The recent overwhelming advances in molecular and cell biology have added enormously to our understanding of the physiological processes involved in bile formation and, by extension, to our comprehension of the consequences of their alteration in cholestatic hepatopathies. The present review addresses in detail this new information by summarizing a number of recent experimental findings on the structural, functional and regulatory aspects of hepatocellular transporter function in acquired cholestasis. This comprises (i) a short overview of the physiological mechanisms of bile secretion, including the nature of the transporters involved and their role in bile formation; (ii) the changes induced by nuclear receptors and hepatocyte-enriched transcription factors in the constitutive expression of hepatocellular transporters in cholestasis, either explaining the primary biliary failure or resulting from a secondary adaptive response; (iii) the post-transcriptional changes in transporter function and localization in cholestasis, including a description of the subcellular structures putatively engaged in the endocytic internalization of canalicular transporters and the involvement of signalling cascades in this effect; and (iv) a discussion on how this new information has contributed to the understanding of the mechanism by which anticholestatic agents exert their beneficial effects, or the manner in which it has helped the design of new successful therapeutic approaches to cholestatic liver diseases.

INTRODUCTION

Cholestasis is a frequent, prominent and often severe manifestation of many liver diseases. It can be defined as an impairment of normal bile flow secondary to structural or biochemical abnormalities of the liver and/or the biliary tree. These alterations may involve: (i) rapid changes in transporter function, e.g. due to drug-induced direct inhibition of relevant transporters or changes in their localization; and (ii) changes in transporter expression, due to inhibition of carrier synthesis or exacerbated degradation. As a consequence of these primary cholestatic alterations affecting primarily hepatocyte bile formation (hepatocellular cholestasis), or in response to an intra- or extra-hepatic blockage of the bile transit (obstructive cholestasis), the liver develops a number of secondary adaptive changes to minimize the detrimental effects of toxic biliary compounds retained as a consequence of the secretory failure. This adaptive response comprises, among other modifications, changes in the expression of both basolateral and canalicular transporters mediated by the co-ordinated activation of a number of nuclear hormone receptors. These regulators act as transcription factors that directly interact with and control the expression of genomic DNA on binding a relevant ligand. These ligands are, more commonly, retained bile salts or conjugated bilirubin, which function as sensors of secretory failure.

The aim of the present review is to give an insight into recent advances in the understanding of the mechanisms of canalicular transporter alterations in acquired cholestasis, with particular emphasis in the structural and functional alterations leading to transport dysfunction. In addition, regulatory aspects involved in the adaptive responses against the detrimental effects of the cholestatic injury will be examined. Finally, we will discuss how certain anticholestatic agents in current therapeutic use may exert beneficial effects, and the mechanistic basis of novel therapeutic strategies based on the information obtained from these basic studies.

PHYSIOLOGICAL MECHANISMS OF BILE FORMATION

Bile formation is an osmotic process driven by the vectorial transport of solutes into bile. This induces passive water movement from blood into bile in response to osmotic gradients, via the water channels AQP (aquaporin) 9 and AQP8 localized at the basolateral and the apical membranes respectively [1,2]. For this to occur, biliary solutes need to be actively concentrated and retained into a confined space, i.e. the bile canaliculus. Only bile salts, glutathione, either reduced (GSH) or oxidized (GSSG) [3] and, perhaps, HCO3 [4] fulfil these requirements. This primary canalicular secretion is modified further by cholangiocytes during its transit along bile ducts. This process is a result of a balance between hormone-dependent secretion of water and electrolytes, and the obligatory absorption of water, electrolytes and organic solutes [5]. Figure 1 shows a schematic representation of the main solutes and transporters relevant to bile flow formation at both the hepatocellular and the bile ductular levels.

Schematic representation of the main transport proteins involved in bile flow generation in both hepatocytes and cholangiocytes

Figure 1
Schematic representation of the main transport proteins involved in bile flow generation in both hepatocytes and cholangiocytes

ATP-dependent transporters are depicted as brown/orange circles. For the sake of simplicity, only rodent transporters are displayed, but human orthologues of all of these transporters have been identified. See the text for details on both rodent and human transporters. Br, bilirubin; BS, bile salts; Glc, glucose; Glu, glutamate; GSH, glutathione; OA, organic anions; VIP, vasoactive intestinal peptide.

Figure 1
Schematic representation of the main transport proteins involved in bile flow generation in both hepatocytes and cholangiocytes

ATP-dependent transporters are depicted as brown/orange circles. For the sake of simplicity, only rodent transporters are displayed, but human orthologues of all of these transporters have been identified. See the text for details on both rodent and human transporters. Br, bilirubin; BS, bile salts; Glc, glucose; Glu, glutamate; GSH, glutathione; OA, organic anions; VIP, vasoactive intestinal peptide.

Bile salts are the predominant organic solutes in bile and the main determinants of bile flow. They are mainly taken up by the NTCP/Ntcp (Na+-taurocholate co-transporting polypeptide; SLC10A1/Slc10a1), driven by the Na+ gradient maintained by the Na+/K+-ATPase pump [6]. A remaining fraction is taken up by a Na+-independent transport system mediated by OATP/Oatp (organic anion-transporting polypeptide) family of transporters, which are non-electrogenic in nature [7]. In addition to conjugated and unconjugated bile salts, OATPs/Oatps accept other amphipathic compounds, such as bilirubin glucuronides and, maybe, unconjugated bilirubin. Four OATPs have been cloned and characterized from human liver, namely OATP1A2 (SLCO1A2/SLC21A3; formerly OATP-A), OATP1B1 (SLC21A6; formerly OATP-C or LST-1), OATP1B3 (SLC21A8; formerly OATP-8) and OATP2B1 (SLC21A9; formerly OATP-B). All but the latter are involved in bile-salt uptake, with OATP1A2 being the most important one [8]. For unconjugated bilirubin [9] and bilirubin monoglucuronides [10], both OATP1B1 and OATP1B3 have been proposed to be involved. Although OATP1B1 was thought to have a far higher efficiency for unconjugated bilirubin than OATP1B3 [10], this finding was not supported by others [11]. Bilirubin diglucuronides appear to be transported only by OATP1B1 [10]. There are three Oatps identified in rats, namely Oatp1a1 (Slc21a1; formerly Oatp1), Oatp1a4 (Slc21a5; formerly Oatp2) and Oatp1b2 (Slc21a10; formerly Oatp4 or Lst-1). Oatp1b2 is the rodent orthologue of both OATP1B1 and OATP1B3 [12]. All of them are involved in the uptake of conjugated and unconjugated bile salts [8,13]. Oatp1a1 and Oatp1a4 are involved in bilirubin monoglucuronide transport [13,14].

After traversing the cell by Fick's diffusion bound to high-affinity cytosolic proteins, monoanionic bile salts are excreted in the canalicular pole by BSEP/Bsep (bile salt export pump; ABCB11), an ABC transporter (ATP-binding-cassette transporter) [15]. In contrast, canalicular efflux of divalent, bipolar sulfated or glucuronidated bile salts is mediated by MRP2/Mrp2 (multidrug resistance-associated protein 2; ABCC2/Abcc2). This carrier is also engaged in the biliary excretion of bilirubin mono- and di-glucuronides [16].

Another key determinant of bile flow is glutathione, both in its reduced (GSH; approx. 80%) and in its oxidized (GSSG) forms [3]. The liver is the main site of glutathione synthesis, and biliary glutathione comes from this intracellular source. A high-affinity electrogenic canalicular carrier for GSH has been functionally characterized, but not cloned [17]. This transporter actively exports GSH into bile, although it can transfer GSSG with lower affinity. On the contrary, GSSG is excreted with higher affinity by Mrp2, which can also transfer GSH, but with low affinity [18].

Another putative candidate to partially determine bile flow is HCO3 [4]; however, it remains questionable whether bile-to-plasma HCO3 gradients can be maintained, due to the high paracellular permeability of this anion [19]. Overall, HCO3 transport involves the sequential participation of three different transporters, namely Na+/K+-ATPase, NHE (Na+/H+ exchanger), localized at the basolateral domain, and AE2/Ae2 (anion exchanger 2; SLC4A2/slc4a2), localized at the apical domain. Na+/K+-ATPase activity maintains plasma-to-cytosol Na+ gradients for basolateral efflux of H+ via NHE. The extruded H+ neutralizes HCO3 in plasma, enabling its passive diffusion as CO2. After conversion of CO2 back into HCO3 by intracellular carbonic anhydrase, the anion is counter-transported with Cl at the canalicular domain by AE2/Ae2.

