Apelin-13: a novel approach to suppressing renin production in RVHT.

Renovascular hypertension (RVHT) is characterized by renal artery stenosis and overactivated renin-angiotensin system (RAS). Apelin, known for its negative modulation of RAS, has protective effects against cardiovascular diseases. The role and mechanisms of the primary active form of apelin, apelin-13, in RVHT are unclear. In this study, male Sprague-Dawley rats were divided into control, two-kidney one-clip (2K1C) model, and 2K1C with apelin-13 treatment groups. Renin expression was analyzed using immunohistochemistry and molecular techniques. Full-length (pro)renin receptor (fPRR) and soluble PRR (sPRR) levels were assessed via Western blotting, and cAMP levels were measured using ELISA. Plasma renin content, plasma renin activity (PRA), angiotensin II (ANG II), and sPRR levels were determined by ELISA. Human Calu-6 and mouse As4.1 cells were used to investigate renin production mechanisms. The 2K1C model exhibited increased systolic blood pressure, plasma renin content, PRA, sPRR, and ANG II levels, while apelin-13 treatment reduced these elevations. Apelin-13 inhibited cAMP production, renin mRNA expression, protein synthesis, and PRR/sPRR protein expression in renal tissue. In Calu-6 cells, cAMP-induced fPRR and site-1 protease (S1P)-derived sPRR expression, which was blocked by cAMP-responsive element-binding protein (CREB) inhibition. Apelin-13 suppressed cAMP elevation, CREB phosphorylation, fPRR/sPRR protein expression, and renin production. Recombinant sPRR (sPRR-His) stimulated renin production, which was inhibited by the PRR decoy peptide PRO20 and S1P inhibitor PF429242. These findings suggest that apelin-13 inhibits plasma renin expression through the cAMP/PKA/sPRR pathway, providing a potential therapeutic approach for RVHT. Understanding the regulation of renin production is crucial for developing effective treatments.NEW & NOTEWORTHY Our research elucidated that apelin-13 inhibits renin production through the cAMP/PKA/soluble (pro)renin receptor pathway, presenting a promising therapeutic approach for renovascular hypertension (RVHT) by targeting renin expression mechanisms. These findings underscore the potential of apelin-13 as a novel strategy to address RVHT.

C3aR Antagonist Alleviates C3a Induced Tubular Profibrotic Phenotype Transition via Restoring PPARα/CPT-1α Mediated Mitochondrial Fatty Acid Oxidation in Renin-Dependent Hypertension.

BACKGROUND: Renin-dependent hypertension with tubulointerstitial injury remains a problem with high prevalence in the clinic. However, whether and how renin participates in tubulointerstitial injury remains incompletely understood. New evidence suggests that renin cleaves C3 into C3a and C3b. In the present study, we aimed to explore the role of renin-mediated C3a/C3a receptor (C3aR) signaling in renin-dependent hypertension-induced kidney injury and illustrate the detailed mechanisms. METHODS: C3a concentration changes in serum from healthy volunteers incubated with recombinant renin were detected by ELISA. C3aR expression in human tubular epithelial cells was evaluated in renal biopsy sections from malignant arteriolonephrosclerosis and benign arteriolonephrosclerosis patients. C3aR changes in human kidney 2 (HK2) cells were detected after the cells were treated with human serum, renin and aliskiren. The C3a analogue and C3aR antagonist SB290157 were used to stimulate HK2 cells to explore the downstream signaling of C3a/C3aR activation. For in vivo studies, two-kidney, one-clipped (2K1C) hypertensive rat model was established to simulate renin-dependent hypertension conditions. C3a and C3aR expression was detected in the clipped kidneys. SB290157 was injected intraperitoneally to block C3a/C3aR signaling in 2K1C rats. RESULTS: The results showed that renin cleaved C3 into C3a and activated C3a/C3aR signaling in tubular epithelial cells (TECs) from both humans and rats. In vitro results demonstrated that C3a/C3aR activation impaired peroxisome proliferator-activated receptor alpha (PPARα)/carnitine palmitoyltransterase-1alpha (CPT-1α)-mediated mitochondrial fatty acid oxidation (Mito FAO) in HK2 cells and induced HK2 cell transition to a profibrotic phenotype, which was inhibited by treatment with the C3aR antagonist SB290157. In vivo results showed that renin mRNA levels, C3a concentrations, C3aR levels and tubulointerstitial fibrosis increased concurrently in the clipped kidney cortex of 2K1C rats. Treatment with the C3aR antagonist SB290157 significantly mitigated the effect of renin induction of C3aR expression and alleviated renin-dependent hypertension-induced tubulointerstitial fibrosis by improving PPARα/CPT-1α-mediated Mito FAO in TECs, as well as inhibiting tubular profibrotic phenotype transition. CONCLUSIONS: Our results prove that renin activates C3a/C3aR signaling to promote renal tubulointerstitial fibrosis by impairing PPARα/CPT-1α-mediated tubular Mito FAO. SB290157 confers a potential therapeutic approach for renin-dependent hypertension-induced kidney injury.

