Renin Receptor Expression Research Articles

PS 04-01] ALISKIREN ENHANCES ANTIFIBROTIC EFFECT OF PARICALCITOL IN OBSTRUCTIVE NEPHROPATHY

Objective: Besides the effect on blood pressure, a previous experiment has reported the renoprotective effect of aliskiren, a direct rennin inhibitor, against unilateral ureteral obstruction (UUO)-induced renal fibrosis and inflammation. Paricalcitol, a vitamin D activator, has also been shown to inhibit renal inflammation and attenuate renal interstitial fibrosis. The aim of this study was to assess any potential additive effects of a therapy combining aliskiren with paricalcitol on reducing renal fibrosis. Design and Method: C57BL6/J mice were treated individually with aliskiren or paricalcitol or their combinations until 7 days after initiation of UUO. Results: In obstructed kidneys of UUO mice, monotherapy with aliskiren or paricalcitol significantly attenuated interstitial fibrosis, collagen deposition and α-smooth muscle actin compared to vehicle-treated UUO control. Levels of renal Nox1 and Nox4 were significantly attenuated with either aliskiren or paricalcitol. The therapy combining aliskiren with paricalcitol showed additive efficacy in inhibition of these parameters. Increased levels of phospho-ERK and phosphor-p38 MAPK in UUO kidneys were also significantly reduced by either aliskiren or paricalcitol treatment alone or in their combinations. Aliskiren seemed to be more potent than paricalcitol in suppressing angiotensin II type 1 receptor expression in obstructed kidneys. Although aliskiren significantly augmented renin expression in UUO kidneys, paricalcitol decreased intrarenal renin expression, and combination therapy with both effectively blocked the renin expression induced by aliskiren. Monotherapy with either aliskiren or paricalcitol significantly reduced the expression of (pro)renin receptor in UUO kidneys and a combination of both resulted in a further reduction of the renal expression of (pro)renin receptor. Conclusions: These results suggest that combination therapy with aliskiren and paricalcitol has a better effect in inhibiting UUO-induced renal fibrosis. This mechanistic basis for this synergy may be attributed to a more profound inhibition of intrarenal RAS activity through interfering with the renin activity by aliskiren and inhibition of renin expression by paricalcitol.

Abstract P201: Enhanced Upregulation of (Pro)renin Receptor Expression by High Salt Intake in the Kidney of Dahl Salt-sensitive Rats

We recently reported that high salt (HS) intake increased the (pro)renin receptor ((P)RR) expression by 3-5 fold in several nephron segments of Sprague-Dawley rats (Peptides 63: 156-162, 2015). The preset study examined the effects of HS intake on the renal (P)RR expression in Dahl-Salt sensitive (DS) rats. Male DS rats were fed a normal salt (NS) diet (0.6%NaCl) and a HS diet (8%NaCl) for 4weeks. A part of the rats fed the HS diet were treated orally with angiotensin II type 1 receptor (AT 1 R) antagonist, candesartan (Can,3mg/kg/day) or mineralocorticoid receptor (MR) antagonist, spironolactone (Spi, 100mg/kg/day). The (P)RR expression in nephron segments was examined by immunoblot and immunohistochemical analyses. HS intake increased the blood pressure, which did not significantly affected by Can or Spi. (P)RR was expressed in the all kidney sections, glomeruli, proximal tubules (PT), medullary thick ascending limbs and inner medullary collecting ducts. HS intake increased the (P)RR expression in the cortex by 22.6 fold (p<0.001) and the PT by 4.9 fold (p<0.01), but did not change it in the other sections or segments. Can inhibited the HS intake-increased (P)RR expression in the cortex by 32% (p<0.05), Spi inhibited it by 89% (p<0.001), but neither drug did not inhibit the HS intake-increased (P)RR expression in the PT. Immunohistochemical analysis also revealed that HS intake increased the (P)RR expression in the PT and distal tubules, and that Can and Spi inhibited the HS intake-increased (P)RR expression in the distal tubules. Additionally, deoxycorticosterone acetate (DOCA, 50mg/kg/week) administered to rats fed the NS diet for 4 weeks increased the (P)RR expression in the cortex by 80% (p<0.001) and distal tubules, but not in the PT. These results indicate that HS intake-increased (P)RR expression is enhanced in the PT and distal tubules of DS rats. The mechanisms of HS intake-increased (P)RR expression may be AT 1 R and MR-dependent manner in the distal tubules, but AT 1 R or MR-independent manner in the PT.

