Optimizing Prolonged (6 h) Normothermic Machine Perfusion of Donor Kidneys (PROPER Study).

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

Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilisation by improving graft quality and enabling viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, RNA interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion in decreasing the imbalance between donor liver demand and supply by enabling the repair and regeneration of damaged donor livers.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

The shortage of donor kidneys is a worldwide problem. One possibility to increase the donor organ pool is to improve the quality of suboptimal donor kidneys. For this reason, an increasing amount of research is being performed on the subject of machine perfusion, a technique during which an organ is connected to a perfusion circuit. Apart from the fact that this technique can be used to bridge the period between donation and actual transplantation, it also provides the opportunity for active interventions to an isolated organ. The first part of my thesis focuses on a cellular intervention during normothermic (37 degrees) machine perfusion with mesenchymal stromal cells. The aim was to determine if it was technically feasible to administer mesenchymal stromal cells, cells with regenerative capacities, during normothermic machine perfusion and what effect these cells had on the donor kidney. It proved to be feasible to administer these cells in such way that a proportion remained detectable after machine perfusion. In a porcine autotransplantation model, mesenchymal stromal cells did not affect early graft function, but further research is necessary to determine if these cells could have a beneficial effect on longer term graft function of the donor kidney. The second part of my thesis focuses on the use of different perfusion solutions and oxygen carriers during normothermic machine perfusion. From these experiments it can be concluded that the composition of the perfusion solution, as well as the choice for a specific oxygen carrier, has a significant impact on kidney function and injury markers during normothermic machine perfusion.

Introduction:Ageing of the general population has led to an increase in the use of suboptimal kidneys from expanded criteria donation after brain death (ECD-DBD) and donation after circulatory death (DCD) donors. However, these kidneys have inferior graft outcomes and lower rates of immediate function. Normothermic machine perfusion (NMP) may improve outcomes of these suboptimal donor kidneys. Previous non-randomized studies have shown the safety of this technique and suggested its efficacy in improving the proportion of immediate functioning kidneys compared to static cold storage (SCS). However, its additional value to hypothermic machine perfusion (HMP), which has already been proved superior to SCS, has not yet been established.Methods and analysis:This single-center, open-label, randomized controlled trial aims to assess immediate kidney function after 120 minutes additional, end-ischemic NMP compared to HMP alone. Immediate kidney function is defined as no dialysis treatment in the first week after transplant. Eighty recipients on dialysis at the time of transplant who receive an ECD-DBD or DCD kidney graft are eligible for inclusion. In the NMP group, the donor kidney is taken of HMP upon arrival in the recipient hospital and thereafter put on NMP for 120 minutes at 37 degrees Celsius followed by transplantation. In the control group, donor kidneys stay on HMP until transplantation. The primary outcome is immediate kidney function.Ethics and dissemination:The protocol has been approved by the Medical Ethical Committee of Erasmus Medical Center (2020-0366). Results of this study will be submitted to peer-reviewed journals.Registration:registered in clinicaltrials.gov (NCT04882254).Highlights:This is the first RCT to compare additional NMP to HMP alone.Extensive sampling will offer in-depth analysis of kidney physiology during NMP.This RCT may help identify biomarkers to predict clinical outcomes during NMP.Biomarkers can help develop NMP as assessment tool for declined kidneys.

