Relation Between Development of Nephropathy and the p22phox C242T and Receptor for Advanced Glycation End Product G1704T Gene Polymorphisms in Type 2 Diabetic Patients

The subjects were 691 unrelated Japanese patients diagnosed with type 2 diabetes according to World Health Organization criteria (11) who had been followed up on a regular basis at the outpatient clinic of Saitama Social Insurance Hospital since 1990. From among these subjects, we recruited all the patients who fulfilled the noted criteria (n = 181) and allocated them to one of two groups: group N, comprised of 108 patients without diabetic nephropathy, and group D, comprised of 73 patients with developing diabetic nephropathy. The stage of nephropathy was defined according to urinary excretion of albumin per gram of creatinine (urinary albumin index; UAI) and categorized as follows: normoalbuminuria, <20 mg/gCr, stage 0; microalbuminuria, 30–300 mg/gCr, stage 1; and macroalbuminuria, >300 mg/gCr, stage 2. Diabetic patients with UAI of 21–29 mg/gCr were assumed to be “borderline” and were therefore excluded from this study. Group N was defined using the following criteria: 1) >10 years after diagnosis of type 2 diabetes and 2) normoalbuminuria throughout the follow-up period. Group D was defined using the following criteria: 1) normoalbuminuria (stage 0) or microalbuminuria (stage 1) at the beginning of follow-up and 2) progression from normoalbuminuria (stage 0) to microalbuminuria (stage 1) or macroalbuminuria (stage 2), or progression from macroalbuminuria (stage 1) to macroalbuminuria (stage 2) during the follow-up period. To exclude patients with nondiabetic renal disease, patients with microscopic hematuria, clinical pyuria, or evidence of bacterial infection in a urine culture were excluded from this study. Diabetic retinopathy was diagnosed by independent diabetic ophthalmologists using standard fundus photographs at least every year; the stage of diabetic retinopathy was classified as simple, preproliferative, or proliferative retinopathy. HbA_1c levels, lipid profiles, blood pressure, and urinary excretion of albumin were measured at least every year. Clinical data, including onset age, BMI at the diagnosis of diabetes, disease duration since the diagnosis of type 2 diabetes, HbA_1c levels, blood pressure, lipid profiles, and the use of medication were obtained from the medical records of the patients. Written informed consent was obtained from each subject after full explanation of the purpose, nature, and risk of all procedures used. The protocol of this study was approved by the ethical review committee of the Saitama Social Insurance Hospital, Saitama, Japan.

Genomic DNA was isolated from peripheral blood leukocytes of the 181 patients, as previously described (12). The C242T transition polymorphism in the NADPH oxidase p22phox gene (GenBank M61107), exon 6 region, was determined by the TaqMan (Applied Biosystems, Tokyo, Japan) PCR method (13). The following primers and probes were included in the reaction: forward primer, 5′-TTC CTC CCT CCC CCA GG-3′; reverse primer, 5′-CCT GGT AAA GGG CCC GAA-3′; T allele−specific probe, 5′-Vic-ACA GAA GTA CAT GAC CG-3′; and C allele−specific probe, 5′-Fam-AGA AGC ACA TGA CCG-3′. The G1704T transversion polymorphism in the RAGE gene (GenBank D28769), intron region, was also determined by TaqMan PCR method. The following primers and probes were included in the reaction: forward primer, 5′-AGG ATG TGA GTG ACC TGG AGA GA-3′; reverse primer, 5′-TCA GCT CCT AGC CTG CCT TTC-3′; T allele−specific probe, 5′-Vic-CCA TAA CTA TCA ACA GGG-3′; and G allele−specific probe, 5′-Fam-CAT AAC TAG CAA CAG GG-3′. PCR was carried out using an ABI Prism 7700 (Applied Biosystems).

The sample size of this study was established on the basis of our pilot study. Regarding p22phox, our pilot studies indicated that the frequency of 242CT + TT would be 0.10 for Group D and 0.28 for Group N. It was calculated that the number of subjects needed to achieve 80% power to detect a difference between the two groups, with a significance level α (chance of a two-sided α error) of 0.05 was 59. Regarding RAGE, pilot studies indicated that the frequency of 1704GT + TT would be 0.30 for Group D and 0.13 for Group N. It was calculated that the number of subjects needed to achieve 80% power to detect a difference between the two groups was 72.

