Association of angiotensin-converting enzyme gene polymorphism (rs1799752) with type 2 diabetes mellitus, hypertension and chronic kidney disease and, its clinical relevance: A preliminary study from South India

Balaji Ramanathan; Gunavathy Nagarajan; Kumaravel Velayutham

doi:10.4103/cdrp.cdrp_6_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 1 | Issue : 2 | Page : 51-57

Association of angiotensin-converting enzyme gene polymorphism (rs1799752) with type 2 diabetes mellitus, hypertension and chronic kidney disease and, its clinical relevance: A preliminary study from South India

Balaji Ramanathan, Gunavathy Nagarajan, Kumaravel Velayutham
Department of Molecular Genetics, Alpha Health Foundation, Madurai, Tamil Nadu, India

Date of Submission	11-Apr-2022
Date of Decision	10-May-2022
Date of Acceptance	24-May-2022
Date of Web Publication	16-Jul-2022

Correspondence Address:
Kumaravel Velayutham
Director . Alpha Health Foundation, Mela Anuppannady, Madurai - 625 009, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cdrp.cdrp_6_22

Abstract

Background: The renin–angiotensin–aldosterone system (RAAS) is important in regulating blood pressure and electrolyte balance. The main effector hormone of the RAAS is angiotensin II, which is generated from angiotensin I in the circulation and in the tissues, mostly as a result of the action of angiotensin-converting enzyme (ACE). The ACE gene has received substantial attention in recent years as a candidate gene for a variety of diseases. Objective: This study was conducted to determine the association of insertion/deletion (I/D) polymorphism of ACE gene in type 2 diabetes mellitus (T2DM), hypertension (HT), and chronic kidney disease (CKD) subjects among South Indian regional population. Methods: A total of 105 subjects participated in this study including 30 T2DM (Group 1), 30 HT (Group 2), 35 CKD (Group 3) patients and 10 controls (Group 4). Blood samples were collected and biochemical investigations were done. Polymerase chain reaction amplification was performed to genotype the DNA. The distribution and allelic frequency of I/D (rs1799752) polymorphism at the 287-base pair Alu repeat sequence in the intron 16 of ACE gene were analyzed using specific primers. Results: The ACE genotypes were distributed as II, 17%; DD, 47%; and ID, 37% in the T2DM group; II, 10%; DD, 50%; and ID, 40% in the HT group; II, 17%; DD, 54%; and ID, 29% in the CKD group; and II, 50%; DD, 20%, and ID, 30% in the control group. The frequency of DD genotype was significantly higher in HT (P = 0.05) and CKD patients (P = 0.05) compared to controls. In codominant model analysis, DD genotype versus II genotype was associated with increased risk of T2DM (odds ratio [OR] = 4.37; 95% confidence interval [CI] = 1.31–14.504), HT (OR = 9.0; 95% CI = 2.23–36.17), and/or CKD (OR = 5.73; 95% CI = 1.906–17.282), respectively. The D allele was more frequent in T2DM (65%), HT (70%), and CKD patients (69%) compared to controls (35%) (P = 0.018, P = 0.005, and P = 0.006, respectively). The D allele was associated with increased risk of T2DM (OR = 3.44; 95% CI = 1.19–9.96), HT (OR = 4.33; 95% CI = 1.48–12.65), and CKD (OR = 4.05; 95% CI = 1.42–11.55). Conclusion: The DD genotype and the D allele of the ACE I/D gene polymorphism can be a risk factor for T2DM, HT, and CKD in South Indian regional population. This result suggests that T2DM and HT patients should be offered analysis to identify defects in ACE I/D polymorphism, which might help to determine the course of CKD disease and aid to choose appropriate antihypertensive therapy with ACE inhibitor/angiotensin receptor blockers.