OS 15-07 ROLE OF (PRO) RENIN RECEPTOR IN THE ACTIVATION OF WNT/ β-CATENIN PATHWAY IN COLON CANCER

Objective: Constitutive activation of Wnt/β-catenin pathway plays an important role in the pathogenesis of colon cancer; however, recent studies have also indicated that elevated Wnt levels are essential. In the present study, we investigated the potential role of (pro) renin receptor in the pathophysiology of colon cancer. Design and Method: (P) RR expression in human colon tissues was measured by immunohistochemistry and Western blot analysis. Human colon cancer cell lines DLD-1 with adenomatous polyposis coli (APC) truncated mutation, and HCT116 withβ-catenin activating mutation, were transfected with scramble siRNA and siRNA against (pro) renin receptor. Results: (Pro) renin receptor expression was significantly increased in colon cancer tissue. (Pro) renin receptor knockdown significantly decreased protein levels of the main components of Wnt/β-catenin pathway (Wnt, phosphorylated-LRP6, active β-catenin and cyclin D1) in both DLD-1 and HCT116 cells. Wnt signaling activity, measured by Tcf/Wnt signaling reporter assay, was also significantly decreased by (pro) renin receptor knockdown. WST-1 assay showed that (pro) renin receptor knockdown markedly reduced cell proliferation activity. Conclusions: These data suggest that (pro) renin receptor promotes colon cancer through Wnt/β-catenin pathway despite of intrinsic its constitutive activation.

Involvement of skeletal renin–angiotensin system and kallikrein–kinin system in bone deteriorations of type 1 diabetic mice with estrogen deficiency

AimsThis study was aimed to investigate the involvement of skeletal renin–angiotensin system (RAS) and kallikrein–kinin system (KKS) in bone deteriorations of mice in response to the combination treatment of estrogen deficiency and hyperglycemia. MethodsThe female C57BL/6J mice were sham-operated or ovariectomized with vehicle or streptozotocin (STZ) treatment. Two weeks later, the biochemistries in serum and urine were determined by standard colorimetric methods or ELISA. The H&E and TRAP staining were performed at the tibial proximal metaphysis. The polymerase chain reaction and immunoblotting were applied for molecular analysis on mRNA and protein expression. ResultsThe mice after treating with ovariectomy and STZ showed the decreased level of serum Ca and the increased level of serum PTH and urine Ca. The H&E staining showed trabecular bone abnormalities as demonstrated by the loss, disconnection and separation of trabecular bone network as well as the loss of chondrocytes and appearance of chondrocyte cluster at growth plate of tibia. The significant increase of matured osteoclast number was shown in group with double treatments. The combination treatment significantly up-regulated mRNA expression of AGT, ACE, renin receptor, MMP-9 and CAII, and protein expression of renin, and decreased the ratio of OPG/RANKL and the expression of bradykinin receptors in bone tissue. ConclusionOvariectomy combined with STZ induction produced more detrimental actions on bone through the activation of local bone RAS and the down-regulation of bradykinin receptors, as compared to the respective single treatment.

