Ralf Kubitz

Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.

BACKGROUND & AIMS Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. METHODS To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. RESULTS Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type-related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. CONCLUSIONS Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.

Expression and function of the bile acid receptor TGR5 in Kupffer cells.

Kupffer cells are resident macrophages in the liver and play a central role in the hepatic response to injury. Bile acids can impair macrophage function leading to decreased cytokine release. TGR5 is a novel, membrane-bound bile acid receptor, and it has been suggested that the immunosuppressive effect of bile acids can be mediated by TGR5. However, the function of TGR5 in Kupffer cells has not been studied and a direct link between TGR5 and cytokine production in macrophages has not been established. The present study demonstrates that TGR5 is localized in the plasma membrane of isolated Kupffer cells and is responsive to bile acids. Furthermore, bile acids inhibited LPS-induced cytokine expression in Kupffer cells via TGR5-cAMP dependent pathways. TGR5-immunoreactivity in Kupffer cells was increased in rat livers following bile-duct ligation, suggesting that TGR5 may play a protective role in obstructive cholestasis preventing excessive cytokine production thereby reducing liver injury.

Regulation of the multidrug resistance protein 2 in the rat liver by lipopolysaccharide and dexamethasone

BACKGROUND & AIMS Endotoxin lipopolysaccharide (LPS) induces cholestasis and down-regulates the multidrug resistance protein 2 (MRP2). This study intends to characterize the short-term effects of LPS on MRP2. METHODS The effects of LPS and dexamethasone on excretion of bromosulphalein (BSP), MRP2 messenger RNA (mRNA) levels, and subcellular MRP2 localization were studied by means of liver perfusion, Northern blots, and confocal microscopy. RESULTS LPS treatment for 3-12 hours decreased biliary BSP excretion (10 micromol/L) by 40%. Hyposmolarity stimulated BSP excretion to control levels 3 hours after LPS injection, but was ineffective after 12 hours or in saline-treated controls. LPS led to a strong decrease of MRP2 mRNA after 12 hours, but not during the first 6 hours. LPS induced the appearance of MRP2 in intracellular vesicles in the immediate vicinity of the canaliculi within 3 hours, and these vesicles were remote from the canaliculi after 6 and 12 hours. The MRP2-containing vesicles did not stain for dipeptidylpeptidase IV (DPPIV). Dexamethasone counteracted the LPS effects on MRP2 mRNA levels, subcellular distribution, and BSP excretion. CONCLUSIONS LPS induces cholestasis due to an early retrieval of MRP2 from the canalicular membrane, whereas down-regulation of MRP2 mRNA is a later event. LPS-induced MRP2 retrieval from the canalicular membrane is not associated with the retrieval of DPPIV, suggestive for selectivity of the process.

Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in caucasians.

A recent genome‐wide study revealed an association between variation in the PNPLA3 gene and liver fat content. In addition, the PNPLA3 single‐nucleotide polymorphism rs738409 (M148I) was reported to be associated with advanced alcoholic liver disease in alcohol‐dependent individuals of Mestizo descent. We therefore evaluated the impact of rs738409 on the manifestation of alcoholic liver disease in two independent German cohorts. Genotype and allele frequencies of rs738409 (M148I) were determined in 1,043 alcoholic patients with or without alcoholic liver injury and in 376 at‐risk drinkers from a population‐based cohort. Relative to alcoholic patients without liver damage (n = 439), rs738409 genotype GG was strongly overrepresented in patients with alcoholic liver cirrhosis (n = 210; OR 2.79; Pgenotype = 1.2 × 10−5; Pallelic = 1.6 × 10−6) and in alcoholic patients without cirrhosis but with elevated alanine aminotransferase levels (n = 219; OR 2.33; Pgenotype = 0.0085; Pallelic = 0.0042). The latter, biochemically defined association was confirmed in an independent population‐based cohort of at‐risk drinkers with a median alcohol intake of 300 g/week (OR 4.75; Pgenotype = 0.040; Pallelic = 0.022), and for aspartate aminotransferase (AST) levels. Frequencies of allele PNPLA3 rs738409(G) in individuals with steatosis and normal alanine aminotransferase (ALT) and AST levels were lower than in alcoholics without steatosis and normal ALT/AST (Pcombined = 0.03). The population attributable risk of cirrhosis in alcoholic carriers of allele PNPLA3 rs738409(G) was estimated at 26.6%. Conclusion: Genotype PNPLA3 rs738409(GG) is associated with alcoholic liver cirrhosis and elevated aminotransferase levels in alcoholic Caucasians. (HEPATOLOGY 2011)

The G‐protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells

Sinusoidal endothelial cells (SEC) constitute a permeable barrier between hepatocytes and blood. SEC are exposed to high concentrations of bile salts from the enterohepatic circulation. Whether SEC are responsive to bile salts is unknown. TGR5, a G‐protein–coupled bile acid receptor, which triggers cAMP formation, has been discovered recently in macrophages. In this study, rat TGR5 was cloned and antibodies directed against the C‐terminus of rat TGR5 were developed, which detected TGR5 as a glycoprotein in transfected HepG2‐cells. Apart from Kupffer cells, TGR5 was detected in SEC of rat liver. SEC expressed TGR5 over the entire acinus, whereas endothelial cells of the portal or central veins were not immunoreactive toward TGR5 antibodies. In isolated SEC, TGR5 mRNA and protein were detected by reverse transcription (RT) PCR, immunofluorescence microscopy, and Western blot analysis. Bile salts increased cAMP in isolated SEC and induced mRNA expression of endothelial NO synthase (eNOS), a known cAMP‐dependent gene. In addition, bile acids activated eNOS by phosphorylation of eNOS at amino acid position 1177. In line with eNOS activation, bile acids induced NO production in liver slices. This is the first report on the expression of TGR5 in SEC. Conclusion: The data suggest that SEC are directly responsive toward specific bile salts. Regulation of eNOS in SEC by TGR5 connects bile salts with hepatic hemodynamics. This is of particular importance in cholestatic livers when bile salt concentrations are increased. (HEPATOLOGY 2007;45:695–704.)