Polycystic kidneys: interaction of notch and renin.

Polycystic kidney disease (PKD) is a developmental disorder, which either manifests in early childhood or later in life, depending on the genetic mutation one harbors. The mechanisms of cyst initiation are not well understood. Increasing literature is now suggesting that Notch signaling may play a critical role in PKD. Activation of Notch signaling is important during nephrogenesis and slows down after development. Deletion of various Notch molecules in the cap mesenchyme leads to formation of cysts and early death in mice. A new study by Belyea et al. has now found that cells of renin lineage may link Notch expression and cystic kidney disease. Here, we use our understanding of Notch signaling and PKD to speculate about the significance of these interactions.

The effect of tannic acid on renal renin-angiotensin signaling pathway in a model of unilateral ureteral obstruction in male Wistar rats.

The purpose of current study was to elucidate polyphenol tannic acid effect on renal function and activity of the renin-angiotensin system after unilateral ureteral obstruction (UUO). Male Wistar rats were divided into three groups of six randomly: 1) Sham, 2) UUO, and 3) UUO + Tannic acid. Rats in the UUO and UUO + Tannic acid groups experienced unilateral ureteral obstruction. In the Sham group, the abdominal cavity was exposed without UUO induction. In the UUO + Tannic acid group, animals received tannic acid (20 mg/kg) intraperitoneally, 6 and 12 h after clamping the left ureter and 6 and 12 h after the right nephrectomy. Blood samples were taken to measure blood urea nitrogen (BUN) and creatinine levels. Kidney tissue samples were obtained for assessment of oxidative stress, inflammatory indices and the levels of renin-angiotensin system components. Tannic acid administration significantly improved UUO-induced kidney dysfunction (serum BUN: 66.42 ± 14.414 mg/dl, p < 0.05; serum creatinine: 1.67 ± 0.258 mg/dl, p < 0.05), oxidative stress (MDA level: 95.29 ± 37.35 µmol/g tissue, p < 0.05; SOD activity: 59.82 ± 13.41 U/g protein, p < 0.01) and inflammation (renal TNF-α: 57.05 ± 15.653 pg/g tissue, p < 0.05; renal IL-6: 117.015 ± 24.076 pg/g tissue, p < 0.001). The treatment caused a reduction in the amount of renal angiotensinogen, renin and ACE genes expression compared to the UUO group (Angiotensinogen: 8.9 ± onefold, p < 0.05, Renin: 6.5 ± 1.14 fold, p < 0.05, ACE: 4.9 ± 0.64 fold, p < 0.05). Angiotensin II type 1 receptor protein levels decreased in the tannic acid-treated rats in comparison with the UUO group (0.61 ± 0.136, p < 0.05). According to the result of the current study, tannic acid considerably attenuated the complications of unilateral ureteral obstruction through renin-angiotensin system modulation. Trial registration: IR.TUMS.MEDICINE.REC.1400.802.

