Total Renin Research Articles

Associations of Vitamin D-Related Biomarkers With Hypertension and the Renin-Angiotensin System in Men and Women.

Vitamin D may prevent the development of hypertension through down-regulation of renin-angiotensin system. However, epidemiologic studies assessing the interrelation of vitamin D-related biomarkers with hypertension are sparse. We examined the prospective associations between vitamin D-related biomarkers and the risk of hypertension in a nested case-control study. In each of the Women's Health Study (WHS) and Physicians' Health Study (PHS) II, 500 incident hypertension cases and 500 age and race-matched controls were randomly selected. Baseline plasma 25(OH)-vitamin D [25(OH)D], parathyroid hormone (PTH), and total renin concentrations were measured. Among controls, 25(OH)D and PTH were inversely correlated, but neither was correlated with total renin. In the crude model, there was a trend of association between increasing quintiles of 25(OH)D and lower risk of hypertension in women, with relative risks and 95% CIs of 1.00, 1.24 (0.84-1.83), 0.82 (0.53-1.25), 0.75 (0.48-1.16), and 0.81 (0.52-1.27) (P, trend: .07). Adjustment for body mass index and other hypertension risk factors eliminated this association (relative risk of 5th quintile: 1.03). No associations were found in men. Baseline PTH and ratio of 25(OH)D to PTH were not associated with the risk of hypertension in women or men. When men and women were included in the same model, vitamin D insufficiency (defined as 25(OH)D <20 ng/mL) also was not associated with an increased risk of hypertension. No interactions were found across subgroups. Our study found no association of baseline plasma 25(OH)D or PTH with the risk of hypertension or total renin concentration in middle-aged and older men and women.

Abstract P221: Soluble (pro)renin Receptor Contributes To 2-kidney, 1-clip-induced Renovascular Hypertension And Ischemic Nephropathy In Mice

The Goldblatt two-kidney, one-clip (2K1C) model features renovascular hypertension and ischemic nephropathy due to overactivated renin-angiotensin system (RAS). Soluble (pro)renin receptor (sPRR), the extracellular domain of (pro)renin receptor (PRR), is primarily generated by site-1 protease (S1P) and implicated as a potential regulator of the RAS and renal function. Here, we examined the function and underlying mechanism of sPRR in 2K1C model by pharmacological inhibition of S1P and genetic mutagenesis of S1P cleavage site. Male 2-mo-old C57BL/6 mice were subjected to the 2K1C procedure or sham operation for 1 month, followed by a 14-day infusion of vehicle or a specific S1P inhibitor PF429242 (PF) (30 mg/kg/day via minipump). The 2K1C procedure induced a 2-fold increase in plasma sPRR, which was reduced by 50% following PF treatment. This was paralleled by improvement of renovascular hypertension (MAP:142.1±1.3 vs. 129.9±2.8mmHg, n=7, p &lt; 0.05) and cardiac hypertrophy (HW/BW:6.3±0.3 vs. 5.0 ± 0.3mg/g, n=7, p &lt; 0.01) accompanied with suppressed cortical and medullary renin activity, active renin content, prorenin content and total renin content in clipped kidney. Meanwhile, cortical and medullary renin mRNA level in clipped kidney was increased 3.8-fold and 5.3-fold, which was reduced by 41% and 55% respectively, following PF treatment. The level of urinary albumin, KIM-1 and NGAL was elevated 5.1-fold, 4.0-fold and 5.7-fold after 2K1C surgery, which was blunted 35%, 49% and 59%, respectively, following PF treatment. Secondly, we employed a novel mouse model with mutagenesis of the cleavage site in PRR (termed as Mutant mice) to verify the role of endogenous sPRR in 2K1C model. Similarly, the hypertensive response and ischemic nephropathy of Mutant mice to 2K1C procedure was blunted (24-day MAP: 2K1C/WT 142.5±2.1 vs. 2K1C/Mutant 128.2±3.2mmHg; 24-day urinary albumin/creatinine: 2K1C/WT 64.9±7.2 vs. 2K1C/Mutant 33.0±1.9mg/g, n=6, p &lt; 0.05), in parallel with attenuated response of systemic and intrarenal RAS. Together, consistent results with the different approaches have established an important role of S1P-derived sPRR in regulation of the RAS and thus the pathogenesis of 2K1C-induced renovascular hypertension and ischemic nephropathy.

