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Volume: 11 Issue: 1 February 2013


Influence of Endothelial Nitric Oxide Synthase Gene Polymorphisms (-786T/C, 4a4b, 894G/T) on Iranian Kidney Transplant Recipients

Objectives: Nitric oxide is a major mediator in vascular biology and regulator of regional blood flow. Its production is catalyzed by the enzyme endothelial nitric oxide synthase. Protective actions of nitric oxide in ischemia and reperfusion are due to its potential as an antioxidant and anti-inflammatory agent, along with its inhibitory effects on cell signaling pathways of nuclear proteins, such as NF-κB. The endothelial nitric oxide synthase gene polymorphisms affect endothelial nitric oxide synthase activity and are associated with endothelial dysfunction. This study sought to examine the association between single nucleotide polymorphisms in endothelial nitric oxide synthase gene (rs 2070744, 27VNTR, and rs1799983) and the development of acute rejection in renal transplant patients.

Materials and Methods: Sixty-six renal transplant recipients (33 patients with an episode of acute rejection and 33 recipients an episode of acute rejection), between June 2010 and March 2011, were included. The polymorphism was determined by simple polymerase chain reaction and polymerase chain reaction-restriction fragment-length polymorphism analysis.

Results: There was only a significant association of endothelial nitric oxide synthase -786T allele and acute rejection (P = .03). Recessive model of T-786C alleles (TT vs TC+CC) and acute rejection confirmed a significant association (odds ratio: 3.12; 95% CI: 0.01-9.83; P = .025). Haplotype CbG was higher in recipients without rejection as compared to rejection group (OR: 0.42, 95% CI: 0.16-1.13; P < .05). Respecting the endothelial nitric oxide synthase gene 894G/T single nucleotide polymorphisms and 27VNTR, no significant association between the allele/genotype and acute rejection was seen.

Conclusion: Recipient endothelial nitric oxide synthase gene polymorphisms do not alter the risk of acute rejection after a renal transplant. Rejection is a complex immunologic event. Therefore, finding associated genetic variants demands a multicentric larger sample size.

Key words : eNOS, Genetic, Haplotype, Renal, Allograft


Nitric oxide (NO) is synthesized from L-arginine, catalyzed by NO- synthase (NOS), and contributes to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. There are 3 different isoforms of NOS; endothelial constitutive NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Endothelial nitric oxide synthase diffuses from the endothelium to the vascular smooth muscle cells, increases the concentration of cGMP, and leads to vascular relaxation.1 It has an important role in regulating vascular tone,1,2 with protective effects by scavenging superoxide radicals. The nNOS produces NO in the central and peripheral nervous system and performs a role in cell communication. The iNOS (which is activated by interferon-gamma [IFN-γ]) is generated by monocytes, macrophages, and neutrophils, and affects the human immune system. In atherosclerosis, diabetes, or hypertension, the eNOS pathways are usually impaired. Ischemia and reperfusion (I/R) injury is a complex inflammatory phenomenon encountered during organ transplant; it induces delayed graft function, primary nonfunctioning graft, and organ loss. It also may cause induction of major histocompatability complex markers with an increase risk of acute and chronic rejection.3

Nitric oxide has protective effects in I/R injury by scavenging hydroxyl radicals and preventing accumulation of free radicals. Vascular smooth muscle relaxations with anti-apoptosis effect are other protective roles. It protects against I/R injury owing to its potential as an antioxidant.3,4 Inhaled NO after an orthotopic liver transplant significantly lowered hepatocyte apoptosis.5

The gene encoding eNOS maps to chromosome 7q35-7q366. Two eNOS polymorphisms, (-786)T/C and the 894G/T (Glu298Asp), have been shown to be associated with altered NOS activity in experimental studies.6,7

The 894T and -786C alleles of the NOS3 gene are significantly associated with both hypertension and cardiovascular disease in renal allograft recipients.8

Yilmaz and associates analyzed the G894T mutation at exon 7 of the eNOS gene among children with chronic allograft nephropathy. They concluded that this polymorphism did not affect long-term renal allograft outcome.9

This study sought to examine the prevalence of the eNOS gene (T-786C, 894 G/T) and a repeat polymorphism (27VNTR) in a group of renal transplant recipients and determine whether these polymorphisms are linked to acute rejection (AR).

