Marco Arrese

The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis

BACKGROUND & AIMS The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein (Mrp2). Mrp2 function is impaired in various experimental models of intrahepatic and obstructive cholestasis, but the underlying molecular mechanisms are unclear. The aim of this study was to investigate these molecular mechanisms. METHODS The effects of endotoxin, ethinylestradiol, and common bile duct ligation (CBDL) on Mrp2 protein, messenger RNA (mRNA) expression, and Mrp2 tissue localization were determined in rat livers by Northern blotting, Western analysis, and tissue immunofluorescence. To assess whether changes were specific for Mrp2, we also examined the expression of canalicular ecto-adenosine triphosphatase (ecto-ATPase) and mdr P-glycoproteins (P-gp). RESULTS All three cholestatic models resulted in a marked decrease in Mrp2 protein (P < 0.01) and its tissue localization at the canalicular membrane. Mrp2 mRNA levels diminished profoundly after endotoxin (P < 0.0005) and CBDL (P < 0.05), but did not change after ethinylestradiol. In contrast to Mrp2, protein expression of ecto-ATPase and P-gp remained unchanged in endotoxin- and ethinylestradiol-treated animals, whereas P-gp levels increased after CBDL (P < 0.05). CONCLUSIONS Down-regulation of Mrp2 expression may explain impaired biliary excretion of amphiphilic anionic conjugates in these models of cholestasis.

Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors.

Sodium-dependent uptake of bile acids across the hepatic basolateral membrane is rapidly and profoundly diminished during sepsis, thus contributing to the pathogenesis of sepsis-associated cholestasis. This effect is mediated by endotoxin or effector cytokines, which reduce expression of several hepatobiliary transporters, including the sodium-dependent bile acid transporter gene, ntcp. We test here the hypothesis that endotoxin treatment leads to impaired binding activity of ntcp promoter trans-acting factors, resulting in reduction of ntcp mRNA expression. After endotoxin administration, ntcp mRNA levels reached their nadir by 16 h, and nuclear run-on assays demonstrated a marked reduction in ntcp gene transcription. At 16 h after treatment, nuclear binding activities of two key factors that transactivate the ntcp promoter, hepatocyte nuclear factor (HNF) 1 and Footprint B binding protein (FpB BP), decreased to 44 and 47% of pretreatment levels, respectively, while levels of the other known ntcp promoter transactivator, signal transducer and activator of transcription 5, were unaffected. In contrast, the universal inflammatory response factors nuclear factor kappaB and activating protein 1 were both upregulated significantly. Examination of nuclear extracts obtained at sequential time points revealed that the maximal decrease in nuclear activities of both HNF1 and FpB BP preceded the nadir of ntcp mRNA expression by 6-10 h. Furthermore, these two nuclear factors returned towards normal levels before the recovery of ntcp mRNA levels observed by 48 h. Since HNF1alpha mRNA levels were unchanged at all time points, HNF1 is likely to be regulated posttranscriptionally by endotoxin. We conclude that the downregulation of ntcp gene expression by endotoxin is mediated at the level of transcription through tandem reductions in the nuclear binding activity of two critical transcription factors. These findings provide new insight into the coordinated downregulation of hepatobiliary transporters during sepsis.

Saccharomyces cerevisiae fungemia after Saccharomyces boulardii treatment in immunocompromised patients.

Saccharomyces cerevisiae is widely used as a probiotic compound. Clinical data suggest that this agent is safe and effective. We report two cases of fungemia caused by S. cerevisiae occurring in immunosuppressed patients treated orally with S. boulardii Molecular typing confirmed clonality in isolate strains from patients and the capsule. Physicians caring for immunosuppressed patients must be aware of this potential serious complication of probiotic use.

Value of Adenosine Deaminase (ada) in Ascitic Fluid for the Diagnosis of Tuberculous Peritonitis: A Meta-analysis

Background and Goals Adenosine deaminase (ADA) levels are used for diagnosing tuberculosis in several locations and although many studies have evaluated ADA levels in ascitic fluid. These studies have defined arbitrary cut-off points creating difficulties in the clinical application of the results. The goals of this study are: to determine the usefulness of ADA levels in ascitic fluid as a diagnostic test for peritoneal tuberculosis (PTB) and define the best cut-off point. Study A systematic review was done on the basis of 2 independent searches. We selected prospective studies that included consecutive patients. Diagnosis of PTB had to be confirmed by bacteriologic or histologic methods and ADA levels determined by the Giusti method. Inclusion/exclusion criteria were applied by 2 independent reviewers. A receiver operating characteristic curve was constructed to establish the optimal cut-off point and the likelihood ratios (LRs) estimated using fixed-effect pooled method. Results Twelve prospective studies were found. Four of them met the inclusion criteria and were thus included in the meta-analysis. They included 264 patients, of which 50 (18.9%) had PTB. ADA levels showed high sensitivity (100%) and specificity (97%) using cut-off values from 36 to 40 IU/L. The included studies were homogeneous. Optimal cut-off point was determined at 39 IU/L, and LRs were 26.8 and 0.038 for values above and below this cut-off. Conclusions This study supports the proposition that ADA determination is a fast and discriminating test for diagnosing PTB with an optimal cut-off value of 39 IU/L.

