Role of epithelial to mesenchymal transition in genetic cholangiopathies related to fibrocystin deficiency

Autosomal recessive polycystic kidney disease (ARPKD), congenital hepatic fibrosis (CHF) and Caroli disease (CD) are a group of genetically related disorders affecting bile ducts and kidney tubules. These diseases are characterized by the progressive formation of fluid-filled cysts and fibrosis in kidneys and liver. Beside the cyst’s mass effect, severe life threatening complications can arise i.e. recurrent cholangitis in the liver and the hazard of the cysts becoming infected or rupture. The most severe clinical consequences however, are, actually, due to the massive portal fibrosis which causes portal hypertension, hypersplenism, and the formation of varices and ascites in the esophagus, increasing the risk for cholangiocarcinoma. ARPKD, CHF and CD are all related to mutations in the PKHD1 gene, acronym for Polycystic Kidney and Hepatic Disease 1. The gene encodes for Fibrocystin (FPC), a protein whose function is still not fully known, normally expressed mainly on the primary cilia of renal tubular and biliary epithelial cells, consistently with the sites primarily affected by the disease, however the mechanistic relationship between a FPC defect and the development of portal fibrosis are still not fully understood. Other cystic diseases are related to defects in ciliary proteins: the Adult Dominant Polycystic Kidney Disease (ADPKD) is caused by mutations in PKD1 or PKD2 encoding respectively for Polycystin 1 and Polycystin2 (PC1 and PC2) and is similarly characterized by cysts formation in kidney and liver, but generally does not present scarring liver fibrosis. The purpose of this project was to investigate the mechanisms leading to portal fibrosis in ARPKD/CHF/CD. In particular our hypothesis was that the defective fibrocystin could activate a program of epithelial mesenchymal transition (EMT) in cholangiocytes that would acquire mesenchymal characteristics, including cells motility and the ability to secrete extracellular matrix proteins, and thus contribute to the development of massive fibrosis in ARPKD. To that end we studied a transgenic mouse harboring a deletion in the Pkhd1 gene (Pkhd1del4/del4 Gallagher et al., 2008), that mimics the human disease ARPKD. Mice defective in Polycystin (Pkd2KO), mmicking ADPKD, and WT mice were used as control. We examined immunophenotipic markers of EMT and compared liver samples from biopsy of healthy and diseased patients and also from the WT mice and the diseases models. We also investigated motility and invasiveness of cholangiocytes isolated from Pkhd1 mice and their profile of cytokines secretion in order to explore their capabilities in epithelial mesenchymal cross-talk. We demonstrated that cholangiocytes in ARPKD display some phenotypic characteristics of mesenchymal cells, suggestive of a partial transition EMT and this may contribute to fibrosis, in association with a hyper-secretive profile of the biliary epithelium that may activate fibrocytes recruited in proximity of biliary cysts