Direct renin inhibitor ameliorates insulin resistance by improving insulin signaling and oxidative stress in the skeletal muscle from post-infarct heart failure in mice

Insulin resistance can occur as a consequence of heart failure (HF). Activation of the renin-angiotensin system (RAS) may play a crucial role in this phenomenon. We thus investigated the effect of a direct renin inhibitor, aliskiren, on insulin resistance in HF after myocardial infarction (MI). MI and sham operation were performed in male C57BL/6J mice. The mice were divided into 4 groups and treated with sham-operation (Sham, n=10), sham-operation and aliskiren (Sham+Aliskiren; 10mg/kg/day, n=10), MI (n=11), or MI and aliskiren (MI+Aliskiren, n=11). After 4 weeks, MI mice showed left ventricular dilation and dysfunction, which were not affected by aliskiren. The percent decrease of blood glucose after insulin load was significantly smaller in MI than in Sham (14±5% vs. 36±2%), and was ameliorated in MI+Aliskiren (34±5%) mice. Insulin-stimulated serine-phosphorylation of Akt and glucose transporter 4 translocation were decreased in the skeletal muscle of MI compared to Sham by 57% and 69%, and both changes were ameliorated in the MI+Aliskiren group (91% and 94%). Aliskiren administration in MI mice significantly inhibited plasma renin activity and angiotensin II (Ang II) levels. Moreover, (pro)renin receptor expression and local Ang II production were upregulated in skeletal muscle from MI and were attenuated in MI+Aliskiren mice, in tandem with a decrease in superoxide production and NAD(P)H oxidase activities. In conclusion, aliskiren ameliorated insulin resistance in HF by improving insulin signaling in the skeletal muscle, at least partly by inhibiting systemic and (pro)renin receptor-mediated local RAS activation, and subsequent NAD(P)H oxidase-induced oxidative stress.

(Pro)renin receptor contributes to regulation of renal epithelial sodium channel.

Recent studies reported increased (Pro)renin receptor (PRR) expression during low-salt intake. We hypothesized that PRR plays a role in regulation of renal epithelial sodium channel (ENaC) through serum and glucocorticoid-inducible kinase isoform 1 (SGK-1)-neural precursor cell expressed, developmentally downregulated 4-2 (Nedd4-2) signaling pathway. Male Sprague-Dawley rats on normal-sodium diet and mouse renal inner medullary collecting duct cells treated with NaCl at 130 mmol/l (normal salt), or 63 mmol/l (low salt) were studied. PRR and α-ENaC expressions were evaluated 1 week after right uninephrectomy and left renal interstitial administration of 5% dextrose, scramble shRNA, or PRR shRNA (n = 6 each treatment). In-vivo PRR shRNA significantly reduced expressions of PRR throughout the kidney and α-ENaC subunits in the renal medulla. In inner medullary collecting duct cells, low salt or angiotensin II (Ang II) augmented the mRNA and protein expressions of PRR (P < 0.05), SGK-1 (P < 0.05), and α-ENaC (P < 0.05). Low salt or Ang II increased the phosphorylation of Nedd4-2. In cells treated with low salt or Ang II, PRR siRNA significantly downregulated the mRNA and protein expressions of PRR (P < 0.05), SGK-1 (P < 0.05), and α-ENaC expression (P < 0.05). We conclude that PRR contributes to the regulation of α-ENaC via SGK-1-Nedd4-2 signaling pathway.

Water Deprivation Increases (Pro)renin Receptor Levels in the Kidney and Decreases Plasma Concentrations of Soluble (Pro)renin Receptor.

