Farnesoid X Receptor Agonists Obeticholic Acid and Chenodeoxycholic Acid Increase Bile Acid Efflux in Sandwich-Cultured Human Hepatocytes: Functional Evidence and Mechanisms

Abstract

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid homeostasis. FXR agonists, obeticholic acid (OCA) and chenodeoxycholic acid (CDCA), increase mRNA expression of efflux transporters in sandwich-cultured human hepatocytes (SCHH). This study evaluated the effects of OCA and CDCA treatment on the uptake, basolateral efflux, and biliary excretion of a model bile acid, taurocholate (TCA), in SCHH. In addition, changes in the protein expression of TCA uptake and efflux transporters were investigated. SCHH were treated with 1 µM OCA, 100 µM CDCA, or vehicle control for 72 hours followed by quantification of deuterated TCA uptake and efflux over time in Ca²⁺-containing and Ca²⁺-free conditions (n = 3 donors). A mechanistic pharmacokinetic model was fit to the TCA mass-time data to obtain estimates for total uptake clearance (CL_Uptake), total intrinsic basolateral efflux clearance (CL_int,BL), and total intrinsic biliary clearance (CL_int,Bile). Modeling results revealed that FXR agonists significantly increased CL_int,BL by >6-fold and significantly increased CL_int,Bile by 2-fold, with minimal effect on CL_Uptake. Immunoblotting showed that protein levels of the basolateral transporter subunits organic solute transporter α and β (OSTα and OSTβ) in FXR agonist-treated SCHH were significantly induced by >2.5- and 10-fold, respectively. FXR agonist-mediated changes in the expression of other TCA transporters in SCHH were modest. In conclusion, this is the first report demonstrating that OCA and CDCA increased TCA efflux in SCHH, which contributed to reduced intracellular TCA concentrations. Increased basolateral efflux of TCA was consistent with increased OSTα/β protein expression in OCA- and CDCA-treated SCHH.

Introduction

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in the homeostasis of bile acids (Lefebvre et al., 2009). Bile acids are signaling molecules that modulate hepatic metabolic and transporter function (Dawson et al., 2009), inflammation (Allen et al., 2011), and lipid, glucose, and energy homeostasis (Staels and Kuipers, 2007; Thomas et al., 2008; Lefebvre et al., 2009). Therefore, FXR is a promising novel drug target to treat metabolic and chronic liver diseases such as nonalcoholic steatohepatitis and primary sclerosing cholangitis (Fiorucci et al., 2007; Jiang et al., 2007; Wagner et al., 2011; Trivedi et al., 2016). The first-in-class FXR agonist, obeticholic acid (OCA), has been approved for the treatment of primary biliary cholangitis. FXR is activated by various bile acids; chenodeoxycholic acid (CDCA) and conjugated CDCA species are the most potent natural agonists. OCA is an analog of CDCA, with 100-fold higher FXR activation potency (the median effective concentration is 99 nM for OCA vs. 8.3 µM for CDCA) (Soisson et al., 2008; Markham and Keam, 2016).

Activation of FXR protects against the toxic accumulation of bile acids by decreasing bile acid synthesis and regulating bile acid transport (Adorini et al., 2012). For example, FXR activation by CDCA increased mRNA expression of bile acid efflux transporters including the bile salt export pump (BSEP) (Yu et al., 2002; Modica et al., 2010), multidrug resistance–associated protein 2 (MRP2) (Modica et al., 2010; Liu et al., 2014), and organic solute transporter (OSTα/β) (Boyer et al., 2006; Modica et al., 2010; Liu et al., 2014) in hepatic cell lines and primary human hepatocytes. Sandwich-cultured human hepatocytes (SCHH) are a physiologically relevant model that maintains transporter function, morphology, and regulatory machinery (LeCluyse et al., 2000; Yang et al., 2016) and therefore, is an ideal system to study the regulation of bile acid transporters after nonacute (>24-hour) treatment of hepatocytes with FXR agonists. Recent studies revealed that OCA and CDCA treatment of SCHH for 72 hours increased mRNA expression of OSTα/β and BSEP with minor changes in MRP3, MRP4, and sodium taurocholate cotransporting polypeptide (NTCP) (Jackson et al., 2016; Zhang et al., 2017).

