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Division of Nephrology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan
a author for correspondence: fax 81-298-53-3202, e-mail sohji-n{at}md.tsukuba.ac.jp
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Introduction |
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 Top Introduction References |
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Several studies have shown that the polymorphisms of the eNOS gene are associated with hemodynamics. Homozygosity of the eNOS4 a allele has been shown to be a risk factor for coronary artery disease among smokers (3). We (4) and Yokoyama et al. (5) demonstrated that a polymorphism of the variable numbers of tandem repeats in intron 4 of the eNOS gene is associated with the progression of nondiabetic end stage renal disease (ESRD), but we failed to demonstrate the association between diabetic renal failure and the polymorphism. The genetic background for the progression of diabetic nephropathy to ESRD is still unclear. Furthermore, in some patients diabetic nephropathy does not show any association with conventional risk factors such as hyperglycemia (6). We speculate that there must be a genetic factor that causes the deterioration of diabetic nephropathy to ESRD. Recently, a Glu298Asp missense polymorphism within exon 7 of the eNOS gene was reported to be associated with essential hypertension, vasospastic angina, and myocardial infarction in a Japanese population (7)(8)(9). In this study, we explore the association between the Glu298Asp mutation and ESRD.
To evaluate whether the Glu298Asp mutation was associated with ESRD, we
examined 159 patients with ESRD undergoing maintenance hemodialysis (96
men and 63 women; mean age, 57.1 years; age range, 17–85 years) and
270 genetically unrelated, apparently healthy control subjects (195 men
and 75 women; mean age, 49.3 years; urine or blood examinations of the
subjects were normal). All patients and controls were Japanese.
Informed consent was obtained from each person enrolled in this study.
The underlying causes of end stage renal failure were chronic
glomerulonephritis (CGN; n = 68), diabetes mellitus (DM;
n = 48), hypertension (n = 17), polycystic kidney disease
(n = 13), lupus nephritis and vasculitis (n = 7), reflux and
obstructive nephropathy (n = 5), and interstitial nephritis
(n = 1; Table 1A
).
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Genomic DNA was extracted from peripheral mononuclear cells with a DNA extraction reagent set (Wako Pure Chemical) and was stored at 4 °C in Tris-EDTA buffer until analysis. To determine the genotypes of eNOS codon 298, we originally designed a set of primers (forward, 5-gaaacggtcgcttcgacgtgct; reverse, 5-ccacccagtcaatccctttg) that produced 140-bp amplified fragments. Approximately 100 ng of genomic DNA was amplified in a total volume of 25 µL containing 2.5 µL of Thermophilic DNA Polymerase Buffer (supplied with Taq polymerase; Promega), 3.0 mmol/L MgCl2, 200 µmol/L deoxynucleotide triphosphates, 0.5 µmol/L each primer, and 1 U of Taq polymerase. The PCR amplifications were 35 cycles performed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems) under the following conditions: denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min. Predenaturation was at 94 °C for 5 min, and final extension was for 7 min. PCR products were then digested by MboI, which produces two fragments 50 and 90 bp in length from alleles with glutamic acid (G) at codon 298 (G, wild type), and by BanII, which produces two fragments 55 and 85 bp in length from alleles with aspartic acid (T, mutant) at the same codon. The digested products were analyzed by 3% agarose gel electrophoresis.
The frequencies of homozygous or heterozygous Glu298Asp mutations (T/T + T/G) were 7.4% (20 of 270) in the control group, 22.0% (35 of 159) in all patients, 22.5% (25 of 111) in the nondiabetic group, 23.5% (16 of 68) in the CGN group, and 20.8% (10 of 48) in the diabetic group. The distribution of Glu298Asp genotypes in controls was in the Hardy-Weinberg equilibrium.
To correct for the contribution of age and sex, we also performed a multiple logistic regression analysis with the genotype at codon 298 of the eNOS gene as the dependent variable, and entered age, sex, and underlying disease (diabetic or nondiabetic renal disease) into the model. Statistical analyses were performed with SPSS, Ver. 6.1 for Macintosh.
Multiple logistic regression analysis revealed that the frequencies of
the Glu298Asp mutation in all patients, in the nondiabetic group, in
the CGN group, and in the diabetic group were significantly higher than
those in the control subjects (P = 0.0001, 0.0019,
0.0021, and 0.0117, respectively). Odds ratios were 2.8–3.2 (Table 1B
).