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

The application of normothermic machine perfusion (NMP) machine technology as an assessment device may help to increase the utility of kidneys from uncontrolled donation after circulatory death (uDCD) donors. Here, we describe a case in which NMP perfusion characteristics were misleading and failed to predict primary nonfunction in 2 kidneys from a uDCD. The donor was a 37-y-old man who died of an intracerebral hemorrhage. Before withdrawal of life supporting treatment the donor had a cardiac arrest. He was rapidly transferred to the operating room and the abdominal organs cold perfused in situ with University of Wisconsin solution at 4°C. The first warm ischemic time was 27 min. Both kidneys appeared very poorly perfused and were further flushed on the back table with 4 L of hyperosmolar citrate solution over a period of 90 min. The appearance of both kidneys improved but they still remained patchy. The kidneys underwent a 1-h period of NMP as previously described.1,2 During NMP, the macroscopic appearance, renal blood flow, and urine output of both kidneys met our published criteria for transplantation2 (Table 1). The kidneys were transplanted into 2 separate nonsensitized recipients (Table 1). Immunological cross-matching was negative for both transplants. Both kidneys had a single renal artery and vein and these were anastomosed to the external iliac vessels. There was no intraoperative hypotension. A baseline biopsy taken from the left kidney 30 min after transplantation showed severe ischemic/reperfusion injury with almost all glomerular and peritubular capillaries filled with sludged red cells. Percutaneous needle core biopsies of both kidneys at day 8 showed infarction of the entire specimen. Computerized tomography angiography demonstrated patency of the main renal arteries and veins but diffuse poor parenchymal enhancement. In the absence of any improvement in renal function, primary nonfunction (PNF) was diagnosed in both kidneys at 3 mo. TABLE 1. - Recipient characteristics, preservation times, and normothermic machine perfusion parameters of the left and right kidney Left kidney Right kidney Recipient characteristics Gender/age Male 44 y Male 43 y Cause of ESRF APCKD Membranous GN Dialysis Predialysis Predialysis eGFR (mL/min) 9 10 HLA mismatch 1-0-1 1-0-1 Preservation times CIT (first) min 177 291 NMP min 67 60 CIT (second) min 158 433 Anastomosis min 47 55 Total CIT min 335 724 NMP parameters Macroscopic appearance 2 1 Mean RBF (mL/min/100g) 65.6 112.8 Total urine output (mL) 45 65 Oxygen consumption (mL/min/g) 36.6 48.0 Pre-NMP Post-NMP Pre-NMP Post-NMP pH 7.42 7.43 7.51 7.53 Potassium (mmol/L) 10.8 15.4 8.4 13.2 Lactate (mmol/L) 10.4 8.2 7.3 7.5 Glucose (mmol/L) 10.6 7.2 10.4 5.2 Recipient characteristics; gender/age, cause of ESRF, dialysis, eGFR, and HLA mismatch. Preservation times; CIT, NMP, second CIT, and anastomosis time. NMP parameters; macroscopic appearance (1, excellent; 2, patchy; 3, poor), mean RBF, total urine output, oxygen consumption after 60 min, prearterial and postarterial NMP levels of pH, potassium, lactate, and glucose. APCKD and membranous GN.APCKD, autosomal dominant polycystic kidney disease; CIT, cold ischemic time; eGFR, estimated glomeruli filtration rate; ESRF, end-stage renal failure; membranous GN, membranous glomerulonephritis; NMP, normothermic machine perfusion; RBF, renal blood flow. Extensive red cell plugging at revascularization led to cortical necrosis and allograft quality assessment by NMP failed to predict PNF. The young age of the donor and the short ischemic times were favorable factors. However, inadequate clearance of the microcirculation after in situ and back-table flushing is a well-described complication of prolonged warm-ischemic injury in uDCD organs.3,4 NMP theoretically offers a more accurate pretransplant viability assessment, because it restores cellular metabolism.2 We have used NMP to successfully assess and salvage kidneys from controlled donation after circulatory death donors because of inadequate clearance of the microcirculation after in situ cooling.2 This is the first report of applying the same principle to uDCD kidneys, and the adverse outcome is admonitory. The failure of NMP to predict PNF in these cases may be related to the duration and nature of the perfusion protocol. A 1-h period of NMP has been the standard in our practice and its effect on delayed graft function is currently being trialed in donation after circulatory death kidneys.5 However, cortical necrosis takes some hours to evolve and a more extended period of NMP might have been more predictive of the outcome. Furthermore, NMP conditions are designed to be protective and could mask the actual level of ischemic injury within a short timeframe. In conclusion, NMP strategies need to be further developed to reliably salvage and assess kidneys with severe ischemic injury complicated by poor clearance of the microvasculature.

