Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study.

BACKGROUND: Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach. METHODS: We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls. FINDINGS: The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68+ and CD15+ innate immune cells, as well as NKp46+ cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models. INTERPRETATION: Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results. FUNDING: OrganX and MSD Avenir.

Comparison between 5 extractions methods in either plasma or serum to determine the optimal extraction and matrix combination for human metabolomics.

BACKGROUND: Although metabolomics continues to expand in many domains of research, methodological issues such as sample type, extraction and analytical protocols have not been standardized, impeding proper comparison between studies and future research. METHODS: In the present study, five solvent-based and solid-phase extraction methods were investigated in both plasma and serum. All these extracts were analyzed using four liquid chromatography coupled with high resolution mass spectrometry (LC-MS) protocols, either in reversed or normal-phase and with both types of ionization. The performances of each method were compared according to putative metabolite coverage, method repeatability and also extraction parameters such as overlap, linearity and matrix effect; in both untargeted (global) and targeted approaches using fifty standard spiked analytes. RESULTS: Our results verified the broad specificity and outstanding accuracy of solvent precipitation, namely methanol and methanol/acetonitrile. We also reveal high orthogonality between methanol-based methods and SPE, providing the possibility of increased metabolome coverage, however we highlight that such potential benefits must be weighed against time constrains, sample consumption and the risk of low reproducibility of SPE method. Furthermore, we highlighted the careful consideration about matrix choice. Plasma showed the most suitable in this metabolomics approach combined with methanol-based methods. CONCLUSIONS: Our work proposes to facilitate rational design of protocols towards standardization of these approaches to improve the impact of metabolomics research.

Tissue Injury Protection: The Other Face of Anticoagulant Treatments in the Context of Ischemia and Reperfusion Injury with a Focus on Transplantation.

Organ transplantation has enhanced the length and quality of life of patients suffering from life-threatening organ failure. Donors deceased after brain death (DBDDs) have been a primary source of organs for transplantation for a long time, but the need to find new strategies to face organ shortages has led to the broadening of the criteria for selecting DBDDs and advancing utilization of donors deceased after circulatory death. These new sources of organs come with an elevated risk of procuring organs of suboptimal quality. Whatever the source of organs for transplant, one constant issue is the occurrence of ischemia-reperfusion (IR) injury. The latter results from the variation of oxygen supply during the sequence of ischemia and reperfusion, from organ procurement to the restoration of blood circulation, triggering many deleterious interdependent processes involving biochemical, immune, vascular and coagulation systems. In this review, we focus on the roles of thrombo-inflammation and coagulation as part of IR injury, and we give an overview of the state of the art and perspectives on anticoagulant therapies in the field of transplantation, discussing benefits and risks and proposing a strategic guide to their use during transplantation procedures.

HEMO2 life® improves renal function independent of cold ischemia time in kidney recipients: A comparison with a large multicenter prospective cohort study.

BACKGROUND: M101 is an extracellular hemoglobin isolated from a marine lugworm and is present in the medical device HEMO2 life®. The clinical investigation OXYOP was a paired kidney analysis (n = 60) designed to evaluate the safety and performance of HEMO2 life® used as an additive to preservation solution in renal transplantation. The secondary efficacy endpoints showed less delayed graft function (DGF) and better renal function in the HEMO2 life® group but due to the study design cold ischemia time (CIT) was longer in the contralateral kidneys. METHODS: An additional analysis was conducted including OXYOP patients and patients from the ASTRE database (n = 6584) to verify that the decrease in DGF rates observed in the HEMO2 life® group may not be due solely to the shorter CIT but also to HEMO2 life® performance. Kaplan-Meier estimate curves of cumulative probability of achieving a creatinine level below 250 µmol/L were generated and compared in both groups. A Cox model was used to test the effect of the explanatory variables (use of HEMO2 life® and CIT). Finally, a bootstrap strategy was used to randomly select smaller samples of patients and test them for statistical comparison in the ASTRE database. RESULTS: Kaplan-Meier estimate curves confirmed the existence of a relation between DGF and CIT and Cox analysis showed a benefit in the HEMO2 life® group regardless of the associated CIT. Boostrap analysis confirmed these results. CONCLUSIONS: The present study suggested that the better recovery of renal function observed among kidneys preserved with HEMO2 life® in the OXYOP study is a therapeutic benefit of this breakthrough innovative medical device.

