Endothelial nitric oxide synthase gene polymorphism in intron 4 affects the progression of renal failure in non-diabetic renal diseases

Ying Wang, Shuichi Kikuchi, Hiromichi Suzuki, Sohji Nagase and Akio Koyama

Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan

Correspondence and offprint requests to: Professor Akio Koyama MD, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1–1–1 Ten-nodai, Tsukuba, Ibaraki 305–8575, Japan.



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Nitric oxide is a very potent regulator of intrarenal haemodynamics and is thought to be an important factor in the deterioration of renal function. Our study sought to verify the hypothesis that endothelial nitric oxide synthase (ecNOS) gene polymorphism in intron 4 might have some relevance to progression in chronic renal failure.

Methods. We studied the frequencies of gene polymorphism of ecNOS intron 4 in patients with end-stage renal disease (302 cases) and compared it with that of healthy subjects (248 cases). ecNOS genotypes were determined by the polymerase chain reaction, followed by agarose gel electrophoresis.

Results. Two alleles of ecNOS intron 4, labelled a and b could be detected; a has four and b has five tandem 27-bp repeats. The frequencies of ecNOS4b/b, ecNOS4b/a, ecNOS4a/a genotypes were 81.0% (201/248), 19.0% (47/248), 0.0% (0/248) in the control group, and 74.8% (226/302), 23.5% (71/302), l.7% (5/302) in all the patients, 72.7% (168/231), 25.1% (58/231), 2.2% (5/231) in the group with end-stage renal diseases, excluding diabetic nephropathy (non-DM group), and 81.7% (58/71), 18.3% (13/71), 0.0% (0/71) in diabetic nephropathy (DM group) respectively. The frequency of the ecNOS4a (ecNOSb/a, and ecNOSa/a) in all the patients and in the non-DM group were significantly higher than that in the control group (P=0.021; P=0.0096 respectively). In contrast, there was no significant difference in the frequencies of ecNOS genotypes between the DM group and the control group (P=0.81).

Conclusion. Among the frequencies of ecNOS intron 4 gene polymorphism, a allele displayed a significantly higher frequency in cases with end-stage renal failure (ESRF) not caused by diabetic nephropathy. ecNOS gene polymorphism in intron 4 appears, therefore, to affect the progression of renal failure in non- diabetic renal diseases, but the same conclusion could not be drawn in diabetic nephropathy.

Keywords: end-stage renal failure; endothelial nitric oxide synthase; gene polymorphism; polymerase chain reaction



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Among the various factors involved in the deterioration of renal function, changes in haemodynamics are thought to be important. Nitric oxide (NO), an extensively studied endothelium-derived relaxing factor, is reported to be a very potent regulator of intrarenal haemodynamics [1,2]. NO is produced from L-arginine by nitric-oxide synthase (NOS). The endothelial NOS (ecNOS) is encoded by a gene located on chromosome 7q35–36 comprising 26 exons that span 21 kilobases (kb) [3,4] and expressed in endothelium. Recently, several studies have shown that the polymorphisms of ecNOS are related to the haemodynamics. Though the highly polymorphic (CA)n repeats in intron 13 and two biallelic markers in intron 18 have been shown to be not associated with essential hypertension [5], Wang et al. [6] showed that homozygosity for the ecNOS4a allele is a risk factor for coronary artery disease among smokers. Meanwhile they identified two alleles in ecNOS intron 4, the larger of which has five tandem 27-bp repeats [GAAGTCTAGACCTGCTGC(A/G) GGGGTGAG]. The first three repeats had A and the last two had G at the 19th base of the 27-bp repeats respectively. However, the smaller one has only four repeats, in which the first two repeats had A and the last two had G at the 19th base of the repeat respectively. They denoted these two alleles as ecNOS4a for four repeats and ecNOS4b for five repeats. Ichihara et al. [7] observed an association between the ecNOS4a allele and myocardial infarction in both a smoking and non-smoking Japanese population. As both coronary lesions and chronic renal failure are basically vascular disorders, we speculate that the gene polymorphism in ecNOS intron 4 might have some relevance to progression in chronic renal failure. To verify this hypothesis, we studied the frequencies of gene polymorphism of ecNOS intron 4 in patients with end-stage renal failure (ESRF), and compared it with that of healthy subjects.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
A total of 302 haemodialysis patients (183 men and 119 women with a mean age of 57.1±14.0 years) were entered in this study; 157 patients (93 men and 64 women) were from the dialysis centre of Tsukuba Gakuen Hospital (Ibaraki, Japan), and 145 patients (90 men and 55 womem) were from Mito Central Hospital (Ibaraki, Japan). In comparison, 248 genetically unrelated healthy Japanese subjects (179 men and 69 women) of mean age 49.3±8.8 years were studied as the control group. All subjects enrolled in the study gave their informed consent. Underlying diseases and other clinical features are shown in Table 1Go. We compared the frequencies of ecNOS gene polymorphism among all patients: the non-DM group (all cases except diabetic nephropathy, n=231), the DM group (underlying disease; diabetic nephropathy, n=71), the CGN group (underlying disease; chronic glomerulonephritis, n=133), and the healthy controls.


