Kidney Tissue Proteome Profiles in Short Versus Long Duration of Delayed Graft Function - A Pilot Study in Donation After Circulatory Death Donors.

Introduction: Delayed graft function (DGF) is often defined as the need for dialysis treatment in the first week after a kidney transplantation. This definition, though readily applicable, is generic and unable to distinguish between "types" of DGF or time needed to recover function that may also significantly affect longer-term outcomes. We aimed to profile biological pathways in donation after circulatory death (DCD) kidney donors that correlate with DGF and different DGF durations. Methods: A total of N = 30 DCD kidney biopsies were selected from the UK Quality in Organ Donation (QUOD) biobank and stratified according to DGF duration (immediate function, IF n = 10; "short-DGF" (1-6 days), SDGF n = 10; "long-DGF" (7-22 days), LDGF n = 10). Samples were matched for donor and recipient demographics and analyzed by label-free quantitative (LFQ) proteomics, yielding identification of N = 3378 proteins. Results: Ingenuity pathway analysis (IPA) on differentially abundant proteins showed that SDGF kidneys presented upregulation of stress response pathways, whereas LDGF presented impaired response to stress, compared to IF. LDGF showed extensive metabolic deficits compared to IF and SDGF. Conclusion: DCD kidneys requiring dialysis only in the first week posttransplant present acute cellular injury at donation, alongside repair pathways upregulation. In contrast, DCD kidneys requiring prolonged dialysis beyond 7 days present minimal metabolic and antioxidant responses, suggesting that current DGF definitions might not be adequate in distinguishing different patterns of injury in donor kidneys contributing to DGF.

Certification Training and Liver Transplant Experience Improves Liver Procurement Outcomes: The Dutch Approach.

BACKGROUND: This study investigates the impact of certification training and liver transplant experience on procurement outcomes of deceased donor liver procurement in the Netherlands. METHODS: Three groups (trainee, certified, and master) were formed, with further subdivision based on liver transplant experience. Three key outcomes-surgical injury, graft discard after injury, and donor hepatectomy duration-were analyzed. RESULTS: There were no significant differences in surgical graft injury in the three groups (trainee, 16.9%; certified, 14.8%; master, 18.2%; P = 0.357; 2011 to 2018). The only predictor for surgical graft injury was donation after cardiac death (odds ratio [OR], 1.49; 95% confidence interval [CI], 1.10-2.02). Of the three groups, the master group had the highest discard rate after surgical injury (trainee, 0%; certified, 1.3%; master, 2.8%; P = 0.013). Master group without liver transplant experience (OR, 3.16; 95% CI, 1.21-8.27) and male donor sex (OR, 3.58; 95% CI, 1.32-9.73) were independent risk factors for discarding livers after surgical injury. Independent predictors for shorter hepatectomy durations included donors older than 50 years (coefficient [Coeff], -7.04; 95% CI, -8.03 to -3.29; P < 0.001), and master group (Coeff, -9.84; 95% CI, -14.37 to -5.31; P < 0.001) and certified group with liver transplant experience (Coeff, -6.54; 95% CI, -10.83 to -2.26; P = 0.003). On the other hand, master group without liver transplant experience (Coeff, 5.00; 95% CI, 1.03-8.96; P = 0.014) and donation after cardiac death (Coeff, 10.81; 95% CI, 8.32-13.3; P < 0.001) were associated with longer hepatectomy durations. CONCLUSIONS: Training and certification in abdominal organ procurement surgery were associated with a reduced discard rate for surgical injured livers and shorter hepatectomy times. The contrast between master group with and without liver transplant experience underscores the need for specialized training in this field.

Renal biopsies from donors with acute kidney injury show different molecular patterns according to the post-transplant function.