New Strategies Protecting from Ischemia/Reperfusion.

This Special Issue aims to highlight new avenues in the management of Ischemia/Reperfusion (I/R) injury [...].

Molecular Markers of Kidney Transplantation Outcome: Current Omics Tools and Future Developments.

PURPOSE OF REVIEW: The emerging field of molecular predictive medicine is aiming to change the traditional medical approach in renal transplantation. Many studies have explored potential biomarker molecules with predictive properties in renal transplantation, issued from omics research. Herein, we review the biomarker molecules of four technologies (i.e., Genomics, Transcriptomics, Proteomics, and Metabolomics) associated with favorable kidney transplant outcomes. RECENT FINDINGS: Several panels of molecules have been associated with the outcome that the majority of markers are related to inflammation and immune response; although. other molecular ontologies are also represented, such as proteasome, growth, regeneration, and drug metabolism. Throughout this review, we highlight the lack of properly validated statistical demonstration. Indeed, the most preeminent molecular panels either remain at the limited size study stage or are not confirmed during large-scale studies. At the core of this problem, we identify the methodological shortcomings and propose a comprehensive workflow for discovery and validation of molecular biomarkers that aims to improve the relevance of these tools in the future. SUMMARY: Overall, adopting a patient management through omics approach could bring remarkable improvement to transplantation success. An increased effort and investment between scientists, medical biologists, and clinicians seem to be the path toward a proper solution.

Preservation of Organs to Be Transplanted: An Essential Step in the Transplant Process.

Organ transplantation remains the treatment of last resort in case of failure of a vital organ (lung, liver, heart, intestine) or non-vital organ (essentially the kidney and pancreas) for which supplementary treatments exist. It remains the best alternative both in terms of quality-of-life and life expectancy for patients and of public health expenditure. Unfortunately, organ shortage remains a widespread issue, as on average only about 25% of patients waiting for an organ are transplanted each year. This situation has led to the consideration of recent donor populations (deceased by brain death with extended criteria or deceased after circulatory arrest). These organs are sensitive to the conditions of conservation during the ischemia phase, which have an impact on the graft's short- and long-term fate. This evolution necessitates a more adapted management of organ donation and the optimization of preservation conditions. In this general review, the different aspects of preservation will be considered. Initially done by hypothermia with the help of specific solutions, preservation is evolving with oxygenated perfusion, in hypothermia or normothermia, aiming at maintaining tissue metabolism. Preservation time is also becoming a unique evaluation window to predict organ quality, allowing repair and/or optimization of recipient choice.

Oxidative Stress Evaluation in Ischemia Reperfusion Models: Characteristics, Limits and Perspectives.

Ischemia reperfusion injury is a complex process consisting of a seemingly chaotic but actually organized and compartmentalized shutdown of cell function, of which oxidative stress is a key component. Studying oxidative stress, which results in an imbalance between reactive oxygen species (ROS) production and antioxidant defense activity, is a multi-faceted issue, particularly considering the double function of ROS, assuming roles as physiological intracellular signals and as mediators of cellular component damage. Herein, we propose a comprehensive overview of the tools available to explore oxidative stress, particularly in the study of ischemia reperfusion. Applying chemistry as well as biology, we present the different models currently developed to study oxidative stress, spanning the vitro and the silico, discussing the advantages and the drawbacks of each set-up, including the issues relating to the use of in vitro hypoxia as a surrogate for ischemia. Having identified the limitations of historical models, we shall study new paradigms, including the use of stem cell-derived organoids, as a bridge between the in vitro and the in vivo comprising 3D intercellular interactions in vivo and versatile pathway investigations in vitro. We shall conclude this review by distancing ourselves from "wet" biology and reviewing the in silico, computer-based, mathematical modeling, and numerical simulation options: (a) molecular modeling with quantum chemistry and molecular dynamic algorithms, which facilitates the study of molecule-to-molecule interactions, and the integration of a compound in a dynamic environment (the plasma membrane...); (b) integrative systemic models, which can include many facets of complex mechanisms such as oxidative stress or ischemia reperfusion and help to formulate integrated predictions and to enhance understanding of dynamic interaction between pathways.

