Pro Renin Receptor Expression Research Articles

Effect of oxidized low-density lipoprotein on the expression of the prorenin receptor in human aortic smooth muscle cells.

The activation of the (pro)renin receptor (PRR) may be potentially active in the development of atherosclerotic plaques independent of angiotensin II. Our previous studies demonstrated that high glucose was able to induce the activation of PRR. The present study was designed to determine the function of oxidized low‑density lipoprotein (ox‑LDL) on the expression of PRR in human aortic smooth muscle cells (HASMCs). Immunofluorescence revealed that PRR was expressed in HASMCs. HASMCs were cultured with 100µg/ml ox‑LDL for 1, 2, 4, 6, 12 and 24h, respectively. Subsequently, HASMCs were cultured with 25, 50, 100, 150, 200 and 300µg/ml ox‑LDL for 6h, respectively. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis revealed that the expression of PRR was markedly upregulated in a time‑ and concentration‑dependent manner, which peaked at 6h and 50µg/ml, then slowly decreased. Therefore, PRR may contribute to the atherogenesis effect induced by ox‑LDL.

DJ-1 regulates the expression of renal (pro)renin receptor via reactive oxygen species-mediated epigenetic modification

BackgroundDJ-1 protein plays multifunctional roles including transcriptional regulation and scavenging oxidative stress; thus, it may be associated with the development of renal disorders. We investigated whether DJ-1 protein regulates the expression of (pro)renin receptor (PRR), a newly identified member of renin–angiotensin system. MethodsThe levels of mRNA and protein were determined by real-time PCR and western blot, respectively. H2O2 production was tested by using fluorescence probe. Histone modification was determined by chromatin immunoprecipitation. ResultsThe expression of PRR was significantly higher in the kidney from DJ-1 knockout mice (DJ-1−/−) compared with wild-type mice (DJ-1+/+). Histone deacetylase 1 recruitment at the PRR promoter was lower, and histone H3 acetylation and RNA polymerase II recruitment were higher in DJ-1−/− than in DJ-1+/+. Knockdown or inhibition of histone deacetylase 1 restored PRR expression in mesangial cells from DJ-1+/+. H2O2 production was greater in DJ-1−/− cells compared with DJ-1+/+ cells. These changes in PRR expression and epigenetic modification in DJ-1−/− cells were induced by H2O2 treatment and reversed completely by addition of an antioxidant reagent. Prorenin-stimulated ERK1/2 phosphorylation was greater in DJ-1−/− than in DJ-1+/+ cells and this was inhibited by a PRR-inhibitory peptide, and by AT1 and AT2 receptor inhibitors. The expression of renal fibrotic genes was higher in DJ-1−/− than in DJ-1+/+ cells and decreased in PRR-knockdown DJ-1−/− cells. ConclusionsWe conclude that DJ-1 protein regulates the expression of renal PRR through H2O2-mediated epigenetic modification. General significanceWe suggest that renal DJ-1 protein may be an important molecule in the acceleration of renal pathogenesis through PRR regulation.

Indoxyl sulfate-induced activation of (pro)renin receptor promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs.ConclusionIS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

Prostaglandin E-prostanoid4 receptor mediates angiotensin II-induced (pro)renin receptor expression in the rat renal medulla.

Angiotensin II (Ang II) stimulates (pro)renin receptor (PRR) expression in the renal collecting duct, triggering the local renin response in the distal nephron. Our recent study provided evidence for involvement of cyclooxygenase-2-prostaglandin E2 pathway in Ang II-dependent stimulation of PRR expression in the collecting duct. Here, we tested the role of E-prostanoid (EP) subtypes acting downstream of cyclooxygenase-2 in this phenomenon. In primary rat inner medullary collecting duct cells, Ang II treatment for 12 hours induced a 1.8-fold increase in the full-length PRR protein expression. To assess the contribution of EP receptor, the cell was pretreated with specific EP receptor antagonists: SC-51382 (for EP1), L-798106 (for EP3), L-161982 (for EP4), and ONO-AE3-208 (ONO, a structurally distinct EP4 antagonist). The upregulation of PRR expression by Ang II was consistently abolished by L-161982 and ONO and partially suppressed by SC-51382 but was unaffected by L-798106. The PRR expression was also significantly elevated by the EP4 agonist CAY10598 in the absence of Ang II. Sprague-Dawley rats were subsequently infused for 1 or 2 weeks with vehicle, Ang II alone, or in combination with ONO. Ang II infusion induced parallel increases in renal medullary PRR protein and renal medullary and urinary renin activity and total renin content, all of which were blunted by ONO. Both tail cuff plethysmography and telemetry demonstrated attenuation of Ang II hypertension by ONO. Overall, these results have established a crucial role of the EP4 receptor in mediating the upregulation of renal medullary PRR expression and renin activity during Ang II hypertension.