Unlike drug-metabolizing enzymes, changes in gene expression of transporters do not always translate to changes in protein expression (Ahlin et al., 2009; Thompson et al., 2017) and/or function (Ohtsuki et al., 2012). This discrepancy may be explained by the prominent role of post-transcriptional regulation of hepatic transporters and the importance of transporter localization in determining transporter function (Wang et al., 2002; Chandra et al., 2005; Zhang et al., 2005; Gu and Manautou, 2010; Schonhoff et al., 2013).

Due to discrepancies between gene expression and transporter function, direct functional evidence for FXR agonist-mediated induction of bile acid efflux transporters (i.e., OSTα/β and BSEP) is lacking. One of the challenges in quantitative assessment of hepatic transporter function is the multiplicity of transporters expressed on the basolateral and canalicular membranes of hepatocytes and the concurrent uptake and efflux of bile acids. Studies showed reduced intracellular accumulation of deuterium-labeled taurocholic acid (d₈-TCA), a prototypical bile acid, after treatment of SCHH with OCA and CDCA (Jackson et al., 2016; Zhang et al., 2017). Since d₈-TCA was added exogenously and the synthesis rate of TCA does not play a role, this change could be due to increased efflux or decreased uptake of d₈-TCA. On the basolateral membrane of human hepatocytes, NTCP and, to a much lesser extent, the organic anion-transporting polypeptides contribute to TCA uptake. MRP3, MRP4, and OSTα/β are responsible for the basolateral efflux of TCA from human hepatocytes to the sinusoidal blood. (Seward et al., 2003; Zhang et al., 2003; Rius et al., 2006; Ballatori et al., 2008; Soroka et al., 2010). To deconvolute these processes, a more sophisticated method such as pharmacokinetic (PK) modeling is needed. To assess the function of the canalicular transporter BSEP, TCA biliary excretion can be quantified in SCHH using B-CLEAR technology (Qualyst Transporter Solutions, Durham, NC).

In this study, mechanistic PK modeling of data obtained from SCHH using B-CLEAR technology and our previously published basolateral efflux protocol (Pfeifer et al., 2013; Yang et al., 2015; Guo et al., 2016) was used to characterize the functional changes in bile acid transporters that occur with FXR activation. Changes in the basolateral uptake clearance, intrinsic basolateral efflux clearance, and intrinsic biliary clearance of an exogenously administered model bile acid, d₈-TCA, were evaluated in SCHH after 72-hour treatment with OCA and CDCA to reflect the transporter function. To identify the transporters that might be responsible for alterations in the overall clearance, the protein expression of TCA uptake and efflux transporters was assessed by immunoblot analysis.

Materials and Methods

Materials.

BioCoat cell culture plates and Matrigel were obtained from BD Biosciences (San Jose, CA). QualGro medium was obtained from Qualyst Transporter Solutions. d₈-TCA was obtained from Toronto Research Chemicals (Toronto, ON, Canada). The Pierce bicinchoninic acid protein assay was obtained from Thermo Fisher Scientific (Waltham, MA).

Primary antibodies for OSTα, OSTβ, BSEP, and NTCP were purchased from Abcam (Cambridge, MA). Primary antibodies for MRP3 and MRP4 were obtained from Cell Signaling Technology (Danvers, MA) and Everest Biotech (Ramona, CA), respectively. The primary antibody for ATPase and all of the secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Other reagents for immunoblot were obtained from Bio-Rad (Hercules, CA) or Thermo Fisher Scientific unless stated otherwise.

Hepatocyte Culture and FXR Agonist Treatment.