Materials and Methods

Sixty-six renal transplant recipients (33 patents with an episode of AR and 33 recipients without an episode of AR), between June 2010 and March 2011, were enrolled. The Ethics Committee of Shiraz University of Medical Sciences approved the protocol, and a written informed consent was obtained from all subjects in accordance with the Helsinki Declaration of 1975. Patients were followed-up for at least 2 months, and episodes of AR were recorded during this time. An AR episode was defined based on clinical or biopsy findings according to Banff criteria.10,11 Clinical rejection was identified as an increase in the serum creatinine level that was ≥ 10% from the baseline value in the absence of infection, obstruction, or evidence of drug toxicity. The clinical characteristics were retrieved from our kidney transplant database. The routine immuno­suppression regimen consisted of cyclosporine or tacrolimus, with mycophenolate mofetil and prednisolone.

Single nucleotide polymorphisms selection
Single nucleotide polymorphisms in the eNOS gene selected for the present study, rs 2070744, 27VNTR, and rs1799983 located at the promoter, intron 4 and exon 7 of the eNOS gene (NOS3). The SNP ID numbers and detailed sequence information are recorded in the dbSNP database.12

Genetic analyses
For genotype analysis, genomic DNA was extracted from buffy coat with the use of DNP DNA isolation kit (Cinagene, Tehran, Iran). Two eNOS SNPs, (-786)T/C and 894G/T, were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously used by Ahluwalia and associates and Wilcox and associates.13,14 The 27VNTR is characterized by presence of either four 27-bp repeats (a allele) or five 27-bp repeats (b allele) and was genotyped using primers as previously used by Kim and associates. Details including primer sequences, PCR conditions, and restriction enzyme with product sizes are presented in Table 1 and Figures 1, 2 and 3.

Statistical analyses
Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 15.0, IBM Corporation, Armonk, NY, USA) and Epi Info Statcalc version 5. Discrete and continuous variables were compared between rejection and nonrejection using the Pearson chi-square test and t test as appropriate. Pearson chi-square test (3 × 2 contingency table) was used to assess the association of SNPs between rejection and nonrejection. The genotypes at their respective loci were coded as recessive (eg, eNOS-786CC vs -786TC + TT) and as dominant models (eg, eNOS -786CC + TC vs TT). Odds ratios and 95% confidence intervals (CIs) for relative risks were calculated. P values were subjected to the Bonferroni correction and considered significant when P value ≤ .05. Haplotype frequencies and Linkage disequilibria were estimated with Arlequin software (version 3.1). Fisher exact probability test was performed to determine significance and risk ratio of the haplotypes between the 2 groups.


In this study, we compared the eNOS T-786C polymorphism in a group of kidney allograft patients with and without AR. Patients included 46 men and 20 women (mean age, 31.35 + 10.2 y). The detailed data of patients’ demographic characteristics and transplant status are shown in Table 2. Statistical analyses of recipient demographic characteristics including donor and recipient age/sex, primary underlying kidney disease, and immunosuppressive regimen showed no differences between the ARs and non-ARs (P > .05). The majority of organs were donated from deceased donors.

Allele and genotype frequencies of the eNOS (-786) T/C and 894G/T SNPs and 27VNTR are given in Table 3. There is only a significant association of eNOS -786T allele and AR (P = .03). Further, recessive model of T-786C alleles and AR confirmed a significant association (odds ratio: 3.12; 95% CI: 0.01-9.83; P = .025) (Table 4).

Haplotype CbG was higher in recipients without rejection as compared to rejection group (OR: 0.42, 95% CI: 0.16-1.13; P < .05) (Table 5). No significant linkage disequilibria was observed among the polymorphisms (-786T/C, 27VNTR (a/b), 894G/T; P > .05).

Respecting genotyping and histologic grade, the distribution of different genotypes is present in Table 6. The major genotype in all groups was (-786T/T, 27VNTR (b/b), 894G/G). There was no association between histologic grade and any SNPs (P > .05).


