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

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.

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

Clinical Science ◽

10.1042/cs19990096 ◽

2000 ◽

Vol 98 (4) ◽

pp. 409 ◽

Cited By ~ 1

Author(s):

Leong L. NG ◽

Sonja JENNINGS ◽

Joan E. DAVIES ◽

Paulene A. QUINN

Keyword(s):

Proximal Tubule ◽

Cell Lines ◽

Spontaneously Hypertensive Rat ◽

Temperature Sensitive ◽

Proximal Tubule Cell ◽

Tubule Cell ◽

Spontaneously Hypertensive ◽

Hypertensive Rat

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Dipeptide-induced Cl−secretion in proximal tubule cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.5.c1623 ◽

1997 ◽

Vol 273 (5) ◽

pp. C1623-C1631 ◽

Cited By ~ 9

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.

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Increased expression of the SA gene in the kidney of the spontaneously hypertensive rat is localized to the proximal tubule

Journal of Hypertension ◽

10.1097/00004872-199412000-00005 ◽

1994 ◽

Vol 12 (12) ◽

pp. 1347???1352 ◽

Cited By ~ 16

Author(s):

Hasmukh R. Patel ◽

Amrik S. Thiara ◽

Kevin P. West ◽

David Lodwick ◽

Nilesh J. Samani

Keyword(s):

Proximal Tubule ◽

Spontaneously Hypertensive Rat ◽

Spontaneously Hypertensive ◽

Hypertensive Rat

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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

AJP Cell Physiology ◽

10.1152/ajpcell.00219.2016 ◽

2017 ◽

Vol 313 (2) ◽

pp. C197-C206 ◽

Cited By ~ 5

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.

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Aberrant ETB receptor regulation of AT1 receptors in immortalized renal proximal tubule cells of spontaneously hypertensive rats

Kidney International ◽

10.1111/j.1523-1755.2005.00440.x ◽

2005 ◽

Vol 68 (2) ◽

pp. 623-631 ◽

Cited By ~ 19

Author(s):

Chunyu Zeng ◽

Zheng Wang ◽

Laureano D. Asico ◽

Ulrich Hopfer ◽

Gilbert M. Eisner ◽

...

Keyword(s):

Proximal Tubule ◽

Spontaneously Hypertensive Rats ◽

Receptor Regulation ◽

Hypertensive Rats ◽

At1 Receptors ◽

Proximal Tubule Cells ◽

Spontaneously Hypertensive ◽

Etb Receptor ◽

Tubule Cells ◽

Renal Proximal Tubule Cells

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

Journal of Cell Science ◽

10.1242/jcs.104.3.695 ◽

1993 ◽

Vol 104 (3) ◽

pp. 695-704 ◽

Cited By ~ 4

Author(s):

N. Cartier ◽

R. Lacave ◽

V. Vallet ◽

J. Hagege ◽

R. Hellio ◽

...

Keyword(s):

Transgenic Mice ◽

Proximal Tubule ◽

Pyruvate Kinase ◽

Cell Lines ◽

Cultured Cells ◽

Simian Virus ◽

T Antigen ◽

Regulatory Sequence ◽

Proximal Tubule Cell ◽

Proximal Tubule Cells

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.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00482.2011 ◽

2012 ◽

Vol 303 (2) ◽

pp. F266-F278 ◽

Cited By ~ 45

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.

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

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.5.f897 ◽

1998 ◽

Vol 274 (5) ◽

pp. F897-F905 ◽

Cited By ~ 18

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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Proximal Tubule–Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis

Journal of the American Society of Nephrology ◽

10.1681/asn.2019030321 ◽

2019 ◽

Vol 30 (12) ◽

pp. 2370-2383 ◽

Cited By ~ 3

Author(s):

Eirini Kefaloyianni ◽

Manikanda Raja Keerthi Raja ◽

Julian Schumacher ◽

Muthu Lakshmi Muthu ◽

Vaishali Krishnadoss ◽

...

Keyword(s):

Proximal Tubule ◽

Reperfusion Injury ◽

Knockout Mice ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Proximal Tubule Cell ◽

Tubule Cell ◽

Proximal Tubule Cells ◽

Kidney Fibrosis ◽

Tubule Cells

BackgroundSustained activation of EGF receptor (EGFR) in proximal tubule cells is a hallmark of progressive kidney fibrosis after AKI and in CKD. However, the molecular mechanisms and particular EGFR ligands involved are unknown.MethodsWe studied EGFR activation in proximal tubule cells and primary tubular cells isolated from injured kidneys in vitro. To determine in vivo the role of amphiregulin, a low-affinity EGFR ligand that is highly upregulated with injury, we used ischemia-reperfusion injury or unilateral ureteral obstruction in mice with proximal tubule cell–specific knockout of amphiregulin. We also injected soluble amphiregulin into knockout mice with proximal tubule cell–specific deletion of amphiregulin’s releasing enzyme, the transmembrane cell-surface metalloprotease, a disintegrin and metalloprotease-17 (ADAM17), and into ADAM17 hypomorphic mice.ResultsYes-associated protein 1 (YAP1)–dependent upregulation of amphiregulin transcript and protein amplifies amphiregulin signaling in a positive feedback loop. YAP1 also integrates signals of other moderately injury-upregulated, low-affinity EGFR ligands (epiregulin, epigen, TGFα), which also require soluble amphiregulin and YAP1 to induce sustained EGFR activation in proximal tubule cells in vitro. In vivo, soluble amphiregulin injection sufficed to reverse protection from fibrosis after ischemia-reperfusion injury in ADAM17 hypomorphic mice; injected soluble amphiregulin also reversed the corresponding protective proximal tubule cell phenotype in injured proximal tubule cell–specific ADAM17 knockout mice. Moreover, the finding that proximal tubule cell–specific amphiregulin knockout mice were protected from fibrosis after ischemia-reperfusion injury or unilateral ureteral obstruction demonstrates that amphiregulin was necessary for the development of fibrosis.ConclusionsOur results identify amphiregulin as a key player in injury-induced kidney fibrosis and suggest therapeutic or diagnostic applications of soluble amphiregulin in kidney disease.

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