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Sample records for cd-induced proximal tubule

  1. Expression of kidney injury molecule-1 (Kim-1) in relation to necrosis and apoptosis during the early stages of Cd-induced proximal tubule injury

    PubMed Central

    Prozialeck, Walter C.; Edwards, Joshua R.; Lamar, Peter C.; Liu, Jie; Vaidya, Vishal S.; Bonventre, Joseph V.

    2009-01-01

    Cadmium (Cd) is a nephrotoxic industrial and environmental pollutant that causes a generalized dysfunction of the proximal tubule. Kim-1 is a transmembrane glycoprotein that is normally not detectable in non-injured kidney, but is up-regulated and shed into the urine during the early stages of Cd-induced proximal tubule injury. The objective of the present study was to examine the relationship between the Cd-induced increase in Kim-1 expression and the onset of necrotic and apoptotic cell death in the proximal tubule. Adult male Sprague-Dawley rats were treated with 0.6 mg (5.36 μmoles) Cd/kg, subcutaneously, 5 days per week for up to 12 weeks. Urine samples were analyzed for levels of Kim-1 and the enzymatic markers of cell death, lactate dehydrogenase (LDH) and alpha-glutathione-S-transferase (α-GST). In addition, necrotic cells were specifically labeled by perfusing the kidneys in situ with ethidium homodimer using a procedure that has been recently developed and validated in the Prozialeck laboratory. Cryosections of the kidneys were also processed for the immunofluorescent visualization of Kim-1 and the identification of apoptotic cells by TUNEL labeling. Results showed that significant levels of Kim-1 began to appear in the urine after 6 weeks of Cd treatment, whereas the levels of total protein, α-GST and LDH were not increased until 8–12 weeks. Results of immunofluorescence labeling studies showed that after 6 weeks and 12 weeks, Kim-1 was expressed in the epithelial cells of the proximal tubule, but that there was no increase in the number of necrotic cells, and only a modest increase in the number of apoptotic cells at 12 weeks. These results indicate that the Cd-induced increase in Kim-1 expression occurs before the onset of necrosis and at a point where there is only a modest level of apoptosis in the proximal tubule. PMID:19371613

  2. Expression of kidney injury molecule-1 (Kim-1) in relation to necrosis and apoptosis during the early stages of Cd-induced proximal tubule injury

    SciTech Connect

    Prozialeck, Walter C. Edwards, Joshua R.; Lamar, Peter C.; Liu, Jie; Vaidya, Vishal S.; Bonventre, Joseph V.

    2009-08-01

    Cadmium (Cd) is a nephrotoxic industrial and environmental pollutant that causes a generalized dysfunction of the proximal tubule. Kim-1 is a transmembrane glycoprotein that is normally not detectable in non-injured kidney, but is up-regulated and shed into the urine during the early stages of Cd-induced proximal tubule injury. The objective of the present study was to examine the relationship between the Cd-induced increase in Kim-1 expression and the onset of necrotic and apoptotic cell death in the proximal tubule. Adult male Sprague-Dawley rats were treated with 0.6 mg (5.36 {mu}mol) Cd/kg, subcutaneously, 5 days per week for up to 12 weeks. Urine samples were analyzed for levels of Kim-1 and the enzymatic markers of cell death, lactate dehydrogenase (LDH) and alpha-glutathione-S-transferase ({alpha}-GST). In addition, necrotic cells were specifically labeled by perfusing the kidneys in situ with ethidium homodimer using a procedure that has been recently developed and validated in the Prozialeck laboratory. Cryosections of the kidneys were also processed for the immunofluorescent visualization of Kim-1 and the identification of apoptotic cells by TUNEL labeling. Results showed that significant levels of Kim-1 began to appear in the urine after 6 weeks of Cd treatment, whereas the levels of total protein, {alpha}-GST and LDH were not increased until 8-12 weeks. Results of immunofluorescence labeling studies showed that after 6 weeks and 12 weeks, Kim-1 was expressed in the epithelial cells of the proximal tubule, but that there was no increase in the number of necrotic cells, and only a modest increase in the number of apoptotic cells at 12 weeks. These results indicate that the Cd-induced increase in Kim-1 expression occurs before the onset of necrosis and at a point where there is only a modest level of apoptosis in the proximal tubule.

  3. Role of proximal tubules in the pathogenesis of kidney disease.

    PubMed

    Nakhoul, Nazih; Batuman, Vecihi

    2011-01-01

    The proximal tubules make up a significant portion of the kidneys; proximal tubule epithelial cells are the most populous cell type in the kidney, and carry out diverse regulatory and endocrine functions where numerous transporters are located. Under normal circumstances, more than two thirds of filtered salt and water, and all filtered bicarbonate is reabsorbed in the proximal tubule. A number of inherited and acquired acid-base and tubule disorders are linked to impaired transporters in the proximal tubule cells. Equally important is the intrinsic immune characteristics of proximal tubule cells that give them the ability to also function as immune responders to a wide range of immunologic, ischemic or toxic injury. It is therefore not surprising that proximal tubule-related phenomena are closely related to the pathogenesis of a vast array of kidney diseases. Many kidney diseases, acute and chronic, first manifest with proximal tubule disorders. Recent insight into molecular characteristics of transport functions in the proximal tubules, and the recognition that proximal tubule cells possess intrinsic immune responses have contributed to an improved understanding of important areas in nephrology, such as Fanconi's syndrome, renal tubular acidosis, phosphate wasting syndromes, Dent's disease, cystinuria and other amino acid transport disorders, acute kidney injury, and the role of proximal tubules in progressive kidney disease. Megalin/ cubilin-mediated endocytosis by proximal tubule cells of increased quantities of filtered proteins (protein overloading) in glomerular diseases appears to evoke cell stress responses resulting in increased inflammatory cytokines leading to tubulointerstitial inflammation and fibrosis. Finally, the proximal tubule may be the site of both active vitamin D synthesis through the action of 1-α-hydroxylase, and the site where erythropoietin synthesis takes place. Thus, proximal tubule injury also contributes to two distressing

  4. Isosmotic volume reabsorption in rat proximal tubule

    PubMed Central

    1980-01-01

    A theoretical model incorporation both active and passive forces has been developed for fluid reabsorption from split oil droplets in rat intermediate and late proximal tubule. Of necessity, simplifying assumptions have been introduced; we have assumed that the epithelium can be treated as a single membrane and that the membrane "effective" HCO3 permeability is near zero. Based on this model with its underlying assumptions, the following conclusions are drawn. Regardless of the presence or absence of active NaCl transport, fluid reabsorption from the split oil droplet is isosmotic. The reabsorbate osmolarity can be affected by changes in tubular permeability parameters and applied forces but is not readily altered from an osmolarity essentially equal to that of plasma. In a split droplet, isosmotic flow need not be a special consequence of active Na transport, is not the result of a particular set of permeability properties, and is not merely a trivial consequence of a very high hydraulic conductivity; isosmotic flow can be obtained with hydraulic conductivity nearly an order of magnitude lower than that previously measured in the rat proximal convoluted tubule. Isosmotic reabsorption is, in part, the result of the interdependence of salt and water flows, their changing in parallel, and thus their ratio, the reabsorbate concentration being relatively invariant. Active NaCl transport can cause osmotic water flow by reducing the luminal fluid osmolarity. In the presence of passive forces the luminal fluid can be hypertonic to plasma, and active NaCl transport can still exert its osmotic effect on volume flow. There are two passive forces for volume flow: the Cl gradient and the difference in effective osmotic pressure; they have an approximately equivalent effect on volume flow. Experimentally, we have measured volume changes in a droplet made hyperosmotic by the addition of 50 mM NaCl; the experimental results are predicted reasonably well by our theoretical model

  5. Isosmotic volume reabsorption in rat proximal tubule.

    PubMed

    Warner, R R; Lechene, C

    1980-11-01

    A theoretical model incorporation both active and passive forces has been developed for fluid reabsorption from split oil droplets in rat intermediate and late proximal tubule. Of necessity, simplifying assumptions have been introduced; we have assumed that the epithelium can be treated as a single membrane and that the membrane "effective" HCO3 permeability is near zero. Based on this model with its underlying assumptions, the following conclusions are drawn. Regardless of the presence or absence of active NaCl transport, fluid reabsorption from the split oil droplet is isosmotic. The reabsorbate osmolarity can be affected by changes in tubular permeability parameters and applied forces but is not readily altered from an osmolarity essentially equal to that of plasma. In a split droplet, isosmotic flow need not be a special consequence of active Na transport, is not the result of a particular set of permeability properties, and is not merely a trivial consequence of a very high hydraulic conductivity; isosmotic flow can be obtained with hydraulic conductivity nearly an order of magnitude lower than that previously measured in the rat proximal convoluted tubule. Isosmotic reabsorption is, in part, the result of the interdependence of salt and water flows, their changing in parallel, and thus their ratio, the reabsorbate concentration being relatively invariant. Active NaCl transport can cause osmotic water flow by reducing the luminal fluid osmolarity. In the presence of passive forces the luminal fluid can be hypertonic to plasma, and active NaCl transport can still exert its osmotic effect on volume flow. There are two passive forces for volume flow: the Cl gradient and the difference in effective osmotic pressure; they have an approximately equivalent effect on volume flow. Experimentally, we have measured volume changes in a droplet made hyperosmotic by the addition of 50 mM NaCl; the experimental results are predicted reasonably well by our theoretical model.

  6. The Proximal Tubule and Albuminuria: Really!

    PubMed Central

    Dickson, Landon E.; Wagner, Mark C.; Sandoval, Ruben M.

    2014-01-01

    Recent data highlight the role of the proximal tubule (PT) in reabsorbing, processing, and transcytosing urinary albumin from the glomerular filtrate. Innovative techniques and approaches have provided exciting insights into these processes, and numerous investigators have shown that selective PT cell defects lead to significant albuminuria, even reaching nephrotic range in animal models. Thus, the mechanisms of albumin reabsorption and transcytosis are undergoing intense study. Working in concert with megalin and cubilin, a nonselective multireceptor complex that predominantly directs proteins for lysosomal degradation, the neonatal Fc receptor (FcRn) located at the brush border of the apical membrane has been implicated as the “receptor” mediating albumin transcytosis. The FcRn pathway facilitates reabsorption and mediates transcytosis by its pH-dependent binding affinity in endosomal compartments. This also allows for selective albumin sorting within the PT cell. This reclamation pathway minimizes urinary losses and catabolism of albumin, thus prolonging its serum half-life. It may also serve as a molecular sorter to preserve and reclaim normal albumin while allowing “altered” albumin to be catabolized via lysosomal pathways. Here, we critically review the data supporting this novel mechanism. PMID:24408874

  7. Developmental Changes in Proximal Tubule Tight Junction Proteins

    PubMed Central

    HADDAD, MAHA; LIN, FANGMING; DWARAKANATH, VANGIPURAM; CORDES, KIMBERLY; BAUM, MICHEL

    2014-01-01

    We demonstrated previously that neonatal proximal tubules have a lower passive paracellular permeability to chloride ions and higher resistance than that of adult proximal tubules. In addition, administration of thyroid hormone to neonates, before the normal maturational increase in serum thyroid hormone levels, prematurely accelerates the developmental increase in chloride permeability to adult levels. To test the hypothesis that there is a maturational change in tight junction proteins and that thyroid hormone mediates these changes, we examined the two known tight junction proteins present in proximal tubules, occludin and claudin 2. Using immunoblot and immunohistochemistry, we demonstrated that claudin 2 has a 4-fold greater abundance in neonatal proximal tubules than in adult tubules. Occludin, however, has a 4-fold greater expression in adult tubules than in neonatal tubules. Administration of thyroid hormone to neonates did not affect claudin 2 expression, occludin expression, or the transepithelial resistance in rat proximal tubule cells in vitro. In conclusion, there are postnatal maturational changes in tight junction proteins. The factors that cause these maturational changes are unknown but unlikely to be due solely to the maturational increase in thyroid hormone. PMID:15585672

  8. Regulation of glomerulotubular balance: flow-activated proximal tubule function.

    PubMed

    Wang, Tong; Weinbaum, Sheldon; Weinstein, Alan M

    2017-03-07

    The purpose of this review is to summarize our knowledge and understanding of the physiological importance and the mechanisms underlying flow-activated proximal tubule transport. Since the earliest micropuncture studies of mammalian proximal tubule, it has been recognized that tubular flow is an important regulator of sodium, potassium, and acid-base transport in the kidney. Increased fluid flow stimulates Na(+) and HCO3(-) absorption in the proximal tubule via stimulation of Na/H-exchanger isoform 3 (NHE3) and H(+)-ATPase. In the proximal tubule, brush border microvilli are the major flow sensors, which experience changes in hydrodynamic drag and bending moment as luminal flow velocity changes and which transmit the force of altered flow to cytoskeletal structures within the cell. The signal to NHE3 depends upon the integrity of the actin cytoskeleton; the signal to the H(+)-ATPase depends upon microtubules. We have demonstrated that alterations in fluid drag impact tubule function by modulating ion transporter availability within the brush border membrane of the proximal tubule. Beyond that, there is evidence that transporter activity within the peritubular membrane is also modulated by luminal flow. Secondary messengers that regulate the flow-mediated tubule function have also been delineated. Dopamine blunts the responsiveness of proximal tubule transporters to changes in luminal flow velocity, while a DA1 antagonist increases flow sensitivity of solute reabsorption. IP3 receptor-mediated intracellular Ca(2+) signaling is critical to transduction of microvillus drag. In this review, we summarize our findings of the regulatory mechanism of flow-mediated Na(+) and HCO3(-) transport in the proximal tubule and review available information about flow sensing and regulatory mechanism of glomerulotubular balance.

  9. Studies of the electrical potential difference in rat proximal tubule.

    PubMed

    Seely, J F; Chirito, E

    1975-07-01

    The electrical potential difference (PD) in the rat proximal convoluted tubule was investigated in vivo as a function of distance from the glomerulus. The PD was found to be invariably negative (up to -4.5 mV) in the earliest segments (less than 0.5 mm from the glomerulus) and rose to positive values (+2 to +4) in the later segments (1 mm beyond the glomerulus). This change in PD correlated with the bubule fluid-to-plasma (TF/P) chloride ratios, which rose from unity in the early segments to approximately 1.3 in the late. Corresponding changes in PD and chloride ratios could be elicited by single-nephron stop-flow techniques in the early segments. Luminal perfusion techniques demonstrated a direct relationship between PD and tubule fluid chloride concentration. Acetazolamide was found to significantly reduce both late proximal PD (less than +2 mV) and TF/P chloride ratios (less than 1.06). Split-drop studies demonstrated that the negative PD in the early proximal tubule was dependent on the presence of glucose and alanine and the absence of a chloride gradient, whereas in the late proximal tubule under the same conditions the PD was not significantly different from zero. In this segment of the nephron the positive PD in free flow appeared to result from the chloride diffusion potential generated by preferential HCO3 reabsorption. These results provide further demonstration of intrinsic differences in the transport properties along the length of the proximal convoluted tubule.

  10. CFTR mediated chloride secretion in the avian renal proximal tubule.

    PubMed

    Laverty, Gary; Anttila, Ashley; Carty, Jenava; Reddy, Varudhini; Yum, Jamie; Arnason, Sighvatur S

    2012-01-01

    In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl(-)-dependent short circuit current (I(SC)) response, consistent with net transepithelial Cl(-) secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl(-) secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I(SC) responses in filter grown cultured monolayers. Bumetanide (10 μM), a specific blocker of NKCC, inhibited forskolin activated I(SC) by about 40%, suggesting that basolateral uptake of Cl(-) is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl(-) conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl(-) gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl(-) current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 μM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl(-) channel to mediate cAMP-activated Cl(-) secretion.

  11. Calcium transport in the rabbit superficial proximal convoluted tubule

    SciTech Connect

    Ng, R.C.; Rouse, D.; Suki, W.N.

    1984-09-01

    Calcium transport was studied in isolated S2 segments of rabbit superficial proximal convoluted tubules. 45Ca was added to the perfusate for measurement of lumen-to-bath flux (JlbCa), to the bath for bath-to-lumen flux (JblCa), and to both perfusate and bath for net flux (JnetCa). In these studies, the perfusate consisted of an equilibrium solution that was designed to minimize water flux or electrochemical potential differences (PD). Under these conditions, JlbCa (9.1 +/- 1.0 peq/mm X min) was not different from JblCa (7.3 +/- 1.3 peq/mm X min), and JnetCa was not different from zero, which suggests that calcium transport in the superficial proximal convoluted tubule is due primarily to passive transport. The efflux coefficient was 9.5 +/- 1.2 X 10(-5) cm/s, which was not significantly different from the influx coefficient, 7.0 +/- 1.3 X 10(-5) cm/s. When the PD was made positive or negative with use of different perfusates, net calcium absorption or secretion was demonstrated, respectively, which supports a major role for passive transport. These results indicate that in the superficial proximal convoluted tubule of the rabbit, passive driving forces are the major determinants of calcium transport.

  12. SGLT2 mediates glucose reabsorption in the early proximal tubule.

    PubMed

    Vallon, Volker; Platt, Kenneth A; Cunard, Robyn; Schroth, Jana; Whaley, Jean; Thomson, Scott C; Koepsell, Hermann; Rieg, Timo

    2011-01-01

    Mutations in the gene encoding for the Na(+)-glucose co-transporter SGLT2 (SLC5A2) associate with familial renal glucosuria, but the role of SGLT2 in the kidney is incompletely understood. Here, we determined the localization of SGLT2 in the mouse kidney and generated and characterized SGLT2-deficient mice. In wild-type (WT) mice, immunohistochemistry localized SGLT2 to the brush border membrane of the early proximal tubule. Sglt2(-/-) mice had glucosuria, polyuria, and increased food and fluid intake without differences in plasma glucose concentrations, GFR, or urinary excretion of other proximal tubular substrates (including amino acids) compared with WT mice. SGLT2 deficiency did not associate with volume depletion, suggested by similar body weight, BP, and hematocrit; however, plasma renin concentrations were modestly higher and plasma aldosterone levels were lower in Sglt2(-/-) mice. Whole-kidney clearance studies showed that fractional glucose reabsorption was significantly lower in Sglt2(-/-) mice compared with WT mice and varied in Sglt2(-/-) mice between 10 and 60%, inversely with the amount of filtered glucose. Free-flow micropuncture revealed that for early proximal collections, 78 ± 6% of the filtered glucose was reabsorbed in WT mice compared with no reabsorption in Sglt2(-/-) mice. For late proximal collections, fractional glucose reabsorption was 93 ± 1% in WT and 21 ± 6% in Sglt2(-/-) mice, respectively. These results demonstrate that SGLT2 mediates glucose reabsorption in the early proximal tubule and most of the glucose reabsorption by the kidney, overall. This mouse model mimics and explains the glucosuric phenotype of individuals carrying SLC5A2 mutations.

  13. Acid-base transport by the renal proximal tubule

    PubMed Central

    Skelton, Lara A.; Boron, Walter F.; Zhou, Yuehan

    2015-01-01

    Each day, the kidneys filter 180 L of blood plasma, equating to some 4,300 mmol of the major blood buffer, bicarbonate (HCO3−). The glomerular filtrate enters the lumen of the proximal tubule (PT), and the majority of filtered HCO3− is reclaimed along the early (S1) and convoluted (S2) portions of the PT in a manner coupled to the secretion of H+ into the lumen. The PT also uses the secreted H+ to titrate non-HCO3− buffers in the lumen, in the process creating “new HCO3−” for transport into the blood. Thus, the PT – along with more distal renal segments – is largely responsible for regulating plasma [HCO3−]. In this review we first focus on the milestone discoveries over the past 50+ years that define the mechanism and regulation of acid-base transport by the proximal tubule. Further on in the review, we will summarize research still in progress from our laboratory, work that addresses the problem of how the PT is able to finely adapt to acid–base disturbances by rapidly sensing changes in basolateral levels of HCO3− and CO2 (but not pH), and thereby to exert tight control over the acid–base composition of the blood plasma. PMID:21170887

  14. ADAM17 substrate release in proximal tubule drives kidney fibrosis

    PubMed Central

    Kefaloyianni, Eirini; Muthu, Muthu Lakshmi; Kaeppler, Jakob; Sun, Xiaoming; Sabbisetti, Venkata; Chalaris, Athena; Rose-John, Stefan; Wong, Eitan; Sagi, Irit; Waikar, Sushrut S.; Rennke, Helmut; Bonventre, Joseph V.

    2016-01-01

    Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates’ receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor–treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis. PMID:27642633

  15. Analysis of standing droplets in rat proximal tubules

    PubMed Central

    1982-01-01

    Volume, osmolality, and concentrations for Na, Cl, and raffinose have been measured as a function of time in standing droplets within rat intermediate and late proximal tubules. Standing droplet reabsorption proceeds without the development of a measurable osmotic difference across the epithelium. After 140 s of tubular exposure, droplet-to- plasma concentration differences are observed for raffinose, Na, and Cl with the observed Na concentration difference, usually referred to as limiting gradient, being approximately 9 mM. It is possible that a smaller or even no limiting difference would be attained with longer exposure times. Previous values measured for the limiting Na concentration in the rat proximal tubule were determined before the attainment of constant concentrations. Assuming that the Na concentration we measured is the limiting value, we estimate that active NaCl transport accounts for a very small fraction, less than 6%, of the volume reabsorption; using an alternative approach of fitting a theoretical model to our experimental data, active NaCl transport is again estimated to account for only 6% of the total reabsorbate. The previous interpretation that a limiting Na concentration gradient constitutes the most direct evidence for active Na transport may be in error; the gradient we measure can be modeled without incorporating active NaCl transport. PMID:7069399

  16. Norepinephrines effect on adenosine transport in the proximal straight tubule

    SciTech Connect

    Barfuss, D.W.; McCann, W.P.; Katholi, R.E.

    1986-03-01

    The effect of norepinephrine on C/sup 14/-adenosine transport in the rabbit proximal tubule (S/sub 2/) was studied. The transepithelial transport of adenosine (0.02 mM0 from lumin to bathing solution was measured by its rate of appearance (J/sub A/) in the bathing solution and by its disappearances (J/sub D/) from the luminal fluid. Norepinephrine (0.24 ..mu..M) was added to the bathing solution after a control flux period. After three samples from the experiment period the tubules were quickly harvested and the cellular concentration of C/sup 14/-adenosine was determined. The high cellular adenosine concentration and th marked difference in adenosine appearance rate in the bathing solution compared to the luminal disappearance rate indicates the absorbed adenosine is trapped in the cells. This trapping may be due to adenosine metabolism or difficulty of crossing the basolateral membrane. Whichever is the case, norepinephrine appears to stimulate movement of adenosine or its metabolites into the bathing solution across the basolateral membrane.

  17. Cell volume regulation in the proximal convoluted tubule.

    PubMed

    Gagnon, J; Ouimet, D; Nguyen, H; Laprade, R; Le Grimellec, C; Carrière, S; Cardinal, J

    1982-10-01

    To evaluate the effect of hyper- and hypotonicity on proximal convoluted tubule (PCT) cell volume, nonperfused PCT were studied in vitro with hypertonic solutions containing sodium chloride, urea, or mannitol (450 mosmol/kg H2O) and with hypotonic low sodium chloride solutions (160 mosmol/kg H2O). When the tubules were subjected to hypertonic peritubular solutions containing NaCl, cell volume immediately decreased by 15.5% and remained constant throughout the experimental period (60 min). With mannitol, the initial decrease was identical to that with NaCl (17.7%), but the PCT volume increased slightly during the experimental period. With urea, the decrease in cell volume was smaller (7%) and transient. In hypotonicity, the PCT swelled rapidly, but this swelling was followed by a rapid regulatory phase in which PCT volume nearly returned to control values after less than 10 min. With a potassium-free peritubular medium or 10(-3) M ouabain, the regulatory phase of hypotonicity completely disappeared, whereas the cells did not maintain their reduced volume in NaCl-induced hypertonicity. These results suggest that Na-K-ATPase plays an important role in the maintenance of a reduced cellular volume in hypertonicity and in the regulatory phase of hypotonicity, probably by an active extrusion of sodium and water from the cell.

  18. Discerning the role of mechanosensors in regulating proximal tubule function.

    PubMed

    Raghavan, Venkatesan; Weisz, Ora A

    2016-01-01

    All cells in the body experience external mechanical forces such as shear stress and stretch. These forces are sensed by specialized structures in the cell known as mechanosensors. Cells lining the proximal tubule (PT) of the kidney are continuously exposed to variations in flow rates of the glomerular ultrafiltrate, which manifest as changes in axial shear stress and radial stretch. Studies suggest that these cells respond acutely to variations in flow by modulating their ion transport and endocytic functions to maintain glomerulotubular balance. Conceptually, changes in the axial shear stress in the PT could be sensed by three known structures, namely, the microvilli, the glycocalyx, and primary cilia. The orthogonal component of the force produced by flow exhibits as radial stretch and can cause expansion of the tubule. Forces of stretch are transduced by integrins, by stretch-activated channels, and by cell-cell contacts. This review summarizes our current understanding of flow sensing in PT epithelia, discusses challenges in dissecting the role of individual flow sensors in the mechanosensitive responses, and identifies potential areas of opportunity for new study.

  19. Isovolumetric regulation of renal proximal tubules in hypotonic medium.

    PubMed

    Lohr, J W

    1990-01-01

    Isolated nonperfused proximal tubules maintained their cell volume at a constant level (isovolumetric regulation, IVR), when osmolality of the bathing medium was gradually decreased from 290 to 190 mosm at 1.5 and 5.0 mosm/min. Hypotonic IVR was blocked by inhibiting the Na(+)-K+ pump with ouabain (10(-4) M) when osmolality was decreased at 1.5 or 5 mosm/min. Concentration-dependent inhibition of cell volume maintenance was observed in the presence of the K+ channel blocker barium (10(-3)-10(-2) M) when osmolality decreased at 5 mosm/min. Quinine (10(-3) M), another K+ channel blocker, also inhibited IVR at osmolality decreases of 1.5 and 5 mosm/min. These results suggest that the maintenance of constant cell volume during gradual hypoosmotic exposure involves mechanisms that depend on intact Na-K-ATPase and the controlled loss of intracellular K+.

  20. Proximal Tubule Cell Hypothesis for Cardiorenal Syndrome in Diabetes

    PubMed Central

    Saito, Akihiko; Kaseda, Ryohei; Hosojima, Michihiro; Sato, Hiroyoshi

    2011-01-01

    Incidence of cardiovascular disease (CVD) is remarkably high among patients with chronic kidney disease (CKD), even in the early microalbuminuric stages with normal glomerular filtration rates. Proximal tubule cells (PTCs) mediate metabolism and urinary excretion of vasculotoxic substances via apical and basolateral receptors and transporters. These cells also retrieve vasculoprotective substances from circulation or synthesize them for release into the circulation. PTCs are also involved in the uptake of sodium and phosphate, which are critical for hemodynamic regulation and maintaining the mineral balance, respectively. Dysregulation of PTC functions in CKD is likely to be associated with the development of CVD and is linked to the progression to end-stage renal disease. In particular, PTC dysfunction occurs early in diabetic nephropathy, a leading cause of CKD. It is therefore important to elucidate the mechanisms of PTC dysfunction to develop therapeutic strategies for treating cardiorenal syndrome in diabetes. PMID:21197105

  1. Conductances, diffusion and streaming potentials in the rat proximal tubule.

    PubMed Central

    De Mello, G B; Lopes, A G; Malnic, G

    1976-01-01

    1. Transtubular potential differences and specific resistances were measured in rat proximal tubules by means of single and double barrelled glass micro-electrodes. 2. Tip localization was made by observation of effective resistance changes measured with double barrelled micro-electrodes upon passage of oil droplets, and by perfusion with choline C1. 3. Mean early proximal p.d.s. of the order of -1 to -2 mV, and late values of +0-5 to +1mV were found. Mean specific resistances ranged from 12 to 15 omega cm2. 4. Diffusion potentials and single ion relative conductances were evaluated, perfusing the lumen with solutions differing only with respect to one salt concentration. Na and K conductances were similar and greater than those of C1. 5. Luminal and peritubular perfusions with hypotonic solutions showed the occurrence of streaming potentials in this structure suggesting the existence of pores lined with negative charges. The effective diameter of these pores appeared to be reduced by hypotonic perfusion, as evidenced by a significant increase in resistance, indicating that the main ion path across this structure is represented by intercellular spaces. PMID:10433

  2. Visualization of Calcium Dynamics in Kidney Proximal Tubules

    PubMed Central

    Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Csohány, Rózsa; Prókai, Ágnes; Kis-Petik, Katalin; Szabó, Attila; Bősze, Zsuzsanna; Bender, Balázs; Tóvári, József; Enyedi, Ágnes; Orbán, Tamás I.

    2015-01-01

    Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin–based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations. PMID:25788535

  3. Autoradiographic Localization of [3H]Gentamicin in the Proximal Renal Tubules of Mice

    PubMed Central

    Kuhar, Michael J.; Mak, Linda L.; Lietman, Paul S.

    1979-01-01

    The site of localization of [3H]gentamicin within mouse kidney is shown to be the proximal renal tubule by coincidence of the radioactivity, as visualized by autoradiography, and the mucopolysaccharide-rich microvilli characteristic of proximal convoluted tubules, as visualized by histochemical staining. Images PMID:426500

  4. Proximal Tubules Have the Capacity to Regulate Uptake of Albumin.

    PubMed

    Wagner, Mark C; Campos-Bilderback, Silvia B; Chowdhury, Mahboob; Flores, Brittany; Lai, Xianyin; Myslinski, Jered; Pandit, Sweekar; Sandoval, Ruben M; Wean, Sarah E; Wei, Yuan; Satlin, Lisa M; Wiggins, Roger C; Witzmann, Frank A; Molitoris, Bruce A

    2016-02-01

    Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level.

  5. Repair of injured proximal tubule does not involve specialized progenitors

    PubMed Central

    Humphreys, Benjamin D.; Czerniak, Suzanne; DiRocco, Derek P.; Hasnain, Wirasat; Cheema, Rabia; Bonventre, Joseph V.

    2011-01-01

    Recently we have established that the kidney tubular epithelium is repaired by surviving epithelial cells. It is not known, however, whether a population of intratubular adult progenitor cells are responsible for this epithelial repair after acute kidney injury. In this study, we used an unbiased DNA analog-based approach that does not rely on candidate markers to track multiple rounds of cell division in vivo. In the proximal tubule, robust thymidine analog incorporation was observed postinjury. Cell division was stochastic and enriched among cells that were injured and dedifferentiated. There was no evidence for the presence of a population of specialized progenitors that repeatedly divide in response to injury. Instead, these results indicate that after injury, new epithelial cells arise from self-duplication of surviving cells, most of which are injured. Because the renal papilla contains DNA label-retaining cells and has been proposed as a stem cell niche, we examined the proliferative behavior of these putative progenitors after ischemia-reperfusion injury. Although label-retaining cells in the renal papilla diminished with time after ischemia-reperfusion injury, they neither proliferated nor migrated to the outer medulla or cortex. Thus, nonlethally injured cells repopulate the kidney epithelium after injury in the absence of any specialized progenitor cell population. PMID:21576461

  6. Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule.

    PubMed

    Christensen, E I; Birn, H

    2001-04-01

    The multiligand, endocytic receptors megalin and cubilin are colocalized in the renal proximal tubule. They are heavily expressed in the apical endocytic apparatus. Megalin is a 600-kDa transmembrane protein belonging to the low-density lipoprotein-receptor family. The cytoplasmic tail contains three NPXY motifs that mediate the clustering in coated pits and are possibly involved in signaling functions. Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is a 460-kDa receptor with no transmembrane domain and no known signal for endocytosis. Because the two receptors bind each other with high affinity and colocalize in several tissues, it is highly conceivable that megalin mediates internalization of cubilin and its ligands. Both receptors are important for normal tubular reabsorption of proteins, including albumin. Among the proteins normally filtered in the glomeruli, cubilin has been shown to bind albumin, immunoglobulin light chains, and apolipoprotein A-I. The variety of filtered ligands identified for megalin include vitamin-binding proteins, hormones, enzymes, apolipoprotein H, albumin, and beta(2)- and alpha(1)-microglobulin. Loss of these proteins and vitamins in the urine of megalin-deficient mice illustrates the physiological importance of this receptor.

  7. Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule

    PubMed Central

    Devuyst, Olivier; Luciani, Alessandro

    2015-01-01

    Abstract The epithelial cells lining the proximal tubules of the kidney reabsorb a large amount of filtered ions and solutes owing to receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Dedifferentiation of proximal tubule cells and dysfunction of receptor-mediated endocytosis characterize Dent’s disease, a rare disorder caused by inactivating mutations in the CLCN5 gene that encodes the endosomal chloride–proton exchanger, ClC-5. The disease is characterized by a massive urinary loss of solutes (renal Fanconi syndrome), with severe metabolic complications and progressive renal failure. Investigations of mutations affecting the gating of ClC-5 revealed that the proximal tubule dysfunction may occur despite normal endosomal acidification. In addition to defective endocytosis, proximal tubule cells lacking ClC-5 show a trafficking defect in apical receptors and transporters, as well as lysosomal dysfunction and typical features of dedifferentiation, proliferation and oxidative stress. A similar but milder defect is observed in mouse models with defective CFTR, a chloride channel that is also expressed in the endosomes of proximal tubule cells. These data suggest a major role for endosomal chloride transport in the maintenance of epithelial differentiation and reabsorption capacity of the renal proximal tubule. Key points The reabsorptive activity of renal proximal tubule cells is mediated by receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Loss-of-function mutations of the endosomal chloride–proton exchanger ClC-5 (Dent’s disease) cause a major trafficking defect in proximal tubule cells, associated with lysosomal dysfunction, oxidative stress and dedifferentiation/proliferation. A similar but milder defect is associated with mutations in CFTR (cystic fibrosis transmembrane conductance regulator). Vesicular chloride transport appears to be important for

  8. Impaired endocytosis in proximal tubule from subchronic exposure to cadmium involves angiotensin II type 1 and cubilin receptors

    PubMed Central

    2013-01-01

    Background Chronic exposure to low cadmium (Cd) levels produces urinary excretion of low molecular weight proteins, which is considered the critical effect of Cd exposure. However, the mechanisms involved in Cd-induced proteinuria are not entirely clear. Therefore, the present study was designed to evaluate the possible role of megalin and cubilin (important endocytic receptors in proximal tubule cells) and angiotensin II type 1 (AT1) receptor on Cd-induced microalbuminuria. Methods Four groups of female Wistar rats were studied. Control (CT) group, vehicle-treated rats; LOS group, rats treated with losartan (an AT1 antagonist) from weeks 5 to 8 (10 mg/kg/day by gavage); Cd group, rats subchronically exposed to Cd (3 mg/kg/day by gavage) during 8 weeks, and Cd + LOS group, rats treated with Cd for 8 weeks and LOS from weeks 5–8. Kidney Cd content, glomerular function (evaluated by creatinine clearance and plasma creatinine), kidney injury and tubular function (evaluated by Kim-1 expression, urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and glucose, and microalbuminuria), oxidative stress (measured by lipid peroxidation and NAD(P)H oxidase activity), mRNA levels of megalin, expressions of megalin and cubilin (by confocal microscopy) and AT1 receptor (by Western blot), were measured in the different experimental groups. Data were analyzed by one-way ANOVA or Kruskal-Wallis test using GraphPad Prism 5 software (Version 5.00). P < 0.05 was considered statistically significant. Results Administration of Cd (Cd and Cd + LOS groups) increased renal Cd content. LOS-treatment decreased Cd-induced microalbuminuria without changes in: plasma creatinine, creatinine clearance, urinary NAG and glucose, oxidative stress, mRNA levels of megalin and cubilin, neither protein expression of megalin nor AT1 receptor, in the different experimental groups studied. However, Cd exposure did induce the expression of the tubular injury marker Kim-1 and decreased

  9. AT1A Angiotensin Receptors in the Renal Proximal Tubule Regulate Blood Pressure

    PubMed Central

    Gurley, Susan B.; Riquier, Anne D. M.; Schnermann, Jurgen; Sparks, Matthew A.; Allen, Andrew M.; Haase, Volker H.; Snouwaert, John N.; Le, Thu H.; McDonough, Alicia A.; Koller, Beverley H.; Coffman, Thomas M.

    2011-01-01

    Summary Hypertension affects more than 1.5 billion people worldwide but the precise cause of elevated blood pressure (BP) cannot be determined in most affected individuals. Nonetheless, blockade of the renin-angiotensin system (RAS) lowers BP in the majority of patients with hypertension. Despite its apparent role in hypertension pathogenesis, the key cellular targets of the RAS that control BP have not been clearly identified. Here we demonstrate that RAS actions in the epithelium of the proximal tubule have a critical and non-redundant role in determining the level of BP. Abrogation of AT1 angiotensin receptor signaling in the proximal tubule alone is sufficient to lower BP, despite intact vascular responses. Elimination of this pathway reduces proximal fluid reabsorption and alters expression of key sodium transporters, modifying pressure-natriuresis and providing substantial protection against hypertension. Thus, effectively targeting epithelial functions of the proximal tubule of the kidney should be a useful therapeutic strategy in hypertension. PMID:21459331

  10. Importance of adenosine triphosphate in phospholipase A2-induced rabbit renal proximal tubule cell injury.

    PubMed Central

    Nguyen, V D; Cieslinski, D A; Humes, H D

    1988-01-01

    The pathogenesis of ischemic renal tubular cell injury involves a complex interaction of different processes, including membrane phospholipid alterations and depletion of high-energy phosphate stores. To assess the role of membrane phospholipid changes due to activation of phospholipases in renal tubule cell injury, suspensions enriched in rabbit renal proximal tubule segments were incubated with exogenous phospholipase A2 (PLA2). Exogenous PLA2 did not produce any significant change in various metabolic parameters reflective of cell injury in control nonhypoxic preparations despite a significant decrease in phosphatidylethanolamine (PE) and moderate increases in lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). In contrast, exogenous PLA2 treatment of hypoxic tubules resulted in a severe degree of cell injury, as demonstrated by marked declines in tubule K+ and ATP contents and significant decreases in tubule uncoupled respiratory rates, and was associated with significant phospholipid alterations, including marked declines in phosphatidylcholine (PC) and PE and significant rises in LPC, LPE, and free fatty acids (FFA). The injurious metabolic effects of exogenous PLA2 on hypoxic tubules were reversed by addition of ATP-MgCl2 to the tubules. The protective effect of ATP-MgCl2 was associated with increases in tubule PC and PE contents and declines in LPC, LPE, and FFA contents. These experiments thus indicate that an increase in exogenous PLA2 activity produces renal proximal tubule cell injury when cell ATP levels decline, at which point phospholipid resynthesis cannot keep pace with phospholipid degradation with resulting depletion of phospholipids and accumulation of lipid by-products. High-energy phosphate store depletion appears to be an important condition for exogenous PLA2 activity to induce renal tubule cell injury. PMID:3417866

  11. Evidence for role of cytosolic free calcium in hypoxia-induced proximal tubule injury.

    PubMed Central

    Kribben, A; Wieder, E D; Wetzels, J F; Yu, L; Gengaro, P E; Burke, T J; Schrier, R W

    1994-01-01

    The role of cytosolic free Ca2+ ([Ca2+]i) in hypoxic injury was investigated in rat proximal tubules. [Ca2+]i was measured using fura-2 and cell injury was estimated with propidium iodide (PI) in individual tubules using video imaging fluorescence microscopy. [Ca2+]i increased from approximately 170 to approximately 390 nM during 5 min of hypoxia. This increase preceded detectable cell injury as assessed by PI and was reversible with reoxygenation. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 100 microM) reduced [Ca2+]i under basal conditions (approximately 80 nM) and during hypoxia (approximately 120 nM) and significantly attenuated hypoxic injury. When [Ca2+]i and hypoxic cell injury were studied concurrently in the same individual tubules, the 10 min [Ca2+]i rise correlated significantly with subsequent cell damage observed at 20 min. 2 mM glycine did not block the rise in [Ca2+]i, yet protected the tubules from hypoxic injury. These results indicate that in rat proximal tubules, hypoxia induces an increase of [Ca2+]i which occurs before cell damage. The protective effect of BAPTA supports a role for [Ca2+]i in the initiation of hypoxic proximal tubule injury. The glycine results, however, implicate calcium-independent mechanisms of injury and/or blockade of calcium-mediated processes of injury such as activation of phospholipases or proteases. Images PMID:8182125

  12. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips

    PubMed Central

    Homan, Kimberly A.; Kolesky, David B.; Skylar-Scott, Mark A.; Herrmann, Jessica; Obuobi, Humphrey; Moisan, Annie; Lewis, Jennifer A.

    2016-01-01

    Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand. PMID:27725720

  13. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips

    NASA Astrophysics Data System (ADS)

    Homan, Kimberly A.; Kolesky, David B.; Skylar-Scott, Mark A.; Herrmann, Jessica; Obuobi, Humphrey; Moisan, Annie; Lewis, Jennifer A.

    2016-10-01

    Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand.

  14. 3D Proximal Tubule Tissues Recapitulate Key Aspects of Renal Physiology to Enable Nephrotoxicity Testing

    PubMed Central

    King, Shelby M.; Higgins, J. William; Nino, Celina R.; Smith, Timothy R.; Paffenroth, Elizabeth H.; Fairbairn, Casey E.; Docuyanan, Abigail; Shah, Vishal D.; Chen, Alice E.; Presnell, Sharon C.; Nguyen, Deborah G.

    2017-01-01

    Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies. PMID:28337147

  15. 3D Proximal Tubule Tissues Recapitulate Key Aspects of Renal Physiology to Enable Nephrotoxicity Testing.

    PubMed

    King, Shelby M; Higgins, J William; Nino, Celina R; Smith, Timothy R; Paffenroth, Elizabeth H; Fairbairn, Casey E; Docuyanan, Abigail; Shah, Vishal D; Chen, Alice E; Presnell, Sharon C; Nguyen, Deborah G

    2017-01-01

    Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.

  16. Proteomic analysis of brush-border membrane vesicles isolated from purified proximal convoluted tubules

    PubMed Central

    Walmsley, Scott J.; Broeckling, Corey; Hess, Ann; Prenni, Jessica

    2010-01-01

    The renal proximal convoluted tubule is the primary site of water, electrolyte and nutrient reabsorption and of active secretion of selected molecules. Proteins in the apical brush-border membrane facilitate these functions and initiate some of the cellular responses to altered renal physiology. The current study uses two-dimensional liquid chromatography/mass spectrometry to compare brush border membrane vesicles isolated from rat renal cortex (BBMVCTX) and from purified proximal convoluted tubules (BBMVPCT). Both proteomic data and Western blot analysis indicate that the BBMVCTX contain apical membrane proteins from cortical cells other than the proximal tubule. This heterogeneity was greatly reduced in the BBMVPCT. Proteomic analysis identified 193 proteins common to both samples, 21 proteins unique to BBMVCTX, and 57 proteins unique to BBMVPCT. Spectral counts were used to quantify relative differences in protein abundance. This analysis identified 42 and 50 proteins that are significantly enriched (p values ≤0.001) in the BBMVCTX and BBMVPCT, respectively. These data were validated by measurement of γ-glutamyltranspeptidase activity and by Western blot analysis. The combined results establish that BBMVPCT are primarily derived from the proximal convoluted tubule (S1 and S2 segments), whereas BBMVCTX include proteins from the proximal straight tubule (S3 segment). Analysis of functional annotations indicated that BBMVPCT are enriched in mitochondrial proteins and enzymes involved in glucose and organic acid metabolism. Thus the current study reports a detailed proteomic analysis of the brush-border membrane of the rat renal proximal convoluted tubule and provides a database for future hypothesis-driven research. PMID:20219825

  17. Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity

    PubMed Central

    Jella, Kishore Kumar; Yu, Ling; Yue, Qiang; Friedman, Daniel; Duke, Billie J.; Alli, Abdel A.

    2016-01-01

    Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells. PMID:27802315

  18. Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity.

    PubMed

    Homma-Takeda, Shino; Kitahara, Keisuke; Suzuki, Kyoko; Blyth, Benjamin J; Suya, Noriyoshi; Konishi, Teruaki; Terada, Yasuko; Shimada, Yoshiya

    2015-12-01

    Renal toxicity is a hallmark of uranium exposure, with uranium accumulating specifically in the S3 segment of the proximal tubules causing tubular damage. As the distribution, concentration and dynamics of accumulated uranium at the cellular level is not well understood, here, we report on high-resolution quantitative in situ measurements by high-energy synchrotron radiation X-ray fluorescence analysis in renal sections from a rat model of uranium-induced acute renal toxicity. One day after subcutaneous administration of uranium acetate to male Wistar rats at a dose of 0.5 mg uranium kg(-1) body weight, uranium concentration in the S3 segment of the proximal tubules was 64.9 ± 18.2 µg g(-1) , sevenfold higher than the mean renal uranium concentration (9.7 ± 2.4 µg g(-1) ). Uranium distributed into the epithelium of the S3 segment of the proximal tubules and highly concentrated uranium (50-fold above mean renal concentration) in micro-regions was found near the nuclei. These uranium levels were maintained up to 8 days post-administration, despite more rapid reductions in mean renal concentration. Two weeks after uranium administration, damaged areas were filled with regenerating tubules and morphological signs of tissue recovery, but areas of high uranium concentration (100-fold above mean renal concentration) were still found in the epithelium of regenerating tubules. These data indicate that site-specific accumulation of uranium in micro-regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney.

  19. SGLT2 Inhibitors: Glucotoxicity and Tumorigenesis Downstream the Renal Proximal Tubule?

    PubMed

    Bertinat, Romina; Nualart, Francisco; Yáñez, Alejandro J

    2016-08-01

    At present, diabetes mellitus is the main cause of end-stage renal disease. Effective glycaemic management is the most powerful tool to delay the establishment of diabetic complications, such as diabetic kidney disease. Together with reducing blood glucose levels, new anti-diabetic agents are expected not only to control the progression but also to restore known defects of the diabetic kidney. Sodium-glucose co-transporter 2 (SGLT2) inhibitors are promising anti-diabetic agents that reduce hyperglycaemia by impairing glucose reabsorption in proximal tubule of the kidney and increasing glucosuria. SGLT2 inhibitors have shown to reduce glucotoxicity in isolated proximal tubule cells and also to attenuate expression of markers of overall kidney damage in experimental animal models of diabetes, but the actual renoprotective effect for downstream nephron segments is still unknown and deserves further attention. Here, we briefly discuss possible undesired effects of enhanced glucosuria and albuminuria in nephron segments beyond the proximal tubule after SGLT2 inhibitor treatment, offering new lines of research to further understand the renoprotective action of these anti-diabetic agents. Strategies blocking glucose reabsorption by renal proximal tubule epithelial cells (RPTEC) may be protective for RPTEC, but downstream nephron segments will still be exposed to high glucose and albumin levels through the luminal face. The actual effect of constant enhanced glucosuria over distal nephron segments remains to be established. J. Cell. Physiol. 231: 1635-1637, 2016. © 2015 Wiley Periodicals, Inc.

  20. The Integrin β1 Subunit Regulates Paracellular Permeability of Kidney Proximal Tubule Cells*

    PubMed Central

    Elias, Bertha C.; Mathew, Sijo; Srichai, Manakan B.; Palamuttam, Riya; Bulus, Nada; Mernaugh, Glenda; Singh, Amar B.; Sanders, Charles R.; Harris, Raymond C.; Pozzi, Ambra; Zent, Roy

    2014-01-01

    Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a “loose” epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a “tight” epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells. PMID:24509849

  1. Autocrine inhibition of Na+/K(+)-ATPase by nitric oxide in mouse proximal tubule epithelial cells.

    PubMed Central

    Guzman, N J; Fang, M Z; Tang, S S; Ingelfinger, J R; Garg, L C

    1995-01-01

    An inducible nitric oxide synthase has recently been described in proximal tubule epithelium. To investigate the effects of proximal tubule NO on Na+/K(+)-ATPase, we induced NO production in mouse proximal tubule epithelial cells by treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) followed by determinations of ouabain-sensitive ATPase activity. Na+/K(+)-ATPase activity decreased after 4 h of LPS/IFN gamma treatment, reaching maximal inhibition after 24 h (34% reduction in activity). The inhibition of Na+/K(+)-ATPase activity by LPS/IFN gamma was prevented by simultaneous incubation with N omega-nitro L-arginine and markedly blunted by removal of L-arginine from the medium. The NO donors sodium nitroprusside and SIN-1 also inhibited Na+/K(+)-ATPase activity to a similar extent than LPS/IFN gamma. However, treatment with 8-pCPT-cGMP only modestly reduced Na+/K(+)-ATPase activity. Interestingly, superoxide dismutase prevented the inhibitory effects of NO on Na+/K(+)-ATPase activity, suggesting a role for peroxynitrite in this inhibition. We conclude that NO generated by mouse proximal tubule epithelial cell iNOS inhibits Na/K ATPase activity in an autocrine fashion and that this inhibition is accompanied by a reduction in Na-dependent solute transport. PMID:7537754

  2. Akt recruits Dab2 to albumin endocytosis in the proximal tubule.

    PubMed

    Koral, Kelly; Li, Hui; Ganesh, Nandita; Birnbaum, Morris J; Hallows, Kenneth R; Erkan, Elif

    2014-12-15

    Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule.

  3. Pyrazinoate transport in the isolated perfused rabbit proximal tubule.

    PubMed

    Besseghir, K; Roch-Ramel, F

    1986-12-01

    The bidirectional tubular transport of pyrazinoate (PZA) was studied in the isolated perfused proximal S2 segment of rabbit kidney. PZA reabsorption was a mechanism of large capacity, temperature-dependent and requiring a normal Na+/K+-ATPase activity. PZA reabsorption was reversibly decreased when lactate was added to the perfusate, indicating that it might occur through the sodium-lactate cotransport. The addition of PAH to the bath had a slight stimulatory effect on PZA reabsorption, suggesting a component of anion exchange in the overall PZA reabsorption. However, SITS added to either the perfusate or the bathing medium induced a non-significant decrease in PZA reabsorption, confirming the minor part of an anion exchange mechanism in this reabsorptive process. PZA reabsorption was not affected by the establishment of a bath-to-lumen H+ gradient, and was only moderately decreased after carbonic anhydrase inhibition by ethoxyzolamide, in opposition to what is known for the reabsorbed anion salicylate. The secretory transport of PZA was saturable and also dependent on a normal Na+/K+-ATPase activity. It is concluded that PZA is bidirectionally transported by facilitated mechanisms in the rabbit proximal S2 segment, one major reabsorptive mechanism appearing to be a sodium-anion cotransport, which might be the sodium-lactate reabsorbing mechanism.

  4. Increased Renal Proximal Convoluted Tubule Transport Contributes to Hypertension in Cyp4a14 Knockout Mice

    PubMed Central

    Quigley, Raymond; Chakravarty, Sumana; Zhao, Xueying; Imig, John D.; Capdevila, Jorge H.

    2009-01-01

    Background/Aims Disrupting the enzyme Cyp4a14 in mice leads to hypertension, which is more severe in the male mice and appears to be due to androgen excess. Because the Cyp4a14 enzyme is located in the proximal tubule of the kidney, we hypothesized that there could be dysregulation of transport in this segment that could contribute to the hypertension. Methods Wild-type (SV/129) mice and mice that had targeted disruption of the Cyp4a14 gene were studied. Proximal convoluted tubules (PCT) from knockout and wild-type mice were dissected and perfused in vitrofor measurement of volume absorption (JV). Expression of the sodium-hydrogen exchanger 3 (NHE3), the predominant transporter responsible for sodium transport in this segment, was measured by immunoblot. Renal vascular (afferent arteriole) responses to angiotensin and endothelin were also measured. Results PCT volume absorption was elevated in tubules from the Cyp4a14 knockout mice as compared to the wild-type mice. Brush border membrane NHE3 expression was almost 2-fold higher in Cyp4a14 knockout mice than in wild-type mice. No difference was found in the afferent arteriolar response. Conclusion Thus, hypertension in the Cyp4a14 knockout mice appears to be driven by excessive fluid reabsorption in the proximal tubule, which is secondary to overexpression of NHE3. PMID:19713718

  5. Mechanisms of adaptation to chronic respiratory acidosis in the rabbit proximal tubule.

    PubMed Central

    Krapf, R

    1989-01-01

    The hyperbicarbonatemia of chronic respiratory acidosis is maintained by enhanced bicarbonate reabsorption in the proximal tubule. To investigate the cellular mechanisms involved in this adaptation, cell and luminal pH were measured microfluorometrically using (2",7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein in isolated, microperfused S2 proximal convoluted tubules from control and acidotic rabbits. Chronic respiratory acidosis was induced by exposure to 10% CO2 for 52-56 h. Tubules from acidotic rabbits had a significantly lower luminal pH after 1-mm perfused length (7.03 +/- 0.09 vs. 7.26 +/- 0.06 in controls, perfusion rate = 10 nl/min). Chronic respiratory acidosis increased the initial rate of cell acidification (dpHi/dt) in response to luminal sodium removal by 63% and in response to lowering luminal pH (7.4-6.8) by 69%. Chronic respiratory acidosis also increased dpHi/dt in response to peritubular sodium removal by 63% and in response to lowering peritubular pH by 73%. In conclusion, chronic respiratory acidosis induces a parallel increase in the rates of the luminal Na/H antiporter and the basolateral Na/(HCO3)3 cotransporter. Therefore, the enhanced proximal tubule reabsorption of bicarbonate in chronic respiratory acidosis may be, at least in part, mediated by a parallel adaptation of these transporters. PMID:2537851

  6. Characteristics of salt and water transport in superficial and juxtamedullary straight segments of proximal tubules.

    PubMed Central

    Kawamura, S; Imai, M; Seldin, D W; Kukko, J P

    1975-01-01

    The purpose of the present studies was to characterize the nature of salt and water transport out of the superficial (SF) and juxtamedullary (JM) straight segments of rabbit proximal tubules as examined by in vitro microperfusion techniques. When the perfusate consisted of a solution simulating ultrafiltrate of plasma, there were no differences between SF and JM straight tubules in either net reabsorption of fluid (SF=0.47 nl/mm per min; JM=0.56 nl/mm per min) or in transtubular potential difference (PD) (SF=-2.1 mV; JM=-1.8 mV). Removal of glucose and alanine from the perfusate had no effect on the magnitude of the PD in either straight segment. Ouabain decreased both the net reabsorptive rates and the PD. Isosmolal replacement of NaCL by Na-cyclamate (a presumed impermeant anion) in the perfusate and the bath caused an increase in luminal negativity in both segments wheras similar substitution of NaCL by choline-CL (nontransported cation) changed the PD TO NEAR ZERO. These studies, therefore, suggest that sodium is transported out of the proximal straight tubules by an active noncoupled process that generates a PD (electrogenic process). When the perfusate consisted of a solution with a high chloride concentration (resulting from greater HCO3 than CI reabsorption in the proximal convoluted tubule), different PDs in SF and JM tubules were generated: SF=+1.6 plus or minus 0.2 mV; JM=-1.3 plus or minus 0.3 mV. This difference in PD was attributed to relative differences in Na and CI permeabilities in these two segments. Electrophysiological and isotopic estimates of the chloride to sodium permeability revealed that the SF tubule is about twice as permeant to chloride than to sodium whereas the JM tubules are approximately twice as permeant to sodium than to chloride. It is concluded that the mechanism of active sodium transport in the straight segment of proximal tubule differs from that of the convoluted segment and that both the SF and JM straight segments differ

  7. Nebivolol Attenuates Maladaptive Proximal Tubule Remodeling in Transgenic Rats

    PubMed Central

    Hayden, Melvin R.; Habibi, Javad; Whaley-Connell, Adam; Sowers, Dilek; Johnson, Megan; Tilmon, Roger; Jain, Deepika; Ferrario, Carlos; Sowers, James R.

    2010-01-01

    Background/Aims The impact of nebivolol therapy on the renal proximal tubular cell (PTC) structure and function was investigated in a transgenic (TG) rodent model of hypertension and the cardiometabolic syndrome. The TG Ren2 rat develops nephropathy with proteinuria, increased renal angiotensin II levels and oxidative stress, and PTC remodeling. Nebivolol, a β1-antagonist, has recently been shown to reduce albuminuria, in part, through reductions in renal oxidative stress. Accordingly, we hypothesized that nebivolol therapy would attenuate PTC damage and tubulointerstitial fibrosis. Methods Young Ren2 (R2-N) and SD (SD-N) rats were treated with nebivolol (10 mg/kg/day) or vehicle (R2-C; SD-C) for 3 weeks. PTC structure and function were tested using transmission electron microscopy and functional measurements. Results Nebivolol treatment decreased urinary N-acetyl-β-D-glucosaminidase, tubulointerstitial ultrastructural remodeling and fibrosis, NADPH oxidase activity, 3-nitrotyrosine levels, and increased megalin and lysosomal-associated membrane protein-2 immunostaining in PTCs. Ultrastructural abnormalities that were improved with therapy included altered canalicular structure, reduced endosomes/lysosomes and PTC vacuoles, basement membrane thickening, and mitochondrial remodeling/fragmentation. Conclusion These observations support the notion that nebivolol may improve PTC reabsorption of albumin and other glomerular filtered small molecular weight proteins in association with the attenuation of oxidative stress, tubulointerstitial injury and fibrosis in this rat model of metabolic kidney disease. PMID:20110666

  8. Modeling oxygen consumption in the proximal tubule: effects of NHE and SGLT2 inhibition

    PubMed Central

    Vallon, Volker; Edwards, Aurélie

    2015-01-01

    The objective of this study was to investigate how physiological, pharmacological, and pathological conditions that alter sodium reabsorption (TNa) in the proximal tubule affect oxygen consumption (QO2) and Na+ transport efficiency (TNa/QO2). To do so, we expanded a mathematical model of solute transport in the proximal tubule of the rat kidney. The model represents compliant S1, S2, and S3 segments and accounts for their specific apical and basolateral transporters. Sodium is reabsorbed transcellularly, via apical Na+/H+ exchangers (NHE) and Na+-glucose (SGLT) cotransporters, and paracellularly. Our results suggest that TNa/QO2 is 80% higher in S3 than in S1–S2 segments, due to the greater contribution of the passive paracellular pathway to TNa in the former segment. Inhibition of NHE or Na-K-ATPase reduced TNa and QO2, as well as Na+ transport efficiency. SGLT2 inhibition also reduced proximal tubular TNa but increased QO2; these effects were relatively more pronounced in the S3 vs. the S1–S2 segments. Diabetes increased TNa and QO2 and reduced TNa/QO2, owing mostly to hyperfiltration. Since SGLT2 inhibition lowers diabetic hyperfiltration, the net effect on TNa, QO2, and Na+ transport efficiency in the proximal tubule will largely depend on the individual extent to which glomerular filtration rate is lowered. PMID:25855513

  9. Water reabsorption capacity of the proximal convoluted tubule: a microperfusion study on rat kidney.

    PubMed Central

    Corman, B; Roinel, N; De Rouffignac, C

    1981-01-01

    1. The differences in the water reabsorption capacity observed from one proximal tubule to another were investigated in vivo by continuous microperfusion. 2. Two to seven loops were punctured along the same tubule. The [3H]inulin, 22Na, [14C]glucose, sodium, chloride and magnesium concentrations as well as the osmolality of the collected samples were studied as a function of the perfused length. 3. With Ringer bicarbonate solution perfused in Saclay Wistar rats, the water reabsorption capacity ranged from 0 to 3 nl . min-1 . mm-1 depending on the tubule. This reabsorption rate was closely correlated with the unidirectional reabsorption flux of sodium, and with the rise in tubular chloride and magnesium concentrations. 4. In Munich Wistar rats with glomeruli accessible at the kidney surface, tubule perfusion with a Ringer bicarbonate solution showed that the highest water reabsorption rates per mm of tubule were found for the perfusion sites closest to the glomerulus; water fluxes were also positively correlated with glucose transport. 5. In a second series of experiments on Saclay rats, perfusion of a Ringer solution containing a high chloride concentration (137 m-equiv/l.) was unable to increase the water reabsorption rate compared to the control perfusion; here again, water fluxes were positively correlated with glucose transport. PMID:7320874

  10. Proximal Tubule Glutamine Synthetase Expression is Necessary for the Normal Response to Dietary Protein Restriction.

    PubMed

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Verlander, Jill W; Weiner, I David

    2017-03-22

    Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and ¬decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of NH4+ and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The current study's purpose was to determine proximal tubule GS's role in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule glutamine synthetase expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.

