Nephron, 2015, 129(2), 104-108 M235T polymorphism in the AGT gene and A/GI8-83 substitution in the REN gene correlate with end stage renal disease Saumya Sarkar1, Vikas Gupta2, Ajay Kumar3, Manoj Chaudhary4, Subhash Diyundi5, Prabodh K Sehajpal6, Kumarasamy Thangaraj7, Singh Rajender1* 1 Division of Endocrinology, Central Drug Research Institute, Lucknow, India Department of Biotechnology, DAV College, Amritsar, Punjab, India 3 Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India 4 Department of Nephrology, Kidney Hospital, Jalandhar, Punjab, India 5 Apeejay Stya University, Gurgaon, India 6 Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, India 7 Centre for Cellular and Molecular Biology, Hyderabad, India * Corresponding author, Dr. Singh Rajender, Division of Endocrinology, Central Drug Research Institute, Lucknow, India, E-mail: [email protected], Phone No. 522-2612411-18 Extn. 4393, Fax: +91-5222771941 Word count: Abstract – 188; Manuscript – 1436 Running title: AGT and REN polymorphisms in ESRD 2 Abstract Background/Aims This study aimed at investigating if M235T polymorphism in the AGT gene and A/GI8-83 polymorphism in the REN gene correlate with the end stage renal disease (ESRD). Methods We analyzed one hundred and seventy three patients having ESRD and three hundred and twenty-nine individuals with normal kidney function for differences in the genotype distribution of AGT-M235T and REN-A/GI8-83 polymorphisms between the two groups. The data for cases and controls were compared using chi square test. Results We found significantly higher levels of serum creatinine and CRP in cases in comparison to controls (P< 0.0001). Data comparison showed significant association of AGT M235T substitution with ESRD in dominant model (p = 0.008) and in comparison of heterozygous substitution against homozygous common genotype (p = 0.005). Similarly, REN A/GI8-83 polymorphism showed a significant difference in the distribution of genotypes between cases and controls (p= < 0.038) such that heterozygous substitution was significantly more common in the ESRD cases in comparison to homozygous common genotype (p = 0.023). Conclusion We conclude that heterozygous substitutions at AGT M235T and REN A/GI8-83 loci correlate significantly with ESRD in a north Indian population. Keywords: End stage renal disease, REN gene, AGT gene, Renin angiotensin system INTRODUCTION Epidemiological investigations on nephropathic patients with diabetes and hypertension have suggested early onset of renal failure and the end stage renal disease (ESRD) .Hypertension and albuminuria are the most potent predictors of bad renal function, which is connected to Nephron, 2015, 129(2), 104-108 alteredRenin-Angiotensin System (RAS) .Angiotensin II,the end product of RAS, is a crucial modulator of primary systems like blood pressure, sodium metabolism, and renal hemodynamic function.Hyperglycemia causes microvascular complications in the kidney, and diabetic nephropathy is the prime causative factor for renal failure and ESRD,which is characterized by Angiotensin II generation .Polymorphisms in the RAS and DNA repair genes may compromise renal capacity and hence the risk of ESRD. Angiotensinogen is a glycoprotein produced by liver and encoded by angiotensinogen gene (AGT). Cleavage of angiotensinogen to produce angiotensin I is mediated by the renin photolytic enzyme that isencoded by the REN gene. Therefore, the level and activity of Angiotensin I can be affected by polymorphisms in the AGT and REN genes.M235T is such a variant of AGT, which results in the substitution of threonine instead of methionine at 235thresidue.Patients homozygous for T allele show significantly higher levels of plasma AGT than those having other genotypes.On the other hand, a common intronic polymorphism in intron eight of the REN gene,A/GI8-83, has never been investigated in the ESRD patients. In the present study, we have chosen a commonly studied and a rarely studied polymorphism in the RAS genes for correlation with ESRD in Indian patients. MATERIALS AND METHODS Subject recruitment and sample collection Cases were recruited from the Guru Nanak Dev Hospital, Amritsar and Kidney Hospital, Jalandhar, INDIA. Patients included in the study had prolonged high levels of serum creatinineand had been advised either for dialysis or renal transplant.The final confirmation of ESRD was based on doctor’s recommendations. One hundred and seventy three patients having ESRD were identified and peripheral blood samples (2-3 ml) were collected in EDTA coated vials, before