Experimental cholestasis


223

SC-435, an Inhibitor of ASBT, Improves Liver Function in a Rat Partial Bile Duct Ligation Model of Cholestasis

Bradley T Keller, Svetlana Nikoulino, Nicolaus Nazarenkov, Bronislovo Gedulin

Lumena Pharmaceuticals' Son Diego, CA

Blocking bile acid absorption in the intestine is an effective approach to reducing the pool of serum bile acids (SBA). Thus, inhibiting the ileal bile acid transporter ASBT is being considered as a new treatment for cholestatic liver diseases. We report the effect of SC-435, a potent, minimally absorbed ASBT inhibitor (ASBTi) on liver function parameters in a rat partial bile duct ligation (pBDL) model of cholestasis. We adapted a previously described mouse pBDL model (Heinrich et al., Surgery 2011) to create a model in HSD rats which displays key characteristics of cholestatic liver disease - markedly elevated serum bile acids and liver function markers. Rats were anesthetized with isoflurane, the common bile duct exposed by midline laparotomy and a short length of PE-10 tubing placed parallel to the bile duct. A ligature of 4-0 silk suture was tied tightly around the duct and tubing after which the tubing was removed resulting in constriction of the duct lumen without complete obstruction. Three days after pBDL surgery, the serum liver alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and ۷-glutamyl transferase (GGT) increased from baseline by 5. 3, 5. 7, 5. 3 and 12. 1-fold respectively. Serum total bilirubin (TBil) levels increased by ≥100-fold. Seven days after surgery, AST and ALT levels had begun to normalize (3. 0 and 1. 7-fold) while ALP, GGT and total bilirubin values remained high at 4. 9, 22 and 103-fold compared to sham controls. This profile was sustained at 14 days post-surgery with elevations of 6. 8-fold for ALP, 15. 5-fold for GGT and 128-fold for TBil. SBA were also dramatically increased by 28.9-fold (30 uM to 873 uM) 7 days after surgery. SC-435 was administered to the test group by once daily oral gavage at 10 mg/kg starting one day prior to pBDL surgery. Seven days after surgery ASBTi treatment had significantly reduced ALP by 58%, GGT by 48%, TBil by 49% and SBA by 52% compared to the untreated control group (p < 0.03 for all parameters). By 14 days post-surgery, ALP was reduced 75%, GGT by 65% and TBil by 67% compared to the untreated control group (p < 0.05 for all parameters). CONCLUSIONS: The pBDL model in HSD rats results in significant increases in SBA and serum liver enzymes from 3 to 14 days after surgery that are characteristic of cholestasis and liver injury. Blocking bile acid recycling with SC-435 prevents dramatic increases in total SBA and liver biomarkers suggesting that an ASBTi may provide a new therapeutic option for the treatment of cholestatic liver disease by decreasing the accumulation of toxic bile acids and reducing the severity of cholestatic liver injury.

Disclosures:

Bradley T. Keller - Employment: Lumena Pharmaceuticals, Rivervest Venture Partners

Bronislava Gedulin - Employment: Lumena Pharmaceuticals

The following people have nothing to disclose: Svetlana Nikoulina, Nicolaus Nazarenkov

224

Nod2 deficiency protects mice from cholestatic liver disease by increasing renal excretion of bile acids

Lirui Wang1, phillipp Hartmann1, Michael Haimerl1, Sai P Botheno2, Yazen Alnouti2, Alan F. Hofmann1, Bernd Schnabl1

1Department of Medicine, University of California Son Diego, Lo Jolla, CA; 2Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE

