J. Zhang1, N. Singh1, W. A. Dorsett-Martin1, C. D. Anderson1, T. M. Earl1 1University Of Mississippi,Division Of Transplant And Hepatobiliary Surgery, Department Of Surgery,Jackson, MS, USA
Introduction: The discrepancy between need and supply of liver allografts has led to alternative strategies to increase the organ pool. One strategy, utilization of donation after cardiac death (DCD) donors, is severely limited by intolerance of the biliary system to warm ischemia resulting in ischemic cholangiopathy which reduces longevity of these allografts. We hypothesize that warm ischemia followed by cold storage and subsequent reperfusion injury results in epithelial to mesenchymal transformation (EMT) and increased extra-cellular matrix production in human cholangiocytes.
Methods: Primary human cholangiocytes were exposed to no treatment (NT), hypoxic storage at 4°C in Histidine-tryptophan-ketoglutarate (HTK) solution for 7 hours (CIR group) or 3 hours hypoxic normothermic storage in PBS followed by 4 hours hypoxic storage in HTK at 4°C (WIR group). Following 7 hours hypoxic storage (CIR and WIR), cholangiocytes were reperfused with media under normoxic, normothermic conditions. Cells were harvested at 24, 72 and 144 hours post-reperfusion. Quantitative real-time PCR for cholangiocyte epithelial marker AQP1, mesenchymal cell markers αSMA and S100α4, and COLA1 was performed at each time point and normalized to house-keeping gene 18S.
Results: One hundred forty-four hours after reperfusion, AQP1 expression is significantly diminished for WIR (WIR 6.2±0.3 vs. NT 210.6±33.8; p<0.001) but not after CIR (CIR 182.4±28.7 vs. NT 210.6±33.8 vs.; p=0.33) and was significantly lower in WIR compared to CIR (WIR 6.2±0.3 vs. CIR 182.4±28.7; p<0.001). Interestingly, following CIR cholangiocytes lost Aqp1 mRNA at 24 and 72 hours but regained it by 144 hours. Mesenchymal marker αSMA mRNA was significantly increased at 144 hours post-reperfusion in cholangiocytes exposed to WIR (NT 2.80±.3 vs. WIR 47.2±2.7; p<0.001), but decreased following CIR (NT 2.8±.3 vs. CIR 1.5±0.6; p=0.02). This difference was not seen at 24 and 72 hours. COL1A1 mRNA increased dramatically at 144 hours following WIR but not after CIR (WIR 130.0±6.0 vs. CIR 5.1±0.3; p<0.001), expression following CIR injury did not differ from NT group at any timepoint. S100α4 trended toward significantly higher expression 144 hours following WIR (WIR 6.0±1.6, CIR 2.8±1.3; p=0.059).
Conclusion: Following warm-ischemia with subsequent cold storage and reperfusion injury human cholangiocytes lose epithelial cell markers, gain mesenchymal cell markers, and have increase expression of collagen 1 mRNA. Further insight into this process may result in better utilization and success of DCD liver allografts.
