Endothelial nitric oxide synthase gene intron 4 polymorphism in patients with end-stage renal disease
Monika Buraczynska,
Piotr Ksiazek,
Wojciech Zaluska,
Teresa Nowicka and
Andrzej Ksiazek
Laboratory for Molecular Diagnostics of Multifactorial Diseases, Department of Nephrology, University Medical School, Lublin, Poland
Correspondence and offprint requests to: Monika Buraczynska, Laboratory for Molecular Diagnostics of Multifactorial Diseases, Department of Nephrology, University Medical School, Dr K. Jaczewskiego 8, 20-954 Lublin, Poland. Email: mjbur{at}asklepios.am.lublin.pl
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Abstract
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Background. Nitric oxide (NO) synthesized by endothelial cell NO synthase (ecNOS) is a potent regulator of intrarenal haemodynamics. A polymorphism in intron 4 of the ecNOS gene is a candidate gene in cardiovascular and renal diseases. We investigated a potential involvement of this polymorphism in chronic renal failure.
Methods. We performed a case-control study involving 706 patients with end-stage renal disease (ESRD) and 321 healthy controls. All subjects were genotyped for the ecNOS4 polymorphism by the polymerase chain reaction followed by agarose gel electrophoresis.
Results. The analysis revealed that the frequencies of the ecNOS4 genotypes were significantly different in ESRD patients, both diabetic and non-diabetic, than in controls. In all dialysis patients for aa, ab and bb genotypes the frequencies were, respectively, 6.5, 35 and 58.5% in the patient group, and 1, 25 and 74% in control subjects. The a allele carriers (aa + ab) were more frequent among ESRD patients than in controls (OR 1.95; 95% CI 1.133.4; P = 0.0031). No significant association was found when hypertensive ESRD patients were compared with normotensive patients. The distribution of genotypes was similar in both subgroups (P = 0.21).
Conclusion. There was a significantly higher frequency of the ecNOS4a allele carriers among ESRD patients, both diabetic and non-diabetic, than in control subjects. This suggests that the ecNOS gene polymorphism may be associated with an increased risk of chronic renal failure.
Keywords: diabetic nephropathy; DNA polymorphism; endothelial nitric oxide synthase; end-stage renal disease; molecular genetics; polymerase chain reaction
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Introduction
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Nitric oxide (NO), an important endothelium derived relaxing factor, is synthesized in the vascular endothelium by the NO synthase. NO is a potent regulator of intrarenal haemodynamics [1]. At the release site it mediates local vasodilatation, antagonizes platelet aggregation and inhibits vascular smooth muscle cell proliferation [2]. In the kidney, NO dilates renal blood vessels and modulates renin secretion [3]. An impairment of NO production causes abnormalities in vascular function in many diseases including human hypertension and renal disease [4,5].
The endothelial constitutive NO synthase (ecNOS), which produces NO from L-arginine, is encoded by a gene located on chromosome 7q35-36, expressed in endothelium [6]. There are two alleles identified in intron 4 of the ecNOS gene. The larger allele, 4b, consists of five tandem 27-bp repeats and the smaller one, 4a, has four repeats [7]. An association of the 4a allele of the ecNOS gene with coronary heart disease and renal disease was reported [4,8,9]. Chronic renal failure is basically a vascular disorder and investigating ecNOS gene polymorphism might shed some light on the pathophysiology of renal disease and progression to end-stage renal disease (ESRD).
The present study attempted to examine a relevance of the ecNOS intron 4 polymorphism to a development and progression of chronic renal failure.
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Subjects and methods
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Patients
The total of 706 unrelated adult individuals on maintenance dialysis (654 haemodialysis and 52 peritoneal dialysis) were involved in this study. All patients were Caucasian of Polish origin. The cause of renal failure was established in 623 patients: glomerulonephritis (n = 239) and interstitial nephritis (n = 109) (biopsy proven in majority of patients), diabetic nephropathy (n = 119), polycystic kidney disease (n = 67), hypertensive nephropathy (n = 50), obstructive nephropathy (n = 29) and other causes (n = 10). From this study group 523 patients (74.2 %) were hypertensive (systolic blood pressure >140 mmHg and diastolic blood pressure >90 mmHg) and receiving antihypertensive treatment. Positive family history in first-degree relatives was reported by 147 patients (21%). The following parameters were measured: creatinine, urea, electrolytes and total cholesterol in serum by routine methods, at the onset of dialysis therapy. Healthy control subjects (n = 321) with no clinical signs of vascular or renal disease and no family history of renal disease were recruited among blood donors and hospital staff.
A written informed consent for genetic studies was obtained from all patients and subjects from the control group. The study protocol was evaluated and approved by the Ethics Committee at the University Medical School in Lublin.
