An AOP-guided in vitro platform for mechanistic screening of platinum-based nephrotoxicity

Mechanistically grounded, human-relevant new approach methodologies (NAMs) are needed to support AOP-informed, animal-free strategies for kidney safety assessment and to improve the prediction of nephrotoxic risk. Here, we developed a mechanistically anchored in vitro assay battery to quantify functional readouts aligned with key events (KEs) central to kidney adverse outcome pathways (AOPs). Using human conditionally immortalised proximal tubule epithelial cells (ciPTECs), we assessed mitochondrial depolarisation, reactive oxygen species (ROS) generation, lysosomal enzyme release, membrane integrity, and cell viability in response to cisplatin, carboplatin, and oxaliplatin. All three compounds produced concentration- and time-dependent effects consistent with their clinical nephrotoxic profiles. Mitochondrial depolarisation emerged as the most sensitive and consistent key event (KE) in ciPTECs, while ROS generation was compound-dependent, robust for cisplatin, moderate for oxaliplatin, and minimal for carboplatin. Later endpoints, including increased β‑N‑acetylglucosaminidase (NAG) activity, lactate dehydrogenase (LDH) release, and loss of cell viability, required higher concentrations to reach significance. These findings identify an apparent sensitivity order among KE-aligned endpoints and provide quantitative data consistent with established mechanisms of nephrotoxicity. These data provide quantitative, KE-aligned response profiles that may support future key event relationship (KER) and quantitative (qAOP) development and demonstrate the utility of an AOP-informed assay battery for mechanistic screening and prioritisation within a focused platinum nephrotoxicity case study.