Background: Nucleotide oligomerization domain 2 (Nod2), a member of the Nod-like receptor (NLR) family of intracellular immune receptors, plays an important role in the defense against bacterial infection through binding to its ligand muramyl dipeptide (MDP). The aim of our study was to test whether Nod2 plays a role in experimental liver fibrosis. Methods and Results: In wild type and Nod2 mice cholestatic liver disease was induced by bile duct ligation for 3 weeks, and toxic liver disease was induced by 12 intraperitoneal injections of carbon tetrachloride. Nod2 deficiency protected mice from cholestatic but not toxin-induced liver injury and fibrosis. Bile duct ligated Nod2-/- showed significantly less liver injury (plasma ALT levels), and less fibrosis (Sirius red staining and collagen α1(I) QPCR); liver inflammation was not changed. To explain this phenotype we initially investigated whether Nod2 deficiency affects bile acid-induced hepatocyte death. Bile acidinduced death in primary mouse hepatocytes was independent of Nod2, suggesting that hepatoprotection from cholestasis was not mediated via Nod2 in hepatocytes. Notably, in bile duct ligated Nod2-/- mice the hepatic bile acid concentration was lower and the urinary concentration was higher than in wild type mice, providing an explanation for the protection of Nod2mice from cholestasis-induced liver injury. Following bile duct ligation Nod2-/- mice the bile acid efflux transporters MRP2 and MRP4 in the kidney were increased. Consistent with this, administration of the Nod2 ligand MDP, caused a decrease in renal mRNA levels of MRP2 and MRP4 in wild type mice, while no inhibitory effect was observed in Nod2 deficient mice. The effect of MDP on renal MRP2 and MRP4 expression was exerted through IL-1 p release, because blocking IL-1 p signaling with the IL-1 receptor antagonist Anakinra abolished MDP-mediated downregulation of MRP2 and MRP4 in vivo. Also, IL-1 p treatment resulted in a marked reduction of MRP2 and MRP4 mRNA expression in a proximal tubular epithelial cell line from normal human kidney and in wild type mice in vivo. We also confirmed that IL-1 p mRNA and protein expression were lower in the kidney of Nod2-/- mice as compared to wild type mice following bile duct ligation for 3 weeks. Conclusion: These findings indicate that Nod2 deficiency protects mice from cholestatic liver injury and fibrosis through enhancing renal excretion of bile acids, which lowers intrahepatic concentrations of bile acids. Thus, the Nod2 appears to be involved in the regulation of renal tubular transport function.

Disclosures:

Alan F. Hofmann - Consulting: Albireo Pharma, Lumena Pharma, Intercept Pharma, GSK; Stock Shareholder: Intercept Pharma

The following people have nothing to disclose: Lirui Wang, Phillipp Hartmann, Michael Haimerl, Sai P. Bathena, Yazen Alnouti, Bernd Schnabl

225

NHERF-1 Knockout Mice Have Lower Hepatic ICAM-1 Expression and Are Protected from Liver Injury Induced by Bile Duct Ligation

Man Li1, Albert Mennone1, Carol J. Soroka1, Kathy M. Harry1, Edward J. Weinmon2, James L. Boyer1

1Internal Medicine/ Yale University School of Medicine, New Haven, CT; 2Department of Medicine, University of Maryland School of Medicine, Baltimore, MD

Recent studies indicate that the intracellular adhesion molecule, ICAM-1, is induced in mouse liver after bile duct ligation (BDL). ICAM-1 plays a key role in neutrophil extravasation across the endothelial barrier as well as neutrophil binding to hepatocytes, two major steps in neutrophil-dependent inflammation which is a predominant inflammatory response associated with liver injury after BDL. ICAM-1 has been shown to interact with ezrin, a member of the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins that also interact with the PDZ protein, Na+/H+ exchanger regulatory factor 1(NHERF-1/EBP50). ERM knockdown reduces ICAM-1 expression in response to the proinflammatory cytokine tumor necrosis factor-a. Aims: To determine whether deficiencies in NHERF-1 may affect hepatic radixin and ICAM-1 expression and, therefore, neutrophil accumulation in the liver after BDL. METHODS: Western blotting was utilized to compare hepatic expression of radixin in wildtype and NHERF-1-/- mice, as well as hepatic expression of ICAM-1 in sham and 7 day bile duct ligated (BDL) wild-type and NHERF-1-/- mice. Liver necrosis and neutrophil infiltration were assessed with H&E and immunohistochemical staining, respectively. Serum and hepatic bile acid levels were measured as well. RESULTS: Compared with wild-type, NHERF-1-/mice had more than 50% reduction in hepatic radixin expression (P<0.001). NHERF-1-/- BDL mice showed significantly lower scores of hepatic necrosis (P<0.01) as well as reduced neutrophil accumulation in the liver compared to the wild-type BDL group. Western blotting demonstrated that ICAM-1was reduced to about 70% of the wild-type in both sham and BDL NHERF-1-/- mouse liver (P<0.05). No significant difference in hepatic bile acid levels was detected between WT and NHERF1-/- mice in both the sham and BDL groups, but serum bile acid levels tended to be decreased in NHERF-1-/- BDL mice. CONCLUSIONS: These findings indicate that NHERF-1 is an important determinant of the expression of hepatic radixin as well as ICAM-1, an essential molecule involved in liver injury associated with neutrophil-dependent inflammation after BDL. While changes in the expression of these proteins can have protective effects in cholestatic liver injury, the protection seems independent of hepatic bile acid concentrations. This study indicates that adhesion molecules are potential therapeutic targets in cholestasis.

