Abstract 601: A Biased AT1 Receptor Agonist Stimulates COX2 Expression In Intercalated Cells

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Daian Chen ◽  
Min Tan ◽  
Johannes Stegbauer ◽  
Marcela Herrera ◽  
Gentzon Hall ◽  
...  
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Collecting Duct ◽  
Intercalated Cells ◽  
Ang Ii ◽  
P38 Inhibitor ◽  
Beneficial Effects ◽  
Enhanced Expression ◽  
Cox2 Expression ◽  
Stimulation Of

We recently discovered a pathway by which angiotensin (Ang) II, acting via angiotensin type 1 receptors (AT1R), triggers expression of cyclo-oxygenase (COX)-2 in intercalated cells (IC) of the collecting duct (CD), generating vasodilator prostaglandins, and attenuating the development of hypertension. We found that AT1R-dependent activation of COX2 could be recapitulated in isolated medullary collecting ducts (IMCDs) and in the C11 clone of the Madin-Darby canine kidney (MDCK) cell line permanently transfected with the AT1AR. This cell line has many features resembling ICs including expression of V-ATPase B1. The robust increase in COX2 expression in C11 cells after AngII was blunted after p38 inhibition (5.1*±0.8 vs. 1.4*±0.4; p=0.014), while PKC and MEK1 inhibition had negligible effects. Ang II activation of AT1R triggers both canonical G-protein and β-arrestin pathways and each pathway may have distinct physiological consequences. In IMCDs, we found that stimulation of COX2 by Ang II requires β-arrestin2, as this response was abrogated in IMCDs from mice lacking β-arrestin2. We next tested the capacity for an AT1R β-arrestin-biased ligand, TRV120023, to activate COX2 in C11 cells. These biased ligands block G-protein signalling like conventional AT1R blockers (ARBs), while stimulating β-arrestin-dependent pathways. TRV120023 caused significant stimulation of COX2 at concentrations of 0.1μM-10μM, indicating that activation of AT1R-linked β-arrestin pathways alone is sufficient to stimulate COX2. Exposure of the IC cell line by TRV also triggered phosphorylation of ERK1/2. The p38 inhibitor SB203580 attenuated TRV-dependent stimulation of ERK1/2 and reduced COX2 expression. In summary, activation of β-arrestin with TRV120023 is sufficient to induce COX2 expression via a pathway involving ERK1/2 and p38 MAPK. This suggests that β-arrestin-biased AT1R agonists might have added beneficial effects compared to conventional ARBs by blocking detrimental G-protein-dependent pathways while preserving beneficial β-arrestin-dependent pathways, such as enhanced expression of COX2.

scholarly journals Collecting duct prorenin receptor knockout reduces renal function, increases sodium excretion, and mitigates renal responses in ANG II-induced hypertensive mice

2017 ◽  
Vol 313 (6) ◽  
pp. F1243-F1253 ◽  
Author(s):  
Minolfa C. Prieto ◽  
Virginia Reverte ◽  
Mykola Mamenko ◽  
Marta Kuczeriszka ◽  
Luciana C. Veiras ◽  
...  
Keyword(s):  
Renal Function ◽  
Collecting Duct ◽  
Ang Ii ◽  
Open Probability ◽  
Sodium Transporters ◽  
Prorenin Receptor ◽  
Active Channels ◽  
The Impact ◽  
Renal Responses ◽  
Stimulation Of

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg−1·min−1), the increases in systolic BP and diastolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CDPRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.

scholarly journals Characterization of anion exchangers in an inner medullary collecting duct cell line.

1998 ◽  
Vol 9 (5) ◽  
pp. 746-754
Author(s):  
G Obrador ◽  
H Yuan ◽  
T M Shih ◽  
Y H Wang ◽  
M A Shia ◽  
...  
Keyword(s):  
Cell Line ◽  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Rat Stomach ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot revealed two immunoreactive bands (approximately 100 and 180 kD in size) in membranes from cultured IMCD cells, rat renal medulla, and freshly isolated IMCD cells. The mobility of the lower molecular weight band was similar to that of AE1 in red blood cell ghosts and kidney homogenate and therefore probably represents AE1. The mobility of the 180-kD band is similar to that for rat stomach and kidney AE2 and therefore probably represents AE2. Selective biotinylation of the apical or basolateral membrane proteins in cultured IMCD cells revealed that both AE1 and AE2 are polarized to the basolateral membrane. Northern blot analysis documented the expression of mRNA for AE1 and AE2 but not AE3. Furthermore, the cDNA sequence of AE1 and AE2 expressed by these cells was found to be virtually identical to that reported for kidney AE1 and rat stomach AE2. It is concluded that this cultured line of rat IMCD cells expresses two members of the anion exchanger gene family, AE1 and AE2, and both of these exchangers probably mediate the electroneutral Cl--dependent HCO3-transport observed in this cell line.

scholarly journals ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load

2019 ◽  
Vol 317 (2) ◽  
pp. F435-F443
Author(s):  
Lydie Cheval ◽  
Naziha Bakouh ◽  
Christine Walter ◽  
Dignê Tembely ◽  
Luciana Morla ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Urinary Excretion ◽  
Secretory Pathway ◽  
Collecting Duct ◽  
Type A ◽  
Intercalated Cells ◽  
Membrane Expression ◽  
Acid Load ◽  
Anp Receptors ◽  
Stimulation Of