Study of the Role of the Tyrosine Kinase Receptor MerTK in the Development of Kidney Ischemia-Reperfusion Injury in RCS Rats.

Renal ischaemia reperfusion (I/R) triggers a cascade of events including oxidative stress, apoptotic body and microparticle (MP) formation as well as an acute inflammatory process that may contribute to organ failure. Macrophages are recruited to phagocytose cell debris and MPs. The tyrosine kinase receptor MerTK is a major player in the phagocytosis process. Experimental models of renal I/R events are of major importance for identifying I/R key players and for elaborating novel therapeutical approaches. A major aim of our study was to investigate possible involvement of MerTK in renal I/R. We performed our study on both natural mutant rats for MerTK (referred to as RCS) and on wild type rats referred to as WT. I/R was established by of bilateral clamping of the renal pedicles for 30' followed by three days of reperfusion. Plasma samples were analysed for creatinine, aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), kidney injury molecule -1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) levels and for MPs. Kidney tissue damage and CD68-positive cell requirement were analysed by histochemistry. monocyte chemoattractant protein-1 (MCP-1), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and histone 3A (H3A) levels in kidney tissue lysates were analysed by western blotting. The phagocytic activity of blood-isolated monocytes collected from RCS or WT towards annexin-V positive bodies derived from cultured renal cell was assessed by fluorescence-activated single cell sorting (FACS) and confocal microscopy analyses. The renal I/R model for RCS rat described for the first time here paves the way for further investigations of MerTK-dependent events in renal tissue injury and repair mechanisms.

A Sodium Oxalate-Rich Diet Induces Chronic Kidney Disease and Cardiac Dysfunction in Rats.

Chronic kidney disease (CKD) is a worldwide public health issue affecting 14% of the general population. However, research focusing on CKD mechanisms/treatment is limited because of a lack of animal models recapitulating the disease physiopathology, including its complications. We analyzed the effects of a three-week diet rich in sodium oxalate (OXA diet) on rats and showed that, compared to controls, rats developed a stable CKD with a 60% reduction in glomerular filtration rate, elevated blood urea levels and proteinuria. Histological analyses revealed massive cortical disorganization, tubular atrophy and fibrosis. Males and females were sensitive to the OXA diet, but decreasing the diet period to one week led to GFR significance but not stable diminution. Rats treated with the OXA diet also displayed classical CKD complications such as elevated blood pressure and reduced hematocrit. Functional cardiac analyses revealed that the OXA diet triggered significant cardiac dysfunction. Altogether, our results showed the feasibility of using a convenient and non-invasive strategy to induce CKD and its classical systemic complications in rats. This model, which avoids kidney mass loss or acute toxicity, has strong potential for research into CKD mechanisms and novel therapies, which could protect and postpone the use of dialysis or transplantation.

High Throughput Proteomic Exploration of Hypothermic Preservation Reveals Active Processes within the Cell Associated with Cold Ischemia Kinetic.