Disclosures:

The following people have nothing to disclose: Man Li, Albert Mennone, Carol J. Soroka, Kathy M. Harry, Edward J. Weinman, James L. Boyer

226

Novel therapeutic implications of modulating β -Catenin during intrahepatic cholestasis

Kari Nejak-Bowen1, Michael Thompson2, Satdarshan (paul) S. Monga1

1Pathology, University of Pittsburgh, pittsburgh, FA; 2Pediatrics, Nationwide Children's Hospital, Columbus, OH

Sustained cholestasis results in injury to the biliary epithelium with subsequent ductular reaction, pericholangitis, stellate cell activation, and fibrogenesis. Such pathologies are commonly associated with diseases such as primary biliary cirrhosis and primary sclersoing cholangitis. The Wnt/p-catenin pathway has been shown to play an important role during bile duct development, but its role in adult bile duct injury and repair remains undetermined. Mice lacking p-catenin in hepatocytes and biliary epithelial cells (Kし)and littermate wild-type (WT) control mice were subjected to bile duct ligation (BDL), a short-term model of acute intrahepatic cholestasis. Intriguingly, while bilirubin levels were comparably enhanced, hepatocyte injury, intrahepatic cholestasis and fibrosis were notably decreased in the KO after BDL. Further analysis yielded a significant decrease in total hepatic bile acids (BA) in the KO, which was associated with suppression of BA synthesis and an increase in apical BA transporter expression. Further, expression of Shp-1 mRNA was dramatically increased in KO after BDL. Hep3B cells transfected with Shp-1 reporter demonstrated a significant increase in activity when treated with p-catenin siRNA in the presence of FXR agonist GW4064. Finally, coprecipitation studies demonstrate a physical association of p-catenin and FXR in the WT that increases after BDL, suggesting an inhibitory function for pcatenin in FXR activation. Lack of p-catenin in K〇 led to enhanced FXR/SHP2 activation both in vitro and in vivo. Thus, loss of p-catenin limits cholestatic injury by modulating BA biosynthesis through regulation of FXR. These findings support an important role of Wnt/p-catenin signaling in bile duct homeostasis and repair and provide novel therapeutic opportunity of modulating p-catenin signaling for alleviating BA-associated hepatic injury during cholestasis.

Disclosures:

Satdarshan (Paul) S. Monga - Consulting: Bristol Myers Squibb, Phase Rx, Merck

The following people have nothing to disclose: Kari Nejak-Bowen, Michael Thompson

227

Secretion of gonadotropin-releasing hormone (GnRH) by cholangiocytes stimulates biliary proliferation via autocrine/paracrine mechanisms

Debolino Roy4, Sharon DoMorrow3, 4, Fanyin Meng1, 3, Julie Venter4, Heather L. Francis1, 3, Laura Hargrove3, Kelly McDaniel5, Syeda H. Afroze4, Paolo Onori2, Eugenio Gaudio2, Shannon S. Glaser1, 3, Gianfranco Alpini1, 3

1Research and Medicine, Central Texas Veterans Health Care System, Texas A&M HSC College of Medicine, Temple, TX; 2Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Universitá, Sapienza Rome, Italy; 3Scott & White Digestive Diseases Research Center, Scott & White, Temple, TX; 4Medicine, Texas A&M HSC COM, Temple, TX; 5Central Texas Veterons Health Core System, Temple, TX

During cholestasis the balance between biliary growth/loss is regulated by neuroendocrine peptides and neurotransmitters by autocrine/paracrine and endocrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone (released from the hypothalamus) regulating reproductive functions in mammals. GnRH also alters the function of extra-pituitary non-reproductive organs such as the kidneys and pancreas. Since no data exists regarding the role of GnRH in regulating biliary homeostasis, we aimed to evaluate if GnRH regulates biliary growth in normal and bile duct ligated (BDL) rats by interacting with GnRH receptor (GnRHR). Methods: The studies were performed in: (i) normal rats treated with saline or GnRH (1 μg/day); and (ii) BDL rats that, immediately after surgery, were treated with non-immune serum or anti-GnRH antibody (300μg/day) for 1 wk. Then, we measured: (i) intrahepatic bile duct mass (IBDM) in liver sections; and CK-19 and PCNA expression in total liver and cholangiocytes; and (ii) serum levels of GnRH by EIA kits. We measured the expression of: (i) GnRH and GnRHR in liver sections and cholangiocytes from normal and BDL rats and biliary lines by immunofluorescence, qPCR or immunoblots; and (ii) the levels of GnRH in the medium of short-term (12 hr) cultures of cholangiocytes from normal and BDL rats and biliary lines by EIA kits. In vitro, the: (i) dose- (10, 50 and 100 nM) and time- (24 to 72 hr) dependent effects of GnRH (in the absence/presence of the GnRHR antagonist, Cetrorelix acetate, 5-10 μM); and (ii) effect of Cetrorelix acetate (5-10 μM) on the proliferation of biliary lines was measured by MTS assays. GnRH expression was transiently knocked-down in biliary lines using siRNA and cell proliferation was assessed by MTS assays. Results: GnRH and GnRHR are expressed by normal bile ducts, cholangiocytes and biliary cell lines. GnRH biliary expression increased after BDL. Cholangiocytes secrete GnRH and, after BDL, GnRH secretion increased. Administration of GnRH to normal rats increased GnRH serum levels, biliary proliferation and IBDM, whereas administration of anti-GnRH antibody to BDL rats reduced biliary proliferation and IBDM. GnRH induced a dosedependent increase in biliary proliferation that was reduced by Cetrorelix acetate. Silencing of GnRH decreased the proliferation of biliary lines. Summary/Conclusion: GnRH stimulates biliary proliferation by autocrine/paracrine mechanisms, which introduces the novel concept that modulation of the GnRH/GnRHR axis may be key for the management of biliary disorders.

