Renin Upregulation Research Articles

#3166 RECOGNITION OF RENAL TUBULAR DYSGENESIS IN ADOLESCENT CKD BY BIALLELIC AGT VARIANTS REQUIRED BROAD GENETIC ANALYSIS

Abstract Background and Aims Autosomal recessive renal tubular dysgenesis (ARRTD) is a rare inherited disorder of renal (tubular) development, clinically characterized by fetal oligo-/anuria leading to oligohydramnion and Potter sequence, resulting in high mortality within the prenatal and neonatal period. Variants in genes encoding components of the renin-angiotensin system (RAS) are causative for this disorder. Herein, we report 2 European families with biallelic variants within AGT and aim to provide novel insides into disease understanding. Method The study was approved by our institutional ethics committee (approval number: 251_18 B). Clinical, histological and pedigree analysis were performed. Exome sequencing of a preselected gene panel (nephroma, 560 genes associated with renal disease) was analysed on the HiSeq System 2500 (Illumina) after enrichment by TWIST human core technology (TWIST Bioscience). Immunohistochemistry staining (IHC) and in-situ hybridization (ISH) for expression of renin were performed on kidney biopsy. In addition, renin expression was determined in primary tubular cells of the index patient by qPCR. Allele frequencies of heterozygous and biallelic predicted deleterious variants were determined by analysis of the Genomics England 100,000 Genomes Project. Results The first family was identified after transition from pediatric to adult nephrology at the University Hospital Erlangen. Initially, the male index patient of consanguineous Turkish descent presented with oligohydramnion in the prenatal period. Directly after birth (32nd gestational week) he suffered from profound hypotension, pulmonary hypoplasia and pulmonary stenosis, as well as third degree acute kidney injury leading to ICU treatment, but no need of hemodialysis. He survived the perinatal period while a decline in renal function was observed over time leading to CKD G3b at the age of 19 years. Exome sequencing revealed a previously reported pathogenic homozygous missense variant within AGT (c.1224G>A, p.Arg375Gln). Kidney biopsy at the age of 14 years revealed tubulointerstitial fibrosis, profound glomerulocystic changes with partly glomerulosclerosis and vasculopathic features. IHC and ISH showed recruitment and strong expression of renin in tubular cells and vessels, where cultured primary tubular cells also showed strong expression of renin. Circulating renin was profoundly elevated, with normal levels of aldosterone. The patient is not hypertensive and receives no antihypertensive medications. The family history revealed at least 2 miscarriages due to oligohydramnion. The second family was identified from the Genomics England 100,000 Genomes Project. The male index patient presented with prenatal bilateral hydronephrosis. He was delivered prematurely around the 35th gestational week. Both kidneys suffered from chronic bilateral urinary reflux leading to CKD G2 at the age of 8 years. Family history revealed one prior miscarriage. Genetic analysis revealed two compound heterozygous missense variants within AGT (c.842A>G, p.Tyr281Cys; c.151T>C, p.Cys51Arg). The heterozygous allele frequency in the Genomics England cohort for AGT predicted deleterious variants was very low. Therefore, we would predict a biallelic hit with the respective disease as an ultra-rare event. Reviewing the literature, to our knowledge 18 individuals are reported to date with different biallelic variants within AGT. From these cases only three survived the perinatal period. It is not reported if these individuals reached adulthood. Conclusion We hereby demonstrate two extremely rare cases, affected by biallelic variants within AGT, that survived the perinatal period and eventually led to chronic kidney disease. Since the phenotypes may be variable, a contemporary approach by broad genetic analysis is the only option to decode the genetic background in individuals with a suspected genetic disorder. Upregulation of renin in tubular cells by inactivation of AGT in the germline may drive tubulointerstitial fibrosis, which is a theoretical concern regarding ongoing targeted pharmacological approaches against AGT for arterial hypertension.

JS-HR-2: HYPERTENSION MANAGEMENT IN CKD: UPDATE FOR BETTER CARDIO-RENAL OUTCOMES

Hypertension in CKD patients is a risk factor for end-stage renal disease, cardiovascular events, and mortality. Thus, the prevention and appropriate management of hypertension in these patients are essential strategies for better cardio-renal outcomes. Recently, novel risk factors for hypertension have been reported. Circulating fibroblast growth factor 21, an endocrine hormone mainly secreted by the liver, is associated with elevated blood pressure (BP) and/or aortic stiffness with renal dysfunction. Furthermore, several promising prognostic markers for cardio-renal outcomes have been suggested. A higher urinary sodium-to-potassium ratio was independently associated with poor renal outcomes in CKD patients. Higher albumin-to-creatinine ratios significantly increased the risk of first stroke or first ischemic stroke. Sodium-glucose cotransporter-2 inhibitors (SGLT2is) and mineralocorticoid receptor (MR) blockers are promising treatment options for better cardio-renal outcomes. The clinical use of SGLT2is has recently expanded to non-diabetic patients with CKD and heart failure as well as diabetic patients. SGLT2is have an antihypertensive effect, and the degree of BP change in patients undergoing SGLT2i therapy depends on the baseline BP; a larger reduction is observed in patients with higher baseline BP, and a minor reduction or slight increase is observed in patients with lower baseline BP. These BP regulation mechanisms may partially depend on body fluid homeostasis by SGLT2is. SGLT2is ameliorate fluid retention through osmotic diuresis and natriuresis, but are also associated with a low rate of hypovolemia, which is evident by the compensatory upregulation of renin and vasopressin levels. These fluid homeostatic mechanisms exerted by SGLT2is may contribute to the stabilization of BP and better cardio-renal outcomes. MR blockers are used in the treatment of essential hypertension and hyperaldosteronism. Recently, a new MR antagonist, finerenone, has been launched as a treatment for CKD with type 2 diabetes. In the FIDELIO-DKD study, finerenone significantly reduced the risk of kidney and cardiovascular events in CKD and type 2 diabetes.