Sex differences in blood pressure, free radicals and plasma cholesterol fractions in Ren-2 transgenic rats of various ages.

Sex-related cardiovascular differences were observed in humans as well as in experimental animals. Our previous study demonstrated a marked sexual dimorphism in blood pressure (BP) of 9-month-old heterozygous transgenic Ren 2 rats (TGR), in which mouse Ren-2 renin gene was inserted into the genome of normotensive Hannover Sprague-Dawley rats (HanSD). We found significantly elevated BP only in male TGR, whereas BP of TGR females was similar to that of HanSD females. The aim of our present study was to compare BP of 3- and 6-month-old heterozygous TGR with age- and sex-matched HanSD under the same conditions as we measured in 9-month-old rats. We also monitored the amount of oxidative stress marker, thiobarbituric acid-reactive substances (TBARS), and a main intracellular antioxidant, reduced glutathione in the heart, kidneys and liver. We also measured plasma triglycerides and cholesterol levels. We found an increased mean arterial pressure in both female and male 3-month-old TGR (172±17 vs. 187±4 mm Hg, respectively) compared to HanSD (115±5 vs. 133±3 mm Hg, respectively) but there was a marked sexual dimorphism of 6 month-old TGR where only males were hypertensive (145±5 mm Hg) while females became normotensive (123±7 mm Hg). We did not find any relationship between BP values and concentrations of TBARS or glutathione or plasma lipid levels. Our results demonstrated that 6-month-old TGR exhibited a marked sexual BP dimorphism, which was not dependent on the abnormalities in oxidative stress or cholesterol metabolism.

Overexpression of notch signaling in renin cells leads to a polycystic kidney phenotype.

Polycystic kidney disease (PKD) is an inherited disorder that results in large kidneys, numerous fluid-filled cysts, and ultimately end-stage kidney disease. PKD is either autosomal dominant caused by mutations in PKD1 or PKD2 genes or autosomal recessive caused by mutations in the PKHD1 or DZIP1L genes. While the genetic basis of PKD is known, the downstream molecular mechanisms and signaling pathways that lead to deregulation of proliferation, apoptosis, and differentiation are not completely understood. The Notch pathway plays critical roles during kidney development including directing differentiation of various progenitor cells, and aberrant Notch signaling results in gross alternations in cell fate. In the present study, we generated and studied transgenic mice that have overexpression of an intracellular fragment of mouse Notch1 ('NotchIC') in renin-expressing cells. Mice with overexpression of NotchIC in renin-expressing cells developed numerous fluid-filled cysts, enlarged kidneys, anemia, renal insufficiency, and early death. Cysts developed in both glomeruli and proximal tubules, had increased proliferation marks, and had increased levels of Myc. The present work implicates the Notch signaling pathway as a central player in PKD pathogenesis and suggests that the Notch-Myc axis may be an important target for therapeutic intervention.

Renin-a in the Subfornical Organ Plays a Critical Role in the Maintenance of Salt-Sensitive Hypertension.

