Pro Renin Receptor Expression Research Articles

Renal tubular (pro)renin receptor deletion does not protect against kidney injury in db/db mice

Background: The (pro)renin receptor (PRR) is a multifunctional protein implicated in blood pressure regulation and kidney fibrosis. Previous studies report enhanced PRR expression in non-diabetic and diabetic kidney disease. In this study, we investigated whether deletion of renal tubular PRR attenuates kidney injury in type 2 diabetes. Methods: Floxed PRR mice were bred with mice expressing Pax8 rtTA and LC1 transgenes and db/db mice (B6.BKS) to obtain renal tubular PRR knockout (KO)-db/db mice. Age matched non-diabetic floxed controls, db/db mice and PRRKO-db/db mice were studied at 16, 20, 26 and 30 weeks of age. To induce PRR deletion, PRRKO-db/db mice were treated with 2 mg/ml doxycycline for 12 days at 8-10 weeks of age. Only male mice were included in this study. Results: All mice survived until the end of the study (N=5-7 mice/group). Compared to controls, db/db mice and PRRKO-db/db mice had higher body weights despite similar food intake throughout the study (p<0.001 by one-way ANOVA). PRRKO-db/db mice had higher urine volume and water intake compared to controls and db/db mice (p <0.01 by ANOVA). Db/db mice had elevated non-fasting blood glucose levels (average: 472 mg/dl at 16 weeks, 292 mg/dl at 30 weeks) while PRR KO-db/db mice had modestly elevated blood glucose levels (average 255 mg/dl at 20 and 30 weeks) relative to controls (average: 150 mg/dl). Urinary albumin excretion was markedly elevated in PRRKO-db/db mice relative to db/db mice and controls throughout the study (950 ug/day vs db/db:145 ug/day, controls: 50 ug/day, p<0.001). At 30 weeks, kidney histology by PAS stain showed minimal tubular injury, glomerulosclerosis or interstitial inflammation among all 3 groups. Interestingly, podocin staining by immunofluorescence was reduced in db/db mice but not PRR-KO db/db mice. Compared to control mice, db/db mice had similar KIM-1, NGAL and collagen-1 mRNA expression in kidney cortex and inner medulla. PRR KO db/db mice had increased cortical KIM-1, NGAL and collagen-I expression compared to control and db/db mice (p<0.01 by ANOVA). Plasma sPRR levels were almost two-fold higher in diabetic mice relative to controls with no difference between db/db mice and PRR KO-db/db mice. Urinary sPRR levels were undetectable in controls and mildly elevated in db/db mice (7.6 ± 0.7 pg/day) and PRR KO-db/db mice (47.6 ± 22.3 pg/ml). No differences in plasma prorenin/renin concentration was noted among the 3 groups while urinary prorenin/renin excretion was higher in PRRKO-db/db mice. Conclusion: Renal tubular deletion of PRR does not protect against kidney injury in type 2 diabetes. It is possible that loss of PRR impairs baseline tubular function which is exacerbated by type 2 diabetes. American Diabetes Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

(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.

Endothelin-1 production via placental (pro)renin receptor in a mouse model of preeclampsia

IntroductionPreeclampsia (PE) pathogenesis is explained by the two-stage disorder theory. However, mechanisms underlying hypertension and proteinuria in PE remain unclear. The role of (pro)renin receptor (PRR) in PE pathology has received special attention. We examined endothelin-1 (ET-1) production via placental PRR in a PE mouse model. MethodsAt 14.5 day-post-coitum (DPC), we performed a reduced uterine perfusion pressure (RUPP) operation, ligating the uterine arteriovenous vessels in female mice. We also infused these mice with a PRR inhibitor, decoy peptide in the handle region of prorenin (HRP) for mice (NH2-RIPLKKMPSV-COOH). At 18.5 DPC, blood, urine, and placenta were collected; fetus and placenta were weighed. We evaluated placental hypoxia using quantitative polymerase chain reaction (PCR), with hypoxia-inducible factor-1α (HIF-1α) as index. We also evaluated PRR, transforming growth factor-β1 (TGF-β1), and ET-1 expression in the placenta using quantitative PCR and western blotting. ET-1 concentration in blood plasma was assessed using enzyme-linked immunosorbent assay. ResultsBlood pressure and proteinuria significantly increased, and fetal and placental weights decreased in RUPP mice. HIF-1α, PRR, TGF-β1, and ET-1 expressions considerably increased in RUPP mice placentas. ET-1 concentration in RUPP mice blood plasma was markedly increased. PRR inhibitor suppressed these changes. DiscussionIn PE model mice that underwent RUPP treatment, placental hypoxia increased PRR and ET-1 expression suggesting a causative relationship between ET-1 and intracellular PRR signaling. RUPP treatment, when combined with HRP, reversed the effect of elevated ET-1 levels in the model. This study may help to elucidate the pathogenesis of PE considering PRR and ET-1.

