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.

A randomized trial of normothermic preservation in liver transplantation.

Liver transplantation is a highly successful treatment, but is severely limited by the shortage in donor organs. However, many potential donor organs cannot be used; this is because sub-optimal livers do not tolerate conventional cold storage and there is no reliable way to assess organ viability preoperatively. Normothermic machine perfusion maintains the liver in a physiological state, avoids cooling and allows recovery and functional testing. Here we show that, in a randomized trial with 220 liver transplantations, compared to conventional static cold storage, normothermic preservation is associated with a 50% lower level of graft injury, measured by hepatocellular enzyme release, despite a 50% lower rate of organ discard and a 54% longer mean preservation time. There was no significant difference in bile duct complications, graft survival or survival of the patient. If translated to clinical practice, these results would have a major impact on liver transplant outcomes and waiting list mortality.

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.

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.

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes.

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, α-actinin-4 and talin-1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin-1 is a specific proteolytic target of calpain-1, we investigated a potential trigger of calpain activation and talin-1 degradation using human ex vivo precision-cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain-1 by transforming growth factor-ß generated proteolytic fragments of talin-1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain-1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies.

Preclinical models versus clinical renal ischemia reperfusion injury: A systematic review based on metabolic signatures.

Despite decennia of research and numerous successful interventions in the preclinical setting, renal ischemia reperfusion (IR) injury remains a major problem in clinical practice, pointing toward a translational gap. Recently, two clinical studies on renal IR injury (manifested either as acute kidney injury or as delayed graft function) identified metabolic derailment as a key driver of renal IR injury. It was reasoned that these unambiguous metabolic findings enable direct alignment of clinical with preclinical data, thereby providing the opportunity to elaborate potential translational hurdles between preclinical research and the clinical context. A systematic review of studies that reported metabolic data in the context of renal IR was performed according to the PRISMA guidelines. The search (December 2020) identified 35 heterogeneous preclinical studies. The applied methodologies were compared, and metabolic outcomes were semi-quantified and aligned with the clinical data. This review identifies profound methodological challenges, such as the definition of IR injury, the follow-up time, and sampling techniques, as well as shortcomings in the reported metabolic information. In light of these findings, recommendations are provided in order to improve the translatability of preclinical models of renal IR injury.

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.

Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model.

BACKGROUND: Remote Ischemic Conditioning (RIC) has been proposed as a therapeutic intervention to circumvent the ischemia/reperfusion injury (IRI) that is inherent to organ transplantation. Using a porcine kidney transplant model, we aimed to decipher the subclinical molecular effects of a RIC regime, compared to non-RIC controls. METHODS: Kidney pairs (n = 8 + 8) were extracted from brain dead donor pigs and transplanted in juvenile recipient pigs following a period of cold ischemia. One of the two kidney recipients in each pair was subjected to RIC prior to kidney graft reperfusion, while the other served as non-RIC control. We designed an integrative Omics strategy combining transcriptomics, proteomics, and phosphoproteomics to deduce molecular signatures in kidney tissue that could be attributed to RIC. RESULTS: In kidney grafts taken out 10 h after transplantation we detected minimal molecular perturbations following RIC compared to non-RIC at the transcriptome level, which was mirrored at the proteome level. In particular, we noted that RIC resulted in suppression of tissue inflammatory profiles. Furthermore, an accumulation of muscle extracellular matrix assembly proteins in kidney tissues was detected at the protein level, which may be in response to muscle tissue damage and/or fibrosis. However, the majority of these protein changes did not reach significance (p < 0.05). CONCLUSIONS: Our data identifies subtle molecular phenotypes in porcine kidneys following RIC, and this knowledge could potentially aid optimization of remote ischemic conditioning protocols in renal transplantation.

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.

Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction.

Assessment of specific ß-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin (INS)-DNA is correlated to ß-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated INS-DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of ß-cell derived unmethylated INS-DNA in cellular lysate. We therefore suggest that this technique can be of value to reliably determine the purity of an islet preparation and may also serve as a measure of the quality of islets prior to transplantation measuring unmethylated INS-DNA as a reflection of the relative amount of lysed ß-cells.

Metabolic needs of the kidney graft undergoing normothermic machine perfusion.