Keywords: Angiotensin-converting enzyme, chronic kidney disease, hypertension, insertion/deletion polymorphism, polymerase chain reaction, type 2 diabetes mellitus

How to cite this article:
Ramanathan B, Nagarajan G, Velayutham K. Association of angiotensin-converting enzyme gene polymorphism (rs1799752) with type 2 diabetes mellitus, hypertension and chronic kidney disease and, its clinical relevance: A preliminary study from South India. Chron Diabetes Res Pract 2022;1:51-7

How to cite this URL:
Ramanathan B, Nagarajan G, Velayutham K. Association of angiotensin-converting enzyme gene polymorphism (rs1799752) with type 2 diabetes mellitus, hypertension and chronic kidney disease and, its clinical relevance: A preliminary study from South India. Chron Diabetes Res Pract [serial online] 2022 [cited 2023 Mar 29];1:51-7. Available from: https://cdrpj.org//text.asp?2022/1/2/51/351259

Introduction

Type 2 diabetes mellitus (T2DM), hypertension (HT), and chronic kidney disease (CKD) are the leading public health burden in India and have been classified as a global epidemic. The global prevalence of T2DM is 10.5%,^[1] HT is 26%,^[2] and CKD is 9.3%.^[3] T2DM is a chronic, metabolic disease characterized by elevated levels of blood glucose, which leads to damage of heart, vasculature, eyes, kidneys, and nerves.^[4] CKD is a major microvascular complication of T2DM and HT, representing the leading cause of end-stage renal disease. Clinical hallmarks of CKD include a progressive increase in urinary albumin excretion and a decline in glomerular filtration rate (GFR).^[5]

The renin–angiotensin–aldosterone system (RAAS) plays a key role in regulating blood pressure and electrolyte balance. Genes encoding components of the RAAS has received substantial attention as a candidate gene for a variety of disease including HT, T2DM, and the risk of developing nephropathy, retinopathy, and neuropathy complications. Among them, the angiotensin-converting enzyme (ACE) gene is the eminent gene in RAAS and an attractive candidate gene possibly playing an active role in the pathogenesis of T2DM, HT, and CKD [Figure 1]. ACE, a metalloproteinase (dipeptidyl carboxypeptidase), converts angiotensin I to angiotensin II and is also a potent vasoconstrictor.^[6] The ACE gene (NC_000017.11) is mapped to chromosome 17q23 and is encoded by a 21-kb gene that consists of 26 exons and 25 introns. The insertion/deletion (I/D) polymorphism (rs1799752) of ACE gene is characterized by the presence (insertion) or absence (deletion) of a 287-bp sequence in intron 16 producing three genotypes (II homozygote, ID heterozygote, and DD homozygote).^[7] By identifying genotype of ACE I/D polymorphism, it would help the physicians for betterment of treatment with suitable hypertensive therapy and to prevent the progression of CKD. Considering the number of factors influencing the beginning, development, and prognosis of the disease, it is necessary to understand the relationship between the environmental and genetic factors related to this disease. Therefore, the present study aims to explore the association and prevalence of ACE gene I/D polymorphism (rs1799752) in T2DM, HT, and CKD patients among South Indian regional population.

Figure 1: Overview of ACEI/D polymorphism in RAAS. Pathophysiological association between hypertension, type 2 diabetes mellitus, and chronic kidney disease. ACEI = Angiotensin-converting enzyme inhibitor, RAAS = Renin–angiotensin aldosterone system

Click here to view

Methods

The present study was carried out by Alpha Health Foundation in association with Alpha Hospital and Research Centre. The proposed study groups were enrolled for the study with clearance from the Institutional Ethical Committee and upon obtaining informed consent from the participants. Patients with either T2DM or HT or CKD or a combination of any of these conditions, age > 18 years, with controlled blood sugar, and willing for the genetic tests were included. Patients with active infections, secondary causes of CKD, malignancies, known autoimmune diseases, uncontrolled blood sugar, and/or psychiatric illness were excluded.

A total of 105 subjects were recruited for this study and they were categorized into four groups. Group 1 consists of 30 T2DM patients, Group 2 consists of 30 HT patients, Group 3 consists of 35 CKD patients, and Group 4 consist of 10 healthy controls. Data were collected from each subject, which included clinical history with the examination. Five milliliters of venous blood sample was collected in an EDTA-coated Vacutainer tube (BD-Becton, Dickinson and Company, India) from the participating subjects. A portion of the blood sample was used for biochemical tests relevant to glucose metabolism, lipid profile, and serum creatinine according to the standard protocols. T2DM was diagnosed according to ADA criteria.^[4] HT was defined by mean systolic blood pressure (SBP) ≥140 mmHg, mean diastolic blood pressure ≥90 mmHg, or taking antihypertensive therapy.^[8] CKD was defined by the abnormalities in the composition of blood/urine or abnormalities in imaging tests either kidney damage or GFR <60 ml/min/1.73 m² for >3 months.^[9]

