Pro)renin Receptor Knockdown Research Articles

(Pro)renin receptor aggravates myocardial pyroptosis in diabetic cardiomyopathy through AMPK-NLRP3 pathway.

As one of the most common complications of diabetes, diabetic cardiomyopathy (DCM) is the main cause of heart failure in patients with diabetes. However, the lack of effective treatments for DCM remains a clinical challenge. (Pro) renin receptor (PRR) is a member of renin angiotensin aldosterone system (RAAS). Here, we aim to determine whether PRR is involved in myocardial pyroptosis in diabetic cardiomyopathy. We established diabetic rats model by intraperitoneal injection of streptozotocin (STZ). PRR overexpression adenovirus or PRR knockdown adenovirus was injected into the tail vein. Western blot, histopathology and immunohistochemistry staining, ELISA and Echocardiography were used to detect cardiac function changes and myocardial injury levels of DCM rats. Primary cardiomyocytes were stimulated with high glucose and PRR overexpression or PRR knockdown was achieved by adenovirus transfection, we also used the inhibitor of AMPK to decrease the activity of AMPK. Western blot, Real-time PCR, Immunofluorescence and ELISA were used to detect the level of PRR and pyroptosis in cardiomyocyte. We found that high glucose increased the expression of PRR in heart. After overexpression of PRR, the expression of the pyroptosis related proteins such as Caspase-1, IL-1β, IL-18, and NLRP3 was significantly increased, the phosphorylation level of AMPK was significantly decreased, and the fibrosis level was significantly increased, thus aggravating the cardiac function injury of DCM. On the contrary, PRR knockdown can alleviate the level of myocardial pyroptosis in DCM and improve cardiac function. The related mechanism was that PRR could inhibit AMPK phosphorylation and promote the activation of NLRP3 inflammasome. PRR aggravated pyroptosis of cardiomyocyte, increased the dysfunction of cardiomyocyte, and may be related to the decrease of AMPK phosphorylation and the overactivation of NLRP3. This may provide new ideas and targets for the treatment of DCM.

(Pro)renin receptor mediates tubular epithelial cell pyroptosis in diabetic kidney disease via DPP4-JNK pathway

Background(Pro)renin receptor (PRR) is highly expressed in renal tubules, which is involved in physiological and pathological processes. However, the role of PRR, expressed in renal tubular epithelial cells, in diabetic kidney disease (DKD) remain largely unknown.MethodsIn this study, kidney biopsies, urine samples, and public RNA-seq data from DKD patients were used to assess PRR expression and cell pyroptosis in tubular epithelial cells. The regulation of tubular epithelial cell pyroptosis by PRR was investigated by in situ renal injection of adeno-associated virus9 (AAV9)-shRNA into db/db mice, and knockdown or overexpression of PRR in HK-2 cells. To reveal the underlined mechanism, the interaction of PRR with potential binding proteins was explored by using BioGrid database. Furthermore, the direct binding of PRR to dipeptidyl peptidase 4 (DPP4), a pleiotropic serine peptidase which increases blood glucose by degrading incretins under diabetic conditions, was confirmed by co-immunoprecipitation assay and immunostaining.ResultsHigher expression of PRR was found in renal tubules and positively correlated with kidney injuries of DKD patients, in parallel with tubular epithelial cells pyroptosis. Knockdown of PRR in kidneys significantly blunted db/db mice to kidney injury by alleviating renal tubular epithelial cells pyroptosis and the resultant interstitial inflammation. Moreover, silencing of PRR blocked high glucose-induced HK-2 pyroptosis, whereas overexpression of PRR enhanced pyroptotic cell death of HK-2 cells. Mechanistically, PRR selectively bound to cysteine-enrich region of C-terminal of DPP4 and augmented the protein abundance of DPP4, leading to the downstream activation of JNK signaling and suppression of SIRT3 signaling and FGFR1 signaling, and then subsequently mediated pyroptotic cell death.ConclusionsThis study identified the significant role of PRR in the pathogenesis of DKD; specifically, PRR promoted tubular epithelial cell pyroptosis via DPP4 mediated signaling, highlighting that PRR could be a promising therapeutic target in DKD.

