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International audience; Aims Totally subcutaneous implantable cardioverter defibrillator (S-ICD) delivers higher shock energy and can have longer time to therapy compared to transvenous implantable cardioverter defibrillator (T-ICD). Aim of the study was to compare time to therapy and to investigate cardiac, cerebral and systemic injuries of S-ICD and T-ICD shocks delivered after ventricular fibrillation (VF) induction. Methods and results Fourteen pigs were randomly implanted with a S-ICD (n = 7) or a T-ICD (n = 7). Five VF episodes were induced in each pig. For each VF episode, up to two shocks could be delivered by the T-ICD or the S-ICD to terminate the arrhythmia. Cardiac, systemic, and cerebral toxicity were monitored. Mean time to therapy was longer in the S-ICD group compared to the T-ICD group (19[18; 23] s vs. 9 [7; 10] s; P = 0.001, respectively). High-sensitivity troponin T levels were significantly higher in the T-ICD group from 1 to 24 h after the procedure (P

The increased number of patients in waiting list for renal transplantation requires the establishment of recommendations regarding graft preservation techniques. The preservation method impacts graft function and survival particularly in case of extended criteria donors. Based on our experience, the aim of this review is to establish a decisional diagram to draw graft management to 5years in relation to donor type and graft quality. Novel biomarkers are necessary to evaluate graft quality. Nuclear magnetic resonance or transcriptomic analyses are promising. Thus, good quality organs will be preserved in static condition associated to hypothermia; while grafts from extended criteria donors need to be assessed early during dynamic perfusion through an evaluation of perfusion solution to discriminate: good organs, with acceptable risks without perfusion conditions modifications; tolerable risk grafts for which it will be recommended to use a supplementation of perfusion solution with oxygen or pharmacologic additives such as mitochondrion protectors or oxygen carriers; and elevated risks graft which will not be used. This diagram based on experimental data needs to be assessed in clinical trials but highlights the crucial role of kidney graft quality assessment for its management and placed dynamic perfusion preservation as the protocol of choice for extended criteria donors.

BackgroundAccumulation of middle-weight uraemic toxins in haemodialysis (HD) patients results in increased morbidity and mortality. Whether medium cut-off HD (MCO-HD) improves removal of middle-weight uraemic toxins remains to be demonstrated.MethodsThis cross-over prospective study included 40 patients randomly assigned to receive either 3 months of MCO-HD followed by 3 months of high-flux HD (HF-HD), or vice versa. The primary endpoint was myoglobin reduction ratio (RR) after 3 months of MCO-HD. Secondary endpoints were the effect of MCO-HD on other middle-weight toxins and protein-bound toxins, and on parameters of nutrition, inflammation, anaemia and oxidative stress.ResultsCompared with HF-HD, MCO-HD provided higher mean RR of myoglobin (36 ± 8 versus 57 ± 13%, P ConclusionsCompared with HF-HD, MCO-HD provides higher myoglobin and other middle molecules RR and is associated with moderate hypoalbuminemia. The potential benefits of this strategy on long-term clinical outcomes deserve further evaluation.

International audience; Static cold storage (SCS) and hypothermic machine perfusion (HMP) are currently standard methods for renal grafts clinical preservation. Both methods are predominantly implemented without the active delivery of oxygen, even for donation after circulatory death-like kidneys. However, even under severe hypothermia (4°C-6°C), kidneys can consume oxygen and produce ATP. What is not established, though, is to what extent and how SCS and HMP compare in terms of oxygen. Using a porcine preclinical model of renal warm ischemia (WI) to compare SCS and HMP methods, we continuously monitored and quantified oxygen level and consumption along preservation; we also determined prepreservation and postpreservation cortical ATP level; values were given as median and [min; max] range. One-hour WI reduced ATP by ∼90% (from 3.3 [1.7; 4.5] mmol/L tissue in Controls). Oxygen consumption (QO2, μmol/min per 100 g) was determined from initial solution PO2 decrease (SCS and HMP) and from arterio-venous difference (HMP). In SCS and HMP, PO2 decreased rapidly (t1/2 ∼1 h) from atmospheric levels to 52.9 [38.0; 65.9] and 8.2 [3.0, 16.0] mmHg, respectively. In HMP, QO2 was 2.7 [0.4; 3.9] versus 0.5 [0.0; 1.3] in SCS (P < 0.05); postpreservation ATP amounted to 5.8 [3.2; 6.5] in HMP versus 0.1 [0.0; 0.2] in SCS. Despite hypothermic conditions in SCS or HMP, donation after circulatory death-like renal grafts require oxygen. Increased oxygen consumption, restored ATP level, and improved histological profile in HMP might explain the established HMP superiority over SCS. These results establish a rational basis for the use of oxygen in hypothermic preservation. Optimal levels required for preservation and graft-type variants remain to be determined.

International audience; Deceased after circulatory death (DCD) donors offer a viable solution to the current organ shortage, particularly the Maastricht Class III (arrest subsequent to cessation of life support in the hospital). Although current results from these donors are very satisfactory, the number of included donors is too low and future expansion of inclusion criteria will likely decrease organ quality, with negative consequences on the complication rate. This donor type thus represents a priority in terms of scientific exploration, so as to study it in controlled settings and prepare for future challenges. Hence, we mimicked the DCD Class III clinical conditions a Large White pig model. Herein, we detail the different strategies attempted to attain our objectives, including technical approaches such as animal positioning and ventilator settings, as well as pharmacological intervention to modulate blood pressure and heart rate. We highlight the best combination of factors to successfully reproduce DCD Class III conditions, with perfusion pressures and functional warm ischemia (hypoperfusion) closely resembling clinical situations. Finally, we detail the functional and histological impacts of these conditions. Such a model could be of critical value to explore novel management alternative for these donors, presenting a uniquely adapted platform for such therapeutics as normothermic regional circulation and/or pharmacological intervention.

The renal cortex drives renal function. Hypoxia/reoxygenation are primary factors in ischemia-reperfusion (IR) injuries, but renal oxygenation per se is complex and awaits full elucidation. Few mathematical models address this issue: none captures cortical tissue heterogeneity. Using agent-based modeling, we develop the first model of cortical oxygenation at the cell-tissue level (RCM), based on first principles and careful bibliographical analysis. Entirely parameterized with Rat data, RCM is a morphometrically equivalent 2D-slice of cortical tissue, featuring peritubular capillaries (PTC), tubules and interstitium. It implements hemoglobin/O2 binding-release, oxygen diffusion, and consumption, as well as capillary and tubular flows. Inputs are renal blood flow RBF and PO2 feeds; output is average tissue PO2 (tPO2). After verification and sensitivity analysis, RCM was validated at steady-state (tPO2 37.7 ± 2.2 vs. 36.9 ± 6 mmHg) and under transients (ischemic oxygen half-time: 4.5 ± 2.5 vs. 2.3 ± 0.5 s in situ). Simulations confirm that PO2 is largely independent of RBF, except at low values. They suggest that, at least in the proximal tubule, the luminal flow dominantly contributes to oxygen delivery, while the contribution of capillaries increases under partial ischemia. Before addressing IR-induced injuries, upcoming developments include ATP production, adaptation to minutes–hours scale, and segmental and regional specification.

