Intrarenal Renin-angiotensin System Research Articles

Urinary angiotensinogen as a surrogate marker predicting the antiproteinuric effects of angiotensin receptor blockers in patients with overt proteinuria: a\xa0multicenter prospective study

BackgroundAlthough urinary angiotensinogen (AGT) and renin reflect intrarenal renin-angiotensin system activity and are enhanced in proteinuric chronic kidney disease, the clinical value of urinary AGT and renin levels during antiproteinuric treatment has yet to be determined. We investigated the clinical usefulness of initial urinary AGT or renin to determine the antiproteinuric effects of angiotensin receptor blockers (ARBs).MethodsThis multicenter, prospective, single-arm study included 205 patients with overt proteinuria (urinary protein/creatinine ratio [uPCR] ≥ 1 mg/mg) enrolled between April 2009 and December 2011. All patients were treated with valsartan. The urinary AGT/creatinine ratio (uAGT/Cr) was measured at the baseline and 24 weeks, and the renin/creatinine ratio (uR/Cr) was measured at the baseline. Fifty-six patients were followed-up for 5 years.ResultsThe mean age was 47.6 years and 51.2% were male. The mean uPCR was 2.32 mg/mg and the mean eGFR was 63.2 mL/min/1.73m2. Natural logarithms (ln) (uAGT/Cr), ln(uR/Cr), and diabetes mellitus were associated with proteinuria decrement (decrease in uPCR ≥1 mg/mg). Ln(uAGT/Cr) was an independent predictor for proteinuria decrement (OR 1.372, 95% CI, 1.068–1.762, P = 0.013). Among the 56 patients followed-up for 5 years, Δln(uAGT/Cr) at 24 weeks was an independent predictor for uPCR < 1 mg/mg at 5 years (OR 0.379, 95% CI, 0.20–0.715, P = 0.003).ConclusionsOur study demonstrates the potential role of both baseline urinary AGT and changes in urinary AGT during the initial 24 weeks as surrogate markers predicting the antiproteinuric effects of ARBs in patients with overt proteinuria.

Renal Androgen and Renin Angiotensin System mRNA Expression in Polycystic Ovary Syndrome

BackgroundPolycystic Ovary Syndrome (PCOS), the most common endocrine disorder in women, is characterized by androgen‐excess and ovarian dysfunction. PCOS is associated with obesity, insulin resistance (IR) and increased blood pressure (BP). Currently therapeutic options to treat PCOS‐associated cardiovascular risk factors are limited. We have previously characterized a rat model of PCOS in which chronic androgen‐excess causes increased body weight (BW), fat mass (FM), IR, BP, and activation of some intrarenal Renin Angiotensin System (RAS) components. Still, the mechanisms responsible for increased BP in women with PCOS remain unclear. In the present study, we tested how chronic androgen‐excess changes the renal mRNA expression profile of the androgen receptor (AR) and RAS components in a model of PCOS.MethodsFour‐week old female SD rats were randomized to subcutaneously receive dihydrotestosterone (DHT, 7.5 mg/90 days) pellets (DHT) or sham surgeries (control) for 90 days (n= 6–10/grp). At the end of the experimental period, food intake (FI), BW, FM measured by Echo‐MRI, and BP using radiotelemetry were assessed in conscious, freely moving animals. Renal mRNA expression was assessed in cortical and medullary tissues separately by RT‐qPCR.ResultsAt the end of the experimental period, DHT rats exhibited higher FI, BW, FM and BP compared to control. In DHT rats, renal cortical mRNA expression of AR, angiotensinogen, AT1R, renin receptor, cathepsin A, aminoendopeptidase A, dipeptidyl peptidase 3, neprilysin, neurolysin, prolylendopeptidase, insulin‐like growth factor 2 receptor were significantly increased. In addition, renal medullary mRNA expression of angiotensin converting enzyme (ACE), angiotensin converting enzyme type 2 (ACE2), angiotensinogen, angiotensin 2 receptor type 1 (AT1R), membrane alanyl aminopeptidase, dipeptidyl peptidase 3, aminopeptidase A, kallikrein 1, neprilysin, neurolysin, prolylendopeptidase, and thimet oligopeptidase were significantly increased. Renal medullary mRNA expression of renin and aminopeptidase B were significantly decreased in DHT rats.Summary and ConclusionsIn summary, hyperandrogenemia leads to upregulation of AR in renal cortex and medulla. The mRNA expression of renal cortical and medullar ACE/AT1R axis components was upregulated in DHT rats. Additionally, mRNA expression of the ACE2 was upregulated mainly in the kidney medulla of DHT rats which could be a compensatory mechanism. This study highlights the effect of androgen‐excess modulating the intrarenal RAS and AR. Treatments focused on targeting RAS system components could be promising therapeutic tools to ameliorate the cardiovascular and renal abnormalities observes in women with PCOS.Support or Funding InformationAHA 0830239N (L.L.Y.C.) and 12SDG8980032 (D.G.R.), EFFERG (L.L.Y.C.), and NIH R21 DK‐113500 (D.G.R.) and P20 GM‐121334 (L.L.Y.C.)

