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Drug-induced cholestasis results in drug discontinuation and market withdrawal, and the prediction of cholestasis risk is critical in the early stages of drug development. Animal tests and membrane vesicle assay are currently being conducted to assess the risk of cholestasis in the preclinical stage. However, these methods have drawbacks, such as species differences with humans and difficulties in evaluating the effects of drug metabolism and other transporters, implying the need for a cholestasis risk assessment system using human hepatocytes. However, human hepatocytes hardly form functional, extended bile canaliculi, a requirement for cholestasis risk assessment. We previously established a culture protocol for functional, extended bile canaliculi formation in human iPSC-derived hepatocytes. In this study, we modified this culture protocol to support the formation of functional, extended bile canaliculi in human cryopreserved hepatocytes (cryoheps). The production of bile acids, which induces bile canaliculi extension, increased time-dependently during bile canaliculi formation using this protocol, suggesting that increased bile acid production may be involved in the extended bile canaliculi formation. We have also shown that our culture protocol can be applied to cryoheps from multiple donors and that bile canaliculi can be formed stably among different culture batches. Furthermore, this protocol enables long-term maintenance of bile canaliculi and scaling down to culture in 96-well plates. We expect our culture protocol to be a breakthrough for in vitro cholestasis risk assessment., (© 2024. The Author(s).)

Hepatocytes produce bile components and secrete them into a lumen, known as a bile canaliculus, that is formed by the apical membranes of adjoining hepatocytes. Bile canaliculi merge to form tubular structures that subsequently connect to the canal of Hering and larger intra- and extrahepatic bile ducts formed by cholangiocytes, which modify bile and enable flow through the small intestine. The major functional requirements for bile canaliculi are the maintenance of canalicular shape to preserve the blood-bile barrier and regulation of bile flow. These functional requirements are mediated by functional modules, primarily transporters, the cytoskeleton, cell-cell junctions, and mechanosensing proteins. I propose here that bile canaliculi behave as robust machines whereby the functional modules act in a coordinated manner to perform the multistep task of maintaining canalicular shape and bile flow. Cholestasis, the general term for aberrant bile flow, stems from drug/toxin-induced or genetic dysregulation of one or more of the protein components in the functional modules. Here, I discuss the interactions between components of the various functional modules in bile canaliculi and describe how these functional modules regulate canalicular morphology and function. I use this framework to provide a perspective on recent studies of bile canalicular dynamics.

The biliary excretion of pharmaceutical and food-related compounds is an important factor for assessing pharmacokinetics and toxicities in humans, and a highly predictive in vitro method for human biliary excretion is required. We have developed a simple in vitro culture method for generating extended and functional bile canaliculi using cryopreserved human hepatocytes. We evaluated the uptake of compounds by hepatocytes and bile canaliculi, and the biliary excretion index (BEI) was calculated. After 21 days of culture, the presence of extended and functional bile canaliculi was confirmed by the uptake of two fluorescent substrates. Positive BEIs were observed for taurocholic acid-d 4 , rosuvastatin, pitavastatin, pravastatin, valsartan, olmesartan, and topotecan (reported biliary-excreted compounds in humans), but no difference in BEI was observed for salicylic acid (a nonbiliary-excreted compound). Furthermore, 8 of 21 food-related compounds with specific structures and reported biliary transporter involvement exhibited positive BEIs. The developed in vitro system was characterized by functional bile canaliculus-like structures, and it could be applied to the prediction of the biliary excretion of pharmaceutical and food-related compounds.

