Expression of sPD-L1 levels in an ex vivo liver perfusion model.

The programmed cell death protein 1 (PD-1) acts as a central inhibitory immune checkpoint receptor. The soluble form of its primary ligand, sPD-L1, was found to be elevated in serum of patients with cancer, infectious diseases and chronic inflammation. So far, the hepatic origin of sPD-L1 has received relatively little attention and is therefore subject of this study in the context of normothermic machine perfusion (NMP) of liver grafts. sPD-L1 concentrations as well as several well-established clinically relevant laboratory parameters were determined in the perfusate of 16 donor liver grafts undergoing normothermic machine perfusion (NMP) up to 30 hours (h). sPD-L1 levels continuously increased during NMP and significantly correlated with markers of hepatic synthesis (cholinesterase), acute-phase proteins (von Willebrand factor, procalcitonin, antithrombin, interleukin-6, fibrinogen) and liver decay markers (gamma-glutamyltransferase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase). Perfusate leukocytes were in the lower reference range, and decreased after 12 h. Mean sPD-L1 levels in the perfusate correlated with donor levels of gamma-glutamyltransferase, alanine aminotransferase, creatinine and blood urea nitrogen. Our study reveals a significant increase of concentration of sPD-L1 following ischemia-reperfusion injury in a hepatic ex vivo model. sPD-L1 concentrations during NMP correlate with established acute-phase proteins and liver cell decay markers suggesting that hepatic sPD-L1 synthesis or shedding increases during the acute phase and cell decay. Furthermore, sPD-L1 correlates with established liver function and synthesis parameters as well as with donor laboratory values and might therefore be a potential biomarker for hepatic function of liver grafts.

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

BACKGROUND: A better understanding of the molecular events during liver normothermic machine perfusion (NMP) is warranted to develop a data-based approach for the identification of biomarkers representative of graft quality and posttransplant outcome. We analysed the dynamic transcriptional changes during NMP and linked them to clinical and biochemical parameters. METHODS: 50 livers subjected to NMP for up to 24 h were enrolled. Bulk RNA sequencing was performed in serial biopsies collected pre and during NMP, and after reperfusion. Perfusate was sampled to monitor liver function. qPCR and immunohistochemistry were performed to validate findings. Molecular profiles were compared between transplanted and non-transplanted livers, and livers with and without early allograft dysfunction. FINDINGS: Pathways related to immune and cell stress responses, cell trafficking and cell regulation were activated during NMP, while cellular metabolism was downregulated over time. Anti-inflammatory responses and genes involved in tissue remodelling were induced at later time-points, suggesting a counter-response to the immediate damage. NMP strongly induced a gene signature associated with ischemia-reperfusion injury. A 7-gene signature corresponds with the benchmarking criteria for transplantation or discard at 6 h NMP (area under curve 0.99). CD274 gene expression (encoding programmed cell-death ligand-1) showed the highest predictive value. LEAP2 gene expression at 6 h NMP correlated with impaired graft function. INTERPRETATION: Assessment of gene expression markers could serve as a reliable tool to evaluate liver quality during NMP and predicts early graft function after transplantation. FUNDING: The research was supported by "In Memoriam Dr. Gabriel Salzner Stiftung", Tiroler Wissenschaftsfond, Jubiläumsfonds-Österreichische Nationalbank and MUI Start grant.

The Predictive Value of Graft Viability and Bioenergetics Testing Towards the Outcome in Liver Transplantation.

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (-0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation.

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.

Interleukin-6 Levels During Normothermic Machine Perfusion Impact Postreperfusion Hemodynamics of Liver Graft Recipients: A Prospective Single-center Observational Study.