Sex Differences in Urinary Angiotensinogen (uAGT) Excretion, Renal Function, and Systolic Blood Pressure in 2‐Kidney, 1‐Clip Hypertensive Rats

Intrarenal renin‐angiotensin system (RAS) activation and tissue injury in nonclipped kidneys of 2‐kidney, 1‐clip (2K1C) hypertensive rats have been characterized in previous studies of male but not female rats. To evaluate possible sex differences in response to renovascular hypertension, urinary angiotensinogen (uAGT) excretion, arterial blood pressure, urinary protein excretion, and renal function were assessed in 2K1C female rats.Female (n=8) and male (n=6) Sprague Dawley rats underwent placement of a 0.2 mm silver clip on the left renal artery to simulate unilateral renal artery stenosis. BP was measured by tail‐cuff plethysmography, and 24‐hour urine volume and water intake were monitored via metabolic cages over 21 days. After this period, renal clearance studies were conducted in anesthetized rats, and urine was collected from each kidney separately over 30 minute intervals. Urine protein concentration was determined by pyrogallol red method, and uAGT was measured by ELISA as an index of intrarenal RAS activity. Inulin and PAH concentrations were measured by spectrophotometry for calculation of glomerular filtration rate (GFR), renal plasma flow (RPF), and renal blood flow (RBF).21 days after renal artery clipping, systolic BP had risen to 176±8 mmHg from a baseline pressure of 120±1 mmHg, and urinary protein excretion increased to 20±5 mg/day in females. uAGT excretion was significantly increased from the baseline of 13 ng/day to 74 ng/day (507±127% increase). The nonclipped kidney showed 90±29% higher uAGT excretion compared to the clipped kidney, consistent with our previous findings in males. GFR and RPF were higher in nonclipped kidneys than in clipped kidneys but were greater than in corresponding measurements in males. GFR, RPF, and RBF were significantly higher in control rats compared to both clipped and nonclipped kidneys. Male rats showed significantly higher increases in uAGT, BP, and protein excretion.The present study demonstrates that BP, uAGT, and urinary protein excretion substantially increase after renal artery clipping in females, but the magnitude of the changes is markedly lower than in males. Additionally, the nonclipped kidney showed higher uAGT excretion compared to the clipped kidney. Although GFR, RPF, and RBF were significantly lower than control values for both clipped and nonclipped kidneys, these values were higher for females than males. These results demonstrated that female rats may be protected against AngII‐mediated renal injury and hypertension.Support or Funding InformationSupported by the ASPiRE Program at Tulane Univ Sch Medicine, New Orleans, LA and the Warren R. Bourgeois III, MD and Usha Ramadhyani Bourgeois, MD Student Research Endowed Fund at Tulane Univ Sch Medicine, New Orleans, LA

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.

ELABELA antagonizes intrarenal renin-angiotensin system to lower blood pressure and protects against renal injury.

Emerging evidence has demonstrated that (pro)renin receptor (PRR)-mediated activation of intrarenal renin-angiotensin system (RAS) plays an essential role in renal handling of Na+ and water balance and blood pressure. The present study tested the possibility that the intrarenal RAS served as a molecular target for the protective action of ELABELA (ELA), a novel endogenous ligand of apelin receptor, in the distal nephron. By RNAscope and immunofluorescence, mRNA and protein expression of endogenous ELA was consistently localized to the collecting duct (CD). Apelin was also found in the medullary CDs as assessed by immunofluorescence. In cultured CD-derived M1 cells, exogenous ELA induced parallel decreases of full-length PRR (fPRR), soluble PRR (sPRR), and prorenin/renin protein expression as assessed by immunoblotting and medium sPRR and prorenin/renin levels by ELISA, all of which were reversed by 8-bromoadenosine 3',5'-cyclic monophosphate. Conversely, deletion of PRR in the CD or nephron in mice elevated Apela and Apln mRNA levels as well as urinary ELA and apelin excretion, supporting the antagonistic relationship between the two systems. Administration of exogenous ELA-32 infusion (1.5 mg·kg-1·day-1, minipump) to high salt (HS)-loaded Dahl salt-sensitive (SS) rats significantly lowered mean arterial pressure, systolic blood pressure, diastolic blood pressure, and albuminuria, accompanied with a reduction of urinary sPRR, angiotensin II, and prorenin/renin excretion. HS upregulated renal medullary protein expression of fPRR, sPRR, prorenin, and renin in Dahl SS rats, all of which were significantly blunted by exogenous ELA-32 infusion. Additionally, HS-induced upregulation of inflammatory cytokines (IL-1β, IL-2, IL-6, IL-17A, IFN-γ, VCAM-1, ICAM-1, and MCP-1), fibrosis markers (TGF-β1, FN, Col1A1, PAI-1, and TIMP-1), and kidney injury markers (NGAL, Kim-1, albuminuria, and urinary NGAL excretion) were markedly blocked by exogenous ELA infusion. Together, these results support the antagonistic interaction between ELA and intrarenal RAS in the distal nephron that appears to exert a major impact on blood pressure regulation.

