Does lithium attenuate the liver damage due to oxidative stress and liver glycogen depletion in experimental common bile duct obstruction?

In extrahepatic cholestasis, the molecular mechanisms of liver damage due to bile acid accumulation remain elusive. In this study, the activation of glutamatergic receptors was hypothesized to be responsible for bile acid-induced oxidative stress and liver damage. Recent evidence showed that lithium, as an N-methyl-d-aspartate receptor (NMDAR) GluN2B subunit inhibitor, may act on the glutamate/NMDAR signaling axis. Guinea pigs were assigned to four groups, as sham laparotomy (SL), bile duct ligated (BDL), lithium-treated SL (SL + Li) and lithium-treated BDL (BDL + Li) groups. Cholestasis-induced liver injury was evaluated by aspartate aminotransferase (AST), alanine transaminase (ALT), interleukin-6 (IL-6), tissue malondialdehyde (MDA), copper­zinc superoxide dismutase and reduced glutathione levels. The liability of glutamate/NMDAR signaling axis was clarified by glutamate levels in both plasma and liver samples, with the production of nitric oxide (NO), as well as with the serum calcium concentrations. Blood glucose, glucagon, insulin levels and glucose consumption rates, in addition to tissue glycogen were measured to evaluate the liver glucose-glycogen metabolism. A high liver damage index (AST/ALT) was calculated in BDL animals in comparison to SL group. In the BDL animals, lithium reduced plasma NO and glutamate in addition to tissue glutamate concentrations, while serum calcium increased. The antioxidant capacities and liver glycogen contents significantly increased, whereas blood glucose levels unchanged and tissue MDA levels decreased 3-fold in lithium-treated cholestatic animals. It was concluded that lithium largely protects the cholestatic hepatocyte from bile acid-mediated damage by blocking the NMDAR-GluN2B subunit.

Bile Modulates Secretion of Incretins and Insulin: A Study of Human Extrahepatic Cholestasis.

OBJECTIVE: Changes in bile flow after bariatric surgery may beneficially modulate secretion of insulin and incretins, leading to diabetes remission. However, the exact mechanism(s) involved is still unclear. Here, we propose an alternative method to investigate the relationship between alterations in physiological bile flow and insulin and incretin secretion by studying changes in gut-pancreatic function in extrahepatic cholestasis in nondiabetic humans. METHODS: To pursue this aim, 58 nondiabetic patients with recent diagnosis of periampullary tumors underwent an oral glucose tolerance test (OGTT), and a subgroup of 16 patients also underwent 4-hour mixed meal tests and hyperinsulinemic-euglycemic clamps. RESULTS: The analysis of the entire cohort revealed a strong inverse correlation between total bilirubin levels and insulinogenic index. When subjects were divided on the basis of bilirubin levels, used as a marker of altered bile flow, subjects with high bilirubin levels displayed inferior glucose control and decreased insulin secretion during the OGTT. Altered bile flow elicited a markedly greater increase in glucagon and glucagon-like peptide 1 (GLP-1) secretion at fasting state, and following the meal, both glucagon and GLP-1 levels remained increased over time. Conversely, Glucose-dependent insulinotropic polypeptide (GIP) levels were comparable at the fasting state, whereas the increase following meal ingestion was significantly blunted with high bilirubin levels. We reveal strong correlations between total bilirubin and glucagon and GLP-1 levels. CONCLUSIONS: Our findings suggest that acute extrahepatic cholestasis determines major impairment in enteroendocrine gut-pancreatic secretory function. The altered bile flow may determine a direct deleterious effect on ß-cell function, perhaps mediated by the impairment of incretin hormone function.

Prostaglandin E1 Preconditioning Attenuates Liver Ischemia Reperfusion Injury in a Rat Model of Extrahepatic Cholestasis.

