miR-145-5p Targets KLF4 to Regulate the SIRT3/GPX4 Axis, Mediating Ferroptosis and Exacerbating Tubular Epithelial...

To investigate whether miR-145-5p regulates high-glucose-induced ferroptosis and injury in renal tubular epithelial cells through the KLF4/SIRT3/GPX4 signaling axis. Ferroptosis, a regulated form of iron-dependent cell death, has been increasingly implicated in DKD pathogenesis. The present investigation was designed to explore the functional significance and underlying molecular mechanisms of the miR-145-5p/KLF4/SIRT3/GPX4 signalling cascade in ferroptotic cell death of renal tubular epithelial cells during DKD.

A high-glucose-stimulated in vitro DKD model was constructed using human renal tubular epithelial cells (HK-2) exposed to 25.0 mmol/L glucose. Gene and protein expression profiles were characterised through RT-qPCR, Western blotting and immunofluorescence staining. Cellular viability, apoptotic rates and ferroptosis-associated biomarkers were quantified using CCK-8 assay, flow cytometric analysis, ELISA and JC-1 mitochondrial probe, respectively. Molecular binding interactions were confirmed through dual luciferase reporter assays and co-immunoprecipitation experiments. Intracellular reduced glutathione (GSH) content and GPX4 enzymatic activity were additionally measured to evaluate the functional status of the antioxidant arm of ferroptosis.

High glucose exposure triggered time-dependent cellular damage and ferroptotic responses in HK-2 cells, characterised by elevated miR-145-5p levels alongside diminished KLF4, SIRT3 and GPX4 expression. Forced expression of miR-145-5p aggravated cellular damage and ferroptotic phenotypes, whilst its functional suppression conferred cytoprotection. Mechanistic analyses demonstrated that miR-145-5p directly engages the 3'-UTR of KLF4 to repress its expression. Restoring KLF4 expression attenuated high-glucose-mediated cellular injury and enhanced SIRT3 and GPX4 levels. Co-immunoprecipitation assays verified a physical protein-protein association between KLF4 and SIRT3. Functionally, HG stimulation reduced intracellular GSH content and GPX4 enzymatic activity. These changes were aggravated by miR-145-5p overexpression but were partially reversed by miR-145-5p inhibition or KLF4 overexpression.

In the context of hyperglycemia, miR-145-5p facilitates ferroptotic cell death in renal tubular epithelial cells through KLF4 suppression, consequently attenuating the SIRT3/GPX4 signalling cascade and worsening DKD-related cellular injury. This regulatory axis may constitute a promising molecular intervention target for DKD treatment.