Canalicular bile flow is modified further during its transit along the bile ducts by both secretory and absorptive processes [20]. Ductular fluid secretion is driven by the secretin-regulated cAMP-dependent output of an HCO3-rich fluid secreted via the Cl/HCO3 exchange system AE2/Ae2. Exchange is driven by the out-to-in Cl concentration gradient, which is maintained by the Cl efflux across the apical membrane via the secretin-activated CFTR (cystic fibrosis transmembrane regulator). As with secretin, VIP (vasoactive intestinal peptide) [21,22] and bombesin [23,24] stimulate ductular HCO3-rich fluid both in rodents and humans. The effects of these peptides is cAMP-independent, at least in rodents [22]. Blood-to-bile water movement at the ductular level is facilitated by constitutive AQP4 in the basolateral membrane, and secretin-stimulated AQP1 in the apical membrane [1,2]. On the other hand, the absorption of ductular water and electrolytes is driven by osmotic gradients created by bile-to-plasma transport of electrolytes and organic solutes. They comprise glutamate, glucose, transported by SGLT1 and GLUT1 at the apical and basolateral domains respectively, and bile salts, taken up by ASBT/Asbt (apical Na+-dependent bile salt transporter; SLC10A2/slc10a2), followed by basolateral extrusion via the export pump MRP3/Mrp3 (ABCC3/Abcc3) and the heterodimeric OSTα–OSTβ/Ostα–Ostβ (where OST/Ost is organic solute transporter) [5,25].

ALTERATIONS IN CANALICULAR TRANSPORTERS IN ACQUIRED CHOLESTASIS

Changes in hepatobiliary transporter expression/activity in acquired cholestasis have been reported to occur at the transcriptional, post-transcriptional or post-translational level. These pathomechanisms result in changes in transporter activity in days, hours or minutes/seconds respectively.

Changes in transporter expression in cholestasis

Decreased expression of hepatobiliary transport systems at the protein level can be the primary cause of the cholestatic injury or, alternatively, the secondary consequence of adaptive changes in transporter expression. In the former case, a clear distinction between the mechanisms that actually cause the secretory failure from the cascade of changes resulting from this initial cholestatic insult is difficult to establish. These secondary adaptive changes can be dissected better in experimental models of obstructive cholestasis (bile duct ligation). In this case, the primary cholestatic insult occurs at an extrahepatic level, so that all of the hepatic changes can be regarded as adaptive. It should be stressed, however, that these animal models cannot be extrapolated unequivocally to human cholestatic conditions, due to differences in transcriptional regulation, transporter specificity and the length of the cholestatic insult (acute in laboratory animal compared with chronic in humans).

Adaptive changes in the expression of transporters and metabolic systems in cholestasis

In several animal models of experimental cholestasis, and in several human cholestatic liver diseases as well, there is a common pattern of adaptive responses involving changes in transporter expression. This response protects the hepatocyte from the retention of toxic biliary compounds, particularly bile salts and bilirubin [26]. The adaptive changes comprise down-regulation of basolateral uptake systems, accompanied by the up-regulation of basolateral extrusion systems (Figure 2). This greatly helps to maintain intracellular concentrations of internalized metabolites, or de-novo-synthesized ones, at a safe low level, despite ongoing secretory failure. This adaptive transporter regulation is assisted further by repression of the bile-salt-synthesizing enzymes Cyp7a1 and Cyp8b1 (where Cyp is cytochrome P450), and the up-regulation of phase I and phase II bile-acid-metabolizing enzymes. Phase I bile-acid-hydroxylating enzymes comprise Cyp3a11 (the rodent homologue of human CYP3A4) and Cyp2b10 (the rodent homologue of human CYP2B6). Phase II-conjugating enzymes are Sult2a1 (where Sult is sulfotransferase) and Ugt2b4 (where Ugt is UDP-glucuronosyltransferase). Bile acid hydroxylation (mainly at 1β, 2β, 4β, 6α, 6β, 22 or 23 positions) results in an increased intracellular pool of less toxic polyhydroxylated bile salts. Most of these transformations facilitate the excretion of bile acids into the bile (sulfation) or into the blood for subsequent urinary elimination (hydroxylation, sulfation and glucuronidation) [27].

Adaptive changes in obstructive cholestasis in the expression of transporters and enzymes involved in bile salt synthesis and bile salt/bilirubin conjugation in the liver, kidney and intestines
Figure 2
Adaptive changes in obstructive cholestasis in the expression of transporters and enzymes involved in bile salt synthesis and bile salt/bilirubin conjugation in the liver, kidney and intestines

Note that, for simplicity, only changes in rodent gene products are displayed. Most of the adaptive changes at the hepatocellular level have been confirmed in patients with cholestasis. On the contrary, most of the adaptive response in ductular epithelia, kidney and intestines described in rodents remains to be confirmed in humans. For details, see the text. Br, unconjugated bilirubin; Br-G, bilirubin glucuronides; CFTR, cystic fibrosis transmembrane regulator; OA, organic anions; BS, bile salts.

Figure 2
Adaptive changes in obstructive cholestasis in the expression of transporters and enzymes involved in bile salt synthesis and bile salt/bilirubin conjugation in the liver, kidney and intestines

Note that, for simplicity, only changes in rodent gene products are displayed. Most of the adaptive changes at the hepatocellular level have been confirmed in patients with cholestasis. On the contrary, most of the adaptive response in ductular epithelia, kidney and intestines described in rodents remains to be confirmed in humans. For details, see the text. Br, unconjugated bilirubin; Br-G, bilirubin glucuronides; CFTR, cystic fibrosis transmembrane regulator; OA, organic anions; BS, bile salts.

Ntcp [2831], Oatp1a1 [28,31,32] and Oatp1b2 [33] protein levels are decreased in several animal models of experimental cholestasis at a transcriptional level [34]. This helps to maintain at a minimum the uptake of potentially toxic compounds arriving to the liver, such as the bile salts reabsorbed by the intestine and bilirubin. On the other hand, Oatp1a4 is up-regulated in bile-duct-ligated mice [33]. Organic substrate transport carried out by Oatp1a4 is potentially bidirectional [35] and, therefore, can reverse its transport direction in conditions of bilirubin conjugate/bile acid overload, thus acting as an alternative export system in cholestasis.

In addition to impairing uptake, the cholestatic insult enhances routes for the basolateral extrusion of organic anions. The basolateral membrane of rodent cholestatic hepatocytes overexpresses several ATP-dependent organic anion efflux pumps belonging to the Mrp family, such as Mrp1 (ABCC1/Abcc1) [34,36,37], Mrp3 (ABCC3/Abcc3) (in rat [28,3843], but not in mouse [44]), Mrp4 (ABCC4/Abcc4) [34,44,45] and Mrp5 (ABCC5/Abcc5) [34,39]. In experimental obstructive cholestasis in rodents, all of these increased expressions occur at a protein level and are, at least in part, transcriptional in nature [34]. These ATP-dependent carriers extrude divalent bile acids (e.g. bile acid sulfoconjugates) and monovalent bile acids (glyco- and tauro-conjugates) in rodents [13]. In humans, specificity and transport mechanisms may vary. For example, MRP3 transports only bile acid glycoconjugates with low affinity [46,47], and MRP4 co-transports bile acids with GSH [48]. In addition, the recently identified OSTα–OSTβ/Ostα–Ostβ, a heterodimeric transporter that exports bile salts and their conjugates back to plasma, is also up-regulated at the transcriptional level in both the bile-duct-ligated mice and in cholestatic patients with PBC (primary biliary cirrhosis) [49]. The relative importance of each of these basolateral extrusion carriers to transport biliary substrates, and their role in protecting the hepatocyte against a cholestatic insult, may be differential. Experiments in Mrp4−/− mice with bile duct ligation revealed that hepatic Mrp4 plays an important role in the adaptive response to obstructive cholestatic liver injury by preferentially improving the extrusion of bile salts into the blood. Indeed, more extensive damage and lower serum bile salt levels occurred in Mrp4−/− mice compared with the wild-type, despite both Mrp3 and Ostα–Ostβ being up-regulated [44]. Conversely, bile-duct-ligated Mrp3−/− mice had no differences compared with the wild-type in both serum bile salt levels and liver histology [43,50]; Mrp3−/− mice had lower serum levels of bilirubin, suggesting a preferential role for Mrp3 in the basolateral extrusion of bilirubin glucuronides.

