scholarly journals Therapeutic concentrations of calcineurin inhibitors do not deregulate glutathione redox balance in human renal proximal tubule cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250996
Author(s):  
Yasaman Ramazani ◽  
Noël Knops ◽  
Sante Princiero Berlingerio ◽  
Oyindamola Christiana Adebayo ◽  
Celien Lismont ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cell Model ◽  
Calcineurin Inhibitors ◽  
Redox Balance ◽  
Solid Organ ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Glutathione Redox

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.

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.

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

2019 ◽  
Vol 30 (12) ◽  
pp. 2370-2383 ◽  
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.

Abstract P169: Antenatal Betamethasone Attenuates the Angiotensin-(1-7)/Nitric Oxide Axis in Adult Male but not Female Renal Proximal Tubule Cells

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Yixin Su ◽  
Victor M Pulgar ◽  
Jianli Bi ◽  
Mark Chappell ◽  
James C Rose
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
Adult Male ◽  
Fetal Programming ◽  
Cell Model ◽  
Specific Effect ◽  
Proximal Tubules ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Na Uptake

Our studies have revealed a sex-specific effect of fetal programming on sodium (Na+) excretion in adult sheep whereby the males exhibit reduced Na+ excretion and an attenuated natriuretic response to Ang-(1-7) as compared to the females. We hypothesize that the renal proximal tubules are a key target for the early programming effects of glucocorticoids exposure to regulate Na+ handling in the adult males. Therefore, we isolated and cultured cortical proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to betamethasone (Beta) or vehicle. Na+ uptake and nitric oxide (NO) were assessed with Sodium Green and DAF fluorescence prior to and following a low dose of Ang-(1-7) (1x10-11 M) in isolated RPTC from sheep at ~1.5 years of age. Data are expressed as % of basal uptake or area under the curve (AUC) for Na+ or % of control for NO. Male Beta RPTC exhibit greater Na+ uptake than male vehicle cells (427±32%, n=13, vs. 315±28%, n=14, p<0.05; however, Beta had no effect on Na+ uptake in the female cells (242±18%, n=9, vs. 250±15%, n=10, p>0.05). Ang-(1-7) inhibited Na+ uptake in RPTC from vehicle male (255±40%) and from both vehicle (191±14%) and Beta (209±11%) females (Figure 1B), but failed to attenuate Na+ uptake in Beta male cells (Figure 1A). Beta exposure also abolished NO stimulation by Ang-(1-7) in male but not female RPTC (Figure 1C). We conclude that an Ang-(1-7)-NO-dependent pathway contributes to the sex-dependent consequences of programming on Na+ regulation in the proximal tubules of the kidney. Moreover, the RPTC retain both the sex and Beta-induced phenotype of the adult and may reflect an appropriate cell model of fetal programming.

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.

scholarly journals Antenatal betamethasone attenuates the angiotensin-(1–7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells

2017 ◽  
Vol 312 (6) ◽  
pp. F1056-F1062 ◽  
Author(s):  
Yixin Su ◽  
Jianli Bi ◽  
Victor M. Pulgar ◽  
Mark C. Chappell ◽  
James C. Rose
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
Cell Model ◽  
Primary Cultures ◽  
Specific Effect ◽  
Renal Tubules ◽  
Proximal Tubule Cells ◽  
Mas Receptor ◽  
Tubule Cells

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 angiotensin-(1–7) [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 percentage of basal uptake or area under the curve for Na+ or percentage 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.

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.

Altered renal proximal tubule cell ion homeostasis during diabetes

1993 ◽  
Vol 51 ◽  
pp. 12-13
Author(s):  
Craig C. Freudenrich ◽  
Daniel Hockett ◽  
Benjamin Kirk ◽  
Kurt Gilliland ◽  
Brian Scherer ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Ion Homeostasis ◽  
Diabetic Rats ◽  
Proximal Tubule Cell ◽  
Sprague Dawley ◽  
Tubule Cell ◽  
Electrolyte Excretion ◽  
Proximal Tubule Cells ◽  
Sprague Dawley Rats ◽  
Renal Tubule Cell

A major characteristic of human and experimental diabetes mellitus is enhanced urinary excretion of electrolytes (e.g. Na, K, Mg, Ca). The increased urinary electrolyte excretion can lead to low serum electrolyte concentrations (e.g. hypomagnesemia, hyponatremia, hypocalcemia, hypokalemia), although this issue remains controversial. Ultimately, elevated urinary electrolyte excretion can result in mineral deficiencies which have important consequences such as retinopathies, convulsions, osteoporosis, arrhythmias, and sudden death ischemia. The increased urinary electrolyte excretion could be due to osmotic diuresis and/or altered renal tubule cell ion homeostasis; however, few studies have addressed whether diabetes alters specific tubule cell ion homeostasis. In this study, we used electron probe x-ray microanalysis (EPXMA) to obtain quantitative microchemical images of proximal tubule cells from diabetic rats so that we could define how subcellular ion homeostasis is altered during diabetes.Adult male Sprague-Dawley rats were housed in individual metabolic cages and urine output was measured daily; peri-orbital plasma samples were collected every 3 days. Urine and plasma ion concentrations (Na, K, Ca, Mg) were assayed by atomic absorption spectroscopy, blood glucose was measured with a commercial glucometer, and urine glucose and ketones were detected with commercial strips.

