Search

Background & Aims: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target., Methods: ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro., Results: The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis., Conclusions: Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Polycystic liver disease (PLD) is a group of genetic disorders with limited treatment options and significant morbidity. Hepatic cysts arise from cholangiocytes exhibiting a hyperproliferative phenotype. Considering that hyperproliferation of many cell types is associated with alterations in autophagy, we hypothesized that autophagy is altered in PLD cholangiocytes, contributes to hepatic cystogenesis, and might represent a potential therapeutic target. We employed functional pathway cluster analysis and next-generation sequencing, transmission electron microscopy, immunofluorescence confocal microscopy, and western blotting to assess autophagy in human and rodent PLD cholangiocytes. A three-dimensional culture model was used to study the effects of molecular and pharmacologic inhibition of autophagy on hepatic cystogenesis in vitro, and the polycystic kidney disease-specific rat, an animal model of PLD, to study the effects of hydroxychloroquine, a drug that interferes with the autophagy pathway, on disease progression in vivo. Assessment of the transcriptome of PLD cholangiocytes followed by functional pathway cluster analysis revealed that the autophagy-lysosomal pathway is one of the most altered pathways in PLD. Direct evaluation of autophagy in PLD cholangiocytes both in vitro and in vivo showed increased number and size of autophagosomes, lysosomes, and autolysosomes; overexpression of autophagy-related proteins (Atg5, Beclin1, Atg7, and LC3); and enhanced autophagic flux associated with activation of the cAMP-protein kinase A-cAMP response element-binding protein signaling pathway. Molecular and pharmacologic intervention in autophagy with ATG7 small interfering RNA, bafilomycin A 1 , and hydroxychloroquine reduced proliferation of PLD cholangiocytes in vitro and growth of hepatic cysts in three-dimensional cultures. Hydroxychloroquine also efficiently inhibited hepatic cystogenesis in the polycystic kidney disease-specific rat., Conclusion: Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. (Hepatology 2018;67:1088-1108)., (© 2017 by the American Association for the Study of Liver Diseases.)

Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein-coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m-tolyl 5-chloro-2-[ethylsulfonyl] pyrimidine-4-carboxylate [SBI-115]), and (3) a combination of SBI-115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA-treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5 -/- ;Pkhd1 del2/del2 mice. Compared to control, expression of TGR5 and Gα s (but not Gα i and Gα q ) proteins was increased 2-fold to 3-fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5-deficient TGR5 -/- ;Pkhd1 del2/del2 mice. TGR5 expression and its colocalization with Gα s were increased ∼2-fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ∼30% in cystic cholangiocytes after treatment with SBI-115 alone and by ∼50% when SBI-115 was combined with pasireotide., Conclusion: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197-1218)., (© 2017 by the American Association for the Study of Liver Diseases.)

In the polycystic liver diseases (PLD), genetic defects initiate the formation of cysts in the liver and kidney. In rodent models of PLD (i.e., the PCK rat and Pkd2(WS25/-) mouse), we have studied hepatorenal cystic disease and therapeutic approaches. In this study, we employed zebrafish injected with morpholinos against genes involved in the PLD, including sec63, prkcsh, and pkd1a. We calculated the liver cystic area, and based on our rodent studies, we exposed the embryos to pasireotide [1 μM] or vitamin K3 [100 μM] and assessed the endoplasmic reticulum (ER) in cholangiocytes in embryos treated with 4-phenylbutyrate (4-PBA). Our results show that (a) morpholinos against sec63, prkcsh, and pkd1a eliminate expression of the respective proteins; (b) phenotypic body changes included curved tail and the formation of hepatic cysts in zebrafish larvae; (c) exposure of embryos to pasireotide inhibited hepatic cystogenesis in the zebrafish models; and (d) exposure of embryos to 4-PBA resulted in the ER in cholangiocytes resolving from a curved to a smooth appearance. Our results suggest that the zebrafish model of PLD may provide a means to screen drugs that could inhibit hepatic cystogenesis.