The phospholipid flippase ATP8B1 is involved in the pathogenesis of Ulcerative Colitis via establishment of intestinal barrier function.

OBJECTIVE: Patients with mutations in ATP8B1 develop Progressive Familial Intrahepatic Cholestasis type 1 (PFIC1), a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. DESIGN: ATP8B1 expression was investigated in intestinal samples of patients with Crohn's Disease (CD) or Ulcerative Colitis (UC) as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with Dextran Sodium Sulphate (DSS) and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knock-down Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. RESULTS: ATP8B1 expression was decreased in UC and DSS-treated mice, and associated with a decreased Tight Junctional pathway transcriptional program. ATP8B1-deficient mice were extremely sensisitve to DSS-induced colitis, evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that associated with affected Claudin-4 (CLDN4) levels and localization.. CLDN4 immunohistochemistry showed a tight-junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. CONCLUSION: ATP8B1 is important in the establishment of the intestinal barrier Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.

Combined inhibition of bile salt synthesis and intestinal uptake reduces cholestatic liver damage and colonic bile salts in mice.

Background & Aims: Intestine-restricted inhibitors of the apical sodium-dependent bile acid transporter (ASBT, or ileal bile acid transporter) are approved as treatment for several inheritable forms of cholestasis but are also associated with abdominal complaints and diarrhoea. Furthermore, blocking ASBT as a single therapeutic approach may be less effective in moderate to severe cholestasis. We hypothesised that interventions that lower hepatic bile salt synthesis in addition to intestinal bile salt uptake inhibition provide added therapeutic benefit in the treatment of cholestatic disorders. Here, we test combination therapies of intestinal ASBT inhibition together with obeticholic acid (OCA), cilofexor, and the non-tumorigenic fibroblast growth factor 15 (Fgf15)/fibroblast growth factor 19 (FGF19) analogue aldafermin in a mouse model of cholestasis. Methods: Wild-type male C57Bl6J/OlaHsd mice were fed a 0.05% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and received daily oral gavage with 10 mg/kg OCA, 30 mg/kg cilofexor, 10 mg/kg ASBT inhibitor (Linerixibat; ASBTi), or a combination. Alternatively, wild-type male C57Bl6J/OlaHsd mice were injected with adeno-associated virus vector serotype 8 (AAV8) to express aldafermin, to repress bile salt synthesis, or to control AAV8. During a 3-week 0.05% DDC diet, mice received daily oral gavage with 10 mg/kg ASBTi or placebo control. Results: Combination therapy of OCA, cilofexor, or aldafermin with ASBTi effectively reduced faecal bile salt excretion. Compared with ASBTi monotherapy, aldafermin + ASBTi further lowered plasma bile salt levels. Cilofexor + ASBTi and aldafermin + ASBTi treatment reduced plasma alanine transaminase and aspartate transaminase levels and fibrotic liver immunohistochemistry stainings. The reduction in inflammation and fibrogenesis in mice treated with cilofexor + ASBTi or aldafermin + ASBTi was confirmed by gene expression analysis. Conclusions: Combining pharmacological intestinal bile salt uptake inhibition with repression of bile salt synthesis may form an effective treatment strategy to reduce liver injury while dampening the ASBTi-induced colonic bile salt load. Impact and Implications: Combined treatment of intestinal ASBT inhibition with repression of bile salt synthesis by farnesoid X receptor agonism (using either obeticholic acid or cilofexor) or by expression of aldafermin ameliorates liver damage in cholestatic mice. In addition, compared with ASBT inhibitor monotherapy, combination treatments lower colonic bile salt load.

