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Sample records for cell scintillation proximity

  1. Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.

    PubMed

    Lerner, C G; Saiki, A Y

    1996-09-05

    DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

  2. Scintillation Proximity Radioimmunoassay Utilizing 125I-Labeled Ligands

    NASA Astrophysics Data System (ADS)

    Udenfriend, Sidney; Diekmann Gerber, Louise; Brink, Larry; Spector, Sydney

    1985-12-01

    A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an 125I-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the 125I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications of the principle to study the kinetics of interaction between receptors and ligands are discussed.

  3. Scintillation proximity radioimmunoassay utilizing 125I-labeled ligands

    SciTech Connect

    Udenfriend, S.; Gerber, L.D.; Brink, L.; Spector, S.

    1985-12-01

    A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an /sup 125/I-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the /sup 125/I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications of the principle to study the kinetics of interaction between receptors and ligands are discussed.

  4. A High Throughput Scintillation Proximity Imaging Assay for Protein Methyltransferases

    PubMed Central

    Ibáñez, Glorymar; Shum, David; Blum, Gil; Bhinder, Bhavneet; Radu, Constantin; Antczak, Christophe; Luo, Minkui; Djaballah, Hakim

    2013-01-01

    Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z′ values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use. PMID:22256970

  5. Identification of DNMT1 Selective Antagonists Using a Novel Scintillation Proximity Assay*

    PubMed Central

    Kilgore, Jessica A.; Du, Xinlin; Melito, Lisa; Wei, Shuguang; Wang, Changguang; Chin, Hang Gyeong; Posner, Bruce; Pradhan, Sriharsa; Ready, Joseph M.; Williams, Noelle S.

    2013-01-01

    A novel scintillation proximity high throughput assay (SPA) to identify inhibitors of DNA methyltransferases was developed and used to screen over 180,000 compounds. The majority of the validated hits shared a quinone core and several were found to generate the reactive oxygen species, H2O2. Inhibition of the production of H2O2 by the addition of catalase blocked the ability of this group of compounds to inhibit DNA methyltransferase (DNMT) activity. However, a related compound, SW155246, was identified that existed in an already reduced form of the quinone. This compound did not generate H2O2, and catalase did not block its ability to inhibit DNA methyltransferase. SW155246 showed a 30-fold preference for inhibition of human DNMT1 versus human or murine DNMT3A or -3B, inhibited global methylation in HeLa cells, and reactivated expression of the tumor suppressor gene RASSF1A in A549 cells. To our knowledge, this work represents the first description of selective chemical inhibitors of the DNMT1 enzyme. PMID:23671287

  6. A High-Throughput Scintillation Proximity-Based Assay for Human DNA Ligase IV

    PubMed Central

    Tseng, Hui-Min; Shum, David; Bhinder, Bhavneet; Escobar, Sindy; Veomett, Nicholas J.; Tomkinson, Alan E.; Gin, David Y.

    2012-01-01

    Abstract Ionizing radiation (IR) and certain chemotherapeutic drugs are designed to generate cytotoxic DNA double-strand breaks (DSBs) in cancer cells. Inhibition of the major DSB repair pathway, nonhomologous end joining (NHEJ), will enhance the cytotoxicity of these agents. Screening for inhibitors of the DNA ligase IV (Lig4), which mediates the final ligation step in NHEJ, offers a novel target-based drug discovery opportunity. For this purpose, we have developed an enzymatic assay to identify chemicals that block the transfer of [α-33P]-AMP from the complex Lig4-[α-33P]-AMP onto the 5′ end of a double-stranded DNA substrate and adapted it to a scintillation proximity assay (SPA). A screen was performed against a collection of 5,280 compounds. Assay statistics show an average Z′ value of 0.73, indicative of a robust assay in this SPA format. Using a threshold of >20% inhibition, 10 compounds were initially scored as positive hits. A follow-up screen confirmed four compounds with IC50 values ranging from 1 to 30 μM. Rabeprazole and U73122 were found to specifically block the adenylate transfer step and DNA rejoining; in whole live cell assays, these compounds were found to inhibit the repair of DSBs generated by IR. The ability to screen and identify Lig4 inhibitors suggests that they may have utility as chemo- and radio-sensitizers in combination therapy and provides a rationale for using this screening strategy to identify additional inhibitors. PMID:22192310

  7. An improved scintillation cell for radon measurements

    NASA Astrophysics Data System (ADS)

    Quindos-Poncela, L. S.; Fernandez, P. L.; Sainz, C.; Arteche, J.; Arozamena, J. G.; George, A. C.

    2003-10-01

    Scintillation cells like "Modified Lucas cells" are widely used for grab-sample measurements of radon in the environment including those developed in the laboratory for evaluation of radon diffusion coefficients of radon barriers, radon emanation powers in small samples of porous materials and radon concentrations in small chambers used for different research studies. In this paper we describe the main characteristics of an improved and practical scintillation cell developed in our laboratory. Experimental efficiency values obtained for different cell volumes ranged between 0.038 and 0.123 cpm/Bq/m 3. The effects of atmospheric pressure on the detection efficiency as a function of the diameter of the cell were also studied. In general, a decrease of 15% was observed when increasing the pressure from 827 to 1013 hPa.

  8. Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

    PubMed

    Meleza, Cesar; Thomasson, Bobbie; Ramachandran, Chidambaram; O'Neill, Jason W; Michelsen, Klaus; Lo, Mei-Chu

    2016-10-15

    Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.

  9. High Throughput Scintillation Proximity Assay for the Identification of FKBP-12 Ligands.

    PubMed

    Graziani; Aldegheri; Terstappen

    1999-01-01

    A high throughput scintillation proximity assay (SPA) was developed to identify novel ligands of FKBP-12, an immunophilin with peptidyl prolyl isomerase (rotamase) activity. Recombinant histidine-tagged FKBP-12 was expressed in Escherichia coli, purified by metal ion affinity chromatography, and immobilized to SPA beads by an antibody that recognizes the histidine tag of the recombinant protein. Using 1 nM [3H] FK506, a well-known macrolid ligand of FKBP-12, specific binding was saturable and accounted for 95% of total binding. Analysis of saturation and homologous displacement isotherms indicated the existence of a single binding site with a Kd value of 1.6 nM. The specificity of [3H] FK506 binding was demonstrated in displacement experiments and showed that rapamycin, another macrolid, was as active as FK506 (IC50 of 3.5 and 3.2 nM, respectively), whereas GPI-1046, a prototype of small molecular compounds with neurotrophic properties and affinity for FKBP-type immunophilins, was more than 1000-fold less active. The high signal-to-noise ratio of 30, together with small standard deviations, makes this novel assay well suited for automated high throughput screening.

  10. Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin.

    PubMed

    Tahir, S K; Kovar, P; Rosenberg, S H; Ng, S C

    2000-07-01

    We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.

  11. Development of a microplate-based scintillation proximity assay for MraY using a modified substrate.

    PubMed

    Solapure, S M; Raphael, P; Gayathri, C N; Barde, S P; Chandrakala, B; Das, K S; De Sousa, S M

    2005-03-01

    MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.

  12. Scintillation proximity assay for inhibitors of Escherichia coli MurG and, optionally, MraY.

    PubMed

    Ravishankar, Sudha; Kumar, Vidya Prasanna; Chandrakala, B; Jha, Ramesh K; Solapure, Suresh M; de Sousa, Sunita M

    2005-04-01

    MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis--penicillin G, moenomycin, and bacitracin--had no effect. Surprisingly, however, the beta-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.

  13. Scintillation Proximity Assay for Inhibitors of Escherichia coli MurG and, Optionally, MraY

    PubMed Central

    Ravishankar, Sudha; Kumar, Vidya Prasanna; Chandrakala, B.; Jha, Ramesh K.; Solapure, Suresh M.; de Sousa, Sunita M.

    2005-01-01

    MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis—penicillin G, moenomycin, and bacitracin—had no effect. Surprisingly, however, the β-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents. PMID:15793120

  14. Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase.

    PubMed

    Takagi, Toshimitsu; Shum, David; Parisi, Monika; Santos, Ruth E; Radu, Constantin; Calder, Paul; Rizvi, Zahra; Frattini, Mark G; Djaballah, Hakim

    2011-09-01

    Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.

  15. A scintillation proximity assay amenable for screening and characterization of DNA gyrase B subunit inhibitors.

    PubMed

    Gevi, Monica; Domenici, Enrico

    2002-01-01

    DNA gyrase is the target of coumarin and cyclothialidine antibacterials, which bind to the B subunit of the enzyme (GyrB). Currently available GyrB inhibitors have not been clinically successful, but their high in vitro potency against DNA gyrase has raised interest in the development of novel noncoumarin antibacterials acting at the same site. We report the development of a simple scintillation proximity assay (SPA) for the study of binding interactions between coumarin or noncoumarin antibacterials and GyrB, which prevents the needs of separation steps and can be run in microtiter plate formats. The assay is based on the detection of the binding of a radioligand, [3H]dihydronovobiocin, to a biotin-labeled 43-kDa fragment of GyrB (biotin-GyrB43), which is captured by streptavidin-coated SPA beads. The typical assay was conducted in 96-well microtiter plates, with final concentration of 10 nM for biotin-GyrB43, 20 nM for [3H]dihydronovobiocin, and 33 microg of SPA beads/well. From saturation experiments, an equilibrium dissociation constant (K(d)) for dihydronovobiocin of 8.10 nM was found. Displacement studies gave 50% inhibitory concentrations (IC(50)) of 42, 64, and 11 nM for novobiocin, dihydronovobiocin, and the cyclothialidine analogue GR122222X, respectively, consistent with previous findings. The assay was found to be robust to dimethyl sulfoxide up to 5% (v/v) and can be used for high-throughput screens of large chemical collections in the search of novel DNA gyrase inhibitors.

  16. Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth

    PubMed Central

    Caballero, Iván; Colmenarejo, Gonzalo; Sanz, Laura M.; Álvarez-Ruiz, Emilio; Gamo, Francisco-Javier; Cid, Concepción

    2016-01-01

    The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [3H]hypoxanthine incorporation assay is considered the “gold standard” assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum. However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [3H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [3H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method. PMID:27458216

  17. Effective Quenchers Are Required to Eliminate the Interference of Substrate: Cofactor Binding in the HAT Scintillation Proximity Assay

    PubMed Central

    Ngo, Liza; Wu, Jiang; Yang, Chao

    2015-01-01

    Abstract Histone acetyltransferases (HATs) mediate the transfer of an acetyl group from the cofactor, acetyl-CoA, to the side chain amino group of specific lysines in diverse protein substrates, most notably nuclear histones. The deregulation of HATs is connected to a number of disease states. Reliable and rapid biochemical assays for HATs are critical for understanding biological functions of protein acetylation, as well as for screening small-molecule inhibitors of HAT enzymes. In this report, we present a scintillation proximity assay (SPA) for the measurement of HAT enzymatic activities. The acetyl donor was [3H]Ac-CoA, and a biotin-modified histone peptide served as the HAT substrate. After the HAT reaction, streptavidin-coated beads were added to induce proximity of acetylated substrate to the scintillant molecules. However, we observed strong nonspecific binding between the cofactor and the histone peptide substrates, which adversely complicated the SPA performance. To prevent this problem, a set of chemical agents were evaluated to eliminate the cofactor–substrate interaction, thus providing reliable SPA readings. With optimization, the SPA showed consistent and robust performance for HAT activity measurement and HAT inhibitor evaluation. Overall, this mix-and-measure assay does not require any washing procedure, can be utilized in the microplate format, and is well suited for high-throughput screening of HAT chemical modulators. PMID:26065557

  18. Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

    PubMed Central

    Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

    2016-01-01

    Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

  19. Effective Quenchers Are Required to Eliminate the Interference of Substrate: Cofactor Binding in the HAT Scintillation Proximity Assay.

    PubMed

    Ngo, Liza; Wu, Jiang; Yang, Chao; Zheng, Yujun George

    2015-05-01

    Histone acetyltransferases (HATs) mediate the transfer of an acetyl group from the cofactor, acetyl-CoA, to the side chain amino group of specific lysines in diverse protein substrates, most notably nuclear histones. The deregulation of HATs is connected to a number of disease states. Reliable and rapid biochemical assays for HATs are critical for understanding biological functions of protein acetylation, as well as for screening small-molecule inhibitors of HAT enzymes. In this report, we present a scintillation proximity assay (SPA) for the measurement of HAT enzymatic activities. The acetyl donor was [(3)H]Ac-CoA, and a biotin-modified histone peptide served as the HAT substrate. After the HAT reaction, streptavidin-coated beads were added to induce proximity of acetylated substrate to the scintillant molecules. However, we observed strong nonspecific binding between the cofactor and the histone peptide substrates, which adversely complicated the SPA performance. To prevent this problem, a set of chemical agents were evaluated to eliminate the cofactor-substrate interaction, thus providing reliable SPA readings. With optimization, the SPA showed consistent and robust performance for HAT activity measurement and HAT inhibitor evaluation. Overall, this mix-and-measure assay does not require any washing procedure, can be utilized in the microplate format, and is well suited for high-throughput screening of HAT chemical modulators.

  20. Radon measurement of natural gas using alpha scintillation cells.

    PubMed

    Kitto, Michael E; Torres, Miguel A; Haines, Douglas K; Semkow, Thomas M

    2014-12-01

    Due to their sensitivity and ease of use, alpha-scintillation cells are being increasingly utilized for measurements of radon ((222)Rn) in natural gas. Laboratory studies showed an average increase of 7.3% in the measurement efficiency of alpha-scintillation cells when filled with less-dense natural gas rather than regular air. A theoretical calculation comparing the atomic weight and density of air to that of natural gas suggests a 6-7% increase in the detection efficiency when measuring radon in the cells. A correction is also applicable when the sampling location and measurement laboratory are at different elevations. These corrections to the measurement efficiency need to be considered in order to derive accurate concentrations of radon in natural gas. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay

    SciTech Connect

    Nelson, N.

    1987-09-01

    A rapid and convenient binding assay for receptors and membrane proteins has been developed. It is based on the binding of /sup 125/I-labeled ligands to membrane proteins adsorbed to polyvinyltoluene plastic scintillation microspheres. Membranes or isolated membrane proteins adsorb to the beads upon mixing, and addition of /sup 125/I-labeled ligand induces photon emission which is proportional to the amount of added receptor or membrane protein. The interaction of acetylcholine receptor with /sup 125/I-labeled alpha-bungarotoxin and antigens with /sup 125/I-labeled antibodies or protein A were used as models to test the system. As little as 1 ng of acetylcholine receptor is detected by the assay and a linear relationship with receptor concentration is observed up to 50 ng of receptor per 250 microliter reaction medium. The effects of detergents, salts, soluble proteins, and neutral membranes were studied. Inclusion of bovine serum albumin up to 1 mg/ml, sodium chloride up to 0.5 M, and membranes up to 10 micrograms/ml cause little or no effect on the assay. Detergents at 10-fold below their critical micelle concentrations had little or no effect on the assay. The pharmacological effects of agonists such as acetylcholine were conveniently studied by following the displacement of the /sup 125/I-labeled ligand. Similarly, the amount of toxin in crude snake venom can be assayed by measuring competition with the labeled toxin. Only a few seconds are required to perform each binding assay.

  2. Modular design of long narrow scintillating cells for ILC detector

    SciTech Connect

    Beznosko, D.; Blazey, G.; Dyshkant, A.; Maloney, J.; Rykalin, V.; Schellpfeffer, J.; /Fermilab

    2005-09-01

    The experimental results for the narrow scintillating elements with effective area about 20 cm{sup 2} are reported. The elements were formed from the single piece of scintillator and were read out via wavelength shifting fibers with the MRS (Metal/Resistor/Semiconductor) photodiodes on both ends of each fiber. The formation of the cells from the piece of scintillator by using grooves is discussed. The cell performance was tested using the radioactive source by measuring the PMT current and a single rate after amplifier and discrimination with threshold at about three photo electrons in each channel and quad coincidences (double coincidences between sensors on each fiber and double coincidences between two neighboring fibers). This result is of high importance for large multi-channel systems, i.e. module may be used as an active element for calorimeter or muon system for the design of the future electron-positron linear collider detector because cell effective area can be smoothly enlarged or reduced (to 4 cm{sup 2} definitely).

  3. Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically inactive (C215S) mutant.

    PubMed

    Skorey, K I; Kennedy, B P; Friesen, R W; Ramachandran, C

    2001-04-15

    Protein tyrosine phosphatases are a class of enzymes that function to modulate tyrosine phosphorylation of cellular proteins and play an essential role in regulating cell function. PTP1B has been implicated in the negative regulation of the insulin signaling pathway by dephosphorylating the activated insulin receptor. Inhibiting this phosphatase and preventing the insulin-receptor downregulation has been suggested as a target for the treatment of Type II diabetes. A high-throughput screen for inhibitors of PTP1B was developed using a scintillation proximity assay (SPA) with GST-- or FLAG--PTP1B((1-320)) and a potent [(3)H]-tripeptide inhibitor. The problem of interference from extraneous oxidizing and alkylating agents which react with the cysteine active-site nucleophile was overcome by the use of the catalytically inactive C215S form of the native enzyme (GST--PTP1B(C215S)). The GST--PTP1B was linked to the protein A scintillation bead via GST antibody. The radiolabeled inhibitor when bound to the enzyme gave a radioactive signal that was competed away by the unknown competitive compounds. Further utility of PTP1B(C215S) was demonstrated by mixing in the same well both the catalytically inactive GST--PTP1B(C215S) and the catalytically active FLAG--CD45 with an inhibitor. Both a binding and kinetic assay was then performed in the same 96-well plate with the inhibition results determined for the PTP1B(C215S) (binding assay) and CD45 (activity assay). In this way inhibitors could be differentiated between the two phosphatases under identical assay conditions in one 96-well assay plate. The use of a mutant to reduce interference in a binding assay and compare with activity assays is also amenable for most cysteine active-site proteases.

  4. Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.

    PubMed

    Mang, Samuel; Bucher, Hannes; Nickolaus, Peter

    2016-01-01

    The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.

  5. A deterministic approach to modeling a scintillator cell for NICADD

    NASA Astrophysics Data System (ADS)

    Barendregt, Alan Carl

    CERN uses many detectors in the particle accelerator to find the building blocks of the universe. One type of detector used is a scintillator cell. These detectors are being optimized by NICADD to have a uniform detection range at the lowest cost. To assist in this endeavor, computer modeling gives the designer the ability to test many designs in a virtual environment prior to making a physical prototype design. Current virtual models for this field have been stochastic, which means the designer will have to repeatedly run the same simulation many times to get rid of the statistical "noise" in the results. In the field of nuclear science engineering, deterministic softwares have proven themselves to be a valid prediction tool for such problems as neutron embrittlement. These models account for the probabilities up front and will provide a single result that can help confirm improvements in design in a single step. This has advantages especially when it comes to comparing two different models. This thesis discusses the method of using TransMED, a deterministic software, to assist NICADD in optimizing scintillator cell design. This thesis provides a road-map on how NICADD can use TransMED in there design work for this project and other anisotripic scattering problems as well.

  6. Calibration of scintillation cells for radon-222 measurements

    NASA Astrophysics Data System (ADS)

    Aakko, Kyllikki; Oksanen, Eero

    The calibration, of a radon-222 measurement system is described. The detector of the system is based on ZnS(Ag) coated scintillation cell. Evacuated cells are used for grab sample measurements of radon-222 in air. Three types of radioactive sources were used to evaluate the calibration coefficient. Standard activities were generated from commercially available solid and liquid radium-226 sources, and from a self-made radon-222 source whose activity was crosschecked by gamma spectrometric measurements. Radium-226 sources are traceable to US National Institute of Standards and Technology reference standards. Sources of error on calibration are discussed. Best accuracy was obtained by gamma spectrometrically crosschecked radon source. Considerable difficulties were encountered with the traditional method of emanating a known activity of radon-222 from a standard liquid radium-226 source. Three separate solid radium-226 sources gave results with rather large deviations. The final error weighted coefficients agree well with international intercalibration results.

  7. Cell Volume Regulation in the Proximal Tubule of Rat Kidney : Proximal Tubule Cell Volume Regulation.

    PubMed

    Edwards, Aurélie; Layton, Anita T

    2017-09-12

    We developed a dynamic model of a rat proximal convoluted tubule cell in order to investigate cell volume regulation mechanisms in this nephron segment. We examined whether regulatory volume decrease (RVD), which follows exposure to a hyposmotic peritubular solution, can be achieved solely via stimulation of basolateral K[Formula: see text] and [Formula: see text] channels and [Formula: see text]-[Formula: see text] cotransporters. We also determined whether regulatory volume increase (RVI), which follows exposure to a hyperosmotic peritubular solution under certain conditions, may be accomplished by activating basolateral [Formula: see text]/H[Formula: see text] exchangers. Model predictions were in good agreement with experimental observations in mouse proximal tubule cells assuming that a 10% increase in cell volume induces a fourfold increase in the expression of basolateral K[Formula: see text] and [Formula: see text] channels and [Formula: see text]-[Formula: see text] cotransporters. Our results also suggest that in response to a hyposmotic challenge and subsequent cell swelling, [Formula: see text]-[Formula: see text] cotransporters are more efficient than basolateral K[Formula: see text] and [Formula: see text] channels at lowering intracellular osmolality and reducing cell volume. Moreover, both RVD and RVI are predicted to stabilize net transcellular [Formula: see text] reabsorption, that is, to limit the net [Formula: see text] flux decrease during a hyposmotic challenge or the net [Formula: see text] flux increase during a hyperosmotic challenge.

  8. WGA-coated yttrium oxide beads enable an imaging-based adenosine 2a receptor binding scintillation proximity assay suitable for high throughput screening.

    PubMed

    Bryant, Robert; McGuinness, Debra; Turek-Etienne, Tammy; Guyer, Deborah; Yu, Liming; Howells, Leighton; Caravano, Joseph; Zhai, Ying; Lachowicz, Jean

    2004-06-01

    Adenosine receptors belong to the superfamily of G protein-coupled receptors and are involved in a variety of physiologic functions. Traditionally, binding assays to detect adenosine 2a (A2a) antagonists and agonists have used filtration methods that are cumbersome to run and not amenable to HTS. We developed scintillation proximity assays (SPA trade mark ) utilizing HEK293 RBHA2AM cell membranes, either wheat germ agglutinin (WGA)-coated yttrium silicate (YSi) or red-shifted yttrium oxide (YO) beads and the A2a-selective radioligand [(3)H]SCH 58261. Both beads gave windows (total binding/nonspecific binding) of >5 and K(d) values of 2-3 nM for the radioligand, in agreement with results obtained by filtration. In contrast, WGA-polyvinyltoluene as well as other bead types had windows of <3 and significant radioligand binding to the uncoated beads. A 384-well WGA-YO bead SPA was optimized utilizing a LEADseeker imaging system and an automated trituration process for dispensing the dense yttrium-based beads. Signals were stable after 4 h, and Z' values were 0.7-0.8. The LEADseeker imaging assay tolerated 2% dimethyl sulfoxide and generated IC(50) values of 3-5 nM for the A2a antagonist CGS 15943, comparable to that obtained by the filtration method. A number of adenosine and xanthine analogues were identified as hits in the Library of Pharmacologically Active Compounds (LOPAC). This imaging-based A2a SPA enables HTS and is a major improvement over the filtration method.

  9. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

  10. Photovoltaic nanocrystal scintillators hybridized on Si solar cells for enhanced conversion efficiency in UV.

    PubMed

    Mutlugun, Evren; Soganci, Ibrahim Murat; Demir, Hilmi Volkan

    2008-03-17

    We propose and demonstrate semiconductor nanocrystal based photovoltaic scintillators integrated on solar cells to enhance photovoltaic device parameters including spectral responsivity, open circuit voltage, short circuit current, fill factor, and solar conversion efficiency in the ultraviolet. Hybridizing (CdSe)ZnS core-shell quantum dots of 2.4 nm in diameter on multi-crystalline Si solar cells for the first time, we show that the solar conversion efficiency is enhanced 2 folds under white light illumination similar to the solar spectrum. Such nanocrystal scintillators provide the ability to extend the photovoltaic activity towards UV.

  11. Metastatic transitional cell carcinoma in proximal humerus of a dog

    PubMed Central

    Malek, Sarah; Murphy, Kimberly A.; Nykamp, Stephanie G.; Allavena, Rachel

    2011-01-01

    Transitional cell carcinoma (TCC) was diagnosed in the proximal humerus of a dog that was presented with persistent right forelimb lameness with no clinical signs of urinary tract involvement. A diagnosis of TCC was made from surgical biopsy of the humeral lesion with subsequent necropsy revealing the prostatic urethra as the primary site of the tumor. PMID:22379204

  12. Shifting scintillator neutron detector

    DOEpatents

    Clonts, Lloyd G; Cooper, Ronald G; Crow, Jr., Morris Lowell; Hannah, Bruce W; Hodges, Jason P; Richards, John D; Riedel, Richard A

    2014-03-04

    Provided are sensors and methods for detecting thermal neutrons. Provided is an apparatus having a scintillator for absorbing a neutron, the scintillator having a back side for discharging a scintillation light of a first wavelength in response to the absorbed neutron, an array of wavelength-shifting fibers proximate to the back side of the scintillator for shifting the scintillation light of the first wavelength to light of a second wavelength, the wavelength-shifting fibers being disposed in a two-dimensional pattern and defining a plurality of scattering plane pixels where the wavelength-shifting fibers overlap, a plurality of photomultiplier tubes, in coded optical communication with the wavelength-shifting fibers, for converting the light of the second wavelength to an electronic signal, and a processor for processing the electronic signal to identify one of the plurality of scattering plane pixels as indicative of a position within the scintillator where the neutron was absorbed.

  13. An in vitro model of renal proximal tubule cell regeneration.

    PubMed

    Kays, S E; Berdanier, C D; Swagler, A R; Lock, E A; Schnellmann, R G

    1993-08-01

    The ability of renal cells to regenerate is critical for the recovery of renal function following injury. Research on the recovery of renal function has been limited by the lack of in vitro models of renal repair. The goal of this study was to develop an in vitro model of renal proximal tubule cell (RPTC) injury and regeneration using primary cultures of rabbit RPTC. Renal proximal tubules were isolated and cultured in hormonally defined DME/F-12 medium at 37 degrees C under 95% air/5% CO2. RPTC were grown to confluency, made quiescent by the removal of insulin and hydrocortisone from the medium for 24-48 hr, and treated with the nephrotoxicant, 1,2-dichlorovinyl-L-cysteine (DCVC). DCVC (100 microM for 2 hr, n = 3-6) resulted in cell injury and the release of nonviable cells from the plate at 24 hr (55% +/- 6% confluency, mean +/- SEM) and 48 hr (37% +/- 7% confluency). Cell monolayers began to regenerate 96 hr after exposure (57% +/- 9% confluency) and continued to regenerate reaching 76% +/- 8% and 84% +/- 1% confluency by 6 and 8 days postexposure. Control cells maintained confluency throughout the experiment. Thus, an in vitro primary cell culture model has been developed in which the cell monolayer regenerates after nephrotoxicant-induced injury. This model may be useful in the study of mechanisms of renal cell injury and repair.

  14. Scintillation proximity assay (SPA) as a new approach to determine a ligand's kinetic profile. A case in point for the adenosine A1 receptor.

    PubMed

    Xia, Lizi; de Vries, Henk; IJzerman, Ad P; Heitman, Laura H

    2016-03-01

    Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a "mix and measure" format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k on and k off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.

  15. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    PubMed

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  16. Development of a high-throughput scintillation proximity assay for the identification of C-domain translational initiation factor 2 inhibitors.

    PubMed

    Delle Fratte, Sonia; Piubelli, Chiara; Domenici, Enrico

    2002-12-01

    Translational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes and has been shown to be essential in Escherichia coli. It stimulates the binding of fMet-tRNA(f)(Met) to the 30S ribosomal subunit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNA(f)(Met) blocked alpha-amino group. IF2 is composed of 3 structural domains: N-domain, whose function is not known; G-domain, which contains the GTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNA(f)(Met). Its activity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 function are not known, and this makes it an ideal target for new drugs with mechanisms of resistance not yet developed. A few assays have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both instances, the assays are based on radioactive detection and do not allow for high throughput because of the need for separation or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecular recognition of fMet-tRNA(f)(Met) by the C-domain. The assay is based on the incubation of biotinyl-IF2 with fMet-tRNA(f)(Met) and the subsequent capture of the radiolabeled complex by streptavidin-coated beads, exploiting the scintillation proximity assay (SPA) technology. The assay has been designed in an automatable, homogeneous, miniaturized fashion suitable for high-throughput screening and is rapid, sensitive, and robust to dimethyl sulfoxide (DMSO) up to 10% v/v. The assay, used to screen a limited chemical collection of about 5000 compounds and a subset of compounds originated by a 2-D substructural search, has shown to be able to detect potential IF2 inhibitors.

  17. 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

  18. Association between proximity to major roads and sputum cell counts

    PubMed Central

    Wallace, Julie; D’silva, Liesel; Brannan, John; Hargreave, Frederick E; Kanaroglou, Pavlos; Nair, Parameswaran

    2011-01-01

    BACKGROUND: Air pollution caused by motor vehicle emissions has been associated with exacerbations of obstructive airway diseases; however, the nature of the resulting bronchitis has not been quantified. OBJECTIVE: To examine whether proximity to major roads or highways is associated with an increase in sputum neutrophils or eosinophils, and to evaluate the effect of proximity to roads on spirometry and exacerbations in patients with asthma. METHODS: A retrospective study of 485 sputum cell counts from patients attending a tertiary chest clinic in Hamilton, Ontario, identified eosinophilic or neutrophilic bronchitis. Patients’ residences were geocoded to the street network of Hamilton using geographic information system software. Associations among bronchitis, lung function, and proximity to major roads and highways were examined using multinomial logistic and multivariate linear regression analyses adjusted for patient age, smoking status and corticosteroid medications. RESULTS: Patients living within 1000 m of highways showed an increased risk of bronchitis (OR 3.8 [95% CI 1.0 to 13.7]; P<0.05), particularly neutrophilic bronchitis (OR 4.7 [95% CI 1.2 to 18.7]; P<0.05) as well as an increased risk of an asthma diagnosis (OR 1.9 [95% CI 1.0 to 3.4]; P<0.05). Patients living within 300 m of a major road were at increased risk for an asthma exacerbation (OR 1.9 [95% CI 1.5 to 15.5]; P<0.01) and lower lung function, particularly in women (P=0.036). CONCLUSION: In patients with airway diseases, living close to a highway or major road was associated with neutrophilic bronchitis, an increased risk of asthma diagnosis, asthma exacerbations and lower lung function. PMID:21369545

  19. 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.

  20. HIV-1 vpr inhibits cytokinesis in human proximal tubule cells

    PubMed Central

    Rosenstiel, Paul; Gruosso, Tina; Letourneau, Audrey; Chan, Justin; LeBlanc, Amanda; Husain, Mohammad; Najfeld, Vesna; Planelles, Vicente; D’Agati, Vivette; Klotman, Mary; Klotman, Paul E.

    2014-01-01

    HIV associated nephropathy (HIVAN) afflicts an estimated 1–3 million people worldwide and is a major cause of morbidity and mortality. Murine transgenic models have demonstrated that expression of HIV-1 genes in kidney cells results in characteristic HIVAN pathology: collapsing FSGS and microcystic tubular disease. While we have gained significant understanding of the podocyte disease, less is known about the tubular epithelial responses to infection. HIV-1 vpr plays an important role in the FSGS of HIVAN particularly in association with nef expression in podocytes. In addition, Vpr is reported to exacerbate tubular pathology. Therefore, we explored the effect of vpr expression on renal tubular epithelial cell function. Proximal tubule epithelial cells (PTEC) were transduced in vitro using a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. HIV-1 vpr expression in cultured PTECs impaired cytokinesis causing cell enlargement and multinucleation. Because the in vitro phenotype was so profound, we re-examined the HIVAN murine model and human HIVAN biopsies to see if similar changes could be seen in vivo. Surprisingly, both the transgenic murine HIVAN model and human HIVAN biopsies showed abundant hypertrophic tubule cells consistent with the in vitro findings. The extent of the tubular cell hypertrophy was particularly impressive and represents a previously unappreciated aspect of the disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells in HIVAN biopsies. This study provides evidence of a new clinical phenotype in HIVAN that may result from Vpr’s ability to impair cytokinesis. PMID:18614999

  1. Angiotensin II induces apoptosis in renal proximal tubular cells.

    PubMed

    Bhaskaran, Madhu; Reddy, Krishna; Radhakrishanan, Neetu; Franki, Nicholas; Ding, Guohua; Singhal, Pravin C

    2003-05-01

    ANG II has been demonstrated to play a role in the progression of tubulointerstial injury. We studied the direct effect of ANG II on apoptosis of cultured rat renal proximal tubular epithelial cells (RPTECs). ANG II promoted RPTEC apoptosis in a dose- and time-dependent manner. This effect of ANG II was attenuated by anti-transforming growth factor (TGF)-beta antibody. Moreover, TGF-beta triggered RPTEC apoptosis in a dose-dependent manner. ANG II also enhanced RPTEC expression of Fas and Fas ligand (FasL); furthermore, anti-FasL antibody attenuated ANG II-induced RPTEC apoptosis. In addition, ANG II increased RPTEC expression of Bax, a cell death protein. Both ANG II type 1 (AT(1)) and type 2 (AT(2)) receptor blockers inhibited ANG II-induced RPTEC apoptosis. SB-202190, an inhibitor of p38 MAPK phosphorylation, and caspase-3 inhibitor also attenuated ANG II-induced RPTEC apoptosis. ANG II enhanced RPTEC heme oxygenase (HO)-1 expression. Interestingly, pretreatment with hemin as well as curcumin (inducers of HO-1) inhibited the ANG II-induced tubular cell apoptosis; conversely, pretreatment with zinc protoporphyrin, an inhibitor of HO-1 expression, promoted the effect of ANG II. These results suggest that ANG II-induced apoptosis is mediated via both AT(1) and AT(2) receptors through the generation of TGF-beta, followed by the transcription of cell death genes such as Fas, FasL, and Bax. Modulation of tubular cell expression of HO-1 has an inverse relationship with the ANG II-induced tubular cell apoptosis.

  2. Mitigation of Memory Effects in Beta Scintillation Cells for Radioactive Gas Detection

    SciTech Connect

    Seifert, Carolyn E; McIntyre, Justin I; Antolick, Kathryn C; Carman, April J; Cooper, Matthew W; Hayes, James C; Heimbigner, Tom R; Hubbard, C W; Litke, Kevin E; Ripplinger, Mike D; Suarez, Reynold

    2005-08-31

    The Automated Radioxenon Sampler/Analyzer (ARSA) developed at PNNL measures the relative concentrations of xenon isotopes using a coincidence system. Previous tests of the ARSA system have shown that latent radioactivity remains in the plastic cells after evacuation of the gases, leading to a “memory effect” in which the background count rate is dependent on the sample history. The increased background results in lower detection sensitivity. Two possible solutions to the memory effect are explored in this work: depositing a thin layer of metal on the plastic cell (“metallization”), and using an inorganic scintillating cell composed of yttrium aluminum perovskite (YAP). In both cases, the presence of inorganic material at the surface is intended to inhibit the diffusion of gases into the cell walls.

  3. Light collection and pulse-shape discrimination in elongated scintillator cells for the PROSPECT reactor antineutrino experiment

    DOE PAGES

    Ashenfelter, J.; Jaffe, D.; Diwan, M. V.; ...

    2015-11-06

    A meter-long, 23-liter EJ-309 liquid scintillator detector has been constructed to study the light collection and pulse-shape discrimination performance of elongated scintillator cells for the PROSPECT reactor antineutrino experiment. The magnitude and uniformity of light collection and neutron-gamma discrimination power in the energy range of antineutrino inverse beta decay products have been studied using gamma and spontaneous fission calibration sources deployed along the cell axis. We also study neutron-gamma discrimination and light collection abilities for differing PMT and reflector configurations. As a result, key design features for optimizing MeV-scale response and background rejection capabilities are identified.

  4. Light collection and pulse-shape discrimination in elongated scintillator cells for the PROSPECT reactor antineutrino experiment

    SciTech Connect

    Ashenfelter, J.; Jaffe, D.; Diwan, M. V.; Dolph, J.; Qian, X.; Sharma, R.; Viren, B.; Zhang, C.

    2015-11-06

    A meter-long, 23-liter EJ-309 liquid scintillator detector has been constructed to study the light collection and pulse-shape discrimination performance of elongated scintillator cells for the PROSPECT reactor antineutrino experiment. The magnitude and uniformity of light collection and neutron-gamma discrimination power in the energy range of antineutrino inverse beta decay products have been studied using gamma and spontaneous fission calibration sources deployed along the cell axis. We also study neutron-gamma discrimination and light collection abilities for differing PMT and reflector configurations. As a result, key design features for optimizing MeV-scale response and background rejection capabilities are identified.

  5. Light collection and pulse-shape discrimination in elongated scintillator cells for the PROSPECT reactor antineutrino experiment

    NASA Astrophysics Data System (ADS)

    Ashenfelter, J.; Balantekin, B.; Band, H. R.; Barclay, G.; Bass, C. D.; Berish, D.; Bowden, N. S.; Bowes, A.; Brodsky, J. P.; Bryan, C. D.; Cherwinka, J. J.; Chu, R.; Classen, T.; Commeford, K.; Davee, D.; Dean, D.; Deichert, G.; Diwan, M. V.; Dolinski, M. J.; Dolph, J.; Dwyer, D. A.; Gaison, J. K.; Galindo-Uribarri, A.; Gilje, K.; Glenn, A.; Goddard, B. W.; Green, M.; Han, K.; Hans, S.; Heeger, K. M.; Heffron, B.; Jaffe, D. E.; Langford, T. J.; Littlejohn, B. R.; Martinez Caicedo, D. A.; McKeown, R. D.; Mendenhall, M. P.; Mueller, P.; Mumm, H. P.; Napolitano, J.; Neilson, R.; Norcini, D.; Pushin, D.; Qian, X.; Romero, E.; Rosero, R.; Saldana, L.; Seilhan, B. S.; Sharma, R.; Sheets, S.; Stemen, N. T.; Surukuchi, P. T.; Varner, R. L.; Viren, B.; Wang, W.; White, B.; White, C.; Wilhelmi, J.; Williams, C.; Wise, T.; Yao, H.; Yeh, M.; Yen, Y. R.; Zangakis, G.; Zhang, C.; Zhang, X.

    2015-11-01

    A meter-long, 23-liter EJ-309 liquid scintillator detector has been constructed to study the light collection and pulse-shape discrimination performance of elongated scintillator cells for the PROSPECT reactor antineutrino experiment. The magnitude and uniformity of light collection and neutron-gamma discrimination power in the energy range of antineutrino inverse beta decay products have been studied using gamma and spontaneous fission calibration sources deployed along the cell axis. We also study neutron-gamma discrimination and light collection abilities for differing PMT and reflector configurations. Key design features for optimizing MeV-scale response and background rejection capabilities are identified.

  6. Registration of Charged Particles by Scintillating Fibers Coupled with μ-CELL SI APDG

    NASA Astrophysics Data System (ADS)

    Basharuli, N.; Bondarenko, G.; Bekenov, B.; Golovin, V.; Petrov, V.; Ponomarev, N.; Grigoriev, E.

    2002-11-01

    Silicon μ-cell Avalanche Photodiode operating in Geiger mode (APDG) was used to detect light produced in scintillating fibers of 1 mm diameter by electrons from a 90Sr-source and by α-particles from a 238Pu-source. This recently developed in mesa-technology square 1 mm2 APDG, consisting of 1370 μ-cells, has enhanced inter-cell optical isolation and individual quenching resistors. It showed at room temperature and low biasing voltages (45-47 V) very high gain (up to 106), low dark counting rates (below 3 × 105sec-1) and high detection efficiency for photons of green light (> 35%). Basic characteristics - internal gain, dark counting rate and average number of detected photoelectrons as a function of bias voltage were measured.

  7. Sex Differences in Renal Proximal Tubular Cell Homeostasis.

    PubMed

    Seppi, Thomas; Prajczer, Sinikka; Dörler, Maria-Magdalena; Eiter, Oliver; Hekl, Daniel; Nevinny-Stickel, Meinhard; Skvortsova, Iraida; Gstraunthaler, Gerhard; Lukas, Peter; Lechner, Judith

    2016-10-01

    Studies in human patients and animals have revealed sex-specific differences in susceptibility to renal diseases. Because actions of female sex hormones on normal renal tissue might protect against damage, we searched for potential influences of the female hormone cycle on basic renal functions by studying excretion of urinary marker proteins in healthy human probands. We collected second morning spot urine samples of unmedicated naturally ovulating women, postmenopausal women, and men daily and determined urinary excretion of the renal tubular enzymes fructose-1,6-bisphosphatase and glutathione-S-transferase-α Additionally, we quantified urinary excretion of blood plasma proteins α1-microglobulin, albumin, and IgG. Naturally cycling women showed prominent peaks in the temporal pattern of urinary fructose-1,6-bisphosphatase and glutathione-S-transferase-α release exclusively within 7 days after ovulation or onset of menses. In contrast, postmenopausal women and men showed consistently low levels of urinary fructose-1,6-bisphosphatase excretion over comparable periods. We did not detect changes in urinary α1-microglobulin, albumin, or IgG excretion. Results of this study indicate that proximal tubular tissue architecture, representing a nonreproductive organ-derived epithelium, undergoes periodical adaptations phased by the female reproductive hormone cycle. The temporally delimited higher rate of enzymuria in ovulating women might be a sign of recurring increases of tubular cell turnover that potentially provide enhanced repair capacity and thus, higher resistance to renal damage.

  8. Development of a scintillation flow-cell detection system for environmental restoration and waste management applications

    SciTech Connect

    DeVol, T.A.; Branton, S.D.; Fjeld, R.A.

    1996-05-01

    A flow-cell detection system was developed utilizing a coincidence circuit and tested with BaF{sub 2}, CaF{sub 2}:Eu and scintillating glass. The coincidence detection system reduced the background from {approximately}200 cps to {approximately}0.5 cps. The detection efficiencies for these cells ranged from 0.38 to 0.66 for {sup 45}Ca beta particles (E{sub max} = 0.257 MeV) and from 0.45 to 0.52 for {sup 233}U alpha particles (E{sub {alpha}} = 4.8 MeV). The minimum detectable activity was calculated for a 30 s count time and determined to be in the range of 1-2 Bq.

  9. Errors in measurements of 222Rn in methane and carbon dioxide using scintillation cells calibrated for 222Rn in air.

    PubMed

    Jenkins, Phillip H; Burkhart, James F; Camley, Robert E

    2014-03-01

    Scintillation cells are used typically for measuring the concentration of (222)Rn in air and are calibrated for that purpose. However, scintillation cells are sometimes used for measuring (222)Rn in natural gas or carbon dioxide. The counting efficiencies of scintillation cells for measurements of (222)Rn in these gases should be different from those for measuring (222)Rn in air because the ranges of alpha particles emitted by (222)Rn and its progeny are greater in methane and smaller in carbon dioxide than in air. If these effects are not taken into consideration, measurements of (222)Rn in natural gas will be biased high and in carbon dioxide will be biased low. The authors previously investigated the effects of barometric pressure on measurements of (222)Rn in air using scintillation cells. A modeling technique was used in a previous study to calculate theoretical errors that would result if atmospheric pressure were not considered. In the current study, the same modeling technique was used to calculate theoretical errors that would be made for measurements of (222)Rn in methane and carbon dioxide if the calibration for (222)Rn in air were used. Results are presented for four types of scintillation cells of varying geometries and for barometric pressures representative of four elevations ranging from sea level to 1,963 m (6,440 feet). These results indicate that the errors introduced by the ranges of the alpha particles in gases different from air can be significant. Depending on the type of cell and the local pressure, a measurement of (222)Rn in methane may be biased high by 2-7%, while a measurement of (222)Rn in CO2 may be biased low by 15-20% if the calibration for (222)Rn in air is used.

  10. Comparative testing of various flow-cell detectors fabricated using CaF{sub 2} solid scintillator

    SciTech Connect

    Kawano, T.; Ohashi, H.; Hamada, Y.; Jamsranjav, E.

    2015-03-15

    A monitoring system based on a flow-cell detector was developed for measuring the tritium concentration in water. The flow-cell detector was fabricated using a granular CaF{sub 2} solid scintillator. This system does not use a liquid scintillation counting system and does not generate radioactive organic liquid waste. Moreover, continuous real-time measurements are possible, in contrast to a liquid scintillation counting system, which requires batch measurements. For further development of the system, four flow-cell detectors were fabricated. They included a single 3-mm-diameter cell, three 3-mm-diameter cells in series, a single 5-mm-diameter cell, and three 5-mm-diameter cells in series. Continuously flowing water containing tritium at various concentrations was passed through the flow cells, and tritium count were measured for 600 and 10000 s. Investigating the relation between the count rate and concentration, the three 5-mm-diameter cells were most sensitive, with a linear relation maintained down to approximately 2 Bq/ml and 10 Bq/ml for 10000- and 600-s measurements, respectively. (authors)

  11. Scintillator material

    DOEpatents

    Anderson, David F.; Kross, Brian J.

    1992-01-01

    An improved scintillator material comprising cerium fluoride is disclosed. Cerium fluoride has been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to known scintillator materials such as thallium-doped sodium iodide, barium fluoride and bismuth germanate. As a result, cerium fluoride is favorably suited for use as a scintillator material in positron emission tomography.

  12. Scintillator material

    DOEpatents

    Anderson, David F.; Kross, Brian J.

    1994-01-01

    An improved scintillator material comprising cerium fluoride is disclosed. Cerium fluoride has been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to known scintillator materials such as thallium-doped sodium iodide, barium fluoride and bismuth germanate. As a result, cerium fluoride is favorably suited for use as a scintillator material in positron emission tomography.

  13. Scintillator material

    DOEpatents

    Anderson, D.F.; Kross, B.J.

    1992-07-28

    An improved scintillator material comprising cerium fluoride is disclosed. Cerium fluoride has been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to known scintillator materials such as thallium-doped sodium iodide, barium fluoride and bismuth germanate. As a result, cerium fluoride is favorably suited for use as a scintillator material in positron emission tomography. 4 figs.

  14. Scintillator material

    DOEpatents

    Anderson, D.F.; Kross, B.J.

    1994-06-07

    An improved scintillator material comprising cerium fluoride is disclosed. Cerium fluoride has been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to known scintillator materials such as thallium-doped sodium iodide, barium fluoride and bismuth germanate. As a result, cerium fluoride is favorably suited for use as a scintillator material in positron emission tomography. 4 figs.

  15. Scintillation detector for carbon-14

    NASA Technical Reports Server (NTRS)

    Knoll, G. F.; Rogers, W. L.

    1971-01-01

    Detector consists of plastic, cylindrical double-wall scintillation cell, which is filled with gas to be analyzed. Thin, inner cell wall is isolated optically from outer (guard) scintillator wall by evaporated-aluminum coating. Bonding technique provides mechanical support to cell wall when device is exposed to high temperatures.

  16. A pathway-centric view of spatial proximity in the 3D nucleome across cell lines

    PubMed Central

    Karathia, Hiren; Kingsford, Carl; Girvan, Michelle; Hannenhalli, Sridhar

    2016-01-01

    In various contexts, spatially proximal genes have been shown to be functionally related. However, the extent to which spatial proximity of genes in a pathway contributes to the pathway’s context-specific activity is not known. Leveraging Hi-C data in six human cell-lines, we show that spatial proximity of genes in a pathway is highly correlated with the pathway’s context-specific expression and function. Furthermore, spatial proximity of pathway genes correlates with interactions of their protein products, and the specific pathway genes that are proximal to one another tend to occupy higher levels in the regulatory hierarchy. In addition to intra-pathway proximity, related pathways are spatially proximal to one another and housekeeping-genes tend to be proximal to several other pathways suggesting their coordinating role. Substantially extending previous works, our study reveals a pathway-centric organization of 3D-nucleome, whereby, functionally related interacting driver genes tend to be in spatial-proximity in a context-specific manner. PMID:27976707

  17. Exosomal Transfer From Human Renal Proximal Tubule Cells To Distal Tubule And Collecting Duct Cells

    PubMed Central

    Gildea, John J.; Seaton, Joscelyn E.; Victor, Ken G.; Reyes, Camellia M.; Wang, Dora Bigler; Pettigrew, Abigail C.; Courtner, Crystal E.; Shah, Neema; Tran, Hanh T.; Van Sciver, Robert E.; Carlson, Julia M.; Felder, Robin A.

    2017-01-01

    Objectives Exosomes are 50-90 nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. Design and methods For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. Results Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multi-vesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. Conclusion Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system. PMID:24976626

  18. Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.

    PubMed

    Gildea, John J; Seaton, Joscelyn E; Victor, Ken G; Reyes, Camellia M; Bigler Wang, Dora; Pettigrew, Abigail C; Courtner, Crystal E; Shah, Neema; Tran, Hanh T; Van Sciver, Robert E; Carlson, Julia M; Felder, Robin A

    2014-10-01

    Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  19. Scintillation Counters

    NASA Astrophysics Data System (ADS)

    Bell, Zane W.

    Scintillators find wide use in radiation detection as the detecting medium for gamma/X-rays, and charged and neutral particles. Since the first notice in 1895 by Roentgen of the production of light by X-rays on a barium platinocyanide screen, and Thomas Edison's work over the following 2 years resulting in the discovery of calcium tungstate as a superior fluoroscopy screen, much research and experimentation have been undertaken to discover and elucidate the properties of new scintillators. Scintillators with high density and high atomic number are prized for the detection of gamma rays above 1 MeV; lower atomic number, lower-density materials find use for detecting beta particles and heavy charged particles; hydrogenous scintillators find use in fast-neutron detection; and boron-, lithium-, and gadolinium-containing scintillators are used for slow-neutron detection. This chapter provides the practitioner with an overview of the general characteristics of scintillators, including the variation of probability of interaction with density and atomic number, the characteristics of the light pulse, a list and characteristics of commonly available scintillators and their approximate cost, and recommendations regarding the choice of material for a few specific applications. This chapter does not pretend to present an exhaustive list of scintillators and applications.

