Cholestatic Patients Research Articles

Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants

The bile salt export pump (BSEP, ABCB11), multidrug resistance protein 3 (MDR3, ABCB4) and the ATPase familial intrahepatic cholestasis 1 (FIC1, ATP8B1) mediate bile formation. This study aimed to determine the contribution of mutations and common variants in the FIC1, BSEP and MDR3 genes to cholestatic disorders of differing disease onset and severity. Coding exons with flanking intron regions of ATP8B1, ABCB11, and ABCB4 were sequenced in cholestatic patients with assumed genetic cause. The effects of new variants were evaluated by bioinformatic tools and 3D protein modeling. In 427 patients with suspected inherited cholestasis, 149 patients carried at least one disease-causing mutation in FIC1, BSEP or MDR3, respectively. Overall, 154 different mutations were identified, of which 25 were novel. All 13 novel missense mutations were disease-causing according to bioinformatics analyses and homology modeling. Eighty-two percent of patients with at least one disease-causing mutation in either of the three genes were children. One or more common polymorphism(s) were found in FIC1 in 35.3%, BSEP in 64.3% and MDR3 in 72.6% of patients without disease-causing mutations in the respective gene. Minor allele frequencies of common polymorphisms in BSEP and MDR3 varied in our cohort compared to the general population, as described by gnomAD. However, differences in ethnic background may contribute to this effect. In a large cohort of patients, 154 different variants were detected in FIC1, BSEP, and MDR3, 25 of which were novel. In our cohort, frequencies for risk alleles of BSEP (p.V444A) and MDR3 (p.I237I) polymorphisms were significantly overrepresented in patients without disease-causing mutation in the respective gene, indicating that these common variants can contribute to a cholestatic phenotype. FIC1, BSEP, and MDR3 represent hepatobiliary transport proteins essential for bile formation. Genetic variants in these transporters underlie a broad spectrum of cholestatic liver diseases. To confirm a genetic contribution to the patients' phenotypes, gene sequencing of these three major cholestasis-related genes was performed in 427 patients and revealed 154 different variants of which 25 have not been previously reported in a database. In patients without a disease-causing mutation, common genetic variants were detected in a high number of cases, indicating that these common variants may contribute to cholestasis development.

Assessment of the Visual Analogue Score in the Evaluation of the Pruritus of Cholestasis.

Background and Aims: A visual analogue score (VAS), based on application of a visual analogue scale, has been widely used to assess pruritus in clinical studies of patients with cholestatic liver disease. A VAS is a numerical score of the severity of the perception of pruritus, and, hence, is inherently subjective. The objective of this study was to assess the reliability of a VAS as an index of pruritus in cholestatic patients.Methods: In 8 patients with chronic pruritus due to primary biliary cholangitis, values for a VAS of pruritus were compared with corresponding measurements of scratching activity, which were generated by a monitoring system specifically designed to quantitate this activity. The relationship between individual values for the VAS and corresponding values for scratching activity during a specific interval immediately preceding the recording of the VAS was examined by determining the Spearman’s rank correlation coefficient.Results: The mean Spearman’s rank correlation coefficient between individual values for the VAS and corresponding mean values for scratching activity was 0.072; the range of these coefficients was −0.04 to 0.26. A VAS of pruritus is an unreliable index of scratching activity, and, hence, of the pathophysiological process responsible for the pruritus of cholestasis.Conclusions: It is concluded that the use of a VAS as a primary quantitative endpoint in trials of the efficacy of potential therapies for the pruritus of cholestasis may be inappropriate.

Increased hepatic ABCA1 transporter is associated with hypercholesterolemia in a cholestatic rat model and primary biliary cholangitis patients.

