Intrarenal RAS Research Articles

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<0.05, n=6), plasma glucose (in mM: C57=7.71±23.9; Aki=23.2±2.3; p<0.01; n=4) and proteinuria (mg/24hrs: C57 18.2±3.1; Aki: 29.4±3.3; p<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<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<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<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<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.

PS-C30-1: URINARY ANGIOTENSINOGEN-MELATONIN RATIO IN GESTATIONAL DIABETES AND PREECLAMPSIA

Objective: A large research portfolio indicates that an activated renal renin-angiotensin system or a deficit on melatonin is associated with several cardiovascular pathologies. In this observational clinical study, we hypothesized that alterations in urinary melatonin or angiotensinogen levels may be altered in two common conditions, preeclampsia and gestational diabetes. Our primary objective was to assess melatonin and angiotensinogen as novel disease biomarkers detectable and quantifiable in the urine of pregnant women with or without pregnancy complications. Design and Methods: This was a concurrent cohort study of pregnant women with selected obstetric pathologies (gestational diabetes, preeclampsia, hypertension and obesity with hypertension). A group of healthy controls was also included. Urinary 6-sulfatoxymelatonin and angiotensinogen were measured by sensitive and specific ELISAs in first morning void urine samples. The patients were included in the cohort consecutively, and the diagnosis was blinded at the level of urine collection. Urinary 6-sulfatoxymelatonin and angiotensinogen levels were investigated in the patients included in the cohort. Results: Urinary levels of angiotensinogen were significantly higher in the gestational diabetes [angiotensinogen/creatinine ratio median (25th, 75th): 0.11 (0.07, 0.18)] and preeclampsia [0.08 (0.06, 0.18)] groups than in those with healthy pregnancy [0.05(0.04, 0.06]; 6-sulfatoxymelatonin levels were significantly lower in the gestational diabetes [ug/h: median (25th, 75th): 0.12(0.08, 0.17)] and preeclampsia [0.12 (0.09, 0.15)] groups than in those with healthy pregnancy [0.20 (0.15, 0.27]. Neither morning void protein/creatinine ratio nor 24-h urine protein estimate were significantly different between the study groups. Conclusion: These results suggest that urinary angiotensinogen levels may indicate an intrarenal RAS activation while melatonin production appears to be defective in gestational diabetes or hypertension. An angiotensinogen/melatonin ratio is suggested as an early biomarker for identification of gestational diabetes or hypertension. This report provides a basis for the potential use of melatonin for the treatment of preeclampsia. A prospective study in a larger number of patients to determine the operative characteristics of these markers as potential diagnostic tests is justified.

Abstract 093: Association Of Preterm Birth And Low Birth Weight With The Circulatory And Urinary Renin-angiotensin System In Youth With Newly Diagnosed Primary Hypertension

Background: While the RAS is altered in youth born preterm and may play a role in development of HTN, this has not been investigated in youth with primary HTN. We hypothesized that lower gestational age (GA) and lower birth weight (BW) would be associated with higher Ang II, lower Ang-(1-7), and higher Ang II/Ang-(1-7) in plasma and urine. Methods: This is a secondary analysis of baseline data from a pilot prospective cohort study of 30 youth aged 5-17 years with newly diagnosed primary HTN per national guidelines. Exclusion criteria were heart or chronic kidney disease and diabetes mellitus. Exposures were GA, BW, and BW z-score on the continuous scale and as binary variables, preterm birth (GA <37 weeks) and low BW (<2500 g). RAS outcomes were Ang II and Ang-(1-7) measured in plasma and urine per well-validated RIAs; urinary values standardized to urine creatinine (Cr). Plasma renin activity and aldosterone were measured per well-validated methods. We estimated associations with unadjusted generalized linear models. Results: Median GA was 39 weeks [IQR 36, 40] with 28% (8/30) born preterm. Mean BW was 3.12 kg ( SD 0.65) with low BW in 17% (5/30). Preterm birth was associated with lower urine Ang-(1-7)/Cr and higher aldosterone/renin ratio and urine Ang II/Ang-(1-7) (Figure). Low BW was associated with higher plasma Ang-(1-7) and urine Ang II/Cr. Conclusion: Early-life risk factors were associated with intrarenal RAS alterations favoring Ang II activation and Ang-(1-7) suppression in youth with primary HTN. Our findings provide novel insight into the mechanistic role that the RAS may play in HTN development in youth born preterm or with low BW that differs from youth born at term who have HTN.

