Journal of Hepatology
Volume 43, Issue 2 , Pages 342-357, August 2005

Enterohepatic transport of bile salts and genetics of cholestasis

Division of Clinical Pharmacology and Toxicology, University Hospital Zurich, Rämistrasse 100, E RAE 09, 8091 Zurich, Switzerland

published online 31 May 2005.

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1. Physiology of enterohepatic circulation and bile formation 

The intestinal conservation mechanism of bile salts is highly efficient. From 20–40g of bile salts excreted daily into bile, only 0.5g are lost through fecal excretion and have to be replaced by de novo bile acid synthesis. This conservation is achieved through enterohepatic circulation of bile salts, which depends on the coordinated action of numerous transporter proteins expressed at the basolateral and apical membrane of liver, biliary and small intestinal epithelial cells. The following paragraphs introduce the major hepatobiliary transport systems involved in hepatobiliary circulation and briefly describe their role in hepatocellular physiology and bile formation. The reader is also referred to several complementary reviews that have appeared recently and that are referenced throughout the manuscript.

1.1. Bile acid synthesis 

Bile acid synthesis starts from cholesterol and can be subdivided into a classical or neutral and an alternative or acidic pathway (reviewed in: [1]). The classical pathway results in the formation of cholic acid and accounts for 90% of bile acid synthesis. It includes the hydroxylation of cholesterol at the 7α and 12α positions via the cytochrome P450 (CYP) enzymes CYP7A1 and CYP8B1, respectively, followed by hydroxylation via mitochondrial CYP27A1 [2], [3]. In contrast, the alternative pathway leads to the formation of chenodeoxycholic acid (CDCA). Here, 7α-hydroxylation is preceded by the formation of different oxysterols, which are further metabolized via CYP7B1 and CYP39A1[4], [5]. Under physiological conditions, 70% of the human bile acid pool is composed of cholic acid and cholic acid metabolites while 30% are constituted by CDCA [6]. After their synthesis, bile acids are conjugated with glycine and taurine, which improves their solubility in the bile fluid and in the intestinal lumen. These conjugated bile acids are present as anionic salts under physiological pH conditions and are therefore called bile salts.

1.2. Hepatocellular excretion of bile salts 

After their synthesis in hepatocytes, conjugated bile salts are secreted into the bile canaliculus via specialized transporter systems expressed at the canalicular membrane of hepatocytes. The canalicular excretion of bile salts constitutes the rate limiting step in bile formation and the first step in the enterohepatic circulation pathway. With the exception of FIC1 (ATP8B1), which is thought to play a role in the regulation of the enterohepatic bile acid pool and in the elimination of hydrophobic substances from the enterohepatic circulation (for review see: [7]), canalicular transporters involved in bile formation belong to different members of the superfamily of ATP-Binding Cassette (ABC) transporters. In the liver, this family includes members of the multidrug resistance (MDR) protein family (ABCB-gene family), the multidrug resistance associated (MRP) protein family (ABCC-gene family) as well as of the family of ABC-half transporters (ABCG-gene family) (Fig. 1).

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  • Fig. 1. 

    Bile salt transporters in human liver, cholangiocytes and small intestine and transcription factors involved in their regulation Efflux transporters (yellow symbol): BSEP: bile salt export pump; MDR: multidrug resistance protein; MRP: multidrug resistance associated protein; ABCG5/8; BCRP; Ostα/Ostβ; Uptake transporters (blue): ASBT: apical sodium dependent bile salt transporter NTCP: sodium-taurocholate cotransporting polypeptide; OATP: organic anion transporting polypeptide; Transcription factors: FXR: Farnesoid X receptor, GR: Glucocorticoid receptor; HNF1a: Human liver factor 1a; SHP-1: Small heterodimeric partner; RAR: Retinoid acid receptor; PPAR: Peroxisome proliferator activated receptor; green lines and red lines designate genes that are activated and inhibited by bile-acid-dependent mechanisms, respectively. Further details are given in the text.

Within the family of multidrug resistance proteins, the bile salt export pump BSEP (ABCB11) and the multidrug resistance protein 3 (MDR3, ABCB4) are two highly conserved members, which are involved in the secretion of cholephilic compounds from the liver cell into the bile canaliculus (reviewed in:[8], [9], [10]). BSEP constitutes the predominant bile salt efflux system of hepatocytes and mediates the cellular excretion of numerous conjugated bile salts such as taurine- or glycine-conjugated cholate, chenodeoxycholate and deoxycholate [11], [12]. MDR3 was shown to function as an ATP-dependent phospholipid flippase, translocating phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane (reviewed in: [13]). Canalicular phospholipids are then solubilized by canalicular bile salts to form mixed micelles, thereby protecting cholangiocytes from the detergent properties of bile salts.

In addition to these processes, MRP2, the only canalicular member of the multidrug resistance associated protein family, mediates the canalicular transport of glucuronidated and sulfated bile salts. MRP2 is the main driving force for bile-salt independent bile flow through canalicular excretion of reduced glutathione. Furthermore, MRP2 transports a wide spectrum of organic anions, including bilirubin-diglucuronide, glutathione-conjugates, leukotriene C4 and divalent bile salt conjugates as well as drug substrates, such as cancer chemotherapeutic agents, uricosurics and antibiotics (reviewed in: [13], [14]).

Only recently, hepatic expression of ABC half transporters could be demonstrated and localized to the canalicular membrane of hepatocytes. The heterodimeric transporter ABCG5/ ABCG8 (ABCG5 and ABCG8) has been identified as the apical transport system involved in the hepatobiliary excretion of plant sterols and cholesterol (reviewed in: [13], [15], [16]). Mutations in ABCG5/G8 have been shown to cause sitosterolemia, a condition which is characterized by increased intestinal absorption and decreased biliary excretion of dietary sterols, leading to hypercholesterolemia and premature atherosclerosis [17]. Overexpression of ABCG5/ABCG8 in transgenic mice led to an increase in biliary cholesterol secretion and a reduction of intestinal absorption of dietary cholesterol, providing strong evidence for ABCG5/ABCG8 being involved in hepatocellular secretion and intestinal efflux of cholesterol [16].

The breast cancer resistance protein BCRP (ABCG2) is another ABC half transporter expressed at the canalicular membrane of hepatocytes, although its highest expression levels were found in mammary epithelium and placenta. In addition to its role in conferring a multidrug resistance phenotype against a variety of xenobiotics, BCRP has recently been shown to in-vitro transport sulfated bile salt conjugates such as taurolithocholate sulfate [18]. BCRP might therefore contribute to the hepatocellular excretion of bile salts.