Regulatory Effect of Vitamin D on Renin Expression at Maternal-Fetal Interface

To investigate the regulatory effect and mechanism of vitamin D on the local renin-angiotensin system at maternal-fetal interface in the pathological process of preeclampsia (PE). The mRNA and protein expression of renin in decidua of normal pregnancy and PE placentas was determined by RT-PCR and Western blot. Normal decidual tissues were treated with active and inactive vitamin D for 48 h in vitro and the expressions of renin and vitamin D deactivating enzyme CYP24A1 were determined by RT-PCR and Western blot. Normal decidual stromal cells and glandular epithelial cells were isolated and purified, and identified by immunocytochemical staining. RT-PCR was used to examine the mRNA of vdr, cyp27 b1, cyp24 a1, and renin in the two types of cells and in decidual tissue, and the mRNA products were subjected to gel electrophoresis. These two cell types were treated with active and inactive vitamin D in vitro and the expressions of renin and vitamin D deactivating enzyme CYP24A1 were determined by RT-PCR and Western blot. Decidual gland epithelial cells were treated with protein kinase A (PKA) activator forskolin or inhibitor H89 to explore the interaction between PKA pathway and vitamin D in the regulation of renin expression. The expression of renin in PE decidua was significantly higher than that of normal control at transcriptional and translational levels ( P<0.05). Vitamin D treatment could significantly down-regulate the expression of renin in normal decidua tissues ( P<0.05), while it significantly up-regulated CYP24A1 expression ( P<0.001). Decidual stromal cells and gland epithelial cells were successfully isolated from decidual tissue. Compared with that in decidual stromal cells, the mRNA level of vitamin D-related molecules in gland epithelial cells was more similar to that in decidual tissue. Active or inactive vitamin D treatment significantly inhibited the expression of renin in glandular epithelial cells ( P<0.05), but the expression of renin in decidual stromal cells was not affected. However, the treatment of active or inactive vitamin D in these two kinds of cells significantly increased the expression of CYP24A1 ( P<0.001). Active vitamin D could significantly inhibit the upregulation of renin by PKA agonist forskolin, and could inhibit the expression of renin through synergy with PKA inhibitor H89. The expression of renin in placental decidua is up-regulated in patients with PE, and the activation of local renin-angiotensin system at the maternal-fetal interface may be involved in the pathogenesis of PE. Vitamin D can specifically down-regulate renin expression in human decidual gland epithelial cells by competing with the PKA pathway. Vitamin D supplementation may have potential value for clinical intervention of PE.

Multi‐omics analysis reveals regulatory mechanisms in chronic cyclosporine A‐induced nephrotoxicity studied a rat model

Chronic calcineurin inhibitor (CNI) nephrotoxicity is a major drawback in current immunosuppressive regimens. In the chronic setting, arteriolar hyalinosis, decreased glomerular filtration rate, interstitial fibrosis and tubular dedifferentiation are the major adverse side effects. Since CsA is still widely used in transplant recipients, particularly in posttransplant diabetes, mechanistic insights into nephrotoxic mechanisms are required. To identify regulatory mechanisms in CsA nephrotoxicity we used quantitative transcriptomic, proteomic and phosphoproteomic methods. We tested the hypothesis that tubulointerstitial pathomechanisms play a predominant role in chronic CNI nephropathy. Whole transciptome RNA‐seq as well as global proteomic and phosphoproteomic methodologies were performed on kidney extracts from normal Wistar rats receiving CsA (25mg/kg b.w./day) or vehicle for 3 weeks. Differentially expressed genes and proteins as well as their phosphorylation status were obtained. Products of interest were tested using Western blot, qPCR, and immunohistochemistry. CsA treatment stimulated genome‐wide alterations in rat kidney according to RNA‐seq data. We identified 342 transcripts upregulated (padj&lt;0.05) which included Ribosome (padj&lt;0,0001) and Oxidative phosphorylation (padj&lt;0,0001) pathways, whereas 331 transcripts were downregulated, with enrichment in genes critical for amino acid metabolism. Data were controlled by the established upregulation of renin and downregulation of calbindin (pdaj&lt;0,05) in global proteomics. KEGG pathway and GO analysis from proteomics largely corresponded to the RNA‐seq results. Upregulated proteins were further related to ECM‐receptor interaction and focal adhesion pathways (padj&lt;0,05). Phosphoproteomics demonstrated functional phosphorylation of components from unfolded protein response pathways, indicating an activation of the integrated stress response upon CsA. Products were verified at the tissue‐ and cell level. In sum, using integrated ‐omics analysis in CsA nephrotoxicity proved to be a powerful approach. Chronic CsA treatment was associated with enhanced energy metabolism and activation of the unfolded protein‐response pathways. A tubulointerstitial focus has been demonstrated. Potential biomarker candidates have been obtained.

