Effets de la température et d'un transporteur naturel d'oxygène au cours de la conservation en transplantation rénale

La méthode de préservation d organes la plus utilisée actuellement en transplantation rénale est la conservation statique en hypothermie. Cependant, ce mode de conservation induit des dommages inhérents aux lésions du syndrome d ischémie/reperfusion (I/R). Cette étude a eu pour objectif d identifier de nouvelles conditions de préservation des greffons, afin de limiter les lésions d I/R, en modulant la température de conservation ou par ajout d un transporteur d oxygène. Nous avons utilisé deux modèles : in vitro avec des cellules endothéliales et in vivo en autotransplantation rénale chez le porc.Les résultats ont confirmé les effets délétères de la conservation à 4C contrairement à des conservations à 19C, 27C et surtout 32C, permettant d obtenir une activité métabolique, une viabilité et une intégrité cellulaire supérieures ainsi qu une diminution des marqueurs de l inflammation et du stress oxydant. Nous avons aussi démontré les bénéfices d'un nouveau transporteur d oxygène, M101, dans deux des solutions de conservation les plus utilisés, UW et HTK. L'utilisation de M101 en conservation statique permet une meilleure reprise de fonction à court terme et une réduction de la fibrose, cause principale de la perte du greffon. Enfin, nous avons montré une conservation des bénéfices de M101 à des doses réduites et déterminé que cette protection était due à une multifonctionnalité de la molécule, combinant un transporteur d oxygène, une activité superoxyde dismutase et une taille importante (permettant de réguler la pression oncotique). Ce travail a montré de nouvelles pistes de réflexion vers une préservation, et donc une qualité, supérieure des organes à transplanter.The most used preservation method in renal transplantation is hypothermic cold storage (CS). However, this method induces damages inherent to the ischemia/ reperfusion (I /R) syndrome.My study was aimed at identifying new grafts preservation conditions, to limit I/R damage, by varying storage temperature or by adding an oxygen carrier.We used two models: in vitro with endothelial cells and in vivo in pig renal autotransplantation. The results confirmed the deleterious effects of 4C storage in contrast to conservations at 19C, 27C and above 32C, resulting in improved metabolic activity, cellular viability and integrity as well as a significant reduction in markers of inflammation and oxidative stress. Then we demonstrated the benefits of a new oxygen carrier, M101, in the two most used preservation solutions, UW and HTK. Indeed, use of M101 in CS protocols improved short-term function recovery and reduced fibrosis development, main cause of graft loss. Finally, we have shown that the benefits of M101 were preserved at lower doses and we determined that this protection was due to a multifunctionality of the molecule, combining oxygen transport, superoxyde dismutase activity and a large size (regulating oncotic pressure). This work permitted the uncovering of new concepts towards improved organ preservation and quality for transplantation.POITIERS-SCD-Bib. électronique (861949901) / SudocSudocFranceF

Conditioning Medicine A new pharmacological preconditioning-based target: from drosophila to kidney transplantation

International audienceOne of the biggest challenges in medicine is to dampen the pathophysiological stress induced by an episode of ischemia. Such stress, due to various pathological or clinical situations, follows a restriction in blood and oxygen supply to tissue, causing a shortage of oxygen and nutrients that are required for cellular metabolism. Ischemia can cause irreversible damage to target tissue leading to a poor physiological recovery outcome for the patient. Contrariwise, preconditioning by brief periods of ischemia has been shown in multiple organs to confer tolerance against subsequent normally lethal ischemia. By definition, preconditioning of organs must be applied preemptively. This limits the applicability of preconditioning in clinical situations, which arise unpredictably, such as myocardial infarction and stroke. There are, however, clinical situations that arise as a result of ischemia-reperfusion injury, which can be anticipated, and are therefore adequate candidates for preconditioning. Organ and more particularly kidney transplantation, the optimal treatment for suitable patients with end stage renal disease (ESRD), is a predictable surgery that permits the use of preconditioning protocols to prepare the organ for subsequent ischemic/reperfusion stress. It therefore seems crucial to develop appropriate preconditioning protocols against ischemia that will occur under transplantation conditions, which up to now mainly referred to mechanical ischemic preconditioning that triggers innate responses. It is not known if preconditioning has to be applied to the donor, the recipient, or both. No drug/target pair has been envisioned and validated in the clinic. Options for identifying new target/drug pairs involve the use of model animals, such as drosophila, in which some physiological pathways, such as the management of oxygen, are highly conserved across evolution. Oxygen is the universal element of life existence on earth. In this review we focus on a very specific pathway of pharmacological preconditioning identified in drosophila that was successfully transferred to mammalian models that has potential application in human health. Very few mechanisms identified in these model animals have been translated to an upper evolutionary level. This review highlights the commonality between oxygen regulation between diverse animals

Gender difference and sex hormone production in rodent renal ischemia reperfusion injury and repair

