Cytokeratins as Targets for Bile Acid-Induced Toxicity
2002; Elsevier BV; Volume: 160; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64868-7
ISSN1525-2191
AutoresPeter Fickert, Michael Trauner, Andrea Fuchsbichler, Cornelia Stumptner, Kurt Zatloukal, Helmut Denk,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoCholestasis is associated with retention of potentially toxic bile acids and profound cytoskeletal alterations of hepatocytes. Given the well-established cytoprotective role of hepatocyte keratins this study aimed to determine the effects of cholestasis on the cytokeratin (CK) intermediate filament network in mouse liver. Mice were subjected to common bile duct ligation or sham operation. Mice were also fed a cholic acid or ursodeoxycholic acid (UDCA)-supplemented diet (0.1%, 0.5%, and 1%) or control diet for 7 days. CK 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction, in situ hybridization, Western blot analysis, and immunofluorescence microscopy. Common bile duct ligation and cholic acid feeding significantly stimulated CK 8 and CK 18 mRNA and protein levels compared to controls, whereas UDCA had no effect. CK overexpression was accompanied by pronounced phosphorylation. Our results show that potentially toxic bile acids induce hepatocytic CK 8 and CK 18 expression and phosphorylation whereas nontoxic UDCA has no effect on CKs. Thus, increased hepatocellular CK expression and phosphorylation in cholestasis may be caused by retention of toxic bile acids and reflect a hepatocellular stress response with potential beneficial effects. Cholestasis is associated with retention of potentially toxic bile acids and profound cytoskeletal alterations of hepatocytes. Given the well-established cytoprotective role of hepatocyte keratins this study aimed to determine the effects of cholestasis on the cytokeratin (CK) intermediate filament network in mouse liver. Mice were subjected to common bile duct ligation or sham operation. Mice were also fed a cholic acid or ursodeoxycholic acid (UDCA)-supplemented diet (0.1%, 0.5%, and 1%) or control diet for 7 days. CK 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction, in situ hybridization, Western blot analysis, and immunofluorescence microscopy. Common bile duct ligation and cholic acid feeding significantly stimulated CK 8 and CK 18 mRNA and protein levels compared to controls, whereas UDCA had no effect. CK overexpression was accompanied by pronounced phosphorylation. Our results show that potentially toxic bile acids induce hepatocytic CK 8 and CK 18 expression and phosphorylation whereas nontoxic UDCA has no effect on CKs. Thus, increased hepatocellular CK expression and phosphorylation in cholestasis may be caused by retention of toxic bile acids and reflect a hepatocellular stress response with potential beneficial effects. Cholestatic liver disorders were found to be associated with profound alterations of all three components of the hepatocyte cytoskeleton, namely microtubules, microfilaments, and cytokeratin (CK) intermediate filaments (IFs).1Trauner M Meier PJ Boyer JL Molecular pathogenesis of cholestasis.N Engl J Med. 1998; 17: 1217-1227Google Scholar, 2Phillips MJ Poucell S Oda M Mechanisms of cholestasis.Lab Invest. 1986; 54: 593-608PubMed Google Scholar, 3Trauner M Fickert P Zollner G Stauber RE Zatloukal K Denk H Krejs GJ Cellular and molecular mechanisms of cholestasis.in: Blum HE Maier KP Sauerbruch T Stadler GA Liver Cirrhosis and Its Development. Kluwer Academic Publishers, Dodrecht2000: 3-20Google Scholar Hydrophobic bile acids accumulate during cholestasis and affect microtubular motors, such as kinesin and dynein, resulting in impaired vesicle movement to the canalicular membrane and reduced bile flow.1Trauner M Meier PJ Boyer JL Molecular pathogenesis of cholestasis.N Engl J Med. 1998; 17: 1217-1227Google Scholar, 2Phillips MJ Poucell S Oda M Mechanisms of cholestasis.Lab Invest. 