  11. Mechanism of NaCl and water reabsorption in the proximal convoluted tubule of rat kidney.

    PubMed Central

    Neumann, K H; Rector, F C

    1976-01-01

    The role of chloride concentration gradients in proximal NaCl and water reabsorption was examined in superficial proximal tubules of the rat by using perfusion and collection techniques. Reabsorptive rates (Jv), chloride concentrations, and transtubular potential difference were measured during perfusion with solutions (A) simulating an ultrafiltrate of plasma; (B) similar to (A) except that 20 meq/liter bicarbonate was replaced with acetate; (C) resembling late proximal fluid (glucose, amino acid, acetate-free, low bicarbonate, and high chloride); and (D) in which glucose and amino acids were replaced with raffinose and bicarbonate was partially replaced by poorly reabsorbable anions (cyclamate,sulfate, and methyl sulfate). In tubules perfused with solutions A and B, Jv were 2.17 and 2.7 nl mm-1 min-1 and chloride concentrations were 131.5 +/- 3.1 and 135 +/- 395 meq/liter, respectively, indicating that reabsorption is qualitatively similar to free-flow conditions and that acetate adequately replaces bicarbonate. With solution C, Jv was 2.10 nl mm-1 min-1 and potential difference was +1.5 +/- 0.2 mV, indicating that the combined presence of glucose, alanine, acetate, and bicarbonate per se is not an absolute requirement. Fluid reabsorption was virtually abolished when tubules were perfused with D solutions; Jv was not significantly different from zero despite sodium and chloride concentrations similar to plasma; chloride concentration was 110.8 +/- 0.2 meq/liter and potential difference was -0.98 mV indicating that chloride was close to electrochemical equilibrium. These results suggest the importance of the chloride gradient to proximal tubule reabsorption in regions where actively reabsorbable solutes (glucose, alanine, acetate, and bicarbonate) are lacking and provide further evidence for a passive model of NaCl and water transport. PMID:993334

  12. Proximal tubule-targeted heme oxygenase-1 in cisplatin-induced acute kidney injury.

    PubMed

    Bolisetty, Subhashini; Traylor, Amie; Joseph, Reny; Zarjou, Abolfazl; Agarwal, Anupam

    2016-03-01

    Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that catalyzes the breakdown of heme to biliverdin, carbon monoxide, and iron. The beneficial effects of HO-1 expression are not merely due to degradation of the pro-oxidant heme but are also credited to the by-products that have potent, protective effects, including antioxidant, anti-inflammatory, and prosurvival properties. This is well reflected in the preclinical animal models of injury in both renal and nonrenal settings. However, excessive accumulation of the by-products can be deleterious and lead to mitochondrial toxicity and oxidative stress. Therefore, use of the HO system in alleviating injury merits a targeted approach. Based on the higher susceptibility of the proximal tubule segment of the nephron to injury, we generated transgenic mice using cre-lox technology to enable manipulation of HO-1 (deletion or overexpression) in a cell-specific manner. We demonstrate the validity and feasibility of these mice by breeding them with proximal tubule-specific Cre transgenic mice. Similar to previous reports using chemical modulators and global transgenic mice, we demonstrate that whereas deletion of HO-1, specifically in the proximal tubules, aggravates structural and functional damage during cisplatin nephrotoxicity, selective overexpression of HO-1 in proximal tubules is protective. At the cellular level, cleaved caspase-3 expression, a marker of apoptosis, and p38 signaling were modulated by HO-1. Use of these transgenic mice will aid in the evaluation of the effects of cell-specific HO-1 expression in response to injury and assist in the generation of targeted approaches that will enhance recovery with reduced, unwarranted adverse effects.

  13. Local pH domains regulate NHE3-mediated Na+ reabsorption in the renal proximal tubule

    PubMed Central

    Burford, James L.; McDonough, Alicia A.; Holstein-Rathlou, Niels-Henrik; Peti-Peterdi, Janos

    2014-01-01

    The proximal tubule Na+/H+ exchanger 3 (NHE3), located in the apical dense microvilli (brush border), plays a major role in the reabsorption of NaCl and water in the renal proximal tubule. In response to a rise in blood pressure NHE3 redistributes in the plane of the plasma membrane to the base of the brush border, where NHE3 activity is reduced. This NHE3 redistribution is assumed to provoke pressure natriuresis; however, it is unclear how NHE3 redistribution per se reduces NHE3 activity. To investigate if the distribution of NHE3 in the brush border can change the reabsorption rate, we constructed a spatiotemporal mathematical model of NHE3-mediated Na+ reabsorption across a proximal tubule cell and compared the model results with in vivo experiments in rats. The model predicts that when NHE3 is localized exclusively at the base of the brush border, it creates local pH microdomains that reduce NHE3 activity by >30%. We tested the model's prediction experimentally: the rat kidney cortex was loaded with the pH-sensitive fluorescent dye BCECF, and cells of the proximal tubule were imaged in vivo using confocal fluorescence microscopy before and after an increase of blood pressure by ∼50 mmHg. The experimental results supported the model by demonstrating that a rise of blood pressure induces the development of pH microdomains near the bottom of the brush border. These local changes in pH reduce NHE3 activity, which may explain the pressure natriuresis response to NHE3 redistribution. PMID:25298526

  14. Cubilin Is Essential for Albumin Reabsorption in the Renal Proximal Tubule

    PubMed Central

    Amsellem, Sabine; Gburek, Jakub; Hamard, Ghislaine; Nielsen, Rikke; Willnow, Thomas E.; Devuyst, Olivier; Nexo, Ebba; Verroust, Pierre J.

    2010-01-01

    Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the “specific” cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases. PMID:20798259

  15. Cubilin is essential for albumin reabsorption in the renal proximal tubule.

    PubMed

    Amsellem, Sabine; Gburek, Jakub; Hamard, Ghislaine; Nielsen, Rikke; Willnow, Thomas E; Devuyst, Olivier; Nexo, Ebba; Verroust, Pierre J; Christensen, Erik I; Kozyraki, Renata

    2010-11-01

    Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.

  16. PKB and megalin determine the survival or death of renal proximal tubule cells.

    PubMed

    Caruso-Neves, Celso; Pinheiro, Ana Acacia S; Cai, Hui; Souza-Menezes, Jackson; Guggino, William B

    2006-12-05

    Renal proximal tubule cells have a remarkable ability to reabsorb large quantities of albumin through megalin-mediated endocytosis. This is an essential process for overall body homeostasis. Overstressing this endocytic system with a prolonged excess of albumin is injurious to proximal tubule cells. How these cells function and protect themselves from injury is unknown. Here, we show that megalin is the sensor that determines whether cells will be protected or injured by albumin. Megalin, through a novel mechanism, binds PKB in a D-3-phosphorylated phospholipid-insensitive manner, anchoring PKB in the luminal plasma membrane. Whereas low doses of albumin are protective, an overload of albumin decreases megalin expression followed by a reduction of plasma membrane PKB, PKB activity, and Bad phosphorylation induced by PKB. The result is albumin-induced apoptosis. These results reveal a model for PKB distribution in the plasma membrane and elucidate mechanisms involved in both the protective and toxic effects of albumin on proximal tubule cells. In addition, our findings suggest a mechanism for the progression of chronic kidney disease to end-stage renal disease.

  17. Effects of chloride channel blockers on hypoxic injury in rat proximal tubules.

    PubMed

    Reeves, W B

    1997-05-01

    These studies examined the pathways and consequences of chloride uptake into proximal tubule cells during in vitro hypoxia. The chloride channel blocker diphenylamine-2-carboxylate (DPC) markedly reduced the degree of hypoxia-induced membrane damage as measured by the release of lactate dehydrogenase (LDH). DPC reduced the release of LDH from hypoxic tubules from 38 +/- 2.7% to 16 +/- 1.7% after 30 minutes of hypoxia (P < 0.001, N = 16) and also reduced 36Cl- uptake by hypoxic tubules. The reduction in LDH release was not associated with better preservation of cell ATP content or with protection against hypoxia-induced DNA damage. Other Cl- channel blockers, such as niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihyrdo-2-methyl-1-oxo-1H-in den-5-yl)oxy] acetic acid (IAA-94) provided even greater protection than DPC and were as effective as 2 mM glycine. The Cl- channel blockers appear to act late in the course of hypoxic injury since DNA damage, an early manifestation of injury, is not prevented by the blockers and since addition of the Cl- channel blocker after the hypoxic injury has begun reduces further membrane damage. These results support the conclusion that transport through Cl- channels contributes to hypoxic cell injury in proximal tubular cells.

  18. The effects of anions on fluid reabsorption from the proximal convoluted tubule of the rat.

    PubMed Central

    Green, R; Greenwood, S L; White, S

    1988-01-01

    1. Fluid reabsorption from surface proximal tubules of the rat was measured in vivo using stationary microperfusion techniques. Reabsorptive rate (Jv) was measured from droplets containing chloride as the main reabsorbable anion and when chloride was substituted by bromide, iodide, nitrate, acetate, isethionate or methylsulphate in either the tubular lumen alone or in both lumen and peritubular capillaries. 2. In tubules with an intact blood supply, droplet volume decreased in a manner best described by a single exponential and substitution of chloride by nitrate or bromide had no effect on Jv. Substitution by iodide or acetate inhibited Jv by approximately 17% but substitution by methylsulphate or isethionate caused droplets to transiently increase in volume before shrinkage which was itself inhibited by approximately 50%. The inhibitory action of isethionate was found to be concentration dependent. 3. Recollection and analysis of droplets which were initially free of chloride, containing either nitrate or isethionate, showed that chloride entered these droplets, but that the initial rate of chloride entry was greater for nitrate than isethionate droplets. 4. When tubules and capillaries were perfused with chloride solutions containing no bicarbonate, Jv was reduced to about 20% of the value when peritubular capillary blood flow was intact. Substituting chloride in the tubular and capillary perfusion revealed a sequence for supporting fluid reabsorption that was identical to that when chloride was substituted in tubule fluid alone: bromide = nitrate greater than iodide = acetate greater than isethionate. Addition of 2.0 mmol l-1 NaCN reduced the reabsorptive flux to zero. 5. The results of this study are consistent with transcellular transport of anions across the proximal tubular epithelium. The pathways for anion transport are likely to involve a series of non-selective mechanisms such as anion exchangers. PMID:3256612

  19. The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells

    PubMed Central

    Li, Yuanli; Cong, Rong; Biemesderfer, Daniel

    2008-01-01

    We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) γ-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by γ-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na+/H+ exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, β-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of γ-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule. PMID:18495814

  20. Differential cytoprotection by glycine against oxidant damage to proximal tubule cells.

    PubMed

    Sogabe, K; Roeser, N F; Venkatachalam, M A; Weinberg, J M

    1996-09-01

    Tert-butyl hydroperoxide (tBHP) injured freshly isolated proximal tubules in an Fe-dependent fashion that was ameliorated by a lipophilic antioxidant, diphenyl-p-phenylenediamine (DPPD), but was only minimally affected by glycine. Menadione-induced injury was Fe-independent and was unaffected by DPPD, but was strongly blocked by glycine. Fe was highly toxic when intracellular loading was facilitated by concomitant treatment with hydroxyquinoline (HQ). This toxicity was blocked by DPPD or chelating the Fe, but not by glycine. All of the lesions were characterized by severe depletion of glutathione and other soluble thiols. Menadione induced large increases in protein associated with the Triton-insoluble cytoskeleton and decreases in protein thiol content, consistent with extensive cross linking, but did not increase thiobarbituric acid reactive substances (TBARS). tBHP and HQ + Fe had either no effect or only moderate, delayed effects on cytoskeletal proteins, but induced substantial increases of TBARS. Glycine did not the alter changes in cytoskeletal proteins, thiols, or TBARS produced by any of the agents. Protection against tBHP toxicity by deferoxamine and DPPD was accompanied by substantial suppression of TBARS accumulation. Superimposition of hypoxia during tBHP exposure reduced TBARS accumulation and restored cytoprotective activity to glycine. Thus, in contrast to its consistently strong cytoprotection against a number of other insults, glycine is only variably cytoprotective against oxidant lesions in freshly isolated proximal tubules. Extensive oxidative crosslinking of proteins is compatible with maintenance of glycine cytoprotection against lethal membrane damage. Fe-induced injury to proximal tubules associated with lipid peroxidation as manifested by TBARS formation is a relatively glycine-insensitive insult.

  1. Modifications of the genital kidney proximal and distal tubules for sperm transport in Notophthalmus viridescens (Amphibia, Urodela, Salamandridae).

    PubMed

    Nicholson, Abbigail E; Siegel, Dustin S

    2014-08-01

    Male salamanders use nephrons from the genital kidney to transport sperm from the testicular lobules to the Wolffian duct. The microstructure of the epithelia of the genital kidney proximal tubule and distal tubule was studied over 1 year in a population of Notophthalmus viridescens from Crawford and Pike counties in central Missouri. Through ultrastructural analysis, we were able to support the hypothesis that the genital kidney nephrons are modified to aid in the transportation of sperm. A lack of folding of the basal plasma membrane, in both the genital kidney proximal and distal tubules when compared to the pelvic kidney proximal and distal tubules, reduces the surface area and thus likely decreases the efficiency of reabsorption in these nephron regions of the genital kidney. Ciliated epithelial cells are also present along the entire length of the genital kidney proximal tubule, but are lacking in the epithelium of the pelvic kidney proximal tubule. The exact function of these cilia remains unknown, but they may aid in mixing of seminal fluids or the transportation of immature sperm through the genital kidney nephrons. Ultrastructural analysis of proximal and distal tubules of the genital kidney revealed no seasonal variation in cellular activity and no mass production of seminal fluids throughout the reproductive cycle. Thus, we failed to support the hypothesis that the cellular activity of the epithelia lining the genital kidney nephrons is correlated to specific events in the reproductive cycle. The cytoplasmic contents and overall structure of the genital and pelvic kidney epithelial cells were similar to recent observations in Ambystoma maculatum, with the absence of abundant dense bodies apically in the epithelial cells lining the genital kidney distal tubule.

  2. Subcellular distribution of folate and folate binding protein in renal proximal tubules

    SciTech Connect

    Sharkey, C.; Hjelle, J.T.; Selhub, J.

    1986-03-01

    High affinity folate binding protein (FBP) found in brush border membranes derived from renal cortices is thought to be involved in the renal conservation of folate. To examine the mechanisms of folate recovery, the subcellular distribution of FBP and /sup 3/H-folate in rabbit renal proximal tubules (PT) was examined using analytical cell fractionation techniques. Tubules contain 3.41 +/- 0.32 picomoles FBP/mg protein (X +/- S.D.; n = 5). Postnuclear supernates (PNS) of PT were layered atop Percoll-sucrose gradients, centrifuged, fractions collected and assayed for various marker enzymes and FBP. Pooled fractions from such gradients were subsequently treated with digitonin and centrifuged in a stoichiometric manner with the activity of the microvillar enzyme, alanylaminopeptidase (AAP); excess FBP distributed with more buoyant particles. Infusion of /sup 3/H-folate into rabbit kidneys followed by tubule isolation and fractionation revealed a time dependent shift in distribution of radiolabel from the AAP-rich gradient fractions to a region containing more buoyant particles; radiolevel was not associated with lysosomal markers. EM-radioautography revealed grains over intracellular vesicles. These results are consistent with the hypothesis that folate is recovered by a process involving receptor-mediated endocytosis or transcytosis.

  3. Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli.

    PubMed

    Sogabe, K; Roeser, N F; Davis, J A; Nurko, S; Venkatachalam, M A; Weinberg, J M

    1996-08-01

    To better define the role of Ca2+ in pathophysiological alterations of the proximal tubule microvillus actin cytoskeleton, we studied freshly isolated tubules in which intracellular free Ca2+ was equilibrated with highly buffered, precisely defined medium Ca2+ levels using a combination of the metabolic inhibitor, antimycin, and the ionophore, ionomycin, in the presence of glycine, to prevent lethal membrane damage and resulting nonspecific changes. Increases of Ca2+ to > or = 10 microM were sufficient to initiate concurrent actin depolymerization, fragmentation of F-actin into forms requiring high-speed centrifugation for recovery, redistribution of villin to sedimentable fractions, and structural microvillar damage consisting of severe swelling and fragmentation of actin cores. These observations implicate Ca(2+)-dependent, villin-mediated actin cytoskeletal disruption in tubule cell microvillar damage under conditions conceivably present during pathophysiological states. However, despite prior evidence for cytosolic free Ca2+ increases of the same order of magnitude and similar structural microvillar alterations, Ca(2+)- and villin-mediated events did not appear to account for the initial microvillar damage that occurs during ATP depletion induced by antimycin alone or hypoxia.

  4. Passive permeability of salicylic acid in renal proximal S2 and S3 tubules

    SciTech Connect

    Chatton, J.Y.; Roch-Ramel, F. )

    1991-03-01

    The role of nonionic diffusion in the transport of salicylic acid across rabbit proximal S2 and S3 segments was investigated using the in vitro isolated perfused tubule technique. The ({sup 14}C) salicylic acid apparent reabsorptive permeability (P'I-b, 10(-5) cm/s) was measured at 19 degrees C with luminal solutions kept at different pH and bath maintained at pH 7.4. In S2 tubules, P'I-b was 25.0 +/- 3.5 when luminal pH was 6.0; P'I-b decreased to 8.1 +/- 1.4 and to 4.4 +/- 1.2 at a luminal pH of 6.5 and 7.0, respectively. In S3 tubules, P'I-b was 17.6 +/- 2.4, 5.3 +/- 1.1 and 3.4 +/- 1.1 at a luminal pH of 6.0, 6.5 and 7.0, respectively. There was a close correlation between P'I-b and the calculated proportion of nonionized salicylic acid present at each pH, indicating that only the nonionized molecule could diffuse in our conditions. We calculated the apparent permeability of nonionic salicylic acid and found 0.248 +/- 0.032 cm/s for S2 and 0.176 +/- 0.022 cm/s for S3 tubules. These calculated permeabilities were independent of pH.

  5. Fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II.

    PubMed

    Cabral, Pablo D; Hong, Nancy J; Hye Khan, Md Abdul; Ortiz, Pablo A; Beierwaltes, William H; Imig, John D; Garvin, Jeffrey L

    2014-03-01

    The proximal nephron reabsorbs 60% to 70% of the fluid and sodium and most of the filtered bicarbonate via Na/H exchanger 3. Enhanced proximal nephron transport is implicated in hypertension. Our findings show that a fructose-enriched diet causes salt sensitivity. We hypothesized that fructose stimulates luminal Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. Na/H exchange was measured in rat proximal tubules as the rate of intracellular pH (pHi) recovery in fluorescent units/s. Replacing 5 mmol/L glucose with 5 mmol/L fructose increased the rate of pHi recovery (1.8±0.6 fluorescent units/s; P<0.02; n=8). Staurosporine, a protein kinase C inhibitor, blocked this effect. We studied whether this effect was because of the addition of fructose or removal of glucose. The basal rate of pHi recovery was first tested in the presence of a 0.6-mmol/L glucose and 1, 3, or 5 mmol/L fructose added in a second period. The rate of pHi recovery did not change with 1 mmol/L but it increased with 3 and 5 mmol/L of fructose. Adding 5 mmol/L glucose caused no change. Removal of luminal sodium blocked pHi recovery. With 5.5 mmol/L glucose, angiotensin II (1 pmol/L) did not affect the rate of pHi recovery (change, -1.1±0.5 fluorescent units/s; n=9) but it increased the rate of pHi recovery with 0.6 mmol/L glucose/5 mmol/L fructose (change, 4.0±2.2 fluorescent units/s; P<0.02; n=6). We conclude that fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. This mechanism is likely dependent on protein kinase C. These results may partially explain the mechanism by which a fructose diet induces hypertension.

  6. Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro

    PubMed Central

    Kolman, Pavel; Pica, Angelo; Carvou, Nicolas; Boyde, Alan; Cockcroft, Shamshad; Loesch, Andrew; Pizzey, Arnold; Simeoni, Mariadelina; Capasso, Giovambattista; Unwin, Robert J.

    2009-01-01

    We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after ∼140 min) into the right jugular vein, and the fluorescence signal (at 650–670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30–40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 ± 0.26, mean ± SE, n = 10), indicating a “priming,” or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 ± 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand β2-glycoprotein. PMID:19261743

  7. Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro.

    PubMed

    Kolman, Pavel; Pica, Angelo; Carvou, Nicolas; Boyde, Alan; Cockcroft, Shamshad; Loesch, Andrew; Pizzey, Arnold; Simeoni, Mariadelina; Capasso, Giovambattista; Unwin, Robert J

    2009-05-01

    We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after approximately 140 min) into the right jugular vein, and the fluorescence signal (at 650-670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 +/- 0.26, mean +/- SE, n = 10), indicating a "priming," or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 +/- 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand beta2-glycoprotein.

  8. Na+-H+ exchanger-1 (NHE1) regulation in kidney proximal tubule.

    PubMed

    Parker, Mark D; Myers, Evan J; Schelling, Jeffrey R

    2015-06-01

    The ubiquitously expressed plasma membrane Na(+)-H(+) exchanger NHE1 is a 12 transmembrane-spanning protein that directs important cell functions such as homeostatic intracellular volume and pH control. The 315 amino acid cytosolic tail of NHE1 binds plasma membrane phospholipids and multiple proteins that regulate additional, ion-translocation independent functions. This review focuses on NHE1 structure/function relationships, as well as the role of NHE1 in kidney proximal tubule functions, including pH regulation, vectorial Na(+) transport, cell volume control and cell survival. The implications of these functions are particularly critical in the setting of progressive, albuminuric kidney diseases, where the accumulation of reabsorbed fatty acids leads to disruption of NHE1-membrane phospholipid interactions and tubular atrophy, which is a poor prognostic factor for progression to end stage renal disease. This review amplifies the vital role of the proximal tubule NHE1 Na(+)-H(+) exchanger as a kidney cell survival factor.

  9. Dopamine and angiotensin type 2 receptors cooperatively inhibit sodium transport in human renal proximal tubule cells.

    PubMed

    Gildea, John J; Wang, Xiaoli; Shah, Neema; Tran, Hanh; Spinosa, Michael; Van Sciver, Robert; Sasaki, Midori; Yatabe, Junichi; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2012-08-01

    Little is known regarding how the kidney shifts from a sodium and water reclaiming state (antinatriuresis) to a state where sodium and water are eliminated (natriuresis). In human renal proximal tubule cells, sodium reabsorption is decreased by the dopamine D(1)-like receptors (D(1)R/D(5)R) and the angiotensin type 2 receptor (AT(2)R), whereas the angiotensin type 1 receptor increases sodium reabsorption. Aberrant control of these opposing systems is thought to lead to sodium retention and, subsequently, hypertension. We show that D(1)R/D(5)R stimulation increased plasma membrane AT(2)R 4-fold via a D(1)R-mediated, cAMP-coupled, and protein phosphatase 2A-dependent specific signaling pathway. D(1)R/D(5)R stimulation also reduced the ability of angiotensin II to stimulate phospho-extracellular signal-regulated kinase, an effect that was partially reversed by an AT(2)R antagonist. Fenoldopam did not increase AT(2)R recruitment in renal proximal tubule cells with D(1)Rs uncoupled from adenylyl cyclase, suggesting a role of cAMP in mediating these events. D(1)Rs and AT(2)Rs heterodimerized and cooperatively increased cAMP and cGMP production, protein phosphatase 2A activation, sodium-potassium-ATPase internalization, and sodium transport inhibition. These studies shed new light on the regulation of renal sodium transport by the dopaminergic and angiotensin systems and potential new therapeutic targets for selectively treating hypertension.

  10. Development and Application of Human Renal Proximal Tubule Epithelial Cells for Assessment of Compound Toxicity

    PubMed Central

    Li, Shuaizhang; Zhao, Jinghua; Huang, Ruili; Steiner, Toni; Bourner, Maureen; Mitchell, Michael; Thompson, David C.; Zhao, Bin; Xia, Menghang

    2017-01-01

    Kidney toxicity is a major problem both in drug development and clinical settings. It is difficult to predict nephrotoxicity in part because of the lack of appropriate in vitro cell models, limited endpoints, and the observation that the activity of membrane transporters which plays important roles in nephrotoxicity by affecting the pharmacokinetic profile of drugs is often not taken into account. We developed a new cell model using pseudo-immortalized human primary renal proximal tubule epithelial cells. This cell line (SA7K) was characterized by the presence of proximal tubule cell markers as well as several functional properties, including transporter activity and response to a few well-characterized nephrotoxicants. We subsequently evaluated a group of potential nephrotoxic compounds in SA7K cells and compared them to a commonly used human immortalized kidney cell line (HK-2). Cells were treated with test compounds and three endpoints were analyzed, including cell viability, apoptosis and mitochondrial membrane potential. The results showed that most of the known nephrotoxic compounds could be detected in one or more of these endpoints. There were sensitivity differences in response to several of the chemicals between HK-2 and SA7K cells, which may relate to differences in expressions of key transporters or other components of nephrotoxicity pathways. Our data suggest that SA7K cells appear as promising for the early detection of renal toxicants.

  11. Megalin and cubilin in proximal tubule protein reabsorption: from experimental models to human disease.

    PubMed

    Nielsen, Rikke; Christensen, Erik Ilsø; Birn, Henrik

    2016-01-01

    Proximal tubule protein uptake is mediated by 2 receptors, megalin and cubilin. These receptors rescue a variety of filtered ligands, including biomarkers, essential vitamins, and hormones. Receptor gene knockout animal models have identified important functions of the receptors and have established their essential role in modulating urinary protein excretion. Rare genetic syndromes associated with dysfunction of these receptors have been identified and characterized, providing additional information on the importance of these receptors in humans. Using various disease models in combination with receptor gene knockout, the implications of receptor dysfunction in acute and chronic kidney injury have been explored and have pointed to potential new roles of these receptors. Based on data from animal models, this paper will review current knowledge on proximal tubule endocytic receptor function and regulation, and their role in renal development, protein reabsorption, albumin uptake, and normal renal physiology. These findings have implications for the pathophysiology and diagnosis of proteinuric renal diseases. We will examine the limitations of the different models and compare the findings to phenotypic observations in inherited human disorders associated with receptor dysfunction. Furthermore, evidence from receptor knockout mouse models as well as human observations suggesting a role of protein receptors for renal disease will be discussed in light of conditions such as chronic kidney disease, diabetes, and hypertension.

  12. Short-term functional adaptation of aquaporin-1 surface expression in the proximal tubule, a component of glomerulotubular balance.

    PubMed

    Pohl, Marcus; Shan, Qixian; Petsch, Thomas; Styp-Rekowska, Beata; Matthey, Patricia; Bleich, Markus; Bachmann, Sebastian; Theilig, Franziska

    2015-06-01

    Transepithelial water flow across the renal proximal tubule is mediated predominantly by aquaporin-1 (AQP1). Along this nephron segment, luminal delivery and transepithelial reabsorption are directly coupled, a phenomenon called glomerulotubular balance. We hypothesized that the surface expression of AQP1 is regulated by fluid shear stress, contributing to this effect. Consistent with this finding, we found that the abundance of AQP1 in brush border apical and basolateral membranes was augmented >2-fold by increasing luminal perfusion rates in isolated, microperfused proximal tubules for 15 minutes. Mouse kidneys with diminished endocytosis caused by a conditional deletion of megalin or the chloride channel ClC-5 had constitutively enhanced AQP1 abundance in the proximal tubule brush border membrane. In AQP1-transfected, cultured proximal tubule cells, fluid shear stress or the addition of cyclic nucleotides enhanced AQP1 surface expression and concomitantly diminished its ubiquitination. These effects were also associated with an elevated osmotic water permeability. In sum, we have shown that luminal surface expression of AQP1 in the proximal tubule brush border membrane is regulated in response to flow. Cellular trafficking, endocytosis, an intact endosomal compartment, and controlled protein stability are the likely prerequisites for AQP1 activation by enhanced tubular fluid shear stress, serving to maintain glomerulotubular balance.

  13. Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis.

    PubMed

    Khan, Shenaz; Abu Jawdeh, Bassam G; Goel, Monu; Schilling, William P; Parker, Mark D; Puchowicz, Michelle A; Yadav, Satya P; Harris, Raymond C; El-Meanawy, Ashraf; Hoshi, Malcolm; Shinlapawittayatorn, Krekwit; Deschênes, Isabelle; Ficker, Eckhard; Schelling, Jeffrey R

    2014-03-01

    Chronic kidney disease progression can be predicted based on the degree of tubular atrophy, which is the result of proximal tubule apoptosis. The Na+/H+ exchanger NHE1 regulates proximal tubule cell survival through interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but pathophysiologic triggers for NHE1 inactivation are unknown. Because glomerular injury permits proximal tubule luminal exposure and reabsorption of fatty acid/albumin complexes, we hypothesized that accumulation of amphipathic, long-chain acyl-CoA (LC-CoA) metabolites stimulates lipoapoptosis by competing with the structurally similar PI(4,5)P2 for NHE1 binding. Kidneys from mouse models of progressive, albuminuric kidney disease exhibited increased fatty acids, LC-CoAs, and caspase-2-dependent proximal tubule lipoapoptosis. LC-CoAs and the cytosolic domain of NHE1 directly interacted, with an affinity comparable to that of the PI(4,5)P2-NHE1 interaction, and competing LC-CoAs disrupted binding of the NHE1 cytosolic tail to PI(4,5)P2. Inhibition of LC-CoA catabolism reduced NHE1 activity and enhanced apoptosis, whereas inhibition of proximal tubule LC-CoA generation preserved NHE1 activity and protected against apoptosis. Our data indicate that albuminuria/lipiduria enhances lipotoxin delivery to the proximal tubule and accumulation of LC-CoAs contributes to tubular atrophy by severing the NHE1-PI(4,5)P2 interaction, thereby lowering the apoptotic threshold. Furthermore, these data suggest that NHE1 functions as a metabolic sensor for lipotoxicity.

  14. Localization of the calcium-regulated citrate transport process in proximal tubule cells.

    PubMed

    Hering-Smith, Kathleen S; Mao, Weibo; Schiro, Faith R; Coleman-Barnett, Joycelynn; Pajor, Ana M; Hamm, L Lee

    2014-06-01

    Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical >basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However, by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles.

  15. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    PubMed

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage.

  16. Complement activation and kidney injury molecule-1-associated proximal tubule injury in severe preeclampsia.

    PubMed

    Burwick, Richard M; Easter, Sarah Rae; Dawood, Hassan Y; Yamamoto, Hidemi S; Fichorova, Raina N; Feinberg, Bruce B

    2014-10-01

    Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case-control study of pregnant women from Brigham and Women's Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, β2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase-associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1.

  17. A Model of Peritubular Capillary Control of Isotonic Fluid Reabsorption by the Renal Proximal Tubule

    PubMed Central

    Deen, W. M.; Robertson, C. R.; Brenner, B. M.

    1973-01-01

    A mathematical model of peritubular transcapillary fluid exchange has been developed to investigate the role of the peritubular environment in the regulation of net isotonic fluid transport across the mammalian renal proximal tubule. The model, derived from conservation of mass and the Starling transcapillary driving forces, has been used to examine the quantitative effects on proximal reabsorption of changes in efferent arteriolar protein concentration and plasma flow rate. Under normal physiological conditions, relatively small perturbations in protein concentration are predicted to influence reabsorption more than even large variations in plasma flow, a prediction in close accord with recent experimental observations in the rat and dog. Changes either in protein concentration or plasma flow have their most pronounced effects when the opposing transcapillary hydrostatic and osmotic pressure differences are closest to equilibrium. Comparison of these theoretical results with variations in reabsorption observed in micropuncture studies makes it possible to place upper and lower bounds on the difference between interstitial oncotic and hydrostatic pressures in the renal cortex of the rat. PMID:4696761

  18. Subapical localization of the dopamine D3 receptor in proximal tubules of the rat kidney.

    PubMed

    Nürnberger, Asja; Räbiger, Marcus; Mack, Andreas; Diaz, Jorge; Sokoloff, Pierre; Mühlbauer, Bernd; Luippold, Gerd

    2004-12-01

    The dopamine D3 receptor (D3R), intensively studied in neuroscience, also plays an important role in the regulation of renal and cardiovascular function. In contrast to functional findings, less information is available on its localization in the kidney. Neither RT-PCR studies nor radioligand binding assays are suitable to selectively determine the distribution of renal D3R at the level of cellular or even subcellular structures. We studied the renal D3R distribution in Sprague-Dawley rats by a polyclonal antiserum directed against an epitope in the third intracytoplasmic loop. D3R immunoreactivity was detected by indirect immunofluorescence and confocal laser scanning microscopy. D3R staining was confined to the renal cortex and occurred in proximal convoluted tubules near or in direct connection with the urinary pole of the glomeruli. The fluorescent spots were restricted to the subapical portion of the proximal tubular cells. Double staining with the F-actin marker phalloidin revealed a localization of the D3R below the brush border region. However, staining by anti-beta1/beta2-adaptins, recognizing clathrin-coated compartments, did not correspond to the distribution of the D3R signal. This is the first description of a D3R accumulation in a cytoplasmic pool in the kidney, probably corresponding to a recycling mechanism or storage compartment.

  19. Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

    PubMed

    Terryn, Sara; Tanaka, Karo; Lengelé, Jean-Philippe; Olinger, Eric; Dubois-Laforgue, Danièle; Garbay, Serge; Kozyraki, Renata; Van Der Smissen, Patrick; Christensen, Erik I; Courtoy, Pierre J; Bellanné-Chantelot, Christine; Timsit, José; Pontoglio, Marco; Devuyst, Olivier

    2016-05-01

    Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of β2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A.

  20. Free-flow reabsorption of glucose, sodium, osmoles and water in rat proximal convoluted tubule.

    PubMed Central

    Bishop, J H; Green, R; Thomas, S

    1979-01-01

    1. Reabsorption of glucose, sodium, total solute (osmoles) and water in the rat proximal tubule (pars convoluta) were studied by free-flow micropuncture at normal (saline-infused), suppressed (saline with phlorizin) and elevated (glucose infusion) glucose reabsorption rates. 2. Phlorizin completely inhibited net glucose reabsorption, approximately halved reabsorption of sodium, total solutes and water, and reduced single nephron glomerular filtration rate (SNGFR). 3. In saline and glucose-infused groups, there were no significant differences between SNGFR nor between reabsorptions (fractional and absolute) of either sodium, total solute or water, which were uniformly distributed along segments assessible to micropuncture. 4. Glucose reabsorptive capacity existed along the entire pars convoluta, with highest reabsorptive rates in convolutions closest to the glomerulus (in saline-infused rats, 90% fractional reabsorption at 2 mm, over 95% at end pars convoluta; in glucose-infused rats, 55 and 90%, respectively). 5. In saline and glucose infused rats, a significant correlation existed between net glucose and sodium reabsorption, but the regression slopes differed and correlations became non-significant when the reabsorptive fluxes were factored by SNGFR. 6. For all groups, the majority of tubular fluid (TF) concentrations of osmoles and sodium were lower than those in plasma (over-all mean TFosm)Posm = 0.973 +/- 0.004, P less than 0.001; TFNa /PNa = 0.964 +/- 0.005, P less than 0.001). 7. Correspondingly, calculated osmolal and sodium concentrations in the reabsorbate were greater than those in plasma, and were significantly correlated with distance to puncture site with maximal values in the most proximal convolutions (for osmolality, approximately +79 m-osmole kg-1 water at 1 mm). PMID:469722

  1. Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells

    PubMed Central

    Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Anwar, Muhammad Ayaz; Choi, Sangdun

    2016-01-01

    Etoposide (ETO) is a commonly used chemotherapeutic drug that inhibits topoisomerase II activity, thereby leading to genotoxicity and cytotoxicity. However, ETO has limited application due to its side effects on normal organs, especially the kidney. Here, we report the mechanism of ETO-induced cytotoxicity progression in human kidney proximal tubule (HK-2) cells. Our results show that ETO perpetuates DNA damage, activates mitogen-activated protein kinase (MAPK), and triggers morphological changes, such as cell and nuclear swelling. When NAC, a well-known reactive oxygen species (ROS) scavenger, is co-treated with ETO, it inhibits an ETO-induced increase in mitochondrial mass, mitochondrial DNA (ND1 and ND4) copy number, intracellular ATP level, and mitochondrial biogenesis activators (TFAM, PGC-1α and PGC-1β). Moreover, co-treatment with ETO and NAC inhibits ETO-induced necrosis and cell swelling, but not apoptosis. Studies using MAPK inhibitors reveal that inhibition of extracellular signal regulated kinase (ERK) protects ETO-induced cytotoxicity by inhibiting DNA damage and caspase 3/7 activity. Eventually, ERK inhibitor treated cells are protected from ETO-induced nuclear envelope (NE) rupture and DNA leakage through inhibition of caspase activity. Taken together, these data suggest that ETO mediates cytotoxicity in HK-2 cells through ROS and ERK pathways, which highlight the preventive avenues in ETO-induced cytotoxicity in kidney. PMID:27666530

  2. Handling of Drugs, Metabolites, and Uremic Toxins by Kidney Proximal Tubule Drug Transporters

    PubMed Central

    Wu, Wei; Bush, Kevin T.; Hoenig, Melanie P.; Blantz, Roland C.; Bhatnagar, Vibha

    2015-01-01

    The proximal tubule of the kidney plays a crucial role in the renal handling of drugs (e.g., diuretics), uremic toxins (e.g., indoxyl sulfate), environmental toxins (e.g., mercury, aristolochic acid), metabolites (e.g., uric acid), dietary compounds, and signaling molecules. This process is dependent on many multispecific transporters of the solute carrier (SLC) superfamily, including organic anion transporter (OAT) and organic cation transporter (OCT) subfamilies, and the ATP-binding cassette (ABC) superfamily. We review the basic physiology of these SLC and ABC transporters, many of which are often called drug transporters. With an emphasis on OAT1 (SLC22A6), the closely related OAT3 (SLC22A8), and OCT2 (SLC22A2), we explore the implications of recent in vitro, in vivo, and clinical data pertinent to the kidney. The analysis of murine knockouts has revealed a key role for these transporters in the renal handling not only of drugs and toxins but also of gut microbiome products, as well as liver-derived phase 1 and phase 2 metabolites, including putative uremic toxins (among other molecules of metabolic and clinical importance). Functional activity of these transporters (and polymorphisms affecting it) plays a key role in drug handling and nephrotoxicity. These transporters may also play a role in remote sensing and signaling, as part of a versatile small molecule communication network operative throughout the body in normal and diseased states, such as AKI and CKD. PMID:26490509

  3. Cell swelling, co-transport activation and potassium conductance in isolated perfused rabbit kidney proximal tubules.

    PubMed Central

    Beck, J S; Potts, D J

    1990-01-01

    1. Isolated, perfused rabbit proximal tubules were used to study the effects of activation of the apical membrane sodium co-transporters, and of the effects of osmotically induced cell swelling, upon cell volume, basolateral membrane potential and apparent partial conductance of potassium. 2. Activation of electrogenic apical sodium co-transport caused a depolarization of the basolateral membrane and a reduction of the basolateral apparent potassium transference number. This was followed by a spontaneous partial recovery of potential and increase in apparent potassium transference number. 3. Stimulation of apical sodium co-transport led to a sustained increase in cell volume. 4. A sustained increase in cell volume (of similar magnitude to that seen after activation of apical membrane sodium co-transporters) was also caused by reduction of bath and perfusate osmolality by removal of 89 mmol l-1 mannitol from both lumen and bath solutions. 5. This reduction in bath and perfusate osmolality also led to a basolateral membrane hyperpolarization and an increase in basolateral apparent potassium transference number. 6. These observations support the possibility that some of the partial recovery of basolateral membrane potential (Vb1) during apical sodium co-transport stimulation is due to a cell volume sensitive change in basolateral potassium conductance. PMID:2213582

  4. Acute leptin exposure reduces megalin expression and upregulates TGFβ1 in cultured renal proximal tubule cells.

    PubMed

    Briffa, Jessica F; Grinfeld, Esther; Mathai, Michael L; Poronnik, Phillip; McAinch, Andrew J; Hryciw, Deanne H

    2015-02-05

    Increased leptin concentrations observed in obesity can lead to proteinuria, suggesting that leptin may play a role in obesity-related kidney disease. Obesity reduces activation of AMP-activated protein kinase (AMPK) and increases transforming growth factor-β1 (TGF-β1) expression in the kidney, leading to albuminuria. Thus we investigated if elevated leptin altered AMPK and TGF-β1 signaling in proximal tubule cells (PTCs). In opossum kidney (OK) PTCs Western blot analysis demonstrated that leptin upregulates TGF-β1 secretion (0.50 µg/ml) and phosphorylated AMPKα (at 0.25, and 0.50 µg/ml), and downregulates megalin expression at all concentrations (0.05-0.50 µg/ml). Using the AMPK inhibitor, Compound C, leptin exposure regulated TGF-β1 expression and secretion in PTCs via an AMPK mediated pathway. In addition, elevated leptin exposure (0.50 µg/ml) reduced albumin handling in OK cells independently of megalin expression. This study demonstrates that leptin upregulates TGF-β1, reduces megalin, and reduces albumin handling in PTCs by an AMPK mediated pathway.

  5. Short term exposure to elevated levels of leptin reduces proximal tubule cell metabolic activity.

    PubMed

    Briffa, Jessica F; Grinfeld, Esther; McAinch, Andrew J; Poronnik, Philip; Hryciw, Deanne H

    2014-01-25

    Leptin plays a pathophysiological role in the kidney, however, its acute effects on the proximal tubule cells (PTCs) are unknown. In opossum kidney (OK) cells in vitro, Western blot analysis identified that exposure to leptin increases the phosphorylation of the mitogen-activated protein kinase (MAPK) p44/42 and the mammalian target of rapamycin (mTOR). Importantly leptin (0.05, 0.10, 0.25 and 0.50 μg/ml) significantly reduced the metabolic activity of PTCs, and significantly decreased protein content per cell. Investigation of the role of p44/42 and mTOR on metabolic activity and protein content per cell, demonstrated that in the presence of MAPK inhibitor U0126 and mTOR inhibitor Ku-63794, that the mTOR pathway is responsible for the reduction in PTC metabolic activity in response to leptin. However, p44/42 and mTOR play no role the reduced protein content per cell in OKs exposed to leptin. Therefore, leptin modulates metabolic activity in PTCs via an mTOR regulated pathway.

  6. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism.

    PubMed

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Lamers, Wouter H; Chaudhry, Farrukh A; Verlander, Jill W; Weiner, I David

    2016-06-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na(+)-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.

  7. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

    PubMed

    Jansen, J; De Napoli, I E; Fedecostante, M; Schophuizen, C M S; Chevtchik, N V; Wilmer, M J; van Asbeck, A H; Croes, H J; Pertijs, J C; Wetzels, J F M; Hilbrands, L B; van den Heuvel, L P; Hoenderop, J G; Stamatialis, D; Masereeuw, R

    2015-11-16

    The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

  8. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

    PubMed Central

    Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

    2015-01-01

    The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

  9. The Endocytic Receptor Megalin and its Associated Proteins in Proximal Tubule Epithelial Cells

    PubMed Central

    De, Shankhajit; Kuwahara, Shoji; Saito, Akihiko

    2014-01-01

    Receptor-mediated endocytosis in renal proximal tubule epithelial cells (PTECs) is important for the reabsorption and metabolization of proteins and other substances, including carrier-bound vitamins and trace elements, in glomerular filtrates. Impairment of this endocytic process results in the loss of such substances and development of proteinuria, which is an important clinical indicator of kidney diseases and is also a risk marker for cardiovascular disease. Megalin, a member of the low-density lipoprotein receptor gene family, is a multiligand receptor expressed in the apical membrane of PTECs and plays a central role in the endocytic process. Megalin interacts with various intracellular adaptor proteins for intracellular trafficking and cooperatively functions with other membrane molecules, including the cubilin-amnionless complex. Evidence suggests that megalin and the cubilin-amnionless complex are involved in the uptake of toxic substances into PTECs, which leads to the development of kidney disease. Studies of megalin and its associated molecules will be useful for future development of novel strategies for the diagnosis and treatment of kidney diseases. PMID:25019425

  10. In vitro safety assessment of food ingredients in canine renal proximal tubule cells.

    PubMed

    Koči, J; Jeffery, B; Riviere, J E; Monteiro-Riviere, N A

    2015-03-01

    In vitro models are useful tools to initially assess the toxicological safety hazards of food ingredients. Toxicities of cinnamaldehyde (CINA), cinnamon bark oil, lemongrass oil (LGO), thymol, thyme oil (TO), clove leaf oil, eugenol, ginger root extract (GRE), citric acid, guanosine monophosphate, inosine monophosphate and sorbose (SORB) were assessed in canine renal proximal tubule cells (CPTC) using viability assay and renal injury markers. At LC50, CINA was the most toxic (0.012mg/ml), while SORB the least toxic (>100mg/ml). Toxicities (LC50) of positive controls were as follows: 4-aminophenol (0.15mg/ml in CPTC and 0.083mg/ml in human PTC), neomycin (28.6mg/ml in CPTC and 27.1mg/ml in human PTC). XYL displayed lowest cytotoxic potency (LC50=82.7mg/ml in CPTC). In vivo renal injury markers in CPTC were not significantly different from controls. The LGO toxicity mechanism was analyzed using qPCR and electron microscopy. Out of 370 genes, 57 genes (15.4%) were significantly up (34, 9.1%) or down (23, 6.2%) regulated, with the most upregulated gene gsta3 (∼200-fold) and the most affected pathway being oxidative stress. LGO induced damage of mitochondria, phospholipid accumulation and lack of a brush border. Viability assays along with mechanistic studies in the CPTC model may serve as a valuable in vitro toxicity screening tool.

  11. Deleting the TGF-β receptor in proximal tubules impairs HGF signaling.

    PubMed

    Nlandu Khodo, Stellor; Neelisetty, Surekha; Woodbury, Luke; Green, Elizabeth; Harris, Raymond C; Zent, Roy; Gewin, Leslie

    2016-03-15

    Transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) play key roles in regulating the response to renal injury but are thought to mediate divergent effects on cell behavior. However, how TGF-β signaling alters the response to HGF in epithelia, the key site of HGF signaling in the injured kidney, is not well studied. Contrary to our expectation, we showed that deletion of the TGF-β type II receptor in conditionally immortalized proximal tubule (PT) cells impaired HGF-dependent signaling. This reduced signaling was due to decreased transcription of c-Met, the HGF receptor, and the TGF-β-dependent c-Met transcription and increased response to HGF in PT cells were mediated by the Notch pathway. The interactions of TGF-β, HGF, and Notch pathways had biologically significant effects on branching morphogenesis, cell morphology, migration, and proliferation. In conclusion, epithelial TGF-β signaling promotes HGF signaling in a Notch-dependent pathway. These findings suggest that TGF-β modulates PT responses not only by direct effects, but also by affecting other growth factor signaling pathways.

  12. Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane

    PubMed Central

    Marks, Joanne; Carvou, Nicolas J C; Debnam, Edward S; Srai, Surjit K; Unwin, Robert J

    2003-01-01

    The mechanism of renal glucose transport involves the reabsorption of filtered glucose from the proximal tubule lumen across the brush border membrane (BBM) via a sodium-dependent transporter, SGLT, and exit across the basolateral membrane via facilitative, GLUT-mediated, transport. The aim of the present study was to determine the effect of streptozotocin-induced diabetes on BBM glucose transport. We found that diabetes increased facilitative glucose transport at the BBM by 67.5 % (P < 0.05) – an effect that was abolished by overnight fasting. Western blotting and immunohistochemistry demonstrated GLUT2 expression at the BBM during diabetes, but the protein was undetectable at the BBM of control animals or diabetic animals that had been fasted overnight. Our findings indicate that streptozotocin-induced diabetes causes the insertion of GLUT2 into the BBM and this may provide a low affinity/high capacity route of entry into proximal tubule cells during hyperglycaemia. PMID:12963802

  13. Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane.

    PubMed

    Marks, Joanne; Carvou, Nicolas J C; Debnam, Edward S; Srai, Surjit K; Unwin, Robert J

    2003-11-15

    The mechanism of renal glucose transport involves the reabsorption of filtered glucose from the proximal tubule lumen across the brush border membrane (BBM) via a sodium-dependent transporter, SGLT, and exit across the basolateral membrane via facilitative, GLUT-mediated, transport. The aim of the present study was to determine the effect of streptozotocin-induced diabetes on BBM glucose transport. We found that diabetes increased facilitative glucose transport at the BBM by 67.5 % (P < 0.05)--an effect that was abolished by overnight fasting. Western blotting and immunohistochemistry demonstrated GLUT2 expression at the BBM during diabetes, but the protein was undetectable at the BBM of control animals or diabetic animals that had been fasted overnight. Our findings indicate that streptozotocin-induced diabetes causes the insertion of GLUT2 into the BBM and this may provide a low affinity/high capacity route of entry into proximal tubule cells during hyperglycaemia.

  14. Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis

    PubMed Central

    Mei, Shuqin; Li, Lin; Wei, Qingqing; Hao, Jielu; Su, Yunchao; Mei, Changlin; Dong, Zheng

    2017-01-01

    Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases. PMID:28317867

  15. Ginsenosides protect apical transporters of cultured proximal tubule cells from dysfunctions induced by h(2)o(2).

    PubMed

    Han, Ho Jae; Yoon, Byung Cheol; Park, Soo Hyun; Park, Ji Yeong; Oh, Young Joon; Lee, Yun Jung; Park, Kwon Moo

    2002-01-01

    Oxidative stress has been implicated as a primary cause of renal failure in certain renal diseases. Indeed, renal proximal tubule is a very sensitive site to oxidative stress and retains functionally fully characterized transporters. It has been reported that ginsenosides have a beneficial effect on diverse diseases including oxidative stress. However, the protective effect of ginsenosides on oxidative stress has not been elucidated in renal proximal tubule cells. Thus, we examined the effect of ginsenosides on oxidative stress-induced alteration of apical transporters and its related mechanism in renal proximal tubule cells. In the present study, hydrogen peroxide (H(2)O(2)) (>10(-5) M) inhibited alpha-methyl-D-glucopyranoside uptake in a dose-dependent manner (p < 0.05). It also inhibited Pi and Na(+) uptake. At a concentration of 20 microg/ml, total ginsenosides significantly reduced H(2)O(2)-induced inhibition of apical transporters. In contrast, protopanaxadiol (PD) and protopanaxatriol (PT) saponins exhibited a less preventive effect than total ginsenosides (p < 0.05). Furthermore, we examined its action mechanism. H(2)O(2) increased lipid peroxide formation, arachidonic acid (AA) release, and Ca(2+) uptake. These effects on H(2)O(2) were significantly prevented by total ginsenosides and PD or PT sanponins. However, total ginsenosides appear to be more protective than PD and PT saponins (p < 0.05). In conclusion, ginsenosides prevented H(2)O(2)-induced inhibition of apical transporters via a decrease in oxidative stress, AA release, and Ca(2+) uptake in primary cultured renal proximal tubule cells.

  16. Local pH domains regulate NHE3-mediated Na⁺ reabsorption in the renal proximal tubule.

    PubMed

    Brasen, Jens Christian; Burford, James L; McDonough, Alicia A; Holstein-Rathlou, Niels-Henrik; Peti-Peterdi, Janos

    2014-12-01

    The proximal tubule Na(+)/H(+) exchanger 3 (NHE3), located in the apical dense microvilli (brush border), plays a major role in the reabsorption of NaCl and water in the renal proximal tubule. In response to a rise in blood pressure NHE3 redistributes in the plane of the plasma membrane to the base of the brush border, where NHE3 activity is reduced. This NHE3 redistribution is assumed to provoke pressure natriuresis; however, it is unclear how NHE3 redistribution per se reduces NHE3 activity. To investigate if the distribution of NHE3 in the brush border can change the reabsorption rate, we constructed a spatiotemporal mathematical model of NHE3-mediated Na(+) reabsorption across a proximal tubule cell and compared the model results with in vivo experiments in rats. The model predicts that when NHE3 is localized exclusively at the base of the brush border, it creates local pH microdomains that reduce NHE3 activity by >30%. We tested the model's prediction experimentally: the rat kidney cortex was loaded with the pH-sensitive fluorescent dye BCECF, and cells of the proximal tubule were imaged in vivo using confocal fluorescence microscopy before and after an increase of blood pressure by ∼50 mmHg. The experimental results supported the model by demonstrating that a rise of blood pressure induces the development of pH microdomains near the bottom of the brush border. These local changes in pH reduce NHE3 activity, which may explain the pressure natriuresis response to NHE3 redistribution.

  17. Proteomic profiling and pathway analysis of the response of rat renal proximal convoluted tubules to metabolic acidosis.

    PubMed

    Schauer, Kevin L; Freund, Dana M; Prenni, Jessica E; Curthoys, Norman P

    2013-09-01

    Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis.

  18. Mechanisms of Cadmium-Induced Proximal Tubule Injury: New Insights with Implications for Biomonitoring and Therapeutic Interventions

    PubMed Central

    Edwards, Joshua R.

    2012-01-01

    Cadmium is an important industrial agent and environmental pollutant that is a major cause of kidney disease. With chronic exposure, cadmium accumulates in the epithelial cells of the proximal tubule, resulting in a generalized reabsorptive dysfunction characterized by polyuria and low-molecular-weight proteinuria. The traditional view has been that as cadmium accumulates in proximal tubule cells, it produces a variety of relatively nonspecific toxic effects that result in the death of renal epithelial cells through necrotic or apoptotic mechanisms. However, a growing volume of evidence suggests that rather than merely being a consequence of cell death, the early stages of cadmium-induced proximal tubule injury may involve much more specific changes in cell-cell adhesion, cellular signaling pathways, and autophagic responses that occur well before the onset of necrosis or apoptosis. In this commentary, we summarize these recent findings, and we offer our own perspectives as to how they relate to the toxic actions of cadmium in the kidney. In addition, we highlight recent findings, suggesting that it may be possible to detect the early stages of cadmium toxicity through the use of improved biomarkers. Finally, some of the therapeutic implications of these findings will be considered. Because cadmium is, in many respects, a model cumulative nephrotoxicant, these insights may have broader implications regarding the general mechanisms through which a variety of drugs and toxic chemicals damage the kidney. PMID:22669569

  19. Hormone-specific regulation of the kidney androgen-regulated gene promoter in cultured mouse renal proximal-tubule cells.

    PubMed Central

    Soler, Montse; Tornavaca, Olga; Solé, Esther; Menoyo, Anna; Hardy, Dianne; Catterall, James F; Vandewalle, Alain; Meseguer, Anna

    2002-01-01

    The kidney androgen-regulated protein (KAP) is specifically expressed and differentially regulated by androgens and tri-iodothyronine (T(3)) in intact mouse early (PCT) and late (PR) proximal-tubule cells. Until now, detailed characterization of the molecular elements mediating androgen-responsive gene expression in the kidney has been hampered by the lack of appropriate cultured cell systems suitable for DNA transfection studies. In the present study we have analysed the hormone-dependent transactivation of the KAP gene promoter in immortalized differentiated PCT and PR proximal-tubule cells derived from L-PK/Tag1 transgenic mice. Transient transfection studies with different KAP promoter constructs indicated that a 224 bp-truncated fragment was sufficient to mediate cell-specific expression of the KAP promoter. Dihydrotestosterone (DHT) stimulated in an androgen-dependent manner the transactivation of KAP in PCT and PR cells, while mutation of a putative androgen-response element (ARE) sequence located at -39 bp from the transcription initiation site abolished the transactivation induced by DHT. Furthermore, insulin-like growth factor 1 (IGF-1), but not T(3), enhanced the androgen-dependent transactivation of KAP in cultured PCT cells. These results demonstrate that the short 224 bp fragment of the KAP promoter is sufficient to drive the proximal-tubule androgen-specific regulated expression of KAP and reveal synergistic interactions between IGF-1 and androgens for KAP regulation in PCT cells. PMID:12030848

  20. Role of proximal tubule in the hypocalciuric response to thiazide of patients with idiopathic hypercalciuria.

    PubMed

    Bergsland, Kristin J; Worcester, Elaine M; Coe, Fredric L

    2013-08-15

    The most common metabolic abnormality found in calcium (Ca) kidney stone formers is idiopathic hypercalciuria (IH). Using endogenous lithium (Li) clearance, we previously showed that in IH, there is decreased proximal tubule sodium absorption, and increased delivery of Ca into the distal nephron. Distal Ca reabsorption may facilitate the formation of Randall's plaque (RP) by washdown of excess Ca through the vasa recta toward the papillary tip. Elevated Ca excretion leads to increased urinary supersaturation (SS) with respect to calcium oxalate (CaOx) and calcium phosphate (CaP), providing the driving force for stone growth on RP. Thiazide (TZ) diuretics reduce Ca excretion and prevent stone recurrence, but the mechanism in humans is unknown. We studied the effect of chronic TZ administration on renal mineral handling in four male IH patients using a fixed three meal day in the General Clinical Research Center. Each subject was studied twice: once before treatment and once after 4-7 mo of daily chlorthalidone treatment. As expected, urine Ca fell with TZ, along with fraction of filtered Ca excreted. Fraction of filtered Li excreted also fell sharply with TZ, as did distal delivery of Ca. Unexpectedly, TZ lowered urine pH. Together with reduced urine Ca, this led to a marked fall in CaP SS, but not CaOx SS. Since CaOx stone formation begins with an initial CaP overlay on RP, by lowering urine pH and decreasing distal nephron Ca delivery, TZ might diminish stone risk both by reducing CaP SS, as well as slowing progression of RP.

  1. Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress.

    PubMed

    Kimura, Tomonori; Takahashi, Atsushi; Takabatake, Yoshitsugu; Namba, Tomoko; Yamamoto, Takeshi; Kaimori, Jun-Ya; Matsui, Isao; Kitamura, Harumi; Niimura, Fumio; Matsusaka, Taiji; Soga, Tomoyoshi; Rakugi, Hiromi; Isaka, Yoshitaka

    2013-11-01

    Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD(+)/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.

  2. MDR1 transporter protects against paraquat-induced toxicity in human and mouse proximal tubule cells.

    PubMed

    Wen, Xia; Gibson, Christopher J; Yang, Ill; Buckley, Brian; Goedken, Michael J; Richardson, Jason R; Aleksunes, Lauren M

    2014-10-01

    Paraquat is a herbicide that is highly toxic to the lungs and kidneys following acute exposures. Prior studies have demonstrated that the organic cation transporter 2 and multidrug and toxin extrusion protein 1 contribute to the urinary secretion of paraquat in the kidneys. The purpose of this study was to determine whether the multidrug resistance protein 1 (MDR1/Mdr1, ABCB1, or P-glycoprotein) also participates in the removal of paraquat from the kidneys and protects against renal injury. Paraquat transport and toxicity were quantified in human renal proximal tubule epithelial cells (RPTEC) that endogenously express MDR1, HEK293 cells overexpressing MDR1, and Mdr1a/1b knockout mice. In RPTEC cells, reduction of MDR1 activity using the antagonist PSC833 or siRNA transfection increased the cellular accumulation of paraquat by 50%. Reduced efflux of paraquat corresponded with enhanced cytotoxicity in PSC833-treated cells. Likewise, stable overexpression of the human MDR1 gene in HEK293 cells reduced intracellular levels of paraquat by 50%. In vivo studies assessed the renal accumulation and subsequent nephrotoxicity of paraquat (10 or 30 mg/kg ip) in wild-type and Mdr1a/1b knockout mice. At 4 h after paraquat treatment, renal concentrations of paraquat in the kidneys of Mdr1a/1b knockout mice were 750% higher than wild-type mice. By 72 h, paraquat-treated Mdr1a/1b knockout mice had more extensive tubular degeneration and significantly greater mRNA expression of kidney injury-responsive genes, including kidney injury molecule-1, lipocalin-2, and NAD(P)H quinone oxidoreductase 1, compared with wild-type mice. In conclusion, MDR1/Mdr1 participates in the elimination of paraquat from the kidneys and protects against subsequent toxicity.

  3. Substrate modulation of fatty acid effects on energization and respiration of kidney proximal tubules during hypoxia/reoxygenation.

    PubMed

    Bienholz, Anja; Al-Taweel, Ahmad; Roeser, Nancy F; Kribben, Andreas; Feldkamp, Thorsten; Weinberg, Joel M

    2014-01-01

    Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.

  4. In vivo study of transepithelial potential difference (TEPD) in proximal convoluted tubules of rat kidney by synchronization modulation electric field.

    PubMed

    Clausell, Mathis; Fang, Zhihui; Chen, Wei

    2014-07-01

    Synchronization modulation (SM) electric field has been shown to effectively activate function of Na(+)/K(+) pumps in various cells and tissues, including skeletal muscle cells, cardiomyocyte, monolayer of cultured cell line, and peripheral blood vessels. We are now reporting the in vivo studies in application of the SM electric field to kidney of living rats. The field-induced changes in the transepithelial potential difference (TEPD) or the lumen potential from the proximal convoluted tubules were monitored. The results showed that a short time (20 s) application of the SM electric field can significantly increase the magnitude of TEPD from 1-2 mV to about 20 mV. The TEPD is an active potential representing the transport current of the Na/K pumps in epithelial wall of renal tubules. This study showed that SM electric field can increase TEPD by activation of the pump molecules. Considering renal tubules, many active transporters are driven by the Na(+) concentration gradient built by the Na(+)/K(+) pumps, activation of the pump functions and increase in the magnitude of TEPD imply that the SM electric field may improve reabsorption functions of the renal tubules.

  5. Diquat induces renal proximal tubule injury in glutathione reductase-deficient mice

    SciTech Connect

    Rogers, Lynette K. . E-mail: rogersl@ccri.net; Bates, Carlton M.; Welty, Stephen E.; Smith, Charles V.

    2006-12-15

    Reactive oxygen species (ROS) have been associated with many human diseases, and glutathione (GSH)-dependent processes are pivotal in limiting tissue damage. To test the hypothesis that Gr1{sup a1Neu} (Neu) mice, which do not express glutathione reductase (GR), would be more susceptible than are wild-type mice to ROS-mediated injury, we studied the effects of diquat, a redox cycling toxicant. Neu mice exhibited modest, dose- and time-dependent elevations in plasma alanine aminotransferase (ALT) activities, 126 {+-} 36 U/l at 2 h after 5 {mu}mol/kg of diquat, but no ALT elevations were observed in diquat-treated C3H/HeN mice for up to 6 h after 50 {mu}mol/kg of diquat. Histology indicated little or no hepatic necrosis in diquat-treated mice of either strain, but substantial renal injury was observed in diquat-treated Neu mice, characterized by brush border sloughing in the proximal tubules by 1 h and tubular necrosis by 2 h after doses of 7.5 {mu}mol/kg. Decreases in renal GSH levels were observed in the Neu mice by 2 h post dose (3.4 {+-} 0.4 vs 0.2 {+-} 0.0 {mu}mol/g tissue at 0 and 50 {mu}mol/kg, respectively), and increases in renal GSSG levels were observed in the Neu mice as early as 0.5 h after 7.5 {mu}mol/kg (105.5 {+-} 44.1 vs 27.9 {+-} 4.8 nmol/g tissue). Blood urea nitrogen levels were elevated by 2 h in Neu mice after doses of 7.5 {mu}mol/kg (Neu vs C3H, 32.8 {+-} 4.1 vs 17.9 {+-} 0.3 mg/dl). Diquat-induced renal injury in the GR-deficient Neu mice offers a useful model for studies of ROS-induced renal necrosis and of the contributions of GR in defense against oxidant-mediated injuries in vivo.

