Integrated network toxicology and experimental validation reveal nephrotoxic effects of acetyl tributyl citrate in HK-2 cells

Acetyl tributyl citrate (ATBC) is an eco-friendly plasticizer widely used in plastic products, yet its potential nephrotoxicity remains unclear. This study integrated network toxicology, molecular docking, and molecular dynamics (MD) simulations to identify putative nephrotoxic targets and mechanisms of ATBC, followed by in vitro validation in HK-2 renal epithelial cells. Network toxicology analysis identified 110 ATBC-related nephrotoxic targets, and protein-protein interaction (PPI) analysis revealed 10 core targets including SRC, HSP90AA1, BCL2, TNF, KRAS, and MAPK1. KEGG analysis showed enrichment in apoptosis, PI3K-Akt, TNF, and MAPK pathways. Molecular docking indicated favorable binding affinities between ATBC and the aforementioned core targets, and 100 ns MD simulations further demonstrated the structural stability of ATBC-protein complexes. In vitro, ATBC exposure significantly decreased HK-2 cell viability, elevated LDH release, enhanced apoptosis, and suppressed proliferation, accompanied by cell cycle arrest and impaired migration and repair. Mechanistically, ATBC activated apoptotic signaling pathways by downregulating BCL2 and upregulating Caspase-3, BAX, FAS, and MAPK1. It also promoted inflammatory responses by increasing the expression of TNFα, IL-6, CXCL2, STAT3, and SRC. Furthermore, ATBC inhibited the PI3K/AKT/mTOR pathway, reduced HSP90AA1 expression, and upregulated KRAS and EGFR, collectively disrupting cellular proliferation, survival, and stress response. Notably, the upregulation of the renal injury biomarker KIM-1 further indicated epithelial tubular damage. These findings suggest that ATBC may induce renal cell injury by promoting apoptosis and inflammation and disrupting survival signaling pathways. This study provides preliminary mechanistic insights into ATBC-induced nephrotoxicity in vitro, offering valuable evidence for its toxicological evaluation and safety regulation.