Diabetic nephropathy is the most common cause of end-stage renal disease (ESRD). Genetic susceptibility plays an important role in the development and progression of diabetic nephropathy. Previous studies have revealed that polymorphisms in the SLC12A3 (solute carrier family 12 member [sodium/chloride] 3) gene, which encodes solute carrier family 12 member 3, might contribute to genetic susceptibility to diabetic nephropathy and essential hypertension. In this study, we examined whether the SLC12A3 gene locus is associated with ESRD resulting from diabetic nephropathy. We genotyped 11 common single nucleotide polymorphisms (SNPs) in the SLC12A3 gene in 177 patients with ESRD due to type 2 diabetes and 184 patients with diabetic retinopathy but with no signs of renal involvement. Three SNPs (g.34372G>A [Arg913Gln], g.39143G>A, and g.41727C>T) were found to be associated with ESRD due to diabetic nephropathy. These three SNPs were in complete linkage disequilibrium. Haplotype 4 in block 2 (18806C, 21822C, 34372A, 39143A, 39240T, 39375C, and 41727T) showed a significant association with ESRD due to type 2 diabetes (P = 0.0028). These results suggest that the SLC12A3 gene locus is associated with ESRD due to diabetic nephropathy.

Diabetic nephropathy is the most common cause of end-stage renal disease (ESRD) (1,2). Strong evidence has been provided by epidemiological (3) and familial (4,5) studies suggesting that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy; however, the causative genes remain elusive. The SLC12A3 (solute carrier family 12 member [sodium/chloride] 3) gene, at chromosome 16q13, which encodes a thiazide-sensitive sodium chloride cotransporter (MIM600968), has been shown to be a new candidate gene for diabetic nephropathy (6,7) and hypertension (8–10). It was shown that loss-of-function mutations in the SLC12A3 gene cause Gitelman’s syndrome, which is inherited as an autosomal-recessive trait and is characterized by low blood pressure due to renal sodium wasting, hypokalemia, metabolic alkalosis, hypocalciuria, and volume depletion (11). On the other hand, substitution of Arg913 to Gln in the SLC12A3 gene (Arg904Gln in the previous report), has been reported to predispose essential hypertension in the Swedish and Japanese (8,9). In addition, blood pressure reduction by thiazides was found to be dependent on genetic variations in the SLC12A3 gene (10). Recently, a genome-wide association study using single nucleotide polymorphisms (SNPs) showed that the gene encoding SLC12A3 (especially Arg913Gln) may contribute to genetic susceptibility to diabetic nephropathy in the Japanese (6). In this study, we investigated the associations between SLC12A3 polymorphisms and ESRD due to type 2 diabetic nephropathy in the Korean population.

We studied 361 patients with type 2 diabetes, comprising 177 patients with ESRD resulting from diabetic nephropathy (the ESRD group) and 184 patients with diabetic retinopathy and duration of diabetes of >15 years but without evidence of renal disease (the control group). The clinical characteristics of these groups are summarized in Table 1. Among the identified 52 polymorphisms in the SLC12A3 gene, as determined by sequencing of 48 Japanese DNA samples (12), we selected four SNPs (g.34372 G>A [Arg913Gln], g.39143 G>A, g.39240 C>T, and g.41727 C>T) that had been previously reported to be associated with diabetic nephropathy (6). We also selected an additional seven common SNPs based on frequency (frequency >0.05 in regulatory region, frequency >0.1 in introns) and linkage disequilibrium (LD) status (12). Two SNPs were in the promoter region, three in exons, and six in introns (Fig. 1A). Haplotype blocks were constructed by using LD patterns among the 11 SNPs genotyped. There was one break in the LD patterns; accordingly, two blocks were found in this study (Fig. 1C). We selected common haplotypes (frequency >0.05) from each block, which accounted for >95% of observed haplotypes (Fig. 1B). The genotype distributions of the 11 SNPs were in Hardy-Weinberg equilibrium.