The utilization of kidneys from donors with acute kidney injury (AKI) is often limited by unpredictable post-transplantation outcomes. The aim of our study was to identify protein mediators implicated in either recovery or failure of these organs. Forty kidney biopsies from donors with (20) and without AKI (20) were selected and then subdivided according to the post-transplant outcome defined as a threshold of 45 ml/min for the eGFR at 1 year from transplantation. Tissue homogenates were analysed by western blot to assess how the levels of 17 pre-selected proteins varied across the four groups. Samples from AKI kidneys with a poor outcome showed a fourfold increase in the levels of PPARg and twofold reduction of STAT1 compared to the other groups (p < 0.05). On the contrary, antioxidant enzymes including TRX1 and PRX3 were increased in the AKI kidneys with a good outcome (p < 0.05). An opposite trend was observed for the detoxifying enzyme GSTp which was significantly increased in the AKI group with poor versus good outcome (p < 0.05). The importance of lipid metabolism (PPARg) and inflammatory signals (STAT1) in the function recovery of these kidneys hints to the therapeutical targeting of the involved pathways in the setting of organ reconditioning.

Red blood cells as oxygen carrier during normothermic machine perfusion of kidney grafts: Friend or foe?

Renal ex vivo normothermic machine perfusion (NMP) is under development as an assessment tool for high-risk kidney grafts and as a means of achieving more physiologically accurate organ preservation. On-going hemolysis has been reported during NMP, as this technique relies on red blood cells for oxygen delivery. In this study, we confirm the occurrence of progressive hemolysis during 6-hour kidney NMP. NMP-associated erythrostasis in the glomeruli and in peri-glomerular vascular networks points to an interaction between the red blood cells and the graft. Continuous hemolysis resulted in prooxidative changes in the perfusate, which could be quenched by addition of fresh frozen plasma. In a cell-based system, this hemolysis induced redox stress and exhibited toxic effects at high concentrations. These findings highlight the need for a more refined oxygen carrier in the context of renal NMP.

No Evidence of Progressive Proinflammatory Cytokine Storm in Brain-dead Organ Donors-A Time-course Analysis Using Clinical Samples.

BACKGROUND: Solid organ transplantation is a cost-effective treatment for end-stage organ failure. Organ donation after brain death is an important source of transplanted organs. Data are limited on the effects of brain injury or donor management on grafts. The consensus view has been that brain death creates a progressively proinflammatory environment. We aimed to investigate time-course changes across a range of cytokines in a donation after brain death cohort of donors who died of intracranial hemorrhage without any other systemic source of inflammation. METHODS: A donor cohort was defined using the UK Quality in Organ Donation biobank. Serum levels of proteins involved in proinflammatory and brain injury pathways (tumor necrosis factor-alpha, interleukin-6, complement C5a, neuron-specific enolase, and glial fibrillary acidic protein) were measured from admission to organ recovery. Moving median analysis was used to combine donor trajectories and delineate a time-course. RESULTS: A cohort of 27 donors with brain death duration between 10 and 30 h was created, with 24 donors contributing to the time-course analysis. We observed no increase in tumor necrosis factor-alpha or interleukin-6 throughout the donor management period. Neuronal injury marker and complement C5a remain high from admission to organ recovery, whereas glial fibrillary acidic protein rises around the confirmation of brain death. CONCLUSIONS: We found no evidence of a progressive rise of proinflammatory mediators with prolonged duration of brain death, questioning the hypothesis of a progressively proinflammatory environment. Furthermore, the proposed approach allows us to study chronological changes and identify biomarkers or target pathways when logistical or ethical considerations limit sample availability.

Impaired O2 unloading from stored blood results in diffusion-limited O2 release at tissues: evidence from human kidneys.