The aim of this study is to explore the hepatoprotective effect of intraportal prostaglandin E1 (PGE1) on liver ischemia reperfusion (IR) injury using an extrahepatic cholestatic model, observing oxidative stress markers, proinflammatory factors, apoptotic marker proteins, and an adhesion molecule. The extrahepatic cholestatic model was induced by common bile duct ligation. After seven days, rats were subjected to ischemia by Pringle maneuver for 15 min, followed by 1, 6, or 24 h of reperfusion. Prostaglandin E1 (PGE group) or normal saline (NS group) was continuously infused from 15 min before liver ischemia to 1 h after reperfusion. After reperfusion, histopathological evaluation of the liver was performed, as were measurements of bilirubin, biochemical enzymes, oxidative stress markers (GSH and MDA), proinflammatory factors (MPO, TNF-α, and IL-1ß), apoptotic marker proteins (Bcl-2 and Bax), and the adhesion molecule (ICAM-1). PGE1 pretreatment attenuated IR injury in extrahepatic cholestatic liver probably by suppressing MDA, MPO, TNF-α, IL-1ß, ICAM-1, and Bax levels and improving GSH and Bcl-2 levels. In conclusion, PGE1 protects extrahepatic cholestatic liver from IR injury by improving hepatic microcirculation and reducing oxidative stress damage, intrahepatic neutrophil infiltration, and hepatocyte apoptosis.

α7-nAChR Knockout Mice Decreases Biliary Hyperplasia and Liver Fibrosis in Cholestatic Bile Duct-Ligated Mice.

α7-nAChR is a nicotinic acetylcholine receptor [specifically expressed on hepatic stellate cells (HSCs), Kupffer cells, and cholangiocytes] that regulates inflammation and apoptosis in the liver. Thus, targeting α7-nAChR may be therapeutic in biliary diseases. Bile duct ligation (BDL) was performed on wild-type (WT) and α7-nAChR-/- mice. We first evaluated the expression of α7-nAChR by immunohistochemistry (IHC) in liver sections. IHC was also performed to assess intrahepatic bile duct mass (IBDM), and Sirius Red staining was performed to quantify the amount of collagen deposition. Immunofluorescence was performed to assess colocalization of α7-nAChR with bile ducts (costained with CK-19) and HSCs (costained with desmin). The mRNA expression of α7-nAChR, Ki-67/PCNA (proliferation), fibrosis genes (TGF-ß1, fibronectin-1, Col1α1, and α-SMA), and inflammatory markers (IL-6, IL-1ß, and TNF-α) was measured by real-time PCR. Biliary TGF-ß1 and hepatic CD68 (Kupffer cell marker) expression was assessed using IHC. α7-nAChR immunoreactivity was observed in both bile ducts and HSCs and increased following BDL. α7-nAChR-/- BDL mice exhibited decreased (i) bile duct mass, liver fibrosis, and inflammation, and (ii) immunoreactivity of TGF-ß1 as well as expression of fibrosis genes compared to WT BDL mice. α7-nAChR activation triggers biliary proliferation and liver fibrosis and may be a therapeutic target in managing extrahepatic biliary obstruction.

Role of farnesoid X receptor in cholestasis.

The nuclear receptor farnesoid X receptor (FXR) plays an important role in physiological bile acid synthesis, secretion and transport. Defects of FXR regulation in these processes can cause cholestasis and subsequent pathological changes. FXR regulates the synthesis and uptake of bile acid via enzymes. It also increases bile acid solubility and elimination by promoting conjugation reactions and exports pump expression in cholestasis. The changes in bile acid transporters are involved in cholestasis, which can result from the mutations of transporter genes or acquired dysfunction of transport systems, such as inflammation-induced intrahepatic cholestasis. The modulation function of FXR in extrahepatic cholestasis is not identical to that in intrahepatic cholestasis, but the discrepancy may be reduced over time. In extrahepatic cholestasis, increasing biliary pressure can induce bile duct proliferation and bile infarcts, but the absence of FXR may ameliorate them. This review provides an update on the function of FXR in the regulation of bile acid metabolism, its role in the pathophysiological process of cholestasis and the therapeutic use of FXR agonists.

Expression of renal Oat5 and NaDC1 transporters in rats with acute biliary obstruction.

