Is coagulation the key to ischemia reperfusion injury treatment? Thrombin inhibition rescues chronic kidney fibrosis in autologous kidney transplantation model
Résumé
Introduction and aims: Chronic kidney fibrosis remains the main issue in kidney transplantation. Ischemia reperfusion injury (IRI) is a key element for the long term condition of the graft. We propose that coagulation, a phenomenon at the center of IRI, is a critical factor in determining the gravity of the injury and represent a determinant target for therapy.
Methods: We studied the effect of Melagatran, a thrombin inhibitor, on the chronic consequences of IRI in an autologous kidney transplantation model, using large white pigs. To mimic non heart beating donor conditions, kidneys underwent warm ischemia for 60 min before being preserved at 4°C for 24 hours using University of Wisconsin solution. Kidneys weretreated with Melagatran (3mg/kg/10min intravenously) 30 min before warm ischemia and preserved in UW to which was added 0.3 mg/L Melagatran.
Results: Treatment with Melagatran during conservation drastically improved survival at 3 month (89% vs. 20% in UW Alone grafts), as confirmed by an amelioration of plasma creatinine (130±6.5 μmol/L vs. 435±47.5 μmol/L in UW Alone, p<0.01), proteinuria (0.30±0.05 vs. 4.16±0.78 g/24h, p<0.01) and by a critical reduction in interstitial fibrosis, measured by Sirius Red staining (6.8±0.2 % vs. 37.3±2.8 %, p<0.01). Mechanistic analysis using proteomic and genomic technologies revealed a profound protective effect of Melagatran: treatment appeared to reduced the expression of TGFß as well as the activation level of TGFß pathway phospho-Smad3 (0.35±0.02 vs. 1.65±0.24 densitometric units, DU, p<0.01), as well as Smad 4 (0.26±0.10 vs. 0.64±0.14 DU, p<0.05) and effector CTGF (0.07±0.02 vs. 0.67±0.19 DU, p<0.05). Moreover, we defined a reduction in fibrinogenesis, evidenced by downregulation of PAI-1 protein (0.09±0.02 vs. 0.29±0.06 DU, p<0.01) as well as gene (0.27±0.05 vs. 7.18±2.14 folds to Control, p<0.001) expression, possibly leading to an lowering of kidney hypoxic injury, which we confirmed by measuring the expression of hypoxia induced factor alpha (HIFα, (0.34±0.17 vs. 6.30±4.23 folds, p<0.05). In addition, Melagatran protected the graft against epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis, as shown by reduction in S100A4 expression (0.59±0.27 vs. 3.42±1.19 folds, p<0.001) and Vimentin staining. Finally, oxidative stress was reduced, as expression of Nox2 (0.43±0.28 vs. 1.59±0.11 folds, p<0.001) and iNOS (0.30±0.11 vs. 2.11±0.25 folds, p<0.001).
Conclusions: We conclude that inhibition of thrombin during kidney graft conservation significantly protected against IRI and reduced chronic graft fibrosis, with a defined positive effect on survival. Coagulation is thus a determining element of IRI, and the development of therapeutic approaches to reduce its impact is critical to the improvement of chronic graft function and patient quality of life.