Transporter certified cryopreserved human hepatocytes [HC3-26, HUM4122C/D, HUM4119C, and HU8246 purchased from XenoTech (Kansas City, KS), Lonza (Basel, Switzerland), and Thermo Fisher Scientific, respectively] were obtained from two Caucasian female donors, one Asian male donor, and one African American female donor, respectively (age range, 30–43 years; body mass index range, 22–39). Hepatocytes were seeded at a density of 0.4–0.5 × 10⁶ cells/well in 24-well BioCoat plates and cultured in a sandwich configuration (overlaid with Matrigel) in QualGro induction medium as previously reported (Swift et al., 2010). On day 2 of culture, SCHH were treated with OCA (1 µM), CDCA (100 µM), or vehicle control for 72 hours. The dose of OCA was selected based on the maximum plasma concentration of OCA at steady state (approximately 0.7–1 µM) (Edwards et al., 2016). In addition, based on the dose-gene expression relationships published previously, the maximal effect on the induction of downstream genes was achieved at 1 µM OCA and 100 µM CDCA (Jackson et al., 2016; Zhang et al., 2017). Previous studies using the same lots of human hepatocytes (Jackson et al., 2016; Zhang et al., 2017), and a study using different lots of human hepatocytes (Yang et al., 2016), demonstrated that neither OCA (1 µM) nor CDCA (100 µM) affected the viability of SCHH. The medium was changed every 24 hours.

d₈-TCA Uptake and Efflux in SCHH.

On day 5 of culture (at the end of 72-hour treatment), uptake of d₈-TCA in Ca²⁺-containing (standard) Hanks’ balanced salt solution (HBSS) and efflux of d₈-TCA in standard and Ca²⁺-free HBSS was performed (n = 3 donors, HC3-26, HUM4122C, and HUM4119C, measured in triplicate), as described previously (Yang et al., 2015; Guo et al., 2016). Briefly, SCHH were preincubated with standard or Ca²⁺-free HBSS for 10 minutes Incubation with Ca²⁺-free HBSS disrupts the tight junctions which seal the canalicular networks, allowing the contents in the bile canaliculi to be released into the medium (B-CLEAR technology) (Liu et al., 1999). After preincubation, SCHH were dosed with 2.5 µM d₈-TCA in standard HBSS containing 4% bovine serum albumin (BSA) for 20 minutes (uptake phase). The presence of BSA (Fraction V; Thomas Scientific, Swedesboro, NJ) during the uptake phase mimics the effect of protein binding in vivo. After the uptake phase, the cells were washed for 1 minute and incubated with fresh protein-free standard or Ca²⁺-free HBSS for 15 minutes (efflux phase). The total amount of d₈-TCA in cells and bile (standard HBSS) and in cells (Ca²⁺-free HBSS) during the uptake phase (2, 5, 10, and 20 minutes) and the efflux phase (2, 5, 10, and 15 minutes), and the total amount of d₈-TCA in the incubation buffer during the efflux phase (2, 5, 10, and 15 minutes) were determined by liquid chromatography–mass spectrometry as reported previously (Guo et al., 2016).

Model-independent parameters describing the overall hepatobiliary disposition of TCA in SCHH were calculated using eqs. 1 and 2 (Liu et al., 1999; Yang et al., 2016) as well as eq. 3.

(1)

(2)

(3)

CL_app,Bile represents the in vitro apparent biliary clearance calculated at 10 minutes during the uptake phase. The biliary excretion index (BEI), determined at 10 minutes during the uptake phase, represents the fraction of compound accumulated in the bile compartment relative to the total accumulation of compound in cells and bile. The buffer recovery index (BRI), determined at 2 minutes during the efflux phase, represents the fraction of compound effluxed from cells and fluxed from bile canaliculi into the buffer relative to the total accumulation in cells at the end of the 20-minute uptake phase.

PK Modeling of TCA Disposition in SCHH.

