New perspectives for the treatment of cholestasis: lessons from basic science applied clinically.
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Animals↗
Biomedical subjects
James L Boyer
Publications and source records attributed to James L Boyer.
Differential effects of hydroxyacetophenone analogues on the transcytotic vesicular pathway in rat liver.
Insertion of transporter proteins into the apical canalicular membrane via vesicular transport is one of several choleretic mechanisms. Based on different choleretic activities of hydroxyacetophenone analogues including 4-mono; 2,6-di and 2,4,6-trihydroxy-acetophenone (MHA, DHA and THA), the present study aims to determine if these compounds stimulated vesicular transport in hepatocytes. Hydroxyacetophenone was continuously infused into the duodenum of the bile fistula rat. Bile flow rate was allowed to stabilize and then followed by an intraportal injection of horseradish peroxidase, a marker of the transcytotic vesicle pathway. MHA which stimulates bile acid independent flow, showed a dose-dependent increase in both the early (paracellular) and late (transcellular) peak of horseradish peroxidase excretion in bile. THA, which stimulates both bile acid dependent flow and bile acid independent flow, did not alter the pattern of horseradish peroxidase excretion into bile. However, DHA, which is more hydrophobic and increases only bile acid dependent flow, decreased the late peak. The stimulating effects of MHA on bile flow and horseradish peroxidase excretion were markedly inhibited by colchicine, suggesting that its choleretic action involves stimulation of exocytosis, as well as increase in paracellular permeability. In contrast, the lack of a stimulatory effect of THA and DHA on biliary horseradish peroxidase excretion suggested that their choleretic action is not associated with vesicular exocytosis. These results demonstrate a variable effect of hydroxyacetophenones on the transcytotic vesicular pathway reflecting different choleretic mechanisms and therapeutic potential.
Acetophenones↗
Hepatic and extrahepatic synthesis and disposition of dinitrophenyl-S-glutathione in bile duct-ligated rats.
The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 micromol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of gamma-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (-39% and -33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S-transferases alpha, mu, and pi, as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP-SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis.
Animals↗
The comparative toxicogenomics database: a cross-species resource for building chemical-gene interaction networks.
Chemicals in the environment play a critical role in the etiology of many human diseases. Despite their prevalence, the molecular mechanisms of action and the effects of chemicals on susceptibility to disease are not well understood. To promote understanding of these mechanisms, the Comparative Toxicogenomics Database (CTD; http://ctd.mdibl.org/) presents scientifically reviewed and curated information on chemicals, relevant genes and proteins, and their interactions in vertebrates and invertebrates. CTD integrates sequence, reference, species, microarray, and general toxicology information to provide a unique centralized resource for toxicogenomic research. The database also provides visualization capabilities that enable cross-species comparisons of gene and protein sequences. These comparisons will facilitate understanding of structure-function correlations and the genetic basis of susceptibility. Manual curation and integration of cross-species chemical-gene and chemical-protein interactions from the literature are now underway. These data will provide information for building complex interaction networks. New CTD features include (1) cross-species gene, rather than sequence, query and visualization capabilities; (2) integrated cross-links to microarray data from chemicals, genes, and sequences in CTD; (3) a reference set related to chemical-gene and protein interactions identified by an information retrieval system; and (4) a "Chemicals in the News" initiative that provides links from CTD chemicals to environmental health articles from the popular press. Here we describe these new features and our novel cross-species curation of chemical-gene and chemical-protein interactions.
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A comparison of gene expression in mouse liver and kidney in obstructive cholestasis utilizing high-density oligonucleotide microarray technology.
AIM: To assess the effects of obstructive cholestasis on a wider range of gene expression using microarray technology. METHODS: Male C57BL/6J mice underwent common bile duct ligation (BDL) and were matched with pair-fed sham-operated controls. After 7 d, the animals were sacrificed and total RNA was isolated from livers and kidneys. Equal amounts of RNA from each tissue were pooled for each group and hybridized to Affymetrix GeneChip MG-U74Av2 containing a total of 12488 probe sets. Data analysis was performed using GeneSpring 6.0 software. Northern analysis and immunofluorescence were used for validation. RESULTS: In sham-operated and BDL mice, 44 and 50% of 12488 genes were expressed in livers, whereas 49 and 51% were expressed in kidneys, respectively. Seven days after BDL, 265 liver and 112 kidney genes with GeneOntology annotation were up-regulated and 113 liver and 36 kidney genes were down-regulated in comparison with sham-operated controls. Many genes were commonly regulated in both tissues and metabolism-related genes represented the largest functional group. CONCLUSION: Following BDL, microarray analysis reveals a broad range of gene alterations in both liver and kidney.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
Retention of structural and functional polarity in cultured skate hepatocytes undergoing in vitro morphogenesis.
