#736 Defective autophagy drives ferroptosis via mitochondrial ROS in diabetic kidney disease

Abstract Background and Aims Ferroptosis is a regulated form of cell death triggered by excessive lipid peroxidation and plays a crucial role in the pathogenesis of acute kidney injury (AKI). While autophagy has been shown to drive ferroptotic cell death in cancer cells, the relationship between ferroptosis and the pathogenesis of diabetic kidney disease (DKD), a condition characterized by defective autophagy, is not clear. Here, we investigated whether autophagy modulates susceptibility to ferroptosis in proximal tubular epithelial cells (PTECs) under DKD conditions. Method We conducted the following investigations: (1) the association of ferroptotic stress and autophagy biomarkers in kidney biopsy samples from patients with DKD , (2) the impact of autophagy on ferroptosis in the context of ischemia-reperfusion (IR) injury toward DKD using ferrostatin-1 (Fer-1) in diabetic mouse models, including streptozotocin-treated type 1 diabetic mice and db/db type 2 diabetic mice, (3) the role of autophagy in ferroptosis in PTECs in vitro, (4) the mechanisms underlying the relationship between autophagy and ferroptosis in PTECs, with a focus on mitochondrial reactive oxygen species (ROS). Results First, analysis of human renal biopsy specimens revealed impaired autophagy and lipid peroxide accumulation, as indicated by increased p62 and 4HNE expression respectively, in PTECs of the diabetic group. Notably, 4HNE expression was positively correlated with p62 accumulation in PTECs. Next, we assessed whether the susceptibility to IR-injury was affected by Atg5 deficiency (Atg5F/F; KAP) in non-diabetic and streptozotocin-treated type 1 diabetic mice. We observed that diabetes and dysregulated autophagy synergistically enhanced IR-induced AKI, which was ameliorated by ferrostatin-1 (Fer-1) treatment. These findings suggest that autophagy deficiency drives ferroptosis induced by IR injury. In db/db type 2 diabetic mice, severe tubular injury was observed following IR injury, but this injury was significantly attenuated by Fer-1 treatment. In addition, rapamycin-mediated improvement of autophagy in db/db mice suppressed IR-induced AKI, demonstrating that improved autophagy attenuates ferroptosis in DKD. Using primary PTECs isolated from Atg5 knockout (Atg5KO) mice, ferroptotic cell death and lipid peroxidation were augmented in Atg5KO cells under erastin (a ferroptosis inducer) treatment. Autophagy deficiency in these cells increased mitochondrial ROS and mitochondrial lipid peroxidation. Importantly, ferroptoric cell death was decreased in Atg5KO Rho0 cells (mitochondrial-DNA/RNA depleted cells), indicating that autophagy protects against ferroptosis by decreasing mitochondrial ROS. Conclusion Defective autophagy enhances susceptibility to ferroptosis in DKD by promoting mitochondrial ROS generation. These findings suggest that therapeutic strategies aimed at improving autophagy may mitigate ferroptosis-related injury in DKD.