Magnetic resonance imaging as a noninvasive adjunct to conventional assessment of functional differences between kidneys in vivo and during ex vivo normothermic machine perfusion.

Normothermic machine perfusion (NMP) is increasingly considered for pretransplant kidney quality assessment. However, fundamental questions about differences between in vivo and ex vivo renal function, as well as the impact of ischemic injury on ex vivo physiology, remain unanswered. This study utilized magnetic resonance imaging (MRI), alongside conventional parameters to explore differences between in vivo and ex vivo renal function and the impact of warm ischemia on a kidney's behavior ex vivo. Renal MRI scans and samples were obtained from living pigs (n = 30) in vivo. Next, kidney pairs were procured and exposed to minimal, or 75 minutes of warm ischemia, followed by 6 hours of hypothermic machine perfusion. Both kidneys simultaneously underwent 6-hour ex vivo perfusion in MRI-compatible NMP circuits to obtain multiparametric MRI data. Ischemically injured ex vivo kidneys showed a significantly altered regional blood flow distribution compared to in vivo and minimally damaged organs. Both ex vivo groups showed diffusion restriction relative to in vivo. Our findings underscore the differences between in vivo and ex vivo MRI-based renal characteristics. Therefore, when assessing organ viability during NMP, it should be considered to incorporate parameters beyond the conventional functional markers that are common in vivo.

The association between hemoglobin levels and renal function parameters during normothermic machine perfusion: A retrospective cohort study using porcine kidneys.

BACKGROUND: Ex vivo normothermic machine perfusion (NMP) is a promising tool for assessing an isolated kidney prior to transplantation. However, there is no consensus on the perfusate's optimal oxygen-carrying capacity to support renal function. To investigate the association of hemoglobin levels with renal function parameters, a retrospective analysis of isolated, normothermically, perfused porcine kidneys was performed. METHODS: Between 2015 and 2021, a total of 228 kidneys underwent 4 h of NMP with perfusates that varied in hemoglobin levels. A generalized linear model was used to determine the association of hemoglobin levels with time-weighted means of renal function markers, such as fractional sodium excretion (FENa) and creatinine clearance (CrCl). Stratified by baseline hemoglobin level (<4.5, 4.5-6, or >6 mmol/L), these markers were modeled over time using a generalized linear mixed-effects model. All models were adjusted for potential confounders. RESULTS: Until a hemoglobin level of around 5 mmol/L was reached, increasing hemoglobin levels were associated with superior FENa and CrCl. Thereafter, this association plateaued. When hemoglobin levels were categorized, hemoglobin <4.5 mmol/L was associated with worse renal function. Hemoglobin levels were neither significantly associated with proteinuria during NMP nor with ATP levels at the end of NMP. Hemoglobin levels >6 mmol/L showed no additional benefits in renal function. CONCLUSION: In conclusion, we found an association between baseline hemoglobin levels and superior renal function parameters, but not injury, during NMP of porcine kidneys. Furthermore, we show that performing a retrospective cohort study of preclinical data is feasible and able to answer additional questions, reducing the potential use of laboratory animals.

Prolonged hypothermic machine perfusion enables daytime liver transplantation - an IDEAL stage 2 prospective clinical trial.

Background: Liver transplantation is traditionally performed around the clock to minimize organ ischemic time. However, the prospect of prolonging preservation times holds the potential to streamline logistics and transform liver transplantation into a semi-elective procedure, reducing the need for nighttime surgeries. Dual hypothermic oxygenated machine perfusion (DHOPE) of donor livers for 1-2 h mitigates ischemia-reperfusion injury and improves transplant outcomes. Preclinical studies have shown that DHOPE can safely extend the preservation of donor livers for up to 24 h. Methods: We conducted an IDEAL stage 2 prospective clinical trial comparing prolonged (≥4 h) DHOPE to conventional (1-2 h) DHOPE for brain-dead donor livers, enabling transplantation the following morning. Liver allocation to each group was based on donor hepatectomy end times. The primary safety endpoint was a composite of all serious adverse events (SAE) within 30 days after transplantation. The primary feasibility endpoint was defined as the number of patients assigned and successfully receiving a prolonged DHOPE-perfused liver graft. Trial registration at: WHO International Clinical Trial Registry Platform, number NL8740. Findings: Between November 1, 2020 and July 16, 2022, 24 patients were enrolled. The median preservation time was 14.5 h (interquartile range [IQR], 13.9-15.5) for the prolonged group (n = 12) and 7.9 h (IQR, 7.6-8.6) for the control group (n = 12; p = 0.01). In each group, three patients (25%; 95% CI 3.9-46%, p = 1) experienced a SAE. Markers of ischemia-reperfusion injury and oxidative stress in both perfusate and recipients were consistently low and showed no notable discrepancies between the two groups. All patients assigned to either the prolonged group or control group successfully received a liver graft perfused with either prolonged DHOPE or control DHOPE, respectively. Interpretation: This first-in-human clinical trial demonstrates the safety and feasibility of DHOPE in prolonging the preservation time of donor livers to enable daytime transplantation. The ability to extend the preservation window to up to 20 h using hypothermic oxygenated machine preservation at a 10 °C temperature has the potential to reshape the landscape of liver transplantation. Funding: University Medical Center Groningen, the Netherlands.

