Exploring the Prognostic Significance of IL10 Variants and Their Mechanistic Regulation in Diabetic Nephropathy

Abstract

IL10 is a very effective anti-inflammatory cytokine. IL10 imbalance is linked to type 2 diabetes mellitus (T2DM) and also to renal hypertrophy, glomerular membrane thickening, and onset of diabetic nephropathy (DN). We aimed to investigate the association of IL10 gene polymorphism (rs1800871T/C, rs1800896A/G) with DN and determine the influence of variants on its expression level and interaction with transcription factors. We genotyped 301 study subjects, comprising 75 DN, 126 T2DM patients, and 100 controls. All were analysed for biochemical assays and genotypic analysis by PCR-RFLP and confirmed by Sanger sequencing. The haplotype analysis was calculated by Chi-square test. mRNA expression and its correlation with variants were assessed by using RT-PCR. Statistical analyses were done by using SPSS and GraphPad software. Screening of transcription factors with IL10 gene variants was performed using the TRANSFAC database, and the variants impact on IL10 molecular interactions was analysed. This study revealed that IL10 gene polymorphism rs1800896 was significantly associated with DN. 'CG' and 'TG' haplotypes were significantly associated with DN. Expression levels of IL10 were upregulated in DN patients. The genetic correlation study shows that IL10 gene expression was downregulated in the rs1800871 alternate variant genotype (CC) while upregulated in the rs1800896 alternate variant genotype (GG). In silico analyses suggest that the binding affinity of transcription factor CEBPA decreases due to the rs1800871 alternate variant, while the affinity of KLF4 increases in the case of the rs1800896 alternate variant. Our in silico results correlated with IL10 expression analysis in respective patient groups. Overall, our findings highlight the role of IL10 gene polymorphism in DN progression in the North Indian population.

Keywords: CEBPA; IL10 gene polymorphism; rs1800871, rs1800896; KLF4; North Indian population; T2DM; diabetic nephropathy; genetic polymorphism.

FIGURE 1

IL10 gene variants rs1800871 and rs1800896 genotyping. (a) 3% Agarose gel showing different genotypes of IL10 rs1800871 T/C, Lane 1 shows marker (100–1000 bp), lane 2 shows TT (206 bp), lane 3, 5, 7 shows CC (185, 21 bp), lane 4,6,8 shows TC (206, 185, 21 bp) digest with MS1I. (b) Frequency of IL10 gene variant rs1800871 in the patient cohort. (c) Chromatogram confirms the presence of all IL10 rs1800871 genotypes. (d) 3% Agarose gel showing different genotypes of IL10 rs1800896 A/G, Lane 3 shows marker (50–1000 bp), lane 2,4 shows AA (323, 67, 61 bp), lane 1,5,7 shows AG (390, 323, 67, 61 bp), lane 8, shows GG (390, 61 bp), digest with Hpyav. (e) Frequency of IL10 gene variant rs1800896 in the patient cohort. (f) Chromatogram confirms the presence of all IL10 rs1800896 genotypes.

FIGURE 2

IL10 expression level and its correlation with genotypes. (a) IL10 mRNA expression level in T2DM and DN compared to HC. (b) IL10 expression level in various TT, TC, CC genotype in rs1800871 variants within T2DM and DN patients. ‘TT’ genotype is considered as a wild‐type. (c) IL10 expression level in various AA, AG, GG genotype in rs1800896 variants within T2DM and DN patients. ‘AA’ genotype is considered as a wild‐type. Statistical significance is indicated as *P < 0.05 and ***P < 0.001.

FIGURE 3

Identification of transcription factors (TF) that are bound on and around the IL10 rs1800871 and 1800896 genomic locations. (a) Schematic diagram of IL10 gene and its promoter region. Both the DNA strands are highlighted as blue arrow. IL10 gene (red colour) is present on the negative strand. Two genetic variations (rs1800871 and rs1800896) investigated here are highlighted together with 10 bases up and downstream as nucleotide sequences on the promoter along with their genomic locations. Both wild and alternate variant nucleotides are shown in red colour. (b) Experimentally validated TFs (green boxes) and TFs bound to the in vivo fragments covering SNPs in the ChIP‐Chip assays (pink boxes) of IL10 promoters are shown. TFs that are further investigated with wild and alternate variant promoter fragments of both the SNPs are enclosed with red boxes. (c) TFs predicted through the Match algorithms in the IL10 promoter fragments (10 bases up and downstream of SNPs) are shown for both wild type and alternate variants of rs1800871 and rs1800896.

FIGURE 4

Best interaction pose of IL10 promoter fragments with associated transcription factors. (a) The superimposed structures of wild and alternate variant IL10 dsDNA fragment with rs1800871 genomic variant in complex with CEBPA transcription factor are shown. (b) The superimposed structures of wild and alternate variant IL10 dsDNA fragment with rs1800896 genomic variant in complex with KLF4 transcription factor are shown. Wild‐type dsDNA fragments are highlighted in green. The transcription factors in complex with the wild‐type dsDNA fragments are shown in blue and with the alternate variant dsDNA fragments are shown in red.