(Pro)renin receptor inhibition attenuated liver steatosis, inflammation, and fibrosis in mice with steatohepatitis.

The (Pro)renin receptor (PRR) is reportedly involved in hepatic lipid metabolism and hepatocyte PRR knockdown protects mice against hepatosteatosis. However, the impact of PRR inhibition on liver inflammation and fibrosis in nonalcoholic steatohepatitis (NASH) remains unclear. Herein, C57BL/6 mice were fed a normal chow diet or fast food diet (FFD) for 24 weeks. Lentivirus-mediated PRR short hairpin RNA (shRNA) or handle region peptide (HRP), a PRR blocker, was administered for PRR inhibition. Mouse primary hepatocytes were cultured with palmitic acid, prorenin, siRNA-targeted PRR, and HRP. In FFD-fed mice, PRR inhibition via lentivirus-mediated PRR knockdown or HRP significantly attenuated liver steatosis, inflammation, and fibrosis. Mechanistically, PRR knockdown or HRP decreased hepatic acetyl-CoA carboxylase (ACC) abundance and upregulated peroxisome proliferator-activated receptor-alpha (PPARα). HRP treatment also decreased hepatic PRR expression. In addition, intrahepatic oxidative stress, apoptosis and inflammatory cell recruitment were ameliorated by PRR knockdown or HRP treatment, along with suppression of proinflammatory cytokine expression. PRR inhibition downregulated the hepatic expression of profibrotic factors, as well as TGF-β1/SMAD3 pathway. In primary mouse hepatocytes, PRR knockdown with siRNA or HRP downregulated cellular ACC and increased PPARα expression. In conclusion, our findings revealed that PRR inhibition attenuated hepatic steatosis, inflammation, and fibrosis in mice with NASH. Accordingly, targeting PRR signaling may serve as a potential treatment for NASH.

High glucose induces trafficking of prorenin receptor and stimulates profibrotic factors in the collecting duct

Growing evidence indicates that prorenin receptor (PRR) is upregulated in collecting duct (CD) of diabetic kidney. Prorenin is secreted by the principal CD cells, and is the natural ligand of the PRR. PRR activation stimulates fibrotic factors, including fibronectin, collagen, and transforming growth factor-β (TGF-β) contributing to tubular fibrosis. However, whether high glucose (HG) contributes to this effect is unknown. We tested the hypothesis that HG increases the abundance of PRR at the plasma membrane of the CD cells, thus contributing to the stimulation of downstream fibrotic factors, including TGF-β, collagen I, and fibronectin. We used streptozotocin (STZ) male Sprague–Dawley rats to induce hyperglycemia for 7 days. At the end of the study, STZ-induced rats showed increased prorenin, renin, and angiotensin (Ang) II in the renal inner medulla and urine, along with augmented downstream fibrotic factors TGF-β, collagen I, and fibronectin. STZ rats showed upregulation of PRR in the renal medulla and preferential distribution of PRR on the apical aspect of the CD cells. Cultured CD M-1 cells treated with HG (25 mM for 1 h) showed increased PRR in plasma membrane fractions compared to cells treated with normal glucose (5 mM). Increased apical PRR was accompanied by upregulation of TGF-β, collagen I, and fibronectin, while PRR knockdown prevented these effects. Fluorescence resonance energy transfer experiments in M-1 cells demonstrated augmented prorenin activity during HG conditions. The data indicate HG stimulates profibrotic factors by inducing PRR translocation to the plasma membrane in CD cells, which in perspective, might be a novel mechanism underlying the development of tubulointerstitial fibrosis in diabetes mellitus.

(Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway.