Intestinal Bacteremia After Liver Transplantation Is a Risk Factor for Recurrence of Primary Sclerosing Cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic progressive pathological process, related to inflammatory bowel disease and subsequent bacterial translocation. Liver transplantation (LT) is the only curative therapy, but outcomes are compromised by recurrence of PSC (rPSC). The aim of the study was to investigate a potential link between intestinal bacteremia, fucosyltransferase-2 (FUT2), and rPSC after LT. METHODS: LT recipients with PSC (n = 81) or without PSC (n = 271) were analyzed for clinical outcomes and positive bacterial blood cultures. A link between bacteremia and the genetic variant of the FUT2 gene was investigated. RESULTS: The incidence of inflammatory bowel disease was significantly higher in PSC recipients but not associated with rPSC. Bacteremia occurred in 31% of PSC recipients. The incidence of rPSC was 37% and was significantly more common in patients with intestinal bacteremia versus no bacteremia (82% versus 30%; P = 0.003). The nonsecretor polymorphism of the FUT2 gene was identified as a genetic risk factor for both intestinal bacteremia and rPSC. Combined FUT2 genotype and intestinal bacteremia in recipients resulted in the highest risk for rPSC (hazard ratio, 15.3; P < 0.001). CONCLUSIONS: Thus, in this article, we showed that bacterial translocation is associated with rPSC after LT and related to the FUT2 nonsecretor status.

Mechanisms of pruritus in cholestasis: understanding and treating the itch.

Pruritus in cholestatic liver diseases can be a major burden and dramatically impair the quality of life of those affected. Here, we provide an update on the latest insights into the molecular pathogenesis of and novel therapeutic approaches for cholestasis-associated itch. Endogenous and exogenous small-molecule pruritogen candidates bind to their receptors on unmyelinated itch C-fibres in the skin. Candidate pruritogens in cholestasis include certain lysophospholipids and sulfated progesterone metabolites, among others, whereas total bile acid or bilirubin conjugates seem unlikely to have a dominant role in the pathogenesis of cholestasis-associated pruritus. Transmission of itch signals via primary, secondary and tertiary itch neurons to the postcentral gyrus and activation of scratch responses offer various targets for therapeutic intervention. At present, evidence-based treatment options for pruritus in fibrosing cholangiopathies, such as primary biliary cholangitis and primary sclerosing cholangitis, are the peroxisome proliferator-associated receptor (PPAR) agonist bezafibrate and the pregnane X receptor (PXR) agonist rifampicin. In pruritus of intrahepatic cholestasis of pregnancy, ursodeoxycholic acid is recommended and might be supported in the third trimester by rifampicin if needed. Alternatively, non-absorbable anion exchange resins, such as cholestyramine, can be administered, albeit with poor trial evidence. Liver transplantation for intolerable refractory pruritus has become an extremely rare therapeutic strategy.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

Systemic ASBT inactivation protects against liver damage in obstructive cholestasis in mice.

Background & Aims: Non-absorbable inhibitors of the apical sodium-dependent bile acid transporter (ASBT; also called ileal bile acid transporter [IBAT]) are recently approved or in clinical development for multiple cholestatic liver disorders and lead to a reduction in pruritus and (markers for) liver injury. Unfortunately, non-absorbable ASBT inhibitors (ASBTi) can induce diarrhoea or may be ineffective if cholestasis is extensive and largely precludes intestinal excretion of bile acids. Systemically acting ASBTi that divert bile salts towards renal excretion may alleviate these issues. Methods: Bile duct ligation (BDL) was performed in ASBT-deficient (ASBT knockout [KO]) mice as a model for chronic systemic ASBT inhibition in obstructive cholestasis. Co-infusion of radiolabelled taurocholate and inulin was used to quantify renal bile salt excretion after BDL. In a second (wild-type) mouse model, a combination of obeticholic acid (OCA) and intestine-restricted ASBT inhibition was used to lower the bile salt pool size before BDL. Results: After BDL, ASBT KO mice had reduced plasma bilirubin and alkaline phosphatase compared with wild-type mice with BDL and showed a marked reduction in liver necrotic areas at histopathological analysis, suggesting decreased BDL-induced liver damage. Furthermore, ASBT KO mice had reduced bile salt pool size, lower plasma taurine-conjugated polyhydroxylated bile salt, and increased urinary bile salt excretion. Pretreatment with OCA + ASBTi in wild-type mice reduced the pool size and greatly improved liver injury markers and liver histology. Conclusions: A reduced bile salt pool at the onset of cholestasis effectively lowers cholestatic liver injury in mice. Systemic ASBT inhibition may be valuable as treatment for cholestatic liver disease by lowering the pool size and increasing renal bile salt output even under conditions of minimal faecal bile salt secretion. Lay summary: Novel treatment approaches against cholestatic liver disease (resulting in reduced or blocked flow of bile) involve non-absorbable inhibitors of the bile acid transport protein ASBT, but these are not always effective and/or can cause unwanted side effects. In this study, we demonstrate that systemic inhibition/inactivation of ASBT protects mice against developing severe cholestatic liver injury after bile duct ligation, by reducing bile salt pool size and increasing renal bile salt excretion.