Renin Feedback Is an Independent Predictor of Outcome in HFpEF.

Drugs which interact with the renin angiotensin aldosterone system (RAAS) aim to reduce the negative effects of angiotensin (Ang) II. Treatment with these drugs anticipate a compensatory up-regulation of renin; however, it has been shown that there is a large variability in circulating plasma renin (PRA), even in patients with optimal medical therapy in patients with heart failure (HF) with reduced ejection fraction (HFrEF). Our aim was to measure plasma renin activity (PRA-S), its response to RAAS inhibitor (RAASi) therapies and its effects on outcome in patients with HF with preserved ejection fraction (HFpEF). For this purpose, 150 HFpEF patients were included into a prospective single-center registry. Equilibrium (eq) angiotensin metabolites were measured from serum samples using mass spectroscopy. PRA-S (eqAng I + eqAng II) was calculated and compared in respect to the primary endpoint defined as all-cause death. PRA-S in patients with RAASi therapy was not significantly higher than in patients without RAASi (p = 0.262). Even after adjusting for confounding factors, PRA-S remained predictive for all-cause death in the multivariable model with a hazard ratio of 2.14 (95%CI 1.20–3.82, p = 0.010). We conclude that high PRA-S is associated with poor prognosis in patients with HFpEF, regardless of RAASi treatment, which could ultimately result in hyperactivated RAAS and consecutive negative effects on the cardiovascular and renal system, leading to poor outcome in patients with HFpEF.

DISTRIBUTION OF RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS) BIOMARKERS IN TREATED HYPERTENSIVE INDIVIDUALS FROM THE GENERAL POPULATION: THE CHRIS STUDY

Objective: Renin-Angiotensin-Aldosterone System (RAAS) is a critical target in hypertension management. However, direct quantification of angiotensin I (AngI) and II (AngII) is challenging due to compound instability, and was never attempted in large population-based cohorts before. Recently, a novel high throughput approach for molecular profiling of the circulating RAAS became available. We aimed at assessing AngI, AngII and aldosterone's distributions in individuals under anti-hypertensive drugs (AHDs) in a real-world setting. Design and method: We selected 500 participants under AHDs and 300 controls from the Cooperative Health Research in South Tyrol (CHRIS) study, a population-based study from an Alpine rural environment, defining age- and sex-matched groups (N = 100 each). Five groups included individuals under AHDs: (i) plain AngI-converting enzyme inhibitor (ACEi); (ii) ACEi+diuretic; (iii) plain AngII type 1 receptor blocker (ARB); (iv) ARB+diuretic; and (v) plain beta-blocker. Three were control groups: (vi) hypertensive without medication (vii) under non-AHDs, (viii) normotensive. Using RAAS Triple-A testing, a novel liquid chromatography combined with tandem mass spectrometry analysis, we simultaneously quantified aldosterone and equilibrium AngI and AngII, and used them to estimate markers of plasma renin activity (PRA-S pmol/L), ace-activity (ACE-S, [pmol/L]/[pmol/L]) and adrenal function (AA2-Ratio, [pmol/L]/[pmol/L]). Results: An evaluation of the first 240 analysed samples revealed specific molecular effects of AHDs, proving the performance of the approach. ACEi and ARB result in compensatory renin up-regulation: PRA-S median (interquartile range) was 54.6 (29.6–86.6) in the pooled controls, 77.4 (49.7–186.4) in ACEi, and 154.6 (55.2–600.4) in ARB (Kruskal-Wallis test p = 0.0001). Using ACEi was associated with suppression of ACE-S, whose median was 5.0 (4.2–6.4) in the pooled controls and 0.3 (0.1–0.6) in ACEi (p &lt; 0.0001). Using ARBs resulted in AA2-Ratio suppression, with a median of 2.3 (1.4–3.9) and 0.7 (0.2–1.7) in the no-medication and ARB groups (p = 0.0001). Combining ACEi or ARB with diuretics was associated with further increase of PRA-S. Data did not support the hypothesis of different aldosterone levels (median = 101.7 pmol/L pooled sample) across groups (p = 0.179). Conclusions: Results point to a potential application of angiotensin-based biomarkers in class-specific drug monitoring, and will be further evaluated in the context of personalized treatment and therapeutic biomarkers.