Increased expression of (pro)renin receptor does not cause hypertension or cardiac and renal fibrosis in mice

Binding of renin and prorenin to the (pro)renin receptor (PRR) increases their enzymatic activity and upregulates the expression of pro-fibrotic genes in vitro. Expression of PRR is increased in the heart and kidney of hypertensive and diabetic animals, but its causative role in organ damage is still unclear. To determine whether increased expression of PRR is sufficient to induce cardiac or renal injury, we generated a mouse that constitutively overexpresses PRR by knocking-in the Atp6ap2/PRR gene in the hprt locus under the control of a CMV immediate early enhancer/chicken beta-actin promoter. Mice were backcrossed in the C57Bl/6 and FVB/N strain and studied at the age of 12 months. In spite of a 25- to 80-fold renal and up to 400-fold cardiac increase in Atp6ap2/PRR expression, we found no differences in systolic blood pressure or albuminuria between wild-type and PRR overexpressing littermates. Histological examination did not show any renal or cardiac fibrosis in mutant mice. This was supported by real-time PCR analysis of inflammatory markers as well as of pro-fibrotic genes in the kidney and collagen in cardiac tissue. To determine whether the concomitant increase of renin would trigger fibrosis, we treated PRR overexpressing mice with the angiotensin receptor-1 blocker losartan over a period of 6 weeks. Renin expression increased eightfold in the kidney but no renal injury could be detected. In conclusion, our results suggest no major role for PRR in organ damage per se or related to its function as a receptor of renin.

Regulation of (pro)renin receptor expression in mIMCD via the GSK-3β-NFAT5-SIRT-1 signaling pathway.

The localization and regulation of (pro)renin receptor (PRR) expression in kidney collecting duct cells are not well established. We hypothesized that low salt (LS) contributes to the regulation of PRR expression in these cells via the GSK-3β-NFAT5-sirtuin1 (SIRT-1) signaling pathway. Mouse inner medullary collecting duct (mIMCD) cells were treated with NaCl at 130 (normal salt; NS), 63 (LS), or 209 mM (high salt; HS) alone or in combination with NFAT5 scrambled small interfering (si) RNA, NFAT5 siRNA, or the SIRT-1 inhibitor EX-527. Compared with NS, LS increased the mRNA and protein expression of PRR by 71% and 69% (P < 0.05), and reduced phosphorylation of GSK-3β by 62% (P < 0.01), mRNA and protein expressions of NFAT5 by 65% and 45% (P < 0.05), and SIRT-1 by 44% and 50% (P < 0.01), respectively. LS also enhanced p65 NF-κB by 102% (P < 0.01). Treatment with HS significantly reduced the mRNA and protein expression of PRR by 32% and 23% (P < 0.05), and increased the mRNA and protein expression of NFAT5 by 39% and 45% (P < 0.05) and SIRT-1 by 51% and 56% (P < 0.05), respectively. HS+NFAT5 siRNA reduced the mRNA and protein expression of NFAT5 by 51% and 35% (P < 0.01) and increased the mRNA and protein expression of PRR by 148% and 70% (P < 0.01), respectively. HS+EX-527 significantly increased the mRNA and protein expression of PRR by 96% and 58% (P < 0.05), respectively. We conclude that expression of PRR in mIMCD cells is regulated by the GSK-3β-NFAT5- SIRT-1 signaling pathway.

COX-2 mediates angiotensin II-induced (pro)renin receptor expression in the rat renal medulla.

