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Increasing evidence shows that antigen-presenting cells (APCs) are involved in the development of inflammation associated to hypertension. However, the potential role of APCs in the modulation of renal sodium transport has not been addressed. We hypothesized that APCs participate in renal sodium transport and, thus, development of high blood pressure in response to angiotensin II plus a high-salt diet. Using transgenic mice that allow the ablation of CD11c high APCs, we studied renal sodium transport, the intrarenal renin–angiotensin system components, blood pressure, and cardiac/renal tissue damage in response to angiotensin II plus a high-salt diet. Strikingly, we found that APCs are required for the development of hypertension and that the ablation/restitution of APCs produces rapid changes in the blood pressure in mice with angiotensin II plus a high-salt diet. Moreover, APCs were necessary for the induction of intrarenal renin–angiotensin system components and affected the modulation of natriuresis and tubular sodium transporters. Consistent with the prevention of hypertension, the ablation of APCs also prevented cardiac hypertrophy and the induction of several indicators of renal and cardiac damage. Thus, our findings indicate a prominent role of APCs as modulators of blood pressure by mechanisms including renal sodium handling, with kinetics that suggest the involvement of tubular cell functions in addition to the modulation of inflammation and adaptive immune response.

The binding of prorenin to the (pro)renin receptor (PRR) stimulates MAPK/ERK1/2, cyclooxygenase-2 (COX-2), NOX-4-dependent production of reactive oxygen species (ROS), transforming growth factor-β (TGF-β), pro-fibrotic factors connecting tissue growth factor (CTGF), and plasminogen activator inhibitor (PAI-I) in collecting duct (CD) cells. However, the specific role of COX-2 and the intracellular signaling pathways involved is not clear. Here, we tested the hypothesis that PRR activation increases profibrotic factors through activation of COX-2-mediated PGE 2 / E prostanoid receptor 4 (EP4), NOX-4/ROS, and Smad pathway. Activation of PRR in M-1cells treated with recombinant prorenin increased ROS production and protein levels of CTGF, PAI-I and TGF-β. Inhibition of MAPK, NOX-4 and COX-2 prevented these effects. Inhibition of MAKP, COX-2 and EP4 also prevented the upregulation of NOX-4. Because TGF-β activates Smad pathway, we further evaluated phosphorylation of Smad 2/3. Either COX-2 inhibition or EP4 antagonism significantly prevented the phosphorylation of Smad 2/3. Importantly, mice chronically infused with recombinant prorenin (100 ng/ml/h) showed increases in CTGF, PAI-I, TGF- β, fibronectin and collagen I expression levels in isolated CD as well as high expression of alpha smooth muscle actin (α-SMA) and interleukin 1 beta mRNA (IL 1β) in renal tissues, which was ameliorated by concomitant COX-2 inhibition. These results indicate that the induction of TGF-β, CTGF, PAI-I and ROS occurs through PRR-dependent activation of MAPK and NOX-4 via a mechanism that depends on COX-2-derived PGE 2 production, activation of EP4, and Smad pathway.

During renin–angiotensin system activation, cyclooxygenase-2 (COX-2)-derived prostaglandins attenuate the pressor and antinatriuretic effects of angiotensin II (AngII) in the renal medulla. The (pro)renin receptor (PRR) is abundantly expressed in the collecting ducts (CD) and its expression is augmented by AngII. PRR overexpression upregulates COX-2 via mitogen-activated kinases/extracellular regulated kinases 1/2 in renal tissues; however, it is not clear whether this effect occurs independently or in concert with AngII type 1 receptor (AT1R) activation. We hypothesized that PRR activation stimulates COX-2 expression independently of AT 1 R in primary cultures of rat renal inner medullary cells. The use of different cell-specific immunomarkers (aquaporin-2 for principal cells, anion exchanger type 1 for intercalated type-A cells, and tenascin C for interstitial cells) and costaining for AT 1 R, COX-2, and PRR revealed that PRR and COX-2 were colocalized in intercalated and interstitial cells whereas principal cells did not express PRR or COX-2. In normal rat kidney sections, PRR and COX-2 were colocalized in intercalated and interstitial cells. In rat renal inner medullary cultured cells, treatment with AngII (100 nmol/L) increased COX-2 expression via AT 1 R. In addition, AngII and rat recombinant prorenin (100 nmol/L) treatments increased extracellular regulated kinases 1/2 phosphorylation, independently. Importantly, rat recombinant prorenin upregulated COX-2 expression in the presence of AT 1 R blockade. Inhibition of mitogen-activated kinases/extracellular regulated kinases 1/2 suppressed COX-2 upregulation mediated by either AngII or rat recombinant prorenin. Furthermore, PRR knockdown using PRR-short hairpin RNA blunted the rat recombinant prorenin-mediated upregulation of COX-2. These results indicate that COX-2 expression is upregulated by activation of either PRR or AT 1 R via mitogen-activated kinases/extracellular regulated kinases 1/2 in rat renal inner medullary cells.

Binding of prorenin or renin to (pro)renin receptor (PRR) activates mitogen activated protein kinases (MAPK)/extracellular regulated kinases 1/2 (ERK 1/2), which have been proposed as mediators in tissue damage. Prorenin, renin, and PRR are overexpressed in the collecting duct (CD) in diabetes and hypertension. Activation of the PRR upregulates profibrotic markers through reactive oxygen species (ROS) formation; however, the exact mechanisms have not been established. We hypothesized that the activation of PRR increases the expression of profibrotic markers through MAPK-dependent ROS formation in CD cells. To address this hypothesis, a mouse renal CD cell line (M-1) was treated with recombinant prorenin plus ROS or MAPK inhibitor to evaluate their effect on the expression of profibrotic markers. Recombinant prorenin increased ROS formation and expression of alpha smooth muscle actin (α-SMA), connecting tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1). Induction of profibrotic factors was blunted by antioxidant p-coumaric acid, and partially prevented by lyophilic antioxidant trolox C or ascorbic acid. Inhibition of MAPK pathway (PD98059) prevented the prorenin-dependent ROS formation and augmentation of profibrotic factors, as well as fibroblast-like phenotype. Knockdown of PRR did not show effects on cell viability in M-1 cells and partially reduced the augmentation of fibronectin, collagen I and fibroblast-like phenotype induced by prorenin treatment. These results suggest that the activation of CD-derived PRR plays a role in the development of tubular damage.

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.

Intrarenal cyclooxygenase-2 (COX-2) activity is increased during activation of the renin-angiotensin-system (RAS) increasing synthesis of prostaglandin E2 (PGE2) and buffering the vasoconstrictor and antinatriuretic effects of angiotensin II (AngII). While AngII upregulates intrarenal COX-2 expression, it remains unclear if this occurs in a time-dependent manner, thereby impacting renal hemodynamics differently during the early and late phases of the development of high blood pressure in AngII-induced hypertension. Male Sprague-Dawley rats were infused with AngII (0.4 μg/min/kg). Systolic blood pressure (SBP), COX-2 expression and PGE2 tissue content and urinary excretion were evaluated at day 3, 7 and 14 of the AngII infusions. In acute studies we evaluated the effects of COX-2 inhibition at day 5-7 and day 14 on renal hemodynamic parameters. Chronic AngII infusions increased SBP from day 7 through 14: 162 ± 5 mmHg; and 198 ± 15 mmHg versus controls: 114 ± 10 mmHg; P

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-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.

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