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl− cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

scholarly journals Aldosterone stimulates vacuolar H+-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

2011 ◽  
Vol 301 (5) ◽  
pp. C1251-C1261 ◽  
Author(s):  
Christian Winter ◽  
Nicole B. Kampik ◽  
Luca Vedovelli ◽  
Florina Rothenberger ◽  
Teodor G. Păunescu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Atpase Activity ◽  
Intracellular Signaling ◽  
Collecting Duct ◽  
Intercalated Cells ◽  
Nongenomic Action ◽  
Kinase C ◽  
Urinary Acidification ◽  
Stimulation Of

Urinary acidification in the collecting duct is mediated by the activity of H+-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H+-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H+-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific Gαq inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca2+ with BAPTA, and blockade of protein kinase C prevented the stimulation of H+-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H+-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H+-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H+-ATPase activity. Thus, the nongenomic modulation of H+-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a Gαq protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H+-ATPase activity and contribute to final urinary acidification.

scholarly journals Mechanisms through which ammonia regulates cortical collecting duct net proton secretion

2002 ◽  
Vol 282 (6) ◽  
pp. F1120-F1128 ◽  
Author(s):  
Amy E. Frank ◽  
Charles S. Wingo ◽  
Peter M. Andrews ◽  
Shana Ageloff ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Tetanus Toxin ◽  
Collecting Duct ◽  
Vesicle Fusion ◽  
Snare Proteins ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Bicarbonate Reabsorption ◽  
Subapical Region ◽  
Stimulation Of

Ammonia stimulates cortical collecting duct (CCD) net bicarbonate reabsorption by activating an apical H+-K+-ATPase through mechanisms that are independent of ammonia's known effects on intracellular pH and active sodium transport. The present studies examined whether this stimulation occurs through soluble N-ethylmaleimide-sensitive fusion attachment receptor (SNARE) protein-mediated vesicle fusion. Rabbit CCD segments were studied using in vitro microperfusion, and transepithelial bicarbonate transport was measured using microcalorimetry. Ammonia's stimulation of bicarbonate reabsorption was blocked by either chelating intracellular calcium with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester or by inhibiting microtubule polymerization with colchicine compared with parallel studies performed in the absence of these inhibitors. An inactive structural analog of colchicine, lumicolchicine, did not alter ammonia's stimulation of bicarbonate reabsorption. Tetanus toxin, a zinc endopeptidase specific for vesicle-associated SNARE (v-SNARE) proteins, prevented ammonia from stimulating net bicarbonate reabsorption. Consistent with the functional evidence for v-SNARE involvement, antibodies directed against a conserved region of isoforms 1–3 of the tetanus toxin-sensitive, vesicle-associated membrane protein (VAMP) members of v-SNARE proteins labeled the apical and subapical region of collecting duct intercalated cells. Similarly, antibodies to NSF protein, a protein involved in activation of SNARE proteins for subsequent vesicle fusion, localized to the apical and subapical region of collecting duct intercalated cells. These results indicate that ammonia stimulates CCD bicarbonate reabsorption through an intracellular calcium-dependent, microtubule-dependent, and v-SNARE-dependent mechanism that appears to involve insertion of cytoplasmic vesicles into the apical plasma membrane of CCD intercalated cells.

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

2015 ◽  
Vol 309 (10) ◽  
pp. F880-F888 ◽  
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.

scholarly journals NS-398 Upregulates Constitutive Cyclooxygenase-2 Expression in the M-1 Cortical Collecting Duct Cell Line

1999 ◽  
Vol 10 (11) ◽  
pp. 2261-2271
Author(s):  
SHAWN FERGUSON ◽  
RICHARD L. HÉBERT ◽  
ODETTE LANEUVILLE
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Cell Line ◽  
Blot Analysis ◽  
Collecting Duct ◽  
Prostaglandin E ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Cox 2 ◽  
Cox 1

Abstract. The cortical collecting duct (CCD) is a major site of intrarenal prostaglandin E2 (PGE2) synthesis. This study examines the expression and regulation of the prostaglandin synthesizing enzymes cyclooxygenase-1 (COX-1) and -2 in the CCD. By indirect immunofluorescence using isoform-specific antibodies, COX-1 and -2 immunoreactivity was localized to all cell types of the murine M-1 CCD cell line. By immunohistochemistry, both COX-1 and COX-2 were localized to intercalated cells of the CCD on paraffin-embedded mouse kidney sections. When COX enzyme activity was measured in the M-1 cells, both indomethacin (COX-1 and -2 inhibitor) and the specific COX-2 inhibitor NS-398 effectively blocked PGE2 synthesis. These results demonstrate that COX-2 is the major contributor to the pool of PGE2 synthesized by the CCD. By Western blot analysis, COX-2 expression was significantly upregulated by incubation with either indomethacin or NS-398. These drugs did not affect COX-1 protein expression. Evaluation of COX-2 mRNA expression by Northern blot analysis after NS-398 treatment demonstrated that the COX-2 protein upregulation occurred independently of any change in COX-2 mRNA expression. These studies have for the first time localized COX-2 to the CCD and provided evidence that the intercalated cells of the CCD express both COX-1 and COX-2. The results also demonstrate that constitutively expressed COX-2 is the major COX isoform contributing to PGE2 synthesis by the M-1 CCD cell line. Inhibition of COX-2 activity in the M-1 cell line results in an upregulation of COX-2 protein expression.

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

Hypertension ◽  
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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