A mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKHD1) gene encoding the protein fibrocystin/polyductin. The aim of our study was to produce a mouse model of ARPKD in which there was no functional fibrocystin/polyductin to study the pathophysiology of cystic and fibrocystic disease in renal and non-renal tissues. Exon 2 of the gene was deleted and replaced with a neomycin resistance cassette flanked by loxP sites, which could be subsequently removed by Cre-lox recombinase. Homozygous Pkhd1(del2/del2) mice were viable, fertile and exhibited hepatic, pancreatic, and renal abnormalities. The biliary phenotype displayed progressive bile duct dilatation, resulting in grossly cystic and fibrotic livers in all animals. The primary cilia in the bile ducts of these mutant mice had structural abnormalities and were significantly shorter than those of wild-type (WT) animals. The Pkhd1(del2/del2) mice often developed pancreatic cysts and some exhibited gross pancreatic enlargement. In the kidneys of affected female mice, there was tubular dilatation of the S3 segment of the proximal tubule (PT) starting at about 9 months of age, whereas male mice had normal kidneys up to 18 months of age. Inbreeding the mutation onto BALBc/J or C57BL/6J background mice resulted in females developing PT dilatation by 3 months of age. These inbred mice will be useful resources for studying the mechanisms underlying the pathogenesis of ARPKD.

Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism.

Bile duct epithelium forms a barrier to the backflow of bile into the liver parenchyma. However, the structure and regulation of the tight junctions in bile duct epithelium is not well understood. In the present study, we evaluated the effect of lipopolysaccharide on tight junction integrity and barrier function in normal rat cholangiocyte monolayers. Lipopolysaccharide disrupts barrier function and increases paracellular permeability in a time- and dose-dependent manner. Lipopolysaccharide induced a redistribution of tight junction proteins, occludin, claudin-1, claudin-4, and zonula occludens (ZO)-1 from the intercellular junctions and reduced the level of ZO-1. Tyrosine kinase inhibitors (genistein and PP2) prevented lipopolysaccharide-induced increase in permeability and subcellular redistribution of ZO-1. Reduced expression of c-Src, TLR4, or LBP by specific small interfering RNA attenuated lipopolysaccharide-induced permeability and redistribution of ZO-1. ML-7, a myosin light chain kinase inhibitor, attenuated LPS-induced permeability. Lipopolysaccharide treatment rapidly increased the phosphorylation of occludin and ZO-1 on tyrosine residues, which was prevented by genistein and PP2. Occludin and ZO-1 were found to be highly phosphorylated on threonine residues in intact cell monolayers. Threonine-phosphorylation of occludin was rapidly reduced by lipopolysaccharide administration. Lipopolysaccharide-induced dephosphorylation of occludin on Thr residues was prevented by genistein and PP2. In conclusion, lipopolysaccharide disrupts the tight junction of a bile duct epithelial monolayer by a c-Src-, TLR4-, LBP-, and myosin light chain kinase-dependent mechanism.

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion.

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

ATP binding cassette transporter gene expression in rat liver progenitor cells.

BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells. The aim of this study was to determine the ABC transporter phenotype of HPCs. METHODS: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx). ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF phi 13 cells and by immunohistochemistry of total liver samples. ABC transporter efflux activity was studied in RLF phi 13 cells by flow cytometry. RESULTS: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3. Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes. Freshly isolated Thy-1 positive cells and cultured RLF phi 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed. Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF phi 13 cells. CONCLUSION: HPCs express high levels of active Mrp1 and Mrp3. These may have a cytoprotective role in conditions of severe hepatotoxicity.

Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia.

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5'-triphosphate, UTP, and UDP each increased Ca. Basolateral addition of adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), but not ATP, to the perifusing bath increased Ca. IBDU perfusion with ATP-gamma-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.

Water movement across rat bile duct units is transcellular and channel-mediated.