1986; 54: 593-608PubMed Google Scholar, 3Trauner M Fickert P Zollner G Stauber RE Zatloukal K Denk H Krejs GJ Cellular and molecular mechanisms of cholestasis.in: Blum HE Maier KP Sauerbruch T Stadler GA Liver Cirrhosis and Its Development. Kluwer Academic Publishers, Dodrecht2000: 3-20Google Scholar, 4Reichen J Krähenbühl Zimmermann H Impact of cholestasis on hepatic function: retention of cholephiles and their potential targets.in: Gentilini P Cholestasis. Elsevier Science, 1994: 167-175Google Scholar Disruption of microfilaments, which normally form a contractile web around the bile canaliculus and regulate tight junction permeability, results in dilated canaliculi, leaky tight junctions, and loss of microvilli. All these changes may not only be consequences of but also contribute to cholestasis.1Trauner M Meier PJ Boyer JL Molecular pathogenesis of cholestasis.N Engl J Med. 1998; 17: 1217-1227Google Scholar, 2Phillips MJ Poucell S Oda M Mechanisms of cholestasis.Lab Invest. 1986; 54: 593-608PubMed Google Scholar, 3Trauner M Fickert P Zollner G Stauber RE Zatloukal K Denk H Krejs GJ Cellular and molecular mechanisms of cholestasis.in: Blum HE Maier KP Sauerbruch T Stadler GA Liver Cirrhosis and Its Development. Kluwer Academic Publishers, Dodrecht2000: 3-20Google Scholar, 4Reichen J Krähenbühl Zimmermann H Impact of cholestasis on hepatic function: retention of cholephiles and their potential targets.in: Gentilini P Cholestasis. Elsevier Science, 1994: 167-175Google Scholar Hepatocytic IFs consisting of CK 8 and CK 18 mechanically support the bile canaliculus (pericanalicular sheath) but their role in bile secretion remains unclear.5Katsuma Y Marceau N Ohla M French SW Cytokeratin intermediate filaments of rat hepatocytes: different cytoskeletal domains and their three-dimensional structure.Hepatology. 1988; 8: 559-568Crossref PubMed Scopus (65) Google Scholar, 6Omary MB Ku N Intermediate filament protein of the liver. Emerging disease association and functions.Hepatology. 1997; 25: 1043-1048Crossref PubMed Scopus (90) Google Scholar Increased density of IFs in experimental obstructive cholestasis in rats results in the thickening of the pericanalicular sheath and has mainly been attributed to increased canalicular pressure as a result of biliary obstruction.7Kawahara H Cadrin M Perry G Autilio-Gambetti L Swierenga SHH Metuzals J Marceau N French SW Role of cytokeratin intermediate filaments in transhepatic transport and canalicular secretion.Hepatology. 1990; 11: 434-448Crossref Scopus (35) Google Scholar, 8Ohta M Marceau N French SW Pathologic changes in the cytokeratin pericanalicular sheath in experimental cholestasis and alcoholic fatty liver.Lab Invest. 1988; 59: 60-74PubMed Google Scholar, 9Song JY Van Noorden CJF Frederiks WM Alterations of hepatocellular intermediate filaments during extrahepatic cholestasis in rat liver.Virchows Arch. 1997; 430: 253-260Crossref PubMed Scopus (5) Google Scholar However, primary biliary cirrhosis and disorders associated with nonmechanical intrahepatic cholestasis, such as alcoholic hepatitis, nonalcoholic steatohepatitis, and Wilson's disease, are also associated with profound cytoskeletal alterations including the formation of Mallory bodies (MBs), which are cytoplasmic inclusions in hepatocytes consisting of CKs and non-CK components.10Gerber MA Orr W Denk H Schaffner F Popper H Hepatocellular hyalin in cholestasis and cirrhosis: its diagnostic significance.Gastroenterology. 1973; 64: 89-98PubMed Scopus (73) Google Scholar, 11Denk H Stumptner C Zatloukal K Mallory body revisited.J Hepatol. 2000; 32: 689-702Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar The CK-IF network has long been considered as being a rather static structure responsible for mechanical stability of cells. More recently, however, the importance of CKs for maintenance of functional integrity of hepatocytes has been demonstrated in several gene knockout mouse models.11Denk H Stumptner C Zatloukal K Mallory body revisited.J Hepatol. 