The bile salt conjugates and the bilirubin glucuronides that are not taken up by the hepatocyte, or that are effluxed into the systemic circulation after uptake, can be eliminated in urine. This alternative excretory route is enhanced under cholestatic conditions [12]. The increased efficiency of the kidney to detoxify these compounds occurs by (i) increased passive glomerular filtration, (ii) repressed transcription, leading to diminished protein levels of Asbt [51], a transporter involved in tubular re-absorption of bile salts, and (iii) the co-ordinated over-expression at the protein level of basolateral Oat1 [52] and of the apical transporter Mrp2 [38,51]; Mrp2 mRNA levels did not increase, suggesting post-transcriptional mechanisms [51]. Similarly, renal Mrp4 which, unlike liver, has an apical localization in kidney, is up-regulated transcriptionally in mouse kidney [53,54], although it is down-regulated at the post-transcriptional level in the rat [45].

The final hepatic fate of the cholephilic compounds under physiological conditions is canalicular transfer, which represents the rate-limiting step in the overall hepatic handling. In obstructive cholestasis, secondary inhibition of the expression of canalicular transporters occurs. These changes can be regarded, however, as beneficial, since this may help to protect bile ducts from mechanical damage by an increase in biliary pressure [53,55] or from cholangiocyte necro-apoptosis induced by toxic biliary solutes [56]. Both Mrp2 protein expression and mRNA levels were considerably impaired in obstructive cholestasis in rats [5759] and humans [60,61]. Bsep expression is less altered by far [59,62]. An adaptive response against this is the enhancement in the bile salt cholehepatic shunt pathway. This mechanism removes bile salts from the biliary space and maintains in operation the hepato-cholangiocyte flux of bile salts, despite ongoing bile duct obstruction. A key ductular bile salt transporter involved in this response is Asbt. Although the expression of this transporter is maintained per cholangiocyte, the number of intrahepatic bile ducts markedly increases in bile-duct-ligated rats (approx. 10-fold after 1 week) [25]. Improvement in Asbt-mediated bile acid transport through the cholangiocyte apical membrane in bile-duct-ligated animals is assisted by a concomitant overexpression of the basolateral bile acid transporters Mrp3 [40] and Ostα–Ostβ [49]; Ostα–Ostβ induction was only confirmed at the mRNA level. Multiplication of bile ducts also induces a proportional increase in the biliary tree death space. This helps to substitute the intestines as the major reservoir of the bile salt pool when bile salt enterohepatic recirculation is impaired [63]. Down-regulation of intestinal Asbt protein levels also occurs. This decreases the amount of bile salts returning to the liver, thus reducing further the liver bile salt load. Adaptive down-regulation of Asbt was observed in bile-duct-ligated rats, the mechanism being post-transcriptional in nature [64].

Commonly, the acquired changes in transporter expression in human cholestatic liver disease are consistent with those occurring in experimental animal models of cholestasis, although the mechanisms accounting for these expression changes may vary. Generally, the contribution of transcriptional mechanisms is more limited, or absent, in humans compared with rodents, with posttranscriptional/post-translational mechanisms being more relevant [13,6567].

As in rodents, the down-regulation of basolateral uptake transport systems is apparent in chronic cholestatic diseases in humans. NTCP and OATP1B1 protein levels decrease in inflammation-induced icteric cholestasis (e.g. cholestatic alcoholic hepatitis) [65], late-stage PBC [66], PFIC2 and PFIC3 (type 2 and 3 progressive familial intrahepatic cholestasis respectively) [68], PSC (primary sclerosing cholangitis) [67] and extrahepatic biliary atresia [69]. OATP1B3 is also down-regulated in PFIC1 (type 1 PFIC) and PFIC2 at the protein level [68]. Similarly, adaptive increments in protein levels of the hepatocellular basolateral export pumps MRP1 [70,71], MRP3 [66,70,71], MRP4 [71,72] and MRP5 [71] have been reported in late-stage PBC. Similarly, MRP3 [73] and MRP4 [68] protein levels were increased in PFIC3. Finally, increases in MRP3 have been reported in both biliary atresia [73] and biliary obstruction, secondary to carcinomas of the biliary tract or pancreas [61]. The mRNA levels of the basolateral extrusion transporter OSTα–OSTβ are also augmented in the liver of patients with advanced PBC [49]. Extensive up-regulation at the mRNA level of OSTα–OSTβ, but not of MRP3 and MRP4, may help to explain why the induction of OSTs is more profound than that of MRP3 and MRP4 in PBC [49]. OATP1B3, which remains highly expressed in liver under cholestatic conditions, behaves as a GSH-dependent symporter, and has recently been proposed to be an additional route for the basolateral extrusion of cholephilic organic anions, including bile salts, in human cholestatic diseases [74]. At the canalicular pole, and similar to the findings in bile-duct-ligated rats, MRP2 expression is decreased at the protein level in cholestatic liver diseases, although the changes are only observed in advanced stages of the disease and are less dramatic in intensity. For example, MRP2 protein expression only decreased in some, but not all, patients with stage IV PBC [75] and remained unaffected in stages I–III PBC [66,75]. Similarly, MRP2 and BSEP mRNA levels fell only in livers from poorly drained patients with obstructive extrahepatic cholestasis, but not in the well-drained ones [61]. Finally, BSEP protein expression was relatively well preserved in patients with extrahepatic biliary atresia, even in the untreated ones [76].

In human cells, other than hepatocytes, the existence of an adaptive response against cholestasis remains largely speculative, although some indications are emerging. Human cholangiocytes actively proliferate in response to many cholestatic conditions, including obstructive cholestasis and PBC [77]. MRP3 protein levels were frequently increased in proliferative cholangiocytes from livers of patients with various forms of cholestasis, as revealed by immunohistochemical studies [70,73]. In the intestine, adaptive down-regulation of ASBT in patients with obstructive cholestasis has been reported [78]. Finally, although all of the transporters involved in the renal response to cholestasis have been identified in human kidney, the occurrence of these adaptive changes in patients with cholestasis remains to be confirmed.

Role of nuclear receptors and hepatocyte-enriched transcription factors in adaptive response in cholestasis

Adaptive changes in expression of hepatocellular transporters are mediated mainly by the co-ordinated modulation of a number of transcription factors, including the nuclear orphan receptors and liver-enriched HNFs (hepatocyte nuclear factors). Most of the information on these mechanisms was obtained from rodent models of cholestasis, by using either specific ligands to activate nuclear receptors or knockout animals with selective deficiency in the synthesis of transcription factors. However, these regulations remain largely unconfirmed in human cholestatic disease. It should be borne in mind that changes involving these regulators are transcriptional in nature and, as stated above, transcriptional mechanisms may be less relevant in humans compared with rodents [13,6567].

The nuclear receptors thought to be involved in feedback or feedforward regulation of carrier genes in cholestasis comprise, among others, FXR (farnesoid X receptor; NR1H4), PXR (pregnane X receptor; NR1I2), CAR (constitutively activated receptor; NR1I3), VDR (vitamin D receptor; NR1I1), LXR (liver X receptor; NR1H3), RARα (retinoic acid receptor α), LRH1 (liver receptor homologue 1; NR5A2), PPARα (peroxisome-proliferator-activated receptor α; NR1C1) and SHP (small heterodimer partner; NR0B2). All except the two latter heterodimerize with RXRα (retinoid X receptor α; NR2B1), enabling high-affinity DNA binding and further activation of gene transcription [12]. The ligands involved in this adaptive response are biliary constituents retained by secretory failure, such as bilirubin [79] and hydrophobic hepatotoxic bile salts (e.g. chenodeoxycholate, deoxycholate and lithocholate) [80,81].

In rodents, FXR-mediated induction of the nuclear repressor SHP inhibits Ntcp expression in experimental obstructive cholestasis [82,83], which surpasses the RXRα/RARα-mediated activation of the Ntcp promoter [83]. The SHP effect may be, at least in part, mediated by a multistep, regulatory cascade involving repression of the hepatocyte-enriched transcription factor HNF4α, which suppresses its activating effect on HNF1α [also known as TCF1 (transcription factor 1)], a potent Ntcp activator [84]. The human SHP promoter region also contains FXR-response elements [85]. The involvement of SHP was, however, challenged by studies showing that Ntcp is down-regulated in bile-duct-ligated SHP−/− mice, suggesting that SHP is not essential for Ntcp repression [86]. As bile salts can repress HNF4α (NR2A1) transcription genes, a direct bile salt inhibition of this transcription factor can help to explain this finding [87]. In humans, the relevance of this latter transcriptional cascade is doubtful, as neither HNF4α, HNF1α nor RXRα/RARα binds the Ntcp promoter, suggesting species differences in NTCP/Ntcp gene regulation [84].

