Pathogenesis Of Bile Duct Injury Research Articles

Cholestatic Liver Disease in Children

Inherited syndromes of intrahepatic cholestasis and biliary atresia are the most common causes of chronic liver disease and the prime indication for liver transplantation in children. Our understanding of the pathogenesis of these diseases has increased substantially by the discovery of genetic mutations in children with intrahepatic cholestasis and the findings that inflammatory circuits are operative at the time of diagnosis of biliary atresia. Building on this solid foundation, recent studies provide new insight into genotype-phenotype relationships and how mutations produce altered bile composition and cholestasis. New evidence exists that although liver transplantation is curative for patients with end-stage liver disease owing to cholestasis, some patients may develop recurrence of cholestasis because of the emergence of autoantibodies that disrupt canalicular function in the new graft. Progress is also evident in biliary atresia, with recent studies identifying candidate modifier genes and directly implicating lymphocytes and inflammatory signals in the pathogenesis of bile duct injury and obstruction.

Endogenous Bile Salts Are Associated With Bile Duct Injury in the Rat Liver Transplantation Model

Nonanastomotic biliary strictures are a serious complication after orthotopic liver transplantation (OLT) and are difficult to cure. We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. A total of 90 Sprague-Dawley rats were divided into three groups: Sham-operated group (Sham, n=30), OLT group with 1 hr donor liver preservation (OLT-1h, n=30), and OLT group with 12 hr donor liver preservation (OLT-12h, n=30). Bile was collected and analyzed biochemically. The histopathologic study was performed to determine the intrahepatic bile duct morphologic changes. Hepatic expressions of bile transporters Ntcp, Bsep, Mdr2, and Oatps were detected. During the first 2 weeks after transplantation, bile salt secretion was not parallel with phospholipid secretion, resulting in high biliary bile salt-to-phospholipid (BS:PL) ratio. The expression of bile transporters was consistent with the change of bile composition. Bile duct injury correlated significantly with bile salt secretion and BS:PL ratio. Moreover, OLT group with longer donor liver preservation time (OLT-12h) had significantly lower Mdr2 messenger RNA/protein level, higher BS:PL ratio, and better correlation between BS:PL ratio and bile duct injury compared with those of OLT-1h. The unparallel secretion of bile salts and phospholipids results in cytotoxic bile formation with high BS:PL ratio after liver transplantation. Longer donor liver preservation time will increase graft bile cytotoxicity. The results of this study suggest that endogenous bile salts play a role in the pathogenesis of bile duct injury after OLT.

The Role of Bile Salt Toxicity in the Pathogenesis of Bile Duct Injury After Non-Heart-Beating Porcine Liver Transplantation

Intrahepatic bile duct strictures are a serious complication after non-heart-beating (NHB) liver transplantation. Bile salt toxicity has been identified as an important factor in the pathogenesis of bile duct injury and cholangiopathies. The role of bile salt toxicity in the development of biliary strictures after NHB liver transplantation is unclear. In a porcine model of NHB liver transplantation, we studied the effect of different periods of warm ischemia in the donor on bile composition and subsequent bile duct injury after transplantation. After induction of cardiac arrest in the donor, liver procurement was delayed for 0 min (group A), 15 min (group B), or more or equal to 30 min (group C). Livers were subsequently transplanted after 4 hr of cold preservation. In the recipients, bile flow was measured, and bile samples were collected daily to determine the bile salt-to-phospholipid ratio. Severity of bile duct injury was semiquantified by using a histologic grading scale. Posttransplantation survival was directly related to the duration of warm ischemia in the donor. The bile salt-to-phospholipid ratio in bile produced early after transplantation was significantly higher in group C, compared with group A and B. Histopathologic condition showed the highest degree of bile duct injury in group C. Prolonged warm ischemia in NHB donors is associated with the formation of toxic bile after transplantation, with a high biliary bile salt-to-phospholipid ratio. These data suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury after NHB liver transplantation.