A 67 years old woman with a Waldenstrom disease was admitted in the intensive care unit for dyspnea and fever. During hospitalization, episodes of undetectable glycemia were observed without any hypoglycemia symptoms. Plasma glucose was determined with the hexokinase method (recommended). From this observation, a literature review on PubMed was performed to investigate similar cases. In patients with protides in excess (e.g. immunoproliferative syndrome), absorption measurements could be disrupted by the precipitation of excess protein (IgM in most cases). Other parameters could be affected: bilirubin, phosphate, HDL cholesterol, GGT, CRP and calcemia. In our case, the main difficulty was to identify the cause of the interference and then correct it. Using a series of dilution, we prevented protide precipitation allowing correct glucose determination. Those interferences are rare, but present a real analytical difficulty. Biologists should be aware of those interferences because of dramatics consequences.

This review focus on kidney organoids derived from pluripotent stem cells, which become a real alternative to the use of in vitro cellular models or in vivo animals models. The comprehension of the key steps involved during kidney embryonic development led to the establishment of protocols enabling the differentiation of pluripotent stem cells into kidney organoids that are highly complex and organized structures, composed of various renal cell types. These mini-organs are endowed with major applications: the possibility to control iPSC genome (by selecting patients with specific disease or by genome editing) allows the generation of kidney organoïds which recapitulate important physiopathological mechanisms such as cyste formation in renal polycystic disease. Kidney organoids can also be used in high-throughput screening to fasten the screening of nephrotoxic/therapeutic compounds. Finally, kidney organoids have a huge interest in the context of tissue repair, which remains for now a challenging goal linked with technological barriers that need still to be overcome.

Background Renal transplantation is increasingly associated with the presence of comorbidity factors such as dyslipidemia which could influence the graft outcome. We hypothesized that hypercholesterolemia could affect vascular repair processes and promote post-transplant renal vascular remodeling through the over-expression of the anti-angiogenic thrombospondin-1 interacting with vascular endothelial growth factor-A levels. Methods We tested this hypothesis in vitro, in vivo and in a human cohort using (1) endothelial cells; (2) kidney auto-transplanted pig subjected (n = 5) or not (n = 6) to a diet enriched in cholesterol and (3) a renal transplanted patient cohort (16 patients). Results Cells exposed to oxidized LDL showed reduced proliferation and an increased expression of thrombospondin-1. In pigs, 3 months after transplantation of kidney grafts, we observed a deregulation of the hypoxia inducible factor 1a—vascular endothelial growth factor-A axis induced in cholesterol-enriched diet animals concomitant with an overexpression of thrombospondin-1 and a decrease in cortical microvessel density promoting vascular remodeling. In patients, hypercholesterolemia was associated with decreased vascular endothelial growth factor-A plasma levels during early follow up after renal transplantation and increased chronic graft dysfunction. Conclusions These results support a potential mechanism through which a high fat-diet impedes vascular repair in kidney graft and suggest the value of controlling cholesterolemia in recipient even at the early stage of renal transplantation. Electronic supplementary material The online version of this article (10.1186/s12967-018-1764-4) contains supplementary material, which is available to authorized users.

International audience; Introduction: Delayed graft function (DGF) has a significant impact on kidney transplantation outcome. One of the underlying pivotal mechanisms is organ preservation and associated hypothermia and biochemical alteration. Area covered: This paper focuses on organ preservation and its clinical consequences and describes 1. A comprehensive presentation of the pathophysiological mechanism involved in delayed graft function development; 2. The impact on endothelial cells and microvasculature integrity and the consequences on transplanted organ outcome; 3. The reassessment of dynamic organ preservation motivated by the growing use of extended criteria donors and the interest in the potential of normothermia; 4. The role of oxygenation during dynamic preservation; and 5. Novel oxygen carriers and their proof of concept in transplantation, among which M101 (HEMO(2)life (R)) is currently the most extensively investigated. Expert opinion: Metabolic disturbances and imbalance of oxygen supply during preservation highlight the importance of providing oxygen. Normothermia, permitted by recent advances in machine perfusion technology, appears to be the leading edge of preservation technology. Several oxygen transporters are compatible with normothermia; however, only M101 also demonstrates compatibility with standard hypothermic preservation.

Ischemia-reperfusion (IR) injury is unavoidable during organ transplantation and impacts graft quality. New paradigms are emerging including preservation at higher temperature than “hypothermia” or “cold”: although 4°C remains largely used for kidney preservation, recent studies challenged this choice. We and others hypothesized that a higher preservation temperature, closer to physiological regimen, could improve organ quality. For this purpose, we used an in vitro model of endothelial cells exposed to hypoxia-reoxygenation sequence (mimicking IR) and an ex vivo ischemic pig kidneys static storage model. In vitro, 19°C, 27°C, and 32°C provided protection against injuries versus 4°C, by reducing cell death, mitochondrial dysfunction, leukocyte adhesion, and inflammation. However, ex vivo, the benefits of 19°C or 32°C were limited, showing similar levels of tissue preservation damage. Ex vivo 4°C-preserved kidneys displayed a trend towards reduced damage, including apoptosis. Macrophage infiltration, tubulitis, and necrosis were increased in the 19°C and 32°C versus 4°C preserved kidneys. Thus, despite a trend for an advantage of subnormothermia as preservation temperature, our in vitro and ex vivo models bring different insights in terms of preservation temperature effect. This study suggests that temperature optimization for kidney preservation will require thorough investigation, combining the use of complementary relevant models and the design of elaborated preservation solution and new technologies.

The growing use of marginal organs for transplantation pushes current preservation methods toward their limits, and the need for improvement is pressing. We previously demonstrated the benefits of M101, a natural extracellular oxygen carrier compatible with hypothermia, for the preservation of healthy renal grafts in a porcine model of autotransplantation. Herein, we use a variant of this preclinical model to evaluate M101 potential benefits both in static cold storage (CS) and in machine perfusion (MP) preservation in the transplantation outcomes for marginal kidneys. In the CS arm, despite the absence of obvious benefits within the first 2 weeks of follow-up, M101 dose-dependently improved long-term function, normalizing creatininemia after 1 and 3 months. In the MP arm, M101 improved short- and long-term functional outcomes as well as tissue integrity. Importantly, we provide evidence for the additivity of MP and M101 functional effects, showing that the addition of the compound further improves organ preservation, by reducing short-term function loss, with no loss of function or tissue integrity recorded throughout the follow-up. Extending previous observations with healthy kidneys, the present results point at the M101 oxygen carrier as a viable strategy to improve current organ preservation methods in marginal organ transplantation.