Cell differentiation and aging accompanied by depletion of the ACE2 protein.

ACE2 was observed as the cell surface receptor of the SARS-CoV-2 virus. Interestingly, we also found ACE2 positivity inside the cell nucleus. The ACE2 levels changed during cell differentiation and aging and varied in distinct cell types. We observed ACE2 depletion in the aortas of aging female mice, similarly, the aging caused ACE2 decrease in the kidneys. Compared with that in the heart, brain and kidneys, the ACE2 level was the lowest in the mouse lungs. In mice exposed to nicotine, ACE2 was not changed in olfactory bulbs but in the lungs, ACE2 was upregulated in females and downregulated in males. These observations indicate the distinct gender-dependent properties of ACE2.Differentiation into enterocytes, and cardiomyocytes, caused ACE2 depletion. The cardiomyogenesis was accompanied by renin upregulation, delayed in HDAC1-depleted cells. In contrast, vitamin D2 decreased the renin level while ACE2 was upregulated. Together, the ACE2 level is high in non-differentiated cells. This protein is more abundant in the tissues of mouse embryos and young mice in comparison with older animals. Mostly, downregulation of ACE2 is accompanied by renin upregulation. Thus, the pathophysiology of COVID-19 disease should be further studied not only by considering the ACE2 level but also the whole renin-angiotensin system.

Fenofibrate prevents iron induced activation of canonical Wnt/\u03b2-catenin and oxidative stress signaling in the retina

Accumulating evidence strongly implicates iron in the pathogenesis of aging and disease. Iron levels have been found to increase with age in both the human and mouse retinas. We and others have shown that retinal diseases such as age-related macular degeneration and diabetic retinopathy are associated with disrupted iron homeostasis, resulting in retinal iron accumulation. In addition, hereditary disorders due to mutation in one of the iron regulatory genes lead to age dependent retinal iron overload and degeneration. However, our knowledge on whether iron toxicity contributes to the retinopathy is limited. Recently, we reported that iron accumulation is associated with the upregulation of retinal and renal renin–angiotensin system (RAS). Evidences indicate that multiple genes/components of the RAS are targets of Wnt/β-catenin signaling. Interestingly, aberrant activation of Wnt/β-catenin signaling is observed in several degenerative diseases. In the present study, we explored whether iron accumulation regulates canonical Wnt signaling in the retina. We found that in vitro and in vivo iron treatment resulted in the upregulation of Wnt/β-catenin signaling and its downstream target genes including renin–angiotensin system in the retina. We confirmed further that iron activates canonical Wnt signaling in the retina using TOPFlash T-cell factor/lymphoid enhancer factor promoter assay and Axin2-LacZ reporter mouse. The presence of an iron chelator or an antioxidant reversed the iron-mediated upregulation of Wnt/β-catenin signaling in retinal pigment epithelial (RPE) cells. In addition, treatment of RPE cells with peroxisome proliferator-activated receptor (PPAR) α-agonist fenofibrate prevented iron-induced activation of oxidative stress and Wnt/β-catenin signaling by chelating the iron. The role of fenofibrate, an FDA-approved drug for hyperlipidemia, as an iron chelator has potentially significant therapeutic impact on iron associated degenerative diseases.

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.

Abstract P3059: ACE S: A NOVEL ANGIOTENSIN BASED MARKER FOR MONITORING PHARMACOLOGIC ACTIVITY OF ACE INHIBITORS IN HYPERTENSIVE PATIENTS

Angiotensin-converting-enzyme-inhibitors (ACEi) are widely used for treating hypertension and cardiovascular diseases and block Angiotensin-II (Ang-II) formation from Angiotensin-I (Ang-I). The therapeutic response to ACE inhibition critically depends on in vivo pharmacologic efficacy and affected by dosing, patient compliance and patient specific compensatory mechanisms, all being relevant topics for the clinical management of hypertension. Samples were collected from 19 hypertensive patients and 7 confirmed primary aldosteronism (PA) patients. Hypertensive patients were investigated at baseline and after 4 weeks on therapy with a single daily dose of perindopril. PA patients were sampled before and after 14 day administration of ramipril. All samples were analysed by LC-MS/MS based RAAS Triple-A testing which involves the calculation of ACE-S, the ratio between plasma equilibrium levels of Ang-II and Ang-I as a novel marker for circulating ACE activity. ACE activity was further measured by analyzing the time dependent conversion of spiked Ang-I (LC-MS/MS) and by performing a clinical colorimetric ACE assay. Treatment adherence was confirmed by quantification of plasma drug levels. ACE-S was significantly lower in all patients on perindopril or ramipril therapy compared to baseline (MEDIANS: 0.35 vs. 2.75 pM/pM, p&lt;0.001). While eqAng-II levels were increased up to 5-fold compared to baseline levels in a subset of patients due to renin up-regulation, ACE-S stayed significantly suppressed in all samples investigated. In contrast, ACE inhibition appeared to be less effective using classical enzymatic approaches assessing ACE activity. The novel angiotensin based marker ACE-S can be used to monitor pharmacologic efficacy of ACE inhibitors independent of their chemical structure. As part of the RAAS Triple-A test that is currently validated in screening for secondary forms of hypertension, ACE-S has the potential to support the clinical management of first-line non-responders in terms of optimizing ACEi dosing and visualizing patient specific compensatory mechanisms, thereby improving the overall control rates in hypertension.