Data are expressed as means ± SD. The statistical significance of differences in mean values was analyzed by Student’s t test or the Mann-Whitney U test. The frequencies of various alleles and genotypes were compared between the groups by χ² or Fisher’s exact test, and multiple logistic regression analysis was performed to calculate the odds ratio and to evaluate the relation between the genotypes and other conventional risk factors. All statistical analyses were performed using Statview Software (version 5.0 for Windows; SAS Institute, Cary, NC).

Table 1 shows the clinical characteristics of the enrolled patients. Clinical data, including HbA_1c levels, lipid profiles, blood pressure, and the use of medication were obtained from the medical records, and retinopathy was assessed in 2001. There were no significant differences in age at onset, disease duration since the diagnosis of type 2 diabetes, BMI at onset, total or LDL cholesterol, or the use of medication between groups N and D, whereas blood pressure, HbA_1c levels, triglyceride levels, and the proportion of subjects with retinopathy were significantly higher in group D than in group N.

The genotype distribution of these genes in each group is shown in Table 2. The genotype frequencies in both groups were in Hardy-Weinberg equilibrium. The frequency of the CC, CT, and TT genotypes of the p22phox gene were 79, 20, and 1% in group N compared with 90, 8, and 2% in group D, respectively. The allelic frequencies of the C and T alleles were 95 and 5% in group D versus 89 and 11% in group N, respectively. Because the frequency of the TT genotype was low, we divided the enrolled subjects into two groups: CC and CT + TT. The frequency of the CT + TT genotypes was significantly higher in group N than in group D (χ² = 4.318, P = 0.0427; 2 × 2). In addition, multiple logistic regression analysis identified the HbA_1c level, systolic blood pressure, and triglyceride level as independent risk factors for the development of diabetic nephropathy (P < 0.005) within the following variables: age at onset, disease duration, BMI at onset, HbA_1c, total cholesterol, triglyceride, retinopathy, systolic blood pressure, and p22phox polymorphism; this was not the case for p22phox CT + TT (P = 0.0991) (data not shown). The frequencies of the GG, GT, and TT genotypes of the RAGE G1704T gene were 87, 12, and 1%, respectively, in group N compared with 74, 23, and 3% in group D. The allelic frequencies of the G and T alleles were 74 and 26% in group D versus 87 and 13% in group N, respectively. Because the frequency of TT was low, we divided the enrolled subjects into two groups: GG and GT + TT. The frequency of the GT + TT genotypes was significantly higher in group D than in group N (χ² = 4.987, P = 0.0313; 2 × 2). Multiple logistic regression analysis identified HbA_1c level and systolic blood pressure as independent risk factors for the development of diabetic nephropathy (P < 0.005), whereas this was not the case for RAGE GT + TT (P = 0.0522) (data not shown).

Next, we analyzed the relation between the development of diabetic nephropathy and the combination of these gene polymorphisms in the patients (Table 3). According to the results in Table 2, we assigned the combination of these genotypes to three levels of predicted risk of developing diabetic nephropathy: high-risk genotype (p22phox CC and RAGE GT + TT), intermediate-risk genotypes (p22phox CC and RAGE GG, p22phox CT + TT and RAGE GT + TT), and low-risk genotype (p22phox CT + TT and RAGE GG). The frequency of p22phox CC and RAGE GT + TT (high-risk genotype) in group D was significantly higher than that in group N (χ² = 10.338, P = 0.0057; 2 × 3). In multiple logistic regression analysis, HbA_1c level, triglyceride level, systolic blood pressure, and the combination of p22phox CC and RAGE GT + TT polymorphisms were shown to be independent variables (P < 0.005) (Table 4). These analyses revealed that the combination of these polymorphisms is a significant factor in the development of diabetic nephropathy.

