Study on the Correlation Between Regulatory Proteins of N 6 -methyladenosine and Oxidative Damage in Cadmium-induced Renal Injury.

As a common environmental heavy metal pollutant, cadmium has been well evidenced to cause kidney damage; yet, the underlying mechanisms are still not fully clarified. In this study, cell viability of human renal tubular epithelial cell (HK-2) was determined by CCK-8 assay after treatment with CdSO 4 . Then, apoptotic morphology of cells was observed by Hoechst staining and level of reactive oxygen species (ROS) was detected by fluorescent probes. Subsequently, mRNA levels of Nrf2, HO-1, m 6 A methyltransferases (METTL3, METTL14, METTL16, WATP), m 6 A demethylases (FTO, ALKBH5), m 6 A methyl-binding proteins (YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2) were detected by real-time polymerase chain reaction (RT-PCR), closely followed by correlation analysis between Nrf2 mRNA levels and m 6 A methyltransferases and demethylases. Lastly, protein expressions of Nrf2, METTL3, and FTO were tested by western blotting assay. The detection results demonstrated that the treatment of CdSO 4 decreased viability while increased apoptosis rate. The Nrf2 mRNA level in CdSO 4 -treated cells was significantly increased when compared with that in the control cells, and the HO-1 mRNA level elevated with the increasing of CdSO 4 concentrations. In addition, mRNA levels of METTL3, METTL14, METTL16, WTAP, FTO, and methyl-binding proteins in CdSO 4 -treated cells were all higher than those in corresponding control cells. Further determination showed that protein expressions of Nrf2, METTL3, and FTO were also upregulation under the treatment of CdSO 4 . Lastly, correlation analysis indicated that mRNA level of Nrf2 was positively correlated with mRNA levels of m 6 A methyltransferases and demethylases. In a word, our results demonstrated that the molecular changes of Nrf2 signaling pathway are correlated with the levels of m 6 A regulatory proteins, suggesting that there may be a regulatory relationship between Nrf2 signaling pathway and m 6 A regulatory proteins in the process of cadmium-induced renal cell cytotoxicity.