Comparison of the effects of insulin and SGLT2 inhibitor on the Renal Renin-Angiotensin system in type 1 diabetes mice

AimsSGLT2 inhibitors have been proposed as an adjunct to insulin therapy for glycemic control in type 1 diabetes (T1D) patients. However, concern has been raised due to an increase in renin–angiotensin-system (RAS) activity reported in a clinical trial in which an SGLT2 inhibitor was added while insulin dose was reduced in T1D patients. We previously reported that insulin inhibits intrarenal angiotensinogen (Agt) gene transcription and RAS activation. We hypothesized that insulin, rather than SGLT2 inhibition might regulate the intrarenal RAS. MethodsWe compared RAS activity in non-diabetic wild type mice, Akita mice (T1D model) and Akita mice treated with insulin or the SGLT2 inhibitor canagliflozin. ResultsTreatment of Akita mice with insulin or canagliflozin produced similar reductions in blood glucose, whereas insulin, but not canagliflozin, reduced elevated systolic blood pressure. Akita mice exhibited increased renal Agt mRNA/protein expression, which was attenuated by insulin, but not by canagliflozin. Furthermore, insulin was more effective than canagliflozin in lowering kidney weight and albuminuria. ConclusionsInsulin, but not canagliflozin, lowers intrarenal RAS activity in Akita mice. Our findings can be of potential clinical importance, especially for T1D patients who are not on RAS inhibitors at the time of adding SGLT2 inhibitors.

Reduction in urinary angiotensinogen levels and improvement of proteinuria by renin-angiotensin system blockade in pediatric chronic kidney disease patients with very low birth weight.

Children with low birth weight (LBW) have an increased risk of developing chronic kidney disease (CKD), and no effective strategies have been established to prevent the progression of CKD in these patients. Urinary angiotensinogen (UAGT) may represent a useful marker of intrarenal renin-angiotensin system (RAS) activation, which has been suggested to play a critical role in the development of hypertension and CKD. Herein, we conducted a prospective study to determine whether RAS blockade is beneficial for suppressing the progression of CKD in children with LBW, using UAGT as a surrogate marker of renal impairment. Nine children with CKD (stages: 1-2) who had very low birth weight (VLBW; < 1500g) were started on RAS blockade with candesartan. We measured blood pressure and laboratory parameters, including urinary concentrations of angiotensinogen, protein, albumin, creatinine (Cr), and estimated glomerular filtration rate (eGFR), before and after candesartan treatment. Birth weight was 712g (range, 536-800g). Age at evaluation was 11.6years (range, 10.3-15.6years). After candesartan treatment for 47.6 ± 25.0months, the UAGT to urinary Cr ratio decreased from 61.9 ± 44.7 to 16.8 ± 14.4μg/g (p= 0.015). The urinary protein to Cr and albumin to Cr ratios also decreased (p= 0.008 and p= 0.012, respectively), whereas there was no significant change in eGFR. RAS blockade reduced UAGT levels and improved proteinuria/albuminuria in children with CKD who had VLBW. Suppression of intrarenal RAS activity may slow the progression of CKD in children with LBW.

CG200745, a Novel HDAC Inhibitor, Attenuates Kidney Fibrosis in a Murine Model of Alport Syndrome.

