Multiple Renin-angiotensin System Research Articles

Shenkang injection improves chronic kidney disease by inhibiting multiple renin-angiotensin system genes by blocking the Wnt/β-catenin signalling pathway.

Chronic kidney disease (CKD) is a major worldwide public health problem. The increase in the number of patients with CKD and end-stage kidney disease requesting renal dialysis or transplantation will progress to epidemic proportions in the next several decades. Although blocking the renin-angiotensin system (RAS) has been used as a first-line standard therapy in patients with hypertension and CKD, patients still progress towards end-stage kidney disease, which might be closely associated with compensatory renin expression subsequent to RAS blockade through a homeostatic mechanism. The Wnt/β-catenin signalling pathway is the master upstream regulator that controls multiple intrarenal RAS genes. As Wnt/β-catenin regulates multiple RAS genes, we inferred that this pathway might also be implicated in blood pressure control. Therefore, discovering new medications to synchronously target multiple RAS genes is necessary and essential for the effective treatment of patients with CKD. We hypothesized that Shenkang injection (SKI), which is widely used to treat CKD patients, might ameliorate CKD by inhibiting the activation of multiple RAS genes via the Wnt/β-catenin signalling pathway. To test this hypothesis, we used adenine-induced CKD rats and angiotensin II (AngII)-induced HK-2 and NRK-49F cells. Treatment with SKI inhibited renal function decline, hypertension and renal fibrosis. Mechanistically, SKI abrogated the increased protein expression of multiple RAS elements, including angiotensin-converting enzyme and angiotensin II type 1 receptor, as well as Wnt1, β-catenin and downstream target genes, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in adenine-induced rats, which was verified in AngII-induced HK-2 and NRK-49F cells. Similarly, our results further indicated that treatment with rhein isolated from SKI attenuated renal function decline and epithelial-to-mesenchymal transition and repressed RAS activation and the hyperactive Wnt/β-catenin signalling pathway in both adenine-induced rats and AngII-induced HK-2 and NRK-49F cells. This study first revealed that SKI repressed epithelial-to-mesenchymal transition by synchronously targeting multiple RAS elements by blocking the hyperactive Wnt/β-catenin signalling pathway.

Captopril, a Renin-Angiotensin System Inhibitor, Attenuates Features of Tumor Invasion and Down-Regulates C-Myc Expression in a Mouse Model of Colorectal Cancer Liver Metastasis.

Simple SummaryApproximately 25% of patients with colorectal cancer will present with or develop colorectal liver metastasis (CRLM). Surgical resection of CRLM offers these patients the best chance of a cure. However, liver resection and the subsequent regenerative response has been linked to tumor recurrence in the liver remnant. The Wnt/β-catenin pathway is one of many pathways common to both post-hepatectomy liver regeneration and tumorigenesis. Wnt signaling modulates multiple genes of the renin-angiotensin system (RAS), and Wnt inhibition can attenuate fibrotic responses and improve cancer outcomes via diverse mechanisms. In this study, we investigate the effects of captopril, a RAS inhibitor (RASi), on the Wnt/β-catenin pathway and phenotypic changes associated with tumor progression in the context of the regenerating liver. We show that RASi induced increased Wnt signaling whilst downregulating features of epithelial-to-mesenchymal transition (EMT). Furthermore, RASi induced significant down-regulation of Wnt target genes, c-myc and cyclin D1, indicating that expression of these genes can be down-regulated by RASi despite the accumulation of stabilized β-catenin.(1) Background: Recent clinical and experimental data suggests that the liver’s regenerative response following partial hepatectomy can stimulate tumor recurrence in the liver remnant. The Wnt/β-catenin pathway plays important roles in both colorectal cancer carcinogenesis and liver regeneration. Studies have shown that the Wnt/β-catenin pathway regulates multiple renin-angiotensin system (RAS) genes, whilst RAS inhibition (RASi) reduces tumor burden and progression. This study explores whether RASi attenuates features of tumor progression in the regenerating liver post-hepatectomy by modulating Wnt/β-catenin signaling. (2) Methods: Male CBA mice underwent CRLM induction, followed one week later by 70% partial hepatectomy. Mice were treated daily with captopril, a RASi, at 250 mg/kg/day or vehicle control from experimental Day 4. Tumor and liver samples were analyzed for RAS and Wnt signaling markers using qRT-PCR and immunohistochemistry. (3) Results: Treatment with captopril reduced the expression of down-stream Wnt target genes, including a significant reduction in both c-myc and cyclin-D1, despite activating Wnt signaling. This was a tumor-specific response that was not elicited in corresponding liver samples. (4) Conclusions: We report for the first time decreased c-myc expression in colorectal tumors following RASi treatment in vivo. Decreased c-myc expression was accompanied by an attenuated invasive phenotype, despite increased Wnt signaling.