  6. Thiazolidinediones enhance sodium-coupled bicarbonate absorption from renal proximal tubules via PPARγ-dependent nongenomic signaling.

    PubMed

    Endo, Yoko; Suzuki, Masashi; Yamada, Hideomi; Horita, Shoko; Kunimi, Motoei; Yamazaki, Osamu; Shirai, Ayumi; Nakamura, Motonobu; Iso-O, Naoyuki; Li, Yuehong; Hara, Masumi; Tsukamoto, Kazuhisa; Moriyama, Nobuo; Kudo, Akihiko; Kawakami, Hayato; Yamauchi, Toshimasa; Kubota, Naoto; Kadowaki, Takashi; Kume, Haruki; Enomoto, Yutaka; Homma, Yukio; Seki, George; Fujita, Toshiro

    2011-05-04

    Thiazolidinediones (TZDs) improve insulin resistance by activating a nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ). However, the use of TZDs is associated with plasma volume expansion through a mechanism that remains to be clarified. Here we showed that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from the renal proximal tubule in vitro and in vivo. TZD-induced transport stimulation is dependent on PPARγ-Src-EGFR-ERK and observed in rat, rabbit and human, but not in mouse proximal tubules where Src-EGFR is constitutively activated. The existence of PPARγ-Src-dependent nongenomic signaling, which requires the ligand-binding ability, but not the transcriptional activity of PPARγ, is confirmed in mouse embryonic fibroblast cells. The enhancement of the association between PPARγ and Src by TZDs supports an indispensable role of Src in this signaling. These results suggest that the PPARγ-dependent nongenomic stimulation of renal proximal transport is also involved in TZD-induced volume expansion.

  7. Angiotensin II counteracts the effects of cAMP/PKA on NHE3 activity and phosphorylation in proximal tubule cells.

    PubMed

    Crajoinas, Renato O; Polidoro, Juliano Z; Carneiro de Morais, Carla P A; Castelo-Branco, Regiane C; Girardi, Adriana C C

    2016-11-01

    Binding of angiotensin II (ANG II) to the AT1 receptor (AT1R) in the proximal tubule stimulates Na(+)/H(+) exchanger isoform 3 (NHE3) activity through multiple signaling pathways. However, the effects of ANG II/AT1R-induced inihibitory G protein (Gi) activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that ANG II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, ANG II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of the cAMP-elevating agent forskolin (FSK), ANG II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity, and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of ANG II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in ANG II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of ANG II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that ANG II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by ANG II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation.

  8. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    SciTech Connect

    Liu, Senyan; Yao, Yunyi; Lu, Shijun; Aldous, Kenneth; Ding, Xinxin; Mei, Changlin; Gu, Jun

    2013-10-01

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity with the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity.

  9. ATP is a coupling modulator of parallel Na,K-ATPase-K-channel activity in the renal proximal tubule.

    PubMed Central

    Tsuchiya, K; Wang, W; Giebisch, G; Welling, P A

    1992-01-01

    A fundamental and essential property of nearly all salt-transporting epithelia is the tight parallel coupling between the magnitude of the K-conductive pathway at the basolateral membrane and the activity of the Na,K-dependent ATPase (Na,K-ATPase). In the present study, we demonstrate that the coupling response in the renal proximal tubule is governed, at least in part, through the interaction between ATP-sensitive K channels and Na,K-ATPase-mediated changes in intracellular ATP levels. First, we identified a K-selective channel at the basolateral membrane, which is inhibited by the cytosolic addition of ATP. Second, conventional microelectrode analysis in the isolated perfused proximal straight tubule revealed that these channels are the major determinant of the macroscopic K conductance so that ATP-mediated changes in the open probability of the K channel could alter the extent of K recycling. Indeed, the increase in the macroscopic K conductance upon stimulation of transcellular Na transport and pump activity was found to be paralleled by a decrease in intracellular ATP. Finally, a causal link between parallel Na,K-ATPase-K-channel activity and ATP was established by the finding that intracellular ATP loading uncoupled the response. With our recent observations that similar ATP-sensitive K channels are expressed abundantly in other epithelia, we postulate that ATP may act as a universal coupling modulator of parallel Na,K-ATPase-K-channel activity. PMID:1321439

  10. Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells.

    PubMed

    Li, Min; Balamuthusamy, Saravanan; Simon, Eric E; Batuman, Vecihi

    2008-07-01

    Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to kappa-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring kappa-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled kappa-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

  11. Uremic Toxins Induce ET-1 Release by Human Proximal Tubule Cells, which Regulates Organic Cation Uptake Time-Dependently.

    PubMed

    Schophuizen, Carolien M S; Hoenderop, Joost G J; Masereeuw, Rosalinde; Heuvel, Lambert P van den

    2015-06-26

    In renal failure, the systemic accumulation of uremic waste products is strongly associated with the development of a chronic inflammatory state. Here, the effect of cationic uremic toxins on the release of inflammatory cytokines and endothelin-1 (ET-1) was investigated in conditionally immortalized proximal tubule epithelial cells (ciPTEC). Additionally, we examined the effects of ET-1 on the cellular uptake mediated by organic cation transporters (OCTs). Exposure of ciPTEC to cationic uremic toxins initiated production of the inflammatory cytokines IL-6 (117 ± 3%, p < 0.001), IL-8 (122 ± 3%, p < 0.001), and ET-1 (134 ± 5%, p < 0.001). This was accompanied by a down-regulation of OCT mediated 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP+) uptake in ciPTEC at 30 min (23 ± 4%, p < 0.001), which restored within 60 min of incubation. Exposure to ET-1 for 24 h increased the ASP+ uptake significantly (20 ± 5%, p < 0.001). These effects could be blocked by BQ-788, indicating activation of an ET-B-receptor-mediated signaling pathway. Downstream the receptor, iNOS inhibition by (N(G)-monomethyl-l-arginine) l-NMMA acetate or aminoguanidine, as well as protein kinase C activation, ameliorated the short-term effects. These results indicate that uremia results in the release of cytokines and ET-1 from human proximal tubule cells, in vitro. Furthermore, ET-1 exposure was found to regulate proximal tubular OCT transport activity in a differential, time-dependent, fashion.

  12. Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

    PubMed Central

    Yamamoto, Takeshi; Takabatake, Yoshitsugu; Kimura, Tomonori; Takahashi, Atsushi; Namba, Tomoko; Matsuda, Jun; Minami, Satoshi; Kaimori, Jun-ya; Matsui, Isao; Kitamura, Harumi; Matsusaka, Taiji; Niimura, Fumio; Yanagita, Motoko; Isaka, Yoshitaka; Rakugi, Hiromi

    2016-01-01

    ABSTRACT Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifen-inducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62- and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases. PMID:26986194

  13. Uremic Toxins Induce ET-1 Release by Human Proximal Tubule Cells, which Regulates Organic Cation Uptake Time-Dependently

    PubMed Central

    Schophuizen, Carolien M. S.; Hoenderop, Joost G. J.; Masereeuw, Rosalinde; van den Heuvel, Lambert P.

    2015-01-01

    In renal failure, the systemic accumulation of uremic waste products is strongly associated with the development of a chronic inflammatory state. Here, the effect of cationic uremic toxins on the release of inflammatory cytokines and endothelin-1 (ET-1) was investigated in conditionally immortalized proximal tubule epithelial cells (ciPTEC). Additionally, we examined the effects of ET-1 on the cellular uptake mediated by organic cation transporters (OCTs). Exposure of ciPTEC to cationic uremic toxins initiated production of the inflammatory cytokines IL-6 (117 ± 3%, p < 0.001), IL-8 (122 ± 3%, p < 0.001), and ET-1 (134 ± 5%, p < 0.001). This was accompanied by a down-regulation of OCT mediated 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP+) uptake in ciPTEC at 30 min (23 ± 4%, p < 0.001), which restored within 60 min of incubation. Exposure to ET-1 for 24 h increased the ASP+ uptake significantly (20 ± 5%, p < 0.001). These effects could be blocked by BQ-788, indicating activation of an ET-B-receptor-mediated signaling pathway. Downstream the receptor, iNOS inhibition by (N(G)‐monomethyl‐l‐arginine) l-NMMA acetate or aminoguanidine, as well as protein kinase C activation, ameliorated the short-term effects. These results indicate that uremia results in the release of cytokines and ET-1 from human proximal tubule cells, in vitro. Furthermore, ET-1 exposure was found to regulate proximal tubular OCT transport activity in a differential, time-dependent, fashion. PMID:26132391

  14. Bicarbonate-water interactions in the rat proximal convoluted tubule. An effect of volume flux on active proton secretion

    PubMed Central

    1984-01-01

    The effect of volume absorption on bicarbonate absorption was examined in the in vivo perfused rat proximal convoluted tubule. Volume absorption was inhibited by isosmotic replacement of luminal NaCl with raffinose. In tubules perfused with 25 mM bicarbonate, as raffinose was increased from 0 to 55 to 63 mM, volume absorption decreased from 2.18 +/- 0.10 to 0.30 +/- 0.18 to -0.66 +/- 0.30 nl/mm X min, respectively, and bicarbonate absorption decreased from 131 +/- 5 to 106 +/- 8 to 91 +/- 13 pmol/mm X min, respectively. This bicarbonate-water interaction could not be attributed to dilutional changes in luminal or peritubular bulk phase bicarbonate concentrations. Inhibition of active proton secretion by acetazolamide abolished the effect of volume flow on bicarbonate absorption, which implies that the bicarbonate reflection coefficient is close to 1 and eliminates the possibility of solvent drag across the tight junction. When the luminal bicarbonate concentration was varied, the magnitude of the bicarbonate-water interaction increased with increasing luminal bicarbonate concentration. The largest interaction occurred at high luminal bicarbonate concentrations, where the rate of proton secretion has been previously shown to be independent of luminal bicarbonate concentration and pH. The results thus suggest that a peritubular and/or cellular compartment exists that limits bicarbonate diffusion, and where pH changes secondary to bicarbonate-water interactions (solute polarization) alter the rate of active proton secretion. PMID:6096481

  15. Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule

    PubMed Central

    Walmsley, Scott J.; Freund, Dana M.

    2012-01-01

    The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH4+ and HCO3− ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMVPCT) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na+-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na+-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMVPCT isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting. PMID:22357915

  16. Proximal tubule-derived Colony Stimulating Factor-1 mediates polarization of renal macrophages and dendritic cells, and recovery in acute kidney injury

    PubMed Central

    Wang, Yinqiu; Chang, Jian; Yao, Bing; Niu, Aolei; Kelly, Emily; Breeggemann, Matthew C.; Abboud Werner, Sherry L.; Harris, Raymond C.; Zhang, Ming-Zhi

    2015-01-01

    Infiltrating cells play an important role in both the development of and recovery from acute kidney injury (AKI). Macrophages and renal dendritic cells are of particular interest because they can exhibit distinctly different functional phenotypes, broadly characterized as proinflammatory (M1) or tissue reparative (M2). Resident renal macrophages and dendritic cells participate in recovery from AKI in response to either ischemia/reperfusion or a model of selective proximal tubule injury induced by diphtheria toxin-induced apoptosis in transgenic mice expressing the human diphtheria toxin receptor on proximal tubule cells. Colony Stimulating Factor-1 (CSF-1) is an important factor mediating the recovery from AKI, and CSF-1 can stimulate macrophage and dendritic cell proliferation and polarization during the recovery phase of AKI. The kidney, and specifically the proximal tubule, is a major source of intrarenal CSF-1 production in response to AKI. We induced selective deletion of proximal tubule CSF-1 to determine its role in expansion and proliferation of renal macrophages and dendritic cells and in recovery from AKI. In both models of AKI, there was decreased M2 polarization, delayed functional and structural recovery and increased tubulointerstitial fibrosis. Thus, intrarenal CSF-1 is an important mediator of macrophage/dendritic cell polarization and recovery from AKI. PMID:26422503

  17. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed Central

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-01-01

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  18. 5-Lypoxygenase Products Are Involved in Renal Tubulointerstitial Injury Induced by Albumin Overload in Proximal Tubules in Mice

    PubMed Central

    Landgraf, Sharon Schilling; Silva, Leandro Souza; Peruchetti, Diogo Barros; Sirtoli, Gabriela Modenesi; Moraes-Santos, Felipe; Portella, Viviane Gomes; Silva-Filho, João Luiz; Pinheiro, Carla Silva; Abreu, Thiago Pereira; Takiya, Christina Maeda; Benjamin, Claudia Farias; Pinheiro, Ana Acacia Sá; Canetti, Claudio; Caruso-Neves, Celso

    2014-01-01

    The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO–/–). The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO–/– mice. The levels of urinary protein observed in the 5-LO–/– mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO–/– mice. LTB4 and LTD4, 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-α and interleukin (IL)-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO–/– mice. However, 5-LO–/– mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload. PMID:25302946

  19. Antenatal Betamethasone Attenuates the Angiotensin-(1-7)/Mas Receptor/Nitric Oxide Axis in Isolated Proximal Tubule Cells.

    PubMed

    Su, Yixin; Bi, Jianli; Pulgar, Victor M; Chappell, Mark C; Rose, James C

    2017-02-22

    We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na(+)) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to Ang-(1-7). The present study determined the Na(+) uptake and nitric oxide (NO) response to low dose Ang-(1-7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as % of basal uptake or area under the curve (AUC) for Na(+) or % of control for NO. Male Beta RPTC exhibited greater Na(+) uptake than male vehicle cells (433±28% vs. 330±26%; p<0.05); however, Beta exposure had no effect on Na(+) uptake in the female cells (255±16% vs. 255±14%; p>0.05). Ang-(1-7) significantly inhibited Na(+) uptake in RPTC from vehicle male (214±11%) and from both vehicle (190±14%) and Beta (209±11%) females, but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1-7) in male, but not female RPTC. Both the Na+ and NO responses to Ang-(1-7) were blocked by Mas receptor antagonist [D-Ala7]-Ang-(1-7). We conclude that the tubular Ang-(1-7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

  20. Interactive toxicity of inorganic mercury and trichloroethylene in rat and human proximal tubules: Effects on apoptosis, necrosis, and glutathione status

    SciTech Connect

    Lash, Lawrence H. . E-mail: l.h.lash@wayne.edu; Putt, David A.; Hueni, Sarah E.; Payton, Scott G.; Zwickl, Joshua

    2007-06-15

    Simultaneous or prior exposure to one chemical may alter the concurrent or subsequent response to another chemical, often in unexpected ways. This is particularly true when the two chemicals share common mechanisms of action. The present study uses the paradigm of prior exposure to study the interactive toxicity between inorganic mercury (Hg{sup 2+}) and trichloroethylene (TRI) or its metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in rat and human proximal tubule. Pretreatment of rats with a subtoxic dose of Hg{sup 2+} increased expression of glutathione S-transferase-{alpha}1 (GST{alpha}1) but decreased expression of GST{alpha}2, increased activities of several GSH-dependent enzymes, and increased GSH conjugation of TRI. Primary cultures of rat proximal tubular (rPT) cells exhibited both necrosis and apoptosis after incubation with Hg{sup 2+}. Pretreatment of human proximal tubular (hPT) cells with Hg{sup 2+} caused little or no changes in GST expression or activities of GSH-dependent enzymes, decreased apoptosis induced by TRI or DCVC, but increased necrosis induced by DCVC. In contrast, pretreatment of hPT cells with TRI or DCVC protected from Hg{sup 2+} by decreasing necrosis and increasing apoptosis. Thus, whereas pretreatment of hPT cells with Hg{sup 2+} exacerbated cellular injury due to TRI or DCVC by shifting the response from apoptosis to necrosis, pretreatment of hPT cells with either TRI or DCVC protected from Hg{sup 2+}-induced cytotoxicity by shifting the response from necrosis to apoptosis. These results demonstrate that by altering processes related to GSH status, susceptibilities of rPT and hPT cells to acute injury from Hg{sup 2+}, TRI, or DCVC are markedly altered by prior exposures.

  1. Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival.

    PubMed

    Abu Jawdeh, Bassam G; Khan, Shenaz; Deschênes, Isabelle; Hoshi, Malcolm; Goel, Monu; Lock, Jeffrey T; Shinlapawittayatorn, Krekwit; Babcock, Gerald; Lakhe-Reddy, Sujata; DeCaro, Garren; Yadav, Satya P; Mohan, Maradumane L; Naga Prasad, Sathyamangla V; Schilling, William P; Ficker, Eckhard; Schelling, Jeffrey R

    2011-12-09

    Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

  2. Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules.

    PubMed

    Vieyra, A; Nachbin, L; de Dios-Abad, E; Goldfeld, M; Meyer-Fernandes, J R; de Moraes, L

    1986-03-25

    Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the

  3. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    SciTech Connect

    Sarró, Eduard; Jacobs-Cachá, Conxita; Itarte, Emilio; Meseguer, Anna

    2012-01-15

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC. Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also identified ER

  4. Superoxide anion production and expression of gp91(phox) and p47(phox) are increased in glomeruli and proximal tubules of cisplatin-treated rats.

    PubMed

    Trujillo, Joyce; Molina-Jijón, Eduardo; Medina-Campos, Omar Noel; Rodríguez-Muñoz, Rafael; Reyes, José Luis; Barrera, Diana; Pedraza-Chaverri, José

    2015-04-01

    The chemotherapeutic drug cisplatin has some side effects including nephrotoxicity that has been associated with reactive oxygen species production, particularly superoxide anion. The major source of superoxide anion is nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. However, the specific segment of the nephron in which superoxide anion is produced has not been identified. Rats were sacrificed 72 h after cisplatin injection (7.5 mg/kg), and kidneys were obtained to isolate glomeruli and proximal and distal tubules. Cisplatin induced superoxide anion production in glomeruli and proximal tubules but not in distal tubules. This enhanced superoxide anion production was prevented by diphenylene iodonium, an inhibitor of NADPH oxidase. Consistently, this effect was associated with the increased expression of gp91(phox) and p47(phox), subunits of NADPH oxidase. The enhanced superoxide anion production in glomeruli and proximal tubules, associated with the increased expression of gp91(phox) and p47(phox), is involved in the oxidative stress in cisplatin-induced nephrotoxicity.

  5. Phosphorylation of rat kidney Na-K pump at Ser938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells.

    PubMed

    Massey, Katherine J; Li, Quanwen; Rossi, Noreen F; Keezer, Susan M; Mattingly, Raymond R; Yingst, Douglas R

    2016-02-01

    How angiotensin (ANG) II acutely stimulates the Na-K pump in proximal tubules is only partially understood, limiting insight into how ANG II increases blood pressure. First, we tested whether ANG II increases the number of pumps in plasma membranes of native rat proximal tubules under conditions of rapid activation. We found that exposure to 100 pM ANG II for 2 min, which was previously shown to increase affinity of the Na-K pump for Na and stimulate activity threefold, increased the amount of the Na-K pump in plasma membranes of native tubules by 33%. Second, we tested whether previously observed increases in phosphorylation of the Na-K pump at Ser(938) were part of the stimulatory mechanism. These experiments were carried out in opossum kidney cells, cultured proximal tubules stably coexpressing the ANG type 1 (AT1) receptor, and either wild-type or a S938A mutant of rat kidney Na-K pump under conditions found by others to stimulate activity. We found that 10 min of incubation in 10 pM ANG II stimulated activity of wild-type pumps from 2.3 to 3.5 nmol K · mg protein(-1) · min(-1) and increased the amount of the pump in the plasma membrane by 80% but had no effect on cells expressing the S938A mutant. We conclude that acute stimulation of Na-K pump activity in native rat proximal tubules includes increased trafficking to the plasma membrane and that phosphorylation at Ser(938) is part of the mechanism by which ANG II directly stimulates activity and trafficking of the rat kidney Na-K pump in opossum kidney cells.

  6. Obesity-induced kidney injury is attenuated by amelioration of aberrant PHD2 activation in proximal tubules

    PubMed Central

    Futatsugi, Koji; Tokuyama, Hirobumi; Shibata, Shinsuke; Naitoh, Makiko; Kanda, Takeshi; Minakuchi, Hitoshi; Yamaguchi, Shintaro; Hayashi, Koichi; Minamishima, Yoji Andrew; Yanagita, Motoko; Wakino, Shu; Itoh, Hiroshi

    2016-01-01

    The involvement of tissue ischemia in obesity-induced kidney injury remains to be elucidated. Compared with low fat diet (LFD)-mice, high fat diet (HFD)-fed mice became obese with tubular enlargement, glomerulomegaly and peritubular capillary rarefaction, and exhibited both tubular and glomerular damages. In HFD-fed mice, despite the increase in renal pimonidazole-positive areas, the expressions of the hypoxia-responsive genes such as Prolyl-hydroxylase PHD2, a dominant oxygen sensor, and VEGFA were unchanged indicating impaired hypoxic response. Tamoxifen inducible proximal tubules (PT)-specific Phd2 knockout (Phd2-cKO) mice and their littermate control mice (Control) were created and fed HFD or LFD. Control mice on HFD (Control HFD) exhibited renal damages and renal ischemia with impaired hypoxic response compared with those on LFD. After tamoxifen treatment, HFD-fed knockout mice (Phd2-cKO HFD) had increased peritubular capillaries and the increased expressions of hypoxia responsive genes compared to Control HFD mice. Phd2-cKO HFD also exhibited the mitigation of tubular damages, albuminuria and glomerulomegaly. In human PT cells, the increased expressions of hypoxia-inducible genes in hypoxic condition were attenuated by free fatty acids. Thus, aberrant hypoxic responses due to dysfunction of PHD2 caused both glomerular and tubular damages in HFD-induced obese mice. Phd2-inactivation provides a novel strategy against obesity-induced kidney injury. PMID:27827416

  7. Sterol carrier protein 2 regulates proximal tubule size in the Xenopus pronephric kidney by modulating lipid rafts.

    PubMed

    Cerqueira, Débora M; Tran, Uyen; Romaker, Daniel; Abreu, José G; Wessely, Oliver

    2014-10-01

    The kidney is a homeostatic organ required for waste excretion and reabsorption of water, salts and other macromolecules. To this end, a complex series of developmental steps ensures the formation of a correctly patterned and properly proportioned organ. While previous studies have mainly focused on the individual signaling pathways, the formation of higher order receptor complexes in lipid rafts is an equally important aspect. These membrane platforms are characterized by differences in local lipid and protein compositions. Indeed, the cells in the Xenopus pronephric kidney were positive for the lipid raft markers ganglioside GM1 and Caveolin-1. To specifically interfere with lipid raft function in vivo, we focused on the Sterol Carrier Protein 2 (scp2), a multifunctional protein that is an important player in remodeling lipid raft composition. In Xenopus, scp2 mRNA was strongly expressed in differentiated epithelial structures of the pronephric kidney. Knockdown of scp2 did not interfere with the patterning of the kidney along its proximo-distal axis, but dramatically decreased the size of the kidney, in particular the proximal tubules. This phenotype was accompanied by a reduction of lipid rafts, but was independent of the peroxisomal or transcriptional activities of scp2. Finally, disrupting lipid micro-domains by inhibiting cholesterol synthesis using Mevinolin phenocopied the defects seen in scp2 morphants. Together these data underscore the importance for localized signaling platforms in the proper formation of the Xenopus kidney.

  8. Effect of bath and luminal potassium concentration on ammonia production and secretion by mouse proximal tubules perfused in vitro.

    PubMed Central

    Nagami, G T

    1990-01-01

    To determine the effects of acute changes in K+ concentration in vitro on ammonia production and secretion by the proximal tubule, we studied mouse S2 segments perfused with and bathed in Krebs-Ringer bicarbonate buffers containing various K+ concentrations. All bath solutions contained L-glutamine as the ammoniagenic substrate. High bath and luminal K+ concentrations (8 mM), but not high luminal K+ concentration alone, inhibited total ammonia production rates by 26%, while low bath and luminal K+ concentrations (2 mM), but not low luminal K+ concentration alone, stimulated total ammonia production rates by 33%. The stimulation of ammonia production by low bath K+ concentration was not observed when L-glutamine was added to the luminal perfusion solution. On the other hand, high luminal K+ concentration stimulated, while low luminal K+ concentration inhibited, net luminal secretion of total ammonia in a way that was: (a) independent of total ammonia production rates, (b) independent of Na(+)-H+ exchange activity, and (c) not due to changes in transepithelial fluxes of total ammonia. These results suggest that luminal potassium concentration has a direct effect on cell-to-lumen transport of ammonia. PMID:2164046

  9. Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat.

    PubMed Central

    Kinoshita, S; Sidhu, A; Felder, R A

    1989-01-01

    The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCT) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of 125I-SCH 23982 (defined by 10 microM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe 125I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific 125I-SCH 23982 binding sites in WKY than in SHR. Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCT of the SHR. Images PMID:2574187

  10. RhoA and MAPK signal transduction pathways regulate NHE1-dependent proximal tubule cell apoptosis after mechanical stretch.

    PubMed

    Bocanegra, Victoria; Gil Lorenzo, Andrea Fernanda; Cacciamani, Valeria; Benardón, María Eugenia; Costantino, Valeria Victoria; Vallés, Patricia G

    2014-10-01

    Mechanical deformation after congenital ureteral obstruction is traduced into biochemical signals leading to tubular atrophy due to epithelial cell apoptosis. We investigated whether Na(+)/H(+) exchanger 1 (NHE1) could be responsible for HK-2 cell apoptosis induction in response to mechanical stretch through its ability to function as a control point of RhoA and MAPK signaling pathways. When mechanical stretch was applied to HK-2 cells, cell apoptosis was associated with diminished NHE1 expression and RhoA activation. The RhoA signaling pathway was confirmed to be upstream from the MAPK cascade when HK-2 cells were transfected with the active RhoA-V14 mutant, showing higher ERK1/2 expression and decreased p38 activation associated with NHE1 downregulation. NHE1 participation in apoptosis induction was confirmed by specific small interfering RNA NHE1 showing caspase-3 activation and decreased Bcl-2 expression. The decreased NHE1 expression was correlated with abnormal NHE1 activity addressed by intracellular pH measurements. These results demonstrate that mitochondrial proximal tubule cell apoptosis in response to mechanical stretch is orchestrated by signaling pathways initiated by the small GTPase RhoA and followed by the opposing effects of ERK1/2 and p38 MAPK phosphorylation, regulating NHE1 decreased expression and activity.

  11. Quantitative Proteomic Analysis of Enriched Nuclear Fractions from BK Polyomavirus-infected Primary Renal Proximal Tubule Epithelial Cells

    PubMed Central

    Justice, Joshua L.; Verhalen, Brandy; Kumar, Ranjit; Lefkowitz, Elliot J.; Imperiale, Michael J.; Jiang, Mengxi

    2016-01-01

    Polyomaviruses are a family of small DNA viruses that are associated with a number of severe human diseases, particularly in immunocompromised individuals. The detailed virus-host interactions during lytic polyomavirus infection are not fully understood. Here we report the first nuclear proteomic study with BK polyomavirus (BKPyV) in a primary renal proximal tubule epithelial cell culture system using stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling coupled with LC-MS/MS. We demonstrated the feasibility of SILAC labeling in these primary cells and subsequently performed reciprocal labeling-infection experiments to identify proteins that are altered by BKPyV infection. Our analyses revealed specific proteins that are significantly up- or down-regulated in the infected nuclear proteome. The genes encoding many of these proteins were not identified in a previous microarray study, suggesting that differential regulation of these proteins may be independent of transcriptional control. Western blotting experiments verified the SILAC proteomic findings. Finally, pathway and network analyses indicated that the host cell DNA damage response signaling and DNA repair pathways are among the cellular processes most affected at the protein level during polyomavirus infection. Our study provides a comprehensive view of the host nuclear proteomic changes during polyomavirus lytic infection and suggests potential novel host factors required for a productive polyomavirus infection. PMID:26354146

  12. Evaluation of “Dream Herb,” Calea zacatechichi, for Nephrotoxicity Using Human Kidney Proximal Tubule Cells

    PubMed Central

    Flynn, Thomas J.; Vohra, Sanah; Wiesenfeld, Paddy; Sprando, Robert L.

    2016-01-01

    A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. “Dream herb,” Calea zacatechichi, has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening of C. zacatechichi for safety especially important. We exposed human proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects of C. zacatechichi relative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support for the need to further scrutinize the safety of this herbal dietary supplement. PMID:27703475

  13. Interleukin-1β suppresses activity of an inwardly rectifying K+ channel in human renal proximal tubule cells.

    PubMed

    Nakamura, Kazuyoshi; Komagiri, You; Kubokawa, Manabu

    2013-09-01

    We investigated the effect of interleukin-1β (IL-1β) on activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells (RPTECs), using the patch-clamp technique and Fura-2 Ca2+ imaging. IL-1β (15 pg/ml) acutely reduced K+ channel activity in cell-attached patches. This effect was blocked by the IL-1 receptor antagonist (20 ng/ml), an inhibitor of phospholipase C, neomycin (300 μM), and an inhibitor of protein kinase C (PKC), GF109203X (500 nM). The Fura-2 Ca2+ imaging revealed that IL-1β increased intracellular Ca2+ concentration even after removal of extracellular Ca2+, which was blocked by an inhibitor of inositol 1,4,5-trisphosphate receptors, 2-aminoethoxydiphenyl borate (2-APB, 1 μM). Moreover, IL-1β suppressed channel activity in the presence of 2-APB without extracellular Ca2+. These results suggest that IL-1β suppresses K+ channel activity in RPTECs through binding to its specific receptor and activation of the PKC pathway even though intracellular Ca2+ does not increase.

  14. Angiotensin II stimulates renal proximal tubule Na(+)-ATPase activity through the activation of protein kinase C.

    PubMed

    Rangel, L B A; Caruso-Neves, C; Lara, L S; Lopes, A G

    2002-08-31

    Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.

  15. Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo

    PubMed Central

    Mattila, Polly E.; Raghavan, Venkatesan; Rbaibi, Youssef; Baty, Catherine J.

    2013-01-01

    The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosphate, glucose, water, and plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell (PTC) function, the organization of the endocytic pathway in these cells remains poorly understood. We have used immunofluorescence and live-cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PTCs isolated from mouse kidney cortex. Cells from the S1 segment could be distinguished from those from more distal PT segments by their robust uptake of albumin and comparatively low expression of γ-glutamyltranspeptidase. Rab11a in these cells is localized to variously sized spherical compartments that resemble the apical vacuoles observed by electron microscopy analysis of PTCs in vivo. These Rab11a-positive structures are highly dynamic and receive membrane and fluid-phase cargo. In contrast, fluid-phase cargoes are largely excluded from Rab11a-positive compartments in immortalized kidney cell lines. The unusual morphology and sorting capacity of Rab11a compartments in primary PTCs may reflect a unique specialization of these cells to accommodate the functional demands of handling a high endocytic load. PMID:24153428

  16. Electrical and freeze-fracture analysis of the effects of ionic cadmium on cell membranes of human proximal tubule cells.

    PubMed Central

    Hazen-Martin, D J; Todd, J H; Sens, M A; Khan, W; Bylander, J E; Smyth, B J; Sens, D A

    1993-01-01

    We previously reported that cell cultures of human proximal tubule (HPT) cells respond to ionic cadmium in a manner consistent with well-defined Cd(2+)-elicited responses reported for in vivo systems. However, one unique finding was that the transepithelial electrical resistance and tight junction sealing strands were altered as a result of Cd2+ exposure at micromolar concentrations. These alterations are reexamined in detail in the present report to determine whether the Cd(2+)-induced alterations are specific alterations in the tight junction structure or reflect a general alteration in the cell membrane. Exhaustive analysis of tight junction sealing strands demonstrated no significant alterations due to Cd2+ exposure, even at the concentration that elicited a significant reduction in transepithelial resistance. Further analysis of intramembrane particle distribution demonstrated a significant increase in apical intramembrane particles, indicating that Cd2+ exposure altered the characteristics of the apical cell membrane. Overall, the results were consistent with evidence of Cd(2+)-induced alteration in the apical cell membrane of the HPT cell. Images Figure 1. Figure 2. Figure 3. a Figure 3. b Figure 3. c Figure 3. d Figure 4. Figure 5. PMID:8137780

  17. Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene.

    PubMed

    Cartier, N; Lacave, R; Vallet, V; Hagege, J; Hellio, R; Robine, S; Pringault, E; Cluzeaud, F; Briand, P; Kahn, A

    1993-03-01

    Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5' regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.

  18. Characterization of FGF23-Dependent Egr-1 Cistrome in the Mouse Renal Proximal Tubule

    PubMed Central

    Portale, Anthony A.; Zhang, Martin Y. H.; David, Valentin; Martin, Aline; Jiao, Yan; Gu, Weikuan; Perwad, Farzana

    2015-01-01

    Fibroblast growth factor 23 (FGF23) is a potent regulator of phosphate (Pi) and vitamin D homeostasis. The transcription factor, early growth response 1 (egr-1), is a biomarker for FGF23-induced activation of the ERK1/2 signaling pathway. We have shown that ERK1/2 signaling blockade suppresses renal egr-1 gene expression and prevents FGF23-induced hypophosphatemia and 1,25-dihydroxyvitamin D (1,25(OH)2D) suppression in mice. To test whether egr-1 itself mediates these renal actions of FGF23, we administered FGF23 to egr-1-/- and wild-type (WT) mice. In WT mice, FGF23 induced hypophosphatemia and suppressed expression of the renal Na/Pi cotransporters, Npt2a and Npt2c. In FGF23-treated egr-1-/- mice, hypophosphatemic response was greatly blunted and Na/Pi cotransporter expression was not suppressed. In contrast, FGF23 induced equivalent suppression of serum 1,25(OH)2D concentrations by suppressing renal cyp27b1 and stimulating cyp24a1 mRNA expression in both groups of mice. Thus, downstream of receptor binding and ERK1/2 signaling, we can distinguish the effector pathway that mediates FGF23-dependent inhibition of Pi transport from the pathway that mediates inhibition of 1,25(OH)2D synthesis in the kidney. Furthermore, we demonstrate that the hypophosphatemic effect of FGF23 is significantly blunted in Hyp/egr-1-/- mice; specifically, serum Pi concentrations and renal Npt2a and Npt2c mRNA expression are significantly higher in Hyp/egr-1-/- mice than in Hyp mice. We then characterized the egr-1 cistrome in the kidney using ChIP-sequencing and demonstrate recruitment of egr-1 to regulatory DNA elements in proximity to several genes involved in Pi transport. Thus, our data demonstrate that the effect of FGF23 on Pi homeostasis is mediated, at least in part, by activation of egr-1. PMID:26588476

  19. A 2D model of axial symmetry for proximal tubule of an average human nephron: indicative results of diffusion, convection and absorption processes

    NASA Astrophysics Data System (ADS)

    Insfrán, J. F.; Ubal, S.; Di Paolo, y. J.

    2016-04-01

    A simplified model of a proximal convoluted tubule of an average human nephron is presented. The model considers the 2D axisymmetric flow of the luminal solution exchanging matter with the tubule walls and the peritubular fluid by means of 0D models for the epithelial cells. The tubule radius is considered to vary along the conduit due to the trans-epithelial pressure difference. The fate of more than ten typical solutes is tracked down by the model. The Navier-Stokes and Reaction-Diffusion-Advection equations (considering the electro-neutrality principle) are solved in the lumen, giving a detailed picture of the velocity, pressure and concentration fields, along with trans-membrane fluxes and tubule deformation, via coupling with the 0D model for the tubule wall. The calculations are carried out numerically by means of the finite element method. The results obtained show good agreement with those published by other authors using models that ignore the diffusive transport and disregard a detailed calculation of velocity, pressure and concentrations. This work should be seen as a first approach towards the development of a more comprehensive model of the filtration process taking place in the kidneys, which ultimately helps in devising a device that can mimic/complement the renal function.

  20. Renal Proximal Tubule Na,K-ATPase is Controlled by CREB Regulated Transcriptional CoActivators as well as Salt Inducible Kinase 1

    PubMed Central

    Taub, Mary; Garamella, Sudha; Kim, Dongwook; Rajkhowa, Trivikram; Cutuli, Facundo

    2015-01-01

    Sodium reabsorption by the kidney is regulated by locally produced natriuretic and anti-natriuretic factors, including dopamine and norepinephrine, respectively. Previous studies indicated that signaling events initiated by these natriuretic and anti-natriuretic factors achieve their effects by altering the phosphorylation of Na,K-ATPase in the renal proximal tubule, and that Protein Kinase A (PKA) and Calcium mediated signaling pathways are involved. The same signaling pathways also control the transcription of the Na,K-ATPase β subunit gene atp1b1 in renal proximal tubule cells. In this report, evidence is presented that 1) both the recently discovered cAMP-Regulated Transcriptional Coactivators (CRTCs), and Salt Inducible Kinase 1 (SIK1) contribute to the transcriptional regulation of atp1b1 in renal proximal tubule (RPT) cells, and 2) that renal effectors including norepinephrine, dopamine, prostaglandins and sodium play a role. Exogenously expressed CRTCs stimulate atp1b1 transcription. Evidence for a role of endogenous CRTCs includes the loss of transcriptional regulation of atp1b1 by a dominant negative CRTC, as well as by a CREB mutant, with an altered CRTC binding site. In a number of experimental systems, SIK phosphorylates CRTCs, which are then sequestered in the cytoplasm, preventing their nuclear effects. Consistent with such a role of SIK in primary RPT cells, atp1b1 transcription increased in the presence of a dominant negative SIK1, and in addition, regulation by dopamine, norepinephrine and monensin was disrupted by a dominant negative SIK1. These latter observations can be explained, if SIK1 is phosphorylated and inactivated in the presence of these renal effectors. Our results support the hypothesis that Na,K-ATPase in the renal proximal tubule is regulated at the transcriptional level via SIK1 and CRTCs by renal effectors, in addition to the previously reported control of the phosphorylation of Na,K-ATPase. PMID:26432356

  1. De novo expression of sodium-glucose cotransporter SGLT2 in Bowman's capsule coincides with replacement of parietal epithelial cell layer with proximal tubule-like epithelium.

    PubMed

    Tabatabai, Niloofar M; North, Paula E; Regner, Kevin R; Kumar, Suresh N; Duris, Christine B; Blodgett, Amy B

    2014-08-01

    In kidney nephron, parietal epithelial cells line the Bowman's capsule and function as a permeability barrier for the glomerular filtrate. Bowman's capsule cells with proximal tubule epithelial morphology have been found. However, the effects of tubular metaplasia in Bowman's capsule on kidney function remain poorly understood. Sodium-glucose cotransporter 2 (SGLT2) plays a major role in reabsorption of glucose in the kidney and is expressed on brush border membrane (BBM) of epithelial cells in the early segment of the proximal tubule. We hypothesized that SGLT2 is expressed in tubularized Bowman's capsule and used our novel antibody to test this hypothesis. Immunohistochemical analysis was performed with our SGLT2 antibody on C57BL/6 mouse kidney prone to have tubularized Bowman's capsules. Cell membrane was examined with periodic acid-Schiff (PAS) stain. The results showed that SGLT2 was localized on BBM of the proximal tubules in young and adult mice. Bowman's capsules were lined mostly with normal brush border-less parietal epithelial cells in young mice, while they were almost completely covered with proximal tubule-like cells in adult mice. Regardless of age, SGLT2 was expressed on BBM of the tubularized Bowman's capsule but did not co-localize with nephrin in the glomerulus. SGLT2-expressing tubular cells expanded from the urinary pole toward the vascular pole of the Bowman's capsule. This study identified the localization of SGLT2 in the Bowman's capsule. Bowman's capsules with tubular metaplasia may acquire roles in reabsorption of filtered glucose and sodium.

  2. Angiotensin II stimulates calcineurin activity in proximal tubule epithelia through AT-1 receptor-mediated tyrosine phosphorylation of the PLC-gamma1 isoform.

    PubMed

    Lea, Janice P; Jin, Shao G; Roberts, Brian R; Shuler, Michael S; Marrero, Mario B; Tumlin, James A

    2002-07-01

    Angiotensin II (AngII) contributes to the maintenance of extracellular fluid volume by regulating sodium transport in the nephron. In nonepithelial cells, activation of phospholipase C (PLC) by AT-1 receptors stimulates the generation of 1,4,5-trisphosphate (IP(3)) and the release of intracellular calcium. Calcineurin, a serine-threonine phosphatase, is activated by calcium and calmodulin, and both PLC and calcineurin have been linked to sodium transport in the proximal tubule. An examination of whether AngII activates calcineurin in a model of proximal tubule epithelia (LLC-PK1 cells) was performed; AngII increased calcineurin activity within 30 s. An examination of whether AngII activates PLC in proximal tubule epithelia was also performed after first showing that all three families of PLC isoforms are present in LLC-PK1 cells. Application of AngII increased IP(3) generation by 60% within 15 s, which coincided with AngII-induced tyrosine phosphorylation of the PLC-gamma1 isoform also observed at 15 s. AngII-induced tyrosine phosphorylation was blocked by the AT-1 receptor antagonist, Losartan. Subsequently, an inhibitor of tyrosine phosphorylation blocked the AngII-induced activation of calcineurin, as did coincubation with an inhibitor of PLC activity and with an antagonist of the AT-1 receptor. It is therefore concluded that AngII stimulates calcineurin phosphatase activity in proximal tubule epithelial cells through a mechanism involving AT-1 receptor-mediated tyrosine phosphorylation of the PLC isoform.

  3. A test of the hypothesis that oxalate secretion produces proximal tubule crystallization in primary hyperoxaluria type I.

    PubMed

    Worcester, Elaine M; Evan, Andrew P; Coe, Fredric L; Lingeman, James E; Krambeck, Amy; Sommers, Andre; Phillips, Carrie L; Milliner, Dawn

    2013-12-01

    The sequence of events by which primary hyperoxaluria type 1 (PH1) causes renal failure is unclear. We hypothesize that proximal tubule (PT) is vulnerable because oxalate secretion raises calcium oxalate (CaOx) supersaturation (SS) there, leading to crystal formation and cellular injury. We studied cortical and papillary biopsies from two PH1 patients with preserved renal function, and seven native kidneys removed from four patients at the time of transplant, after short-term (2) or longer term (2) dialysis. In these patients, and another five PH1 patients without renal failure, we calculated oxalate secretion, and estimated PT CaOx SS. Plasma oxalate was elevated in all PH1 patients and inverse to creatinine clearance. Renal secretion of oxalate was present in all PH1 but rare in controls. PT CaOx SS was >1 in all nonpyridoxine-responsive PH1 before transplant and most marked in patients who developed end stage renal disease (ESRD). PT from PH1 with preserved renal function had birefringent crystals, confirming the presence of CaOx SS, but had no evidence of cortical inflammation or scarring by histopathology or hyaluronan staining. PH1 with short ESRD showed CaOx deposition and hyaluronan staining particularly at the corticomedullary junction in distal PT while cortical collecting ducts were spared. Longer ESRD showed widespread cortical CaOx, and in both groups papillary tissue had marked intratubular CaOx deposits and fibrosis. CaOx SS in PT causes CaOx crystal formation, and CaOx deposition in distal PT appears to be associated with ESRD. Minimizing PT CaOx SS may be important for preserving renal function in PH1.

  4. Angiotensin type 1 receptor resistance to blockade in the opossum proximal tubule cell due to variations in the binding pocket.

    PubMed

    Nistala, Ravi; Andresen, Bradley T; Pulakat, Lakshmi; Meuth, Alex; Sinak, Catherine; Mandavia, Chirag; Thekkumkara, Thomas; Speth, Robert C; Whaley-Connell, Adam; Sowers, James R

    2013-04-15

    Blockade of the angiotensin (ANG) II receptor type 1 (AT(1)R) with angiotensin receptor blockers (ARBs) is widely used in the treatment of hypertension. However, ARBs are variably effective in reducing blood pressure, likely due, in part, to polymorphisms in the ARB binding pocket of the AT(1)R. Therefore, we need a better understanding of variations/polymorphisms that alter binding of ARBs in heterogeneous patient populations. The opossum proximal tubule cell (OKP) line is commonly used in research to evaluate renal sodium handling and therefore blood pressure. Investigating this issue, we found natural sequence variations in the opossum AT(1)R paralleling those observed in the human AT(1)R. Therefore, we posited that these sequence variations may explain ARB resistance. We demonstrate that OKP cells express AT(1)R mRNA, bind (125)I-labeled ANG II, and exhibit ANG II-induced phosphorylation of Jak2. However, Jak2 phosphorylation is not inhibited by five different ARBs commonly used to treat hypertension. Additionally, nonradioactive ANG II competes (125)I-ANG II efficiently, whereas a 10-fold molar excess of olmesartan and the ANG II receptor type 2 blocker PD-123319 is unable to block (125)I-ANG II binding. In contrast, ANG II binding to OKP cells stably expressing rat AT(1A)Rs, which have a conserved AT(1)R-binding pocket with human AT(1)R, is efficiently inhibited by olmesartan. A novel observation was that resistance to ARB binding to opossum AT(1)Rs correlates with variations from the human receptor at positions 108, 163, 192, and 198 within the ARB-binding pocket. These observations highlight the potential utility of evaluating AT(1)R polymorphisms within the ARB-binding pocket in various hypertensive populations.

  5. Expression of VHL Causes Three-Dimensional Morphological Changes in Renal Cells Indicative of Proximal Tubule Differentiation

    PubMed Central

    Chiatar, Shivannah S; Eze, Ogechukwu P; Schoenfeld, Alan R

    2013-01-01

    Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are responsible for the VHL hereditary cancer syndrome, and are associated with the majority of clear cell renal cell carcinomas. In this study, scanning electron microscopy of VHL-negative renal carcinoma cells was utilized to examine the effects of VHL re-expression on the morphology of these cells. Significant differences were observed between the morphology of VHL-negative control cells and those with reintroduced VHL, with VHL expression mediating an apical surface that mounded upward, as opposed to the flat surfaces seen with VHL-negative cells. In long term cultures, rounded VHL-expressing cells grew in clusters on top the monolayer, and microvilli were observed on the apical face of these cells, in a manner suggestive of proximal tubule differentiation. In contrast, VHL-negative cells remained flat and did not develop microvilli in long-term cultures. Since VHL is a key member of an ubiquitin E3 ligase complex whose best known target is hypoxia-inducible factor alpha (HIF-α), we looked at the effects of HIF-α expression on cell morphology. Knockdown of HIF-2α in cells that only express this isoform had no effect on the morphology of the cells. These results indicate that VHL expression directs three dimensional morphological changes in renal cells indicative of differentiation, and while dysregulation of HIF-α may be necessary for tumorigenesis following VHL loss, it is not the major determinant of these VHL-mediated morphological changes. PMID:24308012

  6. Effect of electroneutral luminal and basolateral lactate transport on intracellular pH in salamander proximal tubules

    PubMed Central

    1987-01-01

    We used microelectrodes to examine the effects of organic substrates, particularly lactate (Lac-), on the intracellular pH (pHi) and basolateral membrane potential (Vbl) in isolated, perfused proximal tubules of the tiger salamander. Exposure of the luminal and basolateral membranes to 3.6 mM Lac- caused pHi to increase by approximately 0.2, opposite to the decrease expected from nonionic diffusion of lactic acid (HLac) into the cell. Addition of Lac- to only the lumen also caused alkalinization, but only if Na+ was present. This alkalinization was not accompanied by immediate Vbl changes, which suggests that it involves luminal, electroneutral Na/Lac cotransport. Addition of Lac- to only the basolateral solution caused pHi to decrease by approximately 0.08. The initial rate of this acidification was a saturable function of [Lac-], was not affected by removal of Na+, and was reversibly reduced by alpha-cyano-4-hydroxycinnamate (CHC). Thus, the pHi decrease induced by basolateral Lac- appears to be due to the basolateral entry of H+ and Lac-, mediated by an H/Lac cotransporter (or a Lac-base exchanger). Our data suggest that this transporter is electroneutral and is not present at the luminal membrane. A key question is how the addition of Lac- to the lumen increases pHi. We found that inhibition of basolateral H/Lac cotransport by basolateral CHC reduced the initial rate of pHi increase caused by luminal Lac-. On the other hand, luminal CHC had no effect on the luminal Lac(-)-induced alkalinization. These data suggest that when Lac- is present in the lumen, it enters the cell from the lumen via electroneutral Na/Lac cotransport and then exists with H+ across the basolateral membrane via electroneutral H/Lac cotransport. The net effect is transepithelial Lac- reabsorption, basolateral acid extrusion, and intracellular alkalinization. PMID:3440860

  7. The C-Terminal Fragment of Agrin (CAF), a Novel Marker of Renal Function, Is Filtered by the Kidney and Reabsorbed by the Proximal Tubule

    PubMed Central

    Daryadel, Arezoo; Haubitz, Monika; Figueiredo, Marta; Steubl, Dominik; Roos, Marcel; Mäder, Armin; Hettwer, Stefan

    2016-01-01

    Agrin, a multidomain proteoglycan and neurotrypsin, a neuronal serine protease, are important for forming (neuromuscular) synapses. Proteolytical activity of neurotrypsin produces a C-terminal fragment of agrin, termed CAF, of approximately 22 kDA molecular size which also circulates in blood. The presence of CAF in urine suggests either glomerular filtration or secretion into urine. Blood levels of CAF have been identified as a potential novel marker of kidney function. Here we describe that several nephron segments in the mouse kidney express agrin and neutrotrypsin in addition to the localization of both protein in the glomerulum. Agrin mRNA and protein was detected in almost all nephron segments and mRNA abundance was highest in the inner medullary collecting duct. Neurotrypsin mRNA was mostly detected in the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the inner medullary collecting duct. Moreover, we show that the proximal tubule absorbs injected recombinant CAF by a process shared with receptor-mediated and fluid phase endocytosis. Co-injection of CAF with recombinant human transferrin, a substrate of the receptor-mediated endocytic pathway as well as with FITC-labelled dextran (10 kDa), a marker of fluid phase endocytosis, showed partial colocalization of CAF with both markers. Further colocalization of CAF with the lysosomal marker cathepsin B suggested degradation of CAF by the lysosome in the proximal tubule. Thus, the murine kidney expresses agrin and neurotrypsin in nephron segments beyond the glomerulum. CAF is filtered by the glomerulum and is reabsorbed by endocytosis by the proximal tubule. Thus, impaired kidney function could impair glomerular clearance of CAF and thereby increase circulating CAF levels. PMID:27380275

  8. A retinoid responsive cytokine gene, MK, is preferentially expressed in the proximal tubules of the kidney and human tumor cell lines.

    PubMed Central

    Kitamura, M.; Shirasawa, T.; Mitarai, T.; Muramatsu, T.; Maruyama, N.

    1993-01-01

    The aim of this study was to survey the expression of an embryonic cytokine gene, MK, in the normal organs and neoplastic tissues of adults. Northern analysis showed that MK mRNA was exclusively expressed in the kidney among murine organs including thymus, lung, heart, spleen, liver, and kidney. In situ hybridization analysis revealed that MK expression was localized in the proximal tubules and metaplastic Bowman's epithelium, but not in other nephron segments such as glomeruli, loop of Henle, distal tubules, and collecting ducts. To investigate whether MK expression is a marker of tubular cell lineage, several cell lines originating from renal tubules were tested. No expression of MK was detected in PtK1 and LLC-PK1 cells derived from marsupial and porcine proximal tubules or in MDBK and MDCK cells from bovine and canine distal/collecting tubules. Unexpectedly, the MK gene was expressed in a human renal cell carcinoma line, VMRC-RCW, and the expression was up-regulated in the presence of retinoic acid. To elucidate the involvement of MK in the development of tumors, we further examined its expression in a variety of human neoplastic cell lines: YMB-1-C (breast cancer), EBC-1 (lung squamous cell carcinoma), RERF-LC-OK (lung adenocarcinoma), SBC-3 (lung small cell carcinoma), HSC-2 (mouth squamous cell carcinoma), NUGC-2 (gastric cancer), COLO201 (colon cancer), HepG2 (hepatoma), MIA PaCa-2 (pancreatic cancer), MCAS (ovarian cancer), HeLa (cervical cancer), BeWo (chorionic carcinoma), ITO-II (testicular tumor), T24 (urinary bladder tumor), and G-401 (Wilms' tumor). Strong signals were detected in COLO201, HepG2, ITO-II, T24, G-401, and weaker but distinct signals were detected in YMB-1-C, HSC-2, and MCAS cells. The MK gene was, therefore, widely expressed in neoplastic cells originating from genital organs, intestinal tract, liver, mammary gland, and urinary tract, and the expression was not restricted to adenocarcinomas, but was also observed in other types of

  9. Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease.

    PubMed

    Chintagari, Narendranath Reddy; Nguyen, Julia; Belcher, John D; Vercellotti, Gregory M; Alayash, Abdu I

    2015-03-01

    Sickle cell disease (SCD), a hereditary hemolytic disorder is characterized by chronic hemolysis, oxidative stress, vaso-occlusion and end-organ damage. Hemolysis releases toxic cell-free hemoglobin (Hb) into circulation. Under physiologic conditions, plasma Hb binds to haptoglobin (Hp) and forms Hb-Hp dimers. The dimers bind to CD163 receptors on macrophages for further internalization and degradation. However, in SCD patients plasma Hp is depleted and free Hb is cleared primarily by proximal tubules of kidneys. Excess free Hb in plasma predisposes patients to renal damage. We hypothesized that administration of exogenous Hp reduces Hb-mediated renal damage. To test this hypothesis, human renal proximal tubular cells (HK-2) were exposed to HbA (50μM heme) for 24h. HbA increased the expression of heme oxygenase-1 (HO-1), an enzyme which degrades heme, reduces heme-mediated oxidative toxicity, and confers cytoprotection. Similarly, infusion of HbA (32μM heme/kg) induced HO-1 expression in kidneys of SCD mice. Immunohistochemistry confirmed the increased HO-1 expression in the proximal tubules of the kidney. Exogenous Hp attenuated the HbA-induced HO-1 expression in vitro and in SCD mice. Our results suggest that Hb-mediated oxidative toxicity may contribute to renal damage in SCD and that Hp treatment reduces heme/iron toxicity in the kidneys following hemolysis.

  10. Proximal Tubule Epithelial Cell Specific Ablation of the Spermidine/Spermine N1-Acetyltransferase Gene Reduces the Severity of Renal Ischemia/Reperfusion Injury

    PubMed Central

    Zahedi, Kamyar; Barone, Sharon; Wang, Yang; Murray-Stewart, Tracy; Roy-Chaudhury, Prabir; Smith, Roger D.; Casero, Robert A.; Soleimani, Manoocher

    2014-01-01

    Background Expression and activity of spermidine/spermine N1-acetyltransferase (SSAT) increases in kidneys subjected to ischemia/reperfusion (I/R) injury, while its ablation reduces the severity of such injuries. These results suggest that increased SSAT levels contribute to organ injury; however, the role of SSAT specifically expressed in proximal tubule epithelial cells, which are the primary targets of I/R injury, in the mediation of renal damage remains unresolved. Methods Severity of I/R injury in wt and renal proximal tubule specific SSAT-ko mice (PT-SSAT-Cko) subjected to bilateral renal I/R injury was assessed using cellular and molecular biological approaches. Results Severity of the loss of kidney function and tubular damage are reduced in PT-SSAT-Cko- compared to wt-mice after I/R injury. In addition, animals treated with MDL72527, an inhibitor of polyamine oxidases, had less severe renal damage than their vehicle treated counter-parts. The renal expression of HMGB 1 and Toll like receptors (TLR) 2 and 4 were also reduced in PT-SSAT-Cko- compared to wt mice after I/R injury. Furthermore, infiltration of neutrophils, as well as expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) transcripts were lower in the kidneys of PT-SSAT-Cko compared to wt mice after I/R injury. Finally, the activation of caspase3 was more pronounced in the wt compared to PT-SSAT-Cko animals. Conclusions Enhanced SSAT expression by proximal tubule epithelial cells leads to tubular damage, and its deficiency reduces the severity of renal I/R injury through reduction of cellular damage and modulation of the innate immune response. PMID:25390069

  11. Increased Expression of Rififylin in A < 330 Kb Congenic Strain is Linked to Impaired Endosomal Recycling in Proximal Tubules

    PubMed Central

    Gopalakrishnan, Kathirvel; Kumarasamy, Sivarajan; Yan, Yanling; Liu, Jiang; Kalinoski, Andrea; Kothandapani, Anbarasi; Farms, Phyllis; Joe, Bina

    2012-01-01

    Cell surface proteins are internalized into the cell through endocytosis and either degraded within lysosomes or recycled back to the plasma membrane. While perturbations in endosomal internalization are known to modulate renal function, it is not known whether similar alterations in recycling affect renal function. Rififylin is a known regulator of endocytic recycling with E3 ubiquitin protein ligase activity. In this study, using two genetically similar strains, the Dahl Salt-sensitive rat and an S.LEW congenic strain, which had allelic variants within a < 330 kb segment containing rififylin, we tested the hypothesis that alterations in endosomal recycling affect renal function. The congenic strain had 1.59-fold higher renal expression of rififylin. Transcriptome analysis indicated that components of both endocytosis and recycling were upregulated in the congenic strain. Transcription of Atp1a1 and cell surface content of the protein product of Atp1a1, the alpha subunit of Na+K+ATPase were increased in the proximal tubules from the congenic strain. Because rififylin does not directly regulate endocytosis and it is also a differentially expressed gene within the congenic segment, we reasoned that the observed alterations in the transcriptome of the congenic strain constitute a feedback response to the primary functional alteration of recycling caused by rififylin. To test this, recycling of transferrin was studied in isolated proximal tubules. Recycling was significantly delayed within isolated proximal tubules of the congenic strain, which also had a higher level of polyubiquitinated proteins and proteinuria compared with S. These data provide evidence to suggest that delayed endosomal recycling caused by excess of rififylin indirectly affects endocytosis, enhances intracellular protein polyubiquitination and contributes to proteinuria. PMID:22891072

  12. The lipid composition of high-density lipoprotein affects its re-absorption in the kidney by proximal tubule epithelial cells.

    PubMed Central

    Breznan, Dalibor; Veereswaran, Vasanthi; Viau, France J; Neville, Tracey A-M; Sparks, Daniel L

    2004-01-01

    The kidney is believed to play a major role in the clearance of apoA-I (apolipoprotein A-I) and HDL (high-density lipoprotein) particles from the bloodstream. Proximal tubule epithelial cells of the kidney appear to prevent the loss of these proteins in the urine by re-absorbing them from the urinary filtrate. Experiments were undertaken to investigate the factors that regulate the renal re-absorption of apoA-I and small HDL in a transformed human proximal tubule epithelial (HKC-8) cell line. Fluorescent microscopic studies show that HKC-8 cells can readily bind and take up HDL particles. Intracellular localization of fluorescently labelled native HDL shows its accumulation in endocytotic vesicles, in a perinuclear region after 1 h. Binding studies reveal a saturable cell association of (125)I-HDL with the HKC-8 cell surface after 2 h. HKC-8 cells do not degrade apoA-I or other HDL-apoproteins. The specific cell association of lipid-free apoA-I is approx. 2-fold less than that observed for native HDL. Similarly, reconstituted HDL prepared from HDL-apoproteins and pure phospholipids also exhibits a low cell association with the HKC-8 cells. In contrast, reconstituted HDL prepared with the extracted lipids of HDL and pure apoA-I exhibits an even higher cell association than that observed with the native lipoprotein. A detailed characterization of the major lipid classes in reconstituted HDL shows that only cholesteryl ester increases the cell association of the recombinant particles. These results show that the cholesteryl ester content of HDL may play an important role in the re-absorptive salvage of HDL by the proximal tubule cells of the kidney. PMID:14711371

  13. A mouse model for distal renal tubular acidosis reveals a previously unrecognized role of the V-ATPase a4 subunit in the proximal tubule

    PubMed Central

    Hennings, J Christopher; Picard, Nicolas; Huebner, Antje K; Stauber, Tobias; Maier, Hannes; Brown, Dennis; Jentsch, Thomas J; Vargas-Poussou, Rosa; Eladari, Dominique; Hübner, Christian A

    2012-01-01

    The V-ATPase is a multisubunit complex that transports protons across membranes. Mutations of its B1 or a4 subunit are associated with distal renal tubular acidosis and deafness. In the kidney, the a4 subunit is expressed in intercalated cells of the distal nephron, where the V-ATPase controls acid/base secretion, and in proximal tubule cells, where its role is less clear. Here, we report that a4 KO mice suffer not only from severe acidosis but also from proximal tubule dysfunction with defective endocytic trafficking, proteinuria, phosphaturia and accumulation of lysosomal material and we provide evidence that these findings may be also relevant in patients. In the inner ear, the a4 subunit co-localized with pendrin at the apical side of epithelial cells lining the endolymphatic sac. As a4 KO mice were profoundly deaf and displayed enlarged endolymphatic fluid compartments mirroring the alterations in pendrin KO mice, we propose that pendrin and the proton pump co-operate in endolymph homeostasis. Thus, our mouse model gives new insights into the divergent functions of the V-ATPase and the pathophysiology of a4-related symptoms. PMID:22933323

  14. Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: A {sup 13}C-NMR study

    SciTech Connect

    Renault, Sophie; Faiz, Hassan; Gadet, Rudy; Ferrier, Bernard; Martin, Guy; Baverel, Gabriel; Conjard-Duplany, Agnes

    2010-01-01

    As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-{sup 13}C]-, or L-[2-{sup 13}C]-, or L-[3-{sup 13}C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

  15. Coenzyme Q10 protects renal proximal tubule cells against nicotine-induced apoptosis through induction of p66(shc)-dependent antioxidant responses.