Renal transplant is the treatment of choice for patients with end-stage renal disease. However, episodes of AR have a negative effect on long-term graft survival. The incidence of AR is higher especially the first month after surgery.16 Human leukocyte antigen (HLA) mismatch and antigen-independent factors such as I/R injury, drug toxicity, and infections are factors the contribute to the development of this event. Endothelial cells play an important role in the regulation of vascular remodeling, and NO is critical for the health of vascular endothelium and blood vessels. It is rapidly degraded to the stable end products (eg, nitrite and nitrate) that can be measured in serum and urine.17

Three subtypes of NOS (endothelial, neuronal, and inducible) are expressed in the renal tissue. Endothelial nitric oxide synthase is expressed in the renal vascular endothelium, including afferent and efferent arterioles and thick ascending loop of Henle. Neuronal NOS has been secreted in the juxtaglomerular apparatus, and inducible NOS expression is seen in settings of inflammation and in the inner medullary collecting duct.18-20

Nitric oxide inhibits platelet aggregation and leukocyte adhesion to vascular endothelium and also has antiproliferative effects on vascular smooth muscle cells.3, 4 There are reports about the role of NO in I/R and allograft rejection in liver, heart, kidney, and pancreatic islet cell transplant.21-27 Inhaled NO or NO donor drugs are novel treatments that have been used clinically to diminish I/R.

Intraoperative liposome-mediated gene delivery of eNOS into donor hearts before transplant reduces I/R injury by inhibiting NF-kappaB pathway and the early infiltration of leukocytes, all of which improve graft survival.26, 27 Ishimura and associates found that endothelial expression of eNOS after renal reperfusion is increased and recovery from renal ischemia with improved graft function is enhanced.28

Significant increase in serum NO levels also has been reported during episodes of rejection in renal transplant recipients.27,19,29 Infections and surgical stress had an important role in stimulating NO production after transplant, while drugs such as glucocorticoids or calcineurin inhibitors such as tacrolimus inhibit its production.25 Therefore, NO increases in response to various cytokines that are participate in rejection. The genetic variations in the endothelial nitric oxide synthase gene may influence NO levels and has effects on the inflammatory process. Nakayama and associates believed that the -786T to-C polymorphism is associated with significant variation in eNOS promoter activity.30 The 894G/T polymorphism also is associated with an altered protein sequence and functional effect on the eNOS protein.31 However, recent expression studies have demonstrated no functional difference between G894 (298Glu) and T894 (298Asp).32

Yilmaz and associates studied on G894T mutation at exon 7 of the eNOS gene and correlated it with chronic allograft nephropathy. This polymorphism did not influence long-term renal allograft outcome and is not consider a risk factor for chronic allograft failure.9 Viklický and associates compared the eNOS (G894T) gene polymorphism in patients with preserved graft function with control group and found no differences in allele and genotype distributions between them. There were no links between genotypes, renal function and atherosclerosis risk factors.33 Sezer and associates studied on angiotensin II type 1 receptor (ATR1) and eNOS gene polymorphism in renal transplant patients. They found that bb allele of the eNOS and nonAA allele of ATR1 1166 gene were associated with an anti-inflammatory state and may predict renal outcome in transplant patients.34

In present study, we found association of eNOS -786T allele and AR and higher prevalence of CbG haplotype in recipients without rejection. However, no significant association between each genotype and histologic grade of rejection was identified. Respecting other genotypes and keeping with published articles, we were unable to find any association between genetic findings and AR in kidney grafts. This finding may be due to small sample size that affects the strength of the data. Therefore, identifying associations of genetic variants with complex immunologic event, such as the rejection demand a larger sample size with multicentric investigation.