PGE2 stimulates aldosterone release via cAMP/sPRR/β-catenin pathway in mouse inner medullary collecting duct cells

Increasing evidence showed the generation of aldosterone in the collecting duct (CD), a major site for the synthesis and action of prostaglandin E2 (PGE2). Both aldosterone and PGE2 are stimulated by Na+ depletion or K+ loading or angiotensin II. However, what happens first and the correlation between PGE2 and aldosterone in the CD remains obscure. PGE2 stimulates aldosterone release in the adrenal zona glomerulosa cells via E-prostanoid (EP) receptors by activating the cAMP/cAMP-responsive element-binding pathway. Here, we further tested the hypothesis that the synthesis of aldosterone is stimulated by PGE2 in CD cells and the underlying mechanism. In primary mouse inner medullary CD cells, PGE2 incubation significantly stimulated aldosterone release in parallel with an increase of CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2), (pro)renin receptor (PRR), and prorenin protein expression and upregulation of medium soluble PRR (sPRR), prorenin/renin, and cAMP levels, both of which were attenuated by the EP1 antagonist SC-19220 and the protein kinase C (PKC) inhibitor calphostin C. PGE2-induced release of aldosterone, prorenin/renin, and sPRR was also abolished by the protein kinase A inhibitor H89. Both a PRR antagonist PRO20 and β-catenin inhibitor ICG001 blunted PGE2 or sPRR-induced aldosterone and prorenin/renin secretion. Exogenous sPRR protein incubation not only stimulated the release of aldosterone and prorenin/renin but also reversed PF429242 (an inhibitor of subtilisin kexin isozyme-1, which can block endogenous sPRR generation)-attenuated aldosterone and prorenin/renin secretion. Therefore, we conclude that PGE2 increases CD aldosterone synthesis through the EP1 receptor via the PKC/cAMP/sPRR/β-catenin pathway.

(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.

Nicotine Treatment Resulted in Decreased Protein Expression of Prorenin and Soluble Prorenin Receptor in 3T3‐L1 cells

Over 36.5% of the United States population is affected by obesity, which is a major contributing factor to the rise of hypertension and cardiovascular disease. Furthermore, the CDC reports that over 16 million Americans suffer from nicotine‐related diseases. Nicotine consumption causes an increase in blood pressure and a decrease in adipogenesis. The prorenin receptor (PRR) and its soluble form (sPRR) are newly discovered components of a blood pressure regulatory pathway, the renin angiotensin system (RAS), and are upregulated during times of obesity. Currently, there is no research to understand the contrasting relationship between nicotine and PRR. Therefore, in this present study, we aimed to determine whether nicotine regulates blood pressure through an adipocyte PRR‐dependent.3T3‐L1 cells were grown to confluence and differentiated over 8 days. On day 8, cells were treated with nicotine dose response treatments: vehicle (water), 0.01 µM nicotine, 1 µM nicotine, or 10 µM nicotine. After 24‐hour treatment, cells were collected to measure protein levels of PRR and it soluble form, sPRR. Additionally, gene expression levels of PRR, renin, 18S, furin, and S1P were measured. Media from the cells was collected after 2 hours, 4 hours, and 24 hours of incubation to measure the secretion levels of sPRR into the media.Nicotine significantly decreased PRR protein expression at 1µM (n=2) compared to vehicle treatment (n=6) (vehicle: 1.00 0.37au; 1 µM: 0.46 0.09au; p <0.05). Nicotine did not affect the gene expression of PRR but significantly decreased PRR and sPRR protein levels in a dose response manner compared to vehicle treatment (vehicle: 1.00 0.42au, n=6; 0.01 µM: 0.47 0.18au, n=4, 1uM: 0.32 0.12au, n=2; p <0.05). Nicotine did not change the expression of S1P, and furin, two enzymes involved in PRR cleavage. Finally, renin and angiotensinogen (AGT) increased significantly in differentiated adipocytes treated with nicotine compared to those treated with vehicle. Together our data indicated that nicotine influences local renin angiotensin system by post‐transcriptionally down‐regulating PRR and sPRR protein and by upregulating renin and AGT. Together, these data suggest that nicotine could assert its effect on blood pressure and adipogenesis via an adipocyte‐PRR dependent mechanism.