BACKGROUND: Hemodynamic instability after liver graft reperfusion increases recipient morbidity after liver transplantation. The etiologies of hemodynamic disturbances appear to be multifactorial and are poorly understood. Normothermic machine perfusion (NMP) provides an opportunity to analyze graft quality prior to transplantation. In the present study, we aim to investigate the influence of interleukin-6 (IL-6) levels during NMP on postreperfusion hemodynamics of the recipient. METHODS: Consecutive NMP-liver transplants at a single-center were prospectively analyzed. Perfusate samples were collected at the beginning, after 6 h, and at the end of perfusion and analyzed for IL-6 levels. Mean arterial pressure (MAP) and catecholamine consumption during surgery were recorded. IL-6 levels at the end of NMP were correlated to donor and perfusion characteristics as well as changes in MAP and catecholamine requirements during the anhepatic and reperfusion phase. RESULTS: IL-6 perfusate measurements were assessed in 77 livers undergoing NMP and transplantation. Donor age, sex, cold ischemic time, and NMP time did not correlate with IL-6 levels. Perfusates of donation after circulatory death grafts showed higher IL-6 levels at the end of NMP than donation after brain death grafts. However, IL-6 levels at the end of NMP correlated with catecholamine requirements and MAP in the reperfusion phase. Per log10 increase in IL-6 levels, an increase of 42% points in administered catecholamine dose was observed, despite MAP being decreased by 3.6% points compared to baseline values. CONCLUSIONS: IL-6 levels may be a predictor for recipient hemodynamic instability during liver reperfusion. Larger studies are needed to confirm this finding.

48 h Normothermic Machine Perfusion With Urine Recirculation for Discarded Human Kidney Grafts.

Normothermic machine perfusion (NMP) has reshaped organ preservation in recent years. In this preclinical study, prolonged normothermic perfusions of discarded human kidney grafts were performed in order to investigate perfusion dynamics and identify potential quality and assessment indicators. Five human discarded kidney grafts were perfused normothermically (37°C) for 48 h using the Kidney Assist device with a red-blood-cell based perfusate with urine recirculation. Perfusion dynamics, perfusate and urine composition as well as injury markers were measured and analyzed. Donor age ranged from 41 to 68 years. All but one kidney were from brain dead donors. Perfusions were performed successfully for 48 h with all discarded kidneys. Median arterial flow ranged from 405 to 841 mL/min. All kidneys excreted urine until the end of perfusion (median 0.43 mL/min at the end of perfusion). While sodium levels were consistently lower in urine compared to perfusate samples, this was only seen for chloride and potassium in kidney KTX 2. Lactate, AST, LDH as well as pro-inflammatory cytokines increased over time, especially in kidneys KTX 3 and 4. Ex vivo normothermic perfusion is able to identify patterns of perfusion, biological function, and changes in inflammatory markers in heterogenous discarded kidney grafts.

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.

Bioenergetic and Cytokine Profiling May Help to Rescue More DCD Livers for Transplantation.

The majority of organs used for liver transplantation come from brain-dead donors (DBD). In order to overcome the organ shortage, increasingly donation after circulatory death (DCD) organs are also considered. Since normothermic machine perfusion (NMP) restores metabolic activity and allows for in-depth assessment of organ quality and function prior to transplantation, such organs may benefit from NMP. We herein compare the bioenergetic performance through a comprehensive evaluation of mitochondria by high-resolution respirometry in tissue biopsies and the inflammatory response in DBD and DCD livers during NMP. While livers were indistinguishable by perfusate biomarker assessment and histology, our findings revealed a greater impairment of mitochondrial function in DCD livers after static cold storage compared to DBD livers. During subsequent NMPs, DCD organs recovered and eventually showed a similar performance as DBD livers. Cytokine expression analysis showed no differences in the early phase of NMP, while towards the end of NMP, significantly elevated levels of IL-1ß, IL-5 and IL-6 were found in the perfusate of DCD livers. Based on our results, we find it worthwhile to reconsider more DCD organs for transplantation to further extend the donor pool. Therefore, donor organ quality criteria must be developed, which may include an assessment of bioenergetic function and cytokine quantification.

Liver Transplantation in Recipients With a Positive Crossmatch: A Retrospective Single-Center Match-Pair Analysis.