    PubMed

    Arany, Istvan; Carter, Anthony; Hall, Samuel; Fulop, Tibor; Dixit, Mehul

    2017-02-01

    Chronic nicotine exposure (via smoking, E-cigarettes) increases oxidative stress in the kidney that sensitizes it to additional injury in experimental models and in the renal patient. The pro-apoptotic p66(shc) protein-via serine36 phosphorylation that facilitates its mitochondrial translocation and therein cytochrome c binding-generates oxidative stress that leads to injury of renal proximal tubule cells during chronic nicotine exposure. Coenzyme Q10-a clinically safe antioxidant-has been used against nicotine/smoke extract-associated oxidative stress in various non-renal cells. This study explored the anti-oxidant/anti-apoptotic effect of Coenzyme Q10 on nicotine-induced oxidative stress and its impact on p66shc in cultured rat renal proximal tubule cells (NRK52E). We studied the anti-oxidant effect of 10 µM Coenzyme Q10 using various mutants of the p66shc gene and also determined the induction of selected anti-oxidant entities (antioxidant response element, promoter of the manganese superoxide dismutase gene) in reporter luciferase assay during oxidative stress induced by 200 µM nicotine. Our studies revealed that Coenzyme Q10 strongly inhibits nicotine-mediated production of reactive oxygen species and consequent apoptosis that requires serine36 phosphorylation but not mitochondrial translocation/cytochrome c binding of p66(shc). While both nicotine and Coenzyme Q10 stimulates the p66shc promoter, only nicotine exposure results in mitochondrial translocation of p66(shc). In contrast, the Coenzyme Q10-stimulated and non-mitochondrial p66(shc) activates the anti-oxidant manganese superoxide dismutase promoter via the antioxidant response elements and hence, rescues cells from nicotine-induced oxidative stress and consequent apoptosis.

  16. Heat shock protein 27 expression in human proximal tubule cells exposed to lethal and sublethal concentrations of CdCl2.

    PubMed Central

    Somji, S; Sens, D A; Garrett, S H; Sens, M A; Todd, J H

    1999-01-01

    The expression of hsp 27 mRNA and protein was determined in cultured human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd2+ under both acute and extended conditions. Initial procedures demonstrated that HPT cells display the classic stress response following physical and chemical stress. Heat stress (42.5 degrees C for 1 hr) caused an increase in both hsp 27 mRNA and protein as well as a shift in the protein to a more phosphorylated state. Results were similar when the cells were subjected to chemical stress (exposure to 100 microM sodium arsenite for 4 hr). Acute exposure to 53 microM CdCl2 for 4 hr also resulted in an increase in hsp 27 mRNA and protein and a shift to the more phosphorylated protein isoform. Extended Cd2+ exposure involved continuous treatment with Cd2+ at both lethal and sublethal levels over a 16-day time course. The results of this treatment showed that chronic exposure to Cd2+ failed to increase either hsp 27 mRNA or protein expression in HPT cells, even at lethal Cd2+ concentrations. In fact, hsp 27 protein levels decreased as compared to controls at both lethal and sub-lethal exposure to Cd2+. These findings imply that hsp 27 expression in human proximal tubule cells may have two distinct modes depending on the nature (acute vs. chronic) of the stress. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:10379001

  17. Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

    PubMed Central

    Dugelay, S; Chauvin, M F; Megnin-Chanet, F; Martin, G; Laréal, M C; Lhoste, J M; Baverel, G

    1999-01-01

    Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction. PMID:10477267

  18. Effect of acute acid-base disturbances on the phosphorylation of phospholipase C-γ1 and Erk1/2 in the renal proximal tubule

    PubMed Central

    Skelton, Lara A; Boron, Walter F

    2015-01-01

    The renal proximal tubule (PT) plays a major role in whole-body pH homeostasis by secreting H+ into the tubule lumen. Previous work demonstrated that PTs respond to basolateral changes in [CO2] and [] by appropriately altering H+ secretion—responses blocked by the ErbB inhibitor PD168393, or by eliminating signaling through AT1 angiotensin receptors. In the present study, we analyze phosphorylation of three downstream targets of both ErbBs and AT1: phospholipase C-γ1 (PLC-γ1), extracellular-regulated kinase 1 (Erk1), and Erk2. We expose rabbit PT suspensions for 5 and 20 min to our control (Ctrl) condition (5% CO2, 22 mmol/L , pH 7.40) or one of several conditions that mimic acid-base disturbances. We found that each disturbance produces characteristic phosphorylation patterns in the three enzymes. For example, respiratory acidosis (elevated [CO2], normal []) at 20 min decreases PLC-γ1 phosphorylation at tyrosine-783 (relative to Ctrl). Metabolic acidosis (normal [CO2], decreased []) for 5 min increases Erk1 phosphorylation (p-Erk1) but not p-Erk2, whereas metabolic alkalosis (normal [CO2], elevated []) for 5 min decreases p-Erk1 and p-Erk2. In the presence of CO2/, PD168393 blocks only two of eight induced decreases in phosphorylation. In two cases in which disturbances have no remarkable effects on phosphorylation, PD168393 unmasks decreases and in two others, increases. These drug effects provide insight into the roles of PD168393-sensitive kinases. Our results indicate that PLC-γ1.pY783, p-Erk1, and p-Erk2 in the PT change in characteristic ways in response to acute acid-base disturbances, and thus presumably contribute to the transduction of acid-base signals. PMID:25780091

  19. An angiotensin-(1-7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme.

    PubMed

    Wilson, Bryan A; Cruz-Diaz, Nildris; Marshall, Allyson C; Pirro, Nancy T; Su, Yixin; Gwathmey, TanYa M; Rose, James C; Chappell, Mark C

    2015-03-15

    Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

  20. An angiotensin-(1–7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme

    PubMed Central

    Wilson, Bryan A.; Cruz-Diaz, Nildris; Marshall, Allyson C.; Pirro, Nancy T.; Su, Yixin; Gwathmey, TanYa M.; Rose, James C.

    2015-01-01

    Angiotensin 1–7 [ANG-(1–7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1–7) to ANG-(1–4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313–323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1–7) to ANG-(1–4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min−1·mg−1) compared with the tubules (96 ± 12 fmol·min−1·mg−1) and cortex (107 ± 9 fmol·min−1·mg−1). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1–7) and its endogenous analog [Ala1]-ANG-(1–7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp1]-ANG II, ANG I, and ANG-(1–12). Although the ANG-(1–7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1–7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1–7) tone. PMID:25568136

  1. Expression of the constitutive and inducible forms of heat shock protein 70 in human proximal tubule cells exposed to heat, sodium arsenite, and CdCl(2).

    PubMed Central

    Somji, S; Todd, J H; Sens, M A; Garrett, S H; Sens, D A

    1999-01-01

    We determined the expression of the constitutive (hsc 70) and inducible (hsp 70) forms of heat shock protein 70 mRNA and protein in human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd(+2) under both acute and extended conditions of exposure. The HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (sodium arsenite); hsc 70 mRNA and protein levels were constant or slightly increased, whereas hsp 70 mRNA and protein were greatly elevated. Acute exposure to 53.4 microM CdCl(2) for 4 hr failed to increase either hsc 70 mRNA or protein, a finding similar to that observed under classic conditions of stress. However, under identical conditions of acute exposure to Cd(2+), the expected increase in hsp 70 protein level was suppressed as compared to that found under classic conditions of physical or chemical stress. The decrease in hsp 70 protein level correlated to the reduced expression of mRNA from the hsp 70B gene. The expression of mRNA from the hsp 70A and hsp 70C genes was similar to that found when the cells were treated with heat shock or sodium arsenite. We modeled an extended exposure to Cd(2+) by treating the cells continuously with Cd(2+) at both lethal and sublethal levels over a 16-day time course. Chronic exposure to Cd(2+) failed to increase either hsc 70 mRNA or protein levels in the HPT cells at a nonlethal dosage level and decreased hsc 70 mRNA and protein levels late in the time course of lethal exposure. Under identical conditions, the expression of hsp 70 protein remained at basal levels that were only marginally detectable throughout the time course. Hsp 70A and hsp 70C mRNA levels were unaltered by extended exposure to Cd(2+), and hsp 70B mRNA was not detected during the 16-day time course. Cd(2+) is a poor inducer of hsc 70 and hsp 70 in the proximal tubule under both acute and long-term exposure. These results reinforce the fact that the expression of hsp 70

  2. Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.

    PubMed

    Gorvin, Caroline M; Wilmer, Martijn J; Piret, Sian E; Harding, Brian; van den Heuvel, Lambertus P; Wrong, Oliver; Jat, Parmjit S; Lippiat, Jonathan D; Levtchenko, Elena N; Thakker, Rajesh V

    2013-04-23

    Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis

  3. Transforming growth factor-β1 reduces megalin- and cubilin-mediated endocytosis of albumin in proximal-tubule-derived opossum kidney cells

    PubMed Central

    Gekle, Michael; Knaus, Petra; Nielsen, Rikke; Mildenberger, Sigrid; Freudinger, Ruth; Wohlfarth, Verena; Sauvant, Christoph; Christensen, Erik I

    2003-01-01

    Transforming growth factor (TGF)-β1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial-mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF-β1-induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin-cubilin-dependent receptor-mediated endocytosis. In this study we used a well established cell culture model (proximal-tubule-derived opossum kidney (OK) cells) in order to test the hypothesis that TGF-β1 reduces megalin-cubilin-mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF-β1 led to a time- and dose-dependent downregulation of megalin-cubilin-mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF-β1 action does not involve mitogen-activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF-β1-induced downregulation of megalin-cubilin-mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF-β1. In conclusion our data indicate that enhanced levels of TGF-β1 occurring in various nephropathies can lead to downregulation of megalin-cubilin-dependent endocytosis. Probably, TGF-β1 leads to Smad2- and Smad3-dependent expression of negative regulators of receptor-mediated endocytosis. PMID:14561830

  4. Transforming growth factor-beta1 reduces megalin- and cubilin-mediated endocytosis of albumin in proximal-tubule-derived opossum kidney cells.

    PubMed

    Gekle, Michael; Knaus, Petra; Nielsen, Rikke; Mildenberger, Sigrid; Freudinger, Ruth; Wohlfarth, Verena; Sauvant, Christoph; Christensen, Erik I

    2003-10-15

    Transforming growth factor (TGF)-beta1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial-mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF-beta1-induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin-cubilin-dependent receptor-mediated endocytosis. In this study we used a well established cell culture model (proximal-tubule-derived opossum kidney (OK) cells) in order to test the hypothesis that TGF-beta1 reduces megalin-cubilin-mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF-beta1 led to a time- and dose-dependent downregulation of megalin-cubilin-mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF-beta1 action does not involve mitogen-activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF-beta1-induced downregulation of megalin-cubilin-mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF-beta1. In conclusion our data indicate that enhanced levels of TGF-beta1 occurring in various nephropathies can lead to downregulation of megalin-cubilin-dependent endocytosis. Probably, TGF-beta1 leads to Smad2- and Smad3-dependent expression of negative regulators of receptor-mediated endocytosis.

  5. A SILAC-Based Approach Elicits the Proteomic Responses to Vancomycin-Associated Nephrotoxicity in Human Proximal Tubule Epithelial HK-2 Cells.

    PubMed

    Li, Zhi-Ling; Zhou, Shu-Feng

    2016-01-29

    Vancomycin, a widely used antibiotic, often induces nephrotoxicity, however, the molecular targets and underlying mechanisms of this side effect remain unclear. The present study aimed to examine molecular interactome and analyze the signaling pathways related to the vancomycin-induced nephrotoxicity in human proximal tubule epithelial HK-2 cells using the stable isotope labeling by amino acids in cell culture (SILAC) approach. The quantitative proteomic study revealed that there were at least 492 proteins interacting with vancomycin and there were 290 signaling pathways and cellular functions potentially regulated by vancomycin in HK-2 cells. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, EMT, and ROS generation. These findings suggest that vancomycin-induced proteomic responses in HK-2 cells involvefunctional proteins and pathways that regulate cell cycle, apoptosis, autophagy, and redox homeostasis. This is the first systemic study revealed the networks of signaling pathways and proteomic responses to vancomycin treatment in HK-2 cells, and the data may be used to discriminate the molecular and clinical subtypes and to identify new targets and biomarkers for vancomycin-induced nephrotoxic effect. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for drug-induced renal toxicity.

  6. Characterization of the Mouse and Human Monoacylglycerol O-Acyltransferase 1 (Mogat1) Promoter in Human Kidney Proximal Tubule and Rat Liver Cells

    PubMed Central

    Sankella, Shireesha; Garg, Abhimanyu; Agarwal, Anil K.

    2016-01-01

    Monoacylglycerol acyltransferase 1 (Mogat1) catalyzes the conversion of monoacylglycerols (MAG) to diacylglycerols (DAG), the precursor of several physiologically important lipids such as phosphatidylcholine, phosphatidylethanolamine and triacylglycerol (TAG). Expression of Mogat1 is tissue restricted and it is highly expressed in the kidney, stomach and adipose tissue but minimally in the normal adult liver. To understand the transcriptional regulation of Mogat1, we characterized the mouse and human Mogat1 promoters in human kidney proximal tubule-2 (HK-2) cells. In-silico analysis revealed several peroxisome proliferator response element (PPRE) binding sites in the promoters of both human and mouse Mogat1. These sites responded to all three peroxisome proliferator activated receptor (PPAR) isoforms such that their respective agonist or antagonist activated or inhibited the expression of Mogat1. PPRE site mutagenesis revealed that sites located at -592 and -2518 are very effective in decreasing luciferase reporter gene activity. Chromatin immunoprecipitation (ChIP) assay using PPARα antibody further confirmed the occupancy of these sites by PPARα. While these assays revealed the core promoter elements necessary for Mogat1 expression, there are additional elements required to regulate its tissue specific expression. Chromosome conformation capture (3C) assay revealed additional cis-elements located ~10–15 kb upstream which interact with the core promoter. These chromosomal regions are responsive to both PPARα agonist and antagonist. PMID:27611931

  7. Dietary fat composition influences glomerular and proximal convoluted tubule cell structure and autophagic processes in kidneys from calorie-restricted mice.

    PubMed

    Calvo-Rubio, Miguel; Burón, M Isabel; López-Lluch, Guillermo; Navas, Plácido; de Cabo, Rafael; Ramsey, Jon J; Villalba, José M; González-Reyes, José A

    2016-06-01

    Calorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age-related diseases in a wide range of animals, including non-human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age-related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents.

  8. Axial heterogeneity of bicarbonate, chloride, and water transport in the rat proximal convoluted tubule. Effects of change in luminal flow rate and of alkalemia.

    PubMed Central

    Liu, F Y; Cogan, M G

    1986-01-01

    These studies examined regulation of superficial proximal convoluted tubule (PCT) transport as a function of length. When single nephron glomerular filtration rate (SNGFR) increased from 28.7 +/- 0.7 nl/min in hydropenia to 41.5 +/- 0.4 nl/min in euvolemia, bicarbonate, chloride, and water reabsorption in the early (1st mm) PCT increased proportionally: from 354 +/- 21 peq/mm X min, 206 +/- 55 peq/mm X min, and 5.9 +/- 0.4 nl/mm X min to 520 +/- 12 peq/mm X min, 585 +/- 21 peq/mm X min, and 10.1 +/- 0.4 nl/mm X min, respectively. These high transport rates did not increase further, however, when SNGFR went to 51.2 +/- 0.7 or 50.7 +/- 0.6 nl/min after atrial natriuretic factor or glucagon administration. Anion and water transport rates in the late PCT were lower and exhibited less flow dependence. During chronic metabolic alkalosis, acidification was inhibited in the late but not early PCT. In conclusion, the early PCT is distinguished from the late PCT by having high-capacity, flow-responsive but saturable, anion- and water-reabsorptive processes relatively unaffected by alkalemia. PMID:3782470

  9. Gastrin decreases Na+,K+-ATPase activity via a PI 3-kinase- and PKC-dependent pathway in human renal proximal tubule cells.

    PubMed

    Liu, Tianbing; Konkalmatt, Prasad R; Yang, Yu; Jose, Pedro A

    2016-04-01

    The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes.

  10. Electron microscopic study on the lipid content of intramitochondrial granules in proximal convoluted tubule of guinea pig kidney and their ability to accumulate calcium ions.

    PubMed

    Erkoçak, A

    1977-01-01

    The intramitochondrial dense granules of the kidney proximal tubule fixed with OsO4 are osmiophilic since they are bleached by H2O2 treatment and they disappear after glutaraldehyde fixation alone. Following ethanol extraction and subsequent osmification these granules become invisible but pure aceton treatment does not greatly alter their osmiophilia. The findings suggests that the osmiophilic intramitochondrial granules are rich in phospholipids. When the kidney cortex is incubated in the presence of calcium of acetate, calcium accumulates on the intramitochondrial granules increasing their size and number. The intramitochondrial granules are found more frequently in tissues where the transport of water or ions is big. They contribute to the sodium transport (RIEDEL, BUCHER and ERKOCAK 1968). They are composed mainly of neutral lipids (SANZONE, SWARTZENDRUBER and SNYDER 1970) and phospholipids (WENDEL and BARNARD 1974). They are formed by the precipitation of calcium and other ions (GREENAWALT, ROSSI and LEHNINGER 1964; Peachey 1964). in this present work the structure of dense intramitochondrial granules has been studied regarding electron opaque materials. This way on one hand the lipids and the nucleic acids have been investigated, on the other hand the intramitochondrial granules have been loaded with calcium, a cation showing density in precipitated form and found in great amount into the cell.

  11. The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions

    PubMed Central

    Xu, Peng; Carlson, Julia M.; Gaglione, Robert T.; Bigler Wang, Dora; Kemp, Brandon A.; Reyes, Camellia M.; McGrath, Helen E.; Carey, Robert M.; Jose, Pedro A.; Felder, Robin A.

    2015-01-01

    The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2′-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake. PMID:26447209

  12. Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells.

    PubMed

    Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae

    2012-04-01

    This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.

  13. Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

    PubMed Central

    Suñé, Guillermo; Sarró, Eduard; Puigmulé, Marta; López-Hellín, Joan; Zufferey, Madeleine; Pertel, Thomas; Luban, Jeremy; Meseguer, Anna

    2010-01-01

    Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA. PMID:21085665

  14. The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

    PubMed

    Gildea, John J; Xu, Peng; Carlson, Julia M; Gaglione, Robert T; Bigler Wang, Dora; Kemp, Brandon A; Reyes, Camellia M; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2015-12-01

    The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake.

  15. A novel role for c-Myc in G protein-coupled receptor kinase 4 (GRK4) transcriptional regulation in human kidney proximal tubule cells.

    PubMed

    Gildea, John J; Tran, Hanh T; Van Sciver, Robert E; Bigler Wang, Dora; Carlson, Julia M; Felder, Robin A

    2013-05-01

    The G protein-coupled receptor kinase 4 (GRK4) negatively regulates the dopaminergic system by desensitizing the dopamine-1-receptor. The expressional control of GRK4 has not been reported, but here we show that the transcription factor c-Myc binds to the promoter of GRK4 and positively regulates GRK4 protein expression in human renal proximal tubule cells (RPTCs). Addition of phorbol esters to RPTCs not only increased c-Myc binding to the GRK4 promoter but also increased both phospho-c-Myc and GRK4 expression. The phorbol ester-mediated increase in GRK4 expression was completely blocked by the c-Myc inhibitor, 10074-G5, indicating that GRK4 is downstream of phospho-c-Myc. The autocrine production of angiotensin II (Ang II) in RPTCs increased the phosphorylation and activation of c-Myc and subsequently GRK4 expression. 3-Amino-4-thio-butyl sulfonate, an inhibitor of aminopeptidase A, increased RPTC secretion of Ang II. 3-Amino-4-thio-butyl sulfonate or Ang II increased the expression of both phospho-c-Myc and GRK4, which was blocked by 10074-G5. Blockade of the Ang II type 1 receptor with losartan decreased phospho-c-Myc and GRK4 expression. Both inhibition of c-Myc activity and blockade of Ang II type 1 receptor restored the coupling of dopamine-1-receptor to adenylyl cyclase stimulation in uncoupled RPTCs, whereas phorbol esters or Ang II caused the uncoupling of normally coupled RPTCs. We suggest that the Ang II type 1 receptor impairs dopamine-1-receptor function via c-Myc activation of GRK4. This novel pathway may be involved in the increase in blood pressure in hypertension that is mediated by increased activity of the renin-angiotensin system and decreased activity of the renal dopaminergic system.

  16. The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

    PubMed Central

    Phanish, Mysore K.; Wahab, Nadia A.; Colville-Nash, Paul; Hendry, Bruce M.; Dockrell, Mark E. C.

    2005-01-01

    In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFβ1-Smad (transforming growth factor-β1–Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial–mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFβ are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, α-SMA (α-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFβ1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, α-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFβ1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and α-SMA, and decreased expression of E-cadherin. TGFβ1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in α-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFβ1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis. PMID:16253118

  17. A Human Renal Proximal Tubule Cell Line with Stable Organic Anion Transporter 1 and 3 Expression Predictive for Antiviral-Induced Toxicity.

    PubMed

    Nieskens, Tom T G; Peters, Janny G P; Schreurs, Marieke J; Smits, Niels; Woestenenk, Rob; Jansen, Katja; van der Made, Thom K; Röring, Melanie; Hilgendorf, Constanze; Wilmer, Martijn J; Masereeuw, Rosalinde

    2016-03-01

    Drug-induced nephrotoxicity still hampers drug development, because current translation from in vitro or animal studies to human lacks high predictivity. Often, renal adverse effects are recognized only during clinical stages of drug development. The current study aimed to establish a robust and a more complete human cell model suitable for screening of drug-related interactions and nephrotoxicity. In addition to endogenously expressed renal organic cation transporters and efflux transporters, conditionally immortalized proximal tubule epithelial cells (ciPTEC) were completed by transduction of cells with the organic anion transporter (OAT) 1 or OAT3. Fluorescence-activated cell sorting upon exposure to the OAT substrate fluorescein successfully enriched transduced cells. A panel of organic anions was screened for drug-interactions in ciPTEC-OAT1 and ciPTEC-OAT3. The cytotoxic response to the drug-interactions with antivirals was further examined by cell viability assays. Upon subcloning, concentration-dependent fluorescein uptake was found with a higher affinity for ciPTEC-OAT1 (Km = 0.8 ± 0.1 μM) than ciPTEC-OAT3 (Km = 3.7 ± 0.5 μM). Co-exposure to known OAT1 and/or OAT3 substrates (viz. para-aminohippurate, estrone sulfate, probenecid, furosemide, diclofenac, and cimetidine) in cultures spanning 29 passage numbers revealed relevant inhibitory potencies, confirming the robustness of our model for drug-drug interactions studies. Functional OAT1 was directly responsible for cytotoxicity of adefovir, cidofovir, and tenofovir, while a drug interaction with zidovudine was not associated with decreased cell viability. Our data demonstrate that human-derived ciPTEC-OAT1 and ciPTEC-OAT3 are promising platforms for highly predictive drug screening during early phases of drug development.

  18. Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells

    SciTech Connect

    Wolff, Natascha A.; Lee, Wing-Kee; Abouhamed, Marouan

    2008-07-01

    Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 {mu}M for 24 h) toxicity was significantly attenuated from 27.3 {+-} 3.9% in ARF6-WT to 11.1 {+-} 4.0% in ARF6-DN cells (n = 6, P < 0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0 {+-} 4.6% versus 3.9 {+-} 3.9% of ARF6-WT cells, n = 3, P < 0.01) and/or remained near the PM (89.3 {+-} 5. 6% versus 45.2 {+-} 14.3% of ARF6-WT cells, n = 3, P < 0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells.

  19. Glucosamine-induced Sp1 O-GlcNAcylation ameliorates hypoxia-induced SGLT dysfunction in primary cultured renal proximal tubule cells.

    PubMed

    Suh, Han Na; Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae

    2014-10-01

    The aim of this study is to determine whether GlcN could recover the endoplasmic reticulum (ER) stress-induced dysfunction of Na(+) /glucose cotransporter (SGLT) in renal proximal tubule cells (PTCs) under hypoxia. With the rabbit model, the renal ischemia induced tubulointerstitial abnormalities and decreased SGLTs expression in tubular brush-border, which were recovered by GlcN. Thus, the protective mechanism of GlcN against renal ischemia was being examined by using PTCs. Hypoxia decreased the level of protein O-GlcNAc and the expression of O-GlcNAc transferase (OGT) while increased O-GlcNAcase (OGA) and these were reversed by GlcN. Hypoxia also decreased the expression of SGLTs (SGLT1 and 2) and [(14) C]-α-methyl-D-glucopyranoside (α-MG) uptake which were recovered by GlcN and PUGNAc (OGA inhibitor). Hypoxia enhanced reactive oxygen species (ROS) and then ER stress proteins, glucose-regulated protein 78 (GRP78), and C/EBP-homologous protein (CHOP). However, the expression of GRP78 increased till 6 h and then decreased whereas CHOP increased gradually. Moreover, decreased GRP78 and increased CHOP were reversed by NAC (antioxidant) and GlcN. GlcN ameliorated hypoxia-induced decrease of O-GlcNAc modification of Sp1 but OGT or Sp1 siRNAs blocked the recovery effect of GlcN on SGLT expression and α-MG uptake. In addition, hypoxia-decreased GRP78 and HIF-1α expression was reversed by GlcN but OGT siRNA or Sp1 siRNA ameliorated the effect of GlcN. When PTCs were transfected with GRP78 siRNA or HIF-1α siRNA, SGLT expression and α-MG uptake was decreased. Taken together, these data suggest that GlcN-induced O-GlcNAc modified Sp1 with stimulating GRP78 and HIF-1α activity ameliorate hypoxia-induced SGLT dysfunction in renal PTCs. J. Cell. Physiol. 229: 1557-1568, 2014. © 2014 Wiley Periodicals, Inc.

  20. Lack of formic acid production in rat hepatocytes and human renal proximal tubule cells exposed to chloral hydrate or trichloroacetic acid.

    PubMed

    Lock, Edward A; Reed, Celia J; McMillan, Joellyn M; Oatis, John E; Schnellmann, Rick G

    2007-02-12

    The industrial solvent trichloroethylene (TCE) and its major metabolites have been shown to cause formic aciduria in male rats. We have examined whether chloral hydrate (CH) and trichloroacetic acid (TCA), known metabolites of TCE, produce an increase in formic acid in vitro in cultures of rat hepatocytes or human renal proximal tubule cells (HRPTC). The metabolism and cytotoxicity of CH was also examined to establish that the cells were metabolically active and not compromised by toxicity. Rat hepatocytes and HRPTC were cultured in serum-free medium and then treated with 0.3-3mM CH for 3 days or 0.03-3mM CH for 10 days, respectively and formic acid production, metabolism to trichloroethanol (TCE-OH) and TCA and cytotoxicity determined. No increase in formic acid production in rat hepatocytes or HRPTC exposed to CH was observed over and above that due to chemical degradation, neither was formic acid production observed in rat hepatocytes exposed to TCA. HRPTC metabolized CH to TCE-OH and TCA with a 12-fold greater capacity to form TCE-OH versus TCA. Rat hepatocytes exhibited a 1.6-fold and three-fold greater capacity than HRPTC to form TCE-OH and TCA, respectively. CH and TCA were not cytotoxic to rat hepatocytes at concentrations up to 3mM/day for 3 days. With HRPTC, one sample showed no cytotoxicity to CH at concentrations up to 3mM/day for 10 days, while in another cytotoxicity was seen at 1mM/day for 3 days. In summary, increased formic acid production was not observed in rat hepatocytes or HRPTC exposed to TCE metabolites, suggesting that the in vivo response cannot be modelled in vitro. CH was toxic to HRPTC at millimolar concentrations/day over 10 days, while glutathione derived metabolites of TCE were toxic at micromolar concentrations/day over 10 days [Lock, E.A., Reed, C.J., 2006. Trichloroethylene: mechanisms of renal toxicity and renal cancer and relevance to risk assessment. Toxicol. Sci. 19, 313-331] supporting the view that glutathione derived

  1. Mouse amnionless, which is required for primitive streak assembly, mediates cell-surface localization and endocytic function of cubilin on visceral endoderm and kidney proximal tubules.

    PubMed

    Strope, Sharon; Rivi, Roberta; Metzger, Thomas; Manova, Katia; Lacy, Elizabeth

    2004-10-01

    Impaired primitive streak assembly in the mouse amnionless (amn) mutant results in the absence of non-axial trunk mesoderm, a derivative of the middle region of the primitive streak. In addition, the epiblast of amn mutants fails to increase significantly in size after E7.0, indicating that middle primitive streak assembly is mechanistically tied to the growth of the embryo during gastrulation. Amn, a novel transmembrane protein, is expressed exclusively in an extra-embryonic tissue, visceral endoderm (VE), during the early post-implantation stages. We show that Amn is also expressed in kidney proximal tubules (KPT) and intestinal epithelium, which, like the VE, are polarized epithelia specialized for resorption and secretion. To explore whether Amn participates in the development or function of KPT and intestinal epithelia and to gain insight into the function of Amn during gastrulation, we constructed Amn(-/-) ES cell<-->+/+ blastocyst chimeras. While chimeras form anatomically normal kidneys and intestine, they exhibit variable, selective proteinuria, a sign of KPT malfunction. In humans, AMN has been genetically connected to Cubilin (CUBN), a multi-ligand scavenger receptor expressed by KPT, intestine and yolk sac. Loss of CUBN, the intestinal intrinsic factor (IF)-vitamin B12 receptor, results in hereditary megaloblastic anemia (MGA1), owing to vitamin B12 malabsorption. The recent report of MGA1 families with mutations in AMN suggests that AMN functions in the same pathway as CUBN. We demonstrate that Cubn is not properly localized to the cell surface in Amn(-/-) tissues in the embryo and adult mouse, and that adult chimeras exhibit selective proteinuria of Cubn ligands. This study demonstrates that Amn is an essential component of the Cubn receptor complex in vivo and suggests that Amn/Cubn is required for endocytosis/transcytosis of one or more ligands in the VE during gastrulation to coordinate growth and patterning of the embryo. Furthermore, as AMN is

  2. Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide.

    PubMed

    Caruso-Neves, C; Coelho-Souza, S A; Vives, D; Goes, G; Lara, L S; Lopes, A G

    2002-12-01

    In addition to the (Na(+)+K(+))ATPase another P-ATPase, the ouabain-insensitive Na(+)-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg(2+), MgATP and furosemide on the Na(+)-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na(+)-ATPase at pH 7.0 are: (a) K(0.5) for Na(+)=8.9+/-2.2mM; (b) K(0.5) for MgATP=1.8+/-0.4mM; (c) two sites for free Mg(2+): one stimulatory (K(0.5)=0.20+/-0.06 mM) and other inhibitory (I(0.5)=1.1+/-0.4 mM); and (d) I(0.5) for furosemide=1.1+/-0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na(+) (K(0.5)=19.5+/-0.4) and MgATP (K(0.5)=3.4+/-0.3 mM) but increases the apparent affinity for furosemide (0.18+/-0.02 mM) and Mg(2+) (0.05+/-0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na(+) (K(0.5)=18.7+/-1.5 mM) and furosemide (I(0.5)=3.04+/-0.57 mM) but does not change the apparent affinity to MgATP and Mg(2+). The data presented in this paper indicate that the modulation of the Na(+)-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg(2+) and MgATP complex may play an important role in the Na(+)-ATPase physiological regulatory mechanisms.

  3. Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity: comparison of HK-2, immortalized human proximal tubule epithelial, and primary cultures of human proximal tubular cells

    PubMed Central

    Huang, Johnny X; Kaeslin, Geraldine; Ranall, Max V; Blaskovich, Mark A; Becker, Bernd; Butler, Mark S; Little, Melissa H; Lash, Lawrence H; Cooper, Matthew A

    2015-01-01

    There has been intensive effort to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage and identify injury before significant impairment occurs. Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and human macrophage colony stimulating factor (M-CSF) have been validated as urinary and plasma clinical biomarkers predictive of acute and chronic kidney injury and disease. Similar validation of a high throughput in vitro assay predictive of nephrotoxicity could potentially be implemented early in drug discovery lead optimization to reduce attrition at later stages of drug development. To assess these known in vivo biomarkers for their potential for in vitro screening of drug-induced nephrotoxicity, we selected a panel of nephrotoxic agents and examined their effects on the overexpression of nephrotoxicity biomarkers in immortalized (HK-2) and primary (commercially available and freshly in-house produced) human renal proximal tubule epithelial cells. Traditional cytotoxicity was contrasted with expression levels of KIM-1, NGAL, and M-CSF assessed using ELISA and real-time quantitative reverse transcription PCR. Traditional cytotoxicity assays and biomarker assays using HK-2 cells were both unsuitable for prediction of nephrotoxicity. However, increases in protein levels of KIM-1 and NGAL in primary cells were well correlated with dose levels of known nephrotoxic compounds, with limited correlation seen in M-CSF protein and mRNA levels. These results suggest that profiling compounds against primary cells with monitoring of biomarker protein levels may have potential as in vitro predictive assays of drug-induced nephrotoxicity. PMID:26171227

  4. Proximal Nephron

    PubMed Central

    Zhuo, Jia L.; Li, Xiao C.

    2013-01-01

    The kidney plays a fundamental role in maintaining body salt and fluid balance and blood pressure homeostasis through the actions of its proximal and distal tubular segments of nephrons. However, proximal tubules are well recognized to exert a more prominent role than distal counterparts. Proximal tubules are responsible for reabsorbing approximately 65% of filtered load and most, if not all, of filtered amino acids, glucose, solutes, and low molecular weight proteins. Proximal tubules also play a key role in regulating acid-base balance by reabsorbing approximately 80% of filtered bicarbonate. The purpose of this review article is to provide a comprehensive overview of new insights and perspectives into current understanding of proximal tubules of nephrons, with an emphasis on the ultrastructure, molecular biology, cellular and integrative physiology, and the underlying signaling transduction mechanisms. The review is divided into three closely related sections. The first section focuses on the classification of nephrons and recent perspectives on the potential role of nephron numbers in human health and diseases. The second section reviews recent research on the structural and biochemical basis of proximal tubular function. The final section provides a comprehensive overview of new insights and perspectives in the physiological regulation of proximal tubular transport by vasoactive hormones. In the latter section, attention is particularly paid to new insights and perspectives learnt from recent cloning of transporters, development of transgenic animals with knockout or knockin of a particular gene of interest, and mapping of signaling pathways using microarrays and/or physiological proteomic approaches. PMID:23897681

  5. Changes in gene expression in human renal proximal tubule cells exposed to low concentrations of S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene

    SciTech Connect

    Lock, Edward A. . E-mail: e.lock@ljmu.ac.uk; Barth, Jeremy L.; Argraves, Scott W.; Schnellmann, Rick G.

    2006-10-15

    Epidemiology studies suggest that there may be a weak association between high level exposure to trichloroethylene (TCE) and renal tubule cell carcinoma. Laboratory animal studies have shown an increased incidence of renal tubule carcinoma in male rats but not mice. TCE can undergo metabolism via glutathione (GSH) conjugation to form metabolites that are known to be nephrotoxic. The GSH conjugate, S-(1,2-dichlorovinyl)glutathione (DCVG), is processed further to the cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), which is the penultimate nephrotoxic species. We have cultured human renal tubule cells (HRPTC) in serum-free medium under a variety of different culture conditions and observed growth, respiratory control and glucose transport over a 20 day period in medium containing low glucose. Cell death was time- and concentration-dependent, with the EC{sub 5} for DCVG being about 3 {mu}M and for DCVC about 7.5 {mu}M over 10 days. Exposure of HRPTC to sub-cytotoxic doses of DCVC (0.1 {mu}M and 1 {mu}M for 10 days) led to a small number of changes in gene expression, as determined by transcript profiling with Affymetrix human genome chips. Using the criterion of a mean 2-fold change over control for the four samples examined, 3 genes at 0.1 {mu}M DCVC increased, namely, adenosine kinase, zinc finger protein X-linked and an enzyme with lyase activity. At 1 {mu}M DCVC, two genes showed a >2-fold decrease, N-acetyltransferase 8 and complement factor H. At a lower stringency (1.5-fold change), a total of 63 probe sets were altered at 0.1 {mu}M DCVC and 45 at 1 {mu}M DCVC. Genes associated with stress, apoptosis, cell proliferation and repair and DCVC metabolism were altered, as were a small number of genes that did not appear to be associated with the known mode of action of DCVC. Some of these genes may serve as molecular markers of TCE exposure and effects in the human kidney.

  6. Kinetics of active sodium transport in rat proximal tubules and its variation by cardiac glycosides at zero net volume and ion fluxes. Evidence for a multisite sodium transport system.

    PubMed Central

    Györy, A Z; Lingard, J M

    1976-01-01

    1. Transepithelial Na concentration difference, deltaCNa, across proximal tubules of rat kidney was measured at varying intraluminal Na concentrations (CNainfinity) under conditions of zero net volume and Na flux. Simultaneous stopped-flow intratubular and artificial peritubular capillary perfusion techniques were used together with intratubular raffinose to achieve zero net fluxes. Under these conditions in rat proximal tubules, deltaCNa represents active transport, JactNa, factored by permeability, PNa, plus an electrical factor depending on transepithelial potential difference. 2. The relationship between CNainfinity and deltaCNa appeared sigmoidal with saturation being reached when intratubular Na was above 80 m-mole/kg. In the presence of ouabain (10(-2)M) and scilliroside (10(-3)M) the relationship remained the same. The maximum deltaCNa was reduced by approximately 50% by cardiac glycoside inhibition whereas the half-saturation constant was essentially unchanged. These changes from the control represent simple non-competitive inhibition by the cardiac glycosides. 3. Absence of potential difference (p.d.) measurements precludes exact description of the relation between true active transport and substrate concentration but much evidence indicates that the apparently sigmoid relation in the presence and absence of cardiac glycoside inhibition, would be retained if correction of deltaCNa values were possible. Such results could then be explained if there are at least three or more sites for Na on the pump system, of which at least two are not cardiac glycoside sensitive. They would also unequivocally exclude the presence of a single-site single-pump system or the simple algebraic addition of two such units since the kinetic curves for both would be hyperbolic rather than sigmoidal. PMID:950594

  7. Metabolic alkalosis transition in renal proximal tubule cells facilitates an increase in CYP27B1, while blunting responsiveness to PTH

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Parathyroid hormone (PTH) is the central activator of renal proximal 1-alpha-hydroxylase (CYP27B1), the enzyme responsible for synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Past studies have documented a disruption of CYP27B1 activity in chronic metabolic acidosis in vivo, while simulated ac...

  8. Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules.

    PubMed

    Christensen, Erik I; Devuyst, Olivier; Dom, Geneviève; Nielsen, Rikke; Van der Smissen, Patrick; Verroust, Pierre; Leruth, Michèle; Guggino, William B; Courtoy, Pierre J

    2003-07-08

    Loss of the renal endosome-associated chloride channel, ClC-5, in Dent's disease and knockout (KO) mice strongly inhibits endocytosis of filtered proteins by kidney proximal tubular cells (PTC). The underlying mechanism remains unknown. We therefore tested whether this endocytic failure could primarily reflect a loss of reabsorption by the multiligand receptors, megalin, and cubilin, caused by a trafficking defect. Impaired protein endocytosis in PTC of ClC-5 KO mice was demonstrated by (i) a major decreased uptake of injected 125I-beta 2-microglobulin, but not of the fluid-phase tracer, FITC-dextran, (ii) reduced labeling of endosomes by injected peroxidase and for the endogenous megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and (iii) urinary appearance of low-molecular-weight proteins and the selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels, megalin and cubilin abundance was significantly decreased in kidney extracts of KO mice. Percoll gradients resolving early and late endosomes (Rab5a, Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising lysosomes (acid hydrolases) showed a disappearance of the brush border component for megalin and cubilin in KO mice. Quantitative ultrastructural immunogold labeling confirmed the overall decrease of megalin and cubilin in PTC and their selective loss at the brush border. In contrast, total contents of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected. Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily reflects a trafficking defect of megalin and cubilin in PTC.

  9. Gastrin induces sodium-hydrogen exchanger 3 phosphorylation and mTOR activation via a phosphoinositide 3-kinase-/protein kinase C-dependent but AKT-independent pathway in renal proximal tubule cells derived from a normotensive male human.

    PubMed

    Liu, Tianbing; Jose, Pedro A

    2013-02-01

    Gastrin is natriuretic, but its renal molecular targets and signal transduction pathways are not fully known. In this study, we confirmed the existence of CCKBR (a gastrin receptor) in male human renal proximal tubule cells and discovered that gastrin induced S6 phosphorylation, a downstream component of the phosphatidylinositol 3 kinase (PI3 kinase)-mammalian target of rapamycin pathway. Gastrin also increased the phosphorylation of sodium-hydrogen exchanger 3 (NHE3) at serine 552, caused its internalization, and decreased its expression at the cell surface and NHE activity. The phosphorylation of NHE3 and S6 was dependent on PI3 kinases because it was blocked by 2 different PI3-kinase inhibitors, wortmannin and LY294,002. The phosphorylation of NHE3 and S6 was not affected by the protein kinase A inhibitor H-89 but was blocked by a pan-PKC (chelerythrine) and a conventional PKC (cPKC) inhibitor (Gö6976) (10 μM) and an intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The cPKC involved was probably PKCα because it was phosphorylated by gastrin. The gastrin-mediated phosphorylation of NHE3, S6, and PKCα was via phospholipase C because it was blocked by a phospholipase C inhibitor, U73122 (10 μM). The phosphorylation (activation) of AKT, which is usually upstream of mammalian target of rapamycin in the classic PI3 kinase-AKT-p70S6K signaling pathway, was not affected, suggesting that the gastrin-induced phosphorylation of NHE3 and S6 is dependent on both PI3 kinase and PKCα but not AKT.

  10. The angiotensin receptor type 1-Gq protein-phosphatidyl inositol phospholipase Cbeta-protein kinase C pathway is involved in activation of proximal tubule Na+-ATPase activity by angiotensin(1-7) in pig kidneys.

    PubMed

    Lara, Lucienne S; Correa, Juliana S; Lavelle, Anouchka B; Lopes, Anibal G; Caruso-Neves, Celso

    2008-05-01

    In a previous study, we observed that angiotensin(1-7) (Ang(1-7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1-7) on Na+-ATPase activity through AT1R involves a Gq protein-phosphatidyl inositol-phospholipase Cbeta (PI-PLCbeta) pathway because: (1) the effect was reversed by GDPbetaS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPgammaS, a non-hydrolysable GTP analogue; (3) Ang(1-7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCbeta substrate; and (4) U73122, a specific inhibitor of PI-PLCbeta, abolished Ang(1-7)-induced stimulation of Na+-ATPase activity. Angiotensin(1-7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1-7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1-7) stimulates Na+-ATPase activity through the AT1R-Gq protein-PI-PLCbeta-PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.

  11. Detection and measurement of tubulitis in renal allograft rejection

    NASA Astrophysics Data System (ADS)

    Hiller, John B.; Chen, Qi; Jin, Jesse S.; Wang, Yung; Yong, James L. C.

    1997-04-01

    Tubulitis is one of the most reliable signs of acute renal allograft rejection. It occurs when mononuclear cells are localized between the lining tubular epithelial cells with or without disruption of the tubular basement membrane. It has been found that tubulitis takes place predominantly in the regions of the distal convoluted tubules and the cortical collecting system. The image processing tasks are to find the tubule boundaries and to find the relative location of the lymphocytes and epithelial cells and tubule boundaries. The requirement for accuracy applies to determining the relative locations of the lymphocytes and the tubule boundaries. This paper will show how the different sizes and grey values of the lymphocytes and epithelial cells simplify their identification and location. Difficulties in finding the tubule boundaries image processing will be illustrated. It will be shown how proximate location of epithelial cells and the tubule boundary leads to distortion in determination of the calculated boundary. However, in tubulitis the lymphocytes and the tubule boundaries are proximate.In these cases the tubule boundary is adequately resolved and the image processing is satisfactory to determining relativity in location. An adaptive non-linear anisotropic diffusion process is presented for image filtering and segmentation. Multi-layer analysis is used to extract lymphocytes and tubulitis from images. This paper will discuss grading of tissue using the Banff system. The ability to use computer to use computer processing will be argued as obviating problems of reproducability of values for this classification. This paper will also feature discussion of alternative approaches to image processing and provide an assessment of their capability for improving the identification of the tubule boundaries.

  12. Small heterodimer partner attenuates hydrogen peroxide-induced expression of cyclooxygenase-2 and inducible nitric oxide synthase by suppression of activator protein-1 and nuclear factor-κB in renal proximal tubule epithelial cells.

    PubMed

    Park, Jung Sun; Choi, Hoon In; Bae, Eun Hui; Ma, Seong Kwon; Kim, Soo Wan

    2017-03-01

    The orphan nuclear receptor, small heterodimer partner (SHP), plays a negative regulatory role in innate immune responses and is involved in various inflammatory signaling pathways. In the present study, we aimed to ascertain whether SHP is effective in preventing hydrogen peroxide (H2O2)-induced kidney tubular inflammation and explored the molecular mechanisms underlying the protective effects of SHP. Renal ischemia/reperfusion (I/R) injury was induced in mice by clamping both renal pedicles for 30 min. The effects of H2O2 on cell viability in human renal proximal tubule (HK-2) cells were determined using MTT assays. 2',7'-DCF-DA was used to determine intracellular reactive oxygen species (ROS). SHP, cyclooxygenase-2 (COX-2) levels, and inducible nitric oxide synthase (iNOS) expression levels were determined by semi-quantitative immunoblotting and real-time polymerase chain reaction. In addition, SHP, nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) promoter activities were determined by luciferase assays. SHP mRNA and protein expression levels were reduced, whereas COX-2 and iNOS levels were increased in mice subjected to renal I/R. H2O2 treatment in HK-2 cells decreased cell viability, increased ROS production, and induced COX-2 and iNOS expression. These changes were counteracted by transient transfection with SHP. H2O2 treatment decreased SHP luciferase activity, which was recovered by treatment with the NF-κB inhibitor Bay11-7082, transfection with dominant-negative c-Jun or treatment with N-acetyl cysteine (NAC). AP-1 and NF-κB promoter activities were increased by H2O2 and this increase was blocked by SHP transfection. To conclude, SHP protected HK-2 cells from H2O2-induced tubular injury by inhibition of COX-2 and iNOS through suppression of AP-1 and NF-κB promoter activities.

  13. Ablation of the Stimulatory G Protein α-Subunit in Renal Proximal Tubules Leads to Parathyroid Hormone-Resistance With Increased Renal Cyp24a1 mRNA Abundance and Reduced Serum 1,25-Dihydroxyvitamin D.

    PubMed

    Zhu, Yan; He, Qing; Aydin, Cumhur; Rubera, Isabelle; Tauc, Michel; Chen, Min; Weinstein, Lee S; Marshansky, Vladimir; Jüppner, Harald; Bastepe, Murat

    2016-02-01

    PTH regulates serum calcium, phosphate, and 1,25-dihydroxyvitamin D (1,25(OH)2D) levels by acting on bone and kidney. In renal proximal tubules (PTs), PTH inhibits reabsorption of phosphate and stimulates the synthesis of 1,25(OH)2D. The PTH receptor couples to multiple G proteins. We here ablated the α-subunit of the stimulatory G protein (Gsα) in mouse PTs by using Cre recombinase driven by the promoter of type-2 sodium-glucose cotransporter (Gsα(Sglt2KO) mice). Gsα(Sglt2KO) mice were normophosphatemic but displayed, relative to controls, hypocalcemia (1.19 ±0.01 vs 1.23 ±0.01 mmol/L; P < .05), reduced serum 1,25(OH)2D (59.3 ±7.0 vs 102.5 ±12.2 pmol/L; P < .05), and elevated serum PTH (834 ±133 vs 438 ±59 pg/mL; P < .05). PTH-induced elevation in urinary cAMP excretion was blunted in Gsα(Sglt2KO) mice (2- vs 4-fold over baseline in controls; P < .05). Relative to baseline in controls, PTH-induced reduction in serum phosphate tended to be blunted in Gsα(Sglt2KO) mice (-0.39 ±0.33 vs -1.34 ±0.36 mg/dL; P = .07). Gsα(Sglt2KO) mice showed elevated renal vitamin D 24-hydroxylase and bone fibroblast growth factor-23 (FGF23) mRNA abundance (∼3.4- and ∼11-fold over controls, respectively; P < .05) and tended to have elevated serum FGF23 (829 ±76 vs 632 ±60 pg/mL in controls; P = .07). Heterozygous mice having constitutive ablation of the maternal Gsα allele (E1(m-/+)) (model of pseudohypoparathyroidism type-Ia), in which Gsα levels in PT are reduced, also exhibited elevated serum FGF23 (474 ±20 vs 374 ±27 pg/mL in controls; P < .05). Our findings indicate that Gsα is required in PTs for suppressing renal vitamin D 24-hydroxylase mRNA levels and for maintaining normal serum 1,25(OH)2D.

  14. Nanocapillarity in fullerene tubules

    NASA Astrophysics Data System (ADS)

    Pederson, Mark R.; Broughton, Jeremy Q.

    1992-11-01

    Fullerene tubules are shown to be highly polarizable ``molecular straws'' capable of ingesting dipolar molecules. Local-density-functional calculations on HF molecules within a finite-length tubule, of size 144 atoms, demonstrate this effect. The energy of incarceration is several times the thermal ambient at room temperature. These calculations, now feasible on desktop workstations, open the way to the study of nanoscale capillarity and to, perhaps, precise control over shielding of specific ``guest'' compounds from external electric and magnetic fields.

  15. Bioengineered kidney tubules efficiently excrete uremic toxins

    PubMed Central

    Jansen, J.; Fedecostante, M.; Wilmer, M. J.; Peters, J. G.; Kreuser, U. M.; van den Broek, P. H.; Mensink, R. A.; Boltje, T. J.; Stamatialis, D.; Wetzels, J. F.; van den Heuvel, L. P.; Hoenderop, J. G.; Masereeuw, R.

    2016-01-01

    The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs. PMID:27242131

  16. Lipid tubule growth by osmotic pressure

    NASA Astrophysics Data System (ADS)

    Rangamani, Padmini; Zhang, Di; Orster, George; Shen, Amy

    2013-11-01

    We present here a procedure for growing lipid tubules in vitro. This method allows us to grow tubules of consistent shape and structure and thus can be a useful tool for nano-engineering applications. There are three stages during the tubule growth process: initiation, elongation and termination. Balancing the forces that act on the tubule head shows that the growth of tubules during the elongation phase depends on the balance between osmotic pressure and the viscous drag exerted on the membrane from the substrate and the external fluid. Using a combination of mathematical modeling and experiment, we identify the key forces that control tubule growth during the elongation phase.

  17. Elevated ventricular wall stress disrupts cardiomyocyte t-tubule structure and calcium homeostasis

    PubMed Central

    Frisk, Michael; Ruud, Marianne; Espe, Emil K. S.; Aronsen, Jan Magnus; Røe, Åsmund T.; Zhang, Lili; Norseng, Per Andreas; Sejersted, Ole M.; Christensen, Geir A.; Sjaastad, Ivar; Louch, William E.

    2016-01-01

    Aims Invaginations of the cellular membrane called t-tubules are essential for maintaining efficient excitation–contraction coupling in ventricular cardiomyocytes. Disruption of t-tubule structure during heart failure has been linked to dyssynchronous, slowed Ca2+ release and reduced power of the heartbeat. The underlying mechanism is, however, unknown. We presently investigated whether elevated ventricular wall stress triggers remodelling of t-tubule structure and function. Methods and results MRI and blood pressure measurements were employed to examine regional wall stress across the left ventricle of sham-operated and failing, post-infarction rat hearts. In failing hearts, elevated left ventricular diastolic pressure and ventricular dilation resulted in markedly increased wall stress, particularly in the thin-walled region proximal to the infarct. High wall stress in this proximal zone was associated with reduced expression of the dyadic anchor junctophilin-2 and disrupted cardiomyocyte t-tubular structure. Indeed, local wall stress measurements predicted t-tubule density across sham and failing hearts. Elevated wall stress and disrupted cardiomyocyte structure in the proximal zone were also associated with desynchronized Ca2+ release in cardiomyocytes and markedly reduced local contractility in vivo. A causative role of wall stress in promoting t-tubule remodelling was established by applying stretch to papillary muscles ex vivo under culture conditions. Loads comparable to wall stress levels observed in vivo in the proximal zone reduced expression of junctophilin-2 and promoted t-tubule loss. Conclusion Elevated wall stress reduces junctophilin-2 expression and disrupts t-tubule integrity, Ca2+ release, and contractile function. These findings provide new insight into the role of wall stress in promoting heart failure progression. PMID:27226008

  18. Structural and ultrastructural features of the Malpighian tubules of Dolycoris baccarum (Linnaeus 1758), (Heteroptera: Pentatomidae).

    PubMed

    Özyurt, Nurcan; Amutkan, Damla; Polat, Irmak; Kocamaz, Tenay; Candan, Selami; Suludere, Zekiye

    2016-11-11

    The morphology and ultrastructure of the Malpighian tubules of Dolycoris baccarum were analyzed by scanning (SEM) and transmission (TEM) electron microscopy in order to determine their functional organization. The Malpighian tubules are compared with similar structures of other insects based on cell structure and functional organization. The Malpighian tubules of D. baccarum extend from the midgut-hindgut region of the digestive tract. The Malpighian tubules are divided into two regions: the proximal segment is short and flattened and the distal segment is long, stringy in shape and free in hemolymph. The tubules are generally long and narrow. There is a large number of trachea around the tubules. They consist of a single layer of epithelial cells. It is observed in the TEM observation that the epithelial cells have numerous microvilli at the apical side of the cells. At the basal side of the cells, there is a great number of membrane foldings and mitochondria among them. Besides some spherites, mitochondria, lysosome-like bodies, and large or small granules can be distinguished in the cells. With this study, we aimed to demonstrate the ultrastructure of the Malpighian tubules of D. baccarum and differences or similarities with other species.

  19. Isolation of surface tubules of fowlpox virus.

    PubMed

    Carter, J K; Cheville, N F

    1981-01-01

    Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.

  20. Involvement of Zn Depletion in Cd-Induced Toxicity on Prenatal Bone Formation in Rat.

    PubMed

    Boughammoura, Sana; Chemek, Marouane; Mimouna, Safa Ben; Banni, Mohamed; Messaoudi, Imed

    2017-03-06

    This study explored the potential toxicity of Cd on the Zn bone depletion in prenatal bone formation. Female rats received either tap water, Cd, Zn, or Cd + Zn in their drinking water during gestation, and some markers of bone formation were studied in their fetuses removed at the 20th day of pregnancy (GD20). Cd exposure induced maternal hypozincemia and Zn depletion in the femur of the fetuses. A striking inhibition of bone formation in fetuses, expressed by decreases in femur length, width, and area, by the shortening of diaphysis, and by a decrease in length and area of distal and proximal proliferative zones, was observed in fetuses from Cd-exposed mothers. At the molecular level, Cd caused upregulation of MT-1 and ZIP2 genes and significantly depressed the expression of the ZnT5, colα1, osteocalcin, and ALP genes in the femur. Interestingly, Zn treatment ameliorated the Cd-induced maternal hypozincemia and femoral changes and partially restored the normal histomorphometry of the femur. These results suggest that the observed toxic effects of Cd are, at least in part, mediated by the disruption of maternal Zn metabolism during pregnancy leading to Zn depletion and thus to perturbation of prenatal bone formation.

  1. Assessing Cd-induced stress from plant spectral response

    NASA Astrophysics Data System (ADS)

    Kancheva, Rumiana; Georgiev, Georgi

    2014-10-01

    Remote sensing plays a significant role in local, regional and global monitoring of land covers. Ecological concerns worldwide determine the importance of remote sensing applications for the assessment of soil conditions, vegetation health and identification of stress-induced changes. The extensive industrial growth and intensive agricultural land-use arise the serious ecological problem of environmental pollution associated with the increasing anthropogenic pressure on the environment. Soil contamination is a reason for degradation processes and temporary or permanent decrease of the productive capacity of land. Heavy metals are among the most dangerous pollutants because of their toxicity, persistent nature, easy up-take by plants and long biological half-life. This paper takes as its focus the study of crop species spectral response to Cd pollution. Ground-based experiments were performed, using alfalfa, spring barley and pea grown in Cd contaminated soils and in different hydroponic systems under varying concentrations of the heavy metal. Cd toxicity manifested itself by inhibition of plant growth and synthesis of photosynthetic pigments. Multispectral reflectance, absorbance and transmittance, as well as red and far red fluorescence were measured and examined for their suitability to detect differences in plant condition. Statistical analysis was performed and empirical relationships were established between Cd concentration, plant growth variables and spectral response Various spectral properties proved to be indicators of plant performance and quantitative estimators of the degree of the Cd-induced stress.

  2. Insulin-like growth factor binding protein 7 and tissue inhibitor of metalloproteinases-2: differential expression and secretion in human kidney tubule cells.

    PubMed

    Emlet, David R; Pastor-Soler, Nuria; Marciszyn, Allison; Wen, Xiaoyan; Gomez, Hernando; Humphries, William H; Morrisroe, Seth; Volpe, Jacob K; Kellum, John A

    2017-02-01

    We have characterized the expression and secretion of the acute kidney injury (AKI) biomarkers insulin-like growth factor binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in human kidney epithelial cells in primary cell culture and tissue. We established cell culture model systems of primary kidney cells of proximal and distal tubule origin and observed that both proteins are indeed expressed and secreted in both tubule cell types in vitro. However, TIMP-2 is both expressed and secreted preferentially by cells of distal tubule origin, while IGFBP7 is equally expressed across tubule cell types yet preferentially secreted by cells of proximal tubule origin. In human kidney tissue, strong staining of IGFBP7 was seen in the luminal brush-border region of a subset of proximal tubule cells, and TIMP-2 stained intracellularly in distal tubules. Additionally, while some tubular colocalization of both biomarkers was identified with the injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, both biomarkers could also be seen alone, suggesting the possibility for differential mechanistic and/or temporal profiles of regulation of these early AKI biomarkers from known markers of injury. Last, an in vitro model of ischemia-reperfusion demonstrated enhancement of secretion of both markers early after reperfusion. This work provides a rationale for further investigation of these markers for their potential role in the pathogenesis of acute kidney injury.

  3. Calcium Oxalate Accumulation in Malpighian Tubules of Silkworm (Bombyx mori)

    NASA Astrophysics Data System (ADS)

    Wyman, Aaron J.; Webb, Mary Alice

    2007-04-01

    Silkworm provides an ideal model system for study of calcium oxalate crystallization in kidney-like organs, called Malpighian tubules. During their growth and development, silkworm larvae accumulate massive amounts of calcium oxalate crystals in their Malpighian tubules with no apparent harm to the organism. This manuscript reports studies of crystal structure in the tubules along with analyses identifying molecular constituents of tubule exudate.

  4. Sex differences in proximal and distal nephron function contribute to the mechanism of idiopathic hypercalcuria in calcium stone formers.

    PubMed

    Ko, Benjamin; Bergsland, Kristin; Gillen, Daniel L; Evan, Andrew P; Clark, Daniel L; Baylock, Jaime; Coe, Fredric L; Worcester, Elaine M

    2015-07-01

    Idiopathic hypercalciuria (IH) is a common familial trait among patients with calcium nephrolithiasis. Previously, we have demonstrated that hypercalciuria is primarily due to reduced renal proximal and distal tubule calcium reabsorption. Here, using measurements of the clearances of sodium, calcium, and endogenous lithium taken from the General Clinical Research Center, we test the hypothesis that patterns of segmental nephron tubule calcium reabsorption differ between the sexes in IH and normal subjects. When the sexes are compared, we reconfirm the reduced proximal and distal calcium reabsorption. In IH women, distal nephron calcium reabsorption is decreased compared to normal women. In IH men, proximal tubule calcium reabsorption falls significantly, with a more modest reduction in distal calcium reabsorption compared to normal men. Additionally, we demonstrate that male IH patients have lower systolic blood pressures than normal males. We conclude that women and men differ in the way they produce the hypercalciuria of IH, with females reducing distal reabsorption and males primarily reducing proximal tubule function.

