Hepatocellular bile salt transport : Lessons from cholestasis

MOLECULAR MECHANISMS OF BILE SECRETION Bile secretion starts with the formation of a primary secretion at the bile canalicular level by both bile saltdependent and -independent mechanisms (‘canalicular bile’, which accounts for 75% of daily bile production), followed by modifications along the bile ductules and ducts (25% of bile secretion) (1). Canalicular bile is formed by osmotic filtration of water and electrolytes in response to osmotic gradients generated by active transport systems located at the basolateral and canalicular membrane of hepatocytes. The major determinant of canalicular bile formation is the excretion of bile salts into the canaliculus (‘bile saltdependent’ bile flow). In addition, other (nonbile salt) organic anions and cations, and their conjugates osmotically influence bile flow. Canalicular excretion of glutathione and bicarbonate constitute the major components of the ‘bile salt-independent’ fraction of bile flow, although the contri-

bution of bicarbonate occurs mainly at the level of the bile ductules, particularly when stimulated by hormones and neuropeptides (2).
The hepatocyte is a polarized epithelial cell with distinct features in its basolateral (sinusoidal) and apical (canalicular) plasma membrane domains (3)(4)(5).Two sinusoidal bile salt uptake systems (located at the basolateral membrane) have been cloned in humans (Figure 1) -a sodium (Na + )-dependent/taurocholate cotransporter (NTCP) (6) and a Na + -independent organic anion transporting protein (OATP), which also transports a large variety of other (non-bile salt) organic anions (eg, bromosulfophthalein, estrogen conjugates) and cations (eg, drugs such as ajmalium) (7)(8)(9).Basolateral bile salt transport systems are essential for bile formation because the majority (approximately 95%) of bile salts excreted by the liver are reabsorbed on each pass through the intestine (mainly in the terminal ileum) and are returned to the liver (enterohepatic circulation).Na + -dependent bile salt transport via NTCP is driven by a Na + -potassium (K + )-ATPase, which generates an inwardly directed Na + gradient.Na + -K + -ATPase activity depends in turn on the membrane potential generated by a K + -channel.
The canalicular membrane contains both ATP-dependent and ATP-independent transport systems (4,5,10) (Figure 2).At least four ATP-dependent transport systems (termed 'export pumps') have been identified (Figure 2, left): a multidrug export pump (MDR1) for hydrophobic cationic compounds (eg, anticancer drugs, calcium channel blockers, cyclosporin A, various other drugs) (11); a phospholipid export pump (MDR3) that acts as a phospholipid flippase/translocase (12); a conjugate export pump that is the canalicular isoform of the multidrug-resistanceassociated protein (MRP) and was, therefore, designated as MRP2 (13,14); and a bile salt export pump (BSEP) for monovalent bile salts that has recently been identified as the 'sister of P-glycoprotein' (SPGP) (15,16).The MDR1 and 3 P-glycoproteins are products of the multidrug-resistance (MDR) genes.MRP2 is functionally also known as the canalicular multispecific organic anion transporter (cMOAT) because it mediates the canalicular excretion of a broad range of organic anions, most of which are amphiphilic anionic conjugates with glutathione, glucuronate and sulphate formed by phase II conjugation in the hepatocyte (eg, bilirubin diglucuronide).Canalicular excretion of reduced glutathione also appears to be mediated through MRP2.
In addition to these ATP-dependent transport systems, the canalicular membrane also contains ATP-independent transport systems (Figure 2, right).Canalicular bicarbonate secretion is mediated via the chloride/bicarbonate anion (Cl -/HCO 3 -) exchanger belonging to a family of anion exchangers (AE 1 to 3).AE2 is encountered in various tissues, including the liver and secretory epithelia, and plays a major role in mediating hepatic Cl -/HCO 3 -exchange.The AE2 protein is localized to both bile canaliculi and bile ducts (17).In addition to AE2, bile duct epithelial cells (cholangiocytes) contain several other transport systems for their secretory and absorptive functions, including a chloride ion channel that is the cystic fibrosis transmembrane regulator (CFTR) (2,18,19).The ileal sodiumdependent bile salt transporter has also been identified on the apical domain of large, but not small, cholangiocytes, where it may be involved in the reabsorption of bile salts (20,21).
100D Can J Gastroenterol Vol 14 Suppl D November 2000