In recent studies using freshly isolated rat cholangiocytes, we established that water crosses the cholangiocyte membrane by a channel-mediated mechanism involving aquaporins, a family of water-channel proteins. Our goal was to address the importance of channel-mediated water transport in ductal bile formation by employing a physiologic experimental model, the enclosed, polarized rat intrahepatic bile duct unit (IBDU). Expansion and reduction of luminal areas as a reflection of water movement into and out of IBDUs prepared from livers of normal rats were measured by quantitative computer-assisted image analysis. When enclosed IBDUs were exposed to inward or outward osmotic gradients, their luminal area rapidly increased (approximately 25%) or decreased (approximately 20%) reflecting net water secretion or absorption, respectively. These effects were specifically inhibited by 2 water channel blockers, DMSO and HgCl2. In both instances, beta-mercaptoethanol reversed the inhibitory effects. In the absence of an osmotic gradient, choleretic agents (secretin and forskolin) and a cholestatic hormone (somatostatin) induced a significant increase or decrease of IBDU luminal area by 21% and 22%, respectively. These effects were also inhibited by DMSO and reversed by beta-mercaptoethanol. Under our experimental conditions, DMSO did not interfere with either forskolin-induced cAMP synthesis or the generation of osmotic driving forces via the apical chloride-bicarbonate exchanger. Protamine, an inhibitor of the paracellular pathway, had no effect on hypotonic or forskolin-induced water secretion in IBDUs. These results in a physiologically relevant model of ductal bile formation provide additional support for the concept that osmotically driven and agonist-stimulated water movement into (secretion) and out of (absorption) the biliary ductal lumen is transcellular and water channel-mediated.

Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis.

Chronic inflammation of gastrointestinal tissues is a well-recognized risk factor for the development of epithelial cell-derived malignancies. Although the inflammatory mediators linking chronic inflammation to carcinogenesis are numerous, current information suggests that nitric oxide (NO) contributes to carcinogenesis during chronic inflammation. Inducible nitric oxide synthase (iNOS), expressed by both macrophages and epithelial cells during inflammation, generates the bioreactive molecule NO. In addition to causing DNA lesions, NO can directly interact with proteins by nitrosylation and nitosation reactions. The consequences of protein damage by NO appear to be procarcinogenic. For example, NO inhibits DNA repair enzymes such as human 8-oxodeoxyguanosine DNA glycosylase 1 and blocks apoptosis via nitrosylation of caspases. These cellular events permit DNA damage to accumulate, which is required for the numerous mutations necessary for development of invasive cancer. NO also promotes cancer progression by functioning as an angiogenesis factor. Strategies to inhibit NO generation during chronic inflammation or to scavenge reactive nitrogen species may prove useful in decreasing the risk of cancer development in chronic inflammatory gastrointestinal diseases.

Human Ogg1, a protein involved in the repair of 8-oxoguanine, is inhibited by nitric oxide.

NO-mediated inhibition of base excision DNA repair may potentiate oxidativeDNA damage in cells and could be relevant to carcinogenesis associated with chronic inflammation. Because 8-oxoguanine, a ubiquitous oxidative DNA lesion, is repaired predominantly by human 8-oxoguanine glycosylase (hOgg1), our aim was to determine whether NO directly inhibits its repair activity. Neither induction of NO-generating enzyme inducible NO synthase nor treatment with S-nitroso-N-acetyl-D-L-pencillamine altered expression of hOgg1 in a human cholangiocarcinoma cell line (KMBC). In contrast, both treatments completely inhibited activity of hOgg1 immunoprecipitated from KMBC cells overexpressing hOgg1 and in a cell-free system. Both NO and peroxynitrite were capable of inhibiting hOgg1 activity. Inhibition of hOgg1 protein was characterized by formation of S-nitrosothiol adducts and loss/ejection of zinc ions. Our data indicate that NO, an inflammatory mediator, directly inhibits a key base excision repair enzyme (hOgg1) responsible for base excision repair of 8-oxoguanine. These data support the concept that NO-mediated inhibition of DNA contributes to the mutagenic environment of chronic inflammation.