  20. Responses of proximal tubular cells to injury in congenital renal disease: fight or flight.

    PubMed

    Chevalier, Robert L; Forbes, Michael S; Galarreta, Carolina I; Thornhill, Barbara A

    2014-04-01

    Most chronic kidney disease in children results from congenital or inherited disorders, which can be studied in mouse models. Following 2 weeks of unilateral ureteral obstruction (UUO) in the adult mouse, nephron loss is due to proximal tubular mitochondrial injury and cell death. In neonatal mice, proximal tubular cell death is delayed beyond 2 weeks of complete UUO, and release of partial UUO allows remodeling of remaining nephrons. Progressive cyst expansion develops in polycystic kidney disease (PKD), a common inherited renal disorder. The polycystic kidney and fibrosis (pcy)-mutant mouse (which develops late-onset PKD) develops thinning of the glomerulotubular junction in parallel with growth of cysts in adulthood. Renal insufficiency in nephropathic cystinosis, a rare inherited renal disorder, results from progressive tubular cystine accumulation. In the Ctns knockout mouse (a model of cystinosis), proximal tubular cells become flattened, with loss of mitochondria and thickening of tubular basement membrane. In each model, persistent obstructive or metabolic stress leads ultimately to the formation of atubular glomeruli. The initial "fight" response (proximal tubular survival) switches to a "flight" response (proximal tubular cell death) with ongoing oxidative injury and mitochondrial damage. Therapies should be directed at reducing proximal tubular mitochondrial oxidative injury to enhance repair and regeneration.

  1. Responses of Proximal Tubular Cells to Injury in Congenital Renal Disease: Fight or Flight

    PubMed Central

    Chevalier, Robert L.; Forbes, Michael S.; Galarreta, Carolina I.; Thornhill, Barbara A.

    2013-01-01

    Most chronic kidney disease in children results from congenital or inherited disorders, which can be studied in mouse models. Following 2 weeks of unilateral ureteral obstruction (UUO) in the adult mouse, nephron loss is due to proximal tubular mitochondrial injury and cell death. In neonatal mice, proximal tubular cell death is delayed beyond 2 weeks of complete UUO, and release of partial UUO allows remodeling of remaining nephrons. Progressive cyst expansion develops in polycystic kidney disease (PKD), a common inherited renal disorder. The PCY mutant mouse (which develops late-onset PKD) develops thinning of the glomerulotubular junction in parallel with growth of cysts in adulthood. Renal insufficiency in nephropathic cystinosis, a rare inherited renal disorder, results from progressive tubular cystine accumulation. In the Ctns knock out mouse (a model of cystinosis), proximal tubular cells become flattened, with loss of mitochondria and thickening of tubular basement membrane. In each model, persistent obstructive or metabolic stress leads ultimately to the formation of atubular glomeruli. The initial “fight” response (proximal tubular survival) switches to a “flight” response (proximal tubular cell death) with ongoing oxidative injury and mitochondrial damage. Therapies should be directed at reducing proximal tubular mitochondrial oxidative injury to enhance repair and regeneration. PMID:23949631

  2. p-Cresol mediates autophagic cell death in renal proximal tubular cells.

    PubMed

    Lin, Hsin-Hung; Huang, Chiu-Ching; Lin, Tze-Yi; Lin, Ching-Yuang

    2015-04-02

    Higher serum level of p-cresol (PC) in chronic kidney disease (CKD) patients has been linked with CKD progression. The toxic effect of PC on diverse cells has been reported by prior studies, except for renal tubular cells. Both autophagy and apoptosis contribute to renal tubular cell death, yet evidence of its response to PC is limited and their crosstalk is still unclear. Autophagy is an important cellular process involved in toxin-induced cell death. Renal tubular cell death in tubular injury is thought to be one of the key events causing the progression of CKD. Thus, we treated rat (NRK-52E) and human (HRPTEC) renal proximal tubular cells (RPTC) with PC and found the cell proliferation was significantly decreased. Cell apoptosis was significantly increased and accompanied with the activation of autophagy as evidenced by increases in LC3-II, beclin 1 and Atg 4. We also found an increase of p62 by c-Jun activation. p62 accumulation could mediate the activation of caspase 8-dependent cell apoptosis. Conversely, knockdown of p62 by siRNA of p62 had the opposite effect by arresting LC3-II accumulation and promoting increasing cell viability. We conclude that PC triggered autophagic RPTC death via JNK-mediated p62 accumulation and then activated caspase 8-dependent cell death pathway. PC can be considered as one of the key events causing progression of CKD, which might affect drug disposition in CKD cases.

  3. Residential Proximity to Major Roadways is Associated with Increased Levels of AC133+ Circulating Angiogenic Cells

    PubMed Central

    DeJarnett, Natasha; Yeager, Ray; Conklin, Daniel J.; Lee, Jongmin; O'Toole, Timothy E.; McCracken, James; Abplanalp, Wes; Srivastava, Sanjay; Riggs, Daniel W.; Hamzeh, Ihab; Wagner, Stephen; Chugh, Atul; DeFilippis, Andrew; Ciszewski, Tiffany; Wyatt, Brad; Becher, Carrie; Higdon, Deirdre; Ramos, Kenneth S.; Tollerud, David J.; Myers, John A.; Rai, Shesh N.; Shah, Jasmit; Zafar, Nagma; Krishnasamy, Sathya S.; Prabhu, Sumanth D.; Bhatnagar, Aruni

    2016-01-01

    Objective Previous studies have shown that residential proximity to a roadway is associated with increased cardiovascular disease (CVD) risk. Yet the nature of this association remains unclear, and its impact on individual CVD risk factors has not been assessed. The objective of this study was to determine whether residential proximity to roadways influences systemic inflammation and the levels of circulating angiogenic cells. Approach and Results In a cross-sectional study, CVD risk factors, blood levels of C-reactive protein (CRP), and 15 antigenically-defined circulating angiogenic cell populations were measured in participants (n=316) with moderate to high CVD risk. Attributes of roadways surrounding residential locations were assessed using Geographic Information Systems. Associations between road proximity and cardiovascular indices were analyzed using Generalized Linear Models. Close proximity (<50m) to a major roadway was associated with lower income and higher rates of smoking, but not CRP levels. After adjustment for potential confounders, levels of circulating angiogenic cell in peripheral blood were significantly elevated in people living in close proximity to a major roadway (CD31+/AC133+, AC133+, CD34+/AC133+, and CD34+/45dim/AC133+ cells); and positively associated with road segment distance (CD31+/AC133+, AC133+, and CD34+/AC133+ cells), traffic intensity (CD31+/AC133+ and AC133+ cells), and distance-weighted traffic intensity (CD31+/34+/45+/AC133+ cells). Conclusions Living close to a major roadway is associated with elevated levels of circulating cells positive for the “early” stem marker, AC133+. This may reflect an increased need for vascular repair. Levels of these cells in peripheral blood may be a sensitive index of cardiovascular injury due to residential proximity to roadways. PMID:26293462

  4. Tumor-promoting phorbol esters effect alkalinization of canine renal proximal tubular cells

    SciTech Connect

    Mellas, J.; Hammerman, M.R.

    1986-03-01

    We have demonstrated the presence of specific receptors for tumor-promoting phorbol esters in the plasma membrane of the canine renal proximal tubular cell. These compounds affect proximal tubular metabolism in vitro. For example, we have shown that they inhibit gluconeogenesis in canine renal proximal tubular segments. Tumor-promoting phorbol esters have been shown to effect alkalinization of non-renal cells, by enhancing Na/sup +/-H/sup +/ exchange across the plasma membrane. To determine whether the actions of tumor-promoting phorbol esters in proximal tubular segments might be mediated by a similar process, we incubated suspensions of segments from dog kidney with these compounds and measured changes in intracellular pH using (/sup 14/C)-5,5-dimethoxazoladine-2-4-dione (DMO) and flow dialysis. Incubation of segments with phorbol 12,13 dibutyrate, but not inactive phorbol ester, 4 ..gamma.. phorbol, effected alkalinization of cells within the segments in a concentration-dependent manner. Alkalinization was dependent upon the presence of extracellular (Na/sup +/) > intracellular (Na/sup +/), was prevented by amiloride and was demonstrable in the presence of SITS. Our findings suggest that tumor-promoting esters stimulate the Na/sup +/-H/sup +/ exchanger known to be present in the brush border membrane of the renal proximal tubular cell. It is possible that the stimulation reflects a mechanism by which phorbol esters affect metabolic processes in these cells.

  5. Hybrid scintillators for neutron discrimination

    DOEpatents

    Feng, Patrick L; Cordaro, Joseph G; Anstey, Mitchell R; Morales, Alfredo M

    2015-05-12

    A composition capable of producing a unique scintillation response to neutrons and gamma rays, comprising (i) at least one surfactant; (ii) a polar hydrogen-bonding solvent; and (iii) at least one luminophore. A method including combining at least one surfactant, a polar hydrogen-bonding solvent and at least one luminophore in a scintillation cell under vacuum or an inert atmosphere.

  6. HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells.

    PubMed

    Rosenstiel, Paul E; Gruosso, Tina; Letourneau, Audrey M; Chan, Justin J; LeBlanc, Amanda; Husain, Mohammad; Najfeld, Vesna; Planelles, Vicente; D'Agati, Vivette D; Klotman, Mary E; Klotman, Paul E

    2008-10-01

    Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis.

  7. 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

  8. Morphology of auroral zone radio wave scintillation

    SciTech Connect

    Rino, C.L.; Matthews, S.J.

    1980-08-01

    This paper describes the morphology of midnight sector and morning sector auroral zone scintillation observations made over a two-year period using the Wideband satelite, which is in a sun-synchronous, low-altitude orbit. No definitive seasonal variation was found. The nighttime data showed the highest scintillation ocurrence levels, but significant amounts of morning scintillation were observed. For the most part the scintillation activity followed the general pattern of local magnetic activity. The most prominent feature in the nightime data is a localized amplitude and phase scintillation enhancement at the point where the propagation vector lies within an L shell. A geometrical effect due to a dynamic slab of sheetlike structures in the F region is hypothesized as the source of his enhancement. The data have been sorted by magnetic activity, proximity to local midnight, and season. The general features of the data are in agreement with the accepted morphology of auroral zone scintillation.

  9. Demonstration of neutron detection utilizing open cell foam and noble gas scintillation

    SciTech Connect

    Lavelle, C. M. Miller, E. C.; Coplan, M.; Thompson, Alan K.; Vest, Robert E.; Yue, A. T.; Kowler, A. L.; Koeth, T.; Al-Sheikhly, M.; Clark, Charles W.

    2015-03-02

    We present results demonstrating neutron detection via a closely spaced converter structure coupled to low pressure noble gas scintillation instrumented by a single photo-multiplier tube (PMT). The converter is dispersed throughout the gas volume using a reticulated vitreous carbon foam coated with boron carbide (B{sub 4}C). A calibrated cold neutron beam is used to measure the neutron detection properties, using a thin film of enriched {sup 10}B as a reference standard. Monte Carlo computations of the ion energy deposition are discussed, including treatment of the foam random network. Results from this study indicate that the foam shadows a significant portion of the scintillation light from the PMT. The high scintillation yield of Xe appears to overcome the light loss, facilitating neutron detection and presenting interesting opportunities for neutron detector design.

  10. Giant Cell Tumour of Proximal Phalanx of Ring Finger: Case Report and Review of Literature

    PubMed Central

    Soni, Rishit; Shah, Malkesh; Patel, Amit; Golwala, Paresh

    2016-01-01

    Giant cell tumour (GCT) of bone arising from a phalanx of a finger is extremely rare. Only two percent of all reported GCTs are found in the hand, which show a higher rate of recurrence as compared to those occurring at a more proximal location. Here we report a rare case of giant cell tumour of proximal phalanx of the ring finger in a 20-year-old male, which was treated with extended curettage and bone grafting. After two years of follow-up, the patient was asymptomatic with complete functional recovery and no signs of recurrence. PMID:27900230

  11. Giant Cell Tumor within the Proximal Tibia after ACL Reconstruction.

    PubMed

    Takahashi, Takashi; MacCormick, Lauren; Ellermann, Jutta; Clohisy, Denis; Marette, Shelly

    2016-01-01

    26-year-old female with prior anterior cruciate ligament reconstruction developed an enlarging lytic bone lesion around the tibial screw with sequential imaging over the course of one year demonstrating progression of this finding, which was confirmed histologically to be a giant cell tumor of bone. The lesion originated around the postoperative bed, making the diagnosis challenging during the early course of the presentation. The case demonstrates giant cell tumor which originated in the metaphysis and subsequently grew to involve the epiphysis; therefore, early course of the disease not involving the epiphysis should not exclude this diagnosis.

  12. Giant Cell Tumor within the Proximal Tibia after ACL Reconstruction

    PubMed Central

    Takahashi, Takashi; MacCormick, Lauren; Ellermann, Jutta; Clohisy, Denis; Marette, Shelly

    2016-01-01

    26-year-old female with prior anterior cruciate ligament reconstruction developed an enlarging lytic bone lesion around the tibial screw with sequential imaging over the course of one year demonstrating progression of this finding, which was confirmed histologically to be a giant cell tumor of bone. The lesion originated around the postoperative bed, making the diagnosis challenging during the early course of the presentation. The case demonstrates giant cell tumor which originated in the metaphysis and subsequently grew to involve the epiphysis; therefore, early course of the disease not involving the epiphysis should not exclude this diagnosis. PMID:26981302

  13. Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

    NASA Astrophysics Data System (ADS)

    Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

    2013-11-01

    Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

  14. 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

  15. IgE epitope proximity determines immune complex shape and effector cell activation capacity

    PubMed Central

    Gieras, Anna; Linhart, Birgit; Roux, Kenneth H.; Dutta, Moumita; Khodoun, Marat; Zafred, Domen; Cabauatan, Clarissa R.; Lupinek, Christian; Weber, Milena; Focke-Tejkl, Margarete; Keller, Walter; Finkelman, Fred D.; Valenta, Rudolf

    2016-01-01

    Background IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. Objective To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. Methods We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. Results We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. Conclusions Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex–mediated diseases. PMID:26684291

  16. Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface

    PubMed Central

    Bae, Jungu; Kuroda, Kouichi

    2014-01-01

    Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields. PMID:25304511

  17. Hemoglobin inhibits albumin uptake by proximal tubule cells: implications for sickle cell disease.

    PubMed

    Eshbach, Megan L; Kaur, Amandeep; Rbaibi, Youssef; Tejero, Jesús; Weisz, Ora A

    2017-06-01

    Proximal tubule (PT) dysfunction, including tubular proteinuria, is a significant complication in young sickle cell disease (SCD) that can eventually lead to chronic kidney disease. Hemoglobin (Hb) dimers released from red blood cells upon hemolysis are filtered into the kidney and internalized by megalin/cubilin receptors into PT cells. The PT is especially sensitive to heme toxicity, and tubular dysfunction in SCD is thought to result from prolonged exposure to filtered Hb. Here we show that concentrations of Hb predicted to enter the tubule lumen during hemolytic crisis competitively inhibit the uptake of another megalin/cubilin ligand (albumin) by PT cells. These effects were independent of heme reduction state. The Glu7Val mutant of Hb that causes SCD was equally effective at inhibiting albumin uptake compared with wild-type Hb. Addition of the Hb scavenger haptoglobin (Hpt) restored albumin uptake in the presence of Hb, suggesting that Hpt binding to the Hb αβ dimer-dimer interface interferes with Hb binding to megalin/cubilin. BLAST searches and structural modeling analyses revealed regions of similarity between Hb and albumin that map to this region and may represent sites of Hb interaction with megalin/cubilin. Our studies suggest that impaired endocytosis of megalin/cubilin ligands, rather than heme toxicity, may be the cause of tubular proteinuria in SCD patients. Additionally, loss of these filtered proteins into the urine may contribute to the extra-renal pathogenesis of SCD. Copyright © 2017 the American Physiological Society.

  18. Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

    PubMed Central

    Xiao, Fengxia; Zimpelmann, Joseph; Agaybi, Samih; Gurley, Susan B.; Puente, Lawrence; Burns, Kevin D.

    2014-01-01

    Angiotensin-converting enzyme 2 (ACE2) is highly expressed in the kidney proximal tubule, where it cleaves angiotensin (Ang) II to Ang-(1-7). Urinary ACE2 levels increase in diabetes, suggesting that ACE2 may be shed from tubular cells. The aim of this study was to determine if ACE2 is shed from proximal tubular cells, to characterize ACE2 fragments, and to study pathways for shedding. Studies involved primary cultures of mouse proximal tubular cells, with ACE2 activity measured using a synthetic substrate, and analysis of ACE2 fragments by immunoblots and mass spectrometry. The culture media from mouse proximal tubular cells demonstrated a time-dependent increase in ACE2 activity, suggesting constitutive ACE2 shedding. ACE2 was detected in media as two bands at ∼90 kDa and ∼70 kDa on immunoblots. By contrast, full-length ACE2 appeared at ∼100 kDa in cell lysates or mouse kidney cortex. Mass spectrometry of the two deglycosylated fragments identified peptides matching mouse ACE2 at positions 18-706 and 18-577, respectively. The C-terminus of the 18-706 peptide fragment contained a non-tryptic site, suggesting that Met706 is a candidate ACE2 cleavage site. Incubation of cells in high D-glucose (25 mM) (and to a lesser extent Ang II) for 48–72 h increased ACE2 activity in the media (p<0.001), an effect blocked by inhibition of a disintegrin and metalloproteinase (ADAM)17. High D-glucose increased ADAM17 activity in cell lysates (p<0.05). These data indicate that two glycosylated ACE2 fragments are constitutively shed from mouse proximal tubular cells. ACE2 shedding is stimulated by high D-glucose, at least partly via an ADAM17-mediated pathway. The results suggest that proximal tubular shedding of ACE2 may increase in diabetes, which could enhance degradation of Ang II in the tubular lumen, and increase levels of Ang-(1-7). PMID:24454948

  19. Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells.

    PubMed

    Seow, Ying-ying T; Tan, Michelle G K; Woo, Keng Thye

    2002-07-01

    The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake. Copyright 2002 S. Karger AG, Basel

  20. 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. PMID:28401035

  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. Ankle arthrodesis fusion rates for mesenchymal stem cell bone allograft versus proximal tibia autograft.

    PubMed

    Anderson, John J; Boone, Joshua J; Hansen, Myron; Brady, Chad; Gough, Adam; Swayzee, Zflan

    2014-01-01

    Ankle arthrodesis is commonly used in the treatment of ankle arthritis. The present study compared mesenchymal stem cell (MSC) bone allografts and proximal tibia autografts as adjuncts in performing ankle arthrodesis. A total of 109 consecutive ankle fusions performed from 2002 to 2008 were evaluated retrospectively. Of the 109 fusions, 24 were excluded from the present study, leaving 85 patients who had undergone ankle arthrodesis. Of the 85 patients, 41 had received a proximal tibia autograft and 44, an MSC bone allograft. These 2 groups were reviewed and compared retrospectively at least 2 years postoperatively for the overall fusion rate, interval to radiographic fusion, and interval to clinical fusion. A modified and adjusted American College of Foot and Ankle Surgeons ankle scale was used to measure patient satisfaction. The overall fusion rate was 84.1% in the MSC bone allograft group and 95.1% in the proximal tibia autograft group (p = .158). The corresponding mean intervals to radiographic fusion were 13.0 ± 2.5 weeks and 11.3 ± 2.8 weeks (p ≤ .001). The interval to clinical fusion was 13.1 ± 2.1 weeks and 11.0 ± 1.5 weeks (p ≤ .001) in the MSC bone allograft and proximal tibia autograft group, respectively. No statistically significant difference was found in the fusion rates between the MSC bone allograft and proximal tibia autograft groups. Also, no statistically significant difference was found between the preoperative and postoperative scores using a modified and adjusted American College of Foot and Ankle Surgeons ankle scale between the 2 groups (p = .41 and p = .44, respectively). A statistically significant delay to radiographic and clinical fusion was present in the MSC bone allograft group compared with the proximal tibia autograft group; however, no difference was found in patient satisfaction.

  3. SCINTILLATION SPECTROMETER

    DOEpatents

    Bell, P.R.; Francis, J.E.

    1960-06-21

    A portable scintillation spectrometer is described which is especially useful in radio-biological studies for determining the uptake and distribution of gamma -emitting substances in tissue. The spectrometer includes a collimator having a plurality of apertures that are hexagonal in cross section. Two crystals are provided: one is activated to respond to incident rays from the collimator; the other is not activated and shields the first from external radiation.

  4. Plastic scintillation dosimetry: Optimal selection of scintillating fibers and scintillators

    SciTech Connect

    Archambault, Louis; Arsenault, Jean; Gingras, Luc; Sam Beddar, A.; Roy, Rene; Beaulieu, Luc

    2005-07-15

    Scintillation dosimetry is a promising avenue for evaluating dose patterns delivered by intensity-modulated radiation therapy plans or for the small fields involved in stereotactic radiosurgery. However, the increase in signal has been the goal for many authors. In this paper, a comparison is made between plastic scintillating fibers and plastic scintillator. The collection of scintillation light was measured experimentally for four commercial models of scintillating fibers (BCF-12, BCF-60, SCSF-78, SCSF-3HF) and two models of plastic scintillators (BC-400, BC-408). The emission spectra of all six scintillators were obtained by using an optical spectrum analyzer and they were compared with theoretical behavior. For scintillation in the blue region, the signal intensity of a singly clad scintillating fiber (BCF-12) was 120% of that of the plastic scintillator (BC-400). For the multiclad fiber (SCSF-78), the signal reached 144% of that of the plastic scintillator. The intensity of the green scintillating fibers was lower than that of the plastic scintillator: 47% for the singly clad fiber (BCF-60) and 77% for the multiclad fiber (SCSF-3HF). The collected light was studied as a function of the scintillator length and radius for a cylindrical probe. We found that symmetric detectors with nearly the same spatial resolution in each direction (2 mm in diameter by 3 mm in length) could be made with a signal equivalent to those of the more commonly used asymmetric scintillators. With augmentation of the signal-to-noise ratio in consideration, this paper presents a series of comparisons that should provide insight into selection of a scintillator type and volume for development of a medical dosimeter.

  5. Plastic scintillation dosimetry: Optimal selection of scintillating fibers and scintillators.

    PubMed

    Archambault, Louis; Arsenault, Jean; Gingras, Luc; Sam Beddar, A; Roy, René; Beaulieu, Luc

    2005-07-01

    Scintillation dosimetry is a promising avenue for evaluating dose patterns delivered by intensity-modulated radiation therapy plans or for the small fields involved in stereotactic radiosurgery. However, the increase in signal has been the goal for many authors. In this paper, a comparison is made between plastic scintillating fibers and plastic scintillator. The collection of scintillation light was measured experimentally for four commercial models of scintillating fibers (BCF-12, BCF-60, SCSF-78, SCSF-3HF) and two models of plastic scintillators (BC-400, BC-408). The emission spectra of all six scintillators were obtained by using an optical spectrum analyzer and they were compared with theoretical behavior. For scintillation in the blue region, the signal intensity of a singly clad scintillating fiber (BCF-12) was 120% of that of the plastic scintillator (BC-400). For the multiclad fiber (SCSF-78), the signal reached 144% of that of the plastic scintillator. The intensity of the green scintillating fibers was lower than that of the plastic scintillator: 47% for the singly clad fiber (BCF-60) and 77% for the multiclad fiber (SCSF-3HF). The collected light was studied as a function of the scintillator length and radius for a cylindrical probe. We found that symmetric detectors with nearly the same spatial resolution in each direction (2 mm in diameter by 3 mm in length) could be made with a signal equivalent to those of the more commonly used asymmetric scintillators. With augmentation of the signal-to-noise ratio in consideration, this paper presents a series of comparisons that should provide insight into selection of a scintillator type and volume for development of a medical dosimeter. © 2005 American Association of Physicists in Medicine.

  6. Plastic scintillation dosimetry: optimal selection of scintillating fibers and scintillators.

    PubMed

    Archambault, Louis; Arsenault, Jean; Gingras, Luc; Beddar, A Sam; Roy, René; Beaulieu, Luc

    2005-07-01

    Scintillation dosimetry is a promising avenue for evaluating dose patterns delivered by intensity-modulated radiation therapy plans or for the small fields involved in stereotactic radiosurgery. However, the increase in signal has been the goal for many authors. In this paper, a comparison is made between plastic scintillating fibers and plastic scintillator. The collection of scintillation light was measured experimentally for four commercial models of scintillating fibers (BCF-12, BCF-60, SCSF-78, SCSF-3HF) and two models of plastic scintillators (BC-400, BC-408). The emission spectra of all six scintillators were obtained by using an optical spectrum analyzer and they were compared with theoretical behavior. For scintillation in the blue region, the signal intensity of a singly clad scintillating fiber (BCF-12) was 120% of that of the plastic scintillator (BC-400). For the multiclad fiber (SCSF-78), the signal reached 144% of that of the plastic scintillator. The intensity of the green scintillating fibers was lower than that of the plastic scintillator: 47% for the singly clad fiber (BCF-60) and 77% for the multiclad fiber (SCSF-3HF). The collected light was studied as a function of the scintillator length and radius for a cylindrical probe. We found that symmetric detectors with nearly the same spatial resolution in each direction (2 mm in diameter by 3 mm in length) could be made with a signal equivalent to those of the more commonly used asymmetric scintillators. With augmentation of the signal-to-noise ratio in consideration, this paper presents a series of comparisons that should provide insight into selection of a scintillator type and volume for development of a medical dosimeter.

  7. Distribution of IGF receptors in the plasma membrane of proximal tubular cells

    SciTech Connect

    Hammerman, M.R.; Rogers, S. )

    1987-11-01

    To characterize the distribution of receptors for insulin-like growth factors I and II (IGF I and II) in the plasma membrane of the renal proximal tubular cell, the authors measured binding of {sup 125}I-labeled IGF I and {sup 125}I-labeled IGF II to proximal tubular basolateral and brush-border membranes and characterized IGF I-stimulated phosphorylation of detergent-solubilized membranes. {sup 125}I-IGF bound primarily to a 135,000 relative molecular weight (M{sub r}) protein and IGF II to a 260,000 M{sub r} protein in isolated membranes. Binding of {sup 125}I-IGF I was severalfold greater in basolateral than in brush-border membranes. IGF I-stimulated phosphorylation of the 92,000 M{sub r} {beta}-subunit of its receptors could be demonstrated only in basolateral membranes. These findings are consistent with an asymmetrical distribution of receptors for IGF I in the plasma membrane of the renal proximal tubular cell, localization being primary on the basolateral side. In contrast, binding of {sup 125}I-IGF II to isolated basolateral and brush-border membranes was equivalent, suggesting that receptors for this peptide are distributed more symmetrically in the plasma membrane. The findings suggest that the action of IGF I in proximal tubule are mediated via interaction of circulating peptide with specific receptors in the basolateral membrane. However, the findings established the potential for actions of IGF II to be exerted in proximal tubule via interaction with both basolateral and/or brush-border membrane receptors.

  8. Proximity effect among cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface.

    PubMed

    Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

    PubMed Central

    Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-01-01

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  10. 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

  11. Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells.

    PubMed

    Kowolik, Claudia M; Liang, Shujian; Yu, Ying; Yee, Jiing-Kuan

    2004-08-05

    Primary human renal proximal tubule epithelial cells (RPTECs) are of limited use for basic research and for clinical applications due to their limited lifespan in culture. Here we used two lentivirus vectors carrying the human telomerase (hTERT) and the SV40T antigen (Tag) flanked by loxP sites to reversibly immortalize RPTECs. Transduced RPTEC clones continued to proliferate while retaining biochemical and functional characteristics of primary cells. The clones exhibited contact-inhibited, anchorage- and growth factor-dependent growth and did not form tumors in nude mice, suggesting that the cells were not transformed. Transient Cre expression in these cells led to efficient proviral deletion, upregulation of some renal specific activities, and decreased growth rates. Ultimately, the cells underwent replicative senescence, indicating intact cell cycle control. Thus, reversible immortalization allows the expansion of human RPTECs, leading to large production of RPTECs that retain most tissue-specific properties.

  12. 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.

  13. Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal Cystine Transporter Cystinosin

    PubMed Central

    Van Den Heuvel, Lambertus; Pastore, Anna; Dijkman, Henry; De Matteis, Maria Antonietta; Levtchenko, Elena N.

    2015-01-01

    Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles. PMID:25811383

  14. Gap-junctional hemichannels are activated by ATP depletion in human renal proximal tubule cells.

    PubMed

    Vergara, L; Bao, X; Cooper, M; Bello-Reuss, E; Reuss, L

    2003-12-01

    We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca(2+) removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.

  15. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators.

    PubMed

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel A; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Lo Celso, Cristina; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-Fu; Scadden, David T

    2016-10-06

    Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Proximity-based differential single cell analysis of the niche to identify stem/progenitor cell regulators

    PubMed Central

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Celso, Cristina Lo; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-fu; Scadden, David T

    2016-01-01

    SUMMARY Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on differential single-cell gene expression analysis of mesenchymal osteolineage cells close to and further removed from hematopoietic stem/progenitor cells to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. Amongst the genes which were preferentially expressed in proximal cells, we functionally examined three secreted or cell surface molecules not previously connected to HSPC biology: the secreted RNase Angiogenin, the cytokine IL18 and the adhesion molecule Embigin and discovered that all of these factors are HSPC quiescence regulators. Our proximity-based differential single cell approach therefore reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance understanding of microenvironmental regulation of stem cell function. PMID:27524439

  17. 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.

  18. Ketohexokinase-dependent metabolism of fructose induces proinflammatory mediators in proximal tubular cells.

    PubMed

    Cirillo, Pietro; Gersch, Michael S; Mu, Wei; Scherer, Philip M; Kim, Kyung Mee; Gesualdo, Loreto; Henderson, George N; Johnson, Richard J; Sautin, Yuri Y

    2009-03-01

    Increased consumption of fructose may play an important role in the epidemic of metabolic syndrome and may presage the development of diabetes, cardiovascular disease, and chronic kidney disease. Once in the cell, fructose is phosphorylated by ketohexokinase (KHK), leading to consumption of ATP, formation of AMP, and generation of uric acid through xanthine oxidoreductase (XOR). This study aimed to examine the direct effects of fructose in human kidney proximal tubular cells (HK-2) and whether they are mediated by the fructose metabolism via KHK. At a similar concentration to that observed in peripheral blood after a meal, fructose induced production of monocyte chemotactic protein 1 (MCP-1) and reactive oxygen species in HK-2 cells. Knockdown of KHK by stable transfection with small hairpin RNA demonstrated that these processes were KHK dependent. Several antioxidants, including specific inhibitors of NADPH oxidase and XOR, prevented MCP-1 secretion. We detected XOR mRNA in HK-2 cells and confirmed its activity by identifying uric acid by mass spectrometry. Fructose increased intracellular uric acid, and uric acid induced production of MCP-1 as well. In summary, postprandial concentrations of fructose stimulate redox- and urate-dependent inflammatory mediators in proximal tubular cells.

  19. Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation.

    PubMed

    Phong, Binh L; Avery, Lyndsay; Sumpter, Tina L; Gorman, Jacob V; Watkins, Simon C; Colgan, John D; Kane, Lawrence P

    2015-12-14

    T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation.

  20. Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells

    PubMed Central

    Wagner, Carsten A.; Giebisch, Gerhard; Lang, Florian; Geibel, John P.

    1998-01-01

    Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3− cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl−, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl−, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl− channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl−-dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3− reabsorption along the proximal tubule when tubules are exposed to AII. PMID:9689138

  1. 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.

  2. Far infrared radiation promotes rabbit renal proximal tubule cell proliferation and functional characteristics, and protects against cisplatin-induced nephrotoxicity.

    PubMed

    Chiang, I-Ni; Pu, Yeong-Shiau; Huang, Chao-Yuan; Young, Tai-Horng

    2017-01-01

    Far infrared radiation, a subdivision of the electromagnetic spectrum, is beneficial for long-term tissue healing, anti-inflammatory effects, growth promotion, sleep modulation, acceleration of microcirculation, and pain relief. We investigated if far infrared radiation is beneficial for renal proximal tubule cell cultivation and renal tissue engineering. We observed the effects of far infrared radiation on renal proximal tubules cells, including its effects on cell proliferation, gene and protein expression, and viability. We also examined the protective effects of far infrared radiation against cisplatin, a nephrotoxic agent, using the human proximal tubule cell line HK-2. We found that daily exposure to far infrared radiation for 30 min significantly increased rabbit renal proximal tubule cell proliferation in vitro, as assessed by MTT assay. Far infrared radiation was not only beneficial to renal proximal tubule cell proliferation, it also increased the expression of ATPase Na+/K+ subunit alpha 1 and glucose transporter 1, as determined by western blotting. Using quantitative polymerase chain reaction, we found that far infrared radiation enhanced CDK5R1, GNAS, NPPB, and TEK expression. In the proximal tubule cell line HK-2, far infrared radiation protected against cisplatin-mediated nephrotoxicity by reducing apoptosis. Renal proximal tubule cell cultivation with far infrared radiation exposure resulted in better cell proliferation, significantly higher ATPase Na+/K+ subunit alpha 1 and glucose transporter 1 expression, and significantly enhanced expression of CDK5R1, GNAS, NPPB, and TEK. These results suggest that far infrared radiation improves cell proliferation and differentiation. In HK-2 cells, far infrared radiation mediated protective effects against cisplatin-induced nephrotoxicity by reducing apoptosis, as indicated by flow cytometry and caspase-3 assay.

  3. Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

    SciTech Connect

    Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

    2009-11-15

    Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

  4. Cadmium activates extracellular signal-regulated kinase 5 in HK-2 human renal proximal tubular cells

    SciTech Connect

    Kondo, Mio; Inamura, Hisako; Matsumura, Ken-ichi; Matsuoka, Masato

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cadmium exposure induces ERK5 phosphorylation in HK-2 renal proximal tubular cells. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced ERK5 but not ERK1/2 phosphorylation. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced CREB and c-Fos phosphorylation. Black-Right-Pointing-Pointer ERK5 activation by cadmium exposure may play an anti-apoptotic role in HK-2 cells. -- Abstract: We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the phosphorylation and functionality of extracellular signal-regulated kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, in HK-2 human renal proximal tubular cells. Following exposure to CdCl{sub 2}, ERK5 phosphorylation increased markedly, but the level of total ERK5 was unchanged. ERK5 phosphorylation following CdCl{sub 2} exposure was rapid and transient, similar to the time course of ERK1/2 phosphorylation. Treatment of HK-2 cells with the MAPK/ERK kinase 5 inhibitor, BIX02189, suppressed CdCl{sub 2}-induced ERK5 but not ERK1/2 phosphorylation. The CdCl{sub 2}-induced increase of phosphorylated cAMP response element-binding protein (CREB) and activating transcription factor-1 (ATF-1), as well as the accumulation of mobility-shifted c-Fos protein, were suppressed by BIX02189 treatment. Furthermore, BIX02189 treatment enhanced cleavage of poly(ADP-ribose) polymerase and increased the level of cytoplasmic nucleosomes in HK-2 cells exposed to CdCl{sub 2}. These findings suggest that ERK5 pathway activation by CdCl{sub 2} exposure might induce the phosphorylation of cell survival-transcription factors, such as CREB, ATF-1, and c-Fos, and may exert a partial anti-apoptotic role in HK-2 cells.

  5. 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

  6. PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton

    PubMed Central

    Britton, Graham J; Ambler, Rachel; Clark, Danielle J; Hill, Elaine V; Tunbridge, Helen M; McNally, Kerrie E; Burton, Bronwen R; Butterweck, Philomena; Sabatos-Peyton, Catherine; Hampton-O’Neil, Lea A; Verkade, Paul; Wuelfing, Christoph; Wraith, David Cameron

    2017-01-01

    Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.20003.001 PMID:28112644

  7. hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics.

    PubMed

    Wieser, Matthias; Stadler, Guido; Jennings, Paul; Streubel, Berthold; Pfaller, Walter; Ambros, Peter; Riedl, Claus; Katinger, Hermann; Grillari, Johannes; Grillari-Voglauer, Regina

    2008-11-01

    Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far. Here we have established simple and safe conditions that allow serial passaging of renal proximal tubule epithelial cells (RPTECs) until entry into telomere-dependent replicative senescence. As reported for other cells, senescence of RPTECs is characterized by arrest in G1 phase, shortened telomeres, staining for senescence-associated beta-galactosidase, and accumulation of gamma-H2AX foci. Furthermore, ectopic expression of the catalytic subunit of telomerase (TERT) was sufficient to immortalize these cells. Characterization of immortalized RPTEC/TERT1 cells shows characteristic morphological and functional properties like formation of tight junctions and domes, expression of aminopeptidase N, cAMP induction by parathyroid hormone, sodium-dependent phosphate uptake, and the megalin/cubilin transport system. No genomic instability within up to 90 population doublings has been observed. Therefore, these cells are proposed as a valuable model system not only for cell biology but also for toxicology, drug screening, biogerontology, as well as tissue engineering approaches.

  8. A proximity-dependent assay for specific RNA-protein interactions in intact cells.

    PubMed

    Zhang, Wei; Xie, Mingyi; Shu, Mei-Di; Steitz, Joan A; DiMaio, Daniel

    2016-11-01

    The proximity ligation assay (PLA) is an immune staining method that detects protein-protein interactions in fixed cells. We describe here RNA-PLA, a simple adaptation of this technology that allows the detection of specific RNA-protein interactions in fixed cells by using a DNA oligonucleotide that hybridizes to a target RNA in combination with an antibody that recognizes the protein bound to the target RNA. Stable and transient RNA-protein interactions can be readily detected by generation of a fluorescent signal in discrete compartments in intact fixed cells with high specificity. We demonstrate that this approach requires the colocalization of the binding protein and its RNA target in the same cellular compartment, use of an oligonucleotide complementary to the target RNA, and the presence of a binding site for the protein in the target RNA. © 2016 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. Effect of radiologic contrast media on cell volume regulation in rabbit proximal renal tubules.

    PubMed

    Galtung, H K; Løken, M; Sakariassen, K S

    2001-05-01

    Most radiographic contrast media are hyperosmotic and able to shrink cells with which they are in contact. The authors studied cell volume control in rabbit proximal renal tubules after incubation with three contrast media: iohexol, ioxaglate, and iodixanol. Proximal renal tubules were isolated from rabbit kidneys. The tubules were exposed to Ringer solutions containing 5% vol/vol iohexol (final osmolality, 330 mOsm), ioxaglate (323 mOsm), iodixanol (305 mOsm), or mannitol (control solutions with identical osmolalities), and tubule volumes were monitored. After 2 hours of incubation, the tubules were stimulated with a hyposmotic Ringer solution (165 mOsm). Three groups of 10 experiments were performed. All solutions induced cell shrinkage (8.3%+/-3.8 [standard error] to 15.4%+/-0.5), which was completely or partly reversible in most experiments (volume increase, 44.8%+/-14.7 to 149.9%+/-107.3) but not those with iohexol and iodixanol. With exposure to the hyposmotic solution, the cells swelled by 11.0%+/-1.8 to 39.7%+/-4.8. In general, the tubules that had been exposed to the most hyperosmotic solution swelled the most. Those exposed to contrast media showed less swelling than the mannitol-exposed controls. In all control experiments, the cells exhibited a gradual shrinkage (43.6%+/-28.5 to 87.0%+/-13). This regulatory response was partly inhibited in tubules exposed to iohexol (39.9%+/-15.8 shrinkage) or iodixanol (8.9%+/-15.8) and completely inhibited in those exposed to ioxaglate. Iohexol and ioxaglate exposure also led to a decrease in water permeability. Exposure to hyperosmotic contrast medium tends to induce prolonged cell shrinkage, decrease the water permeability of the cellular plasma membranes, and compromise the ability to regulate cellular volume. These changes seem to reflect both the hyperosmolality of the solutions and their inherent chemical properties.

  10. Proliferation and intracellular pH in cultured proximal tubular cells

    SciTech Connect

    Larsson, S.H.; Fukuda, Y.; Koelare, S.A.; Aperia, A. )

    1990-03-01

    Renal proximal tubule (PT) cells from adult rats will maintain much of their functional characteristics in short-term primary culture. This study examines the growth regulation of these highly differentiated cells with particular reference to cell density, intracellular pH (pHi), and the expression of the Na(+)-H+ exchanger. PT cells were obtained from young adult rats and studied after 48 h in culture. The mitotic rate was determined as the labeling index (LI) after (3H)thymidine autoradiography, and pHi was determined by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein quantitative fluorescence microscopy in single cells. Cells were grown either continuously in serum (S) or were serum deprived after 24 h (D). The cells were nonconfluent and grew in colonies. We defined the two peripheral layers of cells in a colony as peripheral (P) cells and the remaining cells as central (C). In C cells LI/h and pHi were in the range of what has been observed under in vivo conditions. In S condition LI/h was 2.2 +/- 0.3% and in D condition was 0.3 +/- 0.1%. LI was significantly higher in P than in C cells both under S (2.5 +/- 0.4-fold) and D conditions (5.6 +/- 0.8-fold). The rapidly growing P cells had a significantly lower pHi than the growth-retarded C cells both under S (7.25 +/- 0.02 vs. 7.30 +/- 0.01, P less than 0.05) and D conditions (7.21 +/- 0.02 vs. 7.28 +/- 0.01, P less than 0.05).

  11. The effects of colloid solutions on renal proximal tubular cells in vitro.

    PubMed

    Neuhaus, Winfried; Schick, Martin A; Bruno, Raphael R; Schneiker, Bianca; Förster, Carola Y; Roewer, Norbert; Wunder, Christian

    2012-02-01

    Renal failure is a common complication of critically ill patients. Colloids such as hydroxyethyl starch (HES), gelatin, or albumin are regularly used for intravascular volume resuscitation, but there are increasing reports about the nephrotoxic side effects of synthetic colloids in septic patients. Therefore, we investigated the influence of colloids (HES130/0.4 (Voluven®), gelatin (Gelafundin®), human albumin, and the crystalloid Sterofundin® ISO on cell viability of human proximal tubular (HK-2) cells. HK-2 cells were incubated with colloids (0.1%-4%) and with equivalent volumes of the crystalloid solution Sterofundin ISO. After 21 hours, cell viability of HK-2 cells was measured by EZ4U assay (dye XTT). Application of HES130/0.4 decreased cell viability significantly in a concentration-dependent manner (86.80% ± 10.79% by 0.5% HES down to 24.02% ± 4.27% by 4% HES). Human albumin (>1.25%) as well as gelatin (>1%) also showed deleterious effects on HK-2 cells. Interestingly, in lower concentrations, human albumin and the crystalloid solution Sterofundin ISO were cytoprotective in comparison with the NaCl control. In conclusion, synthetic and natural colloids showed a harmful impact on HK-2 cells in higher concentrations without any prior proinflammatory stimulus. HES130/0.4 exhibited the most distinctive harmful impact, whereas the application of crystalloid Sterofundin ISO revealed cytoprotective effects.

  12. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells

    PubMed Central

    Konishi, Satoshi; Gotoh, Shimpei; Tateishi, Kazuhiro; Yamamoto, Yuki; Korogi, Yohei; Nagasaki, Tadao; Matsumoto, Hisako; Muro, Shigeo; Hirai, Toyohiro; Ito, Isao; Tsukita, Sachiko; Mishima, Michiaki

    2015-01-01

    Summary Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1+-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM+ VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a “9 + 2” microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine. PMID:26724905

  13. Na+-H+ exchanger in proximal cells isolated from rabbit kidney. I. Functional characteristics.

    PubMed

    Bidet, M; Tauc, M; Merot, J; Vandewalle, A; Poujeol, P

    1987-11-01

    The purpose of this study was to investigate the characteristics of the Na+-H+ exchange in isolated proximal cells from rabbit kidney cortex. The cells were prepared by mechanical dissociation and sequential passages through nylon meshes. The intracellular pH (pHi) was measured in a bicarbonate-free medium [extracellular pH (pHe) = 7.30], using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Resting pHi was 7.13 +/- 0.04 (n = 11) at 20-22 degrees C. Cells were acid loaded with nigericin in choline solution and H+ efflux, induced by extracellular Na+ (Nae), was calculated using a buffering power of 23.6 +/- 0.6 mmol.1-1.pH unit-1 (n = 19) estimated by NH4Cl exposure. In isolated proximal cells, the Na+-H+ antiporter had an apparent Km for Nae of 86.7 +/- 1.5 mM (n = 4) and was competitively inhibited by amiloride with a Ki of 33.3 +/- 6.4 X 10(-6) M (n = 3). Lowering pHe, inhibited the Na+-H+ exchanger. This inhibition was not purely competitive and the Ki was 40.4 +/- 12.7 nM (n = 3). The Na+-H+ exchange was greatly activated when the cytoplasm was acidified. The intracellular H+ concentration dependence did not follow simple Michaelis-Menten kinetics. Of the different cations tested on pHi recovery, such as Li+, choline+, K+, and tetramethylammonium, only Li+ induced an alkalinization of acidified cells similar to that of Na+. 22Na influx measurements indicated that cellular depletion of Na+ stimulated Na+-H+ exchange. The results permit the conclusion that the isolation procedures did not impair the main features of the Na+-H+ antiporter, at least as compared with those previously described in renal brush-border membrane vesicles or in other cellular systems. The integrity of the transporter in isolated proximal cells would permit the direct study of its hormonal and metabolic control.

  14. Mechanism of increased clearance of glycated albumin by proximal tubule cells

    PubMed Central

    Wagner, Mark C.; Myslinski, Jered; Pratap, Shiv; Flores, Brittany; Rhodes, George; Campos-Bilderback, Silvia B.; Sandoval, Ruben M.; Kumar, Sudhanshu; Patel, Monika; Ashish

    2016-01-01

    Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn)-mediated transcytosis by many cell types, including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of the present study was to address the impact of specific known biological albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it after transcytosis at physiological pH. Equilibrium dissociation constants were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin, we studied glucose and methylgloxal modified albumins (21 days). All had reduced affinity to FcRn at pH 6.0, suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address why modified albumin has reduced affinity, we analyzed the structure of the modified albumins using small-angle X-ray scattering. This analysis showed significant structural changes occurring to albumin with glycation, particularly in the FcRn-binding region, which could explain the reduced affinity to FcRn. These results offer an explanation for enhanced proximal tubule-mediated sorting and clearance of abnormal albumins. PMID:26887834

  15. IL-6 augments angiotensinogen in primary cultured renal proximal tubular cells.

    PubMed

    Satou, Ryousuke; Gonzalez-Villalobos, Romer A; Miyata, Kayoko; Ohashi, Naro; Urushihara, Maki; Acres, Omar W; Navar, L Gabriel; Kobori, Hiroyuki

    2009-11-13

    In human kidneys, the mechanisms underlying angiotensinogen (AGT) augmentation by interleukin 6 (IL-6) are poorly understood and the only information available is in HK-2, immortalized human renal proximal tubular epithelial cells. Therefore, the present study was performed to elucidate the effects of IL-6 on AGT expression in primary cultured human renal proximal tubular epithelial cells (RPTEC) after characterization of HK-2 and RPTEC. RPTEC showed low basal AGT mRNA (11+/-1%) and protein (7.0+/-0.9%) expression, high IL-6 receptor (IL-6R) expression (282+/-17%), and low basal NF-kappaB (43+/-7%) and STAT3 (43+/-7%) activities compared to those in HK-2. In RPTEC, AGT mRNA and protein expressions were enhanced by IL-6 (172+/-31% and 378+/-39%, respectively). This AGT augmentation was attenuated by an IL-6R antibody. STAT3 phosphorylation (366+/-55% at 30min) and translocation were enhanced by IL-6. The AGT augmentation was attenuated by a STAT3 inhibitor. These data indicate that IL-6 increases AGT expression via STAT3 pathway in RPTEC.

  16. 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.

  17. IL-6 Augments Angiotensinogen in Primary Cultured Renal Proximal Tubular Cells

    PubMed Central

    Satou, Ryousuke; Gonzalez-Villalobos, Romer A.; Miyata, Kayoko; Ohashi, Naro; Urushihara, Maki; Acres, Omar W.; Navar, L. Gabriel; Kobori, Hiroyuki

    2009-01-01

    In human kidneys, the mechanisms underlying angiotensinogen (AGT) augmentation by interleukin 6 (IL-6) are poorly understood and the only information available is in HK-2, immortalized human renal proximal tubular epithelial cells. Therefore, the present study was performed to elucidate the effects of IL-6 on AGT expression in primary cultured human renal proximal tubular epithelial cells (RPTEC) after characterization of HK-2 and RPTEC. RPTEC showed low basal AGT mRNA (11±1%) and protein (7.0±0.9%) expression, high IL-6 receptor (IL-6R) expression (282±17%), and low basal NF-κB (43±7%) and STAT3 (43±7%) activities compared to those in HK-2. In RPTEC, AGT mRNA and protein expressions were enhanced by IL-6 (172±31% and 378±39%, respectively). This AGT augmentation was attenuated by an IL-6R antibody. STAT3 phosphorylation (366±55% at 30 min) and translocation were enhanced by IL-6. The AGT augmentation was attenuated by a STAT3 inhibitor. These data indicate that IL-6 increases AGT expression via STAT3 pathway in RPTEC. PMID:19583994

  18. Na sup + -H sup + exchanger in proximal cells isolated from rabbit kidney. I. Functional characteristics

    SciTech Connect

    Bidet, M.; Tauc, M.; Merot, J.; Vandewalle, A.; Poujeol, P. )

    1987-11-01

    The purpose of this study was to investigate the characteristics of the Na{sup +}-H{sup +} exchange in isolated proximal cells from rabbit kidney cortex. The cells were prepared by mechanical dissociation and sequential passages through nylon meshes. The intracellular pH (pH{sub i}) was measured in a bicarbonate-free medium using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Resting pH{sub i} was 7.13 {+-} 0.04. Cells were acid loaded with nigericin in choline solution and H{sup +} efflux, induced by extracellular Na{sup +} (Na{sub e}), was calculated using a buffering power of 23.6 {+-} 0.6 mmol {center dot} l{sup {minus}1} {center dot} pH unit{sup {minus}1} estimated by NH{sub 4}Cl exposure. The intracellular H{sup +} concentration dependence did not follow simple Michaelis-Menten kinetics. Of the different cations tested on pH{sub i} recovery, such as Li{sup +}, choline{sup +}, K{sup +}, and tetramethylammonium, only Li{sup +} induced an alkalinization of acidified cells similar to that of Na{sup +}. {sup 22}Na influx measurements indicated that cellular depletion of Na{sup +} stimulated Na{sup +}-H{sup +} exchange. The results permit the conclusion that the isolation procedures did not impair the main features of the Na{sup +}-H{sup +} antiporter, at least as compared with those previously described in renal brush-border membrane vesicles or in other cellular systems. The integrity of the transporter in isolated proximal cells would permit the direct study of its hormonal and metabolic control.

  19. Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells.

