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

• Published in: Life sciences (1973) 2024-09, Vol.352, p.122839, Article 122839
• Main Authors: Medeot, Anabela C., Boaglio, Andrea C., Salas, Gimena, Maidagan, Paula M., Miszczuk, Gisel S., Barosso, Ismael R., Sánchez Pozzi, Enrique J., Crocenzi, Fernando A., Roma, Marcelo G.
• Format: Article
• Language: English
• Subjects: bile; Bsep; Cholestasis; endocytosis; estradiol; Estradiol 17β-d-glucuronide; excretion; fluorescence; fluorescent antibody technique; hepatocytes; immunoblotting; metabolites; Mrp2; rats; Tauroursodeoxycholate; Ursodeoxycholic acid
• ISSN: 0024-3205; 1879-0631; 1879-0631
• DOI: 10.1016/j.lfs.2024.122839

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. 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. 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. 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. [Display omitted]