Stimulation of ATP secretion in the liver by therapeutic bile acids.

ATP receptors are ubiquitously expressed and are potential targets for the therapy of a number of disorders. However, delivery of ATP or other nucleotides to specific tissues is problematic, and no pharmacological means to stimulate the release of endogenous ATP has been described. We examined the effects of the bile acid ursodeoxycholic acid (UDCA) on ATP release into bile, since this bile acid is the only agent known to be of therapeutic benefit in secretory disorders of the liver, and since its mechanism of action is not established. Both UDCA and its taurine conjugate stimulated secretion of ATP by isolated rat hepatocytes, and produced measurable increases in ATP in bile of isolated rat liver. Perfusion of ATP into microdissected bile-duct segments induced Ca(2+) signalling in bile-duct epithelia, while perfusion of bile acid did not. Thus UDCA may promote bile flow by inducing hepatocytes to release ATP into bile, which then stimulates fluid and electrolyte secretion by bile-duct epithelia downstream via changes in cytosolic Ca(2+). Moreover, these findings demonstrate the feasibility of using pharmacological means to induce secretion of endogenous ATP. Since the liver and other epithelial organs express luminal ATP receptors, these findings more generally suggest that a mechanism exists for pharmacological activation of this paracrine signalling pathway. This strategy may be useful for treatment of cystic fibrosis and other secretory disorders of the liver and other epithelial tissues.

Bcl-2-dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis.

The close association between autoantibodies against pyruvate dehydrogenase-E2 (PDC-E2), a ubiquitous mitochondrial protein, and primary biliary cirrhosis (PBC) is unexplained. Many autoantigens are selectively modified during apoptosis, which has focused attention on apoptotic cells as a potential source of "neo-antigens" responsible for activating autoreactive lymphocytes. Since increased apoptosis of bile duct epithelial cells (cholangiocytes) is evident in patients with PBC, we evaluated the effect of apoptosis on PDC-E2. Autoantibody recognition of PDC-E2 by immunofluorescence persisted in apoptotic cholangiocytes and appeared unchanged by immunoblot analysis. PDC-E2 was neither cleaved by caspases nor concentrated into surface blebs in apoptotic cells. In other cell types, autoantibody recognition of PDC-E2, as assessed by immunofluorescence, was abrogated after apoptosis, although expression levels of PDC-E2 appeared unchanged when examined by immunoblot analysis. Both overexpression of Bcl-2 and depletion of glutathione before inducing apoptosis prevented this loss of autoantibody recognition, suggesting that glutathiolation, rather than degradation or loss, of PDC-E2 was responsible for the loss of immunofluorescence signal. We postulate that apoptotic cholangiocytes, unlike other apoptotic cell types, are a potential source of immunogenic PDC-E2 in patients with PBC.

Quantitative assessment of the rat intrahepatic biliary system by three-dimensional reconstruction.

The anatomical details of the biliary tree architecture of normal rats and rats in whom selective proliferation was induced by feeding alpha-naphthylisothiocyanate (ANIT) were reconstructed in three dimension using a microscopic-computed tomography scanner. The intrahepatic biliary tree was filled with a silicone polymer through the common bile duct and each liver lobe embedded in Bioplastic; specimens were then scanned by a microscopic-computed tomography scanner and modified Feldkamp cone beam backprojection algorithm applied to generate three-dimensional images. Quantitative analysis of bile duct geometry was performed using a customized software program. The diameter of the bile duct segments of normal and ANIT-fed rats progressively decreased with increasing length of the biliary tree. Diameter of bile ducts from ANIT-fed rats (range, 21 to 264 microm) was similar to that of normal rats (22 to 279 microm). In contrast, the number of bile duct segments along the major branch reproducibly doubled, the length of the bile duct segments decreased twofold, and the length of the biliary tree remained unchanged after ANIT feeding. Moreover, the total volume of the biliary tree of ANIT-fed rats was significantly greater (855 microl) than in normal rats (47 microl). Compared with normal rats, the total surface area of the biliary tree increased 26 times after ANIT-induced bile duct proliferation. Taken together, these observations quantitate the anatomical remodeling after selective cholangiocyte proliferation and strongly suggest that the proliferative process involves sprouting of new side branches. Our results may be relevant to the mechanisms by which ducts proliferate in response to hepatic injury and to the hypercholeresis that occurs after experimentally induced bile duct proliferation.