    PubMed

    Al-Rasheed, N M; Meakin, F; Royal, E L; Lewington, A J; Brown, J; Willars, G B; Brunskill, N J

    2004-06-01

    Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy. Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca(2+) was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay. Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3+/-0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1+/-0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca(2+)] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-alpha. Pre-treatment with pertussis toxin abolished PKC-alpha. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pre-treatments. None of these effects is reproduced by scrambled C-peptide. This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.

  20. Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells

    PubMed Central

    Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

    2017-01-01

    Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity. PMID:28139717

  1. Mitogenic action of lysophosphatidic acid in proximal tubular epithelial cells obtained from voided human urine.

    PubMed

    Kumagai, N; Inoue, C N; Kondo, Y; Iinuma, K

    2000-12-01

    Focal tubular cell multiplication at sites on an injured nephron is a critical event in the recovery phase following acute tubular necrosis. During this process, numerous viable tubular cells exfoliate and are shed into the urine. Lysophosphatidic acid (LPA) is generated in the plasma membrane of injured cells and acts as an intercellular mediator of various biological processes, including inflammation, proliferation and repair. In the present study, exfoliated proximal tubule (PT) cells were isolated from human urine and the mitogenic effects of LPA were investigated as a model of repair and proliferation following renal injury. LPA stimulated a 23. 5% increase in DNA synthesis, a 29.4% increase in cell number and an 86.6% decrease in cAMP content. All of these responses were pertussis toxin sensitive, indicating the involvement of G(i)-type G-proteins in LPA signalling. Conversely, the LPA-induced DNA synthesis and the decrease in intracellular cAMP content were insensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), suggesting a mitogenic response via PI3K-independent mechanisms. Furthermore, we detected specific mRNA transcripts for the recently cloned human LPA-receptors, endothelial differentiation gene (Edg)-2 and Edg-4 (Edg-2>Edg-4) by reverse transcription-PCR in PT cells. Our data suggest that LPA may behave as a local growth factor in PT cells following tubular injury.

  2. Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells.

    PubMed

    Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

    2017-01-31

    Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity.

  3. Gene expression profiles of human proximal tubular epithelial cells in proteinuric nephropathies.

    PubMed

    Rudnicki, M; Eder, S; Perco, P; Enrich, J; Scheiber, K; Koppelstätter, C; Schratzberger, G; Mayer, B; Oberbauer, R; Meyer, T W; Mayer, G

    2007-02-01

    In kidney disease renal proximal tubular epithelial cells (RPTEC) actively contribute to the progression of tubulointerstitial fibrosis by mediating both an inflammatory response and via epithelial-to-mesenchymal transition. Using laser capture microdissection we specifically isolated RPTEC from cryosections of the healthy parts of kidneys removed owing to renal cell carcinoma and from kidney biopsies from patients with proteinuric nephropathies. RNA was extracted and hybridized to complementary DNA microarrays after linear RNA amplification. Statistical analysis identified 168 unique genes with known gene ontology association, which separated patients from controls. Besides distinct alterations in signal-transduction pathways (e.g. Wnt signalling), functional annotation revealed a significant upregulation of genes involved in cell proliferation and cell cycle control (like insulin-like growth factor 1 or cell division cycle 34), cell differentiation (e.g. bone morphogenetic protein 7), immune response, intracellular transport and metabolism in RPTEC from patients. On the contrary we found differential expression of a number of genes responsible for cell adhesion (like BH-protocadherin) with a marked downregulation of most of these transcripts. In summary, our results obtained from RPTEC revealed a differential regulation of genes, which are likely to be involved in either pro-fibrotic or tubulo-protective mechanisms in proteinuric patients at an early stage of kidney disease.

  4. Cystinosin-LKG rescues cystine accumulation and decreases apoptosis rate in cystinotic proximal tubular epithelial cells.

    PubMed

    Taranta, Anna; Bellomo, Francesco; Petrini, Stefania; Polishchuk, Elena; De Leo, Ester; Rega, Laura Rita; Pastore, Anna; Polishchuk, Roman; De Matteis, Maria Antonietta; Emma, Francesco

    2017-01-01

    Nephropathic cystinosis is a lysosomal storage disease that is caused by mutations in the CTNS gene encoding a cystine/proton symporter cystinosin and an isoform cystinosin-LKG which is generated by an alternative splicing of exon 12. We have investigated the physiological role of the cystinosin-LKG that is widely expressed in epithelial tissues. We have analyzed the intracellular localization and the function of the cystinosin-LKG conjugated with DsRed (cystinosin-LKG-RFP) in Madin-Darby canine kidney cells (MDCK II) and in proximal tubular epithelial cells carrying a deletion of the CTNS gene (cystinotic PTEC), respectively. Cystinosin-LKG-RFP colocalized with markers of lysosomes, late endosomes and was also expressed on the apical surface of polarized MDCK II cells. Moreover, immune-electron microscopy images of MDCK II cells overexpressing cystinosin-LKG-RFP showed stacked lamellar membranes inside perinuclear lysosomal structures. To study the role of LKG-isoform, we have investigated cystine accumulation and apoptosis that have been described in cystinotic cells. Cystinosin-LKG decreased cystine levels by approximately 10-fold similarly to cystinosin-RFP. The levels of TNFα- and actinomycin D-inducted apoptosis dropped in cystinotic cells expressing LKG-isoform. This effect was also similar to the main isoform. Our results suggest that cystinosin-LKG and cystinosin move similar functional activities in cells.

  5. High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells.

    PubMed

    Zhang, Xiao-Qian; Dong, Jian-Jun; Cai, Tian; Shen, Xue; Zhou, Xiao-Jun; Liao, Lin

    2017-04-11

    Diabetic nephropathy is the primary cause of end-stage renal disease. Apoptosis of tubule epithelial cells is a major feature of diabetic nephropathy. The mechanisms of high glucose (HG) induced apoptosis are not fully understood. Here we demonstrated that, HG induced apoptosis via upregulating the expression of proapoptotic Bcl-2 homology domain 3 (BH3)-only protein Bim protein, but not bring a significant change in the baseline level of autophagy in HK2 cells. The increase of Bim expression was caused by the ugregulation of transcription factors, FOXO1 and FOXO3a. Bim expression initiates BAX/BAK-mediated mitochondria-dependent apoptosis. Silence of Bim by siRNA in HK2 cells prevented HG-induced apoptosis and also sensitized HK2 cells to autophagy during HG treatment. The autophagy inhibitor 3-MA increased the injury in Bim knockdown HK2 cells by retriggering apoptosis. The above results suggest a Bim-independent apoptosis pathway in HK2 cells, which normally could be inhibited by autophagy. Overall, our results indicate that HG induces apoptosis via up-regulation of Bim expression in proximal tubule epithelial cells.

  6. High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells

    PubMed Central

    Zhang, Xiao-Qian; Dong, Jian-Jun; Cai, Tian; Shen, Xue; Zhou, Xiao-Jun; Liao, Lin

    2017-01-01

    Diabetic nephropathy is the primary cause of end-stage renal disease. Apoptosis of tubule epithelial cells is a major feature of diabetic nephropathy. The mechanisms of high glucose (HG) induced apoptosis are not fully understood. Here we demonstrated that, HG induced apoptosis via upregulating the expression of proapoptotic Bcl-2 homology domain 3 (BH3)-only protein Bim protein, but not bring a significant change in the baseline level of autophagy in HK2 cells. The increase of Bim expression was caused by the ugregulation of transcription factors, FOXO1 and FOXO3a. Bim expression initiates BAX/BAK-mediated mitochondria-dependent apoptosis. Silence of Bim by siRNA in HK2 cells prevented HG-induced apoptosis and also sensitized HK2 cells to autophagy during HG treatment. The autophagy inhibitor 3-MA increased the injury in Bim knockdown HK2 cells by retriggering apoptosis. The above results suggest a Bim-independent apoptosis pathway in HK2 cells, which normally could be inhibited by autophagy. Overall, our results indicate that HG induces apoptosis via up-regulation of Bim expression in proximal tubule epithelial cells. PMID:28445931

  7. Effects of cumene hydroperoxide on cellular cation composition in frog kidney proximal tubular cells.

    PubMed

    Petrovic, S; Cemerikic, D

    2000-06-01

    Effects of cumene hydroperoxide were studied on the peritubular membrane potential and cellular cation composition in frog kidney proximal tubular cells. After perfusion of isolated frog kidneys for 30 min with 1.3x10(-4) mol l(-1) cumene hydroperoxide Ringer solution, the peritubular membrane potential gradually declined. The ouabain-like effects were demonstrated on cell Na and K activities after 1 h of perfusion with cumene hydroperoxide. The peritubular apparent transference number for potassium was decreased. Intracellular pH was not altered in the presence of cumene hydroperoxide. Intracellular free Ca(2+) concentration increased slowly and moderately. The concentration of the malondialdehyde in the kidney homogenates, measured as an index of lipid peroxidation, was increased. A previously observable effect of cumene hydroperoxide on the peritubular membrane potential was prevented by oxygen radical scavengers.

  8. Zika virus infects renal proximal tubular epithelial cells with prolonged persistency and cytopathic effects.

    PubMed

    Chen, Jian; Yang, Yi-Feng; Chen, Jun; Zhou, Xiaohui; Dong, Zhaoguang; Chen, Tianyue; Yang, Yu; Zou, Peng; Jiang, Biao; Hu, Yunwen; Lu, Lu; Zhang, Xiaoyan; Liu, Jia; Xu, Jianqing; Zhu, Tongyu

    2017-08-23

    Zika virus (ZIKV) infection can cause fetal developmental abnormalities and Guillain-Barré syndrome in adults. Although progress has been made in understanding the link between ZIKV infection and microcephaly, the pathology of ZIKV, particularly the viral reservoirs in human, remains poorly understood. Several studies have shown that compared to serum samples, patients' urine samples often have a longer duration of ZIKV persistency and higher viral load. This finding suggests that an independent viral reservoir may exist in the human urinary system. Despite the clinical observations, the host cells of ZIKV in the human urinary system are poorly characterized. In this study, we demonstrate that ZIKV can infect renal proximal tubular epithelial cells (RPTEpiCs) in immunodeficient mice in vivo and in both immortalized and primary human renal proximal tubular epithelial cells (hRPTEpiCs) in vitro. Importantly, ZIKV infection in mouse kidneys caused caspase-3-mediated apoptosis of renal cells. Similarly, in vitro infection of immortalized and primary hRPTEpiCs resulted in notable cytopathic effects. Consistent with the clinical observations, we found that ZIKV infection can persist with prolonged duration in hRPTEpiCs. RNA-Seq analyses of infected hRPTEpiCs revealed a large number of transcriptional changes in response to ZIKV infection, including type I interferon signaling genes and anti-viral response genes. Our results suggest that hRPTEpiCs are a potential reservoir of ZIKV in the human urinary system, providing a possible explanation for the prolonged persistency of ZIKV in patients' urine.

  9. Recombinant mussel proximal thread matrix protein promotes osteoblast cell adhesion and proliferation.

    PubMed

    Yoo, Hee Young; Song, Young Hoon; Foo, Mathias; Seo, Eunseok; Hwang, Dong Soo; Seo, Jeong Hyun

    2016-02-16

    von Willebrand factor (VWF) is a key load bearing domain for mamalian cell adhesion by binding various macromolecular ligands in extracellular matrix such as, collagens, elastin, and glycosaminoglycans. Interestingly, vWF like domains are also commonly found in load bearing systems of marine organisms such as in underwater adhesive of mussel and sea star, and nacre of marine abalone, and play a critical load bearing function. Recently, Proximal Thread Matrix Protein1 (PTMP1) in mussel composed of two vWF type A like domains has characterized and it is known to bind both mussel collagens and mammalian collagens. Here, we cloned and mass produced a recombinant PTMP1 from E. coli system after switching all the minor codons to the major codons of E. coli. Recombinant PTMP1 has an ability to enhance mouse osteoblast cell adhesion, spreading, and cell proliferation. In addition, PTMP1 showed vWF-like properties as promoting collagen expression as well as binding to collagen type I, subsequently enhanced cell viability. Consequently, we found that recombinant PTMP1 acts as a vWF domain by mediating cell adhesion, spreading, proliferation, and formation of actin cytoskeleton. This study suggests that both mammalian cell adhesion and marine underwater adhesion exploits a strong vWF-collagen interaction for successful wet adhesion. In addition, vWF like domains containing proteins including PTMP1 have a great potential for tissue engineering and the development of biomedical adhesives as a component for extra-cellular matrix.

  10. Novel human renal proximal tubular cell line for the production of complex proteins.

    PubMed

    Fliedl, Lukas; Manhart, Gabriele; Kast, Florian; Katinger, Hermann; Kunert, Renate; Grillari, Johannes; Wieser, Matthias; Grillari-Voglauer, Regina

    2014-04-20

    Human host cell lines for the production of biopharmaceutical proteins are of interest due to differences in the glycosylation patterns of human and animal cell lines. Specifically, sialylation, which has a major impact on half-life and immunogenicity of recombinant biopharmaceuticals, differs markedly. Here, we established and characterized an immortalized well documented and serum-free host cell line, RS, from primary human renal proximal tubular epithelial cells (RPTEC). In order to test its capacity to produce complex glycosylated proteins, stable recombinant human erythropoietin (rhEpo) producing clones were generated. The clone with highest productivity, RS-1C9 was further characterized and showed stable productivity. Biological activity was observed in in vitro assays and 28% of rhEpo glyco-isoforms produced by RS-1C9 were in range and distribution of the biological reference standard (BRP) isoform, as compared to 11.5% of a CHO based rhEpo. Additionally, cellular α-2,6 sialylation, Galactose-alpha-1,3-galactose (alpha-Gal) and N-glycolylneuraminic acid (NeuGc) patterns compare favourably to CHO cells. While productivity of RS still needs optimization, its amenability to upscaling in bioreactors, its production of glyco-isoforms that will increase yields after down-stream processing of about 2.5 fold, presence of sialylation and lack of Neu5Gc recommend RS as alternative human host cell line for production of biopharmaceuticals. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Expression of xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1.

    PubMed

    Aschauer, Lydia; Carta, Giada; Vogelsang, Nadine; Schlatter, Eberhard; Jennings, Paul

    2015-12-25

    The kidney is a major target for drug-induced injury, primarily due the fact that it transports a wide variety of chemical entities into and out of the tubular lumen. Here, we investigated the expression of the main xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1 at an mRNA and/or protein level. RPTEC/TERT1 cells expressed OCT2, OCT3, OCTN2, MATE1, MATE2, OAT1, OAT3 and OAT4. The functionality of the OCTs was demonstrated by directional transport of the fluorescent dye 4-Di-1-ASP. In addition, P-glycoprotein activity in RPTEC/TERT1 cells was verified by fluorescent dye retention in presence of various P-glycoprotein inhibitors. In comparison to proliferating cells, contact inhibited RPTEC/TERT1 cells expressed increased mRNA levels of several ABC transporter family members and were less sensitive to cyclosporine A. We conclude that differentiated RPTEC/TERT1 cells are well suited for utilisation in xenobiotic transport and pharmacokinetic studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. 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).

  13. 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

  14. Long-term regulation of vacuolar H(+)-ATPase by angiotensin II in proximal tubule cells.

    PubMed

    Carraro-Lacroix, L R; Girardi, A C C; Malnic, G

    2009-09-01

    Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.

  15. 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.

  16. 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

  17. Puerarin protects against cadmium-induced proximal tubular cell apoptosis by restoring mitochondrial function.

    PubMed

    Song, Xiang-Bin; Liu, Gang; Wang, Zhen-Yong; Wang, Lin

    2016-12-25

    Puerarin (PU) is a potent free radical scavenger with a protective effect in nephrotoxin-mediated oxidative damage. Here, we show a novel molecular mechanism by which PU exerts its anti-apoptotic effects in cadmium (Cd)-exposed primary rat proximal tubular (rPT) cells. Morphological assessment and flow cytometric analysis revealed that PU significantly decreased Cd-induced apoptotic cell death of rPT cells. Administration of PU protected cells against Cd-induced depletion of mitochondrial membrane potential (ΔΨm) and lipid peroxidation. Cd-mediated mitochondrial permeability transition pore (MPTP) opening, disruption of mitochondrial ultrastructure, mitochondrial cytochrome c (cyt-c) release, caspase-3 activation and subsequently poly ADP-ribose polymerase (PARP) cleavage could be effectively blocked by the addition of PU. Moreover, up-regulation of Bcl-2 and down-regulation of Bax and hence increased Bcl-2/Bax ratio were observed with the PU administration. In addition, PU reversed Cd-induced ATP depletion by restoring ΔΨm to affect ATP production and by regulating expression levels of ANT-1 and ANT-2 to improve ATP transport. In summary, PU inhibited Cd-induced apoptosis in rPT cells by ameliorating the mitochondrial dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Reversal of radiocontrast medium toxicity in human renal proximal tubular cells by white grape juice extract.

    PubMed

    Andreucci, Michele; Faga, Teresa; Pisani, Antonio; Sabbatini, Massimo; Russo, Domenico; Mattivi, Fulvio; De Sarro, Giovambattista; Navarra, Michele; Michael, Ashour

    2015-03-05

    Radiocontrast media (RCM)-induced nephrotoxicity (CIN) is a major clinical problem accounting for 12% of all hospital-acquired cases of acute kidney injury. The pathophysiology of CIN is not well understood, but direct toxic effects on renal cells have been postulated as contributing to CIN. We have investigated the effect of a white grape (Vitis vinifera) juice extract (WGJe) on human renal proximal tubular (HK-2) cells treated with the radiocontrast medium (RCM) sodium diatrizoate. WGJe caused an increase in phosphorylation of the prosurvival kinases Akt and ERK1/2 in HK-2 cells. Treatment of HK-2 cells with 75 mgI/ml sodium diatrizoate for 2.5h and then further incubation (for 27.5h) after removal of the RCM caused a drastic decrease in cell viability. However, pre-treatment with WGJe, prior to incubation with diatrizoate, dramatically improved cell viability. Analysis of key signaling molecules by Western blotting showed that diatrizoate caused a drastic decrease in phosphorylation of Akt (Ser473), FOXO1 (Thr24) and FOXO3a (Thr32) during the initial 2.5h incubation period, and WGJe pre-treatment caused a reversal of these effects. Further analysis by Western blotting of samples from HK-2 cells cultured for longer periods of time (for up to 27.5h after an initial 2.5h exposure to diatrizoate with or without WGJe pre-treatment) showed that WGJe pre-treatment caused a negative effect on phosphorylation of p38, NF-κB (Ser276) and pERK1/2 whilst having a positive effect on the phosphorylation of Akt, FOXO1/FOXO3a and maintained levels of Pim-1 kinase. WGJe may alleviate RCM toxicity through modulation of signaling molecules that are known to be involved in cell death and cell survival and its possible beneficial effects should be further investigated. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Isoflurane alters proximal tubular cell susceptibility to toxic and hypoxic forms of attack.

    PubMed

    Zager, R A; Burkhart, K M; Conrad, D S

    1999-01-01

    Fluorinated anesthetics can profoundly alter plasma membrane structure and function, potentially impacting cell injury responses. Because major surgery often precipitates acute renal failure, this study assessed whether the most commonly used fluorinated anesthetic, isoflurane, alters tubular cell responses to toxic and hypoxic attack. Mouse proximal tubule segments were incubated under control conditions or with a clinically relevant isoflurane dose. Cell viability (lactate dehydrogenase release), deacylation (fatty acid, such as C20:4 levels), and adenosine triphosphate (ATP) concentrations were assessed under one or more of the following conditions: (a) exogenous phospholipase A2 (PLA2) or C20:4 addition, (b) Ca2+ overload (A23187 ionophore), (c) increased metabolic work (Na ionophore), and (d) hypoxia- or antimycin A-induced attack. Isoflurane's effect on NBD phosphatidylserine uptake (an index of plasma membrane aminophospholipid translocase activity) was also assessed. Isoflurane alone caused trivial deacylation and no lactate dehydrogenase release. However, it strikingly sensitized to both PLA2- and A23187-induced deacylation and cell death. Isoflurane also exacerbated C20:4's direct membrane lytic effect. Under conditions of mild ATP depletion (Na ionophore-induced increased ATP consumption; PLA2-induced mitochondrial suppression), isoflurane provoked moderate/severe ATP reductions and cell death. Conversely, under conditions of maximal ATP depletion (hypoxia, antimycin), isoflurane conferred a modest cytoprotective effect. Isoflurane blocked aminophospholipid translocase activity, which normally maintains plasma membrane lipid asymmetry (that is, preventing its "flip flop"). Isoflurane profoundly and differentially affects tubular cell responses to toxic and hypoxic attack. Direct drug-induced alterations in lipid trafficking/plasma membrane orientation and in cell energy production are likely involved. Although the in vivo relevance of these findings

  20. Mesenchymal stem cell-conditioned medium accelerates regeneration of human renal proximal tubule epithelial cells after gentamicin toxicity.

    PubMed

    Moghadasali, Reza; Mutsaers, Henricus A M; Azarnia, Mahnaz; Aghdami, Nasser; Baharvand, Hossein; Torensma, Ruurd; Wilmer, Martijn J G; Masereeuw, Rosalinde

    2013-07-01

    Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity to regenerate renal tubule epithelia and repair renal function without fusing with resident tubular cells. The goal of the present project was to investigate the role of MSCs secreted cytokines on tubule cell viability and regeneration after a toxic insult, using a conditionally immortalized human proximal tubule epithelial cell (ciPTEC) line. Gentamicin was used to induce nephrotoxicity, and cell viability and migration were studied in absence and presence of human MSC-conditioned medium (hMSC-CM) i.e. medium containing soluble factors produced and secreted by MSCs. Exposure of ciPTEC to 0-3000 μg/ml gentamicin for 24 h caused a significant dose-dependent increase in cell death. We further demonstrated that the nephrotoxic effect of 2000 μg/ml gentamicin was recovered partially by exposing cells to hMSC-CM. Moreover, exposure of ciPTEC to gentamicin (1500-3000 μg/ml) for 7 days completely attenuated the migratory capacity of the cells. In addition, following scrape-wounding, cell migration of both untreated and gentamicin-exposed cells was increased in the presence of hMSC-CM, as compared to exposures to normal medium, indicating improved cell recovery. Our data suggest that cytokines secreted by MSCs stimulate renal tubule cell regeneration after nephrotoxicity. Copyright © 2012 Elsevier GmbH. All rights reserved.

  1. Scintillators and applications thereof

    DOEpatents

    Williams, Richard T.

    2015-09-01

    Scintillators of various constructions and methods of making and using the same are provided. In some embodiments, a scintillator comprises at least one radiation absorption region and at least one spatially discrete radiative exciton recombination region.

  2. Scintillators and applications thereof

    DOEpatents

    Williams, Richard T.

    2014-07-15

    Scintillators of various constructions and methods of making and using the same are provided. In some embodiments, a scintillator comprises at least one radiation absorption region and at least one spatially discrete radiative exciton recombination region.

  3. 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.

  4. 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.

  5. Microparticles released by vascular endothelial cells increase hypoxia inducible factor expression in human proximal tubular HK-2 cells.

    PubMed

    Fernandez-Martínez, Ana Belen; Torija, Ana Valdehita; Carracedo, Julia; Ramirez, Rafael; de Lucio-Cazaña, Francisco Javier

    2014-08-01

    Microparticles are produced by vesiculation of the cell plasma membrane and serve as vectors of cell-to-cell communication. Co-culture experiments have shown that hypoxia-inducible factor-α (HIF-α)-regulated-genes are up-regulated in human renal proximal tubular HK-2 cells by endothelial cell factors which might be transported inside endothelial microparticles (EMP). Here we aimed to study in HK-2 cells the effect of EMP, produced by activated endothelial cells, on HIF-α and HIF-α-regulated vascular endothelial growth factor-A (VEGF-A). EMP, at a concentration much lower than that found in plasma, increased the expression of HIF-α/VEGF-A in a COX-2/EP2 receptor dependent manner. Since the EMP/cells ratio was ∼1/1000, we hypothesized that paracrine mediators produced by HK-2 cells amplified the initial signal. This hypothesis was confirmed by two facts which also suggested that the mediators were conveyed by particles released by HK-2 cells: (i) HIF-α was up-regulated in HK-2 cells treated with the pellet obtained from the conditioned medium of the EMP-treated HK-2 cells. (ii) In transwell experiments, EMP-treated cells increased the expression of HIF-α in untreated HK-2 cells. Interestingly, we detected these cells, particles that were released by EMP-treated HK-2 cells. Depending on the pathological context, activation of HIF-α and VEGF-A signaling in renal tissue/cells may have either beneficial or harmful effects. Therefore, our results suggest that their presence in the urinary space of EMP produced by activated endothelial cells may influence the outcome of a number of renal diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Aldosterone stimulates activity and surface expression of NHE3 in human primary proximal tubule epithelial cells (RPTEC).

    PubMed

    Drumm, Karina; Kress, Theresia R; Gassner, Birgit; Krug, Alexander W; Gekle, Michael

    2006-01-01

    The steroid hormone aldosterone is a major regulator of extracellular volume and blood pressure. Aldosterone effectors are for example the epithelial Na(+) channel (ENaC), the Na(+)-K(+)-ATPase and the proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3). The aim of this study was to investigate whether aldosterone acts directly on proximal tubule cells to stimulate NHE3 and if so whether the EGF-receptor (EGFR) is involved. For this purpose, primary human renal proximal tubule cells were exposed to aldosterone. NHE3 activity was determined from Na(+)- dependent pH-recovery, NHE3 surface expression was determined by biotinylation and immunoblotting. EGFR-expression was assessed by ELISA. pH(i)- measurements revealed an aldosterone-induced increase in NHE3 activity, which was inhibited by the mineralocorticoid receptor blocker spironolactone and by the EGFR-kinase inhibitor AG1478. Immunoprecipitation and immunoblot analysis showed an aldosterone-induced increase in NHE3 surface expression, which was also inhibited by spironolactone and AG1478. Furthermore, aldosterone enhanced EGFR-expression. In conclusion, aldosterone stimulates NHE3 in human proximal tubule cells. The underlying mechanisms include AG1478 inhibitable kinase and are paralleled by enhanced EGFR expression, which could be compatible with EGF-receptor-pathway-dependent surface expression and activity of NHE3 in human primary renal proximal tubule epithelial cells.

  7. Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells.

    PubMed

    Ugarte, Giorgia D; Vargas, Macarena F; Medina, Matías A; León, Pablo; Necuñir, David; Elorza, Alvaro A; Gutiérrez, Soraya E; Moon, Randall T; Loyola, Alejandra; De Ferrari, Giancarlo V

    2015-10-08

    Chromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/β-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that β-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/β-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia.

  8. GIANT CELL TUMOR IN THE PROXIMAL PHALANX WITH PULMONARY METASTASIS: CASE REPORT AND LITERATURE REVIEW

    PubMed Central

    de Medeiros, Frederico Carvalho; de Medeiros, Fernando Carvalho; de Campos Carvalho Lopes, Izabella; de Medeiros, Guilherme Carvalho; de Medeiros, Eduardo Carvalho

    2015-01-01

    This is a case report on a giant cell tumor (GCT) in the proximal phalanx of the third finger of the left hand, with pulmonary metastasis. The patient presented pain in the finger without any previous history of trauma. Clinical examination, radiographic imaging and magnetic resonance imaging were carried out. A histological evaluation was carried out from an incisional biopsy, taking the hypothesis of GCT. The patient underwent amputation of the finger and the diagnosis was confirmed by means of microscopy on the specimen. The patient was followed up because of the risk of lung metastasis, which was shown by radiographic examination and computed tomography on the chest, and thoracotomy was performed. Since then, there has been an improvement in the symptoms that had been reported preoperatively, and no local recurrence or new metastasis has been found. PMID:27027012

  9. High latitude scintillations

    NASA Astrophysics Data System (ADS)

    Basu, Santimay; Basu, Sunanda

    High-latitude phase and amplitude scintillations have been observed with quasi-geostationary polar beacon satellites, high-altitude orbiting GPS satellites, and low-altitude orbiting HiLat and Polar Bear satellites. The scintillation behavior observed in the polar cap, cusp, and nightside auroral oval is described. Consideration is given to the possible mechanisms for the generation of irregularities that cause scintillations. The importance of coordinated multitechnique measurements for scintillation studies is stressed.

  10. Ochratoxin A induced premature senescence in human renal proximal tubular cells.

    PubMed

    Yang, Xuan; Liu, Sheng; Huang, Chuchu; Wang, Haomiao; Luo, Yunbo; Xu, Wentao; Huang, Kunlun

    2017-05-01

    Ochratoxin A (OTA) has many nephrotoxic effects and is a promising compound for the study of nephrotoxicity. Human renal proximal tubular cells (HKC) are an important model for the study of renal reabsorption, renal physiology and pathology. Since the induction of OTA in renal senescence is largely unknown, whether OTA can induce renal senescence, especially at a sublethal dose, and the mechanism of OTA toxicity remain unclear. In our study, a sublethal dose of OTA led to an enhanced senescent phenotype, β-galactosidase staining and senescence associated secretory phenotype (SASP). Cell cycle arrest and cell shape alternations also confirmed senescence. In addition, telomere analysis by RT-qPCR allowed us to classify OTA-induced senescence as a premature senescence. Western blot assays showed that the p53-p21 and the p16-pRB pathways and the ezrin-associated cell spreading changes were activated during the OTA-induced senescence of HKC. In conclusion, our results demonstrate that OTA promotes the senescence of HKC through the p53-p21 and p16-pRB pathways. The understanding of the mechanisms of OTA-induced senescence is critical in determining the role of OTA in cytotoxicity and its potential carcinogenicity. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. 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.

  12. Label retaining cells (LRCs) with myoepithelial characteristic from the proximal acinar region define stem cells in the sweat gland.

    PubMed

    Leung, Yvonne; Kandyba, Eve; Chen, Yi-Bu; Ruffins, Seth; Kobielak, Krzysztof

    2013-01-01

    Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating

  13. Label Retaining Cells (LRCs) with Myoepithelial Characteristic from the Proximal Acinar Region Define Stem Cells in the Sweat Gland

    PubMed Central

    Leung, Yvonne; Kandyba, Eve; Chen, Yi-Bu; Ruffins, Seth; Kobielak, Krzysztof

    2013-01-01

    Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating

  14. Scintillator manufacture at Fermilab

    SciTech Connect

    Mellott, K.; Bross, A.; Pla-Dalmau, A.

    1998-08-01

    A decade of research into plastic scintillation materials at Fermilab is reviewed. Early work with plastic optical fiber fabrication is revisited and recent experiments with large-scale commercial methods for production of bulk scintillator are discussed. Costs for various forms of scintillator are examined and new development goals including cost reduction methods and quality improvement techniques are suggested.

  15. Lead carbonate scintillator materials

    DOEpatents

    Derenzo, Stephen E.; Moses, William W.

    1991-01-01

    Improved radiation detectors containing lead carbonate or basic lead carbonate as the scintillator element are disclosed. Both of these scintillators have been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to other known scintillator materials. The radiation detectors disclosed are favorably suited for use in general purpose detection and in medical uses.

  16. Scintillator materials for calorimetry

    SciTech Connect

    Weber, M.J.

    1994-09-01

    Requirements for fast, dense scintillator materials for calorimetry in high energy physics and approaches to satisfying these requirements are reviewed with respect to possible hosts and luminescent species. Special attention is given to cerium-activated crystals, core-valence luminescence, and glass scintillators. The present state of the art, limitations, and suggestions for possible new scintillator materials are presented.

  17. Scintillator manufacture at Fermilab

    NASA Astrophysics Data System (ADS)

    Mellott, K.; Bross, A.; Pla-Dalmau, A.

    1998-11-01

    A decade of research into plastic scintillation materials at Fermilab is reviewed. Early work with plastic optical fiber fabrication is revisited and recent experiments with large-scale commercial methods for production of bulk scintillator are discussed. Costs for various forms of scintillator are examined and new development goals including cost reduction methods and quality improvement techniques are suggested.

  18. Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells.

    PubMed

    Selbi, W; de la Motte, C A; Hascall, V C; Day, A J; Bowen, T; Phillips, A O

    2006-10-01

    Alteration in the glycosaminoglycan hyaluronan (HA) has been demonstrated in numerous renal diseases. We have demonstrated that renal proximal tubular epithelial cells (PTCs) surround themselves in vitro with HA in an organized pericellular matrix or 'coat', which is associated with cell migration, and also form pericellular HA cable-like structures which modulate PTC-mononuclear leukocytes interactions. The aim of this study was to characterize potential regulatory mechanism in the assembly of PTC-HA into pericellular cables. HA cables are generated by PTCs in the absence of serum. Immunohistochemical analysis demonstrates the incorporation of components of the inter-alpha-inhibitor (IalphaI) family of proteins and versican into HA cables. Addition of an antibody to IalphaI/PalphaI (pre-alpha-inhibitor) inhibits cable formation. In contrast, inhibition of tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) has no effect on cable formation, suggesting that their generation is independent of the known heavy-chain transfer activity of TSG-6. Overexpression of HAS3 is associated with induction of HA cable formation, and also increased incorporation of HA into pericellular coats. Functionally, this resulted in enhanced HA-dependent monocyte binding and cell migration, respectively. Cell surface expression of CD44 and trypsin-released cell-associated HA were increased in HAS3-overexpressing cells. In addition, hyaluronidase (hyal1 and hyal2) and bikunin mRNA expression were increased, whereas PalphaI HC3 mRNA expression was unchanged in the transfected cells. The data demonstrate the importance of IalphaI/PalphaI in cable formation and suggest that expression of HAS3 may be critical for HA cable assembly.

  19. The transcriptome of the Didelphis virginiana opossum kidney OK proximal tubule cell line.

    PubMed

    Eshbach, Megan L; Sethi, Rahil; Avula, Raghunandan; Lamb, Janette; Hollingshead, Deborah J; Finegold, David N; Locker, Joseph D; Chandran, Uma R; Weisz, Ora A

    2017-09-01

    The OK cell line derived from the kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for D. virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The M. domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the D. virginiana OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the M. domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the M. domestica genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells. Copyright © 2017 the American Physiological Society.

  20. Development of bioartificial renal tubule devices with lifespan-extended human renal proximal tubular epithelial cells.

    PubMed

    Sanechika, Noriyuki; Sawada, Kaichiro; Usui, Yukio; Hanai, Kazuya; Kakuta, Takatoshi; Suzuki, Hajime; Kanai, Genta; Fujimura, Satoshi; Yokoyama, Tun Aung; Fukagawa, Masafumi; Terachi, Toshiro; Saito, Akira

    2011-09-01

    The bioartificial renal tubule device is a cell therapy system for renal failure. The major obstacle in the development of the bioartificial renal tubule device is the obtainment of a large number of viable renal tubule cells to seed on the inner surface of hollow fibers. Although our previous studies had used a transformed cell line, they may be dangerous for clinical uses. Therefore, different approaches to amplify renal proximal tubular epithelial cells (RPTEC) in culture without oncogenes, vectors and carcinogens have been required. The limitation of the replicative lifespan of human RPTEC, which is ∼12 population doublings (PDs), was extended by invalidating messenger RNA of cell cycle-related genes with antisense oligonucleotide or small interfering RNA (siRNA). Periodic transfection of siRNA to a tumor suppressor p53 or a cyclin-dependent kinase inhibitor p16(INK4a) extended the lifespan by 33 and 63 PDs, respectively, in 3 months of culture. The siRNA-mediated lifespan extension was controllable because cell division ceased within 2 weeks after the transfection was discontinued. Expressions of γ-glutamyltransferase 1 and glucose transporter 1 were recovered in siRNA-transfected RPTEC cultured on porous membranes. Bioartificial renal tubule devices (0.8 m(2)) constructed with these cells showed reabsorption of water (122.3 ± 4.2 mL/30 min), sodium (18.1 ± 0.7 mEq/30 min) and glucose (121.7 ± 4.4 mg/30 min) after 1 week of circulation. Furthermore, β2-microglobulin and pentosidine were metabolized by RPTEC in mini-devices (65 cm(2)) within 48 h of circulation. These approaches enabled us to yield a high enough number of RPTEC for construction of bioartificial renal tubule devices repeatedly. Lifespan-extended RPTEC could recover their specific characteristics by culturing on porous membranes, and bioartificial renal tubule devices constructed with these cells showed good performances of reabsorption and metabolism. A large number of human renal tubular

  1. 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.

  2. 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

  3. Engineered extracellular matrices with controlled mechanics modulate renal proximal tubular cell epithelialization

    PubMed Central

    Chen, Evan; Putnam, Andrew J.

    2017-01-01

    Acute kidney injury (AKI) is common and associated with significant morbidity and mortality. Recovery from many forms of AKI involves the proliferation of renal proximal tubular epithelial cells (RPTECs), but the influence of the microenvironment in which this recovery occurs remains poorly understood. Here we report the development of a poly(ethylene glycol) (PEG) hydrogel platform to study the influence of substrate mechanical properties on the proliferation of human RPTECs as a model for recovery from AKI. PEG diacrylate based hydrogels were generated with orthogonal control of mechanics and cell-substrate interactions. Using this platform, we found that increased substrate stiffness promotes RPTEC spreading and proliferation. RPTECs showed similar degrees of apoptosis and Yes-associated protein (YAP) nuclear localization regardless of stiffness, suggesting these were not key mediators of the effect. However, focal adhesion formation, cytoskeletal organization, focal adhesion kinase (FAK) activation, and extracellular signal-regulated kinase (ERK) activation were all enhanced with increasing substrate stiffness. Inhibition of ERK activation substantially attenuated the effect of stiffness on proliferation. In long-term culture, hydrogel stiffness promoted the formation of more complete epithelial monolayers with tight junctions, cell polarity, and an organized basement membrane. These data suggest that increased stiffness potentially may have beneficial consequences for the renal tubular epithelium during recovery from AKI. PMID:28715434

  4. Engineered extracellular matrices with controlled mechanics modulate renal proximal tubular cell epithelialization.

    PubMed

    Beamish, Jeffrey A; Chen, Evan; Putnam, Andrew J

    2017-01-01

    Acute kidney injury (AKI) is common and associated with significant morbidity and mortality. Recovery from many forms of AKI involves the proliferation of renal proximal tubular epithelial cells (RPTECs), but the influence of the microenvironment in which this recovery occurs remains poorly understood. Here we report the development of a poly(ethylene glycol) (PEG) hydrogel platform to study the influence of substrate mechanical properties on the proliferation of human RPTECs as a model for recovery from AKI. PEG diacrylate based hydrogels were generated with orthogonal control of mechanics and cell-substrate interactions. Using this platform, we found that increased substrate stiffness promotes RPTEC spreading and proliferation. RPTECs showed similar degrees of apoptosis and Yes-associated protein (YAP) nuclear localization regardless of stiffness, suggesting these were not key mediators of the effect. However, focal adhesion formation, cytoskeletal organization, focal adhesion kinase (FAK) activation, and extracellular signal-regulated kinase (ERK) activation were all enhanced with increasing substrate stiffness. Inhibition of ERK activation substantially attenuated the effect of stiffness on proliferation. In long-term culture, hydrogel stiffness promoted the formation of more complete epithelial monolayers with tight junctions, cell polarity, and an organized basement membrane. These data suggest that increased stiffness potentially may have beneficial consequences for the renal tubular epithelium during recovery from AKI.

  5. Interleukin 2 mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.

    PubMed Central

    Brooimans, R A; Stegmann, A P; van Dorp, W T; van der Ark, A A; van der Woude, F J; van Es, L A; Daha, M R

    1991-01-01

    Previous reports have suggested the production of complement components C4, C2, and factor B by renal tissue. However, the cells involved in production of complement have not been identified. In this study metabolic labeling experiments demonstrated that human proximal tubular epithelial cells (PTEC) synthesize a 180-kD precursor of C3 that is secreted after proteolytic cleavage into a disulphide linked two-chain molecule as found in plasma. C3 present in culture supernatants of PTEC was functionally active, however, during the culture period there was a partial inactivation of the C3 molecule as assessed by hemolytic titration. Recombinant IL-2 enhances the rate of C3 synthesis in a dose-dependent manner reaching maximal stimulation at doses of 200-400 U/ml IL-2. Northern blot analysis demonstrated a 5.2-kb C3 mRNA species present in PTEC that was increased within 24 h of IL-2 treatment. IL-2-induced enhancement of C3 production by PTEC could be neutralized with specific antibodies to IL-2. This study demonstrates that C3 synthesis in PTEC is upregulated by IL-2, the major cytokine produced by activated T cells. Images PMID:1864952

  6. Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

    SciTech Connect

    Lee, Seung-Min; Attieh, Zouhair K.; Son, Hee Sook; Chen, Huijun; Bacouri-Haidar, Mhenia; Vulpe, Chris D.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a

  7. Scintillator reflective layer coextrusion

    DOEpatents

    Yun, Jae-Chul; Para, Adam

    2001-01-01

    A polymeric scintillator has a reflective layer adhered to the exterior surface thereof. The reflective layer comprises a reflective pigment and an adhesive binder. The adhesive binder includes polymeric material from which the scintillator is formed. A method of forming the polymeric scintillator having a reflective layer adhered to the exterior surface thereof is also provided. The method includes the steps of (a) extruding an inner core member from a first amount of polymeric scintillator material, and (b) coextruding an outer reflective layer on the exterior surface of the inner core member. The outer reflective layer comprises a reflective pigment and a second amount of the polymeric scintillator material.

  8. 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.

  9. MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

    PubMed Central

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

    2014-01-01

    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. PMID:25015657

  10. Effect of gentamicin on phospholipid metabolism in cultured rabbit proximal tubular cells

    SciTech Connect

    Ramsammy, L.S.; Josepovitz, C.; Lane, B.; Kaloyanides, G.J.

    1989-01-01

    We examined the hypothesis that the accumulation of phospholipid in cells exposed to gentamicin is due to impaired degradation. Experiments were performed in rabbit proximal tubular cells grown in primary culture. Cells exposed to 10(-3) M gentamicin manifested myeloid body formation and a progressive increase in total phospholipid that by day 6 was 44% higher than that of control cells and reflected increases of phosphatidylinositol of 235%, phosphatidylcholine of 60%, phosphatidylethanolamine of 90%, and phosphatidylserine of 55% above control values. Gentamicin impaired the degradation of these phospholipids. The t1/2 of the phospholipid pool labeled with (3H)myoinositol increased 146% from 1.17 (control) to 2.88 days (gentamicin); the t1/2 of the (3H)choline pool increased 34% from 1.77 to 2.38 days; the t1/2 of the (3H)ethanolamine pool increased 57% from 3.14 to 4.93 days; the t1/2 of the (3H) serine pool increased 37% from 6.30 to 8.63 days. Exposure of cells to gentamicin for 2 days also stimulated increased incorporation of (3H)myoinositol (68%) and (3H)ethanolamine (59%) into phospholipid. The data are consistent with the hypothesis that gentamicin inhibits the activity of lysosomal phospholipases that results in the accumulation of phospholipid within the lysosome in the form of myeloid bodies. Increased phospholipid synthesis may represent a compensatory response to the impaired lysosomal degradation of phospholipid. We postulate that the preferential increase of phosphatidylinositol reflects the capacity of the polycationic gentamicin to interact electrostatically with the anionic phosphoinositides and inhibit their turnover.

  11. Dead cell and side leakage correction for a lead-scintillating fiber electromagnetic calorimeter

    NASA Astrophysics Data System (ADS)

    Zhang, Cheng; Li, Zu-Hao; Tang, Zhi-Cheng; Basegmez du Pree, Suzan; Zhang, Shao-Wen; Wang, Xue-Qiang; Yang, Min; Chen, Guo-Ming; Chen, He-Sheng

    2016-09-01

    The electromagnetic calorimeter (ECAL) of the Alpha Magnetic Spectrometer (AMS-02) is one of the key detectors for dark matter searches. It measures the energies of electrons, positrons and photons and seperates them from hadrons. Currently, there are 5 dead cells in the ECAL, which affect the reconstructed energy of 4.2% of total events in the ECAL acceptance. When an electromagnetic shower axis is close to the ECAL border, due to the side leakage, the reconstructed energy is affected as well. In this paper, methods for dead cells and side leakage corrections for the ECAL energy reconstruction are presented. For events with the shower axis crossing dead cells, applying dead cell correction improves the difference in the reconstructed energy from 12% to 1%, while for events near the ECAL border, with side leakage correction it is improved from 4% to 1%. Supported by National Natural Science Foundation of China(11220101004)

  12. Lack of induction of single-strand breaks in mammalian cells by sodium azide and its proximal mutagen

    SciTech Connect

    Arenaz, P.; Nilan, R.A.; Kleinhofs, A.

    1981-01-01

    The mutagenicity of sodium azide in both higher plants and bacteria is well documented. However, in mammalian cells, research on the effects of azide on gene mutations has produced conflicting results. Furthermore, no research has been conducted on the effects of azide and its proximal mutagen (mutagenic metabolite) on single-strand breaks. The experiments herein were designed to overcome this lack of information on azide mutagenicity and to evaluate the potential hazard of azide exposure to man. Chinese hamster V/sub 79/ cells were treated with either azide or its proximal mutagen(s) for 2 h or 6 h respectively and analyzed by alkaline elution for single-strand breaks. The data showed that there was no effect of either azide or the proximal mutagen(s) on single-strand DNA breaks nor was there any indication that azide or the proximal mutagen(s) induced DNA-protein crosslinks. The data suggest that neither azide nor its proximal mutagen(s) interact directly with DNA and suggest that the methods may be applied to any specified population and dose scenario.

  13. CBAP promotes thymocyte negative selection by facilitating T-cell receptor proximal signaling

    PubMed Central

    Ho, K-C; Chiang, Y-J; Lai, A C-Y; Liao, N-S; Chang, Y-J; Yang-Yen, H-F; Yen, J J-Y

    2014-01-01

    T-cell receptor (TCR)-transduced signaling is critical to thymocyte development at the CD4/CD8 double-positive stage, but the molecules involved in this process are not yet fully characterized. We previously demonstrated that GM-CSF/IL-3/IL-5 receptor common β-chain-associated protein (CBAP) modulates ZAP70-mediated T-cell migration and adhesion. On the basis of the high expression of CBAP during thymocyte development, we investigated the function of CBAP in thymocyte development using a CBAP knockout mouse. CBAP-deficient mice showed normal early thymocyte development and positive selection. In contrast, several negative selection models (including TCR transgene, superantigen staphylococcal enterotoxin B, and anti-CD3 antibody treatment) revealed an attenuation of TCR-induced thymocyte deletion in CBAP knockout mice. This phenotype correlated with a reduced accumulation of BIM upon TCR crosslinking in CBAP-deficient thymocytes. Loss of CBAP led to reduced TCR-induced phosphorylation of proteins involved in both proximal and distal signaling events, including ZAP70, LAT, PLCγ1, and JNK1/2. Moreover, TCR-induced association of LAT signalosome components was reduced in CBAP-deficient thymocytes. Our data demonstrate that CBAP is a novel component in the TCR signaling pathway and modulates thymocyte apoptosis during negative selection. PMID:25393474

  14. T-cell functional regions of the human IL-3 proximal promoter.

    PubMed

    Ryan, G R; Vadas, M A; Shannon, M F

    1994-10-01

    The human interleukin-3 (IL-3) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post-transcriptional level. We have previously shown that treatment of Jurkat T cells with phytohemaglutinin (PHA) and the phorbol ester, PMA, activated transcription initiation from the IL-3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL-3 gene 5' and 3' flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5' flanking region of the IL-3 gene. The promoter region between -173 and -60 contained the strongest activating elements. The transcription factor AP-1 could bind to this positive activator region of the promoter. We also examined the function of the IL-3 CK-1/CK-2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/PHA.

  15. BAG3 regulates ECM accumulation in renal proximal tubular cells induced by TGF-β1.

    PubMed

    Du, Feng; Li, Si; Wang, Tian; Zhang, Hai-Yan; Li, De-Tian; Du, Zhen-Xian; Wang, Hua-Qin; Wang, Yan-Qiu

    2015-01-01

    Previously we have demonstrated that Bcl-2-associated athanogene 3 (BAG3) is increased in renal fibrosis using a rat unilateral ureteral obstruction model. The current study investigated the role of BAG3 in renal fibrosis using transforming growth factor (TGF)-β1-treated human proximal tubular epithelial (HK-2) cells. An upregulation of BAG3 in vitro models was observed, which correlated with the increased synthesis of extracellular matrix (ECM) proteins and expression of tissue-type plasminogen activator inhibitor (PAI)-1. Blockade of BAG3 induction by shorting hairpin RNA suppressed the expression of ECM proteins but had no effect on PAI-1 expression induced by TGF-β1. Forced overexpression of BAG3 selectively increased collagens. TGF-β1-induced BAG3 expression in HK-2 cells was attenuated by ERK1/2 and JNK MAPK inhibitors. In addition, forced BAG3 overexpression blocked attenuation of collagens expression by ERK1/2 and JNK inhibitors. These data suggest that ERK1/2 and JNK signaling events are involved in modulating the expression of BAG3, which would ultimately contribute to renal fibrosis by enhancing the synthesis and deposition of ECM proteins.

  16. 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.

  17. Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells.

    PubMed

    Wilmer, Martijn J; Kluijtmans, Leo A J; van der Velden, Thea J; Willems, Peter H; Scheffer, Peter G; Masereeuw, Rosalinde; Monnens, Leo A; van den Heuvel, Lambertus P; Levtchenko, Elena N

    2011-06-01

    Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.

  18. P-Glycoprotein Induction Ameliorates Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells

    PubMed Central

    Lee, Sun-hyo; Kim, Jin-sun; Ravichandran, Kameswaran; Gil, Hyo-Wook; Song, Ho-yeon; Hong, Sae-yong

    2015-01-01

    The pathogenesis of colistin induced nephrotoxicity is poorly understood. Currently there are no effective therapeutic or prophylactic agents available. This study was aimed to determine the mechanism of colistin induced nephrotoxicity and to determine whether P-glycoprotein (P-gp) induction could prevent colistin induced nephrotoxicity. Colistin induced cell toxicity in cultured human proximal tubular cells in both dose and time dependent manner. Colistin provoked ROS in a dose dependent manner as measured by DCF-DA. To investigate apoptosis, caspase 3/7 activity was determined. Caspase 3/7 activity was increased dose dependently (25, 50, 100 μg/ml) at 6 h. Autophagosome formation was assessed by measuring LC3- II/LC3-I ratio. The ratio of LC3-II to LC3- I was increased at 2 h (25 μg/ml). Suppression of autophagosome formation increased colistin induced nephrotoxicity. The expression of P-gp and the cell toxicity was determined in colistin with or without dexamethasone (P-gp inducer) and verapamil (selective P-gp inhibitor). Colistin itself suppressed the expression of P-gp. P-gp expression and activity decreased colistin induced nephrotoxicity with dexamethasone treatment. In addition induced P-gp transporter was shown to improve the efflux effect on colistin treated HK2 cell line, which was demonstrated by calcein-AM fluorescence accumulation assay. The increased activity could be blocked by N-acetylcysteine. In conclusion, colistin induces nephrotoxicity by suppressing P-gp. Induction of P-gp could ameliorate colistin induced nephrotoxicity by decreasing apoptosis. PMID:26287374

  19. 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.