Urinary Angiotensinogen-Melatonin Ratio in Gestational Diabetes and Preeclampsia.

Background: A large research portfolio indicates that an activated renal renin-angiotensin system or a deficit on melatonin is associated with several cardiovascular pathologies. In this observational clinical study, we hypothesized that alterations in urinary melatonin or angiotensinogen levels may be altered in two common conditions, preeclampsia and gestational diabetes. Our study’s primary objective was to assess melatonin and angiotensinogen as novel disease biomarkers detectable and quantifiable in the urine of pregnant women with or without pregnancy complications. Methods: This was a concurrent cohort study of pregnant women with selected obstetric pathologies (gestational diabetes, preeclampsia, hypertension and obesity with hypertension). A group of healthy controls was also included. Urinary 6-sulfatoxymelatonin and angiotensinogen were measured by sensitive and specific ELISAs in first morning void urine samples. The patients were included in the cohort consecutively, and the diagnosis was blinded at the level of urine collection. Urinary 6-sulfatoxymelatonin and angiotensinogen levels were investigated in the patients included in the cohort. Results: Urinary levels of angiotensinogen were significantly higher in the gestational diabetes [angiotensinogen/creatinine ratio median (25th, 75th): 0.11 (0.07, 0.18)] and preeclampsia [0.08 (0.06, 0.18)] groups than in those with healthy pregnancy [0.05(0.04, 0.06]; 6-sulfatoxymelatonin levels were significantly lower in the gestational diabetes [ug/h: median (25th, 75th): 0.12(0.08, 0.17)] and preeclampsia [0.12 (0.09, 0.15)] groups than in those with healthy pregnancy [0.20 (0.15, 0.27]. Neither morning void protein/creatinine ratio nor 24-h urine protein estimate were significantly different between the study groups. Conclusion: These results suggest that urinary angiotensinogen levels may indicate an intrarenal RAS activation while melatonin production appears to be defective in gestational diabetes or hypertension. An angiotensinogen/melatonin ratio is suggested as an early biomarker for identification of gestational diabetes or hypertension. This report provides a basis for the potential use of melatonin for the treatment of preeclampsia. A prospective study in a larger number of patients to determine the operative characteristics of these markers as potential diagnostic tests is justified.

Effect of intrarenal renin-angiotensin-aldosterone system on renal function in patients after cardiac surgery.

The aim of the study was to investigate the influence of intrarenal RAS on the decrease of renal function in patients undergoing cardiac surgery with cardiopulmonary bypass. This observational study investigated the activation of intrarenal RAS in 24 patients with AKI after cardiac surgery with cardiopulmonary bypass. The activation of intrarenal RAS was determined by urinary angiotensinogen (uAGT), which was measured at 12 hours before surgery, 0 and12 hours after surgery. The results were compared with those of 21 patients without AKI after cardiac surgery with cardiopulmonary bypass. Clinical and laboratory data were collected. Compared with baseline, all patients with cardiac surgery had activation of intrarenal RAS at 0 and 12 hours after surgery. The activation of intrarenal RAS was found significantly higher at both 0 and 12 hours after surgery in AKI group versus non AKI group (6.18 ± 1.93 ng/mL vs 3.49 ± 1.71 ng/mL, 16.38 ± 7.50 ng/mL vs 6.04 ± 2.59 ng/mL, respectively). There was a positive correlation between the activation of RAS at 0 hour after surgery and the decrease of renal function at 48 hours after surgery (r = 0.654, P = .001). These findings suggest that uAGT might be a suitable biomarker for prediction of the occurrence and severity of AKI after cardiac surgery. Inhibition of intrarenal RAS activation might be one the path of future treatment for this type of disease.