Enhanced Intrarenal pro-renin and salt sensitive hypertension in a mouse model of Type-1 diabetes

Hypertension affects up to 50% of patients with type 1 diabetes. Despite aggressive anti-hypertensive therapy, at least 30% of diabetic patients develop chronic kidney disease. Renin is the rate-limiting enzyme in the activation of the RAS and is essential for blood pressure control. Renin is expressed in Juxtaglomerular (JG) cells, as an immature pro-peptide (pro-renin), and then stored as active renin. In diabetic humans, plasma pro-renin is enhanced while renin remains unchanged or low. We hypothesize that intrarenal renin and pro-renin are enhanced in type 1 diabetes, which could potentially contribute to hypertension and diabetic nephropathy. We used the Akita mice strain, one of the few mouse models of diabetes mellitus that shows features of the human disease including hypertension and kidney damage. We found that Akita mice had higher blood pressure as measured by tailcuff (mmHg: C57=109±2.8; Aki=120.6±2.5;p&lt;0.05, n=6), plasma glucose (in mM: C57=7.71±23.9; Aki=23.2±2.3; p&lt;0.01; n=4) and proteinuria (mg/24hrs: C57 18.2±3.1; Aki: 29.4±3.3; p&lt;0.05; n=5) when compared to C57 (control). In addition, plasma renin was not changed in Akita mice (n=6; p= NS). Histological staining of kidney cuts with a renin antibody (recognizes both renin and pro-renin) showed overall decreased total renin expression in Akita mice (O.D C57: 46.6±1.5; Akita=30.7±3.5). Surprisingly, Western Blots of renal cortical lysates and JG cells showed that pro-renin (43-45 kDa) expression was 79±21% higher in male Akita diabetic mice (p&lt;0.05), demonstrating increased intra-renal pro-renin in Akita. The mechanism driving the increase in pro-renin is unknown. We previously showed that ROS potently stimulate renin release from JG cells. We found that Akita mice have increased urinary isoprostanes, supporting higher kidney ROS (ng/24hrs: C57:3.5±0.6 Aki=6.0±0.8 p&lt;0.05; n=5). We measured H202 levels by transducing primary JG cells in culture with the fluorescent H202 protein sensor HyPer and found that JG cells isolated from Akita mice had higher baseline levels of H202 (C57= 100, Akita=117±7%, p&lt;0.05). An activated intrarenal RAS contributes to salt sensitive hypertension, and at least one third of diabetic patients are salt-sensitive. It is not known whether diabetic Akita mice are salt sensitive. Therefore, we tested the effect of a high salt diet on blood pressure in Akita and C57 mice measured by radiotelemetry. High salt increased systolic blood pressure (SBP) in Akita (from 109±5 to 138±6 mmHg, p&lt;0.001) but not in control C57 (from 104±2 to 117±6 mmHg, NS). We conclude that intrarenal renin and pro-renin is enhanced in Akita mice which also exhibit salt sensitive hypertension. The mechanism may involve elevated superoxide and H202 levels within JG cells stimulating pro-renin expression and release. AHA-SDG; NIH-NIDDK-RO3 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renin-Angiotensin-Aldosterone System Activation and Diuretic Response in Ambulatory Patients With Heart Failure

Rationale & ObjectiveHeart failure treatment relies on loop diuretics to induce natriuresis and decongestion, but the therapy is often limited by diuretic resistance. We explored the association of renin-angiotensin-aldosterone system (RAAS) activation with diuretic response.Study DesignObservational cohort.Setting & PopulationEuvolemic ambulatory adults with chronic heart failure were administered torsemide in a monitored environment.PredictorsPlasma total renin, active renin, angiotensinogen, and aldosterone levels. Urine total renin and angiotensinogen levels.OutcomesSodium output per doubling of diuretic dose and fractional excretion of sodium per doubling of diuretic dose.Analytical ApproachRobust linear regression models estimated the associations of each RAAS intermediate with outcomes.ResultsThe analysis included 56 participants, and the median age was 65 years; 50% were women, and 41% were Black. The median home diuretic dose was 80-mg furosemide equivalents. In unadjusted and multivariable-adjusted models, higher levels of RAAS measures were generally associated with lower diuretic efficiency. Higher plasma total renin remained significantly associated with lower sodium output per doubling of diuretic dose (β = −0.41 [−0.76, −0.059] per SD change) with adjustment; higher plasma total and active renin were significantly associated with lower fractional excretion of sodium per doubling of diuretic dose (β = −0.48 [−0.83, −0.14] and β = −0.51 [−0.95, −0.08], respectively) in adjusted models. Stratification by RAAS inhibitor use did not substantially alter these associations.LimitationsSmall sample size; highly selected participants; associations may not be causal.ConclusionsAmong multiple measures of RAAS activation, higher plasma total and active renin levels were consistently associated with lower diuretic response. These findings highlight the potential drivers of diuretic resistance and underscore the need for high-quality trials of decongestive therapy enhanced by RAAS blockade.