Background & AimsActivation of the (pro)renin receptor (PRR) up-regulates the expression of profibrotic genes in the kidney and heart. We aimed to investigate the role of PRR in hepatic fibrogenesis.MethodsHuman hepatic PRR levels were measured in patients with or without liver fibrosis. PRR expression was analyzed in primary mouse hepatic stellate cells (HSCs). Experimental fibrosis was studied in thioacetamide (TAA)-treated or methionine choline-deficient (MCD) diet-fed C57BL/6 mice. Lentivirus-mediated PRR short hairpin RNA was used to knockdown hepatic PRR expression. Lentiviral vectors expressing PRR short hairpin RNA or complementary DNA from the α-smooth muscle actin promoter were used for myofibroblast-specific gene knockdown or overexpression.ResultsPRR is up-regulated in human and mouse fibrotic livers, and in activated HSCs. Hepatic PRR knockdown reduced liver fibrosis by suppressing the activation of HSCs and expression of profibrotic genes in TAA or MCD diet–injured mice without significant changes in hepatic inflammation. Renin and prorenin increased the expression of PRR and production of TGF-β1 in human activated HSC Lieming Xu-2 cells, and knockdown of PRR inactivated Lieming Xu-2 cells with decreased production of transforming growth factor (TGF)-β1 and Mothers against decapentaplegic homolog 3 (Smad3) phosphorylation. Myofibroblast-specific PRR knockdown also attenuated liver fibrosis in TAA or MCD diet–injured mice. Mice with both myofibroblast-specific and whole-liver PRR knockdown showed down-regulation of the hepatic extracellular signal-regulated kinase (ERK)/TGF-β1/Smad3 pathway. Myofibroblast-specific PRR overexpression worsened TAA-induced liver fibrosis by up-regulating the ERK/TGF-β1/Smad3 pathway.ConclusionsPRR contributes to liver fibrosis and HSC activation, and its down-regulation attenuates liver fibrosis through inactivation of the ERK/TGF-β1/Smad3 pathway. Therefore, PRR is a promising therapeutic target for liver fibrosis.

Elevated V-ATPase Activity Following PTEN Loss Is Required for Enhanced Oncogenic Signaling in Breast Cancer.

PTEN loss-of-function contributes to hyperactivation of the PI3K pathway and to drug resistance in breast cancer. Unchecked PI3K pathway signaling increases activation of the mechanistic target of rapamycin complex 1 (mTORC1), which promotes tumorigenicity. Several studies have suggested that vacuolar (H+)-ATPase (V-ATPase) complex activity is regulated by PI3K signaling. In this study, we showed that loss of PTEN elevated V-ATPase activity. Enhanced V-ATPase activity was mediated by increased expression of the ATPase H+ transporting accessory protein 2 (ATP6AP2), also known as the prorenin receptor (PRR). PRR is cleaved into a secreted extracellular fragment (sPRR) and an intracellular fragment (M8.9) that remains associated with the V-ATPase complex. Reduced PTEN expression increased V-ATPase complex activity in a PRR-dependent manner. Breast cancer cell lines with reduced PTEN expression demonstrated increased PRR expression. Similarly, PRR expression became elevated upon PTEN deletion in a mouse model of breast cancer. Interestingly, concentration of sPRR was elevated in the plasma of patients with breast cancer and correlated with tumor burden in HER2-enriched cancers. Moreover, PRR was essential for proper HER2 receptor expression, localization, and signaling. PRR knockdown attenuated HER2 signaling and resulted in reduced Akt and ERK 1/2 phosphorylation, and in lower mTORC1 activity. Overall, our study demonstrates a mechanism by which PTEN loss in breast cancer can potentiate multiple signaling pathways through upregulation of the V-ATPase complex. IMPLICATIONS: Our study contributed to the understanding of the role of the V-ATPase complex in breast cancer cell tumorigenesis and provided a potential biomarker in breast cancer.

(Pro)renin receptor contributes to hypoxia/reoxygenation-induced apoptosis and autophagy in myocardial cells via the beta-catenin signaling pathway.