Foe and friend in the COVID-19-associated acute kidney injury: an insight on intrarenal renin-angiotensin system.

Since the first reported case in December of 2019, the coronavirus disease 2019 (COVID-19) has became an international public health emergency. So far, there are more than 228,206,384 confirmed cases including 4,687,066 deaths. Kidney with high expression of angiotensin-converting enzyme 2 (ACE2) is one of the extrapulmonary target organs affected in patients with COVID-19. Acute kidney injury (AKI) is one of the independent risk factors for the death of COVID-19 patients. The imbalance between ACE2-Ang(1-7)-MasR and ACE-Ang II-AT1R axis in the kidney may contribute to COVID-19-associated AKI. Although series of research have shown the inconsistent effects of multiple common RAS inhibitors on ACE2 expression and enzyme activity, most of the retrospective cohort studies indicated the safety and protective effects of ACEI/ARB in COVID-19 patients. This review article highlights the current knowledge on the possible involvement of intrarenal RAS in COVID-19-associated AKI with a primary focus on the opposing effects of ACE2-Ang(1-7)-MasR and ACE-Ang II-AT1R signaling in the kidney. Human recombinant soluble ACE2 or ACE2 variants with preserved ACE2-enzymatic activity may be the best options to improve COVID-19-associated AKI.

In Vivo Renin Activity Imaging in the Kidney of Progeroid Ercc1 Mutant Mice.

Changes in the renin–angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin–angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/− mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/− mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/− mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/− compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging.

Sodium and glucose cotransporter type 2 inhibitors: a new class of drugs for the treatment of diabetic and non-diabetic nephropathy

Inhibitors of renal sodium-glucose cotransporter type 2 (SGLT2) are a new class of antidiabetic drugs that have recently been introduced into clinical practice for the treatment of patients with type 2 diabetes mellitus. According to CREDENCE study, the inclusion of canagliflozin in drug therapy for patients with type 2 diabetes mellitus not only provides adequate control of blood glucose but also has a pronounced nephroprotective effect, which manifests in a significant reduction in the risk of progression of renal dysfunction in patients with stages 2, 3a and 3b CKD. The identification of nephroprotective effects in SGLT2 inhibitors, which is not related to their antihyperglycemic effect, suggests the possibility of using drugs of this class for drug therapy of patients with CKD of non-diabetic etiology. The review presents the data of clinical studies devoted to elucidating the participation of diuretic action and the associated decrease in blood pressure and venous stasis in the kidneys, improving glomerular hemodynamics and inhibiting the activity of intrarenal RAS in the mechanism of nephroprotective action of these drugs. Large-scale DAPA-CKD and EMPA-KIDNEY studies are currently underway, the results of which will provide information on the clinical efficacy and safety of dapagliflozin and empagliflozin in non-diabetic patients with the impaired renal function of varying severity, including those with stage 4 CKD. Initial data obtained in the DAPA-CKD trial indicated that dapagliflozin, when added to nephroprotective therapy, significantly improves renal outcomes not only in patients with type 2 diabetes but also in patients with CKD of non-diabetic origin, including those with glomerulonephritis, hypertensive nephropathy, and other kidney damage.

(Pro)renin receptor antagonist PRO20 attenuates nephrectomy-induced nephropathy in rats via inhibition of intrarenal RAS and Wnt/β-catenin signaling.