Determination of ecNOS genotype
Genomic DNA was isolated from peripheral blood leukocytes using the method described by Madisen et al. [10] with minor modifications. For PCR amplification, two oligonucleotide primers were used that flank the region of the 27 bp repeat sequence in intron 4 of the ecNOS gene. The forward primer was 5'-AGGCCCTATGGTAGTGCCTTT-3' and the reverse primer was 5'-TCTCTTAGTGCTGTGGTCAC-3'. Genomic DNA (300 ng) was amplified in a final reaction volume of 50 µl, containing 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 µM each dNTP, 1 µM of each primer and 2 U Taq polymerase (all reagents from MBI Fermentas). The reaction mixture was heated to 94°C for 6 min for denaturation and then subjected to 35 cycles at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 2 min. Final extension was at 72°C for 7 min. The PCR products were analysed by electrophoresis in 2.5% agarose gels stained with ethidium bromide.
Statistical analysis
All calculations were performed using SPSS for Windows 5.0. Normally distributed data are presented as means±SD. Genotype distribution and allele frequencies were compared between groups using
2 test and Fisher's exact test, respectively. Student's t-test was used to compare mean times to ESRD between genotypes. Multiple logistic regression analysis was used to assess the relationship between ecNOS gene polymorphism and ESRD.
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Results
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We genotyped 706 patients with ESRD and 321 healthy controls for the ecNOS gene polymorphism. Clinical characteristics of the study group are summarized in Table 1. There were no significant differences between groups with respect to age, gender ratio and total serum cholesterol level. As expected from recruiting only healthy subjects for the control group, patients with ESRD had a higher prevalence of hypertension and diabetes mellitus.
The distribution of genotypes and allele frequencies were compared between patients and controls (Table 2). The genotype frequencies were in agreement with HardyWeinberg equilibrium. Dialysed patients had significantly higher frequency of the aa genotype and ecNOS4a allele (P<0.01) compared with control subjects. The multiple logistic regression analysis with correction for age and sex revealed that the frequency of the ecNOS4a allele carriers was significantly higher in dialysed patients than in healthy subjects (OR 1.95; 95% CI 1.133.4, P = 0.0031) (Table 2).
The genotype and allele frequencies were compared between non-diabetic renal disease patients (n = 587) and those with diabetic nephropathy (n = 119) (Table 3). The frequency of the ecNOS4a allele was higher in diabetic nephropathy patients compared with non-diabetic subjects by means of the
2 test (0.31 vs 0.23, P<0.05). Also, the frequency of the ecNOS4a carriers was higher in diabetic subgroup (51 vs 39%). Compared with the control group, the diabetic nephropathy patients had much higher frequency of the ecNOS4a allele carriers (odds ratio 2.91; 95% CI 1.24.1, P = 0.0017). Also, a higher frequency of carriers was observed in the non-diabetic group than in controls (OR 1.80; 95% CI 1.12.26).
When compared with two other most frequent in ESRD underlying renal diseases, chronic glomerulonephritis (n = 239) and interstitial nephritis (n = 109), diabetic nephropathy patients showed the highest frequency of the ecNOS4a allele (0.31 vs 0.23 and 0.22, respectively, P<0.05) as well as the highest frequency of the 4a allele carriers (51 vs 41 and 39.5%, respectively). All subgroups had a significantly higher frequency of the ecNOS4a allele and percentage of carriers compared with the control group (odds ratio values for diabetic nephropathy, chronic glomerulonephritis and interstitial nephritis were, respectively, OR 2.91; 95% CI 1.24.1, OR 1.93; 95% CI 1.32.4 and OR 1.80; 95% CI 0.92.3) (Table 3).
No significant association was found when hypertensive dialysis patients (n = 484) were compared with normotensive subjects (n = 222). The distribution of three genotypes was similar in both subgroups (P = 0.30) as was the percentage of the ecNOS4a allele carriers (P = 0.21) (data not shown).
To examine the survival bias in evaluating the effect of the ecNOS4 polymorphism on renal disease, we analysed allele and genotype frequencies in subjects shorter than 5 years on dialysis and those dialysed 5 years or longer (data not shown). There were no statistically significant differences between these two groups (P = 0.49), suggesting little or no influence of survival bias on the outcome of the study.
We compared clinical characteristics of carriers of the ecNOS 4a allele (aa + ab genotypes) and non-carriers (bb genotype) (Table 4). Statistically significant differences were observed in male/female ratios and number of subjects with a positive family history of renal disease. Time to ESRD (calculated from the diagnosis of renal disease to initiation of dialysis) was shorter for the carriers of the ecNOS 4a allele (7.2 vs 9.2 years in non-carriers) but the difference did not reach a statistical significance. Among the a allele carriers almost 30% reported family history of renal disease, compared with 15% in non-carriers (P<0.01).
