Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat.
OBJECTIVE
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.
Masyuk
Tatyana V
TV
Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA.
Huang
Bing Q
BQ
Ward
Christopher J
CJ
Masyuk
Anatoliy I
AI
Yuan
David
D
Splinter
Patrick L
PL
Punyashthiti
Rachaneekorn
R
Ritman
Eric L
EL
Torres
Vicente E
VE
Harris
Peter C
PC
LaRusso
Nicholas F
NF
D000818:Animals; D001652:Bile Ducts / Q000378:metabolism* / Q000473:pathology*; D001653:Bile Ducts, Intrahepatic / Q000378:metabolism; D015153:Blotting, Western; D002923:Cilia / Q000378:metabolism / Q000648:ultrastructure*; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D020868:Gene Silencing; D018095:Germ-Line Mutation; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D016253:Microscopy, Immunoelectron; D017044:Polycystic Kidney, Autosomal Recessive / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011922:Rats, Mutant Strains; D011956:Receptors, Cell Surface / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
C479541:PKHD1 protein, rat; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface