Association of endothelial nitric oxide synthase gene polymorphisms with end-stage renal disease: a systematic review and meta-analysis

Abstract

Background and objective: Endothelial nitric oxide synthase (eNOS) is one of the potent regulators of intra renal hemodynamics. Polymorphisms of eNOS gene may be involved in the progression of renal disease, and may be the causative factors that contribute to the deterioration of renal functions. During the past decades, several studies investigated the association of eNOS polymorphisms with the risk of end-stage renal disease (ESRD), but the results remain unclear and the mechanisms are not defined. Our study was designed to examine the role of different eNOS genetic polymorphisms in the progression of ESRD. Materials and methods: Relevant studies were identified through PubMed, Embase, Medline and CNKI (China National Knowledge Infrastructure) database published between January 2000 and November 2013. The association between eNOS polymorphisms and ESRD susceptibility was assessed by pooled odds ratios (ORs) and 95% confidence intervals (95% CI) in fixed or random effects models. Results: Sixteen articles were identified for the analysis of association between eNOS gene polymorphisms and ESRD risk. A total of 2729 patients and 2190 controls for 4b/a, 851 patients and 1171 controls for G894T, and 513 patients and 487 controls for T786C were included in our analysis. Overall, 4a allele of 4b/a polymorphism produced a significant association in the global population (OR = 1.47, 95% CI = 1.05–2.06, p = 0.03) in a random-effect model; T allele of G894T was also significantly associated with ESRD susceptibility in overall populations (OR = 2.12, 95% CI = 1.44–3.12, p = 0.0001). Furthermore, 4a and T carriers were significantly associated with ESRD risk as well. No association was found between T786C polymorphism and ESRD. Conclusion: The evidence accumulated suggested that 4b/a and G894T polymorphisms in the eNOS gene were associated with ESRD susceptibility, indicating that 4a and T allele carriers might become significant genetic molecular markers for the onset of ESRD in overall populations. However, more studies should be performed in the further studies.

• Endothelial nitric oxide synthase
• end-stage renal disease
• gene polymorphism
• meta-analysis

Introduction

End-stage renal disease (ESRD) represents the final stage of chronic kidney disease. It is a worldwide health problem with an irreversible loss of endogenous renal function and with an epidemic extent.1,2 Patients with ESRD experience high rates of morbidity and mortality.3 Studies have showed that incidence and prevalence counts of ESRD in the United States are expected to increase by 44 and 85% from 2000 to 2015, respectively.4 ESRD is a multifactorial disease. Prior studies have identified that proteinuria, calciphylaxis, older age, elevated blood pressure and elevated serum creatinine level may be the independent risk factors for ESRD.5 However, there was rare genetic molecular marker to predict the onset of ESRD. Thus, knowing various genetic factors may be helpful to understand the pathophysiology of the ESRD.

Many genes along with environmental factors may make an individual susceptible for developing ESRD. More recent studies have established endothelial nitric oxide synthase (eNOS) gene polymorphisms as genetic molecular marker for the onset of ESRD in overall populations.6 NOS was first recognized and reported in 1989,7,8 including three NOS isoforms (NOS-1, NOS-2 and NOS-3) that are products of different genes, with different localization, regulation, catalytic properties and inhibitor sensitivity. NOS-3, also called eNOS, is found and mostly expressed in vascular endothelial cells.9 It can inhibit platelet aggregation, leucocyte adhesion and vascular inflammation, control vascular smooth muscle proliferation, stimulate angiogenesis and activate endothelial progenitor cells.10 Three eNOS polymorphisms, the Intron 4 27-bp repeat (4b/a), the G894T (Glu298Asp) missense mutation in exon 7 and the T786C single-nucleotide polymorphism in the promoter region have been studied. T786C reduces eNOS transcription;11 G894T may be associated with a decrease of eNOS activity12 and 4b/a polymorphism has been reported to be associated with decreased plasma nitric oxide (NO) concentrations that may reflect the activity of eNOS.13

These polymorphisms were all reported to be associated with nephropathy. However, the association between ESRD risk and polymorphisms found in eNOS is still controversial. Some studies have found that different genotypes of eNOS were associated with ESRD,14 whereas other reports suggest that they may not be significantly associated with ESRD risk.15 It could be every single study is inadequate in achieving a comprehensive and reliable conclusion. To better address the association between eNOS polymorphisms and ESRD risk, we performed a meta-analysis from all eligible studies based on samples of different ethnicities.