Three individual polymorphisms (g.34372G>A [Arg913Gln], g.39143G>A, and g.41727C>T) of the 11 genotyped SNPs were found to be associated with ESRD caused by diabetic nephropathy (Table 2). Allele frequencies of g.34372G>A (Arg913Gln) and g.41727C>T were found to be significantly different between ESRD patients and control subjects after correction for multiple comparisons (corrected P = 0.033, odds ratio 2.30 [95% CI 1.32–4.00]; corrected P = 0.044, 2.20 [1.27–3.80], respectively; Table 2). These three SNPs were in complete LD (|D′| = 1) and were determinants of haplotype 4 in block 2 (Table 3). An analysis of the frequencies of common haplotypes from each block revealed a significant association between haplotype 4 (18806C, 21822C, 34372A, 39143A, 39240T, 39375C, and 41727T) in block 2 with ESRD due to diabetic nephropathy, even after Bonferroni correction (corrected P = 0.031; Table 3).

In this study, we found that genetic variations of the SLC12A3 gene are associated with ESRD resulting from diabetic nephropathy in the Korean population.

We found that three SNPs (g.34372G>A [Arg913Gln], g.39143G>A, and g.41727C>T) and haplotype 4 (18806C, 21822C, 34372A, 39143A, 39240T, 39375C, and 41727T) in block 2 were significantly associated with ESRD caused by diabetic nephropathy. The variants of three SNPs (34372A, 39143A, and 41727T) were in complete LD and were determinants of haplotype 4 in block 2 (Table 3). Association between haplotype 4 in block 2 and ESRD is more significant than each of three SNPs (g.34372G>A [Arg913Gln], g.39143G>A, and g.41727C>T).

Our results are consistent with the previous observation that the substitution of Arg913 to Gln in the SLC12A3 gene was reported to increase the risk of developing essential hypertension (8,9), considering elevated blood pressure is one of the major risk factors for the development and progression of diabetic nephropathy (13).

However, our results are quite opposite to Japanese data showing that the substitution of Arg913 to Gln is associated with reduced risk of development of diabetic nephropathy (6,7). Although overall frequencies of four SNPs (g.34372 G>A [Arg913Gln], g.39143 G>A, g.39240 C>T, and g.41727 C>T), which had been previously reported to be associated with diabetic nephropathy, in our control group (5, 7, 13, and 6%, respectively) were similar to those of Japanese (8, 9, 12, and 8%, respectively), there were significant differences in the nephropathy group between two studies (12 vs. 3%, 13 vs. 4%, 15 vs. 7%, and 12 vs. 3%, respectively) (6). In addition, the frequency of haplotype 4 in block 2 in the control group (5.4%) was also similar to that of Japanese (6.0%, haplotype 7 + haplotype 9), whereas it was different in the nephropathy group between two studies (11.9 vs 0.9%) (6). At this time, the explanation for the conflicting results between the two studies is unclear, although we selected patients with ESRD due to diabetic nephropathy for nephropathy cases, whereas patients with overt proteinuria and patients undergoing renal replacement therapy were included in the previous report (6).

The use of prevalent ESRD patients as cases may lead to strong survival bias. However, after dividing the patients with ESRD into quartiles by duration of ESRD, we could not find any association of SLC12A3 with the duration of ESRD (data not shown). Therefore, it is less likely that there is a spurious association due to survival bias between SLC12A3 and ESRD attributed to diabetic nephropathy.

At the moment, functional significance of substitution of Arg913 to Gln is unclear, although there was a suggestion that the Arg913Gln in the SLC12A3 gene might be a gain-of-function polymorphism and increase the risk of developing essential hypertension (9). Nevertheless, in this study, we found no association between Arg913Gln and blood pressure in either control subjects or ESRD cases (data not shown). Hence, further biological and/or functional evidence would be needed to confirm the suggestive association of SLC12A3 polymorphisms with ESRD resulting from diabetic nephropathy that has been reported in this study.