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Discussion
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Chronic renal failure is a multifactorial disease with different prevalence and clinical phenotype in different populations. We examined the ecNOS gene polymorphism in intron 4 and found the frequency of the a allele similar to that observed in other European populations [11]. The frequency of the ecNOS 4a allele was in our study significantly higher in dialysed patients than in healthy control subjects. Similarly, the frequency of ecNOS 4a allele carriers was higher in the patient group compared with control subjects (OR 1.95, 95% CI 1.133.4, P = 0.0031). This indicates that the ecNOS 4a allele may be associated with a development of chronic renal failure. Such an association was observed earlier by others. Asakimori et al. [9] found a significantly higher frequency of the ecNOS 4a allele in haemodialysis patients, both non-diabetic and diabetic, and therefore suggested that the polymorphism in intron 4 of the ecNOS gene may have influence on the progression of renal disease. Wang et al. [7] in their study of 302 subjects with end-stage renal disease and 248 healthy controls found a significantly higher frequency of the ecNOS 4a allele in patients with ESRD caused by non-diabetic primary renal diseases. Freedman et al. [12] evaluated the role of four NOS gene polymorphisms in ESRD patients and found that the a allele of the ecNOS 4 polymorphism in the NOS3 gene was associated with all-cause ESRD in probands and their siblings compared with healthy subjects. Results of a study by Morita et al. [13] suggest that the ecNOS gene polymorphism in intron 4 may be involved in the progression of IgAN. Some other studies show contradictory results. Burg et al. [14] analysed a relationship of the ecNOS gene intron 4 polymorphism and the prevalence and course of primary glomerulonephritis in 116 Caucasian patients and found no evidence of the association of this polymorphism with the disease. No association was found between the ecNOS gene polymorphism and diabetic nephropathy in a study involving type 2 diabetic patients [15].
Our study examined large samples of patients and control subjects. Power calculations showed that for 706 ESRD patients and 321 healthy subjects, the study had a power of 93% at a significance level of 0.05 to support hypothesis that polymorphism of the ecNOS gene is associated with susceptibility to chronic renal failure.
There are reports suggesting that the ecNOS 4a allele may be one of the risk factors for diabetic nephropathy in type 1 [16] and type 2 [9,17] diabetes. Our study confirms this hypothesis. The frequency of the ecNOS 4a allele as well as that of carrier genotypes (aa + ab) was significantly higher in diabetic nephropathy patients than in those with non-diabetic renal disease and controls. Comparing clinical profiles of bb homozygotes with that of ecNOS 4a allele carriers, we observed significantly higher proportion of females in the carrier group. This finding requires further study, as we do not have any explanation for it at this point. Also, a family history of renal disease was significantly more frequent among carriers (
30 vs 15% for non-carriers), suggesting the role of ecNOS polymorphism in the disease transmission.
The mechanisms responsible for the association between the ecNOS gene intron 4 polymorphism and renal disease are not known. When we compared hypertensive dialysis patients with normotensive ones, no significant difference was found in the allele frequencies and genotype distribution. Therefore, the observed association cannot be due to hypertension in the ESRD population. It is possible that a defect in NO synthesis affecting NO levels may be involved. Tsukada et al. [18] reported that carriers of the ecNOS 4a allele have 20% lower plasma levels of the NO metabolites than non-carriers. The reduced production of NO by ecNOS may contribute to the progression of glomerular damage via systemic or intraglomerular hypertension. It was found that acute blockade of NO synthesis in mice results in systemic hypertension, renal vasoconstriction and substantial increase in glomerular capillary pressure [19]. Decreased NO production can thus play a role in the pathophysiological condition at the microvascular level and in the progression of glomerular damage leading to renal failure. Diabetic nephropathy is a complication resulting from several metabolic and genetic changes. The ecNOS gene polymorphism could be one of the genetic determinants aggravating diabetic nephropathy. The overproduction of NO is reported in the diabetic kidney and considered to be involved in haemodynamic abnormalities (glomerular hyperfiltration), thus increasing the risk of the progression of diabetic nephropathy. Changes in ecNOS activity and/or enhanced expression of ecNOS due to molecular variant of the ecNOS gene may induce preferential afferent arteriolar dilatation, glomerular enlargement and hyperfiltration [20]. Enhanced endothelial turnover and endothelial dysfunction are main changes observed in advanced diabetic nephropathy [21]. These mechanisms, however, need to be evaluated further for their role in the association of the ecNOS gene polymorphism with diabetic nephropathy.
Our study has some limitations. First, there were relatively small numbers of patients in subsets with different primary renal diseases. This could lead to either underestimating or overestimating the significance of the association of genotype with a disease. Secondly, our control group was not matched for age, although the age range for both groups is very similar. The male:female ratio is 1.35 for ESRD patients and 1.37 for controls. The control subjects were recruited from a low-risk background and all blood donors passed detailed health checkups.