ACE-S

Objective: Angiotensin-converting-enzyme-inhibitors (ACEi) are widely used for treating hypertension and cardiovascular diseases and block Angiotensin-II (Ang-II) formation from Angiotensin-I (Ang-I). The therapeutic response to ACE inhibition critically depends on in vivo pharmacologic efficacy and affected by dosing, patient compliance and patient specific compensatory mechanisms, all being relevant topics for the clinical management of hypertension. Design and method: Samples were collected from 19 hypertensive patients and 7 confirmed primary aldosteronism (PA) patients. Hypertensive patients were investigated at baseline and after 4 weeks on therapy with a single daily dose of perindopril. Two consecutive off-therapy baseline samples separated by 4 hours and two samples on stable therapy (PRE-DOSE, 4 h POST-DOSE) were collected. PA patients were sampled before and after 14 day administration of ramipril. All samples were analysed by LC-MS/MS based RAAS Triple-A testing which involves the calculation of ACE-S, the ratio between plasma equilibrium levels of Ang-II and Ang-I as a novel marker for circulating ACE activity. ACE activity was further measured by analyzing the time dependent conversion of spiked Ang-I (LC-MS/MS) and by performing a clinical colorimetric ACE assay. Treatment adherence was confirmed by quantification of plasma drug levels. Results: ACE-S was significantly lower in all patients on perindopril or ramipril therapy compared to baseline (MEDIANS: 0.35 vs. 2.75 pM/pM, p < 0.001). While eqAng-II levels were increased up to 5-fold compared to baseline levels in a subset of patients due to renin up-regulation, ACE-S stayed significantly suppressed in all samples investigated. In contrast, ACE inhibition appeared to be less effective using classical enzymatic approaches assessing ACE activity, which typically involve a high dilution of the sample in combination with excess of substrates. Conclusions: The novel angiotensin based marker ACE-S can be used to monitor pharmacologic efficacy of ACE inhibitors independent of their chemical structure. As part of the RAAS Triple-A test that is currently validated in screening for secondary forms of hypertension, ACE-S has the potential to support the clinical management of first-line non-responders in terms of optimizing ACEi dosing and visualizing patient specific compensatory mechanisms, thereby improving the overall control rates in hypertension.

Renal iron accelerates the progression of diabetic nephropathy in the HFE gene knockout mouse model of iron overload.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease associated with high mortality worldwide. Increases in iron levels have been reported in diabetic rat kidneys as well as in human urine of patients with diabetes. In addition, a low-iron diet or iron chelators delay the progression of DN in patients with diabetes and in animal models of diabetes. Possible maladaptive mechanisms of organ damage by tissue iron accumulation have not been well studied. We recently reported that iron induced the retinal renin-angiotensin system (RAS) and accelerated the progression of diabetic retinopathy. However, whether iron regulates the systemic RAS is unknown. To explore if iron alters the expression of intrarenal RAS and its role in the progression of DN, we used the high Fe iron (HFE) knockout mouse, a genetic model of systemic iron overload. We found that diabetes upregulated the expression of iron regulatory proteins and augmented tissue iron accumulation in the kidneys of both type 1 and type 2 diabetic mouse models. Iron accumulation in the kidneys of HFE knockout mice was associated with increase in serum and intrarenal renin expression. Induction of diabetes in HFE knockout mice using streptozotocin caused a much higher accumulation of renal iron and accelerated the progression of nephropathy compared with diabetic wild-type mice. Treatment of diabetic mice with the iron chelator deferiprone reversed the renin upregulation and reduced kidney injury. Thus, our results establish a new link between renal iron and RAS activity. Exploring the mechanisms of iron-induced RAS activation further may have a significant therapeutic impact on hypertension and DN.

Effect of SGLT2 inhibitors on body composition, fluid status and renin\u2013angiotensin\u2013aldosterone system in type 2 diabetes: a prospective study using bioimpedance spectroscopy

