Role of the functional Toll-Like receptor-9 promoter polymorphism (-1237T/C) in increased risk of end-stage renal disease: a case-control study

Abstract

Inflammation induced by infectious and noninfectious triggers in the kidney may lead to end stage renal disease (ESRD). Toll-like receptor 9 (TLR-9) a receptor for CpG DNA is involved in activation of immune cells in renal disease and may contribute to chronic inflammatory disease progression through an interleukin-6 (IL-6) dependent pathway. Previous studies indicate that -1237T/C confers regulatory effects on TLR-9 transcription. To date the effect of TLR-9 polymorphisms on ESRD remains unknown. We performed a case-control study and genotyped 630 ESRD patients and 415 controls for -1237T/C, -1486T/C and 1635G/A by real-time PCR assays and assessed plasma concentration of IL-6 by ELISA. Haplotype association analysis was performed using the Haploview package. A luciferase reporter assay and real-time PCR were used to test the function of the -1237T/C promoter polymorphism. A significant association between -1237T/C in TLR-9 and ESRD was identified. The TCA, TTA and CCA haplotype of TLR-9 were associated with ESRD. ESRD patients carrying -1237TC had a higher mean plasma IL-6 level when compared with -1237TT. The TLR-9 transcriptional activity of the variant -1237CC allele is higher than the -1237TT allele. The results indicate that in a Han Chinese population the presence of the C allele of -1237T/C in the TLR-9 gene increases susceptibility towards development of ESRD. In vitro studies demonstrate that -1237T/C may be involved in the development of ESRD through transcriptional modulation of TLR-9.

Figure 1. Effects of TLR-9 -1237T/C genotype on the level of plasma IL-6 in healthy controls and ESRD patients.

IL-6 concentration was quantified by ELISA. Results are expressed as mean ± SEM. The columns represent the mean value and the lines represent standard error of mean. A: Compared to the healthy controls, the ESRD patients showed a significant increase in plasma IL-6 level (P<0.001). B: The difference of mean level of IL-6 between TT (n = 22) and TC (n = 7) carriers was not significant (P = 0.87) in healthy controls. C: The difference of mean level of IL-6 between TT (n = 18) and TC (n = 14) carriers showed significant difference (P = 0.01) in ESRD patients. *P<0.05.

Figure 2. Comparison of PBMC TLR-9 mRNA expression between different genotypes of -1237T/C.

β-actin gene expression was used as an internal control gene. TLR-9 mRNA expression in TT or TC PBMCs in the absence or presence of IL-6 (100 pg/ml) treatment was assessed. Values shown are mean ± SEM. Experiments were performed in triplicate.

Figure 3. Effects of the -1237T/C genotype in TLR-9 promoter on luciferase activity in cultured HEK293 cells.

Luciferase reporters containing a TLR-9 promoter sequence with the wild-type T allele or risk C allele at SNP -1237T/C were transfected into HEK293 cells. The mean ± SEM is given for each construct from three experiments. *P<0.05.