Normothermic machine perfusion (NMP) is emerging as a novel preservation strategy. During NMP, the organ is maintained in a metabolically active state that may not only provide superior organ preservation, but that also facilitates viability testing before transplantation, and ex situ resuscitation of marginal kidney grafts. Although the prevailing perfusion protocols for renal NMP are refined from initial pioneering studies concerning short periods of NMP, it could be argued that these protocols are not optimally tailored to address the putatively compromised metabolic plasticity of marginal donor grafts (i.e., in the context of viability testing and/or preservation), or to meet the metabolic prerequisites associated with prolonged perfusions and the required anabolic state in the context of organ regeneration. Herein, we provide a theoretical framework for the metabolic requirements for renal NMP. Aspects are discussed along the lines of carbohydrates, fatty acids, amino acids, and micronutrients required for optimal NMP of an isolated kidney. In addition, considerations for monitoring aspects of metabolic status during NMP are discussed.

Urine recirculation prolongs normothermic kidney perfusion via more optimal metabolic homeostasis-a proteomics study.

We describe a proteomics analysis to determine the molecular differences between normothermically perfused (normothermic machine perfusion, NMP) human kidneys with urine recirculation (URC) and urine replacement (UR). Proteins were extracted from 16 kidney biopsies with URC (n = 8 donors after brain death [DBD], n = 8 donors after circulatory death [DCD]) and three with UR (n = 2 DBD, n = 1 DCD), followed by quantitative analysis by mass spectrometry. Damage-associated molecular patterns (DAMPs) were decreased in kidney tissue after 6 hours NMP with URC, suggesting reduced inflammation. Vasoconstriction was also attenuated in kidneys with URC as angiotensinogen levels were reduced. Strikingly, kidneys became metabolically active during NMP, which could be enhanced and prolonged by URC. For instance, mitochondrial succinate dehydrogenase enzyme levels as well as carbonic anhydrase were enhanced with URC, contributing to pH stabilization. Levels of cytosolic and the mitochondrial phosphoenolpyruvate carboxykinase were elevated after 24 hours of NMP, more prevalent in DCD than DBD tissue. Key enzymes involved in glucose metabolism were also increased after 12 and 24 hours of NMP with URC, including mitochondrial malate dehydrogenase and glutamic-oxaloacetic transaminase, predominantly in DCD tissue. We conclude that NMP with URC permits prolonged preservation and revitalizes metabolism to possibly better cope with ischemia reperfusion injury in discarded kidneys.

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

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Oxygenated versus standard cold perfusion preservation in kidney transplantation (COMPARE): a randomised, double-blind, paired, phase 3 trial.

BACKGROUND: Deceased donor kidneys are preserved in cold hypoxic conditions. Providing oxygen during preservation might improve post-transplant outcomes, particularly for kidneys subjected to greater degrees of preservation injury. This study aimed to investigate whether supplemental oxygen during hypothermic machine perfusion (HMP) could improve the outcome of kidneys donated after circulatory death. METHODS: This randomised, double-blind, paired, phase 3 trial was done in 19 European transplant centres. Kidney pairs from donors aged 50 years or older, donated after circulatory death, were eligible if both kidneys were transplanted into two different recipients. One kidney from each donor was randomly assigned using permuted blocks to oxygenated hypothermic machine perfusion (HMPO2), the other to HMP without oxygenation. Perfusion was maintained from organ retrieval to implantation. The primary outcome was 12-month estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration equation in pairs of donated kidneys in which both transplanted kidneys were functioning at the end of follow-up. Safety outcomes were reported for all transplanted kidneys. Intention-to-treat analyses were done. This trial is registered with the ISRCTN Registry, ISRCTN32967929, and is now closed. FINDINGS: Between March 15, 2015, and April 11, 2017, 197 kidney pairs were randomised with 106 pairs transplanted into eligible recipients. 23 kidney pairs were excluded from the primary analysis because of kidney failure or patient death. Mean eGFR at 12 months was 50·5 mL/min per 1·73 m2 (SD 19·3) in the HMPO2 group versus 46·7 mL/min per 1·73m2 (17·1) in HMP (mean difference 3·7 mL/min per 1·73m2, 95% CI -1·0 to 8·4; p=0·12). Fewer severe complications (Clavien-Dindo grade IIIb or more) were reported in the HMPO2 group (46 of 417, 11%, 95% CI 8% to 14%) than in the HMP group (76 of 474, 16%, 13% to 20%; p=0·032). Graft failure was lower with HMPO2 (three [3%] of 106) compared with HMP (11 [10%] of 106; hazard ratio 0·27, 95% CI 0·07 to 0·95; p=0·028). INTERPRETATION: HMPO2 of kidneys donated after circulatory death is safe and reduces post-transplant complications (grade IIIb or more). The 12-month difference in eGFR between the HMPO2 and HMP groups was not significant when both kidneys from the same donor were still functioning 1-year post-transplant, but potential beneficial effects of HMPO2 were suggested by analysis of secondary outcomes. FUNDING: European Commission 7th Framework Programme.