Hepatic ATP-binding cassette A1 (ABCA1) transporter is the modulator of intrahepatic cholesterol levels via the efflux of cholesterol into plasma. This study aimed to determine the expression of hepatic ABCA1 levels in a cholestatic rat model and patients with primary biliary cholangitis (PBC). A cholesterol efflux study was conducted with Abca1 knock down using siRNA in WIF9 cells. Cholesterol levels in the ABCA1 siRNA cells in the medium were significantly decreased compared with those in controls (P<0.05). Hepatic ABCA1 mRNA levels were significantly higher in BDL rats than in control rats (P<0.05). Furthermore, the protein expression level of hepatic ABCA1 was also significantly increased by 200% in BDL rats (P<0.05). In PBC patients, expression of hepatic ABCA1 mRNA was 2.2-fold higher than that in controls (P<0.05). The level of hepatic liver X receptor (LXR)β mRNA was correlated with ABCA1 mRNA levels in PBC patients. The expression of hepatic ABCA1 transporter was upregulated in both the cholestatic rat model and PBC patients. Upregulated hepatic ABCA1 may lead to efflux of cholesterol into plasma, thus explaining the mechanism of cholestasis leading to hypercholesterolemia.

Atorvastatin does not protect against ischemia-reperfusion damage in cholestatic rat livers

BackgroundExtrahepatic cholestasis sensitizes the liver to ischemia/reperfusion (I/R) injury during surgery for perihilar cholangiocarcinoma. It is associated with pre-existent sterile inflammation, microvascular perfusion defects, and impaired energy status. Statins have been shown to protect against I/R injury in normal and steatotic mouse livers. Therefore, the hepatoprotective properties of atorvastatin were evaluated in a rat model of cholestatic I/R injury.MethodsMale Wistar rats were subjected to 70% hepatic ischemia (during 30 min) at 7 days after bile duct ligation. Rats were randomized to atorvastatin treatment or vehicle-control in three test arms: (1) oral treatment with 5 mg/kg during 7 days after bile duct ligation; (2) intravenous treatment with 2.5, 5, or 7.5 mg/kg at 24 h before ischemia; and (3) intravenous treatment with 5 mg/kg at 30 min before ischemia. Hepatocellular damage was assessed by plasma alanine aminotransferase (ALT) and histological necrosis.ResultsI/R induced severe hepatocellular injury in the cholestatic rat livers (~10-fold increase in ALT at 6 h after I/R and ~30% necrotic areas at 24 h after I/R). Both oral and intravenous atorvastatin treatment decreased ALT levels before ischemia. Intravenous atorvastatin treatment at 5 mg/kg at 24 h before ischemia was the only regimen that reduced ALT levels at 6 h after reperfusion, but not at 24 h after reperfusion. None of the tested regimens were able to reduce histological necrosis at 24 h after reperfusion.ConclusionPre-treatment with atorvastatin did not protect cholestatic livers from hepatocellular damage after I/R. Clinical studies investigating the role of statins in the protection against hepatic I/R injury should not include cholestatic patients with perihilar cholangiocarcinoma. These patients require (pharmacological) interventions that specifically target the cholestasis-associated hepatopathology.

Lysophosphatidic acid-induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1.

Lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediator by a cheek injection model. Dorsal root ganglion neurons directly respond to LPA depending on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). LPA-induced itch-related behaviours are decreased in TRPA1-knockout (KO), TRPV1KO or TRPA1TRPV1 double KO mice. TRPA1 and TRPV1 channels are activated by intracellular LPA, but not by extracellular LPA following LPA5 receptor activation with an activity of Ca2+ -independent phospholipase A2 and phospholipase D. Intracellular LPA interaction sites of TRPA1 are KK672-673 and KR977-978 (K: lysine, R: arginine). Intractable and continuous itch sensations often accompany diseases such as atopic dermatitis, neurogenic lesions, uremia and cholestasis. Lysophosphatidic acid (LPA) is an itch mediator found in cholestatic itch patients and it induces acute itch and pain in experimental rodent models. However, the molecular mechanism by which LPA activates peripheral sensory neurons remains unknown. In this study, we used a cheek injection method in mice to reveal that LPA induced itch-related behaviours but not pain-related behaviours. The LPA-induced itch behaviour and cellular effects were dependent on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which are important for itch signal transduction. We also found that, among the six LPA receptors, the LPA5 receptor had the greatest involvement in itching. Furthermore, we demonstrated that phospholipase D (PLD) plays a critical role downstream of LPA5 and that LPA directly and intracellularly activates TRPA1 and TRPV1. These results suggest a unique mechanism by which cytoplasmic LPA produced de novo could activate TRPA1 and TRPV1. We conclude that LPA-induced itch is mediated by LPA5 , PLD, TRPA1 and TRPV1 signalling, and thus targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itch interventions.