ABSTRACT: The volume of oxygen drawn from systemic capillaries down a partial pressure gradient is determined by the oxygen content of red blood cells (RBCs) and their oxygen-unloading kinetics, although the latter is assumed to be rapid and, therefore, not a meaningful factor. Under this paradigm, oxygen transfer to tissues is perfusion-limited. Consequently, clinical treatments to optimize oxygen delivery aim at improving blood flow and arterial oxygen content, rather than RBC oxygen handling. Although the oxygen-carrying capacity of blood is increased with transfusion, studies have shown that stored blood undergoes kinetic attrition of oxygen release, which may compromise overall oxygen delivery to tissues by causing transport to become diffusion-limited. We sought evidence for diffusion-limited oxygen release in viable human kidneys, normothermically perfused with stored blood. In a cohort of kidneys that went on to be transplanted, renal respiration correlated inversely with the time-constant of oxygen unloading from RBCs used for perfusion. Furthermore, the renal respiratory rate did not correlate with arterial O2 delivery unless this factored the rate of oxygen-release from RBCs, as expected from diffusion-limited transport. To test for a rescue effect, perfusion of kidneys deemed unsuitable for transplantation was alternated between stored and rejuvenated RBCs of the same donation. This experiment controlled oxygen-unloading, without intervening ischemia, holding all non-RBC parameters constant. Rejuvenated oxygen-unloading kinetics improved the kidney's oxygen diffusion capacity and increased cortical oxygen partial pressure by 60%. Thus, oxygen delivery to tissues can become diffusion-limited during perfusion with stored blood, which has implications in scenarios, such as ex vivo organ perfusion, major hemorrhage, and pediatric transfusion. This trial was registered at www.clinicaltrials.gov as #ISRCTN13292277.

Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions.

PURPOSE: The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate. EXPERIMENTAL DESIGN: Two different kinds of perfusate samples from kidney and liver trials were profiled using liquid chromatography-mass spectrometry (LC-MS/MS). The preparation of clean peptide mixtures for downstream analysis was performed considering different aspects of sample preparation; protein estimation, enrichment, in-gel and urea-based in-solution digestion. RESULTS: In-solution digestion of perfusate allowed identification of the highest number of peptides and proteins with greater sequence coverage and higher confidence data in kidney and liver perfusate. Key pathways identified by gene ontology analysis included complement, coagulation and antioxidant pathways, and a number of biomarkers previously linked to ischemia-reperfusion injury were also observed in perfusate. CONCLUSIONS: This study showed that in-solution digestion is a more efficient method for LC-MS/MS analysis of kidney and liver organ perfusion solutions. This method is also quicker and easier than in-gel digestion, allowing for greater sample throughput, with fewer opportunities for experimental error or peptide loss.

The Effect of Continuous Liver Normothermic Machine Perfusion on the Severity of Histological Bile Duct Injury.

Static Cold Storage (SCS) injures the bile duct, while the effect of Normothermic Machine Perfusion (NMP) is unknown. In a sub-study of the COPE trial on liver NMP, we investigated the impact of preservation type on histological bile duct injury score (BDIS). Transplants with at least one bile duct biopsy, either at end of preservation or 1 h post-reperfusion, were considered. BDIS was determined by assessing peribiliary glands injury, stromal and mural loss, haemorrhage, and thrombosis. A bivariate linear model compared BDIS (estimate, CI) between groups. Sixty-five transplants and 85 biopsies were analysed. Twenty-three grafts were preserved with SCS and 42 with NMP, with comparable baseline characteristics except for a shorter cold ischemic time in NMP. The BDIS increased over time regardless of preservation type (p = 0.04). The BDIS estimate was higher in NMP [8.02 (7.40-8.65)] than in SCS [5.39 (4.52-6.26), p < 0.0001] regardless of time. One patient in each group developed ischemic cholangiopathy, with a BDIS of 6 for the NMP-preserved liver. In six other NMP grafts, BDIS ranged 7-12 without development of ischemic cholangiopathy. In conclusion, BDIS increases over time, and the higher BDIS in NMP did not increase ischemic cholangiopathy. Thus, BDIS may overestimate this risk after liver NMP.

Blood transfusions post kidney transplantation are associated with inferior allograft and patient survival-it is time for rigorous patient blood management.

