N E P H R O L O G Y – O R I G I N A L P A P E R
Endothelial nitric oxide synthase gene intron 4
polymorphism predicts new onset diabetes mellitus
after transplantation in kidney allograft recipients treated
with cyclosporin A
Ihsan Ergu¨n• Kenan Keven•Sule Sengu¨l•
Halil Gu¨rhan Karabulut•Ilhan Kurultak•
Zeki Soypacaci• Bu¨lent Erbay
Received: 13 August 2009 / Accepted: 1 June 2010 / Published online: 18 June 2010 Ó Springer Science+Business Media, B.V. 2010
Abstract
Background Nitric oxide (NO), synthesized from
LS-arginine by the enzyme endothelial nitric oxide
synthase (eNOS), is a potent vasodilator and has been implicated in mediating insulin-induced uptake and metabolism of glucose in skeletal muscle. Polymor-phisms of the eNOS gene have been associated with altered eNOS activity and NO levels. Although several factors have been demonstrated for new onset diabetes mellitus after transplantation (NODAT), determining a genetic susceptibility for all patients requires further study. In our study, we evaluated the relationship between eNOS gene intron 4 polymor-phism and NODAT in kidney allograft recipients.
Methods A total of 82 consecutive patients who received their first kidney transplantation and main-tained graft function for at least a 12-month post-transplant period and who used triple therapy including cyclosporin A (CsA) for maintenance immunosup-pression were included. PCR–RFLP was used for genetic analyses.
Results Nine of 82 patients (11%) developed NODAT. Concerning the prevalence of eNOS intron 4 gene polymorphism, a significantly higher percentage of 4a allele carriers developed NODAT than non-carriers [6/26 (23.1%) versus 3/56 (5.4%), P = 0.02]. Compared with non-diabetics, NODAT patients were older (P = 0.04), had higher rate of hepatitis C (P \ 0.05) and higher body mass index at the time of transplantation (P = 0.03). In regression analyses, having a 4a allele of the eNOS gene intron 4 polymorphism (P = 0.02) and HCV seropositivity (P = 0.03) were found to be inde-pendent risk factors for the development of NODAT.
Conclusions These findings suggest that carrying a 4a allele of the eNOS gene intron 4 polymorphism is associated with NODAT. This may help us to further understand the individual risk for development of NODAT in kidney allograft recipients under CsA treatment.
Keywords Diabetes mellitus Endothelial nitric oxide synthase gene Hepatitis c virus Intron 4 Nitric oxide Notad Ptdm Renal transplantation I. Ergu¨n K. Keven S. Sengu¨l I. Kurultak
Z. Soypacaci B. Erbay
Department of Nephrology, Ankara University School of Medicine, Ankara, Turkey
H. G. Karabulut
Department of Genetics, Ankara University School of Medicine, Ankara, Turkey
I. Ergu¨n (&)
Department of Nephrology, Dr. Rıdvan Ege Hospital, Ufuk University School of Medicine, Balgat, 06100 Ankara, Turkey
e-mail: ihsanerg@yahoo.com; ihsanerg@gmail.com DOI 10.1007/s11255-010-9786-8
Introduction
New onset diabetes mellitus after transplantation (NO-DAT) is a well-known complication in solid organ transplantation recipients and has been documented to occur in 4–25% of kidney allograft recipients [1]. Similar to non-transplant population, NODAT has been reported to be associated with an increased incidence of cardiovascular and infectious complications after kidney transplantation. Moreover, NODAT seems to be an important factor for allograft survival [2]. Recently, Kasiske et al. [3] determined the following as risk factors for NODAT: the type of initial mainte-nance immunosuppression, race, ethnicity, obesity and hepatitis C infection. The incidence of NODAT at 6 months post-transplant was lower with cyclosporin A-microemulsion (CsA-ME) than with tacrolimus [4].
Nitric oxide (NO), synthesized fromL-arginine by
the enzyme eNOS in the vascular endothelium, plays an important role in the regulation of vascular tone, vascular biology and hemostasis. In addition to its action as a vasodilator, NO increases muscle blood flow, facilitates skeletal muscle glucose uptake and aids its subsequent metabolism [5,6].
Duplain et al. showed that eNOS -/- mice had a 60% lower concentration of plasma NO levels and a 40% lower insulin-stimulated glucose uptake than control mice. They concluded that insulin resistance in eNOS -/- mice was related specifically to impaired NO synthesis [7]. Furthermore, in euglycemic hyper-insulinemic clamp studies, decreased glucose infusion and glucose turnover rates were shown in eNOS -/-mice [8]. These findings indicate that eNOS plays a major role in the regulation of insulin sensitivity.