Bile acids initiate cholestatic liver injury by triggering a hepatocyte-specific inflammatory response.

Mechanisms of bile acid-induced (BA-induced) liver injury in cholestasis are controversial, limiting development of new therapies. We examined how BAs initiate liver injury using isolated liver cells from humans and mice and in-vivo mouse models. At pathophysiologic concentrations, BAs induced proinflammatory cytokine expression in mouse and human hepatocytes, but not in nonparenchymal cells or cholangiocytes. These hepatocyte-specific cytokines stimulated neutrophil chemotaxis. Inflammatory injury was mitigated in Ccl2-/- mice treated with BA or after bile duct ligation, where less hepatic infiltration of neutrophils was detected. Neutrophils in periportal areas of livers from cholestatic patients also correlated with elevations in their serum aminotransferases. This liver-specific inflammatory response required BA entry into hepatocytes via basolateral transporter Ntcp. Pathophysiologic levels of BAs induced markers of ER stress and mitochondrial damage in mouse hepatocytes. Chemokine induction by BAs was reduced in hepatocytes from Tlr9-/- mice, while liver injury was diminished both in conventional and hepatocyte-specific Tlr9-/- mice, confirming a role for Tlr9 in BA-induced liver injury. These findings reveal potentially novel mechanisms whereby BAs elicit a hepatocyte-specific cytokine-induced inflammatory liver injury that involves innate immunity and point to likely novel pathways for treating cholestatic liver disease.

The ascending pathophysiology of cholestatic liver disease.

In this review we develop the argument that cholestatic liver diseases, particularly primary biliary cholangitis and primary sclerosing cholangitis (PSC), evolve over time with anatomically an ascending course of the disease process. The first and early lesions are in "downstream" bile ducts. This eventually leads to cholestasis, and this causes bile salt (BS)-mediated toxic injury of the "upstream" liver parenchyma. BS are toxic in high concentration. These concentrations are present in the canalicular network, bile ducts, and gallbladder. Leakage of bile from this network and ducts could be an important driver of toxicity. The liver has a great capacity to adapt to cholestasis, and this may contribute to a variable symptom-poor interval that is often observed. Current trials with drugs that target BS toxicity are effective in only about 50%-60% of primary biliary cholangitis patients, with no effective therapy in PSC. This motivated us to develop and propose a new view on the pathophysiology of primary biliary cholangitis and PSC in the hope that these new drugs can be used more effectively. These views may lead to better stratification of these diseases and to recommendations on a more "tailored" use of the new therapeutic agents that are currently tested in clinical trials. Apical sodium-dependent BS transporter inhibitors that reduce intestinal BS absorption lower the BS load and are best used in cholestatic patients. The effectiveness of BS synthesis-suppressing drugs, such as farnesoid X receptor agonists, is greatest when optimal adaptation is not yet established. By the time cytochrome P450 7A1 expression is reduced these drugs may be less effective. Anti-inflammatory agents are probably most effective in early disease, while drugs that antagonize BS toxicity, such as ursodeoxycholic acid and nor-ursodeoxycholic acid, may be effective at all disease stages. Endoscopic stenting in PSC should be reserved for situations of intercurrent cholestasis and cholangitis, not for cholestasis in end-stage disease. These are arguments to consider a step-wise pathophysiology for these diseases, with therapy adjusted to disease stage. An obstacle in such an approach is that disease stage-defining biomarkers are still lacking. This review is meant to serve as a call to prioritize the development of biomarkers that help to obtain a better stratification of these diseases. (Hepatology 2017;65:722-738).

Ursodeoxycholic Acid Ameliorates Intrahepatic Cholestasis Independent of Biliary Bicarbonate Secretion in Vil2kd/kd Mice.

Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid that possesses many pharmacological effects, including increasing bile flow, changing the hydrophobicity of the bile acid pool, and modulation of the immune response. UDCA has been approved for treating cholestatic liver disease, such as primary biliary cholangitis. However, several unanticipated severe side effects of UDCA are observed in cholestatic patients, and its pharmacological benefits remain controversial. We reported that ezrin-knockdown (Vil2kd/kd) mice exhibited severe hepatic injury because of a functional disorder in bile duct fluidity and alkalinity regulation, resembling human intrahepatic cholestatic disease. Here we used Vil2kd/kd mice as a cholestatic model to investigate the pharmacological effects of UDCA. We investigated the effects of oral and parenteral administration of UDCA on Vil2kd/kd mice. In Vil2kd/kd mice, fed a 0.5% (w/w) UDCA diet for 3 weeks, hepatic injury was exacerbated, although oral administration of a lower dose of UDCA slightly improved hepatic function in Vil2kd/kd mice. On the other hand, intraperitoneal administration of UDCA (50 mg/kg/d) ameliorated hepatic function and markedly reduced periductal fibrosis and cholangiocyte proliferation in Vil2kd/kd mice although biliary pH and HCO3- concentration were not improved. The expression levels of inflammatory and profibrotic genes were also significantly decreased in these mice. Furthermore, UDCA prevented cholangiocytes from hydrophobic bile acid-induced cytotoxicity independent of extracellular pH in in vitro experiments. These results suggest that an appropriate dosage of UDCA can ameliorate the intrahepatic cholestasis in Vil2kd/kd mice without changing the biliary bicarbonate secretion.

Charcoal hemoperfusion in the treatment of medically refractory pruritus in cholestatic liver disease.

Pruritus is a distressing symptom in a considerable proportion of cholestatic patients and a few of them do not respond to conventional treatment. Charcoal hemoperfusion (CH) is an extracorporeal technique that is effective in eliminating protein-bound substances which may have accumulated during cholestasis. Several case reports have shown significant reduction of bilirubin in mechanical jaundice and neonatal hemolytic jaundice. However, the published data of CH for the treatment of refractory pruritus in cholestatic patients are scarce. Procedure code "Charcoal hemoperfusion" (90997) was used to identify patients who received CH at Mayo Clinic, Rochester, from 1 January 2000 to 5 January 2015. Patients who received CH for refractory cholestatic pruritus were retrospectively reviewed. Thirteen patients were identified. A median of 5 (range 1-18) sessions for a total of 20(1-72)h were performed. CH resulted in a significant decrease of pruritus in nine patients (69%). Two patients did not have significant relief and two patients did not pursue further treatments after having adverse reactions during the first session. Median pruritus numerical rating scale significantly decreased from 9/10 (9-10) to 4/10 (0-9) post-treatment (p=0.004). Duration of symptom-free periods ranged from 8 to 90days (median 18days) in six patients who returned for follow-up. Most common adverse reactions were pain, bleeding from the catheter site and fever. CH temporarily improves the severity of medically refractory cholestatic pruritus in some patients. However, the improvement is not sustained and the short duration of benefit should be balanced with the invasive nature of the therapy and the relatively common adverse reactions.

Neutrophil Extracellular Traps Accelerate Cholestatic Liver Injury through Bile Acids in Bile Duct Ligation Mice