Cryptosporidium parvum activates nuclear factor kappaB in biliary epithelia preventing epithelial cell apoptosis.

BACKGROUND & AIMS: Our previous studies have shown that Cryptosporidium parvum induces biliary epithelial cell apoptosis in vivo and causes apoptosis in bystander uninfected biliary epithelia in vitro. We analyzed C. parvum-induced nuclear factor kappa B (NF-kappaB) activation in human biliary epithelial cells and assessed its relevance to epithelial cell apoptosis. METHODS: In vitro models of cryptosporidial infection using a human biliary epithelial cell line were used to assay C. parvum- induced NF-kappaB activation and associated apoptosis. RESULTS: Degradation of I(kappa)B and nuclear translocation of the NF-kappaB family of proteins (p65 and p50) were observed in the biliary epithelial cell cultures directly exposed to the parasite. Activation of NF-kappaB was found only in directly infected cells (but not in bystander uninfected cells). A time-dependent secretion of a known NF-kappaB gene product, interleukin 8, from infected cell cultures was detected. C. parvum-induced biliary epithelial cell apoptosis was limited to bystander uninfected cells. In contrast, inhibition of NF-kappaB activation resulted in apoptosis in directly infected cells and significantly enhanced C. parvum-induced apoptosis in bystander uninfected cells. CONCLUSIONS: These observations support the concept that, while C. parvum triggers host cell apoptosis in bystander uninfected biliary epithelial cells, which may limit spread of the infection, it directly activates the NF-kappaB/I(kappa)B system in infected biliary epithelia thus protecting infected cells from death and facilitating parasite survival and propagation.

High-dose ursodeoxycholic acid as a therapy for patients with primary sclerosing cholangitis.

OBJECTIVES: To assess the tolerability and efficacy of high-dose (25-30 mg/kg per day) ursodeoxycholic acid (UDCA) in patients with primary sclerosing cholangitis (PSC). METHODS: Thirty patients with PSC were enrolled in this pilot study and treated for 1 yr. Changes in the Mayo risk score at 1 yr of treatment and projected survival at 4 yr were compared with that observed in patients randomized to placebo (n = 52) or UDCA (n = 53) at a dose of 13-15 mg/kg per day. RESULTS: A marked improvement in serum alkaline phosphatase activity (1265+/-172 vs 693+/-110 U/L, p < 0.001), AST (161+/-037 vs 77+/-13 U/L, p = 0.001), albumin (4.0+/-0.1 vs 4.2+/-0.1 g/dl, p = 0.03), and total bilirubin (1.6+/-0.3 vs 1.3+/-0.2 mg/dl, p = 0.1) occurred at 1 yr of therapy with high-dose UDCA. Changes in the Mayo risk score after 1 yr of treatment were significantly different among the three groups (p < 0.001), and these changes would be translated into a significantly different expected survival at 4 yr (p = 0.05). This expected survival at 4 yr was significantly different between placebo and the dose of 25-30 mg/kg per day (p = 0.04), but not between placebo and the dose of 13-15 mg/kg per day (p = 0.4). High-dose UDCA was well tolerated. CONCLUSIONS: UDCA at a dose of 25-30 mg/kg per day may be of benefit for patients with PSC, and this regimen deserves further evaluation in a long-term, randomized, placebo-controlled trial.