In addition to these canalicular efflux transporters, the hepatocyte also localizes basolateral excretion systems for bile constituents, which belong to the family of multidrug resistance associated proteins. Five members of this family (MRP1 (ABCC1), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5) and MRP6 (ABCC6)) have been located at the basolateral membrane of hepatocytes. MRP1 is almost absent in normal liver, while MRP3 and MRP4 expression is variable [13]. MRPs have been implicated in the cellular efflux of various organic anions including drug-glutathione, -glucuronide and -sulfate conjugates (MRP1), the efflux of bile salts and bile salt conjugates (MRP3, MRP4), the transport of nucleoside analog drugs such as zidovudine, lamivudine and stavudine (MRP4) and of the cyclic nucleosides cAMP and cGMP as well as methotrexate and the purine analogs 6-mercaptopurine and 6-thioguanine (MRP4 and MRP5) [13]. The efflux of bile salts by MRP4 is coupled to the cotransport of reduced glutathione or S-methyl-glutathione [19].

1.3. Transport of bile salt in cholangiocytes 

Cholangiocytes play an important role in physiological bile secretion and express several transporter systems for secretory and absorptive functions. Uptake of bile salts from canalicular bile into cholangiocytes is mediated by the apical sodium dependent bile salt transporter ASBT (SCL10A2). ASBT belongs to the superfamily of solute carriers and is identical with the gene product expressed in the terminal ileum of small intestine (reviewed in: [20]). Furthermore, the apical uptake of bile salts involves the organic anion transporting polypeptide 1A2 (OATP1A2), which belongs to the OATP superfamily of sodium independent solute transporters (SCLO; former nomenclature: SLC21)) [21], [22], [23].

After their uptake into cholangiocytes, bile salts are effluxed at the basolateral cholangiocyte membrane into the peribiliary plexus via an anion exchange mechanism [24]. From here, bile salts reach the portal circulation and undergo the cholehepatic shunt pathway. MRP3, a basolaterally expressed member of the family of multidrug resistance associated proteins contributes to the efflux of bile salts from cholangiocytes [25]. Moreover, MRP2 was recently localized in gallbladder-derived biliary epithelial cells, where it might contribute to taurocholate homeostasis [25]. In addition, a splicing variant of rat asbt could be localized to the basolateral membrane of cholangiocytes, where it is proposed to function as a bile salt efflux protein. However, the contribution of this truncated protein to bile salt efflux in human cholangiocytes has not been established [26].

1.4. Intestinal bile salt absorption 

The third step in enterohepatic circulation involves intestinal absorption of bile salts, cholesterol and phospholipids from the intestinal lumen of the terminal ileum. The uptake of conjugated bile salts in the terminal ileum is highly efficient and occurs mainly via the apical sodium dependent bile salt transporter ASBT (SLC10A2), which is identical to the transport system expressed in the apical membrane of cholangiocytes [20]. Furthermore, there is evidence that in addition to ASBT, OATP2B1 mediates the transport of taurocholic acid at acidic pH [27]. In addition, sodium independent intestinal uptake of glycine-conjugated bile salts is mediated via Oatp1a5 in rats [28]. The relative contribution of the possible human Oatp1a5 orthologue OATP1A2 to bile salt uptake in human small intestinal cells has not yet been established.

After intracellular transport of bile salts via the ileal bile acid binding protein (I-BABP), bile salts are effluxed at the basolateral membrane of enterocytes into portal circulation. Two carriers have been postulated to be responsible for transporting bile salts across the ileocyte basolateral membrane into the portal circulation. One potential candidate is MRP3, whose high expression in terminal ileum is suggestive for a role in bile salt transport into portal circulation [29]. Only recently, the heteromeric organic solute transporter Ostα/Ostβ has been established as a new candidate basolateral bile salt carrier in mice and may be responsible for bile salt efflux in ileum and other Asbt-expressing tissues [30].

1.5. Hepatocellular uptake of bile salts 

Extraction of bile salts from portal circulation is the last step in the enterohepatic circulation pathway. Basolateral uptake transport is essential for bile formation, since 95% of the biliary excreted bile salts are reabsorbed in the small intestine and reentered into portal circulation. Sodium-dependent and sodium-independent transporter mechanisms mediate the hepatic uptake of endogenous and exogenous (xenobiotic) substances from sinusoidal blood plasma. The sodium dependent pathway is represented by the sodium-taurocholate cotransporting polypeptide NTCP (SLC10A1) [20], which is exclusively expressed at the basolateral membrane of hepatocytes. Substrate specificity of human NTCP is essentially limited to conjugated bile salts and certain sulfated steroids. NTCP accounts for more than 80% of conjugated (i.e. taurocholate and glycocholate) but of less than 50% of unconjugated (i.e. cholate) bile salt uptake [20]. In contrast, the sodium-independent system is represented by different members of the superfamily of organic anion transporting polypeptides (OATP/SLCO) (reviewed in: [21]). The OATP superfamily comprises 9 human members, four of which could be detected in liver with, however, substantial differences in expression levels [21]. Highest expression in liver is found for OATP1B1 (SLCO1B1) and its 80% sequence homologue OATP1B3 (SLCO1B3), which are both predominantly, if not exclusively expressed in the liver. OATPs transport a large variety of albumin-bound amphiphatic organic compounds. With the exception of OATP2B1 (SLCO2B1), whose substrate specificity seems to be limited to bromosulphophtalein (BSP) and steroid sulfates, OATP1A2 (SLCO1A2), OATP1B1 and OATP1B3 exhibit overlapping transport activities for conjugated and unconjugated bile salts, BSP, neutral steroids, steroid sulfates and glucuronides, and selected organic cations [21]. Furthermore, numerous drugs are substrates of OATPs including the antihistamine fexofenadine, opioid peptides, digoxin, the HMG CoA-reductase inhibitor pravastatin, the angiotensin converting enzyme inhibitor enalapril or the antimetabolite methotrexate [21]. In addition, cholecystokinin octapeptide, which is released postprandially from the small intestine is selectively transported by hepatic OATP1B3 [31].

The mechanisms involved in the intracellular movement of bile salts across hepatocytes are not fully elucidated. Under physiological conditions, the majority of bile salts are bound to intracellular binding proteins, while a smaller fraction of unbound bile salts rapidly diffuses through the hepatocyte (reviewed in [32]). In addition, hydrophobic bile salts may be partitioned into intracellular organelles such as endoplasmatic reticulum and Golgi apparatus, especially during episodes of high cellular bile salt loading [32].

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2. Regulation of bile salt transport 

Hepatocellular transport systems are subject to extensive regulation, mainly in response to the intracellular accumulation of bile salts or disease-associated changes in the activation of transcription factors (reviewed in: [33], [34]). While posttranscriptional regulation mechanisms account for rapid changes in bile salt transporter activity, intermediate and long-term changes in transporter expression are achieved through regulation of gene transcription. The coordinated transcriptional and posttranscriptional regulation of hepatocellular transporters involved in hepatic uptake and efflux of bile constituents and of cytochrome P450 enzymes responsible for bile acid synthesis enables the liver to rapidly respond to changes in bile acid homeostasis.