  20. The Replisomes Remain Spatially Proximal throughout the Cell Cycle in Bacteria.

    PubMed

    Mangiameli, Sarah M; Veit, Brian T; Merrikh, Houra; Wiggins, Paul A

    2017-01-01

    The positioning of the DNA replication machinery (replisome) has been the subject of several studies. Two conflicting models for replisome localization have been proposed: In the Factory Model, sister replisomes remain spatially co-localized as the replicating DNA is translocated through a stationary replication factory. In the Track Model, sister replisomes translocate independently along a stationary DNA track and the replisomes are spatially separated for the majority of the cell cycle. Here, we used time-lapse imaging to observe and quantify the position of fluorescently labeled processivity-clamp (DnaN) complexes throughout the cell cycle in two highly-divergent bacterial model organisms: Bacillus subtilis and Escherichia coli. Because DnaN is a core component of the replication machinery, its localization patterns should be an appropriate proxy for replisome positioning in general. We present automated statistical analysis of DnaN positioning in large populations, which is essential due to the high degree of cell-to-cell variation. We find that both bacteria show remarkably similar DnaN positioning, where any potential separation of the two replication forks remains below the diffraction limit throughout the majority of the replication cycle. Additionally, the localization pattern of several other core replisome components is consistent with that of DnaN. These data altogether indicate that the two replication forks remain spatially co-localized and mostly function in close proximity throughout the replication cycle. The conservation of the observed localization patterns in these highly divergent species suggests that the subcellular positioning of the replisome is a functionally critical feature of DNA replication.

  1. The Replisomes Remain Spatially Proximal throughout the Cell Cycle in Bacteria

    PubMed Central

    Mangiameli, Sarah M.; Veit, Brian T.; Wiggins, Paul A.

    2017-01-01

    The positioning of the DNA replication machinery (replisome) has been the subject of several studies. Two conflicting models for replisome localization have been proposed: In the Factory Model, sister replisomes remain spatially co-localized as the replicating DNA is translocated through a stationary replication factory. In the Track Model, sister replisomes translocate independently along a stationary DNA track and the replisomes are spatially separated for the majority of the cell cycle. Here, we used time-lapse imaging to observe and quantify the position of fluorescently labeled processivity-clamp (DnaN) complexes throughout the cell cycle in two highly-divergent bacterial model organisms: Bacillus subtilis and Escherichia coli. Because DnaN is a core component of the replication machinery, its localization patterns should be an appropriate proxy for replisome positioning in general. We present automated statistical analysis of DnaN positioning in large populations, which is essential due to the high degree of cell-to-cell variation. We find that both bacteria show remarkably similar DnaN positioning, where any potential separation of the two replication forks remains below the diffraction limit throughout the majority of the replication cycle. Additionally, the localization pattern of several other core replisome components is consistent with that of DnaN. These data altogether indicate that the two replication forks remain spatially co-localized and mostly function in close proximity throughout the replication cycle. The conservation of the observed localization patterns in these highly divergent species suggests that the subcellular positioning of the replisome is a functionally critical feature of DNA replication. PMID:28114307

  2. Scintillator based beta batteries

    NASA Astrophysics Data System (ADS)

    Rensing, Noa M.; Tiernan, Timothy C.; Shirwadkar, Urmila; O'Dougherty, Patrick; Freed, Sara; Hawrami, Rastgo; Squillante, Michael R.

    2013-05-01

    Some long-term, remote applications do not have access to conventional harvestable energy in the form of solar radiation (or other ambient light), wind, environmental vibration, or wave motion. Radiation Monitoring Devices, Inc. (RMD) is carrying out research to address the most challenging applications that need power for many months or years and which have undependable or no access to environmental energy. Radioisotopes are an attractive candidate for this energy source, as they can offer a very high energy density combined with a long lifetime. Both large scale nuclear power plants and radiothermal generators are based on converting nuclear energy to heat, but do not scale well to small sizes. Furthermore, thermo-mechanical power plants depend on moving parts, and RTG's suffer from low efficiency. To address the need for compact nuclear power devices, RMD is developing a novel beta battery, in which the beta emissions from a radioisotope are converted to visible light in a scintillator and then the visible light is converted to electrical power in a photodiode. By incorporating 90Sr into the scintillator SrI2 and coupling the material to a wavelength-matched solar cell, we will create a scalable, compact power source capable of supplying milliwatts to several watts of power over a period of up to 30 years. We will present the latest results of radiation damage studies and materials processing development efforts, and discuss how these factors interact to set the operating life and energy density of the device.

  3. 'Special K' and a Loss of Cell-To-Cell Adhesion in Proximal Tubule-Derived Epithelial Cells: Modulation of the Adherens Junction Complex by Ketamine

    PubMed Central

    Hills, Claire E.; Jin, Tianrong; Siamantouras, Eleftherios; Liu, Issac K-K; Jefferson, Kieran P.; Squires, Paul E.

    2013-01-01

    Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention. PMID:24009666

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

    SciTech Connect

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

    1993-11-01

    The authors previously reported that cell cultures of human proximal tubule (HPT) cells respond to ionic cadmium in a manner consistent with well-defined Cd[sup 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 Cd[sup 2+] exposure at micromolar concentrations. These alterations are reexamined in detail in the present report to determine whether the Cd[sup 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 Cd[sup 2+] 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 Cd[sup 2+] exposure altered the characteristics of the apical cell membrane. Overall, the results were consistent with evidence of Cd[sup 2+]-induced alteration in the apical cell membrane of the HPT cell.

  5. 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

  6. Effects of SGLT2 inhibition in human kidney proximal tubular cells--renoprotection in diabetic nephropathy?

    PubMed

    Panchapakesan, Usha; Pegg, Kate; Gross, Simon; Komala, Muralikrishna Gangadharan; Mudaliar, Harshini; Forbes, Josephine; Pollock, Carol; Mather, Amanda

    2013-01-01

    Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3.

  7. Recent development in organic scintillators

    NASA Technical Reports Server (NTRS)

    Horrocks, D. L.; Wirth, H. O.

    1969-01-01

    Discussion on recent developments of organic scintillators includes studies of organic compounds that form glass-like masses which scintillate and are stable at room temperature, correlations between molecular structure of organic scintillators and self-quenching, recently developed fast scintillators, and applications of liquid-scintillation counters.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. The renoprotective role of autophagy activation in proximal tubular epithelial cells in diabetic nephropathy.

    PubMed

    Xu, Ying; Liu, Lei; Xin, Wei; Zhao, Xu; Chen, Liyong; Zhen, Junhui; Wan, Qiang

    2015-01-01

    With intensive investigations recently, autophagy is hoped to be a potential therapeutic target to prevent or alleviate diabetic nephropathy (DN). Our previous study revealed that lipotoxicity participated in epithelial-to-mesenchymal transition (EMT) of proximal tubular epithelial cells (PTECs) under diabetic conditions. Based on evidences that autophagy and lipid metabolism are closely related, we investigated autophagy under diabetic conditions and how it contributed in the lipotoxicity and EMT. In high-glucose-cultured PTECs, we found that Beclin1 and LC3-II were elevated, while p62 was decreased. These results indicate that autophagy activity was elevated under diabetic conditions. Autophagy deficiency induced by autophagy inhibitors, chloroquine diphosphate (CQ) and 3-Methyladenine (3-MA), and by Atg5 siRNA transfection exacerbated lipid accumulation and EMT. This supports that the elevated autophagy activity acts as a renoprotective response under diabetic conditions. Treatment of rapamycin, which is a mammalian target of rapamycin (mTOR) receptor-specific inhibitor and a known autophagy activator, attenuated high-glucose-induced lipid accumulation and EMT. The Atg5 silence counteracted the protective effect of rapamycin. The present study deepens our understanding of the role of autophagy in DN, suggesting a complex interplay of autophagy, metabolic pathways, lipotoxicity and EMT.

  13. Suppressive effects of iron on TGF-beta1 production by renal proximal tubular epithelial cells.

    PubMed

    Horino, Taro; Ito, Hiroyuki; Yamaguchi, Takuya; Furihata, Mutsuo; Hashimoto, Kozo

    2005-01-01

    TGF-beta1, which is one of the profibrogenic cytokines, is considered essential for both the tubulointerstitial fibrosis found in chronic kidney diseases and the repair of tissue damage in acute renal injury. Iron plays an important part in inflammatory damage since it supplies cytotoxic hydroxyl radicals. The aim of the present study was to examine the direct effects of iron on TGF-beta1 production and the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress, by human renal proximal tubular epithelial cells (RPTEC). Using human RPTEC, TGF-beta1 expression was studied by immunohistochemical staining, ELISA and RNase protection assays. 8-OHdG expression was evaluated by immunohistochemical staining. Ferric iron suppressed both TGF-beta1 secretion and mRNA expression, and enhanced 8-OHdG expression in RPTEC in a dose-dependent manner. Desferrioxamine, an iron chelator, eliminated the suppressive effect of ferric citrate on TGF-beta1 production. The results suggest that iron may delay the repair of kidney injury during the acute inflammatory phase via a reduction in TGF-beta1 production by RPTEC. Iron chelation may therefore be a useful strategy in the treatment of inflammatory kidney diseases.

  14. Dopamine and Angiotensin Type 2 Receptors Cooperatively Inhibit Sodium Transport in Human Renal Proximal Tubule Cells

    PubMed Central

    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.

    2012-01-01

    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 (RPTCs), sodium reabsorption is decreased by the dopamine D1-like receptors (D1R/D5R) and the angiotensin type 2 receptor (AT2R), while 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 D1R/D5R stimulation increased plasma membrane AT2R 4-fold via a D1R-mediated, cAMP-coupled, and PP2A-dependent specific signaling pathway. D1R/D5R stimulation also reduced the ability of angiotensin II to stimulate phospho-ERK, an effect that was partially reversed by an AT2R antagonist. Fenoldopam did not increase AT2R recruitment in RPTCs with D1Rs uncoupled from adenylyl cyclase, suggesting a role of cAMP in mediating these events. D1Rs and AT2Rs heterodimerized and cooperatively increased cAMP and cGMP production, PP2A 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. PMID:22710646

  15. Surface characteristics of acrylic modified polysulfone membranes improves renal proximal tubule cell adhesion and spreading.

    PubMed

    Teo, Jeremy Choon Meng; Ng, Roderica Rui Ge; Ng, Chee Ping; Lin, Alex Wei Haw

    2011-05-01

    Current polyvinylpyrrolidone-modified polysulfone (PVP-PSU) membranes in haemodialysers do not facilitate the attachment and proliferation of renal proximal tubule cells (RPTCs). For bioartificial kidney (BAK) development expensive extracellular matrices are employed to ensure the PVP-PSU membranes can serve as a substrate for RPTCs. In this study we modified PSU using an acrylic monomer (am-PSU) and polymerization using ultraviolet irradiation. We demonstrated that on adjusting the PSU or acrylic content of the membranes the wettability and surface chemistry were altered, and this affected the amount of fibronectin (Fn) that was adsorbed onto the membranes. Using an integrin blocking assay we ascertained that Fn is an important extracellular matrix component that mediates RPTC attachment. The amount of Fn adsorbed also led to different bioresponses of RPTCs, which were evaluated using attachment and proliferation assays and qualitative quantification of vinculin, focal adhesion kinase, zonula occludens and Na(+)/K(+) ATPase. Our optimized membrane, am-PSU1 (21.4% C-O groups, 19.1% PVP-PSU; contact angle 71.5-80.80, PVP-PSU: 52.4-67.50), supports a confluent monolayer of RPTCs and prevents creatinine and inulin diffusion from the apical to the basal side, meeting the requirements for application in BAKs. However, further in vivo evaluation to assess the full functionality of RPTCs on am-PSU1 is required. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  16. The CpG island in the murine foxl2 proximal promoter is differentially methylated in primary and immortalized cells.

    PubMed

    Tran, Stella; Wang, Ying; Lamba, Pankaj; Zhou, Xiang; Boehm, Ulrich; Bernard, Daniel J

    2013-01-01

    Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5' rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5' flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5' untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone.

  17. The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

    PubMed Central

    Tran, Stella; Wang, Ying; Lamba, Pankaj; Zhou, Xiang; Boehm, Ulrich; Bernard, Daniel J.

    2013-01-01

    Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5’ rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5’ flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5’ untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone. PMID:24098544

  18. Nitro-Arachidonic Acid Prevents Angiotensin II-Induced Mitochondrial Dysfunction in a Cell Line of Kidney Proximal Tubular Cells.

    PubMed

    Sánchez-Calvo, Beatriz; Cassina, Adriana; Rios, Natalia; Peluffo, Gonzalo; Boggia, José; Radi, Rafael; Rubbo, Homero; Trostchansky, Andres

    2016-01-01

    Nitro-arachidonic acid (NO2-AA) is a cell signaling nitroalkene that exerts anti-inflammatory activities during macrophage activation. While angiotensin II (ANG II) produces an increase in reactive oxygen species (ROS) production and mitochondrial dysfunction in renal tubular cells, little is known regarding the potential protective effects of NO2-AA in ANG II-mediated kidney injury. As such, this study examines the impact of NO2-AA on ANG II-induced mitochondrial dysfunction in an immortalized renal proximal tubule cell line (HK-2 cells). Treatment of HK-2 cells with ANG II increases the production of superoxide (O2●-), nitric oxide (●NO), inducible nitric oxide synthase (NOS2) expression, peroxynitrite (ONOO-) and mitochondrial dysfunction. Using high-resolution respirometry, it was observed that the presence of NO2-AA prevented ANG II-mediated mitochondrial dysfunction. Attempting to address mechanism, we treated isolated rat kidney mitochondria with ONOO-, a key mediator of ANG II-induced mitochondrial damage, in the presence or absence of NO2-AA. Whereas the activity of succinate dehydrogenase (SDH) and ATP synthase (ATPase) were diminished upon exposure to ONOO-, they were restored by pre-incubating the mitochondria with NO2-AA. Moreover, NO2-AA prevents oxidation and nitration of mitochondrial proteins. Combined, these data demonstrate that ANG II-mediated oxidative damage and mitochondrial dysfunction is abrogated by NO2-AA, identifying this compound as a promising pharmacological tool to prevent ANG II-induced renal disease.

  19. Subnanosecond Scintillation Detector

    NASA Technical Reports Server (NTRS)

    Hoenk, Michael (Inventor); Hennessy, John (Inventor); Hitlin, David (Inventor)

    2017-01-01

    A scintillation detector, including a scintillator that emits scintillation; a semiconductor photodetector having a surface area for receiving the scintillation, wherein the surface area has a passivation layer configured to provide a peak quantum efficiency greater than 40% for a first component of the scintillation, and the semiconductor photodetector has built in gain through avalanche multiplication; a coating on the surface area, wherein the coating acts as a bandpass filter that transmits light within a range of wavelengths corresponding to the first component of the scintillation and suppresses transmission of light with wavelengths outside said range of wavelengths; and wherein the surface area, the passivation layer, and the coating are controlled to increase the temporal resolution of the semiconductor photodetector.

  20. Measurement of radionuclides using ion chromatography and flow-cell scintillation counting with pulse shape discrimination

    SciTech Connect

    DeVol, T.A.; Fjeld, R.A.

    1995-10-01

    The use of ion chromatography (IC) for radiochemical separations is a well established technique. IC is commonly used in routine environmental monitoring applications as well as in specialized research applications. Typical usage involves the separation of a single radionuclide from the non-radioactive constituents. During the past decade, a limited amount of research has been conducted using automated IC systems in actinide separation applications (e.g.). More recently, separation procedures for common non-gamma emitting activation and fission products were developed utilizing a high performance liquid chromatography (HPLC) system. In addition, a separation procedure for six common actinides has been developed using a HPLC system. These latter systems used on-line flow-cell detectors for quantification of the radioactive constituents of the effluent stream.

  1. 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

  2. Study of equatorial scintillations

    NASA Technical Reports Server (NTRS)

    Pomalaza, J.; Woodman, R.; Tisnado, G.; Nakasone, E.

    1972-01-01

    Observations of the amplitude scintillations produced by the F-region in equatorial areas are presented. The equipment used for conducting the observations is described. The use of transmissions from the ATS-1, ATS-3, and ATS-5 for obtaining data is described. The two principal subjects discussed are: (1) correlation between satellite and incoherent radar observations of scintillations and (2) simultaneous observations of scintillations at 136 MHz and 1550 MHz.

  3. Lead carbonate scintillator materials

    DOEpatents

    Derenzo, S.E.; Moses, W.W.

    1991-05-14

    Improved radiation detectors containing lead carbonate or basic lead carbonate as the scintillator element are disclosed. Both of these scintillators have been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to other known scintillator materials. The radiation detectors disclosed are favorably suited for use in general purpose detection and in medical uses. 3 figures.

  4. Packed Red Blood Cells Are an Abundant and Proximate Potential Source of Nitric Oxide Synthase Inhibition

    PubMed Central

    Zwemer, Charles F.; Davenport, Robertson D.; Gomez-Espina, Juan; Blanco-Gonzalez, Elisa; Whitesall, Steven E.; D'Alecy, Louis G.

    2015-01-01

    Objective We determined, for packed red blood cells (PRBC) and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethylarginine (LNMMA). Background ADMA and LNMMA are near equipotent NOS inhibitors forming blood’s total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined. Methods We measured total (free and protein incorporated) ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis. Results In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM) and LNMMA (58.9 ± 28.9 μM) that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma. Conclusion The compelling physiological ramifications are that regardless of storage age, 1) PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2) PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate

  5. 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.

  6. [Effects of Rhein on the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and angiotensin II in rats].

    PubMed

    Yu, De-Qian; Gao, Yuan; Liu, Xiao-Hong

    2010-04-01

    To explore the effect of Rhein on the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and angiotensin II in rats. Studies were performed on anesthetized SD rats. Renal proximal tubular were gained by microdissection and cultured in RPMI-1640 medium. The cell types were identified by immunocytochemistry. The renal proximal tubular epithelial cells were incubated with high glucose (30 mmol/L) and angiotensin II (10(-7) mol/L) to induce the hypertrophy of cells. To observe the effect of Rhein on hypertrophy induced by high glucose and angiotensin II, renal proximal tubular epithelial cells were cultured with different concentrations of Rhein (30, 15, 5 mg/L) for 72 h, then cell size, 3H-leucine incorporation, and cellular protein content were detected to observe the changes. High glucose (30 mmol/L) and Ang II (10(-7) mol/L) induced hypertrophy of renal proximal tubular epithelial cells result in, cell size, 3H-leucine incorporation and cellular protein content increased significantly. On the contrary, Rhein inhibited the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and Angiotensin II. Rhein 30 mg/L significantly decreased cell size, 3H-leucine incorporation and cellular protein content. Rhein 15 mg/L decreased 3H-leucine incorporation and cellular protein content. Rhein 5 mg/L decreased cellular protein content. Rhein can inhibit the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and Angiotensin II in rats.

  7. The plant-derived natural compound Flavin 7 attenuates oxidative stress in cultured renal proximal tubule cells.

    PubMed

    Ember, Agoston; Clark, Jeb S; Varjas, Timea; Kiss, Istvan; Ember, Istvan; Baliga, Radhakrishna; Arany, Istvan

    2009-01-01

    Cancer therapies and cancer progression can increase oxidative stress that might account for renal toxicity in cancer patients. Flavin 7 (F7) is a natural polyphenol-containing dietary supplement with potential antioxidant activity. Therefore, it might help to attenuate renal toxicity of chemotherapeutics. Cultured mouse renal proximal tubule cells were subjected to H(2)O(2)-mediated oxidative stress. Potential antioxidant effects of F7 were assessed by measuring the production of reactive oxygen species (ROS), mitochondrial depolarization and injury (lactate dehydrogenase release as well as trypan blue exclusion) in cells that were pretreated with F7 prior to treatment with H(2)O(2). F7 pretreatment significantly attenuated H(2)O(2)-induced ROS production, mitochondrial depolarization and consequent injury in renal proximal tubule cells. F7 supplementation might be beneficial for cancer patients in order to prevent renal toxicity of anticancer drug- or cancer progression-related oxidative stress.

  8. Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells.

    PubMed

    Corr, Emma M; Cunningham, Clare C; Helbert, Laura; McCarthy, Geraldine M; Dunne, Aisling

    2017-02-07

    Osteoarthritis (OA) is a chronic debilitating joint disorder of particularly high prevalence in the elderly population. Intra-articular basic calcium phosphate (BCP) crystals are present in the majority of OA joints and are associated with severe degeneration. They are known to activate macrophages, synovial fibroblasts, and articular chondrocytes, resulting in increased cell proliferation and the production of pro-inflammatory cytokines and matrix metalloproteases (MMPs). This suggests a pathogenic role in OA by causing extracellular matrix degradation and subchondral bone remodelling. There are currently no disease-modifying drugs available for crystal-associated OA; hence, the aim of this study was to explore the inflammatory pathways activated by BCP crystals in order to identify potential therapeutic targets to limit crystal-induced inflammation. Primary human macrophages and dendritic cells were stimulated with BCP crystals, and activation of spleen tyrosine kinase (Syk), phosphoinositide-3 kinase (PI3K), and mitogen-activated protein kinases (MAPKs) was detected by immunoblotting. Lipopolysaccharide (LPS)-primed macrophages were pre-treated with inhibitors of Syk, PI3K, and MAPKs prior to BCP stimulation, and cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA). Aa an alternative, cells were treated with synovial fluid derived from osteoarthritic knees in the presence or absence of BCP crystals, and gene induction was assessed by real-time polymerase chain reaction (PCR). We demonstrate that exposure of primary human macrophages and dendritic cells to BCP crystals leads to activation of the membrane-proximal tyrosine kinases Syk and PI3K. Furthermore, we show that production of the pro-inflammatory cytokines interleukin (IL)-1α and IL-1β and phosphorylation of downstream MEK and ERK MAPKs is suppressed following treatment with inhibitors of Syk or PI3K. Finally, we demonstrate that treatment of macrophages with BCP crystals

  9. Intestinal Neuronal Dysplasia-Like Submucosal Ganglion Cell Hyperplasia at the Proximal Margins of Hirschsprung Disease Resections

    PubMed Central

    Swaminathan, Maya; Oron, Assaf P.; Chatterjee, Sumantra; Piper, Hannah; Cope-Yokoyama, Sandy; Chakravarti, Aravinda; Kapur, Raj P.

    2016-01-01

    Intestinal neuronal dysplasia type B (IND) denotes an increased proportion of hyperplastic submucosal ganglia, as resolved histochemically in 15 µm-thick frozen sections. IND has been reported proximal to the aganglionic segment in patients with Hirschsprung disease (HSCR) and is putatively associated with a higher rate of post-surgical dysmotility. We have developed and validated histological criteria to diagnose IND-like submucosal ganglion cell hyperplasia (IND-SH) in paraffin sections, and used the approach to study the incidence and clinical/genetic associations of IND-SH at the proximal margins of HSCR pull-through resection specimens. Full-circumference paraffin sections from the proximal margins of 64 HSCR colonic pull-through specimens and 24 autopsy controls were immunostained for the neuron-specific Hu antigen and nucleated ganglion cells in each submucosal ganglion were counted. In controls, an age-related decline in the relative abundance of “giant” ganglia (≥7 nucleated Hu+ ganglion cells) was observed. A conservative diagnostic threshold for IND-SH (control mean + 3 times the standard deviation) was derived from 15 controls less than 25 weeks of age. No control exceeded this threshold, whereas in the same age range, IND-SH was observed at the proximal margins in 15% (7/46) of HSCR resections, up to 15 cm proximal to the aganglionic segment. No significant correlation was observed between IND-SH and length of or distance from the aganglionic segment, gender, trisomy 21, RET or SEMA3C/D polymorphisms, or clinical outcome, but analysis of more patients with better long-term follow-up will be required to clarify the significance of this histological phenotype. PMID:26699691

  10. Intestinal Neuronal Dysplasia-Like Submucosal Ganglion Cell Hyperplasia at the Proximal Margins of Hirschsprung Disease Resections.

    PubMed

    Swaminathan, Maya; Oron, Assaf P; Chatterjee, Sumantra; Piper, Hannah; Cope-Yokoyama, Sandy; Chakravarti, Aravinda; Kapur, Raj P

    2015-01-01

    Intestinal neuronal dysplasia type B (IND) denotes an increased proportion of hyperplastic submucosal ganglia, as resolved histochemically in 15-μm-thick frozen sections. IND has been reported proximal to the aganglionic segment in patients with Hirschsprung disease (HSCR) and is putatively associated with a higher rate of postsurgical dysmotility. We developed and validated histological criteria to diagnose IND-like submucosal ganglion cell hyperplasia (IND-SH) in paraffin sections and used the approach to study the incidence and clinical and/or genetic associations of IND-SH at the proximal margins of HSCR pull-through resection specimens. Full-circumference paraffin sections from the proximal margins of 64 HSCR colonic pull-through specimens and 24 autopsy controls were immunostained for neuron-specific Hu antigen, and nucleated ganglion cells in each submucosal ganglion were counted. In controls, an age-related decline in the relative abundance of "giant" ganglia (≥7 nucleated Hu-positive [Hu+] ganglion cells) was observed. A conservative diagnostic threshold for IND-SH (control mean ± 3× standard deviation) was derived from 15 controls less than 25 weeks of age. No control exceeded this threshold, whereas in the same age range, IND-SH was observed at the proximal margins in 15% (7 of 46) of HSCR resections, up to 15 cm proximal to the aganglionic segment. No significant correlation was observed between IND-SH and length of or distance from the aganglionic segment, sex, trisomy 21, RET or SEMA3C/D polymorphisms, or clinical outcome, but analysis of more patients, with better long-term follow-up will be required to clarify the significance of this histological phenotype.

  11. 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

  12. Cytotoxicity, metabolism and cellular uptake of the mycotoxin deoxynivalenol in human proximal tubule cells and lung fibroblasts in primary culture.

    PubMed

    Königs, Maika; Lenczyk, Marlies; Schwerdt, Gerald; Holzinger, Hildegard; Gekle, Michael; Humpf, Hans-Ulrich

    2007-10-30

    At the level of the whole animal, the toxic effects of the mycotoxin deoxynivalenol (DON) range from causing diarrhoea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. It also affects the immune system and leads to kidney lesions. Although DON has been tested in different human and animal cell lines for its cytotoxicity, these tests might be limited due to the disadvantages of cell lines (e.g. immortalization, tumour derivation, longtime cultivation) and do not necessarily reflect the response of normal cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human kidney epithelial cells (renal proximal tubule epithelial cells, RPTEC) and human lung fibroblasts (normal human lung fibroblast, NHLF) in primary culture. Cell viability, apoptotic and necrotic cell death, collagens I, III and IV as well as fibronectin secretion were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary cells. A reduction in viability can be observed in both cell types, with fibroblasts reacting more sensitive. Furthermore, DON caused mainly necrotic cell death in kidney cells whereas mainly apoptotic cell death in fibroblasts. DON had no effect on collagen secretion in RPTEC cells. Collagen secretion was partially decreased in NHLF. In both cells, fibronectin secretion was reduced after 5 days of exposure. We also studied the metabolism and the cellular uptake of DON using LC-MS/MS. DON was neither metabolized by proximal tubule cells nor by fibroblasts. DON is incorporated into the cells whereas the intracellular amount of DON in kidney cells is higher than in fibroblasts. No accumulation of DON occurred in the cells.

  13. Cadmium induces phosphorylation and stabilization of c-Fos in HK-2 renal proximal tubular cells

    SciTech Connect

    Iwatsuki, Mamiko; Inageda, Kiyoshi; Matsuoka, Masato

    2011-03-15

    We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the expression and phosphorylation status of members of the Fos family, components of the activator protein-1 transcription factor, in HK-2 human renal proximal tubular cells. Following the exposure to CdCl{sub 2}, the expression of c-fos, fosB, fra-1, and fra-2 increased markedly, with different magnitudes and time courses. The levels of Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2) also increased in response to CdCl{sub 2} exposure. Although the elevation of c-fos transcripts was transient, c-Fos protein levels increased progressively with lower electrophoretic mobility, suggesting stabilization of c-Fos through post-translational modifications. Consistently, we observed phosphorylation of c-Fos at Ser362 and Ser374 in HK-2 cells treated with CdCl{sub 2}. Phosphorylated forms of mitogen-activated protein kinases (MAPKs)-including extracellular signal-regulated protein kinase (ERK), c-Jun NH{sub 2}-terminal kinase, and p38-increased after CdCl{sub 2} exposure, whereas treatment with the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 suppressed the accumulation and phosphorylation of c-Fos. We mutated Ser362 to alanine (S362A), Ser374 to alanine (S374A), and both residues to alanines (S362A/S374A) to inhibit potential phosphorylation of c-Fos at these sites. S374A or double S362A/S374A mutations reduced c-Fos level markedly, but S362A mutation did not. On the other hand, S362A/S374A mutations induced a more pronounced reduction in c-Fos DNA-binding activity than S374A mutation. These results suggest that while Ser374 phosphorylation seems to play a role in c-Fos stabilization, phosphorylation at two C-terminal serine residues is required for the transcriptional activation of c-Fos in HK-2 cells treated with CdCl{sub 2}.

  14. Mannan-binding lectin, a serum collectin, suppresses T-cell proliferation via direct interaction with cell surface calreticulin and inhibition of proximal T-cell receptor signaling.

    PubMed

    Zhao, Na; Wu, Jie; Xiong, Simin; Zhang, Liyun; Lu, Xiao; Chen, Shangliang; Wu, Qifeng; Wang, Hailan; Liu, Ying; Chen, Zhengliang; Zuo, Daming

    2017-06-01

    Mannan binding lectin (MBL), initially reported to activate the complement pathway, is also known to be involved in the pathogenesis of autoimmune diseases. We report a thus far unknown function of MBL as a suppressor of T-cell activation. MBL markedly inhibited T-cell proliferation induced by anti-CD3 and anti-CD28 antibodies. Moreover, the presence of MBL during T-cell priming interfered with proximal T-cell receptor signaling by decreasing phosphorylation of Lck, ZAP-70, and LAT. MBL bound to T cells through interaction between the collagen-like region of MBL and calreticulin (CRT) expressed on the T-cell surface. The neutralizing antibody against CRT abrogated MBL-mediated suppression of T-cell proliferation, suggesting that MBL down-modulates T-cell proliferation via cell surface CRT. We further demonstrated that the feature of MBL-mediated T-cell suppression is shared by other serum collectins (e.g., C1q and collectin 11). The concentrations of MBL correlated negatively with in vivo T-cell activation status in patients with early-stage silicosis. Furthermore, MBL efficiently inhibited activation and proliferation of autoreactive T cells derived from patients with silicosis, indicating that MBL serves as a negative feedback control of the T-cell responses.-Zhao, N., Wu, J., Xiong, S., Zhang, L., Lu, X., Chen, S., Wu, Q., Wang, H., Liu, Y., Chen, Z., Zuo, D. Mannan-binding lectin, a serum collectin, suppresses T-cell proliferation via direct interaction with cell surface calreticulin and inhibition of proximal T-cell receptor signaling. © FASEB.

  15. NKT cell modulates NAFLD potentiation of metabolic oxidative stress-induced mesangial cell activation and proximal tubular toxicity

    PubMed Central

    Alhasson, Firas; Dattaroy, Diptadip; Das, Suvarthi; Chandrashekaran, Varun; Seth, Ratanesh Kumar; Schnellmann, Rick G.

    2015-01-01

    Obesity and nonalcoholic fatty liver disease (NAFLD) are associated with the development and progression of chronic kidney disease. We recently showed that NAFLD induces liver-specific cytochrome P-450 (CYP)2E1-mediated metabolic oxidative stress after administration of the CYP2E1 substrate bromodichloromethane (BDCM) (Seth RK, Das S, Kumar A, Chanda A, Kadiiska MB, Michelotti G, Manautou J, Diehl AM, Chatterjee S. Toxicol Appl Pharmacol 274: 42–54, 2014; Seth RK, Kumar A, Das S, Kadiiska MB, Michelotti G, Diehl AM, Chatterjee S. Toxicol Sci 134:291–303, 2013). The present study examined the effects of CYP2E1-mediated oxidative stress in NAFLD leading to kidney toxicity. Mice were fed a high-fat diet for 12 wk to induce NAFLD. NAFLD mice were exposed to BDCM, a CYP2E1 substrate, for 4 wk. NAFLD + BDCM increased CYP2E1-mediated lipid peroxidation in proximal tubular cells compared with mice with NAFLD alone or BDCM-treated lean mice, thus ruling out the exclusive role of BDCM. Lipid peroxidation increased IL-1β, TNF-α, and interferon-γ. In parallel, mesangial cell activation was observed by increased α-smooth muscle actin and transforming growth factor-β, which was blocked by the CYP2E1 inhibitor diallyl sulphide both in vivo and in vitro. Mice lacking natural killer T cells (CD1d knockout mice) showed elevated (>4-fold) proinflammatory mediator release, increased Toll-like receptor (TLR)4 and PDGF2 mRNA, and mesangial cell activation in the kidney. Finally, NAFLD CD1D knockout mice treated with BDCM exhibited increased high mobility group box 1 and Fas ligand levels and TUNEL-positive nuclei, indicating that higher cell death was attenuated in TLR4 knockout mice. Tubular cells showed increased cell death and cytokine release when incubated with activated mesangial cells. In summary, an underlying condition of progressive NAFLD causes renal immunotoxicity and aberrant glomerular function possibly through high mobility group box 1-dependent TLR4 signaling

  16. Fenofibrate reduces cisplatin-induced apoptosis of renal proximal tubular cells via inhibition of JNK and p38 pathways.

    PubMed

    Thongnuanjan, Penjai; Soodvilai, Sirima; Chatsudthipong, Varanuj; Soodvilai, Sunhapas

    2016-01-01

    Cisplatin is widely used as a standard chemotherapy for solid tumors. The major adverse effect of cisplatin is nephrotoxicity in proximal tubular cells, via oxidative stress, DNA damage, cell apoptosis, and inflammation. The aim of this study was to investigate the pharmacological effect and mechanism of fibrate drugs on cisplatin-induced renal proximal tubular cell death. Cisplatin decreased cell viability of LLC-PK1 and HK-2 cells in a dose-dependent manner. Cisplatin-induced apoptosis was attenuated by co-treatment with fenofibrate while less so with clofibrate and bezafibrate. Fenofibrate's protective effect was not complimented by co-treatment with GW6471, a PPARα antagonist, indicating the protective effect occurred via a PPARα-independent mechanism. Treating cells with cisplatin induced reactive oxygen species (ROS), c-JUN N-terminal kinase (JNK), and p38 kinase (p38), but not extracellular signal-regulated kinase (ERK). Fenofibrate reversed cisplatin-induced JNK and p38 activation, but had no effect on ROS production. The findings suggest fenofibrate's protective effect on cisplatin-induced cytotoxicity is mediated by inhibition of JNK and p38. Moreover, fenofibrate did not alter cisplatin's antitumor effect on cancer cell lines including T84, SW-480, HepG2, and SK-LU-1 cells. Therefore, fenofibrate may be a candidate agent for further development as an adjuvant to cisplatin treatment.

  17. Characterisation of the expression of the Renin-Angiotensin system in primary and immortalised human renal proximal tubular cells.

    PubMed

    Shalamanova, Liliana; Wilkinson, Mark C; McArdle, Frank; Jackson, Malcolm J; Rustom, Rana

    2010-01-01

    Angiotensin II (AngII) is pivotal in the pathogenesis of progressive kidney disease. We have recently shown that AngII induced an increase in markers of oxidative stress, adaptive responses and upregulated stress-related gene expression in immortalised human proximal tubular (HK-2) cells. However, these observed effects of AngII were not mediated solely via AngII type 1 receptor (ATR1). Both HK-2 cells and primary human renal proximal tubular cells (RPTEC) are useful tools to investigate the renin-angiotensin system (RAS), but data on the local expression of the RAS in these cells remain limited. We therefore characterised RAS expression in RPTEC and HK-2 cells. The mRNA and protein expression of RAS in RPTEC and HK-2 cells was examined by RT-PCR, Western blotting and immunoprecipitation. In both cell lines, mRNA for angiotensin-converting enzyme (ACE) and mRNA and protein expression for angiotensinogen, renin, ACE2, ATR1 and ATR4 were detected. Candesartan, a specific ATR1 blocker, effectively blocked the expression of 80% of the stress-related genes that were upregulated in HK-2 cells following exposure to AngII. These data support a role for AngII in mediating oxidative stress via other receptor types stimulated by AngII and confirm that it is possible to investigate ATR4 pathways of potential injury in RPTEC. Copyright © 2010 S. Karger AG, Basel.

  18. Low micromolar concentrations of cadmium and mercury ions activate peritubular membrane K+ conductance in proximal tubular cells of frog kidney.

    PubMed

    Nesovic-Ostojic, Jelena; Cemerikic, Dusan; Dragovic, Simon; Milovanovic, Aleksandar; Milovanovic, Jovica

    2008-03-01

    The present study was designed to investigate the acute effects of extracellular low micromolar concentrations of cadmium and mercury ions on the peritubular cell membrane potential and its potassium selectivity in proximal tubular cells of the frog kidney. Peritubular exposure to 3 micromol/L Cd(2+) or 1 micromol/L Hg(2+) led to a rapid, sustained and reversible hyperpolarization of the peritubular cell membrane, paralleled by an increase in fractional K(+) conductance. Peritubular barium abolished hyperpolarization of the peritubular cell membrane to peritubular 3 micromol/L Cd(2+) or 1 micromol/L Hg(2+). Perfusing the lumen with 10 mmol/L l-alanine plus/minus 3 micromol/L Cd(2+) or Hg(2+) did not modify rapid depolarization and rate of slow repolarization of the peritubular cell membrane potential. In conclusion, low micromolar concentrations of Cd(2+) and Hg(2+) increase K(+) conductive pathway in the peritubular cell membrane and in this way can enhance ability of proximal renal tubular cells to maintain the driving force for electrogenic Na(+) and substrate reabsorption.

  19. Mycobacterium tuberculosis secretory proteins downregulate T cell activation by interfering with proximal and downstream T cell signalling events.

    PubMed

    Sharma, Bhawna; Upadhyay, Rajni; Dua, Bhavyata; Khan, Naim Akhtar; Katoch, Vishwa Mohan; Bajaj, Bharat; Joshi, Beenu

    2015-11-09

    investigate effect of Ag85A and ESAT-6 on TCR- and TCR/CD28- induced upstream and downstream signalling events of T-cell activation in TB patients. This study showed the effect of secretory antigens of M. tuberculosis in the modulation of T cell signalling pathways. This inflection is accomplished by altering the proximal and distal events of signalling cascade which could be involved in T-cell dysfunctioning during the progression of the disease.

  20. 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.

  1. 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.

  2. Thin film scintillators

    NASA Astrophysics Data System (ADS)

    McDonald, Warren; McKinney, George; Tzolov, Marian

    2015-03-01

    Scintillating materials convert energy flux (particles or electromagnetic waves) into light with spectral characteristic matching a subsequent light detector. Commercial scintillators such as yttrium aluminum garnet (YAG) and yttrium aluminum perovskite (YAP) are commonly used. These are inefficient at lower energies due to the conductive coating present on their top surface, which is needed to avoid charging. We hypothesize that nano-structured thin film scintillators will outperform the commercial scintillators at low electron energies. We have developed alternative thin film scintillators, zinc tungstate and zinc oxide, which show promise for higher sensitivity to lower energy electrons since they are inherently conductive. Zinc tungstate films exhibit photoluminescence quantum efficiency of 74%. Cathodoluminescence spectroscopy was applied in transmission and reflection geometries. The comparison between the thin films and the YAG and YAP commercial scintillators shows much higher light output from the zinc tungstate and zinc oxide at electron energies less than 5 keV. Our films were integrated in a backscattered electron detector. This detector delivers better images than an identical detector with commercial YAG scintillator at low electron energies. Dr. Nicholas Barbi from PulseTor LLC, Dr. Anura Goonewardene, NSF Grants: #0806660, #1058829, #0923047.

  3. Differential roles of hydrogen peroxide and hydroxyl radical in cisplatin-induced cell death in renal proximal tubular epithelial cells.

    PubMed

    Baek, Su Mi; Kwon, Chae Hwa; Kim, Jae Ho; Woo, Jae Suk; Jung, Jin Sup; Kim, Yong Keun

    2003-09-01

    Reactive oxygen species (ROS) have been suggested as important mediators of cisplatin-induced acute renal failure in vivo. However, our previous studies have shown that cisplatin-induced cell death in vitro could not be prevented by scavengers of hydrogen peroxide and hydroxyl radical in rabbit renal cortical slices. This discrepancy may be attributed to differential roles of ROS in necrotic and apoptotic cell death. We therefore examined, in this study, the roles of ROS in necrosis and apoptosis induced by cisplatin in primary cultured rabbit proximal tubule. Cisplatin induced necrosis at high concentrations over a few hours and apoptosis at much lower concentrations over longer periods. Necrosis induced by high concentration of cisplatin was prevented by a cell-permeable superoxide scavenger (tiron), hydrogen peroxide scavengers (catalase and pyruvate), and antioxidants (Trolox and deferoxamine), whereas hydroxyl radical scavengers (dimethythiourea and thiourea) did not affect the cisplatin-induced necrosis. However, apoptosis induced by lower concentration of cisplatin was partially prevented by tiron and hydroxyl radical scavengers but not by hydrogen peroxide scavengers and antioxidants. Cisplatin-induced apoptosis was mediated by the signaling pathway that is associated with cytochrome c release from mitochondria and caspase-3 activation. These effects were prevented by tiron and dimethylthiourea but not by catalase. Dimethylthiourea produced a significant protection against cisplatin-induced acute renal failure, and the effect was associated with an inhibition of apoptosis. These results suggest that hydrogen peroxide is involved in the cisplatin-induced necrosis, whereas hydroxyl radical is responsible for the cisplatin-induced apoptosis. The protective effects of hydroxyl radical scavengers are associated with an inhibition of cytochrome c release and caspase activation.

  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. Cytoprotective effects of calbindin-D(28k) against antimycin-A induced hypoxic injury in proximal tubular cells.

    PubMed

    Wu, Ming-Ju; Lai, Li-Wen; Lien, Yeong-Hau H

    2002-06-21

    Intracellular calcium plays an important role on the pathogenesis of hypoxia-induced cellular injury. Calbindin-D(28k), a cytosolic vitamin D-dependent calcium binding protein, can serve as a buffer to limit a surge in intracellular Ca2+ concentration ([Ca2+]i) induced by various stimulations. To evaluate the possible cytoprotective effect of calbindin-D(28k) against hypoxic injury in proximal tubular cells, a plasmid containing calbindin-D(28k) cDNA under the control of CMV immediate-early gene promoter was transfected into the murine proximal tubular epithelial (MCT) cells. The expression of calbindin-D(28k) in the transfected cells was verified with Northern blot analysis, Western blot analysis, and immunofluorescent staining. The non-transfected and transfected MCT cells were subjected to chemical hypoxia induced by antimycin A (10 microM) and glucose deprivation for 30-120 min. The transfection of calbindin-D(28k) reduced lactate dehydrogenase (LDH) release by 41%, 41%, 24%, and 24%, respectively, at 30, 60, 90 and 120 min after hypoxia when compared to the non-transfected cells (all p < 0.05). Cell viability after hypoxic injury was also significantly higher in transfected cells than non-transfected cells. Transfection with the plasmid without calbindin-D(28k) cDNA did not affect LDH release or cell viability after chemical hypoxic injury. [Ca+2]i was measured ratiometrically with fura-2 after exposure to chemical hypoxia. The rate of initial rise in [Ca2+]i and final [Ca+2]i at 30-120 min were significantly lowered in transfected cells. In conclusion, this study demonstrated that transfection of calbindin-D(28k) gene into MCT cells provide protective effects against chemical hypoxic injury probably through its buffering effects on [Ca+2]i.

  6. Stearoyl-CoA Desaturase-1 Protects Cells against Lipotoxicity-Mediated Apoptosis in Proximal Tubular Cells

    PubMed Central

    Iwai, Tamaki; Kume, Shinji; Chin-Kanasaki, Masami; Kuwagata, Shogo; Araki, Hisazumi; Takeda, Naoko; Sugaya, Takeshi; Uzu, Takashi; Maegawa, Hiroshi; Araki, Shin-ichi

    2016-01-01

    Saturated fatty acid (SFA)-related lipotoxicity is a pathogenesis of diabetes-related renal proximal tubular epithelial cell (PTEC) damage, closely associated with a progressive decline in renal function. This study was designed to identify a free fatty acid (FFA) metabolism-related enzyme that can protect PTECs from SFA-related lipotoxicity. Among several enzymes involved in FFA metabolism, we identified stearoyl-CoA desaturase-1 (SCD1), whose expression level significantly decreased in the kidneys of high-fat diet (HFD)-induced diabetic mice, compared with non-diabetic mice. SCD1 is an enzyme that desaturates SFAs, converting them to monounsaturated fatty acids (MUFAs), leading to the formation of neutral lipid droplets. In culture, retrovirus-mediated overexpression of SCD1 or MUFA treatment significantly ameliorated SFA-induced apoptosis in PTECs by enhancing intracellular lipid droplet formation. In contrast, siRNA against SCD1 exacerbated the apoptosis. Both overexpression of SCD1 and MUFA treatment reduced SFA-induced apoptosis via reducing endoplasmic reticulum stress in cultured PTECs. Thus, HFD-induced decrease in renal SCD1 expression may play a pathogenic role in lipotoxicity-induced renal injury, and enhancing SCD1-mediated desaturation of SFA and subsequent formation of neutral lipid droplets may become a promising therapeutic target to reduce SFA-induced lipotoxicity. The present study provides a novel insight into lipotoxicity in the pathogenesis of diabetic nephropathy. PMID:27834856

  7. Cyclic stretch-induced cPLA2 mediates ERK 1/2 signaling in rabbit proximal tubule cells.

    PubMed

    Alexander, Larry D; Alagarsamy, Suganthi; Douglas, Janice G

    2004-02-01

    Recent evidence from this laboratory have demonstrated a critical role of phospholipase A2 (PLA2) and arachidonic acid in angiotensin II type 2 (AT2) receptor-mediated kinase activation in renal epithelium independent of phosphoinositide- specific phospholipase C (PLC) and without the necessity of eicosanoid biosynthesis. In the present study, we investigated whether cyclic stress phosphorylates and activates the mitogen-activated protein kinase (MAPK) pathway and whether PLA2 activation mediates mechanotransduction in renal epithelial cells. The rational for studying kidney epithelial cells relates to their similarity to podocytes, which undergo mechanical stretch related to changes in intraglomerular pressure. To produce strain or stretch, primary cultures of rabbit proximal tubular cell cells are grown in tissue culture wells having a collagen-coated Silastic deformable membrane bottoms and applying vacuum to the well to generate alternating cycles of stretch and relaxation (30 cycles/min). We found that cyclic stretching of rabbit proximal tubular cells caused a time- and intensity-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) in proximal tubular cells as detected by its phosphorylation. In addition, mechanical stretch induced PLA2 activation and a subsequent rapid release of arachidonic acid. Inhibition of PLA2 by mepacrine and methyl arachidonyl fluorophosphonate ketone (AACOCF3) attenuated both arachidonic acid release and ERK 1/2 activation by cyclic stretch, supporting the importance of PLA2 as a mediator of mechanotransduction in renal proximal tubular cells. A requirement for extracellular Ca2+ and stretch-activated Ca2+ channels was also documented. Complete inhibition of ERK 1/2 by PD98059, a MAPK kinase (MEK) inhibitor, did not suppress stretch- induced PLA2 activation and arachidonic acid release, suggesting the later events were upstream of ERK 1/2. Cyclic stretch also caused rapid phosphorylation of the EGF

  8. 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

  9. Na sup + -H sup + exchanger in proximal cells isolated from kidney. II. Short-term regulation by glucocorticoids

    SciTech Connect

    Bidet, M.; Merot, J.; Tauc, M.; Poujeol, P. )

    1987-11-01

    The purpose of this study was to investigate the acute regulation by glucocorticoid of the Na{sup +}-H{sup +} exchanger in isolated renal proximal cells of the rabbit. The changes of intracellular pH (pH{sub i}) were determined in a bicarbonate-free buffer by the use of a fluorescent pH probe that may be trapped intracellularly, 2,7-biscarboxyethyl- 5(6)- carboxyfluorescein (BCECF). The activity of the Na{sup +}-H{sup +} exchanger was estimated by measuring the Na{sup +}-induced H{sup +} efflux in BCECF-loaded cells acid loaded with nigericin in choline medium. The uptake of 1.5 mM {sup 22}Na was also studied in Na{sup +}-depleted cells. Acute application of dexamethasone increased the activity of the Na{sup +}-H{sup +} exchanger. The effect on the kinetics of amiloride-sensitive Na{sup +}-H{sup +} exchange indicated that dexamethasone (DEX) increased the activity by increasing the V{sub max} of the carrier for external sodium and for external H{sup +}. The apparent affinity was not modified either for Na{sup +} or for H{sup +}. The glucocorticoid action was undetectable after pretreatment of cells with actinomycin D or cycloheximide. Acute glucocorticoid activation of the Na{sup +}-H{sup +} exchanger in isolated proximal cells required RNA and protein synthesis and was consistent with an increase in the number of carriers in the membrane.

  10. ZAP-70, CTLA-4 and proximal T cell receptor signaling in cows infected with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Leite, Fernando L; Eslabão, Livia B; Pesch, Bruce; Bannantine, John P; Reinhardt, Timothy A; Stabel, Judith R

    2015-09-15

    Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cell-mediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clinical state. The objective of this study was to investigate the expression of two crucial molecules in T cell function, ZAP-70 (zeta-chain-associated protein of 70 kDa) and CTLA-4 (cytotoxic T-lymphocyte antigen-4), in cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMCs) isolated from control non-infected cows (n=5), and cows in subclinical (n=6) and clinical stages of paratuberculosis (n=6) were cultured alone (medium only), and with concanavalin A, and a whole cell sonicate of MAP for 24, 72 and 144 h to measure the dynamic changes of ZAP-70 and CTLA-4 expression on CD4, CD8, and gamma delta (γδ) T cells. Flow cytometry was also performed to measure ZAP-70 phosphorylation to examine proximal T cell receptor signaling in animals of different disease status. The surface expression of CTLA-4 was increased in animals in subclinical stage of infection while levels of ZAP-70 were decreased in CD4+ T cells of both subclinical and clinical animals, indicating a change in T cell phenotype with disease state. Interestingly, proximal T cell receptor signaling was not altered in infected animals. This study demonstrated changes in crucial signaling molecules in animals infected with MAP, thereby elucidating T cell alterations associated with disease progression.