A mechanistic PK model (scheme depicted in Fig. 1) incorporating linear clearance processes (Yang et al., 2015; Guo et al., 2016) was modified and fit to the TCA total mass-time data in cells and bile, cells, standard HBSS, and Ca²⁺-free HBSS using Phoenix WinNonlin 7.0 software (Certara USA Inc., Princeton, NJ). A mixed proportional and additive error model was used to account for residual error. Parameter estimates from a previous report were used as initial estimates for the control group (Guo et al., 2016). Data from the same hepatocyte donor exposed to different treatments were modeled together. Various model structures were tested. The best-fit model was selected based on Akaike’s information criterion, the precision of parameter estimates, the degree of bias in the residual error, and visual inspection of the predicted curves relative to the observed data. The following kinetic parameters were estimated: total uptake clearance (CL_Uptake), total intrinsic basolateral efflux clearance (CL_int,BL), total intrinsic biliary clearance (CL_int,Bile), and the rate constant describing flux from bile networks into the medium (K_Flux) due to periodic contraction of bile canalicular networks (Oshio and Phillips, 1981; Lee et al., 2010). The mean values and coefficient of variation (CV%) of parameter estimates from the three donors were calculated. Differential eqs. 4–8 were used to describe the changes in the amount of TCA with respect to time in different compartments in this model:

Fig. 1.

Model structure depicting TCA disposition in SCHH in standard HBSS and Ca²⁺-free HBSS. C_u,Cells and C_u,Buffer represent the unbound concentration in the cells and buffer, respectively. X_Bile represents the amount in the bile canalicular compartment.

Mass in standard HBSS:

(4)

Mass in Ca²⁺-free HBSS:

(5)

Mass in Cells:

(6)

Mass in Bile (standard HBSS):

(7)

Mass in Cells+Bile (standard HBSS):

(8)

where C_t,Cells represents the total intracellular concentration and was calculated as X_t,Cells/V_Cells. V_Cells represents cellular volume and was calculated and fixed using the protein content of each hepatocyte preparation and a value of 7.4 μl/mg protein (Pfeifer et al., 2013; Yang et al., 2015). Superscripted plus and minus signs refer to Ca²⁺-containing (standard HBSS) and Ca²⁺-free HBSS, respectively. X_t,Cells, X_t,Cells+Bile, and X_t,Bile represent the total amount in cells, cells and bile, and bile, respectively. C_t,Buffer represents the total concentration in buffer. V_Buffer was set as a constant (0.3 ml). CL_u,Uptake represents unbound uptake clearance. CL_u,int,BL represents unbound intrinsic basolateral efflux clearance. CL_u,int,Bile represents unbound intrinsic biliary clearance. f_u,buffer represents the unbound fraction in buffer containing 4% BSA; f_u,buffer was set at 0.15 (Wolf et al., 2008) during the 20-minute uptake phase and was set at 1 during the 15-minute efflux phase because BSA was present only in the uptake phase. f_u,cell represents the unbound fraction in hepatocytes and was assumed to be 0.15. CL_Uptake was calculated as f_u,buffer × CL_u,Uptake; CL_int,BL and CL_int,Bile were calculated as f_u,cell × CL_u,int,BL and f_u,cell × CL_u,int,Bile, respectively. To mimic the 1-minute wash between the uptake and efflux phase, K_Wash was activated for 1 minute using an if-then statement. K_Wash was fixed at 5 × 10⁴ min⁻¹, which was sufficient to eliminate the d₈-TCA from the buffer compartment. Protein concentrations were measured by the bicinchoninic acid assay.

Immunoblots.

