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

Abstract

Aims: Estradiol 17β-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.

Keywords: Bsep; Cholestasis; Estradiol 17β-d-glucuronide; Mrp2; Tauroursodeoxycholate; Ursodeoxycholic acid.