Background: Patient Blood Management (PBM), endorsed by the World Health Organisation is an evidence-based, multi-disciplinary approach to minimise inappropriate blood product transfusions. Kidney transplantation presents a particular challenge to PBM, as comprehensive evidence of the risk of transfusion is lacking. The aim of this study is to investigate the prevalence of post-transplant blood transfusions across multiple centres, to analyse risk factors for transfusion and to compare transplant outcomes by transfusion status. Methods: This analysis was co-ordinated via the UK Transplant Registry within NHS Blood and Transplant (NHSBT), and was performed across 4 centres. Patients who had received a kidney transplant over a 1-year period, had their transfusion status identified and linked to data held within the national registry. Results: Of 720 patients, 221(30.7%) were transfused, with 214(29.7%) receiving a red blood cell (RBC) transfusion. The proportion of patients transfused at each centre ranged from 20% to 35%, with a median time to transfusion of 4 (IQR:0-12) days post-transplant. On multivariate analysis, age [OR: 1.02(1.01-1.03), p=0.001], gender [OR: 2.11(1.50-2.98), p<0.0001], ethnicity [OR: 1.28(1.28-2.60), p=0.0008], and dialysis dependence pre-transplant [OR: 1.67(1.08-2.68), p=0.02], were associated with transfusion. A risk-adjusted Cox proportional hazards model showed transfusion was associated with inferior 1-year patient survival [HR 7.94(2.08-30.27), p=0.002] and allograft survival [HR: 3.33(1.65-6.71), p=0.0008], and inferior allograft function. Conclusion: RBC transfusions are common and are independently associated with inferior transplant outcomes. We urge that further research is needed to understand the mechanisms behind the outcomes, to support the urgent development of transplant-specific anaemia guidelines.

European Society for Organ Transplantation (ESOT) Consensus Statement on the Role of Pancreas Machine Perfusion to Increase the Donor Pool for Beta Cell Replacement Therapy.

The advent of Machine Perfusion (MP) as a superior form of preservation and assessment for cold storage of both high-risk kidney's and the liver presents opportunities in the field of beta-cell replacement. It is yet unknown whether such techniques, when applied to the pancreas, can increase the pool of suitable donor organs as well as ameliorating the effects of ischemia incurred during the retrieval process. Recent experimental models of pancreatic MP appear promising. Applications of MP to the pancreas, needs refinement regarding perfusion protocols and organ viability assessment criteria. To address the "Role of pancreas machine perfusion to increase the donor pool for beta cell replacement," the European Society for Organ Transplantation (ESOT) assembled a dedicated working group comprising of experts to review literature pertaining to the role of MP as a method of improving donor pancreas quality as well as quantity available for transplant, and to develop guidelines founded on evidence-based reviews in experimental and clinical settings. These were subsequently refined during the Consensus Conference when this took place in Prague.

Perfusate Proteomes Provide Biological Insight Into Oxygenated Versus Standard Hypothermic Machine Perfusion in Kidney Transplantation.

OBJECTIVE: To provide mechanistic insight into key biological alterations in donation after circulatory death kidneys during continuous pefusion we performed mass spectrometry profiling of perfusate samples collected during a phase 3 randomized double-blind paired clinical trial of hypothermic machine perfusion with and without oxygen (COMPARE). BACKGROUND: Despite the clinical benefits of novel perfusion technologies aiming to better preserve donor organs, biological processes that may be altered during perfusion have remained largely unexplored. The collection of serial perfusate samples during the COMPARE clinical trial provided a unique resource to study perfusate proteomic profiles, with the hypothesis that in-depth profiling may reveal biologically meaningful information on how donor kidneys benefit from this intervention. METHODS: Multiplexed liquid chromatography-tandem mass spectrometry was used to obtain a proteome profile of 210 perfusate samples. Partial least squares discriminant analysis and multivariate analysis involving clinical and perfusion parameters were used to identify associations between profiles and clinical outcomes. RESULTS: Identification and quantitation of 1716 proteins indicated that proteins released during perfusion originate from the kidney tissue and blood, with blood-based proteins being the majority. Data show that the overall hypothermic machine perfusion duration is associated with increasing levels of a subgroup of proteins. Notably, high-density lipoprotein and complement cascade proteins are associated with 12-month outcomes, and blood-derived proteins are enriched in the perfusate of kidneys that developed acute rejection. CONCLUSIONS: Perfusate profiling by mass spectrometry was informative and revealed proteomic changes that are biologically meaningful and, in part, explain the clinical observations of the COMPARE trial.

The role of flavin mononucleotide (FMN) as a potentially clinically relevant biomarker to predict the quality of kidney grafts during hypothermic (oxygenated) machine perfusion.