  1. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993;329(27):2002-2012.
    CrossRef - PubMed
  2. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43(2):109-142.
  3. Siriussawakul A, Zaky A, Lang JD. Role of nitric oxide in hepatic ischemia-reperfusion injury. World J Gastroenterol. 2010;16(48):6079-6086.
    CrossRef - PubMed
  4. Phillips L, Toledo AH, Lopez-Neblina F, Anaya-Prado R, Toledo-Pereyra LH. Nitric oxide mechanism of protection in ischemia and reperfusion injury. J Invest Surg. 2009;22(1):46-55.
    CrossRef - PubMed
  5. Effect of Nitric Oxide (N.O.) on Ischemic/Reperfusion Injury During Extended Donor Criteria (EDC) Liver Transplantation. Web site. Accessed May 1, 2012.
  6. Lembo G, De Luca N, Battagli C, et al. A common variant of endothelial nitric oxide synthase (Glu298Asp) is an independent risk factor for carotid atherosclerosis. Stroke. 2001;32(3):735-740.
    CrossRef - PubMed
  7. Colombo MG, Andreassi MG, Paradossi U, et al. Evidence for association of a common variant of the endothelial nitric oxide synthase gene (Glu298-->Asp polymorphism) to the presence, extent, and severity of coronary artery disease. Heart. 2002;87(6):525-528.
    CrossRef - PubMed
  8. Bhandary UV, Tse W, Yang B, Knowles MR, Demaine AG. Endothelial nitric oxide synthase polymorphisms are associated with hypertension and cardiovascular disease in renal transplantation. Nephrology (Carlton). 2008;13(4):348-355. Erratum in: Nephrology (Carlton). 2008;13(7):659. Bhandary, Uswanathan V [corrected to Bhandary, Umanath Vasanth].
    CrossRef - PubMed
  9. Yilmaz E, Mir S, Berdeli A. Endothelial nitric oxide synthase (eNOS) gene polymorphism in early term chronic allograft nephropathy. Transplant Proc. 2009;41(10):4361-4365.
    CrossRef - PubMed
  10. Serón D, Moreso F, Bover J, et al. Early protocol renal allograft biopsies and graft outcome. Kidney Int. 1997;51(1):310-316.
    CrossRef - PubMed
  11. Sis B, Mengel M, Haas M, et al. Banff ‘09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. Am J Transplant. 2010;10(3):464-471.
    CrossRef - PubMed
  12. dbSNP Short Genetic Variations. NCBI Web site. Accessed May 1, 2012.
  13. Ahluwalia TS, Ahuja M, Rai TS, et al. Endothelial nitric oxide synthase gene haplotypes and diabetic nephropathy among Asian Indians. Mol Cell Biochem. 2008;314(1-2):9-17.
    CrossRef - PubMed
  14. Wilcox JN, Subramanian RR, Sundell CL, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 1997;17(11):2479-2488.
    CrossRef - PubMed
  15. Kim IJ, Bae J, Lim SW, et al. Influence of endothelial nitric oxide synthase gene polymorphisms (-786T>C, 4a4b, 894G>T) in Korean patients with coronary artery disease. Thromb Res. 2007;119(5):579-585.
    CrossRef - PubMed
  16. Gjertson DW. Impact of delayed graft function and acute rejection on kidney graft survival. Clin Transpl. 2000:467-480.
  17. Tsikas D. Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids. Free Radic Res. 2005;39(8):797-815.
    CrossRef - PubMed
  18. MacMillan-Crow LA, Crow JP, Kerby JD, Beckman JS, Thompson JA. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci U S A. 1996;93(21):11853-11858.
    CrossRef - PubMed
  19. Chatterjee PK, Hawksworth GM, McLay JS. Cytokine-stimulated nitric oxide production in the human renal proximal tubule and its modulation by natriuretic peptides: A novel immunomodulatory mechanism? Exp Nephrol. 1999;7(5-6):438-448.
    CrossRef - PubMed
  20. Mattson DL, Wu F. Nitric oxide synthase activity and isoforms in rat renal vasculature. Hypertension. 2000;35(1 Pt 2):337-341.
    CrossRef - PubMed
  21. Kuo PC, Alfrey EJ, Abe KY, Huie P, Sibley RK, Dafoe DC. Cellular localization and effect of nitric oxide synthesis in a rat model of orthotopic liver transplantation. Transplantation. 1996;61(2):305-312.