starting of their hemodialysis procedure. Patients’ clinical details were duly filled in a spreadsheet designed for this purpose.Three hundred and twenty-nine blood samples from normal individualsresiding in the surrounding areas of Jalandhar and Amritsar were collected to serveas controls. Non- hypertensive and non-diabetic subjects were included based on a clear renal function history.The study was presented in front of and approved by the Research Degree Committee of the Faculty of Life Sciences, Guru Nanak Dev University, Amritsar. Informed written consent of the participants was obtained. DNA isolation DNA was isolated from the peripheral blood samples according to the phenolchloroform-isoamylalcohol protocol illustrated in our earlier study . The concentration of DNA preparation was determined using spectrophotometric method by reading absorbance at 260 nm, which was followed by preparation of working dilutions (10 ng/μl) in standard TE buffer. The quality of DNA preparation was evaluated by electrophoresis on 1% agarose gel. PCR Amplification and Genetic Analysis The primers for AGT M235T were, forward: 5’CCGTTTGTGCAGGGCCTGGCTCTCT 3’ and reverse: 5’ CAGGGTGCTGTCCACACTGGACCCC 3’ and for REN I8-83 were, forward: 5’ TGAGGTTCGAGTCGGCCCCCT 3’ and reverse: 5’ TGCCCCAAACATGGCCACACAT 3’. PCR composition and temperature conditions are detailed in Supplementary text. The amplicons were purified using Millipore MultiScreen® PCRµ96 Filter Plates and sequenced using BigDye chain termination chemistry on a 3730 DNA analyzer (Applied Biosystems, USA). Nephron, 2015, 129(2), 104-108 Multiple alignment and sequence analyses were done using Auto Assembler Software (Applied Biosystems, USA). Statistical analysis Genotype data for the control group were analyzed for fitness in the Hardy Weinberg equilibrium using online tool available at http://ihg.gsf.de/cgi-bin/hw/hwa1.pl. All other statistical analyses were done using the SPSS software (version 11; SPSS Inc, Chicago, Illinois). Genotypes between cases and controls were compared using 2x3 contingency table. This was followed by genotype comparison using dominant, recessive and co-dominant models.Two sided P values of less than 0.05 (95% level of confidence) were considered significant for statistical inference. RESULTS Demographic details of patients and controls Basic clinical parameters were tabulated and compared between cases and controls (Supplementary Table1). We found significantly higher levels of serum creatinine and CRPin the cases in comparison to controls (P<0.0001). Table 1. Comparison of the AGT M235T genotypes between cases and controls. Analysis model Dominant Recessive Co-dominant Genotype CC CT+TT CC+CT TT CC CT CC TT CT TT Cases (%) 34 (19.65) 139 (80.35) 131 (75.72) 42(24.28) 34 (19.65) 97 (56.07) 34 (19.65) 42 (24.28) 97 (56.07) 42 (24.28) Controls (%) 101 (30.70) 228 (69.30) 250 (75.98) 79 (24.01) 101 (30.70) 149 (45.29) 101 (30.70) 79 (24.01) 149 (45.29) 79 (24.01) Statistical inference p=0.008 OR=1.81 (1.16-2.81) p=1.000 OR=1.01 (0.66-1.55) p=0.005 OR=1.93 (1.21-3.08) p=0.096 OR=1.58 (0.92-2.71) p=0.380 OR=0.82 (0.52-1.28) Genotype comparison AGT M235T substitution increases the risk of ESRD The genotype frequency in the control group followed Hardy-Weinberg Equillibrium. Data comparison using 2x3 contingency table showed a significant difference in the distribution of genotypes between cases and controls (p= 0.019). The frequency of heterozygous genotype was relatively higher in cases in comparison to controls (Table 1). Further comparisons using dominant and recessivemodels showed significant association of this substitution with ESRD in dominant model (p = 0.008) (Table 1).Data comparison in co-dominant models showed significant association of heterozygous genotype with ESRD in comparison to homozygous common genotype (p = 0.005). Therefore, it can be concluded that heterozygous substitution at AGT M235T correlates with ESRD significantly. REN G/AI8-83 substitution increases the risk of ESRD Nephron, 2015, 129(2), 104-108 The genotype frequency in the control group followed the Hardy–Weinberg equilibrium. Genotype comparison using 2x3 contingency table showed a significant difference in the distribution of genotypes between cases and controls (p = 0.038); particularly noticeable was a higher frequency of the heterozygous genotypes in the cases (47.97%) in comparison to controls (36.4%)(Table 2).Further analyses using dominant, recessive, and co-dominant models showed that heterozygous substitutionincreased the risk of ESRD in comparison