Introduction(Pro)renin receptor has emerged as a new member of the renin‐angiotensin system implicated in the pathogenesis of chronic kidney disease (CKD). Herein we report characterization of the therapeutic potential of (pro)renin receptor (PRR) antagonist PRO20 in 5/6 nephrectomy (5/6Nx) rats.MethodsMale Wistar rats underwent 5/6Nx followed by treatment with vehicle or received daily injections of a PRR inhibitor PRO20 (700 μg/kg) via the 3 s.c. Sham group served as a control.ResultsAs compared with the sham control, the 5/6Nx rats exhibited significant increases in proteinuria, glomerulosclerosis, tubular injury, and interstitial inflammation in the remnant kidneys. Treatment with PRO20 significantly attenuated these abnormalities, as evidenced by reduced expression of fibronectin, α‐SMA, collagen 1, TGF‐β1, IL‐6, IL‐8, IL‐1β, MCP‐1 and increased expression of E‐cadherin. Increased urinary/renal levels of renin activity, angiotensinogen (AGT), and Angiotensin II (Ang II) by 5/6Nx, which were all ameliorated by PRO20. Renal PRR, the secreted proteolytic fragment of PRR (sPRR) in renal and urinary, were all elevated in 5/6Nx rats. Moreover, our results revealed that renal Wnt3A and β‐catenin expression were upregulated during 5/6Nx, which were all attenuated by PRO20.ConclusionsOverall we conclude that in vivo antagonism of PRR with PRO20 will improve 5/6Nx‐induced CKD mainly through inhibition of intrarenal RAS and Wnt/β‐catenin signaling pathway.

AT1 receptor blocker, but not an ACE inhibitor, prevents kidneys from hypoperfusion during congestive heart failure in normotensive and hypertensive rats

To provide novel insights into the pathogenesis of heart failure-induced renal dysfunction, we compared the effects of ACE inhibitor (ACEi) and AT1 receptor blocker (ARB) on systemic and kidney hemodynamics during heart failure in normotensive HanSD and hypertensive transgenic (TGR) rats. High-output heart failure was induced by creating an aorto-caval fistula (ACF). After five weeks, rats were either left untreated or treatment with ACEi or ARB was started for 15 weeks. Subsequently, echocardiographic, renal hemodynamic and biochemical measurements were assessed. Untreated ACF rats with ACF displayed significantly reduced renal blood flow (RBF) (HanSD: 8.9 ± 1.0 vs. 4.7 ± 1.6; TGR: 10.2 ± 1.9 vs. 5.9 ± 1.2 ml/min, both P < .001), ACEi had no major RBF effect, whereas ARB completely restored RBF (HanSD: 5.6 ± 1.1 vs. 9.0 ± 1.5; TGR: 7.0 ± 1.2 vs. 10.9 ± 1.9 ml/min, both P < .001). RBF reduction in untreated and ACEi-treated rats was accompanied by renal hypoxia as measured by renal lactate dehydrogenase activity, which was ameliorated with ARB treatment (HanSD: 40 ± 4 vs. 42 ± 3 vs. 29 ± 5; TGR: 88 ± 4 vs. 76 ± 4 vs. 58 ± 4 milliunits/mL, all P < .01). Unlike improvement seen in ARB-treated rats, ACE inhibition didn’t affect urinary nitrates compared to untreated ACF TGR rats (50 ± 14 vs. 22 ± 13 vs. 30 ± 13 μmol/mmol Cr, both P < .05). ARB was more effective than ACEi in reducing elevated renal oxidative stress following ACF placement. A marker of ACEi efficacy, the angiotensin I/angiotensin II ratio, was more than ten times lower in renal tissue than in plasma. Our study shows that ARB treatment, in contrast to ACEi administration, prevents renal hypoperfusion and hypoxia in ACF rats with concomitant improvement in NO bioavailability and oxidative stress reduction. The inability of ACE inhibition to improve renal hypoperfusion in ACF rats may result from incomplete intrarenal RAS suppression in the face of depleted compensatory mechanisms.

Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II-Induced Hypertension in Mice.

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

The Intrarenal RAS Upregulates SGLT2 Expression and SGLT2 Inhibitors Attenuate Angiotensin-II Induced Hypertensive Kidney Injury in Mice

The Intrarenal RAS Upregulates SGLT2 Expression and SGLT2 Inhibitors Attenuate Angiotensin-II Induced Hypertensive Kidney Injury in Mice

Abstract P002: Soluble (pro)renin Receptor Contributes To Angiotensin II-Induced Hypertension In Mice