2.1. Transcriptional regulation 

On a transcriptional level, expression of proteins involved in bile acid synthesis and transport processes is primarily mediated by a group of nuclear hormone receptors that belong to the superfamily of nuclear receptors. Based on their DNA-binding properties, this superfamily can be subdivided into four groups (reviewed in: [35], [36]). Nuclear receptors regulating bile acid synthesis and transporter function mostly belong to the group of class II receptors and include the Farnesoid X Receptor (FXR), the Retinoic Acid Receptor (RAR), the Peroxisome Proliferator-Activated Receptor (PPARα), the Liver X Receptor (LXR) and the Pregnane X Receptor (PXR), the human orthologue of which is the Steroid X Receptor (SXR). All of these class II receptors function as heterodimers with the Retinoid X Receptor (RXR), allowing high-affinity binding to specific DNA elements with subsequent activation of gene transcription (Fig. 1).

2.2. Transcriptional regulation of bile acid synthesis 

Bile salts are ligands for FXR and regulate bile acid synthesis through negative and positive feedback mechanisms, leading to the inhibition of de novo bile acid synthesis and to the production of less toxic bile acid derivatives. FXR is abundantly expressed in the liver where it is activated by different bile acids such as chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid and cholic acid [37], [38]. During states of high hepatocellular bile acid levels, FXR indirectly suppresses cholesterol-7α-hydroxylase (CYP7A1), the rate-limiting step of bile acid synthesis, through induction of the small heterodimeric partner 1 (SHP-1). SHP-1 is another member of the nuclear receptor family strongly expressed in the liver. SHP-1 acts as transcriptional repressor of Liver Receptor Homologue 1 (LRH-1), an orphan nuclear receptor, which is required for constitutive CYP7A1 expression [39], [40], [41]. On the other hand, high circulating cholesterol levels induce CYP7A1 expression levels through ligand-binding of specific cholesterol derivatives to LXR. Ligand activated LXR stimulates CYP7A1 transcription via binding to its specific response element within the CYP7A1 promoter [39], [42].

Besides suppression of CYP1A7, FXR also downregulates the expression of sterol-12α-hydroxylase (CYP8B1) and the sterol-27-hydroxylase (CYP27A1), two other enzymes involved in bile acid synthesis via activation of SHP-1 [43], [44], [45], [46], [47], [48], [49]. In parallel, the gene encoding the enzyme uridine 5′diphosphate-glucuronosyltransferase 2B4 (UGT2B4), which converts hydrophobic bile acids into less toxic glucuronide derivatives is directly upregulated by FXR [50]. Furthermore, bile acid-modifying enzymes, which mediate the conjugation of bile acids to taurine and glycine, are induced by FXR in rat liver [51].

Additionally, the transcriptional activity of PXR and its human counterpart SXR may be stimulated in response to bile salt challenge, thereby possibly enhancing the metabolism of toxic bile salts [52], [53]. The main function of PXR and SXR is the induction of detoxification pathways for various xenobiotics. Accordingly, PXR/SXR induced genes include those encoding CYP3A4 and MDR1, the key enzyme and transporter protein involved in the metabolism and cellular efflux of numerous drugs, respectively, [54], [55], [56], [57]. The effect of rifampicin treatment on pruritus relief in cholestatic disease has been interpreted as an SXR-mediated induction of 6α-hydroxylation of bile acids with subsequent renal excretion of bile salt conjugates [58], [59]. Another nuclear receptor important for hepatic detoxification pathways is the Constitutive Androstane Receptor (CAR). The beneficial effects of treatment with phenobarbital in cholestasis may be due to CAR-mediated induction of hepatic genes involved in bile acid and bilirubin metabolism and excretion [60].

2.3. Transcriptional regulation of hepatocellular bile acid secretion 

FXR also plays a predominant role in the regulation of bile salt transport [61], [62], [63]. Activation of FXR can induce the expression of BSEP and MDR3 [64], [65], [66], [67], thereby increasing bile acid efflux and the formation of mixed micelles in the biliary tree during cholestatic episodes. Thus, FXR mediated up-regulation of canalicular transporter proteins leads to a reduction of the toxic effect of bile salts on hepatocytes and cholangiocytes. In addition, FXR has been shown to induce MRP2 expression in human hepatocytes, which might constitute another compensatory mechanism during cholestasis [68]. In contrast, the alternative bile acid export pumps Mrp3 and Mrp4, which are expressed in the basolateral membrane of hepatocytes, are induced through FXR-independent mechanisms [69]. Studies in mice support the notion that Mrp3 and Mrp4 are induced through a PXR-mediated pathway [65].

2.4. Transcriptional regulation of intestinal bile salt transport 

The apical sodium-dependent bile salt transport system plays a pivotal role in the ileal absorption of bile salts and is negatively regulated by bile salts. In humans the target for this negative regulation by bile salts has been shown to be mediated through RAR/RXR [70]. Furthermore, recent studies suggest that the activation of ASBT in small intestine is also mediated by PPARα [71]. PPARα plays a key function in the regulation of fatty acid metabolism and is activated by fatty acids, eicosanoids, fibrates and NSAIDs [72]. In line with these findings, decreased PPARα activity has been implicated in hypertrigylceridemia and decreased ileal bile salt absorption. Induction of the ASBT gene by direct binding of the Glucocorticoid Receptor (GR) to the ASBT promoter is discussed as one factor that may contribute to the beneficial effect of glucocorticoid treatment in relief of diarrhea in Crohn's disease [73].

2.5. Transcriptional regulation of hepatocellular bile salt uptake transporters 

The basolateral expression of the sodium-taurocholate cotransporting polypeptide NTCP is suppressed during cholestasis through FXR-mediated induction of SHP-1, thereby preventing the hepatocyte from further accumulating toxic bile salts when the hepatocellular bile acid concentrations are already too high [70], [74]. Furthermore, in analogy to ASBT, NTCP seems to be activated by the glucocorticoid receptor [75]. Similarly, the expression of OATP1B1, the predominant sodium-independent bile salt uptake system at the basolateral membrane of human hepatocytes is downregulated during cholestasis through bile acid-mediated activation of SHP-1, which leads to a repression of Hepatocyte Nuclear Factor 1α (HNF1α), the major transcriptional activator of OATP1B1 [76], [77]. Interestingly, hepatic OATP1B3, which constitutes a multispecific basolateral uptake system, is activated through FXR [78]. Although the contribution of OATP1B3 to hepatocellular bile salt uptake remains to be established, its upregulation may compensate for the decreased activity of other transporters, thereby maintaining hepatocellular clearance of xenobiotics during cholestasis.

2.6. Posttranscriptional regulation 

Posttranscriptional regulation mechanisms account for rapid changes in bile salt transporter activity, which might for instance be required during postprandial episodes of high bile salt concentrations in portal blood. Furthermore, changes in liver cell hydration were identified as an independent and potent signal that modifies expression of Mrp2 and Bsep at the canalicular membrane (reviewed in: [79]). Posttranscriptional regulation of transporter function includes the modification of transporter density in the plasma membrane through the recruitment of transporter proteins from intracellular pools and the modification of transporter function by phosphorylation/ dephosphorylation [8], [33], [80].