In humans, a gene located on chromosome 7q35-36, which comprises 26 exons spanning 21 kb, encodes the eNOS. Recently, an eNOS gene polymorphism, a variable number tandem repeat (VNTR) (4 or 5 repeats of 27-bp, respectively) in intron 4 (called eNOS 4a4b) has been shown to be associated with altered plasma nitrite/nitrate levels. Plasma NO levels were signifi-cantly lower in the 4a allele carriers [9].
In this study, we aimed to investigate the potential relationship between intron 4 polymorphism of the eNOS gene and NODAT in a single-center popula-tion of renal transplant recipients treated with triple immunosuppressive protocol including CsA, predniso-lone and mycophenolate mofetil/mycophenolic acid (MMF/MPA).
Patients and methods
This was a cross-sectional study including 126 adult kidney allograft recipients ([18 years) who received their first kidney transplantations between February 2002 and March 2006 in our center. The incidence of NODAT has been shown to be higher in transplant recipients treated with tacrolimus than in those treated with CsA [4] and in our center most of the patients received CsA-based immunosuppression. For this reasons, patients under tacrolimus-based immunosuppression were excluded. Patients under CsA-based triple immunosuppression (predniso-lone ? MMF/MPA) were included. Patients under non-CsA-based immunosuppression (28 patients), those with failed graft (creatinine [ 2.0 mg/dl or graft loss) in 12 months (4 patients), pediatric cases (4 patients), patients with previous diabetes mellitus (4 patients) were excluded. Three patients declined to participate in the study. The study was approved by local Ethics Committee and all patients gave informed consent.
We screened 126 patients, and after exclusion, only 82 patients (63 men and 19 women; mean age: 45 years) fulfilled the inclusion criteria. The onset of NODAT, clinical characteristics, demographic fea-tures (age, sex, dialysis modality and vintage) and laboratory data were obtained from the patients’ charts. In particular, physical examination and labo-ratory values of the patients were recorded at 1-month intervals for 12 months, at 2–3-month intervals for the second year and every 3–6 months thereafter.
Along with a detailed transplantation history and previously identified risk factors for NODAT, the following variables were analyzed: donor source (living or deceased), body mass index (BMI) at the time of transplantation [the ratio of weight to height2 (kg/m2)], number of human leukocyte antigen mis-matches, history of biopsy-proven acute rejection, and comprehensive details of immunosuppressive regimen including dosages, pre-transplantation fast-ing blood glucose levels, hepatitis C antibody status at transplantation and cytomegalovirus (CMV) infec-tion after renal transplantainfec-tion.
Definitions
Diabetes mellitus was diagnosed in accordance with American Diabetes Association/World Health
Organization (ADA/WHO) guidelines as symptoms of diabetes plus any time plasma glucose concentra-tions C200 mg/dL, or finding of two or more consec-utive fasting glycemia [126 mg/dL or 2-h plasma glucose C200 mg/dL during oral glucose tolerance test and the need for insulin or oral anti-diabetic therapy [1].
Immunosuppressive protocols
All patients were on maintenance immunosuppres-sion including prednisolone and CsA (Sandimmune NeoralÒ) with MMF/MPA. Prednisolone was admin-istered at a starting dose of 1 mg/kg and was tapered to 5 mg/day within 6 months. The starting dose of CsA was 6–8 mg/kg/day given in two divided doses, adjusted to keep through levels of 150–250 ng/mL followed by 100–150 ng/mL after a month. MMF or MPA was given at a dose of 2,000 or 1,440 mg/day, respectively. Acute rejection episodes were treated with three boluses of 0.5–1 g intravenous methyl-prednisolone. Total steroid and CsA doses adminis-tered in the first 3 months were calculated for each patient and expressed as per kilogram body weight.
Genetic analysis
Genomic DNA was extracted from peripheral blood leukocytes of all patients by means of standard techniques. eNOS genotypes were determined by polymerase chain reaction (PCR). PCR was performed in 50 ll total volume containing 0.1 lg genomic DNA, 20 pmol of each primer, 0.2 mM of each dNTP, 10 mM Tris, 50 mM KCl, 1.5 mM MgCl2and 1.5 U
Taq polymerase. Oligonucleotide primers (forward primer, 50-AGG CCC TAT GGT AGT GCC TTT-30; reverse primer, 50-TCT CTT AGT GCT GTG GTC AC-30) that flank the region of the 27-bp direct repeat in eNOS intron 4 were used for DNA amplification. PCR conditions were as follows: initial denaturation at 94°C for 2 min and then 35 cycles of 94°C for 1 min, 54°C for 40 s and 72°C for 1 min followed by a final extension at 72°C for 5 min. The genotypes were determined by agarose gel electrophoresis of PCR products in 2.5% agarose gel containing 0.5 lg/ml ethidium bromide staining and ultraviolet transillumi-nation [10].