Inhibitory Potential of Shen-Shuai-Ling Formulation on Renal Interstitial Fibrosis via Upregulation of PLZF.

Background Renal interstitial fibrosis (RIF) is an important cause of kidney disease, which seriously affects people's health. As a traditional Chinese medicine, Shen-Shuai-Ling Formulation (SSLF) has obvious kidney function. However, the therapeutic effect of SSLF on RIF and its molecular mechanism are still unclear. Methods First, the potential targets and pathways of SSLF for RIF were predicted by network pharmacology, and then, the binding of luteolin and target protein to SSLF was verified by molecular docking and Co-IP experiments. Finally, the effects of SSLF and luteolin on PLZF and (Pro) renin receptor (PRR) were verified by western blot and qPCR experiments. Angiotensin (Ang)-1, Ang-2, and transforming growth factor-β (TGF-β) were the indexes of renal interstitial fibrosis. Results Through the drug-active component-target network diagram, we found that luteolin has the most connections, and promyelocytic leukemia zinc finger (PLZF) is the target protein. GO analysis and KEGG pathway analysis of targets were performed using Cytoscape ClueGO. Molecular docking experiments and Co-IP are used to prove that luteolin and PLZF can be combined. Western blot and qPCR results showed that both SSLF and luteolin significantly upregulated the expression of PLZF and decreased the levels of PRR, Ang-1, Ang-2, and TGF-β. The overexpression of PLZF decreased the expression of PRR, the knockdown of PLZF increased the expression of PRR, and the overexpression of PRR decreased the expression of Ang-1, Ang-2, and TGF-β. Conclusions SSLF inhibits PRR and renal interstitial fibers by the upregulation of PLZF levels.

(Pro)renin Receptor Is a Novel Independent Prognostic Marker in Invasive Urothelial Carcinoma of the Bladder.

Simple SummaryThis is a novel description of (Pro)renin receptor (PRR) protein and its prognostic role in invasive urothelial cancer of the bladder. Using a tissue microarray, we investigated PRR expression and other immunohistochemical markers including p53, immune-checkpoint inhibition, and basal and luminal phenotypes in a series of patients with invasive urothelial carcinoma of the bladder treated with radical cystectomy. PRR expression is an independent prognostic marker and could be a potential target in urothelial carcinoma that should be further investigated.(Pro)renin receptor (PRR) is being investigated in several malignancies as it activates pathogenic pathways that contribute to cell proliferation, immunosuppressive microenvironments, and acquisition of aggressive neoplastic phenotypes. Its implication in urothelial cancer (UC) has not been evaluated so far. We retrospectively evaluate the prognostic role of PRR expression in a series of patients with invasive UC treated with radical cystectomy and other clinical and histopathological parameters including p53, markers of immune-checkpoint inhibition, and basal and luminal phenotypes evaluated by tissue microarray. Cox regression analyses using stepwise selection evaluated candidate prognostic factors and disease-specific survival. PRR was expressed in 77.3% of the primary tumors and in 70% of positive lymph nodes. PRR expression correlated with age (p = 0.006) and was associated with lower preoperatively hemoglobin levels. No other statistical association was evidenced with clinical and pathological variables (gender, ASA score, Charlson comorbidity index, grade, pT, pN) or immunohistochemical expressions evaluated (CK20, GA-TA3, CK5/6, CD44, PD-L1, PD-1, B7-H3, VISTA, and p53). PRR expression in primary tumors was associated with worse survival (log-rank, p = 0.008). Cox regression revealed that PRR expression (HR 1.85, 95% CI 1.22–2.8), pT (HR 7.02, 95% CI 2.68–18.39), pN (HR 2.3, 95% CI 1.27–4.19), and p53 expression (HR 1.95, 95% CI 1.1–3.45) were independent prognostic factors in this series. In conclusion, we describe PRR protein and its prognostic role in invasive UC for the first time. Likely mechanisms involved are MAPK/ERK activation, Wnt/β-catenin signaling, and v-ATPAse function.