The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders.

TGR5 (Gpbar‐1) is a plasma membrane‐bound, G protein–coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11‐positive recycling endosome of gallbladder epithelial cells. Furthermore, the TGR5 staining colocalized with the cyclic adenosine monophosphate–regulated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the apical sodium‐dependent bile salt uptake transporter, suggesting a functional coupling of TGR5 to bile acid uptake and chloride secretion. Stimulation with bile acids significantly increased cyclic adenosine monophosphate concentration in human gallbladder tissue. Incubation of gallbladder epithelial cells with a TGR5 agonist led to a rise of N‐(ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide (MQAE)‐fluorescence, suggestive of a decrease in intracellular chloride concentration. The TGR5 agonist–dependent increase in MQAE‐fluorescence was absent in TGR5 knockout mice or in the presence of a CFTR inhibitor, indicating that TGR5 mediates chloride secretion via activation of CFTR. The presence of the receptor in both the plasma membrane and the recycling endosome indicate that TGR5 can be regulated by translocation. Conclusion: The data suggest a role for TGR5 in bile acid–induced fluid secretion in biliary epithelial cells. (HEPATOLOGY 2009.)

Contribution of Variant Alleles of ABCB11 to Susceptibility to Intrahepatic Cholestasis of Pregnancy.

Background: Intrahepatic cholestasis of pregnancy (ICP) has a complex aetiology with a significant genetic component. ABCB11 encodes the bile salt export pump (BSEP); mutations cause a spectrum of cholestatic disease, and are implicated in the aetiology of ICP. Methods: ABCB11 variation in ICP was investigated by screening for five mutant alleles (E297G, D482G, N591S, D676Y and G855R) and the V444A polymorphism (c.1331T>C, rs2287622) in two ICP cohorts (n = 333 UK, n = 158 continental Europe), and controls (n = 261) for V444A. PCR primers were used to amplify and sequence patient and control DNA. The molecular basis for the observed phenotypes was investigated in silico by analysing the equivalent residues in the structure of the homologous bacterial transporter Sav1866. Results: E297G was observed four times and D482G once. N591S was present in two patients; D676Y and G855R were not observed. The V444A polymorphism was associated with ICP (allelic analysis for C vs T: OR 1.7 (95% CI 1.4 to 2.1, p<0.001)). In addition, CC homozygotes were more likely to have ICP than TT homozygotes: OR 2.8 (95% CI 1.7 to 4.4 p<0.0001). Structural analyses suggest that E297G and D482G destabilise the protein fold of BSEP. The molecular basis of V444A and N591S was not apparent from the Sav1866 structure. Conclusions: Heterozygosity for the common ABCB11 mutations accounts for 1% of European ICP cases; these two mutants probably reduce the folding efficiency of BSEP. N591S is a recurrent mutation; however, the mechanism may be independent of protein stability or function. The V444A polymorphism is a significant risk factor for ICP in this population.

Conjugated Bilirubin Triggers Anemia by Inducing Erythrocyte Death

Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency, or liver‐damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca2+ influx, sphingomyelinase activation, formation of ceramide, and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, patients with hyperbilirubinemia had significantly lower erythrocyte and significantly higher reticulocyte counts compared to patients with low bilirubin levels. Conclusion: Bilirubin triggers suicidal erythrocyte death, thus contributing to anemia during liver disease. (Hepatology 2015;61:275–284)

The bile salt export pump (BSEP) in health and disease

The bile salt export pump (BSEP) is the major transporter for the secretion of bile acids from hepatocytes into bile in humans. Mutations of BSEP are associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2), benign recurrent intrahepatic cholestasis type 2 (BRIC-2) and genetic polymorphisms are linked to intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI). Detailed analysis of these diseases has considerably increased our knowledge about physiology and pathophysiology of bile secretion in humans. This review focuses on expression, localization, and function, short- and long-term regulation of BSEP as well as diseases association and treatment options for BSEP-associated diseases.

De novo bile salt transporter antibodies as a possible cause of recurrent graft failure after liver transplantation: A novel mechanism of cholestasis

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations of the bile salt export pump (BSEP [ABCB11]), an ATP‐binding cassette (ABC)‐transporter exclusively expressed at the canalicular membrane of hepatocytes. An absence of BSEP from the canalicular membrane causes cholestasis and leads to liver cirrhosis, which may necessitate liver transplantation in early childhood. We report on the first case of a child with PFIC‐2 suffering from repeated posttransplant recurrence of progressive intrahepatic cholestasis due to autoantibodies against BSEP. These antibodies occurred after transplantation and were detected in the patients serum and at the canalicular membrane of two consecutive liver transplants. The antibodies were reactive toward the first extracellular loop of BSEP, were of high affinity, and inhibited transport activity of BSEP, thus causing severe cholestasis. The patient had three homozygous, missense changes in the BSEP gene. Their combination resulted in the complete absence of BSEP, which explains the lack of tolerance, a prerequisite of autoantibody formation toward BSEP. The findings illustrate a novel disease mechanism due to a new class of functionally relevant autoantibodies resulting in cholestasis and subsequent liver failure. (HEPATOLOGY 2009;50:510–517.)