  5. Tubules of plant reoviruses exploit tropomodulin to regulate actin-based tubule motility in insect vector

    PubMed Central

    Chen, Qian; Zhang, Linghua; Zhang, Yanshuang; Mao, Qianzhuo; Wei, Taiyun

    2017-01-01

    Plant reoviruses are known to exploit virion-packaging tubules formed by virus-encoding non-structural proteins for viral spread in insect vectors. Tubules are propelled by actin-based tubule motility (ABTM) to overcome membrane or tissue barriers in insect vectors. To further understand which insect factors mediate ABTM, we utilized yeast two-hybrid and bimolecular fluorescence complementation assays to test interactions between tubule protein Pns10 of rice dwarf virus (RDV), a plant reovirus, and proteins of its insect vector, the leafhopper Nephotettix cincticeps. Tropomodulin (Tmod), vitellogenin, and lipophorin precursor of N. cincticep displayed positive and strong interaction with Pns10, and actin-associated protein Tmod interacted with Pns10 in pull-down assay and the co-immunoprecipitation system. Further, we determined Pns10 tubules associated with Tmod in cultured cells and midgut of N. cincticep. The expression dynamic of Tmod was consistent with that of Pns10 and the fluctuation of RDV accumulation. Knockdown of Tmod inhibited the Pns10 expression and viral accumulation, thus decreasing the viruliferous rates of leafhopper. These results suggested that Tmod was involved in viral spread by directly interacting with Pns10 tubules, finally promoting RDV infection. This study provided direct evidence of plant reoviruses utilizing an actin-associated protein to manipulate ABTM in insect vectors, thus facilitating viral spread. PMID:28067229

  6. Interpretation of male rat renal tubule tumors.

    PubMed Central

    Rodgers, I S; Baetcke, K P

    1993-01-01

    Based on an analysis of recent scientific studies, a Technical Panel of the U.S. Environmental Protection Agency's (EPA) Risk Assessment Forum recently advised EPA risk assessors against using information on certain male rat renal tubule tumors to assess human risk under conditions specified in a new Forum report. Risk assessment approaches generally assume that chemicals producing tumors in laboratory animals are a potential cancer hazard to humans. For most chemicals, including classical rodent kidney carcinogens such as N-ethyl-N-hydroxyethylnitrosamine, this extrapolation remains appropriate. Some chemicals, however, induce accumulation of alpha 2u-globulin (alpha 2u-g), a low molecular weight protein, in the male rat kidney. The alpha 2u-g accumulation initiates a sequence of events that appears to lead to renal tubule tumor formation. Female rats and other laboratory mammals administered the same chemicals do not accumulate low molecular weight protein in the kidney, and they do not develop renal tubule tumors. Because humans appear to be more like other laboratory animals than like the male rat, in this special situation, the male rat is not a good model for assessing human risk. The Forum report stresses the need for full scrutiny of a substantial set of data to determine when it is reasonable to presume that renal tumors in male rats are linked to a process involving alpha 2u-g accumulation and to select appropriate procedures for estimating human risks under such circumstances. PMID:7517352

  7. Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.

    PubMed

    Watanabe, Sumiyo; Ogasawara, Toru; Tamura, Yoshifuru; Saito, Taku; Ikeda, Toshiyuki; Suzuki, Nobuchika; Shimosawa, Tatsuo; Shibata, Shigeru; Chung, Ung-Il; Nangaku, Masaomi; Uchida, Shunya

    2017-01-01

    Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene

  8. Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments

    PubMed Central

    Watanabe, Sumiyo; Ogasawara, Toru; Tamura, Yoshifuru; Saito, Taku; Ikeda, Toshiyuki; Suzuki, Nobuchika; Shimosawa, Tatsuo; Shibata, Shigeru; Chung, Ung-il; Nangaku, Masaomi; Uchida, Shunya

    2017-01-01

    Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. “adenovirus driven NPT2a-EGFP and endogenous NHE3 protein”, “adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein” and “adenovirus driven AQP2-EGFP and endogenous AQP2 protein”. Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and

  9. Successful treatment of proximal renal tubular acidosis and Fanconi syndrome with vitamin D replacement.

    PubMed

    Ali, Syed Ahsan; Tariq, Muhammad

    2016-01-01

    Proximal renal tubular acidosis (RTA), also known as Type II RTA, is characterized by a defect in the ability to reabsorb bicarbonate (HCO 3 ) in the proximal tubule. It is usually associated with generalized dysfunction of the proximal tubule as part of Fanconi syndrome. Very few case reports in the literature support Vitamin D deficiency as a cause of proximal RTA. We present a case of a young female who presented with proximal RTA and Fanconi syndrome and excellently responded to Vitamin D replacement. Thus, work-up for the etiology of proximal RTA should include Vitamin D levels since replacement of this vitamin in those who are deficient can lead to cure of such patients.

  10. Lengths of nephron tubule segments and collecting ducts in the CD-1 mouse kidney: an ontogeny study.

    PubMed

    Walton, Sarah L; Moritz, Karen M; Bertram, John F; Singh, Reetu R

    2016-11-01

    The kidney continues to mature postnatally, with significant elongation of nephron tubules and collecting ducts to maintain fluid/electrolyte homeostasis. The aim of this project was to develop methodology to estimate lengths of specific segments of nephron tubules and collecting ducts in the CD-1 mouse kidney using a combination of immunohistochemistry and design-based stereology (vertical uniform random sections with cycloid arc test system). Lengths of tubules were determined at postnatal day 21 (P21) and 2 and 12 mo of age and also in mice fed a high-salt diet throughout adulthood. Immunohistochemistry was performed to identify individual tubule segments [aquaporin-1, proximal tubules (PT) and thin descending limbs of Henle (TDLH); uromodulin, distal tubules (DT); aquaporin-2, collecting ducts (CD)]. All tubular segments increased significantly in length between P21 and 2 mo of age (PT, 602% increase; DT, 200% increase; TDLH, 35% increase; CD, 53% increase). However, between 2 and 12 mo, a significant increase in length was only observed for PT (76% increase in length). At 12 mo of age, kidneys of mice on a high-salt diet demonstrated a 27% greater length of the TDLH, but no significant change in length was detected for PT, DT, and CD compared with the normal-salt group. Our study demonstrates an efficient method of estimating lengths of specific segments of the renal tubular system. This technique can be applied to examine structure of the renal tubules in combination with the number of glomeruli in the kidney in models of altered renal phenotype.

  11. Hazard evaluation of chemicals that cause accumulation of alpha 2u-globulin, hyaline droplet nephropathy, and tubule neoplasia in the kidneys of male rats.

    PubMed Central

    Hard, G C; Rodgers, I S; Baetcke, K P; Richards, W L; McGaughy, R E; Valcovic, L R

    1993-01-01

    This review paper examines the relationship between chemicals inducing excessive accumulation of alpha 2u-globulin (alpha 2u-g) (CIGA) in hyaline droplets in male rat kidneys and the subsequent development of nephrotoxicity and renal tubule neoplasia in the male rat. This dose-responsive hyaline droplet accumulation distinguishes CIGA carcinogens from classical renal carcinogens. CIGA carcinogens also do not appear to react with DNA and are generally negative in short-term tests for genotoxicity, CIGA or their metabolites bind specifically, but reversibly, to male rat alpha 2u-g. The resulting complex appears to be more resistant to hydrolytic degradation in the proximal tubule than native, unbound alpha 2u-g. Single cell necrosis of the tubule epithelium, with associated granular cast formation and papillary mineralization, is followed by sustained regenerative tubule cell proliferation, foci of tubule hyperplasia in the convoluted proximal tubules, and renal tubule tumors. Although structurally similar proteins have been detected in other species, including humans, renal lesions characteristic of alpha 2u-g nephropathy have not been observed. Epidemiologic investigation has not specifically examined the CIGA hypothesis for humans. Based on cancer bioassays, hormone manipulation studies, investigations in an alpha 2u-g-deficient strain of rat, and other laboratory data, an increased proliferative response caused by chemically induced cytotoxicity appears to play a role in the development of renal tubule tumors in male rats. Thus, it is reasonable to suggest that the renal effects induced in male rats by chemicals causing alpha 2u-g accumulation are unlikely to occur in humans. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 12. FIGURE 13. PMID:7686485

  12. Proximity fuze

    DOEpatents

    Harrison, Thomas R.

    1989-08-22

    A proximity fuze system includes an optical ranging apparatus, a detonation circuit controlled by the optical ranging apparatus, and an explosive charge detonated by the detonation cirtcuit. The optical ranging apparatus includes a pulsed laser light source for generating target ranging light pulses and optical reference light pulses. A single lens directs ranging pulses to a target and collects reflected light from the target. An optical fiber bundle is used for delaying the optical reference pulses to correspond to a predetermined distance from the target. The optical ranging apparatus includes circuitry for providing a first signal depending upon the light pulses reflected from the target, a second signal depending upon the light pulses from the optical delay fiber bundle, and an output signal when the first and second signals coincide with each other. The output signal occurs when the distance from the target is equal to the predetermined distance form the target. Additional circuitry distinguishes pulses reflected from the target from background solar radiation.

  13. Proximity fuze

    DOEpatents

    Harrison, T.R.

    1987-07-10

    A proximity fuze system includes an optical ranging apparatus, a detonation circuit controlled by the optical ranging apparatus, and an explosive charge detonated by the detonation circuit. The optical ranging apparatus includes a pulsed laser light source for generating target ranging light pulses and optical reference light pulses. A single lens directs ranging pulses to a target and collects reflected light from the target. An optical fiber bundle is used for delaying the optical reference pulses to correspond to a predetermined distance from the target. The optical ranging apparatus includes circuitry for providing a first signal depending upon the light pulses reflected from the target, a second signal depending upon the light pulses from the optical delay fiber bundle, and an output signal when the first and second signals coincide with each other. The output signal occurs when the distance from the target is equal to the predetermined distance from the target. Additional circuitry distinguishes pulses reflected from the target from background solar radiation. 3 figs.

  14. Proximity fuze

    SciTech Connect

    Harrison, T.R.

    1989-08-22

    A proximity fuze system is described. It includes an optical ranging apparatus, a detonation circuit controlled by the optical ranging apparatus, and an explosive charge detonated by the detonation circuit. The optical ranging apparatus includes a pulsed laser light source for generating target ranging light pulses and optical reference light pulses. A single lens directs ranging pulses to a target and collects reflected light from the target. An optical fiber bundle is used for delaying the optical reference pulses to correspond to a predetermined distance from the target. The optical ranging apparatus includes circuitry for providing a first signal depending upon the light pulses reflected from the target, a second signal depending upon the light pulses from the optical delay fiber bundle, and an output signal when the first and second signals coincide with each other. The output signal occurs when the distance from the target is equal to the predetermined distance from the target. Additional circuitry distinguishes pulses reflected from the target from background solar radiation.

  15. Cd-induced Cu deficiency responses in Arabidopsis thaliana: are phytochelatins involved?

    PubMed

    Gielen, Heidi; Vangronsveld, Jaco; Cuypers, Ann

    2017-03-01

    Cadmium (Cd) exposure can disturb the homeostasis of essential elements. In Arabidopsis thaliana, Cd induces a squamosa promoter binding protein-like 7 (SPL7)-dependent Cu deficiency response. We investigated how Cd induces a Cu deficiency response. The Cu deficiency response consists of the active SPL7 transcription factor binding to GTAC motifs in promoters of among others several Cu transporters, a Cu chaperone, and cupro-miRNAs to regulate Cu homeostasis. We demonstrated that the addition of supplemental Cu to Cd-exposed A. thaliana plants diminished the Cu deficiency response in roots, while it even disappeared in leaves. Exposure of plants to Cd in combination with extra Cu reduced Cd levels in both roots and leaves resulting in an improved cellular oxidative state. Furthermore, we demonstrated a role for phytochelatins (PCs) in the Cd-induced Cu deficiency response, because it was reduced in roots of cad1-3 mutant plants exposed to Cd. In conclusion, a working mechanism is provided in which it is suggested that Cd increases PC levels that can complex both Cd and Cu. This results in cellular Cu deficiency and subsequently the activation of SPL7 and hence the induction of the Cu deficiency response.

  16. Excretion of alkaloids by malpighian tubules of insects.

    PubMed

    Maddrell, S H; Gardiner, B O

    1976-04-01

    Nicotine is transported at high rates by Malpighian tubules of larvae of Manduca sexta, Pieris brassicae and Rhodnius prolixus and the transport persists in the absence of alkaloid from the diet. In the fluid-secreting portion of Rhodnius tubules this transport is not coupled to ion transport, nor is it dependent on the physiological state of the animal. The transport, which can occur against a steep electrochemical gradient, shows saturation kinetics with a maximal rate of 700 pmol. min-1 per tubule and is half saturated at 2-3 mM. Nicotine transport independent of ion movements also occurs in the lower resorptive parts of Rhodnius tubules. Both portions of Rhodnius tubules can transport morphine and atropine. These alkaloids and nicotine compete with one naother and are presumed to be carried by the smae transport system. Nicotine transport in Rhodnius was unaffected by organic anions, such as amaranth and benzyl penicillin, or by the organic anion transport inhibitor, probenecid. Fluid secretion in 5-HT-stimulated tubules was reduced by atropine and nicotine, probably by blocking the 5-HT receptors. The Malpighian tubules of adult Calliphora erythrocephala and Musca domestica remove nicotine from bathing solutions, an unknown metabolic accumulating in the tubules. Adult P. brassicae and M. sexta do not exhibit transport of nicotine by their Malpighian tubules.

  17. Nonlinear analysis of lipid tubules by nonlocal beam model.

    PubMed

    Shen, Hui-Shen

    2011-05-07

    Postbuckling, nonlinear bending and nonlinear vibration analyses are presented for lipid tubules. The lipid tubule is modeled as a nonlocal micro/nano-beam which contains small scale effect. The material properties are assumed to be size-dependent. The governing equation is solved by a two-step perturbation technique. The numerical results reveal that the small scale parameter e₀a reduces the postbuckling equilibrium paths, the static large deflections and natural frequencies of lipid tubules. In contrast, it increases the nonlinear to linear frequency ratios slightly for the lipid tubule with immovable end conditions.

  18. Sodium/glucose cotransporter 2 inhibitors and prevention of diabetic nephropathy: targeting the renal tubule in diabetes.

    PubMed

    De Nicola, Luca; Gabbai, Francis B; Liberti, Maria Elena; Sagliocca, Adelia; Conte, Giuseppe; Minutolo, Roberto

    2014-07-01

    Optimal prevention and treatment of chronic kidney disease in diabetes requires implementing therapies that specifically interfere with the pathogenesis of diabetic nephropathy. In this regard, significant attention has been given to alterations of the proximal tubule and resulting changes in glomerular filtration rate. At the onset of diabetes mellitus, hyperglycemia causes increases in proximal tubular reabsorption secondary to induction of tubular growth with associated increases in sodium/glucose cotransport. The increase in proximal reabsorption leads to a decrease in solute load to the macula densa, deactivation of the tubuloglomerular feedback, and increases in glomerular filtration rate. Because glomerular hyperfiltration currently is recognized as a risk factor for progression of kidney disease in diabetic patients, limiting proximal tubular reabsorption constitutes a potential target to reduce hyperfiltration. The recent introduction of sodium/glucose cotransporter 2 (SGLT2) inhibitors opens new therapeutic perspectives for this high-risk patient population. Experimental studies have shown that these new agents attenuate the progressive nature of diabetic nephropathy by blood glucose-dependent and -independent mechanisms. SGLT2 inhibition may prevent glomerular hyperfiltration independent of the effect of lowering blood glucose levels while limiting kidney growth, inflammation, and albuminuria through reductions in blood glucose levels. Clinical data for the potential role of the proximal tubule in the pathophysiology of diabetic nephropathy and the nephroprotective effects of SGLT2 inhibitors currently are limited compared to the more extensive experimental literature. We review the evidence supporting this working hypothesis by integrating the experimental findings with the available clinical data.

  19. Ionic requirements of proximal tubular sodium transport. II. Hydrogen ion.

    PubMed

    Green, R; Giebisch, G

    1975-11-01

    Simultaneous perfusion to proximal convoluted tubules and peritubular capillaries was used to study the effects of different perfusion fluids on sodium reabsorption and hydrogen secretion, which was calculated as bicarbonate reabsorption and titratable acid. Results show that sodium reabsorption was not tightly coupled to hydrogen secretion. Bicarbonate stimulates both sodium reabsorption and hydrogen secretion, but Tris stimulates only sodium reabsorption. Imposing an adverse chloride gradient across the proximal tubule (C1- peritubular greater than C1- luminal) decreased sodium reabsorption but did not diminish hydrogen secretion. Diamox inhibited both net sodium and hydrogen transport. It is concluded that there is not firm linkage between sodium reabsorption and hydrogen secretion and that bicarbonate probably stimulates sodium transport by a number of mechanisms, including an effect on the sodium transport unrelated to its ability to increase hydrogen ion secretion.

  20. Malpighian Tubules as Novel Targets for Mosquito Control

    PubMed Central

    Piermarini, Peter M.; Esquivel, Carlos J.; Denton, Jerod S.

    2017-01-01

    The Malpighian tubules and hindgut are the renal excretory tissues of mosquitoes; they are essential to maintaining hemolymph water and solute homeostasis. Moreover, they make important contributions to detoxifying metabolic wastes and xenobiotics in the hemolymph. We have focused on elucidating the molecular mechanisms of Malpighian tubule function in adult female mosquitoes and developing chemical tools as prototypes for next-generation mosquitocides that would act via a novel mechanism of action (i.e., renal failure). To date, we have targeted inward rectifier potassium (Kir) channels expressed in the Malpighian tubules of the yellow fever mosquito Aedes aegypti and malaria mosquito Anopheles gambiae. Inhibition of these channels with small molecules inhibits transepithelial K+ and fluid secretion in Malpighian tubules, leading to a disruption of hemolymph K+ and fluid homeostasis in adult female mosquitoes. In addition, we have used next-generation sequencing to characterize the transcriptome of Malpighian tubules in the Asian tiger mosquito Aedes albopictus, before and after blood meals, to reveal new molecular targets for potentially disrupting Malpighian tubule function. Within 24 h after a blood meal, the Malpighian tubules enhance the mRNA expression of genes encoding mechanisms involved with the detoxification of metabolic wastes produced during blood digestion (e.g., heme, NH3, reactive oxygen species). The development of chemical tools targeting these molecular mechanisms in Malpighian tubules may offer a promising avenue for the development of mosquitocides that are highly-selective against hematophagous females, which are the only life stage that transmits pathogens. PMID:28125032

  1. Three-dimensional architecture of virus-packed tubule.

    PubMed

    Katayama, Sumie; Wei, Taiyun; Omura, Toshihiro; Takagi, Junichi; Iwasaki, Kenji

    2007-06-01

    When rice dwarf virus (RDV), a member of the Reoviridae family, infects leafhopper cells, formation of protruding tubules composed of nonstructural viral protein Pns 10 can be observed. We examined the three-dimensional (3D) structure of these tubules containing RDV particles using electron tomography. The thin section of RDV-infected leafhopper vector cells in monolayers was subjected to double-tilt tomography. The tomographic 3D map provides a more reliable estimation of the real dimensions of the structure compared with the 2D image of the thin section. Docking of particle models made from atomic coordinates of RDV into the tomogram revealed that the inner diameter of the tubule was close to the outer diameter of the RDV particle. Fourier-transform of the reconstituted tubule image from the purified Pns 10 protein in vitro revealed a helical structure of the tubule.

  2. Proximal Tibial Bone Graft

    MedlinePlus

    ... All Site Content AOFAS / FootCareMD / Treatments Proximal Tibial Bone Graft Page Content What is a bone graft? Bone grafts may be needed for various ... the proximal tibia. What is a proximal tibial bone graft? Proximal tibial bone graft (PTBG) is a ...

  3. Molecular mechanism of kNBC1-carbonic anhydrase II interaction in proximal tubule cells.

    PubMed

    Pushkin, Alexander; Abuladze, Natalia; Gross, Eitan; Newman, Debra; Tatishchev, Sergei; Lee, Ivan; Fedotoff, Olga; Bondar, Galyna; Azimov, Rustam; Ngyuen, Matt; Kurtz, Ira

    2004-08-15

    We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C-terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1-ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C-terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L(958)DDV and D(986)NDD in the wild-type kNBC1-ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1-mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.

  4. Variation in sorbitol accumulation and polyol-pathway activity in cultured human proximal tubule cells.

    PubMed

    Flath, M C; Bylander, J E; Sens, D A

    1992-09-01

    The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications. We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals. When exposed to 27.5 mM glucose in the growth media, cells isolated from approximately 75% of individuals (accumulators) accumulated sorbitol within 1-4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk. Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH. Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media. Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells. Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

  5. The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver.

    PubMed

    Zhang, Runxiang; Yi, Ran; Bi, Yanju; Xing, Lu; Bao, Jun; Li, Jianhong

    2017-01-06

    Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na2SeO3, 2 mg/kg), Cd (CdCl2, 150 mg/kg), or Se + Cd (150 mg/kg of CdCl2 and 2 mg/kg of Na2SeO3) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.

  6. Heterogeneity of T-Tubules in Pig Hearts

    PubMed Central

    Gadeberg, Hanne C.; Bond, Richard C.; Kong, Cherrie H. T.; Chanoit, Guillaume P.; Ascione, Raimondo; Cannell, Mark B.; James, Andrew F.

    2016-01-01

    Background T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in mammalian cardiac myocytes. Although t-tubules were generally considered to be effectively absent in atrial myocytes, recent studies on atrial cells from larger mammals suggest that t-tubules may be more numerous than previously supposed. However, the degree of heterogeneity between cardiomyocytes in the extent of the t-tubule network remains unclear. The aim of the present study was to investigate the t-tubule network of pig atrial myocytes in comparison with ventricular tissue. Methods Cardiac tissue was obtained from young female Landrace White pigs (45–75 kg, 5–6 months old). Cardiomyocytes were isolated by arterial perfusion with a collagenase-containing solution. Ca2+ transients were examined in field-stimulated isolated cells loaded with fluo-4-AM. Membranes of isolated cells were visualized using di-8-ANEPPS. T-tubules were visualized in fixed-frozen tissue sections stained with Alexa-Fluor 488-conjugated WGA. Binary images were obtained by application of a threshold and t-tubule density (TTD) calculated. A distance mapping approach was used to calculate half-distance to nearest t-tubule (HDTT). Results & Conclusion The spatio-temporal properties of the Ca2+ transient appeared to be consistent with the absence of functional t-tubules in isolated atrial myocytes. However, t-tubules could be identified in a sub-population of atrial cells in frozen sections. While all ventricular myocytes had TTD >3% (mean TTD = 6.94±0.395%, n = 24), this was true of just 5/22 atrial cells. Mean atrial TTD (2.35±0.457%, n = 22) was lower than ventricular TTD (P<0.0001). TTD correlated with cell-width (r = 0.7756, n = 46, P<0.0001). HDTT was significantly greater in the atrial cells with TTD ≤3% (2.29±0.16 μm, n = 17) than in either ventricular cells (1.33±0.05 μm, n = 24, P<0.0001) or in atrial cells with TTD >3% (1.65±0.06 μm, n = 5, P<0.05). These

  7. From single molecule to single tubules

    NASA Astrophysics Data System (ADS)

    Guo, Chin-Lin

    2012-02-01

    Biological systems often make decisions upon conformational changes and assembly of single molecules. In vivo, epithelial cells (such as the mammary gland cells) can respond to extracellular matrix (ECM) molecules, type I collagen (COL), and switch their morphology from a lobular lumen (100-200 micron) to a tubular lumen (1mm-1cm). However, how cells make such a morphogenetic decision through interactions with each other and with COL is unclear. Using a temporal control of cell-ECM interaction, we find that epithelial cells, in response to a fine-tuned percentage of type I collagen (COL) in ECM, develop various linear patterns. Remarkably, these patterns allow cells to self-assemble into a tubule of length ˜ 1cm and diameter ˜ 400 micron in the liquid phase (i.e., scaffold-free conditions). In contrast with conventional thought, the linear patterns arise through bi-directional transmission of traction force, but not through diffusible biochemical factors secreted by cells. In turn, the transmission of force evokes a long-range (˜ 600 micron) intercellular mechanical interaction. A feedback effect is encountered when the mechanical interaction modifies cell positioning and COL alignment. Micro-patterning experiments further reveal that such a feedback is a novel cell-number-dependent, rich-get-richer process, which allows cells to integrate mechanical interactions into long-range (> 1mm) linear coordination. Our results suggest a mechanism cells can use to form and coordinate long-range tubular patterns, independent of those controlled by diffusible biochemical factors, and provide a new strategy to engineer/regenerate epithelial organs using scaffold-free self-assembly methods.

  8. An Automated Image Analysis System to Quantify Endosomal Tubulation

    PubMed Central

    Newton, Timothy M.

    2016-01-01

    Recycling of cargos from early endosomes requires regulation of endosomal tubule formation and fission. This regulation is disrupted in cells depleted of the microtubule severing enzyme spastin, causing elongation of endosomal tubules and mis-trafficking of recycling endosomal cargos such as the transferrin receptor. Spastin is encoded by SPAST, mutations in which are the most frequent cause of autosomal dominant hereditary spastic paraplegia, a condition characterised by a progressive loss of lower limb function resulting from upper motor neuron axonopathy. Investigation of molecular factors involved in endosomal tubule regulation is hindered by the need for manual counting of endosomal tubules. We report here the development of an open source automated system for the quantification of endosomal tubules, using ImageJ and R. We validate the method in cells depleted of spastin and its binding partner IST1. The additional speed and reproducibility of this system compared with manual counting makes feasible screens of candidates to further understand the mechanisms of endosomal tubule formation and fission. PMID:28006827

  9. Structure of Lipid Tubules Formed from a Polymerizable Lecithin

    PubMed Central

    Yager, Paul; Schoen, Paul E.; Davies, Carol; Price, Ronald; Singh, Alok

    1985-01-01

    We have studied tubules formed from a polymerizable lipid in aqueous dispersion using freeze-fracture replication and transmission electron microscopy. The polymerizable diacetylenic lecithin 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine converts from liposomes to hollow cylinders, which we call tubules, on cooling through its chain melting phase transition temperature. These tubules differ substantially from cochleate cylinders formed by phosphatidylserines on binding of calcium. The tubules have diameters that range from 0.3 to 1 μm and lengths of up to hundreds of micrometers depending on conditions of formation. The thickness of the walls varies from as few as two bilayers to tens of bilayers in some longer tubules. Their surfaces may be either smooth, gently rippled, or with spiral steps depending on sample preparation conditions, including whether the lipids have been polymerized. The spiral steps may reflect the growth of the tubules by rolling up of flattened liposomes. ImagesFIGURE 1FIGURE 2FIGURE 3FIGURE 4FIGURE 5FIGURE 6FIGURE 7FIGURE 8FIGURE 9 PMID:19431600

  10. Function-informed transcriptome analysis of Drosophila renal tubule

    PubMed Central

    Wang, Jing; Kean, Laura; Yang, Jingli; Allan, Adrian K; Davies, Shireen A; Herzyk, Pawel; Dow, Julian AT

    2004-01-01

    Background Comprehensive, tissue-specific, microarray analysis is a potent tool for the identification of tightly defined expression patterns that might be missed in whole-organism scans. We applied such an analysis to Drosophila melanogaster Malpighian (renal) tubule, a defined differentiated tissue. Results The transcriptome of the D. melanogaster Malpighian tubule is highly reproducible and significantly different from that obtained from whole-organism arrays. More than 200 genes are more than 10-fold enriched and over 1,000 are significantly enriched. Of the top 200 genes, only 18 have previously been named, and only 45% have even estimates of function. In addition, 30 transcription factors, not previously implicated in tubule development, are shown to be enriched in adult tubule, and their expression patterns respect precisely the domains and cell types previously identified by enhancer trapping. Of Drosophila genes with close human disease homologs, 50 are enriched threefold or more, and eight enriched 10-fold or more, in tubule. Intriguingly, several of these diseases have human renal phenotypes, implying close conservation of renal function across 400 million years of divergent evolution. Conclusions From those genes that are identifiable, a radically new view of the function of the tubule, emphasizing solute transport rather than fluid secretion, can be obtained. The results illustrate the phenotype gap: historically, the effort expended on a model organism has tended to concentrate on a relatively small set of processes, rather than on the spread of genes in the genome. PMID:15345053

  11. A bioartificial renal tubule device embedding human renal stem/progenitor cells.

    PubMed

    Sciancalepore, Anna Giovanna; Sallustio, Fabio; Girardo, Salvatore; Gioia Passione, Laura; Camposeo, Andrea; Mele, Elisa; Di Lorenzo, Mirella; Costantino, Vincenzo; Schena, Francesco Paolo; Pisignano, Dario

    2014-01-01

    We present a bio-inspired renal microdevice that resembles the in vivo structure of a kidney proximal tubule. For the first time, a population of tubular adult renal stem/progenitor cells (ARPCs) was embedded into a microsystem to create a bioengineered renal tubule. These cells have both multipotent differentiation abilities and an extraordinary capacity for injured renal cell regeneration. Therefore, ARPCs may be considered a promising tool for promoting regenerative processes in the kidney to treat acute and chronic renal injury. Here ARPCs were grown to confluence and exposed to a laminar fluid shear stress into the chip, in order to induce a functional cell polarization. Exposing ARPCs to fluid shear stress in the chip led the aquaporin-2 transporter to localize at their apical region and the Na(+)K(+)ATPase pump at their basolateral portion, in contrast to statically cultured ARPCs. A recovery of urea and creatinine of (20±5)% and (13±5)%, respectively, was obtained by the device. The microengineered biochip here-proposed might be an innovative "lab-on-a-chip" platform to investigate in vitro ARPCs behaviour or to test drugs for therapeutic and toxicological responses.

  12. Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules.

    PubMed

    Armour, Eric A; Carson, Robert P; Ess, Kevin C

    2012-08-15

    Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.

  13. Association of interstitial nephritis with tubule cell injury and proliferation in NZB/NZW mice.

    PubMed Central

    Hurd, E R; Ziff, M

    1978-01-01

    The relationship between renal interstitial mononuclear cell infiltration and renal tubule cell (TC) proliferation has been examined in (NZB X NZW) F1 hybrid (B/W) and control mice. TC proliferation was measured by tritiated thymidine ([3H]TdR) injection, autoradiographic examination of kidney sections and enumeration of labelled tubule cells. In B/W mice interstitial infiltration began at 5 months and reached a peak at 9 months. Proliferation of TC began at 7 months and also reached a peak at 9 months. Control mice consisting of NZB, CBA/J and C57Bl/6J mice showed no increase with age. When the percentages of labelled TC in areas adjacent to interstitial infiltrates and distant from them were compared, the TC adjacent to infiltrates showed an approximately four-fold greater amount of labelling. Ultrathin light and electron microscopic sections demonstrated TC injury in areas in close proximity to infiltrates. These studies suggest that mononuclear cells in the interstitial infiltrate may be injurious to TC, leading to a sequence of injury and subsequent regeneration of these cells. In diseases in which interstitial nephritis is present, such as Sjögren's syndrome, the interstitial infiltrate may contribute to the observed TC dysfunction. Images FIG. 1 FIG. 2 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 PMID:668188

  14. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.

    PubMed Central

    Wesson, D E

    1989-01-01

    Whether augmented bicarbonate reabsorption by renal tubular epithelium contributes to the maintenance of chloride-deplete metabolic alkalosis is not clear. This study used free-flow micropuncture to investigate bicarbonate reabsorption by surface nephron segments in a rat model of diuretic-induced alkalosis compared to control. The proximal and distal nephron of the alkalotic animals had higher values for both delivered load to and absolute reabsorption from these segments. The proximal tubules of alkalotic and control animals had similar values for the slopes of the linear regression of delivered load vs. reabsorption and for the bicarbonate tubular fluid to plasma (TF/P) ratio at the late proximal tubule. By contrast, the corresponding analysis for the distal segment of alkalotic animals revealed a greater slope (0.98 vs. 0.81, P less than 0.003) and a smaller bicarbonate TF/P ratio at the late distal tubule (0.10 vs. 0.16, P less than 0.006). The data indicate that augmented bicarbonate reabsorption by both the proximal and distal nephron contributes to maintaining the alkalosis of this model. The data suggest primary stimulation of bicarbonate reabsorption in the distal nephron and load-dependent reabsorption in the proximal tubule. PMID:2808701

  15. Dentin tubule numerical density variations below the CEJ

    PubMed Central

    Komabayashi, T.; Nonomura, G.; Watanabe, L.G.; Marshall, G.W.; Marshall, S.J.

    2008-01-01

    Aim To evaluate dentin tubule numerical density variations below the CEJ. Methodology Three human non-carious permanent canines were sectioned parallel to the CEJ to obtain dentin disks 1 mm thick whose surfaces were 1 mm and 2 mm below the CEJ. Each disk was sectioned into quarters resulting in four segment locations: facial, lingual, mesial, and distal. The outer (PDL side) and inner (pulp side) surfaces of the specimens were shaped to expose dentin with SiC papers and polished. Numerical tubule density was determined from SEM images. All data were statistically analyzed using a three-way ANOVA. Results The dentin tubule density (number/mm2) ranged from 13,700 to 32,300. Dentin tubule density was relatively uniform at 1 and 2 mm below the CEJ and increased by a factor of about two from the outer to the inner surface, which was significantly different (P<0.0001). Conclusions The tubule density variations at the cervical root did not present the marked changes. PMID:18786756

  16. Changes in Proximal and Distal Tubular Reabsorption Produced by Rapid Expansion of Extracellular Fluid*

    PubMed Central

    Hayslett, John P.; Kashgarian, Michael; Epstein, Franklin H.

    1967-01-01

    Acute infusions of isotonic saline in the rat cause an increase in glomerular filtration rate and in the excretion of salt and water. The kidney swells, due to expansion of tubular and interstitial volume. Despite the increase in tubular diameter, transit time through the proximal tubules and loops of Henle is decreased, presumably owing to a greatly accelerated rate of tubular flow. Proximal tubular reabsorption, measured in blocked tubules, is inhibited in a way that cannot be ascribed to changes in tubular diameter. The prolongation of proximal reabsorptive half-time is not affected by the administration of aldosterone. It occurs equally in rats chronically loaded with or deprived of salt, and it is therefore not likely that it is influenced by the renal content of renin. In contrast, reabsorption from the distal convoluted tubule is enhanced by saline infusion. This change is observed in segments of tubules blocked with oil and isolated from their glomeruli and thus appears to occur independently of changes in glomerular filtration or tubular flow. Images PMID:6027087

  17. Polyunsaturated Fatty Acids in Lipid Bilayers and Tubules

    NASA Astrophysics Data System (ADS)

    Hirst, Linda S.; Yuan, Jing; Pramudya, Yohannes; Nguyen, Lam T.

    2007-03-01

    Omega-3 polyunsaturated fatty acids (PUFAs) are found in a variety of biological membranes and have been implicated with lipid raft formation and possible function, typical molecules include DHA (Docosahexanoic Acid) and AA (Alphalinoleic Acid) which have been the focus of considerable attention in recent years. We are interested in the phase behavior of these molecules in the lipid bilayer. The addition of lipid molecules with polyunsaturated chains has a clear effect on the fluidity and curvature of the membrane and we investigate the effects the addition of polyunsaturated lipids on bilayer structure and tubule formation. Self-assembled cylindrical lipid tubules have attracted considerable attention because of their interesting structures and potential technological applications. Using x-ray diffraction techniques, Atomic Force Microscopy and confocal fluorescence imaging, both symmetric and mixed chain lipids were incorporated into model membranes and the effects on bilayer structure and tubule formation investigated.

  18. Celastrol ameliorates Cd-induced neuronal apoptosis by targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway.

    PubMed

    Xu, Chong; Wang, Xiaoxue; Gu, Chenjian; Zhang, Hai; Zhang, Ruijie; Dong, Xiaoqing; Liu, Chunxiao; Hu, Xiaoyu; Ji, Xiang; Huang, Shile; Chen, Long

    2017-04-01

    Celastrol, a plant-derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species (ROS). Recently, we have reported that cadmium (Cd) activates c-Jun N-terminal kinase (JNK) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 (PP5), and celastrol prevents Cd-activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS-dependent PP5-JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd-induced expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22(phox) , p40(phox) , p47(phox) , p67(phox) , and Rac1), as well as the generation of ROS in PC12 cells and primary neurons. Also, N-acetyl-l-cysteine, a ROS scavenger, potentiated celastrol's inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd-evoked NOX2-derived ROS. Further research revealed that celastrol was involved in the regulation of PP5 inactivation and JNK/c-Jun activation induced by Cd, as celastrol prevented Cd from reducing PP5 expression, and over-expression of wild-type PP5 or dominant negative c-Jun strengthened celastrol's inhibition of Cd-induced phosphorylation of JNK and/or c-Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX2 with apocynin or silencing NOX2 by RNA interference enhanced the inhibitory effects of celastrol on Cd-induced inactivation of PP5, activation of JNK/c-Jun, ROS, and apoptosis in the cells. Furthermore, we noticed that expression of wild-type PP5 or dominant negative c-Jun, or pretreatment with JNK inhibitor SP600125 reinforced celastrol's suppression of Cd-induced NOX2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd-induced neuronal apoptosis via targeting NOX2-derived

  19. Excretory transport of xenobiotics by dogfish shark rectal gland tubules.

    PubMed

    Miller, D S; Masereeuw, R; Henson, J; Karnaky, K J

    1998-09-01

    Marine elasmobranch rectal gland is a specialized, osmoregulatory organ composed of numerous blind-ended, branched tubules emptying into a central duct. To date, NaCl excretion has been its only described function. Here we use isolated rectal gland tubule fragments from dogfish shark (Squalus acanthias), fluorescent xenobiotics, and confocal microscopy to describe a second function, xenobiotic excretion. Isolated rectal gland tubules rapidly transported the fluorescent organic anion sulforhodamine 101 from bath to lumen. Luminal accumulation was concentrative, saturable, and inhibited by cyclosporin A (CSA), chlorodinitrobenzene, leukotriene C4, and KCN. Inhibitors of renal organic anion transport (probenecid, p-aminohippurate), organic cation transport (tetraethylammonium and verapamil), and P-glycoprotein (verapamil) were without effect. Cellular accumulation of sulforhodamine 101 was not concentrative, saturable, or inhibitable. Rectal gland tubules did not secrete fluorescein, daunomycin, or a fluorescent CSA derivative. Finally, frozen rectal gland sections stained with an antibody to a hepatic canalicular multispecific organic anion transporter (cMOAT or MRP2) showed heavy and specific staining on the luminal membrane of the epithelial cells. We conclude that rectal gland is capable of active and specific excretion of xenobiotics and that such transport is mediated by a shark analog of MRP2, an ATP-driven xenobiotic transporter, but not by P-glycoprotein.

  20. Lipid tubules Formed by Flow-Controlled Hydration

    NASA Astrophysics Data System (ADS)

    Yuan, Jing; Hirst, Linda S.

    2007-03-01

    Self-assembled cylindrical tubules from lipid molecules have attracted considerable attention because of their interesting supramolecular structures and technological applications. Schnur et al. [1] reported the formation of tubular microstructures from a series of diacetylenic phospholipids after liposomes were cooled through their chain melting transition. After that, several methods have been developed to fabricate such unique microstructures mainly by means of deforming preformed Giant unilamellar vesicles. Here we present a simple strategy to construct lipid microtubules through a flow-controlled lipid hydration. Fluorescent microscopy and Confocal Laser Microscopy were used to visualize the formation and the structure of the lipid tubules. Tubules were found to develop following the direction of the dynamic flow with highly parallel alignment. At high flow speeds, partial cross-linking of the lipid tubules was observed. To demonstrate the generality of this method, different types of phospholipids, such as Phosphatidic Acid (PA), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), and Phosphatidylglycerol (PG) were investigated. [1] J.M. Schnur et al, Science, 264, 945 (1994).

  1. Dynamic tubulation of mitochondria drives mitochondrial network formation.

    PubMed

    Wang, Chong; Du, Wanqing; Su, Qian Peter; Zhu, Mingli; Feng, Peiyuan; Li, Ying; Zhou, Yichen; Mi, Na; Zhu, Yueyao; Jiang, Dong; Zhang, Senyan; Zhang, Zerui; Sun, Yujie; Yu, Li

    2015-10-01

    Mitochondria form networks. Formation of mitochondrial networks is important for maintaining mitochondrial DNA integrity and interchanging mitochondrial material, whereas disruption of the mitochondrial network affects mitochondrial functions. According to the current view, mitochondrial networks are formed by fusion of individual mitochondria. Here, we report a new mechanism for formation of mitochondrial networks through KIF5B-mediated dynamic tubulation of mitochondria. We found that KIF5B pulls thin, highly dynamic tubules out of mitochondria. Fusion of these dynamic tubules, which is mediated by mitofusins, gives rise to the mitochondrial network. We further demonstrated that dynamic tubulation and fusion is sufficient for mitochondrial network formation, by reconstituting mitochondrial networks in vitro using purified fusion-competent mitochondria, recombinant KIF5B, and polymerized microtubules. Interestingly, KIF5B only controls network formation in the peripheral zone of the cell, indicating that the mitochondrial network is divided into subzones, which may be constructed by different mechanisms. Our data not only uncover an essential mechanism for mitochondrial network formation, but also reveal that different parts of the mitochondrial network are formed by different mechanisms.

  2. Proximal renal tubular acidosis: a not so rare disorder of multiple etiologies

    PubMed Central

    Haque, Syed K.; Ariceta, Gema; Batlle, Daniel

    2012-01-01

    Proximal renal tubular acidosis (RTA) (Type II RTA) is characterized by a defect in the ability to reabsorb HCO3 in the proximal tubule. This is usually manifested as bicarbonate wastage in the urine reflecting that the defect in proximal tubular transport is severe enough that the capacity for bicarbonate reabsorption in the thick ascending limb of Henle's loop and more distal nephron segments is overwhelmed. More subtle defects in proximal bicarbonate transport likely go clinically unrecognized owing to compensatory reabsorption of bicarbonate distally. Inherited proximal RTA is more commonly autosomal recessive and has been associated with mutations in the basolateral sodium-bicarbonate cotransporter (NBCe1). Mutations in this transporter lead to reduced activity and/or trafficking, thus disrupting the normal bicarbonate reabsorption process of the proximal tubules. As an isolated defect for bicarbonate transport, proximal RTA is rare and is more often associated with the Fanconi syndrome characterized by urinary wastage of solutes like phosphate, uric acid, glucose, amino acids, low-molecular-weight proteins as well as bicarbonate. A vast array of rare tubular disorders may cause proximal RTA but most commonly it is induced by drugs. With the exception of carbonic anhydrase inhibitors which cause isolated proximal RTA, drug-induced proximal RTA is associated with Fanconi syndrome. Drugs that have been recently recognized to cause severe proximal RTA with Fanconi syndrome include ifosfamide, valproic acid and various antiretrovirals such as Tenofovir particularly when given to human immunodeficiency virus patients receiving concomitantly protease inhibitors such as ritonavir or reverse transcriptase inhibitors such as didanosine. PMID:23235953

  3. The relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells.

    PubMed

    Meng, Su-Fang; Mao, Wei-Ping; Wang, Fang; Liu, Xiao-Qian; Shao, Luan-Luan

    2015-11-01

    This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL-68). The expression of LC3B-II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd-exposed WRL-68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL-68 cells. The lysosomal activation was markedly decreased when the cells were co-treated with 3-MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome-lysosome fusion. The colocalization of lysosome-associated membrane protein-2 (LAMP2) and GFP-LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome-lysosome fusion). We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome-lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL-68 cells. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca(2+) stores and the intracellular Ca(2+) channels or pumps were possibly pH-dependent.

  4. Natural history of seminiferous tubule degeneration in Klinefelter syndrome.

    PubMed

    Aksglaede, Lise; Wikström, Anne M; Rajpert-De Meyts, Ewa; Dunkel, Leo; Skakkebaek, Niels E; Juul, Anders

    2006-01-01

    Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates dramatically at the time of puberty with complete hyalinization of the seminiferous tubules, although a few tubules with spermatogenesis may be present in adult life. Activation of the pituitary-gonadal axis at 3 months of age is seen in Klinefelter boys similar to healthy boys. However, the level of testosterone in Klinefelter boys is significantly lower than in controls. After this 'minipuberty', the hormone levels decline to normal prepubertal levels until puberty. In puberty, an initial rise in testosterone, inhibin B, LH and FSH occurs in Klinefelter boys. However, the rise in testosterone levels off and ends at a low-normal level in young adults. Likewise, serum concentration of inhibin B exhibits a dramatic decline to a low, often undetectable level, concomitantly with a rise in FSH, reflecting the degeneration of the seminiferous tubules. Many hypotheses about the underlying mechanism of the depletion of the germ cells in Klinefelter males have been reported and include insufficient supranumerary X-chromosome inactivation, Leydig cell insufficiency and disturbed regulation of apoptosis of Sertoli and Leydig cells. However, at present, the exact mechanism remains unclear. In this article, we summarize current knowledge on the development of the classical endocrinological and histological features of 47,XXY males from fetus to adulthood and review the literature concerning the degeneration of the seminiferous tubules in this syndrome.

  5. Proximal humerus fractures.

    PubMed

    Price, Matthew C; Horn, Pamela L; Latshaw, James C

    2013-01-01

    Proximal humerus fractures are among the most common fractures associated with osteoporosis. With an aging population, incidence of these fractures will only increase. The proximal humerus not only forms the lateral portion of the shoulder articulation but also has significant associations with musculoskeletal and neurovascular structures. As a result, fractures of the proximal humerus can significantly impact not only the function of the shoulder joint, but the health and function of the entire upper extremity as well. Understanding of these fractures, the management options, and associated nursing care, can help reduce morbidity rate and improve functional outcomes.

  6. Locally formed dopamine inhibits Na sup + -K sup + -ATPase activity in rat renal cortical tubule cells

    SciTech Connect

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J. Karolinska Institute, Stockholm )

    1988-10-01

    Dopamine, generated locally from L-dopa, inhibits Na{sup +}-K{sup +}-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na{sup +}-K{sup +}-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate ({dot Q}o{sub 2}) and {sup 86}Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive {dot Q}o{sub 2} or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive {dot Q}o{sub 2} in a concentration-dependent manner, with half-maximal inhibition (K{sub 0.5}) of 5 {times} 10{sup {minus}7} M and a maximal inhibition of 14.1 {plus minus} 1.5% at 10{sup {minus}4}M. L-Dopa also blunted the nystatin-stimulated {dot Q}o{sub 2} in a concentration-dependent manner, indicating the L-dopa directly inhibits Na{sup +}-K{sup +}-ATPase activity and not sodium entry. Ouabain-sensitive {sup 86}Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} and {sup 86}Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na{sup +}-K{sup +}-ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner.

  7. Accelerated recovery of renal mitochondrial and tubule homeostasis with SIRT1/PGC-1α activation following ischemia–reperfusion injury

    SciTech Connect

    Funk, Jason A.; Schnellmann, Rick G.

    2013-12-01

    Kidney ischemia–reperfusion (I/R) injury elicits cellular injury in the proximal tubule, and mitochondrial dysfunction is a pathological consequence of I/R. Promoting mitochondrial biogenesis (MB) as a repair mechanism after injury may offer a unique strategy to restore both mitochondrial and organ function. Rats subjected to bilateral renal pedicle ligation for 22 min were treated once daily with the SIRT1 activator SRT1720 (5 mg/kg) starting 24 h after reperfusion until 72 h–144 h. SIRT1 expression was elevated in the renal cortex of rats after I/R + vehicle treatment (IRV), but was associated with less nuclear localization. SIRT1 expression was even further augmented and nuclear localization was restored in the kidneys of rats after I/R + SRT1720 treatment (IRS). PGC-1α was elevated at 72 h–144 h in IRV and IRS kidneys; however, SRT1720 treatment induced deacetylation of PGC-1α, a marker of activation. Mitochondrial proteins ATP synthase β, COX I, and NDUFB8, as well as mitochondrial respiration, were diminished 24 h–144 h in IRV rats, but were partially or fully restored in IRS rats. Urinary kidney injury molecule-1 (KIM-1) was persistently elevated in both IRV and IRS rats; however, KIM-1 tissue expression was attenuated in IRS rats. Additionally, sustained loss of Na{sup +},K{sup +}–ATPase expression and basolateral localization and elevated vimentin in IRV rats was normalized in IRS rats, suggesting restoration of a differentiated, polarized tubule epithelium. The results suggest that SRT1720 treatment expedited recovery of mitochondrial protein expression and function by enhancing MB, which was associated with faster proximal tubule repair. Targeting MB may offer unique therapeutic strategy following ischemic injury. - Highlights: • We examined recovery of mitochondrial and renal function after ischemia–reperfusion. • SRT1720 treatment after I/R induced mitochondrial biogenesis via SIRT1/PGC-1α. • Recovery of mitochondrial function was

  8. AutoTT: automated detection and analysis of T-tubule architecture in cardiomyocytes.

    PubMed

    Guo, Ang; Song, Long-Sheng

    2014-06-17

    Cardiac transverse (T)-tubules provide a specialized structure for synchronization and stabilization of sarcoplasmic reticulum Ca(2+) release in healthy cardiomyocytes. The application of laser scanning confocal microscopy and the use of fluorescent lipophilic membrane dyes have boosted the discoveries that T-tubule remodeling is a significant factor contributing to cardiac contractile dysfunction. However, the analysis and quantification of the remodeling of T-tubules have been a challenge and remain inconsistent among different research laboratories. Fast Fourier transformation (FFT) is the major analysis method applied to calculate the spatial frequency spectrum, which is used to represent the regularity of T-tubule systems. However, this approach is flawed because the density of T-tubules as well as non-T-tubule signals in the images influence the spectrum power generated by FFT. Preprocessing of images and topological architecture extracting is necessary to remove non-T-tubule noise from the analysis. In addition, manual analysis of images is time consuming and prone to errors and investigator bias. Therefore, we developed AutoTT, an automated analysis program that incorporates image processing, morphological feature extraction, and FFT analysis of spectrum power. The underlying algorithm is implemented in MATLAB (The MathWorks, Natick, MA). The program outputs the densities of transversely oriented T-tubules and longitudinally oriented T-tubules, power spectrum of the overall T-tubule systems, and averaged spacing of T-tubules. We also combined the density and regularity of T-tubules to give an index of T-tubule integrity (TTint), which provides a global evaluation of T-tubule alterations. In summary, AutoTT provides a reliable, easy to use, and fast approach for analyzing myocyte T-tubules. This program can also be applied to measure the density and integrity of other cellular structures.

  9. From cyst to tubule: innovations in vertebrate spermatogenesis.

    PubMed

    Yoshida, Shosei

    2016-01-01

    Although vertebrates share many common traits, their germline development and function exhibit significant divergence. In particular, this article focuses on their spermatogenesis. The fundamental elements that constitute vertebrate spermatogenesis and the evolutionary changes that occurred upon transition from water to land will be discussed. The life-long continuity of spermatogenesis is supported by the function of stem cells. Series of mitotic and meiotic germ cell divisions are 'incomplete' due to incomplete cytokinesis, forming syncytia interconnected via intercellular bridges (ICBs). Throughout this process, germ cells are supported by appropriate microenvironments established primarily by somatic Sertoli cells. In anamniotes (fish and amphibians) spermatogenesis progresses in cysts, in which developing germ cell syncytia are individually encapsulated by Sertoli cells. Accordingly, Sertoli cells undergo turnover with germ cells that they nourish. This mode of cystic spermatogenesis is also observed in nonvertebrates as insects. In amniotes (reptiles, birds, and mammals), however, Sertoli cells do not turn over but comprise a persistent structure of seminiferous tubules. Sertoli cells nourish different stages of germ cells simultaneously in distinct regions of their surface. This function of Sertoli cells is spatiotemporally orchestrated, and the seminiferous epithelial cycle and spermatogenic wave make the seminiferous tubules a high-throughput factory for sperm production. Furthermore, contrary to the organized differentiating cells, undifferentiated spermatogonia that comprise the stem cell compartment exhibit active motion over the basal layer of seminiferous tubules and the frequent breakdown of ICBs. Thus, amniote seminiferous tubules represent a typical facultative (or open) niche environment without a stem cell tethering anatomically defined niche. WIREs Dev Biol 2016, 5:119-131. doi: 10.1002/wdev.204 For further resources related to this article

  10. The Mechanism of Formation of Lipid Tubules from Liposomes

    DTIC Science & Technology

    1988-01-01

    structures about 10 in diameter and as longt as hundreds of micrometers. To elucidate the nature of the conversion process. freeze fracture electron...microscopy was utilized to examine samples that were rapidly quenched during tubule fOrmation. Many transitional structures -Acre obserjed. typically...that form unusual tubular structures [1-61. tain trapped liposomes (Fig. 1). When liposomes The lecithin 1.2-bis( l.l2-tricosadiynoyl)-sn-gly- are

  11. Structure of Lipid Tubules Formed from a Polymerizable Lecithin

    DTIC Science & Technology

    1985-01-01

    Structural errecui cochleate cylinders derives from the fact that when the ntepoodmezoafbsaammrns.I i.Ch’.Sc Ca i chlatd ay fom he hosbatiylsrin. lrge 104...q~ Th ... .... -- STRUCTURE OF LIPID TUBULES FORMED N F ROMN A POLYMIERIZABLE LECITHIN NPAUL YAGER,* PAULL E. SCHOEN,* CAROL DAVIES.: RONALD...phospholipids in aqueous the chemistry and structure of DC:PC. including monitor- dispersion (Bangham et al., 1965), pure tecithins have ing the

  12. Malpighian tubule development in the red flour beetle (Tribolium castaneum).

    PubMed

    King, Benedict; Denholm, Barry

    2014-11-01

    Malpighian tubules (MpTs) are the major organ for excretion and osmoregulation in most insects. MpT development is characterised for Drosophila melanogaster, but not other species. We therefore do not know the extent to which the MpT developmental programme is conserved across insects. To redress this we provide a comprehensive description of MpT development in the beetle Tribolium castaneum (Coleoptera), a species separated from Drosophila by >315 million years. We identify similarities with Drosophila MpT development including: 1) the onset of morphological development, beginning when tubules bud from the gut and proliferate to increase organ size. 2) the tubule is shaped by convergent-extension movements and oriented cell divisions. 3) differentiated tip cells activate EGF-signalling in distal MpT cells through the ligand Spitz. 4) MpTs contain two main cell types - principal and stellate cells, differing in morphology and gene expression. We also describe development of the beetle cryptonephridial system, an adaptation for water conservation, which represents a major modification of the MpT ground plan characterised by intimate association between MpTs and rectum. This work establishes a new model to compare MpT development across insects, and provides a framework to help understand how an evolutionary novelty - the cryptonephridial system - arose during organ evolution.

  13. Capacitive proximity sensor

    DOEpatents

    Kronberg, J.W.

    1994-05-31

    A proximity sensor based on a closed field circuit is disclosed. The circuit comprises a ring oscillator using a symmetrical array of plates that creates an oscillating displacement current. The displacement current varies as a function of the proximity of objects to the plate array. Preferably the plates are in the form of a group of three pair of symmetric plates having a common center, arranged in a hexagonal pattern with opposing plates linked as a pair. The sensor produces logic level pulses suitable for interfacing with a computer or process controller. The proximity sensor can be incorporated into a load cell, a differential pressure gauge, or a device for measuring the consistency of a characteristic of a material where a variation in the consistency causes the dielectric constant of the material to change. 14 figs.

  14. Capacitive proximity sensor

    DOEpatents

    Kronberg, James W.

    1994-01-01

    A proximity sensor based on a closed field circuit. The circuit comprises a ring oscillator using a symmetrical array of plates that creates an oscillating displacement current. The displacement current varies as a function of the proximity of objects to the plate array. Preferably the plates are in the form of a group of three pair of symmetric plates having a common center, arranged in a hexagonal pattern with opposing plates linked as a pair. The sensor produces logic level pulses suitable for interfacing with a computer or process controller. The proximity sensor can be incorporated into a load cell, a differential pressure gauge, or a device for measuring the consistency of a characteristic of a material where a variation in the consistency causes the dielectric constant of the material to change.

  15. The effect of exercise training on transverse tubules in normal, remodeled, and reverse remodeled hearts.

    PubMed

    Kemi, Ole J; Hoydal, Morten A; Macquaide, Niall; Haram, Per M; Koch, Lauren G; Britton, Steven L; Ellingsen, Oyvind; Smith, Godfrey L; Wisloff, Ulrik

    2011-09-01

    The response of transverse (T)-tubules to exercise training in health and disease remains unclear. Therefore, we studied the effect of exercise training on the density and spacing of left ventricle cardiomyocyte T-tubules in normal and remodeled hearts that associate with detubulation, by confocal laser scanning microscopy. First, exercise training in normal rats increased cardiomyocyte volume by 16% (P < 0.01), with preserved T-tubule density. Thus, the T-tubules adapted to the physiologic hypertrophy. Next, we studied T-tubules in a rat model of metabolic syndrome with pressure overload-induced concentric left ventricle hypertrophy, evidenced by 15% (P < 0.01) increased cardiomyocyte size. These rats had only 85% (P < 0.01) of the T-tubule density of control rats. Exercise training further increased cardiomyocyte volume by 8% (P < 0.01); half to that in control rats, but the T-tubule density remained unchanged. Finally, post-myocardial infarction heart failure induced severe cardiac pathology, with a 70% (P < 0.01) increased cardiomyocyte volume that included both eccentric and concentric hypertrophy and 55% (P < 0.01) reduced T-tubule density. Exercise training reversed 50% (P < 0.01) of the pathologic hypertrophy, whereas the T-tubule density increased by 40% (P < 0.05) compared to sedentary heart failure, but remained at 60% of normal hearts (P < 0.01). Physiologic hypertrophy associated with conserved T-tubule spacing (~1.8-1.9 µm), whereas in pathologic hypertrophy, T-tubules appeared disorganized without regular spacing. In conclusion, cardiomyocytes maintain the relative T-tubule density during physiologic hypertrophy and after mild concentric pathologic hypertrophy, whereas after severe pathologic remodeling with a substantial loss of T-tubules; exercise training reverses the remodeling and partly corrects the T-tubule density.

  16. Proximal Hamstring Repair Strength

    PubMed Central

    Harvey, Margaret Ann; Singh, Hardeep; Obopilwe, Elifho; Charette, Ryan; Miller, Suzanne

    2015-01-01

    Background: Proximal hamstring repair for complete ruptures has become a common treatment. There is no consensus in the literature about postoperative rehabilitation protocols following proximal hamstring repair. Some protocols describe bracing to prevent hip flexion or knee extension while others describe no immobilization. There are currently no biomechanical studies evaluating proximal hamstring repairs; nor are there any studies evaluating the effect of different hip flexion angles on these repairs. Hypothesis: As hip flexion increases from 0° to 90°, there will be a greater gap with cyclical loading. Study Design: Controlled laboratory study. Methods: Proximal hamstring insertions were detached from the ischial tuberosity in 24 cadavers and were repaired with 3 single-loaded suture anchors in the hamstring footprint with a Krakow suture technique. Cyclic loading from 10 to 125 N at 1 Hz was then performed for 0°, 45°, and 90° of hip flexion for 1500 cycles. Gap formation, stiffness, yield load, ultimate load, and energy to ultimate load were compared between groups using paired t tests. Results: Cyclic loading demonstrated the least amount of gap formation (P < .05) at 0° of hip flexion (2.39 mm) and most at 90° of hip flexion (4.19 mm). There was no significant difference in ultimate load between hip flexion angles (326, 309, and 338 N at 0°, 45°, and 90°, respectively). The most common mode of failure occurred with knot/suture failure (n = 17). Conclusion: Increasing hip flexion from 0° to 90° increases the displacement across proximal hamstring repairs. Postoperative bracing that limits hip flexion should be considered. Clinical Relevance: Repetitive motion involving hip flexion after a proximal hamstring repair may cause compromise of the repair. PMID:26665049

  17. Traumatic proximal tibiofibular dislocation.

    PubMed

    Burgos, J; Alvarez-Montero, R; Gonzalez-Herranz, P; Rapariz, J M

    1997-01-01

    Proximal tibiofibular dislocation is an exceptional lesion. Rarer still is its presentation in childhood. We describe the clinical case of a 6-year-old boy, the victim of a road accident. He had a tibiofibular dislocation associated with a metaphyseal fracture of the tibia.

  18. Steerable Capacitive Proximity Sensor

    NASA Technical Reports Server (NTRS)

    Jenstrom, Del T.; Mcconnell, Robert L.

    1994-01-01

    Steerable capacitive proximity sensor of "capaciflector" type based partly on sensing units described in GSC-13377 and GSC-13475. Position of maximum sensitivity adjusted without moving sensor. Voltage of each driven shield adjusted separately to concentrate sensing electric field more toward one side or other.

  19. Proximate Analysis of Coal

    ERIC Educational Resources Information Center

    Donahue, Craig J.; Rais, Elizabeth A.

    2009-01-01

    This lab experiment illustrates the use of thermogravimetric analysis (TGA) to perform proximate analysis on a series of coal samples of different rank. Peat and coke are also examined. A total of four exercises are described. These are dry exercises as students interpret previously recorded scans. The weight percent moisture, volatile matter,…

  20. Impaired leaf CO2 diffusion mediates Cd-induced inhibition of photosynthesis in the Zn/Cd hyperaccumulator Picris divaricata.

    PubMed

    Tang, Lu; Ying, Rong-Rong; Jiang, Dan; Zeng, Xiao-Wen; Morel, Jean-Louis; Tang, Ye-Tao; Qiu, Rong-Liang

    2013-12-01

    Mechanisms of cadmium (Cd)-induced inhibition of photosynthesis in the Zn/Cd hyperaccumulator Picris divaricata were investigated using photosynthesis limitation analysis. P. divaricata seedlings were grown in nutrient solution containing 0, 5, 10, 25, 50, or 75 μM Cd for 2 weeks. Total limitations to photosynthesis (TL) increased from 0% at 5 μM Cd to 68.8% at 75 μM Cd. CO2 diffusional limitation (DL) made the largest contribution to TL, accounting for 93-98% of TL in the three highest Cd treatments, compared to just 2-7% of TL attributable to biochemical limitation (BL). Microscopic imaging revealed significantly decreased stomatal density and mesophyll thickness in the three highest Cd treatments. Chlorophyll fluorescence parameters related to photosynthetic biochemistry (Fv/Fm, NPQ, ΦPSII, and qP) were not significantly decreased by increased Cd supply. Our results suggest that increased DL in leaves is the main cause of Cd-induced inhibition of photosynthesis in P. divaricata, possibly due to suppressed function of mesophyll and stomata. Analysis of chlorophyll fluorescence showed that Cd supply had little effect on photochemistry parameters, suggesting that the PSII reaction centers are not a main target of Cd inhibition of photosynthesis in P. divaricata.