In animal models, whole-body cooling reduces end-organ injury after cardiac arrest and other hypoperfusion states. The benefits of cooling in humans, however, are uncertain, possibly because detrimental effects of prolonged cooling may offset any potential benefit. Total liquid ventilation (TLV) provides both ultrafast cooling and rewarming. In previous reports, ultrafast cooling with TLV potently reduced neurological injury after experimental cardiac arrest in animals. We hypothesized that a brief period of rapid cooling and rewarming via TLV could also mitigate multiorgan failure (MOF) after ischemia-reperfusion induced by aortic cross-clamping.Anesthetized rabbits were submitted to 30 minutes of supraceliac aortic cross-clamping followed by 300 minutes of reperfusion. They were allocated either to a normothermic procedure with conventional ventilation (control group) or to hypothermic TLV (33°C) before, during, and after cross-clamping (pre-clamp, per-clamp, and post-clamp groups, respectively). In all TLV groups, hypothermia was maintained for 75 minutes and switched to a rewarming mode before resumption to conventional mechanical ventilation. End points included cardiovascular, renal, liver, and inflammatory parameters measured 300 minutes after reperfusion.In the normothermic (control) group, ischemia-reperfusion injury produced evidence of MOF including severe vasoplegia, low cardiac output, acute kidney injury, and liver failure. In the TLV group, we observed gradual improvements in cardiac output in post-clamp, per-clamp, and pre-clamp groups versus control (53 ± 8, 64 ± 12, and 90 ± 24 vs 36 ± 23 mL/min/kg after 300 minutes of reperfusion, respectively). Liver biomarker levels were also lower in pre-clamp and per-clamp groups versus control. However, acute kidney injury was prevented in pre-clamp, and to a limited extent in per-clamp groups, but not in the post-clamp group. For instance, creatinine clearance was 4.8 ± 3.1 and 0.5 ± 0.6 mL/kg/min at the end of the follow-up in pre-clamp versus control animals (P = .0004). Histological examinations of the heart, kidney, liver, and jejunum in TLV and control groups also demonstrated reduced injury with TLV.A brief period of ultrafast cooling with TLV followed by rapid rewarming attenuated biochemical and histological markers of MOF after aortic cross-clamping. Cardiovascular and liver dysfunctions were limited by a brief period of hypothermic TLV, even when started after reperfusion. Conversely, acute kidney injury was limited only when hypothermia was started before reperfusion. Further work is needed to determine the clinical significance of our results and to identify the optimal duration and timing of TLV-induced hypothermia for end-organ protection in hypoperfusion states.

International audience; The relationship between cold ischaemia time (CIT) and adverse outcome is now acknowledged. However, the underlying mechanisms remain to be defined, which slows the development of adapted therapeutics and diagnostics. We explored the impact of CIT in both preclinical and in vitro models of preservation. We determined that the endoplasmic reticulum (ER) and its stress response (unfolded protein response, UPR) were regulated in close association with CIT; the eIF2α-ATF4 pathway was inhibited early (1-8 h) at the detriment of cell survival, while the ATF6 pathway was activated late (12-24 h) and associated with cell death. The IRE1α-XBP1 branch was activated at reperfusion only if CIT extended beyond 8 h, and had a dual role on cell fate - deleterious through IRE1's RNase activity and beneficial through IRE1α other roles. Finally, the pro-apoptotic factor CHOP was a common target of both ATF6 and IRE1α pathways and was associated with elongated CIT and increased cell death. Microarray analysis of human transplanted kidney confirmed that UPR markers were regulated by CIT and that CHOP was associated with adverse outcome. We show that UPR could be a critical pathway explaining the relationship between CIT and graft outcome, highlighting the potential for UPR-based therapeutics and diagnostics to improve transplantation

International audience; Kidneys from donation after circulatory death (DCD) are highly sensitive to ischemia‐reperfusion injury and thus require careful reconditioning, such as normothermic regional perfusion (NRP). However, the optimal NRP protocol remains to be characterized. NRP was modeled in a DCD porcine model (30 minutes of cardiac arrest) for 2, 4, or 6 hours compared to a control group (No‐NRP); kidneys were machine‐preserved and allotransplanted. NRP appeared to permit recovery from warm ischemia, possibly due to an increased expression of HIF1α‐dependent survival pathway. At 2 hours, blood levels of ischemic injury biomarkers increased: creatinine, lactate/pyruvate ratio, LDH, AST, NGAL, KIM‐1, CD40 ligand, and soluble‐tissue‐factor. All these markers then decreased with time; however, AST, NGAL, and KIM‐1 increased again at 6 hours. Hemoglobin and platelets decreased at 6 hours, after which the procedure became difficult to maintain. Regarding inflammation, active tissue‐factor, cleaved PAR‐2 and MCP‐1 increased by 4‐6 hours, but not TNF‐α and iNOS. Compared to No‐NRP, NRP kidneys showed lower resistance during hypothermic machine perfusion (HMP), likely associated with pe‐NRP eNOS activation. Kidneys transplanted after 4 and 6 hours of NRP showed better function and outcome, compared to No‐NRP. In conclusion, our results confirm the mechanistic benefits of NRP and highlight 4 hours as its optimal duration, after which injury markers appear.

Inflammation is a prominent feature of ischemia-reperfusion injury (IRI), which is characterized by leukocyte infiltration and renal tubular injury. However, signals that initiate these events remain poorly understood. We examined the role of the nuclear alarmin IL-33 in tissue injury and innate immune response triggered by experimental kidney ischemia-reperfusion. In wild-type mice, we found that IL-33 was constitutively expressed throughout the kidney in peritubular and periglomerular spaces, mainly by microvascular endothelial cells, from which it was released immediately during IRI. Compared with wild-type mice, mice lacking IL-33 (IL-33(Gt/Gt)) exhibited reductions in early tubular cell injury and subsequent renal infiltration of IFN-γ/IL-17A–producing neutrophils, with preservation of renal functions. This protection associated with decreased renal recruitment of myeloid dendritic cells, natural killer (NK) cells, and invariant natural killer T (iNKT) cells, the latter of which were reported as deleterious in IRI. Increases in the level of circulating IL-12, a key IL-33 cofactor, and the expression of ST2, an IL-33–specific receptor, on the surface of iNKT cells preceded the IL-33– and iNKT cell–dependent phase of neutrophil infiltration. Furthermore, IL-33 directly targeted iNKT cells in vitro, inducing IFN-γ and IL-17A production. We propose that endogenous IL-33 is released as an alarmin and contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, resulting in neutrophil infiltration and activation at the injury site. Our findings show a novel molecular mediator contributing to innate immune cell recruitment induced by renal ischemia-reperfusion and may provide therapeutic insights into AKI associated with renal transplantation.

International audience; Tubular cells are central targets of ischemia-reperfusion (I/R) injury in kidney transplantation. Inflammation and metabolic disturbances occurring within these cells are deleterious by themselves but also favor secondary events, such as activation of immune response. It is critical to have an in depth understanding of the mechanisms governing tubular cells response to I/R if one wants to define pertinent biomarkers or to elaborate targeted therapeutic interventions. As oxidative damage was shown to be central in the patho-physiological mechanisms, the impact of I/R on proximal tubular cells metabolism has been widely studied, contrary to its effects on expression and activity of membrane transporters of the proximal tubular cells. Yet, temporal modulation of transporters over ischemia and reperfusion periods appears to play a central role, not only in the induction of cells injury but also in graft function recovery. Metabolomics in cell models or diverse biofluids has the potential to provide large pictures of biochemical consequences of I/R. Metabolomic studies conducted in experimental models of I/R or in transplanted patients indeed retrieved metabolites belonging to the pathways known to be particularly affected. Interestingly, they also revealed that metabolic disturbances and transporters activities are in very close mutual interplay. As well as helping to select diagnostic biomarkers, such analyses could also contribute to identify new pharmacological targets and to set up innovative nephroprotective strategies for the future. Even if various therapeutic approaches have been evaluated for a long time to prevent or treat I/R injuries, metabolomics has helped identifying new ones, those related to membrane transporters seeming to be of particular interest. However, considering the very complex and multifactorial effects of I/R in the context of kidney transplantation, all tracks must be followed if one wants to prevent or limit its deleterious consequences

Important progress has been made on cytokine signaling in response to kidney injury in the past decade, especially cytokine signaling mediated by extracellular vesicles (EVs). For example, EVs released by injured renal tubular epithelial cells (TECs) can regulate intercellular communications and influence tissue recovery via both regulating the expression and transferring cytokines, growth factors, as well as other bioactive molecules at the site of injury. The effects of EVs on kidney tissue seem to vary depending on the sources of EVs; however, the literature data are often inconsistent. For example, in rodents EVs derived from mesenchymal stem cells (MSC-EVs) and endothelial progenitor cells (EPC-EVs) can have both beneficial and harmful effects on injured renal tissue. Caution is thus needed in the interpretation of these data as contradictory findings on EVs may not only be related to the origin of EVs, they can also be caused by the different methods used for EV isolation and the physiological and pathological states of the tissues/cells under which they were obtained. Here, we review and discuss our current understanding related to the immunomodulatory function of EVs in renal tubular repair in the hope of encouraging further investigations on mechanisms related to their antiinflammatory and reparative role to better define the therapeutic potential of EVs in renal diseases.