(Pro)renin receptor (PRR) is predominantly expressed in the distal nephron where it is activated by angiotensin II (ANG II), resulting in increased renin activity in the renal medulla thereby amplifying the de novo generation and action of local ANG II. The goal of the present study was to test the role of cycloxygenase-2 (COX-2) in meditating ANG II-induced PRR expression in the renal medulla in vitro and in vivo. Exposure of primary rat inner medullary collecting duct cells to ANG II induced sequential increases in COX-2 and PRR protein expression. When the cells were pretreated with a COX-2 inhibitor NS-398, ANG II-induced upregulation of PRR protein expression was almost completely abolished, in parallel with the changes in medium active renin content. The inhibitory effect of NS-398 on the PRR expression was reversed by adding exogenous PGE2. A 14-day ANG II infusion elevated renal medullary PRR expression and active and total renin content in parallel with increased urinary renin, all of which were remarkably suppressed by the COX-2 inhibitor celecoxib. In contrast, plasma and renal cortical active and total renin content were suppressed by ANG II treatment, an effect that was unaffected by COX-2 inhibition. Systolic blood pressure was elevated with ANG II infusion, which was attenuated by the COX-2 inhibition. Overall, the results obtained from in vitro and in vivo studies established a crucial role of COX-2 in mediating upregulation of renal medullary PRR expression and renin content during ANG II hypertension.

Aliskiren limits abdominal aortic aneurysm, ventricular hypertrophy and atherosclerosis in an apolipoprotein-E-deficient mouse model

Aliskiren is a direct renin inhibitor developed to treat hypertension. Several clinical studies have suggested that aliskiren has beneficial effects on cardiovascular diseases beyond its antihypertensive effect. In the present study, we examined whether aliskiren limits the progression of AAA (abdominal aortic aneurysm), VH (ventricular hypertrophy) and atherosclerosis in an AngII (angiotensin II)-infused mouse model. ApoE-/- (apolipoprotein-E-deficient) mice were infused subcutaneously with AngII (1000ng/kg of body weight per day; 4weeks) to induce AAA and VH. At the completion of the AngII infusion, mice were randomly allocated to three groups to receive vehicle control, low-dose aliskiren (10mg/kg of body weight per day) or high-dose aliskiren (50mg/kg of body weight per day) for 4weeks. Suprarenal aortic diameter assessed by ultrasound was significantly smaller in mice administered aliskiren at days 42 and 56. Aliskiren also significantly reduced the normalized heart weight, ventricular myocyte cell width and aortic arch atherosclerosis. Aliskiren lowered PRR (pro-renin receptor) expression and MAPK (mitogen-activated protein kinase) activity in the suprarenal aorta and heart. Aortic infiltration of T-lymphocytes and macrophages was reduced by aliskiren. In conclusion, aliskiren limits the progression of AAA, VH and atherosclerosis in an AngII-infused mouse model.

Indoxyl Sulfate-Induced Activation of (Pro)Renin Receptor Is Involved in Expression of TGF-β1 and α-Smooth Muscle Actin in Proximal Tubular Cells

Activation of (pro)renin receptor (PRR) is involved in the progression of chronic kidney disease. However, the role of indoxyl sulfate, a uremic toxin, in the activation of PRR is not clear. The present study aimed to clarify the role of indoxyl sulfate in activation of PRR, in relation to renal expression of fibrotic genes. Renal expression of PRR and renin/prorenin was up-regulated in chronic kidney disease rats compared with normal rats, whereas AST-120 suppressed these expression by reducing serum levels of indoxyl sulfate. Furthermore, administration of indoxyl sulfate to normotensive and hypertensive rats increased renal expression of PRR and renin/prorenin. Indoxyl sulfate induced expression of PRR and prorenin in cultured human proximal tubular cells (HK-2 cells). Indoxyl sulfate-induced PRR expression was inhibited by small interfering RNAs of signal transducer and activator of transcription 3 (Stat3) and nuclear factor-κB p65 in proximal tubular cells. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed indoxyl sulfate-induced PRR expression in proximal tubular cells. N-acetylcysteine prevented indoxyl sulfate-induced phosphorylation of Stat3 in proximal tubular cells. PRR small interfering RNA inhibited indoxyl sulfate-induced expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Taken together, indoxyl sulfate-induced up-regulation of prorenin expression and activation of PRR through production of reactive oxygen species and activation of Stat3 and nuclear factor-κB play an important role in the expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Thus, indoxyl sulfate-induced activation of prorenin/PRR might be involved in renal fibrosis.

