Intrahepatic Cholestasis: Summary of an American Association for the Study of Liver Diseases Single-Topic Conference *

"The many, often fantastic, theories advanced to explain jaundice without obstruction have long stood as clear evidence of the need of precise information regarding the formation and excretion of the bile …." A. R. Rich, Bulletin Johns Hopkins Hosp 1930;47:338 An American Association for the Study of Liver Diseases (AASLD)-sponsored Pediatric Single-Topic Conference focused on the category of hepatobiliary disease collectively known as syndromes of intrahepatic cholestasis (September 2004). A single-topic conference that focused on this topic was unique and timely because recent advances in molecular genetics have clearly pointed out new directions for investigation and clinical understanding. These disorders, which are individually rare, are common collectively; they are chronic (by definition) and many cases progress to end-stage disease, requiring liver transplantation. More effective treatment options are needed. Prevention and treatment strategies targeted to this age group will be especially cost-effective. It was the goal of the single-topic conference to help to promote further investigation to identify the mechanisms responsible for this subset of diseases that begin during childhood and lead to ongoing liver dysfunction in children and adults. The ultimate objective was to foster research and collaborative ventures in this important area. The conference brought together leading experts from around the world for presentations that highlighted the newest investigative and therapeutic approaches; the discussions drew needed attention to areas requiring further research. The purpose of this report is to briefly summarize this important single-topic conference. AASLD, American Association for the Study of Liver Diseases; PFIC, progressive familial intrahepatic cholestasis; GCT, giant cell transformation; GGT, γ-glutamyltransferase; BSEP, bile salt export pump; OATP, organic anion transport protein; mRNA, messenger RNA; BRIC, benign recurrent intrahepatic cholestasis; HNF, hepatocyte nuclear factor; TGF, transforming growth factor; NR, nuclear receptor; LCA, lithocholic acid. To place the topic in perspective, William F. Balistreri (Cincinnati Children's Hospital Medical Center) emphasized that in the early 1970s, the differential diagnosis of the neonate with conjugated hyperbilirubinemia (cholestasis) was limited. Biliary atresia accounted for approximately 25% of the cases; a small percentage of cases were considered to be caused by viral infections or were the result of a handful of recognizable metabolic or inherited diseases (i.e.,galactosemia, tyrosinemia, cystic fibrosis).1 The vast majority were designated as idiopathic neonatal hepatitis, clearly a default diagnosis, because the underlying pathophysiology was unknown. What was clear was that although many cases were apparently sporadic, others recurred in families. This led to the speculation that cases of familial neonatal hepatitis represented presumed inborn errors or genetic defects in a fundamental process involved in hepatic metabolic or excretory function. It followed that elucidation of their nature would allow a more clear understanding of liver physiology and also to improved therapy. Indeed, over the past 30 years there has been significant progress in dissecting the components of the idiopathic neonatal hepatitis spectrum. Specifically, substantial progress has been made in subcategorizing the range of hepatobiliary diseases that manifest as intrahepatic cholestasis in infants, children, and even adults.2 A major category is that of genetic intrahepatic cholestasis, a heterogeneous subset of diseases that represents specific disorders of abnormal cell fate, canalicular transport (bile acid or phospholipid), or bile acid synthesis, each with different prognostic implications. There are multiple forms with varying clinical features and with a high degree of variability in presentation and prognosis. Certain progressive, familial forms, as in the group known as progressive familial intrahepatic cholestasis (PFIC), are fatal; however, in patients with syndromic paucity of the ducts (Alagille syndrome), the prognosis is more favorable.2 The current system of nomenclature for the various syndromes in which intrahepatic cholestasis is present is imperfect. A proposed classification scheme is shown in Table 1. These syndromes are of more than theoretical interest; detailed study of affected patients has enhanced our understanding of hepatic excretory function and bile acid metabolism. The pathogenetic basis for these diseases has been defined only partially, and the techniques of molecular genetics have been applied only recently to these disorders. Precise terminology of the intrahepatic cholestatic disorders, based on the documented genetic defect, not only will provide clues