The present study characterized a primary culture model of hepatocytes isolated from the little skate, Leucoraja erinacea, that maintain remarkable structural and functional polarity over 7 days in culture. Skate hepatocytes were isolated as clusters of 3-20 hepatocytes surrounding a bile canaliculus, rather than as single cells. Trypan blue and propidium iodide exclusion was found to be >98%, and the cells maintained high intracellular concentrations of K+, ATP, and reduced glutathione (GSH), and high ratios of ATP/ADP and GSH/GSSG. Glutathione S-transferase activity remained constant, whereas cytochrome P450 activity declined to 16% of initial levels after 7 days. Quantitative RT-PCR analysis revealed that the mRNA levels of several genes remained constant over the 7-day period, whereas Bsep, the canalicular bile salt export pump, levels declined slowly to 30% of initial values. In the presence of dexamethasone, the cells underwent a morphogenesis in which the clusters reannealed into a three-dimensional network of chords. During this morphogenesis, skate hepatocytes clusters maintained a polarized distribution of actin filaments and microtubules, as well as apical and basolateral membrane domains. Polarity of membrane transport systems was confirmed both morphologically, using antibodies raised against Bsep and Mrp2, the canalicular multispecific organic anion transporter, and functionally, by monitoring secretion of the fluorescent organic anions NBD-taurocholate, a Bsep substrate, and fluorescein-methotrexate, an Mrp2 substrate, into the bile canalicular spaces. Overall, the results indicate that in contrast with mammalian hepatocytes, isolated skate hepatocyte clusters retain polarity in culture, and provide an excellent system for investigating long-term effects of drugs and xenobiotics on hepatobiliary functions, and for studying in vitro morphogenesis.
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Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents.
Organic solute transporter (OSTalpha-OSTbeta) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To determine whether OSTalpha-OSTbeta undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OSTalpha and OSTbeta mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ostalpha and Ostbeta also increased in the liver of rats and mice following CBDL. In contrast, expression of Ostalpha and Ostbeta mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ostalpha and Ostbeta compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OSTalpha and OSTbeta mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ostalpha and Ostbeta are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor.
Animals↗
Mrp4-/- mice have an impaired cytoprotective response in obstructive cholestasis.
Mrp4 is a member of the multidrug resistance-associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild-type and Mrp4-/- mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild-type mice. Serum bile acid levels were significantly lower in Mrp4-/- mice than in wild-type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ostalpha-Ostbeta were upregulated in Mrp4-/- mice but were unable to compensate for the loss of Mrp4. High-performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta-muricholic acid ratios were increased twofold in Mrp4-/- mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury.
Animals↗
Radixin is required to maintain apical canalicular membrane structure and function in rat hepatocytes.
BACKGROUND & AIMS: Ezrin-radixin-moesin proteins are cross-linkers between the plasma membrane and actin filaments. Radixin, the dominant ezrin-radixin-moesin protein in hepatocytes, has been reported to selectively tether multidrug-resistance-associated protein 2 to the apical canalicular membrane. However, it remains to be determined if this is its primary function. METHODS: An adenovirus-mediated short interfering RNA (siRNA) was used to down-regulate radixin expression in collagen sandwich-cultured rat hepatocytes and morphologic and functional changes were characterized quantitatively. RESULTS: In control cultures, an extensive bile canalicular network developed with properly localized apical and basolateral transporters that provided for functional excretion of fluorescent cholephiles into the bile canalicular lumina. siRNA-induced suppression of radixin was associated with a marked reduction in the canalicular membrane structure as observed by differential interference contrast microscopy and F-actin staining, in contrast to control cells exposed to adenovirus encoding scrambled siRNA. Indirect immunofluorescence showed that apical transporters (multidrug-resistance-associated protein 2, bile salt export pump, and multidrug-resistance protein 1) dissociated from their normal location at the apical membrane and were found largely associated with Rab11-containing endosomes. Localization of the basolateral membrane transporter, organic anion transporting polypeptide 2 (Oatp2), was not affected. Consistent with this dislocation of apical transporters, the biliary excretion of glutathione-methylfluorescein and cholylglycylamido-fluorescein was decreased significantly in the radixin-deficient cells, but not in the control siRNA cells. CONCLUSIONS: Radixin is essential for maintaining the polarized targeting and/or retaining of canalicular membrane transporters and is a critical determinant of the overall structure and function of the apical membrane of hepatocytes.
Animals↗
FXR: a target for cholestatic syndromes?