At the end of 72-hour treatment (day 5 cultures), SCHH (n = 3 hepatocyte donors, HUM4122D, HC3-26, and HU8246) were washed with PBS, and the membrane protein was extracted using the ProteoExtract Native Membrane Protein Extraction Kit (Millipore Sigma, Burlington, MA). Membrane proteins (12 µg, without boiling) were mixed with loading buffer containing 50 mM dithiothreitol and resolved on NuPAGE 4%–12% Bis-Tris gel or 7% Tris-acetate gel and the proteins were transferred to polyvinylidene fluoride membranes. After blocking in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 hour, blots were incubated with the following primary antibodies diluted in 5% BSA (Sigma-Aldrich, St. Louis, MO): OSTα, (1:500), OSTβ (1:500), MRP3 (1:1000), MRP4 (1:200), BSEP (1:1000), and NTCP (1:1500) overnight at 4°C or with primary antibody for ATPase (1:500) for 3 hours at room temperature. Then the blots were probed with horseradish peroxidase–conjugated anti-rabbit (OSTs, BSEP, MRP3, NTCP, ATPase) or anti-goat IgG secondary antibody (MRP4) for 1 hour at room temperature (1:7000). Signals were detected by using the Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA) or Clarity Max ECL Substrate (Bio-Rad) with a Bio-Rad ChemiDoc XRS+ system. Densitometric analysis was performed using ImageJ 1.6.0 software (National Institutes of Health, Bethesda, MD).

Data Analysis.

Statistical analysis was performed on the clearance values and immunoblot results using GraphPad Prism software (version 6.01; GraphPad Software Inc., La Jolla, CA). One-way analysis of variance with Bonferroni’s test was used to correct for multiple comparisons with the control group.

Results

Uptake and Efflux of d₈-TCA in SCHH.

OCA and CDCA treatment markedly decreased the total amount of TCA in hepatocytes, the hepatocyte plus bile compartment, and the efflux buffer (Fig. 2). The BRI (%), representing the fraction of accumulated TCA in hepatocytes that undergoes efflux into buffer, was increased from 27%–36% in the control to 50%–53% and 47%–78% in OCA- and CDCA-treated SCHH, respectively. The CL_app,Bile of TCA ranged from 0.62 to 1.2 µl/min per milligram protein in control SCHH but was decreased to 0.35–0.81 and 0.097–0.41 µl/min per milligram protein by OCA and CDCA treatment, respectively. However, the BEI (%) in control SCHH (63%–71%) was similar to values after OCA (63%–74%) and CDCA (46%–71%) treatment.

Fig. 2.

Predicted and observed amount of TCA in different matrices from three different SCHH donors. Black, orange, and blue represent the control-, OCA-, and CDCA-treated groups, respectively, for observed (circles) and predicted (solid lines) data. Experimental data represent the mean ± S.D. (triplicate measurements). The fitted mass versus time profiles were generated from eqs. 4–8, and the parameter estimates are reported in Table 1.

PK Modeling of d₈-TCA Disposition in SCHH.

Observed mass-time profiles of TCA from individual donors were plotted against the predicted mass-time profiles generated by using the best-fit parameters based on the mechanistic model (Fig. 2). The estimated clearance values of TCA in individual hepatocyte donors and the mean values calculated from the individual donors are presented in Table 1. The mechanistic model revealed that CL_int,Bile was approximately 3-fold higher than CL_int,BL in the control hepatocytes, consistent with a previous report (Guo et al., 2016). The CV% of the estimated parameters from model fitting were acceptable (<65%; Table 1), indicating good precision of the model fitting. The fold change in each parameter in treated hepatocytes compared with control hepatocytes is shown in Fig. 3. OCA and CDCA treatment significantly increased CL_int,BL by >6-fold and CL_int,Bile by approximately 2-fold. Changes in CL_Uptake were not statistically significant.

TABLE 1.

Parameter estimates of TCA CL_int,BL, CL_int,Bile, CL_Uptake, and K_Flux

Treatment	Donor	CLint,BL		CLint,Bile		CLUptake		KFlux
		Estimate	CV%	Estimate	CV%	Estimate	CV%	Estimate	CV%
		µl/min per milligram protein						min−1
Control	1	0.29	33	0.80	14	1.6	6.4	0.048	7.3
	2	0.25	52	0.80	15	0.95	5.8	0.046	12
	3	0.19	58	0.70	16	1.4	4	0.037	11
	Mean	0.24	N/A	0.77	N/A	1.3	N/A	0.044	N/A
OCA	1	1.7	32	1.3	12	1.9	18	0.048a	N/A
	2	1.6	41	1.3	14	1.04	24	0.046a	N/A
	3	1.4	38	1.5	12	1.54	17	0.037a	N/A
	Mean	1.6	N/A	1.4	N/A	1.5	N/A	0.044a	N/A
CDCA	1	1.7	40	1.1	16	1.03	25	0.048a	N/A
	2	1.6	64	1.4	24	0.54	36	0.046a	N/A
	3	1.3	56	1.5	24	0.79	24	0.037a	N/A
	Mean	1.6	N/A	1.3	N/A	0.78	N/A	0.044a	N/A