<p>Abstract</p> <p>Background</p> <p>Several lines of evidence suggest a protective effect of female sex hormones in several organs subjected to ischemia-reperfusion injury. The aim of the study was to investigate sex hormone production in male rats after a renal ischemia-reperfusion sequence and analyze the influence of gender differences on tissue remodelling during the recovery process.</p> <p>Method</p> <p>Age-matched sexually mature male and female rats were subjected to 60 min of renal unilateral ischemia by pedicle clamping with contralateral nephrectomy and followed for 1 or 5 days after reperfusion. Plasma creatinine, systemic testosterone, progesterone and estradiol levels were determined. Tubular injury, cell proliferation and inflammation, were evaluated as well as proliferating cell nuclear antigen, vimentin and translocator protein (TSPO) expressions by immunohistochemistry.</p> <p>Results</p> <p>After 1 and 5 days of reperfusion, plasma creatinine was significantly higher in males than in females, supporting the high mortality in this group. After reperfusion, plasma testosterone levels decreased whereas estradiol significantly increased in male rats. Alterations of renal function, associated with tubular injury and inflammation persisted during the 5 days post-ischemia-reperfusion, and a significant improvement was observed in females at 5 days of reperfusion. Proliferating cell nuclear antigen and vimentin expression were upregulated in kidneys from males and attenuated in females, in parallel to injury development. TSPO expression was transiently increased in proximal tubules in male rats.</p> <p>Conclusions</p> <p>After ischemia, renal function recovery and tissue injury is gender-dependent. These differences are associated with a modulation of sex hormone production and a modification of tissue remodeling and proliferative cell processes.</p

Analysis of machine perfusion benefits in kidney grafts: a preclinical study

<p>Abstract</p> <p>Background</p> <p>Machine perfusion (MP) has potential benefits for marginal organs such as from deceased from cardiac death donors (DCD). However, there is still no consensus on MP benefits. We aimed to determine machine perfusion benefits on kidney grafts.</p> <p>Methods</p> <p>We evaluated kidney grafts preserved in ViaspanUW or KPS solutions either by CS or MP, in a DCD pig model (60 min warm ischemia + 24 h hypothermic preservation). Endpoints were: function recovery, quality of function during follow up (3 month), inflammation, fibrosis, animal survival.</p> <p>Results</p> <p>ViaspanUW-CS animals did not recover function, while in other groups early follow up showed similar values for kidney function. Alanine peptidase and β-NAG activities in the urine were higher in CS than in MP groups. Oxydative stress was lower in KPS-MP animals. Histology was improved by MP over CS. Survival was 0% in ViaspanUW-CS and 60% in other groups. Chronic inflammation, epithelial-to-mesenchymal transition and fibrosis were lowest in KPS-MP, followed by KPS-CS and ViaspanUW-MP.</p> <p>Conclusions</p> <p>With ViaspanUW, effects of MP are obvious as only MP kidney recovered function and allowed survival. With KPS, the benefits of MP over CS are not directly obvious in the early follow up period and only histological analysis, urinary tubular enzymes and red/ox status was discriminating. Chronic follow-up was more conclusive, with a clear superiority of MP over CS, independently of the solution used. KPS was proven superior to ViaspanUW in each preservation method in terms of function and outcome. In our pre-clinical animal model of DCD transplantation, MP offers critical benefits.</p

FR167653 improves renal recovery and decreases inflammation and fibrosis after renal ischemia reperfusion injury

ObjectiveAcute tubular necrosis (ATN) secondary to induced warm ischemia (WI) results in inflammatory and delayed fibrotic processes and remains a common clinical problem with serious consequences. Because tumor necrosis factor-α (TNF-α) is a prominent proinflammatory factor implicated in the pathophysiology of acute renal ischemia reperfusion injury (IRI), we hypothesized that FR167653 (FR), a potent inhibitor of TNF-α and interleukin-1β production, may reduce IRI.MethodsIRI was induced in male pigs by bilateral clamping of the renal pedicle for 90 minutes (WI90), or unilateral renal clamping (90 minutes) after contralateral nephrectomy (1/2N×90), or unilateral renal clamping without contralateral nephrectomy (WIuni90). FR was administered intravenously 60 minutes before WI (1 mg/kg/h), during WI, and continuously for 3 hours (1 mg/kg/h) during reperfusion in treated groups (FRWI90, FR1/2N×90, or FRWIuni90). Blood and urine samples were collected between day 1 and 3 months after reperfusion for assessment of renal function. Kidneys were excised and renal tissues were collected at 3 months for morphologic and inflammation evaluation and protein analysis. Experimental groups were compared with sham operated (control) and heminephrectomized (Unif) groups without renal ischemia.ResultsThree WI90 animals (43%) and five 1/2N×90 (70%) were euthanized and necropsied at day 7 because of no urine production or poor conditions. Mortality was significantly improved after FR treatment. Survival was 100% in the control, Unif, WIuni90, and FR groups. In Unif groups, FR significantly reduced renal failure and bilateral renal ischemia (P < .05). At 3 months, proteinuria was significantly reduced in FR-treated groups (P < .01). Inflammatory cells count was also dramatically diminished in FR-treated pigs (P < .01 for CD3-positive cells). The second aspect of transient ischemia is the fibrotic process determined at 3 months. FR treatment was characterized by a reduction of renal fibrosis, particularly in Unif groups. TNF-α protein expression was diminished in FR-treated groups.ConclusionThis is the first evidence that FR reduced the early and long-term effect of WI in the severe ischemia model. This effect was particularly marked against fibrosis and inflammation, which would contribute to deterioration of a patient's renal function.Clinical RelevanceAcute ischemia of the kidney is common in the setting of renal artery or aortic surgery. Deterioration in renal function is a common cause of morbidity in patients treated surgically for juxtarenal and suprarenal abdominal aortic aneurysms. FR167653 represents a useful therapeutic approach to prevent renal damage in a planned period of warm ischemia and during suprarenal aortic surgery

