  13. Metabolism, Energetics, and Lipid Biology in the Podocyte – Cellular Cholesterol-Mediated Glomerular Injury

    PubMed Central

    Merscher, Sandra; Pedigo, Christopher E.; Mendez, Armando J.

    2014-01-01

    Chronic kidney disease (CKD) is associated with a high risk of death. Dyslipidemia is commonly observed in patients with CKD and is accompanied by a decrease in plasma high-density lipoprotein, and an increase in plasma triglyceride-rich lipoproteins and oxidized lipids. The observation that statins may decrease albuminuria but do not stop the progression of CKD indicates that pathways other than the cholesterol synthesis contribute to cholesterol accumulation in the kidneys of patients with CKD. Recently, it has become clear that increased lipid influx and impaired reverse cholesterol transport can promote glomerulosclerosis, and tubulointerstitial damage. Lipid-rafts are cholesterol-rich membrane domains with important functions in regulating membrane fluidity, membrane protein trafficking, and in the assembly of signaling molecules. In podocytes, which are specialized cells of the glomerulus, they contribute to the spatial organization of the slit diaphragm (SD) under physiological and pathological conditions. The discovery that podocyte-specific proteins such as podocin can bind and recruit cholesterol contributing to the formation of the SD underlines the importance of cholesterol homeostasis in podocytes and suggests cholesterol as an important regulator in the development of proteinuric kidney disease. Cellular cholesterol accumulation due to increased synthesis, influx, or decreased efflux is an emerging concept in podocyte biology. This review will focus on the role of cellular cholesterol accumulation in the pathogenesis of kidney diseases with a focus on glomerular diseases. PMID:25352833

  14. Targeting reduction of proteinuria in glomerulonephritis: Maximizing the antifibrotic effect of valsartan by protecting podocytes.

    PubMed

    Gu, Chunyan; Zhou, Guangyu; Noble, Nancy A; Border, Wayne A; Cheung, Alfred K; Huang, Yufeng

    2014-06-01

    Although angiotensin (Ang) II blockade has become a standard antifibrotic therapy in kidney disease, the therapeutic efficacy of Ang II blockade is yet to be optimized. Considering the prognostic impact of proteinuria reduction, we hypothesized that titration of Ang II blockade for optimal anti-proteinuric effect would improve renoprotection. One day after induction of Thy 1.1 glomeruonephritis, rats were treated with increasing doses of the Ang II receptor blocker valsartan in drinking water. Six days after disease induction, the therapeutic effect on proteinuria, podocyte injury and glomerular fibrosis was evaluated. Increasing doses of valsartan resulted in increasing reduction of proteinuria. The maximally effective dose of valsartan was determined to be 1000 mg/l, which reduced proteinuria by 80% and maximally reduced glomerular matrix expansion, fibronectin, collagen I and collagen III staining and glomerular mRNAs for TGFß1, PAI-1, FN and collagen I. Notably, valsartan given at this dose prevented podocyte dysfunction by preserving expression of podocin and nephrin and the counter-regulating molecule B7-1 that is involved in podocyte injury. These results support the hypothesis that higher doses of valsartan are required to optimize proteinuria reduction and glomerulosclerosis amelioration. Further, the optimal dose of valsartan also provides an additional therapeutic effect by preventing podocyte dysfunction. PMID:23223090

  15. Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes

    PubMed Central

    Chittiprol, Seetharamaiah; Chen, Phylip; Petrovic-Djergovic, Danica; Eichler, Tad

    2011-01-01

    The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR >> KE, and of doxorubicin was JR ∼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines. PMID:21632959

  16. Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy

    PubMed Central

    Ichimura, Koichiro; Miyazaki, Naoyuki; Sadayama, Shoji; Murata, Kazuyoshi; Koike, Masato; Nakamura, Kei-ichiro; Ohta, Keisuke; Sakai, Tatsuo

    2015-01-01

    Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and foot process, which are simply aligned in this order. When the reconstructed podocytes were viewed from their basal side, the foot processes were branched from a ridge-like prominence, which was formed on the basal surface of the primary process and was similar to the usual foot processes in structure. Moreover, from the cell body, the foot processes were also emerged via the ridge-like prominence, as found in the primary process. The ridge-like prominence anchored the cell body and primary process to the glomerular basement membrane, and connected the foot processes to the cell body and primary process. In conclusion, serial block-face imaging is a powerful tool for clear understanding the three-dimensional architecture of podocytes through its ability to reveal novel structures which were difficult to determine by conventional transmission and scanning electron microscopes alone. PMID:25759085

  17. Balancing Calcium Signals through TRPC5 and TRPC6 in Podocytes

    PubMed Central

    2011-01-01

    Calcium (Ca2+) ions are important mediators of cellular homeostasis owing to their ability to elicit a dynamic, transient, and tightly regulated range of biochemical responses. More than a decade ago, a nonselective, Ca2+-permeable, cationic conductance was identified in podocytes downstream of angiotensin II (Ang II) signaling, but its molecular structure remained elusive. Six years ago, transient receptor potential canonical 6 (TRPC6) mutations were found in families with hereditary FSGS, and TRPC5 and TRPC6 channels are now known as the Ca2+ influx pathways for this previously described, nonselective, cationic current in podocytes. Ang II activation engages this Ca2+ influx to modulate the actin cytoskeleton in podocytes. These discoveries dovetail with previously described regulation of actin dynamics by the Ca2+-activated phosphatase, calcineurin, and the emergence of Rho GTPases as critical regulators of podocyte function in health and disease. Understanding the interconnected signaling regulated by Ca2+ currents offers potential new therapeutic targets and highlights the notion that synergistic therapies targeting multiple levels of biochemistry may be useful in treating proteinuric kidney disease. PMID:21980113

  18. Nerve Growth Factor Mediates a Switch in Intracellular Signaling for PGE2-Induced Sensitization of Sensory Neurons from Protein Kinase A to Epac

    PubMed Central

    Vasko, Michael R.; Habashy Malty, Ramy; Guo, Chunlu; Duarte, Djane B.; Zhang, Yihong; Nicol, Grant D.

    2014-01-01

    We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund's adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation. PMID:25126967

  19. Knockdown of GnT-Va expression inhibits ligand-induced