(Pro)renin receptor (PRR) contributes to regulating many physiological and pathological processes; however, the role of PRR-mediated signaling pathways in myocardial ischemia/reperfusion injury (IRI) remains unclear. In this study, we used an in vitro model of hypoxia/reoxygenation (H/R) to mimic IRI and carried out PRR knockdown by siRNA and PRR overexpression using cDNA in H9c2 cells. Cell proliferation activity was examined by MTT and Cell Counting Kit-8 (CCK-8) assays. Apoptosis-related factors, autophagy markers and beta-catenin pathway activity were assessed by real-time PCR and western blotting. After 24 h of hypoxia followed by 2 h of reoxygenation, the expression levels of PRR, LC3B-I/II, Beclin1, cleaved caspase-3, cleaved caspase-9 and Bax were upregulated, suggesting that apoptosis and autophagy were increased in H9c2 cells. Contrary to the effects of PRR downregulation, the overexpression of PRR inhibited proliferation, induced apoptosis, increased the expression of pro-apoptotic factors and autophagy markers, and promoted activation of the beta-catenin pathway. Furthermore, all these effects were reversed by treatment with the beta-catenin antagonist DKK-1. Thus, we concluded that PRR activation can trigger H/R-induced apoptosis and autophagy in H9c2 cells through the beta-catenin signaling pathway, which may provide new therapeutic targets for the prevention and treatment of myocardial IRI.

Pro-renin receptor suppresses mitochondrial biogenesis and function via AMPK/SIRT-1/ PGC-1α pathway in diabetic kidney.

Abnormal mitochondrial biogenesis and function has been linked to multiple diseases including diabetes. Recently, we demonstrated the role of renal (Pro)renin receptor (PRR) in the dysregulation of mitochondria. We hypothesized that PRR contributes to the reduction of mitochondrial biogenesis and function in diabetic kidney via PGC-1α/AMPK/SIRT-1 signaling pathway. In vivo and in vitro studies were conducted in C57BL/6 mouse and mouse renal mesangial cells (mRMCs). Control and streptozotocin-induced diabetic mice were injected with scramble or PRR shRNA and followed for a period of eight weeks. PRR mRNA and protein expression increased by 44% and 39% respectively (P<0.05) in kidneys of diabetic mice, and in mRMCs exposed to high glucose by 43 and 61% respectively compared to their respective controls. These results were accompanied by reduced mRNA and protein expressions of PGC-1α (67% and 75%), nuclear respiratory factors (NRF-1, 48% and 53%), mitochondrial transcriptional factor A (mtTFA, 56% and 40%), mitochondrial DNA copy number by 75% (all, P<0.05), and ATP production by 54%, respectively in diabetic kidneys and in mRMCs exposed to high glucose. Compared to non-diabetic control mice, PRR knockdown in diabetic mice and in mRMCs, not only attenuated the PRR mRNA and protein expression but also normalized mRNA and protein expressions of PGC-1α, NRF-1, mtTFA, mitochondrial DNA copy number, and ATP production. Treatment with AMPK inhibitor, Compound C, or SIRT-1 inhibitor, EX-527, alone, or combined with PRR siRNA caused marked reduction of mRNA expression of PGC-1α, NRF-1 and mtTFA, and ATP production in mRMCs exposed to high glucose. In conclusion, our study demonstrated the contribution of the PRR to the reduction of mitochondrial biogenesis and function in diabetic kidney disease via decreasing AMPK/SIRT-1/ PGC-1α signaling pathway.

(Pro)renin receptor contributes to renal mitochondria dysfunction, apoptosis and fibrosis in diabetic mice

Recently we demonstrated that increased renal (Pro)renin receptor (PRR) expression in diabetes contributes to development of diabetic kidney disease. However, the exact mechanisms involving PRR activity and diabetic kidney dysfunction are unknown. We hypothesized that PRR is localized in renal mitochondria and contributes to renal fibrosis and apoptosis through oxidative stress-induced mitochondria dysfunction. Controls and streptozotocin-induced diabetic C57BL/6 mice were injected with scramble shRNA and PRR shRNA and followed for a period of eight weeks. At the end of study, diabetic mice showed increased expressions of PRR and NOX4 in both total kidney tissue and renal mitochondria fraction. In addition, renal mitochondria of diabetic mice showed reduced protein expression and activity of SOD2 and ATP production and increased UCP2 expression. In diabetic kidney, there was upregulation in the expressions of caspase3, phos-Foxo3a, phos-NF-κB, fibronectin, and collagen IV and reduced expressions of Sirt1 and total-FOXO3a. Renal immunostaining revealed increased deposition of PRR, collagen and fibronectin in diabetic kidney. In diabetic mice, PRR knockdown decreased urine albumin to creatinine ratio and the renal expressions of PRR, NOX4, UCP2, caspase3, phos-FOXO3a, phos-NF-κB, collagen, and fibronectin, while increased the renal mitochondria expression and activity of SOD2, ATP production, and the renal expressions of Sirt1 and total-FOXO3a. In conclusion, increased expression of PRR localized in renal mitochondria and diabetic kidney induced mitochondria dysfunction, and enhanced renal apoptosis and fibrosis in diabetes by upregulation of mitochondria NOX4/SOD2/UCP2 signaling pathway.