AIM: To examine renal expression of organic anion transporter 5 (Oat5) and sodium-dicarboxylate cotransporter 1 (NaDC1), and excretion of citrate in rats with acute extrahepatic cholestasis. METHODS: Obstructive jaundice was induced in rats by double ligation and division of the common bile duct (BDL group). Controls underwent sham operation that consisted of exposure, but not ligation, of the common bile duct (Sham group). Studies were performed 21 h after surgery. During this period, animals were maintained in metabolic cages in order to collect urine. The urinary volume was determined by gravimetry. The day of the experiment, blood samples were withdrawn and used to measure total and direct bilirubin as indicative parameters of hepatic function. Serum and urine samples were used for biochemical determinations. Immunoblotting for Oat5 and NaDC1 were performed in renal homogenates and brush border membranes from Sham and BDL rats. Immunohistochemistry studies were performed in kidneys from both experimental groups. Total RNA was extracted from rat renal tissue in order to perform reverse transcription polymerase chain reaction. Another set of experimental animals were used to evaluate medullar renal blood flow (mRBF) using fluorescent microspheres. RESULTS: Total and direct bilirubin levels were significantly higher in BDL animals, attesting to the adequacy of biliary obstruction. An important increase in mRBF was determined in BDL group (Sham: 0.53 ± 0.12 mL/min per 100 g body weight vs BDL: 1.58 ± 0.24 mL/min per 100 g body weight, P < 0.05). An increase in the urinary volume was observed in BDL animals. An important decrease in urinary levels of citrate was seen in BDL group. Besides, a decrease in urinary citrate excretion (Sham: 0.53 ± 0.11 g/g creatinine vs BDL: 0.07 ± 0.02 g/g creatinine, P < 0.05) and an increase in urinary excretion of H(+) (Sham: 0.082 ± 0.03 µmol/g creatinine vs BDL: 0.21 ± 0.04 µmol/g creatinine, P < 0.05) were observed in BDL animals. We found upregulations of both proteins Oat5 and NaDC1 in brush border membranes where they are functional. Immunohistochemistry technique corroborated these results for both proteins. No modifications were observed in Oat5 mRNA and in NaDC1 mRNA levels in kidney from BDL group as compared with Sham ones. CONCLUSION: Citrate excretion is decreased in BDL rats, at least in part, because of the higher NaDC1 expression. Using the outward gradient of citrate generated by NaDC1, Oat5 can reabsorb/eliminate different organic anions of pathophysiological importance.

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Gangliocytic Paraganglioma With Atypical Immunohistochemical Features Presenting as Extrahepatic Biliary Obstruction.

Gangliocytic paraganglioma is a rare benign tumor of upper gastrointestinal tract that most commonly involves the second part of duodenum. The tumor is detected incidentally on imaging in most of the cases. However, presentation with extrahepatic biliary obstruction is extremely rare. We recently encountered a 50-year-old male patient who was evaluated for extrahepatic biliary obstruction and was found to have a periampullary mass on imaging. The patient underwent pylorus-preserving pancreaticoduodenectomy along with liver biopsy and hepatoduodenal lymph node dissection. On histopathological examination, a tumor was detected in the periampullary region of duodenum, which was confirmed to be gangliocytic paraganglioma on immunohistochemistry along with atypical histological and immunohistochemical features.

CCN1 induces hepatic ductular reaction through integrin αvß5-mediated activation of NF-κB.

