High Accumulation of Endothelial Nitric Oxide Synthase (ecNOS):A Gene Polymorphism in Patients with End-Stage Renal Disease

Dear Sir,Among the various factors involved in the deterioration of renal function, changes in hemodynamics are thought to be important. Nitric oxide (NO) is an important factor that regulates renal hemodynamics and that is also known to modify inflammatory responses [1, 2]. Therefore, some type of gene polymorphism of NO synthase (NOS) might have some relevance to the progression of chronic renal diseases. If so, there may be a deviation in the specific gene polymorphism in patients with end-stage renal disease as compared with normal subjects. To test this hypothesis, we determined the distribution of gene polymorphism in endothelial nitric oxide synthase (ecNOS) in patients with end-stage renal disease (58 cases; underlying diseases: chronic glomerulonephritis and nephrosclerosis) in comparison with that of healthy subjects (n = 136).The demographic and clinical features of the subjects are shown in table 1. All subjects participated in the study after giving informed consent. To determine the ecNOS genotype of the patients, a genomic DNA fragment from peripheral blood was amplified by polymerase chain reaction. We used oligonucleotide primers that flank the region of the 27-bp direct repeat in intron 4 of the ecNOS gene. The forward primer was 5′-AGGCCCTATGGTAGTGCCTTT-3′, and the reverse primer was 5′-TCTCTTAGTG- CTGTGGTCAC-3′. Each reaction mixture was heated to 94°C for 5 min for denaturation, followed by 35 cycles at 94°C for 1 min, at 56°C for 1 min, and at 72°C for 2 min in a thermal cycler (PC-700; ASTEC, Fukuoka, Japan). The polymerase chain reaction products were separated by electrophoresis in 6% nondenaturing polyacrylamide gel. Fragments were detected by ethidium bromide. The band of 420 bp indicates five repeats (b allele) and the band of 393 bp four repeats (a allele) of 27 bp. We calculated the odds ratios for having ecNOSa genotype. Because there were only 3 subjects who were homozygous for ecNOSa, we considered both homo- and heterozygous subjects for ecNOSa as compared with subjects having only ecNOSb genotype. In calculating odds ratios, their 95% confidence intervals, and two-tailed p values, we used the maximum likelihood estimates and exact methods without mid-p correction. All analyses were performed by Epi-Info version 6.3 (Centers for Disease Control, Atlanta, Ga., USA).The frequencies of ecNOS4b/b, ecNOS4b/a, and ecNOS4a/a were 67.3% (39/58), 31.0% (18/58), and 1.7% (1/58) in the patients and 82.4% (112/136), 16.1% (22/136), and 1.5% (2/136) in the controls, respectively (table 2). The relative risk of chronic renal failure conferred by ecNOSa allele (ecNOSb/a and ecNOSa/a) was 2.27 (95% confidence interval 1.09–5.52, p = 0.018). Although it has also recently become clear that the a allele of ecNOS is a risk factor for coronary artery stenosis [3], there have been no previous reports on the effects of genetic polymorphism on renal failure. If a relative inhibition of NO production occurs in individuals with the a allele, it is possible that this may influence the progress of renal impairment. If this is the case, it might explain why the a allele showed a high incidence in the end-stage renal disease group. This point needs to be clarified in the future.In conclusion, there is a significant increase of a allele of ecNOS in end-stage renal disease patients. The results of this study show that both ecNOS gene polymorphisms may have some relevance to the prevalence of chronic renal diseases, thus indicating the possibility that these polymorphisms may be involved in the pathophysiology of the progression of chronic renal diseases.