Water deprivation activates the renin-angiotensin system. We have hypothesized that the renal expression of (pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, could be changed under dehydration. Moreover, plasma levels of soluble (P)RR (s(P)RR) comprising of the extracellular domain of (P)RR may reflect the renal (P)RR expression. In the present study, we therefore aimed to clarify changes of plasma s(P)RR concentrations and kidney tissue (P)RR levels using rats with dehydration. Male Wister-Kyoto rats were divided into two groups; dehydrated (DH) rats deprived of water for 72 hours with free access to food, and control rats. Plasma s(P)RR concentrations measured by enzyme-linked immunosorbent assay were significantly lower in DH rats (6.94 ± 2.08 ng/mL, mean ± SD, n = 5) than in control (12.54 ± 2.00 ng/mL, n = 5) (p < 0.05). Western blot analysis confirmed lower expression levels of s(P)RR in plasma in DH rats than in control. By contrast, western blot analysis showed higher levels of full-length (P)RR and lower levels of furin (an enzyme responsible for generation of s(P)RR from full-length (P)RR) in the kidney tissues obtained from DH rats compared to control. There was no significant difference in the renal (P)RR mRNA levels between DH rats and control. These findings suggest that water deprivation may elevate the renal full-length (P)RR levels via reducing the expression of furin. Increased full-length (P)RR may contribute to the up-regulation of the renal renin-angiotensin system and the production of concentrated urine under dehydration.

Impairing effects of angiotensin-converting enzyme inhibitor Captopril on bone of normal mice

There are contradicting results about the effects of angiotensin-converting enzyme inhibitors (ACEIs) on bones. This study was aimed to investigate the effect of ACEI, Captopril, on bone metabolism and histology as well as the action of Captopril on skeletal renin–angiotensin system (RAS) and bradykinin receptor pathway in normal male mice. The urine, serum, tibias and femurs from normal control mice and Captopril-treated (10mg/kg) mice were collected for biochemical, histological and molecular analyses after drug administration for eight weeks. The mice after the treatment with Captopril had a significant decrease of serum testosterone level. The histological measurements showed the loss of trabecular bone mass and trabecular bone number, and the breakage of trabecular bone network as well as the changes of chondrocyte zone at epiphyseal plate in Captopril-treated mice. The defect of Captopril on trabecular bone was reflected by the quantitative bio-parameters from micro-CT. The expression of renin receptor and bradykinin B2 receptor (B2R) was significantly up-regulated in tibia of mice upon to the Captopril treatment, which decreased the ratio of OPG/RANKL and the expression of osteoblastic factor RUNX2. Furthermore, Captopril treatment resulted in the increase of pAkt/Akt and pNFκB expression in tibia. The present study revealed the impairing effects of Captopril on bone via interfering with the circulating sex hormone level and B2R pathway, which suggests that the bone metabolism of patients need to be carefully monitored when being prescribed for ACEIs.

Abstract 045: Epigenetic Modification of Brain (pro)renin Receptor Contributes to the Development of Doca-salt Hypertension via Epithelial Sodium Channel

We reported that elevated brain (pro)renin receptor (PRR) expression levels contributes to the development of salt-sensitive hypertension (SSH); however, the underlining mechanism on how PRR is regulated remains unknown. High-salt intake is a major life style epigenetic factors for the SSH. To test our hypothesis that epigenetic mechanism is involved in the regulation of PRR expression in SSH, we used deoxycorticosterone acetate (DOCA)-salt induced hypertension as a model of SSH. C57Bl/6J mice (n=5/group) were implanted with SHAM or DOCA (50mg) pellet and supplied with saline for 3 weeks. Histone modifications on PRR promoter were assessed in the hypothalamus using Chromatin Immunoprecipitation assays at the end of protocol. We found an increase (fold change) in histone 3 lysine 4 trimethylation (H3K4me3) on the PRR promoter in DOCA-salt (2.45±0.36) compared with SHAM treated mice (1.00±0.16, P&lt;0.05). H3K4 methyltransferases (HMT) activity (OD/hour/mg protein), an enzyme complex responsible for H3K4 methylation is increased after DOCA-salt treatment (45±2, P&lt;0.05) compared with SHAM (28±2). To examine the mechanisms of elevated HMT activity during DOCA-salt hypertension, we treated culture neurons (n=3 independent experiments/group) with control (155mM sodium), high salt (170 mM sodium), aldosterone (100 nM), or high salt+aldosterone with or without epithelial sodium channel (ENaC) blocker (amiloride, 100 nM) for 2 hours and test the HMT activity and PRR expression levels. High salt or aldosterone alone has minimal effects on HMT activity and PRR levels. High salt+aldosterone significantly increased HMT activity (41±3 vs. 24±2, P&lt;0.05) and PRR mRNA (1.6±0.1 vs. 1.0±0.1 fold change, P&lt;0.05) levels compare to control. Amiloride attenuated HMT activity (25±4.1, P&lt;0.05) and PRR mRNA levels (0.98±0.1, P&lt;0.05) compared to high salt+aldosterone treatment. In summary, elevated PRR expression is associated with increased HMT activity and H3K4me3 modification on PRR promoter. ENaC blockade attenuates the elevation of HMT activity and PRR levels induced by high salt+aldosterone in culture neurons. We conclude that ENaC may be responsible for epigenetic up-regulation of brain PRR and thus the increase in BP during salt-sensitive hypertension.

