TAUROURSODESOXICOLATE (TUDC) PREVENTS ACTIVATION OF THE PRO-CHOLESTATIC SIGNALLING PATHWAYS, cPKC AND PI3K/Akt, IN ESTRADIOL 17b-D-GLUCURONIDE (E217G)-INDUCED CHOLESTASIS

BOAGLIO, ANDREA; MISZCZUK, GISEL S.; BAROSSO, ISMAEL R.; TOLEDO, FLAVIA D.; CROCENZI, FERNANDO A.; ROMA, MARCELO G.

Londres

Congreso; 49th annual meeting of the European Association for the Study of the Liver; 2014

Background and Aims: E217G induces cholestasis by triggering endocytosis and further intracellular retention of Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy induced cholestasis, associated with high E217G levels, is routinely treated with ursodeoxycholic acid (UDCA). Since protective UDCA action mechanisms in E217G-induced cholestasis have not been elucidated, we ascertained here whether TUDC, the main, active UDCA metabolite, prevents activation of cPKC and PI3K/Akt. Methods: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membranebound and phosphorylated form, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing canalicular vacuolar accumulation (CVA) of their fluorescent substrates, CGamF and GS-MF, respectively. We also studied the preventive mechanisms of TUDC in E217G-induced cholestasis in isolated, perfused rat liver (IPRL), and Bsep/Mrp2 localization by immunofluorescence. Results: E217G (200 mM) activated both cPKC and PI3K/Akt. Pretreatment with TUDC (100 mM) prevented activation of cPKC (−34±4%) and PI3K/Akt (−37±2%); p < 0.05 vs. E217G alone. The E217G-mediated decrease in CVA of cGamF (−59±2%) and GSMF (−55±3%) was partially prevented by TUDC (+72±6%) and (+78±3%); p < 0.05 vs. E217G alone. In IPRL, E217G (2 mmol/liver) induced an acute decrease in bile flow (−59±14%; p < 0.001), which was completely prevented by TUDC (p < 0.001). A similar behaviour was observed for the biliary excretion of the Bsep and Mrp2 substrates, TC and DNP-SG, respectively. Immunofluorescence revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. Conclusions: TUDC restores function and localization of Bsep/Mrp2 as impaired by E217G, by preventing both cPKC and PI3K/Akt activation.