Maintaining Normal Blood Pressure in a Renovascular Stenosis Model of Hypertension in Adult Lewis Rats: Putative Physiological Modulation of the Renin‐Angiotensin System

Prolonged unilateral renal artery stenosis using a Goldblatt Two-Kidney-One Clip (2K1C) technique is a validated scientific approach to inducing experimental hypertension in laboratory animals. This patho-physiological modulation is associated with the activation of the renin-angiotensin system with increased renin and angiotensin II release to initiate hypertension. This mode of experimental hypertension has been demonstrated for many rat strains including Sprague Dawley, Brown Norway and Wistar Kyoto but has not been fully characterized in syngeneic Lewis rats. The objective of this study was to develop and characterize a unilateral renal artery stenosis model of hypertension in adult male Lewis rats using a 2K1C method for hypertension studies in our laboratory. Thirty animals were randomly assigned to two groups to undergo sham or the 2K1C surgical procedure under isoflurane-induced anesthesia; approval was granted by our institutional animal ethics committee (A2404, n =15 per group). Hemodynamic parameters including heart rate; systolic and diastolic blood pressure were monitored weekly for 6 weeks, using a volume-pressure tail cuff and a CODA pressure computer. Plasma concentrations of critical biomarkers of the renin-angiotensin system were quantified using standard ELISA assays and renal histopathological changes were assessed microscopically. Unilateral renal arterial stenosis caused marked renal atrophy, glomerulosclerosis and interstitial fibrosis, but a compensatory renal hypertrophy was observed in contralateral kidneys. Interestingly, unilateral renal arterial stenosis failed to induce hypertension over the six week period. The resistance to develop hypertension in the 2K1C group was associated with a significant decrease in total plasma renin (P = 0.023), an increase in the ratio of plasma angiotensin (1-7) to total renin (P = 0.034) and a decrease in the ratio of angiotensin II to total renin (P = 0.013). There were no changes in plasma electrolytes, glucose, creatinine or urea. These data demonstrate that adult male Lewis rats may have physiologically modulated the activation of the renin-angiotensin system to maintain a homeostatic balance and resist hypertension following unilateral renal artery stenosis.

Sex-specific role of CYP24A1 rs2762939 in the risk of essential hypertension based on the serum vitamin D and total renin concentrations

CYP24A1, Vitamin D 24-hydroxylase catabolizes 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D to 24-hydroxylated vitamin D products. It is widely known that low Vitamin D can lead to increased renal renin and angiotensin II production, consequently elevating blood pressure or development of essential hypertension (EH). We have conducted an investigation on hypertensives and controls to evaluate the association of the gene variant, CYP24A1 rs2762939 and 25(OH)D in an Indian population with EH. On gender-based stratification, with multivariate logistic analysis after adjustment for covariates, the CYP24A1 rs2762939 CC variant showed a higher risk of EH in males (aOR = 3.141, CI 1.164–8.478, P = .024) while females illustrated an inverse association with EH (aOR = 0.398, CI 0.172–0.092, P = .031). The 25(OH)D levels among the three genotypes of hypertensives substantiate these results. Our results clearly suggest that gender, CYP24A1 rs2762939, and Vitamin D status may play a significant role in disease susceptibility towards EH in Indian population.

Inhibition of renin-angiotensin system enzymes by leafy vegetables polyphenol extracts related to fertilizer micro-dosing and harvest time

Increase in human population coupled with continuous rise in oxidative stress-related diseases requires availability of healthier foods like vegetables, which are polyphenol-rich sources. Polyphenols are able to inhibit angiotensin-I converting enzyme (ACE) and renin, thereby regulating blood pressure but this ability can be influenced by nutrient management during plant growth. Fertilizer micro-dosing is a valuable nutrient management option, which increases crop productivity with minimal expenditure of resources. This study assessed the effects of fertilizer micro-dosing and harvest times on inhibition of ACE and renin activities by aqueous polyphenol extracts from Amaranthus cruentus, Ocimum gratissimum and Solanum macrocarpon leaves. Different levels of urea (0, 20, 40 and 60 kg ha(-1)) with cow manure (5 t ha(-1)) or without manure (80 kg ha(-1)) were used to grow the leafy vegetables before they were harvested three times at two (A. cruentus) and four weeks intervals (O. gratissimum and S. macrocarpon). Polyphenols were extracted from leaf powders using water (1:20 ratio) and total phenolic content (TPC), total flavonoids content (TFC), ACE and renin inhibition were assayed according to standard protocols. Two-way analysis of variance and Duncan's test (p<0.05) revealed that TPC (605.70±39.51 to 1024.83±102.89 mg g(-1); gallic acid equivalent) and TFC (149.02±29.12 to 276.19±63.30 mg g(-1); rutin equivalent) were not affected by fertilizer doses and harvest times except TPC from O. gratissimum. Inhibitions of ACE (0.25±0.01 to 8.46±0.42 mg mL(-1)) and renin (-95.41±2.25 to 36.42±0.22%) activities by each specie were significantly affected by fertilizer dose and harvest time. Production with cow manure combined with low or medium doses of urea (20 or 40 kg ha(-1)) followed by collection at the first two harvests are recommended for polyphenol extracts with potential inhibition of renin-angiotensin system enzymes.

(Pro)renin receptor contributes to pregnancy-induced sodium-water retention in rats via activation of intrarenal RAAS and α-ENaC.