to homozygous genotypes(P = 0.023) (Table 2). The frequency of heterozygous plus homozygous substitution was relatively much higher in the cases in comparison to the controls; however, the differences were not statistically significant (p = 0.066).Therefore, it can be concluded that heterozygous substitution at REN G/AI8-83 locus is a significant ESRD risk factor in comparison to homozygous common genotype. Table 2. Comparison of the REN G/AI8-83 genotypes between cases and controls. Analysis model Dominant Recessive Co-dominant Genotype Cases (%) Controls (%) Statistical inference GG GA+AA GG+GA AA GG 80 (46.24) 93 (53.75) 163 (94.2) 10 (5.78) 80 (46.24) 165 (55) 135 (45) 274 (91.4) 26 (8.6) 165 (55) p=0.066 OR=1.42 (0.97-2.07) p=0.254 OR=0.64 (0.30-1.37) GA 83 (47.97) 109 (36.33) GG 80 (46.24) 165 (55) AA 10 (5.78) 26 (8.67) p=0.559 OR=0.79 (0.36-1.72) GA AA 83 (47.97) 10 (5.78) 109 (36.33) 26 (8.67) p=0.083 OR=0.51 (0.23-1.10) p=0.023 OR=1.57 (1.06-2.32) DISCUSSION We found that heterozygous substitutions at both,AGT M235T and REN I8-83, loci were risk factors for nephropathy and ESRD. Among other studies on AGT M235T polymorphism,an investigation on Asian non-insulin dependent diabetes mellitus cohort found no difference in genotypesdistribution of AGT M235T between normal renal function and poor renal function groups .Later, three studies on Chinese populations showed increased risk of diabetic nephropathy in type-2 diabetes mellitus peoplecarrying TT genotype [7-9].Another Asian study showed that the frequency of AGT M235T substitution was significantly higher in diabetes mellitus nephropathy .Similarly, astudy on aEuropean population with type-1 diabetes mellitus found TT genotype to be linked with diabetic nephropathy .Investigation on a Turkish population reported no effect of M235T substitution on the risk of ESRD in diabetic mellitus patients; however, another study on the same population found significant effect of this polymorphism on diabetic nephropathy [12, 13].In a Caucasian population, AGT 235T variant was found to be unrelated to ESRD, but this investigation was undertaken on a small sample size . A German group also reported lack of association between M235T polymorphism and ESRD . Nephron, 2015, 129(2), 104-108 An investigation on a fairly large Caucasian cohortreported no correlation of AGT polymorphism with hypertension, but suggested homozygous rare genotype to be a possible marker for renal failure .Basset et al showed a lack of association between the AGT polymorphism and high blood pressure in patients at the risk of ESRD .Another Korean study found no association of AGT M235T polymorphism with hypertension towards the onset of ESRD .However, astudy on Hungarian ESRD individuals illustrated asignificant correlation of MT (CT) genotype in juvenile hypertension patients with ESRD . A recent meta-analysis suggestedan ethnic specific effect of this polymorphism such thatCaucasians having T allele or TT genotype were more prone to ESRD than Asians and Africans .Since our study population has close affinity with Caucasians than East Asians, we support the hypothesis that M235Tsubstitution may show ethnic specific association with ESRD. REN I8-83 polymorphism has never been investigated in the ESRD cases.We found that REN I8-83 polymorphism correlated with ESRD with a higher risk inthe heterozygous genotypes.Only two studies have correlated this polymorphism with high blood pressure [21, 22]. Further studies on different ethnic populations would provide useful data for reaching consensus about its correlation with ESRD. We conclude that heterozygous substitutions at both thelociinvestigated in this study increase the risk of ESRD in an Indo-European population of India. This is the first report on association of these polymorphisms with ESRD in north Indian population. Literature suggests that M235T substitution may affect the risk of renal failure, but in an ethnic-specific manner. Similarly, heterozygous substitution at REN I8-83 locus increases the risk of ESRD, but it would require further investigations as it has been relatively less investigated. Acknowledgement: CSIR-CDRI Communication No. 8880 References 1. Pontremoli R, Ravera M, Viazzi F, Nicolella C, Berruti V, Leoncini G, Giacopelli F, Bezante GP, Sacchi G, Ravazzolo R, Deferrari G. Genetic polymorphism of the reninangiotensin system and organ damage in essential hypertension. Kidney Int. 2000; 57: 561–569. 2. Parving HH, Osterby R, Ritz E. Diabetic Nephropathy. In The Kidney, 6th Edition, Brenner BM, Levine S, Eds Philadelphia, WB Saunders, 2000: 1731-1773. 