Study on the Correlation Between Regulatory Proteins of N-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. 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, mA methyltransferases (METTL3, METTL14, METTL16, WATP), mA demethylases (FTO, ALKBH5), mA 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 mA 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 decreased viability while increased apoptosis rate. The Nrf2 mRNA level in CdSO-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 concentrations. In addition, mRNA levels of METTL3, METTL14, METTL16, WTAP, FTO, and methyl-binding proteins in CdSO-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. Lastly, correlation analysis indicated that mRNA level of Nrf2 was positively correlated with mRNA levels of mA methyltransferases and demethylases. In a word, our results demonstrated that the molecular changes of Nrf2 signaling pathway are correlated with the levels of mA regulatory proteins, suggesting that there may be a regulatory relationship between Nrf2 signaling pathway and mA regulatory proteins in the process of cadmium-induced renal cell cytotoxicity.