Hypothermic machine perfusion (HMP) provides preservation superior to cold storage and may allow for organ assessment prior to transplantation. Since flavin mononucleotide (FMN) in perfusate has been proposed as a biomarker of organ quality during HMP of donor livers, the aim of this study was to validate FMN as a biomarker for organ quality in the context of HMP preserved kidneys. Perfusate samples (n = 422) from the paired randomised controlled COPE-COMPARE-trial, comparing HMP with oxygenation (HMPO2) versus standard HMP in kidneys, were used. Fluorescence intensity (FI) was assessed using fluorescence spectroscopy (excitation 450nm; emission 500-600nm) and validated by fluorospectrophotometer and targeted liquid chromatography mass spectrometry (LC-MS/MS). Fluorescence intensity (FI)(ex450;em500-600) increased over time during machine perfusion in both groups (p<0.0001). This increase was similar for both groups (p = 0.83). No correlation, however, was found between FI(ex450;em500-600) and post-transplant outcomes, including day 5 or 7 serum creatinine (p = 0.11; p = 0.16), immediate graft function (p = 0.91), creatinine clearance and biopsy-proven rejection at one year (p = 0.14; p = 0.59). LC-MS/MS validation experiments of samples detected FMN in only one perfusate sample, whilst the majority of samples with the highest fluorescence (n = 37/38, 97.4%) remained negative. In the context of clinical kidney HMP, fluorescence spectroscopy unfortunately appears to be not specific and probably unsuitable for FMN. This study shows that FMN does not classify as a clinically relevant predictive biomarker of kidney graft function after transplantation.

Heparin Conjugate Pretreatment of Kidneys From Deceased Donors Before Transplantation: Results From the First-in-human Randomized Phase I Trial.

Pretreating porcine kidneys with Corline Heparin Conjugate (CHC) during hypothermic machine perfusion (HMP) has been shown to reduce preservation injury and improve early kidney function. In this first-in-human phase I study, the safety and tolerability of transplanting CHC-pretreated kidneys were evaluated. Methods: CHC or placebo was added to the preservation solution during HMP of donated kidneys from deceased donors for at least 3 h before transplantation into adult patients. The primary safety endpoint was the number and severity of adverse events (AEs) and serious AEs (SAEs) during the first 30 d after transplantation. Results: In the first 30 d, 66 AEs were reported in 8 patients who received CHC-pretreated kidneys with 39 AEs in 8 patients who received placebo-pretreated kidneys (P = 0.1 in post hoc analysis). The most common AEs were hypertension (CHC, n = 5; placebo, n = 2) and anemia (CHC, n = 5; placebo, n = 2). Most AEs were assessed as mild (58%) or moderate (39%) and not related to treatment (95%). There were 2 SAEs reported in each group. One SAE, considered possibly related to CHC treatment, was a case of severe postprocedural hemorrhage that required reoperation. No patients needed dialysis. There were no observed rejections and no patient deaths. Conclusions: Pretreatment of kidneys with CHC before transplantation was considered safe and tolerable. Efficacy studies are now planned to investigate if CHC can reduce early ischemia-reperfusion injury in humans.

Donor characteristics and their impact on kidney transplantation outcomes: Results from two nationwide instrumental variable analyses based on outcomes of donor kidney pairs accepted for transplantation.

