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.

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.

Low K+ increases Na(+)-K(+)-ATPase alpha- and beta-subunit mRNA and protein abundance in cultured renal proximal tubule cells

1992 ◽  
Vol 263 (2) ◽  
pp. C436-C442 ◽  
Author(s):  
M. J. Tang ◽  
A. A. McDonough
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Mrna Levels ◽  
Proximal Tubule Cells ◽  
Subunit Mrna ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
Low K

Studies from this laboratory demonstrate that LLC-PK1/Cl4 cells, a cultured renal cell line, respond to incubation in low-K+ medium by coordinately increasing abundance of both alpha- and beta-subunits of Na(+)-K(+)-ATPase but increase only beta- and not alpha-mRNA levels (Lescale-Matys et al. J. Biol. Chem. 265: 17935-17940, 1990) and that alpha-abundance is likely increased as a result of increased efficiency of alpha-mRNA translation (L. Lescale-Matys and A. A. McDonough. J. Cell Biol. 111: 311A, 1990). The aim of this report was to determine if nontransformed kidney cells would respond to low K+ in a similar manner. We incubated primary cultures of rat proximal tubule cells in low K+ (0.25 mM) for up to 24 h to address this aim. Na(+)-K(+)-ATPase activity, measured enzymatically, and abundance of alpha- and beta-subunits, measured by immunoblot, were increased significantly and coordinately by 8 h of low K+, and, by 24 h of low K+, these parameters were increased to 2.17 +/- 0.34 (activity), 2.03 +/- 0.21 (alpha), and 2.39 +/- 0.48 (beta)-fold over control. Pretranslationally, beta-mRNA, measured by Northern blot analysis, increased to 1.76 +/- 0.35 after 3 h of low K+ and to 3.4 +/- 0.75-fold over control after 24 h of low K+. The increase in alpha-mRNA was smaller and delayed compared with the beta-mRNA response, but it was sufficient to account for the observed increase in alpha-protein and Na(+)-K(+)-ATPase activity at steady state: alpha-mRNA increased to 1.27 +/- 0.09 after 6 h and to 1.91 +/- 0.41-fold over control after 24 h in low K+. We conclude that the accumulation of sodium pumps in cultured renal proximal tubule cells, unlike LLC-PK1 cells, can be accounted for by increases in both alpha- and beta-subunit mRNA levels.

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.

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

Purinergic-mediated inhibition of Na+-K+-ATPase in proximal tubule cells: elevated cytosolic Ca2+ is not required

1997 ◽  
Vol 272 (4) ◽  
pp. C1169-C1177 ◽  
Author(s):  
W. Jin ◽  
U. Hopfer
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Time Course ◽  
Short Circuit ◽  
Primary Cultures ◽  
Extracellular Atp ◽  
Apical Plasma Membrane ◽  
Similar Time ◽  
Apical Side ◽  
Wistar Kyoto

The involvement of cytosolic Ca2+ concentration ([Ca2+]i) as messenger for the regulation of Na+-K+-ATPase activity was investigated in a renal cell line recently developed by immortalization of early proximal tubule primary cultures from the Wistar-Kyoto rat strain. Na+-K+-ATPase was measured as short-circuit current (Isc) in intact monolayers after permeabilization of the apical plasma membrane with amphotericin B. With symmetrical solutions, Isc quantitatively reflects Na+-K+-ATPase activity as judged by ouabain inhibition and dependence on Na+ and K+. Extracellular ATP (50% effective concentration = 0.32 mM) on the apical side produced acute inhibition of Na+-K+-ATPase-generated Isc of up to 50%. The inhibition peaked within 1 min and lasted approximately 5 min. The potency order was ATP > ADP >> beta,gamma-methyleneadenosine 5'-triphosphate = UTP, consistent with a P2y receptor. Extracellular ATP also stimulated a transient increase in [Ca2+]i. This increase had a similar time course as the inhibition of ATPase and reached a peak change of approximately 120 nM. However, the elevation of [Ca2+]i is not required in the purinergic inhibition of the Na+-K+-ATPase, since, first, increases in [Ca2+]i produced with a Ca2+ ionophore (ionomycin) failed to mimic the purinergic inhibition and, second, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, which abolished the [Ca2+]i elevation, failed to block the purinergic inhibition.

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

Angiotensin (AT1A) receptor-mediated increases in transcellular sodium transport in proximal tubule cells

1998 ◽  
Vol 274 (5) ◽  
pp. F897-F905 ◽  
Author(s):  
Thomas J. Thekkumkara ◽  
Rochelle Cookson ◽  
Stuart L. Linas
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Epithelial Tissue ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Binding Studies ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
And Function ◽  
In Cells

Angiotensin II (ANG II), acting through angiotensin type 1A receptors (AT1A), is important in regulating proximal tubule salt and water balance. AT1A are present on apical (AP) and basolateral (BL) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1A function in epithelial tissue is not well understood, because specific binding of ANG II to intact PTEC has not been found and because a number of isoforms of AT receptors are present in vivo. To overcome this problem, we developed a cell line from opossum kidney (OK) proximal tubule cells, which stably express AT1A( K d = 5.27 nM, Bmax = 6.02 pmol/mg protein). Characterization of nontransfected OK cells revealed no evidence of AT1A mRNA (reverse transcriptase-polymerase chain reaction analysis) or protein (125I-labeled ANG II binding studies) expression. In cells stably expressing AT1A, ANG II binding was saturable, reversible, and regulated by G proteins. Transfected receptors were coupled to increases in intracellular calcium and inhibition of cAMP. To determine the polarity of AT1A expression and function in proximal tubules, transfected cells were grown to confluence on membrane inserts under conditions that allowed selective access to AP or BL surfaces. AT1A were expressed on both AP ( K d = 8.7 nM, Bmax = 3.33 pmol/mg protein) and BL ( K d = 10.1 nM, Bmax = 5.50 pmol/mg protein) surfaces. Both AP and BL AT1Areceptors underwent agonist-dependent endocytosis (AP receptor: t 1/2 = 7.9 min, Ymax = 78.5%; BL receptor: t 1/2 = 2.1 min, Ymax = 86.3%). In cells transfected with AT1A, ANG II caused time- and concentration-dependent increases in transepithelial22Na transport (2-fold over control at 20 min) by increasing Na/H exchange. In conclusion, we have established a stable proximal tubule cell line that expresses AT1A on both AP and BL surfaces, undergoes agonist-dependent receptor endocytosis, and is functional, as evidenced by inhibition of cAMP and increases in cytosolic calcium mobilization and transepithelial sodium movement. This cell line should prove useful for understanding the molecular and biochemical regulation of AT1A expression and function in PTEC.

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