Advanced glycation end products promote ferroptotic injury in human renal tubular epithelial cells through the miR-362-3p/SLC7A11/GPX4 Axis.

Abstract

Diabetic nephropathy (DN) is a major cause of chronic kidney disease, yet the mechanisms underlying tubular epithelial injury remain incompletely understood. Advanced glycation end products (AGEs) contribute to diabetic renal damage, but their role in ferroptosis-associated tubular injury is not fully defined. This study investigated whether AGE-induced injury in human renal tubular epithelial cells (RTECs) involves dysregulation of the miR-362-3p/SLC7A11/GPX4 antioxidant axis. Human RTECs were exposed to AGE to establish an in vitro DN-like model. Cell viability, apoptosis, glutathione (GSH), reactive oxygen species (ROS), lipid peroxidation, and intracellular Fe2+ were assessed using complementary biochemical, fluorescence, and flow-cytometric assays. Ferroptosis-associated injury was evaluated using ferrostatin-1 (Fer-1), with RSL3 included as an independent positive control. Bioinformatic analysis of GSE51674, ENCORI target prediction, dual-luciferase reporter assays, miR-362-3p gain- and loss-of-function studies, and SLC7A11-dependent rescue experiments were used to define pathway causality. AGE exposure reduced RTEC viability, depleted GSH, increased ROS, lipid peroxidation and Fe2+ accumulation, and suppressed SLC7A11/GPX4-associated antioxidant signaling. Fer-1 attenuated these alterations, while RSL3 produced a comparable ferroptosis-associated phenotype. miR-362-3p was identified as a DN-associated SLC7A11-targeting miRNA and was increased in an independent clinical serum cohort. miR-362-3p overexpression aggravated AGE-induced injury, whereas its inhibition restored SLC7A11/GPX4 signaling and reduced ferroptosis-associated stress. Importantly, SLC7A11 knockdown substantially abolished the protective effects of miR-362-3p inhibition. AGE also activated apoptotic signaling, indicating coexistence of multiple regulated injury pathways. Collectively, these findings support a functional miR-362-3p/SLC7A11/GPX4 axis in AGE-induced ferroptosis-associated tubular injury and provide a mechanistic basis for further in vivo and clinical validation.