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The impact of intestinal microflora on serum bilirubin levels
470A
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
1191
1192
BILE ACID AND ORGANIC ANION TRANSPORT IN ENDOTOXEMIC RATS IS PRESERVED BY HEAT STRESS. Ulrich Bolder, Stefan Gabler, Ve-
THE IMPACT OF INTESTINAL MICROFLORA ON SERUM BILIRUBIN LEVELS. Libor Vitek, 1st Med Faculty, Charles Univ, Prague, Praha 2 Czech
rena Kidder, University of Regensburg, Regensburg Germany; Lukas Landmann, University of Basel, Basel Switzerland; Wolfgang E Thasler, Karl-Walter Jauch, University of Regensburg, Regensburg Germany
Republic; Petr Kotal, 1st Medical Faculty, Charles Univ, Prague, Praha 2 Czech Republic; Jiri Malina, Barrandov Medical Ctr, Praha 5 Czech Republic; Johan Fevery, Katholie University Leuven, Leuven Belgium
Background: Previous work has shown transcriptional down regulation of hepatocyte transport proteins for bile acids and organic anions in endotoxemia. As heat stress reduces other sequelae of sepsis, studies were performed to determine whether heat stress would correct deficient bile acid and organic anion transport caused by endotoxin. Methods: Body temperature of SD-rats was elevated to 42 °C for 10 minutes. Lipopolysaccharide (LPS) was injected i.p. at different times after heat stress and maximal transport for cholyltaurine (CT) and sulfobromophthalein (BSP) was measured 12 h after LPS in perfused rat livers (IPRL). Protein levels of the bile acid (ntcp, bsep) and organic anion transporters (oatp, mrp2) were analysed in plasma membrane fractions. mRNA levels of these transporters were also determined. Immunofluorescence studies investigated their distribution in the hepatocyte plasma membrane. Co-immunoprecipitation experiments were performed to study the mode of the chaperoning effects of heat shock proteins. Results: In the IPRL transport of CT and BSP was reduced by endotoxin to 38% and 48% (P ~ 0.01 vs. controls). When heat stress was applied two hours prior to LPS, transport of CT recovered to 93% (NS vs. controls). Transport of BSP was 74% of controls after heat stress followed by LPS (P ~ 0.05 vs. LPS and controls). Longer intervals between heat stress and LPS did not protect transport function. Expression of bile acid and organic anion transporters in pfasma membrane fractions was maintained when heat stress preceded LPS application. In contrast mRNA levels of bile acid transporters were not preserved by heat stress. Transporters showed a decreased fluorescence signal after LPS. FoUowing heat stress and LPS 2 h thereafter, density of transporters was comparable to controls. The protective effects coincided with the expression of heat shock protein 70 and 25 (HSPs). In co-immunopreciptiou studies ntcp and bsep showed direct binding to the induced HSPs. Conclusions: Heat stress preserves bile acid transporters during sepsis by a posttranscriptional mechanism. Direct interaction of HSPs and membrane transporters seems to be involved in the chaperoning effect.
Background: The intestinal microflora plays an important role in the pathogenesis of neonatal jaundice by catalysing the reduction of bilirubin and as such inhibiting the enterohepatic circulation. The present study aimed to investigate the influence of the intestinal microflora on serum bilirubin levels in hyperbilirubinemic Gunn rats. Methods: Five male Gunn rats weighing 250-270 g were studied. After an initial base-line phase I the rats received antibiotics (clindamycine 4 mg/day plus neomycine 30 mg/day) via a gastric tube for the subsequent 4 days (phase II). Finally, intestinal colonization was carried out with a single, bilirubin-reducing strain of Clostridium perfringens isolated from neonatal feces (phase III). Serum bilirubin levels and fecal excretion of bilirubin and urobilinoids were determined at the end of each phase. Results: Antibiotic therapy resulted in vanishing of fecal urobilinoids indicative of the suppression of bilirubin-reduction carried out by the intestinal microflora. Sirnultaneously, serum bilirubin increased dramatically (184.0-+31.8 vs. 298.1-+21.9 umol/1, p~0.005). Intestinal colonization with Clostridium perfringens led to reappearance of urobifinoids in feces (to 26% of the initial excretion) and to a decrease of serum bilirubin (298.1-+21.9 vs. 238.7-+ 17.9 umoVl, p(0.05). Conclnsions: The intestinal microflora greatly affects the serum metabolism of bilirubin. Prolonged use of antibiotics in man may lead to an increase of serum bilirubin levels, while enhancement of its intestinal catabolism may have an opposite effect.
