Comprehensive analysis of mode of action for cadmium-induced renal tubular dysfunction: a case study integrating high-throughput sequencing, targeted cellular assays, and human data

• Two MOAs for Cd-induced renal tubular dysfunction were proposed. • The confidence of MOAs for Cd-induced renal tubular dysfunction was validated. • The perturbation of OPN/PI3K signaling pathway was confirmed as the earliest KE. • The PoD for Cd-induced renal tubular dysfunction was 0.20 μg/g. • This study offers a reference example for MOA-based risk assessment frameworks. Environmental cadmium (Cd) is a toxic heavy metal known to induce renal tubular dysfunction. Although a provisional tolerable monthly intake (PTMI) of 25 μg/kg body weight has been established as a health-based guidance value for Cd exposure, renal damage may still occur at lower exposure levels. This study employed a mode of action (MOA)-based framework to explore more sensitive and reliable toxicological endpoints for Cd-induced renal tubular dysfunction. Differentially expressed genes (DEGs) related to Cd-induced renal injury were identified by integrating data from the GEO and CTD databases. Enrichment analyses were performed using the ’ClusterProfiler’ R package, and protein–protein interaction (PPI) networks were constructed using STRING and Matascape. Two MOA frameworks (MOA-Kidney-1 and MOA-Kidney-2) were proposed, encompassing key events (KEs) such as elevated ROS levels, activation of the p53 signaling pathway, cell cycle arrest, apoptosis, activation of the OPN/PI3K signaling pathway, activation of the NF-κB/NLRP3 signaling pathway, and cellular inflammation/pyroptosis. High-throughput RNA sequencing and human renal tubular epithelial cell-based assays validated the essentiality and dose-/time-response consistency of these KEs. Among them, the OPN/PI3K signaling pathway was identified as the earliest toxicological perturbation, with urinary OPN levels showing a significant positive correlation with renal injury biomarkers in Cd-exposed populations ( p < 0.05). This pathway also demonstrated the lowest point of departure (PoD) concentration of 0.20 μg/g. In summary, our research provides scientific data to support the refinement of health risk assessments for Cd and offers a reference paradigm for MOA-based risk assessment frameworks.