Background: Sepsis-induced acute kidney injury (AKI) is a severe clinical complication characterized by tubular epithelial cell damage and mitochondrial dysfunction. This study aims to elucidate the mechanism by which methylation of CCCTC-binding factor (CTCF) regulates the E1A binding protein p300 (EP300)/methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2 (MTHFD2) axis and, as a result, impacts mitochondrial function during septic tubular injury.

Methods: Human renal proximal tubular epithelial HK-2 cells were treated with lipopolysaccharide (LPS) to mimic septic conditions. The regulatory relationship between CTCF methylation and the EP300/MTHFD2 axis was analyzed using luciferase reporter assays, chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), and methylation-specific PCR (MSP). Cell viability, apoptosis, reactive oxygen species (ROS) levels, and mitochondrial membrane potential were assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, flow cytometry, and JC-1 staining, respectively.

Results: CTCF interacted with EP300 and impacted the expression of MTHFD2 (p < 0.01). Knockdown of CTCF resulted in MTHFD2 downregulation and decreased H3K27ac enrichment at the MTHFD2 promoter (p < 0.001). EP300 regulated MTHFD2 transcription by promoting histone H3 lysine 27 acetylation (H3K27ac) at its promoter (p < 0.05). Functional experiments demonstrated that CTCF silencing exacerbated LPS-induced weakened viability, increased apoptosis, elevated ROS production, and decreased mitochondrial membrane potential (p < 0.001). Notably, EP300 overexpression reversed these pathological changes (p < 0.001). Furthermore, the methylation status of the CTCF promoter was influenced by MTHFD2 expression (p < 0.05).

Conclusion: This in vitro study reveals a novel regulatory axis in which methylation of CTCF modulates the EP300/MTHFD2 pathway, which may contribute to mitochondrial dysfunction in tubular epithelial cells (HK-2) under septic conditions.