Histone deacetylases have been a target of therapy for organ fibrosis. Here, we report the protective effect of CG200745 (CG), a novel histone deacetylase inhibitor, on tubulointerstitial fibrosis in Col4a3−/− mice, a murine model of Alport syndrome. Morphological analyses revealed CG treatment markedly alleviated kidney fibrosis in Col4a3−/− mice at the age of 7 weeks. CG prevented the activation of transforming growth factor β (TGFβ) and its downstream SMAD signaling in the kidney of Col4a3−/− mice. As critical upstream regulators of TGFβ signaling, immunoblotting of whole kidney lysate of Col4a3−/− mice reveled that intra-renal renin–angiotensin system (RAS) was activated with concurrent upregulation of inflammation and apoptosis, which were effectively suppressed by CG treatment. CG suppressed both activation of RAS and up-regulation of TGFβ signals in angiotensin II-stimulated HK-2 cells, a human kidney proximal tubular epithelial cell line. CG inhibited activation of TGFβ-driven signals and fibrosis in NRK-49F cells, a rat kidney fibroblast cell line, under angiotensin II-rich conditions. Collectively, CG was found to be effective both in proximal tubular epithelial cells by inhibiting local RAS and TGFβ signaling activation, as well as in fibroblasts by blocking their transition to myofibroblasts, attenuating renal fibrosis in a murine model of Alport syndrome.

Gut dysbiosis contributes to high fructose-induced salt-sensitive hypertension in Sprague-Dawley rats.

Although it is known that high fructose intake causes salt-sensitive hypertension, the underlying mechanism remains unclear. The aim of this study was to determine whether chronic intake of high fructose coupled with salt (HFS) might alter the structure of the gut microbiota, which contributes to elevated blood pressure. For 8 wk, Sprague-Dawley rats were given 20% fructose in drinking water and 4% sodium chloride in their diet to induce hypertension. A non-absorbable antibiotic vancomycin was used to modify gut microbiota. The 16 S rRNA sequencing for fecal samples was assessed and blood pressure was recorded. Enzyme-linked immunosorbent assay and quantitative polymerase chain reaction were used to examine the renin-angiotensin system in serum, urine, and the kidney. Compared with the control group, HFS feeding resulted in gut dysbiosis by altering the diversity and richness of gut microbiota and decreased the ratio of Firmicutes to Bacteroidetes. Vancomycin reshaped dramatically the HFS-induced dysbiosis. And vancomycin (van) attenuated HFS-increased blood pressure (HFS: 121.3 ± 2.8 mm Hg; HFS-van: 111.1 ± 1.7 mm Hg) and heart rate (HFS: 360.5 ± 9.0 bpm; HFS-van: 318.7 ± 5.6 bpm) as well as the content of angiotensinogen, renin, and angiotensin II in the urine and the angiotensinogen mRNA level in renal cortical tissues. However, HFS-increased triacylglycerol, renin, and angiotensin II in serum were not decreased by vancomycin. The present results demonstrated that gut dysbiosis develops after chronic fructose plus salt intake and contributes to the increase of blood pressure and the activation of the intrarenal renin-angiotensin system. Therefore, targeting gut microbiota provides a helpful therapy method to improve HFS-induced hypertension.

Urinary Angiotensinogen in addition to Imaging Classification in the Prediction of Renal Outcome in Autosomal Dominant Polycystic Kidney Disease.

BackgroundIntrarenal renin-angiotensin system (RAS) is known to play the major role in the development of hypertension and renal progression in autosomal dominant polycystic kidney disease (ADPKD). Urinary angiotensinogen to creatinine ratio (AGT/Cr) was suggested as a novel biomarker to reflect intrarenal RAS activity. This study was performed to evaluate urinary AGT/Cr as a predictive biomarker for renal function decline in addition to imaging classification in a prospective ADPKD cohort.MethodsFrom 2011 to 2016, a total of 364 ADPKD patients were enrolled in the prospective cohort called the KoreaN Cohort Study for Outcomes in Patients With Chronic Kidney Disease (KNOW-CKD). Among them, a total of 207 subjects in chronic kidney disease stage 1–4 with baseline urinary AGT and total kidney volume and subsequent renal function follow-up data over more than 1 year were included in the analysis. Patients were defined as slow progressors (SP) if they are classified as 1A or 1B by imaging classification whereas rapid progressors (RP) if they are classified as 1C–1E. Patients were divided according to AGT/Cr quartiles and annual estimated glomerular filtration rate (eGFR) slope was compared among highest quartile (hAGT group) and the rest of quartiles (lAGT group). Patients were divided into 4 groups to evaluate the predictive value of urinary AGT/Cr in addition to imaging classification: SP/lAGT, SP/hAGT, RP/lAGT, and RP/hAGT. The Cox regression model was used to evaluate the hazard ratio (HR) between groups.ResultsThe mean age was 45.9 years and 88.9% had hypertension. Baseline eGFR was 79.0 ± 28.4 mL/min/1.73 m2 and median height-adjusted total kidney volume was 788.2 (471.2; 1,205.2) mL/m. The patients in the hAGT group showed lower eGFR (72.4 ± 24.8 vs. 81.1 ± 29.2 mL/min/1.73 m2, P = 0.039), lower plasma hemoglobin (13.0 ± 1.4 vs. 13.7 ± 1.6 g/dL, P = 0.007), higher urinary protein to creatinine ratio (0.14 [0.09, 0.38] vs. 0.07 [0.04, 0.12] g/g, P = 0.007) compared to the lAGT group. The hAGT group was an independent risk factor for faster eGFR decline after adjusting for gender, RP, baseline eGFR, and other known risk factors. During median follow-up duration of 4.6 years, a total of 29 renal events (14.0%) occurred. The SP/hAGT group showed significantly higher risk of developing renal outcome compared to SP/lAGT group (HR, 13.4; 95% confidence interval, 1.282–139.324; P = 0.03).ConclusionUrinary AGT/Cr can be a useful predictive marker in the patients with relatively small ADPKD. Various biomarkers should be considered to define RP when implementing novel treatment in the patients with ADPKD.

