RIPK4-p53 interaction drives aflatoxin B1-induced renal mitochondrial apoptosis via Ser15 phosphorylation: A CRISPR-Cas9 mechanistic study.

Aflatoxin B1 (AFB1), recognized as a highly toxic and carcinogenic mycotoxin, contaminates more than 25 % of the global grain supply, thereby presenting a substantial public health threat and posing significant risks to renal health. However, the host factors that mediate the associated damage remain inadequately understood. This study aimed to identify key host factors in AFB1-induced cytotoxicity using a genome-wide CRISPR/Cas9 screen and elucidate the underlying molecular mechanisms. We developed a porcine kidney epithelial (PK15) cell model, followed by knockout validation, CCK-8 assays, qRT-PCR, western blotting, AO-EB staining, flow cytometry, and co-immunoprecipitation to dissect mechanistic pathways. Receptor-Interacting Protein Kinase 4 (RIPK4) was identified as a critical pro-apoptotic factor. RIPK4 knockout increased PK15 cell viability by ~50 % (P < 0.001) and reduced apoptosis by ~44 %(P < 0.001), accompanied by downregulation of APAF1, Cyt-c, cleaved-Caspase-9/-3, and p53 Ser15 phosphorylation, and upregulation of Bcl-2. Mechanistically, RIPK4 directly interacted with p53 via its N-terminal 1-490 aa region, enhancing its phosphorylation and pro-apoptotic activity. In conclusion, RIPK4 promotes AFB1 nephrotoxicity by activating p53-mediated mitochondrial apoptosis, identifying it as a novel therapeutic target. Future studies should validate these findings in vivo models and explore the potential of RIPK4-specific inhibitors for mitigating nephrotoxicity.