NO ASSOCIATION OF GLUTATHIONE S-TRANSFERASE M1 GENE POLYMORPHISM WITH DIABETIC NEPHROPATHY IN JAPANESE TYPE 2 DIABETIC PATIENTS

Abstract

Oxidative stress possibly contributes to the development of diabetic nephropathy. Therefore, the levels of endogenous antioxidants may be one of determinants of the susceptibility to diabetic nephropathy. Glutathione S-transferases (GSTs) can work as one of endogenous antioxidants to protect cells from oxidative stress. The M1 member of GST mu class (GSTM1) is polymorphic and only expressed in 55–60% of Caucasians because of the homozygous deletion of the gene (null genotype). Recent studies have provided evidence that the GSTM1 null genotype, i.e. lack of the GSTM1 activity, is associated with an increased susceptibility to lung cancer and colorectal cancer. The present study was conducted to determine whether the genetic polymorphism influences the development of diabetic nephropathy. We examined 105 patients with diabetic nephropathy and 69 patients without diabetic nephropathy in Japanese type 2 diabetic patients with proliferative diabetic retinopathy. GSTM1 genotyping was performed by polymerase chain reaction. The two patient groups were well matched with regard to age, body mass index and HbA1c. GSTM1 null genotype was observed in 48.6% of patients with nephropathy versus 55.1% of patients without nephropathy. The frequency of GSTM1 null genotype was not significantly higher in the patient group with nephropathy than in the patient group without nephropathy. This study is the first to investigate the association of GSTM1 gene polymorphism with the development of diabetic nephropathy. The present results suggest that GSTM1 null genotype does not contribute to the development of diabetic nephropathy in Japanese type 2 diabetic patients.

• Diabetic nephropathy
• Proliferative diabetic retinopathy
• Type 2 diabetes
• Glutathione S-transferase
• Genetic polymorphism
• Oxidative stress

INTRODUCTION

Recently, hyperglycemia has been reported to cause oxidative stress in type 1 and type 2 diabetic patients [1-3]. The oxidative stress is considered to be one of the major causes in diabetic complications including nephropathy [4-5]. To protect cells from oxidative stress, there are various endogenous antioxidants. Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that play an important role in the cellular detoxification and excretion of numerous physiological and xenobiotic substances [6-8]. GSTs can also work as one of the antioxidants, through their ability to catalyze the conjugation of reduced glutathione with electrophilic compounds including carcinogens, cytotoxic drugs and organic hydroperoxides [6-8] and their glutathione peroxidase activity [9-11]. Therefore, diminished expression of GSTs may result in a reduction in the capacity of defense against the oxidative stress, followed by the development of diabetic nephropathy.

Mammalian cytosolic GSTs can be grouped into three major classes: alpha, mu, and pi based on amino acid sequence homologies, subunit assembly patterns [[12]]. In human kidney, GST mu class is mainly localized in tubuli [[13]]. Human GST mu class is thought to be products of M1, M2, M3, M4, and M5 gene loci [[8]], [14-15]. The M1 member of human GST mu class (GSTM1) is only expressed in 55–60% of Caucasians [16-17]. The genetic locus encoding human GSTM1 is polymorphic, and the absence of the GSTM1 has been ascribed to the homozygous deletion of the gene (null genotype) [[18]].

To determine whether the GSTM1 null genotype which leads to failure to express the GSTM1 is associated with the development of diabetic nephropathy, we investigated the genetic polymorphism in type 2 diabetic patients with nephropathy and those without nephropathy in the present study.

METHODS

Patients

We recruited 105 patients with diabetic nephropathy and 69 patients without diabetic nephropathy from Japanese type 2 diabetic patients with proliferative diabetic retinopathy. The diagnostic criteria of proliferative retinopathy was as follows: the growth of new capillaries and fibrous tissue within the retina and into the vitreous chamber; having received retinal photocoagulation therapy. The diagnostic criteria for diabetic nephropathy was the presence of persistent macroalbuminuria (urinary albumin excretion rate (AER) > 200 μg/min). The patients with normoalbuminuria (AER < 20 μg/min) were regarded as being without diabetic nephropathy. The patients with microalbuminuria (AER between 20 and 200 μg/min), i.e. incipient diabetic nephropathy, were not recruited in this study to assess the contribution of GSTM1 gene polymorphism to the established diabetic nephropathy. Moreover, the patients with urinary tract infection or other renal diseases were excluded from this study. Subjects whose casual blood pressure was > 140/90 mmHg or who were under antihypertensive treatment were defined as being with hypertension.

GSTM1 Genotyping

Genomic DNA was extracted from peripheral white blood cells using a blood DNA kit (QIAGEN, Hilden, Germany). GSTM1 genotyping was performed by polymerase chain reaction (PCR) using the primers from the protocol of Comstock et al. (forward: 5′-CTGCCCTACTTGATTGATGGG-3′ and reverse: 5′-CTGGATTGTAGCAGATCATGC-3′) [[19]]. To confirm that the PCR had worked in subjects homozygous for the GSTM1 gene deletion, the primers described by Frosst et al. (forward: 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ and reverse: 5′-AGGACG-GTGCGGTGAGAGTG-3′) [[20]] were added to amplify a 198-bp fragment of the methylenetetrahydrofolate reductase (MTHFR) gene. The PCR mixture (50 μl) contained 300 ng of genomic DNA, 10 pmol of each primer, 2.5 mM of each deoxynucleotide triphosphate, 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl (pH 8.3) and 2.5 units of Taq DNA polymerase (Takara, Otsu, Shiga, Japan). Amplification was performed in a thermal cycler (MJ Research, Watertown, MA) for 30 cycles with steps of denaturation at 94°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 1 min, followed by final extension at 72°C for 3 min. The PCR products were analyzed by electrophoresis in 2% agarose gels (FMC, Rockland, ME). Subjects with the GSTM1 gene showed a 273-bp fragment, whereas the 273-bp fragment was not identified in subjects homozygous for the GSTM1 gene deletion (Figure 1).