Hepatobiliary Transport: Molecular Mechanisms of Development and Cholestasis

The secretion of bile requires the vectorial transport of organic and inorganic solutes from sinusoidal blood to the canalicular lumen. Hydrostatic forces cannot account for biliary secretion, because secretory pressures within bile ducts exceed that of blood within the sinusoidal space. Instead, the process of bile formation requires active transport across the basolateral membrane, transcellular movement through a variety of mechanisms, and then active transport into the canalicular space between hepatocytes. Separate hepatic and ductular transport mechanisms allow for rapid regulation of bile volume and composition required for changing physiologic needs. The array of transport proteins localized to both poles of the hepatocyte have been characterized physiologically and during development. Many have now been cloned and studied further in transgenic models. The recent identification and characterization of several genes that are mutated in inherited forms of cholestatic liver disease have provided new insight into the normal physiology of bile secretion, the pathophysiology of intrahepatic cholestasis, and an unexpected major role for a novel group of P-type ATPases in human biology and disease.

Protective role of biliary cholesterol and phospholipid lamellae against bile acid-induced cell damage

BACKGROUND/AIMS Bile salts (BS) are cytotoxic agents, but cell damage is not observed in the hepatobiliary system. We hypothesized that biliary lipid vesicles (unilamellae and multilamellae) could have a protective role against BS-induced cytotoxicity. METHODS Biliary lipid lamellar secretion was induced by feeding rats with 0.5% diosgenin. Cytoprotection was assessed in bile duct-obstructed rats and by incubating human erythrocytes with sodium taurocholate. RESULTS Biliary cholesterol concentration increased > 300% in diosgenin-fed rats; electron microscopic examination showed a great abundance of lipid lamellar vesicles in bile and within the canaliculi. After bile duct obstruction, serum hepatic enzyme activities were significantly lower in diosgenin-fed rats. Histologically severe and confluent hepatocellular necrosis was only observed in control rats. Biliary lamellar lipid material significantly reduced the BS-induced hemolytic effect in vitro in a concentration-dependent manner. This protective effect correlated to a progressive decrease in the intermicellar BS concentration. Phosphatidylcholine or cholesterol, alone or as lamellar structures, also showed cytoprotective effect in vitro but always less than native biliary lamellae. CONCLUSIONS These results support the concept that native biliary cholesterol phospholipid lamellae represent an important cytoprotective factor for hepatocytes and biliary epithelial cells against BS-induced damage.

Effect of losartan on early liver fibrosis development in a rat model of nonalcoholic steatohepatitis.

Background and Aim:  Nonalcoholic steatohepatitis (NASH) is a metabolic disorder of the liver that may evolve into fibrosis or cirrhosis. Recent studies have shown reduction of experimental liver fibrosis with the use of angiotensin‐converting‐enzyme inhibitors or angiotensin‐receptor antagonists. The aim of this study was to determine whether losartan can influence the early phase of fibrogenesis in an animal model of NASH.

Molecular analysis of the adaptive response of intestinal bile acid transport after ileal resection in the rat

BACKGROUND & AIMS The apical sodium-dependent bile acid transporter is critical for intestinal reclamation of bile salts. Its expression and activity, along with the ileal lipid-binding protein, were studied before and after intestinal resection in the rat. METHODS The effects of surgical resection and bile acid feeding on the expression of ileal bile acid transport were assessed by a combination of functional (taurocholate uptake into crude brush border membrane vesicles) and molecular assays (Northern and Western blotting). RESULTS Transport, apical sodium-dependent bile acid transporter and ileal lipid-binding protein messenger RNA and protein expression were restricted to the distal 30 cm of ileum. After resection, transport and expression were limited to the remaining portions of this segment. Limited ileal resection increased protein mass and, therefore, transport in the terminal 5 cm of ileum without a specific increase in transporter gene expression. Increased bile acid presentation to the terminal ileum did not induce ileal hyperplasia. Eighty-five percent intestinal resection led to ileal hypertrophy and a specific repression in bile acid transport activity. CONCLUSIONS Native and compensatory bile acid transporter gene expression occur predominantly in the terminal 30 cm of ileum. The specific ileal responses to intestinal resection are dependent on the extent of resection.

Management of nonalcoholic fatty liver disease: An evidence-based clinical practice review

AIM To build a consensus among Chilean specialists on the appropriate management of patients with nonalcoholic fatty liver disease (NAFLD) in clinical practice. METHODS NAFLD has now reached epidemic proportions worldwide. The optimal treatment for NAFLD has not been established due to a lack of evidence-based recommendations. An expert panel of members of the Chilean Gastroenterological Society and the Chilean Hepatology Association conducted a structured analysis of the current literature on NAFLD therapy. The quality of the evidence and the level of recommendations supporting each statement were assessed according to the recommendations of the United States Preventive Services Task Force. A modified three-round Delphi technique was used to reach a consensus among the experts. RESULTS A group of thirteen experts was established. The survey included 17 open-ended questions that were distributed among the experts, who assessed the articles associated with each question. The levels of agreement achieved by the panel were 93.8% in the first round and 100% in the second and third rounds. The final recommendations support the indication of lifestyle changes, including diet and exercise, for all patients with NAFLD. Proven pharmacological therapies include only vitamin E and pioglitazone, which can be used in nondiabetic patients with biopsy-proven nonalcoholic steatohepatitis (the progressive form of NAFLD), although the long-term safety and efficacy of these therapies have not yet been established. CONCLUSION Current NAFLD management is rapidly evolving, and new pathophysiology-based therapies are expected to be introduced in the near future. All NAFLD patients should be evaluated using a three-focused approach that considers the risks of liver disease, diabetes and cardiovascular events.

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 degrees 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.