Cholangiocyte Cilia Research Articles

Omission of citing prior work

LETTERS TO THE EDITOROmission of citing prior workAngela de Souza Otero, and Gabor SzaboAngela de Souza Otero, and Gabor SzaboPublished Online:01 Jun 2007https://doi.org/10.1152/ajpgi.00161.2007MoreSectionsPDF (30 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat to the editor: We write you regarding an article named “Isolation and characterization of cholangiocyte primary cilia,” published by Huang et al. in September of 2006 (1).The Introduction of this article ends with the following sentences: “Despite these findings, our understanding of the functions of the primary cilium and its role in responding to environmental stimuli are still poorly understood. Thus a detailed characterization of ciliary morphology under normal and pathological conditions, the development of approaches for the isolation of primary cilia, and an identification of ciliary-specific proteins are essential steps to further study the functions of these organelles. In the present study, we provide a detailed multidimensional morphological description of cholangiocyte cilia and describe two novel approaches to isolate enriched ciliary fractions from cultured cholangiocytes.”We were deeply disturbed when we read this article and found that Huang et al. failed to refer to published work from our laboratories. Namely, in Mitchell et al. (2) we reported the first protocol for the isolation, as well as the characterization by biochemical methods and electron microscopy, of primary cilia from kidney cells. Our work was published and widely available online since July 13, 2004. More importantly, if one types the key words “isolation/primary/cilium” in Entrez-PubMed, Mitchell et al. is the third article listed. Therefore, it is difficult to understand how Huang et al. were unable to locate and cite peer-reviewed work that is highly relevant to their research subject.Our chief concern is for the effect of this omission on Kimberly P. Mitchell, a young Ph.D. candidate at our department, who has done most of the work, experimental and intellectual, in our article. Kimberly has already two publications and shows great potential as a future researcher and teacher in physiology. However, we fear for her future enthusiasm for an academic career, given that her work does not receive the acknowledgement it fully deserves.We sincerely hope that you can correct this oversight.REFERENCES1 Huang BQ, Masyuk TV, Muff MA, Tietz PS, Masyuk AI, LaRusso NF. Isolation and characterization of cholangiocyte primary cilia. Am J Physiol Gastrointest Liver Physiol 291: G500–G509, 2006.Link | ISI | Google Scholar2 Mitchell KA, Gallagher BC, Szabo G, Otero A de S. NDP kinase moves into developing primary cilia. Cell Motil Cytoskeleton 59: 62–73, 2004.Crossref | PubMed | ISI | Google Scholar Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Video Abstract Volume 292Issue 6June 2007Pages G1813-G1813 Copyright & PermissionsCopyright © 2007 the American Physiological Societyhttps://doi.org/10.1152/ajpgi.00161.2007PubMed17554135History Published online 1 June 2007 Published in print 1 June 2007 Metrics

Cholangiocyte Cilia Detect Changes in Luminal Fluid Flow and Transmit Them Into Intracellular Ca 2+ and cAMP Signaling

Cholangiocytes have primary cilia extending from the apical plasma membrane into the ductal lumen. While the physiologic significance of cholangiocyte cilia is unknown, studies in renal epithelia suggest that primary cilia possess sensory functions. Here, we tested the hypothesis that cholangiocyte cilia are sensory organelles that detect and transmit luminal bile flow stimuli into intracellular Ca2+ ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP) signaling. Scanning electron microscopy, transmission electron microscopy, and immunofluorescent confocal microscopy of rat isolated intrahepatic bile duct units (IBDUs) were used to detect and characterize cholangiocyte cilia. The fluid flow-induced changes in Ca2+ and cAMP levels in cholangiocytes of microperfused IBDUs were detected by epifluorescence microscopy and a fluorescence assay, respectively. In microperfused IBDUs, luminal fluid flow induced an increase in [Ca2+]i and caused suppression of the forskolin-stimulated cAMP increase. The fluid flow-induced changes in [Ca2+]i and cAMP levels were significantly reduced or abolished when cilia were removed by chloral hydrate or when ciliary-associated proteins polycystin-1 (a mechanoreceptor), polycystin-2 (a Ca2+ channel), and the Ca2+-inhibitable adenylyl cyclase isoform 6 were individually down-regulated by small interfering RNAs. Cholangiocyte cilia are sensory organelles containing polycystin-1, polycystin-2, and adenylyl cyclase isoform 6 through which luminal fluid flow affects both [Ca2+]i and cAMP signaling in the cell. The data suggest a new model for regulation of ductal bile secretion involving cholangiocyte cilia.

Isolation and characterization of cholangiocyte primary cilia

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.

Pathophysiology of Cholangiocyte Cilia

Primary cilia are ubiquitous organelles found in most mammalian tissues. Recent studies suggest that they play an important role in epithelial cells, functioning as mechano-, chemo- and/or osmo-sensors. Moreover, morphological and functional ciliary abnormalities contribute to the development of different pathological conditions, in particular to autosomal dominant and recessive polycystic kidney and liver disease. However, information regarding the functions of primary cilia, particularly in the liver, is limited and how cilia respond to environmental stimuli is still poorly understood. Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, each possess an individual cilium (approximately 7 μm in length) extending from the apical membrane into the ductal lumen with a typical 9+0 axoneme structure. They are heterogeneous in length along the biliary tree; i.e., in the large bile ducts, cilia are 2–3 times longer than in small ones. The strategic location of cholangiocyte cilia extending into the bile duct lumen raises questions about their role in sensing and transducting mechanical, chemical and osmotic signals between molecules in bile and cholangiocytes. Thus, development of novel approaches to study and isolate primary cilia are essential to understand the functions of these organelles. In this presentation, we will review the novel models we have developed to study cholangiocyte cilia, including: isolated perfused bile ducts from rat and mouse; cultured rodent cholangiocyte cell systems to evaluate ciliogenesis and cystogenesis; rodent models of cystic kidney and liver disease; and cell biologic techniques to isolate cilia. The application of these in vitro and in vivo techniques to hepatic cyst formation and expansion will also be emphasized.

Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat

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.

25 Fibrocystin, the ARPKD protein, is localized to cholangiocyte cilia and is disrupted in the orthologous rat model, PCK

25 Fibrocystin, the ARPKD protein, is localized to cholangiocyte cilia and is disrupted in the orthologous rat model, PCK