3. Ritz E, Dikow R. Hypertension and antihypertensive treatment of diabetic nephropathy. Nat ClinPractNephrol. 2006; 2: 562–567. 4. Mehri S, Mahjoub S, Farhati A, Bousaada R, Ben Arab S, Baudin B, Hammami M. Angiotensinogen gene polymorphism inacute myocardial infarction patients. J Renin Angiotensin Aldosterone Syst. 2011; 12: 42–47. Nephron, 2015, 129(2), 104-108 5. Thangaraj K, Joshi M B, Reddy AG, Gupta NJ, Chakravarty B, Singh L. CAG repeat expansion in the androgen receptor gene is not associated with male infertility in Indian populations. J Androl. 2002; 23: 815-818. 6. Yoshida H, Kuriyama S, Atsumi Y, Tomonari H, Mitarai T, Hamaguchi A, Kubo H, Kawaguchi Y, Kon V, Matsuoka K, Ichikawa I, Sakai O.. Angiotensin I converting enzyme gene polymorphism in non-insulin dependent diabetes mellitus. Kidney Int. 1996; 50: 657–664. 7. Young RP, Chan JC, Critchley JA, Poon E, Nicholls G, Cockram CS. Angiotensinogen T235 and ACE insertion/deletion polymorphisms associated with albuminuria in Chinese type 2 diabetic patients. Diabetes Care. 1998; 21: 431–437. 8. Wang J, Zhu X, Yang L, Liu Y, Zhou W, Li H. Relationship between angiotensinogen gene M235T variant with diabetic nephropathy in Chinese NIDDM. Chin Med J (Engl). 1999; 112: 797–800. 9. Wu S, Xiang K, Zheng T, Sun D, Weng Q, Zhao H, Li J.. Relationship between the renin-angiotensin system genes and diabetic nephropathy in the Chinese. Chin Med J (Engl). 2000; 113: 437–441. 10. Chang HR, Cheng CH, Shu KH, Chen CH, Lian JD, Wu MY. Study of the polymorphism of angiotensinogen, angiotensin-converting enzyme and angiotensin receptor in type II diabetes with end-stage renal disease in Taiwan. J Chin Med Assoc. 2003; 66: 51–56. 11. Fogarty DG, Harron JC, Hughes AE, Nevin NC, Doherty CC, Maxwell AP. A Molecular variant of angiotensinogen is associated with diabetic nephropathy in IDDM. Diabetes. 1996; 45: 1204-1208. 12. Eroğlu Z, Cetinkalp S, Erdogan M, Kosova B, Karadeniz M, Kutukculer A, Gunduz C, Tetik A, Topcuoglu N, Ozgen AG, Tuzun M. Association of the angiotensinogen M235T and angiotensin-converting enzyme insertion/deletion gene polymorphisms in Turkish type 2 diabetic patients with and without nephropathy. J Diabetes Complications. 2008; 22: 186–190. 13. Reis KA, Ebinç FA, KoçE Demirci H, Erten Y, Güz G, Derici UB, Bali M, Söylemezoğlu O, Arınsoy T, Sindel S. Association of the angiotensinogen M235T and APO e gene polymorphisms in turkish type 2 diabetic patients with and without nephropathy. Ren Fail. 2011; 33: 469–474. 14. Hunley TE, Julian BA, Phillips JA 3rd, Summar ML, Yoshida H, Horn RG, Brown NJ, Fogo A, Ichikawa I, Kon V. Angiotensin converting enzyme gene polymorphism: potential silencer motif and impact on progression in IgA nephropathy. Kidney Int. 1996; 49: 571-577. Nephron, 2015, 129(2), 104-108 15. Schmidt S, Stier E, Hartung R, Stein G, Bahnisch J, Woodroffe AJ, Clarkson AR, Ponticelli C, Campise M, Mayer G, et al. No association of converting enzyme insertion/deletion polymorphism with immunoglobulin A glomerulonephritis. Am J Kidney Dis. 1995; 26: 727-731. 16. Pei Y, Scholey J, Thai K, Suzuki M, Cattran D. Association of angiotensinogen gene T235 variant with progression of immunoglobinA nephropathy in Caucasian patients. J Clin Invest. 1997; 100: 814-820. 17. Basset E, Berthoux P, Cecillon S, Deprle C, Thibaudin D, De Filippis JP, Alamartin E, Berthou F. Hypertension after renal transplantation and polymorphism of genes involved in essential hypertension: ACE, AGT, AT1 R and ecNOS. ClinNephrol. 2002; 57: 192–200. 18. Lee KB, Kim UK. Angiotensinogen and angiotensin II type 1 receptor gene polymorphism in patients with autosomal dominant polycystic kidney disease: Effect on hypertension and ESRD. Yonsei Med J. 2003; 44: 641–647. 19. Papp F, Friedman AL, Bereczki C, Haszon I, Kiss E, Endreffy E, Túri S. Renin– angiotensin gene polymorphism in children with uremia and essential hypertension. PediatrNephrol. 2003; 18: 150–154. 20. Zhou TB, Yin SS, Qin YH.Association of angiotensinogen M235T gene polymorphism with end-stage renal disease risk: a meta-analysis. Mol Bio Rep. 2013; 40: 765-772. 21. Okura T, Kitami Y, Hiwada K. Restriction fragment length polymorphisms of the human renin gene: association study with a family history of essential hypertension. J Hum Hypertens. 1993; 7: 457-461. 22. Frossard PM, Lestringant GG, Elshahat YI, John A, Obineche EN. An MboI two-allele polymorphism may implicate the human renin gene in primary hypertension. Hypertens Res. 1998; 21: 221–225.
© Copyright 2023