Melatonin therapy prevents programmed hypertension and nitric oxide deficiency in offspring exposed to maternal caloric restriction.

Nitric oxide (NO) deficiency is involved in the development of hypertension, a condition that can originate early in life. We examined whether NO deficiency contributed to programmed hypertension in offspring from mothers with calorie-restricted diets and whether melatonin therapy prevented this process. We examined 3-month-old male rat offspring from four maternal groups: untreated controls, 50% calorie-restricted (CR) rats, controls treated with melatonin (0.01% in drinking water), and CR rats treated with melatonin (CR + M). The effect of melatonin on nephrogenesis was analyzed using next-generation sequencing. The CR group developed hypertension associated with elevated plasma asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor), decreased L-arginine, decreased L-arginine-to-ADMA ratio (AAR), and decreased renal NO production. Maternal melatonin treatment prevented these effects. Melatonin prevented CR-induced renin and prorenin receptor expression. Renal angiotensin-converting enzyme 2 protein levels in the M and CR + M groups were also significantly increased by melatonin therapy. Maternal melatonin therapy had long-term epigenetic effects on global gene expression in the kidneys of offspring. Conclusively, we attributed these protective effects of melatonin on CR-induced programmed hypertension to the reduction of plasma ADMA, restoration of plasma AAR, increase of renal NO level, alteration of renin-angiotensin system, and epigenetic changes in numerous genes.

Neuron-Specific (Pro)renin Receptor Knockout Prevents the Development of Salt-Sensitive Hypertension

The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.

Abstract 6: Angiotensin II via Its Type 1 Receptor Up-regulates (Pro)renin Receptor Expression in Doca-salt Hypertension

We previously reported that brain (pro)renin receptor (PRR) expression levels are elevated in deoxycorticosterone acetate (DOCA)-salt induced hypertension; however, the underlining mechanism remains unknown. To address whether angiotensin (Ang II) type 1 receptor (AT1R) signals are involved in the regulation of PRR expression in DOCA-salt hypertension, C57Bl/6J mice were implanted with telemetry transmitters. After two weeks recovery, mice were treated with DOCA (50mg) or SHAM pellet, and supplied with 0.9% saline as drinking solution; at the same time, mice were ICV infused with artificial CSF or AT1R blocker losartan (3mg/kg/day) for 3 weeks. The mean arterial BP was significantly higher in DOCA-salt (132.3±6.3mmHg) compared to SHAM (100.2±2.3mmHg) mice. ICV infusion of losartan prevented the increase in BP (107.4±2.7mmHg) following DOCA-salt treatment. The PRR mRNA levels (fold change) were significantly increased in the paraventricular nucleus (PVN) of DOCA-salt (2.9±0.3) compared with SHAM mice (1.0±0.1, P&lt;0.05). Interestingly, ICV infusion of losartan significantly attenuated PRR mRNA levels in the PVN (1.3±0.1) following DOCA-salt treatment (P&lt;0.05). These data suggest a role of Ang II/AT1R in regulating PRR expression during DOCA-salt hypertension. A transcription factor prediction search showed cAMP response element-binding protein (CREB), activator protein-1 binding protein (AP-1), and NF-kB binding sites on the PRR promoter region. To test whether these Ang II/AT1R downstream transcription factors are involved in PRR regulation, Neuro-2A cells were treated with Ang II (100nM, 2 h) with or without CREB (CBP-CREB interaction inhibitor, 10μM), AP-1 (SR-11302, 10μM), or NF-κB (parthenolide, 10μM) inhibitors. PRR mRNA levels (fold change) were slightly but significantly increased (1.3±0.1 vs. vehicle, P&lt;0.05) following Ang II treatment. CREB (0.8±0.2) or AP-1 (0.7±0.1) inhibitor abolished the increase in PRR mRNA induced by Ang II (P&lt;0.01). The NF-kB inhibitor had no effect on Ang II-induced PRR mRNA elevation. In conclusion, Ang II via AT1R up-regulates PRR mRNA expression in the PVN of DOCA-hypertensive mice possibly through activation of CREB and AP-1 signaling pathways.

