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

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.

Download Full-text

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

AJP Renal Physiology ◽

10.1152/ajprenal.00047.2015 ◽

2015 ◽

Vol 308 (11) ◽

pp. F1268-F1275 ◽

Cited By ~ 13

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.

Download Full-text

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

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.3.c669 ◽

1994 ◽

Vol 266 (3) ◽

pp. C669-C675 ◽

Cited By ~ 41

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.

Download Full-text

Abstract MP11: Decreased Expression Of Ace2 Is Associated Urine Derived Human Renal Proximal Tubule Cells From Inverse Salt Sensitivity Participants Cultured In Low Salt Conditions

Hypertension ◽

10.1161/hyp.78.suppl_1.mp11 ◽

2021 ◽

Vol 78 (Suppl_1) ◽

Author(s):

John J Gildea ◽

Peng Xu ◽

Katie Schiermeyer ◽

Wei Yue ◽

Robin A Felder

Keyword(s):

Proximal Tubule ◽

Salt Sensitivity ◽

Renal Proximal Tubule ◽

Ang Ii ◽

Proximal Tubule Cells ◽

Low Sodium Diet ◽

Low Sodium ◽

Low Salt ◽

Tubule Cells ◽

Renal Proximal Tubule Cells

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. Inverse salt sensitivity (ISS) is the paradoxical increase in blood pressure of individuals to a low sodium diet. Our group previously reported decreased expression of dopamine type 2 receptor (D 2 R), increased expression Aminopeptidase N, and increased Ang II dependent sodium transport in human urine derived renal proximal tubule cells isolated from ISS participants. In an attempt to understand the increased Ang II sensitivity demonstrated in ISS cells, we examined angiotensin converting enzyme 2 (ACE2), a membrane associated enzyme involved in the metabolism of Ang II. Urine derived renal proximal tubule cells grown and immortalized from ISS participants were compared to cells from salt resistant (SR) participants cultured in iso-osmotic media with low salt (LS, 90 mM NaCl) normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl). Cells were incubated in LS, NS, and HS media with and without losartan (LOS,1 μM) overnight (18 hours) and ACE2 expression levels determined by in-cell western blot. A monoclonal antibody specific to an extracellular epitope of ACE2 was used as the primary antibody and an Alexa-647 anti-mouse secondary antibody. ACE2 expression was only reduced in ISS cells in LS condition (28.7±2.1 % reduction, ISS LS vs SR LS, N=4 per group, p<0.05). Addition of losartan completely blocked the decrease in ACE2 expression in low salt conditions in ISS in urine derived human renal proximal tubule cells. No other changes in ACE2 expression were found between ISS and SR in either NS or HS conditions and with or without losartan. In conclusion, a decreased expression of ACE2 in ISS urine proximal tubule cells could explain the previously reported increased sensitivity of ISS cells to Ang II by increasing the half-life of Ang II under low salt conditions.

Download Full-text

Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-κB signal pathways in renal proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00408.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. F534-F541 ◽

Cited By ~ 15

Author(s):

Yu Jin Lee ◽

Ho Jae Han

Keyword(s):

Dna Synthesis ◽

Proximal Tubule ◽

Renal Proximal Tubule ◽

Signal Molecules ◽

Signal Pathways ◽

Proximal Tubule Cells ◽

Intracellular Ca ◽

Tubule Cells ◽

Pkc Inhibitors ◽

Renal Proximal Tubule Cells

It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [3H]thymidine incorporation in a dose (≥3 mg/ml)- and time (≥3 h)-dependent manner, intracellular Ca2+ concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca2+ chelator), 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca2+ chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor-κB (NF-κB) and inhibitor of NF-κB (IκB) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca2+, PKC, EGFR, p44/42 MAPK, or NF-κB signal pathways blocked the BSA-induced incorporation of [3H]thymidine. Consequently, the inhibition of Ca2+, PKC, EGFR, p44/42 MAPKs, or NF-κB blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21WAF/Cip1 and p27Kip1 expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca2+/PKC as well as the EGFR-dependent p44/42 MAPK and NF-κB signal pathways in PTCs.

Download Full-text

The mechanism of angiotensin II binding downregulation by high glucose in primary renal proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00080.2001 ◽

2002 ◽

Vol 282 (2) ◽

pp. F228-F237 ◽

Cited By ~ 22

Author(s):

Soo Hyun Park ◽

Ho Jae Han

Keyword(s):

Proximal Tubule ◽

High Glucose ◽

Renal Proximal Tubule ◽

Channel Blockers ◽

Ang Ii ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Pkc Inhibitors ◽

Renal Proximal Tubule Cells ◽

Tgf Β1

The renin-angiotensin system plays an important role in the development of diabetic nephropathy. However, the mechanism of ANG II receptor regulation in the renal proximal tubule in the diabetic condition has not been elucidated. Thus we investigated the signal pathways involved in high-glucose-induced downregulation of ANG II binding in primary cultured renal proximal tubule cells. Twenty-five millimolar glucose, but not mannitol andl-glucose, induced downregulation of the AT1receptor (AT1R) because of a significant decline in maximal binding with no significant change in the affinity constant. Twenty-five millimolar glucose also decreased AT1R mRNA and protein levels. The 25 mM glucose-induced increase in the formation of lipid peroxides was prevented by antioxidants, protein kinase C (PKC) inhibitors, or L-type calcium channel blockers. These agents also blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. In addition, 25 mM glucose increased transforming growth factor (TGF)-β1 secretion, and anti-TGF-β antibody significantly blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. Furthermore, the 25 mM glucose-induced increase in TGF-β1 secretion was inhibited by PKC inhibitors, L-type calcium channel blockers, or antioxidants. In conclusion, high glucose may induce downregulation of 125I-ANG II binding via a PKC-oxidative stress-TGF-β signal cascade in primary cultured rabbit renal proximal tubule cells.