Static cold storage (SCS) has been the gold standard of organ preservation for decades. Although metabolism is significantly decreased during SCS, there is still anaerobic activity in the organ, leading to an array of adverse intracellular changes.1 Considerable drawbacks of SCS are the limited storage time and challenges in assessing quality and viability of the organ. Ex situ normothermic machine perfusion (NMP) represents a platform that does enable graft assessment before transplantation. During NMP, the organ is kept at body temperature while provided with oxygen and nutrients, creating an optimal environment in which the organ can maintain aerobic metabolic activity. NMP thus provides the opportunity to perform functional assessments aiding in the decision of whether it is safe to transplant the liver. This approach could help to increase organ utilization, especially when using extended criteria donor (ECD) organs. A recent European randomized trial in liver transplantation performed by Nasralla et al2 demonstrated safety and potential benefits of NMP over SCS, providing evidence of ameliorated consequences of ischemia-reperfusion injury. Median duration of NMP in this trial was 9 h (with a maximum of 23 h), using a perfusion device certified for ex situ liver graft preservation up to 24 h. Extending the time of ex situ graft preservation beyond 24 h may, at least in theory, create opportunities for liver repair and regeneration. Prolonged NMP (beyond 24 h), however, requires a more sophisticated machine than the ones that are currently commercially available and should, for example, include a dialysis unit for the removal of waste products.3 Recently, 2 groups have described such an advanced perfusion device that can be used for prolonged NMP for up to a week or even longer, showing preserved viability. However, liver transplants have not been performed.4,5 The Zürich group reported in a recent issue of Nature Biotechnology on the first clinical transplantation of a donor liver after 3 d of ex situ NMP.6 The liver was derived from a 29-y-old patient, who underwent multivisceral transplantation because of an invasive abdominal desmoid fibromatosis associated with chronic intraabdominal abscesses and sepsis (University of Zurich, personal communication). The patient’s liver was offered for domino transplantation but subsequently rejected by all Swiss centers because of an unclassified tumor in segment 1, requiring further pathological analysis. Sepsis originating from chronic intra-abdominal abscesses represented an additional reason not to accept the liver. After hepatectomy, the liver first underwent hypothermic (6–8 °C) oxygenated perfusion for 2 h and 40 min, during which the liver was prepared for NMP. The portal vein (PV), hepatic artery (HA), vena cava inferior, and common bile duct were cannulated, enabling perfusion in a closed system. NMP was performed using the Wyss perfusion machine, a sophisticated perfusion device that was extensively described previously.5 This machine provides oxygenated blood through the HA and PV using physiological pressures. HA hemodynamics were controlled by automated administration of vasodilators and vasoconstrictors. The liver was placed on a silicon mat, mimicking diaphragm movement to prevent pressure injuries. An integrated dialysis unit maintained a physiological electrolyte balance and facilitated the removal of waste products, including urea. The machine was primed with a leukocyte-free red blood cell–based perfusion fluid. Flow rates in the HA were 0.2 to 0.5 L/min, with a mean arterial pressure of 65 mm Hg. PV flows ranged from 0.8 to 0.9 L/min with a pressure between 10 and 12 mm Hg. Timing of transplantation allowed for histopathological diagnostic workup, performed by day 4, after passing a viability assessment that included physiological response to the vasoactive drugs, quality and quantity of bile production, low markers of injury, and inflammation in the perfusate, in addition to acceptable histology. The histological results of the tumor in segment 1 revealed a benign perivascular epithelioma, and broad-spectrum antimicrobial agents were continuously given to treat potential bacterial contamination. During perfusion, lactate levels in the perfusate dropped quickly; there was continuous bile production, and perfusate aspartate transaminase and alanine transaminase concentrations stayed relatively low, as did interleukin 6, indicating indeed an absence of pressure necrosis of the liver and an absent inflammation. After 68 h of NMP, the liver graft was successfully transplanted without signs of postreperfusion syndrome. Peak serum aspartate transaminase and alanine transaminase concentrations in the recipient remained low at 309 and 138 U/L, respectively. The international normalized ratio returned to normal levels by day 4 after transplantation. During NMP, there was very low to no cellular proliferative activity, as shown by the low expression of MIB1/Ki67 and PH3 immunostaining in a liver biopsy. Interestingly, by day 10 after transplantation, a very strong proliferative activity was observed in a protocol biopsy. This observation raises the intriguing question of whether repair on the pump was not necessary or not possible. Another interesting observation was a weight loss of 25% at the end of the NMP, with an equal reduction in hepatocyte volume. It should be noted that this liver graft was derived from a living donor and thus not intrinsically injured by the consequences of brain death or ischemia-reperfusion injury, nor did it need to be repaired before the domino transplantation. This certainly raises some questions on how results reflect repair of a deceased donor liver. Others may argue that prolonged hypothermic oxygenated machine perfusion may also provide safe extension of ex situ graft preservation time7 (Figure 1). From a hepatobiliary injury point of view, many will argue that this domino liver could have been transplanted without NMP. Moreover, the histology of the tumor in segment 1 could also have been obtained before the transplant/donation procedure. Nevertheless, most of us would not have considered this liver primarily for transplantation, and therefore, this graft seemed suited to show the feasibility of prolonged perfusion. The question of whether graft repair and regeneration of a preinjured ECD liver are possible during ex situ machine perfusion, however, remains to be answered.FIGURE 1.: An overview of the different liver preservation methods and the current reported maximum preservation duration. Traditional SCS is safe for up to 12 h. Hypothermic oxygenated perfusion (dual or single; [D]HOPE) is typically performed after SCS for 2 h.8 Prolonged preservation with DHOPE has been reported for up to 20 h.9 “Regular” NMP is possible for up to 24 h.2 Prolonged NMP enables graft preservation for up to 1 wk and maybe even longer.4-6 (D)HOPE, (dual) hypothermic oxygenated machine perfusion; IRI, ischemia-reperfusion injury; NMP, normothermic machine perfusion; SCS, static cold storage.In summary, Clavien et al are the first to show that transplantation of a human liver after 68 h of ex situ NMP can be safe and successful, stimulating further research into long-term liver graft preservation. Prolonged NMP allows us to take the next step in organ preservation and assessment. Moving forward, the ultimate goal will be to enable ex situ recovery and regeneration of preinjured ECD liver grafts to expand the donor pool and decrease waitlist mortality.