Targeted Deletion of Murine CEACAM 1 Activates PI3K-Akt Signaling and Contributes to the Expression of (Pro)Renin Receptor via CREB Family and NF-κB Transcription Factors

The carcinoembryonic antigen-related cell adhesion molecule 1 regulates insulin sensitivity by promoting hepatic insulin clearance. Mice bearing a null mutation of Ceacam1 gene (Cc1(-/-)) develop impaired insulin clearance followed by hyperinsulinemia and insulin resistance, in addition to visceral obesity and increased plasma fatty acids. Because insulin resistance is associated with increased blood pressure, we investigated whether they develop higher blood pressure with activated renal renin-angiotensin system and whether this is mediated, in part, by the upregulation of renal (pro)renin receptor (PRR) expression. Compared with age-matched wild-type littermates, Cc1(-/-) mice exhibited increased blood pressure with increased activation of renal renin-angiotensin systems and renal PRR expression. Cytoplasmic and nuclear immunostaining of phospho-PI3K p85α and phospho-Akt was enhanced in the kidney of Cc1(-/-) mice. In murine renal inner medullary collecting duct epithelial cells with lentiviral-mediated small hairpin RNA knockdown of carcinoembryonic antigen-related cell adhesion molecule 1, PRR expression was upregulated and phosphorylation of PI3K (Tyr508), Akt (Ser473), NF-κB p65 (Ser276), cAMP response element-binding protein/activated transcription factor (ATF)-1 (Ser133), and ATF-2 (Thr71) was enhanced. Inhibiting PI3K with LY294002 or Akt with Akt inhibitor VIII attenuated PRR expression. In conclusion, global null deletion of Ceacam1 caused an increase in blood pressure with increased renin-angiotensin system activation together with upregulation of PRR via PI3K-Akt activation of cAMP response element-binding protein 1, ATF-1, ATF-2, and NF-κB p65 transcription factors.

Mice lacking the gene for Toll Like receptor‐4 (TLR4) had an attenuated blood pressure response to Angiotensin II infusion

Hypertension (HTN) is considered as a low grade inflammatory disease. Previous studies from our lab indicate that brain inflammatory molecules contribute to the development of HTN. Since TLR4 is a major modulator of inflammation, in this study, we explored the role of TLR4 in angiotensin II (ANGII) induced HTN. TLR4 knock‐out (KO) and wild type (WT) mice were implanted with telemetry probes for mean arterial pressure (MAP) measurements. After collecting baseline MAP, osmotic minipump containing ANGII (200ng/kg/min) or saline was implanted for 14 days. Then the brains microdissected for the paraventricular nucleus (PVN), sub fornical organ (SFO), nucleus tractus solatorius (NTS) and ventrolateral medulla (VLM) and examined for the expression of prorenin receptor (PRR) and interleukin(IL)‐1β by RT‐PCR. In WT mice, ANG II infusion increased MAP by day 3 (122±4) and peaked on day 9 (145±2) and remained elevated. This was accompanied by an increase in IL‐1β and PRR in the SFO, PVN, VLM and NTS. In contrast, ANGII infusion in TLR4 KO did not increase MAP (96±2) and remained lower (97±2) when compared with WT+ANGII (144±3). In addition, there was decrease in the expression of PRR and IL‐1β in the brain regions of TLR4 KO after ANGII. These finding indicate that ANGII induced HTN is at least in part mediated by TLR4 expression in the pressor regions of the brain and is associated with increase in PRR and IL‐1β expression. Funding: LSU

Increased Expression of Prorenin Receptor (PRR) in the NTS of Spontaneously Hypertensive Rats (SHR) May Be A Compensatory Mechanism of Hypertension