Abstract MP08: New Function Of Sox6 In Renovascular Hypertension Induced Oxidative Stress And Kidney Damage Through Renin, Prorenin And The (pro)renin Receptor Expression Control.

Introduction: During renovascular hypertension induced by renal artery stenosis (RAStenosis), smooth muscle cells are recruited to produce and release renin compensating for the decrease in renal perfusion. In renovascular hypertension, renin expression increases causing a surge in angiotensin II, oxidative stress and kidney damage. Here we present a new function of the transcription factor Sox6 in kidney injury and oxidative damage during RAStenosis. Methods: We used a mouse model in which Sox6 is knockout specifically in renin expressing cells (Ren1d Cre /Sox6 fl/fl -Sox6-KO). We developed a modified 2-kidney 1-clip (2K1C) Goldblatt model to induce RAStenosis. We measured superoxide production (pmol/mg pr) in kidney using high-performance liquid chromatography in Sox6-KO and Ren1d Cre /Sox6 wt/wt (Sox6-WT) littermates. Renin, prorenin, (pro)renin receptor (PRR) and N-GAL expressions were measured using Western blot using β-actin as housekeeping gene. Western blot band density analysis was performed using ImageJ. Kidney injury was determined by measuring creatinine clearance. Results: We found that Sox6-KO mice exhibit lower expression of renin (0.36 vs. 0.45, SEM= 0.11 n= 11-15, p<0.01), and prorenin (1.25 vs. 2.07, SEM=0.30, n=11-15, p<0.05) and mice are protected against renovascular hypertension (SBP, 114.5 vs. 131.1, SEM=5.31 n=13-15, p<0.01), and kidney injury measuring N-GAL expression (1.3 vs. 2.7, SEM=0.38, n=6-8, p<0.01) and creatinine clearance (2372 vs. 1341, SEM=342.3, n=4-5, p<0.05). Furthermore, we found that the levels of superoxide were significantly lower in Sox6-KO mice compared to Sox6-WT littermates (218 vs 47, SEM=50.6, N=7, P<0.01). The expression of PRR was significantly lower in Sox6-KO compared to Sox6-WT during RAStenosis (1.23 vs. 0.72, SEM=0.33, n=2-5, p<0.01). Conclusions: Our results show that Sox6 depletion in renin expressing cells inhibits the increase in renovascular hypertension and prevents the increase in renin, prorenin, PRR, and N-GAL expression as well as superoxide production, preserving creatinine clearance during RAStenosis. These results suggest that Sox6 has a new function in hypertension and kidney injury induced by RAS.

Abstract MP27: Adipose-derived Human Soluble (Pro)renin Receptor Causes Resistance To Losartan Treatment In High-Fat Diet Male And Female Mice