The (pro)renin receptor (PRR) is a new component of the renin-angiotensin-aldosterone system (RAAS) and regulates renin activity. The objective of the present study was to test potential roles of the renal PRR and intrarenal RAAS in the physiological status of late pregnancy. Late pregnant Sprague-Dawley rats were studied 19-21 days after sperm was observed in vaginal smears. Experiments were performed using age-matched virgin rats and late pregnant rats treated with the specific PRR inhibitor PRO20 (700 μg·kg-1·day-1 sc for 14 days, 3 times/day for every 8 h) or vehicle. The indices of RAAS, including PRR, renin, angiotensin II, and aldosterone levels, were examined by immunoblotting, qRT-PCR, or ELISA. Further analyses of renal epithelial sodium channel (ENaC) expression, sodium-water retention, and plasma volume were performed. We first present evidence for the activation of intrarenal RAAS in late pregnant rats, including increases in urinary renin activity, active and total renin content, and prorenin content, angiotensin II and aldosterone excretion, in parallel with increased renal PRR expression and urinary soluble PRR excretion. Functional evidence demonstrated that PRR antagonism with PRO20 effectively suppressed the indices of intrarenal RAAS in late pregnant rats. In addition, our results revealed that renal α-ENaC expression, sodium-water retention, and plasma volume were elevated during late pregnancy, which were all attenuated by PRO20. In summary, the present study examined the renal mechanism of sodium-water retention and plasma volume expansion in late pregnant rats and identified a novel role of PRR in regulation of intrarenal RAAS and α-ENaC and thus sodium and fluid retention associated with pregnancy.

Enhanced Hydrogen Peroxide and renin release in Primary cultures of Juxtaglomerular (JG) cells from diabetic mice

Diabetes mellitus and its complications represent an increasing burden for healthcare. A large fraction of patients with diabetes mellitus develops diabetic nephropathy. The Akita mice strain is one of the few mouse models of diabetes mellitus that shows features of the human disease including hypertension and kidney damage. Previous reports have shown increased levels of reactive oxygen species in the kidneys of Akita mice. We previously showed that hydrogen peroxide is a potent stimuli for renin release from the juxtaglomerular cells, increasing plasma renin and blood pressure in mice. Thus, we hypothesized that hydrogen peroxide levels are higher in JG cells from Akita mice leading to increased renin release in this strain. Using Western Blot, we found that the NADPH oxidase isoforms NOX1 and NOX4 are expressed, but only NOX1 being upregulated in primary JG cells isolated from Akita mice, compared to JG cells from the control (C57) strain (C57=100%. Akita/NOX1= 147%±10; n=4; p&lt;0.05). Confocal imaging showed the expression of NOX1 and NOX4 in JG cells where they both co‐localized with renin immunolabeling in renin‐containing granules. In addition, the expression of Catalase, which scavenges H202, was reduced in JG cells from diabetic Akita mice (C57=100%; Akita= 72%±7.2 of C57; n=4; p&lt;0.05). We measured H202 levels by transducing JG cells with the fluorescent protein sensor Hyper under control of the renin1 promoter. We found that JG cells isolated from Akita mice had higher baseline levels of H202 (C57= 100, Akita=117±7%, p&lt;0.05). Since we previously found that H202 stimulates renin release, we studied whether JG cells from Akita mice had higher total renin activity or release. For this, we measured baseline renin release to the media during 2 hours from primary JG cells cultured from Akita or control mice. JG cells were then lysed and total renin content measured. We found that in the absence of any stimuli, JG cells from Akita mice had ~2 fold higher renin release (C57= 100%, Akita=219.2±26%; p&lt;0.04, n=6). We did not observe a significant difference in total renin content between strains. Transduction of replicate plates with adenovirus encoding Catalase (AD‐Cat) decreased baseline renin release by 35±8% (p&lt;0.05) in JG cells from Akita mice whereas it did not affect renin release in control JG cells. We conclude that renin cells from diabetic Akita mice have higher endogenous H202 leading to increased renin release. The NADPH isoform NOX1 is enhanced in Akita mice and may be responsible for higher levels of H202 and renin release from JG cells. Our data suggest that direct stimulation of renin release by H202 could be potentially be involved in renal damage in diabetes mellitus.Support or Funding InformationNIH/NIDDK R03DK105300‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 082: On the Significance of Urinary Renin in Diabetic Kidney Disease: A Critical Role of Impaired Proximal Tubular Reabsorption