Angiotensin II regulates brain (pro)renin receptor expression through activation of cAMP response element-binding protein.

We reported that brain (pro)renin receptor (PRR) expression levels are elevated in DOCA-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether ANG II type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplied 0.9% saline as the drinking solution to C57BL/6J mice. Sham pellet-implanted mice that were provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly infused with the AT1R blocker losartan, angiotensin-converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 wk. Intracerebroventricular infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for ANG II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which ANG II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with ANG II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors, as well as CREB knockdown abolished ANG II-induced increases in PRR levels. ANG II also induced PRR upregulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that ANG II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that ANG II acts via AT1R to upregulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter.

Trimethylation of Histone 3 Lysine 4 on Hypothalamic (Pro)renin Receptor Mediates the Development of DOCA‐salt Hypertension.

We reported that brain (pro)renin receptor (PRR) expression is elevated in deoxycorticosterone acetate (DOCA)‐salt hypertension; however, the underlining mechanism remains unknown. To test our hypothesis that epigenetic mechanism is involved in the regulation of PRR expression in DOCA‐salt hypertension, C57Bl/6J mice were implanted with SHAM or DOCA pellet and supplied with saline for 3 weeks. Histone modifications on PRR promoter were assessed in the hypothalamus using Chromatin Immunoprecipitation assays at the end of protocol. We found an increase (fold change) in histone 3 lysine 4 trimethylation (H3K4me3) on the PRR promoter in DOCA‐salt (2.45±0.36) compared with SHAM treated mice (1.00±0.16, P&lt;0.05), which was not altered by ICV infusion of losartan (2.33±0.27). Importantly, we found increased H3K4 methyltransferases (HMT) activity (OD/hour/mg protein), an enzyme complex responsible for H3K4 methylation, after DOCA‐salt treatment (44.54±2.09, P&lt;0.05) compared with SHAM (28.23±2.05). To examine the contribution of H3K4me3 on PRR expression and the increased BP during DOCA‐salt hypertension, mice were treated with DOCA‐salt and ICV infused with either artificial CSF or 5'‐methylthioadenosine (MTA), a specific methyltransferase inhibitor, for 3 weeks. ICV infusion of MTA normalized the increase in H3K4me3 (0.9±0.11), HMT activity (32.3±2.38) compared with ICV aCSF in DOCA‐salt treated mice. ICV infusion of MTA attenuated PRR mRNA levels (1.4±0.1 vs. 2.4±0.2) and BP (123.1±6.3 vs. 138.5±7.4 mmHg) compared with control following DOCA‐salt treatment. In summary, H3K4me3 on PRR promoter is a key epigenetic mechanism for the up‐regulation of brain PRR and the increase in BP during salt‐sensitive hypertension.

Involvement of Renin-Angiotensin System in Damage of Angiotensin-Converting Enzyme Inhibitor Captopril on Bone of Normal Mice.