The nuclear farnesoid X receptor (FXR) plays a pivotal role in maintaining bile acid homeostasis by regulating key genes involved in bile acid synthesis, metabolism and transport, including CYP7A1, UGT2B4, BSEP, MDR3, MRP2, ASBT, I-BABP, NTCP and OSTalpha-OSTbeta in humans. Altered expression or malfunction of these genes has been described in patients with cholestatic liver diseases. This review examines the rationale for the use of FXR ligand therapy in various cholestatic liver disorders and includes potential concerns.
Animals↗
Methotrexate (MTX) plus ursodeoxycholic acid (UDCA) in the treatment of primary biliary cirrhosis.
This placebo-controlled, randomized, multicenter trial compared the effects of MTX plus UDCA to UDCA alone on the course of primary biliary cirrhosis (PBC). Two hundred and sixty five AMA positive patients without ascites, variceal bleeding, or encephalopathy; a serum bilirubin less than 3 mg/dL; serum albumin 3 g/dL or greater, who had taken UDCA 15 mg/kg daily for at least 6 months, were stratified by Ludwig's histological staging and then randomized to MTX 15 mg/m2 body surface area (maximum dose 20 mg) once a week while continuing on UDCA. The median time from randomization to closure of the study was 7.6 years (range: 4.6-8.8 years). Treatment failure was defined as death without liver transplantation; transplantation; variceal bleeding; development of ascites, encephalopathy, or varices; a doubling of serum bilirubin to 2.5 mg/dL or greater; a fall in serum albumin to 2.5 g/dL or less; histological progression by at least two stages or to cirrhosis. Patients were continued on treatment despite failure of treatment, unless transplantation ensued, drug toxicity necessitated withdrawal, or the patient developed a cancer. There were no significant differences in these parameters nor to the time of development of treatment failures observed for patients taking UDCA plus MTX, or UDCA plus placebo. The trial was conducted with a stopping rule, and was stopped early by the National Institutes of Health at the advice of our Data Safety Monitoring Board for reasons of futility. In conclusion, methotrexate when added to UDCA for a median period of 7.6 years had no effect on the course of PBC treated with UDCA alone.
Adult↗
OSTalpha-OSTbeta: a major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia.
The cellular and subcellular localization and mechanism of transport of the heteromeric organic solute transporter (OST) OSTalpha-OSTbeta was examined in human and rodent epithelia. The two subunits of the transporter were expressed together in human small intestine, kidney, and liver, tissues that also express the apical sodium-dependent bile acid uptake transporter ASBT (SLC10A2). Indirect immunofluorescence microscopy localized OSTalpha and OSTbeta to the basolateral membrane of mouse, rat, and human ileal enterocytes, renal proximal tubular cells, and cholangiocytes. Transport in OSTalpha-OSTbeta-expressing Xenopus laevis oocytes was unaffected by depletion of intracellular adenosine triphosphate, or by changes in transmembrane Na(+), K(+), H(+), or Cl(-) concentration gradients. However, the oocytes demonstrated robust substrate efflux and trans-stimulation, indicating that transport occurs by facilitated diffusion. Madin Darby canine kidney cells coexpressing mouse Ostalpha and Ostbeta exhibited enhanced apical to basolateral transport of the major glycine and taurine conjugated bile acid species. In conclusion, the selective localization of OSTalpha and OSTbeta to the basolateral plasma membrane of epithelial cells responsible for bile acid and sterol reabsorption, the substrate selectivity of the transporter, and the facilitated diffusion transport mode collectively indicate that OSTalpha-OSTbeta is a key basolateral transporter for the reabsorption of these important steroid-derived molecules.
Amino Acid Sequence↗
Nuclear receptor ligands: rational and effective therapy for chronic cholestatic liver disease?
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Animals↗
Liver biopsy: evolving role in the new millennium.
Since the origination of the liver biopsy, the technique has evolved into an essential diagnostic tool, with very few complications. In addition to the percutaneous approach, a liver biopsy can also be obtained via transjugular, laparoscopic, or intraoperative approach. While in the early 1960s and 1970s the liver biopsy was used for making a diagnosis in cases of clinically suspected medical liver disease, today it is more often performed to assess disease prognosis and evaluate therapeutic strategies. As a result, indications for the liver biopsy have evolved over the past 2 decades. However with advances in serologic diagnosis of viral/autoimmune hepatitis and laboratory tests for genetic disorders, the role of liver biopsy in certain clinical settings is currently debated. This review discusses the technique, indications, contraindications, and the changing role of liver biopsy in some of the common disorders and the associated controversies.
Biopsy↗
Autoimmune hepatitis type 1: safety and efficacy of prolonged medical therapy.