Sequence variants in the renin-angiotensin system genes are associated with isolated multicystic dysplastic kidney in children.

Multicystic dysplastic kidney (MCDK) is a common form of congenital cystic kidney disease in children. The etiology of MCDK remains unclear. Given an important role of the renin-angiotensin system in normal kidney development, we explored whether MCDK in children is associated with variants in the genes encoding renin-angiotensin system components by Sanger sequencing. The coding regions of renin (REN), angiotensinogen (AGT), ACE, and angiotensin 1 receptor (AGTR1) genes were amplified by PCR. The effect of DNA sequence variants on protein function was predicted with PolyPhen-2 software. 3 novel and known AGT variants were found. 1 variant was probably damaging, 1 was possibly damaging and one was benign. Out of 7 REN variants, 4 were probably damaging and 3 were benign. Of 6 ACE variants, 3 were probably damaging and 3-benign. 3 AGTR1 variants were found. 2 variants were possibly damaging, and one was benign. We report novel associations of sequence variants in REN, AGT, ACE, or AGTR1 genes in children with isolated MCDK in the United States. Our findings suggest a recessive disease model and support the hypothesis of multiple renin-angiotensin system gene involvement in MCDK. Discovery of novel gene variants in renin-angiotensin genes in children with MCDK. Novel possibly damaging gene variants discovered. Multiple renin-angiotensin system gene variants are involved in MCDK.

Wnt/β-catenin regulates blood pressure and kidney injury in rats

Activation of the renin-angiotensin system (RAS) plays a pivotal role in mediating hypertension, chronic kidney and cardiovascular diseases. As Wnt/β-catenin regulates multiple RAS genes, we speculated that this developmental signaling pathway might also participate in blood pressure (BP) regulation. To test this, we utilized two rat models of experimental hypertension: chronic angiotensin II infusion and remnant kidney after 5/6 nephrectomy. Inhibition of Wnt/β-catenin by ICG-001 blunted angiotensin II-induced hypertension. Interestingly, angiotensin II was able to induce the expression of multiple Wnt genes in vivo and in vitro, thereby creating a vicious cycle between Wnt/β-catenin and RAS activation. In the remnant kidney model, renal β-catenin was upregulated, and delayed administration of ICG-001 also blunted BP elevation and abolished the induction of angiotensinogen, renin, angiotensin-converting enzyme and angiotensin II type 1 receptor. ICG-001 also reduced albuminuria, serum creatinine and blood urea nitrogen, and inhibited renal expression of fibronectin, collagen I and plasminogen activator inhibitor-1, and suppressed the infiltration of CD3+ T cells and CD68+ monocytes/macrophages. In vitro, incubation with losartan prevented Wnt/β-catenin-mediated fibronectin, α-smooth muscle actin and Snail1 expression, suggesting that the fibrogenic action of Wnt/β-catenin is dependent on RAS activation. Taken together, these results suggest an intrinsic linkage of Wnt/β-catenin signaling with BP regulation. Our studies also demonstrate that hyperactive Wnt/β-catenin can drive hypertension and kidney damage via RAS activation.

Novel inhibitors of the cellular renin-angiotensin system components, poricoic acids, target Smad3 phosphorylation and Wnt/β-catenin pathway against renal fibrosis.

