Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes

Abstract

The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na(+)-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.

Keywords: HepaRG hepatocytes; hepatobiliary transporters; human hepatocytes; membrane localization; transporter activity.

FIG. 1.

Bile canaliculi networks in HepaRG cell and HH cultures. A, Bile canaliculi structures (arrows) in differentiated HepaRG cells and 4–5 days HHs cultured in sandwich (SCHH) and conventional (CCHH) configuration were observed under phase-contrast microscopy (×20 magnification). B, Pericanalicular F-actin microfilament network after labeling with phalloidin-fluoprobe in HepaRG cells (nuclei stained in using Hoechst dye) and 4–5 days HHs cultured in sandwich and conventional configuration. Imaging was done using Cellomics ArrayScan VTI HCS Reader (ThermoScientific). C, Electron microscopic micrograph of tight junctions surrounding bile canaliculus structures in HepaRG cells (arrows), original magnification × 3000 (bar = 2 μm). H, hepatocyte; BC, bile canaliculus.

FIG. 2.

Bile canaliculi networks in HepaRG cells and primary HHs. Bile canaliculi were labeled using CDFDA, a MRP2 substrate in A, HepaRG cells, SCHHs, and CCHHs. B, The mean surface areas of bile canaliculi networks in HepaRG cells and 4–5 days HHs cultured in sandwich and conventional configuration were estimated by quantifying CDFDA-labeled lumen surfaces using vHCS.scan (V6.2.0) cellomics software (ThermoScientific). Data represent the means ± SD of at least three independent experiments. Data with P < 0.05 is considered significant (*).

FIG. 3.

Bile canaliculi dynamics in HepaRG cells and HHs. Time-lapse imaging of HepaRG cells and 4–5 days HHs cultured in conventional configuration (CCHH). Time-dependent contraction/relaxation activity of bile canaliculi (arrows). Imaging was done using inverted microscope Zeiss Axiovert 200 M and AxioCam MRm.

FIG. 4.

Distribution of main bile acid transporters in HepaRG cells and HHs. Differentiated HepaRG cells and 4–5 days HHs in sandwich (SCHH) and conventional (CCHH) cultures were fixed and incubated with primary antibodies against each of the following hepatobiliary transporters: NTCP, MRP3, BSEP, MRP2, MDR1, and MDR3. Nuclei were labeled using Hoechst. Immunofluorescence images were obtained with a Cellomics ArrayScan VTI HCS Reader (ThermoScientific).

FIG. 5.

Sodium-dependent activity of NTCP in HepaRG cells and HHs. HepaRG cells and 4–5 days HHs in sandwich (SCHH) and conventional (CCHH) cultures were incubated with [³H]-TCA for 30 min in the presence and absence of sodium ions. A, NTCP activity was evaluated through measurement of the radiolabeled substrate TCA accumulated in cellular layers (cells plus bile canaliculi). Uptake of [³H]-TCA in each cell model was expressed relative to the levels found in cells incubated with Na⁺-free buffer, arbitrarily set at a value of 1. B, NTCP activity was evaluated in the presence of Na⁺ as the ratio of total radioactivity measured in cell lysates to total number of hepatocytes in each cell model. Calculations were based on 800 000 hepatocytes in the two primary hepatocyte models and 280 000 HepaRG hepatocytes per well. Results are expressed relative to NTCP activity found in HepaRG hepatocytes, arbitrarily set at a value of 1. Data represent the means ± SD of at least three independent experiments. Data with P < 0.05 are considered significant (*).

FIG. 6.

Functional activity of MRP2 in HepaRG cells and HHs. MRP2 activity was estimated using CDFDA in HepaRG cells and 4–5 days SCHHs and CCHHs. Efflux of fluorescent CDF, a substrate of MRP2, characterized by accumulation of fluorescence into bile canaliculi, was evaluated in standard and Ca²⁺- and Mg²⁺-free buffer in the presence and absence of MK571, an inhibitor of MRP2. Imaging was done using inverted microscope Zeiss Axiovert 200 M and AxioCam MRm.

FIG. 7.

MDRs activity in HepaRG cells. HepaRG cells were incubated for 30 min in the presence or absence of 50 μM verapamil, an inhibitor of MDRs, before 30 min incubation with 7 μM JC-1. A significant intracellular accumulation resulting in the formation of JC-1 dimers characterized by heterogeneous intracytoplasmic staining was observed in HepaRG hepatocytes (+ verapamil). Imaging was done using Cellomics ArrayScan VTI HCS Reader (ThermoScientific).