View this table:
[in this window]
[in a new window]
 
Table 1. Underlying diseases of haemodialysis patients and age of study groups
 
DNA was extracted from peripheral blood using a DNA extraction kit (Wako Pure Chemical, Osaka, Japan) and was stored at 4°C until analysis. ecNOS genotypes were determined by the polymerase chain reaction (PCR) as described by Wang et al. [6]. Briefly, the oligonucleotide primers (the forward primer was 5'-AGGCCCTATGGTAGTGCCTTT -3', and the reverse primer was 5'-TCTCTTAGTGCTGT GGTCAC-3') that flank the region of the 27-bp direct repeat in ecNOS intron 4 were used for DNA amplification. Each reaction mixture was heated to 94°C for 4 min for denaturation and underwent 35 cycles at 94°C for 1 min, finally annealing at 56°C for 1 min with an extension at 72°C for 2 min, and a final extension at 74°C for 7 min. The PCR products were analysed by 3% NuSieve 3:1 agarose gel electrophoresis (FMC BioProducts, Rockland, Maine, USA), and fragments were visualized by ethidium bromide staining and ultraviolet transillumination.

A chi-square test was used to test observed vs expected type frequencies, assuming the Hardy–Weinberg equilibrium. To correct for the contribution of age and sex, we performed multiple logistic regression analysis to explore the relationship between the ecNOS gene polymorphism and ESRF, and also for the effect of ecNOS intron 4 variant as a risk factor for ESRF. The analysis was performed by means of SPSS Advanced Statistics 6.1 for Macintosh (SPSS Japan Inc. Japan). The intron 4 genotypes were coded as dummy variables in which 0 stood for bb, 1 for ba or aa. The odds ratio and 95% confidence interval (CI) were also calculated. A P value of <0.05 was considered statistically significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
PCR analysis of genomic DNA generated fragments of 393 or 420 bp corresponding to the ecNOS4a and ecNOS4b alleles, respectively [6] (Figure 1Go). As few subjects were homozygous for ecNOSa, we considered subjects who were both homo- and heterozygous for ecNOSa for comparison with subjects having only the ecNOSb genotype. The frequencies of ecNOS4b/b, ecNOS4b/a, ecNOS4a/a were 81.0% (201/248), 19.0% (47/248), 0.0% (0/248) in the control group, 74.8% (226/302), 23.5% (71/302), 1.7% (5/302) in all the patients, 72.7% (168/231), 25.1% (58/231), 2.2% (5/23 1) in the non-DM group, 70.7% (94/133), 26.3% (35/133), 3.0% (4/133) in the CGN group and 81.7% (58/71), 18.3% (13/71), 0.0% (0/71) in the DM group respectively (Table 2Go). The genotype frequencies in controls were in agreement with those predicted by the Hardy–Weinberg equilibrium ({chi}2=0.046; df=1; P>0.05).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. Polymorphism of the 27-bp repeat in intron 4 of the ecNOS gene. The direct PCR product was separated by electrophoresis in 3% NuSieve 3:1 agarose gel and visualized by ethidium bromide staining. Lane M, pGEM DNA marker; lane 1, ab heterozygote with fragments of both 420 and 393 bp; lanes 2,3,5, bb homozygote with a fragment of 420 bp; and lane 4, aa homozygote with a fragment of 393 bp.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Frequencies of ecNOS genotype among patients and controls and results of multiple logistic regression analysis
 