Introduction: The mechanisms involved in cholestatic liver injury remain elusive although emerging evidence indicate neutrophils, rather than bile acids (BAs), are the predominant source of damage. Neutrophil extracellular traps (NETs) are double-edge swords that serve to ensnare and kill microbial pathogens but also contribute to excessive inflammation and tissue damage. However, the role of NETs during bile duct ligation (BDL)-induced cholestatic liver injury is largely unknown. Moreover, whether neutrophils and BAs cooperatively participate in this process also needs to be investigated. Our objectives were to localize and determine the magnitude of NETs in the hepatic microvasculature, define the role of NETs on hematological and histological changes, and characterize the role of BAs in the induction of NETs in BDL mice.Methods: BDL was induced in C57BL/6 mice by ligation of the common bile duct. After 3 days, liver tissue was collected and NETs were detected by immunostaining and western blot. Serum myeloperoxidase (MPO)-DNA complexes were measured by ELISA. Cholestatic liver injury was characterized by hematological and histological assessment. NETs were dismantled by DNase I or depleted with peptidylarginine deiminase 4 (PAD4) inhibitor Cl-amidine. The apoptosis of hepatocytes was measured by TUNEL and the induction of chemokines on Kupffer cells was analyzed by quantitative polymerase chain reaction (qPCR). BAs and inflammatory cytokines were utilized collaboratively to induce NETs formation both in vitro and in vivo. Diphenylene iodonium (DPI) was used to inhibit reactive oxygen species (ROS) formation. Plasma was obtained from healthy controls and patients with entrahepatic cholestasis.Results: We found that NETs formed in the sinusoids of cholestatic liver lobes in vivo, which was associated with significantly increased serum MPO-DNA complexes (P<0.01) and tissue levels of citrullinated-histone 3 (P<0.05) in BDL mice compared with sham operated mice. The formation of NETs exacerbated BDL-induced liver injury as evidenced by markedly increased levels of serum alamine transaminase (ALT) and percentage of necrotic tissue area (both P<0.05 vs. sham group, Figure 1). Depleting of NETs with DNase I or Cl-amidine decreased NETs formation, protected hepatocytes and dramatically alleviated cholestatic liver injury, indicating the pathophysiological role of NETs in BDL mice (Figure 1). In addition, NETs promoted the apoptosis of hepatocytes and induced the release of chemokines from Kupffer cells in BDL mice compared to sham group. Both effects could be inhibited by DNase I or Cl-amidine. Moreover, BAs induced mouse neutrophils, primed with inflammatory cytokines, to release NETs in vitro in a dose-dependent manner and mediated by PAD4 and ROS. Intravenously injection of BAs and inflammatory cytokines to healthy mice lead to the formation of NETs and exacerbated liver injury, suggesting that depleting NETs can prevent organ damage. Furthermore, we found NETs markers were significantly increased in the serum of cholestatic patients, correlating positively with increased levels of ALT and BAs.Conclusions: Our results suggest that BAs promote NETs formation via PAD4 and ROS. Development of NETs subsequently initiates inflammatory responses and exacerbates organ damage during BDL-induced cholestatic liver injury, and may therefore serve as a promising therapeutic target in cholestasis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Lipoprotein-X in cholestatic patients causes xanthomas and promotes foam cell formation in human macrophages

Lipoprotein-X (Lp-X) is an abnormal phospholipid-rich lipoprotein found in patients with cholestatic liver disease. Some patients exhibit skin xanthomas and severe hyperlipidemia. We investigated whether Lp-X induces foam cell formation in human-derived macrophages. To compare the atherogenic properties of Lp-X and modified LDL, we isolated Lp-X from 2 patients who had drug-induced cholestasis and xanthoma striata in the interphalangeal folds. We prepared oxidized LDL and acetylated LDL from healthy volunteers for the positive control experiments. When human monocyte-derived macrophages were incubated with these lipoproteins, the isolated Lp-X induced more prominent lipid accumulation than oxidized LDL or acetylated LDL. One case underwent liver biopsy, with the bile ducts showing marked damage, fulfilling the criteria for vanishing bile duct syndrome. The other case was clinically diagnosed as drug-induced hypersensitivity syndrome. In both cases, Lp-X levels decreased markedly and the xanthomas disappeared completely after the improvement of cholestasis. This study indicates that Lp-X induces foam cell formation in human-derived macrophages. Our findings strongly suggest that persistently elevated Lp-X may cause xanthomas.

Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry.

This study aimed at establishing a sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM-MS measurement were also determined. The method was applied to liver samples from ten non-cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM-based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM-MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.

Clinical characteristics of drug-induced liver injury and related risk factors.