The brain renin-angiotensin system plays important roles in blood pressure and cardiovascular regulation. There are two isoforms of prorenin in the brain: the classic secreted form (prorenin/sREN) encoded by renin-a, and an intracellular form (icREN) encoded by renin-b. Emerging evidence indicates the importance of renin-b in cardiovascular and metabolic regulation. However, the role of endogenous brain prorenin in the development of salt-sensitive hypertension remains undefined. In this study, we test the hypothesis that renin-a produced locally in the brain contributes to the pathogenesis of hypertension. Using RNAscope, we report for the first time that renin mRNA is expressed in several regions of the brain, including the subfornical organ (SFO), the paraventricular nucleus of the hypothalamus (PVN), and the brainstem, where it is found in glutamatergic, GABAergic, cholinergic, and tyrosine hydroxylase-positive neurons. Notably, we found that renin mRNA was significantly elevated in the SFO and PVN in a mouse model of DOCA-salt-induced hypertension. To examine the functional importance of renin-a in the SFO, we selectively ablated renin-a in the SFO in renin-a-floxed mice using a Cre-lox strategy. Importantly, renin-a ablation in the SFO attenuated the maintenance of DOCA-salt-induced hypertension and improved autonomic function without affecting fluid or sodium intake. Molecularly, ablation of renin-a prevented the DOCA-salt-induced elevation in NADPH oxidase 2 (NOX2) in the SFO without affecting NOX4 or angiotensin II type 1 and 2 receptors. Collectively, our findings demonstrate that endogenous renin-a within the SFO is important for the pathogenesis of salt-sensitive hypertension.

Structural and dynamic investigation of non-synonymous variations in Renin-AGT complex revealed altered binding via hydrogen-bonding network reprogramming to accelerate the hypertension pathway.

Hypertension is one of the major issues worldwide and one of the main factors involved in heart and kidney failure. Angiotensinogen and renin are key components of the renin-angiotensin-aldosterone system, which plays an indispensable role in hypertension. The aim of this study was to find out the non-synonymous mutations and structure-based mutation-function correlation in the renin-AGT complex and reveal the most deleterious mutations to accelerated hypertension. In the current study, we employed computational modeling and molecular simulation approaches to demonstrate the impact of specific mutations in the REN-AGT interface in hypertension. Computational algorithms, that is, PhD-SNP, PolyPhen-1, MAPP, Sorting Intolerant from Tolerant, Screening of non-acceptable polymorphism, PredictSNP, PolyPhen-2, and Protein Analysis Through Evolutionary Relationships predicted 20 mutations as deleterious in AGT while only five mutations were confirmed as deleterious in the renin protein. Investigation of the bonding analysis revealed that two mutations S107L and V193F in renin altered the hydrogen-bonding paradigm at the interface site. Furthermore, exploration of structural-dynamic behaviors demonstrated by that these mutations also increases the structural stability to regulate the expression of disease pathway. The flexibility index of each residues and structural compactness analysis further validated the findings by portraying the difference in the dynamic behavior in contrast to the wild type. Binding energy calculations based on molecular mechanics/generalized Born surface area methods were used which further established the binding differences between the wild type, S107L, and V193F mutant variants. The total binding energy for wild type, S107L, and V193F was reported to be -27.79, -47.72, and -38.25, respectively. In conclusion, these two mutations increase the binding free energy alongside the docking score to enhance the binding between renin and AGT to overexpress this pathway in a hypertension disease condition. Patients with these mutations may be screened for potential therapeutic intervention.

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.

Overexpression of Renin-B Induces Warburg-like Effects That Are Associated with Increased AKT/mTOR Signaling.

The classical secretory renin-a is known to be involved in angiotensin generation, thereby regulating not only blood pressure, but also promoting oxidative stress as well as apoptotic and necrotic cell death. In contrast, another cytosolic renin isoform named renin-b has been described, exerting protective effects under ischemia-related conditions in H9c2 cardiomyoblasts. Using microarray-based transcriptome analyses, we aimed to identify the signaling pathways involved in mediating cardioprotection in H9c2 cells overexpressing renin-b. By transcriptome profiling, we identified increased gene expression of several genes encoding glycolytic enzymes and glucose transporters, while the transcript levels of TCA-cycle enzymes were decreased. Complementing data from metabolic analyses revealed enhanced glucose consumption and lactate accumulation due to renin-b overexpression. Renin-b overexpression further stimulated AKT/mTOR signaling, where numerous genes involved in this pathway showed altered transcript levels. For AKT, we also detected enhanced phosphorylation levels by means of Western blotting, suggesting an activation of this kinase. Moreover, analysis of the ROS levels identified an increase in ROS accumulation in renin-b-overexpressing cells. Altogether, our data demonstrate that renin-b overexpression induces the metabolic remodeling of H9c2 cells similar to that seen under oxygen deprivation. This metabolic phenotype exerting so-called aerobic glycolysis is also known as the Warburg effect.