Obesity, affecting more that 37% of the US, contributes to hypertension. Despite the use of one or more anti-hypertensive treatments, 48% of the hypertensive population remains with resistant hypertension, which prompts the development for new therapeutic targets. We demonstrated that obesity increased the expression of prorenin receptor (PRR) in the adipose tissue and elevated plasma soluble PRR (sPRR). In addition, the infusion of mouse sPRR increased blood pressure in male mice fed high fat-diet (HF); indicating that adipose-derived sPRR could increase circulating sPRR and contribute to hypertension. However, there is a critical gap in the functional role of human sPRR in obesity-hypertension. In this study, we aim to define whether adipose-derived human sPRR contributes to obesity-hypertension. Human sPRR-Myc-tag transgenic mice were bred with mice expressing adiponectin/Cre to selectively express human sPRR in adipocytes (adi-HsPRR). Adi-HsPRR and control littermate (CTL) male and female mice were fed HF-diet for 20 weeks (N=8-15/group). Body weight was assessed weekly and body composition monthly. Blood pressure was measured by telemetry after 15 weeks of diet. Adipose-derived human sPRR did not significantly elevate body weight or fat mass (Male: CTL.18.3±1.0g; adi-HsPRR. 17.5±0.8g. Female: CTL. 15.6±1.5g; adi-HsPRR. 11.9±1.3g; p>0.05). Systolic blood pressure (SBP) significantly increased in HF-fed male and female mice however; adipose-derived human sPRR did not further elevate SBP (24h SBP. Male: CTL. 136.0±1.7 mmHg; adi-HsPRR: 133.4±1.5mmHg; Female: CTL. 131.9±2.8 mmHg; adi-HsPRR: 130.6±3.1 mmHg; p>0.05). Surprisingly, the anti-hypertensive effect of losartan (Los) to lower blood pressure was significantly reduced in adi-HsPRR male and female mice (Male: CTL. ΔSBP: -12.1±1.5 ΔmmHg; adi-HsPRR: -7.8±0.6 ΔmmHg; Female: CTL. ΔSBP: -13.4±1.1 ΔmmHg; adi-HsPRR: -5.7±2.3 ΔmmHg; p<0.05). In 3T3-L1 cells, sPRR significantly increased phosphorylation of ERK1/2, which was not completely blunted by Los indicating that human sPRR could act as a partial agonist of AT1R or activate ERK1/2 independently of AT1R. Our data suggests that adipose-derived sPRR does not stimulate AT1R-mediated contractility, instead impairs Los efficacy.

Expression of Angiotensin II Receptor 2 in Microcystic Lymphatic Malformation

Objectives: To investigate the presence of components of the renin-angiotensin system (RAS) on the embryonic stem cell (ESC)–like population in microcystic lymphatic malformation (mLM). Methods: mLM tissue samples from 18 patients underwent immunohistochemical staining for RAS components including angiotensinogen, renin, prorenin receptor (PRR), angiotensin-converting enzyme (ACE), ACE2, and angiotensin II receptor 2 (AT2R). Snap-frozen mLM tissues from 6 of the patients were used to confirm protein expression by western blotting for angiotensinogen, PRR, ACE, ACE2, and AT2R. Reverse transcription quantitative polymerase chain reaction was used to detect transcript expression of angiotensinogen, renin, PRR, ACE, ACE2, AT1R, and AT2R in 5 of the mLM tissue samples. Results: Immunohistochemical staining demonstrated expression of AT2R in all, and PRR in 1, while angiotensinogen, renin, ACE, and ACE2 were not observed in any of the 18 mLM samples. Western blotting showed expression of angiotensinogen, PRR, and ACE, but not ACE2 or AT2R in all 6 mLM tissue samples. Reverse transcription quantitative polymerase chain reaction demonstrated transcript expression of angiotensinogen, PRR, ACE, and ACE2 in all, AT1R in 4, AT2R in 2, and renin in 1 of the 5 mLM tissue samples. Immunofluorescence dual-staining in 2 mLM tissue samples demonstrated expression of AT2R on the OCT4+ cells. Conclusion: This study shows expression of angiotensinogen, PRR, ACE. ACE2, AT1R, and AT2R transcripts and AT2R protein, in mLM tissue samples, with AT2R localizing to the OCT4+ ESC-like population. This suggests the ESC-like population may be a novel therapeutic target by modulation of the RAS.

α-Ketoglutarate Upregulates Collecting Duct (Pro)renin Receptor Expression, Tubular Angiotensin II Formation, and Na+ Reabsorption During High Glucose Conditions.