NADPH oxidase is a critical enzyme for superoxide production in phagocytes, vascular smooth muscle cells, and mesangial cells (5). Recently, Guzik and colleagues (14,15) reported that superoxide production in human blood vessels from diabetic patients is mediated by upregulated NADPH oxidase activity through the PKC pathway. P22phox is an essential component of NADPH oxidase, and it has been reported that the C242T polymorphism is associated with vascular superoxide production in human blood vessels from CAD patients (5,6). The C242T polymorphism results in an amino acid polymorphism (His/Tyr) at residue 72, which is located in the putative heme-binding sites (16). Because the histidine residue is considered to be a candidate for the ligand of the heme prosthetic group of cytochrome b, it has been suggested that this polymorphism is directly associated with the function of p22phox (17). Inoue et al. (6) reported that the risk of CAD was lower in individuals carrying the 242T genotype in the Japanese population, whereas other investigations showed conflicting findings in patients with CAD and cerebrovascular disease (18–23). It has also been reported that this polymorphism is associated with significantly lower basal and NADPH-stimulated vascular superoxide production in human blood vessels from patients with atherosclerosis (14). Individuals with the 242T genotype might have lower oxidative stress as a result of lower O₂ production compared with individuals bearing the 242C genotype. In this study, we showed that patients with the 242T genotype have a lower risk of developing diabetic nephropathy. Our result was consistent with that of a previous study of NADPH oxidase function (14). The T allele frequency in control subjects in our study was 0.11, which was similar to that in previous studies of Japanese populations, whereas it was ∼33% of that in Caucasian populations (6,20,23).

AGEs by themselves have been shown to generate ROS and, conversely, the generation of AGEs is enhanced by oxidative stress (24). In vivo and in vitro studies indicate that AGEs play a role in the pathogenesis of diabetic nephropathy and the progression of renal failure (4). It has been reported that aminoguanidine and OPB-9195 are effective in preventing diabetic nephropathy (25,26). AGEs can bind to several binding sites, including RAGE. In RAGE-overexpressing mice, it has been demonstrated that the AGE-RAGE system is essential for the development of diabetic nephropathy (27). The AGE-RAGE interaction in mesangial and endothelial cells causes enhanced formation of oxygen radicals, with subsequent activation of NF-κB and release of pro-inflammatory cytokines and adhesion molecules, leading to collagen gene expression in mesangial cells (28). It has been reported that the A-374T polymorphism is associated with diabetic nephropathy in type 1 diabetic patients with poor metabolic control (4). The G1704T gene polymorphism has been reported to show a significant association with the complication of microvascular dermatoses and antioxidant status (8,9). Subjects bearing the RAGE 1704T polymorphism had significantly lower plasma levels of several antioxidants (total carotenoids, lutein, lycopene, and tocopherol) than those bearing RAGE 1704G, suggesting that this polymorphism may relate to oxidative stress and supporting our present results that patients with the 1704T genotype have a higher risk of developing diabetic nephropathy. The functional impact of the intron site is not clear, but possible mechanisms include alternative splicing, a change in mRNA stability, or a linkage of a nearby coding single nucleotide polymorphism.

We also examined the combination of the p22phox C242T and RAGE G1704T polymorphisms in relation to susceptibility to diabetic nephropathy. We showed that patients with the combination of p22phox CC and RAGE GT + TT genotypes had a significantly higher risk of diabetic nephropathy than those with the combination of p22phox CT + TT and RAGE GG genotypes. In this study, the RAGE G1704T gene polymorphism alone was weakly related to the susceptibility to diabetic nephropathy, but the combination of p22phox and RAGE gene polymorphisms was more significantly related to diabetic nephropathy. Both NADPH oxidase and RAGE are known to be expressed in mesangial cells, and recently, NADPH oxidase was reported to be a central target of RAGE; ROS generated by this mechanism could significantly impact cellular properties (10).

In conclusion, we propose that the combination of NADPH p22phox C242T and RAGE G1704T polymorphisms is associated with the development of diabetic nephropathy. It is obvious that microalbuminuria is the best documented predictor of the development of diabetic nephropathy (29). However, we propose that risk assessment based on gene polymorphisms, such as in this study, is also important. Further prospective studies are needed to clarify whether these polymorphisms can predict the development of diabetic nephropathy. Several studies have suggested that antioxidant treatment such as with ACE inhibitors (30), probucol (30), vitamin E (31), or α-lipoic acid (32) might be beneficial in preventing the development of diabetic nephropathy. It may be useful to identify the risk of the development of diabetic nephropathy and to select a medication such as an antioxidant.