Upregulation of Cortical Renin and Downregulation of Medullary (Pro)Renin Receptor in Unilateral Ureteral Obstruction.

Chronic kidney disease (CKD) is characterized by renal dysfunction, which is a common feature of other major diseases, such as hypertension and diabetes. Unilateral ureteral obstruction (UUO) has been used as a model of CKD in experimental animals and consists of total obstruction of one kidney ureter. The UUO decreases renal blood flow, which promotes the synthesis of renin in the juxtaglomerular apparatus, the first step in renin–angiotensin system (RAS) cascade. RAS induces inflammation and remodeling, along with reduced renal function. However, it remains unknown whether intrarenal RAS (iRAS) is activated in early stages of CKD. Our objective was to characterize different iRAS components in the renal cortex and in the medulla in an early phase of UUO. Male C57BL/6 mice (8–12 weeks old) were subjected to UUO in the left kidney, or to sham surgery, and were euthanized after 7 days (n = 5/group). Renal function, renal inflammatory/remodeling processes, and iRAS expression were evaluated. UUO increased plasma creatinine, right renal hypertrophy (9.08 ± 0.31, P < 0.05 vs. Sham), and tubular dilatation in the left kidney cortex (42.42 ± 8.19µm, P < 0.05 vs. Sham). This correlated with the increased mRNA of IL-1β (1.73 ± 0.14, P < 0.01 vs. Sham, a pro-inflammatory cytokine) and TGF-β1 (1.76 ± 0.10, P < 0.001 vs. Sham, a pro-fibrotic marker). In the renal cortex of the left kidney, UUO increased the mRNA and protein levels of renin (in 35% and 28%, respectively, P < 0.05 vs. Sham). UUO decreased mRNA and protein levels for the (pro)renin receptor in the renal medulla (0.67 ± 0.036 and 0.88 ± 0.028, respectively, P < 0.05 vs. Sham). Our results suggest that modulation of iRAS components depends on renal localization and occurs in parallel with remodeling and pro-inflammatory/pro-fibrotic mechanisms.

Effect of GLP-1 Receptor Agonists in the Cardiometabolic Complications in a Rat Model of Postmenopausal PCOS.

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and ovulatory dysfunction. Women with PCOS have an elevated prevalence of cardiometabolic risk factors that worsen after menopause. Liraglutide (Lira), a glucagon-like peptide-1 receptor agonist, has shown beneficial metabolic effects in small clinic trials in reproductive-age women with PCOS. We have shown that chronic hyperandrogenemia in an experimental model of postmenopausal PCOS is associated with an adverse cardiometabolic profile and upregulation of the intrarenal renin-angiotensin system (RAS). We analyzed the effect of Lira in the cardiometabolic profile, intrarenal RAS, and blood pressure (BP) in postmenopausal PCOS. Four-week-old female Sprague Dawley rats were treated with DHT or placebo for 17 months. Lira administration during the last 3 weeks caused a bigger reduction in food intake, body weight, fat mass, and homeostasis model assessment of insulin resistance index in PCOS than in control rats. Moreover, Lira improved dyslipidemia and elevated leptin levels in PCOS. In contrast, Lira decreased intrarenal expression of RAS components only in the control group. Lira transiently increased heart rate and decreased BP in control rats. However, Lira did not modify BP but increased heart rate in PCOS. The angiotensin-converting-enzyme inhibitor enalapril abolished the BP differences between PCOS and control rats. However, Lira coadministration with enalapril further reduced BP only in control rats. In summary, Lira has beneficial effects for several cardiometabolic risk factors in postmenopausal PCOS. However, hyperandrogenemia blunted the BP-lowering effect of Lira in postmenopausal PCOS. Androgen-induced activation of intrarenal RAS may play a major role mediating increases in BP in postmenopausal PCOS.