Increased expression of renin in the kidney collecting tubule of rodents made diabetic by streptozotocin (STZ) has been well demonstrated but whether this site is the main source of urinary renin is unknown. We wanted to examine the origin and significance of urinary renin in diabetic kidney disease (DKD). Total and active renin was evaluated in urines from people with longstanding type 1 diabetes of more than 25 years, with (n=36) or without DKD (n=38) (eGFR 101 vs. 39 mL/min/1.73m 2 ; p&lt;0.001). Mice given STZ (n=15) or vehicle (n=8) 20 weeks prior to study were also studied. In people with DKD, total renin was markedly increased compared to people without DKD (82 vs. 49 pg/mg Cr; p=0.023). Active renin was also significantly increased in people with DKD compared to people without DKD (3.2 vs. 1.3 pg/mg Cr; p&lt;0.001). In mice with STZ-induced DKD a significant increase in renin was found compared to controls (1093±319 vs. 64±18 pg/mg Cr; p=0.0001). To examine the role of filtration and tubular reabsorption on urinary renin, human active renin was measured in urines from non-diabetic mice infused with human recombinant renin (hrRenin) (n=8), a combination of lysine and hrRenin (n=5) and non-infused controls (n=15). Urines of mice infused with a combination of lysine (a blocker of proximal tubular protein reabsorption) and hrRenin had markedly higher urinary human active renin than those of controls (179±129 vs. 1.6±0.4 pg/mg Cr; p=0.001). The values were also markedly higher than those of mice infused with hrRenin only (4.4±1.1 pg/mg Cr; p=0.003). The effect of lysine was also evaluated in regard to endogenous mouse renin. Urinary mouse renin in mice infused with lysine (n=5) was markedly increased compared to non-infused controls (n=18) (22360±8673 vs. 346±82 pg/mg Cr; p=0.001). In conclusion, in humans with DKD, urine concentrations of both total and active renin are increased. In mice with STZ-induced DKD, urine total renin is also markedly increased. The data further demonstrate that 1) renin is both filterable and reabsorbable in normal mice and 2) the increase of urinary renin in DKD can be attributed largely to impaired reabsorption mainly in the proximal tubule.

Abstract P236: Site-1 Protease-derived Soluble (Pro) Renin Receptor Mediates Angiotensin II-induced Hypertension via Activation of Intrarenal Renin System

We have previously shown that collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) attenuates angiotensin II (Ang II)-induced hypertension, accompanied with reduced soluble PRR (sPRR) that exerts antidiuretic action. Recent preliminary and published results demonstrated site-1portease (S1P) but not furin or ADMA19 as the predominant PPR cleavage enzyme. In the present study, we evaluated involvement of S1P-derived sPRR in Ang II-induced hypertension. By radiotelemetry, CD PRR KO mice exhibited reduced MAP on day 7 of Ang II infusion at 300 ng/kg/min as compared with floxed mice (MAP: 118±5 vs. 137±3 mmHg, N=5, p&lt;0.05). Administration of sPRR-His, a histidine-tagged sPRR, at 120 μg/kg/d via i.v. infusion to CD PRR KO mice for additional 7 days largely restored the sensitivity to Ang II (MAP: 139±6 mmHg in sPRR-His +Ang II group vs. 116±5 mmHg in Ang II group, N = 4, p&lt;0.05). The i.v. infusion was achieved via placement of a catheter in jugular vein with the other end connected to mimipump. In C57/BL6 mice, administration of a S1P inhibitor PF429242 (PF) via mini pump infusion at 30 mg/kg/d for 7 days attenuated Ang II-induced increases in MAP (day 7: 125±5 in Ang II+ PF group vs. 142±3 in Ang II group; N=6, p&lt;0.05), urinary sPRR excretion (27±4 vs. 63±9 pg/24h; N=6, p&lt;0.05). In parallel, urinary renin levels were elevated by Ang II, which was blunted by PF (renin activity: 0.17±0.03 in Ang II+PF vs. 0.80±0.081 in Ang II vs. 0.12±0.016 ng/24h in Control, N=6, p&lt;0.01; active renin content: 15.2±2.7 vs. 236.0±23.2 vs. 5.6±1.3 ng/24h, N=6, p&lt;0.01; prorenin content: 9.6±3.1 vs. 27.8±6.1 vs. 6.2±1.8 ng/24h, N=6, p&lt;0.05; total renin content: 24.8±5.2 vs. 263.8±27.0 vs. 11.8±3.0 ng/24h, N=6, p&lt;0.01). An intravenous infusion of sPRR-His counteracted the blood pressure-lowering effect of PF in Ang II-infused mice (MAP: 147±3 in PF+sPRR vs. 126±4 mmHg in PF; N=4, p&lt;0.05). Together, these results suggest that S1P-derived sPRR contributes to Ang II-induced hypertension through activation of intrarenal renin-angiotensin system.

Abstract P209: Activation of Renal (Pro)Renin Receptor Contributes to High Fuctose-induced Salt Sensitivity

