Polymorphism of angiotensin-converting enzyme (rs4340) and diabetic nephropathy in Caucasians with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a multifactorial chronic metabolic disease characterized by post-prandial hyperglycemia that causes long-term macrovascular or microvascular complications. Microvascular complications are diabetic nephropathy (DN), neuropathy and diabetic retinopathy (DR) [1,2]. Diabetes mellitus (DM) is the most common cause of chronic kidney disease and end-stage renal disease [1,2]. In the pathogenesis of DN several environmental, genetic, and epigenetic factors are involved in complex interactions [3-5].

In DN, there is a major decrease in glomerular filtration rate (GFR) together with a rise in the excretion of proteins in urine [6]. The pathogenesis of DN is related to uncontrolled or chronic hyperglycemia and is characterized by hypertrophy of glomeruli, hyperperfusion, thickening of basement membranes and glomerular hyperfiltration. There is microalbuminuria and subsequently, progressive glomerulosclerosis, but tubulointerstitial fibrosis may occur, eventually leading to reduction in GFR [1,2].

In progression of DM and its complications, many risk factors are involved, e.g., hypertension, dyslipidemia, smoking, obesity, aging and insulin resistance [7,8]. Clinically, non pharmacological interventions such as strict glycemic and blood pressure control, decrease in smoking and in dietary protein intake, have been shown to slow the progression of DN. The most validated clinical strategy for slowing disease progression is therapeutic targeting of the renin-angiotensin aldesterone system (RAAS) [9,10].

Genetic polymorphisms of the RAAS system may affect the progression of DM and its complications, whereby angiotensinogen, angiotensin receptor and angiotensin-converting enzyme (ACE) gene polymorphisms have been implicated in the pathophysiology of DN [11]. Angiotensin-converting enzyme converts angiotensin I into active octapeptide angiotensin II, and inactivates bradykinin via the kalikrein-kininogen system [12].

The ACE insertion/deletion (I/D) gene polymorphism (rs4340) is a 287 bp sequence of DNA in intron 16 of the ACE gene on chromosome 17q23, whereas there are a few polymorphisms that are in a linkage disequilibrium (rs 4341, rs4646994) in some populations [13]. In adults, plasma ACE does not change with age and it is only influenced by environmental or lifestyle factors to a minor extent [15-17]. Compared with rs4340 insertion/insertion (I/I) homozygotes, circulating ACE levels in plasma were found to be nearly 30.0 and 60.0% higher in I/D heterozygotes and deletion/deletion (D/D), homozygotes, respectively [14]. A meta-analysis of studies on glomerulosclerosis reported that the overall frequency of the D allele was 54.0% [13]. Overall frequency of the D allele was unrelated to gender but there were ethnic differences [13].

The ACE I/D gene polymorphism, which correlates with circulating ACE concentration, may be implicated in the etiology of DN, but it has been poorly investigated while giving inconsistent results [18-20]. The present study was undertaken to evaluate the association between rs4340 of the ACE gene and DN in Caucasian patients with T2DM.

We enrolled 651 unrelated Caucasians with T2DM from outpatient clinics of the University Medical Centre Maribor and the General Hospitals in Murska Sobota and Slovenj Gradec, Slovenia. Two hundred and seventy-six patients also had DN (cases) and 375 subjects with T2DM of more than 10 years duration but no clinical signs of DN (controls) were enrolled in the study. Diagnosis of DN was made according to the World Health Organization 1999 diagnostic criteria [16].

We excluded patients with overt nephropathy, poor glycemic control, significant heart failure New York Heart Association classification II-IV (NYHA II-IV), alcoholism, infection and other causes of renal disease. The study was approved by the National Medical Ethics Committee and was performed in compliance with the Helsinki declaration. After informed consent for participation in the study was obtained from all patients, a detailed interview was made.