An understanding of the quantitative relationship between bile canaliculus (BC) dynamics and the disruption of tight junctions (TJs) during drug-induced intrahepatic cholestasis may lead to new strategies aimed at drug development and toxicity testing. To investigate the relationship between BC dynamics and TJ disruption, we retrospectively analyzed the extent of TJ disruption in response to changes in the dynamics of BCs cultured with entacapone (ENT). Three hours after adding ENT, the ZO-1-negative BC surface area ratio became significantly higher (4.1-fold) than those of ZO-1-positive BCs. Based on these data, we calculated slopes of surface area changes, m, of each ZO-1-positive and ZO-1-negative BC. BCs with m ≤ 15 that fell within the 95% confidence interval of ZO-1-positive BCs were defined as ZO-1-positive. To validate this method, we compared the frequency of ZO-1-positive BCs, F Z , with that of BCs with m ≤ 15, F T , in culture using drugs that regulate TJ, or induce intrahepatic cholestasis. F T values were correlated with F Z under all culture conditions (R 2  = .99). Our results indicate that the magnitude of BC surface area changes is a factor affecting TJ disruption, suggesting that maintaining TJ integrity by slowing BC dilation inhibits cell death., (© 2022 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Hepatobiliary imaging is increasingly used by pharmacologists to quantify liver concentrations of transporter-dependent drugs. However, liver imaging does not quantify concentrations in extracellular space, hepatocytes, and bile canaliculi. Our study compared the compartmental distribution of two hepatobiliary substrates gadobenate dimeglumine [BOPTA; 0.08 liver extraction ratio (ER)] and mebrofenin (MEB; 0.93 ER) in a model of perfused rat liver. A gamma counter placed over livers measured liver concentrations. Livers were preperfused with gadopentetate dimeglumine to measure extracellular concentrations. Concentrations coming from bile canaliculi and hepatocytes were calculated. Transporter activities were assessed by concentration ratios between compartments and pharmacokinetic parameters that describe the accumulation and decay profiles of hepatocyte concentrations. The high liver concentrations of MEB relied mainly on hepatocyte and bile canaliculi concentrations. In contrast, the three compartments contributed to the low liver concentrations obtained during BOPTA perfusion. Nonlinear regression analysis of substrate accumulation in hepatocytes revealed that cellular efflux is measurable ∼4 minutes after the start of perfusion. The hepatocyte-to-extracellular concentration ratio measured at this time point was much higher during MEB perfusion. BOPTA transport by multidrug resistance associated protein 2 induced an aquaporin-mediated water transport, whereas MEB transport did not. BOPTA clearance from hepatocytes to bile canaliculi was higher than MEB clearance. MEB did not efflux back to sinusoids, whereas BOPTA basolateral efflux contributed to the decrease in hepatocyte concentrations. In conclusion, our ex vivo model quantifies substrate compartmental distribution and transport across hepatocyte membranes and provides an additional understanding of substrate distribution in the liver. SIGNIFICANCE STATEMENT: When transporter-dependent drugs target hepatocytes, cellular concentrations are important to investigate. Low concentrations on cellular targets impair drug therapeutic effects, whereas excessive hepatocyte concentrations may induce cellular toxicity. With a gamma counter placed over rat perfused livers, we measured substrate concentrations in the extracellular space, hepatocytes, and bile canaliculi. Transport across hepatocyte membranes was calculated. The study provides an additional understanding of substrate distribution in the liver., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Cell polarity and formation of bile canaliculi can be achieved in hepatocytes which are generated from patient-derived induced pluripotent stem cells. This allows for the study of endogenous mutant proteins, patient-specific pathogenesis, and drug responses for diseases where hepatocyte polarity and bile canaliculi play a key role. Here, we describe a step-by-step protocol for the generation of bile canaliculi-forming hepatocytes from induced pluripotent stem cells and their evaluation., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fat accumulation (steatosis) in ballooned hepatocytes alters the expression of membrane transporters in Zucker fatty ( fa/fa ) rats. The aim of the study was to quantify the functions of these transporters and their impact on hepatocyte concentrations using a clinical hepatobiliary contrast agent (Gadobenate dimeglumine, BOPTA) for liver imaging. In isolated and perfused rat livers, we quantified BOPTA accumulation and decay profiles in fa/+ (normal) and fa/fa hepatocytes by placing a gamma counter over livers. Profiles of BOPTA accumulation and decay in hepatocytes were analysed with nonlinear regressions to characterise BOPTA influx and efflux across hepatocyte transporters. At the end of the accumulation period, BOPTA hepatocyte concentrations and influx clearances were not significantly different in fa/+ and fa/fa livers. In contrast, bile clearance was significantly lower in fatty hepatocytes while efflux clearance back to sinusoids compensated the low efflux into canaliculi. The time when BOPTA cellular efflux impacts the accumulation profile of hepatocyte concentrations was slightly delayed (2 min) by steatosis, anticipating a delayed emptying of hepatocytes. The experimental model is useful for quantifying the functions of hepatocyte transporters in liver diseases.

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis., (© 2021 Belicova et al.)

Background & Aims: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2., Methods: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury., Results: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury., Conclusion: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Small-molecule flux in tissue microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely fluorescence loss after photoactivation and intravital arbitrary region image correlation spectroscopy., Approach and Results: The results challenge the prevailing "mechano-osmotic" theory of canalicular bile flow. After active transport across hepatocyte membranes, bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts is diffusion augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts., Conclusions: The liver consists of a diffusion-dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters' folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.

Advances in molecular biology identifying the many carrier-mediated organic anion transporters and advances in microscopy that have provided a more detailed anatomy of the canalicular conduit make updating the concept of osmotically determined canalicular flow possible. For the most part water flow is not transmembrane but via specific pore proteins in both the hepatocyte and the tight junction. These pores independently regulate the rate at which water flows in response to an osmotic gradient and therefore are determinants of canalicular bile acid concentration. Review of the literature indicates that the initial effect on hepatic bile flow of cholestatic agents such as Thorazine and estradiol 17β-glucuronide are on water flow and not bile salt export pump-mediated bile acid transport and thus provides new approaches to the pathogenesis of drug-induced liver injury. Attaining a micellar concentration of bile acids in the canaliculus is essential to the formation of cholesterol-lecithin vesicles, which mostly occur in the periportal region of the canalicular conduit. The other regions, midcentral and pericentral, may transport lesser amounts of bile acid but augment water flow. Broadening the concept of how hepatic bile flow is initiated, provides new insights into the pathogenesis of canalicular cholestasis.