Role of the prorenin receptor in endometrial cancer cell growth.

Endometrial cancer is the most diagnosed gynecological malignancy. Despite numerous scientific advances, the incidence and mortality rate of endometrial cancer continues to rise. Emerging evidence suggests a putative role of the (pro)renin receptor ((P)RR), in the ontogenesis of endometrial cancer. The (P)RR is implicated in breast cancer and pancreatic carcinoma pathophysiology by virtue of its role in proliferation, angiogenesis, fibrosis, migration and invasion. Thus, we aimed to investigate the functional role of the (P)RR in human endometrial cancer. We employed an siRNA-mediated knockdown approach to abrogate (P)RR expression in the endometrial epithelial cell lines; Ishikawa, AN3CA and HEC-1-A and examined cellular proliferation and viability. We also carried out a sophisticated proteomic screen to explore potential pathways via which the (P)RR is acting in endometrial cancer physiology. These data confirmed that the (P)RR is critical for endometrial cancer development, contributing to both its proliferative capacity and in the maintenance of cell viability. This is likely mediated through proteins such as MGA, SLC4A7, SLC7A11 or DHRS2, which were reduced following (P)RR knockdown. These putative protein interactions/pathways, which rely on the presence of the (P)RR, are likely to contribute to endometrial cancer progression and could therefore, represent several novel therapeutic targets for endometrial cancer.

A systematic review of nonsynonymous single nucleotide polymorphisms in the renin-angiotensin-aldosterone system.

In this recent publication review the authors aimed to collect evidence of impact of nonsynonymous single nucleotide polymorphisms (nsSNP) in the renin-angiotensin-aldosterone system on patients' phenotype not only regarding arterial hypertension and its complications, but also the impact on other diseases of interest outside the field of cardiovascular medicine. PubMed database records published between 2017-2020 were searched and all positive case-control studies or positive studies with human DNA were selected. The search identified 104 articles, of which 22 were included on the basis of the inclusion criteria. This paper presents the impact of 44 nsSNPs in panels for genes of renin, angiotensinogen, angiotensin-converting enzyme, angiotensin receptor and aldosterone on the clinical picture of investigated cohorts or on the peptide-protein interactions as consequence of nsSNPs. Genetic variability in nsSNPs of the RAAS is involved in the pathogenesis of arterial hypertension and its complications, and surprisingly also in the pathogenesis of conditions not associated with elevated blood pressure, like neoplasms or inflammatory diseases.

The effect of spironolactone on cardiac and renal fibrosis following myocardial infarction in established hypertension in the transgenic Cyp1a1Ren2 rat.