Furosemide attenuates tubulointerstitial injury and allows functional testing of porcine kidneys during normothermic machine perfusion.

BACKGROUND: Normothermic machine perfusion (NMP) is a promising pretransplant kidney quality assessment platform, but it remains crucial to increase its diagnostic potential while ensuring minimal additional injury to the already damaged kidney. Interventions that alter tubular transport can influence renal function and injury during perfusion. This study aimed to determine whether furosemide and desmopressin affect renal function and injury during NMP. METHODS: Eighteen porcine kidneys (n = 6 per group) were subjected to 30 min of warm ischemia and 4 h of oxygenated hypothermic perfusion before being subjected to 6 h of NMP. Each organ was randomized to receive no drug, furosemide (750 mg), or desmopressin (16 µg) during NMP. RESULTS: Compared with the other groups, the addition of furosemide resulted in significantly increased urine output, fractional excretion of sodium and potassium, and urea clearance during NMP. Urinary neutrophil gelatinase-associated lipocalin levels decreased significantly with furosemide supplementation compared with the other groups. The addition of desmopressin did not result in any significantly different outcome measurements compared with the control group. CONCLUSIONS: This study showed that the addition of furosemide affected renal function while attenuating tubulointerstitial injury during NMP. Therefore, furosemide supplementation may provide renal protection and serve as a functional test for pretransplant kidney viability assessment during NMP.

Bile proteome reveals biliary regeneration during normothermic preservation of human donor livers.

Normothermic machine perfusion (NMP) after static cold storage is increasingly used for preservation and assessment of human donor livers prior to transplantation. Biliary viability assessment during NMP reduces the risk of post-transplant biliary complications. However, understanding of molecular changes in the biliary system during NMP remains incomplete. We performed an in-depth, unbiased proteomics analysis of bile collected during sequential hypothermic machine perfusion, rewarming and NMP of 55 human donor livers. Longitudinal analysis during NMP reveals proteins reflective of cellular damage at early stages, followed by upregulation of secretory and immune response processes. Livers with bile chemistry acceptable for transplantation reveal protein patterns implicated in regenerative processes, including cellular proliferation, compared to livers with inadequate bile chemistry. These findings are reinforced by detection of regenerative gene transcripts in liver tissue before machine perfusion. Our comprehensive bile proteomics and liver transcriptomics data sets provide the potential to further evaluate molecular mechanisms during NMP and refine viability assessment criteria.

Restoration of Bile Duct Injury of Donor Livers During Ex Situ Normothermic Machine Perfusion.

BACKGROUND: End-ischemic ex situ normothermic machine perfusion (NMP) enables assessment of donor livers prior to transplantation. The objective of this study was to provide support for bile composition as a marker of biliary viability and to investigate whether bile ducts of high-risk human donor livers already undergo repair during NMP. METHODS: Forty-two livers that were initially declined for transplantation were included in our NMP clinical trial. After NMP, livers were either secondary declined (n = 17) or accepted for transplantation (n = 25) based on the chemical composition of bile and perfusate samples. Bile duct biopsies were taken before and after NMP and assessed using an established histological injury severity scoring system and a comprehensive immunohistochemical assessment focusing on peribiliary glands (PBGs), vascular damage, and regeneration. RESULTS: Bile ducts of livers that were transplanted after viability testing during NMP showed better preservation of PBGs, (micro)vasculature, and increased cholangiocyte proliferation, compared with declined livers. Biliary bicarbonate, glucose, and pH were confirmed as accurate biomarkers of bile duct vitality. In addition, we found evidence of PBG-based progenitor cell differentiation toward mature cholangiocytes during NMP. CONCLUSIONS: Favorable bile chemistry during NMP correlates well with better-preserved biliary microvasculature and PBGs, with a preserved capacity for biliary regeneration. During NMP, biliary tree progenitor cells start to differentiate toward mature cholangiocytes, facilitating restoration of the ischemically damaged surface epithelium.