Development of a preclinical model of donation after circulatory determination of death for translational application

BACKGROUND: Extracorporeal membranous oxygenation is proposed for abdominal organ procurement from donation after circulatory determination of death (DCD). In France, the national Agency of Biomedicine supervises the procurement of kidneys from DCD, specifying the durations of tolerated warm and cold ischemia. However, no study has determined the optimal conditions of this technique. The aim of this work was to develop a preclinical model of DCD using abdominal normothermic oxygenated recirculation (ANOR). In short, our objectives are to characterize the mechanisms involved during ANOR and its impact on abdominal organs. METHODS: We used Large White pigs weighing between 45 and 55 kg. After 30 minutes of potassium-induced cardiac arrest, the descending thoracic aorta was clamped and ANOR set up between the inferior vena cava and the abdominal aorta for 4 hours. Hemodynamic, respiratory and biochemical parameters were collected. Blood gasometry and biochemistry analysis were performed during the ANOR procedure. RESULTS: Six ANOR procedures were performed. The surgical procedure is described and intraoperative parameters and biological data are presented. Pump flow rates were between 2.5 and 3 l/min. Hemodynamic, respiratory, and biochemical objectives were achieved under reproducible conditions. Interestingly, animals remained hemodynamically stable following the targeted protocol. Arterial pH was controlled, and natremia and renal function remained stable 4 hours after the procedure was started. Decreased hemoglobin and serum proteins levels, concomitant with increased lactate dehydrogenase activity, were observed as a consequence of the surgery. The serum potassium level was increased, owing to the extracorporeal circulation circuit. CONCLUSIONS: Our ANOR model is the closest to clinical conditions reported in the literature and will allow the study of the systemic and abdominal organ impact of this technique. The translational relevance of the pig will permit the determination of new biomarkers and protocols to improve DCD donor management

Impact of Red Blood Cells on Function and Metabolism of Porcine Deceased Donor Kidneys During Normothermic Machine Perfusion

Background. Normothermic machine perfusion (NMP) protocols using blood-based solutions are commonly used in the assessment of kidneys before transplantation. This procedure is, nevertheless, limited by blood availability and warrants the search for alternatives. We compared a blood-based solution with a serum-like preservation solution (Aqix) enriched with colloids with and without red blood cells (RBCs). Methods. Porcine kidneys retrieved from an abattoir were subjected to 30min of warm ischemia, followed by 3h of hypothermic oxygenated machine perfusion at 4 degrees C. Subsequently, kidneys (n=6 per group) were evaluated with NMP for 4h with 5 different solutions: diluted blood, Aqix with BSARBCs, or Aqix with dextran 40RBCs. Results. Throughout NMP, markers of renal function and tubular metabolism were favorable in groups with RBCs. The addition of RBCs resulted in 4- to 6-fold higher oxygen consumption rates. Controls had significantly higher ATP levels post-NMP, exhibited decreased production of oxidative stress markers, and had the highest creatinine clearance. In conclusion, this study shows that the addition of RBCs during NMP reduced renal injury, improved function, and was associated with increased renal metabolism. Conclusions. Although the RBC-BSA-supplemented Aqix solution was also able to support metabolism and renal function, a blood-based perfusion solution remains superior

Controlled oxygenated rewarming up to normothermia for pretransplant reconditioning of liver grafts

Controlled oxygenated rewarming (COR) up to 20 degrees C during ex vivo machine perfusion limits reperfusion-induced tissue injury upon graft implantation. Rewarming up to normothermia might add further benefits and provide better prediction of post-transplantation organ function. The effect of 90 minutes of oxygenated machine perfusion with Aqix RS-I after cold storage combined with gentle rewarming up to 20 degrees C (COR20) or 35 degrees C (COR35) was studied in rat livers and compared with cold storage alone (CS, n = 6, resp). Postpreservation recovery was evaluated upon warm reperfusion using an established in vitro system. COR generally resulted in significantly improved energetic recovery, increased bile flow, less activities alanine aminotransferase (ALT) release, and improved histopathology upon reperfusion as compared to only cold-stored livers, without significant differences between COR20 and COR35. Parameters obtained during COR, especially during COR35, also allowed for prediction of hepatic recovery upon reperfusion. For instance, ulterior bile production upon reperfusion was found closely correlated to bile flow observed already during COR35 (R-2=0.91). COR significantly improved liver quality after static cold storage. Elevation of machine perfusion temperature up to 35 degrees C may prove promising to refine ex vivo evaluation of the graft prior to transplantation