Optimizing Donor Kidneys During Normothermic Machine Perfusion With Sevoflurane: Design of a Research Model.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilisation by improving graft quality and enabling viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, RNA interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion in decreasing the imbalance between donor liver demand and supply by enabling the repair and regeneration of damaged donor livers.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Transcriptomic signatures during normothermic liver machine perfusion correspond with graft quality and predict the early graft function

A comprehensive mechanistic assessment of normothermic machine perfusion (NMP) is an essential step toward identifying biomarkers to assess liver viability. Although some studies have evaluated the effect of NMP on inflammation markers, there are other key pathological mechanisms involved in ischemia/reperfusion injury (IRI) that have not yet been evaluated. Eight human donor livers preserved by NMP were included to analyze IRI during preservation. Concentrations of several biomarkers involved in different biological processes of IRI were measured in the perfusate. Perfusate levels of intercellular adhesion molecule 1, P-selectin, vascular cell adhesion molecule 1, metalloproteinase with thrombospondin motif type 1, member 13, phospholipase A2 group VII, and syndecan-1 progressively increased during NMP. Noteworthy, perfusate lactate levels showed a strong correlation with C-X-C motif chemokine ligand 10 (P = 0.001), intercellular adhesion molecule 1 (P = 0.01), and urokinase plasminogen activator (P = 0.001). Perfusate lactate correlates with the main underlying biological mechanisms occurring in the NMP environment. Moreover, several IRI biomarkers accumulate during NMP, which may limit the extent of the benefits of this technology.

Machine Perfusion as "Comfort Zone": What Are Key Challenges of Liver Viability Assessment Today?

Introduction:Ageing of the general population has led to an increase in the use of suboptimal kidneys from expanded criteria donation after brain death (ECD-DBD) and donation after circulatory death (DCD) donors. However, these kidneys have inferior graft outcomes and lower rates of immediate function. Normothermic machine perfusion (NMP) may improve outcomes of these suboptimal donor kidneys. Previous non-randomized studies have shown the safety of this technique and suggested its efficacy in improving the proportion of immediate functioning kidneys compared to static cold storage (SCS). However, its additional value to hypothermic machine perfusion (HMP), which has already been proved superior to SCS, has not yet been established.Methods and analysis:This single-center, open-label, randomized controlled trial aims to assess immediate kidney function after 120 minutes additional, end-ischemic NMP compared to HMP alone. Immediate kidney function is defined as no dialysis treatment in the first week after transplant. Eighty recipients on dialysis at the time of transplant who receive an ECD-DBD or DCD kidney graft are eligible for inclusion. In the NMP group, the donor kidney is taken of HMP upon arrival in the recipient hospital and thereafter put on NMP for 120 minutes at 37 degrees Celsius followed by transplantation. In the control group, donor kidneys stay on HMP until transplantation. The primary outcome is immediate kidney function.Ethics and dissemination:The protocol has been approved by the Medical Ethical Committee of Erasmus Medical Center (2020-0366). Results of this study will be submitted to peer-reviewed journals.Registration:registered in clinicaltrials.gov (NCT04882254).Highlights:This is the first RCT to compare additional NMP to HMP alone.Extensive sampling will offer in-depth analysis of kidney physiology during NMP.This RCT may help identify biomarkers to predict clinical outcomes during NMP.Biomarkers can help develop NMP as assessment tool for declined kidneys.

Prolonged Normothermic Machine Perfusion: Buying More Time for Liver Graft Assessment and Repair.

The shortage of donor kidneys is a worldwide problem. One possibility to increase the donor organ pool is to improve the quality of suboptimal donor kidneys. For this reason, an increasing amount of research is being performed on the subject of machine perfusion, a technique during which an organ is connected to a perfusion circuit. Apart from the fact that this technique can be used to bridge the period between donation and actual transplantation, it also provides the opportunity for active interventions to an isolated organ. The first part of my thesis focuses on a cellular intervention during normothermic (37 degrees) machine perfusion with mesenchymal stromal cells. The aim was to determine if it was technically feasible to administer mesenchymal stromal cells, cells with regenerative capacities, during normothermic machine perfusion and what effect these cells had on the donor kidney. It proved to be feasible to administer these cells in such way that a proportion remained detectable after machine perfusion. In a porcine autotransplantation model, mesenchymal stromal cells did not affect early graft function, but further research is necessary to determine if these cells could have a beneficial effect on longer term graft function of the donor kidney. The second part of my thesis focuses on the use of different perfusion solutions and oxygen carriers during normothermic machine perfusion. From these experiments it can be concluded that the composition of the perfusion solution, as well as the choice for a specific oxygen carrier, has a significant impact on kidney function and injury markers during normothermic machine perfusion.

