There is considerable interest in understanding the mechanistic relationships between biliary damage and portal fibrosis, the main mechanism of progression in chronic cholangiopathies. Congenital Hepatic Fibrosis (CHF) and Caroli disease (CD) are genetic cholangiopathies caused by mutations in PKHD1, the gene encoding for fibrocystin, characterized by biliary dysgenesis, segmental ductal dilations and progressive portal fibrosis with portal hypertension. In CHF and CD, cholangiocyte dysfunction and portal fibrosis are caused by a genetic defect in the biliary epithelium, rather than by necroinflammatory damage and thus, represent a model disease for elucidating the role of cholangiocytes in portal fibrosis of biliary diseases. We propose to study these mechanisms in a mouse model of CHF/CD, harbouring a deleting mutation in Pkhd1 (Pkhd1del4/del4 mice). Our published and preliminary data have established that in Pkhd1del4/del4 mice biliary fibrosis develops in conjunction with accumulation of a peribiliary cell infiltrate by macrophages and by aSMA-negative, but collagen positive cells like fibrocytes. Furthermore, we have shown Pkhd1del4/del4 cholangiocytes are characterized by an increased PKA-dependent phosphorylation of β-catenin at Ser675, the nuclear translocation of pSer-675-β-catenin and its increased transcriptional activity. ß-catenin interacts with FXR inhibiting its anti-inflammatory signaling and thereby activating NF-kB and the inflammasome-dependent secretion IL-1β and consequently of CXCL1 and CXCL10, that are, in turn, able to attract macrophages. By inhibiting CXCR3, the cognate receptor of CXCL10, or by administration of an FXR agonist (obeticholic acid) macrophage infiltration was significantly reduced as well as cyst growth, spleen size and liver fibrosis. Finally, in Pkhd1del4/del4 cholangiocytes, nuclear shuttling of YAP is a pre-requisite for β-catenin activation and thus, for the expression of the pro-fibrogenic mediators CTGF, CXCL1, and CXCL10. Based on these observations, we propose that, when fibrocystin is defective, the interplay among β-catenin, YAP, FXR signalling regulates the secretion from the biliary epithelium of several chemokines that orchestrate sequential changes in the peribiliary infiltrate and are responsible for the establishment of portal inflammation and fibrosis. To demonstrate this novel hypothesis, we will investigate in specific aim 1 the relationship among YAP, β-catenin and FXR signalling in Pkhd1del4/del4 mice, their role in controlling, cyst growth, inflammation, fibrosis and their relevance as therapeutic targets. While in specific aim 2 we will investigate the nature of the pericystic infiltrate in Pkhd1del4/del4 mice, and its dynamic changes during the establishment of fibrosis and the effects of treatment strategies. These studies will provide a new model for role of cholangiocyte dysfunction in portal fibrosis. Knowledge of the regulatory signaling could lead to new therapeutic strategies.

Narrative Autosomal Recessive Polycystic Kidney Disease (ARPKD), Congenital Hepatic Fibrosis (CHF) and Caroli's Disease (CD), are genetic disorders of the liver and kidney caused by deficiency in fibrocystin, the protein encoded by the PKHD1 gene. In this proposal, we will study the mechanisms of portal fibrosis in a mouse model of CHF/CD. These studies have a strong translational potential and proximity to cure, understanding the mechanism linking cholangiocyte dysfunction to fibrosis will generate therapeutic strategies not only of patients with CHF/CD, but also of those suffering from other chronic liver diseases.