Vasopressin/V2 receptor stimulates renin synthesis in the collecting duct

Renin is synthesized in the principal cells of the collecting duct (CD), and its production is increased via cAMP in angiotensin (ANG) II-dependent hypertension, despite suppression of juxtaglomerular (JG) renin. Vasopressin, one of the effector hormones of the renin-angiotensin system (RAS) via the type 2-receptor (V2R), activates the cAMP/PKA/cAMP response element-binding protein (CREB) pathway and aquaporin-2 expression in principal cells of the CD. Accordingly, we hypothesized that activation of V2R increases renin synthesis via PKA/CREB, independently of ANG II type 1 (AT1) receptor activation in CD cells. Desmopressin (DDAVP; 10−6 M), a selective V2R agonist, increased renin mRNA (∼3-fold), prorenin (∼1.5-fold), and renin (∼2-fold) in cell lysates and cell culture media in the M-1 CD cell line. Cotreatment with DDAVP+H89 (PKA inhibitor) or CREB short hairpin (sh) RNA prevented this response. H89 also blunted DDAVP-induced CREB phosphorylation and nuclear localization. In 48-h water-deprived (WD) mice, prorenin-renin protein levels were increased in the renal inner medulla (∼1.4- and 1.8-fold). In WD mice treated with an ACE inhibitor plus AT1 receptor blockade, renin mRNA and prorenin protein levels were still higher than controls, while renin protein content was not changed. In M-1 cells, ANG II or DDAVP increased prorenin-renin protein levels; however, there were no further increases by combined treatment. These results indicate that in the CD the activation of the V2R stimulates renin synthesis via the PKA/CREB pathway independently of RAS, suggesting a critical role for vasopressin in the regulation of renin in the CD.

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Abstract 475: Vasopressin Type 2 Receptor (v2r) Activation Increases Renin Expression In Renal Collecting Duct Cells.

Hypertension ◽

10.1161/hyp.64.suppl_1.475 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Alexis A Gonzalez ◽

Flavia Cifuentes ◽

Cristobal Ibaceta ◽

Leonardo Zamora ◽

Minolfa C Prieto

Keyword(s):

Cell Line ◽

Collecting Duct ◽

Intracellular Camp ◽

Mrna Levels ◽

Camp Accumulation ◽

Ang Ii ◽

Protein Levels ◽

Renin Synthesis ◽

Renin Mrna ◽

Creb Pathway

Renin synthesis in juxtaglomerular cells (JGC) is mediated by intracellular cAMP accumulation and activation of PKA/CREB pathway. Recent evidence demonstrated that renin is expressed in renal collecting duct (CD) cells. Furthermore, it has been shown that CD renin is augmented in animal models of hypertension and kidney disease, despite the suppressed expression observed in JGC. Vasopressin activates V2R stimulating cAMP/PKA/CREB pathway and aquaporin-2 expression in apical plasma membrane of principal cells of the CD. We hypothesized that activation of V2R increases renin expression in mouse CD cell line M-1 through cAMP/PKA/CREB pathway. Desmopressin (ddAVP, 10-6 mol/L, 6 hrs), a specific V2R agonist, increased renin mRNA, prorenin protein levels in cell lysates and prorenin secretion to the culture media. To determine if this effect was related to PKA pathway, we used the PKA inhibitor H89. Co-treatment with ddAVP + H89 prevented the ddAVP-mediated increase in renin expression. To further confirm if the stimulation of renin synthesis in M-1 cells was mediated by cAMP accumulation, we raised intracellular cAMP levels using forskolin (10-7 mol/L). Forskolin treatment significantly increased renin mRNA and prorenin protein levels as compared to controls. Additionally, ddAVP increased phosphorylated CREB, while H89 blunted this effect. Finally, shRNA against CREB prevented the ddAVP-induced renin synthesis. We additionally confirmed the stimulatory effects of Ang II + ddAVP on renin synthesis in mpkccdc14 cell line, a mouse cortical line composed only by CD principal cells. Tolvaptan (V2R antagonist) reduced the additive effect of Ang II + ddAVP on renin expression. To achieve in vivo relevance we further measured renin mRNA levels in renal inner medullary tissues from mice subjected to 16 hours of water deprivation and controls. Mice water-deprived showed significantly greater renin mRNA levels in the renal inner medulla than controls. These results indicate that the activation of V2R stimulates renin mRNA synthesis and prorenin secretion in M-1 cells via cAMP/PKA/CREB pathway.

