Inhibition of miR-125b-1-3p alleviates acute kidney injury and mitochondrial membrane potential loss by targeting GSTK1

As a type of regulated necrosis driven by iron-dependent lipid peroxidation, ferroptosis critically contributes to acute kidney injury (AKI). Currently, whether miR-125b-1-3p is engaged in this pathological process remains unknown. Here, miR-125b-1-3p was upregulated in kidney tissues from an AKI model (GSE125305), in serum from a small exploratory cohort (15 AKI patients vs 15 controls), and in hypoxia/reoxygenation (H/R)-treated NRK-52E cells and erastin-induced ferroptosis models (n = 3). Suppression of miR-125b-1-3p with an inhibitor attenuated H/R-induced cellular injury and ferroptosis-associated markers, reflected by enhanced cell viability, upregulated GPX4 expression, and decreased levels of MDA, Fe2+, and ROS. Furthermore, ferrostatin-1 (Fer-1) improved cell viability and reduced MDA and Fe2+, mitigating miR-125b-1-3p mimic-induced injury under H/R. In an ischemia/reperfusion (I/R) mouse model (n = 5/group), miR-125b-1-3p inhibition ameliorated acute kidney injury and associated ferroptosis markers, indicated by reduced serum creatinine and BUN, lower tubular injury scores, and attenuation of ferroptosis-related alterations. Mechanistically, bioinformatics and luciferase assays revealed that miR-125b-1-3p directly targets glutathione S-transferase kappa 1 (GSTK1). Circulating GSTK1 mRNA were reduced in AKI patient serum, and GSTK1 expression was decreased in H/R-treated cells, negatively correlating with miR-125b-1-3p. Overexpression of miR-125b-1-3p reduced GSTK1 protein, while inhibition increased it. Importantly, GSTK1 overexpression attenuated miR-125b-1-3p mimic-induced ferroptosis-associated alterations and mitochondrial membrane potential loss in H/R-treated cells, whereas GSTK1 knockdown blunted the protective effects of miR-125b-1-3p inhibition, restoring MDA and Fe2+ accumulation. Our data indicate that miR-125b-1-3p contributes to ferroptosis-associated tubular cell injury partly by targeting GSTK1, highlighting the miR-125b-1-3p/GSTK1 axis as a candidate regulatory pathway involved in AKI.