Association of the genetic polymorphisms of the ACE gene and the eNOSgene with lupus nephropathy in northern Chinese population

Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving environmental, genetic and hormonal elements [1‐3]; also it is a multisystem disease with a variable course and a wide range of clinical manifestations[4], such as lupus nephropathy. Renal injury in SLE is one of the most serious complications and its pathogenesis has not yet been completely clarified [1].

It has been extensively documented that genetic factors play an important role in the development and progression of both SLE and lupus nephropathy [5‐7]. Many studies have showed the critical role of the SNPs of the ACE gene and the eNOS gene in the process of the occurrence and progression of SLE [8‐10].

As is widely known that the reninangiotensin system (RAS) is usually involved in the progression of renal diseases [11]. ACE affects various clinical manifestations through the reninangiotensin system by promoting the formation of angiotensin II and inactivating bradykinin [2]. In human, the ACE gene is located on chromosome 17q22-q24 [12] and is expressed in a wide range of tissues, such as lung, vascular endothelium, kidney, heart and testes [9]. Many studies, including one study in a Chinese population [13], have suggested that a 250 bp insertion/deletion (I/D) polymorphism of the ACE gene was associated with SLE and renal injury [1, 13]. Recent studies have reported that several SNPs (A-5466C, T-3892C, A-240T, CJ237T, G2215A and A2350G) of the ACE gene may affect the risk of certain autoimmune diseases such as essential hypertension, left ventricular hypertrophy, IgA nephropathy, diabetic nephropathy and so on [14‐16]. Therefore, we presume that these SNPs of the ACE gene may also play an important role in the molecular mechanisms of lupus nephropathy.

Nitric oxide (NO) plays an important role in the pathogenesis of SLE, with an elevated level of expression in SLE patients than in healthy controls [17]. NO synthesis is tightly regulated by nitric oxide synthases (NOS), the latter has three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS) [18]. The eNOS gene is located on chromosome 7q35-q36 [19], which is an important factor in the process of immunity reaction and the production of NO. It has been reported that two SNPs (T-786C and G894T) of the eNOS gene were associated with the susceptibility of vascular, infectious and autoimmune diseases, such as resistant hypertension, ischemic stroke, essential hypertension, and lacunar infarction [20‐22]. These SNPs may alter the level of the eNOS gene expression or change the protein product of the gene, and is relevant either to the pathogenesis of SLE or the progression of specific manifestations of diseases such as atherosclerosis and renal complications [23, 24]. Therefore, the eNOS gene is a candidate gene for the analysis of SLE susceptibility [8, 25].

However, the associations between the above mentioned SNPs and the risk of lupus nephropathy have not been evaluated in the Chinese population. The aim of the present study is to investigate the association of these SNPs with lupus nephropathy in a northern Chinese population.

Lupus nephropathy happens at a high frequency in patients with SLE, and it has been suggested to be related to abnormal regulation of the complex system consisting of the RAS and the NO system, both regulating vascular tone and inflammation. These two systems may have a profound effect in the pathogenesis and progression of SLE and the development of lupus nephropathy. Multiple genetic and environmental factors may be implicated in the evolution of lupus nephropathy. Different genetic variants contribute to the SLE phenotype from populations of different genetic backgrounds are becoming increasingly apparent [29]. In recent years, complete genome scans have tried to search for SLE susceptibility loci [30], two candidate genes identified are the ACE gene and the eNOS gene [31], which are inter-related [32]. ACE is a key component of RAS and it converts angiotensin I to II [33]. It inhibits the progression pace of the majority of chronic nephropathies [34]. eNOS is an important isoform of NOS, which plays an important role in autoimmune diseases [23, 35]. Polymorphisms of the eNOS gene are relevant to the pathogenesis of certain diseases correlate with SLE [36]. As a family-based association analysis has already shown an association between ACE G-261T and SLE in a Chinese population [37], we did not test this locus in our study. Six SNPs (A-5466C, T-3892C, A-240T, C1237T, G2215A and A2350G) of the ACE gene and two SNPs (T-786C and G894T) of the eNOS gene have been selected since they may affect the susceptibility to some autoimmune diseases [36].

In this study, significant differences in genotype distribution of two SNPs (A-5466C and A2350G) of the ACE gene were observed between cases and controls. The differences are similar to previous reports [38, 39], and are strong predictors of plasma ACE levels [40, 41]. No association was observed between the allelic frequency distribution of other SNPs and lupus nephropathy, although they have been studied extensively in high prevalence of renal disorders among hypertensive [42, 43]. Our results suggested that ACE -5466C and ACE 2350G allele play a significant protect role in the pathology of renal disease, and ACE -5466A allele in homozygote individuals and ACE 2350A allele in dominant genetic model were risk allele for lupus nephropathy.

The association between some of the haplotypes and lupus nephropathy were shown to be significant. Among them, the haplotypes ACTTAA, ACACGG and ACATGG play protective role in the renal injury in SLE, while haplotypes ATTCGA and CTTCGA were risk factors. To the best of our knowledge, this is the first study focusing on the SNPs of ACE gene and eNOS gene in Chinese population.

NO is an important biological molecule, which has a critical role in many biological systems. It has been identified as a potent mediator of immune and inflammatory response. Studies have shown that the levels of NO are significantly elevated in SLE patients in comparison with controls. A correlation has been found between serum NO and SLE disease activity[17]. For example, the T-786C and its interaction with G894T have impact on the basal nitric oxide activity of renal circulation, essential hypertension and diabetic nephropathy in multi-ethnical population [44, 45]. In this study, the association between the variations of the eNOS gene (G894T) and the presence of nephropathy in SLE was significant, with the eNOS 894T allele play a significant protect role in the pathology of renal injury in SLE, and eNOS 894G allele in dominant genetic model were susceptibility gene for lupus nephropathy. However, no such association was found with the eNOS T-786C.

The above inconsistency could be due in part to the heterogeneity of the ACE gene and the eNOS gene among different ethnic groups [21, 46], since it is often the case that the same SNP has different roles in different populations. Another reason may be the unclear stratification of the patients with the lupus nephropathy, such as first attack or recurrence and the age of onset and so on. Furthermore, environmental factors for cases and controls were not available in this study, which limited the analysis of the interaction between genetic factors and environmental factors, and further the estimation of adjusted ORs. Finally, the sample size for this study was relatively small due to the limited time of the collection of cases, the result of this study need validation.

Although some limitations do exist in this study, ours is an innovative study with some promising findings that contribute to the evaluation of genetic risk factors for lupus nephropathy. The clues provided in this study call for further research, other association studies, as well as functional studies are warranted to prove the association from multiple aspects. For the analysis of the association between these SNPs and the progression of this disease. Further studies, with larger sample sizes, are necessary to determine the contribution of these alleles to lupus nephropathy progression.

Our study shows that two SNPs (-5466C and 2350G) of the ACE gene and the one SNP (G894T) of the eNOS gene were associated with the susceptibility of lupus nephropathy. Five haplotypes (CTTCGA, ACTTAA, ACATGG, ACACGG and ATTCGA) were found to contribute to the risk of lupus nephropathy, suggesting an important role they played in the pathology of lupus nephropathy.