The demand for organs to be transplanted increases pressure on procurement centers, to the detriment of organ quality, increasing complications. New preservation protocols are urgently needed, requiring an in-depth understanding of ischemia-reperfusion mechanisms. We performed a proteomic analysis using LC-MS/MS-TOF data analyzed through R software and Cytoscape's ClueGO application, comparing the proteome of kidney endothelial cells, key cell type, subjected to 3, 6, 12, 19, and 24 h of cold ischemia and 6 h reperfusion. Critical pathways such as energy metabolism, cytoskeleton structure/transport system, and gene transcription/translation were modulated. Important time windows were revealed: a-during the first 3 h, central proteins were upregulated within these pathways; b-the majority of these upregulations were maintained until 12 h cold ischemia time (CIT); c-after that time, the overall decrease in protein expression was observed; d-at reperfusion, proteins expressed in response to cold ischemia were all downregulated. This shows that cold ischemia is not a simple slowing down of metabolism, as deep changes take place within the proteome on major pathways. Time-sensitive expression of key protein reveals possible quality biomarkers as well as potential targets for new strategies to maintain or optimize organ quality.

Molecular Networking for Drug Toxicities Studies: The Case of Hydroxychloroquine in COVID-19 Patients.

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient's plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.

The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model.

The eIF5A hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) was shown to protect from ischemic injuries. We hypothesized that GC7 could be useful for preconditioning kidneys from donors before transplantation. Using a preclinical porcine brain death (BD) donation model, we carried out in vivo evaluation of GC7 pretreatment (3 mg/kg iv, 5 minutes after BD) at the beginning of the 4h-donor management, after which kidneys were collected and cold-stored (18h in University of Wisconsin solution) and 1 was allotransplanted. Groups were defined as following (n = 6 per group): healthy (CTL), untreated BD (Vehicle), and GC7-treated BD (Vehicle + GC7). At the end of 4h-management, GC7 treatment decreased BD-induced markers, as radical oxygen species markers. In addition, GC7 increased expression of mitochondrial protective peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC1α) and antioxidant proteins (superoxyde-dismutase-2, heme oxygenase-1, nuclear factor [erythroid-derived 2]-like 2 [NRF2], and sirtuins). At the end of cold storage, GC7 treatment induced an increase of NRF2 and PGC1α mRNA and a better mitochondrial integrity/homeostasis with a decrease of dynamin- related protein-1 activation and increase of mitofusin-2. Moreover, GC7 treatment significantly improved kidney outcome during 90 days follow-up after transplantation (fewer creatininemia and fibrosis). Overall, GC7 treatment was shown to be protective for kidneys against BD-induced injuries during donor management and subsequently appeared to preserve antioxidant defenses and mitochondria homeostasis; these protective effects being accompanied by a better transplantation outcome.

Microvasculature partial endothelial mesenchymal transition in early posttransplant biopsy with acute tubular necrosis identifies poor recovery renal allografts.

Acute tubular necrosis (ATN), a frequent histopathological feature in the early post-renal transplant biopsy, affects long-term graft function. Appropriate markers to identify patients at risk of no or incomplete recovery after delayed graft function are lacking. In this study, we first included 41 renal transplant patients whose biopsy for cause during the first month after transplantation showed ATN lesions. Using partial microvasculature endothelial (fascin, vimentin) and tubular epithelial (vimentin) to mesenchymal transition markers, detected by immunohistochemistry, we found a significant association between partial endothelial to mesenchymal transition and poor graft function recovery (Spearman's rho = -0.55, P = .0005). Transforming growth factor-ß1 was strongly expressed in these phenotypic changed endothelial cells. Extent of ATN was also correlated with short- and long-term graft dysfunction. However, the association of extensive ATN with long-term graft dysfunction (24 months posttransplant) was observed only in patients with partial endothelial to mesenchymal transition marker expression in their grafts (Spearman's rho = -0.64, P = .003), but not in those without. The association of partial endothelial to mesenchymal transition with worse renal graft outcome was confirmed on 34 other early biopsies with ATN from a second transplant center. Our results suggest that endothelial cell activation at the early phase of renal transplantation plays a detrimental role.

Comparison of the removal of uraemic toxins with medium cut-off and high-flux dialysers: a randomized clinical trial.