The patients enrolled in this study had been on hemodialysis for 9.2
(mean) years in the nondiabetic group, and 4.2 (mean) years in the
diabetic group. To evaluate the survival bias in analyzing the
relationship between the Glu298Asp mutation and progression to chronic
renal failure, we divided the patients into two groups according to the
duration of hemodialysis; among the diabetics, patients who had
experienced >5 years of hemodialysis were placed in the long-term
survival group, whereas patients with hemodialysis <5 years were
placed in the short-term group. A similar division into two groups was
made among the nondiabetic patients: the long-term survival group
(hemodialysis 
10 years) and the short-term group (hemodialysis <10
years). The two groups were compared with regard to the frequencies of
the Glu298Asp mutation by the 
2 test.
Statistical analysis revealed that there were no significant
differences between of the two groups.
In the present study, the Glu298Asp mutation of the eNOS gene was
increased in patients with ESRD (22.0%), whose underlying causes of
chronic renal failure included not only nondiabetic renal diseases
(22.5%) but also DM (20.8%), compared with controls (7.4%; Fig. 1
). The results demonstrate that patients with renal diseases,
including DM, who have the Glu298Asp mutation are at higher risk for
ESRD. This is the first report, as far as we can tell, that refers to
the significance of the Glu298Asp mutation in patients with renal
diseases.
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We could not evaluate the influence of hypertension because most of the patients had received antihypertensive drugs by the time dialysis was initiated. Presence of a family history of hypertension was not significantly different according to the genotype. In previous reports, the relative risk for the Glu298Asp variant was 1.5 in myocardial infarction (9), 2.3 in essential hypertension (7), and 2.73 in coronary spasm (8). The odds ratio in our study [3.2 (CI, 1.8–5.8)] indicates a similarly strong association between ESRD and the Glu298Asp variant, perhaps independent of hypertension or other disorders.
We also evaluated the effects of the Glu298Asp mutation on mortality. Although the frequency of the Glu298Asp mutant in the diabetic long-term survival group was as low as 14.3%, we could not find a statistically significant difference in the frequencies of the Glu298Asp mutation between short-term survival groups and long-term survival groups in both diabetic and nondiabetic patients. Our results may indicate that the Glu298Asp mutation is not a lethal factor but a risk factor for ESRD, although its association with relatively lethal diseases such as myocardial infarction has been reported. Further examination will be necessary.
We did not measure the enzyme activity of the variant. Yoshimura et al.
(8) suggested that according to a computer analysis, the
Glu298Asp mutation might affect enzyme activity because glutamic acid
at codon 298 is in the middle of an 
helix and the mutation produced
a tight turn. Enzyme activities of the polymorphic variants should be
measured to confirm the hypothesis that the polymorphism is responsible
for the progression of renal diseases through decreased endothelial NO
production. However, to date there have been no reports that confirm
the diminished eNOS activity in vivo or in vitro. Tsukada et al.
(10) reported that plasma NOx (nitrate + nitrite)
concentrations of subjects with the eNOS4 a allele of the
variable number of tandem repeats region in intron 4 of the eNOS gene
were significantly lower than those without the a allele.
Although a decrease in serum NOx according to eNOS gene polymorphisms
is suggestive, it is still controversial because serum NOx can vary
according to renal function or dietary intake of nitrite or nitrate
(11). Measurement of the enzyme activity in vivo or in vitro
will solve the controversy.
In conclusion, Glu298Asp mutations of the eNOS gene are significantly increased not only in nondiabetic ESRD but also in ESRD associated with diabetic nephropathy. The Glu298Asp mutation may be a risk factor, but additional studies, such as in vitro measurement of eNOS activity using cultured endothelium, are required.
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Acknowledgments |
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References |
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TopIntroductionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  B. I. Freedman, M. Bostrom, P. Daeihagh, and D. W. Bowden Genetic Factors in Diabetic Nephropathy Clin. J. Am. Soc. Nephrol., November 1, 2007; 2(6): 1306 - 1316. [Abstract] [Full Text] [PDF]
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 Y. Liu, K. P. Burdon, C. D. Langefeld, S. R. Beck, L. E. Wagenknecht, S. S. Rich, D. W. Bowden, and B. I. Freedman T-786C Polymorphism of the Endothelial Nitric Oxide Synthase Gene Is Associated with Albuminuria in the Diabetes Heart Study J. Am. Soc. Nephrol., April 1, 2005; 16(4): 1085 - 1090. [Abstract] [Full Text] [PDF]
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, TG + TT; bars, SD; *, P
<0.01.