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

Abstract

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 (AT 1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT 1R activation through protein kinase C (PKC), but the exact mechanisms are unknown. We hypothesize that ANG II stimulates CD renin synthesis through AT 1R 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-␣ dominant negative, and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca 2ϩ 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-␣, 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.prorenin; hypertension; protein kinase; calcium; adenylyl cyclase-6; gene expression; M-1 cells IN ANGIOTENSIN II -(ANG II) dependent hypertension, the intrarenal ANG II content is greater than can be explained from the levels found in plasma (30, 31). High intrarenal ANG II levels can be partially explained by enhanced ANG II type 1 receptor (AT 1 R)-mediated uptake of ANG II (10). Augmentation of proximal tubule angiotensinogen (AGT) synthesis and secretion is responsible for increased local intratubular ANG II generation (21). The presence of AGT in the urine indicates that AGT traverses the distal nephron segments where it may then be cleaved to ANG I, if an adequate source of renin is available.In addition to the juxtaglomerular cells (JG), renin expression has been described in the proximal tubules, connecting tubules, and cortical and medullary collecting duct (CD) cells from the mouse, rat, and human kidney (39, 44). In contrast to JG cells, in the principal cells of the CD renin is upregulated by ANG II (36), via an AT 1 R-mediated mechanism (37), which is independent of blood pressure (35). In ANG II-dependent hypertensive rats with marked plasma renin activity (PRA) suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid (26). In this model of experimental hypertension, intraluminal conversion of ANG I to ANG II in the CD is supported by the local presence of angiotens...

Prostaglandin E2 Induces Prorenin-Dependent Activation of (Pro)renin Receptor and Upregulation of Cyclooxygenase-2 in Collecting Duct Cells

“…To further confirm the specificity and identity of western blot bands, we used recombinant proteins to clarify the expected molecular weight. As shown in Figure 1C, recombinant prorenin was observed near 45 kDa, whereas renin was detected near 38 kDa; this agrees with our previous reports 9 . Full-length recombinant PRR was detected at 28 kDa.…”

“…It is likely that the effect of PGE 2 might be also mediated by stimulation of EP1 and EP4 receptors and activation of PKC and cAMP respectively, which finally leads to the upregulation or prorenin and renin protein. More recently we reported that actions of AVP require the intact activity of PKC to enhance prorenin and renin protein expression in CD cells 9 . Indeed, PKC potentiates PKA-mediated signals responsible for AQP-2 expression in the plasma membrane in CD cells 30 .…”

“…It has been shown that M-1 CD cells express EP1, EP3, and EP4 17 . We have previously demonstrated that activation of the AT1 receptor increases prorenin and renin expression via cAMP accumulation, which is dependent on PKC activity 9,23 . In the MD, the activation of EP4 receptor enhances cAMP formation and activates the cAMP-CREB pathway and renin synthesis in JG cells 29 .…”

“…Importantly, M-1 cells are composed of principal and intercalated cells which express COX-2 21 EP1, EP3, EP4 17 prorenin-renin, and PRR constitutively 22 which make this cell line a good in vitro model to establish the role of the PRR-COX-2-PGE2 pathway on renin regulation. The M-1 cells were cultured as previously described 9,23 . As shown in Figure 1A, M-1 cells express AQP-2 and can respond to vasopressin (AVP, 10 nM, 4 h) increasing AQP-2 abundance in plasma membranes.…”

“…After systemic RAS activation, angiotensin II (ANG II) stimulates Ca2+ release 5 and protein kinase C (PKC) through type 1 receptor (AT1R) to inhibit renin synthesis in the JG cells 6 . Renin has also been detected in the CD, and its expression increases after ANG II stimulation 7,8 via a mechanism mediated by AT1R and PKC activation 9 . ANG II also increases the expression of the (pro)renin receptor (PRR), which is abundantly expressed in CD intercalated cells 10 .…”