A high-fructose (HF) diet is shown to induce salt-sensitive hypertension but the underlying mechanism remains unclear. The major goal of the present study was to test the role of renal (pro)renin receptor (PRR) in this model. Male Sprague-Dawley rats were randomly divided into the following 4 groups: 1) Control, 2) Fructose, 3) Fructose + PRO20, and 4) Fructose + allopurinol, and the treatments lasted for 3 months. Fructose was added to drinking water (as 20% solution) so was allopurinol (at 30 mg/kg/d). PRO20, an antagonist of (pro)renin receptor, was administered at 700 μg/kg/day via i.p. injections. High fructose (HF) intake induced a 150% increase in renal protein expression of full-length PRR (fPRR), which were attenuated by allopurinol. HF intake also upregulated renal mRNA and protein expression of NHE3 (206% for protein) and NKCC2 (169% for protein) as well as in vivo NKCC2 activity (2-fold increases in 1-h urine volume and UNaV), all of which were nearly completely blocked by PRO20 or allopurinol treatment. HF intake induced &gt;5-fold increases in urinary renin activity, renin content, and total renin content, and a 2-fold increase in urinary AngII, which were suppressed by 60-70% with PRO20 or allopurinol, contrasting to relatively consistent values of these parameters in the plasma, evidence of involvement of intrarenal RAS . At the last week of the experimental period, radiotelemetry was performed to monitor blood pressure during one-week high salt (HS) diet (8% NaCl). The 3-mo HF intake or a 1-wk HS diet alone did not affect mean arterial pressure (MAP), but the combination of the two maneuvers induced a ~10 mm Hg increase of MAP, which was abolished by PRO20 or allopurinol treatment. In cultured human kidney 2 cells, both fructose and uric acid (UA) increased protein expression of soluble PRR (sPRR) in a time- and dose-dependent manner; fructose-induced PRR upregulation was inhibited by allopurinol. Taken together, our data suggest that fructose via UA stimulates renal expression of PRR/sPRR that stimulate NHE3 and NKCC2 expression and intrarenal RAS to induce salt-sensitive hypertension.

Collecting Duct Renin Does Not Mediate DOCA-Salt Hypertension or Renal Injury

Collecting duct (CD)-derived renin is involved in the hypertensive response to chronic angiotensin-II (Ang-II) administration. However, whether CD renin is involved in Ang-II independent hypertension is currently unknown. To begin to examine this, 12 week old male and female CD-specific renin knock out (KO) mice and their littermate controls were subjected to uni-nephrectomy followed by 2 weeks of deoxycorticosterone acetate (DOCA) infusion combined with a high salt diet. Radiotelemetric blood pressure (BP) was similar between KO and control mice at baseline; BP increased in both groups to a similar degree throughout the 2 weeks of DOCA-salt treatment. Urinary albumin excretion and plasma blood urea nitrogen were comparable between the two groups after DOCA-salt treatment. Fibrosis as assessed by Masson’s Trichrome stain/Sirius Red stain and collagen-1 mRNA expression was similar between control and KO mice. Compared to baseline, DOCA-salt treatment decreased plasma renin concentration (PRC), urinary renin excretion and medullary renin mRNA expression in both floxed and CD renin KO mice with no detectable differences between the two groups. Further, in primary culture of rat inner medullary CD, aldosterone treatment did not change renin activity or total renin content. Taken together, these data suggest that CD derived renin does not play a role in DOCA-salt hypertension.

041 - Influence of Local Renal Versus Systemic RAAS Activation on Loop Diuretic Response and Clinical Outcomes in Heart Failure

Introduction: The renin-angiotensin-aldosterone-system (RAAS) is universally recognized as a critical pathophysiologic axis in heart failure (HF). Notably, RAAS activation causes both progression of cardiac and renal dysfunction and is a major driver of renal sodium retention. However, the RAAS system has paracrine as well as endocrine effects and tissue RAAS activity has been shown in many situations to have limited correlation with circulating levels. Urinary renin and urinary angiotensinogen have been shown to correlate well with renal RAAS activity at the tissue level. Our goal was to determine the relative importance of renal versus systemic RAAS activation on renal and cardiac function in addition to survival in a well phenotyped contemporary HF population. Hypothesis: We expect to see cardiorenal parameters correlate differently with local versus systemic RAAS activation. Methods: Heart failure outpatients in the Yale Transition Care Center treated with loop diuretics were prospectively studied. Diuretic efficiency (DE) was defined as total sodium excreted per doubling of the diuretic dose. Total renin and angiotensinogen were determined in plasma and urine using the Meso Scale Discovery (MSD) platform (Meso Scale diagnostics, Gaithersburg, MD, USA). Urine renin and angiotensinogen were indexed to urinary creatinine to account for dilution. Results: In total 164 patients were studied. Cardiac parameters: Ejection fraction correlated with plasma renin (r=-0.17, P = .03) but not urinary renin levels (P = .23). Systolic blood pressure correlated with plasma renin (r=-0.21, P = .007) and plasma angiotensinogen (r = 0.23, P = .003), but did not correlate with urinary markers (P > .58 for both). Renal parameters: eGFR was not correlated with plasma angiotensinogen and only weakly correlated with plasma renin (r = 0.2, P = .01). However, the correlation between urinary RAAS markers and eGFR was substantially stronger (urinary angiotensinogen r=-0.47, P < .0001, urinary renin r=-0.30, P = .001). DE was also more strongly correlated to urinary renin (r=-0.41, P < .0001) than plasma renin (r=-0.29, P < .001) and urinary angiotensinogen (r=-0.29, P = .001) than plasma angiotensinogen (r=-0.17, P = .046). Outcomes: A plasma renin above the median (HR = 4.3, 95% CI 2.1–9.1, P < .0001) or plasma angiotensinogen above the median (HR = 2.1, 95% CI 1.1–4.0, P = .021) were both strongly associated with mortality. However, there was no association between the urinary markers and survival (P > .10 for both). Conclusion: Both systemic and renal RAAS activation appear to be important parameters in HF. However, cardio-renal metrics such as diuretic responsiveness and renal function are more closely related to renal RAAS activity whereas left ventricular function, blood pressure and survival are more strongly linked to plasma RAAS activation

