studies

614A AASLD ABSTRACTS HEPATOLOGY, October, 2015
are also more mature in primary cholangiocytes with Yap deficiency.
YAP activity correlates with Mdr2-/- mice disease progress.
Conclusions: YAP functions to increase bile duct dilation
upon bile duct obstruction through promoting cholangiocyte
E-cadherin junction dynamics. The dilated bile ducts are able
to hold more bile in bile ducts of WT controls, which in turn
reduces hepatocyte damage caused by bile overflow.
Disclosures:
The following authors have nothing to disclose: Quy P. Nguyen, Nan Wu, Tianhao
Zhou, Haibo Bai
814
Glutathione depletion is critical for the early pathogenesis
of biliary atresia (BA) in the toxin-induced BA model
Xiao Zhao 1 , Benjamin J. Wilkins 2 , Kristin Lorent 1 , John R. Porter 3 ,
Rebecca G. Wells 1 , Michael Pack 1 ; 1 Medicine/Division of Gastroenterology,
University of Pennsylvania, Philadelphia, PA; 2 Division
of Anatomic Pathology, Department of Pathology and Laboratory
Medicine, The Children’s Hospital of Philadelphia, Philadelphia,
PA; 3 University of the Sciences, Philadelphia, PA
Biliary atresia (BA) is a complex neonatal cholangiopathy that
is the leading indication for liver transplantation in the pediatric
population. Using an in vivo zebrafish biliary assay, we have
isolated biliatresone, a novel plant isoflavonoid with selective
extrahepatic biliary toxicity that is responsible for BA outbreaks
in newborn Australian livestock. The phenotype induced in
this new BA model is conserved between zebrafish and mammals,
and recapitulates the cardinal features of human BA.
While extrahepatic cholangiocytes are exquisitely sensitive to
biliatresone, hepatocytes and intrahepatic cholangiocytes are
resistant to its deleterious effect. This difference in susceptibility
can potentially be explained by the intrinsic differential capability
of specific liver cell types in mitigating stress. Global
expression profiling of mRNA recovered from larval zebrafish
cholangiocytes and total liver revealed that biliatresone
leads to early up-regulation of the glutathione (GSH) redox
response, suggesting that GSH depletion is an inciting event
in biliatresone-mediated biliary injury. GSH, as a ubiquitous
tripeptide thiol, is a vital cellular antioxidant. These data are
consistent with in vitro assays that confirm the reactivity of biliatresone
with glutathione. Using a specific genetically encoded
GSH redox probe (redox-sensitive GFP, roGFP), we were able
to establish quantitative in vivo mapping of the GSH redox
potential (E GSH
) in hepatocytes and extrahepatic cholangiocyte
(EHC) of the biosensor-transgenic larvae. Interestingly, we
detected endogenous differences in the redox status between
hepatocytes and EHC. Pharmacological manipulation of GSH
redox homeostasis further supported the importance of GSH in
modulating biliatresone-induced injury. This is evidenced by the
temporary attenuation of its biliary toxicity with N-acetylcysteine
(NAC), a GSH precursor. Altogether, these data strongly
suggest redox stress as a contributing factor in biliatresone-induced
injury and differences in intrinsic stress responses can
explain its cell-type specificity. Insufficient antioxidant capacity
of EHC may potentially be critical to the early pathogenesis of
BA.
Disclosures:
The following authors have nothing to disclose: Xiao Zhao, Benjamin J. Wilkins,
Kristin Lorent, John R. Porter, Rebecca G. Wells, Michael Pack
815
The bile acid Ursodeoxycholic acid activates cholangiocyte
TMEM16A Cl- channels
Qin Li, Amal K. Dutta, Charles Kresge, Andrew P. Feranchak;
Department of Pediatric Gastronenterology, Hepatology, University
of Texas Southwestern Medical Center, Dallas, TX
Ursodeoxycholic acid (UDCA) stimulates a bicarbonate-rich
choleresis in part through effects on cholangiocytes. During
cholehepatic shunting, uptake of bile acids at the apical cholangiocyte
membrane is associated with an increase in [Ca2+]
I, Cl- efflux and Cl-/HCO3- exchange, though the cellular
mechanism is unknown. As TMEM16A is a Ca2+-activated Clchannel
in the apical membrane of cholangiocytes (JBC 2011;
286:766-776) the aim of the present study was to determine if
TMEM16A is the target of UDCA-stimulated Cl- secretion and
to identify the regulatory pathway involved. Methods: Studies
were performed in SV40-immortalized mouse cholangiocytes
isolated from the large intrahepatic ducts (MLC) and in vivo
studies were performed in a novel bile duct-cannulated murine
model which we recently developed. This model involves placement
of an indwelling catheter into the bile duct of a live mouse
for measurement of bile flow rate and composition in response
to systemic administration of agonists. [Ca2+]I was measured
by Fura-2, ATP release by luciferase based assay and reported
as arbitrary light units (ALUs), and Cl- currents by patch clamp
techniques. Results: Exposure of MLC to UDCA (100 mM) rapidly
increased [Ca2+]I (increase in Fura-2 fluorescence from
0.65 to 1.05, p ≤0.01), stimulated ATP release (from 331 ± 22
ALUs to 780 ± 106 ALUs, p