Genomic DNA was extracted from the peripheral blood samples using a QIAamp DNA Blood Mini Kit (QIAGEN India Pvt. Ltd., New Delhi, India) according to the manufacturer's protocol. Quantitative and qualitative (260/280-nm absorbance ratio) assessments of the DNA samples were carried out using a nanodrop one spectrophotometer (Thermo Fisher Scientific, USA). Genotyping of ACE gene intron 16 was amplified by polymerase chain reaction (PCR) in a thermocycler (Agilent SureCycler 8800, USA). For amplification, a flanking primer pair 5'-CTGGAGAGCCACTCCCATCCTTTCT-3' and 5'-GACGTGGCCATCA CATTCGTCAGAT-3 (synthesized by IDT-Integrated DNA Technologies, USA) was used. PCR amplification was performed with a 20-μl reaction mixture. Amplification conditions were 95°C for 5 min for initial denaturation, 35 cycles of 94°C for 30 s for denaturation, 65°C for 30 s for annealing of the primers, and 72°C for 1 min for extension, followed by a final extension at 72°C for 7 min. PCR products were separated by 2% agarose gel electrophoresis (Orange, India). The 479-bp products corresponded to the insertion (I) and the 192-bp products to the deletion (D). The PCR products were visualized after electrophoresis using a UV gel documentation system (Medicare, Chennai, India).

Statistical analysis

Clinical characteristics data of all the subjects were expressed as mean ± standard deviation and qualitative data as frequency and percentage. Continuous variable was compared between the case and control groups by using two-tailed Student's t-test and differences in proportions were assessed by the Chi-square test. The genotype frequencies were evaluated among each other, as follows: DD versus ID versus II (codominant model), DD + ID versus II (dominant model), and DD versus ID + II (recessive model), in order to observe any possible codominant, dominant, and recessive effects of the D allele, respectively. Allelic frequencies were calculated by gene-counting method. Risk estimate was done by odds ratio (OR) with 95% confidence interval (95% CI). A level of P < 0.05 was considered statistically significant. All the statistical analysis was carried out using SPSS statistical software, version 26.0, IBM SPSS, version 26 (SPSS, Inc., Chicago Il, USA).

Results

A total of 105 participants, including 10 controls (Group 4) and 95 patients, were included in the study. The baseline characteristics of all the subjects enrolled in this study are shown in [Table 1]. The study included 30 patients with T2DM (Group 1), 30 patients with HT (Group 2), and 35 patients with CKD (Group 3). The mean age of T2DM patients was 49.8 ± 11.7, HT patients was 52.8 ± 11.1, CKD patients was 65.0 ± 13.9, and controls was 49.0 ± 20.0 years. Serum triglycerides were significantly higher in HT (P = 0.001) and CKD (P = 0.01) patients, and random blood sugar was also significantly higher in T2DM (P = 0.001), HT (P = 0.001), and CKD (P = 0.001) patients compared to controls. There was a notable significant difference in SBP (P = 0.028) in HT patients when compared with the control group.

Table 1: Baseline characteristics of the study group

Click here to view

Genotyping results

PCR was performed for analysis of the insertion/deletion polymorphism of the ACE gene [Figure 2]. The genotype distribution of ACE I/D gene polymorphism in T2DM, HT, and CKD patients and controls is presented in [Table 2]. The ACE genotypes were distributed as II, 17%; DD, 47%; and ID, 37% in T2DM (Group 1); II, 10%; DD, 50%; and ID, 40% in HT (Group 2); II, 17%; DD, 54%; and ID, 29% in CKD (Group 3); and II, 50%; DD, 20%; and ID, 30% in control (Group 4) [Figure 3]. The frequency of DD genotype was significantly higher in HT (P = 0.05) and CKD patients (P = 0.05) compared to controls. The DD genotype of the HT and CKD groups was significantly associated with increased risk of HT (OR = 5; 95% CI = 0.93–26.78; P = 0.046) and/or CKD (OR = 4.75; 95% CI = 0.87–25.64; P = 0.05) when compared with controls. The D allele was more frequent in T2DM (65%), HT (70%), and CKD patients (69%) compared to controls (35%) (P = 0.018, P = 0.005, and P = 0.006, respectively). The D allele was associated with increased risk of T2DM (OR = 3.44; 95% CI = 1.19–9.96), HT (OR = 4.33; 95% CI = 1.48–12.65), and CKD (OR = 4.05; 95% CI = 1.42–11.55).