Tubulo-interstitial fibrosis is the final pathway in the progression of chronic kidney disease (CKD) to kidney failure. The renin-angiotensin system (RAS) plays a major role in CKD progression. Hence, we determined the efficacy of novel RAS inhibitors isolated from Poria cocos against renal fibrosis. Effects of three novel tetracyclic triterpenoid compounds, poricoic acid ZC (PZC), poricoic acid ZD (PZD) and poricoic acid ZE (PZE), were investigated on TGFβ1- and angiotensin II (AngII)-treated HK-2 cells and unilateral ureteral obstruction (UUO) in mice. Immunofluorescence staining, quantitative real-time PCR, siRNA, co-immunoprecipitation and Western blot analyses were used to evaluate expression of key molecules in RAS, Wnt/β-catenin and TGFβ/Smad pathways. Addition of the above compounds to culture media and their administration to UUO mice: (i) significantly attenuated epithelial-to-mesenchymal transition and extracellular matrix production in TGFβ1- and AngII-treated HK-2 cells and UUO mice by inhibiting Wnt/β-catenin pathway activation and Smad3 phosphorylation; (ii) selectively inhibited Smad3 phosphorylation by blocking the interaction of TGFBR1 with Smad3; and (iii) specifically inhibited Smad3 activation. PZC and PZD showed a strong inhibitory effect on all RAS components, and PZE showed a strong inhibitory effect on renin. Furthermore, the secolanostane tetracyclic triterpenoids, PZC and PZD, showed a stronger inhibitory effect than the lanostane tetracyclic triterpenoid PZE. Therefore, compounds with secolanostance skeleton showed stronger bioactivity than those with lanostance skeleton. The secolanostane tetracyclic triterpenoids effectively blocked RAS by simultaneously targeting multiple RAS components and lanostane tetracyclic triterpenoids inhibited renin and protected against tubulo-interstitial fibrosis.

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.

Incidence of and risk factors for newly diagnosed hyperkalemia after hospital discharge in non-dialysis-dependent CKD patients treated with RAS inhibitors.

IntroductionRenin-angiotensin system (RAS) inhibitors have been increasingly prescribed due to their beneficial effects on end-organ protection. Iatrogenic hyperkalemia is a well-known life-threatening complication of RAS inhibitor use in chronic kidney disease (CKD) patients. We hypothesized that CKD patients treated with RAS inhibitors frequently develop hyperkalemia after hospital discharge even if they were normokalemic during their hospitalization because their lifestyles change substantially after discharge. The present study aimed to examine the incidence of newly diagnosed hyperkalemia, the timing of hyperkalemia, and its risk factors in CKD patients treated with RAS inhibitors at the time of hospital discharge.MethodsWe retrospectively enrolled patients aged 20 years or older with CKD G3-5 (estimated glomerular filtration rate < 60 mL/min/1.73 m2) and who were treated with RAS inhibitors and discharged from St. Luke’s International Hospital between July 2011 and December 2015. Patients who were under maintenance dialysis or had hyperkalemic events before discharge were excluded. Data regarding the patients’ age, sex, CKD stage, diabetes mellitus status, malignancy status, combined use of RAS inhibitors, concurrent medication, and hyperkalemic events after discharge were extracted from the hospital database. Our primary outcome was hyperkalemia, defined as serum potassium ≥ 5.5 mEq/L. Multiple logistic regression and Kaplan-Meier analyses were performed to identify the risk factors for and the timing of hyperkalemia, respectively.ResultsAmong the 986 patients, 121 (12.3%) developed hyperkalemia after discharge. In the regression analysis, relative to CKD G3a, G3b [odds ratio (OR): 1.88, 95% confidence interval 1.20–2.97] and G4-5 (OR: 3.40, 1.99–5.81) were significantly associated with hyperkalemia. The use of RAS inhibitor combinations (OR: 1.92, 1.19–3.10), malignancy status (OR: 2.10, 1.14–3.86), and baseline serum potassium (OR: 1.91, 1.23–2.97) were also significantly associated with hyperkalemia. The Kaplan-Meier analysis showed that hyperkalemia was most frequent during the early period after discharge, particularly within one month.ConclusionHyperkalemia was frequent during the early period after discharge among previously normokalemic CKD patients who were treated with RAS inhibitors. Appropriate follow-up after discharge should be required for these patients, particularly those with advanced CKD or malignancy status, such as hematological malignancy or late-stage malignancy, and those who are treated with multiple RAS inhibitors.

