5-Aza

This study aimed to evaluate site-specific changes in the methylation of the nephro-protective gene p21 (CDKN1a) after exposure to nephrotoxicants using a targeted bisulfite sequencing approach. A targeted gene bisulfite sequencing (TGBS) method was developed using the Illumina MiSeq platform and Bismark bisulfite mapper. Human embryonic kidney (HEK293) cells and freshly isolated human proximal tubular cells (hPT) were analyzed, and 5-aza-2’-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor, served as a positive control. Methylation differences between human and rat p21 were also investigated. TGBS analysis identified a methylation-sensitive site in the p21 promoter region, sis-inducible element-1 (SIE-1), which regulates p21 expression via transcription factor binding. Treatment with 5-Aza altered methylation at this site, whereas nephrotoxicants cisplatin and bromate (BrO3?) did not, despite increasing p21 protein expression. No SIE-1 site was detected in normal rat kidney cells (NRK), indicating species-specific differences. Cisplatin and BrO3? exposure did not decrease promoter methylation in either HEK293 or NRK cells. These findings reveal species-specific differences in basal p21 promoter methylation and indicate that nephrotoxicant-induced changes in p21 expression are independent of promoter DNA methylation. The study also demonstrates the utility of TGBS for rapid analysis of locus-specific DNA methylation.