  1. Subacute diabetic proximal neuropathy

    NASA Technical Reports Server (NTRS)

    Pascoe, M. K.; Low, P. A.; Windebank, A. J.; Litchy, W. J.

    1997-01-01

    OBJECTIVE: To evaluate the clinical, electrophysiologic, autonomic, and neuropathologic characteristics and the natural history of subacute diabetic proximal neuropathy and its response to immunotherapy. MATERIAL AND METHODS: For the 12-year period from 1983 to 1995, we conducted a retrospective review of medical records of Mayo Clinic patients with diabetes who had subacute onset and progression of proximal weakness. The responses of treated versus untreated patients were compared statistically. RESULTS: During the designated study period, 44 patients with subacute diabetic proximal neuropathy were encountered. Most patients were middle-aged or elderly, and no sex preponderance was noted. The proximal muscle weakness often was associated with reduced or absent lower extremity reflexes. Associated weight loss was a common finding. Frequently, patients had some evidence of demyelination on nerve conduction studies, but it invariably was accompanied by concomitant axonal degeneration. The cerebrospinal fluid protein concentration was usually increased. Diffuse and substantial autonomic failure was generally present. In most cases, a sural nerve biopsy specimen suggested demyelination, although evidence of an inflammatory infiltrate was less common. Of 12 patients who received treatment (with prednisone, intravenous immune globulin, or plasma exchange), 9 had improvement of their conditions, but 17 of 29 untreated patients (59%) with follow-up also eventually had improvement, albeit at a much slower rate. Improvement was usually incomplete. CONCLUSION: We suggest that the entity of subacute diabetic proximal neuropathy is an extensive and severe variant of bilateral lumbosacral radiculoplexopathy, with some features suggestive of an immune-mediated cause. It differs from chronic inflammatory demyelinating polyradiculoneuropathy in that most cases have a more restricted distribution and seem to be monophasic and self-limiting. The efficacy of immunotherapy is unproved

  2. How much can the tubule regenerate and who does it? An open question

    PubMed Central

    Lombardi, Duccio; Becherucci, Francesca; Romagnani, Paola

    2016-01-01

    The tubular compartment of the kidney is the primary site of a wide range of insults that can result in acute kidney injury (AKI), a condition associated with high mortality and an increased risk to develop end-stage renal disease. Nevertheless, kidney function is often quickly recovered after tubular injury. How this happens has only partially been unveiled. Indeed, although it has clearly been demonstrated that regenerated epithelial cells arise from survived intratubular cells, the true entity, as well as the cellular source of this regenerative process, remains mostly unknown. Is whichever proximal tubular epithelial cell able to dedifferentiate and divide to replace neighboring lost tubular cells, thus suggesting an extreme regenerative ability of residual tubular epithelium, or is the regenerative potential of tubular epithelium limited, and mostly related to a preexisting population of intratubular scattered progenitor cells which are more resistant to death? Gaining insights on how this process takes place is essential for developing new therapeutic strategies to prevent AKI, as well as AKI-related chronic kidney disease. The aim of this review is to discuss why the answers to these questions are still open, and how further investigations are needed to understand which is the true regenerative potential of the tubule and who are the players that allow functional recovery after AKI. PMID:26175143

  3. Complexity of glutamine metabolism in kidney tubules from fed and fasted rats.

    PubMed Central

    Vercoutère, Barbara; Durozard, Daniel; Baverel, Gabriel; Martin, Guy

    2004-01-01

    Glutamine is an important renal glucose precursor and energy provider. In order to advance our understanding of the underlying metabolic processes, we studied the metabolism of variously labelled [13C]glutamine and [14C]glutamine molecules and the effects of fasting in isolated rat renal proximal tubules. Absolute fluxes through the enzymes involved, including enzymes of four different cycles operating concomitantly, were assessed by combining mainly the 13C NMR data with an appropriate model of glutamine metabolism. In both nutritional states, unidirectional glutamine removal by glutaminase was partially masked by the concomitant operation of glutamine synthetase; fasting accelerated glutamine removal by increasing flux solely through glutaminase, without changing that through glutamine synthetase. Fasting stimulated net glutamate degradation only by decreasing flux through glutamate dehydrogenase in the reductive amination direction, but surprisingly did not significantly alter complete oxidation of the glutamine carbon skeleton. Finally, gluconeogenesis from glutamine involved not only substantial recycling through the tricarboxylic acid cycle, but also an important anaplerotic flux through pyruvate carboxylase that was accelerated dramatically by fasting. Thus renal glutamine metabolism follows an unexpectedly complex route that is precisely regulated during fasting. PMID:14616091

  4. The dynamin superfamily: universal membrane tubulation and fission molecules?

    PubMed

    Praefcke, Gerrit J K; McMahon, Harvey T

    2004-02-01

    Dynamins are large GTPases that belong to a protein superfamily that, in eukaryotic cells, includes classical dynamins, dynamin-like proteins, OPA1, Mx proteins, mitofusins and guanylate-binding proteins/atlastins. They are involved in many processes including budding of transport vesicles, division of organelles, cytokinesis and pathogen resistance. With sequenced genomes from Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, yeast species and Arabidopsis thaliana, we now have a complete picture of the members of the dynamin superfamily from different organisms. Here, we review the superfamily of dynamins and their related proteins, and propose that a common mechanism leading to membrane tubulation and/or fission could encompass their many varied functions.

  5. Proximity Networks and Epidemics

    NASA Astrophysics Data System (ADS)

    Guclu, Hasan; Toroczkai, Zoltán

    2007-03-01

    We presented the basis of a framework to account for the dynamics of contacts in epidemic processes, through the notion of dynamic proximity graphs. By varying the integration time-parameter T, which is the period of infectivity one can give a simple account for some of the differences in the observed contact networks for different diseases, such as smallpox, or AIDS. Our simplistic model also seems to shed some light on the shape of the degree distribution of the measured people-people contact network from the EPISIM data. We certainly do not claim that the simplistic graph integration model above is a good model for dynamic contact graphs. It only contains the essential ingredients for such processes to produce a qualitative agreement with some observations. We expect that further refinements and extensions to this picture, in particular deriving the link-probabilities in the dynamic proximity graph from more realistic contact dynamics should improve the agreement between models and data.

  6. Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance.

    PubMed

    Tung, Kenneth S K; Harakal, Jessica; Qiao, Hui; Rival, Claudia; Li, Jonathan C H; Paul, Alberta G A; Wheeler, Karen; Pramoonjago, Patcharin; Grafer, Constance M; Sun, Wei; Sampson, Robert D; Wong, Elissa W P; Reddi, Prabhakara P; Deshmukh, Umesh S; Hardy, Daniel M; Tang, Huanghui; Cheng, C Yan; Goldberg, Erwin

    2017-02-20

    Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

  7. Axial tubule junctions control rapid calcium signaling in atria

    PubMed Central

    Brandenburg, Sören; Kohl, Tobias; Williams, George S.B.; Rog-Zielinska, Eva A.; Hebisch, Elke; Dura, Miroslav; Didié, Michael; Nikolaev, Viacheslav O.; Hasenfuss, Gerd; Kohl, Peter; Ward, Christopher W.; Lehnart, Stephan E.

    2016-01-01

    The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon “super-hubs” thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias. PMID:27643434

  8. A dynamic paracellular pathway serves diuresis in mosquito Malpighian tubules.

    PubMed

    Beyenbach, Klaus W

    2012-07-01

    Female mosquitoes gorge on vertebrate blood, a rich nutrient source for developing eggs, but gorging meals increase the risk of predation. Mosquitoes are quick to reduce the flight payload with a potent diuresis. Diuretic peptides of the insect kinin family induce a tenfold reduction in the paracellular resistance of Malpighian tubules and increase the paracellular permeation of Cl(-), the counterion of the transepithelial secretion of Na(+) and K(+). As a result, the transepithelial secretion of NaCl and KCl and water increases. Insect kinins signal the opening of the paracellular pathway via G protein-coupled receptors and the elevation of intracellular [Ca(2+)], which leads to the reorganization of the cytoskeleton associated with the septate junction (SJ). The reorganization may affect the septate junctional proteins that control the barrier and permselectivity properties of the paracellular pathway. The proteins involved in the embryonic formation of the SJ and in epithelial polarization are largely known for ectodermal epithelia, but the proteins that form and mediate the dynamic functions of the SJ in Malpighian tubules remain to be determined.

  9. A conserved domain of alkaline phosphatase expression in the Malpighian tubules of dipteran insects.

    PubMed

    Cabrero, Pablo; Pollock, Valerie P; Davies, Shireen A; Dow, Julian A T

    2004-09-01

    Malpighian (renal) tubules are key components of the insect osmoregulatory system and show correspondingly great diversity in both number and length. Recently, the organisation of the Drosophila melanogaster tubule has been elucidated by enhancer trapping, and an array for functional properties has been shown to align with the functional domains. In Drosophila, there is a lower tubule domain, which coincides with expression of alkaline phosphatase and delineates the absorptive region of the tubule. Here, these observations are extended to three dipteran vectors of disease (Aedes aegypti, Anopheles stephensii and Glossina morsitans) and a non-dipteran out-group, Schistocerca gregaria (Orthoptera). Despite a huge range in cell number and size, alkaline phosphatase was found on the apical surface of the lower 10% of each of the dipteran tubules but nowhere within the orthopteran tubule. An alkaline phosphatase lower tubule domain is thus conserved among Diptera. Cell counts are also provided for each species. As in Drosophila, stellate cells are not found in the lower tubule domain of Anopheles or Aedes tubules, confirming the unique genetic identity of this domain. As previously reported, we failed to find stellate cells in Schistocerca but, remarkably, also failed to find them in Glossina, the dipteran most closely related to Drosophila. The orthodoxy that stellate cells are unique to, and general among, Diptera may thus require revision.

  10. Use of Poly (Amidoamine) Dendrimer for Dentinal Tubule Occlusion: A Preliminary Study

    PubMed Central

    Wang, Tianda; Yang, Sheng; Wang, Lei; Feng, Hailan

    2015-01-01

    The occlusion of dentinal tubules is an effective method to alleviate the symptoms caused by dentin hypersensitivity, a significant health problem in dentistry and daily life. The in situ mineralization within dentinal tubules is a promising treatment for dentin hypersensitivity as it induces the formation of mineral on the sensitive regions and occludes the dentinal tubules. This study was carried out to evaluate the in vitro effect of a whole generation poly(amidoamine) (PAMAM) dendrimer (G3.0) on dentinal tubule occlusion by inducing mineralization within dentinal tubules. Dentin discs were treated with PAMAM dendrimers using two methods, followed by the in vitro characterization using Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), Field emission scanning electron microscopy (FE-SEM) and Energy-Dispersive X-ray Spectroscopy (EDS). These results showed that G3.0 PAMAM dendrimers coated on dentin surface and infiltrated in dentinal tubules could induce hydroxyapatite formation and resulted in effective dentinal tubule occlusion. Moreover, crosslinked PAMAM dendrimers could induce the remineralization of demineralized dentin and thus had the potential in dentinal tubule occlusion. In this in vitro study, dentinal tubules occlusion could be achieved by using PAMAM dendrimers. This could lead to the development of a new therapeutic technique for the treatment of dentin hypersensitivity. PMID:25885090

  11. Functional Coupling of Human Microphysiology Systems: Intestine, Liver, Kidney Proximal Tubule, Blood-Brain Barrier and Skeletal Muscle

    PubMed Central

    Vernetti, Lawrence; Gough, Albert; Baetz, Nicholas; Blutt, Sarah; Broughman, James R.; Brown, Jacquelyn A.; Foulke-Abel, Jennifer; Hasan, Nesrin; In, Julie; Kelly, Edward; Kovbasnjuk, Olga; Repper, Jonathan; Senutovitch, Nina; Stabb, Janet; Yeung, Catherine; Zachos, Nick C.; Donowitz, Mark; Estes, Mary; Himmelfarb, Jonathan; Truskey, George; Wikswo, John P.; Taylor, D. Lansing

    2017-01-01

    Organ interactions resulting from drug, metabolite or xenobiotic transport between organs are key components of human metabolism that impact therapeutic action and toxic side effects. Preclinical animal testing often fails to predict adverse outcomes arising from sequential, multi-organ metabolism of drugs and xenobiotics. Human microphysiological systems (MPS) can model these interactions and are predicted to dramatically improve the efficiency of the drug development process. In this study, five human MPS models were evaluated for functional coupling, defined as the determination of organ interactions via an in vivo-like sequential, organ-to-organ transfer of media. MPS models representing the major absorption, metabolism and clearance organs (the jejunum, liver and kidney) were evaluated, along with skeletal muscle and neurovascular models. Three compounds were evaluated for organ-specific processing: terfenadine for pharmacokinetics (PK) and toxicity; trimethylamine (TMA) as a potentially toxic microbiome metabolite; and vitamin D3. We show that the organ-specific processing of these compounds was consistent with clinical data, and discovered that trimethylamine-N-oxide (TMAO) crosses the blood-brain barrier. These studies demonstrate the potential of human MPS for multi-organ toxicity and absorption, distribution, metabolism and excretion (ADME), provide guidance for physically coupling MPS, and offer an approach to coupling MPS with distinct media and perfusion requirements. PMID:28176881

  12. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    SciTech Connect

    Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  13. Characterization of the Interaction of Staphylococcal Entertoxin B with CD1d Expressed in Human Renal Proximal Tubule Epithelial Cells

    DTIC Science & Technology

    2015-02-04

    typical antigen recognition process that recruits the major histocompatibility complex class II (MHC II) molecules. The immunological importance of CD1d is...underscored by its influences on the performances of natural killer T-cells and thereby mediates the innate and adaptive immune systems. Results...release of many cytokines, and thereby systematically in- fluences the hosts’ adaptive and innate immune systems [35,36]. The involvement of CD1d in

  14. Echosonography with proximity sensors

    NASA Astrophysics Data System (ADS)

    Thaisiam, W.; Laithong, T.; Meekhun, S.; Chaiwathyothin, N.; Thanlarp, P.; Danworaphong, S.

    2013-03-01

    We propose the use of a commercial ultrasonic proximity sensor kit for profiling an altitude-varying surface by employing echosonography. The proximity sensor kit, two identical transducers together with its dedicated operating circuit, is used as a profiler for the construction of an image. Ultrasonic pulses are emitted from one of the transducers and received by the other. The time duration between the pulses allows us to determine the traveling distance of each pulse. In the experiment, the circuit is used with the addition of two copper wires for directing the outgoing and incoming signals to an oscilloscope. The time of flight of ultrasonic pulses can thus be determined. Square grids of 5 × 5 cm2 are made from fishing lines, forming pixels in the image. The grids are designed to hold the detection unit in place, about 30 cm above a flat surface. The surface to be imaged is constructed to be height varying and placed on the flat surface underneath the grids. Our result shows that an image of the profiled surface can be created by varying the location of the detection unit along the grid. We also investigate the deviation in relation to the time of flight of the ultrasonic pulse. Such an experiment should be valuable for conveying the concept of ultrasonic imaging to physical and medical science undergraduate students. Due to its simplicity, the setup could be made in any undergraduate laboratory relatively inexpensively and it requires no complex parts. The results illustrate the concept of echosonography.

  15. A critical synopsis: Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

    NASA Technical Reports Server (NTRS)

    Chuman, L. M.; FINE; COHEN; Saier, M. H.

    1985-01-01

    The kidney forms urine and reabsorbs electrolytes and water. Kidney cell lines and hormone supplemented serum free medium were used for growth. The hormones were insulin, transferrin, vasopressin, cholesterol, prostaglandins, hydrocortisone, and triidothyronine. Epithelial cell lines are polar and form hemicysts. The Madin-Darby canine kidney(MDCK) cell line used is distal tubulelike. LLC-PK sub 1 cells are derived from pig kidneys and have the properties of different kidney segments. The LLC-PK sub 1 cells with proximal tubule properties were maintained in hormone-supplemented serum free medium. Seven factors (the aforementioned homrones and selenium) were needed for growth. Hormone-defined medium supported LLC-PK sub 1 cell growth, allowed transport (as seen by hemicyst formation), and influenced cell morphology. Vasopressin (used for growth and morphology) could be partially replaced by isobutylmethylxanthine or dibutyryl cAMP. The defined medium was used to isolate rabbit proximal tubule kidney epithelial cells free of fibroblasts.

  16. Kidney tubules: intertubular, vascular, and glomerular cross-talk

    PubMed Central

    Ferenbach, David A.; Bonventre, Joseph V.

    2016-01-01

    Purpose of review The kidney mediates the excretion or conservation of water and electrolytes in the face of changing fluid and salt intake and losses. To ultrafilter and reabsorb the exact quantities of free water and salts to maintain euvolemia a range of endocrine, paracrine, and hormonal signaling systems have evolved linking the tubules, capillaries, glomeruli, arterioles, and other intrinsic cells of the kidney. Our understanding of these systems remains incomplete. Recent findings Recent work has provided new insights into the workings of the communication pathways between tubular segments and the glomeruli and vasculature, with novel therapeutic agents in development. Particular progress has also been made in the visualization of tubuloglomerular feedback. Summary The review summarizes our current understanding of pathway functions in health and disease, as well as future therapeutic options to protect the healthy and injured kidney. PMID:27023838

  17. Antimicrobial effect of ozonated water on bacteria invading dentinal tubules.

    PubMed

    Nagayoshi, Masato; Kitamura, Chiaki; Fukuizumi, Takaki; Nishihara, Tatsuji; Terashita, Masamichi

    2004-11-01

    Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. In the present study, we examined the effect of ozonated water against Enterococcus faecalis and Streptcoccus mutans infections in vitro in bovine dentin. After irrigation with ozonated water, the viability of E. faecalis and S. mutans invading dentinal tubules significantly decreased. Notably, when the specimen was irrigated with sonication, ozonated water had nearly the same antimicrobial activity as 2.5% sodium hypochlorite (NaOCl). We also compared the cytotoxicity against L-929 mouse fibroblasts between ozonated water and NaOCl. The metabolic activity of fibroblasts was high when the cells were treated with ozonated water, whereas that of fibroblasts significantly decreased when the cells were treated with 2.5% NaOCl. These results suggest that ozonated water application may be useful for endodontic therapy.

  18. LIPOPROTEIN GRANULES IN THE CORTICAL COLLECTING TUBULES OF MOUSE KIDNEY

    PubMed Central

    Miller, Fritz

    1961-01-01

    The light and, to a lesser extent, the dark cells of the cortical collecting tubules in mouse kidney contain a great number of granules which according to histochemical tests are composed of phospholipids and proteins. These granules are bounded by a triple-layered membrane measuring approximately 75 A across, and contain one or several crystals with a hexagonal or square lattice. These crystals are built up of rod-shaped units, which appear dense after osmium fixation, measure about 48 A in diameter, and are separated by a light interspace of similar dimensions. The mean center-to-center distance of the rods is about 96 A. The structure is explained as a lipoprotein crystallized within a membrane-bounded vacuole. No relationship between these granules and mitochondria was found. The physiological significance of the granules remains unknown. PMID:13770761

  19. Static analysis of masonry kilns built with fictile tubules bricks

    NASA Astrophysics Data System (ADS)

    Olivito, Renato S.; Scuro, Carmelo; Codispoti, Rosamaria

    2016-12-01

    Industrial archeology is a branch that studies all the testimony (tangible and intangible, direct and indirect) related to the process of industrialization since its origins. This technical field is based on an interdisciplinary approach, it has the task of deepening the story, understanding the technological development made by man over the centuries. The present work focused attention on the study and analysis of a masonry kiln, built with the technique of hollow clay fictile tubules. The study, in particular, has been carried out analyzing the stress state caused by the wind on the structure. The kiln is constituted by a particular geometric configuration that develops in height due to the presence of chimney over the dome.

  20. A New Look at Electrolyte Transport in the Distal Tubule

    PubMed Central

    Eladari, Dominique; Chambrey, Régine; Peti-Peterdi, Janos

    2015-01-01

    The distal nephron plays a critical role in the renal control of homeostasis. Until very recently most studies focused on the control of Na+, K+, and water balance by principal cells of the collecting duct and the regulation of solute and water by hormones from the renin-angiotensin-aldosterone system and by antidiuretic hormone. However, recent studies have revealed the unexpected importance of renal intercalated cells, a subtype of cells present in the connecting tubule and collecting ducts. Such cells were thought initially to be involved exclusively in acid-base regulation. However, it is clear now that intercalated cells absorb NaCl and K+ and hence may participate in the regulation of blood pressure and potassium balance. The second paradigm-challenging concept we highlight is the emerging importance of local paracrine factors that play a critical role in the renal control of water and electrolyte balance. PMID:21888509

  1. A new look at electrolyte transport in the distal tubule.

    PubMed

    Eladari, Dominique; Chambrey, Régine; Peti-Peterdi, Janos

    2012-01-01

    The distal nephron plays a critical role in the renal control of homeostasis. Until very recently most studies focused on the control of Na(+), K(+), and water balance by principal cells of the collecting duct and the regulation of solute and water by hormones from the renin-angiotensin-aldosterone system and by antidiuretic hormone. However, recent studies have revealed the unexpected importance of renal intercalated cells, a subtype of cells present in the connecting tubule and collecting ducts. Such cells were thought initially to be involved exclusively in acid-base regulation. However, it is clear now that intercalated cells absorb NaCl and K(+) and hence may participate in the regulation of blood pressure and potassium balance. The second paradigm-challenging concept we highlight is the emerging importance of local paracrine factors that play a critical role in the renal control of water and electrolyte balance.

  2. Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia

    PubMed Central

    Hong, TingTing; Yang, Huanghe; Zhang, Shan-Shan; Cho, Hee Cheol; Kalashnikova, Mariya; Sun, Baiming; Zhang, Hao; Bhargava, Anamika; Grabe, Michael; Olgin, Jeffrey; Gorelik, Julia; Marbán, Eduardo; Jan, Lily Y.; Shaw, Robin M.

    2014-01-01

    Cardiomyocyte T-tubules are important for regulating ionic flux. Bridging Integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is down-regulated in failing hearts. Here we find that cardiac T-tubules normally contain dense protective inner membrane folds that are formed by a cardiac spliced isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased which does not change overall cardiomyocyte morphology, but frees diffusion of local extracellular calcium and potassium ions, prolonging action potential duration, and increasing susceptibility to ventricular arrhythmias. We also find that T-tubule inner folds are rescued only by the BIN1 isoform BIN1+13+17, which promotes N-WASP dependent actin polymerization to stabilize T-tubule membrane at cardiac Z-discs. In conclusion, BIN1+13+17 recruits actin to fold T-tubule membrane, creating a fuzzy space that protectively restricts ionic flux. When BIN1+13+17 is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered and arrhythmias can result. PMID:24836577

  3. Physiologically based pharmacokinetic-pharmacodynamic modeling to predict concentrations and actions of sodium-dependent glucose transporter 2 inhibitor canagliflozin in human intestines and renal tubules.

    PubMed

    Mori, Kazumi; Saito, Ryuta; Nakamaru, Yoshinobu; Shimizu, Makiko; Yamazaki, Hiroshi

    2016-11-01

    Canagliflozin is a recently developed sodium-glucose cotransporter (SGLT) 2 inhibitor that promotes renal glucose excretion and is considered to inhibit renal SGLT2 from the luminal side of proximal tubules. Canagliflozin reportedly inhibits SGLT1 weakly and suppresses postprandial plasma glucose, suggesting that it also inhibits intestinal SGLT1. However, it is difficult to measure the drug concentrations of these assumed sites of action directly. The pharmacokinetic-pharmacodynamic (PK/PD) relationships of canagliflozin remain poorly characterized. Therefore, a physiologically based pharmacokinetic (PBPK) model of canagliflozin was developed based on clinical data from healthy volunteers and it was used to simulate luminal concentrations in intestines and renal tubules. In small intestine simulations, the inhibition ratios for SGLT1 were predicted to be 40%-60% after the oral administration of clinical doses (100-300 mg/day). In contrast, inhibition ratios of canagliflozin for renal SGLT2 and SGLT1 were predicted to be approximately 100% and 0.2%-0.4%, respectively. These analyses suggest that canagliflozin only inhibits SGLT2 in the kidney. Using the simulated proximal tubule luminal concentrations of canagliflozin, the urinary glucose excretion rates in canagliflozin-treated diabetic patients were accurately predicted using the renal glucose reabsorption model as a PD model. Because the simulation of canagliflozin pharmacokinetics was successful, this PBPK methodology was further validated by successfully simulating the pharmacokinetics of dapagliflozin, another SGLT2 inhibitor. The present results suggest the utility of this PBPK/PD model for predicting canagliflozin concentrations at target sites and help to elucidate the pharmacological effects of SGLT1/2 inhibition in humans. Copyright © 2016 John Wiley & Sons, Ltd.

  4. Some Properties of Fuzzy Soft Proximity Spaces

    PubMed Central

    Demir, İzzettin; Özbakır, Oya Bedre

    2015-01-01

    We study the fuzzy soft proximity spaces in Katsaras's sense. First, we show how a fuzzy soft topology is derived from a fuzzy soft proximity. Also, we define the notion of fuzzy soft δ-neighborhood in the fuzzy soft proximity space which offers an alternative approach to the study of fuzzy soft proximity spaces. Later, we obtain the initial fuzzy soft proximity determined by a family of fuzzy soft proximities. Finally, we investigate relationship between fuzzy soft proximities and proximities. PMID:25793224

  5. Some properties of fuzzy soft proximity spaces.

    PubMed

    Demir, İzzettin; Özbakır, Oya Bedre

    2015-01-01

    We study the fuzzy soft proximity spaces in Katsaras's sense. First, we show how a fuzzy soft topology is derived from a fuzzy soft proximity. Also, we define the notion of fuzzy soft δ-neighborhood in the fuzzy soft proximity space which offers an alternative approach to the study of fuzzy soft proximity spaces. Later, we obtain the initial fuzzy soft proximity determined by a family of fuzzy soft proximities. Finally, we investigate relationship between fuzzy soft proximities and proximities.

  6. Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules

    PubMed Central

    Lawrence, Marlon G.; Altenburg, Michael K.; Sanford, Ryan; Willett, Julian D.; Bleasdale, Benjamin; Ballou, Byron; Wilder, Jennifer; Li, Feng; Miner, Jeffrey H.; Berg, Ulla B.; Smithies, Oliver

    2017-01-01

    How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson’s or Alport’s proteinuric syndromes resulting from defects in GBM structural proteins (laminin β2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm. PMID:28246329

  7. Sphingosine 1-Phosphate Receptor-1 Enhances Mitochondrial Function and Reduces Cisplatin-Induced Tubule Injury

    PubMed Central

    Rosin, Diane L.; Chroscicki, Piotr; Lee, Sangju; Dondeti, Krishna; Ye, Hong; Kinsey, Gilbert R.; Stevens, Brian K.; Jobin, Katarzyna; Kenwood, Brandon M.; Hoehn, Kyle L.; Lynch, Kevin R.; Okusa, Mark D.

    2015-01-01

    Sphingosine 1-phosphate (S1P), the natural sphingolipid ligand for a family of five G protein– coupled receptors (S1P1–S1P5Rs), regulates cell survival and lymphocyte circulation. We have shown that the pan-S1PR agonist, FTY720, attenuates kidney ischemia-reperfusion injury by directly activating S1P1 on proximal tubule (PT) cells, independent of the canonical lymphopenic effects of S1P1 activation on B and T cells. FTY720 also reduces cisplatin-induced AKI. Therefore, in this study, we used conditional PT-S1P1-null (PepckCreS1pr1fl/fl) and control (PepckCreS1pr1w/wt) mice to determine whether the protective effect of FTY720 in AKI is mediated by PT-S1P1. Cisplatin induced more renal injury in PT-S1P1-null mice than in controls. Although FTY720 produced lymphopenia in both control and PT-S1P1-null mice, it reduced injury only in control mice. Furthermore, the increase in proinflammatory cytokine (CXCL1, MCP-1, TNF-α, and IL-6) expression and infiltration of neutrophils and macrophages induced by cisplatin treatment was attenuated by FTY720 in control mice but not in PT-S1P1-null mice. Similarly, S1P1 deletion rendered cultured PT cells more susceptible to cisplatin-induced injury, whereas S1P1 overexpression protected PT cells from injury and preserved mitochondrial function. We conclude that S1P1 may have an important role in stabilizing mitochondrial function and that FTY720 administration represents a novel strategy in the prevention of cisplatin-induced AKI. PMID:25145931

  8. Prenatal programming of rat cortical collecting tubule sodium transport.

    PubMed

    Cheng, Chih-Jen; Lozano, German; Baum, Michel

    2012-03-15

    Prenatal insults have been shown to lead to elevated blood pressure in offspring when they are studied as adults. Prenatal administration of dexamethasone and dietary protein deprivation have demonstrated that there is an increase in transporter abundance for a number of nephron segments but not the subunits of the epithelial sodium channel (ENaC) in the cortical collecting duct. Recent studies have shown that aldosterone is elevated in offspring of protein-deprived mothers when studied as adults, but the physiological importance of the increase in serum aldosterone is unknown. As an indirect measure of ENaC activity, we compared the natriuretic response to benzamil in offspring of mothers who ate a low-protein diet (6%) with those who ate a normal diet (20%) for the last half of pregnancy. The natriuretic response to benzamil was greater in the 6% group (821.1 ± 161.0 μmol/24 h) compared with the 20% group (279.1 ± 137.0 μmol/24 h), consistent with greater ENaC activity in vivo (P < 0.05). In this study, we also directly studied cortical collecting tubule function from adult rats using in vitro microperfusion. There was no difference in basal or vasopressin-stimulated osmotic water permeability. However, while cortical collecting ducts of adult offspring whose mothers ate a 20% protein diet had no sodium transport (-1.9 ± 3.1 pmol·mm(-1)·min(-1)), the offspring of rats that ate a 6% protein diet during the last half of pregnancy had a net sodium flux of 10.7 ± 2.6 pmol·mm(-1)·min(-1) (P = 0.01) in tubules perfused in vitro. Sodium transport was measured using ion-selective electrodes, a novel technique allowing measurement of sodium in nanoliter quantities of fluid. Thus we directly demonstrate that there is prenatal programming of cortical collecting duct sodium transport.

  9. Microorganism penetration in dentinal tubules of instrumented and retreated root canal walls. In vitro SEM study

    PubMed Central

    Al-Sulaiman, Alaa; Al-Rasheed, Fellwa; Alnajjar, Fatimah; Al-Abdulwahab, Bander; Al-Badah, Abdulhakeem

    2014-01-01

    Objectives This in vitro study aimed to investigate the ability of Candida albicans (C. albicans) and Enterococcus faecalis (E. faecalis) to penetrate dentinal tubules of instrumented and retreated root canal surface of split human teeth. Materials and Methods Sixty intact extracted human single-rooted teeth were divided into 4 groups, negative control, positive control without canal instrumentation, instrumented, and retreated. Root canals in the instrumented group were enlarged with endodontic instruments, while root canals in the retreated group were enlarged, filled, and then removed the canal filling materials. The teeth were split longitudinally after canal preparation in 3 groups except the negative control group. The teeth were inoculated with both microorganisms separately and in combination. Teeth specimens were examined by scanning electron microscopy (SEM), and the depth of penetration into the dentinal tubules was assessed using the SMILE view software (JEOL Ltd). Results Penetration of C. albicans and E. faecalis into the dentinal tubules was observed in all 3 groups, although penetration was partially restricted by dentin debris of tubules in the instrumented group and remnants of canal filling materials in the retreated group. In all 3 groups, E. faecalis penetrated deeper into the dentinal tubules by way of cell division than C. albicans which built colonies and penetrated by means of hyphae. Conclusions Microorganisms can easily penetrate dentinal tubules of root canals with different appearance based on the microorganism size and status of dentinal tubules. PMID:25383343

  10. In vivo model for microbial invasion of tooth root dentinal tubules

    PubMed Central

    BRITTAN, Jane L; SPRAGUE, Susan V; MACDONALD, Emma L; LOVE, Robert M; JENKINSON, Howard F; WEST, Nicola X

    2016-01-01

    ABSTRACT Objective Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Material and Methods Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF). DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database. Results All root dentine samples with patent tubules showed evidence of bacterial cell invasion (TIF value range from 5.7 to 9.0) to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. Conclusion A novel in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine. PMID:27119760

  11. The postnatal maturation of efferent tubules in the rat: a light and electron microscopy study.

    PubMed

    Francavilla, S; Moscardelli, S; Bruno, B; Barcellona, P S; De Martino, C

    1986-07-01

    The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was investigated by light and transmission electron microscopy, from birth to 50 days of age, when sperms were released from the seminiferous tubules and appeared in the genital duct. At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial cells. During the third week of life, efferent tubules developed specializations for the transport of sperms and fluids, namely the appearance of ciliated elements interspersed among the principal cells of the epithelium, and differentiation of myoid elements in the tubule wall. The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear at the end of the second week of life in the rat, along with the initial secretion of testicular fluid. This suggested that the maturation of efferent tubules is not triggered by the passage of testicular fluid, as surmised for the postnatal differentiation of caput epididymis. The postnatal maturation of efferent tubules was almost complete 35 days after birth. The appearance of sperms in the genital duct of 50-day-old animals was not associated with any remarkable structural change.

  12. Cd-induced oxidative stress and lignification in the roots of two Vicia sativa L. varieties with different Cd tolerances.

    PubMed

    Rui, Haiyun; Chen, Chen; Zhang, Xingxing; Shen, Zhenguo; Zhang, Fenqin

    2016-01-15

    We examined the effects of Cd on growth, lipid peroxidation, reactive oxygen species (ROS) accumulation, antioxidant enzymatic activity, and lignin content in the roots of two varieties of Vicia sativa. Treatment with Cd decreased plant growth and increased ROS and lipid peroxidation levels to a greater extent in the Cd-sensitive variety ZM than in the Cd-tolerant variety L3. Most hydrogen peroxide (H2O2) and superoxide anion (O2(•-)) were accumulated in the cell walls and extracellular spaces in response to Cd treatments. Chemical assays and experiments using inhibitors showed that larger increases in H2O2 and O2(•-) production in ZM than in L3 were probably attributed to elevated Cd-induced nicotinamide adenine dinucleotide-peroxidase (NADH-POD) activity. Cd treatment increased the accumulation of lignin and the guaiacol peroxidase (GPOD) activities in the apoplast more significantly in ZM root than in L3. Howerver, root laccase activity was higher in L3 than in ZM. Thus Cd toxicity induced significant lignification in the roots of V. sativa, and increases in H2O2 accumulation and apoplastic GPOD activity were likely responsible for this effect.

  13. Micro- and nano-tubules built from loosely and tightly rolled up thin sheets.

    PubMed

    Losensky, Luisa; Goldenbogen, Björn; Holland, Gudrun; Laue, Michael; Petran, Anca; Liebscher, Jürgen; Scheidt, Holger A; Vogel, Alexander; Huster, Daniel; Klipp, Edda; Arbuzova, Anna

    2016-01-14

    Tubular structures built from amphiphilic molecules are of interest for nano-sensing, drug delivery, and structuring of oils. In this study, we characterized the tubules built in aqueous suspensions of a cholesteryl nucleoside conjugate, cholesterylaminouridine (CholAU) and phosphatidylcholines (PCs). In mixtures with unsaturated PCs having chain lengths comparable to the length of CholAU, two different types of tubular structures were observed; nano- and micro-tubules had average diameters in the ranges 50-300 nm and 2-3 μm, respectively. Using cryo scanning electron microscopy (cryo-SEM) we found that nano- and micro-tubules differed in their morphology: the nano-tubules were densely packed, whereas micro-tubules consisted of loosely rolled undulated lamellas. Atomic force microscopy (AFM) revealed that the nano-tubules were built from 4 to 5 nm thick CholAU-rich bilayers, which were in the crystalline state. Solid-state (2)H NMR spectroscopy also confirmed that about 25% of the total CholAU, being about the fraction of CholAU composing the tubules, formed the rigid crystalline phase. We found that CholAU/PC tubules can be functionalized by molecules inserted into lipid bilayers and fluorescently labeled PCs and lipophilic nucleic acids inserted spontaneously into the outer layer of the tubules. The tubular structures could be loaded and cross-linked, e.g. by DNA hybrids, and, therefore, are of interest for further development, e.g. as a depot scaffold for tissue regeneration.

  14. Tubulation of Golgi membranes in vivo and in vitro in the absence of brefeldin A.

    PubMed

    Cluett, E B; Wood, S A; Banta, M; Brown, W J

    1993-01-01

    Recent in vivo studies with the fungal metabolite, brefeldin A (BFA), have shown that in the absence of vesicle formation, membranes of the Golgi complex and the trans-Golgi network (TGN) are nevertheless able to extend long tubules which fuse with selected target organelles. We report here that the ability to form tubules (> 7 microns long) could be reproduced in vitro by treatment of isolated, intact Golgi membranes with BFA under certain conditions. Surprisingly, an even more impressive degree of tubulation could be achieved by incubating Golgi stacks with an ATP-reduced cytosolic fraction, without any BFA at all. Similarly, tubulation of Golgi membranes in vivo occurred after treatment of cells with intermediate levels of NaN3 and 2-deoxyglucose. The formation of tubules in vitro, either by BFA treatment or low-ATP cytosol, correlated precisely with a loss of the vesicle-associated coat protein beta-COP from Golgi membranes. After removal of BFA or addition of ATP, membrane tubules served as substrates for the rebinding of beta-COP and for the formation of vesicles in vitro. These results provide support for the idea that a reciprocal relationship exists between tubulation and vesiculation (Klausner, R. D., J. G. Donaldson, and J. Lippincott-Schwartz. 1992. J. Cell Biol. 116:1071-1080). Moreover, they show that tubulation is an inherent property of Golgi membranes, since it occurs without the aid of microtubules or BFA treatment. Finally the results indicate the presence of cytosolic factors, independent of vesicle-associated coat proteins, that mediate the budding/tubulation of Golgi membranes.

  15. Exploring the human mesenchymal stem cell tubule communication network through electron microscopy.

    PubMed

    Valente, Sabrina; Rossi, Roberta; Resta, Leonardo; Pasquinelli, Gianandrea

    2015-04-01

    Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

  16. Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

    SciTech Connect

    Brown, Colin D.A. Sayer, Rachel; Windass, Amy S.; Haslam, Iain S.; Broe, Marc E. de; D'Haese, Patrick C.; Verhulst, Anja

    2008-12-15

    The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.

  17. Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells

    SciTech Connect

    Song, Xiulong Wei, Zhengxi; Shaikh, Zahir A.

    2015-08-15

    Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

  18. Effect of depth and tubule direction on ultimate tensile strength of human coronal dentin.

    PubMed

    Inoue, Satoshi; Pereira, Patricia N R; Kawamoto, Chiharu; Nakajima, Masatoshi; Koshiro, Kenichi; Tagami, Junji; Carvalho, Ricardo M; Pashley, David H; Sano, Hidehiko

    2003-03-01

    The purpose of this study was to evaluate the effect of dentin depth and tubule direction on the ultimate tensile strength (UTS) of human dentin. Dentin slabs of 0.5-mm thickness were trimmed either from the mesial and distal (for specimens with the tubules parallel to the tensile force; parallel group) or from the occlusal and pulpal surfaces (perpendicular group) to reduce the cross-sectional area of the superficial, middle, and deep regions to 0.25 mm2, and subjected to microtensile testing. From SEM photomicrographs of the fractured specimens of the parallel group, the tubule density was investigated. For both parallel and perpendicular groups, superficial dentin showed a significantly higher UTS than deep dentin. The tubule density of superficial dentin was significantly lower than that of middle and deep dentin. When performing the microtensile bond test to deep dentin, it is possible that cohesive failure of dentin can occur at relatively low tensile stresses.

  19. Purine transport by malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster.

    PubMed

    Sullivan, D T; Bell, L A; Paton, D R; Sullivan, M C

    1979-06-01

    Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (pp) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.

  20. A Role for Phospholipase A2 Activity in Membrane Tubule Formation and TGN Trafficking

    PubMed Central

    Schmidt, John A.; Kalkofen, Danielle N.; Donovan, Kirk W.; Brown, William J.

    2015-01-01

    We have investigated the role of phospholipase A2 (PLA2) enzymes in generating membrane tubules at the trans-Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over-expressing kinase dead protein kinase D were inhibited by the PLA2 inhibitors ONO-RS-082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time-lapse imaging revealed for the first time that PLA2 antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA2 enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking. PMID:20874826

  1. Cd-induced production of glomalin by arbuscular mycorrhizal fungus (Rhizophagus irregularis) as estimated by monoclonal antibody assay.

    PubMed

    Malekzadeh, Elham; Aliasgharzad, Nasser; Majidi, Jafar; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal

    2016-10-01

    Glomalin is a specific fungal glycoprotein produced by arbuscular mycorrhizal (AM) fungi belonging to the Glomerales which could efficiently sequestrate heavy metals. The glomalin has been introduced as a heat shock protein and there are evidences that increasing levels of heavy metals could enhance its production. We examined the influence of Cd concentrations on glomalin production by AM fungus, as well as its contribution to the sequestration of Cd in both pot and in vitro culture conditions. Pot experiment was carried out using pure sand with Trifolium repens L. as host plant, mycorrhized by Rhizophagus irregularis and treated with Cd levels of 0, 15, 30, and 45 μM. In vitro experiment was performed in two-compartment plates containing the transformed carrot roots mycorrhized with the same fungus and treated with Cd levels of 0, 0.001, 0.01, and 0.1 mM. The immunoreactive and Bradford reactive glomalin contents in both experiments increased as so raising Cd concentration. Total Cd sequestrated by hyphal glomalin in both cultures was significantly increased as the levels of Cd increased. The highest contents of Cd sequestration in pot (75.78 μg Cd/mg glomalin) and in vitro (11.44 μg Cd/mg glomalin) cultures were recorded at the uppermost levels of Cd, which significantly differed with other levels. Our results suggested that under Cd-induced stress, stimulated production of glomalin by AM fungus may be a protective mechanism against the toxic effect of Cd.

  2. Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish.

    PubMed

    Kitatani, Ryuju; Yamada, Mayu; Kamio, Michiya; Nagai, Hiroshi

    2015-01-01

    A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation.

  3. Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish

    PubMed Central

    Kitatani, Ryuju; Yamada, Mayu; Kamio, Michiya; Nagai, Hiroshi

    2015-01-01

    A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation. PMID:26309256

  4. Resin Adaptation of Radicular Dentin Tubules after Endodontic Instrumentation and Acid Etching.

    DTIC Science & Technology

    1983-02-01

    the manuscript. DISCLAIMERS The statements, opinions, and advertisements in the Journal of Endodontics are solely those of the individual authors...I RD-Ai26 872 RESIN ADAPTATION OF RADICULAR DENTIN TUBULES AFTER / I ENDODONTIC INSTRUMENTATION AND ACID ETCHING(U) WALTER I REED ARMY INST OF...Adaptation to Radicular Dentin Tubules SbisoofpeAfter Endodontic Instrumentation and Acid Etching 1982-1983 6. PERFORMING ORG. REPORTNUMBER -, AUTHOR(a) S

  5. Treatment of dentinal tubules by Nd:YAG laser

    NASA Astrophysics Data System (ADS)

    Chmelíčkova, Hana; Zapletalova, Zdeňka; Peřina, Jan, Jr.; Novotný, Radko; Kubínek, Roman; Stranyánek, Martin

    2005-08-01

    Symptom of cervical dentine hypersensitivity attacks from 10% to 15% of population and causes an uncomfortable pain during contact with any matter. Sealing of open dentinal tubules is one of the methods to reach insensibility. Laser as a source of coherent radiation is used to melt dentine surface layers. Melted dentine turns to hard mass with a smooth, non-porous surface. Simulation of this therapy was made in vitro by means of LASAG Nd:YAG pulsed laser system KLS 246-102. Eighty human extracted teeth were cut horizontally to obtain samples from 2 mm to 3 mm thick. First experiments were done on cross section surfaces to find an optimal range of laser parameters. A wide range of energies from 30 mJ to 210 mJ embedded in 0,3 ms long pulse was tested. Motion in X and Y axes was ensured by a CNC driven table and the pulse frequency 15 Hz was chosen to have a suitable overlap of laser spots. Some color agents were examined with the aim to improve surface absorption. Scanning Electron Microscopy was used to evaluate all samples and provided optimal values of energies around 50 J.cm-2. Next experiments were done with the beam oriented perpendicularly to a root surface, close to the real situation. Optical fibers with the diameter of 0,6 mm and 0,2 mm were used to guide a laser beam to teeth surfaces. Laser processing heads with lens F = 100 mm and F = 50 mm were used. The best samples were investigated by means of the Atomic Force Microscopy.

  6. Angiotensin II Enhances Connecting Tubule Glomerular Feedback (CTGF)

    PubMed Central

    Ren, YiLin; D’Ambrosio, Martin A.; Garvin, Jeffrey L.; Carretero, Oscar A.

    2011-01-01

    Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) causes dilation of the afferent arteriole (Af-Art), a process we call CNT glomerular feedback (CTGF). Angiotensin II (Ang II) stimulates ENaC in the collecting duct via AT1 receptors. We hypothesized that Ang II in the CNT lumen enhances CTGF by activation of AT1 receptors, protein kinase C (PKC) and ENaC. Rabbit Af-Arts and their adherent CNT were microperfused and preconstricted with norepinephrine. Each experiment involved generating two consecutive concentration-response curves by increasing NaCl in the CNT lumen. During the control period, the maximum dilation of the Af-Art was 7.9 ± 0.4 μm, and the concentration of NaCl in the CNT needed to achieve half maximal response (EC50) was 34.7 ± 5.2 mmol/L. After adding Ang II (10−9 mol/L) to the CNT lumen, the maximal response was 9.5 ± 0.7 μm and the EC50 was 11.6 ± 1.3 mmol/L (P=0.01 vs. control). Losartan, an AT1 antagonist (10−6 mol/L) blocked the stimulatory effect of Ang II, PD123319, an AT2 antagonist (10−6 mol/L) did not. The PKC inhibitor staurosporine (10−8 mol/L) added to the CNT inhibited the stimulatory effect of Ang II. The ENaC inhibitor benzamil (10−6 mol/L) prevented both CTGF and its stimulation by Ang II. We concluded that Ang II in the CNT lumen enhances CTGF via activation of AT1, and that this effect requires activation of PKC and ENaC. Potentiation of CTGF by Ang II could help preserve glomerular filtration rate in the presence of renal vasoconstriction. PMID:20696981

  7. Membrane tubule formation by banana-shaped proteins with or without transient network structure

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2016-02-01

    In living cells, membrane morphology is regulated by various proteins. Many membrane reshaping proteins contain a Bin/Amphiphysin/Rvs (BAR) domain, which consists of a banana-shaped rod. The BAR domain bends the biomembrane along the rod axis and the features of this anisotropic bending have recently been studied. Here, we report on the role of the BAR protein rods in inducing membrane tubulation, using large-scale coarse-grained simulations. We reveal that a small spontaneous side curvature perpendicular to the rod can drastically alter the tubulation dynamics at high protein density, whereas no significant difference is obtained at low density. A percolated network is intermediately formed depending on the side curvature. This network suppresses tubule protrusion, leading to the slow formation of fewer tubules. Thus, the side curvature, which is generated by protein–protein and membrane–protein interactions, plays a significant role in tubulation dynamics. We also find that positive surface tensions and the vesicle membrane curvature can stabilize this network structure by suppressing the tubulation.

  8. EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.

    PubMed

    Posey, Avery D; Swanson, Kaitlin E; Alvarez, Manuel G; Krishnan, Swathi; Earley, Judy U; Band, Hamid; Pytel, Peter; McNally, Elizabeth M; Demonbreun, Alexis R

    2014-03-15

    EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.

  9. Self-assembly of helical tubules using a single-tailed surfactant.

    NASA Astrophysics Data System (ADS)

    Lee, Hee-Young; Raghavan, Srinivasa

    2007-03-01

    Hollow micro or nanotubules are an unusual type of self-assembled structure that can be formed in aqueous solution. Such structures could be useful in a variety of applications such as in controlled drug delivery and in electroactive composites. However, these structures are typically formed only by some unusual lipids (i.e., two-tailed amphiphiles) or certain peptides. Here we present a very simple and economical process to make stable tubules by using a single-tailed diacetylenic surfactant in conjunction with an alcohol. The formation of tubules as a function of solution composition and temperature are systematically investigated in this study. The tubules are visualized by optical microscopy, while their detailed structure is seen under TEM. We find that the tubules have helical markings, which is remarkable considering that the precursor molecules are achiral. Our results provide further evidence that molecular chirality is not essential to forming tubules; presumably, tubules can form from achiral molecules by a chiral symmetry-breaking process.

  10. EHD1 mediates vesicle trafficking required for normal muscle growth and tubule development

    PubMed Central

    Posey, Avery D.; Swanson, Kaitlin E.; Alvarez, Manuel G.; Krishnan, Swathi; Earley, Judy E.; Band, Hamid; Pytel, Peter; McNally, Elizabeth M.; Demonbreun, Alexis R.

    2014-01-01

    EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton. PMID:24440153

  11. Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle.

    PubMed

    Demonbreun, Alexis R; Swanson, Kaitlin E; Rossi, Ann E; Deveaux, H Kieran; Earley, Judy U; Allen, Madison V; Arya, Priyanka; Bhattacharyya, Sohinee; Band, Hamid; Pytel, Peter; McNally, Elizabeth M

    2015-01-01

    We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.

  12. Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

    PubMed Central

    Demonbreun, Alexis R.; Swanson, Kaitlin E.; Rossi, Ann E.; Deveaux, H. Kieran; Earley, Judy U.; Allen, Madison V.; Arya, Priyanka; Bhattacharyya, Sohinee; Band, Hamid; Pytel, Peter; McNally, Elizabeth M.

    2015-01-01

    We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy. PMID:26325203

  13. Hirayama Disease with Proximal Involvement

    PubMed Central

    2016-01-01

    Hirayama disease is a slowly progressing benign motor neuron disease that affects the distal upper limb. A 29-year-old man visited the hospital with a 1-year history of weakened left proximal upper limb. He was diagnosed with Hirayama disease 9 years ago, while there was no further progression of the muscle weakness afterward. Atrophy and weakness was detected in proximal upper limb muscles. Magnetic resonance imaging and somatosensory evoked potentials were normal. Needle electromyography showed abnormal findings in proximal upper limb muscles. Our patient had Hirayama disease involving the proximal portion through secondary progression. Clinical manifestation and accurate electromyography may be useful for diagnosis. Rare cases with progression patterns as described here are helpful and have clinical meaning for clinicians. PMID:27550499

  14. Tools for proximal soil sensing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proximal soil sensing (i.e. near-surface geophysical methods) are used to study soil phenomena across spatial scales. Geophysical methods exploit contrasts in physical properties (dielectric permittivity, apparent electrical conductivity or resistivity, magnetic susceptibility) to indirectly measur...

  15. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    SciTech Connect

    Klinefelter, G.R.; Hamilton, D.W.

    1985-11-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-(35S)methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-(35S)methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB(3H)4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.

  16. Maturational changes in connexin 43 expression in the seminiferous tubules may depend on thyroid hormone action

    PubMed Central

    Marchlewska, Katarzyna; Kula, Krzysztof; Walczak-Jedrzejowska, Renata; Kula, Wojciech; Oszukowska, Elzbieta; Filipiak, Eliza; Moszura, Tomasz

    2013-01-01

    Introduction Connexin 43 (Cx43) mediates the effect of thyroid hormone on Sertoli cell maturation in vitro. We investigated the influence of triiodothyronine (T3) administration on Cx43 expression in relation to the progress in seminiferous tubule maturation. Material and methods Male rats were daily injected with 100 µg T3/kg body weight from birth until postnatal day (pnd) 5 (transient treatment – tT3) or until pnd 15 (continuous treatment – cT3) or solvent – control (C). On pnd 16 serum hormone levels, body and testes weight, seminiferous tubule morphometry, Cx43 immunostaining and germ cell degeneration were investigated. Cx43 expression was also assessed in six 50-day-old adult untreated rats. Result tT3 increased 2.6-fold serum level of T3, testes weight, and seminiferous tubule diameter, and induced maturation-like dislocation of Cx43 expression from the apical to the peripheral region of Sertoli cell cytoplasm. In addition, incidence of Cx43-positive tubules declined from 86% in C to 46% after tT3, being similar to the adult value (30% of tubules Cx43-positive). In turn, cT3 increased serum T3 level 12-fold, and decreased body weight. Seminiferous tubules became shortened and distended, Sertoli cell cytoplasm vacuolated, Cx43 expression had minimal intensity and germ cell degeneration increased. Conclusions Cx43 might intermediate a short and transient stimulatory effect of T3 on seminiferous tubule maturation that disappeared together with exposure to the toxic effect of a continuously high level of the hormone. PMID:23515877

  17. Near tubule and intertubular bovine detin mapped at the 250 nm level.

    SciTech Connect

    Stock, S.R.; Veis, A.; Telser, A.; Cai, Z.

    2011-11-01

    In this study, simultaneous diffraction and fluorescence mapping with a (250 nm){sup 2}, 10.1 keV synchrotron X-ray beam investigated the spatial distribution of carbonated apatite (cAp) mineral and elemental Ca (and other cations including Zn) around dentin tubules. In 1 {mu}m thick sections of near-pulp root dentin, where peritubular dentin (PTD) is newly forming, high concentrations of Zn, relative to those in intertubular dentin (ITD), were observed adjacent to and surrounding the tubule lumens. Some but not all tubules exhibited hypercalcified collars (high Ca signal relative to the surrounding ITD), and, when present, the zone of high Ca did not extend around the tubule. Diffraction rings from cAp 00.2 and 11.2 + 21.1 + 30.0 reflections were observed, and cAp was the only crystal phase detected. Profiles of Ca, Zn and cAp diffracted intensities showed the same transitions from solid to tubule lumen, indicating the same cAp content and organization in ITD far from the tubules and adjacent to them. Further, the matching Ca and diffraction profiles demonstrated that all of the Ca is in cAp or that any noncrystalline Ca was uniformly distributed throughout the dentin. Variation of 00.2 and 11.2 + 21.1 + 30.0 diffracted intensity was consistent with the expected biaxial crystallographic texture. Extension of X-ray mapping from near 1 {mu}m resolution to the 250 nm level, performed here for dentin and its tubules, will provide new understanding of other mineralized tissues.

  18. Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle.

    PubMed Central

    Muñoz, P; Rosemblatt, M; Testar, X; Palacín, M; Zorzano, A

    1995-01-01

    1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the

  19. Palladium nanotubes formed by lipid tubule templating and their application in ethanol electrocatalysis.

    PubMed

    Wang, Yinan; Ma, Shenghua; Su, Yingchun; Han, Xiaojun

    2015-04-13

    Palladium nanotubes were fabricated by using lipid tubules as templates for the first time in a controlled manner. The positively charged lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) was doped into lipid tubules to adsorb PdCl4 (2-) on the tubule surfaces for further reduction. The lipid tubule formation was optimized by studying the growing dynamics and ethanol/water ratio. The DOTAP-doped tubules showed pH stability from 0 to 14, which makes them ideal templates for metal plating. The Pd nanotubes are open-ended with a tunable wall thickness. They exhibited good electrocatalytic performance in ethanol. Their electrochemically active surface areas were 6.5, 10.6, and 83.2 m(2)  g(-1) for Pd nanotubes with 77, 101, and 150 nm wall thickness, respectively. These Pd nanotubes have great potential in fuel cells. The method demonstrated also opens up a way to synthesize hollow metal nanotubes.

  20. Transformation of taxol-stabilized microtubules into inverted tubulin tubules triggered by a tubulin conformation switch

    PubMed Central

    Ginsburg, Avi; Kohl, Phillip A.; Li, Youli; Miller, Herbert P.; Wilson, Leslie; Raviv, Uri; Choi, Myung Chul; Safinya, Cyrus R.

    2014-01-01

    Bundles of taxol-stabilized microtubules (MTs) – hollow tubules comprised of assembled αβ-tubulin heterodimers – spontaneously assemble above a critical concentration of tetravalent spermine and are stable over long times at room temperature. Here we report that at concentrations of spermine several-fold higher the MT bundles (BMT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (BITT), the outside surface of which corresponds to the inner surface of the BMT tubules. Using transmission electron microscopy and synchrotron small-angle x-ray scattering, we quantitatively determined both the nature of the BMT to BITT transformation pathway, which results from a spermine-triggered conformation switch from straight to curved in the constituent taxol-stabilized tubulin oligomers, and the structure of the BITT phase, which is formed of tubules of helical tubulin oligomers. Inverted tubulin tubules provide a platform for studies requiring exposure and availability of the inside, luminal surface of MTs to MT-targeted-drugs and MT-associated-proteins. PMID:24441880

  1. The effect of antidiuretic hormone on solute flows in mammalian collecting tubules

    PubMed Central

    Schafer, James A.; Andreoli, Thomas E.

    1972-01-01

    These experiments were intended to evaluate the antidiuretic hormone (ADH)-dependent reflection coefficients of urea, sucrose, and NaCl in cortical and outer medullary collecting tubules isolated from mammalian kidney. In one group of experiments, the ADH-dependent osmotic water flows, when the perfusing solutions contained hypotonic NaCl solutions, were indistinguishable from control observations when either urea or sucrose replaced, in part, NaCl in isotonic bathing solutions (cortical collecting tubules). Similarly, both in cortical and outer medullary collecting tubules exposed to ADH, there was zero net osmotic volume flow when a portion of the NaCl in the bathing and/or perfusing solutions was replaced by either sucrose or urea, so long as the perfusing and bathing solutions were isosmolal. Taken together, these observations suggest that the ADH-dependent reflection coefficients of NaCl, urea, and sucrose, in these tubules, were identical. Since the effective hydrodynamic radii of urea and sucrose are, respectively, 1.8 and 5.2 A, it is likely that σi, for urea, sucrose, and NaCl, was unity. In support of this, the diffusion permeability coefficient (PDi cm sec-1) of urea was indistinguishable from zero. Since the limiting sites for urea penetration were the luminal interfaces of the tubules, these data are consistent with the view that ADH increases diffusional water flow across such interfaces. Images PMID:5057132

  2. Investigation of the Properties of Sand Tubules, a Biomineralization Product, and their Microbial Community.

    PubMed

    Hu, Weilian; Dai, Dehui

    2016-02-01

    Sand tubules, made up of sand grains cemented by microbe-induced calcium carbonate precipitation, have been found in China's Ningxia Province. Sand tubules grow like a tree's roots about 40-60 cm below the surface. The properties of sand tubules and their bacterial community were examined. X-Ray diffraction analysis revealed that the sand tubules were associated with crystalline calcite. Scanning electron microscopy showed that the crystalline solid had a lamellar structure and lacked the presence of cells, suggesting that no bacteria acted as nucleation sites, nor that the crystalline solid was formed by the aggregation of bacteria. Denaturing gradient gel electrophoresis analysis showed 11 of the 12 detectable bands were uncultured bacteria by BLAST analysis in the GenBank database, and the rest were closely related to Paenibacillus sp. (100% identity). By cultivation techniques, the only strain isolated from the sand tubule was suggested to be related to Paenibacillus sp.; no archaea were found. Furthermore, Paenibacillus sp. was demonstrated to induce calcium carbonate precipitation in vitro.

  3. Failed Tubule Recovery, AKI-CKD Transition, and Kidney Disease Progression

    PubMed Central

    Weinberg, Joel M.; Kriz, Wilhelm; Bidani, Anil K.

    2015-01-01

    The transition of AKI to CKD has major clinical significance. As reviewed here, recent studies show that a subpopulation of dedifferentiated, proliferating tubules recovering from AKI undergo pathologic growth arrest, fail to redifferentiate, and become atrophic. These abnormal tubules exhibit persistent, unregulated, and progressively increasing profibrotic signaling along multiple pathways. Paracrine products derived therefrom perturb normal interactions between peritubular capillary endothelium and pericyte-like fibroblasts, leading to myofibroblast transformation, proliferation, and fibrosis as well as capillary disintegration and rarefaction. Although signals from injured endothelium and inflammatory/immune cells also contribute, tubule injury alone is sufficient to produce the interstitial pathology required for fibrosis. Localized hypoxia produced by microvascular pathology may also prevent tubule recovery. However, fibrosis is not intrinsically progressive, and microvascular pathology develops strictly around damaged tubules; thus, additional deterioration of kidney structure after the transition of AKI to CKD requires new acute injury or other mechanisms of progression. Indeed, experiments using an acute-on-chronic injury model suggest that additional loss of parenchyma caused by failed repair of AKI in kidneys with prior renal mass reduction triggers hemodynamically mediated processes that damage glomeruli to cause progression. Continued investigation of these pathologic mechanisms should reveal options for preventing renal disease progression after AKI. PMID:25810494

  4. Cubesat Proximity Operations Demonstration (CPOD)

    NASA Technical Reports Server (NTRS)

    Villa, Marco; Martinez, Andres; Petro, Andrew

    2015-01-01

    The CubeSat Proximity Operations Demonstration (CPOD) project will demonstrate rendezvous, proximity operations and docking (RPOD) using two 3-unit (3U) CubeSats. Each CubeSat is a satellite with the dimensions 4 inches x 4 inches x 13 inches (10 centimeters x 10 centimeters x 33 centimeters) and weighing approximately 11 pounds (5 kilograms). This flight demonstration will validate and characterize many new miniature low-power proximity operations technologies applicable to future missions. This mission will advance the state of the art in nanosatellite attitude determination,navigation and control systems, in addition to demonstrating relative navigation capabilities.The two CPOD satellites are scheduled to be launched together to low-Earth orbit no earlier than Dec. 1, 2015.