Activation of (pro)renin receptor (PRR) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during angiotensin II (AngII) infusion. Soluble PRR (sPRR), the extracellular domain of PRR, is generated by multiple proteases including furin or ADAM19, and recently site-1 protease (S1P). The goal of the present study was to test the role of S1P-derived sPRR mediated AngII-induced hypertension. F1 B6129SF1/J mice were infused for 6 days with control (CTR) or AngII at 300 ng/kg/day alone or in combination with S1P inhibitor PF-429242 (PF) and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated AngII-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histadine-tagged sPRR termed as sPRR-His. Mutagenesis of overlapping recognition site for S1P and furin in PRR for was generated in mice by CRISPR strategy (termed as mutant mice). Mutant mice were fertile and developed normally with a 50% reduction plasma sPRR. These mice exhibited blunted hypertensive response to AngII infusion accompanied with suppressed intrarenal renin levels. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mpkCCD cells exposed for 24 h to AngII, AngII + PF, or AngII + PF + sPRR-His. AngII-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that sPRR derived from S1P or in combination with furin mediates AngII-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

P1002EFFECT OF VITAMIN D ON URINARY ANGIOTENSIOGEN LEVEL IN EARLY DIABETIC NEPHROPATHY

Abstract Background and Aims UrinaryAngiotensinogen (UAGT) is supposed to be a marker of activation of intrarenalreninangiotensin system (RAS) system in early Diabetic Nephropathy (DN). Vitamin D has been studied as a negative regulator of the circulating and tissue RAS activity; so its supplementation may prevent progression of DN. This study was planned to assess the RAS activation and effect of vitamin D supplementation in early diabetic nephropathy progression by estimatingUAGT level. Method A total of 730 Diabetic subjects were screened for early DN based on Inclusion criteria(eGFR ≥ 120ml/min/1.73m2 with unrestricted ACR value eGFR of 60- 120 mL/min/1.73m2 and ACR 30-300mg) and Exclusion criteria(on ACE I or ARB, eGFR&amp;lt;60ml/min/1.73m2, Hypercalcemia (&amp;gt;11.5mg%), UTI, Pregnancy, Hypertension, HbA1c (&amp;gt;8.0 %), vitamin D supplementation during the last 6 month).Of them 103 included subjects were underwent randomization to receive either Cholecalciferol(54) or matching Placebo(49) in a double blind method. All were subjected to routine investigations, urinary albumin: creatinine (UACR), UAGT, serum 25(OH)vitamin D, and intact parathyroid hormone (iPTH) at the zero and six months. A total 40 healthy controls without any comorbidity were also includedfor assessment of same investigations at zero month. Comparative results at zero months of all three groups were analyzed. Results The change in levels of UACR (mg/g), UAGT (ng/ml), vitamin D (ng/ml) and iPTH(pg/ml)in Cholecalciferol group from zero to six month were [Median (IQR)] : 12.50 (-24.82 to -5.95); p&amp;lt;0.001, -6.60 (-20.91 to -2.63);p&amp;lt;0.001, 5.75 (2.07-9.70); p&amp;lt;0.001, -5.35 (-11.55 to -3.67) ;p&amp;lt;0.001, respectively. After six months, median (IQR) of UAGT and UACR levels were significantly lower in the Cholecalciferol group as compared to Placebo group (p&amp;lt;0.001 and p= 0.04 respectively).Significant reduction of UACR (mg/g) and UAGT (ng/ml) level corroborating with increase in 25(OH) vitamin D(ng/ml) levelfrom zero to six months (all three p&amp;lt;0.001). There was significant correlation of vitamin D3 level with UAGT (r value: -0.255 &amp; p value: 0.009) and UACR level (r value: -0.28&amp; p value: &amp;lt;0.01). The median UAGT level was significantly higher in patients with early DN (Cholecalciferol&amp;Placebo Group) than Control Group at zero month (p =0.001). Conclusion Significantly higher UAGT levels in early DN supports role of intrarenal RAS activation. Significant decrease in UAGT level in cholecalciferol group supports beneficial role of vitamin D supplementation in progression of early DN.

Gut microbiota dysbiosis-induced activation of the intrarenal renin-angiotensin system is involved in kidney injuries in rat diabetic nephropathy.