Statistical analyses
The analyses were performed using the Statistical Package for the Social Sciences (SPSS version 11.5 for Windows, Chicago, IL). The data were expressed as medians (minimum–maximum). Average cumulative doses of CsA and prednisolone were given as mean ± standard deviation. Comparisons of the two groups were made with Mann–Whitney U test. Chi-square test was used for non-numerical data. Regression analyses were performed to determine independent variables for NODAT. P value of less than 0.05 was considered significant.
Results
Nine of 82 patients (11%) developed NODAT. Seven were diagnosed in the first year after transplantation, one in the second year and one in the third year. Median time until the diagnosis of NODAT was 5 months (1–35). Four patients were on insulin, and the remaining were on oral antidiabetic therapy. Laboratory values and demographic and clinical features of the NODAT and non-NODAT groups are given in Tables1and2, respectively.
Two alleles of intron 4, labeled 4a (four tandem 27-bp repeats) and 4b (five tandem 27-bp repeats), were detected. The frequencies of eNOS 4b/4b, eNOS 4b/4a and eNOS 4a/4a were 68.2% (56/82), 28.8% (22/82), 4.8% (4/82) in the study group, and 33.3% (3/9), 55.5% (5/9), 11.1% (1/9) in the NODAT group, respectively. The gene frequencies for the study group as a whole were 0.183 (4a allele) and 0.817 (4b allele). The participants were divided into two groups and evaluated in a dominant model on the basis of carrying a 4a allele of the eNOS gene intron 4b/4a polymorphism (aa ? ab vs. bb). Among the frequencies of eNOS intron 4 gene polymorphism, a significantly higher percentage of 4a allele carriers developed NODAT than non-carriers [6/26 (23.1%) allele carriers and 3/56 (5.4%), P = 0.02].
There were no significant differences between the patients with or without NODAT in gender, dialysis modality and vintage, donor sources, number of mismatches, acute rejection episodes, duration of transplantation and the frequency of CMV infection; however, subjects with NODAT were older, had a higher BMI at the time of transplantation and higher
prevalence of hepatitis C virus (HCV) infection, as shown in Table1.
Cumulative CsA [NODAT vs non-NODAT (3.2 ± 0.9 vs. 3.7 ± 0.7 mg/kg/day), P [ 0.05] and corticoste-roid doses [NODAT vs. non-NODAT (0.5 ± 0.15 vs. 0.56 ± 0.13 mg/kg/day), P [ 0.05] in the first 3 months after transplantation were also calculated and there were no significant differences between the groups.
In logistic regression analyses, having 4a allele of eNOS intron 4 gene polymorphism and HCV infec-tion were found to be the independent risk factors for the development of post-transplant DM (Table3).
Discussion
In the present study, the incidence of NODAT was 11%. Carrying a 4a allele of the eNOS gene intron 4
polymorphism and HCV seropositivity were found to be independent risk factors for development of NODAT.
Genetic studies have gained potential interest in evaluating the pathogenesis of insulin resistance and DM in the general population. In a recent study, Galanakis et al. showed a relationship between the eNOS gene a allele and type I and type II DM. They found that having an a allele of eNOS intron 4 polymorphism may represent a genetic susceptibility Table 1 Evaluation of risk
factors: clinical characteristics and demographic features
HD hemodialysis, PD peritoneal dialysis, BMI body mass index
NODAT (n = 9) Non-NODAT (n = 73) P value Age (years) 47 (33–53) 38 (18–65) 0.04 Gender (Male:Female) 7:2 56:17 1.00 Dialysis modality (HD:PD) 8:1 55:16 0.67
Dialysis duration (months) 10 (5–33) 12 (3–81) 0.34 BMI at the time of transplantation (kg/m2) 28.0 (20.0–34.5) 24.4 (17.6–33.9) 0.03 Donor source (Living:Deceased) 8:1 60:13 1.00 Number of mismatches (n) 2 (0–4) 2 (0–5) 0.77 Acute rejection [n(%)] 2 (22%) 14 (19.2%) 0.32 Time since transplantation (months) 34.5 (12–69) 34 (12–68) 1.00 Hepatitis C virus [n(%)] 3 (33.3%) 6 (8.2%) 0.05 Cytomegalovirus [n(%)] 1 (11%) 12 (13%) 0.86
Intron 4 (aa ? ab vs. bb) 6:3 20:53 0.02
Table 2 Laboratory values of the study groups
HDL high-density lipoprotein, LDL low density lipoprotein NODAT (n = 9) Non-NODAT (n = 73) P value
Fasting blood glucose (mg/dl) 105 (85–151) 87 (70–103) 0.001 HbA1C (mg/dL) 6.3 (5.8–8.6) 5.3 (4.2–6.0) 0.001 Creatinine (mg/dL) 1.2 (1.0–2.0) 1.4 (0.8–2.0) 0.84 HDL (mg/dL) 57 (37–69) 55 (23–89) 0.70 LDL (mg/dL) 100 (55–161) 101 (20–208) 0.94 Triglycerides (mg/dL) 179 (63–245) 150 (63–358) 0.42 Uric acid (mg/dL) 7.1 (4.60–9.20) 6.6 (3.9–10.2) 0.46 Homocysteine (mmol/L) 19.4 (12.70–24.66) 18.4 (5.86–133) 0.67 C-reactive protein 3.1 (0.7–16.8) 2.1 (0.3–21.0) 0.10 Hemoglobin (g/dL) 13.8 (11.7–17.2) 13.9 (8.4–17.1) 0.80