International audience; Ischemia reperfusion (IR) lesions are an unavoidable consequence of organ transplantation. Researching new therapeutics against these lesions requires the definition of early mechanisms. The cytoskeleton is composed of 3 types of filaments: microfilaments, intermediate filaments, and microtubules. We aimed to characterize the influence of preservation on their phenotype. In an in vitro model using primary human endothelial cells reproducing the conditions of organ preservation, two aspects were explored: (a) the impact of IR and cold ischemia time on each filament type, evaluating the roles of temperature, solution, and oxygen; and (b) the potential of cytoskeleton-mediated therapy to alleviate cell death. Results showed that intermediary filaments were unaffected, while microfilaments showed radical changes with disappearance of the structure replaced by a disorganized array of nodules; moreover, microtubules almost completely disappeared with time. Furthermore, temperature, and not oxygen deprivation or the solution, was the determining factor of the cytoskeleton's loss of integrity during preservation. Finally, pharmaceutical intervention could indeed preserve fiber structure but did not alter survival. Our work shows that improvement of preservation must include a more adapted temperature before considering oxygen, as it could profoundly improve cytoskeleton organization and thus cell fate. This highlights the importance of this structure for the development of new therapeutics and the definition of graft quality biomarkers.

Despite the high prevalence of diseases involving cardiomyocyte dysfunctions, the available options to study biomechanical properties of cardiomyocytes are limited, especially for human studies. Ra...

Introduction: Ischemia reperfusion (IR) injury is unavoidable during organ transplantation and leads to complications. With the increased use of marginal donors, more sensitive to IR, solutions must be found to improve outcome. We hypothesized that a higher preservation temperature could offer better protection against IR.[br/] Methods: We tested this in an in vitro model of IR using primary endothelial cells and in ex vivo preserved pig kidneys. In both, 24 h preservation in University of Wisconsin solution was used.[br/] Results: In vitro, compared to 4°C, temperatures between 19 and 32°C provided higher protection against cell death (LDH release test), permitting better mitochondrial function (complexes II and V activity tests) and a lower expression of endothelial activation and inflammation markers TLR4, MCP1 and ICAM1. Ex vivo, however, the superiority of 19 or 32°C was lost, as preserved pig kidneys showed similar levels of tissue damage (both tubular dilatation, loss of brush border and endoluminal detachment) during preservation, and at 24 h the 4°C kidneys displayed a trend towards less damage. In addition, the tissular Monocyte/Macrophage infiltration was increased in the 19°C and more in the 32°C temperature conservation as compared to 4°C storage.[br/] Conclusion: Our study shows that although a higher preservation temperature is preferable for cell survival and function, whole organ testing demonstrates that conceptual work needs to be performed to harness the potential of sub-normothermia.

BackgroundDeceased after cardiac death donors represent an important source of organs to reduce organ shortage in transplantation. However, these organs are subjected to more ischaemia–reperfusion injury (IRI). Reducing IRI by targeting coagulation is studied here in an experimental model.MethodsThe effect of an anti-Xa compound (fondaparinux) was evaluated using an autotransplanted kidney model in pigs. Kidneys were clamped for 60 min (warm ischaemia) and then preserved for 24 h at 4°C in University of Wisconsin solution (UW). The anti-Xa compound was injected intravenously before warm ischaemia and used during cold storage, and its effects were compared with those of intravenous injection of unfractionated heparin (UFH) before warm ischaemia and use during cold storage, or use of UW alone during cold storage.ResultsAt 3 months after transplantation, anti-Xa treatment improved recovery of renal function and chronic serum creatinine levels compared with UW and UFH (mean(s.e.m.) 89(4), 250(4) and 217(8) µmol/l respectively). The anti-Xa treatment also reduced fibrosis, and decreased tissue expression of markers of the epithelial–mesenchymal transition compared with UW and UFH. Cleaved protease-activated receptor 2 was overexpressed in the UW group compared with the anti-Xa and UFH groups. Leucocyte infiltrates were decreased in the anti-Xa group compared with the UW and UFH groups. Macrophage invasion was also decreased by anticoagulation treatment.ConclusionPeritransplant anticoagulation therapy was beneficial to graft outcome, in both the acute and chronic phases. Moreover, specific inhibition of coagulation Xa protease further protected kidney grafts, with better recovery and decreased expression of chronic lesion markers. Surgical relevanceThe increasing use of marginal donors highlights the importance of organ quality in transplantation. Renal ischaemia–reperfusion injury (IRI), which includes a deleterious activation of coagulation, plays a central role in determining graft quality and outcome.Using an established porcine renal autotransplantation preclinical model with high clinical relevance, the benefits of anticoagulation therapy using an antifactor Xa molecule were evaluated. Peritransplantion anticoagulation treatment, specifically with an anti-Xa compound, protected marginal kidney grafts, improving functional recovery and reducing chronic lesions.This study demonstrates the benefits of anticoagulation therapy at the time of organ collection, particularly for marginal organs, encountered in cases of extended criteria and deceased after circulatory death donors. This anticoagulation strategy could be an important addition to current donor and organ management protocols in order to limit IRI and improve outcome.

International audience; Protection of endothelial cell function may explain the benefits of nonoxygenated hypothermic machine perfusion (MP) for marginal kidney preservation. However, this hypothesis remains to be tested with a preclinical model. We postulated that MP protects the nitric oxide (NO) signaling pathway, altered by static cold storage (CS), and improves renal circulation recovery compared to CS. The endothelium releases the vasodilator NO in response to flow via either increased endothelial NO synthase (eNOS) expression (KLF2-dependent) or activation of eNOS by phosphorylation (via Akt, PKA or AMPK). Using a porcine model of kidney transplantation, including 1 h of warm ischemia and preserved 24 h by CS or MP (n = 5), we reported that MP did not alter the cortical levels of KLF2 and eNOS at the end of preservation, but significantly increased eNOS activating phosphorylation compared to CS. eNOS phosphorylation appeared AMPK-dependent and was concomitant to an increased NO-dependent vasodilation of renal arteries measured, ex situ, at the end of preservation. In vivo, laser Doppler showed that cortical microcirculation was improved at reperfusion in MP kidneys. In conclusion, we demonstrate for the first time, in a large-animal model, that MP protects the NO signaling pathway, confirming the value of MP for marginal kidney preservation.