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PGE2 upregulates renin through E-prostanoid receptor 1 via PKC/cAMP/CREB pathway in M-1 cells

AJP Renal Physiology ◽

10.1152/ajprenal.00194.2017 ◽

2017 ◽

Vol 313 (4) ◽

pp. F1038-F1049 ◽

Cited By ~ 8

Author(s):

Alexis A. Gonzalez ◽

Nicolas Salinas-Parra ◽

Dan Leach ◽

L. Gabriel Navar ◽

Minolfa C. Prieto

Keyword(s):

Collecting Duct ◽

Fold Increase ◽

Dominant Negative ◽

Ang Ii ◽

Creb Phosphorylation ◽

Ep Receptors ◽

Protein Levels ◽

Renin Synthesis ◽

Ep1 Receptor ◽

Creb Pathway

During the early phase of ANG II-dependent hypertension, tubular PGE2 is increased. Renin synthesis and secretion in the collecting duct (CD) are upregulated by ANG II, contributing to further intratubular ANG II formation. However, what happens first and whether the triggering mechanism is independent of tubular ANG II remain unknown. PGE2 stimulates renin synthesis in juxtaglomerular cells via E-prostanoid (EP) receptors through the cAMP/cAMP-responsive element-binding (CREB) pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2 in CD cells. The M-1 CD cell line expressed EP1, EP3, and EP4 but not EP2. Dose-response experiments, in the presence of ANG II type 1 receptor blockade with candesartan, demonstrated that 10−6 M PGE2 maximally increases renin mRNA (approximately 4-fold) and prorenin/renin protein levels (approximately 2-fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10-fold increase). To evaluate further the signaling pathway involved, we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative. Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation, and augmentation of renin. Knockdown of the EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2 increases CD renin expression through the EP1 receptor via the PKC/cAMP/CREB pathway. Therefore, we conclude that during the early stages of ANG II-dependent hypertension, there is augmentation of PGE2 that stimulates renin in the CD, resulting in increased tubular ANG II formation and further stimulation of renin.

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Collecting duct-specific knockout of renin attenuates angiotensin II-induced hypertension

AJP Renal Physiology ◽

10.1152/ajprenal.00367.2014 ◽

2014 ◽

Vol 307 (8) ◽

pp. F931-F938 ◽

Cited By ~ 35

Author(s):

Nirupama Ramkumar ◽

Deborah Stuart ◽

Sara Rees ◽

Alfred Van Hoek ◽

Curt D. Sigmund ◽

...

Keyword(s):

Angiotensin Ii ◽

Collecting Duct ◽

Plasma Renin ◽

Ang Ii ◽

Water Excretion ◽

Protein Levels ◽

Renin Gene ◽

Induced Hypertension ◽

Renin Mrna ◽

Exon 1

The physiological and pathophysiological significance of collecting duct (CD)-derived renin, particularly as it relates to blood pressure (BP) regulation, is unknown. To address this question, we generated CD-specific renin knockout (KO) mice and examined BP and renal salt and water excretion. Mice containing loxP-flanked exon 1 of the renin gene were crossed with mice transgenic for aquaporin-2-Cre recombinase to achieve CD-specific renin KO. Compared with controls, CD renin KO mice had 70% lower medullary renin mRNA and 90% lower renin mRNA in microdissected cortical CD. Urinary renin levels were significantly lower in KO mice (45% of control levels) while plasma renin concentration was significantly higher in KO mice (63% higher than controls) during normal-Na intake. While no observable differences were noted in BP between the two groups with varying Na intake, infusion of angiotensin II at 400 ng·kg−1·min−1 resulted in an attenuated hypertensive response in the KO mice (mean arterial pressure 111 ± 4 mmHg in KO vs. 128 ± 3 mmHg in controls). Urinary renin excretion and epithelial Na+ channel (ENaC) remained significantly lower in the KO mice following ANG II infusion compared with controls. Furthermore, membrane-associated ENaC protein levels were significantly lower in KO mice following ANG II infusion. These findings suggest that CD renin modulates BP in ANG II-infused hypertension and these effects are associated with changes in ENaC expression.