A1 adenosine receptor knockout mice are protected against acute radiocontrast nephropathy in vivo

2006 ◽  
Vol 290 (6) ◽  
pp. F1367-F1375 ◽  
Author(s):  
H. Thomas Lee ◽  
Michael Jan ◽  
Soo Chan Bae ◽  
Jin Deok Joo ◽  
Farida R. Goubaeva ◽  
...  
Keyword(s):  
Renal Failure ◽  
Acute Renal Failure ◽  
Proximal Tubule ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Proximal Tubule Cells ◽  
Proximal Tubular ◽  
Tubule Cells

The role of renal A1 adenosine receptors (A1AR) in the pathogenesis of radiocontrast nephropathy is controversial. We aimed to further elucidate the role of A1AR in the pathogenesis of radiocontrast nephropathy and determine whether renal proximal tubule A1AR contribute to the radiocontrast nephropathy. To induce radiocontrast nephropathy, A1AR wild-type (WT) or knockout (KO) mice were injected with a nonionic radiocontrast (iohexol, 1.5–3 g iodine/kg). Some A1WT mice were pretreated with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a selective A1AR antagonist) before iohexol injection. A1AR contribute to the pathogenesis of radiocontrast nephropathy in vivo as the A1WT mice developed significantly worse acute renal failure, more renal cortex vacuolization, and had lower survival 24 h after iohexol treatment compared with the A1KO mice. DPCPX pretreatment also protected the A1WT mice against radiocontrast-induced acute renal failure. No differences in renal cortical apoptosis or inflammation were observed between A1WT and A1KO mice. To determine whether the proximal tubular A1AR mediate the direct renal cytotoxicity of radiocontrast, we treated proximal tubules in culture with iohexol with or without 2-chloro- N6-cyclopentyladenosine (a selective A1AR agonist) or DPCPX pretreatment. We also subjected cultured proximal tubule cells overexpressing A1AR or lacking A1AR to radiocontrast injury. Iohexol caused a direct dose-dependent reduction in proximal tubule cell viability as well as proliferation. Neither the A1AR agonist nor the antagonist treatment affected proximal tubule viability or proliferation. Moreover, overexpression or lack of A1AR failed to impact the iohexol toxicity on proximal tubule cells. Therefore, we conclude that radiocontrast causes acute renal failure via mechanisms dependent on A1AR; however, renal proximal tubule A1AR do not contribute to the direct tubular toxicity of radiocontrast.

Angiotensin II AT2 receptors inhibit growth responses in proximal tubule cells

2001 ◽  
Vol 281 (2) ◽  
pp. F300-F308 ◽  
Author(s):  
Joseph Zimpelmann ◽  
Kevin D. Burns
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Receptor Activation ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Phosphotyrosine Phosphatase ◽  
Proximal Tubule Cells ◽  
Transfected Cells ◽  
Mapk Activity ◽  
Tubule Cells

Angiotensin II (ANG II) subtype 2 (AT2) receptors are expressed in the adult kidney, but the effects of AT2 receptor activation are unclear. The proximal tubule cell line LLC-PK1 was transfected with a plasmid containing cDNA for the rat AT2 receptor. In transfected cells, specific binding of 125I-labeled ANG II was detected (dissociation constant = 0.81 nM), with inhibition by the AT2 antagonist PD-123319, and no effect of the AT1 antagonist losartan. ANG II (10−7 M) significantly inhibited mitogen-activated protein kinase (MAPK) activity in transfected cells, associated with decreased phosphorylation of the extracellular signal-related kinases ERK1 and ERK2. ANG II stimulated phosphotyrosine phosphatase activity within 5 min, an effect blocked by PD-123319 and the phosphatase inhibitor vanadate. In transfected cells, ANG II inhibited epidermal growth factor-stimulated [3H]thymidine incorporation, an effect reversed by vanadate. In contrast, vanadate did not block ANG II-stimulated apoptosis of transfected cells. In summary, AT2 receptors in proximal tubule cells inhibit MAPK activity and stimulate phosphotyrosine phosphatase. AT2receptor-induced inhibition of mitogenesis is mediated by phosphatase activation, whereas effects on apoptosis are insensitive to phosphatase inhibition. The data suggest that AT2 receptors inhibit cell growth via distinct signaling pathways in the proximal tubule.

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