Abstract P178: Vitamin D Biomarkers with Risk of Hypertension and Renin-Angiotensin System

Background: Experimental studies suggest that vitamin D may potentially prevent the development of hypertension through down-regulation of the renin-angiotensin system. However, evidence from epidemiologic studies remains limited. Objective: In parallel nested case-control studies of women from the Women’s Health Study (WHS) and men from the Physicians’ Health Study (PHS) with &gt;10 years follow-up, we examined the prospective associations between the circulating biomarkers of vitamin D status with risk of hypertension, as well as the inter-relation between vitamin D biomarkers and renin production. Methods: In each cohort, 500 incident hypertension cases and 500 controls were randomly selected from participants who were free of cardiovascular disease, cancer, and hypertension and provided bloods at baseline. Hypertension cases were identified based on self-reported blood pressure, physician’s diagnosis, or medication use. Controls were matched to cases on gender, age, race, follow-up time, and month of blood draw. Baseline plasma 25(OH)-vitamin D, parathyroid hormone (PTH), and total renin (pro-renin plus active renin) concentrations were measured. Analyses were first performed in each cohort separately and, given the similar results, finally combined for both cohorts. Results: Baseline concentrations of 25(OH)-vitamin D (mean of 26.6 vs. 27.0 ng/mL) and PTH (32.6 vs. 32.2 pg/mL) were similar in hypertension cases and controls. In the crude matched model, the risk of hypertension did not change with 25(OH)-vitamin D or PTH concentrations. Additional adjustment for other known hypertension-related risk factors minimally affected the associations. After adjusting for baseline smoking, alcohol use, exercise, menopausal status and hormone therapy use among women, body mass index, and history of diabetes and hypercholesterolemia, the relative risks of incident hypertension across increasing quintiles of 25(OH)-vitamin D were 1.00, 1.42 (95% CI: 0.94-2.16), 0.94 (95% CI: 0.60-1.48), 0.99 (95% CI: 0.61-1.61), and 1.03 (95% CI: 0.63-1.69) (p, trend, 0.48), and across increasing quintiles of PTH were 1.00, 1.26 (95% CI: 0.83-1.92), 0.91 (95% CI: 0.60-1.40), 1.10 (95% CI: 0.70-1.71), 0.92 (95% CI: 0.59-1.44) (p, trend, 0.50). These associations were similar in men and women. Among controls, plasma 25(OH)-vitamin D was inversely correlated with PTH (Pearson r: -0.24, p&lt;0.0001), while neither 25(OH)-vitamin D (r: -0.02, p=0.53) nor PTH (r: -0.01, p=0.69) was correlated with total renin concentrations. Conclusions: In prospective cohorts of men and women, we found no association between either baseline plasma 25(OH)-vitamin D or PTH with the risk of hypertension, or between either 25(OH)-vitamin D or PTH with total renin concentration. More research is needed to further elucidate the specific effect of vitamin D in the development of hypertension.

Abstract MP01: Vascular versus Tubular Renin: Role in Kidney Development

Renin, the key regulated enzyme of the renin angiotensin system regulates blood pressure and fluid electrolyte homeostasis as well as morphogenesis of the kidney. Classically, renin is synthesized and released from juxtaglomerular cells located in the afferent arterioles at the entrance to the glomeruli. It has also been suggested that renin may also be synthesized by tubular cells. Interestingly, whole body deletion of the renin gene results in striking vascular and tubular abnormalities, hydronephrosis and a urine-concentrating defect. Given the complexity of such phenotype, it has not been possible to discriminate the relative contributions of vascular versus tubular renin. To investigate the relative contribution of renin in the vascular versus tubular compartments during kidney development we deleted renin independently in either compartment by crossing Ren1cfl/fl mice to Foxd1-cre or to Hoxb7-cre mice. We performed blood chemistries, histological analysis, immunostaining for specific cell markers, total kidney renin mRNA quantification and ELISA for renin in plasma. Vascular deletion of renin (Foxd1 lineage) resulted in neonatal mortality that could be rescued with daily injections of saline suggesting that this phenotype is due to an inability to concentrate the urine and improper medullary development. These mice showed absence of renal renin and negligible levels of plasma renin. Histologically, the kidneys had abnormal development of its arterioles, which became progressively thickened by disorganized, concentric hypertrophy of smooth muscle cells and marked hydronephrosis. On the other hand, lack of renin in the collecting ducts (Hoxb7 descendants) did not affect kidney morphology, intra-renal renin distribution, kidney renin mRNA expression or circulating renin during basal conditions or in response to a homeostatic stress such as low sodium diet. We conclude that renin generated in the renal vascular compartment is fundamental for the development and integrity of the kidney whereas the presence of renin in the collecting duct system is dispensable for normal kidney development and cannot compensate for the lack of renin in the vascular compartment. Further, the main source of circulating renin is the kidney vasculature.