Liver cholestatic diseases, which stem from diverse etiologies, result in liver toxicity and fibrosis and may progress to cirrhosis and liver failure. We show that CCN1 (also known as CYR61), a matricellular protein that dampens and resolves liver fibrosis, also mediates cholangiocyte proliferation and ductular reaction, which are repair responses to cholestatic injury. In cholangiocytes, CCN1 activated NF-κB through integrin αvß5/αvß3, leading to Jag1 expression, JAG1/NOTCH signaling, and cholangiocyte proliferation. CCN1 also induced Jag1 expression in hepatic stellate cells, whereupon they interacted with hepatic progenitor cells to promote their differentiation into cholangiocytes. Administration of CCN1 protein or soluble JAG1 induced cholangiocyte proliferation in mice, which was blocked by inhibitors of NF-κB or NOTCH signaling. Knock-in mice expressing a CCN1 mutant that is unable to bind αvß5/αvß3 were impaired in ductular reaction, leading to massive hepatic necrosis and mortality after bile duct ligation (BDL), whereas treatment of these mice with soluble JAG1 rescued ductular reaction and reduced hepatic necrosis and mortality. Blockade of integrin αvß5/αvß3, NF-κB, or NOTCH signaling in WT mice also resulted in defective ductular reaction after BDL. These findings demonstrate that CCN1 induces cholangiocyte proliferation and ductular reaction and identify CCN1/αvß5/NF-κB/JAG1 as a critical axis for biliary injury repair.

SIRT1/PGC-1α signaling protects hepatocytes against mitochondrial oxidative stress induced by bile acids.

Oxidative stress and mitochondrial dysfunction are hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Silent information regulator 1 (SIRT1) attenuates oxidative stress and improves mitochondrial biogenesis in numerous mitochondrial-related diseases; however, a functional role for SIRT1 in chronic liver cholestasis, characterized by increased levels of toxic bile acids, remains unknown. We show decrease in SIRT1 levels and its activity and impairment of mitochondrial biogenesis in the liver of patients with extrahepatic cholestasis. Moreover, we found that glycochenodeoxycholic acid (GCDCA) stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content in L02 cells. Consistent with this finding, GCDCA was found to decrease SIRT1 protein expression and activity, thus promoting the deacetylation of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), a key enzyme involved in mitochondrial biogenesis and function. Conversely, GCDCA-induced mitochondrial injury was efficiently attenuated by SIRT1 overexpression. In summary, these findings indicate that the loss of SIRT1 may play a crucial role in the pathogenesis of liver damage observed in patients with extrahepatic cholestasis. The findings also indicate that genetic supplementation of SIRT1 can ameliorate GCDCA-induced hepatotoxicity through the activation of PGC-1α-dependent mitochondrial biogenesis.

Activation of the renin-angiotensin system stimulates biliary hyperplasia during cholestasis induced by extrahepatic bile duct ligation.

Cholangiocyte proliferation is regulated in a coordinated fashion by many neuroendocrine factors through autocrine and paracrine mechanisms. The renin-angiotensin system (RAS) is known to play a role in the activation of hepatic stellate cells and blocking the RAS attenuates hepatic fibrosis. We investigated the role of the RAS during extrahepatic cholestasis induced by bile duct ligation (BDL). In this study, we used normal and BDL rats that were treated with control, angiotensin II (ANG II), or losartan for 2 wk. In vitro studies were performed in a primary rat cholangiocyte cell line (NRIC). The expression of renin, angiotensin-converting enzyme, angiotensinogen, and angiotensin receptor type 1 was evaluated by immunohistochemistry (IHC), real-time PCR, and FACs and found to be increased in BDL compared with normal rat. The levels of ANG II were evaluated by ELISA and found to be increased in serum and conditioned media of cholangiocytes from BDL compared with normal rats. Treatment with ANG II increased biliary mass and proliferation in both normal and BDL rats. Losartan attenuated BDL-induced biliary proliferation. In vitro, ANG II stimulated NRIC proliferation via increased intracellular cAMP levels and activation of the PKA/ERK/CREB intracellular signaling pathway. ANG II stimulated a significant increase in Sirius red staining and IHC for fibronectin that was blocked by angiotensin receptor blockade. In vitro, ANG II stimulated the gene expression of collagen 1A1, fibronectin 1, and IL-6. These results indicate that cholangiocytes express a local RAS and that ANG II plays an important role in regulating biliary proliferation and fibrosis during extraheptic cholestasis.

Increase of glyoosaminoglycans and metalloprotenases 2 and 9 in liver extracelllular matrix on early stages of extrahepatic cholestaiis / Aumento de glicosaminoglicanos e metaloproteinases 2 e 9 na matriz extracelular de fígado em estágios iniciais de colestase extra-hepática

Context Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Objective Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. Methods Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Results Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Conclusions Cholestasis led to many changes on rats’ liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content. .

