Internalization of canalicular transporters in estradiol 17ß-Dglucuronide-induced cholestasis involves shift from ?raft? to ?non-raft? membrane domains and clathrin-mediated endocytosis

MISZCZUK, GISEL S.; BAROSSO, ISMAEL R.; ANDERMATTEN, ROMINA; LAROCCA, MARÍA CECILIA; MARRONE, JULIETA; MARINELLI, RAUL A.; BOAGLIO, ANDREA C.; SANCHEZ POZZI, ENRIQUE J.; ROMA, MARCELO GABRIEL; CROCENZI, FERNANDO A.

Paris

Congreso; International Liver Congress; 2018

EASL

Background and Aims: Impaired canalicular secretion due to exacerbated endocytosis of canalicular carriers relevant to bile formation, such as BsepandMrp2, main, common pathomechanism in both experimental and human cholestasis. Nevertheless, the mechanisms governing this process are unknown. We studied the involvement of clathrin- and caveolin-mediated endocytosis in estradiol 17β-D-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Method: E17G was administered to isolated rat hepatocyte couplets (IRHC), sandwich-cultured rat hepatocytes (SCRH), isolated and perfused rat livers (IPRL), and whole rats. Clathrin involvement in E17G-induced endocytosis of Bsep and Mrp2 was evaluated in IRHC by using either the inhibitor of clathrin-mediated endocytosis(CME), monodansylcadaverine (MDC), or a K+ depletion protocol, whereas caveolin involvement was evaluated by using the specific inhibitors, filipin and genistein. In SCRH,CME was blocked by knockdown of the clathrin adaptor protein AP2 with specific siRNAs. Bsep/Mrp2 localization was studied in IRHC by immunofluorescence staining followed by confocal microscopy, and statistical comparison of the densitometric profiles along a line perpendicular to the canaliculus (Mann-Whitney test).Colocalization of Mrp2 with the components of CME machinery AP2, Rab5, and clathrin was evaluated in SCRH in confocal images, by using the plugins ?Image calculator? and ?AND?. In liver tissue, distribution of Bsep and Mrp2 between caveolin enriched, ?raft? plasma membrane microdomains and clathrin enriched, ?non-raft? ones was assessed by western blot of these carriers in ?raft? and ?non-raft? fractions obtained by detergent treatment of highly purified plasma membranes.Results: MDC and K+ depletion, but not filipin or genistein, prevented E17G-induced endocytosis and the functional failure of Bsep and Mrp2 in IRHC. Immunofluorescence and confocal microscopy studies showed that,in E17G-treated IRHC,there was a significant increase in colocalization of Mrp2 with clathrin, AP2, and Rab5. Knockdown of AP2 in SCRH completely prevented E17G-induced Bsep and Mrp2 endocytosis. MDC significantly counteracted the impairment in bile flow and biliary output of Bsep and Mrp2 substrates, as well as the endocytosis of these carriers induced by E17G in IPRL. Bsep and Mrp2, which were mostly present in ?raft? membrane domains in control rats, were largely redistributed to ?non-raft? domains in livers from E17G-treated rats, from where they can be readily recruited for CME.Conclusion: Clathrin-mediated endocytosis is involved in the internalization of Bsep and Mrp2 in E17G-induced cholestasis. The shift of these transporters from ?raft? to ?non-raft? lipid domains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.