1193
1194
DOWN-REGULATION OF MRP2 GENE TRANSCRIPTION IN CBDLTREATED RATS IS MEDIATED BY TRANSCRIPTION FACTOR EFIA /
CHANGES IN THE DISTRIBUTION OF HEPATOCELLULAR TRANSPORTERS IN HUMAN END-STAGE CHOLESTATIC LIVER DISEASE. P
YB-1. Andreas Geier, Peter R Mertens, University of Technology Aachen, Aachen Germany; Thomas Gerloff, Humboldt University Berlin, Charite, Berlin Germany; Christoph G Dietrich, Univerity of Technology Aachen, Aachen Germany; Saul J Karpen, Baylor Coil of Medicine, Houston; Guntram Suske, University of Marburg, Marburg Germany; Siegfried Matern, Carsten Gartung, University of Technology Aachen, Aachen Germany
Milkiewicz, E Elias, A Williams, Liver Unit, Queen Elizabeth Hospital, Birmingham United Kingdom; M Milkiewiez, Dept of Physiology, University of Birmingham, Birmingham United Kingdom; A Keogh, Liver Unit, Queen Elizabeth Hospital, Birmingham United Kingdom; S G Hubscher, Dept of Pathology, University of Birmingham, Birmingham United Kingdom
Background: The rat canalicular export pump Mrp2 is down-regulated at the protein and mRNA-level in animal models of cholestasis, e.g. upon treatment with endotoxin and after common bile duct ligation. Retinoid-response elements have been identified as being critical for IL-113dependent suppression of Mrp2 gene expression. Aim:Given the presence of putative binding sites for transcription factor YB-1 (rat EFIA), a major regulator of MDR1 expression, its influence on Mrp2 gene expression was evaluated in an experimental cholestasis model. Methods: Male Sprague-Dawley rats (n~4) were treated with common bile duct ligation (CBDL) or sham operation and hepatic nuclei were isolated after 7 days. DNA binding assays were performed with oligonucleotides harboring the Y-box-element located at -300/-296 bp in the Mrp2 promoter. The rat Mrp2 promoter sequence up to -lkb was cloned into the pGL3basic reporter plasmid and deletion constructs of 733bp, 341bp, 273bp, 220bp and 120bp were generated. Co-transfection of HepG2 cells was performed using calciumphosphate-precipitates. Results:Co-transfection of promoter constructs >220 bps with a YB-1 expression pfasmid resulted in a 50% decrease of reporter activity. Co-introduced SP1 increased promoter activity by 20-50% whereas combined co-transfeetion of YB-1 with SP1 caused a further suppression up to 75% of Mrp2 promoter activity. A potential YB-1 binding motif was identified at -278/-317 bps and confirmed by DNA binding studies using recombinant YB-1, showing preference for single stranded templates compared to double-stranded probes. Furthermore, inclusion of polyclonal anti-YB-1 antibody in the binding reaction resulted in supersfiifts. In CBDD treated rats the nuclear YB-1 binding activity was markedly increased compared to sham controls. Conclusions: YB-1 acts as potent suppressor of Mrp2 gene expression in vitro with concordant changes of binding to a bona fide regulatory element in CBDL-treated rats. SP1 increases YB-1 mediated suppression of the Mrp2 gene, hinting at complex protein interactions underlying Mrp2 gene regulation.