An LPAR 5 -antagonist that reduces nociception and increases pruriception.

Introduction: The G-protein coupled receptor LPAR5 plays a prominent role in LPA-mediated pain and itch signaling. In this study we focus on the LPAR5-antagonist compound 3 (cpd3) and its ability to affect pain and itch signaling, both in vitro and in vivo. Methods: Nociceptive behavior in wild type mice was induced by formalin, carrageenan or prostaglandin E2 (PGE2) injection in the hind paw, and the effect of oral cpd3 administration was measured. Scratch activity was measured after oral administration of cpd3, in mice overexpressing phospholipase A2 ( sPLA 2 tg ), in wild type mice (WT) and in TRPA1-deficient mice (Trpa1 KO). In vitro effects of cpd3 were assessed by measuring intracellular calcium release in HMC-1 and HEK-TRPA1 cells. Results: As expected, nociceptive behavior (induced by formalin, carrageenan or PGE2) was reduced after treatment with cpd3. Unexpectedly, cpd3 induced scratch activity in mice. In vitro addition of cpd3 to HEK-TRPA1 cells induced an intracellular calcium wave that could be inhibited by the TRPA1-antagonist A-967079. In Trpa1 KO mice, however, the increase in scratch activity after cpd3 administration was not reduced. Conclusions: Cpd3 has in vivo antinociceptive effects but induces scratch activity in mice, probably by activation of multiple pruriceptors, including TRPA1. These results urge screening of antinociceptive candidate drugs for activity with pruriceptors.

Differential and organ-specific functions of organic solute transporter α and ß in experimental cholestasis.

Background & Aims: Organic solute transporter (OST) subunits OSTα and OSTß facilitate bile acid efflux from the enterocyte into the portal circulation. Patients with deficiency of OSTα or OSTß display considerable variation in the level of bile acid malabsorption, chronic diarrhea, and signs of cholestasis. Herein, we generated and characterized a mouse model of OSTß deficiency. Methods: Ostß -/- mice were generated using CRISR/Cas9 and compared to wild-type and Ostα -/- mice. OSTß was re-expressed in livers of Ostß -/- mice using adeno-associated virus serotype 8 vectors. Cholestasis was induced in both models by bile duct ligation (BDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding. Results: Similar to Ostα -/- mice, Ostß -/- mice exhibited elongated small intestines with blunted villi and increased crypt depth. Increased expression levels of ileal Fgf15, and decreased Asbt expression in Ostß -/- mice indicate the accumulation of bile acids in the enterocyte. In contrast to Ostα -/- mice, induction of cholestasis in Ostß -/- mice by BDL or DDC diet led to lower survival rates and severe body weight loss, but an improved liver phenotype. Restoration of hepatic Ostß expression via adeno-associated virus-mediated overexpression did not rescue the phenotype of Ostß -/- mice. Conclusions: OSTß is pivotal for bile acid transport in the ileum and its deficiency leads to an intestinal phenotype similar to Ostα -/- mice, but it exerts distinct effects on survival and the liver phenotype, independent of its expression in the liver. Our findings provide insights into the variable clinical presentation of patients with OSTα and OSTß deficiencies. Lay summary: Organic solute transporter (OST) subunits OSTα and OSTß together facilitate the efflux of conjugated bile acids into the portal circulation. Ostα knockout mice have longer and thicker small intestines and are largely protected against experimental cholestatic liver injury. Herein, we generated and characterized Ostß knockout mice for the first time. Ostα and Ostß knockout mice shared a similar phenotype under normal conditions. However, in cholestasis, Ostß knockout mice had a worsened overall phenotype which indicates a separate and specific role of OSTß, possibly as an interacting partner of other intestinal proteins.