Wnt/β-catenin signaling and renin-angiotensin system in chronic kidney disease.

Intrarenal activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of hypertension and chronic kidney diseases (CKD). However, how RAS genes are regulated in vivo was poorly understood until recently. This review focuses on recent findings of the transcriptional regulation of RAS components, as well as their implication in developing novel strategies to treat the patients with CKD. Bioinformatics analyses have uncovered the presence of putative binding sites for T-cell factor/β-catenin in the promoter region of all RAS genes. Both in-vitro and in-vivo studies confirm that Wnt/β-catenin is the master upstream regulator that controls the expression of all RAS components tested, such as angiotensinogen, renin, angiotensin converting enzyme and the angiotensin II type I receptor in the kidney. Targeted inhibition of Wnt/β-catenin, by either small molecule ICG-001 or endogenous Wnt antagonist Klotho, represses RAS activation and ameliorates proteinuria and kidney injury. Blockade of Wnt/β-catenin signaling also normalizes blood pressure in a mouse model of CKD. These recent studies identify Wnt/β-catenin as the master regulator that controls multiple RAS genes, and suggest that targeting this upstream signaling could be an effective strategy for the treatment of patients with hypertension and CKD.

Klotho Ameliorates Kidney Injury and Fibrosis and Normalizes Blood Pressure by Targeting the Renin-Angiotensin System

Loss of Klotho and activation of the renin-angiotensin system (RAS) are common pathological findings in chronic kidney diseases. However, whether these two events are intricately connected is poorly understood. We hypothesized that Klotho might protect kidneys by targeted inhibition of RAS activation in diseased kidneys. To test this hypothesis, mouse models of remnant kidney, as well as adriamycin nephropathy and unilateral ureteral obstruction, were utilized. At 6 weeks after 5/6 nephrectomy, kidney injury was evident, characterized by elevated albuminuria and serum creatinine levels, and excessive deposition of interstitial matrix proteins. These lesions were accompanied by loss of renal Klotho expression, up-regulation of RAS components, and development of hypertension. In vivo expression of exogenous Klotho through hydrodynamic-based gene delivery abolished the induction of multiple RAS proteins, including angiotensinogen, renin, angiotensin-converting enzyme, and angiotensin II type 1 receptor, and normalized blood pressure. Klotho also inhibited β-catenin activation and ameliorated renal fibrotic lesions. Similar results were obtained in mouse models of adriamycin and obstructive nephropathy. In cultured kidney tubular epithelial cells, Klotho dose-dependently blocked Wnt1-triggered RAS activation. Taken together, these results demonstrate that Klotho exerts its renal protection by targeted inhibition of RAS, a pathogenic pathway known to play a key role in the evolution and progression of hypertension and chronic kidney disorders.

Multiple genes of the renin-angiotensin system are novel targets of Wnt/β-catenin signaling.

Activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of CKD and cardiovascular disease. However, current anti-RAS therapy only has limited efficacy, partly because of compensatory upregulation of renin expression. Therefore, a treatment strategy to simultaneously target multiple RAS genes is necessary to achieve greater efficacy. By bioinformatics analyses, we discovered that the promoter regions of all RAS genes contained putative T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding sites, and β-catenin induced the binding of LEF-1 to these sites in kidney tubular cells. Overexpression of either β-catenin or different Wnt ligands induced the expression of all RAS genes. Conversely, a small-molecule β-catenin inhibitor ICG-001 abolished RAS induction. In a mouse model of nephropathy induced by adriamycin, either transient therapy or late administration of ICG-001 abolished established proteinuria and kidney lesions. ICG-001 inhibited renal expression of multiple RAS genes in vivo and abolished the expression of other Wnt/β-catenin target genes. Moreover, ICG-001 therapy restored expression of nephrin, podocin, and Wilms' tumor 1, attenuated interstitial myofibroblast activation, repressed matrix expression, and inhibited renal inflammation and fibrosis. Collectively, these studies identify all RAS genes as novel downstream targets of Wnt/β-catenin. Our results indicate that blockade of Wnt/β-catenin signaling can simultaneously repress multiple RAS genes, thereby leading to the reversal of established proteinuria and kidney injury.