BACKGROUND: Accumulation 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. METHODS: This 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. RESULTS: Compared with HF-HD, MCO-HD provided higher mean RR of myoglobin (36 ± 8 versus 57 ± 13%, P < 0.0001), beta2-microglobulin (68 ± 6 versus 73 ± 15%, P = 0.04), prolactin (32 ± 13 versus 59 ± 11%, P < 0.0001), fibroblast growth factor 23 (20 ± 21 versus 41 ± 22%, P = 0.0002), homocysteine (43 ± 7 versus 46 ± 9%, P = 0.03) and higher median RR of kappa [54 (48-58) versus 70 (63-74)%, P < 0.0001] and lambda free light chain (FLC) [15 (9-22) versus 44 (38-49)%, P < 0.0001]. Mean ± SD pre-dialysis levels of beta2-microglobulin (28.4 ± 5.6 versus 26.9 ± 5.1 mg/L, P = 0.01) and oxidized low-density lipoprote (6.9 ± 4.4 versus 5.5 ± 2.5 pg/mL, P = 0.04), and median (interquartile range) kappa FLC [145 (104-203) versus 129 (109-190) mg/L, P < 0.03] and lambda FLC [106 (77-132) versus 89 (62-125) mg/L, P = 0.002] were significantly lower. Mean albumin levels decreased significantly (38.2 ± 4.1 versus 36.9 ± 4.3 g/L, P = 0.004), without an effect on nutritional status as suggested by unchanged normalized protein catabolic rate and transthyretin level. CONCLUSIONS: Compared 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.

In Vitro/Ex Vivo Models for the Study of Ischemia Reperfusion Injury during Kidney Perfusion.

Oxidative stress is a key element of ischemia-reperfusion injury, occurring during kidney preservation and transplantation. Current options for kidney graft preservation prior to transplantation are static cold storage (CS) and hypothermic machine perfusion (HMP), the latter demonstrating clear improvement of preservation quality, particularly for marginal donors, such as extended criteria donors (ECDs) and donation after circulatory death (DCDs). Nevertheless, complications still exist, fostering the need to improve kidney preservation. This review highlights the most promising avenues of in kidney perfusion improvement on two critical aspects: ex vivo and in vitro evaluation.

Evaluation of Liver Quality after Circulatory Death Versus Brain Death: A Comparative Preclinical Pig Model Study.

The current organ shortage in hepatic transplantation leads to increased use of marginal livers. New organ sources are needed, and deceased after circulatory death (DCD) donors present an interesting possibility. However, many unknown remains on these donors and their pathophysiology regarding ischemia reperfusion injury (IRI). Our hypothesis was that DCD combined with abdominal normothermic regional recirculation (ANOR) is not inferior to deceased after brain death (DBD) donors. We performed a mechanistic comparison between livers from DBD and DCD donors in a highly reproducible pig model, closely mimicking donor conditions encountered in the clinic. DCD donors were conditioned by ANOR. We determined that from the start of storage, pro-lesion pathways such as oxidative stress and cell death were induced in both donor types, but to a higher extent in DBD organs. Furthermore, pro-survival pathways, such as resistance to hypoxia and regeneration showed activation levels closer to healthy livers in DCD-ANOR rather than in DBD organs. These data highlight critical differences between DBD and DCD-ANOR livers, with an apparent superiority of DCD in terms of quality. This confirms our hypothesis and further confirms previously demonstrated benefits of ANOR. This encourages the expended use of DCD organs, particularly with ANOR preconditioning.

Defining the optimal duration for normothermic regional perfusion in the kidney donor: A porcine preclinical study.

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.

Hypercholesterolemia-induced increase in plasma oxidized LDL abrogated pro angiogenic response in kidney grafts.

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.

Oxygen Consumption by Warm Ischemia-Injured Porcine Kidneys in Hypothermic Static and Machine Preservation.

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.