Contexto Colestase produz lesão hepatocelular, infiltração leucocitária, proliferação de células ductulares e fibrose do parênquima hepático por matriz extracelular. Objetivo Analisar os efeitos da ligação do ducto biliar sobre conteúdo de glicosaminoglicanos e atividade de metaloproteinases de matriz (MMP). Métodos Animais (6-8 semanas; n = 40) foram eutanasiados 2, 7 ou 14 dias após ligação do ducto biliar ou falsa ligação. A evolução da doença foi analisada por peso corporal e do fígado, concentrações séricas de bilirrubina direta, globulinas, gama glutamil transpeptidase (GGT), fosfatase alcalina (Alk-P), alanina e aspartato aminotransfesases (ALT e AST), alterações teciduais de mieloperoxidase e metaloproteinases (MMP-9, pro MMP-2 e MMP-2), histopatologia e conteúdo de glicosaminoglicanos. Resultados A colestase causou dano celular com elevação dos níveis séricos de globulinas, GGT, Alk-P, ALT e AST. Houve também infiltração leucocitária observada pelo aumento na atividade de mieloperoxidase nos grupos 7 (P = 0,0064) e 14 dias (P = 0,0002) o que leva ao aumento das lesões no tecido. Ligação do ducto biliar aumentou as atividades de pro MMP-2 (P = 0,0677), MMP-2 (P = 0,0003) e MMP-9 (P<0,0001) aos 14 dias indicando remodelamento da matriz e estabelecimento de processo inflamatório. Animais com ligação do ducto biliar mostraram um aumento do conteúdo de dermatam sulfato e/ou heparam sulfato refletindo a produção de matriz extracelular e aumento de mitose devido a depleção do parênquima hepático. Conclusões Colestase causou várias mudanças no parênquima hepático de ratos, bem como em sua matriz extracelular, com importantes alterações na atividade ...

A nontumorigenic variant of FGF19 treats cholestatic liver diseases.

Hepatic accumulation of bile acids is central to the pathogenesis of cholestatic liver diseases. Endocrine hormone fibroblast growth factor 19 (FGF19) may reduce hepatic bile acid levels through modulation of bile acid synthesis and prevent subsequent liver damage. However, FGF19 has also been implicated in hepatocellular carcinogenesis, and consequently, the potential risk from prolonged exposure to supraphysiological levels of the hormone represents a major hurdle for developing an FGF19-based therapy. We describe a nontumorigenic FGF19 variant, M70, which regulates bile acid metabolism and, through inhibition of bile acid synthesis and reduction of excess hepatic bile acid accumulation, protects mice from liver injury induced by either extrahepatic or intrahepatic cholestasis. Administration of M70 in healthy human volunteers potently reduces serum levels of 7α-hydroxy-4-cholesten-3-one, a surrogate marker for the hepatic activity of cholesterol 7α-hydroxylase (CYP7A1), the enzyme responsible for catalyzing the first and rate-limiting step in the classical bile acid synthetic pathway. This study provides direct evidence for the regulation of bile acid metabolism by FGF19 pathway in humans. On the basis of these results, the development of nontumorigenic FGF19 variants capable of modulating CYP7A1 expression represents an effective approach for the prevention and treatment of cholestatic liver diseases as well as potentially for other disorders associated with bile acid dysregulation.

Increase of glycosaminoglycans and metalloproteinases 2 and 9 in liver extracellular matrix on early stages of extrahepatic cholestasis.

CONTEXT: Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. OBJECTIVE: Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. METHODS: Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. RESULTS: Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. CONCLUSIONS: Cholestasis led to many changes on rats' liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content.

Loss of A(1) adenosine receptor attenuates alpha-naphthylisothiocyanate-induced cholestatic liver injury in mice.