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Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype

AJP Renal Physiology ◽

10.1152/ajprenal.00222.2014 ◽

2015 ◽

Vol 308 (6) ◽

pp. F627-F638 ◽

Cited By ~ 10

Author(s):

Merle L. Gilbert ◽

Linghai Yang ◽

Thomas Su ◽

G. Stanley McKnight

Keyword(s):

Diabetes Insipidus ◽

Apical Membrane ◽

Collecting Duct ◽

Critical Role ◽

Physiological Role ◽

Receptor Activation ◽

Dominant Negative ◽

Regulatory Subunit ◽

Water Excretion ◽

Protein Levels

PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 ( Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

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Abstract 401: Prostaglandin E2 Stimulates Renin Synthesis in Mouse Collecting Duct M-1 Cells via Ep1 Receptor Through Pkc/camp/creb Pathway

Hypertension ◽

10.1161/hyp.60.suppl_1.a401 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Alexis A Gonzalez ◽

Dan Leach ◽

L G Navar ◽

Minolfa C Prieto

Keyword(s):

Prostaglandin E2 ◽

Collecting Duct ◽

Receptor Activation ◽

Fold Change ◽

Dominant Negative ◽

Calphostin C ◽

Renin Synthesis ◽

Renin Mrna ◽

Ep1 Receptor ◽

Ep Receptor

Prostaglandin E2 (PGE2) plays a major role in regulating renin expression and release by the renal juxtaglomerular (JG) cells. Recently it has been demonstrated that PGE2-dependent upregulation of renin in JG cells is mediated by activation of E prostaoind receptor type 4 (EP4) via cAMP accumulation. Renin is also produced by the principal cells of the collecting ducts (CD) and is upregulated during angiotensin II-dependent hypertension. However, the effects of PGE2 on CD renin remain unknown. Four types of receptors have been described in rat and mouse CD, EP1, EP3 and EP4. Here, we tested the hypothesis that renin is upregulated by PGE2 via activation of EP receptors in mouse CD M-1 cells. By immunostaining we confirmed the presence of EP1, EP3 and EP4 receptors, while EP2 was not detected. A dose response treatment with PGE2 showed increased levels of renin mRNA and protein with a maximum response at 1 μmol/L (mRNA: 19.3 ± 3.0; P<0.05; protein: 3.01 ± 0.08 fold change; P<0.05). To assess which EP receptor is involved in the renin upregulation we used specific EP receptor antagonists: ONO-8711 (EP1; 10 nmol/L), L-798106 (EP3; 10 μmol/L) and AH 23848 (EP4; 10 μmol/L). EP1 antagonist suppressed the PGE2-mediated upregulation of collecting duct renin mRNA and protein (mRNA: 1.0 ± 0.2; protein: 0.98 ± 0.13 fold change; P=NS), while EP4 antagonist only partially decreased it (mRNA: 11.2 ± 2.8; P<0.05; protein: 2.81 ± 0.07 fold change; P<0.05). EP3 antagonist exacerbated the PGE2 mediated-upregulation of renin (mRNA: 50.3 ± 6.0; P<0.05; protein: 3.56 ± 0.08; fold change; P<0.05). Because EP1 is a Gq linked receptor that activates PKC, we further assessed the effects of PKC inhibition using calphostin C and a PKCα dominant negative (DN) on renin expression. Calphostin C and PKCα-DN blunted the PGE2-induced renin upregulation. Importantly, the increases in cAMP levels and phosphorylation of the cAMP response element-binding transcription factor (CREB) mediated by PGE2 were also prevented by both treatments. The results indicate that in mouse CD cells, EP1 receptor activation upregulates renin synthesis via PKC/cAMP/CREB, suggesting that the presence of PGE2 in renal medullary tissues may contribute to the stimulation of collecting duct renin.