Prolonged Controlled Oxygenated Rewarming Improves Immediate Tubular Function and Energetic Recovery of Porcine Kidneys During Normothermic Machine Perfusion.

BACKGROUND: Normothermic machine perfusion (NMP) is typically performed after a period of hypothermic preservation, which exposes the kidney to an abrupt increase in temperature and intravascular pressure. The resultant rewarming injury could be alleviated by gradual rewarming using controlled oxygenated rewarming (COR). This study aimed to establish which rewarming rate during COR results in the best protective effect on renal rewarming injury during subsequent NMP. METHODS: Twenty-eight viable porcine kidneys (n = 7/group) were obtained from a slaughterhouse. After these kidneys had sustained 30 min of warm ischemia and 24 h of oxygenated HMP, they were either rewarmed abruptly from 4-8 °C to 37 °C by directly initiating NMP or gradually throughout 30, 60, or 120 min of COR (rate of increase in kidney temperature of 4.46%/min, 2.20%/min, or 1.10%/min) before NMP. RESULTS: Kidneys that were rewarmed during the course of 120 min (COR-120) had significantly lower fractional excretion of sodium and glucose at the start of NMP compared with rewarming durations of 30 min (COR-30) and 60 min (COR-60). Although COR-120 kidneys showed superior immediate tubular function at the start of normothermic perfusion, this difference disappeared during NMP. Furthermore, energetic recovery was significantly improved in COR-30 and COR-120 kidneys compared with abruptly rewarmed and COR-60 kidneys. CONCLUSIONS: This study suggests that a rewarming rate of 1.10%/min during COR-120 could result in superior immediate tubular function and energetic recovery during NMP. Therefore, it may provide the best protective effect against rewarming injury.

Magnetic resonance imaging during warm ex vivo kidney perfusion.

BACKGROUND: The shortage of donor organs for transplantation remains a worldwide problem. The utilization of suboptimal deceased donors enlarges the pool of potential organs, yet consequently, clinicians face the difficult decision of whether these sub-optimal organs are of sufficient quality for transplantation. Novel technologies could play a pivotal role in making pre-transplant organ assessment more objective and reliable. METHODS: Ex vivo normothermic machine perfusion (NMP) at temperatures around 35-37°C allows organ quality assessment in a near-physiological environment. Advanced magnetic resonance imaging (MRI) techniques convey unique information about an organ's structural and functional integrity. The concept of applying magnetic resonance imaging during renal normothermic machine perfusion is novel in both renal and radiological research and we have developed the first MRI-compatible NMP setup for human-sized kidneys. RESULTS: We were able to obtain a detailed and real-time view of ongoing processes inside renal grafts during ex vivo perfusion. This new technique can visualize structural abnormalities, quantify regional flow distribution, renal metabolism, and local oxygen availability, and track the distribution of ex vivo administered cellular therapy. CONCLUSION: This platform allows for advanced pre-transplant organ assessment, provides a new realistic tool for studies into renal physiology and metabolism, and may facilitate therapeutic tracing of pharmacological and cellular interventions to an isolated kidney.

Reducing cold ischemia time by donor liver "back-table" preparation under continuous oxygenated machine perfusion of the portal vein.

INTRODUCTION: Cold ischemia time is a well-known risk factor for the development of non-anastomotic biliary strictures (NAS) after liver transplantation. End-ischemic hypothermic oxygenated machine perfusion (HOPE) of DCD liver grafts reduces the incidence of NAS, and has the potential to reduce cold ischemia times. We hypothesized that if a part of the back-table procedure could be performed under continuous HOPE, cold ischemia times would be reduced. METHODS: In this prospective observational cohort study, all nationwide declined livers that underwent DHOPE-NMP between July 1st 2021 and January 1st 2022 were included. The back-table of ten consecutive high-risk donor livers was performed with ongoing HOPE. Sixty DHOPE-NMP procedures (August 1st 2017-July 1st 2021) with a conventional back-table procedure functioned as a control group. RESULTS: Compared to the control group, this technique led to a decrease in non-oxygenated back-table time from median 74 min (IQR 58-92 min) to median 25 min (IQR 21-31 min), p < .01. Median total cold preservation times were reduced from 279 min (IQR 254-297) to 214 min (IQR 132-254), p < .01. CONCLUSION: Cold ischemia time of liver grafts can be successfully reduced by over one hour by using portal vein only HOPE during back-table preparation.