Some studies have shown that gut microbiota along with its metabolites is closely associated with diabetic mellitus (DM). In this study we explored the relationship between gut microbiota and kidney injuries of early diabetic nephropathy (DN) and its underlying mechanisms. Male SD rats were intraperitoneally injected with streptozotocin to induce DM. DM rats were orally administered compound broad-spectrum antibiotics for 8 weeks. After the rats were sacrificed, their blood, urine, feces, and renal tissues were harvested for analyses. We found that compared with the control rats, DM rats had abnormal intestinal microflora, increased plasma acetate levels, increased proteinuria, thickened glomerular basement membrane, and podocyte foot process effacement in the kidneys. Furthermore, the protein levels of angiotensin II, angiotensin-converting enzyme, and angiotensin II type 1 receptor in the kidneys of DM rats were significantly increased. Administration of broad-spectrum antibiotics in DM rats not only completely killed most intestinal microflora, but also significantly lowered the plasma acetate levels, inhibited intrarenal RAS activation, and attenuated kidney damage. Finally, we showed that plasma acetate levels were positively correlated with intrarenal angiotensin II protein expression (r = 0.969, P < 0.001). In conclusion, excessive acetate produced by disturbed gut microbiota might be involved in the kidney injuries of early DN through activating intrarenal RAS.

Urinary angiotensinogen increases in the absence of overt renal injury in high fat diet-induced type 2 diabetic mice

Aim of the StudyDuring type 2 diabetes (T2D) and hypertension there is stimulation of renal proximal tubule angiotensinogen (AGT), but whether urinary excretion of AGT (uAGT) is an indicator of glomerular damage or intrarenal RAS activation is unclear. We tested the hypothesis that elevations in uAGT can be detected in the absence of albuminuria in a mouse model of T2D. MethodsMale C57BL/6 mice (N = 10) were fed a high fat (HFD; 45% Kcal from fat) for 28 weeks, and the metabolic phenotype including body weight, blood pressures, glucose, insulin, ippGTT, HOMA-IR, and cholesterol was examined. In addition, kidney Ang II content and reactive oxygen species (ROS) was measured along with urinary albumin, creatinine, Ang II, and AGT. ResultsAll parameters consistent with T2D were present in mice after 12–14 weeks on the HFD. Systolic BP increased after 18 weeks in HFD but not NFD mice. Intrarenal ROS and Ang II concentrations were also increased in HFD mice. Remarkably, these changes paralleled the augmentation uAGT excretion (3.66 ± 0.50 vs. 0.92 ± 0.13 ng/mg by week 29; P < 0.01), which occurred in the absence of overt albuminuria. ConclusionsIn HFD-induced T2D mice, increases in uAGT occur in the absence of overt renal injury, indicating that this biomarker accurately detects early intrarenal RAS activation.

Abstract P420: Sexual Difference of Tubular Renin Angiotensin System (RAS) in 2 Kidney 1 Clip Rats

Background: Premenopausal female manifests lower blood pressure compared with age-matched male. Intrarenal RAS is thought to be an important role in hypertension and hypertensive renal disease, while there is rare information about sexual difference in intrarenal RAS. This study was performed to evaluate sexual difference of tubular RAS in 2 kidney 1 clip(2K1C) rat model. Methods: A 2.5-mm clip was inserted into the left renal artery of male and female Spargue-Dawley rats and they were euthanized at 5 week following the operation. Systolic blood pressure(SBP) was measured via the tail-cuff method at 10 day interval. Medullary area for tubular RAS and cardiac tissue was collected for analysis of local RAS expression. Results: At 30 days after clipping operation, SBP and albuminuria was significantly increased in 2K1C male rats (SBP: male 176.75±5.58 vs. female 106.71±8.68 mmHg, p 0.004, albumin/Cr ratio: male 116.93±33.96 vs. female 56.40±13.94 mg/g, p 0.029). Also, left ventricular hypertrophy was observed only in male rats, however cardiac ACE2 and MasR mRNA did not show sexual difference. The clipped kidney(CK) of 2K1C male rat presented worse glomerulosclerosis and more macrophage infiltration. Renin mRNA was more expressed in female CK, but protein expression was rather decreased in female CK and non-clipped kidney(NCK). 2K1C female rats exhibited highly augmented ACE2 and Mas receptor(MasR) in mRNA and protein, but ACE was augmented in mRNA and reduced in protein. Immunohistochemistry showed that tubular renin and ACE was increased in 2K1C male rats, in contrast ACE2 and MasR were increased in 2K1C female rats. Medullary angiotensin II(AngII) was significantly lower in male CK, but angiotensin 1-7(Ang1-7) was higher in female CK but did not show significance (CK AngII: male 201.88±10.40 vs. female 137.01±19.33 fg/mg p 0.030, CK Ang1-7: male 22.21±4.51 vs. female 57.93±19.59 fg/mg p 0.190). Conclusions: Same insult of renal artery stenosis aroused different clinical outcome in 2K1C male and female. Differently activated tubular RAS might influence difference in clinical manifestation. Superiority of nonclassic tubular RAS in female rats could limit the adverse effect of classic RAS under renovascular hypertension.