  5. Structural and functional alterations in Malpighian tubules as biomarkers of environmental pollution: synopsis and prospective.

    PubMed

    Giglio, Anita; Brandmayr, Pietro

    2017-03-06

    Although a number of biomarkers of pollutant exposure have been identified in invertebrate species, little is known about the effect on Malpighian tubules playing an essential role in excretion and osmoregulation. Analyses of structural and functional alterations on this organ can be useful to predict the effects at the organism and population level in monitoring studies of environmental pollution. The aim of the present review is to provide a synthesis of existing knowledge on cellular damages induced by xenobiotics in Malpighian tubules both under laboratory and field conditions. We compared studies of exposure to pesticides and heavy metals as mainly environmental contaminants from anthropogenic activities. This report provided evidence that the exposure to xenobiotics has an effect on this organ and reinforces the need for further research integrating molecular biomarkers with analysis on Malpighian tubules. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Multiple signalling pathways establish cell fate and cell number in Drosophila malpighian tubules.

    PubMed

    Wan, S; Cato, A M; Skaer, H

    2000-01-01

    A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the other, the sibling cell, achaete expression is lost (M. Hoch et al., 1994, Development 120, 3439-3450). In this paper we chart the mechanisms by which achaete expression is differentially maintained in the tip cell lineage to stabilise cell fate. First, wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells. Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Second, Notch signalling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete expression is maintained only in the tip cell. In embryos mutant for Notch tip cells segregate at the expense of sibling cells, whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the developing tubules of embryos lacking Wingless after the critical period for tip cell allocation reveals an additional requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect on the final tubule cell number.

  7. Driven shielding capacitive proximity sensor

    NASA Technical Reports Server (NTRS)

    Vranish, John M. (Inventor); McConnell, Robert L. (Inventor)

    2000-01-01

    A capacitive proximity sensing element, backed by a reflector driven at the same voltage as and in phase with the sensor, is used to reflect the field lines away from a grounded robot arm towards an intruding object, thus dramatically increasing the sensor's range and sensitivity.

  8. Proximity Sensors Make Robot Dexterous

    NASA Technical Reports Server (NTRS)

    Hess, Cliff; Li, Larry C. H.

    1990-01-01

    Control system enables robot hand to grasp objects of varied shapes. Key features of system: reflective proximity sensors furnishing data on position, orientation, and distance of object and software protocol controlling sequence of operations in approaching and grasping objects. Reflected-beam sensing concept applied to simple opposed-jaw industrial grippers as well as to dexterous robot hands.

  9. Formation of lipid/peptide tubules by IAPP and temporin B on supported lipid membranes.

    PubMed

    Kinnunen, Paavo K J; Domanov, Yegor A; Mattila, Juha-Pekka; Varis, Teemu

    2015-12-21

    The conversion of various and to is accelerated by , which are also postulated to represent targets mediating the cytotoxicity of protofibrils. Yet, our understanding of the molecular details governing -catalyzed fibrillogenesis of precursors remains limited. To obtain insight into the intricate interplay of and biophysics we have recently introduced supported bilayers (SLBs) with fluorescent analogs as model biomembranes, observed by time-lapse . Here we demonstrate that human islet () induces within minutes of its application on bilayers the expulsion of numerous flexible tubules from the . Intriguingly, these flexible tubules gradually evolve into a network of straight tubes locally attached to the substrate. Two-color imaging of the and the fluorescently labeled revealed to be distributed along the . Similar linear tubules were observed with the antimicrobial temporin B and the non-amyloidogenic rat , revealing that the above mesoscopic perturbations are not related to formation by the human . Micromanipulation experiments revealed that the linearity of the tubules was caused by tension, stretching the tubules between their points of attachment to the substrate. After longer incubation times, for SLBs containing the oxidatively modified 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (, bearing a terminal carboxyl at the end of the chain) and human (but not the other ) some of the straight transformed into highly regular helices. This is likely to reflect tension originating from an efficient aggregation of the into parallelly aligned bundles, associated with tubes containing the oxidized , possibly together with a concomitant flow of along the tubules to the immobile aggregates attaching the tubules to the substrate, these two processes cause, upon shortening of the linear scaffold, the attached excess tubule to adopt a helical morphology, coiling around the core. The above studies are in line with the multiphasic kinetics of fibrillation in the presence of

  10. Proximity and Anti-proximity effects in nanowires

    NASA Astrophysics Data System (ADS)

    Chan, Moses

    Near a superconductor/normal-metal interface, the leakage of Cooper pairs extends superconducting behavior into the metal. The spatial range of this proximity effect in a normal metal can be as long as 1 µm. However, when a ferromagnet is placed in contact with a superconductor, the Cooper pairs from the superconductor are not expected to survive beyond at most a few nanometers into the ferromagnet. Surprisingly we find when a cobalt nanowire as long as 600 nm is sandwiched between superconducting electrodes, it attains zero resistance at low temperature. For even longer wires, the transition to incomplete superconductivity via this (long range) proximity effect is foreshadowed by a large resistance peak (1). On the other hand when Zn nanowires of 40 nm diameter are contacted by superconducting electrodes, their superconductivity is unexpectedly suppressed (2). Worked supported by Penn State MRSEC.

  11. Molecular Mechanism of Membrane Constriction and Tubulation Mediated by the F-BAR Protein Pacsin/Syndapin

    SciTech Connect

    Wang, Q.; Navarro, M; Peng, G; Molinelli, E; Lin, G; Judson, B; Rajashankar, K; Sondermann, H

    2009-01-01

    Peripheral membrane proteins of the Bin/amphiphysin/Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) family participate in cellular membrane trafficking and have been shown to generate membrane tubules. The degree of membrane bending appears to be encoded in the structure and immanent curvature of the particular protein domains, with BAR and F-BAR domains inducing high- and low-curvature tubules, respectively. In addition, oligomerization and the formation of ordered arrays influences tubule stabilization. Here, the F-BAR domain-containing protein Pacsin was found to possess a unique activity, creating small tubules and tubule constrictions, in addition to the wide tubules characteristic for this subfamily. Based on crystal structures of the F-BAR domain of Pacsin and mutagenesis studies, vesiculation could be linked to the presence of unique structural features distinguishing it from other F-BAR proteins. Tubulation was suppressed in the context of the full-length protein, suggesting that Pacsin is autoinhibited in solution. The regulated deformation of membranes and promotion of tubule constrictions by Pacsin suggests a more versatile function of these proteins in vesiculation and endocytosis beyond their role as scaffold proteins.

  12. Multicopper oxidase-1 is required for iron homeostasis in Malpighian tubules of Helicoverpa armigera.

    PubMed

    Liu, Xiaoming; Sun, Chengxian; Liu, Xiaoguang; Yin, Xinming; Wang, Baohai; Du, Mengfang; An, Shiheng

    2015-10-06

    Multicopper oxidases (MCOs) are enzymes that contain 10 conserved histidine residues and 1 cysteine residue. MCO1 has been extensively investigated in the midgut because this MCO is implicated in ascorbate oxidation, iron homeostasis and immune responses. However, information regarding the action of MCO1 in Malpighian tubules is limited. In this study, Helicoverpa armigera was used as a model to investigate the function of MCO1 in Malpighian tubules. Sequence analysis results revealed that HaMCO1 exhibits typical MCO characteristics, with 10 histidine and 1 cysteine residues for copper ion binding. HaMCO1 was also found to be highly abundant in Malpighian tubules. Temporal expression patterns indicated that HaMCO1 is mainly expressed during larval molting stages. Hormone treatments [the molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH)] revealed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP), and that JH counteracts the action of 20E to activate HaMCO1 transcript expression via its intracellular receptor methoprene-tolerant (Met). HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression. Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules.

  13. A facilitative urea transporter is localized in the renal collecting tubule of the dogfish Triakis scyllia.

    PubMed

    Hyodo, Susumu; Katoh, Fumi; Kaneko, Toyoji; Takei, Yoshio

    2004-01-01

    Reabsorption of filtered urea by the kidney tubule is essential for retaining high levels of urea in body fluids of marine elasmobranchs. To elucidate the mechanisms of urea reabsorption, we examined the distribution of a facilitative urea transporter (UT) in the kidney of the dogfish Triakis scyllia. We isolated a cDNA encoding a UT that is homologous to the facilitative UT cloned from another dogfish species, Squalus acanthias. The Triakis UT mRNA is abundantly expressed in the kidney, while low levels of expression were detected in the brain and liver. In the dogfish kidney, each nephron makes four turns and traverses repeatedly between bundle zone and sinus zone. In the bundle zone, the resulting five tubular segments are arranged in a countercurrent loop fashion. Immunohistochemistry using specific antibodies raised against the cloned UT revealed that, among the nephron segments, the UT is expressed exclusively in the final segment of the bundle zone, i.e. in the collecting tubule of the Triakis kidney. In contrast to the limited localization of UT, the transport enzyme Na+/K+-ATPase is distributed in the basolateral membrane of numerous tubular segments both in the sinus zone and the bundle zone. However, in the collecting tubule, Na+/K+-ATPase immunoreactivity was not detected. The present study suggests that the collecting tubule is responsible for the reabsorption of urea in the marine elasmobranch kidney. Other countercurrent segments may contribute to production of a driving force for facilitative diffusion of urea through the UT.

  14. Evaluation of the Diode laser (810nm,980nm) on dentin tubule diameter following internal bleaching

    PubMed Central

    Kiomarsi, Nazanin; Salim, Soheil; Sarraf, Pegah; Javad-Kharazifard, Mohammad

    2016-01-01

    Background The aim of this study was to evaluate the effect of diode laser irradiation and bleaching materials on the dentinal tubule diameter after laser bleaching. Material and Methods The dentin discs of 40 extracted third molar were used in this experiment. Each disc surface was divided into two halves by grooving. Half of samples were laser bleached at different wavelengths with two different concentrations of hydrogen peroxide. Other half of each disc with no laser bleaching remained as a negative control. Dentin discs were assigned randomly into four groups (n=10) with following hydrogen peroxide and diode laser wavelength specifications; Group 1 (30% - 810 nm), group 2 (30% - 980 nm), group 3 (46% - 810 nm) and group 4 (46% - 980 nm). All specimens were sent for scanning electron microscopic (SEM) analysis in order to measure tubular diameter in laser treated and control halves. Data were analyzed by ANOVA and Tukey test (p<0.05). Results A significant reduction in dentin tubule diameter was observed in groups 1, 2 and 4. There was no significant difference between groups 1 and 2 and between groups 3 and 4 after bleaching. Conclusions The SEM results showed that diode laser was able to reduce dentin tubule diameter and its effect on dentin was dependent on chemical action of bleaching material. Key words:Laser, diode, dentin, tubule, diameter. PMID:27398172

  15. Multicopper oxidase-1 is required for iron homeostasis in Malpighian tubules of Helicoverpa armigera

    PubMed Central

    Liu, Xiaoming; Sun, Chengxian; Liu, Xiaoguang; Yin, Xinming; Wang, Baohai; Du, Mengfang; An, Shiheng

    2015-01-01

    Multicopper oxidases (MCOs) are enzymes that contain 10 conserved histidine residues and 1 cysteine residue. MCO1 has been extensively investigated in the midgut because this MCO is implicated in ascorbate oxidation, iron homeostasis and immune responses. However, information regarding the action of MCO1 in Malpighian tubules is limited. In this study, Helicoverpa armigera was used as a model to investigate the function of MCO1 in Malpighian tubules. Sequence analysis results revealed that HaMCO1 exhibits typical MCO characteristics, with 10 histidine and 1 cysteine residues for copper ion binding. HaMCO1 was also found to be highly abundant in Malpighian tubules. Temporal expression patterns indicated that HaMCO1 is mainly expressed during larval molting stages. Hormone treatments [the molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH)] revealed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP), and that JH counteracts the action of 20E to activate HaMCO1 transcript expression via its intracellular receptor methoprene-tolerant (Met). HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression. Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules. PMID:26437857

  16. Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule.

    PubMed

    Losinno, Antonella D; Sorrivas, Viviana; Ezquer, Marcelo; Ezquer, Fernando; López, Luis A; Morales, Alfonsina

    2016-08-01

    The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis.

  17. Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.

    PubMed

    Jayasinghe, Isuru D; Clowsley, Alexander H; Munro, Michelle; Hou, Yufeng; Crossman, David J; Soeller, Christian

    2015-01-07

    The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

  18. Current understanding ofI sperm-storage tubule (SST) function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Unlike most mammals, birds do not need to synchronize copulation with ovulation. Hens are endowed with tubular structures, the sperm-storage tubules (SST), in their oviducts which the sperm enter and survive for weeks after mating or artificial insemination. Sperm are slowly but continually releas...

  19. Adaptive optics imaging of the outer retinal tubules in Bietti's crystalline dystrophy.

    PubMed

    Battu, R; Akkali, M C; Bhanushali, D; Srinivasan, P; Shetty, R; Berendschot, T T J M; Schouten, J S A G; Webers, C A

    2016-05-01

    PurposeTo study the outer retinal tubules using spectral domain optical coherence tomography and adaptive optics and in patients with Bietti's crystalline dystrophy.MethodsTen eyes of five subjects from five independent families with Bietti's crystalline Dystrophy (BCD) were characterized with best-corrected visual acuity (BCVA), full-field electroretinography, and fundus autofluorescence (FAF). High-resolution images were obtained with the spectral domain optical coherence tomography (SD-OCT) and adaptive optics (AO).ResultsSD-OCT showed prominent outer retinal layer loss and outer retinal tubulations at the margin of outer retinal loss. AO images displayed prominent macrotubules and microtubules with characteristic features in eight out of the 10 eyes. Crystals were present in all ten eyes. There was a reduction in the cone count in all eyes in the area outside the outer retinal tubules (ORT).ConclusionsThis study describes the morphology of the outer retinal tubules when imaged enface on the adaptive optics in patients with BCD. These findings provide insight into the macular structure of these patients. This may have prognostic implications and refine the study on the pathogenesis of BCD.

  20. Fight-or-flight: murine unilateral ureteral obstruction causes extensive proximal tubular degeneration, collecting duct dilatation, and minimal fibrosis.

    PubMed

    Forbes, Michael S; Thornhill, Barbara A; Minor, Jordan J; Gordon, Katherine A; Galarreta, Carolina I; Chevalier, Robert L

    2012-07-01

    Unilateral ureteral obstruction (UUO) is the most widely used animal model of progressive renal disease. Although renal interstitial fibrosis is commonly used as an end point, recent studies reveal that obstructive injury to the glomerulotubular junction leads to the formation of atubular glomeruli. To quantitate the effects of UUO on the remainder of the nephron, renal tubular and interstitial responses were characterized in mice 7 and 14 days after UUO or sham operation under anesthesia. Fractional proximal tubular mass, cell proliferation, and cell death were measured by morphometry. Superoxide formation was identified by nitro blue tetrazolium, and oxidant injury was localized by 4-hydroxynonenol and 8-hydroxydeoxyguanosine. Fractional areas of renal vasculature, interstitial collagen, α-smooth muscle actin, and fibronectin were also measured. After 14 days of UUO, the obstructed kidney loses 19% of parenchymal mass, with a 65% reduction in proximal tubular mass. Superoxide formation is localized to proximal tubules, which undergo oxidant injury, apoptosis, necrosis, and autophagy, with widespread mitochondrial loss, resulting in tubular collapse. In contrast, mitosis and apoptosis increase in dilated collecting ducts, which remain patent through epithelial cell remodeling. Relative vascular volume fraction does not change, and interstitial matrix components do not exceed 15% of total volume fraction of the obstructed kidney. These unique proximal and distal nephron cellular responses reflect differential "fight-or-flight" responses to obstructive injury and provide earlier indexes of renal injury than do interstitial compartment responses. Therapies to prevent or retard progression of renal disease should include targeting proximal tubule injury as well as interstitial fibrosis.

  1. Micropower RF material proximity sensor

    DOEpatents

    McEwan, T.E.

    1998-11-10

    A level detector or proximity detector for materials capable of sensing through plastic container walls or encapsulating materials is disclosed. Thus, it can be used in corrosive environments, as well as in a wide variety of applications. An antenna has a characteristic impedance which depends on the materials in proximity to the antenna. An RF oscillator, which includes the antenna and is based on a single transistor in a Colpitt`s configuration, produces an oscillating signal. A detector is coupled to the oscillator which signals changes in the oscillating signal caused by changes in the materials in proximity to the antenna. The oscillator is turned on and off at a pulse repetition frequency with a low duty cycle to conserve power. The antenna consists of a straight monopole about one-quarter wavelength long at the nominal frequency of the oscillator. The antenna may be horizontally disposed on a container and very accurately detects the fill level within the container as the material inside the container reaches the level of the antenna. 5 figs.

  2. Micropower RF material proximity sensor

    DOEpatents

    McEwan, Thomas E.

    1998-01-01

    A level detector or proximity detector for materials capable of sensing through plastic container walls or encapsulating materials is of the sensor. Thus, it can be used in corrosive environments, as well as in a wide variety of applications. An antenna has a characteristic impedance which depends on the materials in proximity to the antenna. An RF oscillator, which includes the antenna and is based on a single transistor in a Colpitt's configuration, produces an oscillating signal. A detector is coupled to the oscillator which signals changes in the oscillating signal caused by changes in the materials in proximity to the antenna. The oscillator is turned on and off at a pulse repetition frequency with a low duty cycle to conserve power. The antenna consists of a straight monopole about one-quarter wavelength long at the nominal frequency of the oscillator. The antenna may be horizontally disposed on a container and very accurately detects the fill level within the container as the material inside the container reaches the level of the antenna.

  3. An in vivo microperfusion study of distal tubule bicarbonate reabsorption in normal and ammonium chloride rats.

    PubMed Central

    Levine, D Z

    1985-01-01

    For many years it has been thought that distal nephron hydrogen ion secretion can be importantly modulated by factors such as sodium delivery, sodium avidity, and potassium stores. Free flow micropuncture studies have also indicated that the rate of bicarbonate delivery may also alter the rate of bicarbonate reabsorption. The present studies were undertaken to examine possible luminal influences on total CO2 reabsorption in microperfused distal tubules in the rat in vivo. Tubules from normal and acidotic rats were perfused with five solutions in a manner that induced changes in bicarbonate load, sodium and potassium fluxes (JNa, JK), and luminal sulfate concentration. in each collected perfusate, simultaneous analyses were undertaken to determine water reabsorption, Na, and K concentrations using graphite furnace atomic absorption spectroscopy and total CO2 by microcalorimetry. Using factorial analysis of covariance to account for confounding effects on total CO2 flux (JtCO2) such as water reabsorption, distal tubules of acidotic rats reabsorbed CO2 in the range of 50-112 pmol X min-1 X mm-1 X These JtCO2 values were not significantly correlated with HCO3 load, JNa, or JK despite changes in the latter from net reabsorption to net secretion. Distal tubules of rats with normal acid-base status had JtCO2 values which were neither significantly different from zero nor correlated with changes in JK and JNa. Further, doubling the load from 250-500 pmol/min (by doubling the perfusion rate of 25-mM HCO3 solutions) did not stimulate JtCO2 in these normal animals. Accordingly, these acute in vivo microperfusion studies indicate for the first time that neither load nor potassium or sodium fluxes are important modulators of distal tubule bicarbonate reabsorption. PMID:2982915

  4. Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

    PubMed Central

    1985-01-01

    The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway. PMID:2411847

  5. Proximate and polyphenolic characterization of cranberry pomace

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The proximate composition and identification and quantification of polyphenolic compounds in dried cranberry pomace were determined. Proximate analysis was conducted based on AOAC methods for moisture, protein, fat, and ash. Total carbohydrates were determined by the difference method. Polyphenolic ...

  6. Active Targets For Capacitive Proximity Sensors

    NASA Technical Reports Server (NTRS)

    Jenstrom, Del T.; Mcconnell, Robert L.

    1994-01-01

    Lightweight, low-power active targets devised for use with improved capacitive proximity sensors described in "Capacitive Proximity Sensor Has Longer Range" (GSC-13377), and "Capacitive Proximity Sensors With Additional Driven Shields" (GSC-13475). Active targets are short-distance electrostatic beacons; they generate known alternating electro-static fields used for alignment and/or to measure distances.

  7. Specific estrogen-induced cell proliferation of cultured Syrian hamster renal proximal tubular cells in serum-free chemically defined media

    SciTech Connect

    Oberley, T.D.; Lauchner, L.J.; Pugh, T.D.; Gonzalez, A.; Goldfarb, S. ); Li, S.A.; Li, J.J. )

    1989-03-01

    It has long been recognized that the renal proximal tubular epithelium of the hamster is a bona fide estrogen target tissue. The effect of estrogens on the growth of proximal tubule cell explants and dissociated single cells derived from these explant outgrowths has been studied in culture. Renal tubular cells were grown on a PF-HR-9 basement membrane under serum-free chemically defined culture conditions. At 7-14 days in culture, cell number was enhanced 3-fold in the presence of either 17{beta}-estradiol or diethylstilbestrol. A similar 3-fold increase in cell number was also seen at 1 nM 17{beta}-estradiol in subcultured dissociated single tubular cells derived from hamster renal tubular explant outgrowths at 21 days in culture. Concomitant exposure of tamoxifen at 3-fold molar excess in culture completely abolished the increase in cell number seen with 17{beta}-estradiol. The proliferation effect of estrogens on proximal tubular cell growth appears to be species specific since 17{beta}-estradiol did not alter the growth of either rat or guinea pig proximal tubules in culture. In addition, at 7-10 days in culture in the presence of 17{beta}-estradiol, ({sup 3}H)thymidine labeling of hamster tubular cells was enhanced 3-fold. These results clearly indicate that estrogens can directly induce primary epithelial cell proliferation at physiologic concentrations and provide strong additional evidence for an important hormonal role in the neoplastic transformation of the hamster kidney.

  8. Cellular distribution of cell cycle-related molecules in the renal tubules of rats treated with renal carcinogens for 28 days: relationship between cell cycle aberration and carcinogenesis.

    PubMed

    Taniai, Eriko; Hayashi, Hitomi; Yafune, Atsunori; Watanabe, Maiko; Akane, Hirotoshi; Suzuki, Kazuhiko; Mitsumori, Kunitoshi; Shibutani, Makoto

    2012-09-01

    Some renal carcinogens can induce karyomegaly, which reflects aberrant cell division in the renal tubules, from the early stages of exposure. To clarify the cell cycle-related changes during the early stages of renal carcinogenesis, we performed immunohistochemical analysis of tubular cells in male F344 rats treated with carcinogenic doses of representative renal carcinogens for 28 days. For this purpose, the karyomegaly-inducing carcinogens ochratoxin A (OTA), ferric nitrilotriacetic acid, and monuron, and the non-karyomegaly-inducing carcinogens tris(2-chloroethyl) phosphate and potassium bromate were examined. For comparison, a karyomegaly-inducing non-carcinogen, p-nitrobenzoic acid, and a non-carcinogenic non-karyomegaly-inducing renal toxicant, acetaminophen, were also examined. The outer stripe of the outer medulla (OSOM) and the cortex + OSOM were subjected to morphometric analysis of immunoreactive proximal tubular cells. Renal carcinogens, irrespective of their karyomegaly-inducing potential, increased proximal tubular cell proliferation accompanied by an increase in topoisomerase IIα-immunoreactive cells, suggesting a reflection of cell proliferation. Karyomegaly-inducing carcinogens increased nuclear Cdc2-, γH2AX-, and phosphorylated Chk2-immunoreactive cells in both areas, the former two acting in response to DNA damage and the latter one suggestive of sustained G₂. OTA, an OSOM-targeting carcinogen, could easily be distinguished from untreated controls and non-carcinogens by evaluation of molecules responding to DNA damage and G₂/M transition in the OSOM. Thus, all renal carcinogens examined facilitated proximal tubular proliferation by repeated short-term treatment. Among these, karyomegaly-inducing carcinogens may cause DNA damage and G₂ arrest in the target tubular cells.

  9. FLUID AND ION SECRETION BY MALPIGHIAN TUBULES OF LARVAL CHIRONOMIDS, Chironomus riparius: EFFECTS OF REARING SALINITY, TRANSPORT INHIBITORS, AND SEROTONIN.

    PubMed

    Zadeh-Tahmasebi, Melika; Bui, Phuong; Donini, Andrew

    2016-10-01

    Larvae of Chironomus riparius respond to ion-poor and brackish water (IPW, BW) conditions by activating ion uptake mechanisms in the anal papillae and reducing ion absorption at the rectum, respectively. The role that the Malpighian tubules play in ion and osmoregulation under these conditions is not known in this species. This study examines rates of fluid secretion and major cation composition of secreted fluid from tubules of C. riparius reared in IPW, freshwater (FW) and BW. Fluid secretion of tubules from FW and BW larvae was similar but tubules from IPW larvae secrete fluid at higher rates, are more sensitive to serotonin stimulation, and the secreted fluid contains less Na(+) . Therefore in IPW, tubules work in concert with anal papillae to eliminate excess water while conserving Na(+) in the hemolymph. Tubules do not appear to play a significant role in ion/osmoregulation under BW. Serotonin immunoreactivity in the nervous system and gastrointestinal tract of larval C. riparius was similar to that seen in mosquito larvae with the exception that the hindgut was devoid of staining. Hemolymph serotonin titer was similar in FW and IPW; hence, serotonin is not responsible for the observed high rates of fluid secretion in IPW. Instead, it is suggested that serotonin may work in a synergistic manner with an unidentified hormonal factor in IPW. Ion transport mechanisms in the tubules of C. riparius are pharmacologically similar to those of other insects.

  10. The HTV Proximity Communication System

    NASA Astrophysics Data System (ADS)

    Harada, Motoyuki; Takahashi, Tetsuo; Tanaka, Tetsuo

    2002-01-01

    National Space Development Agency of Japan (NASDA) is developing the H-II Transfer Vehicle (HTV) as an unmanned logistic support vehicle for the International Space Station (ISS). The HTV, which is launched by the H-IIA rocket, transports both pressurized and un-pressurized cargoes to the ISS, reloads disposal items from the ISS and performs destructive reentry over ocean area. NASDA plans the first flight of HTV in 2005 for demonstration. The HTV will contribute the ISS assembly and logistic re-supply operations with international commonality. For unmanned vehicle operation, communication link is critical with commanding and control, especially in proximity region to the ISS. As for the HTV operation, NASDA is developing dedicated communication system installed on the Japanese Experiment Module (JEM) of the ISS, which is called the Proximity Communication System (PROX). The HTV receives commands and transmits telemetry data through the PROX in its proximity operation to the ISS. To achieve this, the PROX can communicate with the HTV on its nominal trajectory within 23km of the ISS. Especially within 3km of the ISS, the PROX has capability to perform omni-directional communication to the HTV. The PROX also has GPS receiver and send GPS data to the HTV to support the HTV navigation for relative GPS navigation used in "far" range (500m away from the ISS). In addition to the above fundamental functions, the PROX has a capability of range and range-rate measurement between the ISS and the HTV by the pseudo noise (PN) code epoch and the Doppler shift frequency. This provides a reference data independent of "main" navigation methods (rendezvous sensor navigation or GPS navigation) of the HTV. The PROX also assists the ISS crew with its commanding device, called the Hardware Command Panel (HCP), to issue time-dependent safety-related critical commands for HTV berthing/un-berthing operation. When a failure occurs somewhere on "communication path" from the PROX Base Band

  11. Proximal Biceps in Overhead Athletes.

    PubMed

    Chalmers, Peter N; Verma, Nikhil N

    2016-01-01

    The proximal long head of the biceps tendon and its attachment at the superior glenoid tubercle and labrum are subject to a spectrum of disorders in overhead athletes. Biceps disorders are commonly characterized by intermittent anterior or deep-seated shoulder pain exacerbated by activity. Diagnosis is reached via various physical examination maneuvers; MRI can be uncertain. Nonsteroidal anti-inflammatory medications, targeted ultrasound-guided corticosteroid injections, and supervised physical therapy are the mainstays of nonoperative treatment. Operative treatment, which remains controversial, provides reliable pain relief, restoration of function for activities of daily living, and low complication rates, but return to play can be unpredictable.

  12. Comparative evaluation of NovaMin desensitizer and Gluma desensitizer on dentinal tubule occlusion: a scanning electron microscopic study

    PubMed Central

    Joshi, Surabhi; Gowda, Ashwini Shivananje

    2013-01-01

    Purpose In this study, the effect of calcium sodium phosphosilicate (NovaMin) desensitizing agent, which is a powder-based system, and hydroxyethyl methacrylate and glutaraldehyde (Gluma desensitizer), which is liquid-based system, on dentinal tubule occlusion was analyzed by scanning electron microscope. The effects of the above two along with one control group were compared to determine the more effective method of sealing the dentinal tubules after initial application. Methods Twenty specimens were allocated to each of 3 groups: Control, Gluma desensitizer, and NovaMin. Two additional samples were also prepared and treated with Gluma and NovaMin; these samples were longitudinally fractured. The specimens were prepared from extracted sound human premolars and were stored in 10% formalin at room temperature. The teeth were cleaned of gross debris and then sectioned to provide one to two dentin specimens. The dentin specimens were etched with 6% citric acid for 2 minutes and rinsed in distilled water. Control discs were dried, and the test discs were treated with the desensitizing agents as per the manufacturer's instructions. The discs as well as longitudinal sections were later analyzed under the scanning electron microscope. The proportions of completely occluded, partially occluded, and open tubules within each group were calculated. The ratios of completely and partially occluded tubules to the total tubules for all the groups was determined, and the data was statistically analyzed using nonparametric tests and statistical significance was calculated. Results NovaMin showed more completely occluded tubules (0.545±0.051) while Gluma desensitizer showed more partially occluded tubules (0.532±0.075). The differences among all the groups were statistically significant (P≤ 0.05). Conclusion Both materials were effective in occluding dentinal tubules but NovaMin appeared more promising in occluding tubules completely after initial application. PMID:24455439

  13. Protein Neighbors and Proximity Proteomics.

    PubMed

    Rees, Johanna S; Li, Xue-Wen; Perrett, Sarah; Lilley, Kathryn S; Jackson, Antony P

    2015-11-01

    Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest.

  14. Fibrosis: ultimate and proximate causes

    PubMed Central

    Thannickal, Victor J.; Zhou, Yong; Gaggar, Amit; Duncan, Steven R.

    2014-01-01

    Fibrotic disorders account for an increasing burden of disease-associated morbidity and mortality worldwide. Although numerous risk factors have been recognized, the etiologies of many of these clinical syndromes have not been identified, and they are often termed idiopathic or cryptogenic. Here, we provide an evolutionary perspective on fibrosis aimed at elucidating its etiopathogenesis. By asking the ultimate question of “why” this process evolved in multicellular organisms, we hope to uncover proximate explanations for “how” it causes disease in humans. We posit that physiological fibrosis-like reactions evolved as an essential process in host defense against pathogens and in normal wound healing. Based on this premise, we reason that pathological fibrosis is related to one or more of the following: unidentified infectious or noninfectious antigens, autoimmunity, impaired regenerative responses, and the antagonistically pleiotropic action of genes involved in wound healing or development. The importance of genetic susceptibility, epigenetics, aging, and the modern-day environment are highlighted. Consideration of both ultimate and proximate causation goes beyond philosophical cogitations, as it will better inform pathobiological mechanisms of disease and aid in the prevention and treatment of fibrotic diseases. PMID:25365073

  15. The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia.

    PubMed

    Toye, Ashley M; Parker, Mark D; Daly, Christopher M; Lu, Jing; Virkki, Leila V; Pelletier, Marc F; Boron, Walter F

    2006-10-01

    The human electrogenic renal Na-HCO(3) cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO(3)(-) from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na(+) affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules.

  16. Ash Aggregates in Proximal Settings

    NASA Astrophysics Data System (ADS)

    Porritt, L. A.; Russell, K.

    2012-12-01

    Ash aggregates are thought to have formed within and been deposited by the eruption column and plume and dilute density currents and their associated ash clouds. Moist, turbulent ash clouds are considered critical to ash aggregate formation by facilitating both collision and adhesion of particles. Consequently, they are most commonly found in distal deposits. Proximal deposits containing ash aggregates are less commonly observed but do occur. Here we describe two occurrences of vent proximal ash aggregate-rich deposits; the first within a kimberlite pipe where coated ash pellets and accretionary lapilli are found within the intra-vent sequence; and the second in a glaciovolcanic setting where cored pellets (armoured lapilli) occur within <1 km of the vent. The deposits within the A418 pipe, Diavik Diamond Mine, Canada, are the residual deposits within the conduit and vent of the volcano and are characterised by an abundance of ash aggregates. Coated ash pellets are dominant but are followed in abundance by ash pellets, accretionary lapilli and rare cored pellets. The coated ash pellets typically range from 1 - 5 mm in diameter and have core to rim ratios of approximately 10:1. The formation and preservation of these aggregates elucidates the style and nature of the explosive phase of kimberlite eruption at A418 (and other pipes?). First, these pyroclasts dictate the intensity of the kimberlite eruption; it must be energetic enough to cause intense fragmentation of the kimberlite to produce a substantial volume of very fine ash (<62 μm). Secondly, the ash aggregates indicate the involvement of moisture coupled with the presence of dilute expanded eruption clouds. The structure and distribution of these deposits throughout the kimberlite conduit demand that aggregation and deposition operate entirely within the confines of the vent; this indicates that aggregation is a rapid process. Ash aggregates within glaciovolcanic sequences are also rarely documented. The

  17. CRIP Homologues Maintain Apical Cytoskeleton to Regulate Tubule Size in C. elegans

    PubMed Central

    Tong, Xiangyan; Buechner, Matthew

    2009-01-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of C. elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface. PMID:18384766

  18. CRIP homologues maintain apical cytoskeleton to regulate tubule size in C. elegans.

    PubMed

    Tong, Xiangyan; Buechner, Matthew

    2008-05-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of Caenorhabditis elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface.

  19. Beta-adrenoceptors in kidney tubules of spontaneously hypertensive and normotensive rats

    SciTech Connect

    Struyker-Boudier, H.A.J.; Vervoort-Peters, L.H.T.M.; Rousch, M.J.M.; Smits, J.F.M.; Thijssen, H.H.W.

    1986-01-13

    Beta-adrenoceptor binding characteristics were determined in different fractions of rat kidney tubules using a (/sup 125/Iodo)-(-)-cyanopindolol (ICYP) binding assay. The highest amount of binding sites was found in a fraction containing predominantly distal tubular fragments. In a separate series of experiments the ICYP binding characteristics were compared in whole tubular fractions from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) of different ages. The maximum number of binding sites was significantly higher both in young (3 weeks) and adult (14 weeks) SHR when compared to age-matched WKY. These studies showed the presence of beta-adrenoceptor binding sites in rat kidney tubules and support the potential importance of tubular beta-adrenoceptors in the development of spontaneous hypertension and in the mechanism of antihypertensive action of beta-blockers. 35 references, 1 figure, 3 tables.

  20. Two new species of Nephridiophaga (Zygomycota) in the Malpighian tubules of cockroaches.

    PubMed

    Radek, Renate; Wellmanns, Daniel; Wolf, Anja

    2011-08-01

    The Malpighian tubules of the cockroaches Archimandrita tessellata and Lucihormetica verrucosa were infected with stages of unicellular parasites. Plasmodial stages occurred freely in the lumen and attached to the epithelium of the Malpighian tubules. Vegetative plasmodia contain few or numerous nuclei, while the sporogenic plasmodia (pansporoblasts) have vegetative nuclei in their cytoplasm and in addition endogenously form uninucleate spores. New species of the genus Nephridiophaga were proposed for these two isolates. This decision was made based on the fact that they are not only found in different hosts, but are different in their morphology. For example, the size of the spores and the number of spores per sporogenic plasmodium differed. Light and electron microscopic observations show that the parasites are new members of the genus Nephridiophaga, i.e., Nephridiophaga archimandrita and Nephridiophaga lucihormetica.

  1. Tubulation by amphiphysin requires concentration-dependent switching from wedging to scaffolding

    PubMed Central

    Isas, J. Mario; Ambroso, Mark R.; Hegde, Prabhavati B.; Langen, Jennifer; Langen, Ralf

    2015-01-01

    Summary BAR proteins are involved in a variety of membrane remodeling events, but how they can mold membranes into different shapes remains poorly understood. Using EPR, we find that vesicle binding of the N-BAR protein amphiphysin is predominantly mediated by the shallow insertion of amphipathic N-terminal helices. In contrast, the interaction with tubes involves deeply inserted N-terminal helices together with the concave surface of the BAR domain, which acts as a scaffold. Combined with the observed concentration dependence of tubulation and BAR domain scaffolding, the data indicate that initial membrane deformations and vesicle binding are mediated by insertion of amphipathic helical wedges, while tubulation requires high protein densities at which oligomeric BAR domain scaffolds form. In addition, we identify a pocket of residues on the concave surface of the BAR domain that deeply insert into tube membrane. Interestingly, this pocket harbors a number of disease mutants in the homologous amphiphysin 2. PMID:25865245

  2. Dysferlin at transverse tubules regulates Ca2+ homeostasis in skeletal muscle

    PubMed Central

    Kerr, Jaclyn P.; Ward, Christopher W.; Bloch, Robert J.

    2014-01-01

    The class of muscular dystrophies linked to the genetic ablation or mutation of dysferlin, including Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy (MM), are late-onset degenerative diseases. In lieu of a genetic cure, treatments to prevent or slow the progression of dysferlinopathy are of the utmost importance. Recent advances in the study of dysferlinopathy have highlighted the necessity for the maintenance of calcium handling in altering or slowing the progression of muscular degeneration resulting from the loss of dysferlin. This review highlights new evidence for a role for dysferlin at the transverse (t-) tubule of striated muscle, where it is involved in maintaining t-tubule structure and function. PMID:24639655

  3. Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

    PubMed Central

    Gnemmi, Viviane; Glowacki, François; Pottier, Nicolas; Bouillez, Audrey; Maboudou, Patrice; Zini, Laurent; Leroy, Xavier; Cauffiez, Christelle; Perrais, Michaël; Aubert, Sébastien

    2013-01-01

    Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation. PMID:23799132

  4. Regulation of G-protein signaling via Gnas is required to regulate proximal tubular growth in the Xenopus pronephros

    PubMed Central

    Zhang, Bo; Romaker, Daniel; Ferrell, Nicholas; Wessely, Oliver

    2013-01-01

    In the kidney, proximal tubules are very important for the reabsorption of water, ions and organic solutes from the primary urine. They are composed of highly specialized epithelial cells that are characterized by an elaborate apical brush border to increase transport efficiency. Using the pronephric kidney of Xenopus laevis we discovered that the G-protein modulator cholera toxin resulted in a dramatic reduction of the proximal tubular size. This phenotype was accompanied by changes in the cytoarchitecture characterized by ectopic expression of the distal tubular marker 4A6 and an impairment of yolk platelet degradation. In addition, cholera toxin caused edema formation. However, this phenotype was not due to kidney defects, but rather due to impaired vasculature development. Based on experiments with antisense morpholino oligomers as well as pharmacological agonists and antagonists, we could show that the complex phenotype of cholera toxin in the pronephric kidney was caused by the hyperactivation of a single G-protein alpha subunit, Gnas. This—in turn—caused elevated cAMP levels, triggered a Rapgef4-dependent signaling cassette and perturbed exo- and endocytosis. This perturbation of the secretory pathway by Ctx was not only observed in Xenopus embryos. Also, in a human proximal tubular cell line, cholera toxin or a Rapgef4-specific agonist increased uptake and decreased secretion of FITC-labeled Albumin. Based on these data we propose that the Gnas/cAMP/Rapgef4 pathway regulates the signals inducing the proliferation of proximal tubules to acquire their final organ size. PMID:23352791

  5. Proximal Tubulopathy: Prime Mover and Key Therapeutic Target in Diabetic Kidney Disease.

    PubMed

    Gilbert, Richard E

    2017-04-01

    The current view of diabetic kidney disease, based on meticulously acquired ultrastructural morphometry and the utility of measuring plasma creatinine and urinary albumin, has been almost entirely focused on the glomerulus. While clearly of great importance, changes in the glomerulus are not the major determinant of renal prognosis in diabetes and may not be the primary event in the development of diabetic kidney disease either. Indeed, advances in biomarker discovery and a greater appreciation of tubulointerstitial histopathology and the role of tubular hypoxia in the pathogenesis of chronic kidney disease have given us pause to reconsider the current "glomerulocentric" paradigm and focus attention on the proximal tubule that by virtue of the high energy requirements and reliance on aerobic metabolism render it particularly susceptible to the derangements of the diabetic state. Such findings raise important issues for therapeutic advances specifically targeting the pathophysiological perturbations that develop in this part of the nephron.

  6. Structural alterations in the seminiferous tubules of rats treated with immunosuppressor tacrolimus

    PubMed Central

    Caneguim, Breno H; Cerri, Paulo S; Spolidório, Luís C; Miraglia, Sandra M; Sasso-Cerri, Estela

    2009-01-01

    Background Tacrolimus (FK-506) is an immunosuppressant that binds to a specific immunophilin, resulting in the suppression of the cellular immune response during transplant rejection. Except for some alterations in the spermatozoa, testicular morphological alterations have not been described in rats treated with tacrolimus. In the present study, we purpose to evaluate if the treatment with tacrolimus at long term of follow-up interferes in the integrity of the seminiferous tubules. Methods Rats aging 42-day-old received daily subcutaneous injections of 1 mg/kg/day of tacrolimus during 30 (T-30) and 60 (T-60) days; the rats from control groups (C-30 and C-60) received saline solution. The left testes were fixed in 4% formaldehyde and embedded in glycol methacrylate for morphological and morphometric analyses while right testes were fixed in Bouin's liquid and embedded in paraffin for detection of cell death by the TUNEL method. The epithelial and total tubular areas as well as the stages of the seminiferous epithelium and the number of spermatocytes, spermatids and Sertoli cells (SC) per tubule were obtained. Results In the treated groups, seminiferous tubules irregularly outlined showed disarranged cellular layers and loss of germ cells probably due to cell death, which was revealed by TUNEL method. In addition to germ cells, structural alterations in the SC and folding of the peritubular tissue were usually observed. The morphometric results revealed significant decrease in the number of SC, spermatocytes, spermatids and significant reduction in the epithelial and total tubular areas. Conclusion Tacrolimus induces significant histopathological disorders in the seminiferous tubules, resulting in spermatogenic damage and reduction in the number of Sertoli cells. A careful evaluation of the peritubular components will be necessary to clarify if these alterations are related to the effect of FK-506 on the peritubular tissue. PMID:19243597

  7. Reduced tolerance of immature renal tubules to anoxia by HSF-1 decoy.

    PubMed

    Sreedharan, Rajasree; Riordan, Michael; Wang, Shirley; Thulin, Gunilla; Kashgarian, Michael; Siegel, Norman J

    2005-02-01

    Immature animals demonstrate an amplified heat shock response following a variety of insults compared with that seen in mature animals (M). The potential role of the heat shock response in modulating immature tolerance to injury was compared between rat pups, 10 postnatal days of age (P10), and M. Baseline levels of the heat shock transcription factor (HSF-1) were substantially elevated in P10 compared with M animals. In uninjured P10 pups, HSF-1 level was comparable to that of M animals subjected to 45 min of ischemia. As anticipated, the integrity of suspensions of tubules exposed to anoxia was preserved in P10 animals (23% LDH release) compared with M (40%), P < 0.01. The effect of targeted inhibition of HSF-1 on tubular integrity was studied using a cyclic oligonucleotide decoy. The HSF-1 decoy increased the severity of anoxic injury in P10 pups to a level comparable with M animals. LDH release was 33% in decoy-treated P10 tubules compared with 40% in M. When P10 tubules were treated with scrambled decoy, resistance to anoxia remained intact (24%). The increased vulnerability of the tubular suspension to injury was specific to the HSF-1 decoy and proportional to the dose of decoy applied. This study demonstrates maturation in the abundance of HSF-1 in the immature rat kidney. The loss of resistance of immature tubules to anoxia with specific inhibition of HSF-1 may be due to its effect on the heat shock response or other signaling pathways of critical pathobiological importance in renal cell injury.

  8. Signaling by TGF-betas in tubule cultures of adult rat testis

    PubMed Central

    Chan, Kai-Hui; Galuska, Sebastian P; Kudipudi, Pradeep Kumar; Riaz, Mohammad Assad; Loveland, Kate L; Konrad, Lutz

    2017-01-01

    Although signal transduction of transforming growth factor-betas (TGF-βs) is well characterized in individual cell types, data about TGF-β signaling in a cellular context is still scarce. In this study, we used ex vivo tubule cultures from adult rat testis to investigate TGF-β signaling. We show for the first time in testicular tubules, that TGF-βs also signal via the BMP type I receptors, with ALK2 used by TGF-β1 and ALK3 and ALK6 by TGF-β2. This signal transduction is mediated via Smad3 as well as via Smad1. In contrast, BMPs (BMP2 and BMP7) do not signal via the high-affinity type I and type II TGFβ receptors, TBR1 or TBR2. Furthermore, treatment of tubule cultures with either TGF-β1 or TGF-β2 had profound significant stimulatory effects on secretion of plasminogen activator-1 (PAI-1) through utilization of TGF-β and BMP receptors. Specific inhibitors for either TBR1 or BMP receptors yielded nearly complete inhibition of TGF-β signaling. The TBR1-TBR2 signalosome was detected with Duolink upon stimulation with either TGF-β1 or TGF-β2, predominantly in spermatogenic cells of the adult rat testis, particularly in elongated spermatids. In summary, this examination of intact rat testicular tubules demonstrated for the first time that TGF-βs signal mainly through TBR1 and TBR2 but also use BMP receptors, including for secretion of PAI-1. Whereas ALK2 participates in the TGF-β1-induced TBR1-TBR2 signalosome, ALK3 and ALK6 are involved in signaling of TGF-β2. Detection of the TBR1-TBR2 signalosome in late spermiogenic cells indicates a post-meiotic activity. PMID:28386343

  9. Influence of the direction of tubules on bond strength to dentin.

    PubMed

    Ogata, M; Okuda, M; Nakajima, M; Pereira, P N; Sano, H; Tagami, J

    2001-01-01

    This study investigated the influence of the direction of dentinal tubules on resin-dentin tensile bond strength (mu TBS) using four commercially available bonding systems and observed the resin-dentin interfaces with an SEM. The dentin bonding systems used in this study were Clearfil Liner Bond II (LB, Kuraray), Imperva Fluoro Bond (FB, Shofu), Single Bond (SB, 3M) and One-Step (OS, BISCO). Thirty-six extracted caries-free human molars were used for micro tensile bond testing and eight additional teeth were used for scanning electron microscopy (SEM). The teeth were divided into two groups according to the direction of the dentinal tubules at the resin-dentin interface: a perpendicular group, in which the occlusal enamel was removed perpendicular to the long axis of the tooth, and a parallel group, in which the mesial half of the tooth was removed parallel to the long axis of the tooth, and the coronal dentin surface was used for bonding. After the flat dentin surfaces were polished with #600 silicon carbide paper, each surface was treated with one of the four adhesive systems according to the manufacturer's recommendation, then covered with resin composite (Clearfil AP-X, Kuraray) to provide sufficient bulk for micro-tensile bond testing. After 24 hours in 37 degrees C water, the resin-bonded teeth were serially sliced perpendicular to the adhesive surface, the adhesive interface trimmed to a cross sectional area of 1 mm2 and subjected to tensile forces at a crosshead speed of 1 mm/min. Statistical analysis of the tensile bond strengths were performed using two-way ANOVA and Fisher's PLSD test at 95% level of confidence. The tensile bond strength of the group with tubules parallel to the bonded interface was higher than that of tubules cut perpendicularly. This tendency reached statistical significance using SB and OS.

  10. Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    PubMed Central

    Schulze, Ryan J.; Weller, Shaun G.; Schroeder, Barbara; Krueger, Eugene W.; Chi, Susan; Casey, Carol A.

    2013-01-01

    Lipid droplets (LDs) are lipid storage organelles that in hepatocytes may be catabolized by autophagy for use as an energy source, but the membrane-trafficking machinery regulating such a process is poorly characterized. We hypothesized that the large GTPase Dynamin 2 (Dyn2), well known for its involvement in membrane deformation and cellular protein trafficking, could orchestrate autophagy-mediated LD breakdown. Accordingly, depletion or pharmacologic inhibition of Dyn2 led to a substantial accumulation of LDs in hepatocytes. Strikingly, the targeted disruption of Dyn2 induced a dramatic four- to fivefold increase in the size of autolysosomes. Chronic or acute Dyn2 inhibition combined with nutrient deprivation stimulated the excessive tubulation of these autolysosomal compartments. Importantly, Dyn2 associated with these tubules along their length, and the tubules vesiculated and fragmented in the presence of functional Dyn2. These findings provide new evidence for the participation of the autolysosome in LD metabolism and demonstrate a novel role for dynamin in the function and maturation of an autophagic compartment. PMID:24145164

  11. Stimulation of glutamine metabolism by 3-aminopicolinate in isolated dog kidney-cortex tubules.

    PubMed Central

    Durozard, D; Baverel, G

    1983-01-01

    1. The effects of 3-aminopicolinate, a known hyperglycaemic agent in the rat, on glutamine metabolism were studied in isolated dog kidney tubules. 2. 3-Aminopicolinate greatly stimulated glutamine (but not glutamate) removal and glutamate accumulation from glutamine as well as formation of ammonia, aspartate, lactate, alanine and glucose. 3. The increased accumulation of aspartate from glutamine and glutamate, and the inhibition of glucose synthesis from various non-nitrogenous gluconeogenic substrates, as well as the increased accumulation of malate from succinate, support the proposal that 3-aminopicolinate is an inhibitor rather than a stimulator of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in dog kidney tubules. 4. With glutamine as substrate, the increase in flux through glutamate dehydrogenase (EC 1.4.1.3) could not explain the large increase in glutamine removal caused by 3-aminopicolinate. 5. Inhibition by amino-oxyacetate of accumulation of aspartate and alanine from glutamine caused by 3-aminopicolinate did not prevent the acceleration of glutamine utilization. 6. These data are consistent with a direct stimulation of glutaminase (EC 3.5.1.2) by 3-aminopicolinate in dog kidney tubules. PMID:6134524

  12. Morphometric changes associated with sex and development in the Malpighian tubules of Aedes aegypti.

    PubMed

    de Sousa, R C; Bicudo, H E

    2000-01-01

    The Malpighian tubules of Aedes aegypti showed significant differences in their diameters between male and female larvae, male and female pupae, male larvae and male adults and male pupae and male adults. In every case, female values were greater than in males. Measurements of mean nuclear areas of the principal and stellate cells from Malpighian tubules, taken in males and females during development, showed that this parameter in both types of cell was significantly greater in females than in male larvae, pupae and adult stages. In males, significant differences between developmental stages were observed only in comparison with the nuclear area of larvae and adults in the principal cells, but in females, every comparison between stages showed significant differences except between pupae and adults in stellate cells. The frequency distribution of nuclear area values, in development, for male stellate and principal cells, were mostly concentrated in the first seven classes among the 30 classes considered in every stage, while for females, the frequency dropped drastically in the same classes from larvae to pupae and adults, moving to classes of higher values. Considering the importance of Malpighian tubules in insect physiology, the meaning of the differences detected are discussed on the basis of different metabolic levels, between sexes and developmental stages.

  13. Immune and stress response 'cross-talk' in the Drosophila Malpighian tubule.

    PubMed

    Davies, Shireen-Anne; Overend, Gayle; Sebastian, Sujith; Cundall, Maria; Cabrero, Pablo; Dow, Julian A T; Terhzaz, Selim

    2012-04-01

    The success of insects is in large part due to their ability to survive environmental stress, including heat, cold, and dehydration. Insects are also exposed to infection, osmotic or oxidative stress, and to xenobiotics or toxins. The molecular mechanisms of stress sensing and response have been widely investigated in mammalian cell lines, and the area of stress research is now so vast to be beyond the scope of a single review article. However, the mechanisms by which stress inputs to the organism are sensed and integrated at the tissue and cellular level are less well understood. Increasingly, common molecular events between immune and other stress responses are observed in vivo; and much of this work stems of efforts in insect molecular science and physiology. We describe here the current knowledge in the area of immune and stress signalling and response at the level of the organism, tissue and cell, focussing on a key epithelial tissue in insects, the Malpighian tubule, and drawing together the known pathways that modulate responses to different stress insults. The tubules are critical for insect survival and are increasingly implicated in responses to multiple and distinct stress inputs. Importantly, as tubule function is central to survival, they are potentially key targets for insect control, which will be facilitated by increased understanding of the complexities of stress signalling in the organism.

  14. Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

    PubMed Central

    Maher, Geoffrey J.; McGowan, Simon J.; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O. M.

    2016-01-01

    De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39–90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones. PMID:26858415

  15. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

    PubMed Central

    Halberg, Kenneth A.; Rainey, Stephanie M.; Veland, Iben R.; Neuert, Helen; Dornan, Anthony J.; Klämbt, Christian; Davies, Shireen-Anne; Dow, Julian A. T.

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell–cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border. PMID:27072072

  16. Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes.

    PubMed

    Li, Xiaoheng; Wang, Zhao; Jiang, Zhenming; Guo, Jingjing; Zhang, Yuxi; Li, Chenhao; Chung, Jinyong; Folmer, Janet; Liu, June; Lian, Qingquan; Ge, Renshan; Zirkin, Barry R; Chen, Haolin

    2016-03-08

    Testicular Leydig cells are the primary source of testosterone in males. Adult Leydig cells have been shown to arise from stem cells present in the neonatal testis. Once established, adult Leydig cells turn over only slowly during adult life, but when these cells are eliminated experimentally from the adult testis, new Leydig cells rapidly reappear. As in the neonatal testis, stem cells in the adult testis are presumed to be the source of the new Leydig cells. As yet, the mechanisms involved in regulating the proliferation and differentiation of these stem cells remain unknown. We developed a unique in vitro system of cultured seminiferous tubules to assess the ability of factors from the seminiferous tubules to regulate the proliferation of the tubule-associated stem cells, and their subsequent entry into the Leydig cell lineage. The proliferation of the stem Leydig cells was stimulated by paracrine factors including Desert hedgehog (DHH), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and activin. Suppression of proliferation occurred with transforming growth factor β (TGF-β). The differentiation of the stem cells was regulated positively by DHH, lithium- induced signaling, and activin, and negatively by TGF-β, PDGFBB, and FGF2. DHH functioned as a commitment factor, inducing the transition of stem cells to the progenitor stage and thus into the Leydig cell lineage. Additionally, CD90 (Thy1) was found to be a unique stem cell surface marker that was used to obtain purified stem cells by flow cytometry.

  17. Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation

    PubMed Central

    Inaba, Takehiko; Kishimoto, Takuma; Murate, Motohide; Tajima, Takuya; Sakai, Shota; Abe, Mitsuhiro; Makino, Asami; Tomishige, Nario; Ishitsuka, Reiko; Ikeda, Yasuo; Takeoka, Shinji; Kobayashi, Toshihide

    2016-01-01

    Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cβ1 (PLCβ1) as a new candidate. PLCβ1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCβ1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCβ1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCβ1: plasma membrane remodeling, and in particular, caveolae formation. PMID:27342861

  18. Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae).

    PubMed

    Catae, Aline Fernanda; Roat, Thaisa Cristina; De Oliveira, Regiane Alves; Nocelli, Roberta Cornélio Ferreira; Malaspina, Osmar

    2014-04-01

    Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non-target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC₅₀ (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide.

  19. Diabetic nephropathy induces alterations in the glomerular and tubule lipid profiles1[S

    PubMed Central

    Grove, Kerri J.; Voziyan, Paul A.; Spraggins, Jeffrey M.; Wang, Suwan; Paueksakon, Paisit; Harris, Raymond C.; Hudson, Billy G.; Caprioli, Richard M.

    2014-01-01

    Diabetic nephropathy (DN) is a major life-threatening complication of diabetes. Renal lesions affect glomeruli and tubules, but the pathogenesis is not completely understood. Phospholipids and glycolipids are molecules that carry out multiple cell functions in health and disease, and their role in DN pathogenesis is unknown. We employed high spatial resolution MALDI imaging MS to determine lipid changes in kidneys of eNOS−/− db/db mice, a robust model of DN. Phospholipid and glycolipid structures, localization patterns, and relative tissue levels were determined in individual renal glomeruli and tubules without disturbing tissue morphology. A significant increase in the levels of specific glomerular and tubular lipid species from four different classes, i.e., gangliosides, sulfoglycosphingolipids, lysophospholipids, and phosphatidylethanolamines, was detected in diabetic kidneys compared with nondiabetic controls. Inhibition of nonenzymatic oxidative and glycoxidative pathways attenuated the increase in lipid levels and ameliorated renal pathology, even though blood glucose levels remained unchanged. Our data demonstrate that the levels of specific phospho- and glycolipids in glomeruli and/or tubules are associated with diabetic renal pathology. We suggest that hyperglycemia-induced DN pathogenic mechanisms require intermediate oxidative steps that involve specific phospholipid and glycolipid species. PMID:24864273

  20. Piecewise-Constant-Model-Based Interior Tomography Applied to Dentin Tubules

    DOE PAGES

    He, Peng; Wei, Biao; Wang, Steve; ...

    2013-01-01

    Dentin is a hierarchically structured biomineralized composite material, and dentin’s tubules are difficult to study in situ. Nano-CT provides the requisite resolution, but the field of view typically contains only a few tubules. Using a plate-like specimen allows reconstruction of a volume containing specific tubules from a number of truncated projections typically collected over an angular range of about 140°, which is practically accessible. Classical computed tomography (CT) theory cannot exactly reconstruct an object only from truncated projections, needless to say a limited angular range. Recently, interior tomography was developed to reconstruct a region-of-interest (ROI) from truncated data in amore » theoretically exact fashion via the total variation (TV) minimization under the condition that the ROI is piecewise constant. In this paper, we employ a TV minimization interior tomography algorithm to reconstruct interior microstructures in dentin from truncated projections over a limited angular range. Compared to the filtered backprojection (FBP) reconstruction, our reconstruction method reduces noise and suppresses artifacts. Volume rendering confirms the merits of our method in terms of preserving the interior microstructure of the dentin specimen.« less

  1. Vitality of Enterococcus faecalis inside dentinal tubules after five root canal disinfection methods

    PubMed Central

    Vatkar, Niranjan Ashok; Hegde, Vivek; Sathe, Sucheta

    2016-01-01

    Aim: To compare the vitality of Enterococcus faecalis within dentinal tubules after subjected to five root canal disinfection methods. Materials and Methods: Dentin blocks (n = 60) were colonized with E. faecalis. After 4 weeks of incubation, the dentin blocks were divided into one control and five test groups (n = 10 each). The root canals of test groups were subjected to one of the disinfection methods, namely, normal saline (NS), sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX), neodymium-doped yttrium aluminum garnet (Nd: YAG) laser, and diode laser. The effect of disinfection methods was assessed by LIVE/DEAD BacLight stain under the confocal laser scanning microscopy to determine the “zone of dead bacteria” (ZDB). Mean values were calculated for ZDB and the difference between groups was established. Results: Penetration of E. faecalis was seen to a depth of >1000 μm. Viable bacteria were detected with NS irrigation. NaOCl and CHX showed partial ZDB. When the root canals were disinfected with Nd: YAG and diode lasers, no viable bacteria were found. Conclusion: E. faecalis has the ability to colonize inside dentinal tubules to a depth of >1000 μm. In contrast to conventional irrigants, both Nd: YAG and diode lasers were effective in eliminating the vitality of E. faecalis. NS, NaOCl, and CHX showed viable bacteria remaining in dentinal tubules. PMID:27656064

  2. Optical properties of human radicular dentin: ATR-FTIR characterization and dentine tubule direction influence on radicular post adhesion

    NASA Astrophysics Data System (ADS)

    Quinto, Jose; Zamataro, Claudia B.; Benetti, Carolina; Dias, Derly A.; Blay, Alberto; Zezell, Denise Maria

    2015-06-01

    Knowledge of dental structures is essential for understanding of laser interaction and its consequences during adhesion processes. Tubule density in dentin ranges from 4.900 to 90.000 per mm2, for diameters from 1 to 3 μm. Light propagation inside the tubules is associated with tubules orientation. To the best of our knowledge, there is no previous work in literature characterizing physical-chemical alterations in dentin. The dentin samples were irradiated with a Er,Cr:YSGG Laser at wavelength 2.78 μm, with an energy density of 9.46 J/cm2 , above the ablation threshold. ATRFTIR at wavenumbers 2000 to 700 cm-1 was used to evaluate the differences among third root region and tubules orientation.

  3. Roles of PKC and phospho‑adducin in transepithelial fluid secretion by Malpighian tubules of the yellow fever mosquito

    PubMed Central

    Miyauchi, Jeremy T.; Piermarini, Peter M.; Yang, Jason D.; Gilligan, Diana M.; Beyenbach, Klaus W.