Drug-induced liver injury (DILI) is often undiagnosed or misdiagnosed clinically because of diagnostic difficulties caused by lack of laboratory-specific serological markers. In this study, we comprehensively assessed the clinical characteristics, laboratory indices, hepatotoxic drugs, risk factors and outcomes concerning DILI, and explored the similarities in mechanisms between Chinese and Western drug-induced DILI. Patients with a first diagnosis of DILI and a Roussel Uclaf Causality Assessment Method (RUCAM) score >3 points were enrolled for systematic retrospective study. Their clinical characteristics, clinical classification, risk factors, laboratory indices, hepatotoxic drugs and outcomes were analyzed. Cholestatic patients had the highest alkaline phosphatase (ALP) and prothrombin time activity (PTA) levels (P<0.05). Patients with medication time ≥30 days had significantly higher positive rate of autoantibodies than those with medication time <30 days. Odds ratio values for DILI-related factors such as hepatobiliary diseases, immune dysfunction, diabetes, hypertension, chronic alcohol consumption and age ≥45 years were 6.552, 6.130, 3.774, 2.801, 2.002 and 1.838, respectively. Pathogeneses of Chinese and Western drug-induced DILI may be substantially the same. DILI accompanied with autoantibody positivity may indicate severe liver injury outcome. Hepatobiliary diseases, diabetes and hypertension are likely to increase drug susceptibility, and more prone to cause liver injury.

Liquid chromatography–mass spectroscopy as a tool in the rapid diagnosis of biliary atresia: a pilot study

IntroductionBiliary atresia (BA) is a neonatal obstructive cholangiopathy requiring rapid intervention to prevent end-stage liver failure and death. Low bile acid levels in stool, detectable with high-performance liquid chromatography–mass spectroscopy, may reflect extrahepatic biliary obstruction in cholestasis. HypothesisStool bile acid content can differentiate BA from non-BA forms of cholestasis. MethodsStool samples from four healthy and nine cholestatic patients were collected following internal review board approval. Bile acids were extracted and separated on a 4000-Q-Trap HPLC-MS system. ResultsTotal bile acid content was highest in samples from healthy relative to cholestatic patients: 3354.01 ± 2102.56, 1476.27 ± 1361.07, and 34.29 ± 10.30 μM/mg of stool in healthy, total parenteral nutrition-associated cholestasis, and BA samples, respectively. Mean cholic acid and chenodeoxycholic acid concentrations in healthy samples (2017.5 ± 1413.6 and 876.83 ± 660.60 μM/mg) were higher than in TPN cholestatic samples (93.99±131.55 and 232.34 ± 293.41 μM/mg). The most dramatic reduction in cholic acid and chenodeoxycholic acid was observed in BA samples (0.65 ± 0.47 and 1.22 ± 0.80 μM/mg). ConclusionBile acid content in stool is reduced in cholestatic patients relative to healthy patients with the most dramatic reduction observed in BA-patients.

Enteroendocrine cells are a potential source of serum autotaxin in men

ObjectiveSerum autotaxin (ATX) activity is significantly increased in cholestatic patients. Our study aimed to unravel the source(s) of ATX in cholestasis. Materials and methodsATX activity and protein were measured in sera of healthy (n=33) and cholestatic patients (n=152), including women with intrahepatic cholestasis of pregnancy. ATX mRNA and protein expression were analyzed in various tissues from mice and men. Induction of ATX activity was assessed in mouse models of extrahepatic (bile duct ligation) and intrahepatic cholestasis (Atp8b1G308V/G308V, 0.1% cholate-supplemented diet). ATX clearance in cholestatic and control mice was assessed after intravenous injection of recombinant ATX. Human hepatic clearance was estimated by comparing ATX activity in portal and hepatic vein serum. ResultsSerum ATX activity and ATX protein concentration tightly correlated under all conditions in patients and controls (p<0.0001). In humans Atx mRNA was highly expressed in small intestine, whereas in mice Atx was expressed mainly in brain and placenta but not in small intestine. Extensive ATX protein expression was identified in human, but not murine intestinal enteroendocrine cells. In murine models of cholestasis and cholestatic pregnancy plasma ATX activity was only mildly elevated (up to 2.1-fold). Atx tissue expression and rATX clearance after parenteral administration did not differ between cholestatic and control mice. ConclusionSerum ATX activity during cholestasis and itch is enhanced by increased protein concentration rather than enzymatic induction. In mice, clearance of ATX is not affected by cholestasis. Small intestinal ATX expression by enteroendocrine cells might represent an important source of cholestasis-induced serum ATX activity in men.