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PKC-α-dependent augmentation of cAMP and CREB phosphorylation mediates the angiotensin II stimulation of renin in the collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00155.2015 ◽

2015 ◽

Vol 309 (10) ◽

pp. F880-F888 ◽

Cited By ~ 17

Author(s):

Alexis A. Gonzalez ◽

Liu Liu ◽

Lucienne S. Lara ◽

Camille R. T. Bourgeois ◽

Cristobal Ibaceta-Gonzalez ◽

...

Keyword(s):

Angiotensin Ii ◽

Intracellular Signaling ◽

Collecting Duct ◽

Dominant Negative ◽

Ang Ii ◽

Creb Phosphorylation ◽

Calphostin C ◽

Renin Mrna ◽

Stimulation Of ◽

Creb Pathway

In contrast to the negative feedback of angiotensin II (ANG II) on juxtaglomerular renin, ANG II stimulates renin in the principal cells of the collecting duct (CD) in rats and mice via ANG II type 1 (AT1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT1R activation through protein kinase C (PKC), but the exact mechanisms are unknown. We hypothesize that ANG II stimulates CD renin synthesis through AT1R via PKC and the subsequent activation of cAMP/PKA/CREB pathway. In M-1 cells, ANG II increased cAMP, renin mRNA (3.5-fold), prorenin, and renin proteins, as well as renin activity in culture media (2-fold). These effects were prevented by PKC inhibition with calphostin C, PKC-α dominant negative, and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca2+ depletion impaired ANG II-mediated CREB phosphorylation and upregulation of renin. Adenylate cyclase 6 (AC) siRNA remarkably attenuated the ANG II-dependent upregulation of renin mRNA. Physiological activation of AC with vasopressin increased renin expression in M-1 cells. The results suggest that the ANG II-dependent upregulation of renin in the CD depends on PKC-α, which allows the augmentation of cAMP production and activation of PKA/CREB pathway via AC6. This study defines the intracellular signaling pathway involved in the ANG II-mediated stimulation of renin in the CD. This is a novel mechanism responsible for the regulation of local renin-angiotensin system in the distal nephron.

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Angiotensin II increases fibronectin and collagen I through the β-catenin-dependent signaling in mouse collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00429.2014 ◽

2015 ◽

Vol 308 (4) ◽

pp. F358-F365 ◽

Cited By ~ 31

Author(s):

Catherina A. Cuevas ◽

Alexis A. Gonzalez ◽

Nibaldo C. Inestrosa ◽

Carlos P. Vio ◽

Minolfa C. Prieto

Keyword(s):

Angiotensin Ii ◽

Target Genes ◽

Collecting Duct ◽

Receptor Activation ◽

Collagen I ◽

Ang Ii ◽

Protein Levels ◽

Duct Cells ◽

Collecting Duct Cells ◽

Gsk 3Β

The contribution of angiotensin II (ANG II) to renal and tubular fibrosis has been widely reported. Recent studies have shown that collecting duct cells can undergo mesenchymal transition suggesting that collecting duct cells are involved in interstitial fibrosis. The Wnt/β-catenin signaling pathway plays an essential role in development, organogenesis, and tissue homeostasis; however, the dysregulation of this pathway has been linked to fibrosis. In this study, we investigated whether AT1receptor activation induces the expression of fibronectin and collagen I via the β-catenin pathway in mouse collecting duct cell line M-1. ANG II (10−7M) treatment in M-1 cells increased mRNA, protein levels of fibronectin and collagen I, the β-catenin target genes (cyclin D1 and c-myc), and the myofibroblast phenotype. These effects were prevented by candesartan, an AT1receptor blocker. Inhibition of the β-catenin degradation with pyrvinium pamoate (pyr; 10−9M) prevented the ANG II-induced expression of fibronectin, collagen I, and β-catenin target genes. ANG II treatment promoted the accumulation of β-catenin protein in a time-dependent manner. Because phosphorylation of glycogen synthase kinase-3β (GSK-3β) inhibits β-catenin degradation, we further evaluated the effects of ANG II and ANG II plus pyr on p-ser9-GSK-3β levels. ANG II-dependent upregulation of β-catenin protein levels was correlated with GSK-3β phosphorylation. These effects were prevented by pyr. Our data indicate that in M-1 collecting duct cells, the β-catenin pathway mediates the stimulation of fibronectin and collagen I in response to AT1receptor activation.

