The nuclear bile acid receptor FXR as a novel therapeutic target in cholestatic liver diseases: Hype or hope?


  • Michael Trauner M.D.

    1. Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University Graz, Graz, Austria
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Farnesoid X receptor (FXR) is a bile acid–activated transcription factor that is a member of the nuclear hormone receptor superfamily. FXR-null mice exhibit a phenotype similar to Byler disease, an inherited cholestatic liver disorder. In the liver, activation of FXR induces transcription of transporter genes involved in promoting bile acid clearance and represses genes involved in bile acid biosynthesis. We investigated whether the synthetic FXR agonist GW4064 could protect against cholestatic liver damage in rat models of extrahepatic and intrahepatic cholestasis. In the bile duct ligation and alpha-naphthylisothiocyanate models of cholestasis, GW4064 treatment resulted in significant reductions in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, as well as other markers of liver damage. Rats that received GW4064 treatment also had decreased incidence and extent of necrosis, decreased inflammatory cell infiltration, and decreased bile duct proliferation. Analysis of gene expression in livers from GW4064-treated cholestatic rats revealed decreased expression of bile acid biosynthetic genes and increased expression of genes involved in bile acid transport, including the phospholipid flippase MDR2. The hepatoprotection seen in these animal models by the synthetic FXR agonist suggests FXR agonists may be useful in the treatment of cholestatic liver disease.

Liu Y, Binz J, Numerick MJ, Dennis S, Luo G, Desai B, et al. Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis. J Clin Invest 2003;112:1678–1687. (Reproduced with permission of the Journal of Clinical Investigation.)


The discovery of nuclear receptors for bile acids has revolutionized our understanding of bile secretion and its impairment under pathological conditions (for review, see Karpen1 and Trauner and Boyer2). Many alterations in bile acid transport and metabolism in cholestasis can now be understood as nuclear receptor–mediated action.1, 2 The farnesoid X receptor (FXR) was the first to be identified, and physiologically appears to be far the most relevant nuclear bile acid receptor.3–5 This landmark discovery was soon followed by the insight that other nuclear receptors (e.g., pregnane X receptor [PXR] and vitamin D receptor [VDR]) are also activated by bile acids.6–8

Currently, the pharmacological armamentarium available for the treatment of cholestatic liver diseases is limited. Ursodeoxycholic acid, the major drug approved and used for the treatment of intrahepatic cholestasis, is of limited efficacy.9 Pharmacological ligands for FXR have raised considerable expectations as novel therapeutics agents for cholestatic liver diseases.1, 2 It must be emphasized that ursodeoxycholic acid is not a FXR ligand in vitro,3–5 and most relevant ursodeoxycholic acid effects on hepatobiliary transport in vivo are mediated via a FXR-independent fashion.10

At first glance, FXR seems to be an ideal therapeutic target in cholestasis. First, FXR expression and activity are directly inhibited by several acquired cholestatic injuries (e.g., inflammation).11 Second, hereditary defects of FXR and FXR-dependent genes (e.g., the bile salt export pump, Bsep) may result in cholestasis.2, 12 Third, the absence of FXR results in increased bile acid toxicity in bile acid–challenged FXR knockout mice (FXR−/−).10, 13 Normally, feeding of exogenous bile acids to mice down-regulates hepatic bile acid uptake and endogenous bile acid synthesis and stimulates bile acid export (Fig. 1). FXR−/− mice lack these adaptive changes and subsequently suffer severe liver damage and death when challenged with exogenous bile acids.10, 13 Surprisingly, however, accumulation of endogenous bile acids in bile duct–ligated FXR−/− mice does not increase mortality and liver damage.14

Figure 1.

Hepatic defense mechanisms against bile acid toxicity: potential role of ligand-activated nuclear receptors. Down-regulation of the basolateral Na+/taurocholate cotransporter (Ntcp) and organic anion transporting proteins 1 and 4 (Oatp1,4) limits bile acid uptake. Induction of “orthograde”/canalicular export pumps such as the bile salt export pump (Bsep) for monvalent bile acids and the conjugate export pump (Mrp2) for divalent bile acids, together with induction of “retrograde”/ alternative basolateral efflux pumps (Mrp3, Mrp4) reduces hepatocellular bile acid retention. This is further assisted by down-regulation of bile acid synthetic enzymes (Cyps 7a1 and 8a1) as well as induction of phase I/hydroxylating (Cyp3a11) and phase II/conjugating (sulfotransferase Sult2a1, glucuronosyltransferase Ugt2b4) enzymes, resulting in polyhydroxylated and sulfated/glucuronidated—thus less toxic—bile acids. Many of these changes can now be understood as nuclear receptor–mediated action. Bile acids activate the “classic” nuclear bile acid receptor (FXR), which in turn directly induces Bsep, Mrp2, and Ugt2b4 expression, but indirectly suppresses Ntcp, Cyp7a1, and Cyp8b1 via induction of the transcriptional repressor short heterodimer partner (SHP). Hydrophobic bile acids (e.g., lithocholic acid) activate PXR and VDR, which stimulate expression of phase I and II bile acid detoxification enzymes. PXR also stimulates several transporter genes. The constitutive androstane receptor (CAR) also mediates induction of multiple transporters and enzymes. It is activated by bilirubin, but bile acids have not yet been identified as endogenous constitutive androstane receptor ligands. However, therapeutic drugs can induce hepatobiliary transporters and enzymes via activation of the PXR (e.g., rifampicin) and constitutive androstane receptor (e.g., phenobarbitial). For a review, see Karpen1 and Trauner and Boyer.2 Abbreviations: BA, bile acids; Ntcp, Na+/taurocholate cotransporter; SHP, short heterodimer partner; FXR, farnesoid X receptor; BA-SO3, bile acid sulfates; Bsep, bile salt export pump; BA-OH, polyhydroxylated bile acids; PXR, pregnane X receptor; CAR, constitutive androstane receptor; VDR, vitamin D receptor; Oatp, organic anion transporting protein; Mrp, multidrug resistance-related protein; BA-Glc, bile acid glucuronides.