This study was performed to investigate the effect of angiotensin-converting enzyme inhibitor, captopril, on bone metabolism and histology, and the action of captopril on the components of the skeletal renin-angiotensin system (RAS) and bradykinin receptor in normal male mice. The mice were orally administered captopril (10 mg/kg) for 4 weeks with vehicle-treated mice as normal control. The histology of trabecular bone at the distal femoral end was determined by hematoxylin & eosin, Safranin O and Masson-Trichrome staining. The captopril-treated mice showed a decreased level of testosterone (p<0.05) and procollagen type I N-terminal propeptide (p<0.05) in serum as compared to those in the control group. Captopril has detrimental effects on trabecular bone as demonstrated by the loss of cancellous bone mass and network connections as well as changes to the chondrocytes zone. The expression of angiotensin-converting enzyme (p<0.05), renin receptor (p<0.01), angiotensin II (p<0.05) and bradykinin receptor 2 (p<0.05) was significantly up-regulated following the captopril treatment. Thus, the potential underlying mechanism of the damage of captopril on bone can be attributed the increased activity of local bone RAS and the activation of bradykinin receptor.

Expression of (pro)renin receptor and its upregulation by high salt intake in the rat nephron

A functional receptor for renin and prorenin ((P)RR) was identified as a new component of the renin–angiotensin system. The precise localization of (P)RR in the kidney has not been clarified. The present study was designed to determine the localization of (P)RR in the rat nephron and to investigate the regulation of renal (P)RR expression by high salt (HS) intake. (P)RR mRNA levels in the kidney sections and isolated nephron segments were examined using reverse transcription and polymerase chain reaction (RT-PCR), and (P)RR protein levels were examined by immunoblot and immunohistochemical analyses. Renal (P)RR mRNA and protein levels in rats fed a HS diet for 4 weeks were also compared with those fed a normal salt diet. (P)RR mRNA was expressed in various nephron segments of the cortex and medulla; glomeruli (Glm), proximal tubules (PT), thick ascending limbs (TAL) and collecting ducts (CD). (P)RR protein was highly expressed in the PT, medullary TAL (MTAL) and inner medullary CD (IMCD), and lowly in the preglomerular arterioles (Art) and Glm. HS intake increased (P)RR protein levels in the Glm, PT and tubules of medullary rays. These results indicated that (P)RR is expressed throughout various nephron segments and Art, and that (P)RR protein is expressed predominantly in the PT, MTAL and IMCD. HS intake appears to upregulate the (P)RR expression in the Glm, PT and tubules of medullary rays, suggesting that (P)RR may be involved in the regulation of renal function and HS-induced disorders.

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.

Angiotensin II Increases the Expression of (Pro)Renin Receptor During Low-Salt Conditions

BackgroundEvidence indicates that chronic angiotensin II (Angll) infusion increases (pro)renin receptor ((P)RR) expression in renal inner medullary collecting duct (IMCD) cells. Recently, it has been shown that renal (P)RR expression is augmented during a low-salt (LS) diet. However, the role of Angll in mediating the stimulation of (P) RR during LS conditions is unknown. We hypothesized that Angll mediates the increased expression of (P)RR during low-salt conditions in IMCDs. Methods(P)RR expression and Angll levels were evaluated in Sprague-Dawley rats fed a LS diet (0.03% NaCl) and normal salt (NS; 0.4% NaCl) for 7 days. We examined the effects of sodium reduction (130mM NaCl) and Angll on (P)RR expression in IMCDs isolated in hypertonic conditions (640 mOsmol/L with 280mM NaCl). ResultsPlasma renin activity in LS rats was significantly higher than rats fed with NS (28.1 ± 2.2 versus 6.7 ± 1.1ng AngI·mL–1·hr−1; P < 0.05), as well as renin content in renal cortex and medulla. The (P)RR mRNA and protein levels were higher in medullary tissues from LS rats but did not change in the cortex. Intrarenal AngII was augmented in LS compared with NS rats (cortex: 710 ± 113 versus 277 ± 86 fmol/g, P < 0.05; medulla: 2093 ± 125 versus 1426 ± 126 fmol/g, P < 0.05). In cultured IMCDs, (P)RR expression was increased in response to LS or AngII treatment and potentiated by both treatments (both at 640 mOsmol/L). ConclusionsThese data indicate that (P)RR is augmented in medullary collecting ducts in response to LS and that this effect is further enhanced by the increased intrarenal AngII content.