Diabetes mellitus (DM) causes high glucose (HG) levels in the plasma and urine. The (pro)renin receptor (PRR) is a key regulator of renal Na+ handling. PRR is expressed in intercalated (IC) cells of the collecting duct (CD) and binds renin to promote angiotensin (Ang) II formation, thereby contributing to Na+ reabsorption. In DM, the Kreb's cycle is in a state of suppression in most tissues. However, in the CD, expression of glucose transporters is augmented, boosting the Kreb's cycle and consequently causing α-ketoglutarate (αKG) accumulation. The αKG receptor 1 (OXGR1) is a Gq-coupled receptor expressed on the apical membrane of IC cells of the CD. We hypothesize that HG causes αKG secretion and activation of OXGR1, which increases PRR expression in CD cells. This effect then promotes intratubular AngII formation and Na+ reabsorption. To test this hypothesis, streptozotocin (STZ)-induced diabetic mice were treated with or without montelukast (ML), an OXGR1 antagonist, for 6 days. STZ mice had higher urinary αKG and PRR expression along with augmented urinary AngII levels and Na+ retention. Treatment with ML prevented all these effects. Similarly, primary cultured inner medullary CD cells treated with HG showed increased PRR expression, while OXGR1 antagonist prevented this effect. αKG increases PRR expression, while treatments with ML, PKC inhibition, or intracellular Ca2+ depletion impair this effect. In silico analysis suggested that αKG binds to mouse OXGR1. These results indicate that HG conditions promote increased levels of intratubular αKG and OXGR1-dependent PRR upregulation, which impact AngII formation and Na+ reabsorption.

Role of autophagy and evaluation the effects of microRNAs 214, 132, 34c and prorenin receptor in a rat model of focal segmental glomerulosclerosis

AimsFocal segmental glomerulosclerosis (FSGS) is the common cause of chronic renal disease worldwide. Although there are many etiologic factors which have common theme of podocyte injury conclusive etiology is not clearly understood. In this study, we aimed to explore the role of autophagy in the pathogenesis of podocyte injury, which is the key point in disease progression, and the roles of intrarenal microRNAs and the prorenin receptor (PRR) in the 5/6 nephrectomy and adriamycin nephropathy models of FSGS. Main methodsFor experimental FSGS model, 5/6 nephrectomy and adriamycin nephropathy models were created and characterized in adult Sprague Dawley rats. Microarray analysis was performed on FSGS and control groups that was confirmed by q-RT-PCR. Beclin1, LC3B, PRR, ATG7 and ATG5 expression were evaluated by western blotting and immunohistochemistry. Also, Beclin1 and PRR expression were measured by ELISA. Glomerular podocyte isolation was performed and autophagic activity was evaluated in podocytes before and after transfection with miRNA mimic and antagonists. Key findingsGlomerular expression of Beclin1, LC3B, PRR, ATG7 and ATG5 were significantly lower in the 5/6 nephrectomy than adriamycin nephropathy group and in both groups lower when compared to control groups. Western blot results were consistent with immunohistochemical data. Electron microscopy revealed signs of impaired autophagy in FSGS. Autophagic activity decreased significantly after miR-214, miR-132 and miR-34c mimics and increased after transfection with antagonists. SignificanceThese results showed that the role of autophagic activity and decreased expression of PRR in FSGS pathogenesis and miR-34c, miR-132 and miR-214 could be a potential treatment strategy by regulating autophagy.

Effects of (Pro)renin Receptor on Diabetic Cardiomyopathy Pathological Processes in Rats via the PRR-AMPK-YAP Pathway.

Diabetic cardiomyopathy (DCM) is a common complication associated with diabetes. The (pro)renin receptor (PRR) is an important member of the local tissue renin-angiotensin system and plays a vital role in many cardiovascular diseases. Yes-associated protein (YAP) also plays a crucial role in many cardiovascular diseases. However, the mechanism responsible for the effects of PRR and YAP on DCM remains unclear. The purpose of this study was to determine the role of PRR in the pathological progression of DCM and whether PRR influences the pathological processes of diabetic cardiomyopathy through YAP. We first established diabetic cardiomyopathy rats model, downregulated the expression of PRR, and upregulated and downregulated the expression of YAP. The levels of myocardial inflammation and fibrosis were then measured and cardiac function was evaluated. In vitro, primary rat cardiac fibroblasts (CFs) were cultured with high glucose, with or without transfection with recombinant adenovirus expressing PRR, and GSK621 was used to observe the effect of AMPK. The levels of inflammation and fibrosis were measured in vitro. The results showed that PRR and YAP silencing alleviated myocardial inflammation and fibrosis. GSK621 blocked the effect of PRR on AMPK and YAP and improved CF inflammation and fibrosis. The inhibition of PRR expression offers a new therapeutic strategy for the treatment of DCM. The effects of PRR on the pathological process of DCM in rats may be mediated via the PRR-AMPK-YAP pathway.