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Abstract 214: (pro) Renin Receptor Stimulates the Expression of Fibrotic Genes in Mouse Collecting Ducts Cells via Wnt/β-catenin Signaling, Independently of Angiotensin II

Hypertension ◽

10.1161/hyp.60.suppl_1.a214 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Catherina A Cuevas ◽

Alexis A Gonzalez ◽

Nivaldo C Inestrosa ◽

Carlos P Vio ◽

Minolfa C Prieto

Keyword(s):

Angiotensin Ii ◽

Wnt Signaling ◽

Cyclin D1 ◽

Target Genes ◽

Collecting Duct ◽

Fold Change ◽

Collagen I ◽

Ang Ii ◽

Protein Levels ◽

Prorenin Receptor

The prorenin receptor (PRR) is upregulated in the kidney by high angiotensin II (Ang II) states such as those that occur with AngII-dependent hypertension and low salt diet. The PRR is an accessory protein of the vacuolar H-ATPase, which facilitates Wnt/β-catenin signaling. The Wnt/β-catenin pathway is involved in fibrosis processes. In the present study, we aimed to determine whether the stimulation of PRR in mouse collecting duct M-1 cells induces fibrotic genes independently of Ang II, and if this effect is mediated by activation of Wnt/β-catenin. Both Ang II (10 -7 M) and human recombinant prorenin (hRPr; 2,5 x 10 -8 M) treatments (8 and 16 hours) increased mRNA and protein levels of fibronectin and collagen I (1.5±0.08 and 1.5 ± 0.1 fold change, respectibely; p<0.05); however, the effects of hRPr were elicited earlier. Likewise, Ang II and hRPr stimulated the Wnt target genes, cyclin D1 and c-myc (cyclin D1: 2±0.2 for both; c-myc: 1.4 ± 0.03 and 1.2± 0.002 fold change for Ang II and hRPr, respectively; p<0.001). Ang II type 1 receptor (AT1R) blockade with candesartan (10 -7 M) completely prevented the Ang II-dependent stimulation but not the effects of hRPr on Wnt signaling genes. Upregulation of fibronectin and collagen I genes by Ang II or hRP at 16 h was prevented by Wnt signaling inhibition with Pyrvinium Pamoate (10 -7 M). The data indicate that in M-1 cells, activation of AT1R and PRR stimulate the synthesis of fibrotic genes via Wnt signaling by independent mechanisms.

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Enhancement of renin and prorenin receptor in collecting duct of Cyp1a1-Ren2 rats may contribute to development and progression of malignant hypertension

AJP Renal Physiology ◽

10.1152/ajprenal.00433.2010 ◽

2011 ◽

Vol 300 (2) ◽

pp. F581-F588 ◽

Cited By ~ 29

Author(s):

Minolfa C. Prieto ◽

Dustyn E. Williams ◽

Liu Liu ◽

Kimberly L. Kavanagh ◽

John J. Mullins ◽

...