Figure 1. GSTM1 genotyping. Lane 1: DNA size marker (100-bp DNA Ladder); Lanes 2 and 3: GSTM1 null genotype; Lanes 4 and 5: GSTM1 positive genotype.

Statistical Analysis

Clinical characteristics were compared by Student's t test or Welch's t test after F test for the equality of variances, or Mann-Whitney U test, or chi-square test. Genotype frequency was compared by chi-square test. P value of < 0.05 was regarded as being statistically significant.

RESULTS

Table 1 shows clinical characteristics of subjects in this study. The two patient groups (69 without nephropathy, 105 with nephropathy) were well matched with regard to age, body mass index and HbA1c. As expected, hypertension was observed significantly more often in the patient group with nephropathy. Moreover, serum creatinine concentrations were significantly elevated in the patient group with nephropathy.

Table 1. Clinical Characteristics

	Without nephropathy (n = 69)	With nephropathy (n =105)
Gender (M/F)	32/37	62/43
Age (years)	62 ± 10	60 ± 12
BMI (kg/m^2)	22.8 ± 3.0	22.9 ± 4.9
HbA1c (%)	7.6 ± 2.0	7.8 ± 1.7
Serum creatinine (μmol/L)	62 (44–115)	106 (35–548)^*
Hypertension (n (%))	35 (50.7)	91 (86.7)^*

Data are means ± SD or median (range). BMI, body mass index.

^* p < 0.001.

The genotype frequency of GSTM1 gene is shown in Table 2. The homozygous deletion of the gene, i.e. null genotype, was observed in 48.6% of patients with nephropathy versus 55.1% of patients without nephropathy. The presence of the GSTM1 gene was identified in 51.4% of patients with nephropathy versus 44.9% of patients without nephropathy. The genotype frequency did not differ significantly between the two patient groups (p = 0.401).

Table 2. GSTM1 Genotype Frequency

	Without nephropathy (n =69)	With nephropathy (n =105)	p value
Positive	31 (44.9)	54 (51.4)
Null	38 (55.1)	51 (48.6)	p = 0.401

Data are n (%). ‘Positive’ indicates the presence of the GSTM1 gene. ‘Null’ indicates the homozygous deletion of the GSTM1 gene.

DISCUSSION

We believe that all the subjects of the present study were exposed to hyperglycemia for a long time, since they had proliferative diabetic retinopathy. Nevertheless, part of them did not develop diabetic nephropathy. This suggests that not only hyperglycemia but also genetic factors may contribute to the development of diabetic nephropathy.

Oxidative stress is known to contribute to the development of various pathological events, such as cancer, aging and atherosclerosis. Recently, it has also received considerable attention with respect to the contribution to the development of diabetic complications including nephropathy. To elucidate whether the oxidative stress plays any role in the development of diabetic complications, many researchers have examined the effect of antioxidant supplementation on diabetic complications. In diabetic animals, it has been demonstrated that the supplementation of antioxidants such as vitamin E and glutathione can prevent the development of diabetic complications [21-23]. A recent study has clearly demonstrated that vitamin E treatment can normalize retinal hemodynamic abnormalities and improve renal function in the early stages of type 1 diabetes, resulting in reducing the risks for the development of diabetic complications [[24]]. These lines of evidence suggest that the levels of endogenous antioxidants may be a determinant of the susceptibility to diabetic complications.

The level of GSTM1, one of endogenous antioxidant enzymes, is determined genetically [[18]]. Several studies have demonstrated that GSTM1 homozygous deletion (null) genotype, i.e. a reduced level of GSTM1 activity, is associated with an increased susceptibility to lung cancer [25-26] and colorectal cancer [[17]]. However, the association of this genetic polymorphism with diabetic complications had not been examined previously.

This study is the first to investigate the association of GSTM1 gene polymorphism with the development of diabetic nephropathy. Our results demonstrated that the frequency of GSTM1 null genotype was not significantly higher in the patient group with nephropathy than in the patient group without nephropathy. Therefore, we suggest that the GSTM1 null genotype does not contribute to the development of diabetic nephropathy. However, it has not been examined whether other genetic polymorphisms of GSTs are associated with diabetic nephropathy. Further investigations should be carried out to elucidate their contribution to the development of diabetic nephropathy.

ACKNOWLEDGMENT

We are grateful to Dr. Kyuji Kamoi, Nagaoka Red Cross Hospital; Dr. Takeshi Momotsu, Niigata Shimin Hospital; Dr. Akiko Tsuda, Kido Hospital; Dr. Mutsuko Hayashi, Shironekensei Hospital; and Dr. Yukio Yamada, Shinrakuen Hospital for support of this study.