Tauroursodeoxycholate prevents estradiol 17ß-d-glucuronide-induced cholestasis and endocytosis of canalicular transporters by switching off pro-cholestatic signaling pathways.

AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.

NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis.

The endogenous metabolite of estradiol, estradiol 17ß-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.