2.7. Posttranscriptional regulation of hepatocellular bile salt secretion 

Under basal conditions most of the canalicular transporters involved in bile formation appear to be located intracellularly rather than at the canalicular membrane. The targeting of Bsep, Mdr2 (the rat homologue of human MDR3) and Mrp2 to the canalicular membrane involves the phophoinositide-3-kinase PI3K [81], [82]. In addition, trafficking of Mrp2 to the canalicular membrane is mediated by protein kinase C isoforms, which are activated by UDCA [83], [84]. UDCA also mediates the insertion of transporters in the apical membrane of hepatocytes through activation of p38 mitogen-activated protein kinase and other extracellular signal related kinases [85]. Finally, the canalicular localization of Mrp2 depends on the presence of radixine, which is concentrated at the hepatocyte canalicular membrane and associated with the carboxy-terminal cytoplasmic domain of human MRP2 [86].

2.8. Posttranscriptional regulation of basolateral uptake transporters of bile salts 

cAMP plays an important role in the posttranscriptional regulation of the sodium dependent and independent uptake systems for bile salts. cAMP-mediated dephosphorylation of Ntcp leads to increased retention of Ntcp in the intracellular compartment [87], [88], a process that is controlled by PI3K/ protein kinase B [89]. On the other hand, cAMP alters the function of basolaterally expressed Oatp1a1, probably through a phosphorylation-induced reduction in Vmax [90].

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3. Disease states associated with impaired bile salt transport 

3.1. Hereditary cholestatic diseases 

3.1.1. Familiar intrahepatic cholestasis 

Mutations in canalicular transporter genes have been characterized as cause of progressive and benign forms of familial cholestatic syndromes. These syndromes encompass a heterogeneous group of cholestatic conditions, which share some phenotypic characteristics but differ in their clinical presentation and disease course. While progressive familial intrahepatic cholestasis (PFIC) syndromes are characterized by early-onset cholestasis in the first years of life with progression to end-stage liver disease before adulthood [91], [92], [93], [94], [95], [96], benign recurrent intrahepatic cholestasis (BRIC) usually begins in adulthood and is characterized by recurrent episodes of intrahepatic cholestasis without progressive liver damage [97], [98], [99], [100], [101]. The distinction of progressive and benign forms of cholestasis in the present nomenclature suggests that these two forms of inherited cholestasis are different diseases. However, recent data show that many of the so-called BRIC patients progress over time to more aggressive disease. Together with recent in-vitro phenotyping data this observation indicates that PFIC and BRIC are a continuum of pathophysiologically similar conditions that should be better classified as structural and/or functional canalicular transporter deficiency syndromes. Thus, for example PFIC1 and BRIC1 represent different severity degrees of FIC1 deficiency, while PFIC2 and BRIC2 are both caused by different degrees of BSEP transporter deficiencies. Based on these considerations a more pathophysiologically oriented nomenclature for inherited cholestatic liver diseases is suggested in the following paragraphs.

3.1.2. FIC1 deficiency syndromes (PFIC1, BRIC1) 

Both conditions are caused by a genetic defect of the FIC1 protein in the canalicular membrane of hepatocytes. They are typically associated with normal serum cholesterol and γ-glutamyltranspeptidase (γ-GT) levels, but elevated serum bile salts [92], [97], [98]. The presence of diarrhea and pancreatitis in FIC1 dysfunction points toward a role of FIC1 in extrahepatic tissues, such as for example regulation of intestinal bile salt absorption. While a complete or near complete absence of functional FIC1 in the canalicular membrane is associated with a progressive disease course, residual FIC1 expression and function has to be assumed in the benign forms of the disease (i.e. BRIC1). So far, numerous different mutations in the ATP8B1 gene have been associated with PFIC1 and BRIC1, respectively, none of which has been functionally characterized [92], [97], [99], [102], [103], [104], [105] (Table 1, Table 2).

Table 1. Mutations in FIC1 (ATP1B8) associated with severe FIC1 deficiency syndrome (PFIC1) phenotype
cDNA changeAmino acid changeFIC1 stainingDisease onsetDisease courseIn-vitro phenotypingReferences
Homozygous
IVS+1del3Splice sitendnanand[97]
IVS8+1G>TSplice sitendnanand[97]
IVS21+5G>ASplice sitendnanand[97]
IVS17-1G>AIVS17(−1)ndnanand[97]
IVS18+2T>CIVS18(+2)nd2.5 monthsTx at 6 yearsnd[105]
1660G>AD554N_cndnanand[97], [102]
279G>AA93 splice sitendnanand[97]
3040C>TR1014Xndnanand[97]
380T>CL127P_cndnaTx at 14 yearsnd[97]
841T>AnandnaTx at 5 yearsnd[104]
863T>CL288S_cndnanand[99]
NaR296Cnd1 monthnand[92]
923G>TG308V_cndnanand[99]
1235G>CR412PndnaTx at 18 yearsnd[97]
1367C>TT456M_cndnanand[97]
1587_1589delF529delndnaTx at 4 yearsnd[97]
1604A>TH535L_cndnanand[97]
1993G>TE665Xndnanand[97]
NaI694N_cnd1 monthnand[92]
2124_2125insK709fsndnaTx at 6 yearsnd[97]
2558T>CF853S_cndnanand[97]
2543_2556dupF853fsndnanand[97]
2596_2599dupR867fsndnanand[97]
2674G>AG892R_cndnanand[99]
2788C>TR930Xndnanand[97]
3118G>AG1040R_cndnanand[97]
3140delTL1047fsndnanand[97]
3622_3628delA1208ndnanand[97]
Heterozygous
185_282delnand1 monthnand[92]
1286A>CE429Andnanand[97]
1498T>CY500Hndnanand[97]
2197G>AG733Rndnanand[97]
2097+2T>CSplice sitendnanand[99]
Compound heterozygous
[IVS18+2T>C]+[IVS21+5G>A]Splice sitesndnanand[97]
[IVS3-2A>G]+[1587_1589del]Splice site +F529ndnanand[97]
[556_628del]+[na]na +Q1131Xnd1 monthTx at 6 yearsnd[92]
[614_615ins]+[2854C>T]N205+R952Xndnanand[97]
[1208C>A]+[2373_2374ins]S403Y+P792ndnanand[97], [104]
[1336G>A]+[2271T>A]G446R+Y757Xnd2 monthsTx at 4 yearsnd[105]
[2873delA]+[3069_3070del]N958+Q1023ndnanand[97]
[1982T>C]+[1804C>T]I661T+R602Xndnanand[97]
[1982T>C]+[2854C>T]I661T+R952Xndnanand[97]
[2016delG]+[2788C>T]K672+R930Xndnanand[97]
[2063A>G]+[2674G>A]D688G+G892Rndnanand[97]

Nomenclature was adapted following the suggestions by den Dunnen and Antonarakis [138]. DNA and protein positions correspond to the nucleotide and amino acid changes referenced in the original paper. If not indicated otherwise, reference sequence and/ or accession number is not available. del: deletion; ins: insertion; fs: frameshift; IVS: inversion; dup: duplication; X: stop codon; _c: evolutionary conserved amino acid positions; Tx: liver transplantation; na: not available; nd: not determined.