2000; 32: 689-702Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 12Nam-On K Michie SA Soetikno RM Resurreccion EZ Broome RL Oshima RG Omary MB Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant.J Clin Invest. 1996; 98: 1034-1046Crossref PubMed Scopus (106) Google Scholar, 13Ku N Michie S Oshima RG Omary MB Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant.J Cell Biol. 1995; 131: 1303-1314Crossref PubMed Scopus (144) Google Scholar, 14Ku N Michie SA Soetikno RM Resurreccion EZ Broome RL Omary MB Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice.J Cell Biol. 1998; 143: 2023-2032Crossref PubMed Scopus (86) Google Scholar, 15Liao J Ku N Omary MB Stress, apoptosis, and mitosis induce phosphorylation of human keratin 8 at ser73 in tissues and cultured cells.J Biol Chem. 1995; 272: 17565-17573Crossref Scopus (109) Google Scholar, 16Zatloukal K Stumptner C Lehner M Denk H Baribault H Eshkind LG Franke WW Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies.Am J Pathol. 2000; 156: 1263-1274Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Mutations of CKs in mice are associated with higher susceptibility to hepatotoxins, indicating that CKs may also have important nonmechanical functions, such as defense against toxic injury.12Nam-On K Michie SA Soetikno RM Resurreccion EZ Broome RL Oshima RG Omary MB Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant.J Clin Invest. 1996; 98: 1034-1046Crossref PubMed Scopus (106) Google Scholar, 16Zatloukal K Stumptner C Lehner M Denk H Baribault H Eshkind LG Franke WW Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies.Am J Pathol. 2000; 156: 1263-1274Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar The importance of CK mutations for the pathogenesis of human liver diseases has been emphasized by the recent finding of mutations in the CK 8 gene in patients with cryptogenic cirrhosis.17Ku N Gish R Wright TL Omary MB Keratin 8 mutations in patients with cryptogenic liver disease.N Engl J Med. 2001; 344: 1580-1587Crossref PubMed Scopus (144) Google Scholar In addition, hyperphosphorylation of CKs seems to be involved in cytoprotection against toxic stress.6Omary MB Ku N Intermediate filament protein of the liver. Emerging disease association and functions.Hepatology. 1997; 25: 1043-1048Crossref PubMed Scopus (90) Google Scholar, 11Denk H Stumptner C Zatloukal K Mallory body revisited.J Hepatol. 2000; 32: 689-702Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Liao J Ku N Omary MB Stress, apoptosis, and mitosis induce phosphorylation of human keratin 8 at ser73 in tissues and cultured cells.J Biol Chem. 1995; 272: 17565-17573Crossref Scopus (109) Google Scholar, 18Stumptner C Omary MB Fickert P Denk H Zatloukal K Hepatocyte cytokeratins are hyperphosphorylated at multiple sites in human alcoholic hepatitis and in Mallory bodies.Am J Pathol. 2000; 156: 77-90Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Marked overexpression and hyperphosphorylation of CK 8 and CK 18 were also demonstrated in human alcoholic hepatitis, which is also frequently accompanied by cholestasis.18Stumptner C Omary MB Fickert P Denk H Zatloukal K Hepatocyte cytokeratins are hyperphosphorylated at multiple sites in human alcoholic hepatitis and in Mallory bodies.Am J Pathol. 2000; 156: 77-90Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 19Kenner L Trauner M Fickert P Stauber RE Eferl R Denk H Zatloukal K Overexpression of keratin-8 and -18 mRNA associated with Mallory body formation in patients with alcoholic hepatitis.Hepatology. 