International audience; We describe the results of adding a new biological agent HEMO2life® to a standard preservation solution for hypothermic static lung preservation aiming to improve early functional parameters after lung transplantation. HEMO2life® is a natural oxygen carrier extracted from Arenicola marina with high oxygen affinity developed as an additive to standard organ preservation solutions. Standard preservation solution (Perfadex®) was compared with Perfadex® associated with HEMO2life® and with sham animals after 24 h of hypothermic preservation followed by lung transplantation. During five hours of lung reperfusion, functional parameters and biomarkers expression in serum and in bronchoalveolar lavage fluid (BALF) were measured. After five hours of reperfusion, HEMO2life® group led to significant improvement in functional parameters: reduction of graft vascular resistance (p

Acute kidney injury induced by ischemia/reperfusion is an independent risk factor for chronic kidney disease. Macrophage recruitment plays an essential role during the injury and repair phases after an ischemic episode in the kidney. Here we show that the novel non-steroidal mineralocorticoid receptor antagonist finerenone or selective myeloid mineralocorticoid receptor ablation protects against subsequent chronic dysfunction and fibrosis induced by an episode of bilateral kidney ischemia/reperfusion in mice. This protection was associated with increased expression of M2-antiinflamatory markers in macrophages from finerenone-treated or myeloid mineralocorticoid receptor-deficient mice. Moreover, the inflammatory population of CD11b+, F4/80+, Ly6Chigh macrophages was also reduced. Mineralocorticoid receptor inhibition promoted increased IL-4 receptor expression and activation in the whole kidney and in isolated macrophages, thereby facilitating macrophage polarization to an M2 phenotype. The long-term protection conferred by mineralocorticoid receptor antagonism was also translated to the Large White pig pre-clinical model. Thus, our studies support the rationale for using mineralocorticoid receptor antagonists in clinical practice to prevent transition of acute kidney injury to chronic kidney disease.

International audience; AKI is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig, a model of human AKI. In mice, MR deficiency in smooth muscle cells (SMCs) protected against kidney IR injury. MR blockade by the novel nonsteroidal MR antagonist, finerenone, or genetic deletion of MR in SMCs associated with weaker oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1-GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMCs protected against AKI. Furthermore, genetic deletion of MR in SMCs blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the Large White pig. Altogether, we show that MR antagonism, or deletion of the MR gene in SMCs, limited the renal injury induced by IR through effects on Rac1-mediated MR signaling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI.

Organ transplantation success has seen great improvements in past decades due to progresses in immune-suppressive regiments. However, lack of long-term graft outcome improvement, added to increased marginal donors use, has recently shifted the focus towards the peri-transplant period and particularly ischemia reperfusion injury (IRI). The recent acknowledgment of the link between IRI and outcome, short or long term, has rekindled interest in the study of the physiopathology associated with organ preservation and the methods to optimize it. This article is protected by copyright. All rights reserved.

International audience; As the impact of ischemia reperfusion injury on graft outcome is now well defined, efforts are made towards decreasing these lesions, typically through the improvement of preservation techniques. The use of pharmacological supplements which could be compatible with any preservation solution used by the transplant center and target specific pathways of IR is an interesting strategy to improve graft quality. However, the extensive number of studies showing the benefits a molecule in an animal model of IR without thorough mechanistic determination of the effects of this agent make it difficult to opt for specific pharmaceutical intervention. Herein we expose studies which demonstrate the benefits of several molecules relying on a thorough mechanical analysis of the events occurring during preservation, both at the cellular and the systemic levels. We believe this approach is the most appropriate to truly understand the potential benefits of a molecule and particularly to design a comprehensive pharmaceutical regiment, with several agents acting synergistically against IR, to improve organ preservation and graft outcome.; L’impact de l’ischémie reperfusion sur les résultats de la transplantation rénale étant maintenant bien défini, les efforts se concentrent maintenant sur les moyens de diminuer ces lésions, en particulier par l’amélioration des techniques de préservations.L’utilisation de suppléments pharmacologiques, compatible avec n’importe quelle solution de préservation utilisé par le centre de transplantation et ciblant les voies spécifiques de l’IR est une stratégie intéressante pour améliorer la qualité de la transplant. Cependant, le nombre important d’études montrant les avantages de telle ou telle molécule dans les modèles animaux d’IR sans détermination du mécanisme des effets de cet agent rend difficile le choix définitif pour une intervention pharmaceutique spécifique.Dans cette article, les études qui démontrent les avantages de plusieurs molécules en s’appuyant sur l’analyse des mécanisme impliquées dans les événements survenus pendant la phase de préservation, tant au niveau cellulaire que systémiques sont passées en revue. A notre sens, cette approche est la plus appropriée pour comprendre les avantages potentiels d’une molécule et en particulier de concevoir un régime pharmacologique global, avec plusieurs agents agissant en synergie contre l’Ischémie-reperfusion, pour améliorer la conservation des greffons et les résultats de la greffe transplantation

International audience; During the organ transplantation process, conservation solutions must address responses to the physiologic organ preservation and prevent ischemia-reperfusion injuries. The use of colloids seems beneficial especially for long ischemia time compared to the impermeant molecules used for short time. The colloids family includes molecules as hydroxyethyl starch (HES), albumin, dextran or polyethylene glycol (PEG).In this review, the authors describe the rational for PEG use, its potential immunomodulatory effect and the main results of its experimental and clinical use.; Pendant le processus de transplantation d’organes, les solutions de conservation sont utilisées pour répondre aux modifications physiologiques dues à la conservation et prévenir des lésions d’ischémie-reperfusion. L’utilisation de colloïdes semble bénéfique, surtout en cas de période d’ischémie froide longue par rapport aux molécules utilisées pour des durées d’ischémie froide courtes. La famille des colloïdes comprend des molécules comme l’Hydroxyéthyle Amidon (HES), l’albumine, le dextran ou le polyéthylène-glycol (PEG).Dans cette revue, les auteurs décrivent le rationnel scientifique pour l’utilisation du PEG dans les solutions de conservation, son effet immunomodulateur potentiel ainsi que les principaux résultats de son utilisation expérimentale et clinique.

International audience; Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF‐κB) activation are well‐described elements of IRI. We added cyclodextrin‐complexed curcumin (CDC), a potent antioxidant and NF‐κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial‐to‐mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up‐regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic

The intensity of ischemia-reperfusion injury of the donor organ during the preservation phase and after anastomosis is acknowledged as being a key factor for long-term graft outcome. We previously showed that the addition of 5 g/L of the natural oxygen carrier HEMO2 Life was beneficial for the cold static preservation of kidney grafts in both University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate solutions. Herein, we refined these findings by evaluating HEMO2 Life at various dose levels in UW, both in vitro with endothelial cells and in vivo in a pig kidney autotransplantation preclinical model. We showed in vitro that cells were significantly better preserved with HEMO2 Life in a dose-dependent manner, with benefits in terms of survival, metabolic activity, and cellular integrity. In vivo, serum creatinine measurements at reperfusion confirmed the important benefits of HEMO2 Life treatment on function recovery at the dose levels of 1, 2, and 5 g/L. Likewise, histological analysis of kidney parenchyma biopsies from day 7 confirmed the superiority of HEMO2 Life-supplemented UW over UW alone, and there was no difference between the doses. Three months' follow-up confirmed the trend of the first 2 weeks, with creatinine and fibrosis levels similar to those in pretransplant kidneys.

