Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver

MISZCZUK, GISEL S.; BAROSSO, ISMAEL R.; LAROCCA, MARÍA CECILIA; MARRONE, JULIETA; MARINELLI, RAÚL A.; BOAGLIO, ANDREA C.; SÁNCHEZ POZZI, ENRIQUE J.; ROMA, MARCELO G.; CROCENZI, FERNANDO A.

BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR BASIS OF DISEASE

ELSEVIER SCIENCE BV

Año: 2018 vol. 1864 p. 1072 - 1085

0925-4439

Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporterssuch as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanismsgoverning this process are unknown. We characterized this process in estradiol 17 β-d-glucuronide (E17G)-inducedcholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors ofclathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K⁠+ depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP andMRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC).Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significantincrease in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CMEmachinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-inducedendocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bileflow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, whichwere mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft(clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruitedfor CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of thecanalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raftto non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.