Experimental study of relationship of bile composition imbalance with bile duct injury

To investigate the change of bile composition and its role in bile duct injury after orthotopic liver transplantation (OLT). Rats were randomly divided into 3 groups: group A (sham surgery), group B (OLT with 1 h cold preservation), group C (OLT with 12 h cold preservation). The arterialized rat liver transplantation model with biliary extra-drainage was used in group B and C. Animals were sacrificed at posttransplant 1, 3, 5, 7, 10 and 14 day. Routine bile chemistry and pathological assays were performed. Cold preservation/reperfusion injury (CPRI) could repress the secretion of bile salts and phospholipid. However, in contrast with a rapid increase of bile salt secretion, the biliary secretion of phospholipid recovered more slowly, leading to an abnormal high bile salts/phospholipid ratio early after transplantation. Further analysis suggested that the secretion of bile salts correlated strongly with biochemical and histopathological signs of bile duct injury. CPRI can lead to great changes of graft bile composition, which plays a role in the pathogenesis of bile duct injury following liver transplantation.

Intracellular balance of oxidative stress and cytoprotective molecules in damaged interlobular bile ducts in autoimmune hepatitis and primary biliary cirrhosis: In situ detection of 8‐hydroxydeoxyguanosine and glutathione‐S‐transferase‐pi

Bile duct injury has been thought to be absent in autoimmune hepatitis (AIH), but recent studies have indicated that AIH patients do have bile duct injury. In this study, the intracellular balance of oxidative stress and cytoprotection in biliary epithelial cells was investigated to clarify the pathogenesis of bile duct injury in AIH. The intracellular status of oxidative DNA damage caused by oxidative stress and glutathione, an endogenous cytoprotective molecule, were examined in patients with AIH, primary biliary cirrhosis (PBC), and normal controls by immunostaining of 8-hydroxydeoxyguanosine (8-OHdG) and glutathione-S-transferase-pi. Immunohistochemically, 8-OHdG expression was detected as abundantly in the damaged bile ducts of AIH patients as in PBC patients. Moreover, in AIH, 8-OHdG expression was detected in damaged bile ducts more than in undamaged bile ducts. Glutathione-S-transferase-pi expression was relatively preserved in the damaged bile ducts of AIH patients compared to PBC patients, reflecting preservation of intracellular glutathione. In AIH, oxidative stress and DNA damage may be involved in the pathogenesis of bile duct injury in a manner similar to that found in PBC. However, relatively preserved intracellular glutathione may play a key role in preventing progressive bile duct loss following bile duct injury in AIH.

Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2 +/− mice

Intrahepatic bile duct strictures are a serious complication after orthotopic liver transplantation (OLT). We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. Livers from wild-type mice and mice heterozygous for disruption of the multidrug resistance 2 Mdr2 gene (Mdr2+/-) were transplanted into wild-type recipient mice. Mdr2+/- mice secrete only 50% of the normal amount of phospholipids into their bile, leading to an abnormally high bile salt/phospholipid ratio. In contrast to homozygous Mdr2-/- mice, the Mdr2+/- mice have normal liver histology and function under normal conditions. Two weeks after OLT, bile duct injury and cholestasis were assessed by light and electron microscopy, as well as through molecular and biochemical markers. There were no signs of bile duct injury or intrahepatic cholestasis in liver grafts from wild-type donors. Liver grafts from Mdr2+/- donors, however, had enlarged portal tracts with cellular damage, ductular proliferation, biliostasis, and a dense inflammatory infiltrate after OLT. Parallel to this observation, recipients of Mdr2+/- livers had significantly higher serum transaminases, alkaline phosphatase, total bilirubin, and bile salt levels, as compared with recipients of wild-type livers. In addition, hepatic bile transporter expression was compatible with the biochemical and histological cholestatic profile found in Mdr2+/- grafts after OLT. In conclusion, toxic bile composition, due to a high biliary bile salt/phospholipid ratio, acted synergistically with cold ischemia in the pathogenesis of bile duct injury after transplantation.

Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation

Biliary strictures are a serious cause of morbidity after liver transplantation. We have studied the role of altered bile composition as a mechanism of bile duct injury after human liver transplantation. In 28 liver transplant recipients, bile samples were collected daily posttransplantation for determination of bile composition. Hepatic expression of bile transporters was studied before and after transplantation. Histopathological criteria as well as biliary concentrations of alkaline phosphatase (ALP) and gamma-glutamyltransferase (gamma-GT) were used to quantify bile duct injury. Early after transplantation, bile salt secretion increased more rapidly than phospholipid secretion, resulting in high biliary bile salt/phospholipid ratio (BA/PL). In parallel with this, mRNA levels of the bile salt transporters NTCP and BSEP increased significantly after transplantation, whereas phospholipid translocator MDR3 mRNA levels remained unchanged. Bile duct injury correlated significantly with bile salt secretion and was associated with a high biliary BA/PL ratio. Bile salt secretion after human liver transplantation recovers more rapidly than phospholipid secretion. This results in cytotoxic bile formation and correlates with bile duct injury. These findings suggest that endogenous bile salts have a role in the pathogenesis of bile duct injury after liver transplantation.

Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 ( Abcb4) knockout mice

Background & Aims: Because the mechanisms leading to bile duct damage in sclerosing cholangitis are unknown, we aimed to determine the pathogenesis of bile duct injury in multidrug resistance gene ( Mdr2) ( Abcb4) knockout mice ( Mdr2 −/− ) as a novel model of the disease. Methods: Mdr2 −/− and wild-type controls ( Mdr2 +/+ ) were studied at 2, 4, and 8 weeks of age. Liver histology, ultrastructure, immunofluorescence microscopy (to study inflammatory cells, tight junction protein ZO-1, basement membrane protein laminin, fluorescence-labeled ursodeoxycholic acid), immunohistochemistry (for α-smooth muscle actin, nitrotyrosine), sirius red staining, bacterial cultures of intra-abdominal organs, and polymerase chain reaction (PCR) for Helicobacter bilis DNA were compared between both genotypes. Hepatic cytokine expression was determined by reverse-transcription PCR. Results: Bile ducts of Mdr2 −/− showed disrupted tight junctions and basement membranes, bile acid leakage into portal tracts, induction of a portal inflammatory (CD11b, CD4-positive) infiltrate, and activation of proinflammatory (tumor necrosis factor [TNF]-α, interleukin [IL]-1β) and profibrogenic cytokines (transforming growth factor [TGF]-β1). This resulted in activation of periductal myofibroblasts, leading to periductal fibrosis, separating the peribiliary plexus from bile duct epithelial cells and, finally, causing atrophy and death of the bile duct epithelium. Bacterial translocation was not increased and H. bilis was not detectable in Mdr2 −/− . Conclusions: Sclerosing cholangitis in Mdr2 −/− mice is a multistep process with regurgitation of bile from leaky ducts into the portal tracts, leading to induction of periductal inflammation, followed by activation of periductal fibrogenesis, finally causing obliterative cholangitis owing to atrophy and death of bile duct epithelial cells.

Detection of serum nitrite and nitrate in primary biliary cirrhosis: possible role of nitric oxide in bile duct injury.

The role of nitric oxide synthase (NOS) in autoimmune disease is gaining increased attention because of the relationships between NOS activity and T-lymphocyte subpopulations and, in particular, the influence of NO on cytokine production by Th1 versus Th2 cells. In addition, there is evidence that both the liver and infiltrating hepatic T cells have inducible NOS-2 activity. We studied serum levels of nitrite (NO2-) and nitrate (NO3-) in groups of patients with liver disease secondary to hepatitis B, hepatitis C, autoimmune hepatitis and primary biliary cirrhosis (PBC). Simultaneously, in a nested subpopulation, we studied the liver expression of NOS-2. Interestingly, there was a significant elevation both of nitrite and of nitrate in patients with PBC but not other liver diseases. Despite such increments, there was no correlation of the levels of nitrite and nitrate with sera levels of tumor necrosis factor-alpha, interferon-gamma, alanine aminotransferase, total bilirubin, alkaline phosphatase, gamma-glutamyl transpeptidase, platelet count, IgG, IgM, antimitochondrial antibodies or prothrombin time. These data were extended by demonstrating the expression of NOS-2 by immunohistochemistry in 13/14 patients with PBC, including in 9/14 patient hepatocyte populations and 4/14 bile duct cells. In contrast, NOS-2 expression was noted in hepatitis B and hepatitis C, but only found within mononuclear cells. Our data suggest that NO produced through NOS-2 may play a role in the pathogenesis of bile duct injury in some PBC patients.

Role of glutathione in hepatic bile formation during reperfusion after cold ischemia of the rat liver

Background/Aims: Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. Methods: We examined the effects of cold ischemia (University of Wisconsin solution (4°C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR −-rats, in whom biliary excretion of glutathione is already impaired. Results: Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR −-rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR −-rat livers was not associated with electron microscopic evidence of bile duct damage. Conclusions: We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.