Table 2. Mutations in FIC1 (ATB1B8) associated with benign FIC1 deficiency syndrome (BRIC1)
cDNA changeAmino acid changeFIC1 stainingDisease onsetDisease courseIn-vitro phenotypingReferences
Homozygous
1982T>CI661Tnd5 months to 47 yearsnand[103]
IVS23-3C>ASplice sitendnanand[97]
3722delGG1241fsndnanand[97]
2384delG795_R797delndnanand[99]
Heterozygous
1286A>CE429Andnanand[97]
Compound heterozygous
[208G>A]+[1799G>A]D70N+R600Q_cndnanand[97]
[923G>A] +[1982T>C]G308D_c+I661Tndnanand[97]
[1030A>T]+[1358C>A]I344F+S453Y_cndnanand[97]
[1361A>G]+[2081T>C]D454G_c+I694T_cndnanand[97]
[1798C>T] +[1982T>C]R600W+I661Tndnanand[97]
[1799G>A] +[1982T>C]R600Q+I661Tndnanand[97]
[1882C>T]+[2854C>T]R628W_c+R952Xndnanand[97]
[2674G>A] +[1982T>C]G892R+I661Tndnanand[97]
[3490C>T]+[1982T>C]R1164X+I661Tndnanand[97]
[IVS26+2T>A]+[1982T>C]Splice site +I661Tndnanand[97]
[279G>A]+[1982T>C]A93 splice site +I661Tndnanand[97]

Nomenclature was adapted following the suggestions by DEN Dunnen and Antonaraki [138]s. DNA and protein positions correspond to the nucleotide and amino acid changes referenced in the original paper. If not indicated otherwise, reference sequence and/ or accession number are not available. del: deletion; ins: insertion; X: stop codon; _c: evolutionary conserved amino acid positions; na: not available; nd: not determined.

3.1.3. BSEP deficiency syndromes (PFIC2, BRIC2) 

Both syndromes are caused by mutations in ABCB11, leading to defective BSEP expression and/or function in the canalicular membrane of hepatocytes. So far, about 30 different ABCB11 mutations have been identified as a cause of PFIC2 and BRIC2 syndrome, the majority of which were nonsynonymous changes (Table 3, Table 4) [91], [92], [93], [94], [101]. Some of these mutations, such as for instance the E297G site, have been associated with different clinical phenotypes as they were found in homozygous patients with PFIC2 and BRIC2 syndromes as well as in phenotypically normal heterozygous individuals [91], [92], [93], [94], [101]. In most cases, the impact of these mutations on BSEP expression and function remains to be established. A first attempt to in-vitro phenotype ABCB11 mutations encountered in PFIC2 was performed by Wang and coworkers, who cloned seven nonsynonymous PFIC2 mutations into rat Bsep and expressed the constructs in polarized MDCK cells [106]. Five of the tested mutations prevented the protein from trafficking to the apical membrane and/ or showed abolished taurocholate transport activity, supporting a role of these mutations in promoting intrahepatic cholestasis. However, the D482G mutation, which was found to exhibit decreased function by Wang and coworkers, showed preserved transport activity for taurocholate when cloned into mouse Bsep, indicating the possible impact of species differences on protein function [107]. Recently, in-vitro phenotyping of three novel mutations in human ABCB11 and immunohistochemical characterization of BSEP expression in liver biopsies obtained from two patients with PFIC2 and BRIC2 phenotypes, respectively, could provide direct evidence that these mutations entail a defect in BSEP expression and function, thereby strongly supporting a role of these mutations in causing the observed phenotypes [100]. In the latter report, the extent of in-vitro BSEP dysfunction correlated well with the in-vivo phenotype and allowed an in-vitro distinction between mutations associated with a progressive and a benign course of intrahepatic cholestasis (Table 4).

Table 3. Mutations in BSEP (ABCB11) associated with severe BSEP deficiency syndrome (PFIC2)
cDNA changeAmino acid changeBSEP stainingDisease onsetDisease courseIn-vitro phenotypeReferences
Homozygous
NgG238V_cAbsent1 monthTx at 3 yearsRapidly degraded[94], [106]
890A>GE297G_cndndnaImpaired trafficking and transport[93], [106]
1381A>GK461E_cndndnand[93]
1445A>GD482G_cndndnaImpaired transport[93], [106]
1723C>TR575XAbsent1 monthnand[93]
2944G>AG982R_cndndnaImpaired trafficking and transport[93], [106]
NaG1004D_cnd10 monthnand[92]
NaR1090XAbsent1 monthTx at 4 yearsnd[94]
3457C>TR1153C_cndndnaImpaired trafficking and transport[93], [106]
3767_3768insCT1256fsX1296ndndnand[93]
3803G>AR1268Q_cndndnaImpaired trafficking and transport[93], [106]
Heterozygous
NaS114R(?)Absent4 monthTx at 9 yearsnd[94]
695del (1bp,ng)fsAbsent5 yearTx at 14 yearsnd[94]
908delGR303fsX321ndnanand[93]
NaC336S_cAbsent1 monthTx at 9 yearsNormal trafficking
and transport[94], [106]
1723C>TR575XAbsent1 monthna
2944G>AG982R_cAbsent1 monthTx at 10 yearsImpaired trafficking and transport[94], [106]
3169C>TR1057Xndnanand[93]
Compound heterozygous
[na]+[1145del; 1bp,na]V284L+ fsnd1 monthnand[92]
[na]+[890A>G]R1057X+E297G_cAbsent1 monthTx planednd[94]
[1116G>A]+[1587G>A]V330X+R487Hnd2 monthnand[91]
[1723C>T]+[1907A>G]R575X+E636Gnd5 monthTx at 7 yearsnd[91]
[na]+[1145del; 1bp]V284L_c+ fsnd1 monthnand[94]
[na]+[3213del; 1bp na]S593R_c+ fsAbsent1 yearnand[94]

Nomenclature was adapted following the suggestions by den Dunnen and Antonarakis [138]. DNA and protein positions correspond to the nucleotide and amino acid changes referenced in the original paper. If not indicated otherwise, reference sequence and/ or accession number are not available. del: deletion; ins: insertion; fs: frameshift; X: stop codon; _c: evolutionary conserved amino acid positions; Tx: liver transplantation; na: not available; nd: not determined.