Renal medullary cyclooxygenase-2 and (pro)renin receptor expression during angiotensin II-dependent hypertension

The (pro)renin receptor [(P)RR] upregulates cyclooxygenase-2 (COX-2) in inner medullary collecting duct (IMCD) cells through ERK1/2. Intrarenal COX-2 and (P)RR are upregulated during chronic ANG II infusion. However, the duration of COX-2 and (P)RR upregulation has not been determined. We hypothesized that during the early phase of ANG II-dependent hypertension, membrane-bound (P)RR and COX-2 are augmented in the renal medulla, serving to buffer the hypertensinogenic and vasoconstricting effects of ANG II. In Sprague-Dawley rats infused with ANG II (0.4 μg·min(-1)·kg(-1)), systolic blood pressure (BP) increased by day 7 (162 ± 5 vs. 114 ± 10 mmHg) and continued to increase by day 14 (198 ± 15 vs. 115 ± 13 mmHg). Membrane-bound (P)RR was augmented at day 3 coincident with phospho-ERK1/2 levels, COX-2 expression, and PGE2 in the renal medulla. In contrast, membrane-bound (P)RR was reduced and COX-2 protein levels were not different from controls by day 14. In cultured IMCD cells, ANG II increased secretion of the soluble (P)RR. In anesthetized rats, COX-2 inhibition decreased the glomerular filtration rate (GFR) and renal blood flow (RBF) during the early phase of ANG II infusion without altering BP. However, at 14 days of ANG II infusions, COX-2 inhibition decreased mean arterial BP (MABP), RBF, and GFR. Thus, during the early phase of ANG II-dependent hypertension, the increased (P)RR and COX-2 expression in the renal medulla may contribute to attenuate the vasoconstrictor effects of ANG II on renal hemodynamics. In contrast, at 14 days the reductions in RBF and GFR caused by COX-2 inhibition paralleled the reduced MABP, suggesting that vasoconstrictor COX-2 metabolites contribute to 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.

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.

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.

Aliskiren in early postnatal life prevents hypertension and reduces asymmetric dimethylarginine in offspring exposed to maternal caloric restriction

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is involved in hypertension. We tested whether aliskiren treatment in early postnatal life can reduce ADMA and regulate the renin-angiotensin system to prevent hypertension in rat offspring exposed to maternal caloric restriction (CR). Four groups of 12-week-old male offspring were sacrificed: control, CR, CR+aliskiren, and CR+losartan group. The CR group included offspring from 50% food-restricted maternal rats. The CR+aliskiren and CR+losartan groups were produced by treating CR offspring with oral aliskiren 10 mg/kg/day or losartan 20 mg/kg/day between 2-4 weeks of age, respectively. Blood pressure increased in CR rats, which was prevented by aliskiren or losartan. CR increased plasma ADMA levels, which aliskiren prevented. Renal renin and prorenin receptor (PRR) expression increased in CR rats treated with aliskiren, whereas both were reduced by losartan. Both aliskiren and losartan decreased renal mRNA expression of angiotensinogen, angiotensin II type 2 receptor, and Mas in CR rats. However, aliskiren increased angiotensin II type 2 receptor and Mas protein levels in CR kidneys. Early aliskiren therapy prevents CR-induced hypertension via ADMA reduction, decreases angiotensinogen expression, and increases renal angiotensin II type 2 receptor and Mas protein.