Renin Receptor Activation Research Articles

The influence of prorenin/(pro)renin receptor on progesterone secretion by the bovine corpus luteum

The aim of this study was to evaluate the role of prorenin/(pro)renin receptor activation on luteal progesterone (P4) secretion. Our hypothesis was that the nonproteolytic activation of (pro)renin receptor [P(RR)] is part of the regulatory mechanism responsible for corpus luteum (CL) function. In the first three experiments, prorenin was found to stimulate the production of P4, which is not inhibited by an angiotensin receptor antagonist (saralasin), but rather by a renin/prorenin inhibitor (aliskiren), a MAPK1/3 inhibitor (PD325901) or an EGFR inhibitor (AG1478), which are evidence of nonproteolytic activation of prorenin. Moreover, prorenin induced phosphorylation of MAPK1/3 in luteal cells. Following these in vitro experiments, a sequence of in vivo experiments was performed demonstrating that the intrafollicular injection of aliskiren in preovulatory follicles impaired P4 secretion in cows that ovulated. Furthermore, all profibrotic genes studied were present in the CL and TGFB1 and FN1 mRNA were upregulated from day 5–10 post-ovulation. During luteolysis, REN was downregulated at 48 h, whereas TGFB1 and SERPINE1 were dramatically upregulated in luteal tissue at 12 h after PGF. In summary, these data are evidence that nonproteolytic activation of (P)RR is involved in luteal function.

POS-373 Advanced oxidation protein products activate oxidative stress in vitro via soluble (pro)renin receptor mediated intrarenal renin angiotensin system and Nox4/H2O2 pathway

Advanced oxidation protein products (AOPPs) are newly identified as a novel oxidative stress biomarkers. Previous research has indicated potential associations between AOPPs and activation intrarenal local renin angiotensin system (RAS). However, no known empirical research has focused on exploring relationships between AOPPs and the new member of RAS, (pro) renin receptor (PRR). Aim of this study was to test the role of soluble PRR (sPRR) in AOPPs-induced oxidative stress responses in cultured human renal proximal tubular cell line HK-2 cells. The HK-2 cells were incubated for 24h in the presence of different concentrations of AOPPs, or incubated for different time lengths in the presence of 100 μg/ml of AOPPs. In HK-2 cells treated with PRO20, furin inhibitors, GM6001, PF-429242, sPRR-His, or transfected with PRR siRNA, site-1 protease (S1P) siRNA, Nox4 siRNA prior to exposure to AOPPs, the changes in the protein expressions of PRR/sPRR and Nox4 were examined with Western blotting, mRNA lelvels of PRR, angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R), angiotensinogen (AGT), intercellular adhesion molecule (ICAM1), tumor necrosis factor-α (TNF-α) by using real-time quantitative PCR, the cell medium for renin activity, ACE activity, sPRR, Ang II, H2O2, thiobarbituric acid reactive substances (TBARS) concentrations by using ELISA Assay kits. AOPPs showed time-dependent and dose-independent effects on PRR cleavage produces sPRR. AOPP-albumin treatment induced a >4-times increase in medium sPRR due to enhanced cleavage of PRR but unmodified albumin has no effect. Inhibition of S1P with PF-429242 or siRNA remarkably suppressed AOPPs-induced sPRR production. Surprisingly, this cleavage event was unaffected by inhibition of furin or ADAM19. AOPPs (100 μg/ml) treatment for 24 h induced renin activity and oxidative stress, both of which were attenuated by a PRR decoy inhibitor PRO20 and S1P inhibitor PF429242. What’s more, PF-429242 significantly decreased AOPPs stimulated Nox4 and H2O2 expression and administration of a recombinant sPRR, termed sPRR-His, reversed the effects of S1P inhibition. Taken together, these findings suggest that AOPPs induce oxidative stress via S1P-derived soluble (pro)renin receptor activation intrarenal renin angiotensin system and Nox4/H2O2 pathway in vitro.

Obesity and aging affects skeletal muscle renin-angiotensin system and myosin heavy chain proportions in pre-diabetic Zucker rats.