The Urinary Angiotensinogen to Urinary Albumin Ratio Reflects Whether the Renin-angiotensin System in the Kidney Is Activated due to Filtration of Plasma Angiotensinogen through the Damaged Glomeruli or the Production of Angiotensinogen in the Proximal Tubules.

Objective Urinary angiotensinogen (AGT) is a surrogate marker for intrarenal renin-angiotensin system (RAS) activity that plays an important role in the development of renal damage. Urinary AGT levels are determined by the filtration of plasma AGT through the damaged glomeruli and production of AGT in the proximal tubules. However, the relative merits of the filtration and production of urinary AGT levels in chronic kidney diseases (CKD) have not been clarified. Therefore, we investigated them in CKD patients. Methods We recruited 41 biopsy-proven patients diagnosed with IgA nephropathy (IgAN) in 31, membranous nephropathy (MN) in 5, and tubulointerstitial nephritis (TIN) in 5. The patients taking RAS blockers were excluded. Results The urinary albumin levels in MN patients were significantly higher and those in TIN patients significantly lower than in IgAN patients, and the urinary AGT levels in the MN and TIN patients were significantly higher than those in IgAN patients. Conversely, the urinary AGT-to-urinary albumin (urinary AGT/Alb) ratios were the same for IgAN and MN patients, and those of TIN patients were significantly higher than those of IgAN and MN patients. A multiple linear regression analysis revealed that the urinary AGT/Alb ratios had a significant positive association with IgAN and TIN after adjustments (β=0.75, and p<0.01). Conclusion These data suggest that the origins of urinary AGT may differ according to the etiology of renal damage [i.e. glomerular damage (such as IgAN and MN) or tubulointerstitial damage (such as TIN)], and a higher urinary AGT/Alb ratio, as in TIN, may reflect AGT production in the kidney.

Causes and Consequences of the Dysregulated Maternal Renin-Angiotensin System in Preeclampsia.

A healthy pregnancy outcome depends on the activation of the renin-angiotensin-aldosterone system (RAAS) as a regulated, integrated response to the growing demands of the conceptus. Both the circulating RAAS and the intrarenal renin-angiotensin system (iRAS) play major roles in cardiovascular function and fluid and electrolyte homeostasis. The circulating RAAS becomes dysfunctional in preeclampsia and we propose that dysregulation of the iRAS plays a role in development of the clinical syndrome known as preeclampsia. Experimental studies in animals have shown that placental renin, when released into the maternal circulation, can cause hypertension. We postulate that abnormal placental development is associated with over-secretion of renin and other RAS proteins/angiotensin (Ang) peptides by the placenta/decidua into the maternal circulation. We hypothesise that this is because of increased shedding of exosomes and other placental particles into the maternal circulation that not only contain RAS proteins and peptides but also microRNAs (miRNAs) that target RAS mRNAs, and Ang II type 1 receptor autoantibodies (AT1R-AAs), that are agonists for, and have the same actions as, Ang II. As a result, there is both suppression of the circulating RAAS that is responsible for maintaining maternal homeostasis and activation of the iRAS. Together with altered vascular reactivity to Ang peptides, the iRAS causes hypertension, renal damage and secondary changes in the neurohumoral control of the maternal circulation and fluid and electrolyte balance, which contribute to the pathophysiology of preeclampsia.

Abstract P2048: Epigenetic Modification By Angiotensin II Mediates Augmentation Of Intrarenal Angiotensinogen Expression

