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

CC-4066 therapy delivered to kidneys during cold storage and assessed with normothermic reperfusion is feasible and safe

Introduction: Currently there is an urgent need to translate interventions that may be beneficial to marginal donor kidneys prior to transplant, to improve their quality from bench to bedside. This project investigated the effects of CC-4066, a potent dual inhibitor of cyclophilin proteins A and D, treatment during static cold storage (SCS) in a porcine model of renal ischemia-reperfusion injury (IRI) using Normothermic Reperfusion (NR). Materials and methods: Porcine kidneys and autologous blood were retrieved in pairs from a local abattoir (n = 7). One kidney from each pair was randomly allocated to treatment and one allocated to control and flushed with preservation solution containing CC-4066 or vehicle. After 7 h of SCS kidneys underwent 3 h Normothermic Reperfusion (NR) with autologous whole blood while perfusion characteristics and samples were collected. Results: Perfusion and metabolic parameters showed similar trends and no statistical differences were observed between the groups. IL-6 showed a significant increase over time but no significant difference between groups (p-value 0.009 and 0.14 respectively, two-way ANOVA). Oxygen consumption and lactate levels were similar between groups but there was increased vacuolation on histology in the control group. Conclusions: The addition of CC-4066 during SCS of kidneys is safe and feasible and has no adverse or detrimental effects on perfusion during assessment on NR. There was no difference in cytokine levels although there was a trend towards less vacuolation on histology in the treatment group

Using established biorepositories for emerging research questions: a feasibility study

Background: Proteomics and metabolomics offer substantial potential for advancing kidney transplant research by providing versatile opportunities for gaining insights into the biomolecular processes occurring in donors, recipients, and grafts. To achieve this, adequate quality and numbers of biological samples are required. Whilst access to donor samples is facilitated by initiatives such as the QUOD biobank, an adequately powered biobank allowing exploration of recipient-related aspects in long-term transplant outcomes is missing. Rich, yet unverified resources of recipient material are the serum repositories present in the immunological laboratories of kidney transplant centers that prospectively collect recipient sera for immunological monitoring. However, it is yet unsure whether these samples are also suitable for -omics applications, since such clinical samples are collected and stored by individual centers using non-uniform protocols and undergo an undocumented number of freeze–thaw cycles. Whilst these handling and storage aspects may affect individual proteins and metabolites, it was reasoned that incidental handling/storage artifacts will have a limited effect on a theoretical network (pathway) analysis. To test the potential of such long-term stored clinical serum samples for pathway profiling, we submitted these samples to discovery proteomics and metabolomics. Methods: A mass spectrometry-based shotgun discovery approach was used to obtain an overview of proteins and metabolites in clinical serum samples from the immunological laboratories of the Dutch PROCARE consortium. Parallel analyses were performed with material from the strictly protocolized QUOD biobank. Results: Following metabolomics, more than 800 compounds could be identified in both sample groups, of which 163 endogenous metabolites were found in samples from both biorepositories. Proteomics yielded more than 600 proteins in both groups. Despite the higher prevalence of fragments in the clinical, non-uniformly collected samples compared to the biobanked ones (42.5% vs 26.5% of their proteomes, respectively), these fragments could still be connected to their parent proteins. Next, the proteomic and metabolomic profiles were successfully mapped onto theoretical pathways through integrated pathway analysis, which showed significant enrichment of 79 pathways. Conclusions: This feasibility study demonstrated that long-term stored serum samples from clinical biorepositories can be used for qualitative proteomic and metabolomic pathway analysis, a notion with far-reaching implications for all biomedical, long-term outcome-dependent research questions and studies focusing on rare events

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

Lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular peroxiredoxin 2, and a greater frequency of cells with exofacial peroxiredoxin 2, compared with healthy human lymphocytes

AbstractPeroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood lymphocytes and levels in extracellular fluid in healthy subjects and rheumatoid arthritis patients are poorly described. In the present study, the median intracellular peroxiredoxin 2 protein content of lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with healthy subjects’ lymphocytes. Intracellular peroxiredoxin 3 levels were similar in healthy and rheumatoid arthritis lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca. 8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. Exofacial thioredoxin-1 was also observed. In the total lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17+ve lymphocytes were exofacially peroxiredoxin 2+ve (median, 39%). Prdx2 was also detected in human extracellular fluids. We suggest that crucial inflammatory cell subsets, i.e. interleukin-17+ve T cells, exhibit increased exofacial redox-regulating enzymes and that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic inflammation

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

Peer reviewed: TrueAcknowledgements: The study was accepted for presentation at ESOT Congress, Athens, 2023. We gratefully acknowledge the patients, donors, and donor families as well as all transplant centres that included livers in this trial. We are indebted to Sarah Mertens, Rajeev Kumar, and Bhumika Patel for help with ethics approval, database management, and sample bio banking. We thank the European Commission for their support through the Seventh Framework Programme.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 &amp;lt; 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.</jats:p