PRR expression is significantly increased in the nucleus of the solitary tract (NTS) of SHR compared to WKY controls. This observation led us to hypothesize that increased NTS PRR expression is linked to hypertension. We test this hypothesis by chronic knockdown or overexpression PRR in the NTS using AAV vector mediated gene transfer. Knockdown of PRR by AAV2‐PRR‐shRNA in SHR resulted in a 22 mmHg increase in mean arterial pressure (MAP) (shRNA: 173±5; Control: 151±6 mm Hg) along with shifting the set point of arterial pressure to a higher level. However, no changes were observed on MAP in WKY rats. Consistent with pressor effects of PRR knockdown, overexpression of PRR in SHR by AAV2‐human‐PRR induced a 19 mmHg decrease in MAP (PRR: 148±7; Control: 167±7 mmHg) compared to control animals. Finally, we measured the effect of increased PRR ligand, prorenin on blood pressure. Bilateral acute NTS injection of human renin (2 pmol/side) produced significant greater decreases in MAP and heart rate (HR) in SHR (ΔMAP: −32±5 mmHg, ΔHR; −40±10 bpm) compared to WKY rats (ΔMAP: −8±4 mmHg; ΔHR: −12±7 bpm). These effects in SHR were attenuated (70%) by prorenin handle region peptide, but were not affected by AT1 or AT2 receptor blockers.This study suggests that increased NTS PRR expression may be a compensatory mechanism of hypertension and that it mediates an anti‐hypertensive effect in an Ang II independent mechanism. ( AHA 11SDG7420029)

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.

Direct renin inhibition prevents cardiac dysfunction in a diabetic mouse model: comparison with an angiotensin receptor antagonist and angiotensin-converting enzyme inhibitor

Hyperglycaemia up-regulates intracellular AngII (angiotensin II) production in cardiac myocytes, effects of which are blocked more effectively by renin inhibition than ARBs (angiotensin receptor blockers) or ACEis (angiotensin-converting enzyme inhibitors). In the present study, we determined whether renin inhibition is more effective at preventing diabetic cardiomyopathy than an ARB or ACEi. Diabetes was induced in adult mice for 10 weeks by STZ (streptozotocin). Diabetic mice were treated with insulin, aliskiren (a renin inhibitor), benazeprilat (an ACEi) or valsartan (an ARB) via subcutaneous mini-pumps. Significant impairment in diastolic and systolic cardiac functions was observed in diabetic mice, which was completely prevented by all three RAS (renin-angiotensin system) inhibitors. Hyperglycaemia significantly increased cardiac oxidative stress and circulating inflammatory cytokines, which were blocked by aliskiren and benazeprilat, whereas valsartan was partially effective. Diabetes increased cardiac PRR (prorenin receptor) expression and nuclear translocation of PLZF (promyelocytic zinc finger protein), which was completely prevented by aliskiren and valsartan, and partially by benazeprilat. Renin inhibition provided similar protection of cardiac function to ARBs and ACEis. Activation of PLZF by PRR represented a novel mechanism in diabetic cardiomyopathy. Differential effects of the three agents on oxidative stress, cytokines and PRR expression suggested subtle differences in their mechanisms of action.

Protective effects of saponin on a hypertension target organ in spontaneously hypertensive rats

The present study was undertaken to investigate the protective effects of saponin on a hypertensive target organ (the kidney) in spontaneously hypertensive rats (SHRs) and also to explore the effect of saponin on the renin-angiotensin-aldosterone system (RAAS). A total of 24, 14-week-old SHRs were randomly divided into three groups; the first was administered low-dose saponin, the second with high-dose saponin and the third with a placebo as the control group. An additional eight healthy male Wistar rats were used as the normal group. The blood pressures (BPs) of the rats were determined using an animal BP-6 non-invasive blood pressure tester. Furthermore, the gene expression of TGFB1, collagen I and prorenin receptor (PRR) was determined by quantitative real time (qRT)-PCR. The histopathological and morphological features of the tissue samples were assessed semi-quantitatively. The content of saponin in the renal samples was lower in SHRs than in the normal healthy rats, but the plasma levels of saponin were similar. Mean arterial pressure (MAP) was reduced 5 days subsequent to saponin treatment by 36±3 and 51±4 mmHg in the low- and high-dose saponin groups, respectively. The anti-hypertensive effect of saponin was dose-related during the first 4 weeks of treatment. The gene expression of TGFB1 and collagen I in the renal samples was significantly suppressed in the low- and high-dose saponin groups compared with that in the control group. The gene expression of PRR was significantly and dose-dependently increased in the saponin-treated groups. These findings suggested that saponin reduced systemic BP and blocked the circulating and tissue RAAS.