There is a gap in the knowledge regarding regulation of local renin-angiotensin system (RAS) in skeletal muscle during development of obesity and insulin resistance in vivo. This study evaluates the obesity- and age-related changes in the expression of local RAS components. Since RAS affects skeletal muscle remodelling, we also evaluated the muscle fibre type composition, defined by myosin heavy chain (MyHC) mRNAs and protein content. Gene expressions were determined by qPCR and/or Western blot analysis in musculus quadriceps of 3- and 8-month-old male obese Zucker rats and their lean controls. The enzymatic activity of aminopeptidase A (APA) was determined flourometrically. Activation of renin receptor (ReR)/promyelocytic leukaemia zinc finger (PLZF) negative feedback mechanism was observed in obesity. The expression of angiotensinogen and AT1 was downregulated by obesity, while neutral endopeptidase and AT2 expressions were upregulated in obese rats with aging. Skeletal muscle APA activity was decreased by obesity, which negatively correlated with the increased plasma APA activity and plasma cholesterol. The expression of angiotensin-converting enzyme (ACE) positively correlated with MyHC mRNAs characteristic for fast-twitch muscle fibres. The obesity- and age-related alterations in the expression of both classical and alternative RAS components suggest an onset of a new equilibrium between ACE/AngII/AT1 and ACE2/Ang1-7/Mas at lower level accompanied by increased renin/ReR/PLZF activation. Increased APA release from the skeletal muscle in obesity might contribute to increased plasma APA activity. There is a link between reduced ACE expression and altered muscle MyHC proportion in obesity and aging.

Aortic constriction induces hypertension and cardiac hypertrophy via (pro)renin receptor activation and the PLC‑β3 signaling pathway.

The (pro)renin receptor [(P)RR] serves an important role in cardiovascular complications. However, the precise mechanisms of (P)RR in the heart remain obscure. The authors hypothesized that overexpression of (P)RR would be associated with activation of the relevant signal pathway which could lead to organ injury. The aim of the present study was to test the role of cardiac (P)RR and its potential signaling pathway components including phospholipaseC(PLC), protein kinaseC(PKC), extracellular signal‑regulated kinase(ERK)1/2 and Raf‑1 proto‑oncogene, serine/threonine kinase(Raf‑1). Hypertension and cardiac hypertrophy were induced by partial abdominal aortic ligation in Sprague‑Dawley rats. The expression levels of cardiac (P)RR, PLC‑β3, PKC, ERK1/2 and Raf‑1 were measured following administration of the handle region peptide (HRP) and PLC‑β3 inhibitor U73122. The expression of (P)RR and PLC‑β3 significantly increased in the left ventricle (P<0.05). Levels of PKC‑α, ERK1/2 and Raf‑1 in the heart rose significantly (P<0.05). HRP and U73122 significantly decreased the levels of cardiac (P)RR and PLC‑β3. Furthermore, levels of PKC‑α, ERK1/2 and Raf‑1 were also decreased (P<0.05). Cardiac parameters, blood pressure and plasma Angiotensin(Ang)I and AngII levels were altered significantly (P<0.05). The results demonstrated that hypertension induced by aortic restriction activated the (P)RR in the heart. This action led to hypertension and cardiac hypertrophy via the (P)RR‑PLC‑β3‑PKC‑ERK1/2‑Raf‑1 signaling pathway. These results provide a mechanism by which elevated (P)RR levels in hypertension may contribute to the development of cardiac remodeling.

(Pro)renin receptor activation increases profibrotic markers and fibroblast-like phenotype through MAPK-dependent ROS formation in mouse renal collecting duct cells.

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±0.1nmol/L DCF/μg total protein, P<.05) and expression of profibrotic markers CTGF (149±12%, P<.05), α-SMA (160±20%, P<.05), and PAI-I (153±13%, P<.05) at 10-8 mol/L. Recombinant prorenin-induced phospho ERK 1/2 (p44 and p42) at 10-8 and 10-6 mol/L after 20minutes. 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 24hours 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.

Abstract 095: Human (Pro)renin Receptor Activation Induces an Angiotensin II-Independent Pressor Response Mediated by NADPH Oxidase 4 Activity