Table 3 Regression analyses showing independent variables Variables Significance Exp(B) 95% CI
for Exp(B) Intron 4 (aa ? ba) 0.028 5.803 (1.2–27.8) Hepatitis C virus
seropositivity
factor for diabetes [11]. Rittig et al. [12] also examined the association between NO-dependent vasodilatation in insulin-resistant subjects and showed an indepen-dent association with endothelial function, as indicated by disturbed NO production, in carriers of the intron 4 polymorphism of the eNOS gene. On the basis of these findings, eNOS gene intron 4 [27 bp VNTR (4b/4a)] polymorphism seems to be the suitable genetic vari-ation that may affect the diabetic state. Consistent with these findings, our study documents that carrying the 4a allele of the eNOS gene intron 4 polymorphism influences susceptibility to NODAT.
In some studies, it has been observed that HCV(?) patients have lower plasma insulin and C-peptide levels, as well as decreased insulin responsiveness and increased insulin resistance, when compared with patients without this infection [13,14]. In the general population, there was a higher prevalence of HCV infection in patients with diabetes than in those without diabetes [15]. In HCV(?) renal transplant recipients, a meta-analysis demonstrated a marked increased risk of NODAT [16]. In our study, hepatitis C was associated with NODAT.
Other risk factors consistently reported in the literature include particularly outstanding relevance of calcineurin inhibitor and steroid use, older age and high BMI at the time of transplantation. Both steroids and calcineurin inhibitors have been implicated in the development of NODAT. Van Hooff et al. [17] evaluated the mechanisms of NODAT and concluded that CsA and tacrolimus tend to reduce insulin release, while concomitant use of corticosteroids increases insulin resistance. A dose–response relationship between corticosteroids and NODAT has been dem-onstrated in some studies [17,18] but not in others [19,20]. In our study, differences in mean steroid and CsA doses (mg/kg/day) in the first 3 months were not statistically significant between groups.
In our study, although older age and higher BMI at the time of transplantation appeared to be risk factors for NODAT, they were not found to be independent risk factors. Cosio et al. [2] found a strong correlation between older age ([45 years old) and NODAT. Moreover, Kasiske et al. [3] demonstrated that recipient age was an independent risk factor for developing NODAT, with the relative risk for recip-ients aged 45–59 years determined as 1.92 vs. 2.58 for those aged [60 years. In this regard, our allograft recipients represented a relatively young population,
with median ages of 38 and 47 years in non-diabetics and diabetic patients, respectively. If more patients in their 6th or 7th decade had been included in the study, there likely would have been more diabetes and more significant association between age and development of NODAT.
Obesity defined as BMI C 30 kg/m2 has been shown to be another risk factor for development of NODAT [3]. In our study, patients with NODAT had a higher BMI at the time of transplantation than non-NODAT patients (28.0 vs. 24.4 kg/m2, respectively, P = 0.03). Nonetheless, it was not found to be an independent risk factor. This may due to the low BMI of our population in both groups.
The limitation of our study is the relatively small number of patients and the lack of patients using tacrolimus-, sirolimus- or everolimus-based immu-nosuppressive protocols. However, extrapolating our study to the patients using tacrolimus or sirolimus/ everolimus seems appropriate. Since tacrolimus has been proposed to be more diabetogenic in most studies, genetic susceptibility to diabetes may have a more profound effect in development of NODAT in allograft recipients using tacrolimus.
In conclusion, to the best of our knowledge, this is the first study to show the relationship between the eNOS intron 4 polymorphism and NODAT in kidney allograft recipients. The presence of the 4a allele seems to be an important candidate genetic risk factor for NODAT and this can be important in the prediction as well as prevention of NODAT. Acknowledgments This study was granted and supported by the Turkish Society of Hypertension and Kidney Diseases.
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