Thierry Hauet

Mitochondria as target for antiischemic drugs

The cessation of blood flow followed by a reperfusion period results in severe damages to cell structures. This induces a complex cascade of events involving, more particularly, a loss of energy, an alteration of ionic homeostasis promoting H(+) and Ca(2+) build up and the generation of free radicals. In this context, mitochondria are highly vulnerable and play a predominant role in the cell signaling leading from life to death. This is why, recently, efforts to find an effective therapy for ischemia-reperfusion injury have focused on mitochondria. This review summarizes the pharmacological strategies which are currently developed and the potential mitochondrial targets which could be involved in the protection of cells.

Trophic factor and FR167653 supplementation during cold storage rescue chronic renal injury.

PURPOSEnThe use of organs from deceased after cardiac death and extended criteria donors grew in the last decade. These organs are more sensitive to ischemia-reperfusion injury during transplantation and current preservation protocols do not protect them adequately.nnnMATERIALS AND METHODSnIn an autotransplanted, deceased after cardiac death donor pig kidney model we evaluated the benefits of supplementation with University of Wisconsin solution trophic factors and FR167653, an inhibitor of p38 mitogen-activated protein kinase.nnnRESULTSnSupplemented solution improved renal recovery and limited ischemia-reperfusion injury, particularly when agents were used in conjunction. Long-term benefits were highlighted by decreased renal fibrosis, as determined by Picrosirius staining, and inflammation, as evaluated by renal cell infiltration. Mechanistic evaluation showed decreased expression of epithelial-to-mesenchymal transition markers, a process involved in renal fibrosis development. Tumor necrosis factor-α was markedly decreased in the treated experimental group. Apoptosis was also decreased, accompanied by decreased p38 mitogen-activated protein kinase phosphorylation.nnnCONCLUSIONSnSupplementing the current gold standard kidney preservation protocol with trophic factors and p38 mitogen-activated protein kinase inhibitors markedly increased the quality of grafts in our pig deceased after cardiac death donor model. Hence, this represents a strategy of interest to improve transplantation outcomes.

Renal auto-transplantation promotes cortical microvascular network remodeling in a preclinical porcine model

The vascular network is a major target of ischemia-reperfusion, but has been poorly investigated in renal transplantation. The aim of this study was to characterize the remodeling of the renal vascular network that follows ischemia-reperfusion along with the most highly affected cortex section in a preclinical renal transplantation model. There were two experimental groups. The first was a grafted kidney group consisting of large white pigs for which the left kidney was harvested, cold flushed, preserved for 24 h in the University of Wisconsin’s preservation solution, and then auto-transplanted (n = 5); the right kidney was removed to mimic the situation of human kidney transplantation. The second group (uni-nephrectomized kidney group) consisted of animals that underwent only right nephrectomy, but not left renal transplantation (n = 5). Three months after autotransplantation, the kidneys were studied by X-ray microcomputed tomography. Vessel morphology and density and tortuosity of the network were analyzed using a 3D image analysis method. Cortical blood flow was determined by laser doppler analysis and renal function and tissue injury assessed by plasma creatinine levels and histological analysis. Renal ischemia-reperfusion led to decreased vascular segment volume associated with fewer vessels of less than 30 μm, particularly in the inner cortex:0.79 ± 0.54% in grafted kidneys vs. 7.06 ± 1.44% in uni-nephrectomized kidneys, p < 0.05. Vessels showed higher connectivity throughout the cortex (the arborescence factor of the whole cortex was less in grafted than uni-nephrectomized kidneys 0.90 ± 0.04 vs. 1.07 ± 0.05, p < 0.05, with an increase in the number of bifurcations). Furthermore, cortical blood flow decreased early in kidney grafts and remained low three months after auto-transplantation. The decrease in microvasculature correlated with a deterioration of renal function, proteinuria, and tubular dysfunction, and was associated with the development of fibrous tissue. This work provides new evidence concerning the impact of ischemia-reperfusion injuries on the spectrum of renal vascular diseases and could potentially guide future therapy to preserve microvessels in transplantation ischemia-reperfusion injury.

6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury

Necroptosis is a programmed cell death pathway that has been shown to be of central pathophysiological relevance in multiple disorders (hepatitis, brain and cardiac ischemia, pancreatitis, viral infection and inflammatory diseases). Necroptosis is driven by two serine threonine kinases, RIPK1 (Receptor Interacting Protein Kinase 1) and RIPK3, and a pseudo-kinase MLKL (Mixed Lineage Kinase domain-Like) associated in a multi-protein complex called necrosome. In order to find new inhibitors for use in human therapy, a chemical library containing highly diverse chemical structures was screened using a cell-based assay. The compound 6E11, a natural product derivative, was characterized as a positive hit. Interestingly, this flavanone compound: inhibits necroptosis induced by death receptors ligands TNF-α (Tumor Necrosis Factor) or TRAIL (TNF-Related Apoptosis-Inducing Ligand); is an extremely selective inhibitor, among kinases, of human RIPK1 enzymatic activity with a nM Kd; has a non-ATP competitive mode of action and a novel putative binding site; is weakly cytotoxic towards human primary blood leukocytes or retinal pigment epithelial cells at effective concentrations; protects human aortic endothelial cells (HAEC) from cold hypoxia/reoxygenation injury more effectively than necrostatin-1 (Nec-1) and Nec-1s. Altogether, these data demonstrate that 6E11 is a novel potent small molecular inhibitor of RIPK1-driven necroptosis.