Disclosures:

The following people have nothing to disclose: Debolina Ray, Sharon DeMorrow, Fanyin Meng, Julie Venter, Heather L. Francis, Laura Hargrove, Kelly McDaniel, Syeda H. Afroze, Paolo Onori, Eugenio Gaudio, Shannon S. Glaser, Gianfranco Alpini

228

PPARα positively regulates human MDR3/ABCB4 transcription and increases biliary phosphatidylcholine secretion

Nisanne Ghonem, Meena Ananthanarayanan, Carol J. Soroka, Jomes L. Boyer

Internal Medicine, Liver Center' Yale University' New Haven, CT

BACKGROUND: The multi-drug resistance protein 3 (MDR3/ABCB4, Mdr2 in rodent) is a critical determinant of biliary phosphatidylcholine (PC) secretion. Clinically, mutations and deficiencies in MDR3 result in cholestatic liver injury and MDR3 is a potential therapeutic target. Fibrates are FDAapproved hypolipidemic agents and have been shown to upregulate Mdr2 expression via the nuclear receptor, PPARα. Fenofibrate (FF) can improve liver function in some patients with cholestatic liver disease. AIM: We previously demonstrated that fenofibrate significantly up-regulated MDR3 mRNA and protein expression in primary cultures of human hepatocytes and stimulated MDR3 promoter activity in HepG2 cells (Hepatology, Vol. 56, 4 (Supplement), p. 541A, October 2012). The aim of this study is to determine the mechanism by which fenofibrate regulates human MDR3 gene expression and whether fenofibrate regulates biliary PC secretion. METHODS: In silico analysis of the 5'-upstream region of human MDR3 gene revealed several PPARα response elements (PPRE). The direct binding of PPARα to the human MDR3 promoter was determined by EMSA using the TNT T7 quick-coupled transcription/translation system and by ChIP assays carried out in monolayers of HepG2 cells and primary human hepatocytes cultured in a matrigel sandwich. Site-directed mutagenesis of selected PPREs was performed using a QuikChange Site-Directed Mutagenesis kit. Collagen sandwich cultured rat hepatocytes were used to measure secretion of fluorescent PC into bile canaliculi. RESULTS: Cotransfection of the MDR3 promoter with hPPARa/hRXRa expression plasmids increased luciferase activity by 2-fold in the 6-10 kb upstream region when treated with FF. Targeted mutagenesis of three novel PPREs located at −6775/-6797 bp, −7197/-7219 bp, and −8554/-8576 bp upstream of the transcription start site reduced activation of the MDR3 promoter by FF. EMSA and ChlP assays show direct binding of PPARα to the MDR3 promoter. Treatment of primary rat hepatocytes with FF significantly increased secretion of fluorescent PC into bile canaliculi by 2-fold vs. control (37 ± 9% vs. 18 ± 5. 5%, respectively). CONCLUSIONS: Taken together, our findings indicate that fenofibrate activates MDR3 gene transcription via the binding of PPARa to three novel and functionally critical PPREs in the MDR3 promoter. Fenofibrate treatment further stimulated biliary PC secretion in rat hepatocytes, thereby providing a functional correlate. We have established a molecular mechanism for the beneficial use of fenofibrate therapy in human cholestatic liver disease.

Disclosures:

The following people have nothing to disclose: Nisanne Ghonem, Meena Ananthanarayanan, Carol J. Soroka, James L. Boyer

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