International audience; AIM:Kidney hypoxia can predispose to the development of acute and chronic renal failure in diabetes. Ischaemia-reperfusion injury (IRI) causes inflammation, and diabetes is known to exacerbate this inflammatory response in the kidney, whereas alarmin IL-33 could act as an innate immune mediator during kidney IRI. Thus, the present study examined the impact of genetic IL-33 receptor ST2 deficiency (ST2-/-) on renal IRI in euglycaemic and hyperglycaemic mice.METHODS:Hyperglycaemia was induced with streptozotocin (STZ) in adult male C57BL/6JRj wild-type (WT) mice and ST2-/- mice. Unilateral renal IRI was achieved 3months after STZ treatment by left kidney nephrectomy (non-ischaemic control kidney) and clamping of the right renal artery for 32min in STZ- and vehicle-treated animals. At 24h after reperfusion, renal function and injury were determined by levels of plasma creatinine, blood urea nitrogen (BUN) and histological tubule scores. Also, in a complementary pilot clinical study, soluble ST2 concentrations were compared in diabetics and non-diabetics.RESULTS:Urinary albumin was significantly increased in STZ-induced hyperglycaemic mice, regardless of genotypic background. At 24h post-ischaemia, plasma creatinine, BUN and tubular injury were significantly reduced in ST2-/- mice compared with vehicle-treated WT mice, but this protective effect was lost in the STZ-induced hyperglycaemic ST2-/- animals. Plasma concentrations of soluble ST2 were significantly greater in type 2 diabetes patients vs non-diabetics.CONCLUSION:Our data suggest that the IL-33/ST2 pathway exerts differential effects depending on the glucose environment, opening-up new avenues for future research on alarmins and diabetes in ischaemia-related diseases.

Current organ shortages have led centers to extend the acceptance criteria for organs, increasing the risk for adverse outcomes. Current preservation protocols have not been adapted so as to efficiently protect these organs. Herein, we target oxidative stress, the key mechanism of ischemia reperfusion injury. Vectisol&reg, is a novel antioxidant strategy based on the encapsulation of resveratrol into a cyclodextrin, increasing its bioavailability. We tested this compound as an additive to the most popular static preservation solutions and machine perfusion (LifePort) in a preclinical pig model of kidney autotransplantation. In regard to static preservation, supplementation improved glomerular filtration and proximal tubular function early recovery. Extended follow-up confirmed the higher level of protection, slowing chronic loss of function (creatininemia and proteinuria) and the onset of histological lesions. Regarding machine perfusion, the use of Vectisol&reg, decreased oxidative stress and apoptosis at the onset of reperfusion (30 min post declamping). Improved quality was confirmed with decreased early levels of circulating SOD (Superoxide Dismutase) and ASAT (asparagine amino transferase). Supplementation slowed the onset of chronic loss of function, as well as interstitial fibrosis and tubular atrophy. The simple addition of Vectisol&reg, to the preservation solution significantly improved the performance of organ preservation, with long-term effects on the outcome. This strategy is thus a key player for future multi-drug therapy aimed at ischemia reperfusion in transplantation.

Pancreatic islet transplantation represents an effective therapy with lower morbidity for patients carriers of type 1 diabetes compared to whole pancreas transplantation. Although complete insulin independence is usually not achieved it allows control of glycemia balance reducing the risk of severe hypoglycaemia events and impaired awareness of hypoglycaemia. Recent trials had demonstrated islet transplantation to be more effective than current medical treatment and improvements in outcomes also have been reported after the introduction or modification of current immunosuppressive protocols. The principal disadvantage of this technique is the shortage of pancreatic islets/pancreas , needing several donors for 1 recipient, and the inflammation which affected graft viability. These limitations have led researchers to develop alternative way to assure higher effectiveness such as the development of new cells sources like xenogenic lines (porcine), stem cell-derived β cells or alternative transplantation sites or moreover peculiar techniques to increase cells viability after graft (encapsulation).

International audience; The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of Drosophila during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by N1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

Background Ischemia reperfusion injury (IRI) induced immune response is a critical issue in transplantation. Complement and contact system activation are among its key mechanisms. Study design We investigated the benefits of pre-reperfusion treatment with recombinant human C1INH (rhC1INH), inhibitor of both complement and contact activation, in a pig model of kidney autotransplantation, subjecting the organ to 60 min warm ischemia prior to 24 h static preservation to maximize damage. Results Serum creatinine measurement showed that treated animals recovered glomerular function quicker than the Vehicle group. However, no difference was observed in tubular function recovery, and elevated level of urinary NGal (Neutrophil gelatinase-associated lipocalin) and plasma AST (Aspartate Aminotransferase) were detected, indicating that treatment did not influence IRI-mediated tubular cell necrosis. Regarding chronic graft outcome, rhC1INH significantly prevented fibrosis development and improved function. Immunohistochemistry and western blot showed decreased invasion by macrophages and T lymphocytes, and reduction of epithelial to mesenchymal transition. We determined the effect of treatment on complement activation with immunofluorescence analyses at 30 min post reperfusion, showing an inhibition of C4d deposition and MBL staining in treated animals. Conclusions In this model, the inhibition of complement activation by rhC1INH at reperfusion, while not completely counteracting IRI, limited immune system activation, significantly improving graft outcome on the short and long term. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1013-7) contains supplementary material, which is available to authorized users.

To improve graft preservation and consequently reduce conservation injuries, the composition of preservation solution is of outmost importance. It was demonstrated that the colloid polyethylene glycol (PEG), used in SCOT solution, has protective effects on cell membranes and immunocamouflage properties. The aim of this study was to optimize the concentration and chain length of PEG to improve pancreatic islet preservation and outcome. In a model of murine islet allotransplantation, islets were isolated with SCOT containing various concentrations of PEG 20 kDa or 35 kDa. Better islet yield (IEQ) was obtained with SCOT + PEG at 15–30 g/L versus other PEG concentrations and control CMRL-1066 + 1% BSA solution ( p < 0.05). Allograft survival was better prolonged (up to 20 days) in the groups SCOT + PEG 20 kDa 10–30 g/L compared to PEG 35 kDa (less than 17.8 days) and to control solutions (less than 17.5 days). In terms of graft function recovery, the use of PEG 20 kDa 15–30 g/L induced no primary nonfunction and delayed graft function contrary to CMRL-1066 and other PEG solutions. The use of the extracellular-type solution SCOT containing PEG 20 kDa 15 g/L as colloid could be a new way to optimize graft integrity preservation and allograft outcome.

International audience; Translocator protein (TSPO), formerly known as peripheral-type benzodiazepine receptor (PBR), has been described in several tissues and characterized as one of the main elements of steroidogenesis. However, TSPO is also involved in other pathways and cell functions, such as apoptosis regulation, protein import, membrane biogenesis, cell cycle regulation, oxygen homeostasis and mitochondrial membrane fluidity regulation. In the kidney, TSPO is normally located in the distal parts of the nephron from the thick ascending limb of the loop of Henle to the medullary collecting ducts. However when the kidney is submitted to a stress such as ischemia reperfusion injury there is a defined change in TSPO expression towards more proximal areas of the nephron, and the protein can be detected as high as proximal tubular cells and the Bowman Capsule. As the injury persists, TSPO is also located in invading mononucleated cells, in a pattern reproducing invasion by CD4+ helper Tcells, and in the damaged vessels where TSPO is expressed both in endothelial and smooth muscle cells. Herein we review the potential use of TSPO-directed treatment for ischemia reperfusion injury, particularly regarding pre-conditioning of the organ. We also detail the relationship of proximal TSPO staining with the intensity of the injury, particularly the implication of monomeric (18 kDa) TSPO and its role in hypoxiareoxygenation and apoptosis prevention. The potential implications of the protein with regeneration processes activated in response to injury and their relation with embryogenesis pathways are discussed.