Proteomic analysis of machine perfusion solution from brain dead donor kidneys reveals that elevated complement, cytoskeleton and lipid metabolism proteins are associated with 1-year outcome.

Assessment of donor kidney quality is based on clinical scores or requires biopsies for histological assessment. Noninvasive strategies to identify and predict graft outcome at an early stage are, therefore, needed. We evaluated the perfusate of donation after brain death (DBD) kidneys during nonoxygenated hypothermic machine perfusion (HMP). In particular, we compared perfusate protein profiles of good outcome (GO) and suboptimal outcome (SO) 1-year post-transplantation. Samples taken 15 min after the start HMP (T1) and before the termination of HMP (T2) were analysed using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hierarchical clustering of the 100 most abundant proteins showed discrimination between grafts with a GO and SO at T1. Elevated levels of proteins involved in classical complement cascades at both T1 and T2 and a reduced abundance of lipid metabolism at T1 and of cytoskeletal proteins at T2 in GO versus SO was observed. ATP-citrate synthase and fatty acid-binding protein 5 (T1) and immunoglobulin heavy variable 2-26 and desmoplakin (T2) showed 91% and 86% predictive values, respectively, for transplant outcome. Taken together, DBD kidney HMP perfusate profiles can distinguish between outcome 1-year post-transplantation. Furthermore, it provides insights into mechanisms that could play a role in post-transplant outcomes.

Transplantation outcomes in patients with primary hyperoxaluria: a systematic review.

BACKGROUND: Primary hyperoxaluria type 1 (PH1) is characterized by hepatic overproduction of oxalate and often results in kidney failure. Liver-kidney transplantation is recommended, either combined (CLKT) or sequentially performed (SLKT). The merits of SLKT and the place of an isolated kidney transplant (KT) in selected patients are unsettled. We systematically reviewed the literature focusing on patient and graft survival rates in relation to the chosen transplant strategy. METHODS: We searched MEDLINE and Embase using a broad search string, consisting of the terms 'transplantation' and 'hyperoxaluria'. Studies reporting on at least four transplanted patients were selected for quality assessment and data extraction. RESULTS: We found 51 observational studies from 1975 to 2020, covering 756 CLKT, 405 KT and 89 SLKT, and 51 pre-emptive liver transplantations (PLT). Meta-analysis was impossible due to reported survival probabilities with varying follow-up. Two individual high-quality studies showed an evident kidney graft survival advantage for CLKT versus KT (87% vs. 14% at 15 years, p<0.05) with adjusted HR for graft failure of 0.14 (95% confidence interval: 0.05-0.41), while patient survival was similar. Three other high-quality studies reported 5-year kidney graft survival rates of 48-89% for CLKT and 14-45% for KT. PLT and SLKT yielded 1-year patient and graft survival rates up to 100% in small cohorts. CONCLUSIONS: Our study suggests that CLKT leads to superior kidney graft survival compared to KT. However, evidence for merits of SLKT or for KT in pyridoxine-responsive patients was scarce, which warrants further studies, ideally using data from a large international registry.

Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels.

INTRODUCTION: The lack of approved specific therapeutic agents to treat coronavirus disease (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response. METHODS: Blood samples were collected from 52 individuals with a previous laboratory-confirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. RESULTS: All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). DISCUSSION: Robust associations between nAb titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 nAbs.