Materials and methods

Search strategy and eligibility criteria

A comprehensive search was conducted using the following electronic databases PubMed, Embase, Medline and China National Knowledge Infrastructure (CNKI) for related reports published between January 2000 and November 2013. Articles reporting the association between ESRD and eNOS polymorphisms were identified. The Medical Subject Heading (MeSH) terms “endothelial nitric oxide synthase gene”, “eNOS”, “polymorphism” and “end-stage renal disease” were employed as the searching words. All studies matching the eligibility criteria were retrieved, and references were checked for other relevant publications.

Criteria for article screening

Studies included had to meet all the following criteria: (1) case-control or cohort association studies included at least one of the three polymorphisms, 4b/a, G894T, T786C; (2) based on unrelated individuals; (3) the outcome had to be ESRD; (4) when the same authors reported two or more publications on possibly the same patient populations, only the most recent or complete study was included into this meta-analysis; and (5) the genotype distribution of the control population must be in the Hardy–Weinberg equilibrium (HWE).

Data extraction

Two investigators independently extracted the data and reached a consensus on all of the items. Any disagreement was resolved by discussing with the third expert. Data retrieved from the reports included author, publication year, ethnicity, age, sample size and type of sample for genotyping. Genotype distribution in both cases and controls were also obtained.

Statistical analysis

The pooled odd ratio (OR) with 95% confidence intervals (95% CI) was used to assess the strength of association between eNOS polymorphisms and ESRD. The significance of the pooled OR was determined by the Z-test, and a p value less than 0.05 was considered significant. This study examined the association between eNOS and ESRD risk compared with that for per-allele model (4a vs. 4b for 4b/a, T vs. G for G894T, C vs. T for T786C), dominant model (4aa + 4ba vs. 4bb for 4b/a, TT + GT vs. GG for G894T, CC + TC vs. TT for T786C), recessive model (4aa vs. 4ba + 4bb for 4b/a, TT vs. GT + GG for G894T, CC vs. TC + TT for T786C). The heterogeneity for the included articles was evaluated using Cochran’s Q test and I² statistics. p Value less than 0.1 and I² less than 50% were considered to be statistically significant. When a significant heterogeneity existed across the included studies (I² > 50%), the random effect model was used; when there was no significant heterogeneity across the included studies (I² < 50%), the fixed effect model was used. To assess whether our results were substantially influenced by the presence of any individual study, we conducted a sensitivity analysis by systematically removing each study and recalculating the significance of the result. Begg’s funnel plot was performed to examine the publication bias. Analyses were carried out using the Review manager 5.2 (The Cochrane Collaboration). All tests were two-sided.

Results

Characteristics of studies included

With our search criterion, 36 papers were found. Twenty-two papers concerning association research between eNOS polymorphisms and ESRD were identified. Among those 22 papers, 16 papers met our criteria and six papers were excluded for they did not provide detailed genotype information or a lack of control group data. Of the 16 include studies, two were from China,16,17 two were from Brazil,18,19 one was from France,20 one was from India,21 five were from Japan,22–26 one was from Poland,27 one was from Greece and Cyprus28 one was from Egypt,29 one was from Canada30 and one was from Hungary.31 The major characteristics of the 16 eligible publications are reported in Table 1. The association of eNOS gene polymorphisms in ESRD risk was shown in Table 2.

Table 1. Characteristics of the studies evaluating the effects of eNOS gene polymorphism on ESRD risk.

First-author-published year	Country	Cases				Controls
4b/a polymorphism		Total	4b/4b	4b/4a	4a/4a	Total	4b/4b	4b/4a	4a/4a
Asakimori-2001	Japan	710	541	157	12	189	163	26	0
Lin-2002	China	67	53	13	1	70	64	6	0
Noiri-2002	Japan	185	145	–		304	249	–
Nagase-2003	Japan	302	226	71	5	248	201	47	0
Lamnissou-2004	Greece/Cyprus	361	257	88	16	295	205	80	10
Buraczynska-2004	Poland	706	414	246	46	321	236	81	4
Bellini-2007	Brazil	114	50	50	14	94	60	29	5
Tripathi-2008	India	184	149	30	5	569	398	151	20
Marson-2011	Brazil	100	65	30	5	100	75	24	1
G894T polymorphism		Total	GG	GT	TT	Total	GG	GT	TT
Noiri-2002	Japan	185	136			304	251
de Prado-2002	Canada	91	37	33	21	109	42	46	21
Nagase-2003	Japan	159	124	33	2	270	250	18	2
Tang-2008	China	153	38	46	69	148	55	54	39
Thaha-2008	Japan	100	36	61	3	100	72	26	2
Kerkeni-2009	France	23	5	12	6	120	72	43	5
El-Din and Hamdy-2011	Egypt	40	10	18	12	20	12	7	1
Marson-2011	Brazil	100	43	47	10	100	49	42	9
T786C polymorphism		Total	TT	TC	CC	Total	TT	TC	CC
Asakimori-2002	Japan	168	121	42	5	187	159	28	0
Marson-2011	Brazil	100	39	48	13	100	38	50	12
Zsom-2011	Hungary	245	100	110	35	200	76	92	32

Table 2. Meta-analysis of eNOS gene polymorphisms in ESRD.