Table 4. Mutations in BSEP (ABCB11) associated with benign BSEP deficiency syndrome (BRIC2)
cDNA changeAmino acid changeBSEP stainingDisease onsetAssociated conditionIn-vitro phenotypeReferences
Homozygous
890A>GE297G_cnd2 and 14 monthsCholecystolithiasisnd[101]
1708G>AA570T_cnd1 yearnand[101]
3148C>TR1050C_cNd5 and 24 yearsCholecystolithiasisnd[101]
3383G>AR1128H_cnd5 and 18 yearsCholecystolithiasisnd[101]
Heterozygous
557A>GE186G_cnd4 yearsCholecystolithiasisnd[101]
Compound heterozygous
[890A>G]+[1294G>C]E297G_c+R432T_cnd16 yearsnand[100]
[2767A>G]+[2776G>C]T923P_c+A926P_cnd14 yearsCholecystolithiasisnd[101]
[3148C>T]+[IVS19+1G<A]R1050C_c+ spl sitend2 and 3 yearsCholecystolithiasisnd[101]

Nomenclature was adapted following the suggestions by den Dunnen and Antonarakis [138]. DNA and protein positions correspond to the nucleotide and amino acid changes referenced in the original paper. BSEP accession number: NM_003742; del: deletion; ins: insertion; X: stop codon; _c: evolutionary conserved amino acid positions; Tx: liver transplantation; na: not available; nd: not determined.

3.1.4. MDR3 deficiency syndromes (PFIC3, BRIC3) 

These conditions are related to the defective expression and/or function of MDR3 in the canalicular membrane of hepatocytes. In contrast to the laboratory abnormalities observed in FIC1 and BSEP dysfunctions, patients with MDR3 deficiency syndromes usually display elevated serum γ-GT-levels in the presence of normal or slightly elevated bile acid levels. So far, several different ABCB4 mutations have been associated with PFIC3 disease [95], [108](Table 5). While about half of these mutations resulted in decreased or absent MDR3 expression in the canalicular membrane the impact of the remaining mutations on MDR3 expression remains to be established.

Table 5. Mutations of MDR3 (ABCB4) associated with severe MDR3 deficiency syndrome (PFIC3)
cDNA changeAmino acid changeMDR3 stainingDisease onsetDisease courseIn-vitro phenotypeReferences
Homozygous
111A>GS27XfsAbsent14 months and 2.5 yearsTx at 5 yearsnd[95]
426_432delY132fsX162Absent3 monthsTx at 4 yearsnd[95]
1069G>TS346I_cFaint3 monthsTx at 10 yearsnd[95]
1216A>GE395G_cnd5 yearsTx at 13 yearsnd[95]
1653A>TI541F_cAbsent1 yearsTx at 5 yearsnd[95]
1699T>GL556R_cnd5 monthsTx at 9 yearsnd[95]
1744delTV571fsX587*Absent1 yearsTx at 6 yearsnd[95], [108]
1938C>TQ636Xabsent1 and 2 monthsTx at 3 and 4 yearsnd[95]
1986A>GR652G*nd6 monthsnand[95]
2901C>TR957X*Absent8 monthsTx at 9 yearsnd[95]
Heterozygous
444T>CW138R_cNd1.5 yearsTx at 10 yearsnd[95]
1302A>GT424A_cFaint1 yearsTx at 13 yearsnd[95]
1307G>AV425M_cNormal3 monthsTx at 10 yearsnd[95]
Heterozygous 1986A>GR652GNd1 month to 15 yearsnand[95]
1723A>GD564G_cNd8 monthsnand[95]
2132T>CF711S_cNd13.5 and 20.5 yearsnand[95]
Compound heterozygous
[2975_2984del]+[2979G>A]V981fsX985+G983SNd8 monthsnand[95]

Nomenclature was adapted following the suggestions by DEN Dunnen and Antonarakis [138]. DNA and protein positions correspond to the nucleotide and amino acid changes referenced in the original paper. MDR3 accession number: M23234; del: deletion; ins: insertion; fs: frameshift; X: stop codon; _c: evolutionary conserved amino acid positions; Tx: liver transplantation; na: not available; nd: not determined.

Whether benign forms of MDR3 deficiencies also occur, is presently unclear, since no appropriate assays for in-vitro phenotyping have been developed so far. Nevertheless, a case of benign recurrent form of inherited cholestasis caused by a new ABCB4 splice variant has been reported, indicating that MDR3 deficiencies also represent a continuum of cholestatic liver diseases with different severity degrees (i.e. BRIC3) [109].

3.1.5. MRP2 deficiency syndrome (Dubin–Johnson Syndrome) 

The Dubin–Johnson Syndrome (DJS) is characterized by conjugated hyperbilirubinemia due to inherited dysfunction of MRP2. Hepatic function is preserved in affected patients. Several different mutations have been associated with this condition (Table 6), resulting either in the complete absence of immunohistochemically detectable MRP2 in affected patients or impaired protein maturation and sorting [110], [111], [112], [113], [114], [115], [116], [117], [118], [119]. In-vitro phenotyping of specific mutations in a heterologous expression system revealed that the R1150H and the Q1382R mutation both showed impaired in-vitro transport function, while protein maturation and sorting was not affected. In contrast, the I1173F and R768W mutations cause deficient maturation and impaired sorting leading to inactive transport proteins.

Table 6. Mutations of MRP2 (ABCC2) associated with MRP2 deficiency syndrome (Dubin–Johnson syndrome)
cDNA changeAmino acid changeMRP2 stainingDisease onsetAssociated conditionIn-vitro phenotypeReferences
Homozygous
3517A>TI1173F_cndna–Impaired trafficking and transport[110], [119]
33449G>AR1150H_cndna–Normal trafficking
Impaired transport[110]
2302C>TR768W_cnd51 and 81 years–Rapidly degraded[111], [114], [116]
2272_2439delG758_K813delnd28 years–nd[112]
3196C>TR1066XAbsent20 and 32 years–nd[115], [139], [140]
2002_2068delK635XndChildhood–nd[118]
[1669_1815del;IVS13+2T>A]V557_Q605del; spl sitendna–nd[114]
[2272_2439del;IVS18+2T>C]G758_K813del;spl sitend26, 28 years–nd[114], [116]
4175_4180delR1392_M1393delAbsent15 years–Impaired transport[115], [119]
1901_1968delD634_D656delnd23 years–nd[112]
heterozygous
2302C>TR768W_cnd31 years–Rapidly degraded[111], [114]
2272_2439delG758_K813delnd28 years–nd[114]
compound heterozygous
[IVS18+2T>C]+[4145A>G]spl site +Q1382Rnd24 years–Normal trafficking
Impaired transport[114], [116]
[298C>T]+[3928C>T]R105X(?)+R1310XAbsentChildhood–nd[113]
[IVS13+2T>A]+[3928C>T]K557_Q605del +R1310Xnd7 years–nd[117]
[1901_1968del]+[2026G>C]D634_D656del +G676R_cnd25 years–nd[112]
[2302C>T]+[2272_2439del]R768W_c+G758_K813delnd4 and 7 years–nd[114]

Nomenclature was adapted following the suggestions by den Dunnen and Antonarakis [138]. DNA- and protein positions correspond to the nucleotide and amino acid changes referenced in the original paper. If not indicated otherwise, reference sequence and/ or accession number are not available. del: deletion; ins: insertion; fs: frameshift; X: stop codon; spl site: splice site; _c: evolutionary conserved amino acid positions; Tx: liver transplantation; na: not available; nd: not determined; cell lines.

