STUDY OF MRP2 IMPAIRMENT INDUCED BY CHOLESTATIC AGENTS IN HEPARG CELL LINE

CIRIACI, NADIA; ANDERMATTEN, ROMINA B.; SCHUCK, VIRGINIA S.; BAROSSO, ISMAEL R.; RUIZ, MARÍA LAURA; SANCHEZ POZZI, ENRIQUE J.

Congreso; Reunión Sociedades de Biociencias 2021; 2021

Sociedad Argentina de Investigación Clínica

Human HepaRG is an in vitro alternative to animal models for biliary excretion studies, due to functional bile canaliculi formation and expression of main canalicular transporters, like multidrug resistance-associated protein 2 (Mrp2). HepaRG differentiation toward biliary and hepatocyte phenotypes requires a culture medium containing fetal bovine serum (FBS) and differentiating agent dimethyl sulfoxide (DMSO) to promote polarization.AIMS: To evaluate hepatocyte markers expression and Mrp2 localization in response to culture media supplemented with local FBS, and to study Mrp2 activity impairment by cholestatic agents.METHOD: Proliferation: cells were cultured 2 weeks (manufacturer´s protocol) in medium supplemented with 10% FBS Internegocios S.A.(Bs. As.). Differentiation: cells were cultured 2 additional weeks increasing DMSO concentrations (0.5, 1, 1.7%). Extraction of total mRNA was performed using Trizol following RT-qPCR. Mrp2 and zonula occludens 1 (ZO1) localization were analyzed by confocal immunofluorescence. Cells were treated with estradiol 17ß-d-glucuronide (E17G 200 µM) and taurolithocholate (TLC 2.5 µM) 20 min and Mrp2 activity was evaluated by secretion of fluorescent methylfluorescein (GMF).RESULTS: HepaRG differentiation was verified by hepatocyte transcript levels at day 28 (fold change (FC) to day 6 proliferation) of aldolase FC=83, cytochrome P450 3A4 FC=47, organic anion transporting polypeptide 2B1 FC=5 and Mrp2 FC=3. Confocal images of differentiated cells showed polarized ZO1 hepatocyte-like distribution and canalicular localization of Mrp2. Mrp2-mediated transport of GMF was decreased (% Control) by E17G (43±8*) and TLC (47±3*) *p