Figure 2: Genotyping of ACE I/D, PCR was performed for analysis of the insertion/deletion polymorphism of the ACE gene. PCR amplification of primer specific product that encompasses I/D polymorphism was electrophoretically separated and visualized in 2% agarose gel. II – 495 bp; ID – 495 bp, 192 bp; DD – 192 bp. ACEI = Angiotensin-converting enzyme inhibitor, PCR = Polymerase Chain Reaction

Click here to view

Table 2: Genotypic and allelic frequencies of angiotensin-converting enzyme gene and comparison of odds ratio between study groups

Click here to view

Figure 3: Genotypic and allelic frequency of ACE I/D polymorphism among study groups. ACEI = Angiotensin-converting enzyme inhibitor

Click here to view

The distribution of ACE I/D genotypes and their corresponding ORs were calculated for each variant, as shown in [Table 3]. In codominant model, DD genotype versus II genotype was found to be significantly associated with increased risk of T2DM (OR = 4.37; 95% CI = 1.31–14.50; P = 0.012), HT (OR = 9.0; 95% CI = 2.23–36.17; P = 0.001), and/or CKD (OR = 5.73; 95% CI = 1.90–17.28; P = 0.001). In codominant model analysis, DD genotype versus ID genotype was significantly associated with increased risk of CKD (OR = 2.96; 95% CI = 1.10–7.98; P = 0.028).

Table 3: Distribution of angiotensin-converting enzyme genotypes among study groups

Click here to view

Discussion

T2DM, HT, and CKD are polygenic disorders having multifactorial influence, as evidenced by various disease outcomes modulated by the gene–gene and gene–environment interactions. An inevitable gene of RAAS is ACE and its I/D polymorphism has been frequently reported to be associated with T2DM,^[10] HT,^[11] and CKD^[12] in different ethnic populations. An increased plasma and serum ACE level is genetically determined by I/D polymorphism of the ACE gene. The DD genotype has been commonly revealed to have enhanced the serum ACE levels and activity, whereas II and ID genotypes are associated with low and intermediate levels of ACE, respectively.^[13] The ACE D allele is associated with higher serum ACE levels and increased conversion of angiotensin I (Ang I) to angiotensin II (Ang II).^[14] Elevated serum ACE levels diminish glucose utilization in skeletal muscles during exercise, and ACE inhibitor (ACEI) reduces the ACE levels and increases insulin sensitivity, GLUT-4 synthase, and hexokinase activity, and also suppresses hepatic glucose production.^{[15],[16],[17]} As a result of these previous reports, high ACE activity, namely the ACE DD genotype, seems to increase the risk of impaired glucose metabolism or T2DM.^[18] Ang II elevates blood pressure through its direct vasoconstrictor and sodium-retaining activities. Elevated level of angiotensin II plays an important role in the regulation of GFR and renal blood flow by constricting the efferent and afferent glomerular arterioles.^[19] It may contribute to disease progression of CKD that comprises a group of pathologies such as tubulointerstitial fibrosis, tubular atrophy, mesangial cell expansion, extracellular matrix accumulation, and podocyte apoptosis.^[20] The current guidelines recommend ACEIs/angiotensin receptor blockers (ARBs) as a first-line drug for diabetic hypertensive patients.^[21],[22] ACEI promotes vasodilation by inhibiting angiotensin II formation and degradation of bradykinin and ARB can trigger vasodilation and natriuresis. Apart from the antihypertensive effects, ACEI/ARB has other pleiotropic clinically beneficial effects, such as diminishing sympathetic activity, improving insulin resistance, endothelium function, and plaque stabilization, and is also involved in suppressing ventricular remodeling, atherosclerosis process, thrombosis, and platelet aggregation.^{[23],[24],[25],[26],[27]} Mallat^[28] emphasized that, in patients with evidence of renal disease, guidelines recommended ACEI/ARB-based therapy due to their superior reno-protective effects compared to other antihypertensive classes. However, several studies in diabetic nephropathy patients^{[29],[30],[31]} indicated that ACEIs and ARBs retard renal function deterioration through an antiproteinuric effect that goes beyond the pressure lowering effects.