The multiple logistic regression analysis for correcting the contribution of age and sex revealed that the frequency of the ecNOS4a (ecNOS4b/a and ecNOS4a/a) in all the patients, in the non-DM group and in the CGN group were significantly higher than that in the control group (P=0.021; P=0.0096; P=0.01 respectively). In contrast, there was no significant difference in the frequencies of ecNOS genotypes between the DM group and the control group (P=0.8 1).

The patients of the non-DM group and the DM group had been under the treatment with haemodialysis on an average of 10 and 5 years respectively when this study was performed. In order to examine the survival bias in analysing the relationship between ecNOS intron 4 gene polymorphism and the progression of chronic renal failure, we divided the non-diabetic patients into two groups, one of which had been receiving dialysis for under 10 years and the other for over 10 years, since the mean duration under treatment was 10 years. A similar division into two groups was made regarding diabetic nephropathy, where one group had been on treatment for under 5 years and the other for over 5 years, since the mean duration of haemodialysis was 5 years. We compared the two groups with regard to the frequencies of ecNOS intron 4 gene polymorphism (shown in Table 3Go) by means of the chi-square test. Statistical analysis revealed that there were no significant differences between either of the two groups (P=0.13 in non-diabetic renal diseases; P=0.63 in diabetic nephropathy).


View this table:
[in this window]
[in a new window]
 
Table 3. ecNOS genotype frequencies according to dialysis periods in non-DM and DM group
 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study the frequency of a allele in ecNOS intron 4 was significantly higher in patients from the non-DM group and the CGN group compared with healthy subjects indicating that the ecNOS 4a allele is a risk factor for ESRF in non-diabetic renal diseases. Our data are consistent with the results reported by Yokoyama et al. [8], who recently studied the distribution of ecNOS intron 4 gene polymorphism in 58 cases of patients with ESRF (underlying diseases were nephrosclerosis and chronic glomerulonephritis), and 136 healthy controls. The frequencies of the ecNOS4b/b, ecNOS4b/a, and ecNOS4a/a genotypes were 67.3, 31.0, and 1.7% in the patients and 82.4, 16.1, and 1.5% respectively in the controls in their study. In our study the frequencies of the ecNOS4b/b, ecNOS4b/a, and ecNOS4a/a genotypes were 72.7, 25.1 and 2.2% in non-diabetic renal diseases and 81.0, 19.0 and 0.0% respectively in controls. Both Yokoyama's and our study suggest that ecNOS gene polymorphism in intron 4 might have some relevance to the progression of ESRF in non-diabetic renal diseases. Many studies have been published on the relation between NO and renal failure. A decrease in NO production or an impaired response to NO may contribute to the initiation or maintenance of the increased intraglomerular high-pressure state. One recent study showed that the basal concentration of NO metabolites (nitrate plus nitrite) in the plasma was reduced in individuals with essential hypertension [9]. Tsukada et al. [10] recently reported a strong association between the a allele of the ecNOS gene and the plasma NOx (nitrate and nitrite) levels. The mean plasma NOx level of the subjects who were homozygous for the a allele was nearly 20% lower than in the subjects with the b allele. Although it is disputable whether the NO metabolites in blood are derived entirely from ecNOS in the endothelial cells of blood vessels, it is a fact that plasma NO metabolite levels are different depending on ecNOS gene polymorphism. They concluded, therefore, that the ecNOS gene locus might be responsible for variations in the genetic control of plasma NOx. The molecular mechanism by which ecNOS gene polymorphism acts to affect the progression of ESRF is not known, and it is also unclear whether this polymorphism is a causative variant or a marker of another functional variant. However, the fact that the distribution of the a allele in ecNOS intron 4 showed a significantly higher incidence in the patients with ESRF and plasma NO metabolite levels are reported to be different depending on ecNOS gene polymorphism, suggests that ecNOS gene polymorphism in intron 4 is a useful parameter for studying the relationship between NO and the progression of renal diseases.