    2013-01-01

    The diuretic hormone aedeskinin‑III is known to increase the paracellular Cl- conductance in Malpighian (renal) tubules of the mosquito Aedes aegypti via a G protein-coupled receptor. The increase serves the blood-meal-initiated diuresis and is associated with elevated levels of Ca2+ and phosphorylated adducin in the cytosol of tubule. In the present study we have cloned adducin in Aedes Malpighian tubules and investigated its physiological roles. Immunolabeling experiments are consistent with the association of adducin with the cortical cytoskeleton, especially near the apical brush border of the tubule. An antibody against phosphorylated adducin revealed the transient phosphorylation of adducin 2 min after stimulating tubules with aedeskinin‑III. The PKC inhibitor bisindolylmaleimide‑I blocked the phosphorylation of adducin as well as the electrophysiological and diuretic effects of aedeskinin‑III. Bisindolylmaleimide‑I also inhibited fluid secretion in control tubules. Phorbol 12‑myristate 13‑acetate increased phosphorylated adducin levels in Malpighian tubules, but it inhibited fluid secretion. Thus, the phosphorylation of adducin by PKC alone is insufficient to trigger diuretic rates of fluid secretion; elevated levels of intracellular Ca2+ may also be required. The above results suggest that the phosphorylation of adducin, which is known to destabilize the cytoskeleton, may (1) facilitate the traffic of transporters into the apical brush border supporting diuretic rates of cation secretion and (2) destabilize proteins in the septate junction thereby enabling paracellular anion (Cl‑) secretion at diuretic rates. Moreover, PKC and the phosphorylation of adducin play a central role in control and diuretic tubules, consistent with the dynamic behavior of both transcellular and paracellular transport pathways. PMID:24062972

  4. Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

    PubMed Central

    Schellinger, Jeffrey N.; Rodan, Aylin R.

    2015-01-01

    Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms. PMID:26650886

  5. Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

    PubMed Central

    Fujita, Naonobu; Huang, Wilson; Lin, Tzu-han; Groulx, Jean-Francois; Jean, Steve; Nguyen, Jen; Kuchitsu, Yoshihiko; Koyama-Honda, Ikuko; Mizushima, Noboru; Fukuda, Mitsunori; Kiger, Amy A

    2017-01-01

    Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms. DOI: http://dx.doi.org/10.7554/eLife.23367.001 PMID:28063257

  6. Interaction of the tracheal tubules of Scutigera coleoptrata (Chilopoda, Notostigmophora) with glandular structures of the pericardial septum

    PubMed Central

    Hilken, Gero; Edgecombe, Gregory D.; Müller, Carsten H.G.; Sombke, Andy; Wirkner, Christian S.; Rosenberg, Jörg

    2015-01-01

    Abstract Notostigmophora (Scutigeromorpha) exhibit a special tracheal system compared to other Chilopoda. The unpaired spiracles are localized medially on the long tergites and open into a wide atrium from which hundreds of tracheal tubules originate and extend into the pericardial sinus. Previous investigators reported that the tracheal tubules float freely in the hemolymph. However, here we show for the first time that the tracheal tubules are anchored to a part of the pericardial septum. Another novel finding is this part of the pericardial septum is structured as an aggregated gland on the basis of its specialized epithelium being formed by hundreds of oligocellular glands. It remains unclear whether the pericardial septum has a differently structure in areas that lack a connection with tracheal tubules. The tracheal tubules come into direct contact with the canal cells of the glands that presumably secrete mucous substances covering the entire luminal cuticle of the tracheal tubules. Connections between tracheae and glands have not been observed in any other arthropods. PMID:26257546

  7. Deformed proximity potential for heavy ion reactions

    SciTech Connect

    Baltz, A.J.

    1989-09-01

    The proximity potential is discussed for the inelastic scattering of a spherical nucleus on a deformed nucleus or the mutual interaction of two deformed nuclei. It is shown that the proximity potential is, in general, geometrically more correct than the usual centerline prescription used in inelastic scattering analyses. For the cases where the proximity potential is inadequate a folding model approach is advocated. Techniques to facilitate the coupled channels analysis are presented. 11 refs., 6 figs.

  8. Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion.

    PubMed Central

    Ellison, D H; Velázquez, H; Wright, F S

    1989-01-01

    We studied the effects of dietary NaCl intake on the renal distal tubule by feeding rats high or low NaCl chow or by chronically infusing furosemide. Furosemide-treated animals were offered saline as drinking fluid to replace urinary losses. Effects of naCl intake were evaluated using free-flow micropuncture, in vivo microperfusion, and morphometric techniques. Dietary NaCl restriction did not affect NaCl delivery to the early distal tubule but markedly increased the capacity of the distal convoluted tubule to transport Na and Cl. Chronic furosemide infusion increased NaCl delivery to the early distal tubule and also increased the rates of Na and Cl transport above the rates observed in low NaCl diet rats. When compared with high NaCl intake alone, chronic furosemide infusion with saline ingestion increased the fractional volume of distal convoluted tubule cells by nearly 100%, whereas dietary NaCl restriction had no effect. The results are consistent with the hypotheses that (a) chronic NaCl restriction increases the transport ability of the distal convoluted tubule independent of changes in tubule structure, (b) high rates of ion delivery to the distal nephron cause tubule hypertrophy, and (c) tubule hypertrophy is associated with increases in ion transport capacity. They indicate that the distal tubule adapts functionally and structurally to perturbations in dietary Na and Cl intake. Images PMID:2910903

  9. Range gated strip proximity sensor

    DOEpatents

    McEwan, T.E.

    1996-12-03

    A range gated strip proximity sensor uses one set of sensor electronics and a distributed antenna or strip which extends along the perimeter to be sensed. A micro-power RF transmitter is coupled to the first end of the strip and transmits a sequence of RF pulses on the strip to produce a sensor field along the strip. A receiver is coupled to the second end of the strip, and generates a field reference signal in response to the sequence of pulse on the line combined with received electromagnetic energy from reflections in the field. The sensor signals comprise pulses of radio frequency signals having a duration of less than 10 nanoseconds, and a pulse repetition rate on the order of 1 to 10 MegaHertz or less. The duration of the radio frequency pulses is adjusted to control the range of the sensor. An RF detector feeds a filter capacitor in response to received pulses on the strip line to produce a field reference signal representing the average amplitude of the received pulses. When a received pulse is mixed with a received echo, the mixing causes a fluctuation in the amplitude of the field reference signal, providing a range-limited Doppler type signature of a field disturbance. 6 figs.

  10. Proximal Priority Laser Therapy: PPLT

    NASA Astrophysics Data System (ADS)

    Ohshiro, Toshio

    2004-09-01

    The author has, in the past, classified treatment methods for pain geometrically as point, line, two-dimensional, three-dimensional treatment and has used these over the years. However as a practitioner of western medicine, the author originally treated pain only directed at the painful site, and encountered cases where local treatment did not suffice. The author proved with SPECT and the Rand Phantom that treating the neck which is the midpoint of the brain, the center of the nervous system and the heart, the center of circulation, increased cerebral blood flow and also that laser emitted to neck will reach the spinal chord no matter from where on the neck the laser is emitted. From such research and 25 years of clinical experience, the author has created an anatomy based, systemic treatment method called the Proximal Priority Laser Therapy (PPLT) where not only the cerebral cortex, spinal chord and peripheral nerves are treated but also the tracts of blood vessels and lymph ducts are treated as well. Treatment method and cases are presented herein.

  11. Range gated strip proximity sensor

    DOEpatents

    McEwan, Thomas E.

    1996-01-01

    A range gated strip proximity sensor uses one set of sensor electronics and a distributed antenna or strip which extends along the perimeter to be sensed. A micro-power RF transmitter is coupled to the first end of the strip and transmits a sequence of RF pulses on the strip to produce a sensor field along the strip. A receiver is coupled to the second end of the strip, and generates a field reference signal in response to the sequence of pulse on the line combined with received electromagnetic energy from reflections in the field. The sensor signals comprise pulses of radio frequency signals having a duration of less than 10 nanoseconds, and a pulse repetition rate on the order of 1 to 10 MegaHertz or less. The duration of the radio frequency pulses is adjusted to control the range of the sensor. An RF detector feeds a filter capacitor in response to received pulses on the strip line to produce a field reference signal representing the average amplitude of the received pulses. When a received pulse is mixed with a received echo, the mixing causes a fluctuation in the amplitude of the field reference signal, providing a range-limited Doppler type signature of a field disturbance.

  12. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    SciTech Connect

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Lee, Sang Yong; Han, Myung Kwan; Kim, Duk Hoon; Kim, Won

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  13. Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

    PubMed

    Sabolić, I; Shi, L B; Brown, D; Ausiello, D A; Verkman, A S

    1992-01-10

    A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.

  14. Grouping by proximity in haptic contour detection.

    PubMed

    Overvliet, Krista E; Krampe, Ralf Th; Wagemans, Johan

    2013-01-01

    We investigated the applicability of the Gestalt principle of perceptual grouping by proximity in the haptic modality. To do so, we investigated the influence of element proximity on haptic contour detection. In the course of four sessions ten participants performed a haptic contour detection task in which they freely explored a haptic random dot display that contained a contour in 50% of the trials. A contour was defined by a higher density of elements (raised dots), relative to the background surface. Proximity of the contour elements as well as the average proximity of background elements was systematically varied. We hypothesized that if proximity of contour elements influences haptic contour detection, detection will be more likely when contour elements are in closer proximity. This should be irrespective of the ratio with the proximity of the background elements. Results showed indeed that the closer the contour elements were, the higher the detection rates. Moreover, this was the case independent of the contour/background ratio. We conclude that the Gestalt law of proximity applies to haptic contour detection.

  15. Sugar proximity and human grip strength.

    PubMed

    Quintanar, A F; Hill, T V

    1988-12-01

    In a double-blind experiment with 90 undergraduate students the hypothesis was tested that proximity to sugar reduces human grip strength. An earlier study indicated that sugar proximity does indeed significantly reduce human grip strength. When additional controls were added to this design to reduce random sampling error, the hypothesis was not supported.

  16. Grouping by Proximity in Haptic Contour Detection

    PubMed Central

    Overvliet, Krista E.; Krampe, Ralf Th.; Wagemans, Johan

    2013-01-01

    We investigated the applicability of the Gestalt principle of perceptual grouping by proximity in the haptic modality. To do so, we investigated the influence of element proximity on haptic contour detection. In the course of four sessions ten participants performed a haptic contour detection task in which they freely explored a haptic random dot display that contained a contour in 50% of the trials. A contour was defined by a higher density of elements (raised dots), relative to the background surface. Proximity of the contour elements as well as the average proximity of background elements was systematically varied. We hypothesized that if proximity of contour elements influences haptic contour detection, detection will be more likely when contour elements are in closer proximity. This should be irrespective of the ratio with the proximity of the background elements. Results showed indeed that the closer the contour elements were, the higher the detection rates. Moreover, this was the case independent of the contour/background ratio. We conclude that the Gestalt law of proximity applies to haptic contour detection. PMID:23762364

  17. Proximal Participation: A Pathway into Work

    ERIC Educational Resources Information Center

    Chan, Selena

    2013-01-01

    In a longitudinal case study of apprentices, the term proximal participation was coined to describe the entry process of young people, with unclear career destinations, into the trade of baking. This article unravels the significance of proximal participation in the decision-making processes of young people who enter a trade through initial…

  18. Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function

    PubMed Central

    Wu, Yipin; Baum, Michel; Huang, Chou-Long

    2015-01-01

    Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Barium-sensitive K+ conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. We found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules and decreases fluid secretion and transepithelial K+ flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K+ channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Irk1, Irk2, and Irk3 genes. The role of each of these gene products in renal tubule function is unknown. We found that simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K+ flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K+ flux by ∼50%. Knockdown of any of the three inwardly rectifying K+ channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double-knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na+/K+-ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K+ flux, and 75% of transepithelial K+ transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule and are additive to Na+/K+-ATPase-dependent pathways. PMID:26224687

  19. On the mechanism of parathyroid hormone stimulation of calcium uptake by mouse distal convoluted tubule cells.

    PubMed Central

    Gesek, F A; Friedman, P A

    1992-01-01

    PTH stimulates transcellular Ca2+ absorption in renal distal convoluted tubules. The effect of PTH on membrane voltage, the ionic basis of the change in voltage, and the relations between voltage and calcium entry were determined on immortalized mouse distal convoluted tubule cells. PTH (10(-8) M) significantly increased 45Ca2+ uptake from basal levels of 2.81 +/- 0.16 to 3.88 +/- 0.19 nmol min-1 mg protein-1. PTH-induced 45Ca2+ uptake was abolished by the dihydropyridine antagonist, nifedipine (10(-5) M). PTH did not affect 22Na+ uptake. Intracellular calcium activity ([Ca2+]i) was measured in cells loaded with fura-2. Control [Ca2+]i averaged 112 +/- 21 nM. PTH increased [Ca2+]i over the range of 10(-11) to 10(-7) M. Maximal stimulation to 326 +/- 31 nM was achieved at 10(-8) M PTH. Resting membrane voltage measured with the potential sensitive dye DiO6(3) averaged -71 +/- 2 mV. PTH hyperpolarized cells by 19 +/- 4 mV. The chloride-channel blocker NPPB prevented PTH-induced hyperpolarization. PTH decreased and NPPB increased intracellular chloride, measured with the fluorescent dye SPQ. Chloride permeability was estimated by measuring the rate of 125I- efflux. PTH increased 125I- efflux and this effect was blocked by NPPB. Clamping voltage with K+/valinomycin; depolarizing membrane voltage by reducing extracellular chloride; or addition of NPPB prevented PTH-induced calcium uptake. In conclusion, PTH increases chloride conductance in distal convoluted tubule cells leading to decreased intracellular chloride activity, membrane hyperpolarization, and increased calcium entry through dihydropyridine-sensitive calcium channels. PMID:1522230

  20. Ultrastructure of rat initial collecting tubule. Effect of adrenal corticosteroid treatment.

    PubMed Central

    Stanton, B; Janzen, A; Klein-Robbenhaar, G; DeFronzo, R; Giebisch, G; Wade, J

    1985-01-01

    This study examines the effects of adrenalectomy and physiological replacement of mineralocorticoids and glucocorticoids on the cellular ultrastructure of the rat initial collecting tubule (late distal tubule). Animals were adrenalectomized (ADX) and for 10 d received by osmotic minipump either: vehicle, aldosterone (0.5 micrograms X 100 g-1 X d-1), aldosterone (2.0 micrograms X 100 g-1 X d-1), dexamethasone (1.2 micrograms X 100 g-1 X d-1), or aldosterone (0.5 micrograms X 100 g-1 X d-1) with dexamethasone (1.2 micrograms X 100 g-1 X d-1). Radioimmunoassay revealed that the low dose of aldosterone restored plasma aldosterone to control levels. The higher dose of aldosterone increased plasma levels by threefold. Morphometric techniques were used to measure membrane length of individual principal and intercalated cells in each condition. The basolateral membrane length of principal cells decreased by 35% in ADX animals. Low dose aldosterone replacement (0.5 micrograms X 100 g-1 X d-1) in ADX animals maintained membrane length at control values; at a higher level of aldosterone (2.0 micrograms X 100 g-1 X d-1) membrane length increased by 111% compared with control. Dexamethasone treatment, at a level that restored glomerular filtration rate to normal, had no effect on cellular ultrastructure. Combined aldosterone and dexamethasone replacement had no greater effect on basolateral membrane length than aldosterone alone. The length of the luminal membrane of the principal cell type was not affected by ADX or hormone treatment. Intercalated cell membrane length was not affected by ADX or hormone replacement. Thus, chronic aldosterone levels have an important, selective effect on the basolateral membrane of the principal cell. The correlation between these morphological results and the steroid hormone effects on renal electrolyte excretion, reported in the companion paper (15), suggests that basolateral membrane length is an important factor controlling the rate of

  1. Variations of Dietary Salt and Fluid Modulate Calcium and Magnesium Transport in Renal Distal Tubule

    PubMed Central

    Lee, Chien-Te; Lien, Yeong-Hau H; Lai, Li-Wen; Ng, Hwee-Yeong; Chiou, Terry Ting-Yu; Chen, Hung-Chun

    2014-01-01

    Background The renal distal tubule serves as the fine tuning of renal epithelial calcium transport. Dietary intake of salt and fluid varies day to day and the kidney adapts accordingly to maintain the homeostasis. The alternations in salt and fluid balance affect calcium and magnesium transport in the distal tubule, but the mechanisms are not fully understood. Methods Sprague-Dawley rats were grouped into high salt, low salt and dehydration treatment. Daily intake, water consumption and urine output were recorded. At the end of experiment, blood and urine samples were collected for hormonal and biochemical testes. Genetic analysis, immunoblotting, and immunofluorescence studies were then performed to assess the alterations of calcium and magnesium transport-related molecules. Results High salt treatment increased urinary sodium, calcium and magnesium excretion. Low salt treatment and dehydration were associated with decreased urinary excretion of all electrolytes. High salt treatment was associated with increased intact parathyroid hormone levels. Significant increase in gene expression of TRPV5, TRPV6, calbindin-D28k and TRPM6 was found during high salt treatment while low salt and dehydration diminished the expression. These findings were confirmed with immunofluorescence studies. High salt and low salt intake or dehydration did not cause any significant changes in WNK1, WNK3 and WNK4. Conclusions Alternations in salt and water intake affect renal calcium and magnesium handling. High salt intake increases distal delivery of the divalent cations which upregulates distal tubule calcium and magnesium transport molecules, while the opposite effects are associated with low salt intake or dehydration. PMID:23774784

  2. Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells

    PubMed Central

    Xiao, Fengxia; Zimpelmann, Joseph; Burger, Dylan; Kennedy, Christopher; Hébert, Richard L.; Burns, Kevin D.

    2016-01-01

    Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin (Ang) II to Ang-(1–7), and protects against diabetic renal injury. Soluble ACE2 fragments are shed from the proximal tubule, and appear at high levels in the urine with diabetes. High glucose-induced shedding of ACE2 from proximal tubular cells is mediated by the enzyme “a disintegrin and metalloproteinase-17″ (ADAM17). Here, we investigated the mechanism for constitutive shedding of ACE2. Mouse proximal tubular cells were cultured and ACE2 shedding into the media was assessed by enzyme activity assay and immunoblot analysis. Cells were incubated with pharmacologic inhibitors, or transfected with silencing (si) RNA. Incubation of proximal tubular cells with increasing concentrations of D-glucose stimulated ACE2 shedding, which peaked at 16 mM, while L-glucose (osmotic control) had no effect on shedding. In cells maintained in 7.8 mM D-glucose, ACE2 shedding was significantly inhibited by the pan-protein kinase C (PKC) competitive inhibitor sotrastaurin, but not by an inhibitor of ADAM17. Incubation of cells with the PKC-α and -β1-specific inhibitor Go6976, the PKC β1 and β2-specific inhibitor ruboxistaurin, inhibitors of matrix metalloproteinases-2,-8, and -9, or an inhibitor of ADAM10 (GI250423X) had no effect on basal ACE2 shedding. By contrast, the PKC-δ inhibitor rottlerin significantly inhibited both constitutive and high glucose-induced ACE2 shedding. Transfection of cells with siRNA directed against PKC-δ reduced ACE2 shedding by 20%, while knockdown of PKC-ε was without effect. These results indicate that constitutive shedding of ACE2 from proximal tubular cells is mediated by PKC-δ, which is also linked to high glucose-induced shedding. Targeting PKC-δ may preserve membrane-bound ACE2 in proximal tubule in disease states and diminish Ang II-stimulated adverse signaling. PMID:27313531

  3. Thermal Stress of Supported Lipid Bilayer Induces Formation and Collapse of Uniform Radius Tubules

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Fygenson, Deborah

    2011-03-01

    Supported lipid bilayer (SLB) provides a model system in which to quantitatively investigate fluid bilayer transitions from planar to tubular and tubular to spherical morphologies. Following a small increase in temperature, flexible filaments extrude from a fluid SLB. Individual filaments can reach hundreds of microns in length before spontaneously collapsing into discs. We demonstrate that the filaments are tubular and report the effects of lipid composition and flow-induced tension on their properties. At high ionic strength, the sub-resolution tubules are adsorbed to the SLB, enabling the measurement of their radius to within +/- 5 nm using fluorescence microscopy.

  4. The lamina propria of vertebrate seminiferous tubules: a comparative light and electron microscopic investigation.

    PubMed

    Christl, H W

    1990-01-01

    The lamina propria of the seminiferous tubules was compared by means of light and electron microscopy in specimens obtained from the following vertebrates: Mute swan, northern mallard, blackbird, grey short-tailed opossum, north american opossum, european rabbit, mouse, rat, golden hamster, mini pig, bull, llama, roebuck, horse, coati, cat, dog, java monkey, orang utan. The lamina propria consists of basal lamina, ground substance, collagen fibers, fibroblasts and myofibroblasts. Myofibroblasts are characterized by myofilaments, dense patches and a basal lamina covering their plasmalemma. The layers of myofibroblasts always lie adjacent to the germinal epithelium, while the surrounding fibroblast layers are located peripherally.

  5. Effect of exogenous TGF-β1 on the cadmium-induced nephrotoxicity by inhibiting apoptosis of proximal tubular cells through PI3K-AKT-mTOR signaling pathway.

    PubMed

    Huang, Minyi; Su, Li; Yang, Limin; Zhu, Liangliang; Liu, Zhaowen; Duan, Renyan

    2017-03-22

    Heavy metal polluted soils have been a serious problem for the global ecological balance and people's health. Cadmium (Cd), one of the heavy metals, could induce apoptosis of proximal tubular cells in many experimental models and lead to damage the human kidney. Here, we reported a potent chemokine TGF-β1 which could ameliorate cadmium-induced nephrotoxicity. Interestingly, western blotting and TUNEL staining assays indicated that PI3K-AKT-mTOR signaling pathway was involved in the protective mechanism of TGF-β1 in vitro and in vivo. Moreover, TGF-β1 could alleviate Cd-induced nephrotoxicity by inhibiting apoptosis of proximal tubular cells through detecting the level of caspase 3, 8 and 9. Therefore, up-regulation of exogenous TGF-β1 may be a potential strategy to reverse cadumium-induced nephrotoxicity.

  6. Automated tubule nuclei quantification and correlation with oncotype DX risk categories in ER+ breast cancer whole slide images

    NASA Astrophysics Data System (ADS)

    Romo-Bucheli, David; Janowczyk, Andrew; Romero, Eduardo; Gilmore, Hannah; Madabhushi, Anant

    2016-03-01

    Early stage estrogen receptor positive (ER+) breast cancer (BCa) treatment is based on the presumed aggressiveness and likelihood of cancer recurrence. The primary conundrum in treatment and management of early stage ER+ BCa is identifying which of these cancers are candidates for adjuvant chemotherapy and which patients will respond to hormonal therapy alone. This decision could spare some patients the inherent toxicity associated with adjuvant chemotherapy. Oncotype DX (ODX) and other gene expression tests have allowed for distinguishing the more aggressive ER+ BCa requiring adjuvant chemotherapy from the less aggressive cancers benefiting from hormonal therapy alone. However these gene expression tests tend to be expensive, tissue destructive and require physical shipping of tissue blocks for the test to be done. Interestingly breast cancer grade in these tumors has been shown to be highly correlated with the ODX risk score. Unfortunately studies have shown that Bloom-Richardson (BR) grade determined by pathologists can be highly variable. One of the constituent categories in BR grading is the quantification of tubules. The goal of this study was to develop a deep learning neural network classifier to automatically identify tubule nuclei from whole slide images (WSI) of ER+ BCa, the hypothesis being that the ratio of tubule nuclei to overall number of nuclei would correlate with the corresponding ODX risk categories. The performance of the tubule nuclei deep learning strategy was evaluated with a set of 61 high power fields. Under a 5-fold cross-validation, the average precision and recall measures were 0:72 and 0:56 respectively. In addition, the correlation with the ODX risk score was assessed in a set of 7513 high power fields extracted from 174 WSI, each from a different patient (At most 50 high power fields per patient study were used). The ratio between the number of tubule and non-tubule nuclei was computed for each WSI. The results suggests that for BCa

  7. EVALUATION OF THE RENAL TOXICITY OF HEME PROTEINS AND THEIR DERIVATIVES: A ROLE IN THE GENESIS OF ACUTE TUBULE NECROSIS

    PubMed Central

    Braun, Sheldon R.; Weiss, Frederick R.; Keller, Allen I.; Ciccone, J. Richard; Preuss, Harry G.

    1970-01-01

    This investigation studies the toxicity of heme proteins and/or their break-down products on renal function. Heme proteinemia precedes acute tubule necrosis at a frequency great enough to suggest a causal relationship between the two events. Physiological and metabolic functions of kidney slices are investigated in several models of acute tubule necrosis. Organic acid and organic base transport is depressed earliest. These alterations in tubule function cannot be explained by ischemia or obstruction alone. Heme proteinemia in rats or incubation of renal slices in medium containing heme proteins yields several interesting observations. Neither in vivo or in vitro do hemoglobin and methemoglobin alone produce a depressive effect on the transport systems studied. However, parallel to many clinical situations, when such secondary insults as hypoxia and elevated ammonia concentrations are included in the experimental design, transport functions are depressed. Ferrihemate, a molecule smaller than hemoglobin or methemoglobin, depresses transport function without secondary insults. From these studies it is concluded that heme proteins play a role in tubule dysfunction seen in acute tubule necrosis. A model is presented that collates these data with other factors known to play a part in the pathogenesis of this renal syndrome. PMID:5413325

  8. Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity.

    PubMed

    Carrière, M; Avoscan, L; Collins, R; Carrot, F; Khodja, H; Ansoborlo, E; Gouget, B

    2004-03-01

    Uranium is a naturally occurring heavy metal. Its extensive use in the nuclear cycle and for military applications has focused attention on its potential health effects. Acute exposures to uranium are toxic to the kidneys where they mainly cause damage to proximal tubular epithelium. The purpose of this study was to investigate the biological consequences of acute in vitro uranyl exposure and the influence of uranyl speciation on its cytotoxicity. NRK-52E cells, representative of rat kidney proximal epithelium, were exposed to uranyl-carbonate and -citrate complexes, which are the major complexes transiting through renal tubules after acute in vivo contamination. Before NRK-52E cell exposure, these complexes were diluted in classical or modified cell culture media, which can possibly modify uranyl speciation. In these conditions, uranium cytotoxicity appears after 16 h of exposure. The CI50 cytotoxicity index, the uranium concentration leading to 50% dead cells after 24 h of exposure, is 500 microM (+/-100 microM) and strongly depends on uranyl counterion and cell culture medium composition. Computer modeling of uranyl speciation is reported, enabling one to draw a parallel between uranyl speciation and its cytotoxicity.

  9. In Vitro Ability of a Novel Nanohydroxyapatite Oral Rinse to Occlude Dentine Tubules

    PubMed Central

    Hill, Robert G.; Chen, Xiaohui; Gillam, David G.

    2015-01-01

    Objectives. The aim of the study was to investigate the ability of a novel nanohydroxyapatite (nHA) desensitizing oral rinse to occlude dentine tubules compared to selected commercially available desensitizing oral rinses. Methods. 25 caries-free extracted molars were sectioned into 1 mm thick dentine discs. The dentine discs (n = 25) were etched with 6% citric acid for 2 minutes and rinsed with distilled water, prior to a 30-second application of test and control oral rinses. Evaluation was by (1) Scanning Electron Microscopy (SEM) of the dentine surface and (2) fluid flow measurements through a dentine disc. Results. Most of the oral rinses failed to adequately cover the dentine surface apart from the nHa oral rinse. However the hydroxyapatite, 1.4% potassium oxalate, and arginine/PVM/MA copolymer oral rinses, appeared to be relatively more effective than the nHA test and negative control rinses (potassium nitrate) in relation to a reduction in fluid flow measurements. Conclusions. Although the novel nHA oral rinse demonstrated the ability to occlude the dentine tubules and reduce the fluid flow measurements, some of the other oral rinses appeared to demonstrate a statistically significant reduction in fluid flow through the dentine disc, in particular the arginine/PVM/MA copolymer oral rinse. PMID:26161093

  10. Oriented cube-on-cube nanocrystal assembly of SrTiO3 tubules.

    PubMed

    Zagar, Kristina; Recnik, Aleksander; Ajayan, Pulickel M; Ceh, Miran

    2010-09-17

    We report on an unusual crystallization phenomenon that results in the self-assembly of sub-micron tubules of crystalline SrTiO(3). The deposition of the tubular structures was done in the pores of anodized aluminum oxide templates by the electrophoretic deposition of SrTiO(3) sols and subsequent annealing. Homogeneous nucleation inside the pores produces a critical number of crystallites leading to their self-organization when the nanocrystals reach sizes that equal the mean free distances between the nuclei. Due to steric constraints the crystals start to organize in order to most efficiently fill the available surface of the pore walls. This process leads to the formation of domains containing a large number of idiomorphic SrTiO(3) nano-cubes that are self-aligned into almost perfect cube-on-cube and cube-to-wall registry, which makes up the walls of the tubules. The described mechanism shows the ability of nanocrystals with well defined morphologies to adapt spatial constraints and self-organize into desired geometries.

  11. Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers.

    PubMed

    Kim, A M; Vergara, J L

    1998-10-01

    In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule.

  12. Flagellar cells and ciliary cells in the renal tubule of elasmobranchs.

    PubMed

    Lacy, E R; Luciano, L; Reale, E

    1989-01-01

    Flagella or cilia are present on most epithelial cells in the renal tubule of elasmobranch fishes (little skate, spiny dogfish, smooth dogfish, Atlantic sharpnose, scalloped hammerhead, cow-nosed ray). Flagellar cells, those with numerous flagella ordered in one, two, or more rows on the luminal surface, are shown here for the first time in a vertebrate. The flagellar cells are intercalated among other epithelial cells, each bearing a single cilium, from Bowman's capsule to the third subdivision of the intermediate segment of the nephron. The flagella form undulated ribbons up to 55 microns long. In every ribbon the axis of the central pair of microtubules in the axoneme is oriented parallel to the long axis of the flagellar row. This suggests a beat perpendicular to these two axes. The arrangement of the flagella in ribbons most likely promotes movement of glomerular filtrate down the renal tubule. Cells bearing numerous cilia occur in the large collecting ducts of spiny dogfish but without apparent preferential orientation of the cilia.

  13. Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

    SciTech Connect

    Ecay, T.W.; Valentich, J.D. )

    1991-03-01

    Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of {sup 3}H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.

  14. Examination of transport equations pertaining to permeable elastic tubules such as Henle's loop.

    PubMed Central

    Basmadjian, D; Baines, A D

    1978-01-01

    The transport equations applicable to loops of Henle and similar elastic permeable tubules were re-examined to assess the effect of radial transport resistance in the lumen and tubule geometry on solute transport. Active transport at the wall as well as external gradients equivalent to a 2--1,000-fold concentration increase per centimeter of distance were considered. Wall permeabilities and active transport constants were varied up to 2 . 10(-2) cm/s. It is shown that for conditions applicable to the loop of Henle, resistance to radial solute transfer in the lumen is negligible, both for passive and active transmural transport with concomitant water flux, and that axial dispersion further reduces that resistance. These conclusions apply equally to conical and elliptical geometries likely to arise in loop operation. The validity of Poiseuille's equation for these geometries is discussed. Ii is concluded that the one-dimensional transport equations are a valid representation of loop operation. Images FIGURE 1 PMID:737282

  15. Early effects of aldosterone on Na-K pump in rat cortical collecting tubules

    SciTech Connect

    Fujii, Y.; Takemoto, F.; Katz, A.I. )

    1990-07-01

    Sustained exposure to aldosterone (Aldo) increases the abundance and activity of the Na-K pump in cortical collecting tubules (CCT). However, the onset and mechanism of the early interaction of Aldo with the CCT pump, especially in adrenal-intact animals, are unclear. We evaluated the short-term effects of the hormone on Na-K-adenosinetriphosphatase (ATPase) activity and on ouabain-sensitive 86Rb uptake, a measure of the transporting rate of the pump, in microdissected CCT from adrenal-intact rats. Incubation with Aldo (10(-8) M, 2 h) had no effect on Na-K-ATPase activity (Vmax), whereas it produced at least a twofold increase in 86Rb uptake. This effect was generated by physiological concentrations of the hormone (threshold 10(-10) M; apparent K1/2 approximately 10(-9) M), after a short lag of less than or equal to 30 min. Incubation with Aldo in the presence of amiloride or nystatin or in a Na-free medium (choline chloride) did not prevent the enhanced 86Rb uptake seen after Aldo alone; possible interpretations of these observations are discussed. We conclude that Aldo produces a rapid stimulation of pump function in CCT that precedes its induction of new pump synthesis; the physiological significance of this effect is suggested by its occurrence in tubules from adrenal-intact animals within the time frame and concentration range of the hormone's effects on electrolyte transport.

  16. BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.

    PubMed

    Dennis, Megan K; Mantegazza, Adriana R; Snir, Olivia L; Tenza, Danièle; Acosta-Ruiz, Amanda; Delevoye, Cédric; Zorger, Richard; Sitaram, Anand; de Jesus-Rojas, Wilfredo; Ravichandran, Keerthana; Rux, John; Sviderskaya, Elena V; Bennett, Dorothy C; Raposo, Graça; Marks, Michael S; Setty, Subba Rao Gangi

    2015-05-25

    Hermansky-Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2-deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2-deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.

  17. A conserved amphipathic helix is required for membrane tubule formation by Yop1p

    PubMed Central

    Brady, Jacob P.; Claridge, Jolyon K.; Smith, Peter G.; Schnell, Jason R.

    2015-01-01

    The integral membrane proteins of the DP1 (deleted in polyposis) and reticulon families are responsible for maintaining the high membrane curvature required for both smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, and mutations in these proteins lead to motor neuron diseases, such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain reticulon homology domains (RHDs) that have unusually long hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilize membrane curvature. We have characterized the secondary structure and dynamics of the DP1 family protein produced from the YOP1 gene (Yop1p) and identified a C-terminal conserved amphipathic helix (APH) that, on its own, interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming APHs. Together, these results indicate that APHs play a previously unrecognized role in RHD membrane curvature stabilization. PMID:25646439

  18. Annexin VI is attached to transverse-tubule membranes in isolated skeletal muscle triads.

    PubMed

    Barrientos, G; Hidalgo, C

    2002-07-15

    Annexin VI is a 68-kDa protein of the Annexin family, a group of Ca2+-dependent phospholipid-binding proteins widely distributed in mammalian tissues including skeletal muscle. We investigated a) which membrane system contributes Annexin VI to skeletal muscle triads, and b) whether Annexin VI removal affects triad integrity or function. Annexin VI was present in isolated triads and transverse tubules but not in heavy sarcoplasmic reticulum vesicles, indicating that Annexin VI binds to either free or triad-attached transverse tubules. Extraction with EGTA of Annexin VI from triads did not alter their migration as a single band in sucrose density gradients or their ouabain binding-site density, indicating that triad integrity does not require Annexin VI. Caffeine-induced Ca2+ release kinetics and Ca2+ uptake rates were likewise not affected by Annexin VI removal from triads, suggesting that Annexin VI is not involved in these functions. Annexin VI purified from rabbit skeletal muscle displayed Ca2+-dependent binding to liposomes containing phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Binding saturated at 1/20 molar ratio phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine and was optimal at free [Ca2+] > or = 20 mM. Extraction of Annexin VI from triads did not affect the generation of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, or phosphatidic acid by endogenous lipid kinases, suggesting that despite its capacity to bind to negatively charged phospholipids, Annexin VI does not affect the kinase activities responsible for their generation.

  19. Ability of three desensitizing agents in dentinal tubule obliteration and durability: An in vitro study

    PubMed Central

    Pathan, Azher Banu; Bolla, Nagesh; Kavuri, Sarath Raj; Sunil, Chukka Ram; Damaraju, Bhargavi; Pattan, Sadhiq Khan

    2016-01-01

    Aim: The purpose of this study was to evaluate the effectiveness of three desensitizing agents on dentinal tubule obliteration and their durability in use on the dentinal tubules. Materials and Methods: Sixty specimens were obtained from 30 extracted sound human maxillary first premolars. Each tooth was mesiodistally sectioned to obtain 30 buccal and 30 lingual surfaces, and enamel was removed in order to simulate hypersensitive dentin. Specimens were divided into four groups with 15 specimens each. Group 1 samples were immersed in artificial saliva, Group 2 samples were coated with Vivasens, Group 3 samples were coated with VOCO Admira Protect, and Group 4 samples were coated with Neo Active Apatite suspension. These specimens were examined under scanning electron microscope (SEM) to find out the occluding ability of the respective products. The specimens were brushed to find out their durability for 1 week and 1 month and were examined under SEM. Statistical Analysis: The results were statistically analyzed by analysis of variance (ANOVA) and Tukey's test. Results: Group 1 differed significantly from the Vivasens, Admira, and Neo Active Apatite groups at 5% level of significance (P < 0.05). The Vivasens group differed significantly from the Admira and Neo Active Apatite groups at 5% level of significance (P < 0.05). Conclusion: The Ormocer-based Admira Protect showed the best results. PMID:26957790

  20. BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

    PubMed Central

    Dennis, Megan K.; Mantegazza, Adriana R.; Snir, Olivia L.; Tenza, Danièle; Acosta-Ruiz, Amanda; Delevoye, Cédric; Zorger, Richard; Sitaram, Anand; de Jesus-Rojas, Wilfredo; Ravichandran, Keerthana; Rux, John; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Setty, Subba Rao Gangi

    2015-01-01

    Hermansky–Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2–deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2–deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation. PMID:26008744

  1. A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation

    PubMed Central

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-01-01

    To mediate the degradation of bio-macromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosome storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region, where autophagosomes accumulate, upon autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end directed dynactin-dynein motor, while PI(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PI(3,5)P2-TRPML1-ALG-2-dynein signaling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Collectively, Ca2+ release from lysosomes provides an on-demand mechanism regulating lysosome motility, positioning, and tubulation. PMID:26950892

  2. Elongin C is a Mediator of Notch4 Activity in Human Renal Tubule Cells

    PubMed Central

    Cummins, Timothy D.; Mendenhall, Michael D.; Lowry, Michelle N.; Korte, Erik A.; Barati, Michelle T.; Khundmiri, Syed J.; Salyer, Sarah A.; Klein, Jon B.; Powell, David W.

    2011-01-01

    Notch proteins (Notch 1–4) are a family of trans-membrane cell surface receptors that are converted into transcriptional regulators when activated by interactions with cell surface ligands on adjacent cells. Ligand-binding stimulates proteolytic cleavage of the trans-membrane domain, releasing an active intracellular domain (ICD) that translocates to the nucleus and impacts transcription. In transit, the ICD may interact with regulatory proteins that modulate the expression and transcriptional activity. We have found that Notch4ICD expression is enhanced in the tubule cells of fibrotic kidneys from diabetic mice and humans and identified Notch4ICD interacting proteins that could be pertinent to normal and pathological functions. Using proteomic techniques, several components of the Elongin C complex were identified as candidate Notch4ICD interactors. Elongin C complexes can function as ubiquitin ligases capable of regulating proteasomal degradation of specific protein substrates. Our studies indicate that ectopic Elongin C expression stimulates Notch4ICD degradation and inhibits its transcriptional activity in human kidney tubule HK11 cells. Blocking Elongin C mediated degradation by MG132 indicates the potential for ubiquitin-mediated Elongin C regulation of Notch4ICD. Functional interaction of Notch4ICD and Elongin C provides novel insight into regulation of Notch signaling in epithelial cell biology and disease. PMID:22001063

  3. Effects of movement protein mutations on the formation of tubules in plant protoplasts expressing a fusion between the green fluorescent protein and Cauliflower mosaic virus movement protein.

    PubMed

    Huang, Z; Han, Y; Howell, S H

    2001-08-01

    Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MP(ER2A), a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MP(N6) and GFP-MP(N13), two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MP(WT) and GFP-MP(ER2A) form tubules containing both MP fusions, indicating that although the GFP-MP(ER2A) cannot induce tubule formation, GFP-MP(ER2A) can coassemble or colocalize with CFP-MP(WT) in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.

  4. GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

    PubMed Central

    Wang, Yong; Song, Wei; Li, Shuchun; Guan, Xin; Miao, Shiying; Zong, Shudong; Koide, SS; Wang, Linfang

    2009-01-01

    Background Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules. Results Stable mRHBDD1 knockdown GC-1 cells were sensitive to apoptotic stimuli, PS341 and UV irradiation. In vitro, they survived and proliferated normally. However, they lost the ability to survive and differentiate in mouse seminiferous tubules. Conclusion Our findings suggest that mRHBDD1 may be associated with mammalian spermatogenesis. PMID:19358743

  5. Regulatory Forum Opinion Piece*: Dispelling Confusing Pathology Terminology: Recognition and Interpretation of Selected Rodent Renal Tubule Lesions.

    PubMed

    Seely, John Curtis; Frazier, Kendall S

    2015-06-01

    Renal tubule lesions often prove troublesome for toxicologic pathologists because of the diverse nature and interrelated cell types within the kidney and the presence of spontaneous lesions with overlapping morphologies similar to those induced by renal toxicants. Although there are a number of guidance documents available citing straightforward diagnostic criteria of tubule lesions for the pathologist to refer to, most are presented without further advice on the when to or to the why and the why not of diagnosing one lesion over another. Documents presenting diagnostic perspectives and recommendations derived from an author's experience are limited since guidance documents are generally based on descriptive observations. In this Regulatory Forum opinion piece, the authors attempt to dispel confusing renal tubule lesion terminology in laboratory animal species by suggesting histological advice on the recognition and interpretation of these complex entities.

  6. Promoting proximal formative assessment with relational discourse

    NASA Astrophysics Data System (ADS)

    Scherr, Rachel E.; Close, Hunter G.; McKagan, Sarah B.

    2012-02-01

    The practice of proximal formative assessment - the continual, responsive attention to students' developing understanding as it is expressed in real time - depends on students' sharing their ideas with instructors and on teachers' attending to them. Rogerian psychology presents an account of the conditions under which proximal formative assessment may be promoted or inhibited: (1) Normal classroom conditions, characterized by evaluation and attention to learning targets, may present threats to students' sense of their own competence and value, causing them to conceal their ideas and reducing the potential for proximal formative assessment. (2) In contrast, discourse patterns characterized by positive anticipation and attention to learner ideas increase the potential for proximal formative assessment and promote self-directed learning. We present an analysis methodology based on these principles and demonstrate its utility for understanding episodes of university physics instruction.

  7. A Brief Index for Proximity Searching

    NASA Astrophysics Data System (ADS)

    Téllez, Eric Sadit; Chávez, Edgar; Camarena-Ibarrola, Antonio

    Many pattern recognition tasks can be modeled as proximity searching. Here the common task is to quickly find all the elements close to a given query without sequentially scanning a very large database.

  8. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes.

    PubMed

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi; Danielsen, E Michael

    2003-07-01

    The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid 'rafts'. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo G(M1), and they were sensitive to cholesterol depletion by methyl-beta-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border.

  9. Polarization proximity effect in isolator crystal pairs.

    PubMed

    Linzon, Y; Ferrera, M; Razzari, L; Pignolet, A; Morandotti, R

    2008-12-01

    We experimentally study the polarization dynamics (orientation and ellipticity) of near-infrared light transmitted through magneto-optical yttrium iron garnet isolator crystal pairs using a modified balanced detection scheme. When the pair separation is in the submillimeter range, we observed a proximity effect in which the saturation field is reduced by up to 20%. One-dimensional calculations suggest that the proximity effect originates from magnetostatic interactions between the dipole moments of the isolator crystals.

  10. Proximity sensor system development. CRADA final report

    SciTech Connect

    Haley, D.C.; Pigoski, T.M.

    1998-01-01

    Lockheed Martin Energy Research Corporation (LMERC) and Merritt Systems, Inc. (MSI) entered into a Cooperative Research and Development Agreement (CRADA) for the development and demonstration of a compact, modular proximity sensing system suitable for application to a wide class of manipulator systems operated in support of environmental restoration and waste management activities. In teleoperated modes, proximity sensing provides the manipulator operator continuous information regarding the proximity of the manipulator to objects in the workspace. In teleoperated and robotic modes, proximity sensing provides added safety through the implementation of active whole arm collision avoidance capabilities. Oak Ridge National Laboratory (ORNL), managed by LMERC for the United States Department of Energy (DOE), has developed an application specific integrated circuit (ASIC) design for the electronics required to support a modular whole arm proximity sensing system based on the use of capacitive sensors developed at Sandia National Laboratories. The use of ASIC technology greatly reduces the size of the electronics required to support the selected sensor types allowing deployment of many small sensor nodes over a large area of the manipulator surface to provide maximum sensor coverage. The ASIC design also provides a communication interface to support sensor commands from and sensor data transmission to a distributed processing system which allows modular implementation and operation of the sensor system. MSI is a commercial small business specializing in proximity sensing systems based upon infrared and acoustic sensors.

  11. Proximity operations considerations affecting spacecraft design

    NASA Technical Reports Server (NTRS)

    Staas, Steven K.

    1991-01-01

    Experience from several recent spacecraft development programs, such as Space Station Freedom (SSF) and the Orbital Maneuvering Vehicle (OMV) has shown the need for factoring proximity operations considerations into the vehicle design process. Proximity operations, those orbital maneuvers and procedures which involve operation of two or more spacecraft at ranges of less than one nautical mile, are essential to the construction, servicing, and operation of complex spacecraft. Typical proximity operations considerations which drive spacecraft design may be broken into two broad categories; flight profile characteristics and concerns, and use of various spacecraft systems during proximity operations. Proximity operations flight profile concerns include the following: (1) relative approach/separation line; (2) relative orientation of the vehicles; (3) relative translational and rotational rates; (4) vehicle interaction, in the form of thruster plume impingement, mating or demating operations, or uncontrolled contact/collision; and (5) active vehicle piloting. Spacecraft systems used during proximity operations include the following: (1) sensors, such as radar, laser ranging devices, or optical ranging systems; (2) effector hardware, such as thrusters; (3) flight control software; and (4) mating hardware, needed for docking or berthing operations. A discussion of how these factors affect vehicle design follows, addressing both active and passive/cooperative vehicles.

  12. The T-tubule is a cell-surface target for insulin-regulated recycling of membrane proteins in skeletal muscle.

    PubMed Central

    Muñoz, P; Rosemblatt, M; Testar, X; Palacín, M; Thoidis, G; Pilch, P F; Zorzano, A

    1995-01-01

    (1) In this study we have determined the distribution of various membrane proteins involved in insulin-activated glucose transport in T-tubules and in sarcolemma from rat skeletal muscle. Two independent experimental approaches were used to determine the presence of membrane proteins in T-tubules: (i) the purification of T-tubules free from sarcolemmal membranes by lectin agglutination, and (ii) T-tubule vesicle immunoadsorption. These methods confirmed that T-tubules from rat skeletal muscle were enriched with dihydropyridine receptors and tt28 protein and did not contain the sarcolemmal markers dystrophin or beta 1-integrin. Both types of experiments revealed an abundant content of GLUT4 glucose carriers, insulin receptors and SCAMPs (secretory carrier membrane proteins) in T-tubule membranes. (2) Acute administration in vivo of insulin caused an increased abundance of GLUT4 in T-tubules and sarcolemma. On the contrary, insulin led to a 50% reduction in insulin receptors present in T-tubules and in sarcolemma, demonstrating that insulin-induced insulin receptor internalization affects T-tubules in the muscle fibre. The alteration in the content of GLUT4 and insulin receptors in T-tubules was a consequence of insulin-induced redistribution of these proteins. SCAMPs also redistributed in muscle membranes in response to insulin. They were recruited by insulin from intracellular high-density fractions to intracellular lighter-density fractions and to the cell surface, showing a pattern of insulin-induced cellular redistribution distinct from those of GLUT4 and the insulin receptor. (3) In conclusion, the T-tubule is a cell-surface target for membrane proteins involved in recycling such as SCAMPs or for membrane proteins that acutely redistribute in response to insulin such as GLUT4 or insulin receptors. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8526847

  13. Microtubules Contribute to Tubule Elongation and Anchoring of Endoplasmic Reticulum, Resulting in High Network Complexity in Arabidopsis1[W][OPEN

    PubMed Central

    Hamada, Takahiro; Ueda, Haruko; Kawase, Takashi; Hara-Nishimura, Ikuko

    2014-01-01

    The endoplasmic reticulum (ER) is a network of tubules and sheet-like structures in eukaryotic cells. Some ER tubules dynamically change their morphology, and others form stable structures. In plants, it has been thought that the ER tubule extension is driven by the actin-myosin machinery. Here, we show that microtubules also contribute to the ER tubule extension with an almost 20-fold slower rate than the actin filament-based ER extension. Treatment with the actin-depolymerizing drug Latrunculin B made it possible to visualize the slow extension of the ER tubules in transgenic Arabidopsis (Arabidopsis thaliana) plants expressing ER-targeted green fluorescent protein. The ER tubules elongated along microtubules in both directions of microtubules, which have a distinct polarity. This feature is similar to the kinesin- or dynein-driven ER tubule extension in animal cells. In contrast to the animal case, ER tubules elongating with the growing microtubule ends were not observed in Arabidopsis. We also found the spots where microtubules are stably colocalized with the ER subdomains during long observations of 1,040 s, suggesting that cortical microtubules contribute to provide ER anchoring points. The anchoring points acted as the branching points of the ER tubules, resulting in the formation of multiway junctions. The density of the ER tubule junction positively correlated with the microtubule density in both elongating cells and mature cells of leaf epidermis, showing the requirement of microtubules for formation of the complex ER network. Taken together, our findings show that plants use microtubules for ER anchoring and ER tubule extension, which establish fine network structures of the ER within the cell. PMID:25367857

  14. The swan-neck lesion: proximal tubular adaptation to oxidative stress in nephropathic cystinosis.

    PubMed

    Galarreta, Carolina I; Forbes, Michael S; Thornhill, Barbara A; Antignac, Corinne; Gubler, Marie-Claire; Nevo, Nathalie; Murphy, Michael P; Chevalier, Robert L

    2015-05-15

    Cystinosis is an inherited disorder resulting from a mutation in the CTNS gene, causing progressive proximal tubular cell flattening, the so-called swan-neck lesion (SNL), and eventual renal failure. To determine the role of oxidative stress in cystinosis, histologic sections of kidneys from C57BL/6 Ctns(-/-) and wild-type mice were examined by immunohistochemistry and morphometry from 1 wk to 20 mo of age. Additional mice were treated from 1 to 6 mo with vehicle or mitoquinone (MitoQ), an antioxidant targeted to mitochondria. The leading edge of the SNL lost mitochondria and superoxide production, and became surrounded by a thickened tubular basement membrane. Progression of the SNL as determined by staining with lectin from Lotus tetragonolobus accelerated after 3 mo, but was delayed by treatment with MitoQ (38 ± 4% vs. 28 ± 1%, P < 0.01). Through 9 mo, glomeruli had retained renin staining and intact macula densa, whereas SNL expressed transgelin, an actin-binding protein, but neither kidney injury molecule-1 (KIM-1) nor cell death was observed. After 9 mo, clusters of proximal tubules exhibited localized oxidative stress (4-hydroxynonenal binding), expressed KIM-1, and underwent apoptosis, leading to the formation of atubular glomeruli and accumulation of interstitial collagen. We conclude that nephron integrity is initially maintained in the Ctns(-/-) mouse by adaptive flattening of cells of the SNL through loss of mitochondria, upregulation of transgelin, and thickened basement membrane. This adaptation ultimately fails in adulthood, with proximal tubular disruption, formation of atubular glomeruli, and renal failure. Antioxidant treatment targeted to mitochondria delays initiation of the SNL, and may provide therapeutic benefit in children with cystinosis.

  15. The effects of simulated microgravity on the seminiferous tubules of rats

    NASA Astrophysics Data System (ADS)

    Forsman, Allan D.

    2012-02-01

    Space flight has been shown to have many adverse effects on various systems throughout the body. Because the opportunity to place research animals on board a Space Shuttle or the International Space Station is infrequent, various techniques have been designed to simulate the effects of microgravity in Earth based laboratories. A commonly used technique is known as antiorthostatic suspension, also often referred to as hind limb suspension. In this technique the hind portion of the animal is raised so that its hind limbs are non-weight bearing. This places the animal in roughly a 30° head down tilt position. This results in cephalic fluid shifts similar to those seen in actual space flight. This technique has also been shown to mimic other physiological parameters that are affected during space flight. This study examined testicular tissue from rats subjected to a 7 day antiorthostatic suspension. This tissue was acquired through a tissue sharing program and some of the experimental animals were injected with Interleukin 1 receptor antagonist (IL-1ra) which was hoped to ameliorate some of the effects of antiorthostatic suspension. The injection of IL-1ra was not expected to have any effect on testicular tissue, however this tissue was included in the morphological and statistical analysis to conduct a more complete study. All tissues were embedded in paraffin, sectioned, and stained using standard H&E staining. The tissue was then qualitatively ranked according to the "health" of the seminiferous tubules. Our findings indicate that 7 days of antiorthostatic suspension had adverse effects on the tissue that comprises the walls of the seminiferous tubules. It has long been known that antiorthostatic suspension has deleterious effects on testicular tissue, however this research indicates that these effects occur much faster than indicated by previous researchers. This is a significant finding because it indicates that meaningful earth based studies in this area can be

  16. The effect of dentine location and tubule orientation on the bond strengths between resin and dentine.

    PubMed

    Phrukkanon, S; Burrow, M F; Tyas, M J

    1999-05-01

    This study determined the influence of dentine structure on the micro-tensile bond strengths between resin and dentine of two different dentine adhesive systems (Single Bond, 3M Dental Products, St Paul, MN; MF-102 (experimental self-etching primer), GC Corporation, Tokyo, Japan). The study was separated into two main parts: bond strength measurement and investigation of the bonding interface. Twenty-two human premolars were used for the bond strength measurement. Each tooth was cut vertically, separating the tooth into mesio-distal halves. One half of the tooth was used to bond to a surface perpendicular to the dentinal tubules and other half to bond to a surface parallel to the tubules. For each half, six locations of dentine were bonded. Each material was used in accordance to the manufacturer's directions. Cylindrical hourglass-shaped specimens of 1.2 mm diameter at the bonded interface were manufactured. The bonds were stressed in tension at a crosshead speed of 1 mm/min. Mean bond strengths were compared using LSD, one-way ANOVA, and Student's t-test. The fractured surfaces were examined under a scanning electron microscope, and the frequency of fracture modes was compared using the Kruskal-Wallis and Mann-Whitney U tests. For the investigation of the bonded interface, four teeth were prepared by the same procedure used for the bond test specimens. The bonded interfaces were observed after an acid-base treatment or fracturing across the bonded interface, prior to investigation with a field-emission scanning electron microscope. For Single Bond, the bond strengths for mid-root dentine were significantly lower than for other locations (p < 0.05). For MF-102, there was no significant difference for all locations (p > 0.05). MF-102 bonded well to all locations of dentine while Single Bond showed a porous zone at the base of the hybrid layer. The bonds were not influenced by tubule orientation. The results indicate that the bond for Single Bond may be affected by

  17. Proteomic-Based Insight into Malpighian Tubules of Silkworm Bombyx mori

    PubMed Central

    Liu, Shi-ping; Yi, Qi-ying; Hu, Cui-mei; Wang, Chen; Xia, Qing-you; Zhao, Ping

    2013-01-01

    Malpighian tubules (MTs) are highly specific organs of arthropods (Insecta, Myriapoda and Arachnida) for excretion and osmoregulation. In order to highlight the important genes and pathways involved in multi-functions of MTs, we performed a systematic proteomic analysis of silkworm MTs in the present work. Totally, 1,367 proteins were identified by one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry, and as well as by Trans Proteomic Pipeline (TPP) and Absolute protein expression (APEX) analyses. Forty-one proteins were further identified by two-dimensional gel electrophoresis. Some proteins were revealed to be significantly associated with various metabolic processes, organic solute transport, detoxification and innate immunity. Our results might lay a good foundation for future functional studies of MTs in silkworm and other lepidoptera. PMID:24098719

  18. Culturing immobilized plant cells for the TUBUL space experiments on the DELTA and 12S Missions

    NASA Astrophysics Data System (ADS)

    Sieberer, Björn J.; Emons, Anne Mie C.; Vos, Jan W.

    2007-09-01

    For the TUBUL experiments during the DELTA mission in April 2004 and 12S mission in March/April 2006 on board the Soyuz capsule and the International Space Station we developed a method to culture and chemically fix plant suspension culture cells. The aim of the ten day experiment was to investigate the effect of microgravity on single plant cells. Fully automated experiment cassettes (Plunger Box Units) were developed by Centre for Concepts in Mechatronics (Nuenen, the Netherlands). Tobacco BY- 2 cells were immobilized in a semi- solid agarose matrix that was reinforced by a nylon mesh. This assembly allowed liquid medium refreshment, oxygen supply and chemical fixation, including a post- fixative wash. The method was optimized for post- flight analysis of cell structure, shape and size, cell division, and the microtubule cytoskeleton. The viability of cells in the agarose matrix was similar to cells grown in liquid medium under laboratory conditions, only the stationary growth phase was reached six days later.

  19. Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties ▿

    PubMed Central

    Sánchez-Navarro, Jesús; Fajardo, Thor; Zicca, Stefania; Pallás, Vicente; Stavolone, Livia

    2010-01-01

    Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway. PMID:20130061

  20. Self-organization of engineered epithelial tubules by differential cellular motility

    SciTech Connect

    Mori, Hidetoshi; Gjorevski, Nikolce; Inman, Jamie L; Bissell, Mina J; Nelson, Celeste M

    2009-02-04

    Patterning of developing tissues arises from a number of mechanisms, including cell shape change, cell proliferation, and cell sorting from differential cohesion or tension. Here, we reveal that differences in cell motility can also lead to cell sorting within tissues. Using mosaic engineered mammary epithelial tubules, we found that cells sorted depending on their expression level of the membrane-anchored collagenase matrix metalloproteinase (MMP)-14. These rearrangements were independent of the catalytic activity of MMP14 but absolutely required the hemopexin domain. We describe a signaling cascade downstream of MMP14 through Rho kinase that allows cells to sort within the model tissues. Cell speed and persistence time were enhanced by MMP14 expression, but only the latter motility parameter was required for sorting. These results indicate that differential directional persistence can give rise to patterns within model developing tissues.

  1. Catch-bond behaviour facilitates membrane tubulation by non-processive myosin 1b

    NASA Astrophysics Data System (ADS)

    Yamada, Ayako; Mamane, Alexandre; Lee-Tin-Wah, Jonathan; di Cicco, Aurélie; Prévost, Coline; Lévy, Daniel; Joanny, Jean-François; Coudrier, Evelyne; Bassereau, Patricia

    2014-04-01

    Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour in response to load. In vivo, myosin 1b is required to form membrane tubules at both endosomes and the trans-Golgi network. To establish the link between these two fundamental properties, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes.

  2. Natural tubule clay template synthesis of silver nanorods for antibacterial composite coating.

    PubMed

    Abdullayev, Elshad; Sakakibara, Keita; Okamoto, Ken; Wei, Wenbo; Ariga, Katsuhiko; Lvov, Yuri

    2011-10-01

    Halloysite is naturally available clay mineral with hollow cylindrical geometry and it is available in thousands of tons. Silver nanorods were synthesized inside the lumen of the halloysite by thermal decomposition of the silver acetate, which was loaded into halloysite from an aqueous solution by vacuum cycling. Images of individual ca. 15 nm diameter silver nanorods and nanoparticles were observed with TEM. The presence of silver inside the tubes was also verified with STEM-EDX elemental mapping. Nanorods had crystalline nature with [111] axis oriented ~68° from the halloysite tubule main axis. The composite of silver nanorods encased in clay tubes with the polymer paint was prepared, and the coating antimicrobial activity combined with tensile strength increase was demonstrated. Coating containing up 5% silver loaded halloysite did not change color after light exposure contrary to the sample prepared with loading with unshelled silver nanoparticles. Halloysite tube templates have a potential for scalable manufacturing of ceramic encapsulated metal nanorods for composite materials.

  3. Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

    SciTech Connect

    Backues, Steven K.; Bednarek, Sebastian Y.

    2010-03-19

    The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

  4. Comparative effects of the water accommodated fraction of three oils on mussels. 2. Quantitative alterations in the structure of the digestive tubules.

    PubMed

    Cajaraville, M P; Marigómez, J A; Díez, G; Angulo, E

    1992-05-01

    1. Mussels have been exposed to the water accommodated fraction (WAF) of Ural and Maya crude oils and of a lubricant oil for 91 days and the digestive gland structure has been studied (a) by planimetry, to calculate the mean epithelial thickness (MET), the mean diverticular radius (MDR) and the mean luminal radius (MLR) of the digestive tubules and (b) by subjective tubule grading, to determine the relative (%) occurrence of the different tubule types found in the digestive gland (holding, absorpting, disintegrating, reconstituting and necrotic tubules). 2. Exposure to the 3 types of WAF leads to lowered MET and MET/MDR values, and concomitant higher MLR/MET values. In the case of lubricant- and Ural-WAF exposures, MDR decreases significantly at increasing exposure-doses. 3. Exposure to oil WAFs leads to decreased percentages of holding and absorpting tubules. This decrease is concomitant with an increase of disintegrating tubules in lubricant- and Ural-WAF exposures, and with an increase of regenerating tubules in the Maya-WAF exposure. 4. The statistical significance of the differences found between control and exposed mussels is better at Day 21 than at any other further sampling day (35, 77 and 91). 5. Multiple correlation coefficients between all the percentages of tubule types and each planimetric parameter present an excellent statistical significance.

  5. Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers.

    PubMed Central

    Kim, A M; Vergara, J L

    1998-01-01

    In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule. PMID:9746552

  6. Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

    PubMed Central

    1988-01-01

    Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+- adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane cond