to the pathophysiology but will also help to establish registries, promote development of new treatments, and allow the institution of valid clinical therapeutic trials. Kevin E. Bove (Cincinnati Children's Hospital Medical Center) emphasized that these genetic defects result in lobular cholestasis, variable giant cell transformation (GCT) of liver cells, isolated hepatocyte necrosis, and a highly variable rate of progression to fibrosis. The degree of hepatocellular injury is least in presumed cases of FIC1 deficiency, intermediate in bile salt export pump (BSEP) deficiency, and most destructive in MDR3 deficiency. In routine practice, the diagnosis is tentative and dependent on clinical parameters (i.e., serum γ-glutamyltransferase [GGT] and bile acid level, phenotype), combined with liver biopsy findings using light and electron microscopy.2 He discussed the predominant liver morphological features of the various forms. In FIC1 deficiency, small tidy hepatocytes with minimal ballooning and bland canalicular cholestasis are present; GCT is uncommon; there is little or no cholangiolopathy (periportal ductular metaplasia) with slow progression of portal fibrosis; and an unusual coarse quality of bile in canaliculi is visualized by electron microscopy. In BSEP deficiency, prominent canalicular cholestasis is often zonal (zone 3 > zone 1); prominent clustered balloon cholestasis of hepatocytes is present; GCT is common and persists beyond infancy; perivenular, pericellular, and periportal fibrosis with progression to cirrhosis is present; mild cholangiolopathy is present; normal interlobular bile ducts are present; and delicate wispy nonspecific quality of bile in canaliculi by electron microscopy is evident. In MDR3 deficiency, there is cholestatic hepatitis with GCT in infants and young children, cholangiolopathy, and progressive injury of the interlobular bile ducts in the early stages of disease. In bile acid synthetic defects, Dr. Bove has noted generalized hepatocyte injury with GCT and periportal inflammatory fibrogenic cholangiolitis; the injury and progressive portal fibrosis with fusion or dysmitotic division of immature hepatocytes is attributed to atypical or toxic bile acids present in the liver in conjunction with reduced concentrations of normal bile acids.3 In Alagille syndrome, he has observed that the smallest ductules either fail to form or rapidly regress, with interlobular bile ducts subject to a smoldering cholangiopathy that results in focal proliferation and progressive destruction at a variable rate. The next session focused on potential mechanisms responsible for the clinical and histological features described above. The predominant mechanism in many forms of intrahepatic cholestasis is altered canalicular transport. The bile canaliculus is surrounded by the apical plasma membrane domains of two or three hepatocytes (Fig. 1).4, 5 The composition of the canalicular membrane is distinct from the basolateral membrane and is enriched in cholesterol and sphingomyelin. Canalicular membrane proteins are targeted to this domain via both direct and indirect pathways. The canalicular membrane is enriched in ABC transporters that function as export pumps for bile acids and a variety of organic solutes. James Boyer (Yale University School of Medicine) pointed out that this membrane domain is metabolically active because it contains a number of active ATP-dependent solute transport proteins as well as GPI-anchored proteins like dipeptidyl peptidase IV, GGT, aminopeptidase N, 5′-nucleotidase, and the protein HA-4. Ion and water channels, ion exchangers, skeletal proteins (villin), vesicle fusion proteins (e.g., SNARE, SNAP, Syntaxin, Rabs) and tight-junction proteins are also present. Most of the solute transport-proteins belong to the superfamily of ABC transporters. The BSEP (ABCB11) is one; BSEP mediates the transport of conjugated bile acids (cholic and chenodeoxycholic acid). Like all other plasma membrane proteins, BSEP is synthesized in the endoplasmic reticulum and after glycosylation in the trans-Golgi network travels through the subapical compartment to the canalicular membrane. There is evidence for an intracellular pool of BSEP protein from which BSEP may be recruited on demand. For retraction and internalization of BSEP, interaction with a protein called HAX-1 seems to be important.4 Other apical ABC transporters include MRP2 (ABCC2), which transports glucuronide and glutathione conjugates; MDR3 (ABCB4), a flippase of phosphatidylcholine; MDR1 (ABCB1), a transporter of chemotherapeutic agents; ABCG5G8, a heterodimeric protein that transports sterols; and BCRP breast cancer resistance protein (ABCG2), a transporter of chemotherapeutic agents, carcinogens, and natural products.5-8 Mutations in several of these ABC transporters (MDR3, BSEP, and MRP2) result in clinical disease. The bile canalicular and the major hepatobiliary transport