AIMS: The renin-angiotensin-aldosterone axis plays a key role in mediating cardiac and kidney injury. Mineralocorticoid receptor antagonism has beneficial effects on cardiac dysfunction, but effects are less well quantified in the cardiorenal syndrome. This study investigated cardiac and kidney pathophysiology following permanent surgical ligation to induce myocardial infarction (MI) in hypertensive animals with or without mineralocorticoid receptor antagonism. METHODS: Hypertension was induced in adult male Cyp1a1Ren2 rats. Hypertensive animals underwent MI surgery (n = 6), and were then treated daily with spironolactone for 28 days with serial systolic blood pressure measurements, echocardiograms and collection of urine and serum biochemical data. They were compared to hypertensive animals (n = 4), hypertensive animals treated with spironolactone (n = 4), and hypertensive plus MI without spironolactone (n = 6). Cardiac and kidney tissue was examined for histological and immunohistochemical analysis. RESULTS: MI superimposed on hypertension resulted in an increase in interstitial cardiac fibrosis (p<0.001), renal cortical interstitial fibrosis (p<0.01) and glomerulosclerosis (p<0.01). Increased fibrosis was accompanied by myofibroblast and macrophage infiltration in the heart and the kidney. Spironolactone post-MI, diminished the progressive fibrosis (p<0.001) and inflammation (myofibroblasts (p<0.05); macrophages (p<0.01)) in both the heart and the kidney, despite persistently elevated SBP (182±19 mmHg). Despite the reduction in inflammation and fibrosis, spironolactone did not modify ejection fraction, proteinuria, or renal function when compared to untreated animals post MI. CONCLUSION: This model of progressive cardiorenal dysfunction more closely replicates the clinical setting. Mineralocorticoid receptor blockade at a clinically relevant dose, blunted progression of cardiac and kidney fibrosis with reduction in cardiac and kidney inflammatory myofibroblast and macrophage infiltration. Further studies are underway to investigate the combined actions of angiotensin blockade with mineralocorticoid receptor blockade.

Intermittent Hypoxia Upregulates the Renin and Cd38 mRNAs in Renin-Producing Cells via the Downregulation of miR-203.

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and it is a known risk factor for hypertension. The upregulation of the renin-angiotensin system has been reported in IH, and the correlation between renin and CD38 has been noted. We exposed human HEK293 and mouse As4.1 renal cells to experimental IH or normoxia for 24 h and then measured the mRNA levels using a real-time reverse transcription polymerase chain reaction. The mRNA levels of Renin (Ren) and Cd38 were significantly increased by IH, indicating that they could be involved in the CD38-cyclic ADP-ribose signaling pathway. We next investigated the promotor activities of both genes, which were not increased by IH. Yet, a target mRNA search of the microRNA (miRNA) revealed both mRNAs to have a potential target sequence for miR-203. The miR-203 level of the IH-treated cells was significantly decreased when compared with the normoxia-treated cells. The IH-induced upregulation of the genes was abolished by the introduction of the miR-203 mimic, but not the miR-203 mimic NC negative control. These results indicate that IH stress downregulates the miR-203 in renin-producing cells, thereby resulting in increased mRNA levels of Ren and Cd38, which leads to hypertension.

Kidney Response to Chemotherapy-Induced Heart Failure: mRNA Analysis in Normotensive and Ren-2 Transgenic Hypertensive Rats.

The aim of the present study was to perform kidney messenger ribonucleic acid (mRNA) analysis in normotensive, Hannover Sprague-Dawley (HanSD) rats and hypertensive, Ren-2 renin transgenic rats (TGR) after doxorubicin-induced heart failure (HF) with specific focus on genes that are implicated in the pathophysiology of HF-associated cardiorenal syndrome. We found that in both strains renin and angiotensin-converting enzyme mRNA expressions were upregulated indicating that the vasoconstrictor axis of the renin-angiotensin system was activated. We found that pre-proendothelin-1, endothelin-converting enzyme type 1 and endothelin type A receptor mRNA expressions were upregulated in HanSD rats, but not in TGR, suggesting the activation of endothelin system in HanSD rats, but not in TGR. We found that mRNA expression of cytochrome P-450 subfamily 2C23 was downregulated in TGR and not in HanSD rats, suggesting the deficiency in the intrarenal cytochrome P450-dependent pathway of arachidonic acid metabolism in TGR. These results should be the basis for future studies evaluating the pathophysiology of cardiorenal syndrome secondary to chemotherapy-induced HF in order to potentially develop new therapeutic approaches.

Rivaroxaban attenuates cardiac hypertrophy by inhibiting protease-activated receptor-2 signaling in renin-overexpressing hypertensive mice.