Drug-induced hepatocellular cholestasis leads to altered bile flow. Bile is propelled along the bile canaliculi (BC) by actomyosin contractility, triggered by increased intracellular calcium (Ca 2+ ). However, the source of increased intracellular Ca 2+ and its relationship to transporter activity remains elusive. We identify the source of the intracellular Ca 2+ involved in triggering BC contractions, and we elucidate how biliary pressure regulates Ca 2+ homeostasis and associated BC contractions. Primary rat hepatocytes were cultured in collagen sandwich. Intra-canalicular Ca 2+ was measured with fluo-8; and intra-cellular Ca 2+ was measured with GCaMP. Pharmacological modulators of canonical Ca 2+ -channels were used to study the Ca 2+ -mediated regulation of BC contraction. BC contraction correlates with cyclic transfer of Ca 2+ from BC to adjacent hepatocytes, and not with endoplasmic reticulum Ca 2+ . A mechanosensitive Ca 2+ channel (MCC), Piezo-1, is preferentially localized at BC membranes. The Piezo-1 inhibitor GsMTx-4 blocks the Ca 2+ transfer, resulting in cholestatic generation of BC-derived vesicles whereas Piezo-1 hyper-activation by Yoda1 increases the frequency of Ca 2+ transfer and BC contraction cycles. Yoda1 can recover normal BC contractility in drug-induced hepatocellular cholestasis, supporting that Piezo-1 regulates BC contraction cycles. Finally, we show that hyper-activating Piezo-1 can be exploited to normalize bile flow in drug-induced hepatocellular cholestasis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies., Competing Interests: The authors of this manuscript have read the journal's policy and have the following competing interests: CY, YI, AY, YY, YK, YO, YK, YI, CT are paid employees of PhoenixBio Co., Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials. PXB-mice and PXB-cells are products of PhoenixBio Co., Ltd.

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The mechanisms of organ size control remain poorly understood. A key question is how cells collectively sense the overall status of a tissue. We addressed this problem focusing on mouse liver regeneration. Using digital tissue reconstruction and quantitative image analysis, we found that the apical surface of hepatocytes forming the bile canalicular network expands concomitant with an increase in F-actin and phospho-myosin, to compensate an overload of bile acids. These changes are sensed by the Hippo transcriptional co-activator YAP, which localizes to apical F-actin-rich regions and translocates to the nucleus in dependence of the integrity of the actin cytoskeleton. This mechanism tolerates moderate bile acid fluctuations under tissue homeostasis, but activates YAP in response to sustained bile acid overload. Using an integrated biophysical-biochemical model of bile pressure and Hippo signaling, we explained this behavior by the existence of a mechano-sensory mechanism that activates YAP in a switch-like manner. We propose that the apical surface of hepatocytes acts as a self-regulatory mechano-sensory system that responds to critical levels of bile acids as readout of tissue status., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Liver is pivotal in organism metabolism. This organ is receiving nutriments from the portal vein and then storing, metabolizing, distributing in the circulation or excreting excess and xenobiotics in bile. Liver architecture and hepatocyte polarization are crucial to achieve these functions. To study these mechanisms in details, relevant cell culture systems are required, which is not the case with standard 2D cell culture. Besides, primary hepatocytes rapidly de-differenciate making them inefficient in forming physiological system. Herein, we used an hepatoma-derived cell line to produce matrix-free hepatic spheroids and developed an integrated structural cell biology methodology by combining light sheet fluorescence microscopy and 3D electron microscopy to study their function and structure. Within these spheroids, hepatocytes polarize and organize to form bile canaliculi active for both organics and inorganics excretion. Besides, live imaging revealed the high dynamic of actin networks in basal membranes compared to their high stability in the apical pole that constitutes bile canaliculi. Finally, the first structure of active bile canaliculi was solved at nm resolution and showed the very high density of microvilli coming from all cells constituting the canaliculus. Therefore, this study is the first comprehensive and in-depth functional and structural study of bile canaliculi in a physiological-relevant context.

ATP-binding cassette (ABC) transporters are the members of the efflux pumps that are responsible for the removal of cytotoxic substances by active transport. ABCB11, the bile salt efflux pump of hepatocytes, coordinates cellular excretion of numerous conjugated bile salts into the bile canaliculi, whereas ABCB4 acts as an ATP-dependent floppase translocating phosphatidylcholine from the inner to the outer leaflet of the bile canalicular membrane. Loss of functional ABCB11 and ABCB4 proteins causes early-onset refractory cholestasis or cholangiopathy. In this study, we investigated the expression and localization pattern of ABCB11 and ABCB4 using immunohistochemistry and RNA profiling in liver samples from patients with different types and stages of chronic cholestatic liver disease, with emphasis on primary sclerosing cholangitis (PSC), compared to a variety of cholestatic and noncholestatic hepatopathies. Therefore, ABCB11 and ABCB4 expressions were investigated on formalin-fixed and paraffin-embedded (FFPE) material in a patient cohort of total 43 patients with or without cholestatic liver diseases, on protein level using immunohistochemistry and on RNA level using nanoString technology. Intriguingly, our results demonstrated increased expression of ABCB11 and ABCB4 on protein as well as RNA level in PSC, and the expression pattern correlated with disease progression. We concluded from our study that patients with PSC demonstrate altered expression levels and pattern of ABCB11 and ABCB4 which correlated with disease progression; thereby, ABCB11 and ABCB4 analysis may be a useful tool for assessment of disease stages in PSC., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2019 Cornelia Thoeni et al.)