Mean values of these three individual estimates were calculated. Estimates and the corresponding CV% values were obtained by fitting the model to the time-course data from individual SCHH preparations (Fig. 2) based on the model scheme depicted in Fig. 1. Donors 1, 2, and 3 represent HUM4119C, HUM4122C, and HC3-26, respectively. N/A, not applicable.

^aK_Flux values in treated SCHH were fixed to the values in control SCHH from the same hepatocyte donor.

Fig. 3.

Fold change in CL_int,BL, CL_int,Bile, and CL_Uptake in OCA- and CDCA-treated SCHH compared with the control. Data represent the mean ± S.D. (n = 3 hepatocyte donors, as detailed in Table 1). **P < 0.01; ***P < 0.001 (treated vs. control).

Protein Expression of TCA Transporters.

To investigate which transporters might have contributed to the changes in TCA disposition, immunoblot analysis of major bile acid transporters was performed using SCHH from three hepatocyte donors. As shown in Fig. 4, OCA treatment increased the average expression of OSTα and OSTβ to 260% and 1100% of the control, respectively. CDCA treatment increased the average expression of OSTα and OSTβ to 280% and 1300% of the control, respectively. The average BSEP expression was modestly upregulated to 185% and 165% of the control by OCA and CDCA. Changes in MRP3, MRP4, and NTCP protein expression were negligible (<25% changes). The changes in OSTα and OSTβ expression were statistically significant, whereas the changes in other proteins were inconclusive.

Fig. 4.

Effect of OCA and CDCA treatment of SCHH on the protein expression of OSTα, OSTβ, MRP3, MRP4, BSEP, and NTCP. (A) Immunoblots from three independent studies using three hepatocyte donors are shown. ATPase was used as the loading control for each blot. Donors 1, 2, and 3 represent HU8246, HUM4122D, and HC3-26, respectively. Each protein was analyzed on a separate membrane except that BSEP and NTCP shared the same membrane and the same ATPase bands. (B) Quantitative analyses conducted by calculating the relative densities of each protein normalized by the density of ATPase (mean ± S.D. expressed as the percentage of the control; n = 3 hepatocyte donors). **P < 0.01; ***P < 0.001 (treated vs. control).

Discussion

To our knowledge, this study is the first direct demonstration of functional induction of bile acid efflux transporters in response to FXR agonists, revealed by mechanistic PK modeling results. The combination of mechanistic PK modeling and molecular analysis provided a comprehensive data set for understanding the mechanisms of OCA- and CDCA-mediated alterations in hepatic bile acid transporters. Increases in the basolateral efflux clearance and biliary clearance were consistent with a pronounced upregulation of OSTβ protein expression and a smaller increase in BSEP protein expression, respectively (Fig. 5).

Fig. 5.

The effect of FXR agonists on bile acid clearance and protein expression of bile acid transporters in SCHH. Different colors represent the fold change in clearance values (arrowheads; CL_Uptake, CL_int,BL, and CL_int,Bile) and protein levels of transporters (circles; OSTα/β, MRP3, MRP4, BSEP, and NTCP).