Dell'omo et al. found that the D allele in hypertensive patients poses a higher risk for microalbuminuria and treatment with ACEIs produces a greater reduction in microalbuminuria in hypertensive patients homozygous for the I allele.^[32] Heidari et al. have reported that blood pressure was significantly reduced in response to ACEIs (lisinopril and enalapril) in male hypertensive subjects with DD genotype.^[33] ACEIs are found to be effective in subjects with DD genotype resulting in increased ACE serum levels and ACE activity which enhances inhibition.^[34],[35] In addition, ARBs have been reported to be associated with a significant decrease in blood pressure in individuals with II genotype compared to DD or ID genotypes.^[36] However, the individuals with DD genotype showed a better response to ACEIs.^[33] The data obtained from the current study substantiate the association between ACE I/D polymorphism and T2DM, HT, and CKD. Our results corroborate with the earlier reports of the association between D allele and T2DM,^[37],[38] HT,^[39],[40] and CKD^[41],[42] in different ethnic populations. Our study has demonstrated a remarkable difference in the prevalence of DD genotype and D allele of ACE gene in T2DM, HT, and CKD patients than in controls. The results of this study support the hypothesis that the DD genotype has a strong association with T2DM, HT, and CKD and that ACE polymorphism plays an important role in the pathogenesis of these diseases. Limitation of our current study includes a small sample size and nonestimation of serum ACE levels. We could not analyze the effect of overlap among the groups, on the association between genotype and disease due to the small sample size. A detailed multicenter, prospective study with ACEI/ARB will strengthen our results and enlighten the importance of ACE I/D polymorphism in prediction of CKD progression and reno-protective effect of RAS blockade in HT patients.

