Co-exposure to benzoic acid and acesulfame K synergistically induces nephrotoxicity through the CTNNB1/STAT3 axis

Benzoic acid (BA) and acesulfame K (ACE) are commonly co-present food additives, yet their combined nephrotoxic risk remains poorly understood. Employing network toxicology, molecular simulations, and in vitro assays, we systematically evaluated their individual and combined effects on nephrotoxicity. Computational predictions indicated that both compounds exhibited nephrotoxic propensity, with shared targets enriched in pathways such as "response to xenobiotic stimulus" and metabolic disruption. Protein-protein interaction network analysis identified CTNNB1 and STAT3 as the respective core targets for BA and ACE, with molecular simulations confirming their stable binding. In vitro experiments showed that BA alone inhibited HK2 cell proliferation, and downregulated β-catenin (encoded by CTNNB1). ACE alone did not significantly suppress proliferation but activated STAT3 signaling. Notably, combined exposure produced a marked synergistic anti-proliferative effect, attributable to a dual mechanism: BA impaired renal tubular repair capacity by suppressing the CTNNB1/β-catenin pathway, while ACE exacerbated inflammatory and injury responses via STAT3 activation, thereby simultaneously impairing repair and exacerbating damage in the kidney. This study is the first to reveal that BA and ACE have the potential to synergistically induce nephrotoxicity via the CTNNB1/STAT3 signaling axis, providing new scientific evidence for the systematic safety assessment of mixed food-additive exposures.