This study demonstrated the advantages of PK modeling in assessing changes in concurrent clearance pathways in a whole cell system. Quantitative assessment of basolateral bile acid efflux transporter function is challenging, since plasma or medium bile acid concentrations reflect the net effect of synthesis and metabolism as well as uptake and efflux. A recent report quantified the transport kinetics of a bile acid tracer [N-methyl-¹¹C] cholylsarcosine, which is similar to glycocholic acid, in human blood and liver using positron emission tomography (Orntoft et al., 2017). Based on a kinetic model, the rate constant for basolateral efflux of [N-methyl-¹¹C] cholylsarcosine was higher in cholestatic patients compared with healthy subjects. Similarly, in this mechanistic PK modeling study, concurrent clearance pathways of TCA in hepatocytes were deconvoluted; CL_int,BL and CL_int,Bile of TCA were increased by FXR agonist treatment. This information may have been overlooked by merely calculating model-independent parameters. Although BRI showed a 2-fold increase, indicating increased basolateral efflux, it underestimated the magnitude of change compared with the model-estimated 6-fold increase in CL_int,BL. After FXR agonist treatment, BEI remained the same and CL_app,Bile decreased, whereas model-estimated CL_int,Bile increased by 2-fold. In this case, BEI and CL_app,Bile measured at a single time point failed to reflect the modest changes in biliary excretion because these model-independent parameters are also affected by basolateral uptake and efflux.

The FXR agonist-induced changes in transporter protein expression (greater induction of OSTβ and OSTα than BSEP) were consistent with the increases in TCA clearance (greater increase in CL_int,BL than CL_int,Bile), which would be expected for plasma membrane-localized proteins. In addition, alterations in protein expression agreed with previously published gene expression data in SCHH. OCA treatment (1 µM, 72 hours) increased OSTα, OSTβ, and BSEP mRNA in SCHH by 6.4-, 43-, and 4.6-fold, respectively (Zhang et al., 2017), while CDCA treatment (100 µM, 72 hours) increased OSTα, OSTβ, and BSEP mRNA by 3.1-, 21-, and 2.2-fold, respectively (Jackson et al., 2016). No marked changes in the mRNA expression of MRP3, MRP4, or NTCP were observed in previous studies (Jackson et al., 2016; Zhang et al., 2017), consistent with the present protein expression data. Therefore, the increase in bile acid efflux was likely due to the induction of OSTβ and to a lesser extent to OSTα induction. Protein expression of NTCP remained unchanged in FXR agonist-treated SCHH, consistent with no statistically significant changes in CL_Uptake in CDCA-treated SCHH.

OSTα/β is expressed in many other organs, including the intestine, kidneys, and testis, and it can transport a variety of endogenous compounds in addition to bile acids, such as estrone 3-sulfate, prostaglandin E, dehydroepiandrosterone 3-sulfate, and drugs like digoxin (Soroka et al., 2010). Therefore, upregulation of OSTα and OSTβ may affect the disposition of various endogenous and exogenous compounds and may have significant physiologic implications and affect pharmacotherapy. The heterodimeric OSTα/β is a facilitated transporter that can translocate organic solutes across the basolateral membrane in either direction in vitro (Ballatori et al., 2005). In vivo, it is the primary bile acid efflux transporter in the intestine (Soroka et al., 2010). Lower expression of OSTα and OSTβ in transfected COS cells decreased OSTα/β-mediated transport (Sultan et al., 2017). OSTβ is required for the maturation and stability of OSTα (Dawson et al., 2010). The baseline gene expression level of OSTα is 3- to 7-fold higher than OSTβ in human liver tissue (Ballatori et al., 2005, 2009). Since heterodimerization of the two subunits is required for transport function, greater induction of OSTβ might be needed for maximal function of OSTα/β. Like other basolateral efflux transporters, OSTα/β is expressed at much lower levels in healthy human hepatocytes. Under cholestatic conditions, basolateral efflux transporters are often upregulated to serve as alternate excretory pathways for bile acids (Zollner et al., 2003; Chai et al., 2012). In this study, OCA and CDCA treatment both increased the CL_int,BL of TCA more than 6-fold relative to control, resulting in clearance values that were slightly greater than the CL_int,Bile. An increase in CL_int,BL may exert a hepatoprotective effect by decreasing the hepatocellular accumulation of bile acids.