Genotype	Q test	OR (95% CI)	p	pHeterogeneity	Model
4b/a polymorphism
4a vs. 4b	8	1.47 (1.05, 2.06)	0.03	<0.0001	R
4aa + 4ba vs. 4bb	9	1.42 (1.02, 1.97)	0.04	<0.0001	R
4aa vs. 4ba + 4bb	8	2.39 (1.59, 3.61)	<0.0001	0.12	F
G894T polymorphism
T vs. G	8	2.12 (1.44, 3.12)	0.0001	0.0002	R
TT + TG vs. GG	9	2.27 (1.48, 3.48)	0.0002	0.0004	R
TT vs. TG + GG	8	2.00 (1.44, 2.78)	<0.0001	0.13	F
T786C polymorphism
C vs. T	3	1.23 (0.74, 2.05)	0.42	0.005	R
CC + TC vs. TT	3	1.22 (0.69, 2.15)	0.49	0.02	R
CC vs. TC + TT	3	1.06 (0.69, 1.63)	0.78	0.19	F

Notes: R, random-effect model; F, fixed-effect model.

Association of the eNOS 4b/a gene polymorphism with ESRD risk

In this meta-analysis, nine studies were considered eNOS 4b/a gene polymorphisms with ESRD risk, including 2729 cases and 2190 controls. As shown in Figure 1, we found that eNOS 4a allele was associated with an increased risk of ESRD when compared with the 4b allele in global populations (OR = 1.47, 95% CI = 1.05–2.06, p = 0.03) in a random-effect model. Under dominant model, a similar significant result between 4a genotype (4aa + 4ba) and the risk of ESRD in comparison with eNOS 4bb (OR = 1.42, 95% CI = 1.02–1.97, p = 0.04) in a random-effect model (Figure 2). Under the recessive genetic model, 4aa genotype was significantly associated with an increased risk of ESRD in the global populations under the homozygote genetic model (OR = 2.39, 95% CI = 1.59–3.61, p < 0.0001) in a fixed model.

Figure 1. Forest plot on the association between 4a allele carriers and ESRD risk.

Figure 2. Forest plot on the association between 4a genotype and ESRD.

Association of the eNOS-894 polymorphism with ESRD risk

A total of eight studies were included in association investigation of eNOS-894 polymorphism and ESRD risk. There were 851 patients and 1171 controls. The result showed a weak association between the T allele and ESRD risk compared with the G allele in the global population (OR = 2.12, 95% CI = 1.44–3.12, p = 0.0001) in the random-effect model (Figure 3). Similar association was found in the dominant model (OR = 2.27, 95% CI = 1.48–3.48, p = 0.0002). As shown in Figure 4, significant association between eNOS-894 TT and the risk of ESRD was found in the global population in the recessive genetic model (OR = 2.00, 95% CI = 1.44–2.78, p < 0.0001).

Figure 3. Association between T allele and ESRD risk in overall population.

Figure 4. Association of T genotype with ESRD risk in overall populations.

Association of the eNOS-789 polymorphisms with ESRD risk

Only three studies were included in association investigation of eNOS-786 polymorphism and ESRD risk. As shown in Table 2, there was no association between the C allele and ESRD risk compared with the T allele in the global population (OR = 1.23, 95% CI = 0.74–2.05, p = 0.42). No association betweene NOS-786 CC and the risk of ESRD was found in the global population in the dominant model (OR = 1.22, 95% CI = 0.69–2.15, p = 0.49) and in the recessive genetic model (OR = 1.06, 95% CI = 0.69–1.63, p = 0.78).

Sensitivity analyses and publication bias

A single study included in the meta-analysis was deleted each time to reflect the influence of the individual data set to the pooled ORs. For 4b/4a polymorphism, if Lamnissou’s and Tripathi’s studies were excluded, no heterogeneity was found between each included studies (I² = 0), and the corresponding pooled ORs were not materially changed. This procedure confirmed the stability of our overall result.

Egger’s test was conducted to assess the publication bias of the literature. The shape of funnel plots did not reveal any evidence of funnel plot asymmetry. As shown in Figure 5, the statistical results still did not show publication bias.

Figure 5. Publication bias for the analysis of association of eNOS Glu298Asp gene polymorphism and ESRD susceptibility for overall populations.