Background. Little is known about the expression of hepatocyte transport proteins in human liver diseases. In particular, data are lacking on the distribution of bile acids transporters in end-stage cholestatic liver disorders. Such data are important to provide further insight into the pathogenesis of cholestasis. Aim. To study the distribution and expression by immunohlstochemlstry and Western blotting of the following transport proteins: NTCP, MRP2, MRP3, MDR3 in normal livers and in end-stage cholestatic liver disease (PBC). Methods. Cholestatic liver tissues were obtained from 5 patients with end-stage PBCwho underwent liver transplantation in our centre. Normal liver tissues were obtained from 4 donor livers which underwent trimming before pediatric transplantation. Results. In normal livers, all four transporters showed uniform immunoreactivity in hepatocytes. MDR3 and MRP2 were expressed on biliary canaliculi, whereas NTCP and MRP3 had a membranous distribution. Bile ducts and ductules also stained positivelyfor MRP3, but not the other proteins. In PBC livers there was a redistribution of NTCP, MRP3 and MDR3. NTCP was downregulated in the peripheral areas of cirrhotic nodules and upregulated in the central areas. MRP3 showed the opposite pattern and was upregulated peripherally and downregulated in the central areas of cirrhotic nodules. There was also immunoreactivity for MRP3 in bile ducmles at the periphery of cirrhotic septa. Canalicular immunoreactivity for MDR3, like MRP3, was stronger in the peripheral areas. In addition there was strong expression of MDR3 on the basolateral membranes of periseptal hepatocytes, a pattern not seen in normal livers. MRP2 showed a similar pattern of distribution in normal and PBC livers. In contrast to MDR3, there was no basolateral expression of MRP2 in PBC livers. Western blotting analysis did not show any significant changes in the overall expression of NTCP, MRP2 and MRP3. MDR3 was upregulated in PBC livers by 66% (P<0.005). Conclusions. Adaptation to end-stage cholestasis by the human hepatocyte appears to be a response to the local micro-environment and differs markedly according to localisation within the PBC cirrhotic nodule. In periseptal hepatocytes there is: (i)downregulation of bile acid uptake by NTCP, which may protect hepatocytes from the further uptake of toxic bile acids; (ii) up-regulation of the multi-organic anion transporter MRP3 which facilitates the extrusion of potentially hepatotoxic biliary organic anions and (iii) adaptation of the basolateral membrane to express the biliary canalicular transport protein MDR3. The latter may enable the basolateral membrane to carry out functions normally occurring at the biliary canalicular membrane, and may also be an important component of the ductular metaplasia that occurs in chronic cholestatic liver diseases such as PBC.
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
1191
1192
BILE ACID AND ORGANIC ANION TRANSPORT IN ENDOTOXEMIC RATS IS PRESERVED BY HEAT STRESS. Ulrich Bolder, Stefan Gabler, Ve-
THE IMPACT OF INTESTINAL MICROFLORA ON SERUM BILIRUBIN LEVELS. Libor Vitek, 1st Med Faculty, Charles Univ, Prague, Praha 2 Czech
rena Kidder, University of Regensburg, Regensburg Germany; Lukas Landmann, University of Basel, Basel Switzerland; Wolfgang E Thasler, Karl-Walter Jauch, University of Regensburg, Regensburg Germany
Republic; Petr Kotal, 1st Medical Faculty, Charles Univ, Prague, Praha 2 Czech Republic; Jiri Malina, Barrandov Medical Ctr, Praha 5 Czech Republic; Johan Fevery, Katholie University Leuven, Leuven Belgium
Background: Previous work has shown transcriptional down regulation of hepatocyte transport proteins for bile acids and organic anions in endotoxemia. As heat stress reduces other sequelae of sepsis, studies were performed to determine whether heat stress would correct deficient bile acid and organic anion transport caused by endotoxin. Methods: Body temperature of SD-rats was elevated to 42 °C for 10 minutes. Lipopolysaccharide (LPS) was injected i.p. at different times after heat stress and maximal transport for cholyltaurine (CT) and sulfobromophthalein (BSP) was measured 12 h after LPS in perfused rat livers (IPRL). Protein levels of the bile acid (ntcp, bsep) and organic anion transporters (oatp, mrp2) were analysed in plasma membrane fractions. mRNA levels of these transporters were also determined. Immunofluorescence studies investigated their distribution in the hepatocyte plasma membrane. Co-immunoprecipitation experiments were performed to study the mode of the chaperoning effects of heat shock proteins. Results: In the IPRL transport of CT and BSP was reduced by endotoxin to 38% and 48% (P ~ 0.01 vs. controls). When heat stress was applied two hours prior to LPS, transport of CT recovered to 93% (NS vs. controls). Transport of BSP was 74% of controls after heat stress followed by LPS (P ~ 0.05 vs. LPS and controls). Longer intervals between heat stress and LPS did not protect transport function. Expression of bile acid and organic anion transporters in pfasma membrane fractions was maintained when heat stress preceded LPS application. In contrast mRNA levels of bile acid transporters were not preserved by heat stress. Transporters showed a decreased fluorescence signal after LPS. FoUowing heat stress and LPS 2 h thereafter, density of transporters was comparable to controls. The protective effects coincided with the expression of heat shock protein 70 and 25 (HSPs). In co-immunopreciptiou studies ntcp and bsep showed direct binding to the induced HSPs. Conclusions: Heat stress preserves bile acid transporters during sepsis by a posttranscriptional mechanism. Direct interaction of HSPs and membrane transporters seems to be involved in the chaperoning effect.
