Interleukin-10 gene promoter variants and susceptibility to diabetic nephropathy; a meta-analysis

1Department of Zoology, Guru Ghasidas Vishwavidyalaya, Bilaspur, 495009 (CG), India 2Section on Cellular Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA 3Departamento de Biología Molecular y Genomica, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, México

Introduction: Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD) in diabetes patients. There is ample evidence that the inflammatory pathways are central to both diabetes and DN. Several studies that examined the link between the interleukin-10 (IL10) polymorphisms and DN risk yielded conflicting results. Objectives: The purpose of this meta-analysis is to evaluate the associations between IL10 promoter polymorphisms and DN risk. Methods: A bibliographic search was carried out on PubMed, Google scholar and Web of Science from the beginning until July 30, 2020. Association between IL10 promoter variants (-1082 A>G, -819 C>T and -592 C>A) and DN risk were assessed by considering diabetes without nephropathy (DWN) as well as healthy controls. Data were retrieved and the pooled odds ratio (OR) with 95% confidence interval (CI) was calculated. Results: For the IL10 -1082 A> G analysis, a total of 4 studies with DWN controls (682 cases and 529 controls) and 5 studies with healthy controls (1025 cases and 1625 controls) were considered. For the IL10 -819 C> T analysis, a total of three studies with DWN controls (9619 cases and 445 controls) and 5 studies with healthy controls (1005 cases and 1537 controls) were considered. For the IL10 -592 C> T analysis, a total of 5 studies with DWN controls (819 cases and 645 controls) and 5 studies with healthy controls (1005 cases and 1537 controls) were considered. In addition, there was no evidence of publication bias for IL10 promoter variants. No substantial association was observed between IL10 promoter variants and DN risk. Conclusion: Our study signifies that polymorphisms of IL10 -1082 A>G, -819 C>T and -592 C>A are not linked with DN risk.

Introduction
Diabetic nephropathy (DN) causes serious health problems and is a leading cause of morbidity and mortality. The occurrence of DN is relatively high among type 2 diabetes mellitus (T2DM) patients representing huge health and economic burden (1). DN is the leading cause of chronic kidney disease (CKD), which leads to end-stage renal disease (ESRD). DN is characterised by micro albuminuria, loss of glomerular filtration rate to progressive CKD in patients with long standing diabetes (2). Several lines of research revealed that the DN is a complex disorder involving both genetic and environmental components (3). Diabetic kidney disease (diabetic nephropathy) is induced by inadequate glycaemic control in diabetic patients (4). Insulin resistance caused by hyperinsulaemia in diabetic patients leading to inflammation (5). Increasing evidences demonstrated that the inflammatory pathways are central to both diabetes and DN (6). The association of inflammatory mediator's such as interleukins, tumor necrosis factor-α, and macrophage chemotactic protein-1 has been documented in the literature (7).
Interleukin-10 (IL-10) is one of the important regulators of inflammation during diverse pathological conditions. Interleukin-10 is mainly produced by macrophages, B-cells, dendritic cells, monocytes, mast cells, neutrophils and eosinophils (8). IL-10 is an anti-inflammatory cytokine, which involved in progression of T2DM (9). Interleukin-10 levels are significantly corelated with the development of T2DM as well as DN. IL-10 is encoded by the IL10 gene, which is located on chromosome 1 and has several polymorphic variants that determine its expression. Three IL10 gene variants (-1082 A>G, -819 C>T and -592 C>A) have been studied for their association with DN (10). However, there is no consensus regarding the association of these polymorphism with DN in different populations, due to ethnic differences and less sample sizes used in the studies. In order to assess the exact role of these variants we conducted a meta-analysis by pooling the data from previous association studies.

Literature search
Articles assessing the association between IL10 gene variants and DN were retrieved from PubMed, Google Scholar, and Web of Science. The keywords used for retrieving the literature include diabetic nephropathy, interleukin-10 or IL10, -1082 A>G, -819 C>T and -592 C>A, polymorphism or variants. Specific inclusion and exclusion criteria were adapted to select the papers for data extraction. The inclusion criteria include 1) case control studies analysing IL10 variants and DN, 2) Comparison group having either healthy controls or diabetes without nephropathy (DWN), 3) Studies having genotype data in both DN and comparison groups for estimating odds ratio and 95% confidence interval. Studies that do not follow the above criteria were excluded from the study. From each article genotypic data of both cases and control (healthy controls and/or DWN) were extracted and tabulated.

Statistical analysis
The strength of the association between the IL10 promoter variants (-1082 A>G, -819 C>T and -592 C>A) and DN was assessed for all studies. The crude odds ratios (ORs) and their corresponding 95% confidence interval (CI) limits were calculated. The presence of heterogeneity was evaluated with the Cochran's Q test and inconsistency I 2 statistics. Based on the extent of heterogeneity, fixed effects model or random effects model were adopted for pooled analysis. The association between IL-10 polymorphisms and DN was analysed in dominant, recessive, and allelic genetic models. To assess the robustness of the study, sensitivity analysis was performed by excluding each study once and estimating the OR for the rest of the studies. Publication bias was measured by drawing a funnel plot and Egger's linear regression method.