A similar FXR-mediated repression of the SHP/HNF4α/HNF1α cascade has been proposed to be a primary mediator of the transcriptional impairment of Oatp1a1 and Oatp1b2 in rodent models of cholestasis [13,87]. Interestingly, OATP1B1, the gene of the human orthologue of Oatp1b2, contains an HNF1α-binding site in its promoter region [88], suggesting that this latter mechanism may be operative in patients with cholestasis [89]. In addition, PXR and CAR up-regulate the Na+-independent bile-acid uptake transporter Oatp1a4 [33].

CAR is one of the nuclear receptors involved in the up-regulation of Mrp3 and Mrp4 in rodents, thus enhancing basolateral export pathways in these animal models [9092]. Mrp3, but not Mrp4, overexpression is assisted by PXR [90,93] and LRH1 [94]. In this latter case, another factor mediating bile-acid-dependent induction would be required, as bile acids are not LRH1 ligands. In contrast, induction of Mrp3 and Mrp4 are FXR-independent [53,95,96]. Furthermore, FXR appears to inhibit Mrp4 in rodent cholestatic liver, but CAR overrides this inhibitory effect [97]. In humans, the mechanisms of MRP3 induction may be different to rodents, as a CAR cis-binding element could not be identified in the human MRP3 promoter [98]. PXR-mediated up-regulation of human MRP3 is an alternative possibility, as ligand-activated PXR has also been shown to induce MRP3/Mrp3 expression in human hepatoma cell lines [93]. LRH1-induced MRP3 expression is also possible, as the MRP3 promoter contains a binding element for LRH1 [99]. Finally, MRP3 overexpression may also involve a decrease in the expression of RXRα/RARα in human cholestasis. This nuclear receptor down-regulates MRP3 expression by abrogating the activity of the transcription factor Sp1 on the MRP3 gene promoter [98].

FXR activates the basolateral bile-salt-exporting carrier OSTα–OSTβ/Ostα–Ostβ, both in healthy rodents [96] and in cholestatic rodents and humans [49]. Similarly, FXR transactivates OATP1B3, but not other OATP isoforms [100]. Differential activation of OATP1B3 may be related to its bile-salt-exporting function at the basolateral domain [74].

In the canalicular pole, bile-salt-activated induction of FXR is responsible for the maintenance of Bsep expression under these cholestatic conditions by up-regulating the otherwise decreased Bsep expression. This regulatory role for FXR has been only confirmed in a rodent model of obstructive cholestasis [53]; however, the BSEP (ABCB11) gene also contains an IR1 (inverted repeat 1) element in it's promoter regions that directly binds to the FXR/RXRα heterodimer upon activation by bile acids, resulting in enhanced transactivation of the BSEP gene [101103].

Unlike Bsep, Mrp2 expression is extensively repressed in cholestatic liver [57]. Obstructive cholestasis is accompanied by systemic release of pro-inflammatory cytokines [104]. The decrease in Mrp2 content in this condition appears to be due to a cytokine [IL (interleukin) 1β]-dependent impairment in RARα expression [105,106]. Cytokines also induce re-localization of the nuclear receptor partner RXRα from the nucleus to cytosol, a finding shown in LPS (lipopolysaccharide)-induced cholestasis [107]. If the latter event also occurs in obstructive cholestasis, this may contribute further to the reduced occurrence of RARα/RXRα heterodimerization and that of other nuclear receptors that transactivate MRP2/Mrp2, such as FXR, PXR and CAR [108].

Nuclear receptors also mediate adaptive changes in enzymes involved in bile salt synthesis and bile salt/bilirubin detoxification [27,109,110]. Most of these regulations have been observed in rodent models of cholestasis, and the involvement of these regulatory mechanisms in patients with cholestasis has been inferred, but not determined, from studies in patients without cholestasis receiving specific nuclear receptor ligands for therapy or from studies in human cells lines.

FXR (via the SHP-mediated repression of LHR1 [85]) and PXR [80,111] inhibit de novo bile salt synthesis by repressing the transcription of the genes encoding Cyp7a1, the rate-limiting enzyme in the ‘classic’ bile salt biosynthetic pathway. In primary human hepatocytes, activation of PXR by rifampicin promotes PXR interaction with HNF4α and further CYP7A1 inhibition [112]. Cyp8b1, which controls the dihydroxy-to-trihydroxy bile acid synthesis ratio, was also repressed, thus regulating the bile salt hydrophobicity index. A similar down-regulation of both Cyp7a1 and Cyp8b1 occurs after PPARα activation in rodents [113,114].

PXR and CAR enhance the expression of Cyp3a-mediated bile salt hydroxylation in rodents [80,92,111,115], and this also appears to apply to humans. Indeed, a response element in the human CYP3A promoter has been identified for PXR [116] and CAR [117], and ligands of both nuclear receptors stimulate CYP3A expression in human hepatocytes [118]. Interestingly, administration of the PXR ligand rifampicin stimulates CYP3A4 expression in otherwise healthy patients with cholesterol gallstones [119]. HNF4α appears to be critically involved in PXR- and CAR-mediated transcriptional activation of CYP3A in humans. A specific cis-acting element in the CYP3A4 gene enhancer has been identified that confers HNF4α binding, thus permitting PXR- and CAR-mediated gene activation [120]. Finally, a role for LXR in CYP3A4 induction has been proposed recently [121].

Rodent Ugt1a1 (UDP-glucuronosyltransferase 1A1)-mediated bilirubin glucuronoconjugation is also activated by PXR and CAR [90,92]. The PXR activator rifampicin [122] and the CAR activator phenobarbital [123] both induced human UGT1A1 protein expression.

CAR [90], and to a lesser extent FXR [124], PXR [125,126], VDR [127] and LXR [128], activate SULT2A1/Sult2a1. This enzyme enhances bile acid sulfation and further renal excretion of the sulfated bile salts. Finally, both FXR [129] and PPARα [130] increase bile acid glucuronidation by transactivating human UGT2B4.

Changes in transporter expression as a primary cause of cholestasis

Changes in transporter expression in acquired hepatocellular cholestasis may represent the primary causal factor of cholestatic dysfunction. Although adaptive changes similar to those occurring in obstructive cholestasis can also take place to protect the hepatocyte against secondary manifestations of the cholestatic insult, these adaptive changes may further aggravate the primary secretory failure by reversing hepatocellular polarity.

Many acquired non-obstructive cholestatic disorders involve primary changes in hepatocellular transporter expression (hepatocellular cholestasis). They can be divided in two large subgroups, namely (i) inflammation-induced cholestasis, which have cytokines as the common deleterious effector, and (ii) ‘pure’ (bland) cholestasis, which is caused by drugs or hormones. It should, however, be borne in mind that cholestatic diseases with features of both obstructive and hepatocellular cholestasis also exist. For example, PBC [75] and PSC [67], two prototypical chronic cholestatic liver disease in humans, develop bile duct obstruction by cholangiodestruction, but inflammation-induced release of cytokines is also critically involved in the progression of the disease [131,132].

Inflammation-induced cholestasis

Pro-inflammatory cytokines produced as a consequence of either infectious or non-infectious diseases are potent inducers of intrahepatic cholestasis by acting as potent inhibitors of transporter expression [133]. Among the multiple pathologies in which inflammation-induced cholestasis is involved, sepsis-induced cholestasis is the prototypical one [134,135]. The cholestatic complication of this disease has been associated with the action of the bacterial wall component LPS, and the consequent hepatic release of the inflammatory cytokines IL1β, TNFα (tumour necrosis factor α) and IL6 [136]. Therefore LPS administration to rodents has been widely used as an experimental model to characterize sepsis-induced cholestasis. This model was also helpful in understanding the cholestatic mechanisms of cytokines. This information is relevant to human cholestatic diseases other than sepsis, as cytokines are critical mediators of alcoholic, viral, autoimmune and drug-induced cholestatic hepatitis [133].

Protein expression of both Bsep and Mrp2 are impaired in experimental sepsis in rodents [37,137,138], although the alteration of Bsep appears to be less pronounced [62]. In rats, Mrp2, but not Bsep, expression is modulated transcriptionally [37,137], whereas, in humans, down-regulation of both BSEP and MRP2 is post-transcriptional in nature, indicating marked species differences [137].

The decrease in canalicular transporter expression in LPS-induced cholestasis in rats is accompanied by transcriptional down-regulation of Ntcp [139,140] and several Oatps [136,139,140]. Up-regulation of Mrp1 and Mrp3 was also reported [37,140], although the up-regulation of Mrp3 was not observed by others [136,141]. At least part of these alterations occur in human liver slices exposed to LPS, in particular those involving NTCP transcriptional down-regulation [137]. This event correlated inversely with TNFα and IL1β levels [137].