BackgroundSGLT2-inhibitors are potent antihyperglycemic drugs for patients with type 2 diabetes and have been shown to reduce body weight. However, it is unclear which body compartments are reduced and to what extent.MethodsIn this longitudinal observational study, we analyzed the body composition of 27 outpatients with type 2 diabetes mellitus during the first week and up to 6 months after initiation of treatment with SGLT2-inhibitors (n = 18 empagliflozin, n = 9 dapagliflozin) using bioimpedance spectroscopy (BCM, Fresenius). Fluid status of hypertensive patients taking medication with hydrochlorothiazide (n = 14) and healthy persons (n = 16) were analyzed for comparison.ResultsAt 6 months, HbA1c decreased by 0.8% (IQR 2.3; 0.4), body weight and BMI by 2.6 kg (1.5; 9.3) and 0.9 kg/m2 (0.4; 3.3), respectively. Bioimpedance spectroscopy revealed significant decrease in adipose tissue mass and fat tissue index while lean tissue parameters remained stable. Overhydration (OH) and extracellular water (ECW) decreased by − 0.5 L/1.73 m2 (− 0.1; − 0.9) and − 0.4 L/1.73 m2 (− 0.1; − 0.8) at day 3, respectively, and returned to the initial value after 3 and 6 months. Plasma renin activity increased by 2.1-fold (0.5; 3.6) at 1 month and returned to the initial level at month 3 and 6. Fluid status of patients with SGLT2 inhibitors after 6 months showed no difference from that of hypertensive patients taking hydrochlorothiazide or healthy persons.ConclusionsBody weight reduction under the treatment with SGLT2-inhibitors is caused by reduction of adipose tissue mass and transient loss of extracellular fluid, which is accompanied by upregulation of renin–angiotensin–aldosterone system (RAAS). Permanent loss of extracellular water does not occur under SGLT2 inhibition.

A11374 RAAS Triple A-Testing

Objectives: RAAS Triple-A testing is a novel mass spectrometry based approach providing a comprehensive biochemical evaluation of the circulating renin-angiotensin-system (RAS) on the basis of equilibrium angiotensin levels and circulating aldosterone levels. Methods: RAS-Equilibrium-Analysis combines the robustness and accuracy of LC-MS/MS based quantification with the versatility of serum sampling to generate a highly accurate readout containing multiple layers of information regarding the biochemical features of the circulating RAAS. Equilibrium Angiotensin I (Ang I), Angiotensin II (Ang II) and Aldosterone were simultaneously quantified in 500 μl of standard collected serum samples from healthy volunteers or hypertensive patients receiving different anti-hypertensive first-line therapies. Results: ACE inhibitor therapy resulted in a significant reduction of the Ang II-to-Ang I-Ratio in equilibrium analysis, which was accompanied by an up-regulation of renin. Surprisingly, PRA showed a high correlation with the sum of equilibrium Ang I and Ang II, which was independent of ACE inhibitor treatment. While the ARR was strongly suppressed in the presence of ACE inhibitor treatment, the Aldosterone-to-Angiotensin II-Ratio (AA2-Ratio) was not affected, suggesting superior applicability in screening for primary aldosteronism (PA). Conclusion: RAAS Triple-A testing is a mass spectrometry based multiplex assay combining Ang I, Ang II and Aldosterone to novel angiotensin based surrogate markers for renin and ACE activity drawing a comprehensive picture of a patient's “RAAS Status” while further delivering a novel ACE independent screening test for PA, the AA2-Ratio, which might pave the way for patient screening hypertensive patients without the need of withdrawing anti-hypertensive therapies.

Abstract P251: Angiotensin Based Biomarkers for Molecular Profiling and Drug Efficacy Monitoring in Hypertension

RAAS Triple-A testing is a novel mass spectrometry based approach providing a comprehensive biochemical evaluation of the circulating renin-angiotensin-system (RAS) on the basis of equilibrium angiotensin levels and circulating aldosterone levels. In contrast to previous technologies involving complex sampling procedures, RAS-Equilibrium-Analysis combines the robustness and accuracy of LC-MS/MS based quantification with the versatility of serum sampling to generate a highly accurate readout containing multiple layers of information regarding the biochemical features of the circulating RAAS. Equilibrium Angiotensin I (Ang I), Angiotensin II (Ang II) and Aldosterone were simultaneously quantified in 500μl of standard collected serum samples from healthy volunteers or hypertensive patients receiving different anti-hypertensive first-line therapies. Stable-isotope labeled internal standards were used to control for analyte recovery. Following analyte extraction, samples were subjected to UPLC-MS/MS analysis and diagnostic ratios were calculated. ACE inhibitor therapy resulted in a significant reduction of the Ang II-to-Ang I-Ratio in equilibrium analysis, which was accompanied by an up-regulation of renin, as expected. Surprisingly, PRA showed a high correlation with the sum of equilibrium Ang I and Ang II, which was independent of ACE inhibitor treatment. While the ARR was strongly suppressed in the presence of ACE inhibitor treatment, the Aldosterone-to-Angiotensin II-Ratio (AA2-Ratio) was not affected, suggesting superior applicability in screening for primary aldosteronism (PA). RAAS Triple-A testing is a mass spectrometry based multiplex assay combining Ang I, Ang II and Aldosterone to diagnostic values that draw a comprehensive picture of a patient’s "RAAS Status". While the sum of Ang I and Ang II serves as a strong PRA surrogate marker, ACE activity and ACE inhibitor therapy efficacy can be monitored using the Ang II/Ang I-Ratio. On top, the AA2-Ratio serves as an advanced diagnostic marker for PA that might pave the way for patient screening without the need of withdrawing anti-hypertensive therapies.