  11. Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells.

    PubMed

    Hryciw, Deanne H; Ekberg, Jenny; Lee, Aven; Lensink, Ingrid L; Kumar, Sharad; Guggino, William B; Cook, David I; Pollock, Carol A; Poronnik, Philip

    2004-12-31

    Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

  12. Segmented scintillation antineutrino detector

    DOEpatents

    Reyna, David

    2017-05-09

    The various technologies presented herein relate to incorporating a wavelength-shifting material in a scintillator to facilitate absorption of a first electromagnetic particle (e.g., a first photon) having a first wavelength and subsequent generation and emission of a second electromagnetic particle (e.g., a second photon) having a second wavelength. The second electromagnetic particle can be emitted isotropically, with a high probability that the direction of emission of the second electromagnetic particle is disparate to the direction of travel of the first electromagnetic particle (and according angle of incidence). Isotropic emission of the second electromagnetic particle enables the second electromagnetic particle to be retained in the scintillator owing to internal reflection. Accordingly, longer length scintillators can be constructed, and accordingly, the scintillator array has a greater area (and volume) over which to detect electromagnetic particles (e.g., antineutrinos) being emitted from a nuclear reaction.

  13. Mitochondrial Fission Increases Apoptosis and Decreases Autophagy in Renal Proximal Tubular Epithelial Cells Treated with High Glucose.

    PubMed

    Lee, Wen-Chin; Chiu, Chien-Hua; Chen, Jin-Bor; Chen, Chiu-Hua; Chang, Hsueh-Wei

    2016-11-01

    The aim of this study was to examine the effect of mitochondrial morphogenesis changes on apoptosis and autophagy of high-glucose-treated proximal tubular epithelial cells (HK2). Cell viability, apoptosis, and mitochondrial morphogenesis were examined using crystal violet, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and mitotracker staining, respectively. High glucose inhibited cell viability and induced mitochondrial fission in HK2 cells. After depleting mitofusin 1 (MFN1), the MFN1(-) HK2 cells (fission type) became more susceptible to high-glucose-induced apoptosis and mitochondrial fragmentation observed by TUNEL and mitotracker assays. In siMFN2 HK2 cells (fission type), mitochondria were highly fragmented (>80% fission rate) with or without high-glucose treatment; however, siFIS1 (mitochondrial fission protein 1) HK2 cells (fusion type) exhibited little fragmentation (<13%). High-glucose treatment induced autophagy, characterized by the formation of autophagosome and microtubule-associated protein light chain 3 (LC3) B-II, as observed by transmission electron microscopy and western blotting, respectively. LC3B-II levels decreased in both MFN1(-) and siMFN2 HK2 cells, but increased in siFIS1 HK2 cells. Moreover, autophagy displays a protective role against high-glucose-induced cell death based on cotreatment with autophagy inhibitors (3-methyladenine and chloroquine). Mitochondrial fission may increase apoptosis and decrease autophagy of high-glucose-treated HK2 cells.

  14. Renal expression of the C3a receptor and functional responses of primary human proximal tubular epithelial cells.

    PubMed

    Braun, Michael C; Reins, Rose Y; Li, Tong-Bin; Hollmann, Travis J; Dutta, Ranjan; Rick, Wetsel A; Teng, Ba-Bie; Ke, Baozhen

    2004-09-15

    Although complement activation and deposition have been associated with a variety of glomerulopathies, the pathogenic mechanisms by which complement directly mediates renal injury remain to be fully elucidated. Renal parenchymal tissues express a limited repertoire of receptors that directly bind activated complement proteins. We report the renal expression of the receptor for the C3 cleavage product C3a, a member of the anaphylatoxin family. C3aR is highly expressed in normal human and murine kidney, as demonstrated by immunohistochemistry and in situ hybridization. Its distribution is limited to epithelial cells only, as glomerular endothelial and mesangial cells showed no evidence of C3aR expression. The C3aR is also expressed by primary renal proximal tubular epithelial cells in vitro as demonstrated by FACS, Western blot, and RT-PCR. In vitro C3aR is functional in terms of its capacity to bind 125I-labeled C3a and generate inositol triphosphate. Finally, using microarray analysis, four novel genes were identified and confirmed as transcriptionally regulated by C3aR activation in proximal tubular cells. These studies define a new pathway by which complement activation may directly modulate the renal response to immunologic injury.

  15. Scintillating pad detectors

    SciTech Connect

    Adams, D.; Baumbaugh, B.; Borcherding, F.

    1996-12-31

    We have been investigating the performance of scintillating pad detectors, individual small tiles of scintillator that are read out with wavelength-shifting fibers and visible light photon counters, for application in high luminosity colliding beam experiments such as the D0 Upgrade. Such structures could provide {open_quotes}pixel{close_quotes} type readout over large fiducial volumes for tracking, preshower detection and triggering.

  16. 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.

  17. Angiotensin II receptor blocker inhibits tumour necrosis factor-alpha-induced cell damage in human renal proximal tubular epithelial cells.

    PubMed

    Kagawa, Toru; Takao, Toshihiro; Horino, Taro; Matsumoto, Reiko; Inoue, Kousuke; Morita, Tatsuhito; Hashimoto, Kozo

    2008-06-01

    We investigated the effect of angiotensin II (AII) type 1 (AT1) and angiotensin II type 2 (AT2) receptor blockers on tumour necrosis factor alpha (TNF-alpha)-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). The lactate dehydrogenase (LDH) and N-acetyl-beta-glucosaminidase (NAG) release into the medium after TNF-alpha treatment in RPTEC were determined using modified commercial procedures. In addition, the levels of caspase 3/7 activity in RPTEC were measured after TNF-alpha treatment with AlphaTau1 or AT2 receptor blockers. Finally we investigated the change of p22phox protein levels after TNF-alpha with AlphaTau1 or AT2 receptor blockers in RPTEC. Tumour necrosis factor alpha (10(-8) mol/L) significantly increased LDH and NAG release into the medium from RPTEC. AlphaTau1 receptor blockers, olmesartan and valsartan (10(-9)-10(-6) mol/L) showed a significant reduction on TNF-alpha-induced LDH and NAG release in RPTEC. AT2 receptor blocker, PD123319 (10(-7)-10(-5) mol/L) also decreased TNF-alpha-induced LDH and NAG release in RPTEC. Blockade of both AlphaTau1 and AT2 receptor indicated additional reduction on TNF-alpha-induced LDH and NAG release. TNF-alpha (10(-8) mol/L) treatment showed small but significant increases of caspase 3/7 activity in RPTEC, and AT1 and AT2 receptor blockers (10(-8) mol/L) comparably decreased TNF-alpha-induced caspase 3/7 activity. Significant increases of p22phox protein levels were observed in TNF-alpha-treated group in RPTEC. However, only AlphaTau1 (10(-8) mol/L) but not AT2 (10(-5) mol/L) receptor blocker significantly decreased TNF-alpha-induced p22phox protein levels. The present study demonstrates that TNF-alpha induces renal tubular cell damage in RPTEC and AT1/AT2 receptor blockers showed cytoprotective effects probably via at least partly different mechanism.

  18. MIF-2/D-DT enhances proximal tubular cell regeneration through SLPI- and ATF4-dependent mechanisms.

    PubMed

    Ochi, Akinobu; Chen, Dong; Schulte, Wibke; Leng, Lin; Moeckel, Nickolas; Piecychna, Marta; Averdunk, Luisa; Stoppe, Christian; Bucala, Richard; Moeckel, Gilbert

    2017-09-01

    Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic actions that is produced by several organs and cell types. Depending on the target cell and the inflammatory context, MIF can engage its two component receptor complex CD74 and CD44 and the chemokine receptors CXCR2/4. MIF is constitutively expressed in renal proximal tubular cells, stored in intracellular preformed pools, and released at a low rate. Recently, a second MIF-like protein (i.e., MIF-2/D-DT) has been characterized in mammals. Our study was aimed at examining the role of MIF-2/D-DT, which mediates tissue protection in the heart, in tubular cell regeneration from ischemia-reperfusion injury. We found that Mif-/-, Mif-2-/-, and Cd74-/- mice had significantly worse tubular injury compared with wild-type (WT) control mice and that treatment with MIF-2/D-DT significantly improved recovery of injured epithelial cells. RNAseq analysis of kidney tissue from the ischemia-reperfusion injury model revealed that MIF-2/D-DT treatment stimulates secretory leukocyte proteinase inhibitor (SLPI) and cyclin D1 expression. MIF-2/D-DT additionally activates of eukaryotic initiation factor (eIF) 2α and activating transcription factor (ATF) 4, two transcription factors involved in the integrated stress response (ISR), which is a cellular stress response activated by hypoxia, nutrient deprivation, and oxygen radicals. MIF-2/D-DT also inhibited apoptosis and induced autophagy in hypoxia-treated mouse proximal tubular (MPT) cells. These results indicate that MIF-2/D-DT is an important factor in tubular cell regeneration and may be of therapeutic utility as a regenerative agent in the clinical setting of ischemic acute kidney injury. Copyright © 2017 the American Physiological Society.

  19. Cytotoxicity of Portuguese Propolis: The Proximity of the In Vitro Doses for Tumor and Normal Cell Lines

    PubMed Central

    Falcão, Soraia; Queiroz, Maria João R. P.; Ferreira, Isabel C. F. R.

    2014-01-01

    With a complex chemical composition rich in phenolic compounds, propolis (resinous substance collected by Apis mellifera from various tree buds) exhibits a broad spectrum of biological activities. Recently, in vitro and in vivo data suggest that propolis has anticancer properties, but is the cytoxicity of propolis specific for tumor cells? To answer this question, the cytotoxicity of phenolic extracts from Portuguese propolis of different origins was evaluated using human tumor cell lines (MCF7—breast adenocarcinoma, NCI-H460—non-small cell lung carcinoma, HCT15—colon carcinoma, HeLa—cervical carcinoma, and HepG2—hepatocellular carcinoma), and non-tumor primary cells (PLP2). The studied propolis presented high cytotoxic potential for human tumor cell lines, mostly for HCT15. Nevertheless, excluding HCT15 cell line, the extracts at the GI50 obtained for tumor cell lines showed, in general, cytotoxicity for normal cells (PLP2). Propolis phenolic extracts comprise phytochemicals that should be further studied for their bioactive properties against human colon carcinoma. In the other cases, the proximity of the in vitro cytotoxic doses for tumor and normal cell lines should be confirmed by in vivo tests and may highlight the need for selection of specific compounds within the propolis extract. PMID:24982911

  20. Distribution of CD105 and CD166 positive cells in the proximal epiphysis of developing rat humerus.

    PubMed

    Ozbey, Ozlem; Sahin, Zeliha; Acar, Nuray; Ustunel, Ismail

    2010-11-01

    The expression of cell surface receptors, CD105 and CD166, are characteristic of mesenchymal stem cells in cartilage. However, there is limited data regarding their immunolocalization in the cartilage of developing rat epiphysis. The purpose of this study was to determine the presence of CD105 and CD 166 positive cells in the proximal epiphysis of developing rat humerus and specify their zonal distribution with age. The tissues of rat humerus were taken on embryonic day 15 (E15), embryonic day 19 (E19), postnatal day 10 (PN10), postnatal day 20 (PN20) and adult rats and studied for the immunolocalization of CD105 and CD166. Our results showed that CD105 and CD166 positive cells were scattered in early stages of development of humerus epiphysis. For E15, only the hypertrophic zone was positive, whereas for E19 almost all zones of the epiphysis were positively stained for these markers. For PN10 and PN20, the CD105 and CD166 positive cells were mainly localized on the surface of the articular cartilage. In adult articular cartilage the CD105 and CD166 positive cells were localized in the superficial and transitional zones and in the upper regions of the deep zone. Our study provides evidence that in the developing cartilage tissue the localization of CD105 and CD166 positive cells is both dynamic and stage dependent, which may imply the existence of stem cell-like cells in cartilage from an early age to adult.

  1. Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects.

    PubMed

    Hervé, Cécile; Rogowski, Artur; Blake, Anthony W; Marcus, Susan E; Gilbert, Harry J; Knox, J Paul

    2010-08-24

    Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.

  2. Calcium Dobesilate Prevents Diabetic Kidney Disease by Decreasing Bim and Inhibiting Apoptosis of Renal Proximal Tubular Epithelial Cells.

    PubMed

    Cai, Tian; Wu, Xiao-Yun; Zhang, Xiao-Qian; Shang, Hong-Xia; Zhang, Zhong-Wen; Liao, Lin; Dong, Jian-Jun

    2017-04-01

    Apoptosis of renal proximal tubular epithelial cells (PTECs) plays a vital role in the pathogenesis and progression of diabetic kidney disease (DKD). Calcium dobesilate is a vascular protective compound used for treatment of diabetic retinopathy and chronic venous insufficiency. The aim of this study was to determine whether calcium dobesilate can protect PTECs from glucose-induced apoptosis and the potential mechanism of this effect. It is indicated that high glucose promoted abnormal apoptosis of HK2 cells, which was inhibited by treatment of calcium dobesilate, while Bim expression decreased in response to calcium dobesilate in high-glucose-treated HK2 cells. These findings confirmed the therapeutic effects of calcium dobesilate on DKD and emphasized the importance of it as a potentially crucial drug in treatment of DKD.

  3. Ionospheric Scintillation Explorer (ISX)

    NASA Astrophysics Data System (ADS)

    Iuliano, J.; Bahcivan, H.

    2015-12-01

    NSF has recently selected Ionospheric Scintillation Explorer (ISX), a 3U Cubesat mission to explore the three-dimensional structure of scintillation-scale ionospheric irregularities associated with Equatorial Spread F (ESF). ISX is a collaborative effort between SRI International and Cal Poly. This project addresses the science question: To what distance along a flux tube does an irregularity of certain transverse-scale extend? It has been difficult to measure the magnetic field-alignment of scintillation-scale turbulent structures because of the difficulty of sampling a flux tube at multiple locations within a short time. This measurement is now possible due to the worldwide transition to DTV, which presents unique signals of opportunity for remote sensing of ionospheric irregularities from numerous vantage points. DTV spectra, in various formats, contain phase-stable, narrowband pilot carrier components that are transmitted simultaneously. A 4-channel radar receiver will simultaneously record up to 4 spatially separated transmissions from the ground. Correlations of amplitude and phase scintillation patterns corresponding to multiple points on the same flux tube will be a measure of the spatial extent of the structures along the magnetic field. A subset of geometries where two or more transmitters are aligned with the orbital path will be used to infer the temporal development of the structures. ISX has the following broad impact. Scintillation of space-based radio signals is a space weather problem that is intensively studied. ISX is a step toward a CubeSat constellation to monitor worldwide TEC variations and radio wave distortions on thousands of ionospheric paths. Furthermore, the rapid sampling along spacecraft orbits provides a unique dataset to deterministically reconstruct ionospheric irregularities at scintillation-scale resolution using diffraction radio tomography, a technique that enables prediction of scintillations at other radio frequencies, and

  4. Receptor-Associated Protein Blocks Internalization and Cytotoxicity of Myeloma Light Chain in Cultured Human Proximal Tubular Cells

    PubMed Central

    Sengul, Sule; Erturk, Sehsuvar; Khan, Altaf M.; Batuman, Vecihi

    2013-01-01

    Background Free light chains (LCs) are among the many ligands that bind to cubilin/megalin for endocytosis via the clathrin-dependent endosomal/lysosomal pathway. Receptor associated protein (RAP), is a 39 kDA high-affinity, chaperone-like ligand for megalin that assists in the proper folding and functioning of megalin/cubilin. Although RAP is known to inhibit ligand binding to megalin/cubilin, its effect on LC endocytosis has not been shown directly. Methods and Principal Findings We investigated whether RAP can block the endocytosis of LC in cultured human proximal tubule cells and whether this can prevent LC cytotoxicity. Immunofluorescence microscopy and flow cytometry showed that fluorescently labeled LC endocytosis was markedly inhibited in HK-2 cells pretreated with human RAP. The effect of RAP was dose-dependent, and was predominantly on endocytosis as it had no effect on the small acid-washable fraction of LC bound to cell membrane. RAP significantly inhibited LC induced cytokine production and phosphorylation of ERK1/2 and p38 MAPK. Prolonged exposure to LC for 48 h resulted in epithelial-to-mesenchymal transformation in HK-2 cells as evidenced by marked reduction in the expression of the epithelial cell marker E-cadherin, and increased the expression of the mesenchymal marker α-SMA, which was also prevented by RAP in the endocytosis medium. Conclusions RAP inhibited LC endocytosis by ∼88% and ameliorated LC-induced cytokine responses and EMT in human PTCs. The results not only provide additional evidence that LCs endocytosis occurs via the megalin/cubilin endocytic receptor system, but also show that blocking LC endocytosis by RAP can protect proximal tubule cells from LC cytotoxicity. PMID:23894629

  5. Surface preparation and coupling in plastic scintillator dosimetry.

    PubMed

    Ayotte, Guylaine; Archambault, Louis; Gingras, Luc; Lacroix, Frédéric; Beddar, A Sam; Beaulieu, Luc

    2006-09-01

    One way to improve the performance of scintillation dosimeters is to increase the light-collection efficiency at the coupling interfaces of the detector system. We performed a detailed study of surface preparation of scintillating fibers and their coupling with clear optical fibers to minimize light loss and increase the amount of light collected. We analyzed fiber-surface polishing with aluminum oxide sheets, coating fibers with magnesium oxide, and the use of eight different coupling agents (air, three optical gels, an optical curing agent, ultraviolet light, cyanoacrylate glue, and acetone). We prepared 10 scintillating fiber and clear optical fiber light guide samples to test different coupling methods. To test the coupling, we first cut both the scintillating fiber and the clear optical fiber. Then, we cleaned and polished both ends of both fibers. Finally, we coupled the scintillating fiber with the clear optical fiber in either a polyethylene jacket or a V-grooved support depending on the coupling agent used. To produce more light, we used an ultraviolet lamp to stimulate scintillation. A typical series of similar couplings showed a standard deviation in light-collection efficiency of 10%. This can be explained by differences in the surface preparation quality and alignment of the scintillating fiber with the clear optical fiber. Absence of surface polishing reduced the light collection by approximately 40%, and application of magnesium oxide on the proximal end of the scintillating fiber increased the amount of light collected from the optical fiber by approximately 39%. Of the coupling agents, we obtained the best results using one of the optical gels. Because a large amount of the light produced inside a scintillator is usually lost, better light-collection efficiency will result in improved sensitivity.

  6. Surface preparation and coupling in plastic scintillator dosimetry

    SciTech Connect

    Ayotte, Guylaine; Archambault, Louis; Gingras, Luc; Lacroix, Frederic; Beddar, A. Sam; Beaulieu, Luc

    2006-09-15

    One way to improve the performance of scintillation dosimeters is to increase the light-collection efficiency at the coupling interfaces of the detector system. We performed a detailed study of surface preparation of scintillating fibers and their coupling with clear optical fibers to minimize light loss and increase the amount of light collected. We analyzed fiber-surface polishing with aluminum oxide sheets, coating fibers with magnesium oxide, and the use of eight different coupling agents (air, three optical gels, an optical curing agent, ultraviolet light, cyanoacrylate glue, and acetone). We prepared 10 scintillating fiber and clear optical fiber light guide samples to test different coupling methods. To test the coupling, we first cut both the scintillating fiber and the clear optical fiber. Then, we cleaned and polished both ends of both fibers. Finally, we coupled the scintillating fiber with the clear optical fiber in either a polyethylene jacket or a V-grooved support depending on the coupling agent used. To produce more light, we used an ultraviolet lamp to stimulate scintillation. A typical series of similar couplings showed a standard deviation in light-collection efficiency of 10%. This can be explained by differences in the surface preparation quality and alignment of the scintillating fiber with the clear optical fiber. Absence of surface polishing reduced the light collection by approximately 40%, and application of magnesium oxide on the proximal end of the scintillating fiber increased the amount of light collected from the optical fiber by approximately 39%. Of the coupling agents, we obtained the best results using one of the optical gels. Because a large amount of the light produced inside a scintillator is usually lost, better light-collection efficiency will result in improved sensitivity.

  7. 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

  8. Proximal and distal regulation of the HYAL1 gene cluster by the estrogen receptor α in breast cancer cells

    PubMed Central

    Edjekouane, Lydia; Benhadjeba, Samira; Jangal, Maïka; Fleury, Hubert; Gévry, Nicolas; Carmona, Euridice; Tremblay, André

    2016-01-01

    Chromosomal and genome abnormalities at the 3p21.3 locus are frequent events linked to epithelial cancers, including ovarian and breast cancers. Genes encoded in the 3p21.3 cluster include HYAL1, HYAL2 and HYAL3 members of hyaluronidases involved in the breakdown of hyaluronan, an abundant component of the vertebrate extracellular matrix. However, the transcriptional regulation of HYAL genes is poorly defined. Here, we identified the estrogen receptor ERα as a negative regulator of HYAL1 expression in breast cancer cells. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL1 gene expression in human breast tumors. ChIP-Seq analysis identified several ERα binding sites within the 3p21.3 locus, supporting the role of estrogen as an upstream signal that diversely regulates the expression of 3p21.3 genes at both proximal and distal locations. Of these, HYAL1 was repressed by estrogen through ERα binding to a consensus estrogen response element (ERE) located in the proximal promoter of HYAL1 and flanked by an Sp1 binding site, required to achieve optimal estrogen repression. The repressive chromatin mark H3K27me3 was increased at the proximal HYAL1 ERE but not at other EREs contained in the cluster, providing a mechanism to selectively downregulate HYAL1. The HYAL1 repression was also specific to ERα and not to ERβ, whose expression did not correlate with HYAL1 in human breast tumors. This study identifies HYAL1 as an ERα target gene and provides a functional framework for the direct effect of estrogen on 3p21.3 genes in breast cancer cells. PMID:27764788

  9. Chlorotrifluoroethylcysteine interaction with rabbit proximal tubule cell basolateral membrane organic anion transport and apical membrane amino acid transport.

    PubMed

    Groves, C E; Morales, M N

    1999-11-01

    The interaction of the cysteine conjugate S-(1-chloro-1,2,2, -trifluoroethyl)-L-cysteine (CTFC) with organic anion and amino acid transport in the basolateral and apical membranes was examined with rabbit renal proximal tubule suspensions and primary cultures of rabbit renal proximal tubule cells. The apparent K(i) for CTFC inhibition of the 1-min uptake of [(3)H]p-aminohippurate in tubule suspensions was 105+/-3 microM and suggests that CTFC interacts with basolateral organic anion transport. Also, the addition of 1 mM CTFC decreased the secretion and intracellular accumulation of fluorescein by approximately 70 to 75%. The addition of 1 mM CTFC to the apical compartment decreased the reabsorption and intracellular accumulation of the amino acid [(3)H]phenylalanine by approximately 60 to 70%. Similar to CTFC, saturating concentrations of the organic anion [(3)H]p-aminohippurate and the amino acid phenylalanine reduced by approximately 75% fluorescein secretion and [(3)H]phenylalanine reabsorption, respectively, by approximately 60 to 70%. Thus, the cysteine conjugate CTFC appears to be a potent inhibitor of basolateral organic anion and apical amino acid transepithelial transport. In contrast to its effects on apical phenylalanine uptake, CTFC had no effect on the basal uptake of [(3)H]phenylalanine by primary cultures. The presence of CTFC in the external bath did trans-stimulate the efflux of fluorescein and [(3)H]phenylalanine across the basal and apical membrane in tubule suspensions or primary cultures, respectively, grown on plastic. Collectively, these data demonstrate that CTFC interacts with, and is transported by, two anatomically and functionally distinct transporters, the basolateral organic anion and apical neutral amino acid pathways, in the rabbit renal proximal tubule cell.

  10. 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.

  11. Indoxyl sulfate downregulates expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells.

    PubMed

    Ng, Hwee-Yeong; Yisireyili, Maimaiti; Saito, Shinichi; Lee, Chien-Te; Adelibieke, Yelixiati; Nishijima, Fuyuhiko; Niwa, Toshimitsu

    2014-01-01

    Renin-angiotensin system (RAS) plays a pivotal role in chronic kidney disease (CKD). Angiotensin converting enzyme-related carboxypeptidase 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor axis counteracts the deleterious actions of Ang II. ACE2 exerts its actions by cleaving Ang II into Ang-(1-7) which activates Mas receptor. This study aimed to determine if the expression of Mas receptor is altered in the kidneys of CKD rats, and if indoxyl sulfate (IS), a uremic toxin, affects the expression of Mas receptor in rat kidneys and cultured human proximal tubular cells (HK-2 cells). The expression of Mas receptor was examined in the kidneys of CKD and AST-120-treated CKD rats using immunohistochemistry. Further, the effects of IS on Mas receptor expression in the kidneys of normotensive and hypertensive rats were examined. The effects of IS on the expression of Mas receptor and phosphorylation of endothelial nitric oxide synthase (eNOS) in HK-2 cells were examined using immunoblotting. CKD rats showed reduced renal expression of Mas receptor, while AST-120 restored its expression. Administration of IS downregulated Mas receptor expression in the kidneys of normotensive and hypertensive rats. IS downregulated Mas receptor expression in HK-2 cells in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR), and signal transducer and activator of transcription 3 (Stat3) inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. N-acetylcysteine, an antioxidant, also inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. Ang-(1-7) attenuated IS-induced transforming growth factor-β1 (TGF-β1) expression. Mas receptor expression is reduced in the kidneys of CKD rats. IS downregulates renal expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells. IS-induced downregulation of Mas receptor might be involved in upregulation of TGF-β1 in proximal tubular

  12. 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

  13. GAIP, GIPC and Galphai3 are concentrated in endocytic compartments of proximal tubule cells: putative role in regulating megalin's function.

    PubMed

    Lou, Xiaojing; McQuistan, Tammie; Orlando, Robert A; Farquhar, Marilyn Gist

    2002-04-01

    Megalin is the most abundant endocytic receptor in the proximal tubule epithelium (PTE), where it is concentrated in clathrin-coated pits (CCPs) and vesicles in the brush border region. The heterotrimeric G protein alpha subunit, Galphai3, has also been localized to the brush border region of PTE. By immunofluorescence GIPC and GAIP, components of G protein-mediated signaling pathways, are also concentrated in the brush border region of PTE and are present in megalin-expressing cell lines. By cell fractionation, these signaling molecules cosediment with megalin in brush border and microvillar fractions. GAIP is found by immunoelectron microscopy in CCPs, and GIPC is found in CCPs and apical tubules of endocytic compartments in the renal brush border. In precipitation assays, GST-GIPC specifically binds megalin. The concentration of Galphai3, GIPC, and GAIP with megalin in endocytic compartments of the proximal tubule, where extensive endocytosis occurs, and the interaction between GIPC and the cytoplasmic tail of megalin suggest a model whereby G protein-mediated signaling may regulate megalin's endocytic function and/or trafficking.

  14. Simvastatin Inhibits Epithelial-to-Mesenchymal Transition Through Induction of HO-1 in Cultured Renal Proximal Tubule Cells.

    PubMed

    Clark, Jeb S; Carter, Anthony J; Dixit, Mehul; Arany, Istvan

    2016-01-01

    Studies have shown that simvastatin (SIM) inhibits epithelial-mesenchymal transition (EMT), a key step in fibrosis, and activates the anti-fibrotic heme oxygenase-1 (HO-1) gene in renal proximal tubule cells independent of its lipid-lowering. We tested the hypothesis that SIM inhibits EMT via HO-1-dependent suppression of reactive oxygen species (ROS) release. Renal proximal tubule cells were treated with either 10 μM SIM or 10 ng/ml transforming growth factor-β1 (TGFβ1) or with their combination and promoter activity of the alpha-smooth muscle actin (α-SMA) gene, stress fiber formation (markers of EMT), as well as ROS production were determined. HO-1 was manipulated via genetic and pharmacologic means. SIM prevented TGFβ1-dependent EMT and ROS production. Inhibition/knockdown of HO-1 reversed, while induction/overexpression of HO-1 emulated beneficial effects of SIM. SIM, via HO-1, suppresses TGFβ1-dependent ROS production and, hence, EMT. Further evaluation of the anti-fibrotic nature of SIM in the kidney would be useful in the treatment of chronic kidney disease. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  15. Scintillator Measurements for SNO+

    NASA Astrophysics Data System (ADS)

    Kaptanoglu, Tanner; SNO+ Collaboration

    2016-03-01

    SNO+ is a neutrino detector located 2km underground in the SNOLAB facility with the primary goal of searching for neutrinoless double beta decay. The detector will be filled with a liquid scintillator target primarily composed of linear alkyl benzene (LAB). As charged particles travel through the detector the LAB produces scintillation light which is detected by almost ten thousand PMTs. The LAB is loaded with Te130, an isotope known to undergo double beta decay. Additionally, the LAB is mixed with an additional fluor and wavelength shifter to improve the light output and shift the light to a wavelength regime in which the PMTs are maximally efficient. The precise scintillator optics drastically affect the ultimate sensitivity of SNO+. I will present work being done to measure the optical properties of the SNO+ scintillator cocktail. The measured properties are used as input to a scintillation model that allows us to extrapolate to the SNO+ scale and ultimately predict the sensitivity of the experiment. Additionally, I will present measurements done to characterize the R5912 PMT, a candidate PMT for the second phase of SNO+ that provides better light collection, improved charge resolution, and a narrower spread in timing.

  16. 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

  17. Spatial Proximity to Fibroblasts Impacts Molecular Features and Therapeutic Sensitivity of Breast Cancer Cells Influencing Clinical Outcomes.

    PubMed

    Marusyk, Andriy; Tabassum, Doris P; Janiszewska, Michalina; Place, Andrew E; Trinh, Anne; Rozhok, Andrii I; Pyne, Saumyadipta; Guerriero, Jennifer L; Shu, Shaokun; Ekram, Muhammad; Ishkin, Alexander; Cahill, Daniel P; Nikolsky, Yuri; Chan, Timothy A; Rimawi, Mothaffar F; Hilsenbeck, Susan; Schiff, Rachel; Osborne, Kent C; Letai, Antony; Polyak, Kornelia

    2016-11-15

    Using a three-dimensional coculture model, we identified significant subtype-specific changes in gene expression, metabolic, and therapeutic sensitivity profiles of breast cancer cells in contact with cancer-associated fibroblasts (CAF). CAF-induced gene expression signatures predicted clinical outcome and immune-related differences in the microenvironment. We found that fibroblasts strongly protect carcinoma cells from lapatinib, attributable to its reduced accumulation in carcinoma cells and an elevated apoptotic threshold. Fibroblasts from normal breast tissues and stromal cultures of brain metastases of breast cancer had similar effects as CAFs. Using synthetic lethality approaches, we identified molecular pathways whose inhibition sensitizes HER2(+) breast cancer cells to lapatinib both in vitro and in vivo, including JAK2/STAT3 and hyaluronic acid. Neoadjuvant lapatinib therapy in HER2(+) breast tumors lead to a significant increase of phospho-STAT3(+) cancer cells and a decrease in the spatial proximity of proliferating (Ki67(+)) cells to CAFs impacting therapeutic responses. Our studies identify CAF-induced physiologically and clinically relevant changes in cancer cells and offer novel approaches for overcoming microenvironment-mediated therapeutic resistance. Cancer Res; 76(22); 6495-506. ©2016 AACR.

  18. Scintillator plate calorimetry

    SciTech Connect

    Price, L.E.

    1990-01-01

    Calorimetry using scintillator plates or tiles alternated with sheets of (usually heavy) passive absorber has been proven over multiple generations of collider detectors. Recent detectors including UA1, CDF, and ZEUS have shown good results from such calorimeters. The advantages offered by scintillator calorimetry for the SSC environment, in particular, are speed (<10 nsec), excellent energy resolution, low noise, and ease of achieving compensation and hence linearity. On the negative side of the ledger can be placed the historical sensitivity of plastic scintillators to radiation damage, the possibility of nonuniform response because of light attenuation, and the presence of cracks for light collection via wavelength shifting plastic (traditionally in sheet form). This approach to calorimetry is being investigated for SSC use by a collaboration of Ames Laboratory/Iowa State University, Argonne National Laboratory, Bicron Corporation, Florida State University, Louisiana State University, University of Mississippi, Oak Ridge National Laboratory, Virginia Polytechnic Institute and State University, Westinghouse Electric Corporation, and University of Wisconsin.

  19. Human umbilical vein endothelial cells accelerate oxalate-induced apoptosis of human renal proximal tubule epithelial cells in co-culture system which is prevented by pyrrolidine dithiocarbamate.

    PubMed

    Sarıca, Kemal; Aydin, Hasan; Yencilek, Faruk; Telci, Dilek; Yilmaz, Bayram

    2012-10-01

    Oxalate is the most common component of kidney stones and elevated urinary levels induce renal tubular cell toxicity and death which is essential for crystal attachment. Endothelial cells, in some studies have been shown to regulate certain functions of renal proximal tubule cells. The aim of this study was to evaluate the effect of endothelial cells on tubular cell apoptosis in a co-culture system mimicking the in vivo renal physiological settings. The human umbilical vein endothelial cells (HUVEC) and human renal proximal tubule epithelial cells (RPTEC) were exposed to increasing concentrations (0-1.0 mM) of oxalate with or without 10 μM PDTC pretreatment for 24 h. In HUVEC, RPTEC and HUVEC-RPTEC co-cultures, the cell viability was measured using the WST-1 assay and cell death with the TUNEL analysis using the flow cytometry. The treatment of RPTECs with oxalate lead to 8.9-26.2% cell death which was reduced to 0-1.6% with the PDTC pretreatment. The death rate of RPTECs was significantly increased by 15-19% at different oxalate concentrations when co-cultured with HUVECs. In contrast, cell viability was not substantially altered in PDTC pretreated RPTECs that were co-cultured with HUVECs. Apoptosis was the way of cell death as similar rate of apoptosis was observed in cell culture systems. Although cell viability of RPTECs was further reduced when co-cultured with HUVECs, it was restored with the pretreatment of PDTC. This is the first study focusing on the role of endothelial cells on RPTEC apoptosis following hyperoxaluria.

  20. Photon scattering effects in heterogeneous scintillator systems

    SciTech Connect

    Ross, H.H.

    1989-01-01

    This paper describes a new experimental approach that reveals the individual contributions of sample geometry and scattering phenomena in heterogeneous flow-cell detectors. The experimental detector responses obtained using scintillating polystyrene beads with optically smooth surfaces are compared with those obtained using similar beads with highly diffuse surfaces. These comparisons are carried out for both alpha- and beta-emitting nuclides. The experimental detection efficiencies are compared to Monte Carlo simulations of the detection process. Also, a new technique will be described for the fabrication of scintillating beads. 7 refs., 8 figs., 2 tabs.

  1. Bright Lu2O3:Eu thin-film scintillators for high-resolution radioluminescence microscopy

    PubMed Central

    Sengupta, Debanti; Miller, Stuart; Marton, Zsolt; Chin, Frederick; Nagarkar, Vivek

    2015-01-01

    We investigate the performance of a new thin-film Lu2O3:Eu scintillator for single-cell radionuclide imaging. Imaging the metabolic properties of heterogeneous cell populations in real time is an important challenge with clinical implications. We have developed an innovative technique called radioluminescence microscopy, to quantitatively and sensitively measure radionuclide uptake in single cells. The most important component of this technique is the scintillator, which converts the energy released during radioactive decay into luminescent signals. The sensitivity and spatial resolution of the imaging system depend critically on the characteristics of the scintillator, i.e. the material used and its geometrical configuration. Scintillators fabricated using conventional methods are relatively thick, and therefore do not provide optimal spatial resolution. We compare a thin-film Lu2O3:Eu scintillator to a conventional 500 μm thick CdWO4 scintillator for radioluminescence imaging. Despite its thinness, the unique scintillation properties of the Lu2O3:Eu scintillator allow us to capture single positron decays with over fourfold higher sensitivity, a significant achievement. The thin-film Lu2O3:Eu scintillators also yield radioluminescence images where individual cells appear smaller and better resolved on average than with the CdWO4 scintillators. Coupled with the thin-film scintillator technology, radioluminescence microscopy can yield valuable and clinically relevant data on the metabolism of single cells. PMID:26183115

  2. Bright Lu2 O3 :Eu Thin-Film Scintillators for High-Resolution Radioluminescence Microscopy.

    PubMed

    Sengupta, Debanti; Miller, Stuart; Marton, Zsolt; Chin, Frederick; Nagarkar, Vivek; Pratx, Guillem

    2015-07-16

    The performance of a new thin-film Lu2 O3 :Eu scintillator for single-cell radionuclide imaging is investigated. Imaging the metabolic properties of heterogeneous cell populations in real time is an important challenge with clinical implications. An innovative technique called radioluminescence microscopy has been developed to quantitatively and sensitively measure radionuclide uptake in single cells. The most important component of this technique is the scintillator, which converts the energy released during radioactive decay into luminescent signals. The sensitivity and spatial resolution of the imaging system depend critically on the characteristics of the scintillator, that is, the material used and its geometrical configuration. Scintillators fabricated using conventional methods are relatively thick and therefore do not provide optimal spatial resolution. A thin-film Lu2 O3 :Eu scintillator is compared to a conventional 500 μm thick CdWO4 scintillator for radioluminescence imaging. Despite its thinness, the unique scintillation properties of the Lu2 O3 :Eu scintillator allow us to capture single-positron decays with fourfold higher sensitivity, which is a significant achievement. The thin-film Lu2 O3 :Eu scintillators also yield radioluminescence images where individual cells appear smaller and better resolved on average than with the CdWO4 scintillators. Coupled with the thin-film scintillator technology, radioluminescence microscopy can yield valuable and clinically relevant data on the metabolism of single cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. 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.

  4. Roles of ZIP8, ZIP14, and DMT1 in transport of cadmium and manganese in mouse kidney proximal tubule cells.

    PubMed

    Fujishiro, Hitomi; Yano, Yu; Takada, Yukina; Tanihara, Maya; Himeno, Seiichiro

    2012-07-01

    Chronic exposure to cadmium causes preferential accumulation of cadmium in the kidney, leading to nephrotoxicity. In the process of renal cadmium accumulation, the cadmium bound to a low-molecular-weight metal-binding protein, metallothionein, has been considered to play an important role in reabsorption by epithelial cells of proximal tubules in the kidney. However, the role and mechanism of the transport of Cd(2+) ions in proximal tubule cells remain unclear. Zinc transporters such as Zrt, Irt-related protein 8 (ZIP8) and ZIP14, and divalent metal transporter 1 (DMT1) have been reported to have affinities for Cd(2+) and Mn(2+). To examine the roles of these metal transporters in the absorption of luminal Cd(2+) and Mn(2+) into proximal tubule cells, we utilized a cell culture system, in which apical and basolateral transport of metals can be separately examined. The uptake of Cd(2+) and Mn(2+) from the apical side of proximal tubule cells was inhibited by simultaneous addition of Mn(2+) and Cd(2+), respectively. The knockdown of ZIP8, ZIP14 or DMT1 by siRNA transfection significantly reduced the uptake of Cd(2+) and Mn(2+) from the apical membrane. The excretion of Cd(2+) and Mn(2+) was detected predominantly in the apical side of the proximal tubule cells. In situ hybridization of these transporters revealed that ZIP8 and ZIP14 are highly expressed in the proximal tubules of the outer stripe of the outer medulla. These results suggest that ZIP8 and ZIP14 expressed in the S3 segment of proximal tubules play significant roles in the absorption of Cd(2+) and Mn(2+) in the kidney.

  5. Surround inhibition of mammalian AII amacrine cells is generated in the proximal retina

    PubMed Central

    Bloomfield, Stewart A; Xin, Daiyan

    2000-01-01

    Intracellular recordings were obtained from neurons in the superfused retina-eyecup preparation of the rabbit under dark-adapted conditions. Neurotransmitter agonists and antagonists were applied exogenously via the superfusate to dissect the synaptic pathways pharmacologically and thereby determine those pathways responsible for the generation of the on-centre/off-surround receptive fields of AII amacrine cells. Application of the metabotropic glutamate receptor agonist, APB, reversibly blocked both the on-centre and off-surround responses of AII cells. These data were consistent with the idea that both the centre- and surround-mediated responses are derived from inputs from the presynaptic rod bipolar cells. Whereas rod bipolar cells showed on-receptive fields ≈100 μm across, we found no evidence for an antagonistic off-surround response using light stimuli which effectively elicited the off-surrounds of AII amacrine cells. These results indicated that the surrounds of AII cells are not derived from rod bipolar cell inputs. Application of the ionotropic glutamate receptor antagonists CNQX or DNQX enhanced the on-centre responses of AII cells but attenuated the off-surround responses. These data indicated that the centre- and surround-mediated responses could not both be derived from signals crossing the rod bipolar-to-AII cell synapse. Application of the glycine antagonist, strychnine, had only minor and variable effects on AII cell responses. However, the GABA antagonists picrotoxin and bicuculline enhanced the on-centre response but attenuated or completely blocked the off-surround response of AII cells. The GABA antagonists had no effect on the responses of horizontal cells indicating that their effects on AII cell responses reflected actions on inner retinal circuitry rather than feedback circuitry in the outer plexiform layer. Application of the voltage-gated sodium channel blocker TTX enhanced the on-centre responses of AII cells but attenuated or

  6. 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. Published by Elsevier Inc.

  7. Exposure of cultured human proximal tubular cells to cadmium, mercury, zinc and bismuth: toxicity and metallothionein induction.

    PubMed

    Rodilla, V; Miles, A T; Jenner, W; Hawksworth, G M

    1998-08-14

    The kidney, in particular the proximal convoluted tubule, is a major target site for the toxic effects of various metals. However, little is known about the early effects of these metals after acute exposure in man. In the present study we have evaluated the toxicity of several inorganic metal compounds (CdCl2, HgCl2, ZnCl2, and Bi(NO3)3) and the induction of metallothionein by these compounds in cultured human proximal tubular (HPT) cells for up to 4 days. The results showed that bismuth was not toxic even at the highest dose (100 microM) used, while zinc, cadmium and mercury exhibited varying degrees of toxicity, zinc being the least toxic and mercury the most potent. A significant degree of interindividual variation between the different isolates used in these experiments was also observed. All metals used in the present study induced MT, as revealed by immunocytochemistry. All metals showed maximal induction between 1 and 3 days after treatment. Although a certain amount of constitutive MT was present in the cultures, the intensity of the staining varied with time in culture and between the different isolates studied. No correlation could be made between the intensity of the staining in control cultures (indicating total amount of constitutive MT) and the susceptibility of a given isolate to metal toxicity. Furthermore, no correlation could be made between metal-induced MT and the susceptibility of a given isolate to that particular metal.

  8. Scintillator-fiber charged-particle track-imaging detector

    NASA Technical Reports Server (NTRS)

    Binns, W. R.; Israel, M. H.; Klarmann, J.

    1983-01-01

    A scintillator-fiber charged-particle track-imaging detector has been developed using a bundle of square cross-section plastic scintillator fiber optics, proximity focused onto an image intensified Charge Injection Device (CID) camera. Detector to beams of 15 MeV protons and relativistic Neon, Manganese, and Gold nuclei have been exposed and images of their tracks are obtained. This paper presents details of the detector technique, properties of the tracks obtained, and range measurements of 15 MeV protons stopping in the fiber bundle.

  9. 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.

  10. Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay.

    PubMed

    Bahjat, Mahnoush; Bloedjes, Timon A; van der Veen, Amélie; de Wilde, Guus; Maas, Chiel; Guikema, Jeroen E J

    2017-06-09

    The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

  11. Toxicological significance of renal Bcrp: Another potential transporter in the elimination of mercuric ions from proximal tubular cells

    SciTech Connect

    Bridges, Christy C. Zalups, Rudolfs K.; Joshee, Lucy

    2015-06-01

    Secretion of inorganic mercury (Hg{sup 2+}) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg{sup 2+} was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg{sup 2+}. To further characterize the role of Bcrp in the handling of mercuric ions and in the induction of Hg{sup 2+}-induced nephropathy, Sprague–Dawley and Bcrp knockout (bcrp{sup −/−}) rats were exposed intravenously to a non-nephrotoxic (0.5 μmol·kg{sup −1}), a moderately nephrotoxic (1.5 μmol·kg{sup −1}) or a significantly nephrotoxic (2.0 μmol·kg{sup −1}) dose of HgCl{sub 2}. In general, the accumulation of Hg{sup 2+} was greater in organs of bcrp{sup −/−} rats than in Sprague–Dawley rats, suggesting that Bcrp may play a role in the export of Hg{sup 2+} from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp{sup −/−} rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla, was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2. - Highlights: • Bcrp may mediate transport of mercury out of proximal tubular cells. • Hg-induced nephropathy was more severe in Bcrp knockout rats. • Bcrp and Mrp2 may differ in their ability to transport Hg.

  12. Effect of radiologic contrast material on cell volume regulation in proximal renal tubules from trout (Salmo trutta).

    PubMed

    Galtung, H K; Løken, M; Sakariassen, K S

    2000-11-01

    Most radiographic contrast media (CM) are hyperosmotic and pose an osmotic threat to cells they are in contact with. To study these effects at the cellular level, cell volume regulatory mechanisms were observed in proximal renal tubules following exposure to the CM iohexol, ioxaglate, and iodixanol. Isolated renal tubules from trout (Salmo trutta) were exposed to 5% vol/vol iohexol (326 mOsm), ioxaglate (314 mOsm), or iodixanol (300 mOsm) or mannitol (to achieve the same osmolalities), and cell volume changes were observed videometrically. Iohexol and ioxaglate solutions induced a rapid shrinkage (12%-13%) not followed by cell volume regulation. Without CM (same osmolality), the cells shrank 11% but then showed a 77%-88% volume recovery. This reswelling was inhibited by 55% with the Na+, K+, Cl- symporter inhibitor bumetanide (50 micromol/L). Iodixanol did not significantly affect cell volume. Tubules preincubated with CM or mannitol were then stimulated with a hypoosmotic Ringer solution (160 mOsm) resulting in a 26%-36% cellular volume increase. Compared with results of experiments without mannitol and CM, preexposure to iohexol or ioxaglate almost completely inhibited the expected regulatory shrinkage phase, while previous exposure to hyperosmotic solutions with mannitol reduced the shrinkage response by 40%-53%. In this system, the hyperosmotic iohexol and ioxaglate cause cell shrinkage followed by an impaired cell volume regulatory response. Exposure to these two CM also inhibits cell volume regulation on hypoosmotic stimulation. The isosmotic iodixanol has no such effects. These changes appear to some extent to be a result of the CM's degree of hyperosmolality, but this property alone does not explain these findings.

  13. A study of disequilibrium between 220Rn and 216Po for 220Rn measurements using a flow-through Lucas scintillation cell.

    PubMed

    Sathyabama, N; Datta, D; Gaware, J J; Mayya, Y S; Tripathi, R M

    2014-01-01

    Lucas-type scintillation cells (LSCs) are commonly used for rapid measurements of (220)Rn concentrations in flow-through mode in field and for calibration experiments in laboratories. However, in those measurements, equilibrium between (220)Rn and (216)Po is generally assumed and two alpha particles are considered to be emitted per (220)Rn decay due to very short half-life of (216)Po. In this paper, a small, yet significant disequilibrium existing between (220)Rn and (216)Po has been examined and shown that less than two alpha particles are actually emitted per (220)Rn decay in the cell when flow is maintained. A theoretical formula has been derived for the first time for a correction factor (CF) to be applied to this measured concentration to account for the disequilibrium. The existence of this disequilibrium has been verified experimentally and is found to increase with the increase in the ratio of flow rate to cell volume. The reason for the disequilibrium is attributed to the flushing out of (216)Po formed in the cell before its decay due to the flow. Uncertainties in measured concentrations have been estimated and the estimated CF values have been found to be significant for the flow rates considered above 5 dm(3) min(-1) for a cell of volume 0.125 dm(3). The calculated values of the CF are about 1.055 to 1.178 in the flow rate range of 4 to 15 dm(3) min(-1) for the cell of volume 0.125 dm(3), while the corresponding experimental values are 1.023 to 1.264. This is a systematic error introduced in (220)Rn measurements using a flow-through LSC, which can be removed either by correct formulation or by proper design of a measurement set-up.

  14. Scintillator Waveguide For Sensing Radiation

    DOEpatents

    Bliss, Mary; Craig, Richard A.; Reeder; Paul L.

    2003-04-22

    The present invention is an apparatus for detecting ionizing radiation, having: a waveguide having a first end and a second end, the waveguide formed of a scintillator material wherein the therapeutic ionizing radiation isotropically generates scintillation light signals within the waveguide. This apparatus provides a measure of radiation dose. The apparatus may be modified to permit making a measure of location of radiation dose. Specifically, the scintillation material is segmented into a plurality of segments; and a connecting cable for each of the plurality of segments is used for conducting scintillation signals to a scintillation detector.

  15. Scintillator requirements for medical imaging

    SciTech Connect

    Moses, William W.

    1999-09-01

    Scintillating materials are used in a variety of medical imaging devices. This paper presents a description of four medical imaging modalities that make extensive use of scintillators: planar x-ray imaging, x-ray computed tomography (x-ray CT), SPECT (single photon emission computed tomography) and PET (positron emission tomography). The discussion concentrates on a description of the underlying physical principles by which the four modalities operate. The scintillator requirements for these systems are enumerated and the compromises that are made in order to maximize imaging performance utilizing existing scintillating materials are discussed, as is the potential for improving imaging performance by improving scintillator properties.

  16. Making maxillary barbels with a proximal-distal gradient of Wnt signals in matrix-bound mesenchymal cells

    PubMed Central

    Figueroa, Francisco; Singer, Susan S.; LeClair, Elizabeth E.

    2015-01-01

    a proximal-distal gradient along the appendage. This documents a novel mechanism of vertebrate appendage outgrowth. Similar genetic signals and cell behaviors may be responsible for the independent and repeated evolution of barbel structures in other fish species. PMID:26492827

  17. Polysiloxane scintillator composition

    DOEpatents

    Walker, James K.