    CrossRef - PubMed
  22. Stoica SC, Satchithananda DK, Atkinson C, Charman S, Goddard M, Large SR. Heat shock protein, inducible nitric oxide synthase and apoptotic markers in the acute phase of human cardiac transplantation. Eur J Cardiothorac Surg. 2003;24(6):932-939.
    CrossRef - PubMed
  23. Stevens RB, Ansite JD, Mills CD, et al. Nitric oxide mediates early dysfunction of rat and mouse islets after transplantation. Transplantation. 1996;61(12):1740-1749.
    CrossRef - PubMed
  24. Dedeoglu IO, Feld LG. Decreased urinary excretion of nitric oxide in acute rejection episodes in pediatric renal allograft recipients. Transplantation. 1996;62(12):1936-1938.
    CrossRef - PubMed
  25. Albrecht EW, van Goor H, Tiebosch AT, Moshage H, Tegzess AM, Stegeman CA. Nitric oxide production and nitric oxide synthase expression in acute human renal allograft rejection. Transplantation. 2000;70(11):1610-1616.
    CrossRef - PubMed
  26. Khanafer A, Ilham MA, Namagondlu GS, et al. Increased nitric oxide production during acute rejection in kidney transplantation: a useful marker to aid in the diagnosis of rejection. Transplantation. 2007;84(5):580-586.
    CrossRef - PubMed
  27. Bellos JK, Perrea DN, Theodoropoulou E, Vlachos I, Papachristodoulou A, Kostakis AI. Clinical correlation of nitric oxide levels with acute rejection in renal transplantation. Int Urol Nephrol. 2011;43(3):883-890.
    CrossRef - PubMed
  28. Ishimura T, Fujisawa M, Isotani S, Iijima K, Yoshikawa N, Kamidono S. Endothelial nitric oxide synthase expression in ischemia-reperfusion injury after living related-donor renal transplantation. Transpl Int. 2002;15(12):635-640.
    CrossRef - PubMed
  29. Joles JA, Vos IH, Gröne HJ, Rabelink TJ. Inducible nitric oxide synthase in renal transplantation. Kidney Int. 2002;61(3):872-875.
    CrossRef - PubMed
  30. Nakayama M, Yasue H, Yoshimura M, et al. T-786-->C mutation in the 5’-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999;99(22):2864-2870.
    CrossRef - PubMed
  31. Mount PF, Power DA. Nitric oxide in the kidney: functions and regulation of synthesis. Acta Physiol (Oxf). 2006;187(4):433-446.
    CrossRef - PubMed
  32. Fairchild TA, Fulton D, Fontana JT, Gratton JP, McCabe TJ, Sessa WC. Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)-->Asp variant of human endothelial nitric-oxide synthase. J Biol Chem. 2001;276(28):26674-26679.
    CrossRef - PubMed
  33. Viklický O, Hubácek JA, Vítko S, et al. G-protein beta-3-subunit and eNOS gene polymorphism in transplant recipients with long-term renal graft function. Kidney Blood Press Res. 2002;25(4):245-249.
    CrossRef - PubMed
  34. Sezer S, Uyar M, Akcay A, et al. Endothelial nitric oxide synthase and angiotensin II type 1 receptor gene polymorphisms can influence chronic inflammatory state in renal transplant patients. Transplant Proc. 2005;37(2):776-778.
    CrossRef - PubMed

Volume : 11
Issue : 1
Pages : 21 - 26
DOI : 10.6002/ect.2012.0040

PDF VIEW [265] KB.

From the 1Shiraz Transplant Research Center, 2Organ Transplant Center, Shiraz University of Medical Sciences, Shiraz, Iran
Acknowledgements: N.A., M.H.A., and B.G. participated in research design. All authors participated in the writing of the paper. M.H. participated in data analysis. We are also grateful to Transplant Research Center, Shiraz University of Medical Sciences, for funding and support this project. None of the authors have any conflicts of interest to declare.
Corresponding author: Negar Azarpira, MD, Organ Transplant Research Center, Zand Street, Nemazi Hospital, Postal Code Number: 7193711351, Shiraz University of Medical Sciences, Shiraz, Iran
Phone: +98 711 6276211
Fax: +98 711 627621