Inappropriately activated intrarenal renin-angiotensin system, including proximal tubular angiotensinogen (AGT) augmentation, contributes to the progression of hypertension and associated kidney injury. It has been demonstrated that interleukin 6 (IL-6) is required for angiotensin II (Ang II)-induced AGT augmentation in renal proximal tubular cells (PTC). However, molecular mechanisms remain unelucidated. Recent studies suggest that Ang II downregulates cardiac class II histone deacetylases (HDAC), which exhibit protective effects against hypertension related tissue injury. Furthermore, HDAC9 (a class II HDAC) has been shown to act as an epigenetic repressor of proximal tubular AGT. Accordingly, the present study investigated the hypothesis that Ang II decreases HDAC9 levels, thereby enabling IL-6 to promote AGT expression in PTC. Ang II (400 ng/kg/min) was chronically perfused to male mice (N=7) for 14 days to investigate the regulation of intrarenal HDAC9. Moreover, cultured human PTC (hPTC) were used to create wild-type, HDAC9 overexpression, and HDAC9 knockdown by shRNA models and were treated with 100 nM Ang II, 10 ng/ml IL-6, or both for 24 hours. Chronic Ang II infusion elevated blood pressure and suppressed renal cortical HDAC9 levels. A decrease in HDAC9 was also observed in Ang II-treated hPTC. Treatment of hPTC with both Ang II and IL-6 increased AGT mRNA levels (1.50±0.10, ratio to the control, N=6), whereas individual treatments with Ang II or IL-6 did not alter AGT expression levels as previously demonstrated. This upregulation of AGT expression by Ang II and IL-6 co-stimulation was attenuated by the overexpression of HDAC9 in hPTC (1.10±0.08, ratio to the control). Importantly, AGT expression was enhanced by IL-6 without Ang II (2.31±0.08 by IL-6 and 2.27±0.15 by Ang II+IL-6, ratio to the control) in cells receiving the HDAC9 gene knockdown, suggesting that HDAC9 down-regulation mediates IL-6-induced AGT upregulation in hPTC. These results indicate that Ang II-induced epigenetic modification, such as the suppression of intrarenal HDAC9, results in increased accessibility of IL-6-derived transcription factors to the AGT promoter and consequent augmentation of intrarenal AGT expression in Ang II-dependent hypertension.

Olmesartan Attenuates Kidney Fibrosis in a Murine Model of Alport Syndrome by Suppressing Tubular Expression of TGFβ.

Despite the wide use of angiotensin II receptor blockers in the treatment of Alport syndrome (AS), the mechanism as to how angiotensin II receptor blockers prevent interstitial fibrosis remains unclear. Here, we report that treatment of olmesartan effectively targets the feedback loop between the renin–angiotensin system (RAS) and transforming growth factor β (TGFβ) signals in tubular epithelial cells and preserves renal angiotensin-converting enzyme 2 (ACE2) expression in the kidney of Col4a3–/– mice, a murine model of experimental AS. Morphology analyses revealed amelioration of kidney fibrosis in Col4a3–/– mice by olmesartan treatment. Upregulation of TGFβ and activation of its downstream in Col4a3–/– mice were attenuated by olmesartan in Col4a3–/– mice. Intriguingly, TGFβ expression was preferentially upregulated in damaged tubular epithelial cells in Col4a3–/– mice. Concurrent upregulation of TNFα-converting enzyme and downregulation of ACE2 suggested RAS activation in Col4a3–/– mice, which was prevented by olmesartan. Mechanistically, olmesartan suppressed TGFβ-induced RAS activation in tubular epithelial cells in vitro. Collectively, we concluded that olmesartan effectively suppresses the progression of tubulointerstitial fibrosis in AS by interrupting RAS-TGFβ feedback loop to counterbalance intrarenal RAS activation.

Decreased Ang-(1–7) and Downregulated Intrarenal RAS May Contribute to the Direct Podocyte Injury With Proteinuria in Preeclampsia

The mechanisms of proteinuria development in preeclampsia (PE) are still enigmatic. Renin-angiotensin system (RAS) components may play a role. Maternal serum and urinary concentrations of angiotensin-(1-7) [Ang-(1-7)], angiotensin II (Ang II), and angiotensinogen in women with PE (n - 14), gestational hypertension (n - 14), and normal pregnancy were quantified. The alteration in these concentrations was used to evaluate their relationships with podocyturia and proteinuria in PE. In addition, the podocytes cultured in vitro were interfered in serum of preeclamptic and normotensive pregnant women, with or without Ang- (1-7). The morphologic change in podocyte was observed using a microscope. The changes in podocyte-specific proteins (nephrin, CD2-associated protein [CD2AP]), the cytoskeletal protein F-actin, the tight junction protein (ZO-1), and Mas receptor (MasR) were examined by immunofluorescence. Western blot was used to examine the expression and variation of MasR. We found that the concentrations of RAS components were associated with prepartal urinary podocyte number, random urine albumin/creatinine ratio, blood pressure, and renal function. The expression of nephrin, F-actin, ZO-1, and MasR on podocytes interfered in serum of PE was significantly decreased compared to normal control and normal pregnant serum group in vitro, yet their expression was significantly increased after coculture by 10-6 mol/L Ang-(1-7) and the preeclamptic serum. The expression of CD2AP had no significant difference. We concluded that decreased Ang-(1-7) and downregulated intrarenal RAS contributed to the direct podocyte injury with proteinuria in PE.

Intrarenal Renin-Angiotensin System Involvement in the Pathogenesis of Chronic Progressive Nephropathy-Bridging the Informational Gap Between Disciplines.

