Brief Communication

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Saudi J Kidney Dis Transpl 2017;28(3):552-557 © 2017 Saudi Center for Organ Transplantation

Saudi Journal of Kidney Diseases and Transplantation

Brief Communication Aldosterone Synthase Gene is Not a Major Susceptibility Gene for Progression of Chronic Kidney Disease in Patients with Autosomal Dominant Polycystic Kidney Disease Gnanasambandan Ramanathan1, Ramprasad Elumalai2, Soundararajan Periyasamy2, Bhaskar V. K. S. Lakkakula1,3 Departments of 1Biomedical Sciences and 2Nephrology, Sri Ramachandra University, Chennai, Tamil Nadu, 3Research Division, Sickle Cell Institute Chhattisgarh, Raipur, Chhattisgarh, India ABSTRACT. Autosomal dominant polycystic kidney disease (ADPKD) is the most common heritable kidney disease and is characterized by bilateral renal cysts. Hypertension is a frequent cause of chronic kidney disease (CKD) and mortality in patients with ADPKD. The aldosterone synthase gene polymorphisms of the renin-angiotensin-aldosterone system have been extensively studied as hypertension candidate genes. The present study is aimed to investigate the potential modifier effect of CYP11B2 gene on the progression of CKD in ADPKD. One hundred and two ADPKD patients and 106 healthy controls were recruited based on Ravine inclusion and exclusion criteria. The three tag-SNPs within CYP11B2 gene (rs3802230, rs4543, and rs4544) were genotyped using FRET-based KASPar method. Cochran–Armitage trend test was used to assess the potential associations between these polymorphisms and CKD stages. Mantel– Haenszel stratified analysis was used to explore confounding and interaction effects of these polymorphisms. Of the three tag-SNPs genotyped, rs4544 polymorphism was monomorphic and rs3802230 deviated Hardy–Weinberg equilibrium. The CYP11B2 tag-SNPs did not show significant association with ADPKD or CKD. Further, these polymorphisms did not exhibit confounding effect on the relationship between CKD progression and hypertension. Our results suggest that aldosterone synthase gene is not a major susceptibility gene for progression of CKD in South Indian ADPKD patients. Correspondence to: Dr. Bhaskar V. K. S. Lakkakula, Research Division, Sickle Cell Institute Chhattisgarh, Raipur - 492 001, Chhattisgarh, India. E-mail: [email protected]

Introduction Autosomal dominant polycystic kidney disease (ADPKD) is a late-onset multisystem disorder characterized by bilateral renal cysts that affects an estimated 4–6 million people worldwide.1 ADPKD is a genetically heterogeneous


Aldosterone synthase gene in ADPKD

disorder resulting from mutations of PKD1 and PKD2, which have been proposed to account for 83.3% and 16.7% of cases, respectively.2 Patients with PKD1 mutations are associated with more severe disease and earlier mean age at the onset of end-stage renal disease (ESRD) than those with mutations in PKD2.3 ADPKD causes renal and extrarenal manifestations and accounts for approximately 4.7% of cases of ESRD in the United States.4 Although cyst formation as well as subsequent cyst expansion and growth are the main causes of complications in ADPKD, little is known about the additional environmental and genetic factors that may modify disease severity.5 Extreme inter- and intra-familial variations in the severity of the phenotype suggest the involvement of modifiers.6 Modifiable phenotypic traits such as serum high-density lipoprotein-cholesterol, urine sodium excretion, and 24-h urine osmolality are linked to clinical heterogeneity in ADPKD.7 Hypertension occurs approximately in 60–70% of ADPKD individuals and leads to ESRD by 60 years of age.8 The renin-angiotensinaldosterone system is the key regulator for the development of hypertension and has been implicated as a mechanism for hypertension. Hypertensive ADPKD patients showed higher serum aldosterone levels compared to the normotensive ADPKD patients.9 The aldosterone synthase (CYP11B2) enzyme plays a key role in the process of aldosterone synthesis in the adrenal glands and its expression is regulated by angiotensin II and potassium.10 The gene coding for CYP11B2 is located on chromosome 8q22 and contains nine exons. CYP11B2 gene harbors several polymorphic variants that either completely inactivate the encoded CYP11B2 enzyme or significantly impair its synthesis.11 There are a few studies assessing the association between CYP11B2 polymorphisms and renal function, which revealed inconsistent and inconclusive results.12,13 To our knowledge, no study has analyzed the distribution of CYP11B2 gene polymorphisms and relationships with the progression of chronic kidney disease (CKD) in ADPKD patients. In