The binding of prorenin to the (pro)renin receptor (PRR) induces non-proteolytic activation of prorenin and generation of angiotensin II (Ang II). PRR activation can also induce Ang II-independent signaling pathways. However, whether Ang II-independent signaling pathways are critical for blood pressure (BP) regulation is not known. To address this question, we created transgenic mice that overexpress the human PRR (hPRR) selectively in neurons (Syn-hPRR). Activated human prorenin (hPRO) cannot cleave endogenous mouse angiotensinogen to generate Ang II. Therefore, administration of hPRO to Syn-hPRR mice can be used to examine Ang II-independent PRR signaling in BP regulation. Intracerebroventricular (ICV) infusion of hPRO increases BP in Syn-hPRR mice (ΔMAP 23 ± 4.6, n = 4) but has no effect on wildtype (WT) mice (ΔMAP 2 ± 0.8, n = 6). The hPRO-induced pressor response in Syn-hPRR mice is unaffected by co-infusion with the Ang II type 1 receptor blocker losartan (ΔMAP 19 ± 5.2, n = 8), suggesting that the response is independent of Ang II. Interestingly, co-infusion with an inhibitor of the reactive oxygen species-generating enzyme NADPH oxidase (NOX), diphenyleneiodonium, nearly abolishes the hPRO-induced pressor response in Syn-hPRR mice (ΔMAP 4.7 ± 1.0, n = 4), indicating that NOX activity is required. Additionally, we find that basal NOX activity is enhanced in the Syn-hPRR hypothalamus relative to WT mice (1.4 fold change). We next examined which NOX isoform is responsible for the hPRO-induced pressor response and enhanced activity. NOX4 mRNA levels are greater (2.7 ± 0.6 fold change), but NOX1 (1.2 ± 0.3 fold change) and NOX2 (1.2 ± 0.3 fold change) mRNA levels are not different, in the hypothalamus of Syn-hPRR compared to WT mice (n = 3). Adenovirus-mediated delivery of NOX2, NOX4, or a scrambled sequence shRNA was ICV injected in Syn-hPRR mice. After 7 days, we found that treatment with NOX2 (ΔMAP 20 ± 5.2) or scrambled (ΔMAP 23 ± 3.2) shRNA had no effect on the hPRO-induced pressor response (n = 5). However, the hPRO-induced increase in BP is attenuated in Syn-hPRR mice injected with NOX4 shRNA (ΔMAP 5.9 ± 2.8). Together, these data indicate that NOX4 mediates the Ang II-independent pressor response to activation of the human (pro)renin receptor in Syn-hPRR mice.

Inhibiting (pro)renin receptor-mediated p38 MAPK signaling decreases hypoxia/reoxygenation-induced apoptosis in H9c2 cells.

The (pro)renin receptor is a new molecular member of the renin-angiotensin system and participates in regulating many physiological and pathological processes. However, the role of (pro)renin receptor-mediated signaling pathways in myocardial ischemic/reperfusion injury remains unclear. In this study, we hypothesized that p38 mitogen-activated protein kinase (MAPK) signaling pathway activation by the (pro)renin receptor had effects on myocardial apoptosis induced by ischemia/reperfusion. This analysis was performed using a hypoxia/reoxygenation model in H9c2 cells to mimic ischemia/reperfusion injury. The H9c2 rat cardiomyocyte cell line was subjected to 2 h of hypoxia followed by 6 h of reoxygenation. The (pro)renin receptor, caspase 3, and phosphorylated p38 MAPK protein expression levels were analyzed by Western blot. After 2 h of hypoxia followed by 6 h of reoxygenation, apoptosis increased in H9c2 cells; the (pro)renin receptor, caspase 3, and phosphorylated p38 MAPK protein expressions were upregulated. siRNA silencing of the (pro)renin receptor significantly decreased p38 MAPK phosphorylation. siRNA silencing of the (pro)renin receptor and treatment with the p38MAPK inhibitor SB203580 significantly decreased the hypoxia/reoxygenation-induced apoptosis and caspase 3 protein expression in H9c2 cells. Furthermore, we found that the role of the (pro)renin receptor was independent of angiotensin II (Ang II). Thus, we concluded that (pro)renin receptor activation could trigger hypoxia/reoxygenation-induced apoptosis in H9c2 cells, partially through the p38 MAPK/caspase 3 signaling pathway, independent of Ang II. Therefore, this study may provide new therapeutic targets for myocardial ischemic/reperfusion injury prevention, and further in vivo studies are needed.

Abstract 602: RAS-induced Hypertension Is Primarily Mediated By (Pro)renin Receptor Activation At The Paraventricular Nucleus Of The Hypothalamus.

Studies have shown that renin-angiotensin-system (RAS)-induced inflammation at the paraventricular nucleus of the hypothalamus (PVN) contributes to hypertension development by increasing sympathetic outflow and elevating blood pressure. Recent data suggest that PRR activation increases pro-inflammatory cytokine (PIC) production in an Ang II-independent manner helping sustain kidney inflammation, retinal disease and diabetic neuropathy. Astrocytes house all the components of the RAS and help regulate the immune response within the brain. However, the role of (pro)renin (PRO) receptor (PRR) activation at the PVN on blood pressure regulation and astrocyte activation has not been evaluated. We show that at the PVN, 12 weeks old spontaneously hypertensive rats (SHRs) have significantly (p &lt; 0.05) higher PRR mRNA levels than normotensive Wistar Kyoto (WKY) and Sprague Dawley (SD) rats (1.6 ± 0.3 vs. 0.96 ± 0.4 &amp; 1.0 ± 0.2, SHR, WKY and SD respectively). In situ-hybridization data using RNA Scope, confirmed these results as qualitative analysis from coronal sections containing the PVN from SD and WKY rats showed lower PRR signal when compared to those from SHRs. Our data shows that bilateral downregulation of the PRR, using a short-hairpin RNA (shRNA) vector construct, at the PVN of SHRs (n = 5) reduces blood pressure compared to control animals (n = 5) with PRR levels intact (146.1 ± 6.6 vs. 133.3 ± 9.0 mmHG; p &lt; 0.05). Similarly, PRR downregulation at the PVN can also reduce the pressor response triggered by sub-cutaneous Ang II infusion (200ng/kg/min) in SD rats (143 ± 13.2 vs. 114.8 ± 13.2 mmHG, p &lt; 0.05; control vs. shRNA animals, respectively). In both experimental designs, blood pressure decreases were not accompanied by changes in heart rate. Our results also show lower glial fibrillary acidic protein (GFAP) and interleukin (IL)-1β mRNA levels at the PVN in shRNA-injected animals and using the Image J tool we show a trend for astroglial area fraction in the PVN to decrease. These changes are in concert with our in vitro data which show that PRO increases PICs levels and astrogliosis in cultured astrocytes. Our findings show that at the PVN, PRR activation is key in RAS-induced hypertension and potential mechanisms may include astrocyte- and neuroimmune activation.