Prolonged preservation by hypothermic machine perfusion facilitates logistics in liver transplantation: A European observational cohort study.

A short period (1-2 h) of hypothermic oxygenated machine perfusion (HOPE) after static cold storage is safe and reduces ischemia-reperfusion injury-related complications after liver transplantation. Machine perfusion time is occasionally prolonged for logistical reasons, but it is unknown if prolonged HOPE is safe and compromises outcomes. We conducted a multicenter, observational cohort study of patients transplanted with a liver preserved by prolonged (≥4 h) HOPE. Postoperative biochemistry, complications, and survival were evaluated. The cohort included 93 recipients from 12 European transplant centers between 2014-2021. The most common reason to prolong HOPE was the lack of an available operating room to start the transplant procedure. Grafts underwent HOPE for a median (range) of 4:42 h (4:00-8:35 h) with a total preservation time of 10:50 h (5:50-20:50 h). Postoperative peak ALT was 675 IU/L (interquartile range 419-1378 IU/L). The incidence of postoperative complications was low, and 1-year graft and patient survival were 94% and 88%, respectively. To conclude, good outcomes are achieved after transplantation of donor livers preserved with prolonged (median 4:42 h) HOPE, leading to a total preservation time of almost 21 h. These results suggest that simple, end-ischemic HOPE may be utilized for safe extension of the preservation time to ease transplantation logistics.

Sequential hypothermic and normothermic machine perfusion enables safe transplantation of high-risk donor livers.

Ex situ normothermic machine perfusion (NMP) is increasingly used for viability assessment of high-risk donor livers, whereas dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia-reperfusion injury. We aimed to resuscitate and test the viability of initially-discarded, high-risk donor livers using sequential DHOPE and NMP with two different oxygen carriers: an artificial hemoglobin-based oxygen carrier (HBOC) or red blood cells (RBC). In a prospective observational cohort study of 54 livers that underwent DHOPE-NMP, the first 18 procedures were performed with a HBOC-based perfusion solution and the subsequent 36 procedures were performed with an RBC-based perfusion solution for the NMP phase. All but one livers were derived from extended criteria donation after circulatory death donors, with a median donor risk index of 2.84 (IQR 2.52-3.11). After functional assessment during NMP, 34 livers (63% utilization), met the viability criteria and were transplanted. One-year graft and patient survival were 94% and 100%, respectively. Post-transplant cholangiopathy occurred in 1 patient (3%). There were no significant differences in utilization rate and post-transplant outcomes between the HBOC and RBC group. Ex situ machine perfusion using sequential DHOPE-NMP for resuscitation and viability assessment of high-risk donor livers results in excellent transplant outcomes, irrespective of the oxygen carrier used.

Prolonged dual hypothermic oxygenated machine preservation (DHOPE-PRO) in liver transplantation: study protocol for a stage 2, prospective, dual-arm, safety and feasibility clinical trial.

INTRODUCTION: End-ischaemic preservation of a donor liver by dual hypothermic oxygenated machine perfusion (DHOPE) for 2 hours prior to transplantation is sufficient to mitigate ischaemia-reperfusion damage and fully restore cellular energy levels. Clinical studies have shown beneficial outcomes after transplantation of liver grafts preserved by DHOPE compared with static cold storage. In addition to graft reconditioning, DHOPE may also be used to prolong preservation time, which could facilitate logistics for allocation and transplantation globally. METHODS AND ANALYSIS: This is a prospective, pseudo-randomised, dual-arm, IDEAL-D (Idea, Development, Exploration, Assessment, Long term study-Framework for Devices) stage 2 clinical device trial designed to determine safety and feasibility of prolonged DHOPE (DHOPE-PRO). The end-time of the donor hepatectomy will determine whether the graft will be assigned to the intervention (16:00-3:59 hour) or to the control arm (4:00-15:59 hour). In total, 36 livers will be included in the study. Livers in the intervention group (n=18) will undergo DHOPE-PRO (≥4 hours) until implantation the following morning, whereas livers in the control group (n=18) will undergo regular DHOPE (2 hours) prior to implantation. The primary endpoint of this study is a composite of the occurrence of all (serious) adverse events during DHOPE and up to 30 days after liver transplantation. ETHICS AND DISSEMINATION: The protocol was approved by the Medical Ethical Committee of Groningen, METc2020.126 in June 2020, and the study was registered in the Netherlands National Trial Registry (https://www.trialregister.nl/) prior to initiation. TRIAL REGISTRATION NUMBER: NL8740.