1998; 28: A564Abstract Full Text PDF Scopus (103) Google Scholar Therefore, the aim of this study was to investigate whether bile acids play a causal role in overexpression and phosphorylation of CK 8 and CK 18. For this purpose we studied expression and phosphorylation of CKs in different mouse models associated with elevated bile acid levels. Male Swiss Albino mice (strain Him OF1 SPF) were obtained from the Institute of Laboratory Animal Research, University of Vienna School of Medicine, Himberg, Austria, housed with a 12-hour light-dark cycle and permitted ad libitum consumption of water and a standard mouse diet (Marek, Vienna, Austria). Experiments were performed with 2-month-old mice weighing 25 to 30 g. The experiments described in this article were approved by the local ethics committee and followed the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" (National Academy of Sciences) as published by the National Institutes of Health (NIH publication 86-23, revised 1985). Cholic acid (CA) was obtained from Aldrich (Steinheim, Germany), ursodeoxycholic acid (UDCA) was kindly provided by the Falk Foundation (Freiburg, Germany). All surgical procedures were performed under sterile conditions. To study the effects of obstructive cholestasis on hepatic CK expression, the common bile duct was ligated under general anesthesia (10 mg Avertin intraperitoneally) close to the liver hilum immediately below the bifurcation and dissected between the ligatures as described previously.20Trauner M Arrese M Soroka CJ Anathanarayanan M Koeppel TA Schlosser SF Suchy FJ Keppler D Boyer JL The rat canalicular conjugate export pump (Mrp2) is downregulated in intrahepatic and obstructive cholestasis.Gastroenterology. 1997; 113: 255-264Abstract Full Text PDF PubMed Scopus (467) Google Scholar Cholecystectomy was performed after ligation of the cystic duct. Controls underwent a sham operation with exposure, but without ligation of the common bile duct and removal of the gallbladder. The livers were excised under general anesthesia 3 and 7 days after surgery, respectively. Aliquots of liver tissue were frozen in liquid nitrogen for molecular analysis and immunohistochemistry or fixed in 4% neutral buffered formaldehyde solution and paraffin-embedded for light microscopy and in situ hybridization. Serum samples from each mouse were stored at −70°C for analysis of AST/ALT, alkaline phosphatase, and total bile acid levels. To study the differential effects of bile acids on CK expression and phosphorylation, mice were fed a diet supplemented with toxic CA or hydrophilic nontoxic UDCA in different concentrations (0.1%, 0.5%, or 1%) for 7 days.21Van Nieuwkerk CM Oude Elferink RP Groen AK Ottenhoff R Tytgat GN Dingemans KP Van Den Bergh Weerman MA Offerhaus GJ Effects of ursodeoxycholate and cholate feeding on liver diseases in FVB mice with disrupted mdr2 P-glycoprotein gene.Gastroenterology. 1996; 111: 165-171Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar mRNA copy numbers for CK 8, CK 18, and glyceraldehyde-3-phosphate dehydrogenase were determined by competitive reverse transcriptase-polymerase chain reaction as previously described by Zatloukal and colleagues.16Zatloukal K Stumptner C Lehner M Denk H Baribault H Eshkind LG Franke WW Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies.Am J Pathol. 2000; 156: 1263-1274Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Labeled sense and antisense transcripts were synthesized in a 20-μl reaction mixture containing 1 μg of linearized plasmid, 10× transcription buffer (0.4 mol/L Tris-HCl, pH 8.0, 60 mmol/L MgCl2, 20 mmol/L spermidine, 100 mmol/L dithiothreitol), 20 mmol/L dithiothreitol, rNTP-labeling mixture (1 mmol/L each of ATP, CTP, GTP; Boehringer Mannheim, Mannheim, Germany), 40 U Inhibit-ACE (5′→3′ Inc, Boulder, CO), 120 μCi α-35S UTP (Amersham, Buckinghamshire, UK), and 20 U of either SP6 or T7 RNA polymerase (Boehringer Mannheim). After an incubation period of 2 hours at 37°C, DNA was digested with 2 U DNase (RNase-free, Boehringer Mannheim) for 10 minutes at 37°C and the reaction was stopped with 2 μl of 0.5 mol/L ethylenediaminetetraacetic acid (EDTA), pH 