Conclusion

In the present study, it was observed that the D allele of ACE gene was significantly associated with T2DM, HT, and CKD as compared to controls. These results suggest that T2DM/HT patients should be offered analysis for defects in ACE I/D polymorphism, and if present, their risk of CKD progression may be high. Identifying the ACE, I/D gene polymorphism could aid to choose an appropriate antihypertensive therapy like ACEIs or angiotensin-receptor blockers. The clinicians could identify groups of patients, with the susceptible ACE genotypes that need intensive monitoring to prevent both onset and the development of diseases. Further, large-scale studies in the regional population may offer better insight on the analyzed susceptible gene variants.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	International Diabetes Federation. IDF Diabetes Atlas, 10th edn. Brussels, Belgium: International Diabetes Federation; 2021.
2.	Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. Global burden of hypertension: Analysis of worldwide data. Lancet 2005;365:217-23.
3.	Cockwell P, Fisher LA. The global burden of chronic kidney disease. Lancet 2020;395:662-4.
4.	Gavin JR, Alberti KG, Davidson MB, DeFronzo RA, Drash A, Gabbe SG, et al. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 2000;23:S4-20.
5.	Giunti S, Barit D, Cooper ME. Mechanisms of diabetic nephropathy: Role of hypertension. Hypertension 2006;48:519-26.
6.	Urata H, Healy B, Stewart RW, Bumpus FM, Husain A. Angiotensin II-forming pathways in normal and failing human hearts. Circ Res 1990;66:883-90.
7.	Ramanathan B, Murugan J, Velayutham K. Pilot study on evaluation and determination of the prevalence of Polycystic Ovarian Syndrome (PCOS) associated gene markers in the South Indian population. Indian J Endocrinol Metab 2021;25:551-8.
8.	Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr., et al. Seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. Hypertension 2003;42:1206-52.
9.	Levey AS, Coresh J, Bolton K, Culleton B, Harvey KS, Ikizler TA, et al. K/DOQI clinical practice guidelines for chronic kidney disease: Evaluation, classification, and stratification. Am J Kidney Dis 2002;39:i-ii+S1-266.
10.	Qian Y, Kehr B, Halldórsson BV. PopAlu: Population-scale detection of Alu polymorphisms. PeerJ 2015;3:e1269.
11.	Cowie CC, Port FK, Wolfe RA, Savage PJ, Moll PP, Hawthorne VM. Disparities in incidence of diabetic end-stage renal disease according to race and type of diabetes. N Engl J Med 1989;321:1074-9.
12.	Mallamaci F, Zuccalà A, Zoccali C, Testa A, Gaggi R, Spoto B, et al. The deletion polymorphism of the angiotensin-converting enzyme is associated with nephroangiosclerosis. Am J Hypertens 2000;13:433-7.
13.	Settin A, Elbaz R, Abbas A, Abd-Al-Samad A, Noaman A. Angiotensin-converting enzyme gene insertion/deletion polymorphism in Egyptian patients with myocardial infarction. J Renin Angiotensin Aldosterone Syst 2009;10:96-100.
14.	Ueda S, Elliott HL, Morton JJ, Connell JM. Enhanced pressor response to angiotensin I in normotensive men with the deletion genotype (DD) for angiotensin-converting enzyme. Hypertension 1995;25:1266-9.
15.	Dietze GJ, Henriksen EJ. Angiotensin-converting enzyme in skeletal muscle: Sentinel of blood pressure control and glucose homeostasis. J Renin Angiotensin Aldosterone Syst 2008;9:75-88.
16.	Sayed-Tabatabaei FA, Oostra BA, Isaacs A, van Duijn CM, Witteman JC, Scheen AJ. Prevention of type 2 diabetes mellitus through inhibition of the Renin-Angiotensin system. Drugs 2004;64:2537-65.
17.	Barbalić M, Skarić-Jurić T, Cambien F, Barbaux S, Poirier O, Turek S, et al. Gene polymorphisms of the renin-angiotensin system and early development of hypertension. Am J Hypertens 2006;19:837-42.
18.	Zhou JB, Yang JK, Lu JK, An YH. Angiotensin-converting enzyme gene polymorphism is associated with type 2 diabetes: A meta-analysis. Mol Biol Rep 2010;37:67-73.
19.	Heyeraas KJ, Aukland K. Interlobular arterial resistance: Influence of renal arterial pressure and angiotensin II. Kidney Int 1987;31:1291-8.
20.	Sun HJ, Wu ZY, Cao L, Zhu MY, Liu TT, Guo L, et al. Hydrogen sulfide: Recent progression and perspectives for the treatment of diabetic nephropathy. Molecules 2019;24:2857.
21.	Marathe PH, Gao HX, Close KL. American Diabetes Association standards of medical care in diabetes 2017. J Diabetes 2017;9:320-4.
22.	Williams B, Mancia G, Spiering W, Agabiti Rosei E, Azizi M, Burnier M, et al. 2018 ESC/ESH Guidelines for the management of arterial hypertension: The Task Force for the management of arterial hypertension of the European Society of Cardiology (ESC) and the European Society of Hypertension (ESH). Eur Heart J 2018;39:3021-104.