The purpose of this minireview is to show that a new paradigm is developing regarding hepatic bile flow. The focus thus far has been on carrier-mediated transport of bile acids and other solutes, such as glutathione, which create an osmotic gradient for the transcellular and paracellular flow of water into canaliculi. In addition to the physicochemical properties of bile acids, which govern the osmotic gradient, data now exist showing that the tight junctions governing paracellular water flow and Aquaporin-8 water channels governing transcellular water flow are regulated independently. Thus, the rate of water flow into the canaliculus in response to bile acid transport is variable and determines canalicular bile acid concentration, which affects the production and solubilization of cholesterol-lecithin vesicles. These new considerations modify thinking regarding the occurrence of cholestasis and its progression and reorient the design of experimental studies that can distinguish the different determinants of bile flow. SIGNIFICANCE STATEMENT: The paradigm that water flow into the canaliculus is determined only by the rate of carrier-mediated transport has been challenged recently by the changes that occur in hepatic bile composition in the Claudin-2 knockout mouse and with the cholestatic effect of estradiol 17 β -d-glucuronide. Thus, a respective reduction in paracellular or transcellular canalicular water flow, probably via Aquaporin 8, has no significant effect on bile acid excretion., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Background & Aims: Hepatocyte polarity is essential for the development of bile canaliculi and for safely transporting bile and waste products from the liver. Functional studies of autologous mutated proteins in the context of the polarized hepatocyte have been challenging because of the lack of appropriate cell models. The aims of this study were to obtain a patient-specific hepatocyte model that recapitulated hepatocyte polarity and to employ this model to study endogenous mutant proteins in liver diseases that involve hepatocyte polarity., Methods: Urine cell-derived pluripotent stem cells, taken from a patient with a homozygous mutation in ATP7B and a patient with a heterozygous mutation, were differentiated towards hepatocyte-like cells (hiHeps). HiHeps were also derived from a patient with MEDNIK syndrome., Results: Polarized hiHeps that formed in vivo-like bile canaliculi could be generated from embryonic and patient urine cell-derived pluripotent stem cells. HiHeps recapitulated polarized protein trafficking processes, exemplified by the Cu 2+ -induced redistribution of the copper transporter protein ATP7B to the bile canalicular domain. We demonstrated that, in contrast to the current dogma, the most frequent yet enigmatic Wilson disease-causing ATP7B-H1069Q mutation per se did not preclude trafficking of ATP7B to the trans-Golgi Network. Instead, it prevented its Cu 2+ -induced polarized redistribution to the bile canalicular domain, which could not be reversed by pharmacological folding chaperones. Finally, we demonstrate that hiHeps from a patient with MEDNIK syndrome, suffering from liver copper overload of unclear etiology, showed no defect in the Cu 2+ -induced redistribution of ATP7B to the bile canaliculi., Conclusions: Functional cell polarity can be achieved in patient pluripotent stem cell-derived hiHeps, enabling, for the first time, the study of the endogenous mutant proteins, patient-specific pathogenesis and drug responses for diseases where hepatocyte polarity is a key factor., Lay Summary: This study demonstrates that cells that are isolated from urine can be reprogrammed in a dish towards hepatocytes that display architectural characteristics similar to those seen in the intact liver. The application of this methodology to cells from patients diagnosed with inherited copper metabolism-related liver diseases (that is, Wilson disease and MEDNIK syndrome) revealed unexpected and novel insights into patient mutation-specific disease mechanisms and drug responses., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

To mimic (human) cholestasis in vitro requires multiple triggers to establish a diseased phenotype. However, this is currently not simulated by existing in vitro models. Therefore, there is a high need for multicellular systems similar to the human physiology. In such an in vitro model, cell-cell interactions and intact bile canaliculi with functional bile flow should be present and preserved during long-term culture. Precision-cut liver slices represent an ex vivo tissue culture technique that replicates most of the multicellular characteristics of a whole liver in vivo. This chapter describes the preparation and culturing of (human) precision-cut liver slices. Furthermore, a protocol to use the precision-cut liver slices technique to predict drug-induced cholestatic liver injury is described.

Cholestasis remains a major challenge in drug-induced liver injury, and therefore warrants identification of chemical entities that may lead to cholestasis. Recent advances in cell culture methods enable 3D spheroid models to remain viable for much longer periods of time than conventional sandwich cultures of primary human hepatocytes while maintaining native tissue-like functionality, such as drug metabolism activity, receptor signaling functionality, and physiological relevance. These spheroid models enable us to study repeated exposure effects associated with chemicals and their metabolites that may ultimately progress to cholestasis and liver injury. HepaRG cells cultured as spheroids are viable for more than 4 weeks with cytochrome P450 enzymatic activities comparable to ranges observed in freshly isolated/cryopreserved suspensions of primary human hepatocytes. HepaRG spheroids form bile canalicular structures with potential application as a model to study biliary excretion processes and intrahepatic obstruction of bile flow, leading to hepatocellular damage and death. In this chapter, we describe methods to culture 3D spheroids of HepaRG cells with extensive bile canalicular structures/networks, image transport of bile acid (cholyl-lysyl-fluorescein) to the bile canaliculi, and measure cholestatic drug-induced cytotoxicity.