Abstract 079: Long-Lasting RNAi Therapeutics Targeting Angiotensinogen Induces a Robust and Durable Antihypertensive Effect

All angiotensin stems from angiotensinogen (AGT). A single dose of small interfering ribonucleic acids (siRNA) targeting AGT may provide long-lasting blood pressure reductions, as it would abolish angiotensin generation. Here we assessed efficacy of AGT siRNA in spontaneously hypertensive rats (SHRs). SHRs were treated for 4 weeks with vehicle, siRNA (10 mg/kg; s.c. every 2 weeks), valsartan (31 mg/kg/day; oral), captopril (100 mg/kg/day; oral), valsartan+siRNA, or captopril+valsartan (all groups n=8). Mean arterial pressure (MAP) was measured via radiotelemetry. Baseline MAP was 137±2 mmHg. ΔMAP was largest after valsartan+siRNA (-67±3 mmHg; P&lt;0.01 vs. captopril+valsartan), followed by captopril+valsartan, captopril, siRNA and valsartan (-55±4, -24±2, -14±1, and -9±2 mmHg, respectively). Valsartan+siRNA reduced cardiac hypertrophy the most (P&lt;0.05 vs. captopril+valsartan). After 4 weeks, siRNA lowered AGT by 98.6%, which increased to 99.9% in combination with valsartan. All treatments increased renin, the highest rise occurring after valsartan+siRNA. Yet, only valsartan+siRNA lowered angiotensin II. No treatment altered aldosterone. Plasma K + tended to increase in all groups, significance being reached only in the valsartan+siRNA group. Both types of dual blockade attenuated normal growth from the second week of treatment onwards. In conclusion, due to renin upregulation, circulating angiotensin II remained intact even with only 1.4% of AGT left, relative to pretreatment. Consequently, AGT siRNA caused a similar antihypertensive effect as valsartan and captopril. Importantly, when combining siRNA+valsartan, angiotensin II collapsed, and blood pressure decreased synergistically. Given the potential for low dosing frequency suggested by this study, this novel treatment may address medication adherence problems in patients with resistant hypertension and further development is warranted.

Up-regulation of renal renin–angiotensin system and inflammatory mechanisms in the prenatal programming by low-protein diet: beneficial effect of the post-weaning losartan treatment

Previous studies have shown that the renin-angiotensin system (RAS) is affected by adverse maternal nutrition during pregnancy. The aim of this study was to investigate the effects of a maternal low-protein diet on proinflammatory cytokines, reactive oxygen species and RAS components in kidney samples isolated from adult male offspring. We hypothesized that post-weaning losartan treatment would have beneficial effects on RAS activity and inflammatory and oxidative stress markers in these animals. Pregnant Sprague-Dawley rats were fed with a control (20% casein) or low-protein diet (LP) (6% casein) throughout gestation. After weaning, the LP pups were randomly assigned to LP and LP-losartan groups (AT1 receptor blockade: 10 mg/kg/day until 20 weeks of age). At 20 weeks of age, blood pressure levels were higher and renal RAS was activated in the LP group. We also observed several adverse effects in the kidneys of the LP group, including a higher number of CD3, CD68 and proliferating cell nuclear antigen-positive cells and higher levels of collagen and reactive oxygen species in the kidney. Further, our results revealed that post-weaning losartan treatment completely abolished immune cell infiltration and intrarenal RAS activation in the kidneys of LP rats. The prevention of augmentation of angiotensin (Ang II) concentration abolished inflammatory and fibrotic events, indicating that Ang II via the AT1 receptor is essential for pathological initiation. Our results suggest that the prenatal programming of hypertension is dependent on the up-regulation of local RAS and presence of immune cells in the kidney.

Novel RAS inhibitor 25-O-methylalisol F attenuates epithelial-to-mesenchymal transition and tubulo-interstitial fibrosis by selectively inhibiting TGF-β-mediated Smad3 phosphorylation