Background: The intestinal microflora plays an important role in the pathogenesis of neonatal jaundice by catalysing the reduction of bilirubin and as such inhibiting the enterohepatic circulation. The present study aimed to investigate the influence of the intestinal microflora on serum bilirubin levels in hyperbilirubinemic Gunn rats. Methods: Five male Gunn rats weighing 250-270 g were studied. After an initial base-line phase I the rats received antibiotics (clindamycine 4 mg/day plus neomycine 30 mg/day) via a gastric tube for the subsequent 4 days (phase II). Finally, intestinal colonization was carried out with a single, bilirubin-reducing strain of Clostridium perfringens isolated from neonatal feces (phase III). Serum bilirubin levels and fecal excretion of bilirubin and urobilinoids were determined at the end of each phase. Results: Antibiotic therapy resulted in vanishing of fecal urobilinoids indicative of the suppression of bilirubin-reduction carried out by the intestinal microflora. Sirnultaneously, serum bilirubin increased dramatically (184.0-+31.8 vs. 298.1-+21.9 umol/1, p~0.005). Intestinal colonization with Clostridium perfringens led to reappearance of urobifinoids in feces (to 26% of the initial excretion) and to a decrease of serum bilirubin (298.1-+21.9 vs. 238.7-+ 17.9 umoVl, p(0.05). Conclnsions: The intestinal microflora greatly affects the serum metabolism of bilirubin. Prolonged use of antibiotics in man may lead to an increase of serum bilirubin levels, while enhancement of its intestinal catabolism may have an opposite effect.
1193
1194
DOWN-REGULATION OF MRP2 GENE TRANSCRIPTION IN CBDLTREATED RATS IS MEDIATED BY TRANSCRIPTION FACTOR EFIA /
CHANGES IN THE DISTRIBUTION OF HEPATOCELLULAR TRANSPORTERS IN HUMAN END-STAGE CHOLESTATIC LIVER DISEASE. P
YB-1. Andreas Geier, Peter R Mertens, University of Technology Aachen, Aachen Germany; Thomas Gerloff, Humboldt University Berlin, Charite, Berlin Germany; Christoph G Dietrich, Univerity of Technology Aachen, Aachen Germany; Saul J Karpen, Baylor Coil of Medicine, Houston; Guntram Suske, University of Marburg, Marburg Germany; Siegfried Matern, Carsten Gartung, University of Technology Aachen, Aachen Germany
Milkiewicz, E Elias, A Williams, Liver Unit, Queen Elizabeth Hospital, Birmingham United Kingdom; M Milkiewiez, Dept of Physiology, University of Birmingham, Birmingham United Kingdom; A Keogh, Liver Unit, Queen Elizabeth Hospital, Birmingham United Kingdom; S G Hubscher, Dept of Pathology, University of Birmingham, Birmingham United Kingdom
Background: The rat canalicular export pump Mrp2 is down-regulated at the protein and mRNA-level in animal models of cholestasis, e.g. upon treatment with endotoxin and after common bile duct ligation. Retinoid-response elements have been identified as being critical for IL-113dependent suppression of Mrp2 gene expression. Aim:Given the presence of putative binding sites for transcription factor YB-1 (rat EFIA), a major regulator of MDR1 expression, its influence on Mrp2 gene expression was evaluated in an experimental cholestasis model. Methods: Male Sprague-Dawley rats (n~4) were treated with common bile duct ligation (CBDL) or sham operation and hepatic nuclei were isolated after 7 days. DNA binding assays were performed with oligonucleotides harboring the Y-box-element located at -300/-296 bp in the Mrp2 promoter. The rat Mrp2 promoter sequence up to -lkb was cloned into the pGL3basic reporter plasmid and deletion constructs of 733bp, 341bp, 273bp, 220bp and 120bp were generated. Co-transfection of HepG2 cells was performed using calciumphosphate-precipitates. Results:Co-transfection of promoter constructs >220 bps with a YB-1 expression pfasmid resulted in a 50% decrease of reporter