Early Alterations of Bile Canaliculi Dynamics and the Rho Kinase/Myosin Light Chain Kinase Pathway Are Characteristics of Drug-Induced Intrahepatic Cholestasis

Abstract

Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.

Functional polarization of human hepatoma HepaRG cells in response to forskolin

“…BC formed in HepaRG cell cultures appear as characteristic refractive structures, corresponding to bright/white objects on phase-contrast photographs, as previously reported 21 , whereas cells appear as dense/black elements, containing nuclei recognizable by their round/delimited shape and differential density. After capturing images using an Axiovert 40 C phase contrast microscope (Carl Zeiss, Le Pecq, France) and adjusting brightness parameters to optimally distinguish between white and black densities, bright objects reflecting BC as well as round/delimited nuclei-related objects were segmented by adjusting the shape and area parameters to exclude non-corresponding objects, using a dedicated in-house macro-program based on ImageJ 1.48 software 22 .…”

Penicillinase-resistant antibiotics induce non-immune-mediated cholestasis through HSP27 activation associated with PKC/P38 and PI3K/AKT signaling pathways

“…Disruption of BC dynamics by certain drugs is associated with occurrence of cholestasis injury 24 . If few cholestatic drugs such as cyclosporine A 31, 32 and chlorpromazine 33 , induced constriction of BC associated with irreversible severe damage and cell death, others covering a wider range of cholestatic compounds, induced dilatation 24, 34 . Importantly, BC dilatation is observed in patients suffering from cholestasis 35, 36 .…”

A dynamic mathematical model of bile acid clearance in HepaRG cells

“…In addition to drug-mediated inhibition of hepatocellular transport function, the inter-individual variability of transporter expression and function are altered by pre-existing hepatic diseases and genetic factors, which are believed to contribute to the development of drug-induced risk of cholestasis in susceptible individuals (Trauner et al, 1998). Recently, some of us reported that cholestatic drugs caused alteration of bile canaliculi dynamics associated with impairment of the Rhokinase/ myosin light chain kinase signaling pathway using human HepaRG cells (Burbank et al, 2016;Sharanek et al, 2016).…”

“…A validation of the model was further achieved by use of two prototypical cholestatic drugs, cyclosporine A (CsA) and chlorpromazine (CPZ), previously shown to exhibit differences in the mechanisms of apical and basolateral transporter inhibition (Antherieu et al, 2013;Sharanek et al, 2014) and their effect on BC dynamics (Burbank, et al 2016).…”