Knockout of the secretin/secretin receptor axis delays regeneration of small and large cholangiocytes by enhanced senescence following 70\% partial hepatectomy (PH)

Limited data exists regarding the neuroendocrine factors that regulate the renewal of the biliary tree after PH. The secretin (SCT)/secretin receptor (SR) axis is normally expressed only by large cholangiocytes. However, during the damage of large bile ducts small cholangiocytes (more resistant to injury) replenish the large damaged ducts by de novo acquisition of large biliary phenotypes. Cellular senescence is a state of irreversible cell cycle arrest involved in biliary diseases including primary sclerosing cholangitis and primary biliary cirrhosis. Large, senescent cholangiocytes are the target cells in the cholestatic models of bile duct ligation and MDR2 KO (PSC). Hypothesis: in hepatectomized SCT/SR double knockout (DKO) mice there is reduced regeneration of small and large bile ducts due to enhanced senescence. Methods: The studies were performed in normal wild type (WT) and SCT/SR DKO mice at 0, 3, 6 hours and 1, 3, 7, and 14 days after sham or 70% PH. Liver injury was evaluated by: (i) H&E staining in liver sections; and (ii) measurement of serum levels of transaminases. We evaluated biliary proliferation by: (i) immunohistochemistry (IHC) for PCNA and intrahepatic bile duct mass (IBDM) by CK-19 IHC in liver sections; and (ii) qPCR for PCNA and CK-19 in total liver samples and small and large cholangiocytes. Cellular senescence was evaluated by SA-β-Gal staining in liver sections and qPCR for P18, PAI-1 and CCl2 in total liver samples and small and large cholangiocytes. Results: Small cholangiocytes from PH WT mice displayed less senescent features compared to large cholangiocytes. In PH WT mice, small less senescent cholangiocytes regenerate as early as 1 hr and rebuild IBDM within 1 day of PH, whereas large regenerate as early as 6 hr and rebuild IBDM at a lower rate (after 3-7 days of PH). Large cholangiocytes from DKO PH animals displayed increased expression of senescent markers relative to PH WT mice. In DKO PH mice, there was reduced regeneration time (by PCNA) and regrowth (by CK-19) of small and large bile ducts concomitant with enhanced expression of senescence markers (p18, PAI-1 and CCL2) in large cholangiocytes as well as in small cholangiocytes that display normally low levels of senescence. In SCT/SR double KO mice, at day 14 of PH IBDM returned at only 50% of the original biliary mass (0.238 ± 0.014, sham, vs. 0.128 ± 0.007, SCT/SR KO, p<0.05). There was liver damage and enhanced transaminases levels (more evident at day 14) in PH SCT/SR double KO compared to WT mice. Conclusion: Regression of biliary senescence (by upregulation of SCT/SR axis) may be a key approach for accelerating biliary repair post liver transplantation.