SMAD4 Inhibits HO-1–Driven Ferroptosis Through Transcriptional Repression in Sepsis-Associated Acute Kidney Injury

Background: Sepsis-associated acute kidney injury (SA-AKI) is a frequent and severe complication in critically ill patients, yet effective targeted therapies are lacking. Ferroptosis has been implicated in various forms of organ injury, but its role in SA-AKI and underlying regulatory mechanisms remain unclear. Methods: A SA-AKI mouse model was established using cecal ligation and puncture (CLP). Renal histopathology, kidney function assays, and spatial proteomics were employed to assess ferroptosis activation. In vivo and in vitro models were subjected to lipopolysaccharide (LPS) stimulation to evaluate ferroptosis-related markers, including reactive oxygen species (ROS), lipid peroxidation, ferrous iron levels, and mitochondrial membrane potential. DNA pull-down coupled with mass spectrometry identified potential upstream regulators of HO-1. Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) and dual-luciferase reporter assays were used to validate transcriptional regulation by SMAD4. Functional studies assessed the impact of SMAD4 on HO-1 expression, ferroptosis, and renal function. Results: Ferroptosis was markedly activated during SA-AKI progression. LPS stimulation induced significant ROS accumulation, lipid peroxidation, elevated ferrous iron levels, mitochondrial membrane potential disruption, and robust upregulation of heme oxygenase-1 (HO-1). SMAD4 was identified as a transcriptional repressor of HO-1. ChIP-qPCR and dual-luciferase assays confirmed SMAD4 binding to the HO-1 promoter and suppression of its transcription. SMAD4 overexpression reduced HO-1 expression, alleviated ferroptosis, and improved renal function in both in vivo and in vitro models. Conclusions: SMAD4 mitigates ferroptosis by transcriptionally repressing HO-1, exerting a protective effect in SA-AKI. This study identifies a novel SMAD4–HO-1 regulatory axis and suggests a potential therapeutic target for sepsis-induced kidney injury. Antioxid. Redox Signal. 00, 000–000.