An Additive Effect of Endothelial Nitric Oxide Synthase Gene Polymorphisms Contributes to the Severity of Atherosclerosis in Patients on Dialysis

AJH 2007; 20:758 –763 An Additive Effect of Endothelial Nitric Oxide Synthase Gene Polymorphisms Contributes to the Severity of Atherosclerosis in Patients on Dialysis Belinda Spoto, Francesco A. Benedetto, Alessandra Testa, Giovanni Tripepi, Francesca Mallamaci, Renke Maas, Rainer H. Boeger, and Carmine Zoccali Background: Reduced synthesis of nitric oxide (NO) is considered a major proatherogenic mechanism in patients with end-stage renal disease (ESRD), but genetic factors impinging on this mechanism have been little studied in this population. Methods: We tested the relationship between carotid intima–media thickness (IMT) and three endothelial NO synthase (eNOS) polymorphisms (G894T, T-786C, and 27-bp repeat in intron 4) in an ethnically and geographically homogeneous group of 147 patients with ESRD. Results: The IMT was significantly thicker (P ⫽ .01) in patients with the TT genotype (G894T polymorphism) than in patients with TG or GG genotypes, and a similar association was observed for the T-786C polymorphism (P ⫽ .02). These relationships remained statistically significant (P ⫽ .02 and .01), also in multivariate models including traditional and emerging risk factors for athero- sclerosis. Moreover, there was a direct association between the number of risk alleles and IMT (no risk allele: 0.97 ⫾ 0.22 mm, 1 risk allele: 1.03 ⫾ 0.20 mm, 2 risk alleles: 1.07 ⫾ 0.22 mm, ⱖ3 risk alleles: 1.23 ⫾ 0.36 mm, P ⬍ .001) that remained statistically significant in a multiple regression model. Conclusions: In patients on dialysis the risk alleles of G894T and T-786C polymorphisms of the eNOS gene are associated with carotid atherosclerosis. The additive effect of the two polymorphisms may contribute to the severity of atherosclerosis independently of other risk factors and of endogenous substances that influence the NO synthesis in this population. Am J Hypertens 2007;20:758 –763 © 2007 American Journal of Hypertension, Ltd. Key Words: Asymmetric dimethylarginine, carotid atherosclerosis, endothelial nitric oxide synthase, intima– media thickness, polymorphism. therosclerosis is a slow, progressive disease of the arterial system that originates in the innermost vascular layer, the endothelium. This disease is a partly heritable disorder, but the genes promoting atherogenesis are still largely unknown. Nitric oxide (NO) is considered as an important atheroprotective substance, and the gene that encodes the endothelial nitric oxide synthase (eNOS) enzyme is one of the most studied genes among those potentially related to atherosclerosis. Several polymorphic regions have been identified in this gene, but only three of them have been solidly associated with an increased risk of cardiovascular disease,1 namely, a single A nucleotide polymorphism (SNP) located in the 5=-flanking region of the gene (T-786C) associated with coronary spasm and carotid artery stenosis,2,3 a G894T polymorphism associated with a variety of atherosclerotic complications,1 and a polymorphism in intron 4, which was linked to coronary artery disease.4 Of note, it was reported that the T-786C polymorphism is associated with carotid plaque presence,5 as well as with mortality in nondiabetic Japanese patients on dialysis.6 Patients with end-stage renal disease (ESRD) have an exceedingly high prevalence of cardiovascular complications, and it is well known that traditional risk factors only Received December 21, 2006. First decision January 21, 2007. Accepted February 14, 2007. From the CNR-IBIM, Institute of Biomedicine, Clinical Epidemiology and Physiopathology of Renal Diseases and Hypertension & Division of Nephrology (BS, AT, GT, FM, CZ) and Cardiology Unit, Morelli Hospital (FAB), Reggio Calabria, Italy; and Clinical Pharmacology Unit, Department of Pharmacology, University Hospital Hamburg-Eppendorf (RM, RHB), Hamburg-Eppendorf, Germany. This study was supported by grants from the Regione Calabria. Address correspondence and reprint requests to Prof. Carmine Zoccali, CNR-IBIM, Istituto di Biomedicina, Epidemiologia Clinica e Fisiopatologia, delle Malattie Renali e dell’Ipertensione Arteriosa, c/o Ki Point—Gransial Srl, Via Filippini, 85, 89125 Reggio Calabria, Italy; e-mail: carmine. 0895-7061/07/$32.00 doi:10.1016/j.amjhyper.2007.02.009 © 2007 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc. AJH–July 2007–VOL. 20, NO. 7 ENDOTHELIAL NITRIC OXIDE SYNTHASE POLYMORPHISMS AND ATHEROSCLEROSIS in part explain the high cardiovascular (CV) morbidity and mortality in this population. We have recently reported a significant association between the eNOS G894T polymorphism and both intima–media thickness (IMT) in the carotid arteries7 and cardiovascular mortality in a group of patients on dialysis,8 suggesting that this polymorphism may be responsible for a genetically determined modulation of the atherosclerotic process in ESRD. Because the susceptibility to atherosclerosis is dictated by a polygenic mode of inheritance and by environmental exposures, considering the combination of eNOS gene variants, rather than a single polymorphism, may better our understanding of the genetic component of this disease. With this background in mind, we investigated whether an additive effect among the three eNOS polymorphisms exists and whether this effect is associated with vascular damage in a wellcharacterized series of ESRD patients. Methods The protocol was approved by the local ethics committee, and informed consent was obtained from each participant. Patients One hundred forty-seven (89 men and 58 women, all white) patients on dialysis (101 on hemodialysis [HD] and 46 on chronic ambulatory peritoneal dialysis [CAPD]), who had been on regular dialysis treatment (RDT) for at least 6 months and who were free of overt infections (fever, infected vascular access, peritonitis, or exit site infection), were recruited for the study. Hemodialysis patients were being treated three times a week with standard bicarbonate dialysis (Na 138 mmol/L, HCO3 5 mmol/L, K 1.5 mmol/L, Ca 1.25 mmol/L, Mg 0.75 mmol/L) either with cuprophan or semisynthetic membranes. The average urea Kt/V (fractional urea clearance) in these patients was 1.28 ⫾ 0.30. The remaining 46 patients were on CAPD (weekly Kt/V 1.66 ⫾ 0.33). All HD patients were virtually anuric (24-h urine volume ⬍200 mL/d), whereas a minority of CAPD patients (n ⫽ 6) had a 24-h diuresis ⬎200 mL/d. Twenty-two patients were diabetics and 70 were habitual smokers (22 ⫾ 18 cigarettes/d). Eighty-one patients were on antihypertensive drugs (54 on monotherapy with angiotensin-converting enzyme [ACE] inhibitors, angiotensin I [Ang-I] antagonists, calcium channel blockers, ␣- and ␤-blockers, and the remaining 27 on double or triple therapy with various combinations of these drugs). Seventy-one patients were on treatment with erythropoietin. Patient characteristics are detailed in Table 1. Carotid Ultrasonography Carotid IMT was measured by ultrasonographic (...truncated)