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Étude des effets potentiels du récepteur MerTK sur le développement des lésions liées à une ischémie/reperfusion rénale chez le rat

Abstract : Ischemia reperfusion is characterized by two distinct phases: a cessation then a return of blood supply and may occur in some pathological situations as well as during organ transplantation. This sequence of events can induce cell death, formation of cell debris and microparticles. Apoptotic bodies and microparticles are characterized by the exposure of phosphatidylserine on the outer membrane bilayer. Ischemia reperfusion-induced cell damage lead to the activation of inflammation and to leukocyte recruitment through the release of cytokines/chemokines. Recruited macrophages may phagocyte cellular debris and microparticles. It has previously been established that the MerTK tyrosine kinase receptor, expressed by several cell types including macrophages, plays a major role in the phagocytosis process. The vitamin K-dependent anticoagulant factor Protein S is the MerTK receptor ligand. It binds by its C-terminus to MerTK receptor on the surface of macrophages and by its N-terminus to phosphatidylserins exposed by apoptotic bodies and microparticles bridging thereby the phagocytes (macrophages) to the substrate (phosphatidylserine exposing bodies). The objective of our study is to characterize the role of MerTK in both the development of lesions induced by renal ischemia reperfusion and in post-ischemic repair. We used a model of RCS (Royal College of Surgeons) rats characterized by a defective MerTK due to a natural mutation in the MerTK gene. Simulation of renal ischemia reperfusion is performed by clamping the artery and renal vein. The blood and kidneys are collected and, using ELISA, Western Blot and biochemical assays, markers of kidney function and lesions, inflammation and leukocyte recruitment are determined. Histological lesions on renal sections are monitored by HES-PAS staining, and the location and quantification of leukocyte recruitment were assessed by immunohistochemistry technique. We also used flow cytometry to quantify microparticles of platelet, endothelial and leukocyte origin in plasma. Finally, we studied the possible role of MerTK in phagocytosis in vitro, by co-incubation of apoptotic bodies with monocytes/macrophages, derived from the blood of MerTK-deficient RCS rats or control rats normally expressing this receptor.Our work has made it possible to develop a technique for detecting microparticles by double labelling, with an antibody specific to cell origin and annexin V, a molecule that marks all microparticles by binding to phosphatidylserine. The quantification of these microparticles shows an increase, post-surgery, of platelet, endothelial and leukocyte origin. The measurements of creatinine, a marker of renal function, as well as those of inflammatory markers and histological lesions did not show significant differences between any of the renal ischemia reperfusion groups. Nevertheless, macrophage infiltration 3 days after ischemia reperfusion seems more important in RCS rats than in control rats. In addition, in vitro phagocytosis did not show any significant difference between RCS rats and their control. Our study suggests minimal involvement of the MerTK receptor in the development and repair of lesions during a sequence of renal ischemia reperfusion. Nevertheless, this work has enabled us to characterize and quantify the microparticles generated following renal ischemia reperfusion by techniques that could subsequently be used in the evaluation of biomarkers of renal lesions.
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Thomas Pelé. Étude des effets potentiels du récepteur MerTK sur le développement des lésions liées à une ischémie/reperfusion rénale chez le rat. Médecine humaine et pathologie. Université de Poitiers, 2019. Français. ⟨NNT : 2019POIT1404⟩. ⟨tel-02618405⟩

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