The water channel aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cyclic AMP.

We previously found that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway. Recently, it has been reported that mRNA for the water channel, aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected an N-glycosylated band of approximately 34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt(2)cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt(2)cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt(2)cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.

Nitric oxide-mediated inhibition of DNA repair potentiates oxidative DNA damage in cholangiocytes.

BACKGROUND & AIMS: Chronic inflammation, a risk factor for the development of bile duct cancer, induces inducible nitric oxide synthase (iNOS) with nitric oxide (NO) generation, which promotes oxidative damage of DNA, a process that probably is important in the initiation and progression of malignancies. Because inhibition of DNA repair is required for accumulation of oxidative DNA lesions, our aim was to determine if NO also inhibits repair of oxidative DNA damage. METHODS: A cholangiocarcinoma cell line and a cholangiocyte cell line were transfected with iNOS. RESULTS: Extracts from transfected but not untransfected cells were unable to repair 8-oxodeoxyguanine (8-oxodG); this effect was irreversible because addition of dithiothreitol to cell extracts had no effect. NO inhibition of 8-oxodG repair was blocked by NO scavengers but not by peroxynitrite scavengers or inhibitors of the soluble guanylyl cyclase/protein kinase G pathway. NO also potentiated hydrogen peroxide-induced DNA damage. Finally, immunohistochemistry in human liver samples uniformly demonstrated de novo expression of iNOS and the presence of 3-nitrotyrosine and 8-oxodG formation in the biliary epithelia of 30 patients with primary sclerosing cholangitis (a premalignant disease of the biliary tract) compared with controls. CONCLUSIONS: Collectively, these data implicate NO-mediated inhibition of 8-oxodG base excision DNA repair processes as a mechanism potentiating DNA damage in human inflammatory diseases involving the biliary tract.

Upregulation of secretin receptors on cholangiocytes after bile duct ligation.

Secretin not only increases ductular bile secretion in vivo in rats after bile duct ligation (BDL) [1], but also increases cAMP levels and stimulates exocytosis in isolated cholangiocytes [2]. Although we have previously reported that secretin receptor mRNA was upregulated in cholangiocytes after BDL [3], the cholangiocyte secretin receptor has not been functionally characterized or quantified after BDL. In this work, we used a novel, photolabile and biologically active analogue of secretin to quantify and characterize secretin receptors on cholangiocytes isolated from normal and BDL rats. The cholangiocyte secretin receptor bound radioligand with high affinity and in a rapid, reversible, and temperature-dependent manner. While receptors on cholangiocytes from normal and BDL rats were functionally and biochemically identical, receptor density on cholangiocytes was increased 5-fold following BDL. The combination of increased cell number with increased functional secretin receptors per cell is due to the fact that cholangiocyte hyperplasia represents a reactive response to a cholestatic condition and this effort on the part of the organism to maintain bile secretion, explains the increased hormone-responsive choleresis observed after BDL and may reflect an adaptive response of the organism to cholestasis.

Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.

BACKGROUND & AIMS: We report a novel approach to study biliary water, bile acid, and HCO(3)(-) transport: the microperfusion of intrahepatic bile duct units (IBDUs) isolated from normal rat liver. METHODS: To study water transport, IBDUs were perfused in vitro with a membrane-impermeant fluorescent volume marker, fluorescein sulfonate; net water movement (J(v)) and osmotic water permeability (P(f)) were then calculated. To study solute transport, IBDUs were perfused with taurocholic acid (TCA) and bile acid uptake was calculated from the concentrations of TCA in the perfused and collected solutions. To study ion transport, IBDUs were perfused with the cell-impermeant pH-sensitive dye BCECF dextran; luminal pH was determined from fluorescence excitation ratios. RESULTS: When inward (secretory) or outward (absorptive) osmotic gradients were established across IBDUs, water movement was observed from bath to lumen (i.e., secretion) and from lumen to bath (i.e., absorption). The perfused IBDUs absorbed TCA in a saturable, sodium-dependent manner; in addition, TCA absorption was blocked in a dose-dependent fashion by S0960, a specific inhibitor of the Na(+)/bile acid cotransporter. Addition of forskolin to HCO(3)(-)-containing (but not HCO(3)(-)-free) bath buffer resulted in lumen alkalinization reflecting HCO(3)(-) transport into the lumen of perfused IBDUs. CONCLUSIONS: The results provide direct functional evidence that intrahepatic bile ducts both secrete and absorb water in response to osmotic gradients, actively absorb bile acid, and transport HCO(3)(-).