Chronic progressive nephropathy (CPN) is the most commonly encountered spontaneous background finding in laboratory rodents. Various theories on its pathogenesis have been proposed, but there is a paucity of data regarding specific mechanisms or physiologic pathways involved in early CPN development. The current CPN mechanism of action for tumorigenesis is largely based on its associated increase in tubular cell proliferation without regard to preceding subcellular degenerative changes. Combing through the published literature from multiple biology disciplines provided insight into the preceding cellular events. Mechanistic pathways involved in the progressive age-related decline in rodent kidney function and several key inflexion points have been identified. These critical pathway factors were then connected using data from renal models from multiple rodent strains, other species, and mechanistic work in humans to form a cohesive picture of pathways and protein interactions. Abundant data linked similar renal pathologies to local events involving hypoxia (hypoxia-inducible factor 1α), altered intrarenal renin-angiotensin system (RAS), oxidative stress (nitric oxide), and pro-inflammatory pathways (transforming growth factor β), with positive feedback loops and downstream effectors amplifying the injury and promoting scarring. Intrarenal RAS alterations seem to be central to all these events and may be critical to CPN development and progression.

Intrarenal Renin-Angiotensin-System Dysregulation after Kidney Transplantation

Angiotensin-converting enzyme inhibitors (ACEis) are beneficial in patients with chronic kidney disease (CKD). Yet, their clinical effects after kidney transplantation (KTx) remain ambiguous and local renin-angiotensin system (RAS) regulation including the ‘classical’ and ‘alternative’ RAS has not been studied so far. Here, we investigated both systemic and kidney allograft-specific intrarenal RAS using tandem mass-spectrometry in KTx recipients with or without established ACEi therapy (n = 48). Transplant patients were grouped into early (<2 years), intermediate (2–12 years) or late periods after KTx (>12 years). Patients on ACEi displayed lower angiotensin (Ang) II plasma levels (P < 0.01) and higher levels of Ang I (P < 0.05) and Ang-(1–7) (P < 0.05) compared to those without ACEi independent of graft vintage. Substantial intrarenal Ang II synthesis was observed regardless of ACEi therapy. Further, we detected maximal allograft Ang II synthesis in the late transplant vintage group (P < 0.005) likely as a consequence of increased allograft chymase activity (P < 0.005). Finally, we could identify neprilysin (NEP) as the central enzyme of ‘alternative RAS’ metabolism in kidney allografts. In summary, a progressive increase of chymase-dependent Ang II synthesis reveals a transplant-specific distortion of RAS regulation after KTx with considerable pathogenic and therapeutic implications.

Evidence of Augmented Intrarenal Angiotensinogen Associated With Glomerular Swelling in Gestational Hypertension and Preeclampsia: Clinical Implications.

BackgroundAGT (angiotensinogen) synthesis occurs in renal proximal tubular epithelial cells, independent from systemic AGT, as a component of the intrarenal renin–angiotensin system. We investigated urinary AGT, as a biomarker for renin–angiotensin system activation, and electrolyte concentrations, in relation to glomerular volume, as a proxy for glomerular endotheliosis in renal biopsy tissue from pregnant normotensive control and hypertensive women.Methods and ResultsUrine samples were collected from normotensive control (n=10), gestational hypertensive (n=6), and pre‐eclamptic (n=16) women at the time a renal biopsy was obtained. Samples were collected from Lund University Hospital between November 1999 and June 2001. Urinary AGT, potassium, and sodium were measured, normalized to urinary creatinine. Mean glomerular volume was estimated from biopsy sections. AGT protein expression and localization were assessed in renal biopsies by immunohistochemistry. Urinary AGT concentrations were higher in hypertensive pregnancies (median, gestational hypertension: 11.3 ng/mmol [interquartile range: 2.8–13.6]; preeclampsia: 8.4 ng/mmol [interquartile range: 4.2–29.1]; normotensive control: 0.6 ng/mmol [interquartile range: 0.4–0.8]; P<0.0001) and showed a positive relationship with estimated mean glomerular volume. Urinary potassium strongly correlated with urinary AGT (P<0.0001). Although numbers were small, AGT protein was found in both glomeruli and proximal tubules in normotensive control but was present only in proximal tubules in women with hypertensive pregnancy.ConclusionsThis study shows that pregnant women with gestational hypertension or preeclampsia have increased urinary AGT and potassium excretion associated with signs of glomerular swelling. Our data suggest that the kidneys of women with hypertensive pregnancies and endotheliosis have inappropriate intrarenal renin–angiotensin system activation, which may contribute toward the pathogenesis of hypertension and renal injury.