In our study, the frequencies of ecNOS gene polymorphism showed no significant difference between the DM group and healthy subjects. The mechanisms behind diabetic nephropathy have not yet been fully elucidated and several hypotheses have been proposed, including impaired vasodilatory function. In fact, an abnormal renal vasomotor tone is present in the early stage of diabetes mellitus [11]. NO, a potent vascular regulator, has been extensively studied as a factor in the pathogenesis and progression of diabetic nephropathy. Several in vitro studies have concluded that diabetic animals have a decreased production and/or responsiveness to NO, and this decreased NO activity in diabetes may have relevance to the renal haemodynamic changes associated with this disease [12,13]. On the other hand, measurements made in vivo indicate an increase in endogenous NO production in diabetic rats [1416]. Overproduction of NO may play a significant role in the genesis of diabetic hyperfiltration and hyperperfusion [1417], which are related to glomerular hypertrophy with mesangial expansion and increased intracapillary pressure [18]. As for the role of the endothelium and NO in the control of renal haemodynamics in diabetic nephropathy, this remains disputable. In our study we failed to detect an association between ecNOS gene polymorphism and the progression of diabetic nephropathy.

This polymorphism is reportedly related to coronary heart diseases [6,7], and might affect other vascular diseases that are lethal. The subjects of the study have been dialysed for relatively long periods. Therefore we examined the survival bias. The frequencies of ecNOS intron 4 gene polymorphism in the long-dialysis period group did not differ from those in the short period group for both non-diabetes and diabetes. It appears, therefore, that neither death nor survival are factors in estimating the role of gene polymorphism in disease progression.

In conclusion, among the frequencies of ecNOS intron 4 gene polymorphism, the a allele showed a significantly higher incidence in the cases with ESRF except those caused by diabetic nephropathy. It would appear that ecNOS gene polymorphism in intron 4 affects the progression of renal failure in non-diabetic renal diseases, but the same conclusion could not be drawn in diabetic nephropathy and we cannot entirely exclude a possibility that ecNOS gene polymorphism might play a role in the progression of diabetic nephropathy. In diabetics, there might be several deteriorative factors besides this polymorphism.