Symptom patterns in the daily life of PSC patients.

BACKGROUND & AIMS: Patients with primary sclerosing cholangitis (PSC) may suffer from complaints such as pruritus, right upper abdominal quadrant pain (RUQ-A) and fatigue. However, the severity of these complaints, daily and/or seasonal patterns and other factors of influence in PSC are largely unknown. The aim of this study is to assess daily symptoms and patterns thereof in PSC patients in their natural setting. METHODS: A mobile application was designed according to the experience sampling method. Push notifications with a response time of max 4 h were sent during tiers of 3 months. Questions comprised VAS scales on degree of pruritus, fatigue, RUQ-A, time of the day these symptoms were worst, as well as time of intake of medication. Linear mixed modelling was used to identify patient- and external factors associated with pruritus, fatigue and RUQ-A pain. RESULTS: A total of 6713 questionnaires were completed by 137 patients. Fatigue was the most prevalent symptom among PSC patients being reported in a striking 71% of measurements, followed by pruritus (38%). Both increased during the day and were associated with longer disease duration. A highly significant correlation between pruritus and day temperature was observed (ρ = -0.14, p = .000), and itch was generally worse during winter (p = .000). Patient preference for the tool was high. CONCLUSION: Pruritus and fatigue are prevalent symptoms in the daily life of PSC patients and show a distinct diurnal pattern. This may have implications for efficient dosing of anti-pruritic agents. The level of pruritus is highly correlated with day temperature, which may have several implications.

Long-Term Effects of Biliverdin Reductase a Deficiency in Ugt1-/- Mice: Impact on Redox Status and Metabolism.

Accumulation of neurotoxic bilirubin due to a transient neonatal or persistent inherited deficiency of bilirubin glucuronidation activity can cause irreversible brain damage and death. Strategies to inhibit bilirubin production and prevent neurotoxicity in neonatal and adult settings seem promising. We evaluated the impact of Bvra deficiency in neonatal and aged mice, in a background of unconjugated hyperbilirubinemia, by abolishing bilirubin production. We also investigated the disposal of biliverdin during fetal development. In Ugt1-/- mice, Bvra deficiency appeared sufficient to prevent lethality and to normalize bilirubin level in adults. Although biliverdin accumulated in Bvra-deficient fetuses, both Bvra-/- and Bvra-/-Ugt1-/- pups were healthy and reached adulthood having normal liver, brain, and spleen histology, albeit with increased iron levels in the latter. During aging, both Bvra-/- and Bvra-/-Ugt1-/- mice presented normal levels of relevant hematological and metabolic parameters. Interestingly, the oxidative status in erythrocytes from 9-months-old Bvra-/- and Bvra-/-Ugt1-/- mice was significantly reduced. In addition, triglycerides levels in these 9-months-old Bvra-/- mice were significantly higher than WT controls, while Bvra-/-Ugt1-/- tested normal. The normal parameters observed in Bvra-/-Ugt1-/- mice fed chow diet indicate that Bvra inhibition to treat unconjugated hyperbilirubinemia seems safe and effective.

On the Mechanisms of Biliary Flux.