(Pro)renin receptor-mediated myocardial injury, apoptosis, and inflammatory response in rats with diabetic cardiomyopathy

Excessive activation of the renin-angiotensin system (RAS) in diabetic cardiomyopathy (DCM) provokes a series of structural and functional abnormalities, and causes ventricular remodeling and heart failure in diabetes. (Pro)renin receptor (PRR) is a component of the RAS and has been reported to be up-regulated in some cardiovascular diseases. Furthermore, PRR blockade in some cardiovascular diseases, such as myocardial infarction and hypertension, has been demonstrated to reverse their pathogenesis. However, there have been few studies about the function of PRR in the pathogenesis of DCM. In this study, we hypothesized that PRR is involved in the pathogenesis of DCM and mediates myocardial injury in DCM. To explore the role of PRR in DCM, we evaluated the effects of PRR overexpression and knockdown on the DCM phenotype in vivo and in vitro The results show that PRR overexpression exacerbates myocardial injury and the inflammatory response in rats with DCM. Conversely, PRR knockdown alleviates myocardial fibrosis, apoptosis, and the inflammatory response, reversing the cardiac dysfunction in rats with DCM. In cell experiments, PRR overexpression also up-regulated the protein expression of collagen I and fibronectin, aggravated the inflammatory response, and increased the production of reactive oxygen species, whereas PRR knockdown had the opposite effect. Thus, PRR mediates myocardial injury, apoptosis, and the inflammatory response, likely through a PRR/extracellular signal-regulated kinase/reactive oxygen species pathway.

Prorenin receptor (PRR)-mediated NADPH oxidase (Nox) signaling regulates VEGF synthesis under hyperglycemic condition in ARPE-19 cells

The stimulation of angiotensin II (Ang II), the effector peptide of renin–angiotensin system, has been reported to increase the expression of vascular endothelial growth factor (VEGF) through the activation of the Ang II type 1 receptor (AT1R). In this study, we investigated whether hyperglycemia (HG, 33 mM glucose) in ARPE-19 cells could promote the expression of VEGF independently of Ang II through prorenin receptor (PRR), via an NADPH oxidase (Nox)-dependent mechanism. ARPE-19 cells were treated with the angiotensin converting enzyme (ACE) inhibitor perindopril to block the synthesis of Ang II. Treatment with HG induced VEGF expression in ARPE-19 cells, which was attenuated by pretreatment with the inhibitors of Nox, but not those of nitric oxide synthase, xanthine oxidase and mitochondrial O2 synthesis. In addition, Nox-derived and H2O2 signaling in the regulation of VEGF was determined by using both polyethylene glycol (PEG)-catalase (CAT) and PEG-superoxide dismutase (SOD). We demonstrated that small interfering RNA (siRNA)-mediated knockdown of PRR, Nox2 and Nox4 significantly reduced the HG-induced stimulation of VEGF. On the other hand, Nox4 overexpression significantly potentiated PRR-induced stimulation of VEGF under hyperglycemia in ARPE-19 cells. Furthermore, Nox4 was shown to be associated with enhanced activities of ERK1/2 and NF-κB (p65), indicating their involvement in PRR-induced activation of VEGF under HG in ARPE-19 cells. Our results support the hypothesis that Nox4-derived reactive oxygen species (ROS) signaling is implicated in the hyperglycemia-induced increase of VEGF expression through PRR in ARPE-19 cells. However, further work is needed to evaluate the role of PRR and Nox-s in HG-induced stimulation of VEGF in vivo.

