Identification of new drug targets to prevent ischemia-induced bile toxicity using a human biliary organoid model

F.J.M. Roos, M. Bijvelds, H. de Jonge, H.J. Metselaar, K. Burka, J.N.M. IJzermans, M.M.A. Verstegen, L.J.W. van der Laan

Wednesday 13 march 2019

16:20 - 16:30h at Koningin Máximazaal

Categories: Best abstracts, Parallelsessie

Parallel session: Parallelsessie VIII – Best abstracts II

Background: Ischemic cholangiopathy (IC) is the most severe complication after liver transplantation (LT). Hypoxia during transplantation might accelerate bile toxicity in cholangiocytes, due to insufficient protection by CFTR-related bicarbonate (NH₃) secretion and thereby contribute to IC development. Liver-derived organoids (LDOs) resemble cholangiocyte-like cells which could be suitable models for testing this hypothesis. We investigated if LDOs have functional cholangiocyte transport-channels and could serve as a model for hypoxia-related biliary injury as well as be used for IC related drug-discovery purposes.

Methods: LDOs, cultured from donor livers reserved for LT, were analyzed on gene- and protein level for cholangiocyte-specific transporters (CFTR and AE2). Channel functionality was tested using an Ussing-chamber assay in 2D-grown organoids (n=42). Forskolin (cAMP-activator) added to the apical side of the cells, initiated CFTR activation which was specifically inhibited by GlyH. Hypoxic conditions were achieved by nitrogen gas (95%N₂/5%CO­₂) exposure. To study bile-related toxicity, undiluted bile was added under oxygen and hypoxic conditions and cell death was analyzed. Finally, compounds were tested for the ability to abrogate the hypoxic-induced inhibition of CFTR.

Results: CFTR was expressed in all LDOs on both gene (qPCR) and protein (Western blot) level. Moreover CFTR could be activated by Forskolin in the Ussing chamber set-up. CFTR activity was lower when measured under hypoxic conditions compared to oxygen (1.66± 0.45 vs. 4.18 ± 0.48, p=0.005). Furthermore, a significant decrease in activity was observed when the same 2D-organoids were switched from oxygenated to hypoxic conditions (8.00 ±1.19 vs. 5.89±1.26, p=0.02). Further experiments showed that NH₃is the driving factor when CFTR is activated in LDOs, suggesting that during hypoxia less NH₃is excreted into the bile. When 2D-grown LDOs were exposed to bile, it resulted in more cell death in hypoxic versus oxygen conditions (31.2%±4.32 vs. 19.18±4.81, p=0.04). Most importantly, addition of compound C (cAMP-inhibitor) was able to rescue CFTR activity under hypoxic conditions.

Conclusions: LDOs provide an excellent model to study cholangiocyte-transporters. We demonstrate that hypoxia inhibits CFTR-related bicarbonate secretion and cAMP-inhibitor compound C can restore this. This encourages further clinical studies to test whether cAMP-inhibitors can prevent hypoxia-related biliary injury during graft preservation and after LT.