Down-regulation of Mrp2, Ntcp and Oatps in rodents may be due, in part, to repressed transcriptional expression of both RXRα/RARα and HNF1α [142], which are transactivators of these transporters. LPS-induced cytokine release may play a key role [141,142]. Indeed, depletion of Kuppfer cells, the main hepatic source of cytokines, counteracted the decrease induced by LPS in the binding activity of the transcription factors RXRα/RARα and HNF1α, and the associated decrease in Ntcp expression in rats [143]. Cytokines released after LPS administration to rodents also reduce the binding activity of RXRα/RARα by inducing a shift in RXRα from the nucleus into the cytosol, which induces the removal of the heterodimeric partner from the nucleus [107]. Phosphorylation of RXRα by JNK (c-Jun N-terminal kinase) after LPS-induced stimulation of Kupffer cells [144] has been proposed to trigger this re-localization [107]. JNK activation has been also proposed to be a key determinant in the decreased binding activity of HNF1α. This effect appears to be secondary to a decrease in HNF4α promoter activity and protein expression, with JNK-mediated HNF4α phosphorylation having a critical role [145].

Selective inhibition of different cytokines in LPS-induced cholestasis in vivo revealed that IL1β is the major cytokine involved in these effects [146], whereas IL6 only plays a crucial role during the later stages of endotoxaemia [147]. However, mechanisms independent of cytokines, and not involving the repression of RXRα/RARα and HNF1α, may also occur [146]. For example, LPS-induced PXR activation has been shown to contribute to Mrp2 down-regulation in mice [148].

Alterations in hepatobiliary transport systems have been reported in inflammation-induced acute icteric cholestasis caused by cholestatic alcoholic hepatitis, cholestatic autoimmune hepatitis and drug-induced hepatitis [65]. A decreased expression of basolateral uptake transporters (NTCP and OATP2) and canalicular export pumps (BSEP and MRP2) was reported. This was accompanied by low mRNA levels of NTCP and OATP2 and, to a lesser extent, of BSEP. In contrast, MRP2 down-regulation appears to occur by post-transcriptional mechanisms. Finally, MRP3 expression is maintained, which may protect hepatocytes from further accumulation of potentially toxic biliary constituents.

‘Pure’ (bland) cholestasis

Causes of ‘pure’ (bland) cholestasis include hormones and hormone derivatives, such as oestrogens, C17-substituted testosterone and anabolic steroids, as well as a large number of cholestatic drugs [149]. Among the several cholestatic pathologies belonging to the latter group, oestrogen-induced cholestasis in rats and its clinical correlate, obstetric cholestasis, were studied with more detail in terms of hepatocellular transporter expression and these will be focused upon below.

Oestrogens have been proposed to be a key causal factor in intrahepatic cholestasis in susceptible women during pregnancy [150], or following administration of oral contraceptives and postmenopausal replacement therapy [151]. Owing to this relevant clinical correlate, cholestasis induced by oestrogens in rodents is a common experimental model of hepatocellular cholestasis. The cholestatic injury is produced either over the course of a few days by administration of EE (17α-ethynyloestradiol), a model oestrogen, or within minutes by E217G (oestradiol 17β-D-glucuronide), an oestrogen metabolite.

In the EE-induced cholestatic model, Bsep expression is impaired slightly [62,152], whereas Mrp2 expression is decreased to a higher degree at the post-transcriptional level [57]. EE also induces transcriptional down-regulation of Ntcp [32,152,153] and Oatps [32,153,154], together with the up-regulation of Mrp3 [42,58,154]. The nuclear receptor ERα (oestrogen receptor α) has been recently proposed to mediate, at least in part, EE-induced cholestasis and the changes in transporter expression induced by the cholestatic agent [155]. Indeed, ERα−/− mice lack the EE-induced repression of the mRNA expression of Ntcp, Oatp1a1, Oatp1a4, Bsep and bileacid-biosynthesis enzymes (Cyp7a1, Cyp7b1 and Cyp8b1) [155]. On the contrary, other nuclear receptors, such us ERβ, FXR, SHP, PXR and CAR, appear not to be involved [155]. However, a role for FXR or SHP in other species cannot be ruled out. Unlike this study in mice, EE down-regulates FXR and up-regulates SHP, via ERα, in rats [156,157]. In addition, an oestrogen-induced decrease in nuclear binding activity of Ntcp transactivators, such as HNF1α, C/EBP (CAAT/enhancer binding protein) and PXR, has also been proposed to play a role in the down-regulation of Ntcp [153]. Finally, an indirect mechanism by which oestrogens influence the secretion of pituitary hormones which, in turn, modify liver gene expression, has been proposed recently [157]. In summary, EE induces changes in bile acid metabolism and transport through: (i) an interaction with nuclear receptors, such as ERα, FXR and SHP, (ii) a indirect interaction with pituitary (or pituitary-dependent) hormones, and (iii) as yet unidentified nuclear-receptor- and pituitary-independent mechanisms.

Although oestrogens are thought to be key causal factors in obstetric cholestasis [150], the extent to which experimental EE-induced cholestasis in rats resembles obstetric cholestasis during pregnancy is unknown. Differences can be anticipated, however, since women who develop obstetric cholestasis appear to have a genetically linked susceptibility to the disease, and are exposed to high levels of other hormones apart from oestrogens (e.g., progesterone, prolactin and placental lactogens) [13,150]. Owing to this complexity, animal models of pregnancy-induced cholestasis are currently unavailable, but the pattern of expression of transporters in normal pregnant rats may provide some clues. As in EE-induced cholestasis, transcriptional down-regulation of Ntcp was apparent in pregnant rats, a finding attributed to the down-regulation of HNF1α and decreased promoter binding of RXRα/RARα [158]. This phenomenon was, however, not observed by others [159]. The limited, if any, decrease in Ntcp expression might be attributed to the simultaneous stimulation of Ntcp transcription by prolactin, an effect involving the binding of Stat5 (signal transducer and activator of transcription 5) to the Ntcp promoter [13,13,160]. Unlike in EE-induced cholestasis, in which all Oatps were moderately down-regulated, Oatp1a1 remains unchanged in pregnant rats, whereas Oatp1a4 is only marginally decreased at both the protein and mRNA levels [159]. Although a counter-stimulatory effect of prolactin was not confirmed for Oatp1a1 and Oatp1a4, the hormone up-regulates Oatp1b2 expression via the Stat5 signal transduction pathway [161]. The expression of basolateral export pumps in pregnant rats also differs from that in EE-induced cholestasis. Expression of both Mrp1 and Mrp3 is decreased at a post-transcriptional and transcriptional level respectively [162]. Mrp6 mRNA levels are also decreased [162]. Finally, at the canalicular pole, Mrp2 expression was extensively reduced at a post-transcriptional level, whereas Bsep expression is not impaired. Bsep expression may be sustained by the selective counter-stimulatory effect of prolactin on Bsep expression [159]. Although moderate when compared with the changes occurring in a ‘pure’ oestrogen model of cholestasis, the alterations in transporter expression observed in pregnant rats suggest that pregnancy represents an oestrogen-linked cholestatic-prone condition that, when overlapping with subclinical genetically linked alterations in bile formation in susceptible women, may lead to overt cholestasis.

Manipulation of the expression of hepatocellular transporters as a therapeutic strategy in cholestasis

The identification of a critical role for nuclear receptors as mediators of the adaptive changes in hepatocellular transporter expression in cholestasis has raised expectation about the employment of specific optimized ligands of these nuclear receptors for the treatment of cholestatic disorders [163165]. These new therapeutic approaches are aimed not only at restoring the normal plasma-to-bile excretory route by stimulating defective transporter expression, but also at enabling alternative elimination routes. This knowledge has also provided new rationales for the understanding of the beneficial effects of well-established anticholestatic agents, which have been used in an empirical manner in the past.

Among the anticholestatic agents, UDC (ursodeoxycholate) is the prototypical one. Feeding UDC to normal rodents stimulated hepatocellular expression of both Mrp2 and Bsep by transcriptional and post-transcriptional mechanisms respectively [166]. Expression at both mRNA and protein levels of both of the basolateral efflux pumps Mrp3 [167] and Mrp4 [96] was increased, whereas that of Oatp1a1 was decreased (Figure 3). On the other hand, expression of the uptake transporter Ntcp [166] and the basolateral export transporter Ostα–Ostβ [96] remained unaffected. Feeding UDC also stimulates the protein expression of Mrp2 [167] and Mrp4 [96] efflux pumps in mice kidney, and that of Mrp2 [167], Mrp3 [167] and Mrp4 [96] in mouse intestines. Intestinal Ostα–Ostβ mRNA was also elevated [96]. Finally, the induction of bile-salt-hydroxylating enzymes [95] and repression of bile-salt-synthesizing enzymes [96] also occurs after UDC administration. Taken together, this response enhances the overall capacity of the body to detoxify toxic biliary constituents.