3.2. Acquired cholestatic disease 

3.2.1. Primary biliary cirrhosis and primary sclerosing cholangitis 

Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are chronic inflammatory hepatic disorders, slowly progressing to end stage liver failure in most of the affected patients. In PBC, the inflammatory process affects predominantly the small bile ducts, while in PSC inflammation, fibrosis and obstruction of large and medium sized intra- and extrahepatic ductuli is predominant [120], [121]. Whether a genetically determined functional disturbance of BSEP and MDR3 might play a role for the pathogenesis of PBC and PSC was addressed in a recent study, where the genetic variability and haplotype structure of ABCB11 and ABCB4 in PBC and PSC patients was compared to a large healthy Caucasian population [122]. In this study, no differences in overall variant segregation or haplotype structure could be detected between healthy Caucasian individuals and PBC and PSC patients, respectively. These findings are supported by a recent work of Rosmorduc and coworkers, who did not find evidence for a major role of ABCB4 genetic variation in the pathogenesis of PSC. On the other hand, two non-synonymous ABCB11 variants were specific to PBC patients (G620D and A1228V) and one to PSC patients (S194P) (Table 7), while four and one ABCB4 mutations were specific to PBC (L73V, D243A, K435T and K1251Q) and PSC (R545C), respectively (Table 8). Furthermore, one ABCB4 and one ABCB11 haplotype were significantly more frequent in the patient cohort as compared to healthy controls, while another ABCB11 haplotype revealed an association with higher Mayo Risk Scores [122]. As for multifactorial and complex traits like PBC and PSC the effect of a specific haplotype is expected to be small an association between certain haplotypes and disease pathogenesis cannot be ruled out. Furthermore, it cannot be excluded that some of the rare and population-specific haplotypes might have an impact on BSEP or MDR3 function, although no strong pattern of disease-associated haplotypes could be observed in the PBC and the PSC collectives.

Table 7. Mutations of BSEP (ABCB11) associated with aquired forms of cholestasis
CDNA changeAmino acid changeBSEP stainingDisease phenotypeAssociated conditionIn-vitro phenotypeReferences
Heterozygous
580T>CS194P_cndPSC–nd[136]
779G>AG260D_cndPBC–nd[136]
1772A>GN519S_cndICP–nd[109]
3683C>TA1228V_cndPBC–nd[136]

Nomenclature was adapted following the suggestions by Dunnen and Antonarakis [138]. DNA and protein positions correspond to the nucleotide and aminO acid changes referenced in the original paper. BSEP accession number: NM_003742 del: deletion; ins: insertion; _c: evolutionary conserved amino acid positions; ICP: intrahepatic cholestasis of pregnancy; PBC: primary biliary cirrhosis; PSC: primary sclerosing cholangitis.

Table 8. Mutations in MDR3 (ABCB4) associated with acquired forms of cholestasis
cDNA changeAmino acid changeMDR3 stainingDisease onsetDisease courseIn-vitro phenotypingReferences
Homozygous
959C>TS320F_cndICP, cholelithiasis,nand[109], [131]
1772T>AL591Q_cndCholelithiasisnand[131]
3481C>TP1161S_cndCholelithiasisnand[131]
Heterozygous
217C>GL73V_cndPBCnand[136]
495T>AF165I_cndCholelithiasisnand[131]
523A>GT175A_cndICP, cholelithiasisnand[96], [131]
728A>CD243A_cndPBCnand[136]
902T>CM301T_cndCholelithiasisnand[131]
1007_1015delL341XndCholelithiasisnand[131]
1007_1015insD355XndCholelithiasisnand[131]
1304A>CK435T_cndPBCnand[136]
1327insAD447XndCholelithiasis, ICPnand[96]
1584G>CE528DndCholelithiasisnand[131]
NaG535D_creducedICP, cholelithiasis,nand[141]
1633C>TR545C_cndPSCnand[136]
NaA546D_cndICPnaImpaired trafficking normal transport[136]
1712delTV571fsX587*ndICPnand[108]
1954A>GR652G*ndICPnand[95]
1973G>AY658XndCholelithiasisnand[131]
2285G>AG762E_cndICPnand[109]
2363G>TR788E_cndCholelithiasisnand[131]
2270_2273insTK793XndCholelithiasisnand[131]
2901C>TR957X*ndICPnand[95]
3481C>TP1161S_cndCholelithiasisnand[131]
3751A>CG1251Q_cndPBCnand[136]
IVS15+6T>CSplice sitendPBCnand[136]
IVS21+1G>ASplice sitendICPnand[109]
IVS25+5G>CSplice sitendICPnand[109]
IVS25-3C>Gsplice sitendICPnand[109]
IVS26+53A>GSplice sitendICPrecurrent cholestasisnd[109]

Nomenclature was adapted following the suggestions by den Dunnen and Antonarakis [138]. DNA and protein positions correspond to the nucleotide and amino acid changes referenced in the original paper. If not indicated otherwise, reference sequence and/ or accession number are not available. del: deletion; ins: insertion; _c: evolutionary conserved amino acid positions; Tx: liver transplantation; na: not available; ICP: intrahepatic cholestasis of pregnancy; PBC: primary biliary cirrhosis; PSC: primary sclerosing cholangitis; *heterozygous mother of PFIC3 child; na: not available; nd: not determined.

3.2.2. Intrahepatic cholestasis of pregnancy 

Intrahepatic cholestasis of pregnancy (ICP) is characterized by the occurrence of transient cholestasis in pregnant women typically occurring in the third trimester of pregnancy, when the circulating levels of estrogens are high [108], [122], [123]. ICP is associated with pruritus and biochemical cholestasis of varying severity and constitutes a risk factor for prematurity and intrauterine fetal death. A genetic predisposition has been suspected based upon the strong regional clustering, the higher prevalence in female family members of patients with ICP and the susceptibility of ICP-patients to develop intrahepatic cholestasis under other hormonal challenges such as oral contraception [95]. A possible pathogenic role of MDR3 dysfunction was deduced from the observation that female members of a large consanguineous family with one affected PFIC3 patient experienced typical recurrent episodes of ICP [108]. This led to the hypothesis that the heterogeneous state for an MDR3 gene defect represents a genetic predisposition in these women. Recently, the extent of genetic variation in ABCB4 was studied in a large collective of unrelated Caucasian women with ICP and the extent of genetic variation was compared to that observed in healthy pregnant control women [124]. In this collective, 47% of ICP patients had elevated γ-GT levels and 77% of these patients carried ICP-specific ABCB4 mutations, including three newly detected splicing consensus mutation (intron 21: G(+1)A, intron 25: G(+5)C and C(-3)G) and 2 non-synonymous variants in highly conserved regions of the protein (S320F and G762E) (Table 8)[124]. Although in-vitro phenotyping of these MDR3 mutations remains to be done, the observed correlations between ABCB4 mutations and clinical disease course might indicate that at least some forms of ICP represent true benign forms of MDR3 deficiency syndromes (i.e. BRIC3).

