Na+ -taurocholate cotransporting polypeptide inhibition has hepatoprotective effects in cholestasis in mice.
ABSTRACT: Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+ -Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1-G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC-fed, Atp8b1-G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC-fed and Atp8b1-G308V mice. NTCP-inhibition increased plasma BS levels from 604±277 to 1746±719 ?m in DDC-fed mice, 432±280 to 762±288 ?m in Atp8b1-G308V mice and from 522±130 to 3625±378 ?m in BDL mice. NTCP-inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP-inhibition reduced biliary BS output in DDC-fed and Atp8b1-G308V mice by ?50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. CONCLUSION:NTCP-inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP-inhibition were well tolerated. NTCP-inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018).
Project description:Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium-taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B-mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP-binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8-/- mice. Stimulatory effects of NTCP inhibition were maintained in Sr-b1-/- mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high-density lipoprotein-derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.
Project description:Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so-called bile infarcts that correspond to Charcot-Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon-based imaging of BDL mice was performed with fluorescent bile salts (BS) and non-BS organic anion analogues. Key findings were followed up by matrix-assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1-3 days after BDL, BS concentrations in bile increased and single-cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a "domino effect" of further death events of neighboring hepatocytes. Bile infarcts provided a trans-epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS-overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in.
Project description:Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism by modulating farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1-overexpressing (SIRToe ) and hepatocyte-specific SIRT1-KO (knockout) mice (SIRThep-/- ) were subjected to bile duct ligation (BDL) and were fed with a 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA (24-norursodeoxycholic acid) was tested in BDL/SIRToe mice. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL, and Mdr2 knockout mice (Mdr2-/- ) animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRToe mice showed exacerbated parenchymal injury whereas SIRThep-/- mice evidenced a moderate improvement after BDL and 0.1% DDC feeding. Likewise, hepatocytes isolated from SIRToe mice showed increased apoptosis in response to bile acids, whereas a significant reduction was observed in SIRThep-/- hepatocytes. Importantly, the decrease, but not complete inhibition, of SIRT1 exerted by norUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRToe mice. Interestingly, both SIRT1 overexpression and hepatocyte-specific SIRT1 depletion correlated with inhibition of FXR, whereas modulation of SIRT1 by NorUDCA associated with restored FXR signaling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Fine-tuning expression of SIRT1 is essential to protect the liver from cholestatic liver damage.
Project description:The Na(+) -taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1(-/-) hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1(-/-) mice, explained by increased Asbt and Ost?/? expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1(-/-) hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1(-/-) mice.The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation.
Project description:Background & aims:The sodium taurocholate co-transporting polypeptide (NTCP) is the entry receptor for the hepatitis B and delta virus (HBV/HDV) and the main hepatic uptake transporter of conjugated bile acids. Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, blocks HBV/HDV infection and inhibits NTCP-mediated bile acid uptake. In humans this increases systemic bile acid levels, which remain elevated for hours even after Myrcludex B is cleared from the circulation. Here, we investigated the dynamics of Myrcludex B-induced NTCP-mediated bile acid transport inhibition in mice and if/how the duration of this effect relates to NTCP protein turnover. Methods:Plasma bile acids were determined in Myrcludex B-treated OATP1a/1b-deficient mice. In vitro, plasma membrane-resident NTCP was labeled with biotin or fluorescein isothiocyanate (FITC)-labeled Myrcludex B and traced in time using hNTCP-overexpressing U2OS cells. Förster resonance energy transfer by fluorescent lifetime imaging microscopy was used to investigate whether Myrcludex B can transfer to newly synthesized NTCP. Results:Conjugated bile salt levels in plasma peaked 4 h after subcutaneous Myrcludex B administration. After 24 h, plasma bile salt levels were completely normalized, in line with restored NTCP-mediated bile acid transport in vitro. Biotin-labeled NTCP disappeared faster than Myrcludex B-FITC, with almost 40% of FITC signal remaining after 24 h. FITC fluorescence lifetime was strongly decreased upon expression of DY547-labeled acyl carrier protein-tagged NTCP, demonstrating transfer of pre-bound Myrcludex B-FITC to newly formed NTCP. Conclusions:The dynamics of NTCP protein turnover and Myrcludex B-induced plasma bile salt elevations are similar, suggesting that the Myrcludex B:NTCP interaction is very long-lived. Nevertheless, Myrcludex B is not completely degraded together with NTCP and can transfer to newly synthesized NTCP. Lay summary:The experimental drug Myrcludex B binds the sodium taurocholate co-transporting polypeptide (NTCP), the viral entry receptor for the hepatitis B and D virus (HBV/HDV), and thereby prevents infection, but also inhibits hepatic bile salt uptake leading to transiently elevated bile salt levels. This study describes that while the normalization of plasma bile salt levels likely depends on the protein turnover rate of NTCP, Myrcludex B partly escapes co-degradation with NTCP by transferring from one NTCP molecule to another. This is of importance to the HBV/HDV research field as it provides a potential explanation for the distinct kinetics and dose-dependence of Myrcludex B's effects on viral infection versus bile salt transport.