Download Full-text

Abstract 362: G Protein-Linked Receptor Kinase 4 is Palmitoylated and Acetylated and Regulates Subcellular Localization in Human Renal Proximal Tubule Cells

Hypertension ◽

10.1161/hyp.60.suppl_1.a362 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

John J Gildea ◽

Christine L Hou ◽

Huang Zhenyue ◽

Robin A Felder

Keyword(s):

Plasma Membrane ◽

Proximal Tubule ◽

G Protein ◽

Renal Proximal Tubule ◽

Receptor Kinase ◽

Cytoplasmic Fraction ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Renal Proximal Tubule Cells ◽

First Time

G protein-linked receptor kinase 4 (GRK4) is a kinase expressed in human renal proximal tubule cells (RPTC). Three single nucleotide polymorphisms in this gene are associated with hyper-phosphorylation and inactivation of the dopamine-1 receptor (D1R), and are associated with both hypertension and salt sensitivity. GRK4 is a prime therapeutic target for hypertension so we sought to discover potential post-translational modifications that could affect both localization and function. We identified two unique posttranslational modifications of GRK4 that we hypothesize are associated with protein localization. GRK4 is found at the plasma membrane (PM), in the cytoplasm and in the nucleus. We determined that GRK4 is both acetylated and palmitoylated. Using cell fractionation and immunoprecipitation, we determined that GRK4 was found in the nucleus, but acetylated GRK4 was only found in the cytoplasmic fraction using six different human RPTC cell lines (>10 fold n=6, p<0.01). We previously reported that PM GRK4 internalized following fenoldopam (FEN, dopamine agonist) stimulation, but the internalization only occurred in normally coupled human renal proximal tubule cells (nRPTC), not uncoupled cells (uRPTC). GRK4 is palmitoylated at the C-Terminal end of the protein (cystein 563, GGCPalmLTMVP). Using the palmitic acid tracer, 17-octadecynoic acid (17-ODYA) and Huisgen’s cycloaddition reaction (Click Reaction Chemistry) and biotin-azide for detection, we determined that GRK4 is depalmitoylated in a FEN (1μM, 30 min) and cAMP (forskolin, 10 μM, 30 min.) dependent manner. The amount of palmitoylated GRK4 was decreased following treatment with FEN (28.4%±3.6, n=3, p<0.01 vs VEH) or forskolin (35.3%±6.7, n=3, p<0.05 vs VEH) in nRPTC. No significant change in GRK4 palmitoylation was observed in uRPTC. The depalmitoylation of GRK4 correlates with its change in plasma membrane localization in both nRPTC and uRPTC. In conclusion we have determined for the first time that GRK4 is acetylated and the acetylated GRK4 is found only in the cytoplasmic fraction. In addition we have determined for the first time that GRK4 is depalmitoylated in nRPTC (not in uRPTC) when stimulated with FEN and this effect was mimicked by increasing intracellular cAMP using forskolin.

Download Full-text

Signaling cascade of ANG II-induced inhibition of α-MG uptake in renal proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00217.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. F634-F642 ◽

Cited By ~ 16

Author(s):

Ho Jae Han ◽

Soo Hyun Park ◽

Yun Jung Lee

Keyword(s):

Proximal Tubule ◽

Renal Proximal Tubule ◽

Receptor Blocker ◽

Dependent Manner ◽

Ang Ii ◽

Proximal Tubule Cells ◽

Tk Inhibitors ◽

Tubule Cells ◽

Renal Proximal Tubule Cells ◽

Mapk Inhibitor

ANG II and Na+-glucose cotransporter have been reported to be associated with the onset of diverse renal diseases. However, the effect of ANG II on Na+-glucose cotransporter activity was not elucidated. The effects of ANG II on α-methyl-d-[14C]glucopyranoside (α-MG) uptake and its related signal pathways were examined in the primary cultured rabbit renal proximal tubule cells (PTCs). ANG II (>2 h; >10-9 M) inhibited α-MG uptake in a time- and concentration-dependent manner and decreased the protein level of Na+-glucose cotransporters, the expression of which was abrogated by both actinomycin D and cycloheximide exposure. ANG II-induced inhibition of α-MG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD-123319, an ANG II type 2 receptor blocker. ANG II-induced inhibition of α-MG uptake was blocked by genistein, herbimycin A [tyrosine kinase (TK) inhibitors], mepacrine, and AACOCF3 (phospholipase A2 inhibitors), suggesting the role of TK phosphorylation and arachidonic acid (AA). Indeed, ANG II increased AA release, which was blocked by losartan or TK inhibitors. The effects of ANG II on AA release and α-MG uptake also were abolished by staurosporine and bisindolylmaleimide I (protein kinase C inhibitors) or PD-98059 (p44/42 MAPK inhibitor), but not SB-203580 (p38 MAPK inhibitor), respectively. Indeed, ANG II increased p44/42 MAPK activity. ANG II-induced activation of p44/42 MAPK was blocked by staurosporine. In conclusion, ANG II inhibited α-MG uptake via PKC-MAPK-cPLA2 signal cascade through the AT1 receptor in the PTCs.

Download Full-text