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,
Genetic polymorphisms of the RAAS system may affect the progression of DM and its complications, whereby angiotensinogen, angiotensin receptor and angiotensin-converting enzyme (
The ACE insertion/deletion (I/D) gene polymorphism (rs4340) is a 287 bp sequence of DNA in intron 16 of the
The
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.
Total serum cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), triglycerides (TGs), serum cystatin C, fasting-serum glucose, serum glycemic Hb (Hb A1c), serum urea, serum creatinine were determined by standard biochemical methods. Albumin-to-creatinine ratio was also determined for each patient in three urine samples, according to the diagnostic criteria. To assess the kidney function, we used the modification diet in renal disease (MDRD) equation and serum-cystatin C [21,22].
Genomic DNA was extracted from 100 µL of whole blood using a Qiagen isolation kit (Qiagen GmbH, Hilden, Germany) following the blood and body fluid spin »V3« protocol. The protocol was supported by five different reagents (QIAgene DNA Blood Mini Kit; Qiagen GmbH): buffer AL, 96.0% ethanol, buffer AW1, buffer AW2, buffer AE and appropriate amount of proteases (285 µL of proteases/200 µL of blood). From 200 µL of blood, 3-12 mg of genomic DNA,
The protocol for rs4340 polymorphism of the
Statistical analyses were conducted with the use of the Statistical Package for the Social Sciences program for Windows, version 20 (SPSS Inc., Chicago, IL, USA). Continuous clinical data were compared by unpaired Student’s
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 A1c, fasting-serum glucose, serum urea, serum creatinine, and urine albumin/creatinine ratio.
Clinical and laboratory characteristics of case and control groups The average value for Hb A1c. Comparison of men who are DN+ Comparison of men who are DN+ Comparison of men who are DN+ Comparison of men who are DN+ Comparison women who are DN+ Comparison women who are DN+ Comparison women who are DN+ Comparison of men who are DN+ Comparison of men who are DN+ Comparison of men who are DN+ Comparison of men who are DN+ Comparison women who are DN+ Comparison women who are DN+ Comparison women who are DN+ Comparison women who are DN+ DN+: cases; DN–: controls; T2DM: type 2 diabetes mellitus; Hb: hemoglobin; eGFR: estimated glomerular filtration rate; MDRD: modification diet in renal disease; HDL: high-density lipoproteins; LDL: low-density lipoproteins; TG: triglycerides. The values represent mean ± SD (standard deviation); statistically significant results are bold.Number 276 375 Gender (% males) 59.1 52.4 0.1 Age (years) 64.75 ± 9.15 63.75 ± 8.0 0.13 Duration of T2DM (years) 14.71 ± 7.97 14.60 ± 6.73 0.84 Duration of hypertension (years) 12.23 ± 9.88 10.52 ± 8.22 Systolic blood pressure (mmHg) 155.27 ± 18.92 149.84 ± 19.63 Diastolic blood pressure (mmHg) 84.87 ± 11.63 84.06 ± 11.42 0.36 Body mass index 31.30 ± 4.68 30.77 ± 5.00 0.23 Active smokers (%) 6.6 8.9 0.31 Serum Hb A1c (%) 7.98 ± 1.38 7.65 ± 1.14 Serum-fasting glucose (mmol/L) 9.03 ± 2.76 8.51 ± 2.53 Hb (g/dL) 13.94 ± 1.49 13.94 ± 1.29 0.99 Serum urea (mmol/L) 7.35 ± 3.73 6.26 ± 1.91 Serum creatinine (µmol/L): 93.13 ± 58.21 78.44 ± 20.15 Males 101.57 ± 61.84 84.28 ± 19.93 Females 79.70 ± 49.21 71.91 ± 18.35 eGFR (MDRD equation; mL/min.): 72.60 ± 19.74 75.22 ± 15.16 0.22 Males 71.97 ± 19.45 77.66 ± 14.33 Females 74.31 ± 20.72 72.45 ± 15.69 0.13 Serum cystatin C (mg/L) 0.95 ± 0.48 0.78 ± 0.21 Serum total cholesterol (mmol/L) 4.62 ± 1.17 4.55 ± 0.99 0.42 Serum HDL cholesterol (mmol/L) 1.23 ± 0.35 1.26 ± 0.36 0.29 Serum LDL cholesterol (mmol/L) 2.59 ± 0.95 2.57 ± 0.80 0.73 Serum TG cholesterol (mmol/L) 2.08 ± 1.60 1.83 ± 1.24 0.04 Urine albumin/creatinine ratio (g/mol); sample #1 27.49 ± 55.46 1.57 ± 3.05 Urine albumin/creatinine ratio (g/mol); sample #2 23.13 ± 39.34 1.60 ± 3.67 Urine albumin/creatinine ratio (g/mol); sample #3 23.36 ± 42.49 1.62 ± 2.49
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 (
Distribution of the rs4340 genotypes and alleles in DN+ patients (cases) and in DN– controls. PHWE: values were computed using Pearson’s goodness-of-fit χ2 (1 df) test.rs4340 DD genotype (%) 90 (32.8) 115 (30.7) ID genotype (%) 143 (51.7) 169 (45.0) 0.02 II genotype (%) 43 (25.5) 91 (24.3) D allele (%) 323 (58.5) 399 (53.2) 0.06 I allele (%) 229 (41.5) 351 (46.8) PHWE 0.3 0.07
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 A1c, 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).
Association of rs4340 polymorphisms with DN in Caucasians with logistic regression. OR: odds ratio; 95% CI: 95% confidence interval.rs4340 Codominant DD 90 (32.8%) 115 (30.7%) 1.03 (0.39-2.72)/0.9 0.80 (0.29-2.25)/0.7 ID 143 (51.7%) 169 (45.0%) 1.54 (0.61-3.90)/0.4 1.32 (0.49-3.53)/0.6 II 43 (15.5%) 91 (24.3) Reference Reference
In our cross-sectional study we found no association between rs4340 of the
Wide inter-ethnic differences have been reported for allele/genotype frequencies of the I/D polymorphism of the
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
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