Ferroptosis is an iron-dependent regulated cell death pathway increasingly implicated in diabetic kidney disease (DKD). This study sought to determine whether miR-145-5p regulates high-glucose-induced ferroptosis and injury in renal tubular epithelial cells via modulation of the KLF4/SIRT3/GPX4 signaling cascade. The aim was to define functional significance and molecular mechanisms of the miR-145-5p/KLF4/SIRT3/GPX4 axis in ferroptotic cell death during DKD.
An in vitro DKD model was established using human renal proximal tubular epithelial HK-2 cells exposed to 25.0 mmol/L glucose to simulate hyperglycemia. Gene expression and protein abundance were assessed using RT-qPCR, Western blotting and immunofluorescence. Cell viability and apoptotic rates were measured by CCK-8 assay and flow cytometry respectively. Ferroptosis-associated readouts included ELISA-based measurements of malondialdehyde (MDA), Fe2+, reactive oxygen species (ROS) and ATP, JC-1 mitochondrial membrane potential probe, intracellular reduced glutathione (GSH) quantification, and GPX4 enzymatic activity assay.
Molecular binding and interactions were tested by dual-luciferase reporter assays to probe miRNA–3'-UTR binding and by co-immunoprecipitation (co-IP) to evaluate protein–protein association between KLF4 and SIRT3. miR-145-5p was experimentally manipulated through overexpression and inhibition; KLF4 expression was restored via overexpression constructs in selected experiments. All experimental groups were repeated in triplicate as reported.
Exposure to high glucose produced time-dependent cellular injury in HK-2 cells. High-glucose (HG) conditions led to progressive reductions in cell viability and increases in apoptosis across measured time points. Concurrently, RT-qPCR and Western blot analyses documented an increase in miR-145-5p expression and declines in KLF4, SIRT3, and GPX4 transcript and protein levels over time. HG-treated cells showed decreases in intracellular GSH content and GPX4 enzymatic activity, consistent with impaired antioxidant capacity.
Forced overexpression of miR-145-5p in HG-treated HK-2 cells exacerbated cellular injury: viability fell and apoptosis increased relative to controls. Overexpression also intensified ferroptosis-related changes including higher MDA, Fe2+, and ROS levels and further reductions in ATP, GSH and GPX4 activity. By contrast, inhibition of miR-145-5p conferred measurable cytoprotection under HG conditions, partially reversing the biochemical and functional markers of ferroptosis and improving cell survival metrics.
Dual-luciferase reporter assays demonstrated that miR-145-5p directly binds the 3'-UTR of KLF4, leading to repression of KLF4 expression. Restoration of KLF4 expression attenuated HG-mediated cellular injury and increased levels of SIRT3 and GPX4, indicating that KLF4 positively influences the downstream antioxidant axis. Co-immunoprecipitation experiments provided evidence of a physical protein–protein association between KLF4 and SIRT3, supporting a mechanistic link through which KLF4 may regulate SIRT3 function.
HG stimulation induced multiple ferroptotic phenotypes in HK-2 cells: elevations in lipid peroxidation (MDA), labile iron (Fe2+) and ROS, with declines in cellular ATP. JC-1 mitochondrial assays indicated loss of mitochondrial membrane potential under HG conditions, consistent with mitochondrial dysfunction during ferroptosis. Importantly, intracellular reduced GSH and GPX4 enzymatic activity — key elements of the canonical ferroptosis defense — were decreased by HG exposure; these deficits were worsened by miR-145-5p overexpression and ameliorated by miR-145-5p inhibition or KLF4 restoration.
Functionally, reintroducing KLF4 into HG-exposed HK-2 cells reduced apoptotic and ferroptotic phenotypes and restored SIRT3 and GPX4 expression. These changes correlated with partial recovery of intracellular GSH and GPX4 activity, indicating that KLF4 lies upstream of the SIRT3/GPX4 antioxidant axis and that its suppression by miR-145-5p contributes to the vulnerability of tubular epithelial cells to ferroptotic death under hyperglycemic stress.
The data support a model in which hyperglycemia upregulates miR-145-5p, which directly targets KLF4 to suppress its expression. Loss of KLF4 is associated with reduced SIRT3 and GPX4, weakening cellular antioxidant defenses (GSH/GPX4) and promoting ferroptosis in renal tubular epithelial cells. Experimentally manipulating miR-145-5p or restoring KLF4 mitigated these effects in vitro, indicating that the miR-145-5p/KLF4/SIRT3/GPX4 axis may represent a molecular target for interventions aimed at reducing ferroptotic tubular injury in DKD.
The authors conclude that miR-145-5p facilitates ferroptotic cell death in renal tubular epithelial cells by suppressing KLF4, thereby attenuating the SIRT3/GPX4 signaling cascade and exacerbating DKD-related cellular injury. They propose that targeting this regulatory axis could offer therapeutic benefit in DKD.
The authors declared no conflicts of interest.