Background & Aims: Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative., Methods: We focused on claudin-3 (Cldn3), a paracellular barrier-forming tight junction protein whose expression in mouse liver decreases with age. Since Cldn3-knockout mice exhibited gallstone diseases, we used them to assess the causal relationship between paracellular phosphate ion permeability and the core formation of cholesterol gallstones., Results: In the liver of Cldn3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose caused cholesterol gallstone disease in these mice., Conclusion: We revealed that in the hepatobiliary system, Cldn3 functions as a paracellular barrier for phosphate ions, to help maintain biliary ion homeostasis. We provide in vivo evidence that elevated phosphate ion concentrations play a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease under cholesterol overdose., Lay Summary: Herein, we reveal a new mechanism for cholesterol gallstone formation, in which increased paracellular phosphate ion permeability across hepatobiliary epithelia causes calcium phosphate core formation and cholesterol gallstones. Thus, altered phosphate ion metabolism under cholesterol overdose plays a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease., (Copyright © 2018. Published by Elsevier B.V.)

Cholestasis represents a major subtype of drug-induced liver injury and novel preclinical models for its prediction are needed. Here we used primary human hepatocytes (PHH) from different donors in 2D-sandwich (2D-sw) and/or 3D-spheroid cultures to study inter-individual differences in the response towards cholestatic hepatotoxins after short-term (48-72 hours) and long-term repeated exposures (14 days). The cholestatic liabilities of drugs were determined by comparing cell viability upon exposure to the highest non-cytotoxic drug concentration in the presence and absence of a non-cytotoxic concentrated bile acid mixture. In 2D-sw culture, cyclosporine A and amiodarone presented clear cholestatic liabilities in all four PHH donors tested, whereas differences in the susceptibility of the various PHH donors towards the cholestatic toxicity of bosentan, chlorpromazine and troglitazone were observed. In PHH from one donor, the cholestatic liabilities of chlorpromazine and troglitazone could only be detected after long-term repeated exposures when maintained in 3D-spheroid culture, but not after short-term exposures in either 2D-sw or 3D-spheroid culture, suggesting that cholestatic hepatotoxicity may require time to develop. In conclusion, inter-individual susceptibility exists towards drug-induced cholestasis, which depends on the compound as well as the exposure time., (Copyright © 2018 Elsevier B.V. All rights reserved.)

With recent advances in liver imaging, the estimation of liver concentrations is now possible following the injection of hepatobiliary contrast agents and radiotracers. However, how these images are generated remains partially unknown. Most experiments that would be helpful to increase this understanding cannot be performed in vivo . For these reasons, we investigated the liver distribution of the magnetic resonance (MR) contrast agent gadobenate dimeglumine (Gd-BOPTA, MultiHance®, Bracco Imaging) in isolated perfused rat livers (IPRLs). In IPRL, we developed a new set up that quantifies simultaneously the Gd-BOPTA compartment concentrations and the transfer rates between these compartments. Concentrations were measured either by MR signal intensity or by count rates when the contrast agent was labelled by [ 153 Gd]. With this experimental model, we show how the Gd-BOPTA hepatocyte concentrations are modified by temperature and liver flow rates. We define new pharmacokinetic parameters to quantify the canalicular transport of Gd-BOPTA. Finally, we present how transfer rates generate Gd-BOPTA concentrations in rat liver compartments. These findings better explain how liver imaging with hepatobiliary radiotracers and contrast agents is generated and improve the image interpretation by clinicians.

Background: Neonates on long-term parenteral nutrition (PN) may develop parenteral nutrition-associated liver disease (PNALD). Aluminum (Al) is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3⁻6 days, were placed into two groups. The high Al group ( n = 8) received PN with 63 µg/kg/day of Al, while the low Al group ( n = 7) received PN with 24 µg/kg/day of Al. Serum samples for total bile acids (TBA) were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry were studied by transmission electron microscopy (TEM) and ImageJ software analysis. Results: The canalicular space was smaller and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow.

Alternative models explaining the biliary lipid secretion at the canalicular membrane of hepatocytes exist: successive lipid extraction by preformed bile salt micelles, or budding of membrane fragments with formation of mixed micelles. To test the feasibility of the latter mechanism, we developed a mathematical model that describes the formation of lipid microdomains in the canalicular membrane. Bile salt monomers intercalate into the external hemileaflet of the canalicular membrane, to form a rim to liquid disordered domain patches that then pinch off to form nanometer-scale mixed micelles. Model simulations perfectly recapitulate the measured dependence of bile salt-dependent biliary lipid extraction rates upon modulation of the membrane cholesterol (lack or overexpression of the cholesterol transporter Abcg5-Abcg8) and phosphatidylcholine (lack of Mdr2, also known as Abcb4) content. The model reveals a strong dependence of the biliary secretion rate on the protein density of the membrane. Taken together, the proposed model is consistent with crucial experimental findings in the field and provides a consistent explanation of the central molecular processes involved in bile formation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