Background: Donor-characteristics and donor characteristics-based decision algorithms are being progressively used in the decision process whether or not to accept an available donor kidney graft for transplantation. While this may improve outcomes, the performance characteristics of the algorithms remains moderate. To estimate the impact of donor factors of grafts accepted for transplantation on transplant outcomes, and to test whether implementation of donor-characteristics-based algorithms in clinical decision-making is justified, we applied an instrumental variable analysis to outcomes for kidney donor pairs transplanted in different individuals. Methods: This analysis used (dis)congruent outcomes of kidney donor pairs as an instrument and was based on national transplantation registry data for all donor kidney pairs transplanted in separate individuals in the Netherlands (1990-2018, 2,845 donor pairs), and the United Kingdom (UK, 2000-2018, 11,450 pairs). Incident early graft loss (EGL) was used as the primary discriminatory factor. It was reasoned that a scenario with a dominant impact of donor variables on transplantation outcomes would result in high concordance of EGL in both recipients, whilst dominance of asymmetrical outcomes could indicate a more complex scenario, involving an interaction of donor, procedural and recipient factors. Findings: Incidences of congruent EGL (Netherlands: 1·2%, UK: 0·7%) were slightly lower than the arithmetical (stochastic) incidences, suggesting that once a graft has been accepted for transplantation, donor factors minimally contribute to incident EGL. A long-term impact of donor factors was explored by comparing outcomes for functional grafts from donor pairs with asymmetrical vs. symmetrical outcomes. Recipient survival was similar for both groups, but a slightly compromised graft survival was observed for grafts with asymmetrical outcomes in the UK cohort: (10-years Hazard Ratio for graft loss: 1·18 [1·03-1·35] p<0·018); and 5 years eGFR (48·6 [48·3-49·0] vs. 46·0 [44·5-47·6] ml/min in the symmetrical outcome group, p<0·001). Interpretation: Our results suggest that donor factors for kidney grafts deemed acceptable for transplantation impact minimally on transplantation outcomes. A strong reliance on donor factors and/or donor-characteristics-based decision algorithms could result in unjustified rejection of grafts. Future efforts to optimize transplant outcomes should focus on a better understanding of the recipient factors underlying transplant outcomes. Funding: None.

In situ normothermic regional perfusion versus ex situ normothermic machine perfusion in liver transplantation from donation after circulatory death.

In situ normothermic regional perfusion (NRP) and ex situ normothermic machine perfusion (NMP) aim to improve the outcomes of liver transplantation (LT) using controlled donation after circulatory death (cDCD). NRP and NMP have not yet been compared directly. In this international observational study, outcomes of LT performed between 2015 and 2019 for organs procured from cDCD donors subjected to NRP or NMP commenced at the donor center were compared using propensity score matching (PSM). Of the 224 cDCD donations in the NRP cohort that proceeded to asystole, 193 livers were procured, resulting in 157 transplants. In the NMP cohort, perfusion was commenced in all 40 cases and resulted in 34 transplants (use rates: 70% vs. 85% [p = 0.052], respectively). After PSM, 34 NMP liver recipients were matched with 68 NRP liver recipients. The two cohorts were similar for donor functional warm ischemia time (21 min after NRP vs. 20 min after NMP; p = 0.17), UK-Donation After Circulatory Death risk score (5 vs. 5 points; p = 0.38), and laboratory Model for End-Stage Liver Disease scores (12 vs. 12 points; p = 0.83). The incidence of nonanastomotic biliary strictures (1.5% vs. 2.9%; p > 0.99), early allograft dysfunction (20.6% vs. 8.8%; p = 0.13), and 30-day graft loss (4.4% vs. 8.8%; p = 0.40) were similar, although peak posttransplant aspartate aminotransferase levels were higher in the NRP cohort (872 vs. 344 IU/L; p < 0.001). NRP livers were more frequently allocated to recipients suffering from hepatocellular carcinoma (HCC; 60.3% vs. 20.6%; p < 0.001). HCC-censored 2-year graft and patient survival rates were 91.5% versus 88.2% (p = 0.52) and 97.9% versus 94.1% (p = 0.25) after NRP and NMP, respectively. Both perfusion techniques achieved similar outcomes and appeared to match benchmarks expected for donation after brain death livers. This study may inform the design of a definitive trial.

Kidney Normothermic Machine Perfusion Can Be Used as a Preservation Technique and a Model of Reperfusion to Deliver Novel Therapies and Assess Inflammation and Immune Activation.