Since the late 1950s, transport of bile in the liver has been described by the "osmotic concept," according to which bile flows into the canaliculi toward the ducts, countercurrent to the blood flow in the sinusoids. However, because of the small size of canaliculi, it was so far impossible to observe, let alone to quantify this process. Still, "osmotic canalicular flow" was a sufficient and plausible explanation for the clearance characteristics of a wide variety of choleretic compounds excreted in bile. Imaging techniques have now been established that allow direct flux analysis in bile canaliculi of the intact liver in living organisms. In contrast to the prevailing osmotic concept these analyses strongly suggest that the transport of small molecules in canalicular bile is diffusion dominated, while canalicular flow is negligibly small. In contrast, with the same experimental approach, it could be shown that in the interlobular ducts, diffusion is augmented by flow. Thus, bile canaliculi can be compared to a standing water zone that is connected to a river. The seemingly subtle difference between diffusion and flow is of relevance for therapy of a wide range of liver diseases including cholestasis and NAFLD. Here, we incorporated the latest findings on canalicular solute transport, and align them with extant knowledge to present an integrated and explanatory framework of bile flux that will undoubtedly be refined further in the future.

Molecular regulation of the hepatic bile acid uptake transporter and HBV entry receptor NTCP.

Transporters expressed by hepatocytes and enterocytes play a critical role in maintaining the enterohepatic circulation of bile acids. The sodium taurocholate cotransporting polypeptide (NTCP), exclusively expressed at the basolateral side of hepatocytes, mediates the uptake of conjugated bile acids. In conditions where bile flow is impaired (cholestasis), pharmacological inhibition of NTCP-mediated bile acid influx is suggested to reduce hepatocellular damage due to bile acid overload. Furthermore, NTCP has been shown to play an important role in hepatitis B virus (HBV) and hepatitis Delta virus (HDV) infection by functioning as receptor for viral entry into hepatocytes. This review provides a summary of current molecular insight into the regulation of NTCP expression at the plasma membrane, hepatic bile acid transport, and NTCP-mediated viral infection.

Cholestasis-Associated Pruritus and Its Pruritogens.

Pruritus is a debilitating symptom of various cholestatic disorders, including primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and inherited progressive familial intrahepatic cholestasis (PFIC). The molecular mechanisms leading to cholestasis-associated pruritus are still unresolved and the involved pruritogens are indecisive. As a consequence of pruritus, patients suffer from sleep deprivation, loss of daytime concentration, auto-mutilation and sometimes even suicidal ideations. Current guideline-approved therapy of cholestasis-associated pruritus includes stepwise administration of several medications, which may alleviate complaints in some, but not all affected patients. Therefore, also experimental therapeutic approaches are required to improve patients' quality of life. This article reviews the current state of research on pruritogens and their receptors, and shortly discusses the most recent experimental therapies.

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation.

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.

Efficacy of AAV8-hUGT1A1 with Rapamycin in neonatal, suckling, and juvenile rats to model treatment in pediatric CNs patients.

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (hUGT1A1) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-hUGT1A1, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs.

Hepatobiliary acid-base homeostasis: Insights from analogous secretory epithelia.

Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical stress. Bicarbonate is a key component of human bile and impaired biliary bicarbonate secretion is associated with liver damage. Major efforts have been undertaken to gain insight into acid-base homeostasis in cholangiocytes and more can be learned from analogous secretory epithelia. Extrahepatic examples include salivary and pancreatic duct cells, duodenocytes, airway and renal epithelial cells. The cellular machinery involved in acid-base homeostasis includes carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors. This pH-regulatory system is orchestrated by protein-protein interactions, the establishment of an electrochemical gradient across the plasma membrane and bicarbonate sensing of the intra- and extracellular compartment. In this review, we discuss conserved principles identified in analogous secretory epithelia in the light of current knowledge on cholangiocyte physiology. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system.