A primary amino-functionalized methyl methacrylate-based statistical copolymer is covalently coupled with retinoic acid (RA) and a fluorescent dye (DY590) in order to investigate the feasibility of the RA containing polymeric nanoparticles for Raman imaging studies and to study the possible selectivity of RA for hepatic stellate cells via intravital microscopy. Cationic nanoparticles are prepared by utilizing the nanoprecipitation method using modified polymers. Raman studies show that RA functional nanoparticles can be detectable in all tested cells without any need of additional label. Moreover, intravital microscopy indicates that DY590 is eliminated through the hepatobiliary route but not if used as covalently attached tracing molecule for nanoparticles. However, it is a suitable probe for sensitive detection of polymeric nanoparticles., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: We studied histologic changes of bile canalicular-ductule networks in the future liver remnant (FLR) while associating liver partition and portal vein occlusion for staged hepatectomy (ALPPS), since little is known about regeneration of these networks during the relatively short interval between procedures in ALPPS., Methods: Bile canalicular-ductule networks were examined in specimens from eight patients treated with ALPPS and six patients undergoing hepatectomy following portal vein embolization (PVE). Expression of multidrug resistance-1 (MDR1), a membrane transporter in bile canaliculi (BC), was analyzed immunohistochemistcally. Morphologic changes of BC and tight junctions (TJs) adjoining BC were also assessed electron microscopically., Results: Extrapolated kinetic growth of the FLR was greater during ALPPS (17.2 ± 6.8 mL/day) than after PVE (6.3 ± 3.4 mL/day; p = 0.005), and continuity of the MDR1-positive bile canalicular networks was less evident in ALPPS than PVE (p < 0.001). Electron microscopically, no significant difference was evident in numbers of BC or BC lumen size between the two groups; however, development of microvilli in BC was poorer in the ALPPS group than in the PVE group (p < 0.001). TJ/desmosome complexes were shorter in the ALPPS group (0.69 ± 0.52 μm) than in the PVE group (1.09 ± 0.50 μm; p < 0.001), and leaky TJs were seen more frequently in the ALPPS group (64.9 vs. 23.6%; p = 0.001)., Conclusions: Regeneration of bile canalicular-ductule networks in the FLR was poorer in ALPPS than PVE, which may be associated with prolonged cholestasis following final hepatectomy in ALPPS.

Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). We used an E-iPSC-line to model hepatogenesis in vitro. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4α and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. We use E-iPSCs for modeling gene regulatory networks associated with human hepatogenesis and gastrulation in general.

In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca 2+ -dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38 MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38 MAPK , and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 μM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38 MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38 MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.

Several endothelin receptor antagonists (ERAs) have been developed for the treatment of pulmonary arterial hypertension (PAH). Some of them have been related to clinical cases of hepatocellular injury (sitaxentan [SIT]) and/or cholestasis (bosentan [BOS]). We aimed to determine if ambrisentan (AMB) and macitentan (MAC), in addition to BOS and SIT, could potentially cause liver damage in man by use of human HepaRG cells. Our results showed that like BOS, MAC-induced cytotoxicity and cholestatic disorders characterized by bile canaliculi dilatation and impairment of myosin light chain kinase signaling. Macitentan also strongly inhibited taurocholic acid and carboxy-2',7'-dichlorofluorescein efflux while it had a much lower inhibitory effect on influx activity compared to BOS and SIT. Moreover, these three drugs caused decreased intracellular accumulation and parallel increased levels of total bile acids (BAs) in serum-free culture media. In addition, all drugs except AMB variably deregulated gene expression of BA transporters. In contrast, SIT was hepatotoxic without causing cholestatic damage, likely via the formation of reactive metabolites and AMB was not hepatotoxic. Together, our results show that some ERAs can be hepatotoxic and that the recently marketed MAC, structurally similar to BOS, can also cause cholestatic alterations in HepaRG cells. The absence of currently known or suspected cases of cholestasis in patients suffering from PAH treated with MAC is rationalized by the lower therapeutic doses and Cmax, and longer receptor residence time compared to BOS., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Cholesterol gallstone disease (CGD) is one of the most common gastrointestinal diseases. Lithogenic hepatic bile secretion precedes the formation of cholesterol gallstones. Constitutive androstane receptor (CAR), a member of nuclear family, plays an important role in cholesterol and bile acid metabolism. To examine whether activation of CAR can prevent cholesterol gallstone formation, we treated C57BL6/J mice maintained on a lithogenic diet with CAR agonist 1,4-bis-[2-(3, 5-dichlorpyridyloxy)] benzene and performed bile duct cannulation to study the dynamics of biliary lipids. We report that activation of CAR decreases the biliary cholesterol concentration and prevents CGD formation. The lower biliary cholesterol level was largely attributed to suppressed Abcg5 and Abcg8 expression in CAR-activated mice. CAR activation also promoted cholesterol conversion into bile acids by increasing the expression of Cyp7a1, a rate-limiting enzyme in bile acid biosynthesis. Activation of CAR enhanced bile acid re-absorption via increasing the expression of bile acid transporters Asbt and Ostβ in the ileum. The hepatic steatosis was also improved in the liver of CAR-activated mice. Furthermore, activation of CAR protected the mice against the liver X receptor α-sensitized CGD through suppressing the expression of Abcg5/8. Collectively, CAR plays an important role in maintaining the homeostasis of cholesterol, bile acids, and triglycerides levels, and it might be a promising therapeutic target for preventing or treating CGD., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.