(Pro)renin Receptor Is an Amplifier of Wnt/β-Catenin Signaling in Kidney Injury and Fibrosis.

The (pro)renin receptor (PRR) is a transmembrane protein with multiple functions. However, its regulation and role in the pathogenesis of CKD remain poorly defined. Here, we report that PRR is a downstream target and an essential component of Wnt/β-catenin signaling. In mouse models, induction of CKD by ischemia-reperfusion injury (IRI), adriamycin, or angiotensin II infusion upregulated PRR expression in kidney tubular epithelium. Immunohistochemical staining of kidney biopsy specimens also revealed induction of renal PRR in human CKD. Overexpression of either Wnt1 or β-catenin induced PRR mRNA and protein expression in vitro Notably, forced expression of PRR potentiated Wnt1-mediated β-catenin activation and augmented the expression of downstream targets such as fibronectin, plasminogen activator inhibitor 1, and α-smooth muscle actin (α-SMA). Conversely, knockdown of PRR by siRNA abolished β-catenin activation. PRR potentiation of Wnt/β-catenin signaling did not require renin, but required vacuolar H+ ATPase activity. In the mouse model of IRI, transfection with PRR or Wnt1 expression vectors promoted β-catenin activation, aggravated kidney dysfunction, and worsened renal inflammation and fibrotic lesions. Coexpression of PRR and Wnt1 had a synergistic effect. In contrast, knockdown of PRR expression ameliorated kidney injury and fibrosis after IRI. These results indicate that PRR is both a downstream target and a crucial element in Wnt signal transmission. We conclude that PRR can promote kidney injury and fibrosis by amplifying Wnt/β-catenin signaling.

Abstract P195: Prorenin Receptor Activation Increases Profibrotic Markers and Fibroblast Like Phenotype Through MAPK-dependent ROS Formation in Mouse Renal Collecting Duct 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.

Adipocyte (Pro)Renin-Receptor Deficiency Induces Lipodystrophy, Liver Steatosis and Increases Blood Pressure in Male Mice.

Adipose tissue dysfunction related to obesity is overwhelmingly associated with increased risk of developing cardiovascular diseases. In the setting of obesity, (pro)renin receptor (PRR) is increased in adipose tissue of mice. We sought to determine the physiological consequences of adipocyte-PRR deficiency using adiponectin-Cre mice. We report a unique model of adipocyte-PRR-deficient mice (PRR(Adi/Y)) with almost no detectable white adipose tissues. As a consequence, the livers of PRR(Adi/Y) mice were enlarged and demonstrated a marked accumulation of lipids. Adipocyte-specific deficiency of PRR increased systolic blood pressure and the concentration of soluble PRR in plasma. To determine whether adipocyte-PRR was involved in the development of obesity-induced hypertension, mice were fed a low-fat or a high-fat diet for 16 weeks. Adipocyte-PRR-deficient mice were resistant to diet-induced obesity. Both high-fat- and low-fat-fed PRR(Adi/Y) mice had elevated insulin levels. Interestingly, adipocyte-PRR deficiency improved glucose tolerance in high-fat-fed PRR(Adi/Y) mice. In response to feeding either low-fat or high-fat diets, systolic blood pressure was greater in PRR(Adi/Y) mice than in control mice. High-fat feeding elevated soluble PRR concentration in control and PRR(Adi/Y) mice. In vitro knockdown of PRR by siRNA significantly decreased mRNA abundance of PPARγ (peroxisome proliferator-activated receptor gamma), suggesting an important role for PRR in adipogenesis. Our data indicate that adipocyte-PRR is involved in lipid homeostasis and glucose and insulin homeostasis, and that soluble PRR may be a predictor of metabolic disturbances and play a role in systolic blood pressure regulation.

Regulation of growth hormone secretion by (pro)renin receptor.