    1992-01-01

    A plastic scintillator useful for detecting ionizing radiation comprising a matrix which comprises an optically transparent polysiloxane having incorporated therein at least one ionizing radiation-hard fluor capable of converting electromagnetic energy produced in the polysiloxane upon absorption of ionizing radiation to detectable light.

  18. Polysiloxane scintillator composition

    DOEpatents

    Walker, J.K.

    1992-05-05

    A plastic scintillator useful for detecting ionizing radiation comprising a matrix which comprises an optically transparent polysiloxane having incorporated therein at least one ionizing radiation-hard fluor capable of converting electromagnetic energy produced in the polysiloxane upon absorption of ionizing radiation to detectable light.

  19. Boron loaded scintillator

    DOEpatents

    Bell, Zane William [Oak Ridge, TN; Brown, Gilbert Morris [Knoxville, TN; Maya, Leon [Knoxville, TN; Sloop, Jr., Frederick Victor; Sloop, Jr., Frederick Victor [Oak Ridge, TN

    2009-10-20

    A scintillating composition for detecting neutrons and other radiation comprises a phenyl containing silicone rubber with carborane units and at least one phosphor molecule. The carbonate units can either be a carborane molecule dispersed in the rubber with the aid of a compatibilization agent or can be covalently bound to the silicone.

  20. An equatorial scintillation model

    NASA Astrophysics Data System (ADS)

    Fremouw, E. J.; Robins, R. E.

    1985-09-01

    Radiowave scintillation in the presence of natural and/or high altitude nuclear disturbances has the potential to disrupt numerous transionospheric radio and radar systems. This report develops a model characterizing the plasma density irregularities that produce scintillation in the naturally disturbed equatorial F layer. The model has been incorporated into Program WBMOD along with subroutines for computing both link geometry and scintillation indices, the latter by means of phase screen diffraction theory. The model is based on similarly extensive analysis of Wideband data from two equatorial stations. It describes irregularities at an effective height of 350 km that are isotropic across the geomagnetic field and elongated by a factor of 50 along the field and whose one dimensional spatial power spectrum obeys a single regime power law with a (negative) spectral index of 1.5. The height-integrated spectral strength of the irregularities is modeled as a function of solar epoch (sunspot number), the angle between the sunset terminator and the geomagnetic field line through the equatorial F layer point in question (a measure of seasonal and longitudinal variation), time after E-layer sunset on that field line, and the F-layer magnetic apex latitude of the point. The report also highlights a factor missing from complete characterization of the joint seasonal/longitudinal variation of scintillation, thought to depend upon thermospheric neutral winds.

  1. SCINTILLATION EXPOSURE RATE DETECTOR

    DOEpatents

    Spears, W.G.

    1960-11-01

    A radiation detector for gamma and x rays is described. The detector comprises a scintillation crystal disposed between a tantalum shield and the input of a photomultiplier tube, the crystal and the shield cooperating so that their combined response to a given quantity of radiation at various energy levels is substantially constant.

  2. Quenching equation for scintillation

    NASA Astrophysics Data System (ADS)

    Kato, Takahisa

    1980-06-01

    A mathematical expression is postulated showing the relationship between counting rate and quenching agent concentration in a liquid scintillation solution. The expression is more suited to a wider range of quenching agent concentrations than the Stern-Volmer equation. An estimation of the quenched correction is demonstrated using the expression.

  3. ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer

    PubMed Central

    2012-01-01

    Background ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. Results It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. Conclusion This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease. PMID:22550998

  4. Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell.

    PubMed

    Rodman, J S; Kerjaschki, D; Merisko, E; Farquhar, M G

    1984-05-01

    The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule

  5. 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

  6. Potentiation by nitric oxide of cyclosporin A and FK506-induced apoptosis in renal proximal tubule cells.

    PubMed

    Hortelano, S; Castilla, M; Torres, A M; Tejedor, A; Boscá, L

    2000-12-01

    Proximal tubular epithelial cells (PTEC) exhibit a high sensitivity to undergo apoptosis in response to proinflammatory stimuli and immunosuppressors and participate in the onset of several renal diseases. This study examined the expression of inducible nitric oxide (NO) synthase after challenge of PTEC with bacterial cell wall molecules and inflammatory cytokines and analyzed the pathways that lead to apoptosis in these cells by measuring changes in the mitochondrial transmembrane potential and caspase activation. The data show that the apoptotic effects of proinflammatory stimuli mainly were due to the expression of inducible NO synthase. Cyclosporin A and FK506 inhibited partially NO synthesis. However, both NO and immunosuppressors induced apoptosis, probably through a common mechanism that involved the irreversible opening of the mitochondrial permeability transition pore. Activation of caspases 3 and 7 was observed in cells treated with high doses of NO and with moderate concentrations of immunosuppressors. The conclusion is that the cooperation between NO and immunosuppressors that induce apoptosis in PTEC might contribute to the renal toxicity observed in the course of immunosuppressive therapy.

  7. Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells.

    PubMed

    Limonciel, Alice; Wilmes, Anja; Aschauer, Lydia; Radford, Robert; Bloch, Katarzyna M; McMorrow, Tara; Pfaller, Walter; van Delft, Joost H; Slattery, Craig; Ryan, Michael P; Lock, Edward A; Jennings, Paul

    2012-11-01

    Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

  8. Transcriptional regulation of NHE3 and SGLT1 by the circadian clock protein Per1 in proximal tubule cells

    PubMed Central

    Solocinski, Kristen; Richards, Jacob; All, Sean; Cheng, Kit-Yan; Khundmiri, Syed J.

    2015-01-01

    We have previously demonstrated that the circadian clock protein period (Per)1 coordinately regulates multiple genes involved in Na+ reabsorption in renal collecting duct cells. Consistent with these results, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. The proximal tubule is responsible for a majority of Na+ reabsorption. Previous work has demonstrated that expression of Na+/H+ exchanger 3 (NHE3) oscillates with a circadian pattern and Na+-glucose cotransporter (SGLT)1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of the present study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3 but not SGLT2 in the renal cortex of mice. Per1 small interfering RNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA suggested that the effects of Per1 on NHE3 and SGLT1 expression occurred at the level of transcription. Per1 and the circadian protein CLOCK were detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na+-K+-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney. PMID:26377793

  9. Toxicological Significance of Renal Bcrp: Another Potential Transporter in the Elimination of Mercuric Ions from Proximal Tubular Cells

    PubMed Central

    Bridges, Christy C.; Zalups, Rudolfs K.; Joshee, Lucy

    2015-01-01

    Secretion of inorganic mercury (Hg2+) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg2+ was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg2+. To further characterize the role of Bcrp in the handling of mercuric ions and in the induction of Hg2+-induced nephropathy, Sprague-Dawley and Bcrp knockout (bcrp−/−) rats were exposed intravenously to a non-nephrotoxic (0.5 μmol • kg−1), a moderately nephrotoxic (1.5 μmol • kg−1) or a significantly nephrotoxic (2.0 μmol • kg−1) dose of HgCl2. In general, the accumulation of Hg2+ was greater in organs of bcrp−/− rats than in Sprague-Dawley rats, suggesting that Bcrp may play a role in the export of Hg2+ from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp−/− rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2. PMID:25868844

  10. Hypoxia response and VEGF-A expression in human proximal tubular epithelial cells in stable and progressive renal disease.

    PubMed

    Rudnicki, Michael; Perco, Paul; Enrich, Julia; Eder, Susanne; Heininger, Dorothea; Bernthaler, Andreas; Wiesinger, Martin; Sarközi, Rita; Noppert, Susie-Jane; Schramek, Herbert; Mayer, Bernd; Oberbauer, Rainer; Mayer, Gert

    2009-03-01

    Proteinuria, inflammation, chronic hypoxia, and rarefaction of peritubular capillaries contribute to the progression of renal disease by affecting proximal tubular epithelial cells (PTECs). To study the transcriptional response that separates patients with a stable course from those with a progressive course of disease, we isolated PTECs by laser capture microdissection from cryocut tissue sections of patients with proteinuric glomerulopathies (stable n=20, progressive n=11) with a median clinical follow-up of 26 months. Gene-expression profiling and a systems biology analysis identified activation of intracellular vascular endothelial growth factor (VEGF) signaling and hypoxia response pathways in progressive patients, which was associated with upregulation of hypoxia-inducible-factor (HIF)-1alpha and several HIF target genes, such as transferrin, transferrin-receptor, p21, and VEGF-receptor 1, but downregulation of VEGF-A. The inverse expression levels of HIF-1alpha and VEGF-A were significantly superior in predicting clinical outcome as compared with proteinuria, renal function, and degree of tubular atrophy and interstitial fibrosis at the time of biopsy. Interactome analysis showed the association of attenuated VEGF-A expression with the downregulation of genes that usually stimulate VEGF-A expression, such as epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and HIF-2alpha. In vitro experiments confirmed the positive regulatory effect of EGF and IGF-1 on VEGF-A transcription in human proximal tubular cells. Thus, in progressive but not in stable proteinuric kidney disease, human PTECs show an attenuated VEGF-A expression despite an activation of intracellular hypoxia response and VEGF signaling pathways, which might be due to a reduced expression of positive coregulators, such as EGF and IGF-1.

  11. Activation of liver X receptor inhibits OCT2-mediated organic cation transport in renal proximal tubular cells.

    PubMed

    Wongwan, Teerasak; Kittayaruksakul, Suticha; Asavapanumas, Nithi; Chatsudthipong, Varanuj; Soodvilai, Sunhapas

    2017-07-25

    Liver X receptor (LXR) is transcriptional factor that plays an important role in the regulation of energy metabolism such as cholesterol, lipid, and glucose metabolism as well as membrane transporters and channels. Using both in vitro and in vivo models, LXR regulation of the expression and function of renal organic cation transporter 2 (OCT2) was observed. Synthetic LXR agonist (GW3965) and endogenous LXR agonist (22R-hydroxycholesterol) significantly reduced the uptake of (3)H-MPP(+), a prototypic substrate of OCT2, in both OCT2- Chinese hamster ovary K1 and human renal proximal tubular cells (RPTEC/TERT1). GW3965 decreased transport activity of OCT2 via a reduction of the maximal transport rate of MPP(+) without affecting transporter affinity. The inhibitory effect of GW3965 was attenuated by co-treatment with LXR antagonist (fenofibrate) indicating the inhibition was LXR-dependent mechanism. In addition, co-treatment with a retinoic X receptor (RXR) ligand, 9-cis retinoic acid enhanced the inhibitory effect of GW3965, indicating negative regulation of OCT2 transport activity by the LXR/RXR complex. Treatment RPTEC/TERT1 cells with GW3965 significantly reduced OCT2 protein expression without changing mRNA expression. In parallel, the effect of LXR activation on OCT2 function was investigated in intact mouse kidney. Treating mice with 50 mg/kg BW T0901317 for 14 days significantly decreased (3)H-MPP(+) uptake into renal cortical slices, correlating with decreased OCT2 protein expression in renal cortex without changes in mRNA expression levels. Taken together, LXR/RXR activation downregulates the protein expression and function of OCT2 in renal proximal tubule, suggesting LXR might affect the total profile of renal excretion of cationic compounds.

  12. Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

    PubMed Central

    Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne; Patel, Sanjeevkumar R.; Shisheva, Assia

    2016-01-01

    The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34–deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux. PMID:26825532

  13. 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

  14. Investigating the Anisotropic Scintillation Response in Organic Crystal Scintillator Detectors

    NASA Astrophysics Data System (ADS)

    Schuster, Patricia Frances

    This dissertation presents several studies that experimentally characterize the scintillation anisotropy in organic crystal scintillators. These include measurements of neutron, gamma-ray and cosmic muon interactions in anthracene, a historical benchmark among organic scintillator materials, to confirm and extend measurements previously available in the literature. The gamma-ray and muon measurements provide new experimental confirmation that no scintillation anisotropy is present in their interactions. Observations from these measurements have updated the hypothesis for the physical mechanism that is responsible for the scintillation anisotropy concluding that a relatively high dE/dx is required in order to produce a scintillation anisotropy. The directional dependence of the scintillation output in liquid and plastic materials was measured to experimentally confirm that no scintillation anisotropy correlated to detector orientation exists in amorphous materials. These observations confirm that the scintillation anisotropy is not due to an external effect on the measurement system, and that a fixed, repeating structure is required for a scintillation anisotropy. The directional dependence of the scintillation output in response to neutron interactions was measured in four stilbene crystals of various sizes and growth-methods. The scintillation anisotropy in these materials was approximately uniform, indicating that the crystal size, geometry, and growth method do not significantly impact the effect. Measurements of three additional pure crystals and two mixed crystals were made. These measurements showed that 1) the magnitude of the effect varies with energy and material, 2) the relationship between the light output and pulse shape anisotropy varies across materials, and 3) the effect in mixed materials is very complex. These measurements have informed the hypothesis of the mechanism that produces the directional dependence. By comparing the various relationships

  15. Effects of amyloid-β plaque proximity on the axon initial segment of pyramidal cells.

    PubMed

    León-Espinosa, Gonzalo; DeFelipe, Javier; Muñoz, Alberto

    2012-01-01

    The output of cortical pyramidal cells reflects the balance between excitatory inputs of cortical and subcortical origin, and inhibitory inputs from distinct populations of cortical GABAergic interneurons, each of which selectively innervate different domains of neuronal pyramidal cells (i.e., dendrites, soma and axon initial segment [AIS]). In Alzheimer's disease (AD), the presence of amyloid-β (Aβ) plaques alters the synaptic input to pyramidal cells in a number of ways. However, the effects of Aβ plaques on the AIS have still not been investigated to date. This neuronal domain is involved in input integration, as well as action potential initiation and propagation, and it exhibits Ca2+- and activity-dependent structural plasticity. The AIS is innervated by GABAergic axon terminals from chandelier cells, which are thought to exert a strong influence on pyramidal cell output. In the AβPP/PS1 transgenic mouse model of AD, we have investigated the effects of Aβ plaques on the morphological and neurochemical features of the AIS, including the cisternal organelle, using immunocytochemistry and confocal microscopy, as well as studying the innervation of the AIS by chandelier cell axon terminals. There is a strong reduction in GABAergic terminals that appose AIS membrane surfaces that are in contact with Aβ plaques, indicating altered inhibitory synapsis at the AIS. Thus, despite a lack of gross structural alterations in the AIS, this decrease in GABAergic innervation may deregulate AIS activity and contribute to the hyperactivity of neurons in contact with Aβ plaques.

  16. 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.

  17. Live-Cell Labeling of Specific Protein Glycoforms by Proximity-Enhanced Bioorthogonal Ligation

    PubMed Central

    Robinson, Peter V.; de Almeida-Escobedo, Gabriela; de Groot, Amber E.; McKechnie, Julia L.

    2016-01-01

    Reagents for detecting post-translational modifications in the context of their protein scaffold are powerful tools, but are challenging to develop for glycosylated epitopes. We describe a strategy for detecting protein-specific glycosylation through the use of cyclooctyne-aptamer conjugates. These molecules selectively ligate to azidosugar-labeled glycans exclusively on a target protein on live cells. We characterized aptamer conjugates against two different cell surface glycoproteins and show that these reagents are amenable to detecting protein sialoforms by mass spectrometry, Western blotting, and flow cytometry. Given the abundance of aptamers that bind cell surface targets, we expect this technology will be a useful platform for investigating the roles of protein-specific glycosylation in various cellular contexts. PMID:26280358

  18. Melamine induces Ca(2+)-sensing receptor activation and elicits apoptosis in proximal tubular cells.

    PubMed

    Yiu, Allen J; Ibeh, Cliff-Lawrence; Roy, Sanjit K; Bandyopadhyay, Bidhan C

    2017-07-01

    Melamine causes renal tubular cell injury through inflammation, fibrosis, and apoptosis. Although melamine affects the rise in intracellular Ca(2+) concentration ([Ca(2+)]i), reactive oxygen species (ROS) production, and proapoptotic pathway activation, the mechanism of upstream Ca(2+) signaling is unknown. Because melamine has some structural similarities with l-amino acids, which endogenously activate Ca(2+)-sensing receptors (CSR), we examined the effect of melamine on CSR-induced Ca(2+) signaling and apoptotic cell death. We show here that melamine activates CSR, causing a sustained Ca(2+) entry in the renal epithelial cell line, LLC-PK1. Moreover, such CSR stimulation resulted in a rise in [Ca(2+)]i, leading to enhanced ROS production. Furthermore, melamine-induced elevated [Ca(2+)]i and ROS production caused a dose-dependent increase in apoptotic (by DAPI staining, DNA laddering, and annexin V assay) and necrotic (propidium iodide staining) cell death. Upon examining the downstream mechanism, we found that transforming growth factor β1 (TGF-β1), which increases extracellular matrix genes and proapoptotic signaling, was also upregulated at lower doses of melamine, which could be due to an early event inducing apoptosis. Additionally, cells exposed to melamine displayed a rise in pERK activation and lactate dehydrogenase release resulting in cytotoxicity. These results offer a novel insight into the molecular mechanisms by which melamine exerts its effect on CSR, causing a sustained elevation of [Ca(2+)]i, leading to ROS generation, fibronectin production, proapoptotic pathway activation, and renal cell damage. Together, these results thus suggest that melamine-induced apoptosis and/or necrosis may subsequently result in acute kidney injury and promote kidney stone formation.

  19. Overexpression of RGPR-p117 enhances regucalcin gene expression in cloned normal rat kidney proximal tubular epithelial cells.

    PubMed

    Sawada, Natsumi; Yamaguchi, Masayoshi

    2005-12-01

    A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. Whether overexpression of RGPR-p117 can modulate gene expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells was investigated. NRK52E cells (wild-type) or HA-RGPR-p117/phCMV2-transfected NRK52E cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Proliferation of NRK52E cells (wild-type) was not significantly altered by overexpression of HA-RGPR-p117. The expression of rat regucalcin, alpha-fetoprotein, albumin, glucokinase, 11beta-hydroxy-steroid dehydrogenase, phosphoenolpyruvate carboxykinase, which contains TTGGC motif in the promoter region of their genes, was seen in NRK52E cells (wild-type) by using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, regucalcin mRNA levels were significantly enhanced in transfectants. The expression of p21 or glycero-aldehyde-3-phosphate dehydrogenase mRNA was not significantly changed in transfectants. The results of Western blot analysis showed that regucalcin protein was significantly increased in transfectants. The enhancement of regucalcin mRNA expression in transfectants was significantly suppressed in the presence of staurosporine (10(-10) M), an inhibitor of protein kinase C. This enhancement was not significantly changed in the presence of dibucaine (10(-8) M), PD98059 (10(-8) M) or vanadate (10(-6) M). This study demonstrates that overexpression of RGPR-p117 enhances the expression of regucalcin mRNA and its protein level in NRK52E cells. RGPR-p117 may play a role as a transcriptional factor.

  20. Bilirubin activates transcription of HIF-1α in human proximal tubular cells cultured in the physiologic oxygen content.

    PubMed

    Kim, Sung Gyun; Ahn, Shin-Young; Lee, Eun Seong; Kim, Sejoong; Na, Ki Young; Chae, Dong-Wan; Chin, Ho Jun

    2014-09-01

    The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1α protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1α was increased by 1.69±0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1α expression by bilirubin. HIF-1α expression decreased by 10 µM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1α concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1α mRNA expression. In coonclusion, bilirubin enhances HIF-1α transcription as well as the up-regulation of HIF-1α protein translation through the attenuation of ROS and subunits of NADPH oxidase.

  1. Model of very fast (>75 Hz) network oscillations generated by electrical coupling between the proximal axons of cerebellar Purkinje cells

    PubMed Central

    Traub, Roger D; Middleton, Steven J; Knöpfel, Thomas; Whittington, Miles A

    2009-01-01

    Very fast oscillations (VFO, >75 Hz) occur transiently in vivo, in the cerebellum of mice genetically modified to model Angelman syndrome, and in a mouse model of fetal alcohol syndrome. We recently reported VFO in slices of mouse cerebellar cortex (Crus I and II of ansiform and paramedian lobules), either in association with gamma oscillations (~40 Hz, evoked by nicotine), or in isolation (evoked by nicotine in combination with GABAA receptor blockade). The experimental data suggest a role for electrical coupling between Purkinje cells (blockade of VFO by drugs reducing gap junction conductance, and spikelets in some Purkinje cells); and the data suggest the specific involvement of Purkinje cell axons (because of field oscillation maxima in the granular layer). We show here that a detailed network model (1,000 multicompartment Purkinje cells) replicates the experimental data, when gap junctions are located on the proximal axons of Purkinje cells, provided sufficient spontaneous firing is present. Unlike other VFO models, most somatic spikelets do not correspond to axonal spikes in the parent axon, but reflect spikes in electrically coupled axons. The model predicts gating of VFO frequency by gNa inactivation, and experiments prolonging this inactivation time constant, with β-pompilidotoxin, are consistent with this prediction. The model also predicts that cerebellar VFO can be explained as an electrically coupled system of axons which are not intrinsic oscillators: the electrically uncoupled cells do not individually oscillate (in the model), and axonal firing rates are much lower in the uncoupled state than in the coupled state. PMID:18973579

  2. Scintillator-fiber charged particle track-imaging detector

    NASA Technical Reports Server (NTRS)

    Binns, W. R.; Israel, M. H.; Klarmann, J.

    1983-01-01

    A scintillator-fiber charged-particle track-imaging detector was developed using a bundle of square cross section plastic scintillator fiber optics, proximity focused onto an image intensified charge injection device (CID) camera. The tracks of charged particle penetrating into the scintillator fiber bundle are projected onto the CID camera and the imaging information is read out in video format. The detector was exposed to beams of 15 MeV protons and relativistic Neon, Manganese, and Gold nuclei and images of their tracks were obtained. Details of the detector technique, properties of the tracks obtained, and preliminary range measurements of 15 MeV protons stopping in the fiber bundle are presented.

  3. Direct Contacts Between Extracellular Membrane-Proximal Domains are Required for VEGF Receptor Activation and Cell Signaling

    SciTech Connect

    Yang, Y.; Xie, P; Opatowsky, Y; Schlessinger, J

    2010-01-01

    Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 {angstrom}. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.

  4. Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle

    PubMed Central

    Radford, Robert; Slattery, Craig; Jennings, Paul; Blacque, Oliver; Pfaller, Walter; Gmuender, Hans; Van Delft, Joost; Ryan, Michael P.

    2012-01-01

    The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO3) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO3 resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO3 exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO3 cause significant deciliation in a model of the proximal tubule. With KBrO3, this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO3 exposure. PMID:22262483

  5. Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle.

    PubMed

    Radford, Robert; Slattery, Craig; Jennings, Paul; Blacque, Oliver; Blaque, Oliver; Pfaller, Walter; Gmuender, Hans; Van Delft, Joost; Ryan, Michael P; McMorrow, Tara

    2012-04-15

    The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO(3)) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO(3) resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO(3) exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO(3) cause significant deciliation in a model of the proximal tubule. With KBrO(3), this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO(3) exposure.

  6. The Influence of Trocar Fenestration and Volume on Connective Tissue Progenitor Cells (Stem Cells) in Arthroscopic Bone Marrow Aspiration From the Proximal Humerus.

    PubMed

    Voss, Andreas; McCarthy, Mary Beth; Singh, Hardeep; Beitzel, Knut; DiVenere, Jessica; Cote, Mark P; Hoberman, Alexander R; Nowak, Michael; Imhoff, Andreas B; Mazzocca, Augustus D

    2017-06-01

    To evaluate the number of connective tissue progenitor cells (CTPs) and nucleated cells obtained during bone marrow aspiration (BMA) from the proximal humerus using either a fenestrated or a nonfenestrated trocar and determine differences in varying amounts of aspiration volume. The first hypothesis was that the number of CTPs extracted with the fenestrated trocar would be greater due to its potential to extract more cells through its fenestrations. The second hypothesis was that using consecutive aspirations with either trocar would provide a consistent number of CTPs and nucleated cells throughout the aspiration with no significant decrease of cells at the end. Patients were eligible for inclusion if they underwent primary or revision arthroscopic rotator cuff surgery, were between 18 and 75 years of age, and signed the informed consent. Between January 2011 and September 2013, 24 patients underwent BMA from the proximal humerus during arthroscopic surgery. They were grouped according to which of 3 different trocars were used for aspiration: (1) nonfenestrated, (2) fenestrated trocar A, and (3) fenestrated trocar H. Four consecutive 12 mL double syringes were used for each aspiration: 1 (0-12 mL), 2 (12-24 mL), 3 (24-36 mL), and 4 (36-48 mL). One milliliter was removed from each syringe (nonconcentrated BMA). The remainder of the BMA was then spun using a centrifuge. BMA and concentrated BMA were brought to the laboratory, counted for nucleated cells (million cells/mL BMA) and cultured for 7 days to obtain colony-forming units (CTPs/million cells). No significant differences were observed in tubes 1 to 4 in the number of nucleated cells in the nonconcentrated and concentrated BMA using the nonfenestrated trocar compared with the fenestrated trocars A and H (all P > .05), except for concentrated BMA tube 3 (P = .014) and tube 4 (P = .003). Nonconcentrated and concentrated BMA from tubes 1 to 4 had a significantly higher CTP prevalence using the

  7. 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.

  8. 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.

  9. An efficient and simple method for measuring (226)Ra using the scintillation cell in a delayed coincidence counting system (RaDeCC).

    PubMed

    Waska, Hannelore; Kim, Seolwon; Kim, Guebuem; Peterson, Richard N; Burnett, William C

    2008-12-01

    A delayed coincidence counter (RaDeCC), developed to determine ultra-low levels of (223)Ra (half life = 11.1 days) and (224)Ra (half life = 3.6 days) in seawater, was adapted to measure (226)Ra (half life = 1622 years). After pre-concentration of Ra from seawater onto MnO(2)-coated fiber we show in this study that the (226)Ra activity can be determined using the RaDeCC's ability to record alpha decay of its daughters as total counts. For sufficient ingrowth of (222)Rn, the Mn-fiber is hermetically sealed in a column for a few days. Then, the ingrown (222)Rn is circulated through the RaDeCC air-loop system followed by shutting down of the pump and closure of the scintillation cell for equilibration. Counting may be completed within a few hours for seawater samples. Sample measurements with this method agreed well with data obtained using gamma-ray spectrometry. This proves that a set of Ra isotopes ((223)Ra, (224)Ra, and (226)Ra), commonly used for geophysical studies such as mixing rates of different water masses and submarine groundwater discharge, can be efficiently and rapidly measured using the RaDeCC.

  10. Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in Jurkat cells.

    PubMed

    Szabo, M; Czompoly, T; Kvell, K; Talaber, G; Bartis, D; Nemeth, P; Berki, T; Boldizsar, F

    2012-02-01

    Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling.

  11. In vitro membrane reconstitution of the T cell receptor proximal signaling network

    PubMed Central

    Hui, Enfu; Vale, Ronald D.

    2014-01-01

    T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45, and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over a ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that coclustering TCR-Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs. PMID:24463463

  12. Inhibition of D4 Dopamine Receptors on Insulin Receptor Expression and Effect in Renal Proximal Tubule Cells.

    PubMed

    Zhang, Ye; Ren, Hongmei; Lu, Xi; He, Duofen; Han, Yu; Wang, Hongyong; Zeng, Chunyu; Shi, Weibin

    2016-04-22

    Ion transport in the renal proximal tubule (RPT), which is increased in essential hypertension, is regulated by numerous hormones and humoral factors, including insulin and dopamine. Activation of dopamine receptor inhibits sodium reabsorption, whereas activation of insulin receptor increases sodium reabsorption in RPTs, and hyperinsulinemic animals and patients have defective renal dopaminergic system. We presume that there is an inhibition of D4 receptor on insulin receptor expression and effect, and the regulation is lost in spontaneously hypertensive rats (SHRs). Insulin receptor expression was determined by immunoblotting, and Na(+)-K(+)-ATPase activity was detected in both Wistar-Kyoto (WKY) and SHR RPT cells. Stimulation of D4 receptor with PD168077 decreased expression of insulin receptors, which was blocked in the presence of the calcium-channel blocker, nicardipine (10(-6) mol/L per 24 hours), in cell culture medium without calcium or in the presence of inositol 1,4,5-trisphosphate (IP3) receptor blocker (2-aminoethyl diphenylborinate [2-ADB]; 10(-6) mol/L per 24 hours), indicating that extracellular calcium entry and calcium release from the endoplasmic reticulum were involved in the signal pathway. Stimulation of the insulin receptor stimulated Na(+)-K(+)-ATPase activity, whereas pretreatment with PD168077 for 24 hours decreased the inhibitory effects of insulin receptor on Na(+)-K(+)-ATPase activity in WKY cells. However, in SHR cells, inhibition of D4 receptor on insulin receptor expression and effect were lost. Activation of D4 receptor inhibits insulin receptor expression in RPT cells from WKY rats. The aberrant inhibition of D4 receptor on insulin receptor expression and effect might be involved in the pathogenesis of essential hypertension. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  13. 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.

  14. SOCS3 overexpression inhibits advanced glycation end product-induced EMT in proximal tubule epithelial cells.

    PubMed

    Yu, Lin; Zhang, Ying; Zhang, Huimin; Li, Yingtao

    2017-06-01

    Diabetic nephropathy (DN) is among the most severe complications of diabetes mellitus, and may lead to end-stage renal disease. Sustained exposure to advanced glycation end products (AGEs) typically causes renal tubular epithelial cells (TECs) to suffer from an epithelial-to-mesenchymal transition (EMT). However, there remains no consensus regarding the mechanism underlying the cause of EMT in TECs as induced by AGEs. In the present study, we investigated the promotion of EMT in TECs by AGEs, and the activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. In addition, we constructed a recombinant adenovirus (Ad) that overexpressed suppressor of cytokine signaling 3 (SOCS3), and examined the regulatory role of SOCS3 in the activation of JAK/STAT signaling and the promotion of EMT in TECs. The results demonstrated that AGE-bovine serum albumin (BSA) treatment significantly promoted the expression of EMT-associated proteins, while reducing the expression of the epithelial cell marker, E-cadherin. Furthermore, the Ad-mediated SOCS3 overexpression markedly inhibited the AGE-BSA-induced JAK2/STAT3 activation; phosphorylated JAK2 and phosphorylated STAT3 expression levels were reduced by the Ad-SOCS3 infection, compared with the control Ad (Ad-con) infection, in HK-2 cells subject to AGE-BSA. Moreover, the overexpression of SOCS3 markedly inhibited the AGE-BSA-promoted EMT in HK-2 cells. AGE-BSA-promoted EMT-associated proteins, such as α-smooth muscle actin and collagen I, were reduced by the Ad-SOCS3 virus infection, in contrast to the Ad-con virus infection. Furthermore, reduced E-cadherin expression was reversed by the Ad-SOCS3 virus infection, in contrast to the Ad-con virus infection, in epithelial HK-2 cells. In conclusion, the present study confirmed the inhibitory role of SOCS3 in the AGE-induced EMT in renal TECs, implying the protective role of SOCS3 in DN.

  15. 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

  16. Composite scintillator screen

    DOEpatents

    Zeman, Herbert D.

    1994-01-01

    A scintillator screen for an X-ray system includes a substrate of low-Z material and bodies of a high-Z material embedded within the substrate. By preselecting the size of the bodies embedded within the substrate, the spacial separation of the bodies and the thickness of the screen, the sensitivity of the screen to X-rays within a predetermined energy range can be predicted.

  17. An Equatorial Scintillation Model

    DTIC Science & Technology

    1985-09-30

    been incor- porated into Program WBMOD along with subroutines for computing both link geometry and scintillation indices, the latter by means of...phase4screen diffraction theory. , Earlier versions of WBMOD , which are operational at USAF Global Weather Central and at several other user locations...which has been incorporated in WBMOD Version 8DI, is based on similarly extensive analysis of Wideband data from two equatorial stations. It describes

  18. The HK-2 human renal proximal tubule cell as a model for GRK4-mediated dopamine-1 receptor uncoupling

    PubMed Central

    Gildea, John J.; Shah, Ishan; Weiss, Ryan; Casscells, Nicholas D.; McGrath, Helen E.; Zhang, Jin; Felder, Robin A.

    2012-01-01

    HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of “normal” RPTCs. We recently discovered that HK-2 cells are uncoupled from dopamine-1 receptor (D1R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms (SNPs) may be responsible for the D1R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 SNPs, measuring D1-like receptor agonist (fenoldopam)stimulated cAMP accumulation, quantifying D1R inhibition of sodium transport, and testing the ability of GRK4 siRNA to reverse the D1R/AC uncoupling. We compared HK-2 to 2 normally coupled human RPTC cell lines (nRPTC) and 2 uncoupled RPTC cell lines (uRPTC). The HK-2 cell line was found to have 4 out of 6 potential GRK4 SNPs known to uncouple the D1R from AC (namely R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the two nRPTC cell lines (FEN 2.02±0.05-fold and 2.33±0.19-fold over control, P<0.001, N=4), but not in the two uncoupled or HK-2 cell lines. GRK4 siRNA rescued the fenoldopam-mediated AC stimulation in the uncoupled cells, including HK-2. The expected fenoldopam -mediated inhibition of sodium hydrogen exchanger type 3 was absent in HK-2 (N=6) and uRPTCs (N=6), but was observed in the two nRPTCs (−25.41±4.7% and −27.36±2.70% (P<0.001, N=6)), which express wild-type GRK4. Despite the fact that HK-2 cells retain many functional characteristics of RPTCs, they are not normal from the perspective of dopaminergic function. PMID:20660820

  19. A 350 bp region of the proximal promoter of Rds drives cell-type specific gene expression

    PubMed Central

    Cai, Xue; Conley, Shannon M.; Cheng, Tong; Al-Ubaidi, Muayyad R.; Naash, Muna I.

    2010-01-01

    RDS (retinal degeneration slow) is a photoreceptor-specific tetraspanin protein required for the biogenesis and maintenance of rod and cone outer segments. Mutations in the Rds gene are associated with multiple forms of rod- and cone-dominant retinal degeneration. To gain more insight into the mechanisms underlying the regulation of this gene the identification of regulatory sequences within the promoter of Rds was undertaken. A 3.5kb fragment of the 5′ flanking region of the mouse Rds gene was isolated and binding sites for Crx, Otx2, Nr2e3, RXR family members, Mef2C, Esrrb, NF1, AP1, and SP1 in addition to several E-boxes, GC-boxes and GAGA-boxes were identified. Crx binding sequences were conserved in all mammalian species examined. Truncation expression analysis of the Rds promoter region in Y-79 retinoblastoma cells showed maximal activity in the 350bp proximal promoter region. We also show that inclusion of more distal fragments reduced promoter activity to the basal level, and that the promoter activities are cell-type and direction specific. Co-transfection with Nrl increased promoter activity, suggesting that this gene positively regulates Rds expression. Based on these findings, a relatively small fragment of the Rds promoter may be useful in future gene transfer studies to drive gene expression in photoreceptors. PMID:20447394

  20. Targeted Covalent Inhibition of Grb2-Sos1 Interaction through Proximity-Induced Conjugation in Breast Cancer Cells.

    PubMed

    Yu, Yongsheng; Nie, Yunyu; Feng, Qian; Qu, Jiale; Wang, Rui; Bian, Liming; Xia, Jiang

    2017-05-01

    Targeted covalent inhibitors of protein-protein interactions differ from reversible inhibitors in that the former bind and covalently bond the target protein at a specific site of the target. The site specificity is the result of the proximity of two reactive groups at the bound state, for example, one mild electrophile in the inhibitor and a natural cysteine in the target close to the ligand binding site. Only a few pharmaceutically relevant proteins have this structural feature. Grb2, a key adaptor protein in maintaining the ERK activity via binding Sos1 to activated RTKs, is one: the N-terminal SH3 domain of Grb2 (Grb2(N-SH3)) carries a unique solvent-accessible cysteine Cys(32) close to its Sos1-binding site. Here we report the design of a peptide-based antagonist (a reactive peptide) that specifically binds to Grb2(N-SH3) and subsequently undergoes a nucleophilic reaction with Cys(32) to form a covalent bond thioether, to block Grb2-Sos1 interaction. Through rounds of optimization, we eventually obtained a dimeric reaction reactive peptide that can form a covalent adduct with endogenous Grb2 protein inside the cytosol of SK-BR-3 human breast cancer cells with pronounced inhibitory effect on cell mobility and viability. This work showcases a rational design of Grb2-targeted site-specific covalent inhibitor and its pronounced anticancer effect by targeting Grb2-Sos1 interaction.

  1. 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.

  2. Effect of betamethasone on Na-K-ATPase activity and basal and lateral cell membranes in proximal tubular cells during early development.

    PubMed

    Igarashi, Y; Aperia, A; Larsson, L; Zetterström, R

    1983-08-01

    The mechanism by which betamethasone induces Na-K-ATPase activity in developing tissue was studied in homogenates of proximal tubular cells from 10-day-old rats. A significant increase in Na-K-ATPase activity occurred after 5 micrograms . 100 g-1 . 12 h-1 X 2 beta-methasone and a maximal increase after 15-60 micrograms . 100 g-1 . 12 h-1 X 2. Following a single dose of 60 micrograms . 100 g-1 betamethasone Na-K-ATPase activity increased significantly after 16 h and maximally after 24-30 h. The 16-h time lag suggests that betamethasone does not act only directly on Na-K-ATPase synthesis. Betamethasone 60 micrograms . 100 g-1 increases Na-K-ATPase activity significantly in kidneys in which glomerular filtration rate is reduced by ureteral ligation, but the increase is significantly less pronounced than in kidneys with intact ureters, suggesting that the induction is not mediated only by alterations in sodium supply. Twenty-four hours after 10-60 micrograms . 100 g-1 betamethasone there was no significant increase in glucose-6-phosphatase and Mg-ATPase activity in 10-day-old rats or in Na-K-ATPase activity in 40-day-old rats. The basal and lateral cell membranes of the proximal tubular cells were not significantly increased 24 h after 60 micrograms . 100 g-1 betamethasone. Accordingly, structural development is not a prerequisite for enzymatic differentiation.

  3. Targeting superoxide dismutase to renal proximal tubule cells inhibits nephrotoxicity of cisplatin and increases the survival of cancer-bearing mice.

    PubMed

    Nishikawa, M; Nagatomi, H; Nishijima, M; Ohira, G; Chang, B J; Sato, E; Inoue, M

    2001-10-10

    Because cis-diamminedichloroplatinum(II) (cisplatin) which generates reactive oxygen species induces renal dysfunction, administration of a large dose for killing cancer cells is highly limited. We recently synthesized a cationic superoxide dismutase (SOD) (hexamethylenediamine-conjugated SOD, AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Treatment of Ehrlich ascites tumor cells (EATC)-bearing mice with cisplatin sufficient for killing tumor cells increased their motality. The motality of cisplatin-treated EATC-bearing mice was markedly decreased by AH-SOD. These results suggest that targeting SOD to renal proximal tubule cells might permit the administration of high doses of cisplatin and related anticancer agents without causing renal injury.

  4. Characterization of the interaction of staphylococcal enterotoxin B with CD1d expressed in human renal proximal tubule epithelial cells.

    PubMed

    Hammamieh, Rasha; Chakraborty, Nabarun; Lin, Yixin; Shupp, Jeffrey W; Miller, Stacy-Ann; Morris, Sam; Jett, Marti

    2015-02-04

    Participation of renal cells in the pathogenesis of staphylococcal enterotoxin B (SEB) is critical for late cleansing and sequestration of the antigens facilitated by CD1d mediated antigen sensing and recognition. This is a noted deviation from the 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. Using diffraction-based dotReady™ immunoassays, the present study showed that SEB directly and specifically conjugated to CD1d. The specificity of the conjugation between SEB and CD1d expressed on human renal proximal tubule epithelial cells (RPTEC) was further established by selective inhibition of CD1d prior to its exposure to SEB. We found that SEB induced the expression of CD1d on the cell surface prompting a rapid conjugation between them. The mRNA transcripts encoding CD1d remained elevated potentially after completing the antigen cleansing process. Molecular targets associated with the delayed pathogenic response have essential therapeutic values. Particularly in the event of bioterrorism, the caregivers are typically able to intervene much later than the toxic exposures. Given circumstances mandate a paradigm shift from the conventional therapeutic strategy that counts on targeting the host markers responding to the early assault of pathogens. We demonstrated the role of CD1d in the late stage of pathogen recognition and cleansing, and thereby underscored its clinical potential in treating bioweaponizable antigens, such as Staphylococcal enterotoxin B (SEB).

  5. V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells.

    PubMed

    Cao, Xueqin; Yang, Qiongqiong; Qin, Jing; Zhao, Shili; Li, Xiaoyan; Fan, Jinjin; Chen, Wenfang; Zhou, Yi; Mao, Haiping; Yu, Xueqing

    2012-05-01

    The ubiquitous vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na(+)-independent pH recovery was significantly faster after an NH(4)Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-β1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.

  6. Antioxidant treatment induces transcription and expression of transforming growth factor beta in cultured renal proximal tubular cells.

    PubMed

    Wolf, G; Hannken, T; Schroeder, R; Zahner, G; Ziyadeh, F N; Stahl, R A

    2001-01-19

    Transforming growth factor beta (TGF-beta) plays an important role in the development of tubulointerstitial fibrosis in chronic renal disease. We were interested whether interference with oxygen radicals may modulate TGF-beta expression. Unexpectedly, we discovered that diphenylene iodine (DIP), an inhibitor of NADP(H) oxidase, induces a robust increase in TGF-beta transcript expression in cultured mouse proximal tubular cells (MCT cells). A similar increase was seen with EUK-8, a synthetic salen-manganese complex with high oxyradical scavenger activities. This induction of TGF-beta1 mRNA was paralleled by increasing protein expression. Transient transfection of MCT cells with a reporter construct in which murine TGF-beta1 enhancer/promoter elements were cloned in front of the luciferase gene, revealed that DIP, EUK-8, and Tiron all stimulated transcription of the TGF-beta1 gene whereas exogenous H2O2 suppressed transcription. Antisense oligonucleotides against p22phox, but not sense oligonucleotides, also increased transcriptional activity of TGF-beta1. Mutagenesis of Sp1 binding sites in the mouse TGF-beta1 enhancer/promoter abolished the stimulatory effect of the antioxidants. Gel shift experiments revealed that DIP as well as EUK-8 activated binding of nuclear proteins to Sp1 consensus sequence. Our data provide evidence that TGF-beta1 transcription is negatively regulated in MCT cells under basal conditions by NADP(H) oxidase-mediated oxygen radicals. Thus, antioxidant therapy may increase local synthesis of TGF-beta1 in the tubulointerstitium.

  7. New scintillator and waveshifter materials

    NASA Astrophysics Data System (ADS)

    Zheng, H.; Baumbaugh, B.; Gerig, A.; Hurlbut, C.; Kauffman, J.; Marchant, J.; Pla-Dalmau, A.; Reynolds, K.; Ruchti, R.; Warchol, J.; Wayne, M.

    1998-11-01

    Experimental applications requiring fast timing and/or high efficiency position and energy measurements typically use scintillation materials. Scintillators utilized for triggering, tracking, and calorimetry in colliding beam detectors are vulnerable to the high radiation fields associated with such experiments. We have begun an investigation of several fluorescent dyes which might lead to fast, efficient, and radiation resistant scintillators. Preliminary results of spectral analysis and efficiency are presented.

  8. 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

  9. A functional study of proximal goat β-casein promoter and intron 1 in immortalized goat mammary epithelial cells.

    PubMed

    Kung, M H; Lee, Y J; Hsu, J T; Huang, M C; Ju, Y T

    2015-06-01

    Goat β-casein (CSN2) promoter has been extensively used to derive expression of recombinant therapeutic protein in transgenic goats; however, little direct evidence exists for signaling molecules and the cis-elements of goat CSN2 promoter in response to lactogenic hormone stimulation in goat mammary epithelial cells. Here, we use an immortalized caprine mammary epithelial cell line (CMC) to search for evidence of the above. Serial 5'-flanking regions deleted of promoter and intron 1 in goat CSN2 (-4,047 to +2,054) driven by firefly luciferase reporter gene were constructed and applied to measure promoter activity in CMC. The intron 1 region (+393 to +501) significantly decreased basal activity of the promoter. This finding contradicts other studies of the role of intron 1. The signal transducer and activator of transcription (STAT)5a played a significant role in activating promoter activity by prolactin stimulation. Hydrocortisone enhanced and prolonged the activity of STAT5a and promoter in CMC, but was independent of the glucocorticoid receptor response element. The minimum length of the CSN2 promoter segment in response to lactogenic stimulation was confirmed by 5' serial deletions. A cis-element located from -300 to -90 in proximal goat CSN2 promoter that is absent in bovine and human CSN2 promoter was newly identified. We demonstrated the presence of a STAT5a binding site (-102 to -82) and preservation of the guanosine nucleotide at position -90 based on responses to the presence of lactogenic hormone using internal deletions and point mutations of the predicted STAT5a binding site, and chromatin immunoprecipitation assay. Together, these findings demonstrate that the proximal -300 bp of goat CSN2 promoter containing the STAT5a binding site (-102 to -82) is the response element for lactogenic hormone stimulation. Additionally, intron 1 may be required for tissue or developmental stage-specific expression in mammary gland. The role of the far-distal regions of

  10. Neutron crosstalk between liquid scintillators

    SciTech Connect

    Verbeke, J. M.; Prasad, M. K.; Snyderman, N. J.

    2015-05-01

    We propose a method to quantify the fractions of neutrons scattering between liquid scintillators. Using a spontaneous fission source, this method can be utilized to quickly characterize an array of liquid scintillators in terms of crosstalk. The point model theory due to Feynman is corrected to account for these multiple scatterings. Using spectral information measured by the liquid scintillators, fractions of multiple scattering can be estimated, and mass reconstruction of fissile materials under investigation can be improved. Monte Carlo simulations of mono-energetic neutron sources were performed to estimate neutron crosstalk. A californium source in an array of liquid scintillators was modeled to illustrate the improvement of the mass reconstruction.

  11. Neutron crosstalk between liquid scintillators

    NASA Astrophysics Data System (ADS)

    Verbeke, J. M.; Prasad, M. K.; Snyderman, N. J.

    2015-09-01

    A method is proposed to quantify the fractions of neutrons scattering between liquid scintillators. Using a spontaneous fission source, this method can be utilized to quickly characterize an array of liquid scintillators in terms of crosstalk. The point model theory due to Feynman is corrected to account for these multiple scatterings. Using spectral information measured by the liquid scintillators, fractions of multiple scattering can be estimated, and mass reconstruction of fissile materials under investigation can be improved. Monte Carlo simulations of mono-energetic neutron sources were performed to estimate neutron crosstalk. A californium source in an array of liquid scintillators was modeled to illustrate the improvement of the mass reconstruction.

  12. PLASTIC SCINTILLATOR FOR RADIATION DOSIMETRY.

    PubMed

    Kim, Yewon; Yoo, Hyunjun; Kim, Chankyu; Lim, Kyung Taek; Moon, Myungkook; Kim, Jongyul; Cho, Gyuseong

    2016-09-01

    Inorganic scintillators, composed of high-atomic-number materials such as the CsI(Tl) scintillator, are commonly used in commercially available a silicon diode and a scintillator embedded indirect-type electronic personal dosimeters because the light yield of the inorganic scintillator is higher than that of an organic scintillator. However, when it comes to tissue-equivalent dose measurements, a plastic scintillator such as polyvinyl toluene (PVT) is a more appropriate material than an inorganic scintillator because of the mass energy absorption coefficient. To verify the difference in the absorbed doses for each scintillator, absorbed doses from the energy spectrum and the calculated absorbed dose were compared. From the results, the absorbed dose of the plastic scintillator was almost the same as that of the tissue for the overall photon energy. However, in the case of CsI, it was similar to that of the tissue only for a photon energy from 500 to 4000 keV. Thus, the values and tendency of the mass energy absorption coefficient of the PVT are much more similar to those of human tissue than those of the CsI. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. 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.

  14. The Impact of Geographic Proximity to Transplant Center on Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation

    PubMed Central

    Abou-Nassar, Karim E.; Kim, Haesook T.; Blossom, Jeff; Ho, Vincent T.; Soiffer, Robert J.; Cutler, Corey S.; Alyea, Edwin P.; Koreth, John; Antin, Joseph H.; Armand, Philippe

    2011-01-01

    BACKGROUND Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) need access to specialized care. We hypothesized that access to the transplant center after HSCT may be challenging for patients living in geographically distant areas, and that this would have an adverse effect on their outcome. METHODS We analyzed 1912 adult patients who underwent allogeneic HSCT at DF/BWCC between 1996 and 2009 and who resided within 6 hours driving time of the institution. Driving time from primary residence to DF/BWCC based on zipcode was determined using geographic information systems. RESULTS The median driving time (range) to DF/BWCC was 72 (2-358) minutes. When patients were stratified by driving time quartile, overall survival (OS) after HSCT was similar in the first year but worse after 1 year in patients in the top quartile (≥160 minutes driving time). In a landmark analysis of the 909 patients alive and free of disease at 1 year, 5-yr OS was 76% and 65% for patients in the 1st (≤40 minutes) and 4th (≥160 minutes) quartiles, respectively (p=0.027). This was confirmed in a multivariable analysis. The difference appeared to be mostly due to an increase in non-relapse mortality. The number of visits to the transplant center between day 100 and 365 after HSCT declined significantly with increasing driving time to the transplant center, which was independently associated with worse survival. CONCLUSION Long driving time to the transplant center is associated with worse OS in patients alive and disease-free one year after HSCT, independently of other patient-, disease- and HSCT related variables. This may be in part related to the lower frequency of post-HSCT visits in patients living farther away. PMID:21906576

  15. Short and long term gene expression variation and networking in human proximal tubule cells when exposed to cadmium

    PubMed Central

    2013-01-01

    Cadmium (Cd2+) is a known nephrotoxin causing tubular necrosis during acute exposure and potentially contributing to renal failure in chronic long-term exposure. To investigate changes in global gene expression elicited by cadmium, an in-vitro exposure system was developed from cultures of human renal epithelial cells derived from cortical tissue obtained from nephrectomies. These cultures exhibit many of the qualities of proximal tubule cells. Using these cells, a study was performed to determine the cadmium-induced global gene expression changes after short-term (1 day, 9, 27, and 45 μM) and long-term cadmium exposure (13 days, 4.5, 9, and 27 μM). These studies revealed fundamental differences in the types of genes expressed during each of these time points. The obtained data was further analyzed using regression to identify cadmium toxicity responsive genes. Regression analysis showed 403 genes were induced and 522 genes were repressed by Cd2+ within 1 day, and 366 and 517 genes were induced and repressed, respectively, after 13 days. We developed a gene set enrichment analysis method to identify the cadmium induced pathways that are unique in comparison to traditional approaches. The perturbation of global gene expression by various Cd2+ concentrations and multiple time points enabled us to study the transcriptional dynamics and gene interaction using a mutual information-based network model. The most prominent network module consisted of INHBA, KIF20A, DNAJA4, AKAP12, ZFAND2A, AKR1B10, SCL7A11, and AKR1C1. PMID:23369406

  16. 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.