systems. The basolateral membrane of the hepatocyte contains the solute carrier systems (organic anion transport proteins [OATPs]), whereas the ATP-dependent ABC transporters are mainly located in the canalicular membrane. During cholestasis, multidrug resistance proteins MRP3 and MRP4, two organic anion transporting systems, become expressed at the basolateral membrane. MRP3 mainly transports conjugates such as bilirubin glucuronide; MRP4 transports bile acids together with glutathione. Transport proteins, water and electrolyte channels, and exchangers at the bile duct epithelium indicate that bile composition is modified at this level. The function of transporters in the basolateral membrane is quite different from those at the apical membrane. Uptake from the portal blood into the liver does not require active transport; electroneutrality is sufficient. Some of these transporters are exchangers that mediate organic anion uptake in exchange for bicarbonate or glutathione. These proteins belong to the superfamily of solute carriers (organic anion transport proteins [OATPs]) named OATP-1 (Slc21a1), OATP-2 (Slc21a5), OATP-4 (Slc21a6), and OATP-8 (Slc21a8). They have broad and partly overlapping substrate specificity. NTCP (Slc10a1) is an exception; it transports only bile acids and is a sodium–bile acid cotransporting protein.7 Solute carriers reach the basolateral membrane via a direct sorting route from the trans-Golgi. Their expression is dependent on transcriptional and post-transcriptional regulatory mechanisms (Fig. 2). MRP3 (Abcc3) and MRP4 (Abcc4) in the basolateral membrane act as efflux pumps when canalicular secretion is impaired. MRP4 is a cotransporter of bile acids and glutathione, whereas MRP3 mainly transports conjugates such as bilirubin monoglucuronide and diglucuronide. These transporters have a low expression in normal liver but are upregulated in cholestatic conditions.9, 10 Much remains to be learned about the mechanisms of canalicular membrane formation in development and its maintenance in health and disease. Post-transcriptional regulation of canalicular transporters. Apical transport proteins cycle between the apical membrane and a subapical compartment (SAC). On hypoosmotic conditions and/or stimulation by cyclic-AMP, protein kinase C, phosphatidylinositol-3-kinase, or MAP-kinase, transport proteins are inserted into the apical membrane. On hyperosmotic conditions, LPS administration, or bile duct ligation, they are retrieved into a SAC. The regular half-life of a transport protein is governed by proteosomal degradation after ubiquination. Drugs may influence the activity of transporters by direct interference with their activity. They can do this from the cytoplasmic side (cis-inhibition) or from the canalicular lumenal side (trans-inhibition). Modified from Trauner and Boyer.5 Frederick J. Suchy (Mount Sinai Medical Center, New York) discussed the ontogeny and regulation of these bile acid transporters. At birth, hepatic excretory function is immature, the enterohepatic circulation is inefficient, and bile flow is reduced; immediately after birth, serum bile acid levels are elevated.11 Ntcp and Bsep messenger RNA (mRNA) expression is not fully developed until 1 week after birth (rats). Ntcp protein expression closely follows this mRNA expression. Bsep protein expression is somewhat delayed and is not fully developed until 4 weeks after birth. The same holds true for Mrp2 mRNA and protein.12 The genes encoding these proteins are under transcriptional control of the nuclear hormone receptors RXR, FXR, PXR, CAR, RAR, LXR, and LRH. These are transcription factors that translocate from the cytoplasm to the nucleus on binding a relevant ligand. Bile acids bind to FXR, drugs to CAR and PXR, retinoic acid to RARα,β,γ, and oxysterols to LXRα,β. LRH-1 is an orphan receptor with no known ligand. 9-Cis-retinoic acid binds to RXRα,β,γ. In the nucleus, they interact as heterodimers with palindromic response elements in the promotor regions of target genes. RXRα,β,γ are obligate partners.13 What has been said about the ontogeny of Ntcp, Bsep, and Mrp2 is also true for the nuclear hormone receptors; their expression is significantly less than adult values until 1 week after birth.14 Because these receptors play an important role in protecting the liver against substrate overload, it holds that in the first week of life, the neonate is particularly susceptible to cholestasis and liver damage.2 The exact mechanisms governing the ontogeny of transport proteins and metabolizing enzymes is not known. A complex interaction of nuclear hormone receptors and other transcription factors is likely to play a role. It is clear that nuclear hormone receptors do not act alone but are part of a complex of coactivating proteins. Coactivator-associated arginine methyltransferase (CARM1) acts as a coactivator of FXR, activating BSEP. FXR:RXR and CARM1 are needed for full