Hemodynamics and metabolic parameters in normothermic kidney preservation are linked with donor factors, perfusate cells, and cytokines

Kidney transplantation is the best renal-replacement option for most patients with end-stage renal disease. Normothermic machine preservation (NMP) of the kidney has been studied extensively during the last two decades and implemented in clinical trials. Biomarker research led to success in identifying molecules with diagnostic, predictive and therapeutic properties in chronic kidney disease. However, perfusate biomarkers and potential predictive mechanisms in NMP have not been identified yet. Twelve discarded human kidneys (n = 7 DBD, n = 5 DCD) underwent NMP for up to 24 h. Eight were perfused applying urine recirculation (URC), four with replacement of urine (UR) using Ringer's lactate. The aim of our study was to investigate biomarkers (NGAL, KIM-1, and L-FABP), cells and cytokines in the perfusate in context with donor characteristics, perfusate hemodynamics and metabolic parameters. Cold ischemia time did not correlate with any of the markers. Perfusates of DBD kidneys had a significantly lower number of leukocytes after 6 h of NMP compared to DCD. Arterial flow, pH, NGAL and L-FABP correlated with donor creatinine and eGFR. Arterial flow was higher in kidneys with lower perfusate lactate. Perfusate TNF-α was higher in kidneys with lower arterial flow. The cytokines IL-1β and GM-CSF decreased during 6 h of NMP. Kidneys with more urine output had lower perfusate KIM-1 levels. Median and 6-h values of lactate, arterial flow, pH, NGAL, KIM-1, and L-FABP correlated with each other indicating a 6-h period being applicable for kidney viability assessment. The study results demonstrate a comparable cytokine and cell profile in perfusates with URC and UR. In conclusion, clinically available perfusate and hemodynamic parameters correlate well with donor characteristics and measured biomarkers in a discarded human NMP model

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

Abstract 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

Ex situ hypothermic perfusion of nonhuman primate pancreas: A feasibility study

International audiencePancreatic static cold storage (SCS) is the gold-standard method for pancreas preservation. Our main objective was to evaluate feasibility of hypothermic perfusion (HP) of nonhuman primates' pancreases for potential organ transplantation. Seven baboon pancreases were tested. Animals were included in a study approved by the French Research Ministry of Health. Two groups were compared: the control group (n = 2) was preserved using conventional SCS for 24-h and the perfusion group (n = 5) used HP for 24-h, with three different perfusion pressures (PP): 15 (n = 3), 20 (n = 1), and 25 mm Hg (n = 1). In the control group, focal congestion of islets was observed after 6-h. At 24-h, ischemic necrosis and multifocal congestion also occurred. In the HP group, at 15 mm Hg PP, multifocal congestion of islets was present at 24-h. At 20 mm Hg PP, no ischemic necrosis was found after 6-h. At 12-h and 24-h, focal congestion of islets was seen. At 25 mm Hg PP, focal congestion of islets appeared after 12-h. Immunostaining for insulin, glucagon, and somatostatin was normal and similar in controls and perfused pancreas transplants even after 24-h. Apoptosis index represented by cleaved caspase 3 activity, was less than 1% in perfusion and control groups, even after 24-h. HP of nonhuman primate pancreas is feasible and not deleterious as far as 24-h compared to SCS. SCS for more than 12-h was harmful for the transplants. Systolic perfusion pressure between 15-20 mm Hg did not cause any pathological injury of the tested organs

Ischemia-Reperfusion Injuries Assessment during Pancreas Preservation

International audienceMaintaining organ viability between donation and transplantation is of critical importance for optimal graft function and survival. To date in pancreas transplantation, static cold storage (SCS) is the most widely practiced method of organ preservation. The first experiments in ex vivo perfusion of the pancreas were performed at the beginning of the 20th century. These perfusions led to organ oedema, hemorrhage, and venous congestion after revascularization. Despite these early hurdles, a number of factors now favor the use of perfusion during preservation: the encouraging results of HMP in kidney transplantation, the development of new perfusion solutions, and the development of organ perfusion machines for the lung, heart, kidneys and liver. This has led to a resurgence of research in machine perfusion for whole organ pancreas preservation. This review highlights the ischemia-reperfusion injuries assessment during ex vivo pancreas perfusion, both for assessment in pre-clinical experimental models as well for future use in the clinic. We evaluated perfusion dynamics, oedema assessment, especially by impedance analysis and MRI, whole organ oxygen consumption, tissue oxygen tension, metabolite concentrations in tissue and perfusate, mitochondrial respiration, cell death, especially by histology, total cell free DNA, caspase activation, and exocrine and endocrine assessment