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Polycystic liver disease (PLD) is a group of genetic disorders with limited treatment options and significant morbidity. Hepatic cysts arise from cholangiocytes exhibiting a hyperproliferative phenotype. Considering that hyperproliferation of many cell types is associated with alterations in autophagy, we hypothesized that autophagy is altered in PLD cholangiocytes, contributes to hepatic cystogenesis, and might represent a potential therapeutic target. We employed functional pathway cluster analysis and next-generation sequencing, transmission electron microscopy, immunofluorescence confocal microscopy, and western blotting to assess autophagy in human and rodent PLD cholangiocytes. A three-dimensional culture model was used to study the effects of molecular and pharmacologic inhibition of autophagy on hepatic cystogenesis in vitro, and the polycystic kidney disease-specific rat, an animal model of PLD, to study the effects of hydroxychloroquine, a drug that interferes with the autophagy pathway, on disease progression in vivo. Assessment of the transcriptome of PLD cholangiocytes followed by functional pathway cluster analysis revealed that the autophagy-lysosomal pathway is one of the most altered pathways in PLD. Direct evaluation of autophagy in PLD cholangiocytes both in vitro and in vivo showed increased number and size of autophagosomes, lysosomes, and autolysosomes; overexpression of autophagy-related proteins (Atg5, Beclin1, Atg7, and LC3); and enhanced autophagic flux associated with activation of the cAMP-protein kinase A-cAMP response element-binding protein signaling pathway. Molecular and pharmacologic intervention in autophagy with ATG7 small interfering RNA, bafilomycin A 1 , and hydroxychloroquine reduced proliferation of PLD cholangiocytes in vitro and growth of hepatic cysts in three-dimensional cultures. Hydroxychloroquine also efficiently inhibited hepatic cystogenesis in the polycystic kidney disease-specific rat., Conclusion: Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. (Hepatology 2018;67:1088-1108)., (© 2017 by the American Association for the Study of Liver Diseases.)

Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs, using precise temporal exposure to key biliary morphogens, and we characterized the cells, using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in three-dimensional culture. In vivo, the cells engraft within mouse liver, following retrograde intrabiliary infusion. In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modeling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies.

Background & Aims: In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes., Methods: Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats., Results: Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts., Conclusions: Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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.

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.

Background Information: We have previously showed that: (i) cholangiocytes contain AQP1 (aquaporin 1) water channels sequestered in intracellular vesicles; and (ii) upon stimulation with choleretic agonists such as secretin or dibutyryl-cAMP (dbcAMP), the AQP1 vesicles move via microtubules to the apical cholangiocyte membrane to facilitate osmotically driven, passive water movement (i.e. ductal bile secretion). The aim of the present study was to determine which proteins and mechanisms regulate AQP1 trafficking in cholangiocytes., Results: Using polarized cultured NMCs (normal mouse cholangiocytes) or NRCs (normal rat cholangiocytes) and affinity-purified antibodies, we performed immunofluorescent confocal microscopy on fixed cells or immunoblotting on cell lysates for actin, tubulin, kinesin and dynein, proteins known to regulate intracellular vesicle trafficking. By immunostaining, the appropriate orientation of the actin (i.e. sub-apical) and tubulin (i.e. generalized) cytoskeleton was apparent; kinesin and dynein displayed a homogeneous punctate distribution. Immunoblotting showed kinesin and dynein to be present in both cholangiocyte lysates and in isolated AQP1-containing vesicles. We utilized real-time fluorescence confocal microscopy of NMCs transfected with a GFP (green fluorescent protein)-AQP1 fusion construct in the presence and absence of dbcAMP., Conclusions: Our results provide additional insights into the potential molecular mechanisms of ductal bile secretion.

Although accumulating evidence supports an active role for host cells during Cryptosporidium parvum invasion of epithelia, our knowledge of the underlying parasite-specific processes triggering such events is limited. In an effort to better understand the invasion strategy of C. parvum, we characterized the presence and distribution of the apical organelles (micronemes, dense granules, and rhoptry) through the stages of attachment to, and internalization by, human biliary epithelia, using serial-section electron microscopy. Novel findings include an apparent organized rearrangement of micronemes upon host cell attachment. The apically segregated micronemes were apposed to a central microtubule-like filamentous structure, and the more distal micronemes localized to the periphery and apical region of the parasite during internalization, coinciding with the formation of the anterior vacuole. The morphological observations presented here extend our understanding of parasite-specific processes that occur during attachment to, and internalization by, host epithelial cells.

To explore the mechanisms by which Cryptosporidium parvum infects epithelial cells, we performed a detailed morphological study by serial electron microscopy to assess attachment to and internalization of biliary epithelial cells by C. parvum in an in vitro model of human biliary cryptosporidiosis. When C. parvum sporozoites initially attach to the host cell membrane, the rhoptry of the sporozoite extends to the attachment site; both micronemes and dense granules are recruited to the apical complex region of the attached parasite. During internalization, numerous vacuoles covered by the parasite's plasma membrane are formed and cluster together to establish a preparasitophorous vacuole. This preparasitophorous vacuole comes in contact with host cell membrane to form a host cell-parasite membrane interface, beneath which an electron-dense band begins to appear within the host cell cytoplasm. Simultaneously, host cells display membrane protrusion along the edge of the host cell-parasite membrane interface, resulting in the formation of a mature parasitophorous vacuole that completely covers the parasite. During internalization, vacuole-like structures appear in the apical complex region of the attached sporozoite, which bud out into host cells. A tunnel directly connecting the parasite to the host cell cytoplasm forms during internalization and remains when the parasite is totally internalized. Immunoelectron microscopy showed that sporozoite-associated proteins were localized along the dense band and at the parasitophorous vacuole membrane. These morphological observations provide evidence that secretion of parasite apical organelles and protrusion of host cell membrane play an important role in the attachment and internalization of host epithelial cells by C. parvum.

Background & Aims: Recent studies have showed that proteins associated with polycystic kidney disease (PKD) are expressed in cilia, linking this organelle and cyst formation in the kidney, but involvement of cilia in PKD-related biliary cystogenesis has not been shown. We investigated: (1) the expression of fibrocystin (a product of PKHD1, the autosomal-recessive PKD [ARPKD] gene) in cholangiocyte cilia; (2) biliary cyst formation in an orthologous rat model, PCK; and (3) the effect of Pkhd1 mutation on ciliary structure., Methods: Biliary cystogenesis was assessed by microcomputed tomography. Fibrocystin expression in cholangiocytes of isolated intrahepatic bile ducts (IBDUs) and liver cysts was analyzed by confocal and immunoelectron microscopy, and ciliary structure and length by scanning and transmission electron microscopy. Small interfering RNAs (siRNA) were used to examine the effect of fibrocystin loss on ciliary structure., Results: The biliary tree in the PCK rat was distorted markedly, showing multiple bile duct dilatation and focal budding. In normal IBDUs, each cholangiocyte had a single cilium that expressed fibrocystin. In contrast, cilia in the PCK rat were abnormal with bulbous extensions and diminished length, and were devoid of fibrocystin. In cholangiocytes of normal IBDUs, specific siRNA reduced Pkhd1 messenger RNA by 80%, the length of cilia by 41%, and fibrocystin ciliary expression to an undetectable level., Conclusions: Our results indicate that fibrocystin is expressed in cholangiocyte cilia and that disruption of Pkhd1 by a germ line mutation in the PCK rat or by siRNA in IBDUs results in abnormalities in ciliary morphology and possibly biliary cystogenesis.