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 glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner

2015 ◽  
Vol 308 (11) ◽  
pp. F1268-F1275 ◽  
Author(s):  
Yixin Su ◽  
Jianli Bi ◽  
Victor M. Pulgar ◽  
Jorge Figueroa ◽  
Mark Chappell ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Fetal Programming ◽  
Specific Effect ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
Female Sheep ◽  
Male Sheep ◽  
In Cells

We have shown a sex-specific effect of fetal programming on Na+ excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na+ uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and female sheep, we measured Na+ uptake under basal conditions and after stimulation with ANG II. To gain insight into the mechanisms involved, we also measured nitric oxide (NO) levels, ANG II receptor mRNA levels, and expression of Na+/H+ exchanger 3. Basal Na+ uptake increased more in cells from Beta-exposed male sheep than in cells from vehicle-exposed male sheep (400% vs. 300%, P < 0.00001). ANG II-stimulated Na+ uptake was also greater in cells from Beta-exposed males. Beta exposure did not increase Na+ uptake by RPTCs from female sheep. NO production was suppressed more by ANG II in RPTCs from Beta-exposed males than in RPTCs from either vehicle-exposed male or female sheep. Our data suggest that one site of the sex-specific effect of Beta-induced fetal programming in the kidney is the RPTC and that the enhanced Na+ uptake induced by antenatal Beta in male RPTCs may be related to the suppression of NO in these cells.

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.

scholarly journals Porcine myosin-VI: characterization of a new mammalian unconventional myosin.

1994 ◽  
Vol 127 (2) ◽  
pp. 425-440 ◽  
Author(s):  
T Hasson ◽  
M S Mooseker
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Actin Binding ◽  
Proximal Tubule Cell ◽  
Myosin Vi ◽  
Proximal Tubule Cells ◽  
Unconventional Myosin ◽  
Tubule Cells ◽  
The Difference

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC-PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.

Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis

1994 ◽  
Vol 266 (3) ◽  
pp. C669-C675 ◽  
Author(s):  
J. R. Schelling ◽  
S. L. Linas
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Receptor Internalization ◽  
Ang Ii ◽  
Na Transport ◽  
Cellular Mechanisms ◽  
Transport Pathways ◽  
Proximal Tubule Cells ◽  
Receptor Mediated Endocytosis ◽  
Tubule Cells

Angiotensin II (ANG II) receptors are present on apical and basolateral surfaces of proximal tubule cells. To determine the cellular mechanisms of proximal tubule ANG II receptor-mediated Na transport, apical-to-basolateral 22Na flux was measured in cultured proximal tubule cells. Apical ANG II caused increases in 22Na flux (maximum response: 100 nM, 30 min). Basolateral ANG II resulted in 22Na flux that was 23-56% greater than 22Na flux observed with equimolar apical ANG II. Apical ANG II-induced 22Na flux was prevented by preincubation with amiloride, ouabain, and the AT1 receptor antagonist losartan. Because apical ANG II signaling was previously shown to be endocytosis dependent, we questioned whether endocytosis was required for ANG II-stimulated proximal tubule Na transport as well. Apical (but not basolateral) ANG II-dependent 22Na flux was inhibited by phenylarsine oxide, an agent which prevents ANG II receptor internalization. In conclusion, apical and basolateral ANG II caused proximal tubule Na transport. Apical ANG II-dependent Na flux was mediated by AT1 receptors, transcellular transport pathways, and receptor-mediated endocytosis.

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.

scholarly journals Intracellular ANG II induces cytosolic Ca2+ mobilization by stimulating intracellular AT1 receptors in proximal tubule cells

2006 ◽  
Vol 290 (6) ◽  
pp. F1382-F1390 ◽  
Author(s):  
Jia L. Zhuo ◽  
Xiao C. Li ◽  
Jeffrey L. Garvin ◽  
L. Gabriel Navar ◽  
Oscar A. Carretero
Keyword(s):  
Proximal Tubule ◽  
Biological Effects ◽  
Physiological Role ◽  
Ang Ii ◽  
Cellular Mechanisms ◽  
Proximal Tubule Cells ◽  
Intracellular Ca ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
First Time

Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([Ca2+]i) and, if so, elucidated the cellular mechanisms involved. Changes in [Ca2+]i responses were studied by fluorescence imaging using the Ca2+ indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (AT1) receptors were blocked by losartan (10 μM). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [Ca2+]i that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [Ca2+]i that peaked at 30 s (basal: 2.2 ± 0.3 vs. ANG II: 14.9 ± 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular Ca2+ with EGTA (2 mM) did not alter microinjected ANG II-induced [Ca2+]i responses (Ca2+ free + ANG II: 12.3 ± 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular Ca2+ stores or with U-73122 to inhibit phospholipase C (1 μM each) markedly attenuated microinjected ANG II-induced [Ca2+]i responses. Combined microinjection of ANG II and losartan abolished [Ca2+]i responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [Ca2+]i by stimulating intracellular AT1 receptors and releases Ca2+ from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function.

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