Coagulation in kidney graft ischemia reperfusion injury: protective effect of thrombin inhibition against chronic kidney graft inflammation and tubular atrophy
Résumé
Introduction and aims: Recent advances in the domains of immunosupression and patient care have not substantially impacted the rate of chronic kidney graft failure. Ischemia reperfusion injury (IRI) is believe to be a pivotal factor in the long term well being of the graft and the next problematic to solve in the quest for long lasting kidney transplants. We formulate the hypothesis that coagulation is a major pathway of IRI, and thus that targeting coagulation during conservation will improve the graft chronically.
Methods: We used a large white pig autologous kidney transplantation model, with Melagatran treatment in the peri-conservation period to inhibit thrombin. Kidneys undergo warm ischemia for 60 min before being harvested and preserved at 4°C for 24 hours using University of Wisconsin solution, mimicking non heart beating donor conditions.
Results: There was a direct positive impact of Melagatran treatment on tubular atrophy (23.81.1 vs. 51.32.0% in UW alone, p<0.001) and a protection of functional parameters such as GFR (103.03.2 vs. 44.02.5mL/min in UW alone, p<0.001) and proteinuria (0.40.1 vs. 3.50.5g/24h in UW Alone, p<0.001).We determined that Melagatran treatment significantly reduced kidney inflammation at 3 month, as evidenced by immunohistochemical score reduction in CD3+ (6.34±2.17 vs 16.55±5.80 cells by field, in UW Alone, p<0.001).In addition, exposure to Melagatran decreased endothelial cell activation, shown by a reduction in expression of Thrombospondin (0.17±0.05 vs. 3.26±0.31 Folds to Control, p<0.001), Pselectin (0.36±0.16 vs. 2.98±0.76 folds, p<0.001), as well as complement activation shown by C3 (0.43±0.28 vs. 3.39±0.53 folds, p<0.001).Furthermore, RT-PCR analysis determined that markers of Th1 immunity, such as IFNγ (0.29±0.06 vs. 2.18±0.19 folds, p<0.001), TNFα (0.86±0.44 vs. 7.17±4.10 folds, p<0.001) and IL-2 (0.56±0.21 vs. 2.23±0.73 folds, p<0.01) were significantly reduces in Melagatran-treated grafts. A similar finding was found with markers of Th2 immune response, with IL-1Rn (2.79±1.24 vs. 748.95±410.22 folds, p<0.01) and IL-10 (1.88±0.26 vs. 0.20±0.07 folds, p<0.001), as well as Th17 marker IL-17 (4.58±2.60 vs. 0.68±0.13 folds, p<0.01).This reduction in immune invasion and activation was concomitant with a significant increase in regeneration marker VEGF protein expression (0.82±0.11 vs. 0.26±0.20 Densitometric unit, p<0.01) as well as a reduction of Notch 4 mRNA expression (0.61±0.38 vs. 5.90±1.82 folds, p<0.001).
Conclusions: In summary, inhibition of Thrombin during preservation of a NHBD graft was determinant in preserving the tubular integrity and function through a protective effect against pro-inflammatory cytokines and chemokines expression and a concomitant reduction of immune cell invasion. Coagulation thus appears as a keystone of IRI and a critical target for the development of therapeutic strategies to improve kidney transplant patient's quality of life.