Study on the Correlation Between Regulatory Proteins of N6-methyladenosine and Oxidative Damage in Cadmium-induced Renal Injury

Abstract

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 CdSO₄. 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, m⁶A methyltransferases (METTL3, METTL14, METTL16, WATP), m⁶A demethylases (FTO, ALKBH5), m⁶A 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 m⁶A 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 CdSO₄ decreased viability while increased apoptosis rate. The Nrf2 mRNA level in CdSO₄-treated cells was significantly increased when compared with that in the control cells, and the HO-1 mRNA level elevated with the increasing of CdSO₄ concentrations. In addition, mRNA levels of METTL3, METTL14, METTL16, WTAP, FTO, and methyl-binding proteins in CdSO₄-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 CdSO₄. Lastly, correlation analysis indicated that mRNA level of Nrf2 was positively correlated with mRNA levels of m⁶A methyltransferases and demethylases. In a word, our results demonstrated that the molecular changes of Nrf2 signaling pathway are correlated with the levels of m⁶A regulatory proteins, suggesting that there may be a regulatory relationship between Nrf2 signaling pathway and m⁶A regulatory proteins in the process of cadmium-induced renal cell cytotoxicity.

Keywords: Cadmium sulfate; N6-methyladenosine; Nuclear factor-erythroid 2-related factor 2; Oxidative damage; Renal injury.