JAK2/STAT3-dependent regulation of MDM4/MDM2-p53 signaling in methotrexate-induced ferroptosis and nephrotoxicity

Methotrexate (MTX), a cornerstone therapeutic agent for malignancies and autoimmune diseases, is clinically constrained by its severe nephrotoxic effects. Although oxidative stress and apoptosis have been implicated in MTX-induced nephrotoxicity, the precise molecular mechanisms underlying this process remain incompletely characterized. This study investigates ferroptosis as a novel pathological contributor to MTX-induced nephrotoxicity and evaluates therapeutic interventions targeting the JAK2/STAT3-MDM4/MDM2 signaling axis. Through integrated approaches including RNA sequencing, lentiviral-mediated knockdown experiments (MTX: IC₂₀ 38 μM), and a rat model of MTX (20 mg/kg)-induced acute kidney injury, we demonstrated that MTX treatment upregulated MDM4 expression, activated the JAK2/STAT3 signaling pathway, and enhanced MDM4/MDM2 heterodimer formation, thereby suppressing p53 and contributing to ferroptotic cell death. Importantly, either the knockdown of MDM4 or pharmacological inhibition of JAK2/STAT3 signaling pathway with JSI-124 partially attenuated MTX-induced ferroptosis, improved renal function indicators, and attenuated histopathological damage in vivo. Our findings demonstrate that MTX mediates phosphorylation-dependent activation of the JAK2/STAT3 pathway, which facilitates MDM4/MDM2 interaction to induce ferroptosis-associated nephrotoxicity. These findings support a role for JAK2/STAT3-MDM4/MDM2 signaling in MTX-induced ferroptosis and suggest that targeted inhibition of this axis may represent a potential nephroprotective strategy.