VOC-Based Probes, a New Set of Analytical Tools to Monitor Patient Health from Blood Sample. Proof of Concept on Tracking COVID-19 Infection.

Induced volatolomics is an emerging field that holds promise for many biomedical applications including disease detection and prognosis. In this pilot study, we report the first use of a cocktail of volatile organic compounds (VOCs)-based probes to highlight new metabolic markers allowing disease prognosis. In this pilot study, we specifically targeted a set of circulating glycosidases whose activities could be associated with critical COVID-19 illness. Starting from blood sample collection, our approach relies on the incubation of VOC-based probes in plasma samples. Once activated, the probes released a set of VOCs in the sample headspace. The dynamic monitoring of the signals of VOC tracers enabled the identification of three dysregulated glycosidases in the initial phase after infection, for which preliminary machine learning analyses suggested an ability to anticipate critical disease development. This study demonstrates that our VOC-based probes are a new set of analytical tools that can provide access to biological signals until now unavailable to biologists and clinicians and which could be included in biomedical research to properly construct multifactorial therapy algorithms, necessary for personalized medicine.

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.

Molecular Frontiers in Transplantation-Induced Ischemia-Reperfusion Injury.

This Special Issue aims to summarize the most up-to-date research on ischemia-reperfusion and organ transplantation [...].

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.

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.

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.

Efficacy of the natural oxygen transporter HEMO2 life® in cold preservation in a preclinical porcine model of donation after cardiac death.

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.

Rapid or Slow Time to Brain Death? Impact on Kidney Graft Injuries in an Allotransplantation Porcine Model.

The use of donors deceased after brain death (DBD) with extended criteria in response to the shortage of grafts leads to the removal of more fragile kidneys. These grafts are at greater risk of not being grafted or delayed function. A better knowledge of the pathophysiology of DBDs would improve this situation. There is a difference between the results from animal models of DBD and the clinical data potentially explained by the kinetics of brain death induction. We compared the effect of the induction rate of brain death on the recovery of post-transplant renal function in a pig model of DBD followed by allografts in nephrectomized pigs. Resumption of early function post-transplant was better in the rapidly generated brain death group (RgBD) and graft fibrosis at three months less important. Two groups had identical oxidative stress intensity but a greater response to this oxidative stress by SIRT1, PGC1-α and NRF2 in the RgBD group. Modulation of mechanistic target of rapamycin (mTOR) stimulation by NRF2 would also regulate the survival/apoptosis balance of renal cells. For the first time we have shown that an allostatic response to oxidative stress can explain the impact of the rapidity of brain death induction on the quality of kidney transplants.

Vectisol Formulation Enhances Solubility of Resveratrol and Brings Its Benefits to Kidney Transplantation in a Preclinical Porcine Model.

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® 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® 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® 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.

The Optimal PEG for Kidney Preservation: A Preclinical Porcine Study.

University of Wisconsin (UW) solution is not optimal for preservation of marginal organs. Polyethylene glycol (PEG) could improve protection. Similarly formulated solutions containing either 15 or 20 g/L PEG 20 kDa or 5, 15 and 30 g/L PEG 35 kDa were tested in vitro on kidney endothelial cells, ex vivo on preserved kidneys, and in vivo in a pig kidney autograft model. In vitro, all PEGs provided superior preservation than UW in terms of cell survival, adenosine triphosphate (ATP) production, and activation of survival pathways. Ex vivo, tissue injury was lower with PEG 20 kDa compared to UW or PEG 35 kDa. In vivo, function recovery was identical between UW and PEG 35 kDa groups, while PEG 20 kDa displayed swifter recovery. At three months, PEG 35 kDa 15 and 30 g/L animals had worse outcomes than UW, while 5 g/L PEG 35 kDa was similar. PEG 20 kDa was superior to both UW and PEG 35 kDa in terms of function and fibrosis development, with low activation of damage pathways. PEG 20 kDa at 15 g/L was superior to 20 g/L. While in vitro models did not discriminate between PEGs, in large animal models of transplantation we showed that PEG 20 kDa offers a higher level of protection than UW and that longer chains such as PEG 35 kDa must be used at low doses, such as found in Institut George Lopez (IGL1, 1g/L).

Inhibition of complement improves graft outcome in a pig model of kidney autotransplantation.

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.

Role of warm ischemia on innate and adaptive responses in a preclinical renal auto-transplanted porcine model.