Juxtaglomerular apparatus hyperplasia under dual angiotensin blockade. A footprint of adequate RAS inhibition or a concern for renal fibrosis?

BackgroundDual renin-angiotensin system blockade with angiotensin-converting enzyme inhibitors and angiotensin receptor blockers has been advocated to minimize proteinuria. However, recent trials have questioned the renal safety of this approach. Our understanding on the molecular effects of dual blockade in humans is incomplete.Case presentationWe present a patient with corticoid resistant nephrotic syndrome who developed marked juxtaglomerular apparatus hyperplasia and renin expression in the context of dual angiotensin system blockade.ConclusionsAlthough renin may have profibrotic effects mediated by (pro)renin receptor activation, this report raises questions on the potential consequences of local renin activation on chronic kidney disease in patients with dual angiotensin blockade.

Prorenin engages the (pro)renin receptor like renin and both ligand activities are unopposed by aliskiren

Inhibition of (pro)renin receptor activation was demonstrated to inhibit or even abolish the development of end-organ damage in animal models. The new renin inhibitor, aliskiren, markedly increases the plasma concentration of the (pro)renin receptor ligands prorenin and renin in patients. The effects of prorenin and of renin inhibitors on the signal transduction cascade of the (pro)renin receptor are currently unknown. Our results indicate that renin and prorenin were equally potent in (pro)renin receptor activation by decreasing (pro)renin receptor mRNA, increasing phosphatidylinositol-3 kinase p85alpha mRNA and augmenting viable cell number, respectively. These effects of renin and prorenin are both abolished using small-interfering RNA against the (pro)renin receptor or its adaptor promyelocytic zinc finger protein. The renin inhibitor aliskiren did not inhibit the renin-induced or prorenin-induced activation of the (pro)renin receptor. This is the first report demonstrating equal ligand activities of both, renin and prorenin, on the (pro)renin receptor - promyelocytic zinc finger protein-phosphatidylinositol-3 kinase-p85alpha pathway. The failure of aliskiren to inhibit the noncatalytic effects of renin and prorenin may be of clinical relevance considering the increase in plasma concentrations of (pro)renin under aliskiren treatment.

The mysterious handle region peptide does not block cellular (pro)renin receptor activation

The mysterious handle region peptide does not block cellular (pro)renin receptor activation

Renin-stimulated TGF-β1 expression is regulated by a mitogen-activated protein kinase in mesangial cells

Recent evidence indicates that renin itself might be profibrotic, independent of angiotensin II; however, the signaling system by which renin exerts a direct effect is not known. We tested the hypothesis that renin receptor activation, in turn, activates the extracellular-signal regulated kinase 1 and 2 (ERK1/2) of the mitogen-activated protein kinase system in mesangial cells. Recombinant rat renin induced a rapid phosphorylation of ERK1/2 and subsequent cell proliferation in a dose- and time-dependent manner. ERK1/2 activation by renin addition was not altered by angiotensin-converting enzyme inhibition or angiotensin receptor blockade. An ERK kinase inhibitor significantly reduced the renin-induced ERK1/2 phosphorylation and the subsequent increase in transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor-1 mRNA expression. A small-inhibiting RNA, siRNA, against the renin receptor completely blocked ERK1/2 activation by rat renin. We conclude that renin induces ERK1/2 activation though a receptor-mediated, angiotensin II-independent mechanism in mesangial cells. This renin-activated pathway triggers cell proliferation along with TGF-beta1 and plasminogen activator inhibitor-1 gene expression. This system may play an important role in the overall profibrotic actions of renin.