LaRusso, Nicholas F.

Cholangiocytes are the target of a group of chronic liver diseases termed the “cholangiopathies,” in which cholangiocytes react to exogenous and endogenous insults, leading to disease initiation and progression. In primary sclerosing cholangitis (PSC), the focus of this review, the cholangiocyte response to genetic or environmental insults can lead to a heterogeneous response; that is, a subpopulation acquires a ductular reactive and proliferative phenotype, while another subpopulation undergoes senescence and growth arrest. Both ductular reactive cholangiocytes and senescent cholangiocytes can modify the periductal microenvironment through their ability to secrete various cytokines, chemokines, and growth factors, initiating and perpetuating inflammatory and profibrotic responses. This review discusses the similarities and differences, the interrelationships, and the potential pathogenic roles of these reactive proliferative and senescent cholangiocyte subpopulations in PSC.

Primary sclerosing cholangitis (PSC) is a pathogenically complex, chronic, fibroinflammatory disorder of the bile ducts without known etiology or effective pharmacotherapy. Emerging in vitro and in vivo evidence support fundamental pathophysiologic mechanisms in PSC centered on enterohepatic circulation. To date, no studies have specifically interrogated the chemical footprint of enterohepatic circulation in PSC. Herein, we evaluated the metabolome and lipidome of portal venous blood and bile obtained at the time of liver transplantation in patients with PSC (n = 7) as compared to individuals with noncholestatic, end-stage liver disease (viral, metabolic, etc. (disease control, DC, n = 19)) and to nondisease controls (NC, living donors, n = 12). Global metabolomic and lipidomic profiling was performed on serum derived from portal venous blood (portal serum) and bile using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and differential mobility spectroscopy-mass spectroscopy (DMS-MS; complex lipid platform). The Mann–Whitney U test was used to identify metabolites that significantly differed between groups. Principal-component analysis (PCA) showed significant separation of both PSC and DC from NC for both portal serum and bile. Metabolite set enrichment analysis of portal serum and bile demonstrated that the liver-disease cohorts (PSC and DC) exhibited similar enrichment in several metabolite categories compared to NC. Interestingly, the bile in PSC was uniquely enriched for dipeptide and polyamine metabolites. Finally, analysis of patient-matched portal serum and biliary metabolome revealed that these biological fluids were more homogeneous in PSC than in DC or NC, suggesting aberrant bile formation and enterohepatic circulation. In summary, PSC and DC patients exhibited alterations in several metabolites in portal serum and bile, while PSC patients exhibited a unique bile metabolome. These specific alterations in PSC are amenable to hypothesis testing and, potentially, therapeutic pharmacologic manipulation.

Improved methods are needed to risk stratify and predict outcomes in patients with primary sclerosing cholangitis (PSC). Therefore, we sought to derive and validate a prediction model and compare its performance to existing surrogate markers. The model was derived using 509 subjects from a multicenter North American cohort and validated in an international multicenter cohort (n = 278). Gradient boosting, a machine‐based learning technique, was used to create the model. The endpoint was hepatic decompensation (ascites, variceal hemorrhage, or encephalopathy). Subjects with advanced PSC or cholangiocarcinoma (CCA) at baseline were excluded. The PSC risk estimate tool (PREsTo) consists of nine variables: bilirubin, albumin, serum alkaline phosphatase (SAP) times the upper limit of normal (ULN), platelets, aspartate aminotransferase (AST), hemoglobin, sodium, patient age, and number of years since PSC was diagnosed. Validation in an independent cohort confirms that PREsTo accurately predicts decompensation (C‐statistic, 0.90; 95% confidence interval [CI], 0.84‐0.95) and performed well compared to Model for End‐Stage Liver Disease (MELD) score (C‐statistic, 0.72; 95% CI, 0.57‐0.84), Mayo PSC risk score (C‐statistic, 0.85; 95% CI, 0.77‐0.92), and SAP <1.5 × ULN (C‐statistic, 0.65; 95% CI, 0.55‐0.73). PREsTo continued to be accurate among individuals with a bilirubin <2.0 mg/dL (C‐statistic, 0.90; 95% CI, 0.82‐0.96) and when the score was reapplied at a later course in the disease (C‐statistic, 0.82; 95% CI, 0.64‐0.95). Conclusion: PREsTo accurately predicts hepatic decompensation (HD) in PSC and exceeds the performance among other widely available, noninvasive prognostic scoring systems.

Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6‐targeting miRNAs, miR‐433 and miR‐22, are down‐regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin‐5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin‐5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin‐5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. Conclusion: These data suggest that down‐regulated Exportin‐5 impairs the nuclear export of miR‐433 and miR‐22 precursor forms, causing a decrease in levels of mature miR‐433 and miR‐22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).

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[Formula: see text]) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca[Formula: see text] that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5′-triphosphate, UTP, and UDP each increased Ca[Formula: see text]. Basolateral addition of adenosine 5′- O-(3-thiotriphosphate) (ATP-γ-S), but not ATP, to the perifusing bath increased Ca[Formula: see text]. IBDU perfusion with ATP-γ-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.

To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 μM) to block Na+channels, exposure of the apical membrane to ATP significantly increased the short-circuit current ( Isc) from 18.2 ± 5.9 to 52.8 ± 12.7 μA ( n = 18). The response to ATP is mediated by basolateral-to-apical Cl−transport because it is inhibited by 1) the Cl−channel blockers 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 μM) in the apical chamber, 2) the K+channel blocker Ba2+(5 mM), or 3) the Na+-K+-2Cl−cotransport inhibitor bumetanide (200 μM) in the basolateral chamber. Other nucleotides stimulated an increase in Iscwith a rank order potency of UTP = ATP = adenosine 5′-O-(3)-thiotriphosphate, consistent with P2upurinergic receptors. ADP, AMP, 2-methylthioadenosine 5′-triphosphate, and adenosine had no effect. A cDNA encoding a rat P2ureceptor (rP2uR) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl−secretion through activation of P2ureceptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.

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.

Aquaporin-1 (AQP1) water channels are present in the apical and basolateral plasma membrane domains of bile duct epithelial cells, or cholangiocytes, and mediate the transport of water in these cells. We previously reported that secretin, a hormone known to stimulate ductal bile secretion, increases cholangiocyte osmotic water permeability and stimulates the redistribution of AQP1 from an intracellular vesicular pool to the cholangiocyte plasma membrane. Nevertheless, the target plasma membrane domain (i.e., basolateral or apical) for secretin-regulated trafficking of AQP1 in cholangiocytes is unknown, as is the functional significance of this process for the secretion of ductal bile. In this study, we used primarily an in vivo model (i.e., rats with cholangiocyte hyperplasia induced by bile duct ligation) to address these issues. AQP1 was quantitated by immunoblotting in apical and basolateral plasma membranes prepared from cholangiocytes isolated from rats 20 min after intravenous infusion of secretin. Secretin increased bile flow (78%, P < 0.01) as well as the amount of AQP1 in the apical cholangiocyte plasma membrane (127%, P < 0.05). In contrast, the amount of AQP1 in the basolateral cholangiocyte membrane and the specific activity of an apical cholangiocyte marker enzyme (i.e., γ-glutamyltranspeptidase) were unaffected by secretin. Similar observations were made when freshly isolated cholangiocytes were directly exposed to secretin. Immunohistochemistry for AQP1 in liver sections from secretin-treated rats showed intensified staining at the apical region of cholangiocytes. Pretreatment of rats with colchicine (but not with its inactive analog β-lumicolchicine) inhibited both the increases of AQP1 in the cholangiocyte plasma membrane (94%, P < 0.05) and the bile flow induced by secretin (54%, P < 0.05). Our results in vivo indicate that secretin induces the microtubule-dependent insertion of AQP1 exclusively into the secretory pole (i.e., apical membrane domain) of rat cholangiocytes, a process that likely accounts for the ability of secretin to stimulate ductal bile secretion.