8.0. Unincorporated nucleotides were removed using a MicroSpin S-200 HR column (Pharmacia, Uppsala, Sweden). Fifty μl of hydrolysis buffer (80 mmol/L NaHCO3, 120 mmol/L Na2CO3, 120 mmol/L dithiothreitol) were added to 50 μl of the eluted sample and hydrolysis was performed at 60°C for 45 minutes to obtain an average sample size of 150 bp. After addition of 5 μl of stopping solution (0.2 mol/L Na acetate, 10 mmol/L dithiothreitol, 1% glacial acetic acid) the sample was precipitated with LiCl/isopropanol. The washed pellet was resuspended in 100 μl of 50% formamide containing 25 mmol/L dithiothreitol. Four-μm paraffin sections mounted on silanized glass slides were deparaffinized and postfixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at room temperature. After rinsing with Tris-buffered saline (TBS) (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl) sections were treated with 0.2 mol/L of HCl for 10 minutes. After washing in TBS sections were incubated in 20 μg/ml of proteinase K (Sigma Chemical Company, St. Louis, MO) in TBS containing 2 mmol/L of CaCl2 for 15 minutes at 37°C. The reaction was stopped with TBS for 5 minutes at 4°C. After treatment with 0.5% acetic anhydride in 100 mmol/L Tris, pH 8.0, for 10 minutes, sections were rinsed with TBS, dehydrated in graded ethanol, and air-dried. For hybridization the labeled sample (1 × 106cpm per section) was diluted in 50 μl of hybridization buffer containing 12.5 mmol/L phosphate buffer, pH 6.8, 12.5 mmol/L Tris, 0.4 mol/L NaCl, 3 mmol/L EDTA, 1.25× Denhardt, 50% formamide, 12.5% dextran sulfate, 0.1 mol/L dithiothreitol, 100 nmol/L S-rATP (Boehringer Mannheim), 60 ng t-RNA, and 30 ng poly(A). The sections were hybridized with the diluted probe overnight at 50°C in a humid chamber containing 2× standard saline citrate (0.3 mol/L NaCl, 30 mmol/L Na citrate, pH 7.0) and 50% formamide. Thereafter, sections were washed with formamide washing buffer (10 mmol/L phosphate buffer, pH 6.8, 10 mmol/L Tris-HCl, pH 7.7, 0.3 mol/L NaCl, 5 mmol/L EDTA, 0.1× Denhardt, 0.07% β-mercaptoethanol, and 50% formamide) at 45°C once for 1 hour and once for 2 hours, followed by washing in 10 mmol/L Tris-HCl, pH 7.4, 0.5 mol/L NaCl, 2.5 mmol/L EDTA, and 0.07% β-mercaptoethanol two times for 15 minutes. After RNase A treatment (20 μg/ml, Boehringer Mannheim) in the same buffer for 30 minutes at 37°C, washing was continued overnight in the formamide washing buffer at 37°C. On the next day there was a high-stringent wash in 2× standard saline citrate and 0.07% β-mercaptoethanol for 30 minutes at 45°C and in 0.1× standard saline citrate and 0.07% β-mercaptoethanol for 30 minutes at 45°C. After dehydration in graded ethanol, air-dried sections were coated with Ilford K2 photoemulsion (Ilford Ltd., Mobberly, Cheshire, UK). After 1 to 3 weeks of exposure slides were developed with Kodak D19 developer (Eastman Kodak, Rochester, NY) and counterstained with hematoxylin and eosin. Snap-frozen liver tissue was homogenized in buffer containing 10 mmol/L NaH2PO4, pH 7.4, 5% sodium dodecyl sulfate, and 10% β-mercaptoethanol. Protein concentration was determined using the Bradford method.22Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (224852) Google Scholar Samples (20 μg protein per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.23Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (218007) Google Scholar Equal loading amounts of protein were confirmed by Coomassie blue staining. For immunoblotting proteins were electrotransferred to nitrocellulose membranes (BioRad, Hercules, CA).24Towbin H Staehelin T Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci. 1979; 76: 1350-1354Crossref Scopus (47743) Google Scholar After blocking with 3% nonfat milk in PBS, CKs were detected using a monoclonal mouse antibody against CK 8 (Ks 8.7; Progen, Heidelberg, Germany) in a dilution of 1:500 and a monoclonal mouse antibody to CK 18 (K18.04, Progen) in a dilution of 1:1000. In addition, β-actin was detected using a monoclonal mouse anti-β-actin antibody (Sigma) in a dilution of 1:5000. After washing in PBS, blots were incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO, Glostrup, Denmark) in a dilution of 1:1000. Specific binding was detected using the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham) and exposing the blots to Trimax XDA Plus films (3M Imation, White City, OR). Band intensities were determined with a Docu Gel V video densitometer (MWG-Biotech, Munich, Germany) and RFLP-Scan Software (Scanalytics, Billerica, MA). Accuracy of the ECL method for quantification of CK protein levels was determined by standard curves (not shown). To study alterations of the IF network, immunofluorescence microscopy was performed using the polyclonal rabbit CK antibody 50K160 recognizing CK 8 and CK 1825Hutter H Zatloukal K Winter G Stumptner C Denk H Disturbance of keratin homeostasis in griseofulvin-intoxicated mouse liver.Lab Invest. 1993; 69: 576-582PubMed Google Scholar, 26Zatloukal K Denk H Spurej G Lackinger E Preisegger KH Franke WW High molecular weight component of Mallory bodies detected by monoclonal antibody.Lab Invest. 1990; 62: 427-434PubMed Google Scholar as well as the monoclonal mouse anti-K7 (Monosan, Uden, Netherlands) and anti-K19 (Amersham) recognizing CK 7 and CK 19, respectively. In addition, the monoclonal antibody LJ4 (kindly provided by Bishr Omary, Palo Alto, CA) directed against CK 8 phosphorylated at Ser 79 was used.16Zatloukal K Stumptner C Lehner M Denk H Baribault H Eshkind LG Franke WW Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies.Am J Pathol. 2000; 156: 1263-1274Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Immunofluorescent specimens were analyzed with a MRC 600 (BioRad) laser-scanning confocal device attached to a Zeiss Axiophot microscope. The fluorescent images were collected using the confocal photomultiplier tube as full frame (768 × 512 pixels). For dual labeling, separate excitation wavelengths (488 nm for fluorescein isothiocyanate; 568 nm for tetramethylrhodamine B isothiocyanate) from a krypton/argon ion laser were used. At the time of harvesting, mouse livers were fixed in 4% neutral buffered formaldehyde solution and embedded in paraffin. Hematoxylin and eosin-stained sections were coded and examined by two pathologists (HD, KZ), who were blinded in regard to the treatment groups. Serum biochemistry (ALT and AST) was performed by routine clinical chemistry testing on a Hitachi 717 analyzer (Boehringer Mannheim). Alkaline phosphatase and total serum bile acid levels were determined for assessment of the degree of cholestasis. For determination of total serum bile acid levels a commercially available 3α-hydroxysteroid dehydrogenase assay (Merck, Darmstadt, Germany) was used. Tests were performed in duplicate (Table 1, Table 2).Table 1Serum Liver Enzymes after CBDLGroupASTALTAPBAControl202 ± 6038 ± 740 ± 205 ± 1CBDL, 3 day672 ± 290*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).414 ± 200*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).1196 ± 270*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).1131 ± 413*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).CBDL, 7 day581 ± 42*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).399 ± 57*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).1360 ± 608*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).288 ± 61*P < 0.05 mice challenged with CBDL versus sham-operated mice (control).Values are means ± SD. AST, aspartate aminotransferase (U/L); ALT, alanine aminotransferase (U/L); AP, alkaline phosphatase (U/L); BA, bile acids (μMol/L).