Abstract P084: Oxidative Stress Preconditioning Is Required for Leptin to Stimulate Renin Release From Mouse Juxtaglomerular Cells

Leptin is enhanced in animal models of obesity. The role of leptin in hypertension is unclear with some studies showing antihypertensive actions and others showing a pro-hypertensive role. In normotensive, non-obese, animals, infusion of leptin induces natriuresis. However, in animal models of enhanced oxidative stress, obesity, or activated renin-angiotensin system, infusion of leptin tends to increase blood pressure. Renin is a critical mediator of angiotensin-II formation, and thus blood pressure control. However, the direct effect of leptin on the release of renin from juxtaglomerular (JG) cells has not been studied. We hypothesize that under normal conditions leptin does not affect renin release whereas during high oxidative stress leptin stimulates renin release. We isolated primary cultures of mouse juxtaglomerular (JG) cells. After treatment with different agonists, renin released to the supernatant was measured by radioimmunoassay. We first tested the direct effect of murine leptin on renin release. We found that 1 hr treatment with leptin (0.1-1 uM) decreased basal renin by 20 ± 5% (p&lt;0.05; n=8). Treatment of JG cells for longer periods (4 and 24 hrs) did not affect renin release (n=6) or total renin expression (n=4). We then tested whether oxidative stress modified the effect of leptin on renin release. For this we pre-incubated JG cells with 10 uM hydrogen peroxide (H2O2) for 1 hr. After this, the medium was removed and H2O2 was completely washed out from JG cells followed by treatment with vehicle (cont) or 10 uM leptin. We found that, in cells pre-treated with H2O2, leptin increased renin release by 49.2 ±16 %(P&lt;0.05 vs vehicle). By Western blot, we detected the expression of the leptin receptor in lysates from JG cells. We concluded that under normal conditions leptin inhibits renin release from JG cells. However, after exposure to H2O2, leptin stimulates renin release. Our data suggest the hypothesis that oxidative stress reverses the inhibitory effect of leptin on renin release and supports a pro-hypertensive role for leptin during chronic inflammatory conditions that induces oxidative stress.

Maternal insulin-like growth factor 1 and 2 differentially affect the renin–angiotensin system during pregnancy in the guinea pig

ObjectiveInsulin-like growth factors (IGFs) are known to interact with the renin–angiotensin system (RAS). We previously demonstrated that administration of IGF1 to guinea pigs in early to mid pregnancy promotes placental function and fetal growth in mid to late gestation. Early administration of IGF2 had sustained, but not acute, effects on these parameters and also on placental structural differentiation. Here, we aimed to determine whether the IGFs interact with the placental RAS in early to mid gestation to modulate placental development and increase fetal growth and survival, and if IGF2 binding the IGF2R is implicated in the sustained effects of IGF2 treatment. DesignAt day 20 of pregnancy, guinea pigs were infused with 1mg/kg/day of IGF1, IGF2, Leu27IGF2 or vehicle for 18days and sacrificed on either day 62 (late pregnancy) or during the infusion period on day 35 (early–mid pregnancy). Placental structure at day 35 was analyzed using morphometric technique and expression of RAS genes in the placenta and placental and plasma renin activity were measured at both time points. ResultsCompared with vehicle at day 35 of gestation, IGF1 infusion reduced the total midsagittal cross-sectional area of the placenta (−17%, p=0.02) and the labyrinth area (−22%, p=0.014) but did not alter the labyrinth volume nor labyrinth:interlobium ratios. IGF2 treatment did not affect placental structure.IGF1 did not alter placental mRNA for any of the RAS genes quantified at day 35 (AGTR1, ACE, AGT, TGFB1) but increased TGFB1 expression by more than 16-fold (p=0.005) at day 62. IGF2 increased placental expression of AGTR1 (+88%, p=0.03) and decreased AGT (−73%, p=0.01) compared with the vehicle-treated group at day 35, and both IGF2 and Leu27IGF2 increased expression of TGFB1 at day 62 by 9-fold (p=0.016) and 6-fold (p=0.019) respectively.Both IGFs increased the ratio of active:total placental renin protein (+22% p=0.026 p=0.038) compared to vehicle compared to vehicle at day 35 but not 62. At day 62, IGF2-treated mothers showed a marked increase in total plasma renin (+495%) and active renin (+359%) compared to vehicle but decreased the ratio of active to total renin by 41% (p=0.042). Leu27IGF2-treated animals had higher levels of placental active renin (+73%, p=0.001) and total renin (+71%, p=0.001) compared with the vehicle control. ConclusionsThe data obtained in the current study suggest the potential for alternate roles for the induction of the RAS after IGF treatment. IGF1 and 2 treatments increase the activation of prorenin to renin in the placenta, possibly due to increased protease activity. In addition, IGF2 treatment in early pregnancy may enhance the maternal adaptation to pregnancy through stimulation of renin in the kidney. The sustained effects on placental differentiation and function after IGF2 treatment suggest therapeutic potential for exogenous administration of IGFs in improving pregnancy outcomes.

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.

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.