Primary cilia are essential for biliary regeneration following hypoxic injury

BACKGROUND AND AIMS: Biliary complications (BC) are one of the most common complications following orthotopic liver transplantation (OLT). Up to 25% of OLT recipients will develop BC, a major factor determining long term patient survival. BC have been associated with pre-transplant cold storage, hypoxia and insufficient regeneration of biliary epithelial cells (BECs) post OLT. BECs have primary cilia (PC), unique organelles that are crucial to sense the extracellular environment and regulate cell proliferation. In this study we investigate the impact of PC on regeneration in the setting of BC post OLT. METHODS: Human biopsies were used to study the structure/ function of PC in liver transplant recipients with (N=7) and without BC (N=12). We developed novel murine models of liver pre-transplantation stages, where we can study the role of PC in BECs, using conditional ablation of PC (K19CreERT Kif3aflox/flox mouse model). Lastly, Tubastatin A was used to stabilise PC and promote BEC regeneration, in a combination of in vitro and in vivo models of cold storage. RESULTS: BECs` PC are shortened prior to OLT in livers that later develop BC (p=0.006). We identify hypoxia as the main molecular mechanism responsible for this damage during cold-storage conditions. Hypoxia induced shortening/ loss of PC triggers the onset of cellular senescence, impairing the regenerative capacity of BECs in vitro and in vivo. We also explore how hypoxia-independent genetic ablation of PC induces cellular senescence, indicating the presence of a feedback loop that negatively impacts the regenerative response of BECs following OLT. Inhibition of cellular senescence (using p21-/- mice or by administration of senolytics) preserves PC during cold storage (p=0.0004), improving BEC regeneration. We finally show how stabilisation of PC during cold storage improves BEC proliferation in vitro (p=0.0005). CONCLUSIONS: pre-transplantation hypoxic conditions trigger loss of PC in BECs, impairing biliary regeneration through cellular senescence. Our results indicate that PC represent a potential novel therapeutic target to improve biliary regeneration and prevent BC development during liver transplantation