Overexpression of FTO inhibits ferroptosis induced by OGD/R in mouse renal tubular epithelial cells

Abstract Ischemia-reperfusion (I/R) is recognized as one of the major causes of acute kidney injury (AKI), yet its underlying mechanism remains unclear. This study aimed to investigate the molecular mechanism by which FTO alleviates AKI through activating the NRF2/SLC7A11/GPX4 pathway to inhibit ferroptosis. AKI model induced by renal I/R in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) cell model were established, and FTO was overexpressed in the cells and treated with NRF2 inhibitory ML-385 and ferroptosis activator. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), serum creatinine (Scr), and blood urea nitrogen (BUN) in serum and cell supernatant were detected by ELISA. Western blot was performed to detect the protein expression of m6A modification enzymes (METTL3, METTL14, WTAP, FTO, ALKBH5) and ferroptosis-related pathway proteins (NRF2, SLC7A11, GPX4) in both in vivo and in vitro models. RT-qPCR was used to measure the mRNA levels of KIM1 and NGAL in cells. Subsequently, FTO-overexpressing lentivirus was transfected into OGD/R-induced cells, and cell viability, apoptosis rate, Fe 2+ concentration, and levels of oxidative stress markers (ROS and MDA) were detected. Finally, Western blot was used to assess the protein expression of NRF2, SLC7A11, and GPX4 in each group. Compared with the control group, the protein expression of METTL3, METTL14, WTAP, and ALKBH5 was significantly upregulated, while FTO was markedly downregulated in both in vivo and in vitro models of renal injury, with FTO showing the most prominent expression difference ( P < 0.05). The levels of TNF-α, IL-6, IL-1β, Scr, and BUN were significantly increased in the in vitro and in vivo models ( P < 0.05). In the OGD/R group, compared with the control group, KIM1 and NGAL mRNA levels were significantly increased, accompanied by decreased cell viability, increased apoptosis rate, elevated levels of Fe 2+ , ROS, and MDA, and reduced SOD activity ( P < 0.05). Meanwhile, the protein levels of NRF2, SLC7A11, and GPX4 were also significantly decreased ( P < 0.05). No significant differences were observed between the OGD/R group and the empty vector-overexpressing group. However, FTO overexpression significantly reversed all these aforementioned phenomena ( P < 0.05). Compared with the OE-FTO group, ML-385 and erastin could significantly weaken the protective effect of overexpressed FTO ( P < 0.05). FTO overexpression inhibits ferroptosis by activating the NRF2/SLC7A11/GPX4 pathway and reduces OGD/ R-induced cell injury. FTO may be a potential target for regulating ferroptosis in AKI.