expression of BSEP mRNA in Hep G2 cells.15 Given the complexity of the structure and function of the bile canaliculus and hepatic excretory systems, it has long been postulated that specific inborn molecular defects could lead to cholestasis. A large number of genes mutated in forms of intrahepatic cholestasis have been identified (Table 2)16-40; however, many as-yet unidentified cholestasis genes likely exist. Laura Bull (University of California San Francisco, San Francisco General Hospital) described genetic approaches to decipher disorders such as intrahepatic cholestasis. These approaches complement functional studies of hereditary diseases. Bull emphasized that a key feature of genetic mapping studies is that they permit identification of the genetic etiology of a disease, even if the identity of the disease gene could not have been predicted. Thus, such studies can facilitate our understanding of disease etiology. For example, it is unlikely that FIC1/ATP8B1 would have been easily identified as a cholestasis gene using a functional, rather than whole-genome screening, approach. Bull has carried out genetic mapping studies to sift through the approximately 3.2 gigabasepairs of DNA in a single copy of the genome to identify mutations that result in disease.41 Roderick Houwen (Wilhelmina Children's Hospital, Utrecht, The Netherlands) discussed the clinical relevance of FIC1 (ATP8B1), the gene involved in various forms of intrahepatic cholestasis associated with FIC1 deficiency (PFIC type 1, Byler's disease, Greenland familial cholestasis, and benign recurrent intrahepatic cholestasis [BRIC] type 1).42, 43 Both PFIC type 1 and BRIC type 1 are autosomal recessive diseases. Nonsense, frameshift, and deletional mutations cause PFIC type 1, whereas missense and splice site mutations cause BRIC type 1. However, identical mutations can cause both PFIC type 1 and BRIC type 1. Houwen emphasized that homozygosity for the common I661T mutation can also occur in healthy persons. This indicates that modifier genes and environmental influences play a role in the expression of PFIC type 1 and BRIC type 1. The pathophysiology of these diseases is not well understood. FIC1 encodes an aminophospholipid translocase that has been proposed to flip phosphatidylserine and phosphatidylethanolamine from the outer to the inner layer of the plasma membrane. How a defect of this protein leads to cholestasis is unknown, and studies carried out on FIC1 knockout mice have failed to elucidate the mechanism.44 FIC1 protein expression in the intestine exceeds that in the liver, and therefore defects in both intestine and liver may be involved in the pathogenesis of these diseases. Increased intestinal absorption of bile acids, decreased canalicular secretion of bile acids, or both may be implicated. Partial biliary diversion, in which the gallbladder is externalized via a stoma using a loop of small intestine, is of clinical benefit in approximately half of the patients with PFIC type 1.45 The bile acid pool is reduced via diversion of bile, but how this leads to amelioration of cholestasis is unknown. Richard Thompson (Guy's, King's & St Thomas' School of Medicine, London) discussed BSEP (ABCB11), the major canalicular bile acid transporter. Mutations of the BSEP gene lead to deficiency of canalicular BSEP expression and variable degrees of intrahepatic cholestasis (also known as PFIC type 2 and BRIC type 2). As in FIC1 deficiency (PFIC type 1 and BRIC type 1), serum GGT activity in these patients is not elevated despite the cholestasis. A great number of mutations have been described in patients with PFIC type 2.18, 46 As in PFIC type 1, severe mutations lead to a phenotype of unremitting cholestasis from birth onward. Missense mutations have been shown to cause a milder disease not unlike BRIC; follow-up studies of this group of patients has not been long enough to know if this disease runs the same benign course as BRIC type 1. Thompson also reported eight patients with PFIC type 2 in whom malignancy developed: five with hepatocellular carcinoma, one with hepatoblastoma, and two with cholangiocarcinoma. The pathogenesis of PFIC type 2 seems clear; deficiency of the major canalicular bile salt transporter leads to severe cholestasis. Therefore, it was surprising that Bsep knockout mice are viable and in fact have a very mild phenotype.47, 48 Studies on these mice made it clear that mice have to or bile acids, or for example, bile acids are that can be via have the to the expression of basolateral MRP4 as a hepatic of PFIC type 2 is mainly followed by liver in many The role of biliary diversion has to be patients well to this particularly those with the or Medical Center, The Netherlands) was involved in the of as a translocase that from the inner to the outer layer of the canalicular Mutations of the MDR3 gene lead to the formation of bile with a normal bile acid The high of bile acids in