Strategies in Organ Preservation-A New Golden Age.

Background/Objectives: APOL1 renal-risk variants (RRVs) are of increasing relevance to kidney disease and transplant outcomes. It is currently understood that the presence of RRVs in donors negatively impacts kidney allograft survival in an autosomal recessive pattern of inheritance. Less well known is the interplay between ischemia and alternative allograft preservation methods, such as normothermic machine perfusion (NMP), on APOL1 gene expression. To investigate this, we examined the effects of APOL1 RRVs on APOL1 gene expression in ischemic donor kidneys and compared the differences in cytokine and APOL1 expression patterns between the alternative preservation methods, static cold storage (CS) and NMP. Methods: Non-utilized deceased donor kidney pairs from donors of African ancestry were procured from Mid-America Transplant after being deemed unsuitable for kidney transplant. Samples were collected from each donor kidney pair and DNA was extracted for APOL1 genotyping. APOL1 RRVs G1 (rs73885319) (rs60910145) and G2 (rs71785313) were identified by Sanger sequencing. From each pair, one kidney underwent 6 h NMP (n = 3) and the contralateral kidney 6 h of CS (n = 3) following the initial CS. Renal perfusion and biochemical, and histologic parameters were recorded. NMP was directly compared with CS using paired donor kidneys using NMP with allogeneic red blood cells, followed by assessment of perfusion, biochemical, and histologic parameters, in addition to gene expression. Results: Donor genotyping identified kidney pairs as heterozygous for the G1 RRV (G1/G0), homozygous for the G0 allele (G0/G0), and homozygous for the G2 RRV (G2/G2), respectively. All kidneys were successfully reperfused, with mRNA transcript levels of APOL1-related genes subsequently measured. Significant differences in APOL1 gene expression were observed among all three groups of kidneys. In paired kidneys from baseline to hour 6 of NMP, mRNA expression varied significantly between G1/G0 and G2/G2 homozygous pairs (p = 0.002) as well as between the G0/G0 and G2/G2 pairs (p = 0.002). APOL1 expression shifted by a significantly higher-fold change of 2.4 under NMP conditions in the G2/G2 genotype (p < 0.001). The inflammatory cytokine marker IFN-γ was also significantly upregulated in the G2/G2 genotype kidney, in both CS and NMP groups (p = 0.001). Other related genes such as KIM-1 were upregulated by a change of 3.9-fold in the NMP group for the G2/G2 kidney. Conclusion: Donor kidney pairs with the high-risk APOL1 genotype, especially G2/G2, show increased APOL1 expression and inflammation, particularly under NMP conditions. NMP enables detection of genotype-specific molecular changes in an ischemic reperfusion injury model, supporting its potential to improve donor kidney assessment before transplantation.

Normothermic machine perfusion (NMP) aims to reduce ischemia-reperfusion injury in donor livers and its clinical manifestation, early allograft dysfunction (EAD) by maintaining perfusion and oxygenation. However, there is limited data on which NMP perfusate biomarkers might be associated with such EAD and the role of perfusate hemoglobin has not been assessed. We performed a pilot retrospective analysis of adult donor livers undergoing NMP between 2020 and 2022 at our center. NMP was commenced at the recipient hospital after initial static cold storage. All NMP circuits were primed in the same manner according to the manufacturer's instructions. Livers were stratified by initial perfusate hemoglobin below (≤5.2 mmol/L) or above (>5.2 mmol/L) the median. The association between hemoglobin levels and EAD or recipient peak transaminase levels was assessed. Among 23 livers, eight were considered unsuitable for transplantation, leaving 15 livers for assessment. Higher initial hemoglobin was associated with a lower risk of EAD (0% vs. 55.6%, p = 0.04). Perfusate hemoglobin decreased after NMP initiation (p = 0.003) and negatively correlated with recipient peak transaminase levels (ALT: ρ = -0.72, p = 0.002; AST: ρ = -0.79, p < 0.001). Consistently, higher hemoglobin livers also demonstrated lower perfusate liver enzymes. Perfusate hemoglobin levels decreased during NMP, and lower perfusate hemoglobin levels were associated with a higher incidence of EAD and higher levels of liver injury markers. Maintaining higher hemoglobin levels during NMP may help reduce ischemia-reperfusion injury and prevent or attenuate EAD. Larger prospective studies are needed to validate the findings of this pilot study.