Expression of Components of the Renin-Angiotensin System by Cancer Stem Cells in Renal Clear Cell Carcinoma.

This study investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cells (CSCs) we have recently demonstrated in renal clear cell carcinoma (RCCC). Fifteen RCCC tissue samples underwent immunohistochemical staining for components of the RAS: renin, pro-renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), and angiotensin II receptor 2 (AT2R). Immunofluorescence co-staining or double immunohistochemical staining of these components of the RAS with stemness-associated markers OCT4 or KLF4 was performed on two of the samples. Protein and transcript expression of these components of the RAS in six RCCC tissue samples was investigated using western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, angiotensin II receptor 1 (AT1R) was investigated using RT-qPCR only. Immunohistochemical staining demonstrated expression of renin, PRR, and ACE2 in 11, 13, and 13 out of 15 RCCC samples, respectively, while AT2R was expressed in all 15 samples. ACE was detected in the endothelium of normal vasculature only. Double immunohistochemical staining demonstrated localization of ACE2, but not renin, to the KLF4+ CSCs. Immunofluorescence staining showed localization of PRR and AT2R to the OCT4+ CSCs. Western blotting confirmed protein expression of all components of the RAS except renin. RT-qPCR demonstrated transcript expression of all components of the RAS including AT1R, but not AT2R, in all six RCCC tissue samples. This study demonstrated expression of PRR, ACE2, and AT2R by the CSCs within RCCC. Further studies may lead to novel therapeutic targeting of CSCs by manipulation of the RAS in the treatment of this aggressive cancer.

Silencing of Central (Pro)renin Receptor Ameliorates Salt-Induced Renal Injury in Chronic Kidney Disease

Aims: A high-salt diet can aggravate oxidative stress, and renal fibrosis via the brain and renal renin-angiotensin system (RAS) axis in chronic kidney disease (CKD) rats. (Pro)renin receptor (PRR) plays a role in regulating RAS and oxidative stress locally. However, whether central PRR regulates salt-induced renal injury in CKD remains undefined. Here, we hypothesized that the reduction of central PRR expression could ameliorate central lesions and thereby ameliorate renal injury in high-salt-load CKD rats. Results: We investigated RAS, sympathetic nerve activity, oxidative stress, inflammation, and tissue injury in subfornical organs and kidneys in high-salt-load 5/6 nephrectomy CKD rats after the silencing of central PRR expression by intracerebroventricular lentivirus-RNAi. We found that the sympathetic nerve activity was reduced, and the levels of inflammation and oxidative stress were decreased in both brain and kidney. Renal injury and fibrosis were ameliorated. To explore the mechanism by which central inhibition of PRR expression ameliorates kidney damage, we blocked central MAPK/ERK1/2 and PI3K/Akt signaling pathways as well as angiotensin converting enzyme 1-angiotensin II-angiotensin type 1 receptors (ACE1-Ang II-AT1R) axis. Salt-induced overexpression of renal RAS, inflammation, oxidative stress, and fibrosis in CKD rats were prevented by central blockade of the pathways. Innovation: This study provides new insights into the mechanisms underlying salt-induced kidney damage. Targeting central PRR or PRR-mediated signaling pathway may be a novel strategy for the treatment of CKD. Conclusions: These results suggested that the silencing of central PRR expression ameliorates salt-induced renal injury in CKD through Ang II-dependent and -independent pathways.

