Abstract

Background: Iron-catalyzed formation of reactive oxygen species (ROS) increases after APAP overdose and triggers the mitochondrial permeability transition (MPT). Previous studies show that iron translocation from lysosomes into mitochondria by MCU in hepatocytes promotes the MPT after APAP. Kupffer cells are liver resident macrophages that are involved in uptake, processing, and export of iron. Here, our Aim was to investigate and compare the roles of MCU in hepatocytes and Kupffer cells in APAP hepatotoxicity.

Methods: Hepatocyte specific MCU knockout mice (hsMCU KO) and Kupffer cell specific MCU knockout mice (ksMCU KO) were created by individual crosses of floxed MCU mice with Alb-Cre mice and Clec4f-Cre-tdTomato mice, respectively. MCU deficiency in hepatocytes and Kupffer cells was confirmed by Western blot or immunocytochemistry. hsMCU KO, ksMCU KO, and widetype (WT) mice were treated with 300 mg/kg APAP or vehicle. Liver injury was assessed by histology and serum ALT at 24 h.

Results: Western blots confirmed the absence of MCU protein expression in isolated hepatocytes from hsMCU KO mice. MCU protein remained expressed in the kidneys of hsMCU KO mice. The absence of MCU protein expression in Kupffer cells of ksMCU KO mice was confirmed by immunocytochemistry. In hsMCU WT mice, overdose of APAP increased serum ALT (9398 U/L) and centrilobular necrosis (55%) at 24 h, indicating severe liver injury. By contrast in hsMCU KO mice, ALT and hepatic necrosis were decreased to 4104 U/L and 24%, respectively. However, ALT and hepatic necrosis between ksMCU KO and the corresponding WT mice at 24 h after overdose APAP were not significantly different. CYP2E1 expression in hsMCU KO mice was similar to WT mice, indicating the protection against APAP-induced hepatic injury in MCU deficient hepatocytes was not the result of decreased APAP metabolism by CYP2E1.

Conclusion: The core protein of the mitochondrial calcium uniporter complex, MCU, specifically located in hepatocytes, but not in Kupffer cells, plays a central role in APAP-induced hepatotoxicity.