BackgroundAssessment and treatment of severe ischemia-reperfusion-injury (IRI) remains an unmet challenge in kidney transplantation. Normothermic machine perfusion (NMP) recapitulates IRI ex situ, but there is limited understanding of the transcriptional pathways, and the associated cellular landscape, driving IRI during NMP and determining its severity. Such knowledge is essential for therapeutic targeting and organ resuscitation during machine perfusion.MethodsUsing tissue obtained at the time of NMP from kidneys subsequently transplanted as part of a randomized controlled trial, we undertook in-depth transcriptomic analyses comparing kidneys suffering severe IRI, (manifesting clinically as the development of delayed graft function (DGF)), to kidneys with mild IRI (defined by immediate graft function, IGF) post-transplantation.ResultsWe validated upregulation of previously described pro-inflammatory and immune transcriptomic pathways, including TNFa via NFkB signaling, Allograft Rejection and Inflammatory Response. Going further, we identified innate immune system driven processes at the core of the transcriptional signature in kidneys suffering severe IRI, such as recruitment and migration of myeloid leucocytes, macrophage activation, phagocytosis and inflammasome activation. Deconvolution using single-cell-RNAseq data showed kidneys with severe IRI and post-transplant DGF were enriched for pro-inflammatory mononuclear phagocytes, myofibroblasts and fibroblasts, but depleted of tubuloepithelial, cell signatures. These transcriptional findings were recapitulated in tissue biopsies obtained during NMP from an external cohort comparing kidneys with high acute tubular injury and severe IRI to kidneys with low acute tubular injury and mild IRI; these kidneys were histologically similar to the DGF/IGF kidneys, respectively.DiscussionTogether, our study characterizes the transcriptional signature of severe IRI during NMP, suggesting the role of pro-inflammatory innate/pro-fibrotic cells in this process. We describe a transcriptomic signature that may support future prospective therapeutic trials as a potential efficacy endpoint, and highlight potential cellular targets for therapeutic intervention during NMP in an era of precision medicine.

BackgroundThe 5-year graft survival rate of donor kidneys transplanted in the Eurotransplant Senior Program (ESP) is only 47 per cent. Normothermic machine perfusion (NMP) may be a new preservation technique that improves graft outcome. This pilot study aimed to assess safety and feasibility of this technique within the ESP.MethodsRecipients were eligible for inclusion if they received a donor kidney within the ESP. Donor kidneys underwent 2 h of oxygenated NMP with a red cell-based solution at 37°C, additional to standard-of-care preservation (non-oxygenated hypothermic machine perfusion). The primary outcome was the safety and feasibility of NMP. As a secondary outcome, graft outcome was investigated and compared with that in a historical group of patients in the ESP and the contralateral kidneys.ResultsEleven patients were included in the NMP group; the function of eight kidneys could be compared with that of the contralateral kidney. Fifty-three patients in the ESP, transplanted consecutively between 2016 and 2018, were included as controls. No adverse events were noted, especially no arterial thrombosis or primary non-function of the transplants. After 120 min of oxygenated NMP, median flow increased from 117 (i.q.r. 80–126) to 215 (170–276) ml/min (P = 0.001). The incidence of immediate function was 64 per cent in the NMP group and 40 per cent in historical controls (P = 0.144). A significant difference in graft outcome was not observed.DiscussionThis pilot study showed NMP to be safe and feasible in kidneys transplanted in the ESP. A well powered study is warranted to confirm these results and investigate the potential advantages of NMP on graft outcome.

Fifteen donor kidneys were subjected to HMP followed by 2 h of NMP before transplantation. NMP perfusate was collected at 3 time points (0, 1, 2 h) for the measurements of prorenin/renin, erythropoietin (EPO), and vitamin D, and urine samples were collected at 1 h and 2 h for urodilatin measurement. Fifteen HMP perfusate samples were collected for the same measurements. Kidneys on NMP secreted significantly more prorenin, renin, EPO, and active vitamin D than during HMP. EPO and vitamin D secretion remained stable during 2 h of NMP, whereas the prorenin release rate increased and renin release rate decreased after 1 h. Donation after brain death kidneys secreted more vitamin D and less EPO during NMP than donation after circulatory death kidneys. Twelve donor kidneys produced urine during NMP and released detectable levels of urodilatin. Kidneys exhibited a large variation in hormone release rates. No significant differences were found in hormone release capacity between delayed graft function (DGF) and non-DGF kidneys, and no significant correlations were found between hormone release rates and the duration of DGF or 1-mo posttransplant serum creatinine levels. Human transplant kidneys display endocrine activity during NMP. To explore whether correlations exist between hormone release rates and posttransplant kidney function, large numbers of kidneys are required.