  17. Caveolin-1 and Dopamine-Mediated Internalization of NaKATPase in Human Renal Proximal Tubule Cells

    PubMed Central

    Gildea, John J.; Israel, Jonathan A.; Johnson, Andrew K.; Zhang, Jin; Jose, Pedro A.; Felder, Robin A.

    2010-01-01

    In moderate sodium-replete states, dopamine 1–like receptors (D1R/D5R) are responsible for regulating >50% of renal sodium excretion. This is partly mediated by internalization and inactivation of NaKATPase, when associated with adapter protein 2. We used dopaminergic stimulation via fenoldopam (D1-like receptor agonist) to study the interaction among D1-like receptors, caveolin-1 (CAV1), and the G protein– coupled receptor kinase type 4 in cultured human renal proximal tubule cells (RPTCs). We compared 2 groups of RPTCs, 1 of cell lines that were isolated from normal subjects (nRPTCs) and a second group of cell lines that have D1-like receptors that are uncoupled (uncoupled RPTCs) from adenylyl cyclase second messengers. In nRPTCs, fenoldopam increased the plasma membrane expression of D1R (10.0-fold) and CAV1 (1.3-fold) and markedly decreased G protein– coupled receptor kinase type 4 by 94±8%; no effects were seen in uncoupled RPTCs. Fenoldopam also increased the association of adapter protein 2 and NaKATPase by 53±9% in nRPTCs but not in uncoupled RPTCs. When CAV1 expression was reduced by 86.0±8.5% using small interfering RNA, restimulation of the D1-like receptors with fenoldopam in nRPTCs resulted in only a 7±9% increase in association between adapter protein 2 and NaKATPase. Basal CAV1 expression and association with G protein– coupled receptor kinase type 4 was decreased in uncoupled RPTCs (58±5% decrease in association) relative to nRPTCs. We conclude that the scaffolding protein CAV1 is necessary for the association of D1-like receptors with G protein– coupled receptor kinase type 4 and the adapter protein 2-associated reduction in plasma membrane NaKATPase. PMID:19752292

  18. Dominance of the proximal coordinate frame in determining the locations of hippocampal place cell activity during navigation

    PubMed Central

    Siegel, Jennifer J.; Neunuebel, Joshua P.; Knierim, James J.

    2007-01-01

    The place-specific activity of hippocampal cells provides downstream structures with information regarding an animal's position within an environment, and perhaps the location of goals within that environment. In rodents, recent research has suggested that distal cues primarily set the orientation of the spatial representation, whereas the boundaries of the behavioral apparatus determine the locations of place activity. The current study was designed to address possible biases in some previous research that may have minimized the likelihood of observing place activity bound to distal cues. Hippocampal single-unit activity was recorded from 6 freely moving rats as they were trained to perform a tone-initiated place preference task on an open field platform. To investigate whether place activity was bound to the room- or platform-based coordinate frame (or both), the platform was translated within the room at an “early” and at a “late” phase of task acquisition (Shift 1 and Shift 2). At both time points, CA1 and CA3 place cells demonstrated room-associated and/or platform-associated activity, or remapped in response to the platform-shift. Shift 1 revealed place activity that reflected an interaction between a dominant platform-based (proximal) coordinate frame and a weaker room-based (distal) frame, as many CA1 and CA3 place fields shifted to a location intermediate to the two reference frames. Shift 2 resulted in place activity that became more strongly bound to either the platform- or room-based coordinate frame, suggesting the emergence of two independent spatial frames of reference (with many more cells participating in platform-based than room-based representations). PMID:17959742

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

    PubMed Central

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

    2013-01-01

    Blockade of the angiotensin (ANG) II receptor type 1 (AT1R) 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 AT1R. 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 AT1R paralleling those observed in the human AT1R. Therefore, we posited that these sequence variations may explain ARB resistance. We demonstrate that OKP cells express AT1R mRNA, bind 125I-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 125I-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 125I-ANG II binding. In contrast, ANG II binding to OKP cells stably expressing rat AT1ARs, which have a conserved AT1R-binding pocket with human AT1R, is efficiently inhibited by olmesartan. A novel observation was that resistance to ARB binding to opossum AT1Rs 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 AT1R polymorphisms within the ARB-binding pocket in various hypertensive populations. PMID:23389452

  20. 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.

  1. 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.

  2. 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.

  3. Macrophage-stimulating protein attenuates gentamicin-induced inflammation and apoptosis in human renal proximal tubular epithelial cells

    SciTech Connect

    Lee, Ko Eun; Kim, Eun Young; Kim, Chang Seong; Choi, Joon Seok; Bae, Eun Hui; Ma, Seong Kwon; Kim, Kyung Keun; Lee, Jong Un; Kim, Soo Wan

    2013-05-10

    Highlights: •MSP/RON system is activated in rat kidney damaged by gentamicin. •MSP inhibits GM-induced cellular apoptosis and inflammation in HK-2 cells. •MSP attenuates GM-induced activation of MAPKs and NF-κB pathways in HK-2 cells. -- Abstract: The present study aimed to investigate whether macrophage-stimulating protein (MSP) treatment attenuates renal apoptosis and inflammation in gentamicin (GM)-induced tubule injury and its underlying molecular mechanisms. To examine changes in MSP and its receptor, recepteur d’origine nantais (RON) in GM-induced nephropathy, rats were injected with GM for 7 days. Human renal proximal tubular epithelial (HK-2) cells were incubated with GM for 24 h in the presence of different concentrations of MSP and cell viability was measured by MTT assay. Apoptosis was determined by flow cytometry of cells stained with fluorescein isothiocyanate-conjugated annexin V protein and propidium iodide. Expression of Bcl-2, Bax, caspase-3, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), IκB-α, and mitogen-activated protein kinases (MAPKs) was analyzed by semiquantitative immunoblotting. MSP and RON expression was significantly greater in GM-treated rats, than in untreated controls. GM-treatment reduced HK-2 cell viability, an effect that was counteracted by MSP. Flow cytometry and DAPI staining revealed GM-induced apoptosis was prevented by MSP. GM reduced expression of anti-apoptotic protein Bcl-2 and induced expression of Bax and cleaved caspase 3; these effects and GM-induced expression of COX-2 and iNOS were also attenuated by MSP. GM caused MSP-reversible induction of phospho-ERK, phospho-JNK, and phospho-p38. GM induced NF-κB activation and degradation of IκB-α; the increase in nuclear NF-κB was blocked by inhibitors of ERK, JNK, p-38, or MSP pretreatment. These findings suggest that MSP attenuates GM-induced inflammation and apoptosis by inhibition of the MAPKs

  4. In situ detection of estrogen receptor dimers in breast carcinoma cells in archival materials using proximity ligation assay (PLA).

    PubMed

    Iwabuchi, Erina; Miki, Yasuhiro; Ono, Katsuhiko; Onodera, Yoshiaki; Suzuki, Takashi; Hirakawa, Hisashi; Ishida, Takanori; Ohuchi, Noriaki; Sasano, Hironobu

    2017-01-01

    Estrogen receptor (ER) is required for carcinoma cell proliferation in the great majority of breast cancer and also functions as a dimer. ER dimeric proteins have been largely identified by BRET/FRET analyses but their in situ visualization have not yet been reported. Recently, in situ Proximity Ligation Assay (PLA) has been developed as the methods detecting protein interactions in situ. Therefore, in this study we firstly demonstrated the dimerization of ERα in breast carcinoma cell lines and tissues using PLA. The human breast carcinoma cell lines MCF-7, T-47D and MDA-MB-231 were used in this study. Cells were treated with ER agonist or antagonist and fixed in 4% PFA, and ER dimers were subsequently detected using PLA. The evaluation of ER dimers in breast carcinoma cell lines were quantified by measuring the area of dots localized in the nuclei using image analysis. We also firstly demonstrated the visualization of ER dimer patterns in 10% formalin-fixed paraffin-embedded tissues of breast cancer using PLA technique. Estradiol (E2) administration induced ERα homodimers in the nuclei of MCF-7 and T-47D but not in ER-negative MDA-MB-231. 4-OH tamoxifen also induced ERα homodimers but the subcellular localization of these ERα homodimers was predominant in cytoplasm instead of the nuclei induced by E2 treatment. ICI182,780 treatment did decrease the number of formation of ERα homodimers in MCF-7. In breast cancer patients, ERα PLA score was significantly correlated positively with ERα- or PgR (progesterone receptor) immunohistochemical scores and inversely with Ki-67-labeling index, respectively. We also demonstrated the ERα/β heterodimer as well as ERα homodimers in both breast carcinoma cell lines and surgical pathology specimens. In summary, we did firstly succeed in the visualization of ER dimeric proteins using PLA method. The evaluation of ER dimer patterns could provide pivotal information as to the prediction of response to endocrine therapy of

  5. 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

  6. Hypoxia reduces constitutive and TNF-{alpha}-induced expression of monocyte chemoattractant protein-1 in human proximal renal tubular cells

    SciTech Connect

    Li Xuan; Kimura, Hideki . E-mail: hkimura@fmsrsa.fukui-med.ac.jp; Hirota, Kiichi; Sugimoto, Hidehiro; Yoshida, Haruyoshi

    2005-10-07

    Chronic hypoxia has been reported to be associated with macrophage infiltration in progressive forms of kidney disease. Here, we investigated the regulatory effects of hypoxia on constitutive and TNF-{alpha}-stimulated expression of monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal renal tubular cells (HPTECs). Hypoxia reduced constitutive MCP-1 expression at the mRNA and protein levels in a time-dependent fashion for up to 48 h. Hypoxia also inhibited MCP-1 up-regulation by TNF-{alpha}. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression resulted mainly from a decrease in the transcription but not the mRNA stability. Immunoblot and immunofluorescence analyses revealed that treatment with hypoxia or an iron chelator, desferrioxamine, induced nuclear accumulation of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in HPTECs. Desferrioxamine mimicked hypoxia in the reduction of MCP-1 expression. However, overexpression of a dominant negative form of HIF-1{alpha} did not abolish the hypoxia-induced reduction of MCP-1 expression in HPTECs. These results suggest that hypoxia is an important negative regulator of monocyte chemotaxis to the renal inflamed interstitium, by reducing MCP-1 expression partly via hypoxia-activated signals other than the HIF-1 pathway.

  7. GPS Scintillation Analysis.

    DTIC Science & Technology

    2007-11-02

    Rev. 2-89) Prescribed by ANSI Std. Z39-1 298-102 TABLE OF CONTENTS 1. INTRODUCTION 1 2. GPS COMPARISON WITH ALL-SKY IMAGES OVER AGUA VERDE...Depletions from 1 October 1994 2 3. GPS data from Agua Verde, Chile on the night of 1 October 1994 3 4. PL-SCINDA display of GPS ionospheric...comparison of GPS measurements with GOES8 L-band scintillation data, are discussed. 2. GPS COMPARISON WITH ALL-SKY IMAGES OVER AGUA VERDE, CHILE As

  8. 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.

  9. 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

  10. Development of intrinsic IPT scintillator

    SciTech Connect

    Bross, A.D.

    1989-07-31

    We report on the development of a new polystyrene based plastic scintillator. Optical absorption, fluorescence and light output measurements are presented. Preliminary results of radiation damage effects are also given and compared to the effects on a commercial plastic scintillator, NE 110. 6 refs., 12 figs.

  11. Development of radiation hard scintillators

    NASA Astrophysics Data System (ADS)

    Markley, F.; Davidson, M.; Keller, J.; Foster, G.; Pla-Dalmau, A.; Harmon, J.; Biagtan, E.; Schueneman, G.; Senchishin, V.; Gustfason, H.

    1993-11-01

    The authors have demonstrated that the radiation stability of scintillators made from styrene polymer is very much improved by compounding with pentaphenyl trimethyl trisiloxane (DC 705 vacuum pump oil). The resulting scintillators are softer than desired, so they decided to make the scintillators directly from monomer where the base resin could be easily crosslinked to improve the mechanical properties. They can now demonstrate that scintillators made directly from the monomer, using both styrene and 4-methyl styrene, are also much more radiation resistant when modified with DC705 oil. In fact, they retain from 92% to 95% of their original light output after gamma irradiation to 10 Mrads in nitrogen with air annealing. When these scintillators made directly from monomer are compared with scintillators of the same composition made from polymer the latter have much higher light outputs. They commonly reach 83% while those made from monomer give only 50% to 60% relative to the reference, BC408. When oil modified scintillators using both p-terphenyl and tetra phenyl butadiene are compared with identical scintillators except that they use 3 hydroxy-flavone as the only luminophore the radiation stability is the same. However the 3HF system gives only 30% as much light as BC408 instead of 83% when both are measured with a green extended Phillips XP2081B phototube.

  12. Extruding plastic scintillator at Fermilab

    SciTech Connect

    Anna Pla-Dalmau; Alan D. Bross; Victor V. Rykalin

    2003-10-31

    An understanding of the costs involved in the production of plastic scintillators and the development of a less expensive material have become necessary with the prospects of building very large plastic scintillation detectors. Several factors contribute to the high cost of plastic scintillating sheets, but the principal reason is the labor-intensive nature of the manufacturing process. In order to significantly lower the costs, the current casting procedures had to be abandoned. Since polystyrene is widely used in the consumer industry, the logical path was to investigate the extrusion of commercial-grade polystyrene pellets with dopants to yield high quality plastic scintillator. This concept was tested and high quality extruded plastic scintillator was produced. The D0 and MINOS experiments are already using extruded scintillator strips in their detectors. An extrusion line has recently been installed at Fermilab in collaboration with NICADD (Northern Illinois Center for Accelerator and Detector Development). This new facility will serve to further develop and improve extruded plastic scintillator. This paper will discuss the characteristics of extruded plastic scintillator and its raw materials, the different manufacturing techniques and the current R&D program at Fermilab.

  13. Development of radiation hard scintillators

    SciTech Connect

    Markley, F.; Davidson, M.; Keller, J.; Foster, G.; Pla-Dalmau, A.; Harmon, J.; Biagtan, E.; Schueneman, G.; Senchishin, V.; Gustfason, H.; Rivard, M.

    1993-11-01

    The authors have demonstrated that the radiation stability of scintillators made from styrene polymer is very much improved by compounding with pentaphenyltrimethyltrisiloxane (DC 705 vacuum pump oil). The resulting scintillators are softer than desired, so they decided to make the scintillators directly from monomer where the base resin could be easily crosslinked to improve the mechanical properties. They can now demonstrate that scintillators made directly from the monomer, using both styrene and 4-methyl styrene, are also much more radiation resistant when modified with DC705 oil. In fact, they retain from 92% to 95% of their original light output after gamma irradiation to 10 Mrads in nitrogen with air annealing. When these scintillators made directly from monomer are compared with scintillators of the same composition made from polymer the latter have much higher light outputs. They commonly reach 83% while those made form monomer give only 50% to 60% relative to the reference, BC408. When oil modified scintillators using both p-terphenyl and tetraphenylbutadiene are compared with identical scintillators except that they use 3 hydroxy-flavone as the only luminophore the radiation stability is the same. However the 3HF system gives only 30% as much light as BC408 instead of 83% when both are measured with a green extended Phillips XP2081B phototube.

  14. A large liquid scintillator detector for a long baseline neutrino oscillation experiment

    NASA Astrophysics Data System (ADS)

    Border, P.; Cushman, P.; Heller, K.; Maxam, D.; Nelson, J. K.; Ruddick, K.; Rusack, R.; Schwienhorst, R.; Berg, T.; Chase, T.; Hansen, M.; Bower, C.; Hatcher, R.; Heinz, R.; Miller, L.; Mufson, S.

    2001-05-01

    We present the concept and design of a liquid scintillator detector for a long-baseline neutrino oscillation experiment. Neutrinos interact in 2.5 cm thick steel plates alternating with 2.0 cm thick planes of liquid scintillator. The scintillator is contained in multicell PVC extrusions containing individual 2 cm×3 cm cells up to 8 m long. Readout of the scintillation light is via wavelength-shifting fibers which transport light to pixellated photodetectors at one end of the cells.

  15. Cobalt Chloride Induces Expression and Function of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Renal Proximal Tubular Epithelial Cell Line HK-2.

    PubMed

    Nishihashi, Katsuki; Kawashima, Kei; Nomura, Takami; Urakami-Takebayashi, Yumiko; Miyazaki, Makoto; Takano, Mikihisa; Nagai, Junya

    2017-01-01

    The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.

  16. YES, a Src family kinase, is a proximal glucose-specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet β cells.

    PubMed

    Yoder, Stephanie M; Dineen, Stacey L; Wang, Zhanxiang; Thurmond, Debbie C

    2014-04-18

    Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

  17. YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells*

    PubMed Central

    Yoder, Stephanie M.; Dineen, Stacey L.; Wang, Zhanxiang; Thurmond, Debbie C.

    2014-01-01

    Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling. PMID:24610809

  18. Safety and feasibility of cell-based therapy of autologous bone marrow-derived mononuclear cells in plate-stabilized proximal humeral fractures in humans.

    PubMed

    Seebach, Caroline; Henrich, Dirk; Meier, Simon; Nau, Christoph; Bonig, Halvard; Marzi, Ingo

    2016-11-15

    Local implantation of ex vivo concentrated, washed and filtrated human bone marrow-derived mononuclear cells (BMC) seeded onto β-tricalciumphosphate (TCP) significantly enhanced bone healing in a preclinical segmental defect model. Based on these results, we evaluated in a first clinical phase-I trial safety and feasibility of augmentation with preoperatively isolated autologous BMC seeded onto β-TCP in combination with angle stable plate fixation for the therapy of proximal humeral fractures as a potential alternative to autologous bone graft from the iliac crest. 10 patients were enrolled to assess whether cell therapy with 1.3 × 10(6) autologous BMC/ml/ml β-TCP, collected on the day preceding the definitive surgery, is safe and feasible when seeded onto β-TCP in patients with a proximal humeral fracture. 5 follow-up visits for clinical and radiological controls up to 12 weeks were performed. β-tricalciumphosphate fortification with BMC was feasible and safe; specifically, neither morbidity at the harvest site nor at the surgical wound site were observed. Neither local nor systemic inflammation was noted. All fractures healed within the observation time without secondary dislocation. Three adverse events were reported: one case each of abdominal wall shingles, tendon loosening and initial screw perforation, none of which presumed related to the IND. Cell therapy with autologous BMC for bone regeneration appeared to be safe and feasible with no drug-related adverse reactions being described to date. The impression of efficacy was given, although the study was not powered nor controlled to detect such. A clinical trial phase-II will be forthcoming in order to formally test the clinical benefit of BMC-laden β-TCP for PHF patients. Trial registration The study was registered in the European Clinical Trial Register as EudraCT No. 2012-004037-17. Date of registration 30th of August 2012. Informed consent was signed from all patients enrolled.

  19. Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells.

    PubMed

    Carranza, Andrea; Musolino, Patricia L; Villar, Marcelo; Nowicki, Susana

    2008-12-01

    The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.

  20. AID-targeting and hypermutation of non-immunoglobulin genes does not correlate with proximity to immunoglobulin genes in germinal center B cells.

    PubMed

    Gramlich, Hillary Selle; Reisbig, Tara; Schatz, David G

    2012-01-01

    Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this "collateral damage" model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.

  1. Discrete scintillator coupled mercuric iodide photodetector arrays for breast imaging

    SciTech Connect

    Tornai, M.P.; Levin, C.S.; Hoffman, E.J.

    1996-12-31

    Multi-element (4x4) imaging arrays with high resolution collimators, size matched to discrete CsI(Tl) scintillator arrays and mercuric iodide photodetector arrays (HgI{sub 2} PDA) are under development as prototypes for larger 16 x 16 element arrays. The compact nature of the arrays allows detector positioning in proximity to the breast to eliminate activity not in the line-of-sight of the collimator, thus reducing image background. Short collimators, size matched to {le}1.5 x 1.5 mm{sup 2} scintillators show a factor of 2 and 3.4 improvement in spatial resolution and efficiency, respectively, compared to high resolution collimated gamma cameras for the anticipated compressed breast geometries. Monte Carlo simulations, confirmed by measurements, demonstrated that scintillator length played a greater role in efficiency and photofraction for 140 keV gammas than cross sectional area, which affects intrinsic spatial resolution. Simulations also demonstrated that an increase in the ratio of scintillator area to length corresponds to an improvement in light collection. Electronic noise was below 40 e{sup -} RMS indicating that detector resolution was not noise limited. The high quantum efficiency and spectral match of prototype unity gain HgI{sub 2} PDAs coupled to 1 x 1 x 2.5 mm{sup 3} and 2 x 2 x 4 mm{sup 3} CsI(Tl) scintillators demonstrated energy resolutions of 9.4% and 8.8% FWHM at 140 keV, respectively, without the spectral tailing observed in standard high-Z, compound semi-conductor detectors. Line spread function measurements matched the scintillator size and pitch, and small, complex phantoms were easily imaged.

  2. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment

    PubMed Central

    Bender, Ruben R.; Muth, Anke; Schneider, Irene C.; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J.

    2016-01-01

    Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs. PMID:27281338

  3. Proton recoil scintillator neutron rem meter

    DOEpatents

    Olsher, Richard H.; Seagraves, David T.

    2003-01-01

    A neutron rem meter utilizing proton recoil and thermal neutron scintillators to provide neutron detection and dose measurement. In using both fast scintillators and a thermal neutron scintillator the meter provides a wide range of sensitivity, uniform directional response, and uniform dose response. The scintillators output light to a photomultiplier tube that produces an electrical signal to an external neutron counter.

  4. TGF-β1 stimulates movement of renal proximal tubular epithelial cells in a three-dimensional cell culture via an autocrine TGF-β2 production.

    PubMed

    Luo, Deyi; Guan, Qiunong; Wang, Kunjie; Nguan, Christopher Y C; Du, Caigan

    2017-01-01

    TGF-βs are multifunctional cytokines, but their roles in human renal homeostasis are not fully understood. This study investigated the role of TGF-β1 in the movement of human renal proximal tubular epithelial cells (PTECs) in a three-dimensional (3D) model. HKC-8 cells, a human PTEC line, were grown in a 3D collagen culture system. Cell movement was observed under a microscope. The gene expression was examined using PCR Arrays or qRT-PCR, and protein levels by Western blot. Here, we showed that the tight junction structure formed between adjacent cells of a HKC-8 cell colony in 3D cultures, and TGF-β1 stimulated their movement, evidenced by the appearance of fingerlike pseudopodia in the leader cells at the edge of the colonies. The cell movement of these human PTECs was correlated with up-regulation of both MMP2 and MMP9 and down-regulation or inactivation of PLAUR and PTK2B. Analysis of TGF-β signaling targets confirmed autocrine production of TGF-β2 and its cleaving enzyme furin as well as SNAI1 by TGF-β1stimulation. Knockdown of TGF-β2 expression disrupted TGF-β1-stimulated PTEC invasiveness, which was correlated with the down-regulation of MMP2 and MMP9. In conclusion, the activation of TGF-β receptor autocrine signaling by up-regulated TGF-β2 may play a pivotal role in TGF-β1-induced human PTEC movement, which could be mediated at least by both MMP2 and MMP9. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients.

    PubMed

    Kim, Phillip; Liu, Xinjun; Lee, Tani; Liu, Limin; Barham, Robert; Kirkland, Richard; Leesman, Glen; Kuller, Anne; Ybarrondo, Belen; Ng, Shi-Chung; Singh, Sharat

    2011-12-15

    The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment. We have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity. CEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over

  6. Liquid scintillator tiles for calorimetry

    NASA Astrophysics Data System (ADS)

    Amouzegar, M.; Belloni, A.; Bilki, B.; Calderon, J.; De Barbaro, P.; Eno, S. C.; Hatakeyama, K.; Hirschauer, J.; Jeng, G. Y.; Pastika, N. J.; Pedro, K.; Rumerio, Paolo; Samuel, J.; Sharp, E.; Shin, Y. H.; Tiras, E.; Vishnevskiy, D.; Wetzel, J.; Yang, Z.; Yao, Y.; Youn, S. W.

    2016-11-01

    Future experiments in high energy and nuclear physics may require large, inexpensive calorimeters that can continue to operate after receiving doses of 50 Mrad or more. The light output of liquid scintillators suffers little degradation under irradiation. However, many challenges exist before liquids can be used in sampling calorimetry, especially regarding developing a packaging that has sufficient efficiency and uniformity of light collection, as well as suitable mechanical properties. We present the results of a study of a scintillator tile based on the EJ-309 liquid scintillator using cosmic rays and test beam on the light collection efficiency and uniformity, and some preliminary results on radiation hardness.

  7. Liquid scintillator tiles for calorimetry

    SciTech Connect

    Amouzegar, M.; Belloni, A.; Bilki, B.; Calderon, J.; Barbaro, P. De; Eno, S. C.; Hatakeyama, K.; Hirschauer, J.; Jeng, G. Y.; Pastika, N. J.; Pedro, K.; Rumerio, Paolo; Samuel, J.; Sharp, E.; Shin, Y. H.; Tiras, E.; Vishnevskiy, D.; Wetzel, J.; Yang, Z.; Yao, Y.; Youn, S. W.

    2016-11-28

    Future experiments in high energy and nuclear physics may require large, inexpensive calorimeters that can continue to operate after receiving doses of 50 Mrad or more. Also, the light output of liquid scintillators suffers little degradation under irradiation. However, many challenges exist before liquids can be used in sampling calorimetry, especially regarding developing a packaging that has sufficient efficiency and uniformity of light collection, as well as suitable mechanical properties. We present the results of a study of a scintillator tile based on the EJ-309 liquid scintillator using cosmic rays and test beam on the light collection efficiency and uniformity, and some preliminary results on radiation hardness.

  8. High energy resolution plastic scintillator

    NASA Astrophysics Data System (ADS)

    van Loef, Edgar V.; Feng, Patrick; Markosyan, Gary; Shirwadkar, Urmila; Doty, Patrick; Shah, Kanai S.

    2016-09-01

    In this paper we present results on a novel tin-loaded plastic scintillator. We will show that this particular plastic scintillator has a light output similar to that of BGO, a fast scintillation decay (< 10 ns), exhibits good neutron/gamma PSD with a Figure-of-Merit of 1.3 at 2.5 MeVee cut-off energy, and excellent energy resolution of about 12% (FWHM) at 662 keV. Under X-ray excitation, the radioluminescence spectrum exhibits a broad band between 350 and 500 nm peaking at 420 nm which is well-matched to bialkali photomultiplier tubes and UV-enhanced photodiodes.

  9. Scintillator materials containing lanthanum fluorides

    DOEpatents

    Moses, William W.

    1991-01-01

    An improved radiation detector containing a crystalline mixture of LaF.sub.3 and CeF.sub.3 as the scintillator element is disclosed. Scintillators made with from 25% to 99.5% LaF.sub.3 and the remainder CeF.sub.3 have been found to provide a balance of good stopping power, high light yield and short decay constant that is equal to or superior to other known scintillator materials, and which may be processed from natural starting materials containing both rare earth elements. The radiation detectors disclosed are favorably suited for use in general purpose detection and in positron emission tomography.

  10. Ionospheric scintillation studies

    NASA Technical Reports Server (NTRS)

    Rino, C. L.; Freemouw, E. J.

    1973-01-01

    The diffracted field of a monochromatic plane wave was characterized by two complex correlation functions. For a Gaussian complex field, these quantities suffice to completely define the statistics of the field. Thus, one can in principle calculate the statistics of any measurable quantity in terms of the model parameters. The best data fits were achieved for intensity statistics derived under the Gaussian statistics hypothesis. The signal structure that achieved the best fit was nearly invariant with scintillation level and irregularity source (ionosphere or solar wind). It was characterized by the fact that more than 80% of the scattered signal power is in phase quadrature with the undeviated or coherent signal component. Thus, the Gaussian-statistics hypothesis is both convenient and accurate for channel modeling work.

  11. Drift scintillation meter

    NASA Astrophysics Data System (ADS)

    1984-03-01

    This is the final report for the subject contract under which The University of Texas at Dallas (UTD) built, tested and delivered an engineering model and three flight versions of the Drift Scintillation Meter (DSM) to the Air Force Geophysics Laboratory for flight on the Air Force DMSP satellites. The report is divided into three sections. Section 1 contains the instrument description and theory of operation. Section 2 contains a description of planned spacecraft-level instrument testing, stimulation requirements and instrument handling and safety. Section 3 contains an instrument interconnection diagram and a list of the schematics, drawings, parts lists and wiring lists that describe the as-built configuration of the instrument. This documentation is available in the R&D Equipment Information Reports that were submitted to AFGL after each instrument delivery.

  12. Properties of scintillator solutes

    SciTech Connect

    Fluornoy, J.M.

    1998-06-01

    This special report summarizes measurements of the spectroscopic and other properties of the solutes that were used in the preparation of several new liquid scintillators developed at EG and G/Energy Measurements/Santa Barbara Operations (the precursor to Bechtel Nevada/Special Technologies Laboratory) on the radiation-to-light converter program. The data on the individual compounds are presented in a form similar to that used by Prof. Isadore Berlman in his classic handbook of fluorescence spectra. The temporal properties and relative efficiencies of the new scintillators are presented in Table 1, and the efficiencies as a function of wavelength are presented graphically in Figure 1. In addition, there is a descriptive glossary of the abbreviations used herein. Figure 2 illustrates the basic structures of some of the compounds and of the four solvents reported in this summary. The emission spectra generally exhibit more structure than the absorption spectra, with the result that the peak emission wavelength for a given compound may lie several nm away from the wavelength, {lambda}{sub avg}, at the geometric center of the emission spectrum. Therefore, the author has chosen to list absorption peaks, {lambda}{sub max}, and emission {lambda}{sub avg} values in Figures 3--30, as being most illustrative of the differences between the compounds. The compounds, BHTP, BTPB, ADBT, and DPTPB were all developed on this program. P-terphenyl, PBD, and TPB are commercially available blue emitters. C-480 and the other longer-wavelength emitters are laser dyes available commercially from Exciton Corporation. 1 ref., 30 figs.

  13. Effects of angiotensin II type 1 receptor blocker on albumin-induced cell damage in human renal proximal tubular epithelial cells.

    PubMed

    Takao, Toshihiro; Horino, Taro; Kagawa, Toru; Matsumoto, Reiko; Inoue, Kousuke; Taguchi, Takafumi; Morita, Tatsuhito; Iwasaki, Yasumasa; Hashimoto, Kozo; Terada, Yoshio

    2009-01-01

    Proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. We investigated the effect of angiotensin II type 1 receptor blockers (ARBs) on albumin-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). The N-acetyl-beta-D-glucosaminidase (NAG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the medium after albumin treatment with ARBs were determined by commercially available kits. The levels of p22(phox) protein in RPTEC were measured using Western blotting after albumin treatment with ARBs. Angiotensin II concentrations in cell media and cell lysates were assayed with a commercially available kit. Human albumin (0.1-10 mg/ml) dose-dependently increased NAG release and olmesartan or valsartan (10(-9)-10(-7) mol/l) showed a significant reduction on albumin (1 mg/ml)-induced NAG release in RPTEC. Albumin treatment (1 mg/ml) showed significant increases in p22(phox) protein levels in RPTEC and ARBs significantly decreased albumin-induced p22(phox) protein levels. Significant increases in 8-OHdG levels were observed in the albumin (1 mg/ml)-treated group and ARBs markedly reduced albumin-induced 8-OHdG levels in RPTEC. Human albumin dose-dependently increased angiotensin II concentrations in both cell media and lysates. These observations suggest renal tubular cell-protective properties of ARBs related to decreased oxidative stress during proteinuria. Copyright (c) 2008 S. Karger AG, Basel.

  14. Long-term effects of ochratoxin A on fibrosis and cell death in human proximal tubule or fibroblast cells in primary culture.

    PubMed

    Schwerdt, Gerald; Holzinger, Hildegard; Sauvant, Christoph; Königs, Maika; Humpf, Hans-Ulrich; Gekle, Michael

    2007-03-22

    Ochratoxin A (OTA) is a mycotoxin produced by several fungi which grow on human food source material. Consumption of OTA is almost unavoidable. The consumption leads to low but detectable amounts of OTA in human blood. Risk assessment of OTA is based on studies performed either in animals or cultured cells. So far, mainly cell lines of different origin were used. To be as close as possible to the situation in humans with respect to the experimental setup, we studied the effect of OTA in human proximal tubule cells (RPTEC) and human fibroblasts in primary culture. OTA was administered at concentrations ranging from 0.3 nmol/l up to 10 micromol/l for time periods up to 14 days. Apoptotic and necrotic cell death, collagen I, III, IV and fibronectin secretion as well as NF-kappaB activation were studied. Under our experimental conditions OTA exerted comparable effects on caspase-3 activity and necrosis in both cell types, however RPTEC were more sensitive (order of 10). Surprisingly, very low concentrations of OTA (0.3-10nM) led to cell hypertrophy during prolonged exposure (14 days). RPTEC but not fibroblasts responded with an increase of NF-kappaB activity and collagen III as well as fibronectin secretion underlining the profibrotic action of OTA in the kidney. Collagen I and IV secretion was only slightly changed. The results presented here give good reasons to re-asses the risk of OTA consumption leading to low blood concentrations which have so far been considered harmless.

  15. Customized Knee Prosthesis in Treatment of Giant Cell Tumors of the Proximal Tibia: Application of 3-Dimensional Printing Technology in Surgical Design

    PubMed Central

    Luo, Wenbin; Huang, Lanfeng; Liu, He; Qu, Wenrui; Zhao, Xin; Wang, Chenyu; Li, Chen; Yu, Tao; Han, Qing; Wang, Jincheng; Qin, Yanguo

    2017-01-01

    Background We explored the application of 3-dimensional (3D) printing technology in treating giant cell tumors (GCT) of the proximal tibia. A tibia block was designed and produced through 3D printing technology. We expected that this 3D-printed block would fill the bone defect after en-bloc resection. Importantly, the block, combined with a standard knee joint prosthesis, provided attachments for collateral ligaments of the knee, which can maintain knee stability. Material/Methods A computed tomography (CT) scan was taken of both knee joints in 4 patients with GCT of the proximal tibia. We developed a novel technique – the real-size 3D-printed proximal tibia model – to design preoperative treatment plans. Hence, with the application of 3D printing technology, a customized proximal tibia block could be designed for each patient individually, which fixed the bone defect, combined with standard knee prosthesis. Results In all 4 cases, the 3D-printed block fitted the bone defect precisely. The motion range of the affected knee was 90 degrees on average, and the soft tissue balance and stability of the knee were good. After an average 7-month follow-up, the MSTS score was 19 on average. No sign of prosthesis fracture, loosening, or other relevant complications were detected. Conclusions This technique can be used to treat GCT of the proximal tibia when it is hard to achieve soft tissue balance after tumor resection. 3D printing technology simplified the design and manufacturing progress of custom-made orthopedic medical instruments. This new surgical technique could be much more widely applied because of 3D printing technology. PMID:28388595

  16. 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.

  17. Global transcription regulation by DNA topoisomerase I in exponentially growing Saccharomyces cerevisiae cells: activation of telomere-proximal genes by TOP1 deletion.

    PubMed

    Lotito, Luca; Russo, Alessandra; Chillemi, Giovanni; Bueno, Susana; Cavalieri, Duccio; Capranico, Giovanni

    2008-03-21

    To establish the cellular functions of DNA topoisomerase I-B (Top1p) at a global level, we have determined the expression profiles and histone modification patterns affected by TOP1 gene deletion (DeltaTOP1) in Saccharomyces cerevisiae. In exponentially growing cells, DeltaTOP1 specifically increases transcription of telomere-proximal genes and decreases glucose utilization and energy production pathways. Immunoprecipitation data demonstrate that Top1p can bind to and is catalytically active at telomeric DNA repeats, and that both DeltaTOP1 and an inactive Y727F Top1p mutant increase H4 histone acetylation at telomere-proximal regions. Interestingly, while the Y727F mutation has no influence on enzyme recruitment to chromatin sites, it has a marked effect on H4 K16 acetylation at subtelomeric regions. The Top1p mutation also increases H3 histone K4 dimethylation, which has been associated with gene transcription, at 3' termini of subtelomeric genes. No major effect of DeltaTOP1 or mutation was detected on Sir3p recruitment; however, DeltaTOP1 has an effect on transcript levels of genes known to regulate telomeric silencing. Thus, the findings indicate that Top1p activity can favor both a repressed chromatin organization and a reduced gene expression level at telomere-proximal regions in yeast. As telomere-proximal regions are known to be enriched for stress-activated genes, our findings show that Top1p can optimize transcript levels for cell growth in exponentially growing cells under a synthetic medium with glucose.

  18. 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 Central

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

    2015-01-01

    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 Ser938 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 Ser938 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. PMID:26582472

  19. Characterizing Daytime GHZ Scintillation at Equatorial Regions Using Gnss Radio Occultation Measurements

    NASA Astrophysics Data System (ADS)

    Seif, A.; Zhang, K.; Tsunoda, R. T.; Abdullah, M.; Carter, B. A.; Norman, R.; Wu, S.

    2015-12-01

    Ionospheric scintillation of radio waves can behave differently at different locations with a strong diurnal dependence; particularly in the equatorial regions. Ionospheric scintillations at gigahertz (GHz) frequencies have been observed during both daytime and nighttime. It is believed that daytime scintillation is associated with blanketing sporadic E (Esb), whereas nighttime scintillation is attributed to F layer irregularities. Scintillation events associated with Esbduring daytime are of our primary interest. Recent studies show that in the ionosphere, electron density profiles from Global Navigation Satellite System (GNSS) Radio Occultation (RO) provide valuable information to help better understand the physics of the ionosphere. In particular, GNSS RO observations of GHz scintillation in the proximity of the E-layer have been interpreted as being caused by sporadic E. In this paper the characteristics of daytime scintillations at 1.5 GHz recorded simultaneously from two stations (i) Universiti Kebangsaan Malaysia (UKM) (2.55°N, 101.461°E; dip latitude 5.78°S), and (ii) Langkawi (6.19°N, 99.51°E; dip latitude 1.90°S) during November and December 2010 are analyzed. The characteristics of daytime GHz scintillation and its relationship with E region irregularities at equatorial regions are investigated. Ground-based scintillation and Total Electron Content (TEC) data recorded by the GSV4004 receivers were utilized in combination with the amplitude scintillation measurements in terms of GPS C/A code SNR fluctuations during a ground-based GPS and space-borne GNSS RO experiment at the two equatorial stations. Scintillation activity was found to be more prominent at UKM. Moreover, strong scintillation with the S4 index exceeding 0.6 has only been observed at UKM, while at Langkawi the scintillation intensity (S4 index) did not exceed 0.3. Signal-to-noise measurements obtained from GNSS RO indicate that daytime scintillations are very likely caused by Esb. Our

  20. Scintillator fiber optic long counter

    DOEpatents

    McCollum, Tom; Spector, Garry B.

    1994-01-01

    A flat response position sensitive neutron detector capable of providing neutron spectroscopic data utilizing scintillator fiber optic filaments embedded in a neutron moderating housing having an open end through which neutrons enter to be detected.

  1. Scintillator fiber optic long counter

    DOEpatents

    McCollum, T.; Spector, G.B.

    1994-03-29

    A flat response position sensitive neutron detector capable of providing neutron spectroscopic data utilizing scintillator fiber optic filaments embedded in a neutron moderating housing having an open end through which neutrons enter to be detected is described. 11 figures.

  2. Photon statistics in scintillation crystals

    NASA Astrophysics Data System (ADS)

    Bora, Vaibhav Joga Singh

    Scintillation based gamma-ray detectors are widely used in medical imaging, high-energy physics, astronomy and national security. Scintillation gamma-ray detectors are eld-tested, relatively inexpensive, and have good detection eciency. Semi-conductor detectors are gaining popularity because of their superior capability to resolve gamma-ray energies. However, they are relatively hard to manufacture and therefore, at this time, not available in as large formats and much more expensive than scintillation gamma-ray detectors. Scintillation gamma-ray detectors consist of: a scintillator, a material that emits optical (scintillation) photons when it interacts with ionization radiation, and an optical detector that detects the emitted scintillation photons and converts them into an electrical signal. Compared to semiconductor gamma-ray detectors, scintillation gamma-ray detectors have relatively poor capability to resolve gamma-ray energies. This is in large part attributed to the "statistical limit" on the number of scintillation photons. The origin of this statistical limit is the assumption that scintillation photons are either Poisson distributed or super-Poisson distributed. This statistical limit is often dened by the Fano factor. The Fano factor of an integer-valued random process is dened as the ratio of its variance to its mean. Therefore, a Poisson process has a Fano factor of one. The classical theory of light limits the Fano factor of the number of photons to a value greater than or equal to one (Poisson case). However, the quantum theory of light allows for Fano factors to be less than one. We used two methods to look at the correlations between two detectors looking at same scintillation pulse to estimate the Fano factor of the scintillation photons. The relationship between the Fano factor and the correlation between the integral of the two signals detected was analytically derived, and the Fano factor was estimated using the measurements for SrI2:Eu, YAP

  3. Neutron crosstalk between liquid scintillators

    DOE PAGES

    Verbeke, J. M.; Prasad, M. K.; Snyderman, N. J.

    2015-05-01

    We propose a method to quantify the fractions of neutrons scattering between liquid scintillators. Using a spontaneous fission source, this method can be utilized to quickly characterize an array of liquid scintillators in terms of crosstalk. The point model theory due to Feynman is corrected to account for these multiple scatterings. Using spectral information measured by the liquid scintillators, fractions of multiple scattering can be estimated, and mass reconstruction of fissile materials under investigation can be improved. Monte Carlo simulations of mono-energetic neutron sources were performed to estimate neutron crosstalk. A californium source in an array of liquid scintillators wasmore » modeled to illustrate the improvement of the mass reconstruction.« less

  4. Relationship between expression of drug-resistance factors and drug sensitivity in normal human renal proximal tubular epithelial cells in comparison with renal cell carcinoma.

    PubMed

    Asakura, Tadashi; Imai, Akiko; Ohkubo-Uraoka, Noriko; Kuroda, Mayuko; Iidaka, Yoko; Uchida, Kumiko; Shibasaki, Toshiaki; Ohkawa, Kiyoshi

    2005-09-01

    The relationship between the expression level of putative drug resistance factors and sensitivity to anticancer drugs in human normal renal proximal tubule epithelial cells (RPTEC) and 3 kinds of renal cell carcinoma (RCC) cells, VMRC-RCW (RCW), OS-RC-2 (OS2), TUHR14TKB (14TKB), was examined. RPTEC exhibited high expression of P-glycoprotein (Pgp), gamma-glutamyl cysteine synthetase (gammaGCS) and cis-diamminedichloroplatinum (II) (CDDP) resistance-related gene 9 (CRR9), low expression of vacuolar ATPase (V-ATPase) and no expression of multidrug resistance-associated protein 1 (MRP1). 14TKB exhibited high expression of gammaGCS and CRR9, low expression of Pgp and V-ATPase, and no expression of MRP1. OS2 showed high expression of CRR9, low expression of Pgp, gammaGCS and MRP1, and no expression of V-ATPase. RCW exhibited high expression of Pgp, MRP1 and CRR9 and low expression of gammaGCS and V-ATPase. The level of expression of the resistance factors varied among the cells. GST activity and GST-pi expression level of each cell were correlated, and there were high levels in OS2 and RPTEC. When the cytotoxicity of anticancer drugs against each cell was measured at 96 h, the sensitivity to CDDP and Doxorubicin (DXR) in RPTEC and RCW was lower than that in the other cells. Sensitivity to DXR was enhanced by treatment with the Pgp inhibitor, Verapamil, in proportion to the Pgp expression level, and the sensitivity to CDDP was increased by the gammaGCS inhibitor, Buthionine sulfoximine, in proportion to the gammaGCS expression level (corresponding to GSH content). Although a significant increase in sensitivity to CDDP was not observed by treatment of RCC with the V-ATPase inhibitor, Bafilomycin, the sensitivity to DXR in Bafilomycin-treated cells increased about 2-fold. However, no relation between drug sensitivity and V-ATPase expression was observed. The features (such as degree of resistance) varied among the RCC cell lines manifesting many resistance factors or to

  5. About NICADD extruded scintillating strips

    SciTech Connect

    Dyshkant, A.; Beznosko, D.; Blazey, G.; Chakraborty, D.; Francis, K.; Kubik, D.; Lima, J.G.; Rykalin, V.; Zutshi, v.; Baldina, E.; Bross, A.; Deering, P.; Nebel, T.; Pla-Dalmau, A.; Schellpfeffer, J.; Serritella, C.; Zimmerman, J.; /Fermilab

    2005-04-01

    The results of control measurements of extruded scintillating strip responses to a radioactive source Sr-90 are provided, and details of strip choice, preparation, and method of measurement are included. About four hundred one meter long extruded scintillating strips were measured at four different points. These results were essential for prototyping a tail catcher and muon tracker for a future international electron positron linear collider detector.

  6. 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 ...

  7. Incarceration of a large cell neuroendocrine carcinoma arising from the proximal stomach with an organoaxial gastric volvulus through an esophageal hiatal hernia: report of a case.

    PubMed

    Iso, Yukihiro; Tagaya, Nobumi; Nemoto, Takehiko; Kita, Junji; Sawada, Tokihiko; Kubota, Keiichi

    2009-01-01

    An 86-year-old woman was admitted to the hospital to undergo an examination for tarry stools. Laboratory tests showed hypoproteinemia and renal dysfunction. Upper gastrointestinal endoscopy demonstrated a type 5 tumor located in the upper body of the stomach. An upper gastrointestinal series and computed tomography revealed an organoaxial gastric volvulus and the dislocation of the proximal stomach through an esophageal hiatal hernia. The preoperative diagnosis was the incarceration of a gastric carcinoma arising from the proximal stomach with an organoaxial gastric volvulus through an esophageal hiatal hernia. A total gastrectomy and hernia repair were performed. A microscopic examination of the surgical specimen revealed a gastric large cell neuroendocrine carcinoma (GLCNEC). The patient was discharged 22 days after the surgery. Although the prognosis of GLCNEC is significantly worse than that of a conventional adenocarcinoma, the patient was doing well without recurrence at 15 months after surgery. The details of this case are reported with some bibliographical comments.

  8. Cadherin Expression, Vectorial Active Transport, and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells

    PubMed Central

    Slusser, Andrea; Bathula, Chandra S.; Sens, Donald A.; Somji, Seema; Sens, Mary Ann; Zhou, Xu Dong; Garrett, Scott H.

    2015-01-01

    Background Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells. Methods Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells. Results It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells. Conclusions The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype. PMID:25803827

  9. 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

  10. Extruded plastic scintillator including inorganic powders

    DOEpatents

    Bross, Alan D.; Mellott, Kerry L.; Pla-Dalmau, Anna

    2006-06-27

    A method for producing a plastic scintillator is disclosed. A plurality of nano-sized particles and one or more dopants can be combined with a plastic material for the formation of a plastic scintillator thereof. The nano-sized particles, the dopant and the plastic material can be combined within the dry inert atmosphere of an extruder to produce a reaction that results in the formation of a plastic scintillator thereof and the deposition of energy within the plastic scintillator, such that the plastic scintillator produces light signifying the detection of a radiative element. The nano-sized particles can be treated with an inert gas prior to processing the nano-sized particles, the dopant and the plastic material utilizing the extruder. The plastic scintillator can be a neutron-sensitive scintillator, x-ray sensitive scintillator and/or a scintillator for the detection of minimum ionizing particles.

  11. The role of Krüppel-like factor 4 in transforming growth factor-β-induced inflammatory and fibrotic responses in human proximal tubule cells.

    PubMed

    Mreich, Ellein; Chen, Xin-Ming; Zaky, Amgad; Pollock, Carol A; Saad, Sonia

    2015-06-01

    Krüppel-like factor 4 (KLF4) is known to mitigate inflammation in several cell types. Using human proximal tubule cells, the present study aimed to investigate the role of KLF4 in regulating transforming growth factor (TGF)-β₁ induced inflammatory and fibrotic responses. Human kidney proximal tubular cells were exposed to high glucose, or TGF-β₁ and KLF4 expressions were determined. Cells were then transfected with empty vector or KLF4 and exposed to 2-ng/mL TGF-β₁ for up to 72 h. Inflammatory proteins (macrophage migration inhibitory factor and monocyte chemoattractant protein-1) and pro-fibrotic proteins (fibronectin and collagen IV) were measured after 72 h by enzyme-linked immunosorbent assay and western blot, respectively. To determine the relevance to in vivo models of chronic kidney disease, KLF4 protein expression in streptozotocin-induced diabetic mice was determined. Krüppel-like factor 4 messenger RNA (mRNA) levels were significantly reduced in high glucose-treated human kidney proximal tubular cells. High glucose increased TGF-β₁ mRNA expression, which significantly increased migration inhibitory factor and monocyte chemoattractant protein-1 protein secretion. Transforming growth factor-β₁ significantly increased fibronectin and collagen IV protein expression. The overexpression of KLF4 significantly reduced TGF-β-mediated increases in migration inhibitory factor and monocyte chemoattractant protein-1 but had no effect on TGF-β-mediated fibronectin and collagen IV mRNA and protein expression. The levels of KLF4 mRNA were significantly reduced in the diabetic kidney, and diabetic animals had a significant reduction in renal tubular expression of KLF4 proteins. This data suggest that KLF4 reduces inflammation induced by TGF-β₁, suggesting a therapeutic role for KLF4 in diabetic nephropathy. © 2015 Wiley Publishing Asia Pty Ltd.