Clinical Implications of (Pro)renin Receptor (PRR) Expression in Renal Tumours

(1) Background: Renal cancer is one of the most frequent malignancies in Western countries, with an unpredictable clinical outcome, partly due to its high heterogeneity and the scarcity of reliable biomarkers of tumour progression. (Pro)renin receptor (PRR) is a novel receptor of the renin–angiotensin system (RAS) that has been associated with the development and progression of some solid tumours by RAS-dependent and -independent mechanisms. (2) Methods: In this study, we analysed the immunohistochemical expression of PRR at the centre and border in a series of 83 clear-cell renal cell (CCRCCs), 19 papillary (PRCC) and 7 chromophobe (ChRCC) renal cell carcinomas, and the benign tumour renal oncocytoma (RO, n = 11). (3) Results: PRR is expressed in all the tumour subtypes, with higher mean staining intensity in ChRCCs and ROs. A high expression of PRR at the tumour centre and at the infiltrative front of CCRCC tissues is significantly associated with high grade, tumour diameter, local invasion and stage, and with high mortality risk by UCLA integrated staging system (UISS) scale. (4) Conclusions: These findings indicate that PRR is associated with the development and progression of renal tumours. Its potential as a novel biomarker for RCC diagnosis/prognosis and as a promising therapeutic target should be taken into account in the future.

Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System.

We investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cell (CSC) subpopulations in metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC). Immunohistochemical staining demonstrated expression of prorenin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all cases and angiotensinogen in 14 cases; however, renin and ACE2 were not detected in any of the 20 mHNcSCC tissue samples. Western blotting showed protein expression of angiotensinogen in all six mHNcSCC tissue samples, but in none of the four mHNcSCC-derived primary cell lines, while PRR was detected in the four cell lines only. RT-qPCR confirmed transcripts of angiotensinogen, PRR, ACE, and angiotensin II receptor 1 (AT1R), but not renin or AT2R in all four mHNcSCC tissue samples and all four mHNcSCC-derived primary cell lines, while ACE2 was expressed in the tissue samples only. Double immunohistochemical staining on two of the mHNcSCC tissue samples showed expression of angiotensinogen by the SOX2+ CSCs within the tumor nests (TNs), and immunofluorescence showed expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the peritumoral stroma (PTS). ACE was expressed on the endothelium of the tumor microvessels within the PTS. We demonstrated expression of angiotensinogen by CSCs within the TNs, PRR, and AT2R by the CSCs within the TNs and the PTS, in addition to ACE on the endothelium of tumor microvessels in mHNcSCC.

(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.

CBMS-12 Pro renin receptor antibody regulates glioblastoma stemness

Objective: Glioblastoma multiforme (GBM) is characterized by a strong self-renewal potential and poor differentiated state. We previously reported that (pro)renin receptor (PRR) was a potential target for glioma therapy by silencing the gene of PRR. Here, we have developed the monoclonal antibody of PRR and examined their effects on GBM. Materials and methods: We performed immunohistochemical analysis to detect the protein expression of PRR and SOX-2 in human sample of 56 gliomas. We used human glioma cell lines (U251MG and U87MG) and glioma stem cell line (MGG23) in vitro study. PRR antibody was designed to target the extracellular domain of the PRR with the rat lymph node method. Expression of the Wnt signaling components and stem marker (SOX-2, Oct3/4) in human glioma cell lines and glioma stem cell line treated with PRR antibody were measured using Western blotting. The effects of PRR antibody on cell proliferation, sphere formation, apoptosis, invasion were also examined. Subcutaneous xenografts with U87MG were induced in nude mice. Results: PRR expression showed a positive correlation with SOX-2 expression in glioma samples. Treatment with PRR antibody significantly reduced expression of Wnt signaling components and stem marker. We observed that PRR antibody significantly reduced cell proliferation and decreased sphere formation. Furthermore, PRR antibody suppressed invasion and induced apoptosis. In a subcutaneous U87MG xenograft model, systemic administration of the PRR antibody significantly reduced the size of the tumor volume. Conclusion: PRR has important role for the maintenance of stem cells and contribute to stem cell proliferation. PRR antibody inhibits cell proliferation and cell invasion and induces apoptosis. Treatment with PRR antibody could be an attractive therapeutic strategy for GBM.