Liu and colleagues15 investigated the effects of the FXR agonist GW4064 and tauroursodeoxycholic acid (TUDCA) as clinical comparator in α-naphthylisothiocyanate (ANIT)-treated and common bile duct ligated (CBDL) rats as models of intrahepatic and extrahepatic cholestasis, respectively. Biochemical markers of liver injury and cholestasis, liver histology, and messenger RNA expression of several key hepatocellular transport systems, rate-limiting enzymes of bile acid synthesis and proapoptotic receptors were studied. In both models, GW4064 but not TUDCA (pre)treatment significantly reduced biochemical and histological liver injury. Surprisingly, reductions in serum levels of bile acids and bilirubin–the two major biliary constituents–were only observed in the ANIT model, but not the CBDL model. The authors attributed the observed hepatoprotective effects of GW4064 to induction of canalicular transporter messenger RNA levels (mainly of the phospholipid export pump Mdr2, to a lesser extent of Bsep and the conjugate export pump Mrp2), suppression of enzymes involved in bile acid synthesis (Cyp7a1 and 8b1), and reduction of proapoptotic receptors (Fas, TRAIL2/DR5). Given the established role of FXR in the regulation of these transporters and enzymes1, 2, 13 (Fig. 1), these results may not come as too much of a surprise. Unfortunately, bile acid composition, protein, and functional data are entirely lacking.

Moreover, there are other concerns. First, the chosen ANIT setting may not be the ideal model for studying cholestasis. At the early time point (2 days after single ANIT administration) investigated in the present study, massive hepatocellular necrosis rather than true cholestasis was present, as is evident from the provided microphotographs and massively elevated serum lactate dehydrogenase levels. Most notably, destruction of ducts resulting in ductopenia (and more closely reflecting clinically relevant conditions such as primary biliary cirrhosis) is not seen in this acute setting but only with chronic ANIT administration.16 Because rats were pretreated with GW4064 before ANIT, it cannot be excluded that potential effects on ANIT metabolism and transport could also have contributed to the findings. Second, TUDCA (dissolved in corn oil and injected intraperitoneally) may have been biologically ineffective. In contrast to previous studies in CBDL rats,17 TUDCA did not ameliorate bile duct proliferation. Critical data such as TUDCA serum levels were not provided. Therefore, GW4064 and TUDCA should only be compared very cautiously.

When interpreting potential therapeutic effects of GW4064, it has to be kept in mind that both ANIT and CBDL injury alone profoundly affect transporter and enzyme expression, changes which may reflect intrinsic adaptive mechanisms helping the liver to cope with cholestasis (see Fig. 1).2 One key question must be whether or not GW4064 is capable of further adding to these already existing adaptive changes or–even more important–whether or not it is capable of recruiting novel detoxification and elimination pathways. As such, the alternative hepatocellular bile acid overflow system Mrp3 (facilitating alternative/retrograde bile acid elimination from liver; see Fig. 1) was stimulated by both injuries, but further induction was not achieved by GW4064. Unfortunately, the effects on Mrp4–another major basolateral bile acid overflow system18 (see Fig. 1)–were not investigated. Moreover, key phase I (hydroxylation) and phase II (conjugation) enzymes involved in bile acid detoxification and partly already known to be regulated by FXR19 (see Fig. 1), as well as renal transporters participating in alternative bile acid elimination,2 were not studied. Surprisingly, GW4064 treatment reversed down-regulation of Na+/taurocholate cotransporter and organic anion transporting protein 1 after ANIT, effects which would be predicted to counteract the intrinsic protective suppression of hepatic bile acid uptake (see Fig. 1). No further repression of Na+/taurocholate cotransporter or organic anion transporting protein 1 was observed in CBDL rats treated with GW4064. Regrettably, the biologically more interesting organic anion transporting protein isoforms (Oatp2 and Oatp4)2 (see Fig. 1) were not investigated.