BackgroundTubulo-interstitial fibrosis (TIF) is the common pathway in the chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) is a major contributor to the TIF by the increased myofibroblasts. Renin-angiotensin system (RAS) is critical mediator on EMT in progressive CKD. Angiotensin II (ANG) mediates EMT and causes TIF by stimulating transforming growth factor-β1 (TGF-β1). RAS activation could further activate TGF-β1. Inhibition of the RAS is one of the most powerful therapies for progressive CKD. 25-O-methylalisol F (MAF) is a new tetracyclic triterpenoid compound isolated from the Alismatis rhizoma, which is extensively used for anti-hypertensive, diuretic and anti-hyperlipidemic effects. MethodsInhibitory effect of MAF on EMT is investigated in both TGF-β1- and ANG-induced tubular epithelial cells (NRK-52E) and fibroblasts (NRK-49F). Western blot analysis, qRT-PCR, siRNA, immunofluorescence staining and co-immunoprecipitation techniques were used to evaluate the inhibition of MAF on EMT and further revealed the intervention effects on RAS, TGF-β/Smad and Wnt/β-catenin pathways. ResultsMAF treatment significantly inhibited TGF-β1 and ANG-induced expressions of collagen I, fibronectin, α-SMA, vimentin and E-cadherin at both mRNA and protein levels in the NRK-52E and NRK-49F cells. The action mechanism revealed that MAF significantly ameliorated upregulation of angiotensinogen, renin, ACE and AT1R expressions. Further, MAF attenuated upregulation of Smad3 phosphorylation and downregulation of Smad7, but did not affect the phosphorylation of Smad2, PI3K, ERK1/2 and p38 expressions and Smad4 expression in NRK-52E cells. Co-immunoprecipitation analysis indicated that MAF selectively blocked the combination of Smad3 with TGFβRI and Smad3 with SARA without interfering with the Smad2, TGFβRI and SARA interaction. Additionally, MAF suppressed the expressions of Wnt1 and β-catenin as well as its downstream target Snail1, Twist, MMP-7, PAI-1 and FSP1 expressions in NRK-52E cells. ConclusionsMAF simultaneously targeted multiple RAS components and it was a novel RAS inhibitor. MAF inhibited EMT by Smad3-specific signaling in the TGF-β/Smad-dependent pathway and Wnt/β-catenin pathway. MAF has an important effect on crosstalk between the TGF-β/Smad and Wnt/β-catenin pathway in EMT process by activation of RAS.

Tamm-Horsfall protein/uromodulin deficiency elicits tubular compensatory responses leading to hypertension and hyperuricemia.

Expression of Tamm-Horsfall protein (THP or uromodulin) is highly restricted to the kidney thick ascending limb (TAL) of loop of Henle. Despite the unique location and recent association of THP gene mutations with hereditary uromodulin-associated kidney disease and THP single nucleotide polymorphisms with chronic kidney disease and hypertension, the physiological function(s) of THP and its pathological involvement remain incompletely understood. By studying age-dependent changes of THP knockout (KO) mice, we show here that young KO mice had significant salt and water wasting but were partially responsive to furosemide, due to decreased luminal translocation of Na-K-Cl cotransporter 2 (NKCC2) in the TAL. Aged THP KO mice were, however, markedly oliguric and unresponsive to furosemide, and their NKCC2 was localized primarily in the cytoplasm as evidenced by lipid raft floatation assay, cell fractionation, and confocal and immunoelectron microscopy. These aged KO mice responded to metolazone and acetazolamide, known to target distal and proximal tubules, respectively. They also had marked upregulation of renin in juxtaglomerular apparatus and serum, and they were hypertensive. Finally, the aged THP KO mice had significant upregulation of Na-coupled urate transporters Slc5a8 and Slc22a12 as well as sodium-hydrogen exchanger 3 (NHE3) in the proximal tubule and elevated serum uric acid and allantoin. Collectively, our results suggest that THP deficiency can cause progressive disturbances in renal functions via initially NKCC2 dysfunction and later compensatory responses, resulting in prolonged activation of the renin-angiotensin-aldosterone axis and hyperuricemia.

Poricoic acid ZA, a novel RAS inhibitor, attenuates tubulo-interstitial fibrosis and podocyte injury by inhibiting TGF-β/Smad signaling pathway

BackgroundThe pathogenesis of tubulo-interstitial fibrosis and glomerulosclerosisis was characterized by cellular hypertrophy, extracellular matrix accumulation and podocyte detachment. Poricoic acid ZA (PZA) is a tetracyclic triterpenoid compound extracted from the surface layer of Poria cocos (LPC), which have been used extensively for diuretic and renoprotective effects. MethodsThe anti-fibrotic effect of PZA is investigated in HK-2 cells and podocytes induced by TGF-β1 and angiotensin II (ANGII). qRT-PCR, siRNA, immunofluorescence staining, co-immunoprecipitation and Western blot analyses are used to evaluate the expression of RAS signaling, TGF-β/Smad pathway, epithelial-to-mesenchymal transition (EMT) and podocyte markers. ResultsPZA restores the mRNA and protein expression of EMT in HK-2 cells. Specific TGF-β1-siRNA efficiently blocks ANGII-induced protein expression of TGF-β1 and further inhibits activated Smad signaling. PZA significantly attenuates up-regulation of angiotensinogen, renin, ACE and AT1. Further, PZA reverses up-regulation of TGFβRII and suppresses Smad proteins. Simultaneously, PZA inhibits the protein interaction of TGF-β receptor and Smads and PZA also inhibits activated RAS and TGF-β/Smad signaling cascade and up-regulates protein expression of podocyte markers and mitigates podocyte injury. ConclusionsThis study demonstrated the beneficial role of PZA in renal fibrosis and podocyte injury. Our study highlighted that PZA inhibits RAS and further suppresses TGF-β/Smad pathway through inhibiting Smad2/3 phosphorylation via blocking Smad2/3-TGFβRI protein interaction. PZA is implicated in activation of RAS/TGF-β/Smad axis in HK-2 cells and podocytes. PZA could be considered as a novel RAS inhibitor for treating CKD.