The marked induction of OSTα, OSTβ, and, to a lesser extent, BSEP, but not MRP3, MRP4, or NTCP, as shown by immunoblotting, could be explained by the differential regulatory mechanisms of these transporters. SLC51A, SLC51B (genes encoding OSTα and OSTβ, respectively), and ABCB11 (gene encoding BSEP) are direct target genes for FXR (Ananthanarayanan et al., 2001; Yu et al., 2002; Landrier et al., 2006; Lee et al., 2006; Lefebvre et al., 2009). The FXR pathway was functional in SCHH and activated by OCA (1 µM) and CDCA (100 µM) treatment, as shown by the following changes in other FXR target genes: decreased gene expression of CYP7A1, the synthesizing enzyme for bile acids, and increased gene expression of a small heterodimer partner (Jackson et al., 2016; Zhang et al., 2017), similar to findings reported by Liu et al. (2014). However, MRP3 is regulated by the retinoic acid receptor, pregnane X receptor, constitutive androstane receptor, and liver receptor homolog-1 (Inokuchi et al., 2001; Teng et al., 2003; Chen et al., 2007; Geier et al., 2007). MRP4 is regulated primarily by the constitutive androstane receptor (Assem et al., 2004; Geier et al., 2007). Although rat Ntcp is a target gene of FXR (Denson et al., 2001), gene expression of human NTCP was minimally affected by FXR agonists. Gene expression of NTCP remained unchanged in human liver slices after CDCA treatment (100 µM, 24 hours) (Jung et al., 2007) and in SCHH after CDCA (100 µM, 72 hours) and OCA treatment (1 µM, 72 hours) (Jackson et al., 2016; Zhang et al., 2017). A slight decrease (30%) in NTCP mRNA was reported in human hepatocytes after CDCA treatment (100 µM, 48 hours) (Liu et al., 2014). Our results, together with these literature reports, showed that NTCP protein expression and function do not appear to be markedly affected by OCA and CDCA treatment of SCHH at the concentrations used in our study.

One assumption of the current PK model was that K_Flux was the same across different treatment groups. K_Flux is dependent on the contraction of bile canaliculi driven by the actin network (Watanabe et al., 1985). Since there is no evidence that FXR regulates actin function, K_Flux was fixed to control values to avoid overparameterization.

In conclusion, FXR agonists OCA and CDCA increased bile acid efflux in SCHH, which contributed to reduced hepatocellular concentrations of d₈-TCA. OSTα/β appeared to be the major transporter responsible for the increase in intrinsic basolateral efflux clearance of d₈-TCA. These cellular effects, together with the suppression of bile acid synthesis (Jackson et al., 2016; Zhang et al., 2017), provide the mechanistic rationale for FXR as a therapeutic target for the treatment of cholestatic diseases by reducing bile acid burden in hepatocytes. These results will enhance our understanding of the FXR-dependent mechanisms of OCA and its impact on bile acid homeostasis.

Abbreviations

BEI

biliary excretion index

BRI

buffer recovery index

BSA

bovine serum albumin

BSEP

bile salt export pump

CDCA

chenodeoxycholic acid

CL_app,Bile

apparent biliary clearance

CL_int,Bile

intrinsic biliary clearance

CL_int,BL

intrinsic basolateral efflux clearance

CL_Uptake

uptake clearance

CV%

coefficient of variation

FXR

farnesoid X receptor

HBSS

Hanks’ balanced salt solution

MRP

multidrug resistance–associated protein

NTCP

sodium taurocholate cotransporting polypeptide

OCA

obeticholic acid

OST

organic solute transporter

PK

pharmacokinetic

SCHH

sandwich-cultured human hepatocytes

TCA

taurocholic acid

Authorship Contributions

Participated in research design: Guo, Edwards, K. R. Brouwer, K. L. R. Brouwer.

Conducted experiments: Guo.

Performed data analysis: Guo, LaCerte, Edwards, K. R. Brouwer, K. L. R. Brouwer.

Wrote or contributed to the writing of the manuscript: Guo, Edwards, K. R. Brouwer, LaCerte, K. L. R. Brouwer.