Bile Acid-Induced Suicidal Erythrocyte Death

Background/Aims: In nucleated cells, bile acids may activate cation channels subsequently leading to entry of Ca²⁺. In erythrocytes, increase of cytosolic Ca²⁺ activity triggers eryptosis, the suicidal death of erythrocytes characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis is triggered by bile duct ligation, an effect partially attributed to conjugated bilirubin. The present study explored, whether bile acids may stimulate eryptosis. Methods: Phosphatidylserine exposing erythrocytes have been identified utilizing annexin V binding, cell volume estimated from forward scatter, cytosolic Ca²⁺ activity determined using Fluo-3 fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Results: The exposure of human erythrocytes to glycochenodesoxycholic (GCDC) and taurochenodesoxycholic (TCDC) acid was followed by a significant decrease of forward scatter and significant increase of Fluo-3 fluorescence, ceramide abundance as well as annexin V binding. The effect on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca²⁺. Conclusion: Bile acids stimulate suicidal cell death, an effect paralleled by and in part due to Ca²⁺ entry and ceramide. The bile acid induced eryptosis may in turn lead to accelerated clearance of circulating erythrocytes and, thus, may contribute to anemia in cholestatic patients.

Management of symptom complexes in primary biliary cholangitis

Primary biliary cholangitis (PBC) is a rare autoimmune liver disease that increasingly is diagnosed at early stages of disease. Although survival has improved, a majority suffers from one or more symptom complexes associated with PBC. Much remains unknown about the mechanisms, significance, or effective treatment of these symptoms. The present review will outline the latest understanding and management of the most common symptoms of PBC. Patients rate fatigue as the most impactful on their quality of life, yet its specificity to PBC remains unsettled. Both central and peripheral nervous system mechanisms have been proposed, but remain enigmatic. Lysophosphatidic acid and G-protein-coupled bile acid receptor 1 have emerged as potential targets for cholestatic-specific pathways of pruritus. Fibrates have also shown promise as a new class of antipruritic in cholestatic patients. PBC and Sjogren's syndrome share many demographic and histological features, but the clinical significance of their association is not well understood. Common symptoms in PBC carry an increasing share of the disease burden as patients live longer, yet existing therapies are frequently ineffective or poorly tolerated. Targeted therapies may emerge as the molecular pathways of PBC-associated symptoms are unraveled.

Interleukin-18 Down-Regulates Multidrug Resistance-Associated Protein 2 Expression through Farnesoid X Receptor Associated with Nuclear Factor Kappa B and Yin Yang 1 in Human Hepatoma HepG2 Cells.

Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.

Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis

Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and ε were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis.

Testicular immunohistochemical and Ultrastructural changes associated with chronic cholestasis in rats: Effect of ursodeoxycholic acid.

Testicular atrophy has been commonly reported in patients with chronic liver diseases. Ursodeoxycholic acid is the most widely used drug for the treatment of many liver diseases. However, its effect on testicular ultrastructure associated with chronic cholestasis has never been studied. Thus, this study aimed to assess how chronic obstructive jaundice affects the testicular ultrastructure and whether it affects the androgen receptor or the proliferating cell nuclear antigen. The role of ursodeoxycholic acid was also investigated. Cholestasis was induced by bile duct ligation. Samples were collected 4weeks postoperative. Testicular changes were assessed using immunohistochemistry and transmission electron microscopy. Chronic cholestasis resulted in testicular atrophy evidenced by shrinkage and deformation of seminiferous tubules, thickening of peritubular boundaries, vacuolation, disorganization of germ cells, and maturation arrest. This was accompanied by decreased immunoreactivity of androgen receptors and proliferating cell nuclear antigen. Administration of ursodeoxycholic acid improved the testicular morphology and reversed cholestasis-induced immunohistochemical and ultrastructural changes. Ursodeoxycholic acid can improve the testicular ultrastructure and restore the spermatogenic process in rats with chronic cholestasis. These findings support the clinical application of ursodeoxycholic acid in cholestatic patients especially those with hypogonadism.