Beneficial effects of the anticholestatic therapeutic agent UDC on the expression of transporters and enzymes involved in bile salt synthesis, and bile salt and bilirubin metabolism in the liver, kidney and intestines in normal rodents
Figure 3
Beneficial effects of the anticholestatic therapeutic agent UDC on the expression of transporters and enzymes involved in bile salt synthesis, and bile salt and bilirubin metabolism in the liver, kidney and intestines in normal rodents

At the hepatocellular level, UDC stimulates the expression of the canalicular transporters Bsep and Mrp2 and the basolateral efflux pumps Mrp3 and Mrp4. Expression of Ntcp, Oatp1 and Ostα–Ostβ remain unaffected. The increased expression of Bsep has been attributed to UDC-induced activation of FXR, with the remaining responses presumably involving other nuclear factors, such as PXR. UDC also induces beneficial changes in bile salt metabolism, particularly the repression of bile acid synthesis from cholesterol and the stimulation of phase I bile acid metabolism, leading to more hydroxylated, less toxic, bile salts, which can be more easily sulfated/glucuronidated and exported further via urine. This latter process is also stimulated by UDC by up-regulating both Mrp2 and Mrp4 efflux pumps in the kidney. Finally, UDC up-regulates Mrp2, Mrp3 and Mrp4 efflux pumps in the intestine. Overexpression of the latter two efflux pumps contributes to preserve enterohepatic bile salt recirculation. Note that the Figure shows the effects of UDC on healthy rodents. The extent to which these mechanisms are in operation in patients with cholestasis is uncertain. See the text for further details.

Figure 3
Beneficial effects of the anticholestatic therapeutic agent UDC on the expression of transporters and enzymes involved in bile salt synthesis, and bile salt and bilirubin metabolism in the liver, kidney and intestines in normal rodents

At the hepatocellular level, UDC stimulates the expression of the canalicular transporters Bsep and Mrp2 and the basolateral efflux pumps Mrp3 and Mrp4. Expression of Ntcp, Oatp1 and Ostα–Ostβ remain unaffected. The increased expression of Bsep has been attributed to UDC-induced activation of FXR, with the remaining responses presumably involving other nuclear factors, such as PXR. UDC also induces beneficial changes in bile salt metabolism, particularly the repression of bile acid synthesis from cholesterol and the stimulation of phase I bile acid metabolism, leading to more hydroxylated, less toxic, bile salts, which can be more easily sulfated/glucuronidated and exported further via urine. This latter process is also stimulated by UDC by up-regulating both Mrp2 and Mrp4 efflux pumps in the kidney. Finally, UDC up-regulates Mrp2, Mrp3 and Mrp4 efflux pumps in the intestine. Overexpression of the latter two efflux pumps contributes to preserve enterohepatic bile salt recirculation. Note that the Figure shows the effects of UDC on healthy rodents. The extent to which these mechanisms are in operation in patients with cholestasis is uncertain. See the text for further details.

The effects of UDC in rodents were only partially reproduced in humans. When administered to otherwise healthy patients with cholesterol gallstones, UDC stimulated post-transcriptionally the expression of BSEP and MRP4, but not MRP2, MRP3, OATP1B1 or bile-salt-metabolizing enzymes in liver [168]. However, the situation in patients with cholestasis may be far more complex. For example, a positive correlation was observed between the hepatic enrichment of UDC in liver of UDC-treated patients with PBC and the hepatic levels of OATP1B1 and MRP2, whereas BSEP levels had no correlation [66]. This suggests that, unlike healthy subjects, patients with PBC lack UDC-stimulatory mechanisms of BSEP overexpression. Furthermore, UDC would improve MRP2 or OATP1B1 levels only when their expression is impaired by the disease.

Some of the effects induced by UDC in rodents appear to involve the activation of nuclear factors. With the exception of the induction of Bsep by UDC, which appears to be FXR-dependent in mice [167], the remaining responses to UDC are FXR-independent and may involve, in part, other nuclear factors, probably PXR [95]. As most of these effects occur spontaneously in the cholestatic liver, UDC administration to bile-duct-ligated mice only have a limited protective response [13]. More specific and effective therapies using newly synthesized ligands that bind FXR with higher affinity, e.g. GW4064 and 6-ethyl chenodeoxycholic acid, are being actively tested; they might counteract the reduced expression and activity of FXR in cholestasis, particularly in cholestasis induced by pro-inflammatory cytokines (e.g. endotoxaemia) [169], pregnancy [170] or oestrogens [156,157]. For example, the FXR agonist 6-ethyl chenodeoxycholic acid has beneficial effects in oestrogen-induced cholestasis [154], a pathology that occurs with a decrease in FXR mRNA levels [157]. This FXR ligand increases the expression of Mrp2 and Bsep in EE-induced cholestasis and reduces the transcriptional expression of Cyp7a1, Cyp8b1 and Ntcp [154]. Although FXR-mediated stimulation of Bsep expression can be beneficial in hepatocellular cholestasis with impaired Bsep transport, it can be detrimental in obstructive cholestasis, where pressure in the obstructed bile ducts can build up to hazardous levels. This explains why FXR−/− mice are more resilient to obstructive cholestasis [97] and that UDC induces the FXR-mediated harmful effects in bile-duct-ligated animals [55]. As most of the relevant acquired cholestasis in humans, such as PBC and PSC, develops an obstructive/cholangiodestructive component, this factor may narrow the therapy window for treatments with FXR ligands [171].

Apart from improving the expression of key transporters in certain cholestatic diseases, in others UDC largely enhances the activity of transporters without affecting expression. For example, EE impairs Mrp2 expression and the biliary excretion of Mrp2 substrates, but UDC co-administration largely restored this alteration without counteracting the Mrp2 down-regulation [172]. The same holds true for the anticholestatic hepatoprotector silymarin [173]. This might involve a direct positive interaction of the anticholestatic agents either with a putative regulatory site of the transporter or with its lipid microenvironment.

The capability of UDC to induce beneficial changes in bile acid metabolism in rodents, particularly repression of Cyp7a1 and stimulation of Cyp3a11 (the rodent homologue of human CYP3A4), has proved to be limited in humans, but can be complemented with simultaneous administration of PXR agonists, such as rifampicin [119]. Rifampicin, which has also been used for a long time in the treatment of cholestatic diseases, preferentially stimulated beneficial changes in bile acid metabolism [119]. This drug also enhances bilirubin-conjugating capability (via UGT1A1), bilirubin excretion (via MRP2) and bile acid/bilirubin glucuronide retrograde exportation (via MRP3) [119]. It should be highlighted again that these stimulatory effects have been tested in patients without cholestasis, but these mechanism may be different in patients with cholestasis. If confirmed under cholestatic conditions, similar complementary effects of UDC with CAR agonists, such as phenobarbital or 6,7-dimethylesculetin (the main active component of a herbal concoction used in traditional Yin–Chin medicine to treat neonatal jaundice) are to be expected, as CAR co-ordinately regulates a similar group of detoxifying genes to those induced by PXR (see above). In line with this, FXR, PXR and CAR agonists protected against hepatic bile acid toxicity in a complementary manner in rats [92,115]. This preliminary encouraging information supports the implementation of clinical trials using combinations of specific ligands targeting multiple but complementary nuclear receptors.

Post-translational alterations of hepatobiliary transporters

Bile flow can be impaired by certain cholestatic agents within hours or even minutes, a time period incompatible with changes in transporter expression at the protein level. These acute cholestatic changes account, for example, for the rapid cholestatic effects induced by drugs in patients [174]. Moreover, certain chronic cholestatic conditions do not involve changes in transporter expressions of a suitable magnitude to explain the extent of bile flow impairment, and anticholestatic agents may have beneficial effects without improving transporter expression. In these cases, alterations in transporter intrinsic activity or dynamic localization may play a crucial role [175].