BACKGROUND: Deceased after cardiac arrest donor are an additional source of kidney graft to overcome graft shortage. Deciphering the respective role of renal warm and cold ischemia is of pivotal interest in the transplantation process. METHODS: Using a preclinical pig model of renal auto-transplantation, we investigated the consequences of warm and cold ischemia on early innate and adaptive responses as well as graft outcome. Kidneys were subjected to either 60 min-warm ischemia (WI) or auto-transplanted after cold storage for 24 h at 4°C (CS), or both conditions combined (WI+CS). Renal function, immune response and cytokine expression, oxidative stress and cell death were investigated at 3 h, 3 and 7 days (H3, D3 and D7) after reperfusion. At 3 months, we focused on cell infiltration and tissue remodelling. RESULTS: WI + CS induced a delayed graft function linked to higher tubular damage. Innate response occurred at D3 associated to a pro-oxidative milieu with a level dependent on the severity of ischemic injury whereas adaptive immune response occurred only at D7 mainly due to CS injuries and aggravated by WI. Graft cellular death was an early event detected at H3 and seems to be one of the first ischemia reperfusion injuries. These early injuries affect graft outcome on renal function, cells infiltration and fibrosis development. CONCLUSIONS: The results indicate that the severe ischemic insult found in kidneys from deceased after cardiac arrest donor affects kidney outcome and promotes an uncontrolled deleterious innate and adaptive response not inhibited 3 months after reperfusion.

Benefits of active oxygenation during hypothermic machine perfusion of kidneys in a preclinical model of deceased after cardiac death donors.

BACKGROUND: Deceased after cardiac death donors (DCDs) represent a valuable source of organs; however, preventing poor outcome is difficult, even with the use of machine perfusion (MP). It is of paramount importance to improve this method. We proposed to evaluate the benefits of active oxygenation during kidney graft hypothermic MP using a novel perfusion machine: Kidney Assist (KA). METHODS: We used a pig model of DCD transplantation in Large White pigs. Cold preservation was performed by conventional non-oxygenated MP (KAnoO2) or oxygenated MP (KA). RESULTS: In the first 2 wk post-transplant, KA grafts displayed a lower serum creatinine peak and a faster return to normal levels compared with KAnoO2 animals, translating into a smaller area under the curve. Urinary neutrophil gelatinase-associated lipocalin levels and serum aspartate amino transferase levels were lower in KA animals compared with the non-oxygenated group. These correlated with better chronic function. Longer follow-up of the animals (3 mo) permitted evaluation of chronic outcome lesions. Interstitial fibrosis was reduced in the KA group, and these kidneys also displayed significantly lower levels of vimentin staining. Further histologic investigation also showed a trend toward decreased chronic inflammation in kidneys preserved with oxygen. CONCLUSIONS: This new MP system is efficient in preserving DCD kidneys, greatly enhancing the capacity of the graft to withstand preservation stress and improving outcome. Oxygen delivery during preservation is thus valuable for highly damaged organs and offers an important therapeutic tool for transplant teams faced with decreased quality of donor organs.

[Ischemia reperfusion control: the key of kidney graft outcome]. / L'ischémie reperfusion - Acteur essentiel du devenir du greffon rénal.

During the transplantation procedure, ischemia reperfusion is an inevitable situation characterized by specific pathophysiological processes, which ultimately act synergistically to create injuries in the graft. These injuries are involved in early graft dysfunctions which promote chronic dysfunction and compromise graft outcome. Progresses in immunosuppressive drug regimens now place ischemia reperfusion injury control at the forefont for innovative therapeutic strategy to improve the quality of the graft. This review details these different processes and its consequences on renal graft function underlying the interest of novel therapeutic strategy.

[Organ conservation and transportation modalities]. / Modalités de conservation et de transport des organes.

ORGAN CONSERVATION AND TRANSPORTATION MODALITIES. Organ preservation in transplantation is an essential step in the graft journey between the donor and the recipient. The modalities of preservation have become a major element in this process given the evolution of donors in terms of age and associated comorbidities. This situation has led to the evolution of preservation in terms of the composition of solutions and perfusion technologies for all organs. Several concepts have thus emerged with extracellular type composition and the contribution of new molecules such as high molecular weight polyethylene glycols. The evolution also concerns new techniques such as normothermic abdominal circulation and perfusion machines with the use of hypothermia or normothermia and the oxygenation of the medium. Finally, new molecules are available to the teams and other concepts such as perfusion, evaluation and rehabilitation units.

MODALITÉS DE CONSERVATION ET DE TRANSPORT DES ORGANES. La conservation des organes est une étape essentielle dans le parcours du greffon entre le donneur et le receveur. Les modalités de conservation sont devenues un élément majeur dans ce processus compte tenu de l'évolution de l'âge des donneurs et des comorbidités associées. Cette situation a conduit à faire évoluer la conservation en matière de composition des solutions et de technologie de perfusion, et cela pour tous les organes. Plusieurs concepts ont ainsi émergé, avec la composition de type extracellulaire et l'apport de nouvelles molécules comme les polyéthylènes glycols de haut poids moléculaire. Les progrès concernent aussi de nouvelles techniques, comme la circulation régionale normothermique et les machines de perfusion avec l'utilisation de l'hypothermie ou de la normothermie et l'oxygénation du milieu. Enfin, de nouvelles molécules sont à disposition des équipes, et d'autres concepts se développent, comme les unités de perfusion, d'évaluation et de réhabilitation.