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this study, we investigated the involvement of the CYP11B2 gene polymorphisms on the progression of CKD in ADPKD. Materials and Methods Patients A total of 102 South Indian patients with ADPKD, including 55.88% of men, were recruited from the Department of Nephrology, Sri Ramachandra University, Chennai, between February 2000 and June 2014. The diagnosis of ADPKD was made based on previously described Ravine ultrasound criteria.14 From the serum creatinine levels of each patient, estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease study formula. Further, among the ADPKD patients, CKD was defined according to the Kidney Disease Outcomes Quality Initiative criteria for stages of CKD, and the patients were divided into two stages such as early stage (CKD stages 1–3) and advanced stage (CKD stages 4 and 5) using eGFR.15 After thorough screening, 106 healthy unrelated individuals without family history of polycystic kidney disease or any other kidneyrelated complications were included as controls. All controls were nondiabetic, normotensive, and aged between 30 and 60 years. A written informed consent was obtained from all the participants and it was documented. The study was approved by the Institutional Ethical Committee of Sri Ramachandra University, Chennai, India. CYP11B2 tag-SNPs (rs3802230, rs4543, and rs4544) were ascertained from genotyped SNPs in a Gujarati Indians in Houston population in the HapMap Project phase II with a minor allele frequency ≥0.05 and linkage disequilibrium patterns with r2 ≥0.8 as a cutoff (www.hapmap.org). We obtained a 3 mL sample of peripheral blood from all the participants, and genomic DNA was isolated according to the standard procedure.16 The KASPar SNP Genotyping Method (KBioscience, Herts., UK) that uses fluorescent resonance energy transfer (FRET) was adopted for genotyping.17


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Hardy–Weinberg equilibrium was tested for each of the SNPs based on the genotyping of ADPKD patients and healthy controls. Genotypic associations of SNPs between ADPKD and controls were tested by Cochran–Armitage trend test. Among the ADPKD patients, Cochran–Armitage trend test was used to assess the potential associations between these polymorphisms and CKD stages. Further, multivariate logistic regression analysis was performed to adjust for the presence of multiple risk factors. Mantel–Haenszel Chi-square test was performed to evaluate the influence of different genotypes on the relationship between different CKD stages and hypertension. All statistical analyses were performed using Statistical Package for the Social Science (SPSS) version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Results Baseline laboratory characteristics of the study participants are documented in Table 1. The mean age of the control group was 53.27 ± 12.43 years and of the ADPKD group was 46.89 ± 11.38 years. Of the three tag-SNPs, rs4544 polymorphism was monomorphic and was excluded from further analysis. The genotype frequency data for rs3802230 and rs4543 polymorphisms among controls and the ADPKD group are summarized in Table 2. The distribution of CYP11B2 genotypes between the control and ADPKD groups was not statistically significant for both polymorphisms

(Table 2). Among ADPKD patients, 54 individuals (53%) showed early-stage CKD with a mean age of 51.8 ± 4.8 years and 48 patients (47%) showed advanced-stage CKD with a mean age of 35.8 ± 6.6 years. Further, these CYP11B2 genotypes were not significantly different between early- and advanced-stage CKD groups (Table 3). Both rs3802230 and rs4543 polymorphisms did not exhibit confounding effect on the relationship between CKD progression and hypertension (Table 4). Discussion In the present study, we investigated the association between the CYP11B2 tag-SNPs and CKD progression in ADPKD patients. The CYP11B2 tag-SNPs did not show any significant association with ADPKD. None of the polymorphisms were associated with the advancement of CKD in ADPKD patients. Further, these polymorphisms did not exhibit confounding effect on the relationship between CKD progression and hypertension. Initial studies using clinical and animal models have demonstrated that the excess aldosterone levels were associated with proteinuria, glomerulosclerosis, and glomerular filtration rate.18 In corroboration with this, aldosterone receptor blockade remarkably decreased proteinuria and retarded the progression to CKD.19 Increased plasma aldosterone levels were observed in hypertensive ADPKD patients compared with normotensive patients.9 Our results are supported by few case–con-

Table 1. Laboratory characteristics of the study patients and controls. Variables Control (n=106) ADPKD (n=102) Age (years) 53.9±12.8 46.9±11.6 Hb (g/dL) 11.7±2.6 10.7±2.1 BUN (mg/dL) 13.4±7.0 28.4±24.8 Creatinine (mg/dL) 0.95±0.21 3.02±2.58 Sodium (mmol/L) 136.5±5.8 137.1±8.7 Potassium (mmol/L) 3.96±0.88 4.49±2.13 Chloride (mmol/L) 101.1±6.1 101.3±11.8 Bicarbonate (mmol/L) 24.5±3.7 22.9±4.2 Calcium (mg/dL) 10.2±1.4 7.7±1.1 eGFR (mL/min/1.73 m2) 82.6±20.7 43.8±34.7 Hb: Hemoglobin, BUN: Blood urea nitrogen, eGFR: estimated glomerular filtration Autosomal dominant polycystic kidney disease.

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