Characteristics of the included studies
The bibliographic search strategy and the selection process of the articles for this meta-analysis are presented in Figure  1. A total of 10 articles that analysed DWN controls or healthy controls or both were included in the pooled analysis (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Out of these only 5 articles studied all theree IL10 promoter variants (-1082 A>G, -819 C>T and -592 C>A). For IL10 -1082 A>G pooled analysis, four studies including 682 cases and 529 DWN controls and 5 studies including 1025 cases and 1625 healthy controls were included. For IL10 -819 C>T pooled analysis, three studies including 619 cases and 445 DWN controls and 5 studies including 1005 cases and 1537 healthy controls were included. For IL10 -592 C>A pooled analysis, five studies including 819 cases and 645 DWN controls and 5 studies including 1005 cases and 1537 healthy controls were included. The genotype distribution in both cases and controls for each study was included in Table 1.

Heterogeneity
The association between IL10 promoter variants and DN risk did not exhibit considerable heterogeneity between studies in all three genetic models (Table 2). Nevertheless, for the association between -1082 A>G, -592 C>A   Table 2).

Association of the IL10 promoter variants and DN risk
The results of this study showed that there was no significant association between IL10 promoter variants and risk of DN in all three genetic comparison models when DWN patients used as controls (Table 2). However, IL10 -819 C>T showed a trend for protective effect towards DN risk when we used DWN as controls, which is not statistically significant. Further, no significant association was observed between IL10 promoter variants and risk of DN when healthy individuals used as controls ( Table 2).

Results of sensitivity analysis and publication bias
Sensitivity analysis performed by omitting each study showed that individual studies did not affect the pooled effects estimates for these IL-10 promoter variants, indicating that the results were statistically reliable. The funnel plot shapes were found to be symmetrical for all three IL-10 promoter variants, indicating the absence of publication bias. Egger's linear regression test also showed that there was no evidence of publication bias for all three IL10 promoter variants in all three genetic models (P> 0.050; Table 2). However for IL10 -592 C>T significant publication bias was found in recessive model when used DWN controls and dominant model when used healthy control for comparison (Table 2).

Discussion
The present meta-analysis has shown that there is no significant association between IL-10 gene promoter variants and the risk of diabetic nephropathy. A trend of protective effect for DN was found for -819 C>T polymorphism in a comparison using DWN as controls. No significant heterogeneity between studies was noted for all three promoter polymorphisms. In addition, there was no evidence of publication bias for IL-10 promoter variants were detected. Our meta-analysis is consistent with previous meta-analysis in which a trend for protective effect was documented for -1082 A>G and/or -819 C>T (10,21). Another meta-analysis demonstrated significantly increased risk of DN with -1082 A>G polymorphism (22). Several in vivo and in vitro studies have shown that the IL-10 gene expression and IL10-induced signalling pathways are important for the regulation and maintenance of normal kidney function (23). Further abnormal IL-10 expression has been linked to occurrence and progression of various kidney disorders (24). Increased serum levels of IL10 is associated with the severity of nephropathy (25). As an anti-inflammatory cytokine, IL-10 plays a major role in inhibiting a synthesis of pro inflammatory cytokines and modulate the inflammation in the body. Long standing DM and inflammatory conditions are associated with DN (26). Inflammation and subsequent tissue remodeling can increase oxidative stress and initiate kidney damage leading to DN (27,28). Therefore, selective targeting of IL10 expression and pathways associated with IL-10 may be one of the therapeutic approaches for treating many of the kidney diseases (24). Abnormal IL10 expressions may contribute to diabetes induced kidney disease. Studies in animal models suggested contradictory results in terms of its association with renal functioning (29,30).
IL-10 levels are correlated with the polymorphism in promoter of IL-10 gene (31). Subsequent studies demonstrated that the IL-10 secretion is mainly depending on the haplotypes of IL10 locus (32). Further, it is also documented that the ethnicity influences the allele frequency distribution of cytokine polymorphisms (33). Several functional polymorphisms of IL10 gene have been assessed for their association with the risk of DN, however the results are contradictory. Main reasons for the discrepancies are criteria used for characterisation of DN, study design, very low sample sizes and ethnicity. In conclusion, we suggest that IL10 gene promoter variants are not associated with the development of DN.

Authors' contribution
BVKSL conceived the study. GM and SG collected data. GM, SG, TPKR and BVKSL analysed and prepared the manuscript. TPKR and BVKSL critically revised the manuscript. All authors approved the manuscript.

Conflicts of interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical considerations
Ethical issues (including plagiarism, data fabrication, double publication) have been completely observed by the authors.