Coagulation is an important pathway in the pathophysiology of ischemia-reperfusion injuries. In particular, deceased after circulatory death (DCD) donors undergo a no-flow period, a strong activator of coagulation. Hence, therapies influencing the coagulation cascade must be developed. We evaluated the effect of a new highly specific and effective anti-Xa/Ila molecule, with an integrated innovative antidote site (EP217609), in a porcine preclinical model mimicking injuries observed in DCD donor kidney transplantation. Kidneys were clamped for 60 minutes (warm ischemia), then flushed and preserved for 24 hours at 4 degrees C in University of Wisconsin (UW) solution (supplemented or not). EP217609-supplemented UW solution (UW-EP), compared with unfractionated heparin-supplemented UW solution (UW-UFH) or UW alone (UW). A mechanistic investigation was conducted in vitro: addition of EP217609 to endothelial cells during hypoxia at 4 degrees C in the UW solution inhibited thrombin generation during reoxygenation at 37 degrees C in human plasma and reduced tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 messenger RNA cell expressions. In vivo, function recovery was markedly improved in the UW-EP group. Interestingly, levels of thrombin-anti thrombin complexes (reflecting thrombin generation) were reduced 60 minutes after reperfusion in the UW-EP group. In addition, 3 months after transplantation, lower fibrosis, epithelial-mesenchymal transition, inflammation, and leukocyte infiltration were observed. Using this new dual anticoagulant, anti-Xa/lla activity during kidney flush and preservation is protected by reducing thrombin generation at revascularization, improving early function recovery, and decreasing chronic lesions. Such an easy-to-deploy clinical strategy could improve marginal graft outcome.

Objectifs Le phenomene d’ischemie-reperfusion (IR) est un processus central implique dans la dysfonction aigue et chronique du greffon renal. L’objectif de cette etude etait de caracteriser les cellules souches mesenchymateuses du tissu adipeux (TA) porcin (pASC) et leurs capacites a ameliorer la reprise de fonction des greffons dans des conditions mimant la situation des donneurs decedes apres arret cardiaque (DDAC). Methodes La morphologie, la proliferation, le phenotype en cytometrie de flux et le profil metabolique en resonance magnetique nucleaire (RMN) des pASC ont ete determines. Leur resistance a une sequence d’hypoxie-reoxygenation (HR) a ete evaluee en analysant leur viabilite cellulaire et leur profil metabolique en RMN. La faisabilite, les resultats fonctionnels et histologiques d’une injection autologue de 10 6 pASC/kg dans l’artere renale de 3 greffons renaux autotransplantes (apres 1 h d’ischemie chaude et 24 h de conservation a 4 °C dans la solution UW et nephrectomie controlaterale – groupe pASC-AUTO) ont ete compares a un groupe de porcs autotransplantes sans injection de pASC – groupe AUTO-T. Resultats La technique d’extraction cellulaire est reproductible et permet d’obtenir suffisamment de pASC ayant les caracteristiques de cellules souches mesenchymateuses. Leur profil metabolique en RMN n’etait pas modifie avec les passages, caracterisant la stabilite des lignees. Leur viabilite apres HR etait superieure a 70 %. L’injection de 10 6 pASC/kg est realisable 15 jours apres le prelevement du TA. A j7, la creatininemie moyenne du groupe pASC-AUTO etait de 298 ± 33,8 versus 997,6 ± 170,6 μmol/L pour le groupe AUTO-T ( p = 0,04). La reprise de fonction etait significativement amelioree et les lesions histologiques reduites dans le groupe pASC-AUTO. Conclusion L’injection de pASC a la reperfusion des greffons dans un modele porcin mimant la situation des DDAC semble ameliorer la reprise de fonction et limiter les lesions tubulaires liees a l’ischemie-reperfusion des greffons a j7. Ces potentialites therapeutiques devront etre confirmees par des etudes complementaires a la fin du suivi de 6 animaux a 3 mois.

What's known on the subject? and What does the study add? Machine perfusion of kidney graft improves short term outcome. However, the technique still requires optimization, as the rate of delayed graft function remains high. Machine perfusion of kidney graft has significant benefits on chronic outcome in terms of interstitial fibrosis and kidney function. Improvement of the technique is possible using alternative perfusion fluids, such as fourth generation preservation solutions containing polyethylene glycol. OBJECTIVES • To assess the effect of machine perfusion (MP) on renal function recovery vs kidney preservation in static cold storage (CS), in a large animal preclinical model. • To assess whether MP benefits are dependent on the preservation solution used. METHODS • Using an established autotransplantation pig kidney model associated with a contralateral nephrectomy we studied the impact of MP against the deleterious effects of warm ischaemia (WI; 60 min), then 22 h of cold ischaemia using MP or static CS, followed by autotransplantation. • We used Berzer MP solution (MPS), recommended for MP, and Institut Georges Lopez preservation solution (IGL-1), designed for CS. • The pigs were divided into four study groups: MPS-CS: static CS with MPS (n = 7); MPS-MP: renal perfusion with MPS using the Waters Medical Systems (Rochester, MN, USA) RM3 pulsatile machine (n = 7); IGL-CS: static CS with IGL-1 solution (n = 7); IGL-MP: renal perfusion with IGL-1 solution (n = 7). • The effect of ischaemia was determined using different variables: pig survival; plasma creatinine; proteinuria; oxidative stress; tubular sodium reabsorption rate; and tissue damage at 1 month. RESULTS • Pig survival was higher in MP and IGL groups compared to MPS-CS. • Plasma creatinine levels did not differ among the groups, but proteinuria assay showed significant benefits for the MP vs static CS groups. • Histological evaluation of kidney grafts showed more injury in the CS groups than in the MP groups. • Urinary measurement of tubular enzyme activity differed substantially among the groups, highlighting the benefits of MP in maintaining brush border integrity. CONCLUSIONS • In our model reproducing the conditions of deceased after cardiac arrest donors we show that MP decreases the risk of renal dysfunction and preserves kidney parenchyma. • A non-invasive urinary enzyme assay can provide valuable information on graft integrity. • The preservation solution used is important as the wrong solution can decrease the benefits of MP.

RESUME L’ischemie reperfusion (IR) est un episode deletere, incontournable de la transplantation d’organes. La conservation hypothermique permet de la limiter en reduisant l’activite metabolique des organes conserves. Cependant, la conservation en hypothermie induit un syndrome physiopathologique complexe responsable de plusieurs processus lesionnels. Les consequences sont importantes sur le devenir a court terme en retardant le fonctionnement des organes greffes et a long terme en participant a l’apparition de lesions chroniques.

International audience; Background. Ischemia reperfusion injury (IRI) is inherent to transplantation, and correlates with negative outcome. Limiting IRI requires new preservation. Fourth generation solutions are emerging, using new colloid based on polyethylene glycol (PEG) polymers and extracellular ionic composition. We evaluated their eventual benefits for optimal resistance to IRI and improved outcome. Methods. Using primary cell culture and a preclinical pig model of low-mismatch kidney allograft transplantation, not requiring immunosuppression, we compared the following solutions: UW (University of Wisconsin), high potassium with hydroxyethyl starch, gold standard in preservation; IGL-1 (Institute George Lopez-1), low potassium solution using PEG (35 kDa, 1 g/L); and SCOT (Solution de Conservation des Organes et Tissus), plasma-like ionic composition, containing PEG 20 kDa (30 g/L). Results. In vitro, SCOT-preserved cells had better viability and less necrosis. In vivo, SCOT-grafts had better function recovery, with limited histological injury compared with the other solutions. During the 3 months follow-up, we found low innate and adaptative immune response in SCOT organs, whereas other groups presented high rate of invasion and antigen presentation. SCOT-preserved kidneys showed low fibrosis, transforming growth factor-beta expression and apoptosis compared with the other groups. These differences impacted survival at 3 months, which was low in UW (20%) and IGL-1 (40%) groups, whereas it remained high for SCOT animals (80%, P < 0.05 to UW). Conclusions. In conclusion, plasma-like ionic composition and nontoxic molecule PEG provide high resistance against IRI and optimize graft outcome. Such solutions could be invaluable for the use of fragile organs, such as from extended criteria or deceased after cardiac death donors.