Trauner et al
Figure 1) Basolateral transport systems of hepatocytes.Two sinusoidal bile salt (BS -) uptake systems have been cloned, a sodium (Na + )-dependent/taurocholate cotransporter (NTCP) and a Na + -independent organic anion transporting protein (OATP), which also transports a large variety of other (nonbile salt) organic anions (eg, bromosulfophthalein [BSP], estrogen conjugates) and cations (eg, ajmalium).Na + -dependent bile salt transport via NTCP is driven by a Na + -potassium (K + )-ATPase that generates an inwardly directed Na + -gradient.Na + -K + -ATPase activity in turn depends on the membrane potential generated by a K + -channel

Figure 2) Canalicular transport systems of hepatocytes. Four ATPdependent transport systems (export pumps) have been identified (left): a multidrug export pump (MDR1) for hydrophobic cationic compounds (eg, anticancer drugs, calcium channel blockers, cyclosporin A, various other drugs); a phospholipid export pump (MDR3) that acts as a 'phospholipid flippase'; a conjugate export pump (MRP2/cMOAT) that mediates the canalicular excretion of various amphiphilic conjugates formed by phase II conjugation (eg, bilirubin diglucuronide); and a bile salt export pump (BSEP) that is the 'sister of P-glycoprotein' (SPGP). In addition, the canalicular membrane also contains ATP-independent transport systems (right). Canalicular bicarbonate secretion is mediated via the chloride/bicarbonate anion (Cl -/HCO3 -) exchanger isoform 2 (AE2). Cl -/HCO3 -exchange via AE2 is driven by a chloride ion channel
MOLECULAR MECHANISMS OF CHOLESTASIS Cholestasis may result either from a functional defect in bile formation at the level of the hepatocyte (hepatocellular cholestasis) or from an impairment in bile secretion and flow at the level of bile ductules or ducts (ductular/ductal cholestasis).By using molecular probes for hepatobiliary transport systems in experimental and clinical forms of cholestasis, decreased or even absent expression of transporter proteins has been shown, which may explain the impairment of transport functions with subsequent reduction in bile flow and the development of cholestasis (22).Other mechanisms (not discussed here) that may also contribute to cholestasis are disruption of the cytoskeleton and vesicle transport that normally determine the hepatocyte's secretory polarity, impairment of signal transduction pathways that normally coordinates cell functions in the hepatic lobule via gap junctions and stimulate bile canalicular contractions, and defects in tight junctional structures that lead to the dissipation of osmotic gradients via 'leaky' paracellular pathways (22)(23)(24)(25).This review focuses on molecular alterations of hepatocellular transport systems in cholestasis.
Several experimental models of cholestasis have been used to study the mechanisms of human cholestatic liver diseases, such as sepsis-induced cholestasis (endotoxin [lipopolysaccharide (LPS)]-treated rats), oral contraceptive-induced cholestasis or cholestasis of pregnancy (ethinylestradiol [EE]-treated rats) and extrahepatic biliary obstruction (common bile duct ligation [CBDL]) (26,27).Administration of LPS, EE or CBDL to rats results in a marked reduction of mRNA and protein levels of the basolateral NTCP and OATP, as well as MRP2 and BSEP (28)(29)(30)(31)(32)(33)(34)(35)(36).Decreased expression of these transporters may explain impaired hepatocellular uptake and canalicular excretion of bile salts and other (nonbile salt) organic anions (eg, bilirubin diglucuronide) in cholestasis.In contrast, the expression of other transport systems such as the MDR1 at the canalicular membrane and MRP-isoforms at the (baso)lateral membrane (MRP1 and 3) increases following CBDL and LPS administration (35)(36)(37)(38)(39). Upregulation of these transport systems in cholestasis may be a compensatory mechanism that prevents further accumulation of potentially toxic biliary constituents within cholestatic hepatocytes.
Recent findings suggest that the downregulation of transporter gene expression occurs primarily at the transcriptional level (28,33), and that the reduction in gene transcription may be due to alterations in the quantity or function of regulatory nuclear transcription factors (33).For example, LPS administration in vivo reduces the activity of critical, liver-specific transcription factors (eg, hepatocyte nuclear factor 1) that normally regulate the NTCP promoter, thus providing a molecular mechanism for decreased NTCP gene transcription in a rat model of sepsis-induced cholestasis (33).
