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...

During the early phase of ANG II-dependent hypertension, tubular PGE 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. PGE 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 PGE 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 M PGE 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 PGE-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 PGE 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 PGE that stimulates renin in the CD, resulting in increased tubular ANG II formation and further stimulation of renin.

Recent studies suggested that activation of the PRR upregulates profibrotic markers through reactive oxygen species (ROS) formation; however, the exact mechanisms have not been investigated in CD cells. We hypothesized that activation of the PRR increases the expression of profibrotic markers through MAPK-dependent ROS formation in CD cells. Mouse renal CD cell line (M-1) was treated with recombinant prorenin plus ROS or MAPK inhibitors and PRR-shRNA to evaluate their effect on the expression of profibrotic markers. PRR immunostaining revealed plasma membrane and intracellular localization. Recombinant prorenin increases ROS formation (6.0 ± 0.5 vs. 3.9 ± 0.1 nM DCF/μg total protein, P<0.05) and expression of profibrotic markers CTGF (149 ± 12%, P<0.05), α-SMA (160 ± 20%, P<0.05), and PAI-I (153 ± 13%, P<0.05) at 10-8 M. Recombinant prorenin induced phospho ERK 1/2 (p44 and p42) at 10-8 and 10-6 M after 20 min. Prorenin-dependent ROS formation and augmentation of profibrotic factors were blunted by ROS scavengers (trolox, p-coumaric acid, ascorbic acid), the MEK inhibitor PD98059 and PRR transfections with PRR-shRNA. No effects were observed in the presence of antioxidants alone. Prorenin-induced upregulation of collagen I and fibronectin was blunted by ROS scavenging or MEK inhibition independently. PRR-shRNA partially prevented this induction. After 24 h prorenin treatment M-1 cells undergo to epithelial mesenchymal transition phenotype, however MEK inhibitor PD98059 and PRR knockdown prevented this effect. These results suggest that PRR might have a significant role in tubular damage during conditions of high prorenin-renin secretion in the CD.

Background Prostaglandin E2 (PGE2) regulates renin expression in renal juxtaglomerular cells. PGE2 acts through E-prostanoid (EP) receptors in the renal collecting duct (CD) to regulate sodium and water balance. CD cells express EP1 and EP4, which are linked to protein kinase C (PKC) and protein kinase A (PKA) downstream pathways, respectively. Previous studies showed that the presence of renin in the CD, and that PKC, and PKA pathways activate its expression. The (pro)renin receptor (PRR) is also expressed in CD cells and its activation enhances cyclooxygenase-2 (COX-2) through extracellular signal–regulated kinase (ERK). We hypothesized that PGE2 stimulates prorenin and renin synthesis leading to subsequent activation of PRR and upregulation of COX-2. Methods We used a mouse M-1 CD cell line that expresses EP1, EP3, and EP4 but not EP2. Results PGE2 (10−6 M) treatment increased prorenin and renin protein levels at 4 and 8 h. No differences were found at 12 h post PGE2 treatment. Phospho-ERK was significantly augmented after 12 h. COX-2 expression was decreased after 4 h of PGE2 treatment, but increased after 12 h. Interestingly, the full-length form of the PRR was upregulated only at 12 h. PGE2 mediated phospho-ERK and COX-2 upregulation was suppressed by PRR silencing. Conclusions Our results suggest that PGE2 induces biphasic regulation of COX-2 through renin-dependent PRR activation via EP1 and EP4 receptors. PRR-mediated increases in COX-2 expression may enhance PGE2 synthesis in CD cells serving as a buffer mechanism in conditions of activated renin angiotensin system.

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