In contrast to ABCB4, evidence for a pathogenic involvement of hereditary ABCB11 mutations in ICP is controversial. A possible role of BSEP in the development of pregnancy-associated cholestasis was first reported in one mother with an affected PFIC2 child [95]. Biochemical workup of ICP-patients allows the differentiation between high and low γ-GT forms of ICP, suggesting the involvement of different transporters. While high γ-GT values were present in the majority of ICP-patients with an ABCB4 mutation, genetic BSEP dysfunction was postulated in the low γ-GT cases [108], [122]. A recent study investigating the association of two intragenic ABCB11 marker SNPs in Finnish patients with ICP and unaffected controls suggested that ABCB11 might be a susceptibility gene for pregnancy-associated intrahepatic cholestasis [125]. However, genetic analysis of flanking markers for ABCB11 and ABCB4 in 16 individuals from two Finnish ICP families could not find such an association [126], which is in line with another recent study that did not find evidence supporting a strong role of BSEP mutations in the pathogenesis of pregnancy-associated cholestasis [124]. In this study, only one nonsynonymous ABCB11 mutation was specific for the ICP patients (Table 7). On the other hand, the V444A polymorphism in exon 13 of the ABCB11 gene was observed more frequently in ICP patients than in controls [124]. Recent data indicate that the A-allele is associated with decreased hepatic BSEP expression in healthy human liver tissue [127] As Bsep has been shown to be trans-inhibited by estradiol 17β-glucuronide [128] and several progesterone metabolites [129], lower canalicular BSEP expression might constitue a risk factor for the development of cholestasis during states of high circulating estrogen and progesteron levels thereby defining an individual's susceptibility to develop pregnancy-associated cholestasis.

3.2.3. Gallstone disease 

Gallstone disease is one of the most common and costly of all digestive diseases with a prevalence of up to 17% in Caucasian women. Cholesterol containing gallstones are the major form of gallstones and supersaturation of bile with cholesterol is therefore a prerequisite for gallstone formation [131]. An involvement of ABCB4 mutations in the pathogenesis of cholesterol gallstone disease was based upon the observation that intrahepatic biliary lithiasis or gallbladder lithiasis was found in children with a PFIC3 phenotype as well as in some of their unaffected parents [95]. From a pathophysiological point of view, ABCB4 mutations might lead to changes in biliary phospholipid concentration, which in turn promotes a decrease in the biliary solubilization of cholesterol. In a study conducted to investigate the possible role of ABCB4 genetic variation in gallstone disease, homo- and heterozygous carriers of ABCB4 mutation were found among patients with symptomatic intrahepatic sludge and gallbladder cholesterol stones [96], [131] (Table 8). Furthermore, the biliary cholesterol to phospholipid ratio was abnormally elevated in these patients while the cholesterol saturation index was increased [96]. Interestingly gallstone-related symptomatology recurred after cholecystectomy in patients with ABCB4 mutations. Furthermore, some of these patients developed cholestasis during pregnancy or after starting oral contraception [96].

3.2.4. Drug-induced cholestasis 

Inhibition of BSEP function by drugs is an important mechanism of drug-induced cholestasis, leading to the hepatic accumulation of bile salts and subsequent liver cell damage. Several drugs have been implicated in BSEP inhibition. Most of these drugs, such as rifampicin, cyclosporine, glibenclamide or troglitazone directly cis-inhibit ATP-dependent taurocholate transport in a competitive manner, while estrogen and progesterone metabolites indirectly trans-inhibits Bsep after secretion into the bile canaliculus by Mrp2 [128], [129], [132], [133]. Alternatively, drug-mediated stimulation of MRP2 can promote cholestasis by changing bile composition. It could recently be shown that bosentan stimulates and significantly increases Mrp2-dependent bilirubin excretion and bile salt-independent bile flow, while biliary lipid secretion was profoundly inhibited and uncoupled from bile salt secretion [134]. As inhibition of biliary lipid secretion was not seen in Mrp2-deficient TR- rats, it was suggested that the MRP2-mediated translocation of organic anions across the canalicular membrane is a prerequisite for the occurrence of this effect. It can be speculated, whether Mrp2 induced choleresis dilutes bile salts in the bile canaliculi below the concentration required for solubilization of phosphatidylcholine and cholesterol, leading to a clinical phenotype comparable to MDR3 dysfunction. Consequently, decreased biliary phospholipid secretion could also be explained by a physicochemical disequilibrium in bile composition. In addition it has been speculated whether direct drug binding to MDR3 could adversely affect phospholipid or toxin secretion under conditions of stress [135]. Genetically determined decreased MDR3 expression and function might therefore result in impaired biliary phospholipid secretion under therapy with MDR3 inhibitors such as cyclosporine A, verapamil or vinblastine [135].

The functional and clinical impact of genetic variations in ABCB11, ABCB4 and other canalicular transporter genes for the development of drug induced liver injury remains to be delineated. Systematic genetic screenings in healthy individuals identified several common polymorphisms in the promoter as well as in the coding regions of these genes [136], [137]. Some polymorphisms such as the V444A polymorphism in ABCB11 or the C1515Yin ABCC2 were associated with decreased hepatic expression of these proteins [127]. Whether this observation constitutes a risk factor for the development of drug-induced cholestasis is currently under investigation. It can be expected that in the future the functional characterization of genetic variations in these and possibly other canalicular transporter genes will allow to identify those that might predispose to drug-induced liver injury.

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4. Conclusions 

From the examples delineated in this article it is apparent that dysfunction of bile salt transporters is one of the leading causes of cholestasis. The intracellular accumulation of toxic bile constituents initiates a cascade of different FXR-mediated feedback loops, leading to the upregulation of proteins involved in hepatic bile secretion, while hepatocellular uptake of bile salts and bile acid synthesis are inhibited. Since the elucidation of the genetic basis of hereditary cholestatic syndromes it has become increasingly evident that genetics are a major determinant of hepatocellular transporter function and expression thereby determining an individual's susceptibility to develop cholestasis. However, there are still a number of cholestatic liver diseases for which the underlying genetic defect has not yet been determined. A special challenge will be the elucidation of the possible role of genetic defects in hepatobiliary transporters for acquired cholestatic syndromes, such as drug-induced cholestasis, since the number of drug-induced liver injury has further increased during the last ten years and represents an important clinical problem with significant morbidity and mortality.

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PII: S0168-8278(05)00307-7

doi:10.1016/j.jhep.2005.03.017

Journal of Hepatology
Volume 43, Issue 2 , Pages 342-357, August 2005

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