* P < 0.05 mice challenged with CBDL versus sham-operated mice (control). Open table in a new tab Table 2Serum Liver Enzymes in Bile Acid-Fed MiceGroupASTALTAPBAControl203 ± 5332 ± 743 ± 154 ± 0.8Cholate 0.1%226 ± 80254 ± 76*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39233 ± 31*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.3911 ± 6*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39Cholate 0.5%268 ± 83287 ± 87*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39387 ± 2*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.3937 ± 19*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39Cholate 1%360 ± 168*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39313 ± 9*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39402 ± 61*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.3958 ± 36*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39UDCA 1%148 ± 1535 ± 5101 ± 5391 ± 35*P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39Values are means ± SD. AST, aspartate aminotransferase (U/L); ALT, alanine aminotransferase (U/L); AP, alkaline phosphatase (U/L); BA, bile acids (μMol/L).* P < 0.05 bile acid fed mice versus standard diet-fed mice (control); animals have also been used for a separate study and their biochemical data have been reported previously.39Fickert P Zollner G Fuchsbichler A Pojer C Zenz R Stumptner C Pojer C Zenz R Lammert F Stieger B Meier PJ Zatloukal K Denk H Trauner M Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transport expression in mouse liver.Gastroenterology. 2001; 120: 170-183Abstract Full Text PDF PubMed Scopus (229) Google Scholar Open table in a new tab Values are means ± SD. AST, aspartate aminotransferase (U/L); ALT, alanine aminotransferase (U/L); AP, alkaline phosphatase (U/L); BA, bile acids (μMol/L). Values are means ± SD. AST, aspartate aminotransferase (U/L); ALT, alanine aminotransferase (U/L); AP, alkaline phosphatase (U/L); BA, bile acids (μMol/L). In each group five animals were studied at each time point. Data are reported as arithmetic means ± SEM. Statistical analysis was performed using Student's t-test as appropriate, or analysis of variance with posttesting when three or more groups were compared. A P value <0.05 was considered significant. Obstructive cholestasis (CBDL) for 3 days led to biliary type of hepatocytic necroses (resembling bile infarcts), predominantly in acinar zones 1 and 2 that were infiltrated by variable numbers of neutrophils. Interlobular bile ducts were elongated with dilated lumina and irregular epithelium. The surrounding portal tissue was edematous and infiltrated by neutrophils. No pronounced ductular reaction was observed. Necroses were more prominent in 7-day-ligated than in 3-day-ligated mouse livers. In agreement with the histological findings serum transaminases, alkaline phosphatase, and serum bile acid levels were significantly elevated in comparison to controls (for details see Table 1). CBDL resulted in a significant increase of CK 8 and CK 18 mRNA levels after 3 and 7 days (Figure 1A). CK 8 and CK 18 proteins were also increased after 3 and 7 days of CBDL (Figure 1B) compared to sham-operated animals, whereas β-actin expression remained constant (Figure 1B). After CBDL, increased CK 8 mRNA was observed particularly in hepatocytes surrounding bile infarcts (Figure 1C), in hepatocytes in acinar zone 1, and also to a minor degree in bile duct epithelial cells (Figure 1D) as revealed by in situ hybridization. Increased CK 8 and CK 18 protein expression after CBDL was also detected by immunofluorescence microscopy (Figure 1, E and F). CBDL resulted in an increased density of the cytoplasmic IF network predominantly around bile canaliculi (Figure 1F, arrowheads). Because neoexpression of CK 7 and CK 19 has previously been described in hepatocytes in human cholestatic liver disease,27Van Eyken P Sciot R Desmet VJ A cytokeratin immunohistochemical study of cholestatic liver disease: evidence that hepatocytes can express 'bile duct-type' cytokeratins.Histopathology. 1989; 15: 125-135Crossref PubMed
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