Dipeptide-induced Cl−secretion in proximal tubule cells

1997 ◽  
Vol 273 (5) ◽  
pp. C1623-C1631 ◽  
Author(s):  
Wenwu Jin ◽  
Ulrich Hopfer
Keyword(s):  
Proximal Tubule ◽  
Spontaneously Hypertensive Rat ◽  
Intact Cell ◽  
Ph Dependence ◽  
Primary Cultures ◽  
Electrical Current ◽  
Proximal Tubule Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Wistar Kyoto

During a survey of dipeptides that might be transported by the renal PEPT2 transporter in proximal tubule cells, we discovered that acidic dipeptides could stimulate transient secretory anion current and conductance increases in intact cell monolayers. The stimulatory effect of acidic dipeptides was observed in several proximal tubule cell lines that have been recently developed by immortalization of early proximal tubule primary cultures from the Wistar-Kyoto and spontaneously hypertensive rat strains and humans, suggesting that this phenomenon is a characteristic of proximal tubule cells. The electrical current induced in intact monolayers by Ala-Asp, a representative of these acidic dipeptides, must represent Cl− secretion rather than Na+ or H+ absorption, because 1) it was Na+ independent, 2) it showed a pH dependence different from that of the PEPT2 cotransporter, and 3) it correlated with an Ala-Asp-induced increase in Cl− conductance of the apical membrane in basolaterally amphotericin B-permeabilized monolayers. The secretory current could be inhibited by stilbene disulfonates, but not diphenylamine-2-carboxylates, suggesting a non-cystic fibrosis transmembrane conductance regulator type of Cl− conductance. The effect of Ala-Asp was dose dependent, with an apparent 50% effective concentration of ∼1 mM. Ala-Asp also produced intracellular acidification, suggesting that acidic dipeptides are also substrates for an H+-peptide cotransporter.

Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat

2000 ◽  
Vol 98 (4) ◽  
pp. 409-418 ◽  
Author(s):  
Leong L. NG ◽  
Sonja JENNINGS ◽  
Joan E. DAVIES ◽  
Paulene A. QUINN
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Western Blotting ◽  
Spontaneously Hypertensive Rat ◽  
Proximal Tubule Cell ◽  
Permissive Temperature ◽  
Proximal Tubule Cells ◽  
Spontaneously Hypertensive ◽  
Hypertensive Rat ◽  
Tubule Cells

Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na+/H+ exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar–Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 °C, but growth is abolished at the restrictive temperature of 39 °C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE-694-sensitive H+ efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0±2.8 and 39.9±5.7 mmol·min-1·l-1 respectively; P < 0.001; n = 6). H+ efflux due to non-Na+-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of 32P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers.

Glucocorticoid upregulates Na-K-ATPase alpha- and beta-mRNA via an indirect mechanism in proximal tubule cell primary cultures

1995 ◽  
Vol 268 (5) ◽  
pp. F862-F867 ◽  
Author(s):  
Y. C. Lee ◽  
H. H. Lin ◽  
M. J. Tang
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Time Course ◽  
Primary Cultures ◽  
Control Level ◽  
Proximal Tubule Cell ◽  
Protein Abundance ◽  
Mrna Levels ◽  
Proximal Tubule Cells ◽  
Tubule Cells

Adrenalectomy leads to the decline in the levels of renal Na-K-adenosinetriphosphatase (Na-K-ATPase) alpha- and beta-subunit protein and mRNA. Both alpha- and beta-mRNA, however, return to the control level within 1 h after corticosterone administration. Whether or not glucocorticoid acts directly on a specific segment of nephron to upregulate Na-K-ATPase has not been determined. Studies were undertaken in an attempt to elucidate this problem. Using primary cultures of renal proximal tubules, we found that 24-h treatment with dexamethasone augmented Na-K-ATPase activity and induced coordinate increase of alpha- and beta-protein and mRNA abundance dependent on the doses in the range of 10(-8) to 10(-6) M. We further demonstrated that 24-h incubation of dexamethasone (10(-7) M) enhanced Na-K-ATPase activity by 58 +/- 14%, alpha- and beta-protein abundance by 70 +/- 18 and 51 +/- 10%, and alpha- and beta-mRNA levels by 87 +/- 12 and 62 +/- 11%, respectively. The time course studies revealed that significant increase of Na-K-ATPase activity and alpha and beta-protein abundance was reached within 4 hr of dexamethasone treatment. Pretreatment of cultured proximal tubule cells with cycloheximide (20 micrograms/ml) completely inhibited dexamethasone-induced increase of Na-K-ATPase alpha- and beta-mRNA. Our results indicate that dexamethasone upregulates Na-K-ATPase in proximal tubule cells via pretranslational mechanisms, which may be mediated by proteins.

Nephrotoxicity Testing In Vitro: The Current Situation

1997 ◽  
Vol 25 (5) ◽  
pp. 497-503
Author(s):  
Jean-Paul Morin ◽  
Marc E. De Broe ◽  
Walter Pfaller ◽  
Gabriele Schmuck
Keyword(s):  
Proximal Tubule ◽  
Culture Media ◽  
Task Force ◽  
Primary Cultures ◽  
Phenotypic Expression ◽  
Transepithelial Transport ◽  
Specific Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

scholarly journals Loss of NHERF-1 expression prevents dopamine-mediated Na-K-ATPase regulation in renal proximal tubule cells from rat models of hypertension: aged F344 rats and spontaneously hypertensive rats

2017 ◽  
Vol 313 (2) ◽  
pp. C197-C206 ◽  
Author(s):  
Michelle T. Barati ◽  
Corey J. Ketchem ◽  
Michael L. Merchant ◽  
Walter B. Kusiak ◽  
Pedro A. Jose ◽  
...  
Keyword(s):  
Animal Models ◽  
Proximal Tubule ◽  
Spontaneously Hypertensive Rats ◽  
Hypertensive Rats ◽  
Proximal Tubule Cells ◽  
Spontaneously Hypertensive ◽  
Sodium Hydrogen Exchanger ◽  
Tubule Cells ◽  
Wistar Kyoto ◽  
F344 Rats

Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria

2009 ◽  
Vol 37 (3) ◽  
pp. 127-132 ◽  
Author(s):  
Karen L. Price ◽  
Sally-Anne Hulton ◽  
William G. van’t Hoff ◽  
John R. Masters ◽  
Gill Rumsby
Keyword(s):  
Proximal Tubule ◽  
Primary Cultures ◽  
Primary Hyperoxaluria ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

2012 ◽  
Vol 303 (2) ◽  
pp. F266-F278 ◽  
Author(s):  
Šárka Lhoták ◽  
Sudesh Sood ◽  
Elise Brimble ◽  
Rachel E. Carlisle ◽  
Stephen M. Colgan ◽  
...  
Keyword(s):  
Er Stress ◽  
Proximal Tubule ◽  
Lipid Accumulation ◽  
Cell Injury ◽  
Apoptotic Cell ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca2+-independent phospholipase A2 (iPLA2β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.

Cyclosporin A and vehicle toxicity in primary cultures of rabbit renal proximal tubule cells

1990 ◽  
Vol 183 (6) ◽  
pp. 2438
Author(s):  
P.P. Sokol ◽  
L.C. Capodagli ◽  
M. Dixon ◽  
P.D. Holohan ◽  
C.R. Ross ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cyclosporin A ◽  
Primary Cultures ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells
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