the of may this bile toxic to hepatocytes and to the bile duct and mice with a canalicular expression severe hepatocyte and bile duct portal and periportal and bile duct The of this disease is and intrahepatic biliary and intrahepatic cholestasis of In the cholestatic that is associated with MDR3 mutations is called MDR3 deficiency or PFIC type In to PFIC type 1 and PFIC type PFIC type 3 is by serum GGT A number of PFIC type 3 with MDR3 well to Certain forms of intrahepatic cholestasis have been to altered the mechanisms and features of these of the biliary system were The most common cause of chronic cholestasis during childhood associated with specific features is the Alagille There is variability in the of affected patients may present with of five major chronic cholestasis from a paucity of interlobular bile such as or more severe as in of defects, and other and unique In the and system may be The disease is often manifest as neonatal cholestasis, with and prominent in of hepatic and are responsible for most and The genetic defect in patients with the Alagille has been shown to in mutations of the Children's Hospital of how mutations in the gene with the variable in patients with the Alagille the identification of mutations of the of in affected several studies have the to which mutations with disease to the of genes that cell proteins that interact with to cell during To genetic techniques have mutations in to of with and in other However, are These mutations are out the and include gene protein mutations by and and missense Mutations are in more than of the cases; although most mutations occur in the domain of the there is no predominant it to genetic in the of patients with the Alagille At the molecular level, gene seems to be the mechanism of relevant missense which for to of all lead to intracellular defects, with of the protein in the the membrane at the the understanding of the molecular of the for mutations has been with no between mutations and the expression in patients with the Alagille is the high of the mutation with a specific the that specific mutations may have in the mutations that with hepatic have not been This an important role for the in the hepatobiliary system is by findings of abnormal of the intrahepatic bile ducts in For example, mice with for the gene and of its receptor a phenotype to the Alagille of gene expression leading to in the was recently shown to also result in abnormal development of the intrahepatic biliary Frederick and (University of at recent molecular that play key regulatory in the of the biliary in to intrahepatic bile ducts have been in mice with as discussed In to the biliary system have been in encodes the protein which is expressed in the biliary epithelium expression is by the however, in to the intrahepatic phenotype when the is in of results in gallbladder and severe of bile the distinct of the intrahepatic and biliary systems, the molecular are at least in by common as by the of mice the of genes encoding of two of transcription The first is the hepatocyte nuclear factors and a of the of is expressed in intrahepatic biliary and gallbladder the gene was in development of the was biliary the gallbladder not and bile ducts were by an structure that the liver to the is known to control the expression of The functional between these factors and relevance to biliary development were further by the findings of paucity of small intrahepatic bile of intrahepatic bile and abnormal gallbladder and cystic duct in mice These findings led to the that the is for biliary It is likely that the of strategies to other transcription factors that are and expressed in the biliary such as the will further how in to promote biliary The group of transcription factors is the known to in a broad range of such as and In the hepatobiliary the is expressed in and in the intrahepatic and bile The of this expression to biliary development in gene of the gene was to and were to have normal intrahepatic bile but or of the gene in development of intrahepatic bile because of the of to biliary cell the to genes in and at different of development has the of the molecular of the biliary system (Table The of an understanding of the molecular basis of biliary development to clinical is highlighted by the direct of the with the Alagille In the of these in children with syndromes of intrahepatic and cholestasis will their potential role as cause or of disease understanding of the disorders of intrahepatic cholestasis will require from and expression. (Cincinnati Children's Hospital Medical Center) discussed the of molecular through gene to study liver and in specific clinical To more than such studies have focused on such as liver and viral studies have also been carried out on the topic of cholestasis in both and this has potential in the genetic basis of disease as well as the transcriptional regulation of it was emphasized that very is to the very