Unlabelled: The adenoviral gene transfer of human aquaporin-1 (hAQP1) water channels to the liver of 17α-ethinylestradiol-induced cholestatic rats improves bile flow, in part by enhancing canalicular hAQP1-mediated osmotic water secretion. To gain insight into the mechanisms of 17α-ethinylestradiol cholestasis improvement, we studied the biliary output of bile salts (BS) and the functional expression of the canalicular BS export pump (BSEP; ABCB11). Adenovector encoding hAQP1 (AdhAQP1) or control vector was administered by retrograde intrabiliary infusion. AdhAQP1-transduced cholestatic rats increased the biliary output of major endogenous BS (50%-80%, P < 0.05) as well as that of taurocholate administered in choleretic or trace radiolabel amounts (around 60%, P < 0.05). Moreover, liver transduction with AdhAQP1 normalized serum BS levels, otherwise markedly elevated in cholestatic animals. AdhAQP1 treatment was unable to improve BSEP protein expression in cholestasis; however, its transport activity, assessed by adenosine triphosphate-dependent taurocholate transport in canalicular membrane vesicles, was induced by 90% (P < 0.05). AdhAQP1 administration in noncholestatic rats induced no significant changes in either biliary BS output or BSEP activity. Canalicular BSEP, mostly present in raft (high cholesterol) microdomains in control rats, was largely found in nonraft (low cholesterol) microdomains in cholestasis. Considering that BSEP activity directly depends on canalicular membrane cholesterol content, decreased BSEP presence in rafts may contribute to BSEP activity decline in 17α-ethinylestradiol cholestasis. In AdhAQP1-transduced cholestatic rats, BSEP showed a canalicular microdomain distribution similar to that of control rats, which provides an explanation for the improved BSEP activity., Conclusion: Hepatocyte canalicular expression of hAQP1 through adenoviral gene transfer promotes biliary BS output by modulating BSEP activity in estrogen-induced cholestasis, a novel finding that might help us to better understand and treat cholestatic disorders. (Hepatology 2016;64:535-548)., (© 2016 by the American Association for the Study of Liver Diseases.)

Following the injection of hepatobiliary contrast agents, MRI detects all molecules included in a region of interest but cannot estimate true concentrations in sinusoids, interstitium, hepatocytes or bile canaliculi. The aim of the study was to measure true concentrations in hepatocytes and to show how transfer rates across sinusoidal and canalicular membranes generate these concentrations. We perfused livers isolated from normal rats with 200 μM Gd-DTPA and Gd-BOPTA and measured clearances from sinusoids to liver and from hepatocytes to bile canaliculi or back to interstitium. We detected Gd-BOPTA with a gamma probe and determined true concentrations in each liver compartment knowing their liver volumes. No pharmacokinetic modelling was applied. Gd-BOPTA clearance from sinusoids to liver (2.5 ± 0.4 mL/min) was 50 times higher than that of Gd-DTPA (0.05 ± 0.02 mL/min) when portal flow rate was 30 mL/min (p < 0.0001). Gd-BOPTA clearance from sinusoids to liver was always superior to hepatocyte clearance, and hepatocyte Gd-BOPTA concentrations measured by the probe increased over time. Gd-BOPTA concentrations reached 439 ± 83 μM in hepatocytes and 15 × 700 ± 3100 μM in bile canaliculi, while concentrations in sinusoids were 200 μM. Gd-BOPTA true concentrations in hepatocytes depend on the simultaneous clearances from sinusoids to hepatocytes and from hepatocytes to bile canaliculi and back to sinusoids. The study better defines how signal intensities are generated when hepatobiliary contrast agents are injected in clinical imaging. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.

The Cu(+) pump ATP7B plays an irreplaceable role in the elimination of excess Cu(+) by the hepatocyte into the bile. The trafficking and site of action of ATP7B are subjects of controversy. One current proposal is that an increase in intracellular Cu(+) results in the translocation of ATP7B to the lysosomes and excretion of excess Cu(+) through lysosomal-mediated exocytosis at the bile canaliculus. Here, we show that ATP7B is transported from the trans-Golgi network (TGN) to the bile canaliculus by basolateral sorting and endocytosis, and microtubule-mediated transcytosis through the subapical compartment. Trafficking ATP7B is not incorporated into lysosomes, and addition of Cu(+) does not cause relocalization of lysosomes and the appearance of lysosome markers in the bile canaliculus. Our data reveal the pathway of the Cu(+)-mediated transport of ATP7B from the TGN to the bile canaliculus and indicates that the bile canaliculus is the primary site of ATP7B action in the elimination of excess Cu(.), (© 2016. Published by The Company of Biologists Ltd.)

Estradiol-17β-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.