Abstract P426: Suppression of Intrarenal Ras With a 20-hydroxyeicosatetraenoic Acid Antagonist Attenuates Ang II-dependent Malignant Hypertension and Reverses Established Ang-II-dependent Hypertension in Cyp1a1-ren-2 Transgenic Rats

Rationale and Objective: Interplay of 20-HETE and ANG II might be a factor in pathophysiology of many forms of experimental hypertension. We hypothesized that intrarenal 20-HETE potentiates prohypertensive actions of ANG II in Cyp1a1-Ren-2 transgenic rats (TGR), a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of a new, orally active 20-HETE antagonist (SOLA) in this model. Methods: Treatment with SOLA (10 mg.kg -1 .day - 1 in drinking water) was started either simultaneously with induction of hypertension (early treatment) by indol-3-carbinol or 10 days later, during established hypertension (late treatment). Systolic blood pressure (SBP, measured by radiotelemetry) was monitored continuously and indices of renal and cardiac injury, and kidney 20-HETE and ANG II levels were determined at the end of experiments. Results: In TGR with induced hypertension, early SOLA treatment reduced SBP elevation (to 161±3 vs. 199±3 mmHg in induced TGR, p&lt;0.001), reduced albuminuria (16±2 vs. 35±3 mg/24 h, p&lt;0.002), glomerulosclerosis index (0.12±0.01 vs. 0.32±0.02, p&lt;0.001) and cardiac hypertrophy (left ventricle weight (mg)/tibial length ratio: 16.9±0.4 vs 20.6±0.5, p&lt;0.02). TGR with induced hypertension exhibited elevated intrarenal 20-HETE levels (15.2±0.03 vs. 9.2±0.02 μg/g in noninduced rats, p&lt;0.01); however, the elevation was not altered by SOLA treatment. Hypertensive TGR showed also augmented kidney ANG II levels (405±30 vs. 51±2 fmol/g in noninduced rats, p&lt;0.001). SOLA treatment significantly lowered kidney ANG II (95±19). Remarkably, in TGR with established hypertension, late SOLA treatment also decreased SBP (from 187±4 to 158±4 mmHg, p&lt;0.002) and exhibited cardio- and renoprotective effects in addition to a marked suppression of kidney ANG II levels (72±12 fmol/g). Conclusion: In hypertensive TGR the new orally active 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent experimental malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal RAS activation by 20-HETE is important in pathophysiology of this hypertension form

PP.17.01] ACTIVATION OF RENIN-ANGIOTENSIN SYSTEM STIMULATES THE ORGANIC CATION TRANSPORTER 2 AND INCREASES CISPLATIN TOXICITY