A positive crossmatch (XM+) is considered a contraindication to solid abdominal organ transplantation except liver transplantation (LT). Conflicting reports exist regarding the effects of XM+ on post-transplant outcomes. The goal of this retrospective single-center analysis is to evaluate the influence of XM+ on relevant outcome parameters such as survival, graft rejection, biliary and arterial complications. Forty-nine adult patients undergoing LT with a XM+ between 2002 and 2017 were included. XM+ LT recipients were matched 1:2 with crossmatch negative (XM-) LT recipients based on the balance of risk (BAR) score. Patient and graft survival were compared using Kaplan-Meier survival analysis and the log-rank test. Comparative analysis of clinical outcomes in XM+ and XM- groups were conducted. Patient and graft survival were similar in XM+ and XM- patients. Rejection episodes did not differ either. Recipients with a strong XM+ were more likely to develop a PCR+ CMV infection. A XM+ was not associated with a higher incidence of biliary or arterial complications. Donor age, cold ischemia time, PCR+ CMV infection and a rejection episode were associated with the occurrence of ischemic type biliary lesions. A XM+ has no effects on patient and graft survival or other relevant outcome parameters following LT.

ESOT Consensus Platform for Organ Transplantation: Setting the Stage for a Rigorous, Regularly Updated Development Process.

The European Society for Organ Transplantation (ESOT) has created a platform for the development of rigorous and regularly updated evidence based guidelines for clinical practice in the transplantation field. A dedicated Guideline Taskforce, including ESOT-council members, a representative from the Centre for Evidence in Transplantation, editors of the journal Transplant International has developed transparent procedures to guide the development of guidelines, recommendations, and consensus statements. During ESOT's first Consensus Conference in November 2022, leading experts will present in-depth evidence based reviews of nine themes and will propose recommendations aimed at reaching a consensus after public discussion and assessment by an independent jury. All recommendations and consensus statements produced for the nine selected topics will be published including the entire evidence-based consensus-finding process. An extensive literature review of each topic was conducted to provide final evidence and/or expert opinion.

Mitochondrial respiration during normothermic liver machine perfusion predicts clinical outcome.

BACKGROUND: Reliable biomarkers for organ quality assessment during normothermic machine perfusion (NMP) are desired. ATP (adenosine triphosphate) production by oxidative phosphorylation plays a crucial role in the bioenergetic homeostasis of the liver. Thus, detailed analysis of the aerobic mitochondrial performance may serve as predictive tool towards the outcome after liver transplantation. METHODS: In a prospective clinical trial, 50 livers were subjected to NMP (OrganOx Metra) for up to 24.ßh. Biopsy and perfusate samples were collected at the end of cold storage, at 1.ßh, 6.ßh, end of NMP, and 1.ßh after reperfusion. Mitochondrial function and integrity were characterized by high-resolution respirometry (HRR), AMP, ADP, ATP and glutamate dehydrogenase analysis and correlated with the clinical outcome (L-GrAFT score). Real-time confocal microscopy was performed to assess tissue viability. Structural damage was investigated by histology, immunohistochemistry and transmission electron microscopy. FINDINGS: A considerable variability in tissue viability and mitochondrial respiration between individual livers at the end of cold storage was observed. During NMP, mitochondrial respiration with succinate and tissue viability remained stable. In the multivariate analysis of the 35 transplanted livers (15 were discarded), area under the curve (AUC) of LEAK respiration, cytochrome c control efficiency (mitochondrial outer membrane damage), and efficacy of the mitochondrial ATP production during the first 6.ßh of NMP correlated with L-GrAFT. INTERPRETATIONS: Bioenergetic competence during NMP plays a pivotal role in addition to tissue injury markers. The AUC for markers of outer mitochondrial membrane damage, ATP synthesis efficiency and dissipative respiration (LEAK) predict the clinical outcome upon liver transplantation. FUNDING: This study was funded by a Grant from the In Memoriam Dr. Gabriel Salzner Stiftung awarded to SS and the Tiroler Wissenschaftsfond granted to TH.

Impact of the COVID-19 pandemic on the kidney community: lessons learned and future directions.