   Acknowledgments
 
We are deeply grateful to Dr Burton D. Cohen for his valuable criticism in preparing the manuscript. This study was supported in part by the Scientific Research Funds of the Ministry of Education, Science and Culture of Japan, and in part by Japan–China Sasagawa Medical Scholarship Foundation. We are also most grateful to Dr H. Kikuchi in Tskuba Gakuen Hospital and Dr M. Narita in Mito Central Hospital for collecting blood samples for this study.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Zate R, De Nucci G. Effects of acute nitric oxide inhibition on rat glomerular microcirculation. Am J Physiol 1991; 261: F360–363[Abstract/Free Full Text]
  2. Tolins JP, Palmer RMJ, Moncada S, Raij L. Role of endothelium-derived relaxing factor in regulation of renal hemodynamic responses. Am J Physiol 1990; 258: H655–H662[Abstract/Free Full Text]
  3. Miyahara K, Kawamoto T, Sase K et al. Cloning and structural characterization of the human endothelial nitric-oxide-synthase gene. Eur J Biochem 1994; 233: 719–726
  4. Nadaud S, Bonnardeaux A, Lathrop M, Soubrier F. Gene structure polymorphism and mapping of the human endothelial nitric oxide synthase gene. Biochem Biophys Res Commun 1994; 198: 1027–1033[ISI][Medline]
  5. Bonnardeaux A, Nadaud S, Charru A, Jeunemaitre X, Corvol P, Soubrier F. Lack of evidence for linkage of the endothelial cell nitric oxide synthase gene to essential hypertension. Circulation 1995; 91: 96–102[Abstract/Free Full Text]
  6. Wang XL, Sim AS, Badenhop RF, Mccredie RM, Wilcken DEL. A smoking dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nature Med 1996; 2: 41–45[ISI][Medline]
  7. Ichihara S, Yamada Y, Fujimura T, Nakashima N, Yokota M. Association of a polymorphism of the endothelial constitutive nitric oxide synthase gene with myocardial infarction in the Japanese population. Am J Cardiol 1998; 81: 83–86[ISI][Medline]
  8. Yokoyama K, Tsukada T, Matsuoka H, Hara S, Yamada A, Kawaguchi Y. High accumulation of endothelial nitric oxide synthase (ecNOS): a gene polymorphism in patients with end-stage renal disease. Nephron 1998; 79: 360–361[ISI][Medline]
  9. Node K, Kitakaze M, Yoshikawa H, Kosaka H, Hori M. Reduced plasma concentrations of nitrogen oxide in individuals with essential hypertension. Hypertension 1997; 30: 405–408[Abstract/Free Full Text]
  10. Tsukada T, Yokoyama K, Arai T et al. Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun 1998; 245: 190–193[ISI][Medline]
  11. Miller JA, Floras JS, Zinman B, Skorecki KL, Logan AG. Abnormalities in the renal and vascular responses to LBNP in hummans with early diabetes. Am J Physiol 1994; 266: R442–450[Abstract/Free Full Text]
  12. Wang YX, Brooks DP, Edwards RM. Attenuated glomerular cGMP production and renal vasodilation in streptozotocin-induced diabetic rats. Am J Physiol 1993; 264: R952–956[Abstract/Free Full Text]
  13. Taylor PD, Mccarthy AL, Thomas CR, Poston L. Endothelium-dependent relaxation and noradrenaline sensitivity in mesenteric resistance arteries of streptozotocin-induced diabetic rats. Br J Pharmacol 1992; 107: 393–399[Abstract]
  14. Tolins JP, Shultz PJ, Raij L, Brown DM, Mauer SM. Abnormal renal hemodynamic response to reduced renal perfusion pressure in diabetic rats: role of NO. Am J Physiol 1993; 265: F886–895[Abstract/Free Full Text]
  15. Bank N, Aynedjian HS. Role of EDRF (nitric oxide) in diabetic renal hyperfiltration. Kidney Int 1993; 43: 1306–1312[ISI][Medline]
  16. Komers R, Allen TJ, Cooper ME. Role of endothelium-derived nitric oxide in the pathogenesis of the renal hemodynamic changes of experimental diabetes. Diabetes 1994; 43: 1190–1197[Abstract]
  17. Mattar AL, Fujihara CK, Ribeiro MO, De Nucci G, Zatz R. Renal effects of acute and chronic nitric oxide inhibition in experimental diabetes. Nephron 1996; 74: 136–143[ISI][Medline]
  18. Steffes MW, Østerby R, Chavers B, Mauer SM. Mesangial expansion as a central mechanism for loss of kidney function in diabetic patients. Diabetes 1989; 38: 1077–1081[Abstract]
Received for publication: 3.12.98
Accepted in revised form: 21. 5.99