Renal Normothermic Machine Perfusion: The Road Toward Clinical Implementation of a Promising Pretransplant Organ Assessment Tool.

The increased utilization of high-risk renal grafts for transplantation requires optimization of pretransplant organ assessment strategies. Current decision-making methods to accept an organ for transplantation lack overall predictive power and always contain an element of subjectivity. Normothermic machine perfusion (NMP) creates near-physiological conditions, which might facilitate a more objective assessment of organ quality before transplantation. NMP is rapidly gaining popularity, with various transplant centers developing their own NMP protocols and renal viability criteria. However, to date, no validated sets of on-pump viability markers exist nor are there unified NMP protocols. This review provides a critical overview of the fundamentals of current renal NMP protocols and proposes a framework to approach further development of ex vivo organ evaluation. We also comment on the potential logistical implications of routine clinical use of NMP, which is a more complex procedure compared with static cold storage or even hypothermic machine perfusion.

Magnetic resonance imaging assessment of renal flow distribution patterns during ex vivo normothermic machine perfusion in porcine and human kidneys.

Acceptance criteria of deceased donor organs have gradually been extended toward suboptimal quality, posing an urgent need for more objective pre-transplant organ assessment. Ex vivo normothermic machine perfusion (NMP) combined with magnetic resonance imaging (MRI) could assist clinicians in deciding whether a donor kidney is suitable for transplantation. Aim of this study was to characterize the regional distribution of perfusate flow during NMP, to better understand how ex vivo kidney assessment protocols should eventually be designed. Nine porcine and 4 human discarded kidneys underwent 3 h of NMP in an MRI-compatible perfusion setup. Arterial spin labeling scans were performed every 15 min, resulting in perfusion-weighted images that visualize intrarenal flow distribution. At the start of NMP, all kidneys were mainly centrally perfused and it took time for the outer cortex to reach its physiological dominant perfusion state. Calculated corticomedullary ratios based on the perfusion maps reached a physiological range comparable to in vivo observations, but only after 1 to 2 h after the start of NMP. Before that, the functionally important renal cortex appeared severely underperfused. Our findings suggest that early functional NMP quality assessment markers may not reflect actual physiology and should therefore be interpreted with caution.

[Viability testing of discarded donor livers; More livers fit for transplantation because of machine perfusion]. / Testen van de vitaliteit van afgekeurde donorlevers.

In the Netherlands, the average quality of donor organs continues to decrease as a result of the increasing life expectancy, as well as higher incidence of obesity, diabetes mellitus, and other comorbidities in the general population. Storing donor organs on ice, the current standard practice, appears to be inadequate for preserving these high-risk donor livers. Consequently, an increasing number of donor organs is discarded for transplantation. Over the past years, a novel dynamic preservation technique using a machine perfusion device has been developed. Preservation by machine perfusion enables optimisation and viability testing of high-risk donor livers prior to liver transplantation. A large proportion of initially declined donor livers appeared to be suitable for transplantation after evaluation during machine perfusion. Testing of donor livers has led to a significant increase in the number of liver transplantations performed in the Netherlands. This technique has the potential for use in other organs, such as kidney allografts.

MRI for diagnosis of post-renal transplant complications: current state-of-the-art and future perspectives.

Kidney transplantation has developed into a widespread procedure to treat end stage renal failure, with transplantation results improving over the years. Postoperative complications have decreased over the past decades, but are still an important cause of morbidity and mortality. Early accurate diagnosis and treatment is the key to prevent renal allograft impairment or even graft loss. Ideally, a diagnostic tool should be able to detect post-transplant renal dysfunction, differentiate between the different causes and monitor renal function during and after therapeutic interventions. Non-invasive imaging modalities for diagnostic purposes show promising results. Magnetic resonance imaging (MRI) techniques have a number of advantages, such as the lack of ionizing radiation and the possibility to obtain relevant tissue information without contrast, reducing the risk of contrast-induced nephrotoxicity. However, most techniques still lack the specificity to distinguish different types of parenchymal diseases. Despite some promising outcomes, MRI is still barely used in the post-transplantation diagnostic process. The aim of this review is to survey the current literature on the relevance and clinical applicability of diagnostic MRI modalities for the detection of various types of complications after kidney transplantation.