Ischaemia-reperfusion injury (IRI) is an inevitable process in transplantation and results in inflammation and immune system activation. Alpha-1 antitrypsin (AAT) has anti-inflammatory properties. Normothermic machine perfusion (NMP) can be used to deliver therapies and may help in assessing the effects of IRI and immunity. This study investigated the effects of AAT on IRI and inflammation in pig kidneys when administered during preservation, followed by normothermic reperfusion (NR) with autologous whole blood, as a surrogate for transplant. Two different models were used to deliver AAT or placebo to paired slaughterhouse pig kidneys: Model 1: 7-h static cold storage (SCS) + 3-h NR (n = 5 pairs), where either AAT (10 mg/ml) or placebo was delivered in the flush following retrieval; Model 2: 4-h SCS + 3-h NMP + 3-h NR (n = 5 pairs), where either AAT or placebo was delivered during NMP. Injury markers and cytokines levels were analysed in the perfusate, and heat shock protein 70 KDa (HSP-70) was analysed in biopsies. AAT delivered to kidneys showed no adverse effects on perfusion parameters. HSP-70 fold changes were significantly lower in the AAT group during NMP (P < 0.01, paired t-test) but not during NR. Interleukin-1 receptor antagonist (IL-1ra) fold changes were significantly higher in the AAT group during NR model 1 (p < 0.05, two-way ANOVA). In contrast to the AAT group, significant upregulation of interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) between t = 90 min and t = 180 min and interleukin-8 (IL-8) between baseline and t = 90 min was observed in the control group in NR model 2 (p < 0.05, Tukey's multiple comparison test). However, overall inflammatory cytokines and injury markers showed similar levels between groups. Delivery of AAT to pig kidneys was safe without any detrimental effects. NMP and NR provided excellent methods for comparison of inflammation and immune activation in the delivery of a novel therapy.

Assessment of Mitochondrial Function and Oxygen Consumption Measured During Ex Vivo Normothermic Machine Perfusion of Injured Pig Kidneys Helps to Monitor Organ Viability.

Donor kidney assessment may improve organ utilisation. Normothermic Machine Perfusion (NMP) has the potential to facilitate this advance. The mechanism of action is not yet determined and we aimed to assess mitochondrial function during NMP. Anaesthetised pigs (n = 6) had one kidney clamped for 60 min. The healthy contralateral kidney was removed and underwent NMP for 8 h (healthy control (HC), n = 6). Following 60 min warm ischaemia the injured kidney underwent HMP for 24 h, followed by NMP for 8 h (n = 6). Mitochondria were extracted from fresh tissue for analysis. Injured kidneys were analysed as two separate groups (IMa, n = 3 and IMb, n = 3). Renal resistance was higher (0.39ï, ± 0.29 vs. 1.65ï, ± 0.85; p = 0.01) and flow was lower (55ï, ± 28 vs. 7ï, ± 4; p = 0.03) during HMP in IMb than IMa. NMP blood flow was higher in IMa versus IMb (2-way ANOVA; p < 0.001) After 60 min NMP, O2 consumption was significantly lower in IMb versus IMa (p ≤ 0.002). State-3 respiration was significantly different between the groups (37ï, ± 19 vs. 24ï, ± 14 vs. 10ï, ± 8; nmolO2/min/mg; p = 0.049). Lactate levels were significantly lower in IMa versus IMb (p = 0.028). Mitochondrial respiration levels during NMP may be suggestive of kidney viability. Oxygen consumption, renal blood flow and lactate can differentiate severity of kidney injury during NMP.

The Effect of Hypothermic Machine Perfusion to Ameliorate Ischemia-Reperfusion Injury in Donor Organs.

Hypothermic machine perfusion (HMP) has become the new gold standard in clinical donor kidney preservation and a promising novel strategy in higher risk donor livers in several countries. As shown by meta-analysis for the kidney, HMP decreases the risk of delayed graft function (DGF) and improves graft survival. For the liver, HMP immediately prior to transplantation may reduce the chance of early allograft dysfunction (EAD) and reduce ischemic sequelae in the biliary tract. Ischemia-reperfusion injury (IRI), unavoidable during transplantation, can lead to massive cell death and is one of the main causes for DGF, EAD or longer term impact. Molecular mechanisms that are affected in IRI include levels of hypoxia inducible factor (HIF), induction of cell death, endothelial dysfunction and immune responses. In this review we have summarized and discussed mechanisms on how HMP can ameliorate IRI. Better insight into how HMP influences IRI in kidney and liver transplantation may lead to new therapies and improved transplant outcomes.