23.	James PA, Oparil S, Carter BL, Cushman WC, Dennison-Himmelfarb C, Handler J, et al. 2014 evidence-based guideline for the management of high blood pressure in adults: Report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA 2014;311:507-20.
24.	Schuh JR, Blehm DJ, Frierdich GE, McMahon EG, Blaine EH. Differential effects of renin-angiotensin system blockade on atherogenesis in cholesterol-fed rabbits. J Clin Invest 1993;91:1453-8.
25.	Schiffrin EL, Park JB, Intengan HD, Touyz RM. Correction of arterial structure and endothelial dysfunction in human essential hypertension by the angiotensin receptor antagonist losartan. Circulation 2000;101:1653-9.
26.	Ido A, Hasebe N, Takeuchi T, Kikuchi K. Effects of Temocapril and olmesartan on myocardial sympathetic nervous activity and fatty acid metabolism in rats with chronic β-adrenergic stimulation. J Cardiovas Pharmacol 2003;41:S133-7.
27.	Yamaguchi K, Ura N, Murakami H, Togashi N, Hyakukoku M, Higashiura K, et al. Olmesartan ameliorates insulin sensitivity by modulating tumor necrosis factor-alpha and cyclic AMP in skeletal muscle. Hypertens Res 2005;28:773-8.
28.	Mallat SG. What is a preferred angiotensin II receptor blocker-based combination therapy for blood pressure control in hypertensive patients with diabetic and non-diabetic renal impairment? Cardiovasc Diabetol 2012;11:32.
29.	Atkins RC, Briganti EM, Lewis JB, Hunsicker LG, Braden G, de Crespigny PJ, et al. Proteinuria reduction and progression to renal failure in patients with type 2 diabetes mellitus and overt nephropathy. Am J Kidney Dis 2005;45:281-7.
30.	Lea J, Greene T, Hebert L, Lipkowitz M, Massry S, Middleton J, et al. The relationship between magnitude of proteinuria reduction and risk of end-stage renal disease: Results of the African American study of kidney disease and hypertension. Arch Intern Med 2005;165:947-53.
31.	de Zeeuw D, Remuzzi G, Parving HH, Keane WF, Zhang Z, Shahinfar S, et al. Proteinuria, a target for renoprotection in patients with type 2 diabetic nephropathy: Lessons from RENAAL. Kidney Int 2004;65:2309-20.
32.	Dell'omo G, Penno G, Pucci L, Lucchesi D, Fotino C, Del Prato S, et al. ACE gene insertion/deletion polymorphism modulates capillary permeability in hypertension. Clin Sci (Lond) 2006;111:357-64.
33.	Heidari F, Vasudevan R, Mohd Ali SZ, Ismail P, Etemad A, Pishva SR, et al. Association of insertion/deletion polymorphism of angiotensin-converting enzyme gene among Malay male hypertensive subjects in response to ACE inhibitors. J Renin Angiotensin Aldosterone Syst 2015;16:872-9.
34.	Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990;86:1343-6.
35.	Danser AJ, Schalekamp MA, Bax WA, van den Brink AM, Saxena PR, Riegger GA, et al. Angiotensin-converting enzyme in the human heart: Effect of the deletion/insertion polymorphism. Circulation 1995;92:1387-8.
36.	Kurland L, Melhus H, Karlsson J, Kahan T, Malmqvist K, Ohman KP, et al. Angiotensin converting enzyme gene polymorphism predicts blood pressure response to angiotensin II receptor type 1 antagonist treatment in hypertensive patients. J Hypertens 2001;19:1783-7.
37.	Goossens GH. The renin-angiotensin system in the pathophysiology of type 2 diabetes. Obes Facts 2012;5:611-24.
38.	Alami HE, Ghazal H, Abidi O, Al Idrissi N, Wakrim L, Naamane A, et al. Relationship between insertion/deletion (I/D) polymorphism of angiotensin converting enzyme (ACE) gene and susceptibility to type 2 diabetes mellitus in the Middle East and North Africa Region: A meta-analysis. Diabetes Metab Syndr 2022;7:102386.
39.	Cardoso RL, Nogueira AR, Salis LH, Ürményi TP, Silva R, Moura-Neto RS, et al. The association of ACE gene D/I polymorphism with cardiovascular risk factors in a population from Rio de Janeiro. Braz J Med Biol Res 2008;41:512-8.
40.	Liu M, Yi J, Tang W. Association between angiotensin converting enzyme gene polymorphism and essential hypertension: A systematic review and meta-analysis. J Renin Angiotensin Aldosterone Syst 2021;22:1470320321995074.
41.	Shanmuganathan R, Kumaresan R, Giri P. Prevalence of angiotensin converting enzyme (ACE) gene insertion/deletion polymorphism in South Indian population with hypertension and chronic kidney disease. J Postgrad Med 2015;61:230-4. [PUBMED] [Full text]
42.	Deepashree GA, Ramprasad E, Jayakumar M, Paul SF, Gnanasambandan R. ACE ID gene polymorphism contributes to chronic kidney disease progression but not NOS3 gene among type 2 diabetes with nephropathy patients. Endocr Metab Sci 2021;4:100100.