These experimental findings in rat models of cholestasis are confirmed by data obtained from clinical forms of cho-lestasis.For example, mRNA levels of the basolateral bile salt transporter NTCP are decreased in patients with extrahepatic biliary atresia, which subsequently increase if complete biliary drainage by portoenterostomy (Kasai procedure) is performed (40).On the other hand, MDR1 and MDR3 mRNA levels are increased in patients with obstructive cholestasis (41).Expression of the Cl - /HCO 3 -anion exchanger isoform AE2 is reduced in the livers of patients with primary biliary cirrhosis (42,43).Because Cl -/HCO 3 exchange activity contributes to the secretion of both canalicular and ductular bile, decreased hepatic expression of AE2 can lead to impaired bile flow.Upregulation of AE2 mRNA and protein levels has been reported in primary biliary cirrhosis patients treated with ursodeoxycholic acid (UDCA), indicating that the improvement of hepatobiliary excretory function under UDCA treatment may be, in part, mediated by the stimulation of gene expression of defective hepatobiliary transport systems (42,43).UDCA may also increase the number of transport proteins contained in the canalicular membrane by stimulating vesicular exocytosis and targeting transporters to the canalicular membrane (44,45).Future studies need to investigate whether UDCA also stimulates the expression of hepatobiliary transport systems for the canalicular excretion of bile salts and conjugated bilirubin (eg, BSEP, MRP2).
In addition to acquired alterations of hepatobiliary transport systems, hereditary mutations of transporter genes can also result in cholestasis (5,22) (Figure 3).Progressive familial intrahepatic cholestasis (PFIC) is a severe type of cholestatic liver disease found in infants and children that is inherited in an autosomal recessive fashion (46,47).Three types (PFIC 1-3) have been identified.PFIC-1 (also known as Byler disease) is characterized by low gamma-glutamyl transpeptidase (γ-GT) serum levels, elevated serum bile salts, normal serum cholesterol and low biliary bile salt levels.This form of PFIC has been mapped to chromosome 18q21-22 (48).Benign recurrent intrahepatic cholestasis (BRIC), a recurrent cholestatic disorder in adults, also known as Summerskill syndrome, has also been mapped to chromosome 18q21-22 (49), indicating that there may be a 'familial cholestasis gene' that is responsible for both PFIC-1 and BRIC.Recently, a gene encoding a 'P-type ATPase' (FIC1) likely to be involved in the enterohepatic circulation of bile salts and mutated in both PFIC-1 and BRIC has been described (50).The mutations were found in different regions of the FIC1 gene, possibly explaining the different phenotypic appearances of PFIC-1 and BRIC.PFIC-2 is similar phenotypically to PFIC-1, but is different genetically because the gene locus is on chromosome 2q24 (51).PFIC-2 is caused by mutations in the BSEP/SPGP gene (16), resulting in the absence of the canalicular BSEP in the liver of these patients (52).
In contrast to PFIC-1 and PFIC-2, the third subtype, PFIC-3, is characterized by high γ-GT serum levels, as well as bile duct proliferation and inflammatory infiltrates in portal areas.This type of cholestasis is caused by mutations of the MDR3 gene that results in the absence of canalicular MDR3 and a marked reduction of biliary phospholipid levels (53,54).Because phospholipids in bile normally protect bile ductular epithelial cells from bile salt toxicity by the formation of mixed micelles, the marked reduction or even absence of biliary phospholipids may explain bile duct injury in these patients (Figure 4).UDCA may benefit some patients with PFIC-3 by exerting its protective effect from the biliary lumen, presumably by counteracting the toxic effects of other (more hydrophobic) bile salts in bile (55).PFIC-3 provides an important link between a hepatocellular (canalicular) transport defect and the developement of cholangiopathies.Many human neonatal and adult cholangiopathies and cholestatic syndromes are being re-evaluated for possible MDR3 defects (56).Of note, patients with primary biliary cirrhosis have normal MDR3 mRNA levels (57), suggesting that decreased MDR3 gene expression is not involved in the pathogenesis of this vanishing bile duct syndrome.Heterozygotes for hereditary transporter mutations may have an increased susceptibility to exogenous choles-tatic injuries (eg, drugs, hormones).Of interest, some mothers of PFIC-3 patients had recurrent episodes of cholestasis during pregnancy (54).
Dubin-Johnson syndrome is caused by mutations of the human MRP2 gene that results in the absence of MRP2 in the liver (58,59).Although patients with Dubin-Johnson syndrome are usually hyperbilirubinemic rather than cholestatic, this is yet an another important example of how a mutation of a hepatocellular transporter gene can impair bile excretory function.This syndrome is characterized by abnormal biliary excretion of various endogenous and exogenous substances (eg, bilirubin diglucuronide, bromosulphophthalein conjugates, oral cholecystographic agents) that are normally excreted by MRP2 (60).
Cholangiocytes are also the primary cellular target in various cholestatic diseases.Mutations of the CFTR gene result in the impairment of ductal Cl -and water secretion.This defect is associated with mucus obstrucion of intrahepatic bile ducts, and can lead to focal areas of biliary fibrosis and cirrhosis.The molecular mechanisms of cystic fibrosis and the pathophysiology of immune-mediated, infectious and drug-induced cholangiopathies have recently been reviewed elsewhere (18,19,61,62).