The coronavirus disease 2019 (COVID-19) pandemic has disproportionately affected patients with kidney disease, causing significant challenges in disease management, kidney research and trainee education. For patients, increased infection risk and disease severity, often complicated by acute kidney injury, have contributed to high mortality. Clinicians were faced with high clinical demands, resource shortages and novel ethical dilemmas in providing patient care. In this review, we address the impact of COVID-19 on the entire spectrum of kidney care, including acute kidney injury, chronic kidney disease, dialysis and transplantation, trainee education, disparities in health care, changes in health care policies, moral distress and the patient perspective. Based on current evidence, we provide a framework for the management and support of patients with kidney disease, infection mitigation strategies, resource allocation and support systems for the nephrology workforce.

Normothermic machine perfusion of kidneys: current strategies and future perspectives.

PURPOSE OF REVIEW: This review aims to summarize the latest original preclinical and clinical articles in the setting of normothermic machine perfusion (NMP) of kidney grafts. RECENT FINDINGS: Kidney NMP can be safely translated into the clinical routine and there is increasing evidence that NMP may be beneficial in graft preservation especially in marginal kidney grafts. Due to the near-physiological state during NMP, this technology may be used as an ex-vivo organ assessment and treatment platform. There are reports on the application of mesenchymal stromal/stem cells, multipotent adult progenitor cells and microRNA during kidney NMP, with first data indicating that these therapies indeed lead to a decrease in inflammatory response and kidney injury. Together with the demonstrated possibility of prolonged ex-vivo perfusion without significant graft damage, NMP could not only be used as a tool to perform preimplant graft assessment. Some evidence exists that it truly has the potential to be a platform to treat and repair injured kidney grafts, thereby significantly reducing the number of declined organs. SUMMARY: Kidney NMP is feasible and can potentially increase the donor pool not only by preimplant graft assessment, but also by ex-vivo graft treatment.

Hyperspectral Imaging as a Tool for Viability Assessment During Normothermic Machine Perfusion of Human Livers: A Proof of Concept Pilot Study.

Normothermic machine perfusion (NMP) allows for ex vivo viability and functional assessment prior to liver transplantation (LT). Hyperspectral imaging represents a suitable, non-invasive method to evaluate tissue morphology and organ perfusion during NMP. Liver allografts were subjected to NMP prior to LT. Serial image acquisition of oxygen saturation levels (StO2), organ hemoglobin (THI), near-infrared perfusion (NIR) and tissue water indices (TWI) through hyperspectral imaging was performed during static cold storage, at 1h, 6h, 12h and at the end of NMP. The readouts were correlated with perfusate parameters at equivalent time points. Twenty-one deceased donor livers were included in the study. Seven (33.0%) were discarded due to poor organ function during NMP. StO2 (p < 0.001), THI (p < 0.001) and NIR (p = 0.002) significantly augmented, from static cold storage (pre-NMP) to NMP end, while TWI dropped (p = 0.005) during the observational period. At 12-24h, a significantly higher hemoglobin concentration (THI) in the superficial tissue layers was seen in discarded, compared to transplanted livers (p = 0.036). Lactate values at 12h NMP correlated negatively with NIR perfusion index between 12 and 24h NMP and with the delta NIR perfusion index between 1 and 24h (rs = -0.883, p = 0.008 for both). Furthermore, NIR and TWI correlated with lactate clearance and pH. This study provides first evidence of feasibility of hyperspectral imaging as a potentially helpful contact-free organ viability assessment tool during liver NMP.

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.

Forty-eight hours of normothermic kidney preservation applying urine recirculation.

Kidney transplantation is limited due to the organ scarcity and the large discrepancy between transplantable organs and patients on the waiting list. Ex-situ normothermic kidney preservation has been studied extensively as a tool to enlarge the donor pool and to enable viability assessment. Urine recirculation, for volume control, was applied to perfuse a discarded human kidney for 48 hours. Long-term kidney NMP was feasible under stable hemodynamic conditions with a physiological acid-base-balance and an intact histological morphology of the organ.