Objective: The intrarenal RAS is characterized by high levels of angiotensin II (AII) and wide distribution of angiotensin receptors (ATR). There is indirect data showing cross-talk between the RAS and the human organic cation transporter 2 (hOCT2). This transporter is highly expressed in the basolateral membrane of proximal tubules cells and plays an important role for uptake, accumulation, and toxicity of cisplatin. Therefore, we aimed to investigate whether AII is able to modulate cisplatin toxicity in Madin-Darby canine kidney (MDCK) cells. Design and method: The uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+, 20 μM) added to the basolateral compartment of MDCK cells stably expressing hOCT2 growing on filters was measured in the presence of 1 nM AII alone or with the AT1R blocker losartan. Following 1 hr incubation at 37C, cells were solubilized and the intracellular ASP+ concentration was measured with a microfluorimeter. Next, cell monolayers were incubated with 0.1 mM cisplatin alone or with 1 nM AII and blocker combinations. During and after incubation with different solutions trans-epithelial electrical resistance (TEER) and cell viability of MDCK cell monolayers was evaluated. All values are given as mean ± SEM. Results: Incubation with 1 nM AII significantly stimulated hOCT2 mediated ASP+ uptake (ASP+ concentration in lysates of control and AII stimulated cells: 1.8 ± 0.4 and 2.2 ± 0.2 μmol/g protein, n=7–13, respectively). This effect was abolished under 1 μM losartan. Following incubation with 0.1 mM cisplatin TEER decreased to values similar of no-cell control. AII had no additional effect on the loss of electrical resistance. However, cell viability decreased more drastically when cisplatin was applied together with AII. This effect was abolished in the presence of losartan (cisplatin alone - 46.6 ± 1.4% vs with AII - 39.2 ± 2% of control experiments which were set as 100%, p < 0.05). Application of cisplatin together with cimetidine and AII lessened the toxicity (49.1 ± 1.3% of control, p < 0.05). Conclusions: We propose that in MDCK cell system AII augments cisplatin toxicity and that this effect is related to stimulated hOCT2 function.

Sodium butyrate suppresses Angiotensin II‐induced hypertension by inhibition of renal (pro)renin receptor and intrarenal RAS

BackgroundButyrate, one of Short‐chain fatty acids, is end product produced from the fermentation of complex carbohydrates by the gut microbiota. Recently, sodium butyrate (NaBu) is found to play protective roles in a number of chronic diseases. However, it is still unclear whether NaBu processes a therapeutic potential in hypertension. The present study was aimed to investigate the role of NaBu in Ang II‐induced hypertension and further explore the underlying mechanism.MethodsAng II (200 ng/min) was infused in uninephrectomized Sprague‐Dawley rats plus intramedullary infusion of NaBu (1μg/kg·min) or not for 14 days. Blood pressure were monitored by the telemetry system. The renal tissues and serum samples as well as the last 24‐hour urine samples were collected to examine renal injury and regulation of (pro)renin receptor (PRR) and renin.ResultsIntramedullary infusion of NaBu in Sprague‐Dawley rats lowered the Ang II‐induced mean arterial pressure from 128.8 ± 6.2 mmHg to 108.0 ± 3.9 mmHg (P&lt;0.01). This was paralleled with improvement of Ang II‐induced renal injury, including urinary albumin (μg/24h: Ctrl 270.7±49.2 vs AngII 1012.0±219.2 vs AngII‐NaBu 449.4±73.3, P&lt;0.05), glomerulosclerosis (GDI: Ctrl 1.48±0.08 vs AngII 2.70±0.06 vs AngII‐NaBu 1.68±0.08, P&lt;0.01), and renal fibrosis (α‐SMA protein: Ctrl 1.00±0.06 vs AngII 1.38±0.05 vs AngII‐NaBu 0.64±0.05, P&lt;0.01), as well as the expression of inflammatory mediators TNF‐α and IL‐6. The renal protein expression of (pro)renin receptor, urinary excretion of soluble (pro)renin receptor and renin were all increased by AngII infusion but decreased by NaBu treatment. In cultured IMCD cells, NaBu treatment attenuated AngII‐induced expression of PRR and renin.ConclusionThese results demonstrated NaBu exerts antihypertensive action likely via suppressing PRR‐mediated intrarenal RAS.Support or Funding InformationThis work was supported by National Natural Science Foundation of China Grant No. 91439205, No. 31330037 and No. 81600322; Fundamental Research Funds of Sun Yat‐sen University 16ykpy47; Veterans Affairs Merit Review, and National Institutes of Health Grant DK094956. T. Yang is Research Career Scientist in the Department of Veterans Affairs.