SUMMARY AND CONCLUSIONS
Hereditary mutations in transporter genes or exposure to substances that cause cholestatic injury, such as drugs, hormones or proinflammatory cytokines, result in the decreased or even absent expression of the basolateral and canalicular transport systems (Figure 5).These molecular changes may explain the impaired hepatocellular uptake, and excretion of bile salts and other organic anions in cholestasis.The increasing information on the molecular regulation of hepatobiliary transport systems should bring new insights into the pathophysiology and treatment of human cholestatic liver diseases.Since submission of this manuscript, additional members of the OATP gene family

Figure 3 )
Figure 3) Hereditary defects of hepatocellular transport systems.Mutations of transporter genes can cause congenital cholestasis or hyperbilirubinemia.Chromosomal localizations of cholestatic or hyperbilirubinemic syndromes are given on the right.BESP Bile salt export pump; cMOAT Cationic multispecific organic anion transporter; GGT Gammaglutamyl transpeptidase; MDR Multidrug resistance; MRP MDRassociated protein; PFIC Progressive familial intrahepatic cholestasis; SPGP Sister of P-glycoprotein

Figure 4 )Figure 5 )
Figure 4) Congenital MDR3 defect in a subtype of progressive familial intrahepatic cholestasis (PFIC-3).Left Under normal conditions phospholipids (PL) in bile protect bile ductular epithelial cells from bile salt (BS -) toxicity by the formation of mixed micelles.Right Mutations of the MDR3 gene result in decreased biliary phospholipid levels (broken line).Bile salts are still excreted and cause bile duct injury (cholangitis) further downstream