Rivaroxaban (Riv), a direct factor Xa (FXa) inhibitor, exerts anti-inflammatory effects in addition to anticoagulation. However, its role in cardiovascular remodeling is largely unknown. We tested the hypothesis that Riv attenuates the progression of cardiac hypertrophy and fibrosis induced by continuous activation of the renin-angiotensin system (RAS) in renin-overexpressing hypertensive transgenic (Ren-Tg) mice. We treated 12-week-old male Ren-Tg and wild-type (WT) mice with a diet containing Riv (12 mg/kg/day) or a regular diet for 4 weeks. After this, FXa in plasma significantly increased in Ren-Tg mice compared with WT mice, and Riv inhibited this increase. Left ventricular wall thickness (LVWT) and the area of cardiac fibrosis evaluated by Masson's trichrome staining were greater in Ren-Tg mice than in WT mice, and Riv decreased them. Cardiac expression levels of the protease-activated receptor (PAR)-2, tumor necrosis factor-α, transforming growth factor (TGF)-ß1, and collagen type 3 α1 (COL3A1) genes were all greater in Ren-Tg mice than in WT mice, and Riv attenuated these increases. To investigate the possible involvement of PAR-2, we treated Ren-Tg mice with a continuous subcutaneous infusion of 10 µg/kg/day of the PAR-2 antagonist FSLLRY for 4 weeks. FSLLRY significantly decreased LVWT and cardiac expression of PAR-2, TGF-ß1, and COL3A1. In isolated cardiac fibroblasts (CFs), Riv or FSLLRY pretreatment inhibited the FXa-induced increase in the phosphorylation of extracellular signal-regulated kinases. In addition, Riv or FSLLRY inhibited FXa-stimulated wound closure in CFs. Riv exerts a protective effect against cardiac hypertrophy and fibrosis development induced by continuous activation of the RAS, partly by inhibiting PAR-2.

Vitamin D decreases silencer methylation to downregulate renin gene expression.

Renin, encoded by REN, is an essential enzyme in the renin-angiotensin aldosterone system (RAAS) which is responsible for the maintenance of blood pressure homeostasis. Transcriptional regulation of REN has been linked to enhancer-promoter crosstalk, cAMP response element-binding protein (CREB), the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and a less well-characterized intronic silencer element. We hypothesized that in addition to these, differential DNA methylation is linked to REN expression and influenced by 1,25(OH)2D3. REN expressing cells (HEK293) were used to elucidate the effect of 1,25(OH)2D3 on REN methylation and expression as quantified by methylation-sensitive qPCR and RT-qPCR, respectively. In vitro 1,25(OH)2D3 supplementation (10 nM) induced significant hypomethylation of the REN silencer (P < 0.050), which was linked to a significant reduction in REN expression (P < 0.010) but had no effect on enhancer methylation. In addition, 1,25(OH)2D3 increased VDR (P < 0.05), as well as TET1 (P < 0.05) expression, suggesting an association between 1,25(OH)2D3 and DNA methylation. Thus, it appears that the silencer element, which is controlled by DNA methylation and influenced by 1,25(OH)2D3, plays an essential role in regulating REN expression.

Activation of (pro)renin by (pro)renin receptor in extracellular vesicles from osteoclasts.

The (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin. To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the "handle region peptide" from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.

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.

Confined placental mosaicism involving multiple de novo copy number variants associated with fetal growth restriction: A case report.

The presence of multiple large (>1 Mb) copy number variants (CNVs) in non-malignant tissue is rare in human genetics. We present a liveborn male with a birth weight below the first percentile associated with placental mosaicism involving eight 2.4-3.9 Mb de novo duplications. We found that the duplications likely co-localized to the same cells, were mosaic in the placenta, and impacted maternal and paternal chromosomes. In addition, 27.4 Mb and 240 genes were duplicated in affected cells, including candidate placental genes KISS1 and REN. We ruled out involvement of homologous recombination-based mechanisms or an altered epigenome in generating the CNVs. This case highlights the diversity of genetic abnormalities in the human placenta and the gaps in our knowledge of how such errors arise.