Transporter inhibition by cholestatic drugs

A number of cholestatic agents cis-inhibit ATP-dependent Bsep-mediated bile salt transport in isolated rat liver canalicular membrane vesicles, mostly in a competitive manner [174]. These agents include cyclosporin A [176], glibenclamide [176], rifamycin [176], rifampicin [176], bosentan [177,178] and thiazolidinedione derivatives [179,180]. This effect was confirmed for human BSEP with some of these compounds [178,181]. Flutamide was also shown to cis-inhibit BSEP [182]. On the other hand, some cholestatic compounds may induce trans-inhibition of BSEP/Bsep. For example, E217G has been suggested to trans-inhibit Bsep in rat liver, as it requires an intact Mrp2-mediated E217G-transporting activity to exert its cholestatic effect [176,183]. This has been confirmed further in Xenopus laevis oocytes co-expressing rat Bsep, the inhibition mechanism being non-competitive in nature [183]. However, the relevance of this mechanism in the intact liver is doubtful, as experimental manoeuvres that increase the biliary E217G concentration attenuate E217G-induced cholestasis [184,185], and others that dilute the cholestatic compound in bile are not protective at all [186]. The dependency of E217G-induced cholestasis on intact Mrp2 activity, which indeed occurs in vivo [187], can be explained alternatively by the Mrp2 inhibitory modulation of Bsep transport activity via, for example, protein–protein interactions.

PM4-S (5α-pregnan-3α-ol-20-one sulfate), a progesterone metabolite that accumulates in the serum of women with pregnancy-induced cholestasis, has also been shown to trans-inhibit rat Bsep in X. laevis oocytes co-expressing rat Bsep in a non-competitive manner [183]. This suggests that this metabolite impairs bile flow in the isolated perfused rat liver, while being excreted at concentrations higher than the Ki for Bsep trans-inhibition [183].

Changes in transporter localization

Impairment of transporter activity in cholestasis can also occur by endocytic internalization of relevant canalicular transporters, which re-localize into intracellular vesicular structures, presumably the subapical endosomal compartment. If maintained with time in chronic cholestatic conditions, this may lead to delivery of the protein to the lysosomal compartment, followed by degradation [188]. This may contribute to the decreased post-transcriptional expression of canalicular transporters shown to occur in different cholestatic diseases.

Endocytic internalization of the canalicular transporters Mrp2 and Bsep occurs in several models of experimental cholestasis, including bile duct ligation [188], hyperosmotic perfusion [189,190] and oxidative challenge [191193]. Administration of cholestatic compounds, such as LPS [190], E217G [194,195], TLC (taurolithocholate) [196,197] and cyclosporine A [198], also induces this effect. In addition, Mrp2 internalization has been reported to occur in post-cold ischaemic rat liver, a phenomenon dependent on Kupffer cell activation, with thromboxane A2 being the most likely mediator [199]. Together with endocytic internalization, abnormal expression of this canalicular transporter in the apical end of the lateral membrane was also shown to occur under sustained E217G-induced cholestasis. This was attributed to mis-sorting of endocytosed transporter-containing vesicles localized at the pericanalicular area to the closer lateral membrane domain [200].

Changes in localization of canalicular export pumps have been also shown to occur in human cholestatic hepatopathies, including PBC [201], obstructive extrahepatic cholestasis [61,202], PSC [202], autoimmune hepatitis [202] and acute intrahepatic cholestasis induced by drugs, such as antibiotics, tiopronin, chlorpromazine, NSAIDs (non-steroidal anti-inflammatory drugs) and antidepressants [202204]. In the latter case, redistribution of Mrp2 occurs towards the basolateral membrane rather than into intracellular vesicles [204].

The mechanism by which endocytosis of canalicular transporters occurs remains largely unknown, although it is known that the process is microtubule-independent [205]. Alterations of actin cytoskeletal integrity onset transporter endocytosis [206], although retrieval of canalicular transporters can also occur with intact actin organization [195,197]. Proteins involved in the cross-linking of actin filaments with plasma membrane proteins, such as the ERM (ezrin/radixin/moesin) family of proteins, may be altered in these cases, without any apparent impairment of actin organization [207,208]. Interestingly, a deficient localization of radixin at the canalicular membrane occurs in patients with PBC, which is associated with retrieval of MRP2, MRP1, MDR3 and BSEP, together with co-localization of these carriers with radixin in intracellular vesicles [201]. This finding was confirmed for MRP2 in other human cholestatic diseases, such as obstructive jaundice, PSC, autoimmune hepatitis and drug-induced liver injury [202].

Accumulating evidence indicates that changes in canalicular transporter localization occurring in cholestasis depend upon activation of critical signalling pathways (Figure 4). One of the pathways involves ‘classical’ Ca2+-dependent PKC (protein kinase C) isoforms (mainly PKCα in hepatocytes). Selective activation of Ca2+-dependent PKCs induces cholestasis and retrieval of Bsep from the canalicular membrane and cholestasis in the isolated perfused rat liver [209]. PKC activation also induces redistribution of Mrp2 from the canalicular to the basolateral membrane in HepG2 cells [210]. A critical participation of Ca2+-dependent PKCs in the retrieval of Bsep and the associated bile salt secretory failure in t-BOOH (t-butylhydroperoxide)-induced oxidative stress has been reported recently by our group [193]. However, the kind of canalicular protein that is internalized under oxidative stress conditions and the signalling molecule(s) involved appear to depend on the pro-oxidant agent employed. For example, the oxidizing compound ethacrynic acid, which does not translocate ‘classical’ but rather ‘novel’ PKC isoforms, internalizes Mrp2 selectively without affecting Bsep [192]. The ‘novel’ PKC isoform PKCϵ is also activated in TLC-induced cholestasis and has been suggested to be involved in TLC-induced cholestatic effect [211]. This phenomenon occurs in a PI3K (phosphoinositide 3-kinase)-dependent manner, as products of PI3K are potent activators of PKCϵ [212].

Post-translational alterations in hepatobiliary transporters in cholestasis
Figure 4
Post-translational alterations in hepatobiliary transporters in cholestasis

Cholestatic drugs may cis- or trans-inhibit BSEP/Bsep or induce endocytic retrieval of canalicular transporters. Cyclosporin A, glibenclamide, rifamycin, rifampicin, bosentan, thiazolidinedione derivatives and flutamide all cis-inhibit either or both Bsep and BSEP, whereas PM4-S and E217G trans-inhibit the transporter in a non-competitive manner. Alternatively, E217G inhibition can be explained by Mrp2 inhibitory modulation of Bsep transport activity via protein–protein interaction, while E217G is being transported by Mrp2. A number of cholestatic manoeuvres in rats also lead to endocytic internalization of canalicular transporters into the subapical compartment (SAC); this may lead to delivery to the lysosomal compartment, followed by degradation. Canalicular transporter retrieval has also been confirmed in several human cholestatic diseases (see text for details). Several signalling pathways have been proposed to mediate this retrieval in experimental models of cholestasis. Activation of PKCϵ, when activated by ethacrynic acid or TLC, induces transporter internalization in rats. In addition, activation of PKCα in t-BOOH-induced oxidative stress in rats leads to transporter retrieval as well. Experimental therapeutic manoeuvres may prevent the retrieval of the transporters or accelerate exocytic re-insertion. Increases in intracellular cAMP levels induced by administration of the permeant cAMP analogue dibutyryl cAMP or by the phosphodiesterase inhibitor silibinin prevents internalization and accelerates re-insertion, via cytosolic Ca2+ elevation, in rat cholestatic hepatocytes. On the other hand, TUDC prevents transporter endocytosis, probably via ERK- and p38 MAPK-dependent pathways.

Figure 4
Post-translational alterations in hepatobiliary transporters in cholestasis

Cholestatic drugs may cis- or trans-inhibit BSEP/Bsep or induce endocytic retrieval of canalicular transporters. Cyclosporin A, glibenclamide, rifamycin, rifampicin, bosentan, thiazolidinedione derivatives and flutamide all cis-inhibit either or both Bsep and BSEP, whereas PM4-S and E217G trans-inhibit the transporter in a non-competitive manner. Alternatively, E217G inhibition can be explained by Mrp2 inhibitory modulation of Bsep transport activity via protein–protein interaction, while E217G is being transported by Mrp2. A number of cholestatic manoeuvres in rats also lead to endocytic internalization of canalicular transporters into the subapical compartment (SAC); this may lead to delivery to the lysosomal compartment, followed by degradation. Canalicular transporter retrieval has also been confirmed in several human cholestatic diseases (see text for details). Several signalling pathways have been proposed to mediate this retrieval in experimental models of cholestasis. Activation of PKCϵ, when activated by ethacrynic acid or TLC, induces transporter internalization in rats. In addition, activation of PKCα in t-BOOH-induced oxidative stress in rats leads to transporter retrieval as well. Experimental therapeutic manoeuvres may prevent the retrieval of the transporters or accelerate exocytic re-insertion. Increases in intracellular cAMP levels induced by administration of the permeant cAMP analogue dibutyryl cAMP or by the phosphodiesterase inhibitor silibinin prevents internalization and accelerates re-insertion, via cytosolic Ca2+ elevation, in rat cholestatic hepatocytes. On the other hand, TUDC prevents transporter endocytosis, probably via ERK- and p38 MAPK-dependent pathways.

