Effects of Proinflammatory Cytokines on Rat Organic Anion Transporters During Toxic Liver Injury and Cholestasis

Abstract

Hepatobiliary transporters are down–regulated in toxic and cholestatic liver injury. Cytokines such as tumor necrosis factor α (TNF–α) and interleukin 1β (IL–1β) are attributed to mediate this regulation, but their particular contribution in vivo is still unknown. Thus, we studied the molecular mechanisms by which Ntcp, Oatp1, Oatp2, and Mrp2 are regulated by proinflammatory cytokines during liver injury. Rats were injected intraperitoneally with either carbon tetrachloride or endotoxin. Inactivation of TNF–α and IL–1β was achieved by repetitive intraperitoneal injection of etanercept and anakinra, respectively. Messenger RNA (mRNA) levels of transporters and binding activities as well as nuclear protein levels of Ntcp, Oatp2, and Mrp2 transactivators were determined 20 to 24 hours later. In contrast to IL–1β, TNF–α inactivation alone fully prevented down–regulation of Ntcp, Oatp1, and Oatp2 mRNA as well as reduced binding activity of hepatocyte nuclear factor 1 (HNF–1) in CCl₄–induced toxic injury. In endotoxemia, down–regulation of Mrp2, and partially in case of Ntcp, could be prevented by IL–1β but not TNF–α blockade. However, inactivation of either cytokine led to preservation of HNF1 and partially of retinoid X receptor/retinoic acid receptor (RXR/RAR) binding activity. No effect of anticytokines was seen on pregnane X receptor (PXR) and constitutive androstane receptor (CAR) binding activity as well as nuclear protein mass. In conclusion, TNF–α represents the master cytokine responsible for HNF1–dependent down–regulation of Ntcp, Oatp1, and Oatp2 in CCl₄–induced toxic liver injury. IL–1β predominates in a complex signaling network of Ntcp and Mrp2 regulation in cholestatic liver injury. In contrast to in vitro studies, HNF1 and RXR/RAR–independent mechanisms appear to be more important in regulation of Mrp2 and Ntcp gene expression in endotoxemia.