In vivo regulation of renal expression of (pro)renin receptor by a low-sodium diet

Effects of low salt (LS) on (pro)renin receptor (PRR) expression are not well established. We hypothesized that LS enhances renal PRR expression via the cGMP-protein kinase G (PKG) signaling pathway. Sprague-Dawley rats were fed a normal-salt (NS) or LS diet associated with intrarenal cortical administration of vehicle (V), the nitric oxide (NO) synthase inhibitor nitro-l-arginine methyl ester (l-NAME), the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP), the cGMP analog 8-bromoguanosine (8-Br)-cGMP, the guanylyl cyclase inhibitor 1H-[1, 2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or a PKG inhibitor (PKGi) for 6 days via osmotic minipump. We evaluated the effects of each treatment on renal interstitial fluid (RIF) levels of nitrate/nitrite and cGMP and renal PRR expression. There were no significant changes in blood pressure with any of the treatments. Urinary sodium excretion was significantly lower in rats given a LS diet. Compared with NS + V, RIF nitrate/nitrite and cGMP levels increased in LS + V rats. In NS groups, RIF nitrate/nitrite and cGMP levels did not change with l-NAME, ODQ, or PKGi and increased in response to SNAP. 8-Br-cGMP increased RIF cGMP but not RIF nitrate/nitrite. In LS groups, RIF nitrate/nitrite decreased with l-NAME and did not change with ODQ or PKGi whereas RIF cGMP decreased with l-NAME, ODQ, and PKGi. PRR mRNA and protein increased in LS + V. In NS rats, PRR mRNA and protein increased in response to 8-Br-GMP and were not affected by any of other treatments. In LS rats, PRR mRNA and protein decreased significantly in response to l-NAME, ODQ, and PKGi. We conclude that LS intake enhances renal expression of PRR via cGMP-PKG signaling pathway.

Abstract 249: High Salt and Aldosterone Increase (pro)renin Receptor Expression Through Enac α Activation in Neuronal Cells

Activation of the brain renin-angiotensin-aldosterone system plays an essential role in hyperactivity of the sympathetic nervous system during salt sensitive hypertension. We previously reported an up-regulation of (pro)renin receptor (PRR) expression levels in DOCA-salt hypertensive mice brain. However, the mechanism by which PRR is regulated in DOCA-salt hypertension remains unknown. We hypothesize that aldosterone increases PRR expression by activation of epithelial sodium channel (ENaC). To test this hypothesis, we used neuro-2A cell line from brain hypothalamus which expresses all ENaC α, β, and γ subunits, as well as mineralocorticoid receptor (MR). Cells were incubated in normal salt (NS, 142 mM) or high salt (HS, 160 mM) culture medium for 1, 3, or 5 days, with or without the addition of aldosterone (1μM) for 2 hours. Interestingly, the combination of HS (5 days) with aldosterone significantly increased PRR mRNA levels (fold change) (3.28 ± 0.11) compared to NS (1.00 ± 0.01), HS (1.23 ± 0.01), or aldosterone (1.40 ± 0.01) alone in Neuro-2A cells. The PRR expression levels (1.253 ± 0.006; 1.89 ± 0.002) were significantly attenuated by ENaC inhibition (amiloride, 10μM) or MR inhibition (eplernone, 10μM) respectively (P&lt;0.05), suggesting that ENaC and MR activation mediates the up-regulation of PRR expression in neuronal cells. Furthermore, HS increased ENaC α (2.42 ± 0.01), while decreased ENaC β (0.05 ± 0.01), and ENaC γ (0.03 ± 0.01) mRNA expression levels compared to NS treatment (1.00 ±0.01) in Neuro-2A cells suggesting an increase in synthesis rate of α subunit following HS treatment. In summary, HS and aldosterone increase PRR expression levels via activation of ENaC. ENaC α may be the regulating subunit in the assembly of functional ENaC in neuronal cells. We conclude that regulation of PRR expression by high salt and aldosterone may be the critical step to activate angiotensinergic sympatho-excitatory pathways leading to salt sensitive hypertension.