Experimental therapeutic approaches based upon the modulation of post-translational alterations of transporters in cholestasis

Direct transporter inhibition by cholestatic drugs is, in most cases, reversed by withdrawing the drug, due to the reversible nature of the phenomenon [213]. Similarly, mechanisms involving changes in transporter localization can be spontaneously reversed if the cholestatic insult is transient. For example, canalicular transporters that are internalized by a rapid bolus administration of E217G are spontaneously re-targeted to the canalicular membrane by a microtubule-dependent mechanism within 2 h [214]. Some experimental therapeutic approaches have been designed to prevent transporter internalization and/or accelerate this re-insertion in order to avoid irreversible consequences of sustained internalization, as outlined below.

cAMP

cAMP is a second messenger that stimulates hepatocellular exocytic pathways [215] and partially prevents the impairment of bile flow and internalization of ABC transporters in E217G- and TLC-induced cholestasis. Reduction in bile flow and activity of Bsep [195] and Mrp2 [194] in the acute phase of E217G-induced cholestasis is partially prevented by the membrane-permeant cAMP analogue dibutyryl cAMP. More significantly, dibutyryl cAMP shortened the spontaneous recovery to normal bile flow, Mrp2 function and Mrp2 localization [194]. A similar acceleration of the re-insertion of endocytosed transporters has also been described by us for Bsep in TLC-induced cholestasis [197]. In the IRHC (isolated rat hepatocyte couplet) model, a preventive effect of dibutyryl cAMP was observed on both E217G- [195,216] and TLC- [197,216] induced Bsep relocation. In this case, however, prevention by dibutyryl cAMP was complete. This protective effect was significantly blocked by the Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)], but not by the PKA (protein kinase A) inhibitor KT5720, suggesting the involvement of Ca2+ or Ca2+-dependent signalling pathways in the protective effect of dibutyryl cAMP. A similar protective effect in terms of the signalling modulators involved was afforded by silibinin, the active component of the hepatoprotector silymarin. This was most probably due to the ability of silibinin to inhibit cAMP phosphodiesterase, thus increasing endogenous cAMP levels [216].

TUDC (tauroUDC)

TUDC, the naturally occuring taurine conjugate of UDC, stimulates exocytosis in the cholestatic liver [217] and counteracts endocytic internalization of both Bsep [218] and Mrp2 [196] in TLC-induced cholestasis. The Ca2+-sensitive PKC isoform PKCα has been proposed to mediate the anticholestatic effects of TUDC against TLC-induced cholestasis [196]. This is in apparent contradiction with more recent findings that PKCα is cholestatic rather than hepatoprotective [209]. In previous studies, pan-specific PKC inhibitors were used, and the biological response evoked by the interplay between different PKC isoforms may be different from that evoked by just one of them. Furthermore, TUDC activates the members of the MAPK (mitogen-activated protein kinase) family ERK (extracellular-signal-regulated kinase) [219] and p38 MAPK [220], and the cholestatic effect of PKCα may be overridden by the choleretic effects of these signal transduction pathways.

CONCLUDING REMARKS AND FUTURE DIRECTIONS

The recent enormous progress in molecular biology has prompted the identification and characterization of several novel ATP-dependent and ATP-independent hepatocellular transporters, including many critically involved in bile flow generation. This new information has been quickly transferred to the liver physiopathology area, providing an extensive understanding of the alterations accounting for cholestatic diseases at the molecular level.

In acquired hepatocellular cholestasis, these alterations may involve early changes in the dynamic localization of canalicular transporters, followed by long-term transcriptional or post-transcriptional factors affecting transporter synthesis. As a consequence of these primary alterations, an adaptive response occurs to minimize the detrimental effects of biliary compounds retained due to the secretory failure. This includes decreased uptake, increased biotransformation to less toxic metabolites, decreased expression of canalicular transporters and enhanced expression of alternative basolateral pumps, which export these metabolites into the blood for further renal excretion. This adaptive response is promoted by the co-ordinated expression of a complex network of nuclear receptors and hepatocyte-enriched transcription factors, whose activity is modulated by biliary metabolites retained by the cholestatic failure. These adaptive changes have been largely characterized in animal models of experimental cholestasis. The occurrence of a similar protective response in human cholestatic hepatopathies is becoming increasingly apparent, but species differences in the mechanisms of action (e.g. post-transcriptional in humans compared with transcriptional in rodents) appears to exist and will require characterization in each case.

This adaptive spontaneous mechanism is, however, not always sufficient to prevent hepatocellular damage and therapeutic intervention is often required. A number of newly designed therapeutic strategies are currently being explored to enhance adaptability by targeted activation of nuclear receptors with high-affinity specific ligands. Furthermore, the recognition of the involvement of these transcription factors in the anticholestatic effects of remedies classically used in the treatment of cholestatic disease (e.g. UDC and rifampicin) has added a rationale to their usage and extended their applications. The development of new ‘tailor made’ strategies for each hepatopathy by targeting specific nuclear receptors according to the particular deficiency is a key future challenge for both basic and clinical hepatologists. This goal will require characterization of the impairments in transporter and/or nuclear receptor expression in each human cholestatic disease. The finding of more selective potent nuclear receptor ligands for an appropriate gene control will be also an area of active research in a near future.

Finally, a number of compounds have been tested in animal models of cholestasis to prevent/revert the endocytic internalization of canalicular transporters that occurs shortly after cholestasis. These experimental therapeutic strategies are based either on the well-recognized capability of the therapeutic agents to favour exocytic carrier re-insertion (e.g. dibutyryl cAMP and TUDC) or to counteract potentially cholestatic signalling pathways (e.g. PKC or PI3K inhibitors). Taking into account that post-transcriptional changes involving transporter retrieval are increasingly recognized in cholestatic disease in humans and that they may account for post-transcriptional changes in this hepatopathy, this strategy may effectively complement the effect of inducers of carrier expression by co-operatively assuring proper localization and constitutive expression. Whether such a concerted beneficial action will occur effectively in patients with cholestatic hepatopathies remains to be ascertained, and this will represent a challenge for clinical researchers in the way to explore better therapeutic alternatives in cholestatic liver disease.

Abbreviations

     
  • ABC transporter

    ATP-binding-cassette transporter

  •  
  • AE2/Ae2

    anion exchanger 2

  •  
  • AQP

    aquaporin

  •  
  • ASBT/Asbt

    apical Na+-dependent bile salt transporter

  •  
  • BSEP/Bsep

    bile salt export pump

  •  
  • CAR

    constitutively activated receptor

  •  
  • CYP/Cyp

    ctyochrome P450

  •  
  • E217G

    oestradiol 17β-D-glucuronide

  •  
  • EE

    17α-ethynyloestradiol

  •  
  • ER

    oestrogen receptor

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • FXR

    farnesoid X receptor

  •  
  • HNF

    hepatocyte nuclear factor

  •  
  • IL

    interleukin

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LPS

    lipopolysaccharide

  •  
  • LRH1

    liver receptor homologue 1

  •  
  • LXR

    liver X receptor

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MRP/Mrp

    multidrug resistance-associated protein

  •  
  • NHE

    Na+/H+ exchanger

  •  
  • NTCP/Ntcp

    Na+-taurocholate co-transporting polypeptide

  •  
  • OATP/Oatp

    organic anion-transporting polypeptide

  •  
  • OST/Ost

    organic solute transporter

  •  
  • PBC

    primary biliary cirrhosis

  •  
  • PFIC

    progressive familial intrahepatic cholestasis

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PKC

    protein kinase C

  •  
  • PPARα

    peroxisome-proliferator-activated receptor α

  •  
  • PSC

    primary sclerosing cholangitis

  •  
  • PXR

    pregnane X receptor

  •  
  • RARα

    retinoic acid receptor α

  •  
  • RXRα

    retinoid X receptor α

  •  
  • SHP

    small heterodimer partner

  •  
  • Stat5

    signal transducer and activator of transcription 5

  •  
  • SULT/Sult

    sulfotransferase

  •  
  • t-BOOH

    t-butylhydroperoxide

  •  
  • TLC

    taurolithocholate

  •  
  • TNFα

    tumour necrosis factor α

  •  
  • UDC

    ursodeoxycholate

  •  
  • TUDC

    tauroUDC

  •  
  • UGT/Ugt

    UDP-glucuronosyltransferase

  •  
  • VDR

    vitamin D receptor

This work was supported by CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas; PIP 6442), and ANPCyT (Agencia Nacional de Promoción Científica y Tecnológica; PICT 05-26115), Argentina.

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