Project description:The secretin/secretin receptor (SR) axis is up-regulated by proliferating cholangiocytes during cholestasis. Secretin stimulates biliary proliferation by down-regulation of let-7a and subsequent up-regulation of the growth-promoting factor, nerve growth factor (NGF). It is not known whether the secretin/SR axis plays a role in subepithelial fibrosis observed during cholestasis. Our aim was to determine the role of the secretin/SR axis in activation of biliary fibrosis in animal models and human primary sclerosing cholangitis (PSC). Studies were performed in wild-type (WT) mice with bile duct ligation (BDL), BDL SR(-/-) mice, or Mdr2(-/-) mouse models of cholestatic liver injury. In selected studies, the SR antagonist (Sec 5-27) was used to block the secretin/SR axis. Biliary proliferation and fibrosis were evaluated as well as secretion of secretin (by cholangiocytes and S cells), expression of markers of fibrosis, transforming growth factor-?1 (TGF-?1), transforming growth factor-?1 receptor (TGF-?1R), let-7a, and downstream expression of NGF. Correlative studies were performed in human control and PSC liver tissue biopsies, serum, and bile. SR antagonist reduced biliary proliferation and hepatic fibrosis in BDL WT and Mdr2(-/-) mice. There was decreased expression of let-7a in BDL and Mdr2(-/-) cholangiocytes that was associated with increased NGF expression. Inhibition of let-7a accelerated liver fibrosis was attributed to cholestasis. There was increased expression of TGF-?1 and TGF-?1R. Significantly higher expression of secretin, SR, and TGF-?1 was observed in PSC patient liver samples compared to healthy controls. In addition, there was higher expression of fibrosis genes and remarkably decreased expression of let-7a and increased expression of NGF compared to the control.The secretin/SR axis plays a key role in regulating the biliary contribution to cholestasis-induced hepatic fibrosis. (Hepatology 2016;64:865-879).
Project description:The Na+ -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7?-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids.NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).
Project description:Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to have a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR-21-/- mice underwent Sham or bile duct ligation (BDL) for 1 week, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and small mothers against decapentaplegic 7 (Smad-7) expression. In vitro, immortalized murine biliary cell lines (IMCLs) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. In addition, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers transforming growth factor-?1 and ?-smooth muscle actin. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared with control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury, miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis.
Project description:Cholestasis-induced accumulation of bile acids in the liver leads to farnesoid X receptor (FXR)-mediated transcriptional down-regulation of the bile acid importer Na+-taurocholate cotransporting protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the bile acid transporter NTCP. ER stress was induced using thapsigargin or subtilase cytotoxin in human osteosarcoma (U2OS) and human hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular bile acid uptake was determined using radiolabeled taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of thapsigargin, and effects of ER stress on NTCP messenger RNA (mRNA) and protein were measured by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis. Effects of cholestasis on NTCP and ER stress were assessed in response to 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding or bile duct ligation in FXR-/- mice after 7 or 3 days, respectively. Novel NTCP-interacting proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co-immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP protein expression, plasma membrane abundance, and NTCP-mediated bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with calnexin, an ER chaperone. In mice, expression of both NTCP and calnexin was reduced by thapsigargin or cholestasis-induced ER stress. Calnexin down-regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress-induced down-regulation of calnexin provides an additional mechanism to dampen NTCP-mediated bile acid uptake and protect hepatocytes against bile acid overload during cholestasis.
Project description:Alterations in apical junctional complexes (AJCs) have been reported in genetic or acquired biliary diseases. The vitamin D nuclear receptor (VDR), predominantly expressed in biliary epithelial cells in the liver, has been shown to regulate AJCs. The aim of our study was thus to investigate the role of VDR in the maintenance of bile duct integrity in mice challenged with biliary-type liver injury. Vdr(-/-) mice subjected to bile duct ligation (BDL) displayed increased liver damage compared to wildtype BDL mice. Adaptation to cholestasis, ascertained by expression of genes involved in bile acid metabolism and tissue repair, was limited in Vdr(-/-) BDL mice. Furthermore, evaluation of Vdr(-/-) BDL mouse liver tissue sections indicated altered E-cadherin staining associated with increased bile duct rupture. Total liver protein analysis revealed that a truncated form of E-cadherin was present in higher amounts in Vdr(-/-) mice subjected to BDL compared to wildtype BDL mice. Truncated E-cadherin was also associated with loss of cell adhesion in biliary epithelial cells silenced for VDR. In these cells, E-cadherin cleavage occurred together with calpain 1 activation and was prevented by the silencing of calpain 1. Furthermore, VDR deficiency led to the activation of the epidermal growth factor receptor (EGFR) pathway, while EGFR activation by EGF induced both calpain 1 activation and E-cadherin cleavage in these cells. Finally, truncation of E-cadherin was blunted when EGFR signaling was inhibited in VDR-silenced cells.Biliary-type liver injury is exacerbated in Vdr(-/-) mice by limited adaptive response and increased bile duct rupture. These results indicate that loss of VDR restricts the adaptation to cholestasis and diminishes bile duct integrity in the setting of biliary-type liver injury.