AIM: To assess the efficacy and safety of prolonged medical therapy for up to four decades in a cohort of patients with AIH. METHODS: Forty-two patients were followed long term in the Yale Liver Clinics who met the criteria of 'definite autoimmune hepatitis' as defined by the International Autoimmune Hepatitis Group. Records were reviewed for the dosage of immunosuppression, rate of relapse, steroid side effects, current status of liver function tests and evidence for cirrhosis and its complications. RESULTS: Mean follow-up was 16 years and ranged from 7 to 43 years. The median follow-up was 13.5 years. Steroid withdrawal resulted in a mean of 1.78 relapses/patient (range, 0-8). All but six patients responded well to prednisone and azathioprine and alanine aminotransferases were completely normal in 29 (81%) at last exam. Five patients have discontinued medication. Steroid side effects have been minimal (weight gain in eight, osteoporosis in three) except for one patient who recovered successfully from cryptococcal meningitis and another with aseptic necrosis of the hip. Progression to cirrhosis occurred in 54% with evidence of esophageal varices in 37% but none developed hepatocellular carcinoma. Only one patient has received a liver transplant, while five others are currently listed because of symptoms of ascites, encephalopathy or bleeding from esophageal varices. CONCLUSIONS: AIH can be managed effectively over three to four decades with low-dose immunosuppression resulting in essentially normal lifestyles and minimal side effects. Liver transplantation with an increased risk of rejection and graft failure in this group can be avoided for long periods in most of these patients.
Adult↗
The maintenance and generation of membrane polarity in hepatocytes.
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Animals↗
Down-regulation of the organic cation transporter 1 of rat liver in obstructive cholestasis.
The liver plays a major role in biotransformation and elimination of various therapeutic agents and xenobiotics, many of which are organic cations and substrates of the organic cation transporter 1 (Oct1, Slc22a1). Oct1 is expressed at the basolateral membranes of hepatocytes and proximal renal tubules. Although Oct1 is the major uptake mechanism in hepatocytes for many pharmaceutical compounds, little is known about the effects of liver injury on this process. Our aim was to investigate the effects of obstructive cholestasis on Oct1 expression and function in liver and kidney. The effects of bile duct ligation (BDL) on Oct1 protein, messenger RNA (mRNA) expression, and tissue localization were determined in rat liver and kidney with Western analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunofluorescence. To assess Oct1 function, the model substrate tetraethylammonium ([(14)C]TEA) was administered intravenously to BDL and control rats and distribution of radioactivity was determined. Oct1 protein significantly decreased in cholestatic livers to 42.1 +/- 17.7% (P <.001), 15.5 +/- 4.7% (P <.05), and 8.6 +/- 2.7% (P <.05) of controls after 3, 7, and 14 days, respectively, but not in kidneys. Hepatic Oct1 mRNA decreased to 77.2 +/- 12.7%, 40.7 +/- 8.1% (P <.05), and 50.3 +/- 7.5% (P <.05) 3, 7, and 14 days after BDL, respectively. Tissue immunofluorescence corroborated these data. Hepatic accumulation of [(14)C]TEA in 14-day BDL rats was reduced to 29.6 +/- 10.9% of controls (P <.0005). In conclusion, obstructive cholestasis down-regulates Oct1 and impairs Oct1-mediated uptake in rat liver, suggesting that hepatic uptake of small cationic drugs may be impaired in cholestatic liver injury.
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Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat.
BACKGROUND/AIMS: Multidrug resistance-associated protein 4 (Mrp4, ABCC4) transports cyclic nucleotides, anti-retroviral compounds, and sulfated bile acids. Mrp4 expression is increased in farnesyl/bile acid receptor knockout mice. Our aim was to investigate Mrp4 expression and function in rat liver and kidney in obstructive cholestasis. METHODS: Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham-surgery. Animals were sacrificed after 3, 7, and 14 days and tissues were harvested for Western blot analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunohistochemistry. RESULTS: Western blot analysis revealed a progressive, more than seven-fold increase (P < 0.05) of Mrp4 expression in cholestatic livers, 14 days after BDL. In contrast, Mrp4 in 14-day BDL kidneys decreased to 26+/-4% of controls (P < 0.005). Immunohistochemistry localized Mrp4 to the basolateral hepatocyte membrane and corroborated its hepatic up-regulation after BDL. Real-time RT-PCR demonstrated no major changes of Mrp4 mRNA levels in liver and kidney after BDL. Cyclic adenosine monophosphate, an MRP4 substrate, was increased in plasma and urine, consistent with these findings. CONCLUSIONS: Obstructive cholestasis in rats results in progressive up-regulation of Mrp4 protein in liver but down-regulation in kidney. The absence of corresponding changes in Mrp4 mRNA suggests posttranscriptional mechanisms as predominant regulators of Mrp4 expression in BDL rats.
Animals↗