Recombinant Thrombomodulin Alpha Protects Kidney Graft from Damage after Long Cold Ischemia Time in a Rat Kidney Transplant Model

Abstract

Objectives Thrombomodulin (TM) extensively expresses on the endothelial cells in the steady state and is known to prevent hypercoagulation via combining with thrombin and inactivating its procoagulant activity. TM also has anti-inflammatory effects. In inflammatory situation including ischemia reperfusion injury TM is known to decrease its expression on the endothelial cells. The purpose of this study is to investigate whether perfusate solution saturated with recombinant TM alpha (rTM) can protect the kidney grafts from the damage after long cold ischemia time. Methods We employed a rat syngeneic kidney transplant model using Lewis strain. Donor kidney grafts were preserved in a cold UW perfusate solution saturated with rTM (Group A, n=7), or not (Group B, n=8) for 24 hours. Then kidney transplantation (Lewis to Lewis) was performed. Blood and urine samples were sequentially collected (post operative day 1, 2, and 7) to measure creatinine clearance. Neutrophil gelatinase-associated lipocalin (NGAL), an acute kidney injury marker, was also measured by ELISA. Kidney graft samples in an acute phase (2 hours after operation) were collected to investigate the grade of tubular damage. TUNEL assay by immunofluorescence was performed. Immunohistochemistry (IHC) for TM was also performed. Results IHC revealed that the expression of TM on kidney grafts after 24-hour cold ischemia was extensively reduced compared with those after 0-hour ischemia. Creatinine clearance (ml/min/kg) in Group A was significantly ameliorated compared with that in Group B: mean (SD), 95% CI of Group A vs Group B; 0.68 (0.33), 0.41-0.96 vs 0.23 (0.13), 0.11-0.35 on POD 1; 1.22 (0.40), 0.86-1.59 vs 0.59 (0.30), 0.31-0.86 on POD 2; and 2.63 (1.41), 1.45-3.81 vs 1.17 (0.69), 0.47-1.87 on POD 7; p=0.02, two-way repeated measured ANOVA. Furthermore, serum levels of NGAL (mg/ml) in Group A were significantly lower than those in Group B: mean (SD), 95% CI of Group A vs Group B; 6.5 (1.7), 0.70-4.71 vs 9.1 (1.1), 7.8-10.4 on POD 1; and 4.3 (2.7), 1.5-7.1 vs 8.9 (1.8), 6.6-11.1 on POD 2; p=0.021, two-way repeated measured ANOVA. Fewer damaged tubular cells and apoptosis positive cells at a very acute phase were observed in Group A (Figure 1), compared with in Group B (Figure 2). Conclusion Perfusion with rTM significantly attenuates kidney graft damage after long cold ischemia time by abrogating apoptosis in tubular cells