Norcantharidin induces nephrotoxicity by disrupting the ATF4/CTH/H2S pathway and promoting apoptosis

Norcantharidin (NCTD) induces nephrotoxicity via mitochondria-dependent apoptosis. • The nephrotoxicity of NCTD involves inhibition of the ATF4/CTH/H 2 S pathway. • NCTD suppresses ATF4-driven CTH expression, reducing H 2 S production. • NCTD reduces H 2 S, impairing Drp1 S-sulfhydration and mitochondrial function. • ATF4, CTH, and H 2 S are potential therapeutic targets for NCTD-induced kidney injury. Norcantharidin serves as a therapeutic agent in the clinical care of malignant neoplasms. However, recent safety alerts issued in China in June 2025 highlighted its association with renal injury. Given that the mechanisms underlying NCTD-induced nephrotoxicity have yet to be completely defined, this work was designed to explore the impact of the ATF4/CTH/H 2 S pathway on nephrotoxicity triggered by NCTD. Our data revealed that exposure to NCTD triggered mitochondria-dependent apoptosis in renal cells, alongside decreased H 2 S levels, ROS accumulation, compromised mitochondrial integrity, and caspase-3 activation. Mechanistically, NCTD inhibited both ATF4 protein levels and its transcriptional function, resulting in downregulation of CTH and a subsequent reduction in H 2 S production. H 2 S deficiency impaired S-sulfhydration of Drp1, enhanced the Drp1-VDAC1 interaction, promoted mPTP opening, and ultimately triggered apoptosis. H 2 S supplementation rescued these defects by restoring Drp1 S-sulfhydration, but this protective effect was completely abolished by DTT, confirming the specificity of S-sulfhydration. Furthermore, overexpression of ATF4 or CTH alleviated NCTD-triggered apoptosis via H 2 S restoration, whereas knockdown of ATF4 or CTH exacerbated cytotoxicity. Critically, CTH knockdown abolished the protective effect of ATF4 overexpression, while CTH overexpression remained functional independently of ATF4. In conclusion, our findings established that NCTD induced nephrotoxicity primarily through the inhibition of the ATF4/CTH/H 2 S axis, which disrupted Drp1-mediated mitochondrial homeostasis. These results identified H 2 S restoration as a promising therapeutic strategy to mitigate NCTD-induced kidney injury.