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)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis.Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K+ depletion, butnot the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis ofBSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocytecouplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC,there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essentialmembers of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completelyprevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and theimpairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEPand MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largelyfound in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can bereadily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for theinternalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of thesetransporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosedunder cholestatic conditions