Combination Therapies to Treat Hypertensive Patients with CKD

The need for combination therapy in hypertensive patients with chronic kidney disease (CKD) should require no elaboration. This is particularly true when considering the need to achieve the lower blood pressure goals (&lt;130/80 mmHg) recommended by most guidelines for patients with CKD. Mean number of antihypertensives required to achieve this blood pressure goal in renal outcome trials is between 3.5 and 4 agents. However, the benefit of inhibitors of renin angiotensin system (RAS) shown in multiple renal outcome trials makes their inclusion in the antihypertensive regimen in hypertensive patients with CKD obvious. Adequate diuresis is required to achieve blood pressure goal. In addition, in the ALLHAT trial, the thiazide‐type diuretic, chlorthalidone, was found to be at least as effective as the ACE inhibitor, lisinopril, in slowing progression of renal disease regardless of diabetic status (including the metabolic syndrome) and stage of kidney disease. Although calcium channel blockers in renal outcome trials have been shown to be less effective than RAS inhibitors in slowing the progression of CKD, their efficacy in blood pressure lowering generally requires their inclusion in the regimen. In addition, no loss of renal protection is seen when they are combined with RAS inhibitors. In those with estimated GFR &lt; 40 ml/min/1.73m2, loop diuretics should replace thiazide‐type diuretics in antihypertensive drug combinations, though indapamide and metolazone may retain some efficacy if volume status is not an issue. While some earlier studies suggested additive benefit of multiple RAS inhibitors on renal outcomes, recent large renal outcome trials have failed to confirm this benefit.

Regulation of multiple renin-angiotensin system genes by Sry.

We demonstrated that the Sry gene complex on the spontaneously hypertensive rat (SHR) Y chromosome is a candidate locus for hypertension that accounts for the SHR Y chromosome blood pressure effect. All rat strains examined to date share six Sry loci, and a seventh Sry locus (Sry3) appears to be unique to SHR male rats. Previously, we showed that Sry1 increased activity of the tyrosine hydroxylase promoter in transfected PC12 cells, and Sry1 delivered to adrenal gland of Wistar-Kyoto (WKY) rats increased blood pressure and sympathetic nervous system activity. The objective of this study was to determine whether renin-angiotensin system genes participate in Sry-mediated effects. Sry expression vectors were co-transfected into CHO cells with luciferase reporter constructs containing promoters of angiotensinogen (Agt -1430/+22), renin (Ren -1050/-1), angiotensin-converting enzyme (ACE) (ACE -1677/+21) and ACE2 (ACE2 -1091/+83). Sry1, Sry2 and Sry3 differentially upregulated activity of the promoters of angiotensinogen, renin and ACE genes and downregulated ACE2 promoter activity. The largest effect was seen with Sry3, which increased activity of angiotensinogen promoter by 1.7-fold, renin promoter by 1.3-fold, ACE promoter by 2.6-fold and decreased activity of ACE2 promoter by 0.5-fold. The effect of Sry1 on promoter activity was significantly less than that of Sry3. Sry2 activated promoters at a significantly lower level than Sry1 did. The result of either an additive effect of Sry regulation of multiple genes in the renin-angiotensin system or alterations in expression of a single gene could favor increased levels of Ang II and decreased levels of Ang-(1-7). These actions of Sry could result in increased blood pressure in males and contribute to sex differences in blood pressure.