Keyword(s):

Gene Expression ◽

Collecting Duct ◽

Normal Diet ◽

Malignant Hypertension ◽

Soluble Form ◽

Transgenic Rat ◽

Ang Ii ◽

Transgenic Rats ◽

Protein Levels ◽

Prorenin Receptor

To determine whether in the transgenic rat model [TGR(Cyp1a1Ren2)] with inducible ANG II-dependent malignant hypertension changes in the activation of intrarenal renin-angiotensin system may contribute to the pathogenesis of hypertension, we examined the gene expression of angiotensinogen (AGT) in renal cortical tissues and renin and prorenin receptor [(P)RR] in the collecting duct (CD) of the kidneys from Cyp1a1Ren2 rats ( n = 6) fed a normal diet containing 0.3% indole-3-carbinol (I3C) for 10 days and noninduced rats maintained on a normal diet (0.6% NaCl diet; n = 6). Rats induced with I3C developed malignant hypertension and exhibited alterations in the expression of renin and (P)RR expressed by the CD cells. In the renal medullary tissues of the Cyp1a1Ren2 transgenic rats with malignant hypertension, renin protein levels in CD cells were associated with maintained renin content and lack of suppression of the endogenous Ren1c gene expression. Furthermore, these tissues exhibited increased levels of (P)RR transcript, as well as of the protein levels of the soluble form of this receptor, the s(P)RR. Intriguingly, although previous findings demonstrated that urinary AGT excretion is augmented in Cyp1a1Ren2 transgenic rats with malignant hypertension, in the present study we did not find changes in the gene expression of AGT in renal cortical tissues of these rats. The data suggest that upregulation of renin and the s(P)RR in the CD, especially in the renal medullary tissues of Cyp1a1Ren2 transgenic rats with malignant hypertension, along with the previously demonstrated increased availability of AGT in the urine of these rats, may constitute a leading mechanism to explain elevated formation of kidney ANG II levels in this model of ANG II-dependent hypertension.

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Increased AQP2 targeting in primary cultured IMCD cells in response to angiotensin II through AT1 receptor

AJP Renal Physiology ◽

10.1152/ajprenal.00090.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. F340-F350 ◽

Cited By ~ 78

Author(s):

Yu-Jung Lee ◽

In-Kyung Song ◽

Kyung-Jin Jang ◽

Jakob Nielsen ◽

Jørgen Frøkiær ◽

...

Keyword(s):

Plasma Membrane ◽

Angiotensin Ii ◽

Collecting Duct ◽

Receptor Activation ◽

Ang Ii ◽

Sprague Dawley ◽

Water Reabsorption ◽

Sprague Dawley Rats ◽

Medullary Collecting Duct ◽

Camp Levels

Vasopressin and angiotensin II (ANG II) play a major role in renal water and Na+ reabsorption. We previously demonstrated that ANG II AT1 receptor blockade decreases dDAVP-induced water reabsorption and AQP2 levels in rats, suggesting cross talk between these two peptide hormones ( Am J Physiol Renal Physiol 288: F673–F684, 2005). To directly address this issue, primary cultured inner medullary collecting duct (IMCD) cells from male Sprague-Dawley rats were treated for 15 min with 1) vehicle, 2) ANG II, 3) ANG II + the AT1 receptor blocker candesartan, 4) dDAVP, 5) ANG II + dDAVP, or 6) ANG II + dDAVP + candesartan. Immunofluorescence microscopy revealed that 10−8 M ANG II or 10−11 M dDAVP ( protocol 1) was associated with increased AQP2 labeling of the plasma membrane and decreased cytoplasmic labeling, respectively. cAMP levels increased significantly in response to 10−8 M ANG II and were potentiated by cotreatment with 10−11 M dDAVP. Consistent with this finding, immunoblotting revealed that this cotreatment significantly increased expression of phosphorylated AQP2. ANG II-induced AQP2 targeting was blocked by 10−5 M candesartan. In protocol 2, treatment with a lower concentration of dDAVP (10−12 M) or ANG II (10−9 M) did not change subcellular AQP2 distribution, whereas 10−12 M dDAVP + 10−9 M ANG II enhanced AQP2 targeting. This effect was inhibited by cotreatment with 10−5 M candesartan. ANG II-induced cAMP accumulation and AQP2 targeting were inhibited by inhibition of PKC activity. In conclusion, ANG II plays a role in the regulation of AQP2 targeting to the plasma membrane in IMCD cells through AT1 receptor activation and potentiates the effect of dDAVP on AQP2 plasma membrane targeting.

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