(Pro)renin receptor (PRR) has a single transmembrane domain that co-purifies with the vacuolar H+-ATPase (V-ATPase). In addition to its role in cellular acidification, V-ATPase has been implicated in membrane fusion and exocytosis via its Vo domain. Results from the present study show that PRR is expressed in pituitary adenoma cells and regulates growth hormone (GH) release via V-ATPase-induced cellular acidification. Positive PRR immunoreactivity was detected more often in surgically resected, growth hormone-producing adenomas (GHomas) than in nonfunctional pituitary adenomas. GHomas strongly expressing PRR showed excess GH secretion, as evidenced by distinctly high plasma GH and insulin-like growth factor-1 levels, as well as an elevated nadir GH in response to the oral glucose tolerance test. Suppression of PRR expression in rat GHoma-derived GH3 cells using PRR siRNA resulted in reduced GH secretion and significantly enhanced intracellular GH accumulation. GH3 treatment with bafilomycin A1, a V-ATPase inhibitor, also blocked GH release, indicating mediation via impaired cellular acidification of V-ATPase. PRR knockdown decreased Atp6l, a subunit of the Vo domain that destabilizes V-ATPase assembly, increased intracellular GH, and decreased GH release. To our knowledge, this is the first report demonstrating a pivotal role for PRR in a pituitary hormone release mechanism.

Nucleus of the Solitary Tract (Pro)Renin Receptor-Mediated Antihypertensive Effect Involves Nuclear Factor-κB-Cytokine Signaling in the Spontaneously Hypertensive Rat

The importance of the (pro)renin receptor (PRR) in the function of the central nervous system is increasingly evident because PRR seems to play a role in neuronal control of cardiovascular function. PRR expression is elevated in the nucleus of the solitary tract (NTS) of spontaneously hypertensive rats (SHR). In this study, we tested the hypothesis that altered activity of PRR in the NTS is linked to hypertension. Eight weeks of chronic knockdown of the NTS PRR, using recombinant adeno-associated virus type 2 (AAV2)-PRR-small hairpain RNA (shRNA)-mediated gene transduction, caused a significant increase in mean arterial pressure (MAP) in the SHR (shRNA, 173±5; Control, 151±6 mm Hg) but not in Wistar Kyoto rats (shRNA, 108±7; Control, 106±6 mm Hg). The MAP elevation in the SHR was associated with decreased inflammatory markers tumor necrosis factor-α, interleukin-6, C-C motif ligand 5, and their transcription factor, nuclear factor-κB. Consistent with the pressor effects of the PRR knockdown, acute bilateral NTS injection of human renin (2 pmol/side) decreased MAP and heart rate (HR) in SHR (ΔMAP, -38±4 mm Hg; Δheart rate, -40±10 bpm), with negligible responses in Wistar Kyoto rats (ΔMAP, -4±3 mm Hg; Δheart rate, -12±7 bpm). These effects in SHR were attenuated (80%) by prorenin handle region peptide but were not affected by angiotensin II type 1 or angiotensin II type 2 receptor blockers. Finally, PRR activation in SHR neuronal cultures by prorenin activated nuclear factor-κB and increased mRNA levels of interleukin-1β (250-fold), tumor necrosis factor-α (32-fold), interleukin-6 (35-fold), C-C motif ligand 5 (12-fold), and interleukin-10 (7-fold) in a nuclear factor-κB-dependent but angiotensin II type 1 receptor-independent manner. Therefore, NTS PRR mediates antihypertensive effects via an angiotensin II-independent mechanism in SHR, which involves stimulation of the nuclear factor-κB-cytokine signaling pathway.

Brain-Targeted (Pro) Renin Receptor Knockdown Modulates Body Fluid Homeostasis

Objectives: The (pro) renin receptor (PRR) is highly expressed in the brain and is involved in the central regulation of blood pressure. However, the role of the brain PRR in regulating body fluid homeostasis in hypertension remains unclear. We hypothesized that the brain PRR knockdown modulates water intake, urine, and urinary sodium excretion in the context of angiotensin II (Ang II)-induced hypertension. Methods and Results: Brain PRR was knocked down in non-transgenic (NT) normotensive and human reninangiotensinogen double-transgenic (RA) mice by intracerebroventricular (ICV) injection of adeno-associated virus expressing short hairpin RNA targeting the PRR (AAV-PRR-shRNA). Water and food intake, and urinary excretion were recorded using metabolic cages. At baseline, RA mice exhibited higher water intake, food intake, urine excretion, urinary sodium excretion and potassium excretion compared to NT mice. PRR knockdown in the brain significantly decreased water and food intake, and urinary potassium and sodium excretion in RA mice, but had no such effects in NT mice. PRR knock down also decreased reactive oxygen species generation and plasma Ang II concentration in RA mice. Conclusion: PRR knockdown modulates body fluid homeostasis in hypertensive RA mice, suggesting that the brain PRR plays a role in regulating body fluid homeostasis during Ang II-dependent hypertension.