The demographic and clinical data of the case and control groups are shown in Table 1. There were no significant differences between the groups with respect to age, sex, duration of diabetes, diastolic blood pressure, body mass index (BMI), smoking status, duration of DR, serum hemoglobin (Hb), estimated glomerular filtration rate (eGFR), TG and total HDL, LDL and cholesterol levels. However, statistically significant differences were observed in the duration of hypertension, systolic blood pressure, Hb A_1c, fasting-serum glucose, serum urea, serum creatinine, and urine albumin/creatinine ratio.

Table 1

Clinical and laboratory characteristics of case and control groups

Differences in parameters reflecting renal function (serum creatinine, cystatin C, eGFR and urine albumin/ creatinine ratio) confirmed chronic kidney disease in the DN+ group. Cystatin C was a better marker for the assessment of renal function than eGFR (MDRD equation mL/ min.). Cystatin C was significantly higher in the DN+ group (p <0.001) than in the control group. Univariate analysis demonstrated a statistically significant difference in genotype distribution in rs4340 genotypes (Table 2).

Table 2

Distribution of the rs4340 genotypes and alleles in DN+ patients (cases) and in DN– controls.

We used logistic regression analysis to evaluate whether this single nucleotide polymorphism (SNP) was independently associated with DN after adjusting for duration of hypertension, systolic blood pressure, cardiovascular disease, DR, diabetic foot, Hb A_1c, serum fasting glucose, serum urea, serum creatinine, serum cystatin C, urine albumin/creatinine ratio (g/mol), and found no statistically significant association of rs4340 with DN (Table 3).

Table 3

Association of rs4340 polymorphisms with DN in Caucasians with logistic regression.

In our cross-sectional study we found no association between rs4340 of the ACE gene and DN in Caucasians with T2DM. Conflicting reports have been made in different populations on the association between the I/D polymorphism in the ACE gene (rs4340) and DN. Our findings are in accordance with the study in French subjects with T2DM, but they differ from most studies performed in Chinese, Japanese and Indian populations, as well as in two meta-analyses in Asian and Arab patients with T2DM [13,19,20,23,25-29].

Wide inter-ethnic differences have been reported for allele/genotype frequencies of the I/D polymorphism of the ACE gene (rs4340), and these differences have been assumed to be responsible for the contradictory results among association studies in subjects with DN [13,19,20,23-26]. An association between rs4340 and DN was demonstrated in Asian populations (Japan, South India) [20,23] but not in Iran [31] and Malaysia [32]. A meta-analysis has demonstrated that the ACE I/D gene polymorphism affected the development of DN, i.e., that the D allele was a risk factor for DN, and I allele was a protective factor against DN in T2DM in an Asian population [26,33]. Another meta-analysis [27] reported that ACE I/D gene polymorphism (rs4340) was associated with T2DM in an Arab population. Moreover, the D/D genotype (rs4340) was reported to be an independent risk factor for renal end points in Chinese patients with T2DM [24]. Wang et al. [34] have recently demonstrated an association between the D allele (rs4340) and glomerular filtration impairment in Chinese patients with T2DM.

There were only a few reports in Caucasians [25,28, 29]. In a large case-control study, which enrolled patients in France, Finland and Denmark, it was shown that the rs4340 was associated with DN in subjects with type 1 diabetes mellitus (T1DM) [29]. Additionally, the D allele (rs4340) was reported to be an independent risk factor for both the onset and the progression of DN in T1DM patients [30]. Moreover, Yu et al. [25] reported that the DD genotype (rs4340) was associated with the end-stage renal disease in DN patients with T2DM. Contrary to the association between rs4340 and DN in T1DM, Hadjadj et al. [28] were not able to demonstrate an association between the rs4340 and DN in French subjects with T2DM, whereas so far, there have been no reports of rs4341 and DN in Caucasians.

A limitation of our study was the use of cross-sectional data in the analyses, restricting the possibility of causal inferences from our data. Another limitation of the study was the sample size. The strength of our cross-sectional study was a rather large community-based sample of Caucasians with T2DM, and the detailed phenotypic characterization of the subjects with DN. In conclusion, rs4340 of the ACE gene was not demonstrated to be a potential genetic marker for DN in Caucasians with T2DM.