Purpose: The aim of this study was to analyze the importance of donor factors and especially the potential role of hemodynamic management in regard to delayed graft function in paired kidney recipient patients after renal transplantation and to analyze the urine of organ donors by proton-nuclear magnetic resonance spectroscopy to identify urine markers potentially correlated with delayed graft function in recipient patients. Methods: A prospective multicenter epidemiologic study was conducted. A logistic regression model taking into account paired data was used. Results: Data from 72 donors and the 144 corresponding paired recipients were analyzed. Univariate analysis showed that age of donor, previous history of tobacco, ischemic cause of brain death, norepinephrine infusion, and recipient age were the risk factors for delayed graft function. After adjusting for correlated outcome data and controlling for other potential prognostic factors, 3 variables remained significantly associated with outcome: donor age (odds ratio [OR], 10.7), hemodynamic status (OR, 0.167), and hydroxyl-ethyl starch infusion (OR, 0.135). Proton-nuclear magnetic resonance analysis evidenced 3 metabolites of interest in donors (trimethylamine-N-oxide, citrate, and lactate). However, these peaks were not correlated the clinical parameters in donors. Conclusions: Paired analysis of kidney transplantation emphasizes the important role of factor donor associated with delayed graft function in recipient. Thus, a particular attention should be paid to the hemodynamic management of donor.

Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is an 18-kDa drug- and cholesterol-binding protein localized to the outer mitochondrial membrane and implicated in a variety of cell and mitochondrial functions. To determine the role of TSPO in ischemia-reperfusion injury (IRI), we used both in vivo and in vitro porcine models: an in vivo renal ischemia model where different conservation modalities were tested and an in vitro model where TSPO-transfected porcine proximal tubule LLC-PK1cells were exposed to hypoxia and oxidative stress. The expression of TSPO and its partners in steroidogenic cells, steroidogenic acute regulatory protein (StAR) and cytochrome P-450 side chain cleavage CYP11A1, as well as the impact of TSPO overexpression and exposure to TSPO ligands in vitro in hypoxia-ischemia conditions were investigated. Hypoxia induced caspase activation, reduction of ATP content, and LLC-PK1cell death. Transfection and overexpression of TSPO rescued the cells from the detrimental effects of hypoxia and reoxygenation. Moreover, TSPO overexpression was accompanied by a reduction of H2O2-induced necrosis. TSPO drug ligands did not affect TSPO-mediated functions. In vivo, TSPO expression was modulated by IRI and during regeneration particularly in proximal tubule cells, which do not express this protein at the basal level. Under the same conditions, StAR and CYP11A1 protein and gene expression was reduced without apparent relation to TSPO changes. Pregnenolone was identified and measured in the pig kidney. Pregnenolone synthesis was not affected by the experimental conditions used. Taken together, these results indicate that changes in TSPO expression in kidney regenerating tissue could be important for renal protection and maintenance of kidney function.

International audience; The storage conditions of the donor kidney may influence the deleterious consequences of ischemia/reperfusion (IR), which remains a major source of complications in clinical practice. Delayed graft function (DGF), seen in 20% to 50% of transplanted cadaver kidneys, is a major risk factor affecting early and long-term graft survival, patient management, and costs of transplantation. Cold preservation plays a key role in this process and is based on hypothermia and high potassium solutions. In this review, the authors focused on the major molecular mechanisms of cold storage (CS) injury at the cellular level, which have been recently evidenced with modern biochemical and cell biologic methods. These newly uncovered aspects of cold preservation injury are often not fully addressed by preservation solutions in current clinical practice. The role of new molecules such as polyethylene glycol (PEG) is presented and their properties are analyzed in the organ preservation context. PEG improves organ function recovery and reduces inflammation and fibrosis development in several models. Because organs shortage is also a real public health problem, organs from non-heart beating donors or marginal donors are now used to expand pool of organs. As a consequence, the development of better organ preservation methods remains a major target and deserves scientific consideration.

Acute renal failure (ARF) is often the consequence of an ischemia-reperfusion injury (IRI) and associated with high mortality. Warm ischemia (WI) is a crucial factor of tissue damage, and tissue destruction led by ischemia-reperfusion (I/R) can impact the early and long-term functional outcome. Trimetazidine (TMZ) is an anti-ischemic drug. Previously, we already verified its protective effect on a cold-ischemic pig kidney model by directly adding TMZ into the preservation solution (Faure JP, Baumert H, Han Z, Goujon JM, Favreau F, Dutheil D, Petit I, Barriere M, Tallineau C, Tillement JP, Carretier M, Mauco G, Papadopoulos V, Hauet T. Biochem Pharmacol 66: 2241–2250, 2003; Faure JP, Petit I, Zhang K, Dutheil D, Doucet C, Favreau F, Eugene M, Goujon JM, Tillement JP, Mauco G, Vandewalle A, Hauet T. Am J Transplant 4: 495–504, 2004). In this study, we aimed to study the potential effect of TMZ pretreatment (5 mg/kg iv 24 h before WI) on the injury caused by WI for 45, 60, and 90 min and reperfusion in a WI pig kidney model. Compared with sham-operated (control) and uninephrectomized animals (UNX), TMZ pretreatment significantly reduced deleterious effects after 45 min, and particularly 60 and 90 min, of WI by improving the recovery of renal function and minimizing the inflammatory response commonly prevalent in ischemic kidney injury. Compared with controls (control group and UNX group), it was observed that 1) hypoxia-inducible factor-1 (HIF-1α) expression occurred earlier and with a higher intensity in the TMZ-treated groups; 2) the reduction of IRI during the first week following reperfusion was correlated with an earlier and greater expression of stathmin, which is involved in the process of tubular repair; and 3) the tubulointerstitial fibrosis was reduced, particularly after 60 and 90 min of WI. In conclusion, TMZ made the warm-ischemic kidneys more resistant to the deleterious impact of a single episode of I/R and reduced early and long-term subsequent damage.

International audience

In renal transplantation, live donor kidney grafts are associated with optimum success rates due to the shorter period of ischemia during the surgical procedure. The current shortage of donor organs for adult patients has caused a shift towards deceased donors, often with co-morbidity factors, whose organs are more sensitive to ischemia-reperfusion injury, which is unavoidable during transplantation. Donor management is pivotal to kidney graft survival through the control of the ischemia-reperfusion sequence, which is known to stimulate numerous deleterious or regenerative pathways. Although the key role of endothelial cells has been established, the complexity of the injury, associated with stimulation of different cell signaling pathways, such as unfolded protein response and cell death, prevents the definition of a unique therapeutic target. Preclinical transplant models in large animals are necessary to establish relationships and kinetics and have already contributed to the improvement of organ preservation. Therapeutic strategies using mesenchymal stem cells to induce allograft tolerance are promising advances in the treatment of the pediatric recipient in terms of reducing/withdrawing immunosuppressive therapy. In this review we focus on the different donor management strategies in kidney graft conditioning and on graft preservation consequences by highlighting the role of endothelial cells. We also propose strategies for preventing ischemia-reperfusion, such as cell therapy.