Magnetic resonance cholangiography in patients with biliary disease: its role in primary sclerosing cholangitis.

BACKGROUND/AIM: Magnetic resonance cholangiography (MRC) is a non-invasive diagnostic procedure whose role in the management of patients with primary sclerosing cholangitis (PSC) is unclear. The aim of this study was to determine the usefulness of MRC in the evaluation of the biliary tree in patients with suspected biliary disease, and in particular, PSC. METHODS: MRC and invasive cholangiography (ERCP or PTC) were both performed in 73 patients, (33 male, 40 female, mean age 56 years) with clinical and/or biochemical evidence of cholestasis. Images were interpreted by two radiologists unaware of the results of other studies. RESULTS: Forty-two patients (58%) had benign biliary disease, including 23 patients (32%) with PSC; 9 patients (12%) had malignant biliary disease; and 22 patients (30%) had a normal biliary tree. Diagnostic quality images were obtained in 73/73 (100%) of MRC, and in 70/73 (96%) of invasive cholangiography (68 ERCP's, 2 PTC's) procedures. Using ERCP/PTC findings as the reference standard, MRC had an accuracy greater than 90% in the diagnosis of normal bile ducts, biliary dilatation, biliary obstruction, bile duct stones, and PSC. Using the final diagnosis, MRC had an overall diagnostic accuracy of 90% in the detection of biliary disease compared to 97% for invasive cholangiography. Additional diagnostic/therapeutic interventions were performed during ERCP in 73% of patients with PSC and in 43% of patients without PSC (p=0.02). CONCLUSIONS: MRC has excellent diagnostic accuracy in the presence of biliary disease. Because of its noninvasive nature, MRC may have advantages over invasive cholangiography when diagnosis is the major goal of the procedure.

A pilot study of pentoxifylline for the treatment of primary sclerosing cholangitis.

OBJECTIVE: There is no effective therapy for patients with primary sclerosing cholangitis (PSC). Rats with experimental small bowel bacterial overgrowth develop hepatobiliary injury similar to PSC. The hepatobiliary injury results from peptidoglycan-polysaccharide-mediated activation of Kupffer cells, release of cytokines such as tumor necrosis factor (TNF-alpha), and is prevented by pentoxifylline. Our aims were to assess the safety and effects of pentoxifylline on symptoms and biochemical liver tests in patients with PSC. METHODS: A total of 20 patients with clinical, cholangiographic, and histological features of PSC of varying severity were treated with pentoxifylline sustained release (SR) tablets (400 mg q.i.d.) for < or = 1 yr. Serum alkaline phosphatase, aspartate aminotransferase, and bilirubin were monitored every 3 months for 1 year; serum TNF-alpha and TNF receptor subtypes I and II were assessed at baseline and 1 year. RESULTS: Of 20 patients, 16 tolerated pentoxifylline and completed the study. Two patients were withdrawn because of severe nausea, and two patients were noncompliant with medication and withdrew. Pentoxifylline did not significantly alter symptoms of fatigue or pruritus, serum liver tests, serum TNF-alpha or TNF receptor levels. CONCLUSIONS: In the current regimen, pentoxifylline alone does not significantly improve symptoms or liver tests in patients with PSC.