Discussion

ESRD is a major health problem associated with very high morbidity and mortality. Reducing the incidence of ESRD is widely recognized as a major public health goal. Recently, many potential risk factors have been suggested, such as angiotensin converting enzyme (ACE), uncoupling protein (UCP2), eNOS.32–34 However, data on the risk factors for the pathogenesis of ESRD were insufficient. The goal of this study was to mainly evaluate the prognostic value of eNOS gene polymorphisms and the susceptibility of ESRD.

In the present study, a significant association between ESRD risk and eNOS 4b/a, Glu298Asp polymorphisms was observed in the global population. No association was found between eNOS T708C polymorphism and ESRD risk. According to those results, we conclude that eNOS 4b/a and Glu298Asp would be considered as effective genetic factors, which contributed to the pathology of ESRD.

Nitric oxide (NO), an important intracellular messenger molecule, plays a major role in maintaining homeostasis.35 After being released into endothelial cells, NO diffuses rapidly through cell membranes and relaxes neighboring vascular smooth muscle cells through the production of guanosine 3′,5′-cyclic monophosphate (cGMP).36 A single-nucleotide polymorphism (SNP) in the promoter region (T-786C), a SNP in exon 7 (Glu298Asp) and the variable number of tandem repeats (VNTR) in intron 4 have received most of the attention, because of their association with cardiovascular and renal diseases.

In the 27-bp repeat of intron 4, two alleles have been identified, the larger one, eNOS 4b, has five tandem 27-bp repeats, and the smaller, eNOS 4a, has four repeats.37 Although the precise molecular effects of this polymorphism have not been elucidated, there is biochemical evidence of reduced eNOS expression and enzymatic activity associated with this eNOS 27-bp VNTR.38 Tsukada et al.39 reported that eNOS gene polymorphisms correlate with the circulating NO concentrations, where homozygotes eNOS 4bb and eNOS 4aa exhibit the highest and the lowest NO levels, respectively. Moreover, eNOS 4a allele was associated with the pathogenesis of ESRD. Bellini et al.19 demonstrated a strong correlation between eNOS 4a polymorphism and ESRD risk, while Marson et al.18 found no significant differences in haplotype distribution between eNOS 4a polymorphism and ESRD risk. Our result showed that the frequency of the 4a allele in eNOS intron 4 was significantly higher in patients than in controls (OR = 1.47, 95% CI = 1.05–2.06, p = 0.03), and the frequency of eNOS 4a carrier genotypes (eNOS 4aa and 4ab) was also higher in patients than in healthy controls (p = 0.04), indicating that the eNOS 4a allele may be one of the risk factors for ESRD in ESRD patients.

G894T polymorphism is located in exon 8, which leads to a single amino acid substitution from glutamate to aspartate at the 298 site. The eNOS Glu298Asp variation is fairly widespread in the human population, with 35–40% prevalence of the T allele in Caucasians and less prevalence in African Americans and Asians.40 It is an important gene mutation of eNOS, and Glu298Asp gene polymorphism includes GG (Glu/Glu), GT (Glu/Asp) and TT (Asp/Asp) genotypes, and G (Glu) and T (Asp) alleles. Shear is a key modulator of NOS3 function in vivo and association with caveolae is important for the control of NOS3 protein activity.41 The disease, states most consistently implicating of this SNP, has typically been shown to involve endothelial dysfunction.41 The present epidemiologic study shows that the eNOS Glu298Asp gene polymorphism has been implicated in the etiology of ESRD. However, the available evidence reported to date is weak, due to the sparseness of data or disagreements among studies. In the past years, there were some meta-analyses to explore the association of eNOS Glu298Asp gene polymorphism with the susceptibility of some diseases. Su et al.42 showed significant associations between eNOS Glu298Asp polymorphisms and idiopathic recurrent pregnancy loss. Yu et al.43 found that the eNOS Glu298Asp polymorphism is not associated with a significant increased risk of pre-eclampsia. In our study, we found that T allele was associated with ESRD susceptibility in overall populations (OR = 2.12, 95% CI = 1.44–3.12, p = 0.0001), and GG genotype might play a protective role against ESRD susceptibility in overall populations.

T786C polymorphism is located in the promoter of the eNOS gene at the eNOS-786 site. Molecular exploration indicated that the replacement of thymidine by cytosine is able to cause a reduced promoter activity, which leads to a compromised production of the eNOS enzyme in vivo.44 Our study showed no association between T786C polymorphism and ESRD risk.

In conclusion, an increased susceptibility of eNOS gene 4a (4b/a) and T (G894T) variant carriers to ESRD was observed in the present meta-analysis. GG and 4bb genotype might play a protective role against ESRD susceptibility in overall populations. However, more case-control association studies on larger, stratified populations are required to further elaborate the role of eNOS gene polymorphisms in ESRD susceptibility in different ethnicities.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.