Cholestasis has limited therapeutic options and is associated with high morbidity and mortality. The A(1) adenosine receptor (A(1)AR) was postulated to participate in the pathogenesis of hepatic fibrosis induced by experimental extrahepatic cholestasis; however, the contribution of A(1)AR to intrahepatic cholestatic liver injury remains unknown. Here, we found that mice lacking A(1)AR were resistant to alpha-naphthyl isothiocyanate (ANIT)-induced liver injury, as evidenced by lower serum liver enzyme levels and reduced extent of histological necrosis. Bile acid accumulation in liver and serum was markedly diminished in A(1)AR(-/-) mice compared with wild-type (WT) mice. However, biliary and urinary outputs of bile acids were significantly enhanced in A(1)AR(-/-) mice. In the liver, mRNA expression of genes related to bile acid transport (Bsep and Mdr2) and hydroxylation (Cyp3a11) was increased in A(1)AR(-/-) mice. In the kidney, A(1)AR deficiency prevented the decrease of glomerular filtration rate caused by ANIT. Treatment of WT mice with A(1)AR antagonist DPCPX also protected against ANIT hepatotoxicity. Our results indicated that lack of A(1)AR gene protects mice from ANIT-induced cholestasis by enhancing toxic biliary constituents efflux through biliary excretory route and renal elimination system and suggested a potential role of A(1)AR as therapeutic target for the treatment of intrahepatic cholestasis.

Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis.

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

The effects of chitosan and low dose dexamethasone on extrahepatic cholestasis after bile duct ligation in Wistar rats.

UNLABELLED: Inflammation and oxidative stress are important pathways in the development of liver fibrosis following biliary obstruction. AIM: To evaluate the effects of low dose dexamethasone and chitosan, a natural compound with no side-effects, on liver damage caused by bile duct ligation in rats. MATERIALS AND METHODS: Fifty female Wistar rats, randomly and equally divided in 5 groups: I (SHAM) underwent only laparotomy, II (BDL) with bile duct ligation, III (DEX) 0.125 mg/kg dexamethasone i.m. daily, IV (CS) 1 mg/kg chitosan by gavage and group V (DEX+CS), both substances. After six days, the following parameters were assessed from liver homogenates: malondialdehyde (MDA), protein carbonyls (PC), reduced glutathione (GSH), total SH groupings, nitric oxide (NO), and from plasma: MDA, γ-glutamyltranspeptidase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TB). A histopathological examination was performed using some of the elements of the Knodell Histological Activity Index. RESULTS: BDL significantly increases the levels of MDA, liver enzymes, and the necro-inflammatory score compared to the sham group and it decreases the antioxidant capacity. DEX protects against lipid peroxidation and improves the antioxidant capacity, but it is not able to protect the hepatocytes. Chitosan significantly decreases (p<0.05) the levels of MDA (0.07±0.01 vs 0.10±0.01 nmoles/mg protein BDL group, p=0.027) and also ALT, TB, GGT and reduces liver necrosis and inflammation (2.75±0.95 vs 1±0, p<0.05). Both CS and DEX reduce the level of NO significantly. CONCLUSION: BDL induces severe oxidative stress damage after six days already. Chitosan proved very efficient in protecting the hepatocytes against oxidative stress, a fact supported by the histological findings.

Protective effects of thymoquinone against cholestatic oxidative stress and hepatic damage after biliary obstruction in rats.

The aim of this study was to examine the preventive and therapeutic effects of thymoquinone (TQ) against cholestatic oxidative stress and liver damage in common bile duct ligated rats. A total of 24 male Sprague-Dawley rats were divided into three groups: control, bile duct ligation (BDL) and BDL + received TQ; each group contain 8 animals. The rats in TQ treated groups were given TQ (50 mg/kg body weight) once a day orally for 2 weeks starting 3 days prior to BDL operation. To date, no more biochemical and histopathological changes on common bile duct ligated rats by TQ treatment have been reported. The application of BDL clearly increased the tissue hydroxyproline (HP) content, malondialdehyde (MDA) levels and decreased the antioxidant enzyme [superoxide dismutase (SOD), glutathione peroxidase (GPx)] activities. TQ treatment significantly decreased the elevated tissue HP content, and MDA levels and raised the reduced of SOD, and GPx enzymes in the tissues. The changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with TQ attenuated alterations in liver histology. The immunopositivity of alpha smooth muscle actin and proliferating cell nuclear antigen in BDL were observed to be reduced with the TQ treatment. The present study demonstrates that oral administration of TQ in bile duct ligated rats maintained antioxidant defenses and reduces liver oxidative damage and ductular proliferation. This effect of TQ may be useful in the preservation of liver function in cholestasis.

Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat.

Cholestatic patients often present with clinical features suggestive of adrenal insufficiency. In the bile duct-ligated (BDL) model of cholestasis, the hypothalamic-pituitary-adrenal (HPA) axis is suppressed. The consequences of this suppression on cholangiocyte proliferation are unknown. We evaluated 1) HPA axis activity in various rat models of cholestasis and 2) effects of HPA axis modulation on cholangiocyte proliferation. Expression of regulatory molecules of the HPA axis was determined after BDL, partial BDL, and α-naphthylisothiocyanate (ANIT) intoxication. The HPA axis was suppressed by inhibition of hypothalamic corticotropin-releasing hormone (CRH) expression by central administration of CRH-specific Vivo-morpholinos or by adrenalectomy. After BDL, the HPA axis was reactivated by 1) central administration of CRH, 2) systemic ACTH treatment, or 3) treatment with cortisol or corticosterone for 7 days postsurgery. There was decreased expression of 1) hypothalamic CRH, 2) pituitary ACTH, and 3) key glucocorticoid synthesis enzymes in the adrenal glands. Serum corticosterone and cortisol remained low after BDL (but not partial BDL) compared with sham surgery and after 2 wk of ANIT feeding. Experimental suppression of the HPA axis increased cholangiocyte proliferation, shown by increased cytokeratin-19- and proliferating cell nuclear antigen-positive cholangiocytes. Conversely, restoration of HPA axis activity inhibited BDL-induced cholangiocyte proliferation. Suppression of the HPA axis is an early event following BDL and induces cholangiocyte proliferation. Knowledge of the role of the HPA axis during cholestasis may lead to development of innovative treatment paradigms for chronic liver disease.

Attenuated liver fibrosis after bile duct ligation and defective hepatic stellate cell activation in neural cell adhesion molecule knockout mice.

AIM: Neural cell adhesion molecule (N-CAM) is expressed by activated hepatic stellate cells (HSC), portal fibroblasts, cholangiocytes and hepatic progenitor cells during liver injury. Its functional role in liver disease and fibrogenesis is unknown. The aim of this study was to investigate the role of N-CAM in liver fibrogenesis. METHODS: To induce fibrosis, N-CAM knockout mice and wild-type controls were subjected to bile duct ligation (BDL) or repeated carbon tetrachloride (CCl(4) ) injections. Fibrosis was quantified by hydroxyproline, immunhistochemistry staining and image analysis. Protein levels were determined with immunoblotting. HSCs were isolated by ultracentrifugation in a Larcoll gradient and thereafter in vitro stimulated with recombinant transforming growth factor (TGF)-ß1. RESULTS: Two weeks after BDL, wild-type mice had developed pronounced liver fibrosis while N-CAM-/- mice had less such alterations. N-CAM-/- mice had less deposition of collagen and fibronectin seen in immunhistochemistry. The protein levels of fibronectin were higher in the liver from the wild type, while laminin were unaltered. CCl(4) -treated N-CAM-/- and wild-type mice showed no significant difference in the extent of liver fibrosis or the expression levels of the above-mentioned genes. HSC isolated from N-CAM-/- mice showed declined levels of smooth muscle actin and desmin after stimulation in vitro with TGF-ß1. CONCLUSIONS: Loss of N-CAM results in decreased hepatic collagen and fibronectin deposition in mice subjected to BDL, but not in animals exposed to repeated CCl(4) injections. HSC isolated from N-CAM null mice show impaired activation in vitro. This indicates a role of N-CAM in cholestatic liver disease and HSC activation.