Background: Sepsis-associated acute kidney injury (SA-AKI) is a critical clinical complication with high mortality, involving ferroptosis and mitochondrial dysfunction, although the regulatory mechanisms remain incompletely defined. E3 ubiquitin ligases play key roles in post-translational regulation of cell death pathways, but their specific involvement in SA-AKI-associated ferroptosis is poorly understood. This study aimed to investigate the role and mechanism of the E3 ubiquitin ligase mind bomb 2 (MIB2) in SA-AKI, focusing on its regulation of ferroptosis and mitochondrial function via glutathione peroxidase 4 (GPX4).

Methods and Materials: An SA-AKI model was established by treating HK-2 cells with different concentrations of lipopolysaccharide (LPS). Cell viability was detected using the cell counting kit-8 (CCK-8) assay. The mRNA and protein expression levels of MIB2 and GPX4 were detected using reverse-transcription polymerase chain reaction (RT-qPCR) and Western blotting (WB). The interaction between MIB2 and GPX4 was verified by co-immunoprecipitation (Co-IP). An MIB2 knockdown model was constructed by means of sh-MIB2 transfection. The MIB2 knockdown model treated with ras-selective lethal small molecule 3 (RSL3), a GPX4 inhibitor, was subjected to the detection of reactive oxygen species (ROS), glutathione (GSH), Fe²⁺, and malondialdehyde (MDA) by means of flow cytometry and assay kits. To evaluate mitochondrial function, the mitochondrial morphology, membrane potential, and adenosine triphosphate (ATP) content were analyzed via transmission electron microscopy, JC-1 staining, and chemiluminescence.

Results: LPS reduced the viability of HK-2 cells in a manner dependent on both concentration and time, significantly increased the mRNA and protein levels of MIB2 (p < 0.001), and simultaneously decreased the mRNA and protein levels of GPX4 (p < 0.001). Co-IP confirmed a direct interaction between MIB2 and GPX4. Knocking down MIB2 could significantly reverse the down-regulation of GPX4 induced by LPS (p < 0.001), and reduced the levels of intracellular reactive oxygen species (ROS), Fe²⁺ and MDA, accompanied by glutathione (GSH) depletion and lipid ROS accumulation. MIB2 knockdown also improved mitochondrial morphological aberrations (increased proportion and length of intact cristae), increased mitochondrial membrane potential and ATP content, and enhanced cell viability (p < 0.001). Treatment with RSL3 could reverse the up-regulation effect of MIB2 knockdown on GPX4 and the protective effects on ferroptosis and mitochondrial function.

Conclusions: The E3 ubiquitin ligase MIB2 induces ferroptosis and mitochondrial dysfunction in HK-2 cells by binding to GPX4 and promoting its degradation, thus exacerbating SA-AKI. MIB2 knockdown can alleviate these pathological processes by stabilizing GPX4.