activity. Co-introduced SP1 increased promoter activity by 20-50% whereas combined co-transfeetion of YB-1 with SP1 caused a further suppression up to 75% of Mrp2 promoter activity. A potential YB-1 binding motif was identified at -278/-317 bps and confirmed by DNA binding studies using recombinant YB-1, showing preference for single stranded templates compared to double-stranded probes. Furthermore, inclusion of polyclonal anti-YB-1 antibody in the binding reaction resulted in supersfiifts. In CBDD treated rats the nuclear YB-1 binding activity was markedly increased compared to sham controls. Conclusions: YB-1 acts as potent suppressor of Mrp2 gene expression in vitro with concordant changes of binding to a bona fide regulatory element in CBDL-treated rats. SP1 increases YB-1 mediated suppression of the Mrp2 gene, hinting at complex protein interactions underlying Mrp2 gene regulation.
Background. Little is known about the expression of hepatocyte transport proteins in human liver diseases. In particular, data are lacking on the distribution of bile acids transporters in end-stage cholestatic liver disorders. Such data are important to provide further insight into the pathogenesis of cholestasis. Aim. To study the distribution and expression by immunohlstochemlstry and Western blotting of the following transport proteins: NTCP, MRP2, MRP3, MDR3 in normal livers and in end-stage cholestatic liver disease (PBC). Methods. Cholestatic liver tissues were obtained from 5 patients with end-stage PBCwho underwent liver transplantation in our centre. Normal liver tissues were obtained from 4 donor livers which underwent trimming before pediatric transplantation. Results. In normal livers, all four transporters showed uniform immunoreactivity in hepatocytes. MDR3 and MRP2 were expressed on biliary canaliculi, whereas NTCP and MRP3 had a membranous distribution. Bile ducts and ductules also stained positivelyfor MRP3, but not the other proteins. In PBC livers there was a redistribution of NTCP, MRP3 and MDR3. NTCP was downregulated in the peripheral areas of cirrhotic nodules and upregulated in the central areas. MRP3 showed the opposite pattern and was upregulated peripherally and downregulated in the central areas of cirrhotic nodules. There was also immunoreactivity for MRP3 in bile ducmles at the periphery of cirrhotic septa. Canalicular immunoreactivity for MDR3, like MRP3, was stronger in the peripheral areas. In addition there was strong expression of MDR3 on the basolateral membranes of periseptal hepatocytes, a pattern not seen in normal livers. MRP2 showed a similar pattern of distribution in normal and PBC livers. In contrast to MDR3, there was no basolateral expression of MRP2 in PBC livers. Western blotting analysis did not show any significant changes in the overall expression of NTCP, MRP2 and MRP3. MDR3 was upregulated in PBC livers by 66% (P<0.005). Conclusions. Adaptation to end-stage cholestasis by the human hepatocyte appears to be a response to the local micro-environment and differs markedly according to localisation within the PBC cirrhotic nodule. In periseptal hepatocytes there is: (i)downregulation of bile acid uptake by NTCP, which may protect hepatocytes from the further uptake of toxic bile acids; (ii) up-regulation of the multi-organic anion transporter MRP3 which facilitates the extrusion of potentially hepatotoxic biliary organic anions and (iii) adaptation of the basolateral membrane to express the biliary canalicular transport protein MDR3. The latter may enable the basolateral membrane to carry out functions normally occurring at the biliary canalicular membrane, and may also be an important component of the ductular metaplasia that occurs in chronic cholestatic liver diseases such as PBC.