In summary, the a allele of the ecNOS intron 4 gene polymorphism showed a significantly higher frequency in ESRD patients, both non-diabetic and diabetic. These results suggest that the ecNOS gene polymorphism can serve as a useful genetic marker for evaluation of susceptibility to chronic renal failure. However, the interactions between this genetic predisposition and environmental factors require further studies.
Conflict of interest statement. None declared.
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References
|
---|
- Tolins JP, Palmer RM, Moncada S, Raij L. Role of endothelium-derived relaxing factor in regulation of renal hemodynamic responses. Am J Physiol 1990; 258: 655662
- Schmidt HH, Walter U. NO at work. Cell 1994; 78: 919925[ISI][Medline]
- Kone BC, Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol 1997; 272: F561F578[ISI][Medline]
- Miyamoto Y, Saito Y, Kajiyama N et al. Endothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension 1998; 32: 38[Abstract/Free Full Text]
- Schmidt RJ, Baylis C. Total nitric oxide production is low in patients with chronic renal disease. Kidney Int 2000; 58: 12611266[CrossRef][ISI][Medline]
- 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: 10271033[CrossRef][ISI][Medline]
- Wang Y, Kikuchi S, Suzuki H, Nagase S, Koyama A. Endothelial nitric oxide synthase gene polymorphism in intron 4 affects the progression of renal failure in non-diabetic renal diseases. Nephrol Dial Transplant 1999; 14: 29892902
- Hibi K, Ishigami T, Tamura K et al. Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction. Hypertension 1998; 32: 521526[Abstract/Free Full Text]
- Asakimori Y, Yorioka N, Yamamoto I et al. Endothelial nitric oxide synthase intron 4 polymorphism influences the progression of renal disease. Nephron 2001; 89: 219223[CrossRef][ISI][Medline]
- Madisen L, Hoar LD, Holroyd CD, Crisp M, Hodes ME. DNA banking: the effect of storage of blood and isolated DNA on integrity of DNA. Am J Med Genet 1987; 27: 379390[ISI][Medline]
- Pulkkinen A, Viitanen L, Kareinen A, Lehto S, Vauhkonen I, Laakso M. Intron 4 polymorphism of the endothelial nitric oxide synthase gene is associated with elevated blood pressure in type 2 diabetic patients with coronary heart disease. J Mol Med 2000; 78: 372379[CrossRef][ISI][Medline]
- Freedman BI, Yu H, Anderson PJ, Roh BH, Rich SS, Bowden BW. Genetic analysis of nitric oxide and endothelin in end-stage renal disease. Nephrol Dial Transplant 2000; 15: 17941800[Abstract/Free Full Text]
- Morita T, Ito H, Suehiro T et al. Effect of a polymorphism of endothelial nitric oxide synthase gene in Japanese patients with IgA nephropathy. Clin Nephrol 1999; 52: 203209[ISI][Medline]
- Burg M, Menne J, Ostendorf T, Kliem V, Floege J. Gene-polymorphisms of angiotensin converting enzyme and endothelial nitric oxide synthase in patients with primary glomerulonephritis. Clin Nephrol 1997; 48: 205211[ISI][Medline]
- Fujita H, Narita T, Meguro H et al. Lack of association between an ecNOS gene polymorphism and diabetic nephropathy in type 2 diabetic patients with proliferative diabetic retinopathy. Horm Metab Res 2000; 32: 8083[ISI][Medline]
- Zanchi A, Moczulski DK, Hanna LS, Wantman M, Warram JH, Krolewski AS. Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric oxide synthase gene polymorphism. Kidney Int 2000; 57: 405413[CrossRef][ISI][Medline]
- Neugebauer S, Baba T, Watanabe T. Association of the nitric oxide synthase gene polymorphism with an increased risk for progression to diabetic nephropathy in type 2 diabetes. Diabetes 2000; 49: 500503[Abstract]
- 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: 190193[CrossRef][ISI][Medline]
- Zate R, De Nucci G. Effects of acute nitric oxide inhibition on rat glomerular microcirculation. Am J Physiol 1991; 261: F360F363[ISI][Medline]
- Sugimoto H, Shikata K, Matsuda M et al. Increased expression of endothelial cell nitric oxide synthase (ecNOS) in afferent and glomerular endothelial cells is involved in glomerular hyperfiltration of diabetic nephropathy. Diabetologia 1998; 41: 14261434[CrossRef][ISI][Medline]
- Lorenzi M, Cagliero E. Pathology of endothelial and other vascular cells in diabetes mellitus: call for data. Diabetes 1991; 40: 653659[Abstract]
Received for publication: 22. 4.03
Accepted in revised form: 28.11.03