“On the canalicular side”, however, GW4064 was able to induce Mdr2 beyond the already induced levels observed in ANIT and CBDL and stimulated expression of Mrp2 and Bsep, which remained unaffected by ANIT or CBDL alone. Liu and coworkers15 reasoned that this ability to induce canalicular bile acid transporters may account for many of the beneficial effects of GW4064 in cholestasis. Although stimulation of canalicular bile flow may be conceptually appropriate in primarily canalicular cholestasis (e.g., some forms of hereditary and drug-induced cholestasis), it is more difficult to envision a beneficial effect in the presence of ductular and ductal obstruction such as CBDL. It may be simplistic to expect amelioration of cholestasis through mere stimulation of bile flow in the presence of unrelieved biliary obstruction. Rather, recent studies have emphasized that stimulation of bile flow in the presence of an unresolved obstruction may even aggravate liver injury.20 Unfortunately, many clinically relevant cholestatic liver diseases (e.g., primary sclerosing cholangitis) have a significant obstructive component which, however, may evolve slowly in contrast to acute biliary obstruction induced by CBDL and thus may permit more time for adaptation and pharmaceutical interventions. Most importantly, future therapeutic approaches should be tested in more appropriate animal models which better resemble those clinical conditions we aim to treat pharmaceutically.20

A key question is how stimulation of canalicular transporters by GW4064 could contribute to the observed hepatoprotective effects in the presence of biliary obstruction. In this situation, stimulation of canalicular bile acid excretion via Bsep could only be beneficial when followed by immediate reabsorption of bile acids (i.e., “cholehepatic shunting”) from obstructed ducts (e.g., via the apical Na+-dependent bile salt transporter), followed by their renal elimination. To allow such conclusions, expression of cholangiocellular and renal tubular bile acid transporters and urinary bile acid excretion should have have been studied.2 Nevertheless, the impressive increase of Mdr2 may be a key to understanding the observed beneficial effects, because stimulation of biliary phospholipid excretion could counteract bile acid (stasis)-induced bile duct injury by rendering bile less toxic,2, 20 thus protecting the ducts in both CBDL and ANIT models. Similarly, it is attractive to speculate that stimulation of glutathione efflux via induced Mrp2 could also limit bile duct damage by providing a better antioxidative stress defense within the bile duct lumen and epithelium.

It cannot be excluded that down-regulation of FXR in cholestasis could also exert a hepatoprotective effect by interrupting bile acid excretion (via the FXR-dependent canalicular export pumps Bsep and Mrp2) into obstructed ducts. Lower serum bile acid levels and the absence of bile infarcts in CBDL FXR−/− mice further argues for such potential advantages of FXR suppression.14 Gene-selective agonists or even (partial) antagonists of FXR may serve to better target therapy to wanted and “unwanted” FXR effects in cholestasis.1, 2, 21 The surprising finding that cholestasis in bile duct–ligated FXR−/ − mice did not increase mortality and liver damage14 may at least be partially attributed to recruitment of additional–and obviously FXR-independent–adaptive changes in hepatic and renal bile acid transport and metabolism, including induction of hepatic and renal bile acid overflow systems (e.g., Mrp3 and Mrp4) and induction of bile acid detoxification (e.g., hydroxylation via Cyp3a11), changes that altogether may facilitate renal bile acid elimination in cholestasis.2 Other nuclear bile acid receptors such as PXR, VDR, and possibly the constitutive androstane receptor may also be involved in stimulating such complementary adaptive responses.22 Future therapeutic strategies may be aimed at these nuclear receptors and their target genes, including (1) alternative bile acid phase I and II detoxification systems, (2) alternative/basolateral overflow systems in hepatocytes that allow escape of these metabolites into plasma, and (3) renal transport systems for the clearance of these metabolites from plasma (see Fig. 1). Of note, several agents long used empirically to treat cholestasis and its complications (e.g., pruritus) are in fact ligands for nuclear receptors (e.g., rifampcin for PXR; phenobarbital for the constitutive androstane receptor).1, 2

Despite some conceptual and methodological limitations, this interesting study by Liu and colleagues indicates an important new direction in the treatment of cholestasis. This concept needs to be refined by the use of more gene-selective agonists and combination approaches targeting both regular/“orthograde” (FXR-dependent) and alternative/“retrograde” (FXR-independent) pathways of bile acid transport and metabolism. FXR will not remain the only key to treating intrahepatic cholestasis. Although the current hype about nuclear receptors also raises unrealistic expectations, there are strong reasons to hope that nuclear receptor ligands will play a major role in the treatment of cholestatic disorders in the near future. Other nuclear receptors (e.g., peroxisome proliferator-activated receptor) and their ligands may already have shown us the way to go in liver disease.23