Background & Aims: Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis., Methods: We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells., Results: Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and ε were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation., Conclusions: Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The somatostatin analog octreotide can lead to hyperbilirubinemia without evidence of liver injury. Here we investigate whether octreotide inhibits the main sinusoidal/canalicular bilirubin carriers and whether it is a transport substrate. Octreotide showed the most potent inhibitory effect toward OATP1B1-mediated transport and weaker inhibition for OATP1B3- and MRP2-mediated transport. Octreotide had no effect on OATP2B1-mediated transport. Octreotide inhibited [(3)H]estradiol-17-β-glucuronide (E17βG) influx mediated by OATP1B1, 1B3, and multidrug resistance-associated protein 2 (MRP2) in a concentration-dependent manner, and the IC50 values were computed to be 23 μM (95% confidence interval [CI] 18-29), 68 μM (95% CI 50-91), and 116.6 μM (95% CI 74.5-182.4), respectively. The interaction between octreotide and OATP1B1 was further studied. Inhibition of [(3)H]E17βG OATP1B1-mediated transport was purely competitive with no changes in maximum transport capacity (Vmax) and a twofold Km increase when the influx kinetics of [(3)H]E17βG were measured in the presence of octreotide (8.8 ± 3.1 versus 4.4 ± 1.2 μM, P = 0.03). The inhibition constant (Ki) of octreotide for the transport of [(3)H]E17βG was calculated at 33.5 ± 5.5 μM. Uptake of radiolabeled octreotide by OATP1B1-CHO cells was higher than in wild-type CHO cells and nonlabeled octreotide at the extracellular compartment was able to trans-stimulate the OATP1B1-mediated efflux of intracellular [(3)H]E17βG, suggesting that octreotide is a substrate of OATP1B1. In summary, this study shows interaction of octreotide on the human hepatocellular bilirubin transporters OATP1B1, OATP1B3, and MRP2, notably OATP1B1. These findings are in line with the clinical observation that a fraction of patients under treatment with octreotide exhibit hyperbilirubinemia., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Many substances are hepatotoxic due to their ability to cause intrahepatic cholestasis. Therefore, there is a high demand for in vitro systems for the identification of cholestatic properties of new compounds. Primary hepatocytes cultivated in collagen sandwich cultures are known to establish bile canaliculi which enclose secreted biliary components. Cholestatic compounds are mainly known to inhibit bile excretion dynamics, but may also alter canalicular volume, or hepatocellular morphology. So far, techniques to assess time-resolved morphological changes of bile canaliculi in sandwich cultures are not available. In this study, we developed an automated system that quantifies dynamics of bile canaliculi recorded in conventional time-lapse image sequences. We validated the hepatocyte sandwich culture system as an appropriate model to study bile canaliculi in vitro by showing structural similarity measured as bile canaliculi length per hepatocyte to that observed in vivo. Moreover, bile canalicular excretion kinetics of CMFDA (5-chloromethylfluorescein diacetate) in sandwich cultures resembled closely the kinetics observed in vivo. The developed quantification technique enabled the quantification of dynamic changes in individual bile canaliculi. With this technique, we were able to clearly distinguish between sandwich cultures supplemented with dexamethasone and insulin from control cultures. In conclusion, the automated quantification system offers the possibility to systematically study the causal relationship between disturbed bile canalicular dynamics and cholestasis.

Aim: To study whether transfer of blood between the right gastroepiploic artery and gastroduodenal artery could lessens the damage to bile canaliculi., Methods: Forty male Bama miniature pigs were divided into four groups as follows: a control group, two hepatic artery ischemia groups (1 h and 2 h), and a hepatic artery bridging group. The hemodynamics of the hepatic artery in the hepatic artery bridging group was measured using color Doppler ultrasound. Morphological changes in the bile canaliculus were observed by transmission electron microscopy. Cofilin, heat shock protein 27 and F-actin expression was detected by immunohistochemistry, Western blot, and real-time polymerase chain reaction. Terminal deoxynucleotidyl transferase-mediated nick end-labeling method was used to evaluate liver injury., Results: The hemodynamics was not changed in the hepatic artery bridging group. The microvilli in the bile canaliculus were impaired in the two hepatic artery ischemia groups. The down-regulation of cofilin and F-actin and up-regulation of heat shock protein 27 were observed in the two hepatic artery ischemia groups, while there were no significant differences between the control group and hepatic artery bridging group., Conclusion: Hepatic artery ischemia aggravates damage to bile canaliculi, and this damage can be diminished by a hepatic artery bridging duct.

The biological significance of STK17A, a serine/threonine kinase, in the liver is not known. We analyzed STK17A expression in HepG2 cells and human liver tissue. Accordingly, we investigated whether STK17A could help in identifying earlier changes during the evolution of chronic rejection (CR) after liver transplantation. RT-PCR and immunofluorescence were used to analyze STK17A expression in HepG2 cells. Antibody microarray was performed using human liver samples from CR and healthy donors. Immunohistochemistry was used to verify the clinical utility of STK17A on sequential biopsies for the subsequent development of CR. A novel short isoform of STK17A was found in HepG2 cells. STK17A was localized in the nuclei and bile canaliculi in HepG2 cells and human livers. Microarray of STK17A revealed its decrease in failed liver allografts by CR. During the evolution of CR, the staining pattern of bile canalicular STK17A gradually changed from diffuse linear to focal intermittent. The focal intermittent staining pattern was observed before the definite diagnosis of CR. In conclusion, the present study was the first to find localization of STK17A in normal bile canaliculi. Abnormal expression and localization of STK17A were associated with CR of liver allografts since the early stage of the rejection process.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)