In conclusion, we found that SNPs and haplotypes of the SLC12A3 gene, especially Arg913Gln, are significantly associated with ESRD caused by diabetic nephropathy in the Korean population.

We studied 361 unrelated patients with type 2 diabetes comprising two groups according to the following criteria: 1) the control group (n = 184): patients with diabetic retinopathy and a duration of diabetes >15 years but with no sign of renal involvement, i.e., a urinary albumin-to-creatinine ratio <30 mg/g and with a creatinine clearance (using the Cockroft equation [14]) of >60 ml/min per m2; or 2) the ESRD group (n = 177): patients with diabetic retinopathy and ESRD due to type 2 diabetes, as indicated by a creatinine clearance rate of <15 ml/min per m2 or being under renal replacement therapy. We excluded subjects with ESRD with any one of the following criteria: 1) no retinopathy, 2) a consistent hematuria history before renal function deterioration, 3) no history of proteinuria before renal function deterioration, 4) reduced kidney size, and 5) evidence of other systemic or primary glomerular diseases. The patients with ESRD were recruited from four university hospitals and three dialysis clinics, and the control subjects were recruited from two hospital clinics from June 2003 to July 2004. All subjects enrolled in this study were ethnic Koreans. Type 2 diabetes was diagnosed using World Health Organization criteria (15). Subjects with positive GAD antibodies and an age at diagnosis of type 2 diabetes of <30 years, as well as those who started insulin treatment within 1 year of diagnosis and were ketosis prone, were excluded. The institutional review board of the Clinical Research Institute at Seoul National University Hospital approved the study protocol, and informed consent for genetic analysis was obtained from each subject.

Genotyping for SNPs in the SLC12A3 gene.

Since there is a close genetic relationship between Koreans and Japanese (16,17), we used Japanese sequencing data to select common polymorphisms in the SLC12A3 gene (12). We selected four SNPs (g.34372 G>A [Arg913Gln], g.39143 G>A, g.39240 C>T, and g.41727 C>T) that had been previously reported to be associated with diabetic nephropathy (6), and we also selected seven additional common SNPs based on frequency (frequency >0.05 in regulatory region, frequency >0.1 in introns) and LD status (r2 > 0.5) among the identified polymorphisms in the SLC12A3 gene, as determined by sequencing of 48 Japanese DNA samples (12) (Fig. 1A). The 11 polymorphisms were genotyped by fluorescence polarization detection or single-base extension (18). For genotyping polymorphic sites, amplifying primers and probes were designed for TaqMan (19). Primer sets for the amplification and sequencing analysis of SLC12A3 gene were designed based on GenBank sequences (ref. genome seq. NC_000016, released on 29 December 2004). Information regarding the primers used is available on our website (http://www.snp-genetics.com/reference/SLC12A3.doc). The TaqMan method and single-base extension both achieved a >98% success rate for genotype.

Statistics.

Differences in genotype frequencies were compared using the χ2 test. We used Bonferroni correction for the adjustment of multiple comparisons (11 independent tests in genotype and 11 independent tests in haplotype). For association analyses between SNPs in SLC12A3 and blood pressure, we adjusted for age, sex, and BMI using a general linear regression procedure. Haplotype structures were visualized by Haploview software (20). Haplotype frequencies and P values of haplotype associations were analyzed by the algorithm developed by Schaid et al. (haplo.score) (21). A P value <0.05 was considered statistically significant.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

C.A. and K.S.P. jointly supervised this project.

This work was supported by a grant from the Korean Health 21 R & D Project, Korean Ministry of Health & Welfare (00-PJ3-PG6-GN07-001).

We thank Drs. Sung Kyun Kim, Woo Kyung Jung, Ji-Eun Oh, Eung Taek Kang, Joong Geon Lee, and Jin Cheol Kim for help in recruiting study subjects.

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