A nationwide evaluation of deceased donor kidney transplantation indicates detrimental consequences of early graft loss.

Early graft loss (EGL) is a feared outcome of kidney transplantation. Consequently, kidneys with an anticipated risk of EGL are declined for transplantation. In the most favorable scenario, with optimal use of available donor kidneys, the donor pool size is balanced by the risk of EGL, with a tradeoff dictated by the consequences of EGL. To gauge the consequence of EGL we systematically evaluated its impact in an observational study that included all 10,307 deceased-donor kidney transplantations performed in The Netherlands between 1990 and 2018. Incidence of EGL, defined as graft loss within 90 days, in primary transplantation was 8.2% (699/8,511). The main causes were graft rejection (30%), primary nonfunction (25%), and thrombosis or infarction (20%). EGL profoundly impacted short- and long-term patient survival (adjusted hazard ratio; 95% confidence interval: 8.2; 5.1-13.2 and 1.7; 1.3-2.1, respectively). Of the EGL recipients who survived 90 days after transplantation (617/699) only 440 of the 617 were relisted for re-transplantation. Of those relisted, only 298 were ultimately re-transplanted leading to an actual re-transplantation rate of 43%. Noticeably, re-transplantation was associated with a doubled incidence of EGL, but similar long-term graft survival (adjusted hazard ratio 1.1; 0.6-1.8). Thus, EGL after kidney transplantation is a medical catastrophe with high mortality rates, low relisting rates, and increased risk of recurrent EGL following re-transplantation. This implies that detrimental outcomes also involve convergence of risk factors in recipients with EGL. The 8.2% incidence of EGL minimally impacted population mortality, indicating this incidence is acceptable.

Subclinical effects of remote ischaemic conditioning in human kidney transplants revealed by quantitative proteomics.

BACKGROUND: Remote ischaemic conditioning (RIC) is currently being explored as a non-invasive method to attenuate ischaemia/reperfusion injuries in organs. A randomised clinical study (CONTEXT) evaluated the effects of RIC compared to non-RIC controls in human kidney transplants. METHODS: RIC was induced prior to kidney reperfusion by episodes of obstruction to arterial flow in the leg opposite the transplant using a tourniquet (4 × 5 min). Although RIC did not lead to clinical improvement of transplant outcomes, we explored whether RIC induced molecular changes through precision analysis of CONTEXT recipient plasma and kidney tissue samples by high-resolution tandem mass spectrometry (MS/MS). RESULTS: We observed an accumulation of muscle derived proteins and altered amino acid metabolism in kidney tissue proteomes, likely provoked by RIC, which was not reflected in plasma. In addition, MS/MS analysis demonstrated transient upregulation of several acute phase response proteins (SAA1, SAA2, CRP) in plasma, 1 and 5 days post-transplant in RIC and non-RIC conditions with a variable effect on the magnitude of acute inflammation. CONCLUSIONS: Together, our results indicate sub-clinical systemic and organ-localised effects of RIC.

A systematic review to identify whether perfusate biomarkers produced during hypothermic machine perfusion can predict graft outcomes in kidney transplantation.

There is good evidence to support the use of hypothermic machine perfusion (HMP) over static cold storage as the favoured preservation method for deceased donor kidneys. However, the utility of HMP as a tool to assess the viability of kidneys for transplant is unclear. There is a need to determine whether perfusate biomarkers produced during HMP can predict post-transplant outcomes and assess the suitability of organs for transplantation. Three different databases (MEDLINE, Embase, Transplant Library) were screened to 31 May 2019. Articles were included if a relationship was reported between one or more perfusate biomarkers and post-transplant outcomes. Studies were assessed and graded for methodological quality and strength of evidence. Glutathione S-transferase was the most promising biomarker for predicting delayed graft function, but its predictive ability was at best moderate. Analysis of primary nonfunction rates was challenging due to low occurrence rates and small sample sizes. Existing studies are limited in quality and have not yielded biomarkers for kidneys undergoing HMP that are able to predict post-transplant outcomes with sufficient accuracy to support routine clinical use. Further studies with larger samples and more robust methodology are needed. (PROSPERO registration: CRD42019121161).