Angiotensin II–Independent Upregulation of Cyclooxygenase-2 by Activation of the (Pro)Renin Receptor in Rat Renal Inner Medullary Cells

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.

Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor

The discovery of a specific prorenin receptor (PRR) suggests a biological function of prorenin that is independent of angiotensin I production. In the present study, we investigated the role of PRR on smooth muscle cell (SMC) migration. PRR was expressed in human mammary arteries and in cultured human aortic SMCs. Prorenin induced SMC migration in a dose-dependent manner, as assessed by Boyden chamber chemotaxis assay, and increased SMC random motility, as determined by video microscopy. The prorenin decoy peptide inhibited SMC migration in response to prorenin, and knockdown of PRR by small interfering RNA completely inhibited the migratory response to prorenin, demonstrating that the chemotactic action of prorenin is mediated by the PRR. Prorenin induced cytoskeleton reorganization and lamellipodia formation and increased the intracellular levels of both RhoA-GTP and Rac1-GTP through PRR. These effects were required for SMC migration, because the suppression by small interfering RNA of either Rac1 or RhoA GTP-bound forms completely blocked the PRR-mediated chemotactic effect. Prorenin also induced the formation of larger focal adhesions and cleavage of the focal adhesion kinase (pp125(FAK)) into two main fragments with molecular weights of 50 and 90 kDa. The generation of these two fragments of pp125(FAK) was reduced by the calpain inhibitor ALLN, which also inhibited SMC migration in response to prorenin. These results demonstrate that prorenin is a chemotactic factor for human aortic SMCs expressing PRR. This effect is elicited through reorganization of the cytoskeleton and focal adhesion, activation of RhoA and Rac1, and calpain-mediated cleavage of pp125(FAK).

NTS (pro)renin receptor (PRR)‐mediated antihypertensive effect involves NF‐KappaB‐cytokine signaling in the spontaneously hypertensive rats (SHR)

Increased PRR expression in the NTS of SHR, coupled with the evidence on role of the brain inflammation in cardiovascular functions, has led us to hypothesize that the NTS PRR may play a part in blood pressure (BP) regulation by modulation of the brain inflammatory pathways. Acute NTS injections of renin (0.02–2 pmol) resulted in a significantly greater dose‐dependent decrease in systolic BP (ΔSBP; −12±5 to −32±5 mmHg) and heart rate (ΔHR; −13±7 to −40±10 bpm) in the SHR compared to the WKY rats (ΔSBP: −3±1 to−8±4 mmHg; ΔHR: −2±4 to −12±7 bpm). These effects were not blocked by the AT1R or AT2R blocker. Consistent with this, chronic PRR knockdown with AAV2‐PRR‐shRNA in the SHR NTS caused an increase in mean arterial pressure (shRNA: 173±5; Control: 151±6 mmHg), and decreases in baroreflex bradycardia (shRNA: 0.26±0.04; Control: 0.44±0.04) and parasympathetic tone (shRNA: 31±4; Control: 49±6). This was accompanied by a decrease in the inflammatory markers TNFa, IL6 and Ccl5, and their transcription factors, NF‐κB and AP‐1. Finally, PRR activation in the SHR brain neuronal cultures activated NF‐κB, and increased the mRNA of IL1β (250 fold), TNFα (32 fold), IL6 (35 fold) and Ccl5 (12 fold) in an NF‐κB‐ and AP1‐dependent, but AT1R/AT2R‐independent manner. Therefore, the NTS PRR mediates Ang II‐independent antihypertensive effects by stimulation of NF‐κB/AP‐1/cytokine signaling (AHA 11SDG7420029)