| dc.description.abstract | Autosomal recessive polycystic kidney disease (ARPKD) is a rare genetic disorder that occurs in 1:20,000 live births. Patients present with a broad spectrum of symptoms involving the kidneys, liver, and pancreas. Renal manifestations are characterized by the presence of cysts that are derived from dilated collecting ducts. All patients with ARPKD develop some degree of congenital hepatic fibrosis (CHF) at birth. CHF is characterized by bile duct dilation resulting in eventual development of cysts and pericystic fibrosis in the liver. Accompanying cyst growth and fibrosis, recent reports suggest that inflammation is also present, and likely contributes to disease pathogenesis and/or progression. Therefore, three interrelated processes form a ‘pathogenic triumvirate’ in CHF/ARPKD: cell proliferation (cyst growth), fibrosis, and inflammation. Aside from management of symptoms, and liver and/or kidney transplant, no effective pharmacologic therapies exist for CHF/ARPKD. Therefore, the goal of this research was to explore additional mechanisms of hepatic cyst and pericystic fibrosis progression in CHF/ARPKD to identify new points of therapeutic intervention. We first characterized the gene expression in CHF/ARPKD by performing RNA-seq in whole liver from Sprague Dawley (SD) and PCK rats at postnatal day (PND) 15, 20, 30, and 90. Upstream regulator and pathway analysis of differentially expressed genes was subsequently conducted using Ingenuity Pathways Analysis (IPA). We found the number of differentially expressed genes was increased in PCK rats over time. Among the top genes upregulated in PCK rats, Cd44, the most well-described receptor for the extracellular matrix glycosaminoglycan hyaluronan, and Tead4, a transcription factor required for regulation of genes downstream of the Hippo-kinase- yes-associated protein pathway, were consistently upregulated from PND 15 to PND 90. This led us to investigate the role of hyaluronan and yes associate protein (YAP) in CHD/ARPKD. Upstream regulator analysis predicted activation of several pro-proliferative and pro-inflammatory transcription factors in PCK rats including Ctnnb (catenin beta-1), Mtpn (myotrophin), Tcf7l2 (transcription factor 7 like 2), and several Stat (signal transducer and activator of transcription) family members (z score ≥ 2). Further, PCK rat livers exhibited inhibition of Smad7, an inhibitory SMAD, which is expected to result in a pro-fibrotic effect by preventing the Smad7-induced negative regulation of TGF-β/SMAD signaling. Additionally, a significant suppression of Hnf4a, the master regulator of hepatic differentiation, was also observed in the PCK rat livers. Mast cells (MCs) are immune cells involved in many liver diseases and release mediators such as histamine from preformed granules found in their cytoplasm. In fact, previously published data demonstrate that histamine induces proliferation of cholangiocytes, which are the precursors of the cyst wall epithelial cells (CWEC) in CHF. We observed MC infiltration in the hepatic periportal areas, but not in the kidneys, in polycystic kidney (PCK) rats. Therefore, we hypothesized that MCs contribute to hepatic cyst growth in PCK rats. To test this hypothesis, we treated PCK rats with one of two different mast cell stabilizers, cromolyn sodium (CS) or ketotifen, or saline from PND 15 to PND 30. We confirmed that CS and ketotifen treatment decreased MC degranulation in liver. Interestingly, we observed an increase in liver/body weight ratio after CS and ketotifen treatment paralleled by a significant increase in cyst size. In contrast, we saw a decreased kidney/body weight ratio paralleled by a significant decrease in individual cyst size after CS treatment. We excluded the direct effect of CS on cyst growth by treating isolated cyst wall epithelial cells (CWECs) and a mouse model (no MC observed) of ARPKD with CS. Taken together, these data demonstrate that hepatic and renal cysts are differentially regulated by MC granule contents in PCK rats. RNA-seq analysis suggested that TEA Domain Transcription Factor 4 (Tead4), a transcription factor in Hippo signaling pathway, is upregulated in PCK rats. Hippo signaling pathway is a conserved signaling that plays critical roles in liver size control and cell proliferation. Our preliminary data suggested that YAP, the downstream effector of the Hippo signaling pathway, was increased in proliferating CWECs in PCK rats and in human ARPKD patients. Consistently, there was increased expression of YAP target genes, Ccnd1 (cyclin D1) and Ctgf (connective tissue growth factor), in PCK rat livers. Extensive expression of YAP and its target genes was also detected in human CHF/ARPKD liver samples. Therefore, we hypothesized that YAP plays a role in CWEC proliferation in CHF/ARPKD. We inhibited YAP activity pharmacologically using verteporfin and genetically using short hairpin (sh) RNA in primary liver CWECs. We found that CWEC proliferation was significantly reduced. These data indicate that increased YAP activity, possibly through dysregulation of the Hippo signaling pathway, is associated with hepatic cyst growth in CHF/ARPKD. In RNA-seq analysis, we found that Cd44 was upregulated in PCK rats from PND 15 to PND 90. Hyaluronan (HA), a CD44 ligand, is a ubiquitous, anionic glycosaminoglycan present in the extracellular matrix (ECM). HA is implicated in liver injury, inflammation, and fibrogenesis. Our study showed an increased HA level in livers from PCK rats and polycystic liver disease patients relative to healthy controls. HA accumulation could be due to an increased Has1 (a HA synthase) expression and decreased expression of HA degrading enzymes. Therefore, we hypothesized that HA plays a pathogenic role in progression of CHF/ARPKD. Future studies can be performed to test the role of HA in CHF/ARPKD by genetically deleting Has enzymes or the HA receptor, CD44, in PCK rats or in Pkhd1 mutant mice Taken together, CHF/ARPKD is a hepatobiliary disease that is regulated by a ‘pathogenic triumvirate’ (cell proliferation/cyst growth, inflammation, fibrosis). My studies aimed to discover novel mechanisms driving CHF/ARPKD and investigated how MC, and YAP, regulated the ‘pathogenic triumvirate’ in CHF/ARPKD. Moreover, we identified and upregulation of HA and Cd44 in PCK rats the significance of which can be explored in future studies. Overall, these studies have uncovered several new avenues for additional research into the mechanisms of cell proliferation/cyst growth, inflammation, and fibrosis, in CHF/ARPKD and from which future therapeutic strategies can be developed. | |