  12. 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

  13. BMP-7 fails to attenuate TGF-beta1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells.

    PubMed

    Dudas, Paul L; Argentieri, Rochelle L; Farrell, Francis X

    2009-05-01

    In rodent models of chronic renal disease bone morphogenetic protein-7 (BMP-7) has been shown to halt disease progression and promote recovery. Subsequent studies utilizing immortalized rodent renal cell lines showed that BMP-7 was renoprotective by antagonizing TGF-beta1-stimulated epithelial-to-mesenchymal transition (EMT). The present study sought to determine if BMP-7 prevents TGF-beta1-induced EMT in primary (RPTEC) and immortalized (HK-2) human proximal tubule epithelial cells. EMT was determined by quantitative real-time PCR analysis of e-cadherin, vimentin, CTGF and TGF-beta1 transcript expression and immunocytochemical analysis of ZO-1 and alpha-smooth muscle actin (alpha-SMA) protein expression following TGF-beta1 treatment in RPTEC and HK-2 cells. In RPTEC and HK-2 cells, TGF-beta1 significantly reduced e-cadherin expression and significantly increased vimentin, CTGF and TGF-beta1 expression. TGF-beta1 also diminished ZO-1 immunoreactivity and increased alpha-SMA expression in confluent cell monolayers. Co-incubation of TGF-beta1 with an anti-TGF-beta1 neutralizing antibody substantially reduced the cytokine's effects, which indicated EMT in these cells was inhibitable. Co-administration of BMP-7 over a broad concentration range (0.01-100 microg/ml) with TGF-beta1 failed to attenuate EMT in RPTEC or HK-2 cells, as demonstrated by no inhibition of altered e-cadherin, vimentin, CTGF and TGF-beta1 expression and no restoration of ZO-1 immunoreactivity. Furthermore, when BMP-7 was applied to proximal tubule cells alone, it also decreased e-cadherin expression and increased vimentin, CTGF and TGF-beta1 expression. Additionally, BMP-7 failed to induce the mesenchymal-to-epithelial transition (MET) in NRK-49F rat renal fibroblasts. BMP-7 did however prevent TGF-beta1-mediated e-cadherin downregulation in TCMK-1 mouse renal tubular epithelial cells. BMP-7 activity was routinely confirmed by examining BMP-7-induced phosphorylation of SMADs 1/5/8, BMP-7 regulation

  14. Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4+ T cell induced injury in human renal proximal tubular epithelial cells

    PubMed Central

    Chung, Byung Ha; Kim, Bo-Mi; Doh, Kyoung Chan; Cho, Mi-La

    2017-01-01

    Background The aim of this study was to investigate the protective effect of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on effector CD4+ T cells or on inflammatory cytokine-induced injury in human renal proximal tubular epithelial cells (HRPTEpiC). Methods First, we investigated the effect of 1,25(OH)2D3 on CD4+ T cell proliferation. Second, we examined the effect of 1,25(OH)2D3 on inflammatory cytokine secretion or fibrosis in HRPTEpiC induced by inflammatory cytokines or activated CD4+ T cells using ELISA and real-time PCR. Lastly, we compared urine inflammatory-cytokine (IL-6, IL-8) or KIM-1 levels in kidney transplant recipients low serum 25-hydroxyvitamin D (25(OH)D) group (< 20 ng/mL) (n = 40) and normal 25(OH)D group (n = 50). Results Pre-incubation with 1,25(OH)2D3 significantly reduced the percentages of Th1 and Th17 cells compared to that of Th0 condition (P < 0.05 for each). In contrast, 1,25(OH)2D3 increased the proportion of Th2 and Treg cells in a dose-dependent manner (P < 0.05 for each). Treatment of HRPTEpiC with inflammatory cytokines (TNF-α, IL-17, and TGF-β) or effector CD4+ T cells resulted in increased production of IL-6, IL-8, or KIM-1 from HRPTEpiC in a dose-dependent manner. However, treatment with 1,25(OH)2D3 significantly reduced the level of these cytokines (P < 0.05 for all). Western blot analysis demonstrated that the mTOR/STAT3/ERK pathway was downregulated by 1,25(OH)2D3 in HRPTEpiC. Furthermore, the concentrations of urine IL-6/creatinine (P < 0.05) and Kim-1/creatinine (P < 0.05) were higher in the low 25(OH)D group than in the normal 25(OH)D group in kidney transplant recipients. Conclusion The results of this study suggests that vitamin D may have a significant role in the regulation of inflammation in allograft tissue in kidney transplant recipients. Trial registration All participants provided written informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of

  15. Possible roles of tumor necrosis factor-α and angiotensin II type 1 receptor on high glucose-induced damage in renal proximal tubular cells.

    PubMed

    Takao, Toshihiro; Horino, Taro; Matsumoto, Reiko; Shimamura, Yoshiko; Ogata, Koji; Inoue, Kousuke; Taniguchi, Yoshinori; Taguchi, Takafumi; Terada, Yoshio

    2015-02-01

    Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22(phox) protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30 mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22(phox) protein levels compared with those in regular glucose medium (5.6 mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22(phox) protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.

  16. Cytosolic Ca2+ deregulation and blebbing after HgCl2 injury to cultured rabbit proximal tubule cells as determined by digital imaging microscopy.

    PubMed Central

    Smith, M W; Phelps, P C; Trump, B F

    1991-01-01

    Acute injury to renal proximal tubule cells has previously been shown to result in elevated cytosolic Ca2+ ([Ca2+]i), blebbing, and eventual cell death. In this study, digital imaging fluorescence microscopy was used to evaluate these changes in response to HgCl2 treatment of cultured rabbit proximal tubular cells. Monolayer cells loaded with fura-2 were treated with 10, 50, or 100 microM HgCl2 in both 1.37 mM CaCl2-containing and nominally Ca(2+)-free (less than 5 microM) Hanks' balanced salt solution. [Ca2+]i was estimated by measuring the ratio of fluorescent image pairs (collected at 340- and 380-nm excitation), morphological changes were observed by phase-contrast microscopy, and viability was assessed by trypan blue exclusion. After exposure of cells to 10 microM HgCl2, [Ca2+]i initially increased about 2-fold by 5 min; after 50 or 100 microM HgCl2, [Ca2+]i rapidly rose 2- to 3-fold, peaked at 1-3 min, and then generally decreased slightly. In nominally Ca(2+)-free (less than 5 microM) medium, [Ca2+]i stabilized, but in 1.37 mM Ca(2+)-containing medium, [Ca2+]i continued to slowly rise, often reaching levels of fura-2 saturation. The rate and extent of blebbing and the rate of cell death were increased in the presence of 1.37 mM Ca2+. These results show that sustained elevations of [Ca2+]i precede both cell blebbing and cell death and that when these elevations are limited by removing extracellular Ca2+ the amount of blebbing is reduced and cell viability is prolonged. Images PMID:2052574

  17. Serum level of proximal renal tubular epithelial cell-binding immunoglobulin G in patients with lupus nephritis.

    PubMed

    Yap, D Y H; Yung, S; Zhang, Q; Tang, C; Chan, T M

    2016-01-01

    In vitro data showed that immunoglobulin G (IgG) from lupus nephritis (LN) patients could bind to proximal renal tubular epithelial cells (PTEC), but the clinical relevance of such binding remained unclear. Binding of IgG and subclasses to PTEC was measured by cellular ELISA (expressed as OD index) in 189 serial serum samples from 23 Class III/IV ± V LN patients who had repeated renal flares (48 during renal flares, 141 during low level disease activity (LLDA)), and compared with 64 patients with non-lupus glomerular diseases (NLGD) and 23 healthy individuals. Total IgG PTEC-binding index was 0.34 ± 0.16, 0.29 ± 0.16, 0.62 ± 0.27 and 0.83 ± 0.38 in healthy controls, NLGD, LN patients during LLDA, and LN patients during nephritic flare, respectively (p < 0.001, LLDA vs. renal flare; p < 0.001, healthy controls or NLGD vs. LN during LLDA or renal flare). PTEC-binding index for IgG1 was 0.09 ± 0.05, 0.16 ± 0.12, 0.44 ± 0.34 and 0.71 ± 0.46 for the corresponding groups (p < 0.001, LLDA vs. renal flare; p < 0.001, healthy controls or NLGD vs. LN during LLDA or renal flare). Sixteen of 48 episodes (33.3%) of nephritic flare showed persistent PTEC-binding IgG seropositivity for more than 9.4 ± 3.1 months, despite clinical response to immunosuppressive treatment. Total IgG and IgG1 PTEC-binding correlated with anti-dsDNA level (r = 0.34 and 0.52, respectively, p < 0.001 for both), and inversely with C3 level (r = -0.26 and -0.50, respectively, p = 0.002 and<0.001). Sensitivity/specificity of PTEC-binding index in detecting renal flares was 45.8%/80.1% for total IgG (ROC AUC 0.630, p = 0.007) and 87.5%/35.5% for IgG1 (ROC AUC 0.615, p = 0.018). IgG1 PTEC-binding index correlated with tubulo-interstitial inflammation score in renal biopsy from corresponding patients. Our data suggested that total IgG and IgG1 PTEC-binding index in serum of LN patients correlate with serological activity, and in combination could predict renal flares. The correlation between IgG1

  18. CBFB and MYH11 in inv(16)(p13q22) of Acute Myeloid Leukemia Display Close Spatial Proximity in Interphase Nuclei of Human Hematopoietic Stem Cells

    PubMed Central

    Weckerle, Allison B.; Santra, Madhumita; Ng, Maggie C.Y.; Koty, Patrick P.; Wang, Yuh-Hwa

    2013-01-01

    To gain a better understanding of the mechanism of chromosomal translocations in cancer, we investigated the spatial proximity between CBFB and MYH11 genes involved in inv(16)(p13q22) found in acute myeloid leukemia patients. Previous studies have demonstrated a role for spatial genome organization in the formation of tumorigenic abnormalities. The non-random localization of chromosomes and, more specifically, of genes appears to play a role in the mechanism of chromosomal translocations. Here, two-color fluorescence in situ hybridization and confocal microscopy were used to measure the interphase distance between CBFB and MYH11 in hematopoietic stem cells, where inv(16)(p13q22) is believed to occur, leading to leukemia development. The measured distances in hematopoietic stem cells were compared to mesenchymal stem cells, peripheral blood lymphocytes and fibroblasts, as spatial genome organization is determined to be cell-type specific. Results indicate that CBFB and MYH11 are significantly closer in hematopoietic stem cells compared to all other cell types examined. Furthermore, the CBFB-MYH11 distance is significantly reduced compared to CBFB and a control locus in hematopoietic stem cells, although separation between CBFB and the control is ~70% of that between CBFB and MYH11 on metaphase chromosomes. Hematopoietic stem cells were also treated with fragile site-inducing chemicals since both genes contain translocation breakpoints within these regions. However, treatment with fragile site-inducing chemicals did not significantly affect the interphase distance. Consistent with previous studies, our results suggest that gene proximity may play a role in the formation of cancer-causing rearrangements, providing insight into the mechanism of chromosomal abnormalities in human tumors. PMID:21638519

  19. Tofogliflozin, A Highly Selective Inhibitor of SGLT2 Blocks Proinflammatory and Proapoptotic Effects of Glucose Overload on Proximal Tubular Cells Partly by Suppressing Oxidative Stress Generation.

    PubMed

    Ishibashi, Y; Matsui, T; Yamagishi, S

    2016-03-01

    Ninety percent of glucose filtered by the glomerulus is reabsorbed by a sodium-glucose cotransporter 2 (SGLT2), which is mainly expressed on S1 and S2 segment of renal proximal tubules. Since SGLT-2-mediated glucose reabsorption is increased under diabetic conditions, selective inhibition of SGLT2 is a potential therapeutic target for the treatment of diabetes. We have recently shown that an inhibitor of SGLT2 has anti-inflammatory and antifibrotic effects on experimental diabetic nephropathy partly by suppressing advanced glycation end products formation and oxidative stress generation in the kidney. However, the direct effects of SGLT2 inhibitor on tubular cell damage remain unclear. In this study, we investigated the effects of tofogliflozin, a highly selective inhibitor of SGLT2 on oxidative stress generation, inflammatory and proapoptotic reactions in cultured human proximal tubular cells exposed to high glucose. Tofogliflozin dose-dependently suppressed glucose entry into tubular cells. High glucose exposure (30 mM) for 4 and 24 h significantly increased oxidative stress generation in tubular cells, which were suppressed by the treatment of tofogliflozin or an antioxidant N-acetylcysteine (NAC). Monocyte chemoattractant protein-1 (MCP-1) gene expression and apoptotic cell death were induced by 4 h- and 8 day-exposure to high glucose, respectively, both of which were also blocked by tofogliflozin or NAC. The present study suggests that SGLT2-mediated glucose entry into tubular cells could stimulate oxidative stress and evoke inflammatory and proapoptotic reactions in this cell type. Blockade of glucose reabsorption in tubular cells by SGLT2 inhibitor might exert beneficial effects on tubulointerstitial damage in diabetic nephropathy.

  20. A deletion in the proximal untranslated pX region of human T-cell leukemia virus type II decreases viral replication but not infectivity in vivo.

    PubMed

    Cockerell, G L; Rovnak, J; Green, P L; Chen, I S

    1996-02-01

    The function of untranslated (UT) nucleotide sequences in the proximal portion of the pX region of the human T-cell leukemia virus (HTLV) family of retroviruses remains enigmatic. Previous studies have shown that these sequences are not necessary for the expression of viral proteins or for the induction, transmission, or maintenance of the transformed cell type in vitro. To determine the effect of the UT region in vivo, separate groups of rabbits were inoculated with lethally irradiated, stable clones of the human B-lymphoblastoid cell line, 729, transfected with either a full-length wild-type HTLV-II clone (pH6neo) or a mutant clone containing a 324-bp deletion in the proximal UT portion of pX (pH6neo delta UT[6661-6984]), or nontransfected 729 cells. All rabbits inoculated with either wild-type or pX-deleted HTLV-II developed a similar profile and titer of serum antibodies against HTLV-II antigens, as determined by Western immunoblots, by 4 weeks postinoculation (PI). Antibody titers, as determined by enzyme immunoassay, were similar between the two groups of rabbits and increased over the 18-week period of study. All rabbits were killed at 18 weeks PI, and spleen, peripheral blood lymphocytes (PBMC), bone marrow, and mesenteric lymph node were assayed for HTLV-II tax/rex sequences by quantitative polymerase chain reaction. Virus was detected in all tissues tested from all rabbits inoculated with 729pH6neo cells containing wild-type HTLV-II, which contained between 1.4 and 0.3 mean copies of provirus per cell. In contrast, the distribution and number of provirus copies were more limited in rabbits inoculated with 729pH6neo delta UT(6661-6984) cells containing UT-deleted HTLV-II; in most tissues, there was a fivefold to sevenfold reduction in mean provirus copies per cell as compared with rabbits inoculated with wild-type HTLV-II. All rabbits inoculated with control 729 cells remained negative for HTLV-II infection, as determined by the same techniques. It was

  1. Molecular origins of scintillation in organic scintillators (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Feng, Patrick; Mengesha, Wondwosen; Myllenbeck, Nicholas

    2016-09-01

    Organic-based scintillators are indispensable materials for radiation detection owing to their high sensitivity to fast neutrons, low cost, and tailorable properties. There has been a recent resurgence of interest in organic scintillators due to exciting discoveries related to neutron discrimination and gamma-ray spectroscopy, which represent capabilities previously thought not possible in these materials. I will discuss our development of crystalline and polymer-based scintillators for these applications. Structure-property relationships related to intermolecular interactions and host-guest electronic exchange will be discussed in the context of energy-transfer pathways relevant to scintillation. An emphasis will be placed on the rational design of these materials, as guided by first principles and DFT calculations. Two related topics will be discussed: 1) Incorporation of organometallic triplet-harvesting additives to plastic scintillator matrices to confer a 'two-state' (singlet and triplet) luminescence signature to different types of ionizing radiation. This approach relies upon energetic and spatial overlap between the donor and acceptor excited states for efficient electronic exchange. Key considerations also include synthetic modification of the luminescence spectra and kinetics, as well as the addition of secondary additives to increase the recombination efficiency. 2) Design of organotin-containing plastic scintillators as a route towards gamma-ray spectroscopy. Organometallic compounds were selected on the basis of distance-dependent quenching relationships, phase compatibility with the polymer matrix, and the gamma-ray cross sections. This approach is guided by molecular modeling and radiation transport modeling to achieve the highest possible detection sensitivity luminescence intensity.

  2. FNAL-NICADD extruded scintillator

    SciTech Connect

    Beznosko, D.; Bross, A.; Dyshkant, A.; Pla-Dalmau, A.; Rykalin, V.; /Northern Illinois U.

    2005-09-01

    The possibility to produce a scintillator that satisfies the demands of physicists from different science areas has emerged with the installation of an extrusion line at Fermi National Accelerator Laboratory (FNAL). The extruder is the product of the fruitful collaboration between FNAL and Northern Illinois Center for Accelerator and Detector Development (NICADD) at Northern Illinois University (NIU). The results from the light output, light attenuation length and mechanical tolerance indicate that FNAL-NICADD scintillator is of high quality. Improvements in the extrusion die will yield better scintillator profiles and decrease the time needed for initial tuning. This paper will present the characteristics of the FNAL-NICADD scintillator based on the measurements performed. They include the response to MIPs from cosmic rays for individual extruded strips and irradiation studies where extruded samples were irradiated up to 1 Mrad. We will also discuss the results achieved with a new die design. The attractive perspective of using the extruded scintillator with MRS (Metal Resistive Semiconductor) photodetector readout will also be shown.

  3. Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells.

    PubMed

    McGoldrick, Trevor A; Lock, Edward A; Rodilla, Vicente; Hawksworth, Gabrielle M

    2003-07-01

    Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)- l-cysteine (DCVC), S-(1,2,2-trichlorovinyl)- l-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)- l-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50-1,000 microM, the order of toxicity being DCVC>TCVC>TFEC=CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 microM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 microM) with AOAA (250 microM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 microM), an inhibitor of gamma-glutamyl transferase (gamma-GT), partially protected against DCVG (500 microM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by gamma-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached

  4. Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes.

    PubMed

    Debaize, Lydie; Jakobczyk, Hélène; Rio, Anne-Gaëlle; Gandemer, Virginie; Troadec, Marie-Bérengère

    2017-01-01

    Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients' cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.

  5. Radiopure metal-loaded liquid scintillator

    SciTech Connect

    Rosero, Richard; Yeh, Minfang

    2015-08-17

    Metal-loaded liquid scintillator plays a key role in particle and nuclear physics experiments. The applications of metal ions in various neutrino experiments and the purification methods for different scintillator components are discussed in this paper.

  6. Radiopure Metal-Loaded Liquid Scintillator

    SciTech Connect

    Rosero, Richard; Yeh, Minfang

    2015-03-18

    Metal-loaded liquid scintillator plays a key role in particle and nuclear physics experiments. The applications of metal ions in various neutrino experiments and the purification methods for different scintillator components are discussed in this paper.

  7. Advances in scintillators for medical imaging applications

    NASA Astrophysics Data System (ADS)

    van Loef, Edgar V.; Shah, Kanai S.

    2014-09-01

    A review is presented of some recent work in the field of inorganic scintillator research for medical imaging applications, in particular scintillation detectors for Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET).

  8. Liquid scintillator tiles for calorimetry

    DOE PAGES

    Amouzegar, M.; Belloni, A.; Bilki, B.; ...

    2016-11-28

    Future experiments in high energy and nuclear physics may require large, inexpensive calorimeters that can continue to operate after receiving doses of 50 Mrad or more. Also, the light output of liquid scintillators suffers little degradation under irradiation. However, many challenges exist before liquids can be used in sampling calorimetry, especially regarding developing a packaging that has sufficient efficiency and uniformity of light collection, as well as suitable mechanical properties. We present the results of a study of a scintillator tile based on the EJ-309 liquid scintillator using cosmic rays and test beam on the light collection efficiency and uniformity,more » and some preliminary results on radiation hardness.« less

  9. Scintillating glass fiber neutron senors

    SciTech Connect

    Abel, K.H.; Arthur, R.J.; Bliss, M.

    1994-04-01

    Cerium-doped lithium-silicate glass fibers have been developed at Pacific Northwest Laboratory (PNL) for use as thermal neutron detectors. By using highly-enriched {sup 6} Li , these fibers efficiently capture thermal neutrons and produce scintillation light that can be detected at the ends of the fibers. Advantages of scintillating fibers over {sup 3}He or BF{sub 3} proportional tubes include flexibility in geometric configuration, ruggedness in high-vibration environments, and less detector weight for the same neutron sensitivity. This paper describes the performance of these scintillating fibers with regard to count rates, pulse height spectra, absolute efficiencies, and neutron/gamma discrimination. Fibers with light transmission lengths (1/e) of greater than 2 m have been produced at PNL. Neutron sensors in fiber form allow development of a variety of neutron detectors packaged in previously unavailable configurations. Brief descriptions of some of the devices already produced are included to illustrate these possibilities.

  10. Unitary scintillation detector and system

    DOEpatents

    McElhaney, Stephanie A.; Chiles, Marion M.

    1994-01-01

    The invention is a unitary alpha, beta, and gamma scintillation detector and system for sensing the presence of alpha, beta, and gamma radiations selectively or simultaneously. The scintillators are mounted in a light-tight housing provided with an entrance window for admitting alpha, beta, and gamma radiation and excluding ambient light from the housing. Light pulses from each scintillator have different decay constants that are converted by a photosensitive device into corresponding differently shaped electrical pulses. A pulse discrimination system identifies the electrical pulses by their respective pulse shapes which are determined by decay time. The identified electrical pulses are counted in separate channel analyzers to indicate the respective levels of sensed alpha, beta, and gamma radiations.

  11. Unitary scintillation detector and system

    DOEpatents

    McElhaney, S.A.; Chiles, M.M.

    1994-05-31

    The invention is a unitary alpha, beta, and gamma scintillation detector and system for sensing the presence of alpha, beta, and gamma radiations selectively or simultaneously. The scintillators are mounted in a light-tight housing provided with an entrance window for admitting alpha, beta, and gamma radiation and excluding ambient light from the housing. Light pulses from each scintillator have different decay constants that are converted by a photosensitive device into corresponding differently shaped electrical pulses. A pulse discrimination system identifies the electrical pulses by their respective pulse shapes which are determined by decay time. The identified electrical pulses are counted in separate channel analyzers to indicate the respective levels of sensed alpha, beta, and gamma radiations. 10 figs.

  12. Fracture-resistant lanthanide scintillators

    DOEpatents

    Doty, F Patrick [Livermore, CA

    2011-01-04

    Lanthanide halide alloys have recently enabled scintillating gamma ray spectrometers comparable to room temperature semiconductors (<3% FWHM energy resolutions at 662 keV). However brittle fracture of these materials upon cooling hinders the growth of large volume crystals. Efforts to improve the strength through non-lanthanide alloy substitution, while preserving scintillation, have been demonstrated. Isovalent alloys having nominal compositions of comprising Al, Ga, Sc, Y, and In dopants as well as aliovalent alloys comprising Ca, Sr, Zr, Hf, Zn, and Pb dopants were prepared. All of these alloys exhibit bright fluorescence under UV excitation, with varying shifts in the spectral peaks and intensities relative to pure CeBr.sub.3. Further, these alloys scintillate when coupled to a photomultiplier tube (PMT) and exposed to .sup.137Cs gamma rays.

  13. Development of radiation hard scintillators

    NASA Astrophysics Data System (ADS)

    Markley, F.; Woods, D.; Pla-Dalmau, A.; Foster, G.; Blackburn, R.

    1993-01-01

    Substantial improvements have been made in the radiation hardness of plastic scintillators. Cylinders of scintillating materials 2.2 cm in diameter and 1 cm thick have been exposed to 10 Mrads of gamma rays at a dose rate of 1 Mrad/h in a nitrogen atmosphere. One of the formulations tested showed an immediate decrease in pulse height of only 4% and has remained stable for 12 days while annealing in air. By comparison a commercial PVT scintillator showed an immediate decrease of 58% and after 43 days of annealing in air it improved to a 14% loss. The formulated sample consisted of 70 parts by weight of Dow polystyrene, 30 pbw of pentaphenyltrimethyltrisiloxane (Dow Corning DC 705 oil), 2 pbw of p-terphenyl, 0.2 pbw of tetraphenylbutadiene, and 0.5 pbw of UVASIL299LM from Ferro.

  14. Scintillation-Hardened GPS Receiver

    NASA Technical Reports Server (NTRS)

    Stephens, Donald R.

    2015-01-01

    CommLargo, Inc., has developed a scintillation-hardened Global Positioning System (GPS) receiver that improves reliability for low-orbit missions and complies with NASA's Space Telecommunications Radio System (STRS) architecture standards. A software-defined radio (SDR) implementation allows a single hardware element to function as either a conventional radio or as a GPS receiver, providing backup and redundancy for platforms such as the International Space Station (ISS) and high-value remote sensing platforms. The innovation's flexible SDR implementation reduces cost, weight, and power requirements. Scintillation hardening improves mission reliability and variability. In Phase I, CommLargo refactored an open-source GPS software package with Kalman filter-based tracking loops to improve performance during scintillation and also demonstrated improved navigation during a geomagnetic storm. In Phase II, the company generated a new field-programmable gate array (FPGA)-based GPS waveform to demonstrate on NASA's Space Communication and Navigation (SCaN) test bed.

  15. Interstellar scintillations of pulsar radiation.

    PubMed

    Lang, K R

    1969-12-12

    Time fluctuations in the intensity of pulsed radiation from CP 0834, CP 1133, AP 1237, and CP 1919 have been investigated. Power spectra, modulation indices, frequency distributions, and decorrelation frequencies are consistent with scintillation theory. If it is assumed that these scintillations are due to irregularities in the interstellar medium that travel at a velocity of 20 kilometers per second, the irregularities have a scale size on the order of 10(4) kilometers and a distance from the earth of approximately 70 parsecs. These interstellar scintillations would not have been observed if the apparent angular diameters of the pulsars were larger than 0.3 X 10(-5) second of arc, and they would cause even a point radio source to have an apparent angular diameter of approximately 10(-3) second of arc at 318 megahertz.

  16. Development of radiation hard scintillators

    SciTech Connect

    Markley, F.; Woods, D.; Pla-Dalmau, A.; Foster, G. ); Blackburn, R. )

    1992-05-01

    Substantial improvements have been made in the radiation hardness of plastic scintillators. Cylinders of scintillating materials 2.2 cm in diameter and 1 cm thick have been exposed to 10 Mrads of gamma rays at a dose rate of 1 Mrad/h in a nitrogen atmosphere. One of the formulations tested showed an immediate decrease in pulse height of only 4% and has remained stable for 12 days while annealing in air. By comparison a commercial PVT scintillator showed an immediate decrease of 58% and after 43 days of annealing in air it improved to a 14% loss. The formulated sample consisted of 70 parts by weight of Dow polystyrene, 30 pbw of pentaphenyltrimethyltrisiloxane (Dow Corning DC 705 oil), 2 pbw of p-terphenyl, 0.2 pbw of tetraphenylbutadiene, and 0.5 pbw of UVASIL299LM from Ferro.

  17. RNA Whole-Mount In Situ Hybridization Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

    PubMed

    Roussis, Ioannis M; Myers, Fiona A; Scarlett, Garry P

    2017-03-03

    Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to the study of RNA regulation (rISH-PLA). The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single-cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogeneous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos. © 2017 by John Wiley & Sons, Inc.

  18. Nanophosphor composite scintillator with a liquid matrix

    DOEpatents

    McKigney, Edward Allen; Burrell, Anthony Keiran; Bennett, Bryan L.; Cooke, David Wayne; Ott, Kevin Curtis; Bacrania, Minesh Kantilal; Del Sesto, Rico Emilio; Gilbertson, Robert David; Muenchausen, Ross Edward; McCleskey, Thomas Mark

    2010-03-16

    An improved nanophosphor scintillator liquid comprises nanophosphor particles in a liquid matrix. The nanophosphor particles are optionally surface modified with an organic ligand. The surface modified nanophosphor particle is essentially surface charge neutral, thereby preventing agglomeration of the nanophosphor particles during dispersion in a liquid scintillator matrix. The improved nanophosphor scintillator liquid may be used in any conventional liquid scintillator application, including in a radiation detector.

  19. High speed pulse digitization in driftless gas scintillation proportional counters

    SciTech Connect

    Valero, J.; Smith, A.; Peacock, A.

    1988-06-01

    In a driftless gas scintillation proportional counter the observed light burst profile provides a wealth of information both about the incident photon and the transit of the electron cloud through the scintillation region. The different features of such profiles are difficult to separate with conventional analog electronics. In this work the authors apply the technique of high speed pulse digitization to the problem. They show that by this technique not only can the performance of the instrument be improved (e.g. background rejection) but valuable data can be obtained regarding the design of both the gas cell and the analysis electronics.

  20. Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation

    PubMed Central

    Hung, Victoria; Lam, Stephanie S; Udeshi, Namrata D; Svinkina, Tanya; Guzman, Gaelen; Mootha, Vamsi K; Carr, Steven A; Ting, Alice Y

    2017-01-01

    The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP’s ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes. DOI: http://dx.doi.org/10.7554/eLife.24463.001 PMID:28441135

  1. Scintillator Cosmic Ray Super Telescope

    NASA Astrophysics Data System (ADS)

    González, L. X.; Valdés-Galicia, J. F.; Matsubara, Y.; Nagai, Y.; Itow, Y.; Sako, T.; López, D.; Mitsuka, G.; Munakata, K.; Kato, C.; Yasue, S.; Kosai, M.; Tsurusashi, M.; Nakamo, Y.; Shibata, S.; Takamaru, H.; Kojima, H.; Tsuchiya, H.; Watanabe, K.; Koi, T.; Fragoso, E.; Hurtado, A.; Musalem, O.

    2013-04-01

    The Scintillator Cosmic Ray Super Telescope (SciCRST) is a new experiment to detect solar neutrons, and also it is expected to work as a muon and cosmic ray detector. The SciCRST consist of 14,848 plastic scintillator bars, and it will be installed at the top of Sierra Negra volcano, Mexico, 4580 m.a.s.l. We use a prototype, called as miniSciBar, to test the hardware and software of the final experiment. In this paper, we present the status and details of the experiment, and results of the prototype.

  2. Hygroscopicity Evaluation of Halide Scintillators

    SciTech Connect

    Zhuravleva, M; Stand, L; Wei, H; Hobbs, C. L.; Boatner, Lynn A; Ramey, Joanne Oxendine; Burger, Arnold; Rowe, E; Bhattacharya, P.; Tupitsyn, E; Melcher, Charles L

    2014-01-01

    A collaborative study of relative hygroscopicity of anhydrous halide scintillators grown at various laboratories is presented. We have developed a technique to evaluate moisture sensitivity of both raw materials and grown crystals, in which the moisture absorption rate is measured using a gravimetric analysis. Degradation of the scintillation performance was investigated by recording gamma-ray spectra and monitoring the photopeak position, count rate and energy resolution. The accompanying physical degradation of the samples exposed to ambient atmosphere was photographically recorded as well. The results were compared with ben

  3. 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

  4. Composite scintillators for detection of ionizing radiation

    DOEpatents

    Dai, Sheng [Knoxville, TN; Stephan, Andrew Curtis [Knoxville, TN; Brown, Suree S [Knoxville, TN; Wallace, Steven A [Knoxville, TN; Rondinone, Adam J [Knoxville, TN

    2010-12-28

    Applicant's present invention is a composite scintillator having enhanced transparency for detecting ionizing radiation comprising a material having optical transparency wherein said material comprises nano-sized objects having a size in at least one dimension that is less than the wavelength of light emitted by the composite scintillator wherein the composite scintillator is designed to have selected properties suitable for a particular application.

  5. Characteristics of High Latitude Ionosphere Scintillations

    NASA Astrophysics Data System (ADS)

    Morton, Y.

    2012-12-01

    As we enter a new solar maximum period, global navigation satellite systems (GNSS) receivers, especially the ones operating in high latitude and equatorial regions, are facing an increasing threat from ionosphere scintillations. The increased solar activities, however, also offer a great opportunity to collect scintillation data to characterize scintillation signal parameters and ionosphere irregularities. While there are numerous GPS receivers deployed around the globe to monitor ionosphere scintillations, most of them are commercial receivers whose signal processing mechanisms are not designed to operate under ionosphere scintillation. As a result, they may distort scintillation signal parameters or lose lock of satellite signals under strong scintillations. Since 2008, we have established and continuously improved a unique GNSS receiver array at HAARP, Alaska. The array contains high ends commercial receivers and custom RF front ends which can be automatically triggered to collect high quality GPS and GLONASS satellite signals during controlled heating experiments and natural scintillation events. Custom designed receiver signal tracking algorithms aim to preserve true scintillation signatures are used to process the raw RF samples. Signal strength, carrier phase, and relative TEC measurements generated by the receiver array since its inception have been analyzed to characterize high latitude scintillation phenomena. Daily, seasonal, and solar events dependency of scintillation occurrence, spectral contents of scintillation activities, and plasma drifts derived from these measurements will be presented. These interesting results demonstrate the feasibility and effectiveness of our experimental data collection system in providing insightful details of ionosphere responses to active perturbations and natural disturbances.

  6. Photonic crystal scintillators and methods of manufacture

    DOEpatents

    Torres, Ricardo D.; Sexton, Lindsay T.; Fuentes, Roderick E.; Cortes-Concepcion, Jose

    2015-08-11

    Photonic crystal scintillators and their methods of manufacture are provided. Exemplary methods of manufacture include using a highly-ordered porous anodic alumina membrane as a pattern transfer mask for either the etching of underlying material or for the deposition of additional material onto the surface of a scintillator. Exemplary detectors utilizing such photonic crystal scintillators are also provided.

  7. Synthesis of plastic scintillation microspheres: Evaluation of scintillators

    NASA Astrophysics Data System (ADS)

    Santiago, L. M.; Bagán, H.; Tarancón, A.; Garcia, J. F.

    2013-01-01

    The use of plastic scintillation microspheres (PSm) appear to be an alternative to liquid scintillation for the quantification of alpha and beta emitters because it does not generate mixed wastes after the measurement (organic and radioactive). In addition to routine radionuclide determinations, PSm can be used for further applications, e.g. for usage in a continuous monitoring equipment, for measurements of samples with a high salt concentration and for an extractive scintillation support which permits the separation, pre-concentration and measurement of the radionuclides without additional steps of elution and sample preparation. However, only a few manufacturers provide PSm, and the low number of regular suppliers reduces its availability and restricts the compositions and sizes available. In this article, a synthesis method based on the extraction/evaporation methodology has been developed and successfully used for the synthesis of plastic scintillation microspheres. Seven different compositions of plastic scintillation microspheres have been synthesised; PSm1 with polystyrene, PSm2 with 2,5-Diphenyloxazol(PPO), PSm3 with p-terphenyl (pT), PSm4 with PPO and 1,4-bis(5-phenyloxazol-2-yl) (POPOP), PSm5 pT and (1,4-bis [2-methylstyryl] benzene) (Bis-MSB), PSm6 with PPO, POPOP and naphthalene and PSm7 with pT, Bis-MSB and naphthalene. The synthesised plastic scintillation microspheres have been characterised in terms of their morphology, detection capabilities and alpha/beta separation capacity. The microspheres had a median diameter of approximately 130 μm. Maximum detection efficiency values were obtained for the PSm4 composition as follows 1.18% for 3H, 51.2% for 14C, 180.6% for 90Sr/90Y and 76.7% for 241Am. Values of the SQP(E) parameter were approximately 790 for PSm4 and PSm5. These values show that the synthesised PSm exhibit good scintillation properties and that the spectra are at channel numbers higher than in commercial PSm. Finally, the addition of

  8. The expression of RNA helicase DDX5 is transcriptionally upregulated by calcitriol through a vitamin D response element in the proximal promoter in SiHa cervical cells.

    PubMed

    González-Duarte, Ramiro José; Cázares-Ordoñez, Verna; Díaz, Lorenza; Ortíz, Víctor; Larrea, Fernando; Avila, Euclides

    2015-12-01

    The DEAD box RNA helicase DDX5 is a multifunctional protein involved in the regulatory events of gene expression. Herein, we presented evidence indicating that DDX5 is transcriptionally upregulated by calcitriol, the hormonal form of vitamin D3. In silico analysis revealed the presence of two putative vitamin D response elements (VDREs) in the DDX5 promoter region. Using luciferase reporter assays, we demonstrated that the DDX5 promoter containing these putative VDREs significantly increased the luciferase activity in vitamin D receptor (VDR)-positive SiHa cells upon calcitriol treatment. Electrophoretic mobility shift assays showed the ability of VDR and retinoid X receptor to interact only with the most proximal VDRE, while chromatin immunoprecipitation analysis confirmed the occupancy of this VDRE by the VDR. Finally, we demonstrated that calcitriol significantly increased both DDX5 mRNA and protein in SiHa cells. In summary, this study shows that DDX5 gene is transcriptionally upregulated by calcitriol through a VDRE located in its proximal promoter. Given the importance of DDX5 as a master regulator of differentiation programs, our study suggests that the pro-differentiating properties of calcitriol may be related with the induction of DDX5.

  9. Sp7/Osterix induces the mouse pro-α2(I) collagen gene (Col1a2) expression via the proximal promoter in osteoblastic cells.

    PubMed

    Yano, Hiroyuki; Hamanaka, Ryoji; Nakamura-Ota, Miki; Adachi, Sawako; Zhang, Juan Juan; Matsuo, Noritaka; Yoshioka, Hidekatsu

    2014-09-26

    Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amounts of other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process in which many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-rich sequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 gene containing multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site specifically decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its specific siRNA showed that this G/C-rich sequence is responsible for the specific expression in osteoblastic cells. Consistent with these data, Sp7/Osterix bound to the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.

  10. 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.

  11. Low dose ouabain stimulates NaK ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells.

    PubMed

    Ketchem, Corey J; Conner, Clayton D; Murray, Rebecca D; DuPlessis, Madalyn; Lederer, Eleanor D; Wilkey, Daniel; Merchant, Michael; Khundmiri, Syed J

    2016-11-01

    Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of NaK ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of NaK ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1μg/kg body wt./day) for 9days and increased angiotensin II levels in cell culture media after 24h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10pM stimulated NKA-mediated (86)Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. 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

  13. 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

  14. The stress response of human proximal tubule cells to cadmium involves up-regulation of haemoxygenase 1 and metallothionein but not cytochrome P450 enzymes.

    PubMed

    Boonprasert, Kanyarat; Satarug, Soisungwan; Morais, Christudas; Gobe, Glenda C; Johnson, David W; Na-Bangchang, Kesara; Vesey, David A

    2016-05-13

    Enzymes of the cytochrome P450 (CYP) super-family are implicated in cadmium (Cd) -induced nephrotoxicity, however, direct evidence is lacking. This study investigated the endogenous expression of various CYP proteins together with the stress-response proteins, heme oxygenase-1 (HO-1) and metallothionein (MT) in human kidney sections and in cadmium-exposed primary cultures of human proximal tubular epithelial cells (PTC). By immunohistochemistry, the CYP members 2B6, 4A11 and 4F2 were prominently expressed in the cortical proximal tubular cells and to a lesser extent in distal tubular cells. Low levels of CYPs 2E1 and 3A4 were also detected. In PTC, in the absence of Cd, CYP2E1, CYP3A4, CYP4F2 and MT were expressed, but HO-1, CYP2B6 and CYP4A11 were not detected. A range of cadmium concentrations (0-100μM) were utilized to induce stress conditions. MT protein was further induced by as little as 0.5μM cadmium, reaching a 6-fold induction at 20μM, whereas for HO-1, a 5μM cadmium concentration was required for initial induction and at 20μM cadmium reached a 15-fold induction. The expression of CYP2E1, CYP3A4, and CYP4F2 were not altered by any cadmium concentrations tested at 48h. Cadmium caused a reduction in cell viability at concentrations above 10μM. In conclusion although cultured PTC, do express CYP proteins, (CYP2E1, CYP3A4, and CYP4F2), Cd-induced cell stress as indicted by induction of HO-1 and MT does not alter expression of these CYP proteins at 48h. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. A novel segmented-scintillator antineutrino detector

    NASA Astrophysics Data System (ADS)

    Abreu, Y.; Amhis, Y.; Arnold, L.; Ban, G.; Beaumont, W.; Bongrand, M.; Boursette, D.; Buhour, J. M.; Castle, B. C.; Clark, K.; Coupé, B.; Cucoanes, A. S.; Cussans, D.; De Roeck, A.; D'Hondt, J.; Durand, D.; Fallot, M.; Fresneau, S.; Ghys, L.; Giot, L.; Guillon, B.; Guilloux, G.; Ihantola, S.; Janssen, X.; Kalcheva, S.; Kalousis, L. N.; Koonen, E.; Labare, M.; Lehaut, G.; Mermans, J.; Michiels, I.; Moortgat, C.; Newbold, D.; Park, J.; Petridis, K.; Piñera, I.; Pommery, G.; Popescu, L.; Pronost, G.; Rademacker, J.; Reynolds, A.; Ryckbosch, D.; Ryder, N.; Saunders, D.; Shitov, Yu. A.; Schune, M.-H.; Scovell, P. R.; Simard, L.; Vacheret, A.; Van Dyck, S.; Van Mulders, P.; van Remortel, N.; Vercaemer, S.; Waldron, A.; Weber, A.; Yermia, F.

    2017-04-01

    The next generation of very-short-baseline reactor experiments will require compact detectors operating at surface level and close to a nuclear reactor. This paper presents a new detector concept based on a composite solid scintillator technology. The detector target uses cubes of polyvinyltoluene interleaved with 6LiF:ZnS(Ag) phosphor screens to detect the products of the inverse beta decay reaction. A multi-tonne detector system built from these individual cells can provide precise localisation of scintillation signals, making efficient use of the detector volume. Monte Carlo simulations indicate that a neutron capture efficiency of over 70 % is achievable with a sufficient number of 6LiF:ZnS(Ag) screens per cube and that an appropriate segmentation enables a measurement of the positron energy which is not limited by γ-ray leakage. First measurements of a single cell indicate that a very good neutron-gamma discrimination and high neutron detection efficiency can be obtained with adequate triggering techniques. The light yield from positron signals has been measured, showing that an energy resolution of 14%/√E(MeV) is achievable with high uniformity. A preliminary neutrino signal analysis has been developed, using selection criteria for pulse shape, energy, time structure and energy spatial distribution and showing that an antineutrino efficiency of 40% can be achieved. It also shows that the fine segmentation of the detector can be used to significantly decrease both correlated and accidental backgrounds.

  16. 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.

  17. High density scintillating glass proton imaging detector

    NASA Astrophysics Data System (ADS)

    Wilkinson, C. J.; Goranson, K.; Turney, A.; Xie, Q.; Tillman, I. J.; Thune, Z. L.; Dong, A.; Pritchett, D.; McInally, W.; Potter, A.; Wang, D.; Akgun, U.

    2017-03-01

    In recent years, proton therapy has achieved remarkable precision in delivering doses to cancerous cells while avoiding healthy tissue. However, in order to utilize this high precision treatment, greater accuracy in patient positioning is needed. An accepted approximate uncertainty of +/-3% exists in the current practice of proton therapy due to conversions between x-ray and proton stopping power. The use of protons in imaging would eliminate this source of error and lessen the radiation exposure of the patient. To this end, this study focuses on developing a novel proton-imaging detector built with high-density glass scintillator. The model described herein contains a compact homogeneous proton calorimeter composed of scintillating, high density glass as the active medium. The unique geometry of this detector allows for the measurement of both the position and residual energy of protons, eliminating the need for a separate set of position trackers in the system. Average position and energy of a pencil beam of 106 protons is used to reconstruct the image rather than by analyzing individual proton data. Simplicity and efficiency were major objectives in this model in order to present an imaging technique that is compact, cost-effective, and precise, as well as practical for a clinical setting with pencil-beam scanning proton therapy equipment. In this work, the development of novel high-density glass scintillator and the unique conceptual design of the imager are discussed; a proof-of-principle Monte Carlo simulation study is performed; preliminary two-dimensional images reconstructed from the Geant4 simulation are presented.

  18. Extruded plastic scintillator for MINERvA

    SciTech Connect

    Pla-Dalmau, Anna; Bross, Alan D.; Rykalin, Victor V.; Wood, Brian M.; /NICADD, DeKalb

    2005-11-01

    An extrusion line has recently been installed at Fermilab in collaboration with NICADD (Northern Illinois Center for Accelerator and Detector Development). This new facility will serve to further develop and improve extruded plastic scintillator. Since polystyrene is widely used in the consumer industry, the logical path was to investigate the extrusion of commercial-grade polystyrene pellets with dopants to yield high quality plastic scintillator. The D0 and MINOS experiments are already using extruded scintillator strips in their detectors. A new experiment at Fermilab is pursuing the use of extruded plastic scintillator. A new plastic scintillator strip is being tested and its properties characterized. The initial results are presented here.

  19. Method of making a scintillator waveguide

    DOEpatents

    Bliss, Mary; Craig, Richard A.; Reeder, Paul L.

    2000-01-01

    The present invention is an apparatus for detecting ionizing radiation, having: a waveguide having a first end and a second end, the waveguide formed of a scintillator material wherein the therapeutic ionizing radiation isotropically generates scintillation light signals within the waveguide. This apparatus provides a measure of radiation dose. The apparatus may be modified to permit making a measure of location of radiation dose. Specifically, the scintillation material is segmented into a plurality of segments; and a connecting cable for each of the plurality of segments is used for conducting scintillation signals to a scintillation detector.

  20. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  1. SNO+ Scintillator Purification and Assay

    SciTech Connect

    Ford, R.; Vazquez-Jauregui, E.; Chen, M.; Chkvorets, O.; Hallman, D.

    2011-04-27

    We describe the R and D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O{sub 2}, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed ''natural'' radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  2. Complex Dynamics of Equatorial Scintillation

    NASA Astrophysics Data System (ADS)

    Piersanti, Mirko; Materassi, Massimo; Forte, Biagio; Cicone, Antonio

    2017-04-01

    Radio power scintillation, namely highly irregular fluctuations of the power of trans-ionospheric GNSS signals, is the effect of ionospheric plasma turbulence. The scintillation patterns on radio signals crossing the medium inherit the ionospheric turbulence characteristics of inter-scale coupling, local randomness and large time variability. On this basis, the remote sensing of local features of the turbulent plasma is feasible by studying radio scintillation induced by the ionosphere. The distinctive character of intermittent turbulent media depends on the fluctuations on the space- and time-scale statistical properties of the medium. Hence, assessing how the signal fluctuation properties vary under different Helio-Geophysical conditions will help to understand the corresponding dynamics of the turbulent medium crossed by the signal. Data analysis tools, provided by complex system science, appear to be best fitting to study the response of a turbulent medium, as the Earth's equatorial ionosphere, to the non-linear forcing exerted by the Solar Wind (SW). In particular we used the Adaptive Local Iterative Filtering, the Wavelet analysis and the Information theory data analysis tool. We have analysed the radio scintillation and ionospheric fluctuation data at low latitude focusing on the time and space multi-scale variability and on the causal relationship between forcing factors from the SW environment and the ionospheric response.

  3. Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with alpha3beta1 integrin.

    PubMed

    Park, Sun-Woo; Bae, Jong-Sup; Kim, Ki-San; Park, Sun-Hee; Lee, Byung-Heon; Choi, Je-Yong; Park, Jae-Yong; Ha, Sung-Woo; Kim, Yong-Lim; Kwon, Tae-Hwan; Kim, In-San; Park, Rang-Woon

    2004-06-30

    Betaig-h3 (betaig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of betaig-h3 is responsive to TGF-Beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating betaig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of betaig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of betaig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed betaig-h3 and the expression was dramatically induced by exogenous TGF-Beta1 treatment. betaig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known alpha3beta1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in betaig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that alpha3beta1 integrin mediates the adhesion and migration of RPTECs on betaig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that betaig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with alpha3beta1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.

  4. 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.

  5. CBFB and MYH11 in inv(16)(p13q22) of acute myeloid leukemia displaying close spatial proximity in interphase nuclei of human hematopoietic stem cells.

    PubMed

    Weckerle, Allison B; Santra, Madhumita; Ng, Maggie C Y; Koty, Patrick P; Wang, Yuh-Hwa

    2011-09-01

    To gain a better understanding of the mechanism of chromosomal translocations in cancer, we investigated the spatial proximity between CBFB and MYH11 genes involved in inv(16)(p13q22) found in patients with acute myeloid leukemia. Previous studies have demonstrated a role for spatial genome organization in the formation of tumorigenic abnormalities. The nonrandom localization of chromosomes and, more specifically, of genes appears to play a role in the mechanism of chromosomal translocations. Here, two-color fluorescence in situ hybridization and confocal microscopy were used to measure the interphase distance between CBFB and MYH11 in hematopoietic stem cells (HSCs), where inv(16)(p13q22) is believed to occur, leading to leukemia development. The measured distances in HSCs were compared with mesenchymal stem cells, peripheral blood lymphocytes, and fibroblasts, as spatial genome organization is determined to be cell-type specific. Results indicate that CBFB and MYH11 are significantly closer in HSCs compared with all other cell types examined. Furthermore, the CBFB-MYH11 distance is significantly reduced compared with CBFB and a control locus in HSCs, although separation between CBFB and the control is ∼70% of that between CBFB and MYH11 on metaphase chromosomes. HSCs were also treated with fragile site-inducing chemicals because both the genes contain translocation breakpoints within these regions. However, treatment with fragile site-inducing chemicals did not significantly affect the interphase distance. Consistent with previous studies, our results suggest that gene proximity may play a role in the formation of cancer-causing rearrangements, providing insight into the mechanism of chromosomal abnormalities in human tumors.

  6. Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region

    PubMed Central

    Englert, Neal A.; Turesky, Robert J.; Han, Weiguo; Bessette, Erin E.; Spivack, Simon D.; Caggana, Michele; Spink, David C.; Spink, Barbara C.

    2014-01-01

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)n repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)n was n = 4>5≫6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis. PMID:22728919

  7. Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

    PubMed

    Englert, Neal A; Turesky, Robert J; Han, Weiguo; Bessette, Erin E; Spivack, Simon D; Caggana, Michele; Spink, David C; Spink, Barbara C

    2012-09-01

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.

  8. The pro-oxidant gene p66shc increases nicotine exposure-induced lipotoxic oxidative stress in renal proximal tubule cells.

    PubMed

    Arany, Istvan; Hall, Samuel; Reed, Dustin K; Dixit, Mehul

    2016-09-01

    Nicotine (NIC) exposure augments free fatty acid (FFA) deposition and oxidative stress, with a concomitant increase in the expression of the pro-oxidant p66shc. In addition, a decrease in the antioxidant manganese superoxide dismutase (MnSOD) has been observed in the kidneys of mice fed a high‑fat diet. The present study aimed to determine whether the pro‑oxidant p66shc mediates NIC‑dependent increases in renal oxidative stress by augmenting the production of reactive oxygen species (ROS) and suppressing the FFA‑induced antioxidant response in cultured NRK52E renal proximal tubule cells. Briefly, NRK52E renal proximal tubule cells were treated with 200 µM NIC, 100 µM oleic acid (OA), or a combination of NIC and OA. The expression levels of p66shc and MnSOD were modulated according to genetic method