BackgroundThe 5-year graft survival rate of donor kidneys transplanted in the Eurotransplant Senior Program (ESP) is only 47 per cent. Normothermic machine perfusion (NMP) may be a new preservation technique that improves graft outcome. This pilot study aimed to assess safety and feasibility of this technique within the ESP.MethodsRecipients were eligible for inclusion if they received a donor kidney within the ESP. Donor kidneys underwent 2 h of oxygenated NMP with a red cell-based solution at 37°C, additional to standard-of-care preservation (non-oxygenated hypothermic machine perfusion). The primary outcome was the safety and feasibility of NMP. As a secondary outcome, graft outcome was investigated and compared with that in a historical group of patients in the ESP and the contralateral kidneys.ResultsEleven patients were included in the NMP group; the function of eight kidneys could be compared with that of the contralateral kidney. Fifty-three patients in the ESP, transplanted consecutively between 2016 and 2018, were included as controls. No adverse events were noted, especially no arterial thrombosis or primary non-function of the transplants. After 120 min of oxygenated NMP, median flow increased from 117 (i.q.r. 80–126) to 215 (170–276) ml/min (P = 0.001). The incidence of immediate function was 64 per cent in the NMP group and 40 per cent in historical controls (P = 0.144). A significant difference in graft outcome was not observed.DiscussionThis pilot study showed NMP to be safe and feasible in kidneys transplanted in the ESP. A well powered study is warranted to confirm these results and investigate the potential advantages of NMP on graft outcome.

Ann Ogbemudia, Julien Branchereau (Joint first authors), Gabriella Hakim, Fungai Dengu, FaysalEl-Gilani, John Mulvey, Kaithlyn Rozenberg, Thomas Prudhomme, Letizia Lo Faro, James Hunter,Paul Johnson, Rutger Ploeg and Peter Friend&#x0D; &#x0D; Objective Static cold storage (SCS) is the standard method for pancreas preservation but does not facilitate objective organ assessment prior to transplantation. Normothermic machine perfusion (NMP) has been used to test other abdominal and thoracic organs’ function and viability in transplantation settings. Our aim was to develop a NMP protocol specific for pancreases and then investigate its potential as an organ assessment strategy.&#x0D; Method 8 porcine pancreases were procured in conditions replicating donation after circulatory death with warm ischaemia time of 25 minutes. After 3 hours of static cold storage (SCS) the pancreases were divided into 3 experimental groups 1) the feasibility group (n=2) that underwent 2.5 hours of NMP 2) the SCS group (n = 2) that underwent an additional 6 hours of SCS prior to assessment on NMP for an hour and 3) the Oxygenated Hypothermic Machine Perfusion (oxyHMP) group (n = 4) that underwent 6 hours of oxyHMP followed by 1-hour assessment on NMP. The NMP protocol used autologous, leucodepleted blood delivered at a mean arterial pressure of 40mmHg with a temperature of 37oC. At timed intervals during NMP, perfusate samples were collected for gas analysis and perfusion parameters were recorded.&#x0D; Results The feasibility group was used to develop the NMP protocol and demonstrated stable perfusion parameters throughout NMP. Compared to the SCS group the oxyHMP group demonstrated better average perfusion characteristics with lower resistances, higher flow rates, lower mean lactate levels and physiological pH. The oxyHMP group maintained normal macroscopic appearances during NMP. At the end of NMP the SCS group had an average 32% weight increase compared to the oxyHMP group that were found to have a 17% weight reduction.&#x0D; Conclusion Normothermic machine perfusion of whole pancreases is feasible after cold preservation and potentially useful as an assessment strategy. Furthermore, it demonstrated that oxygenated HMP may be beneficial for pancreas preservation compared to SCS.

Background/Objectives: APOL1 renal-risk variants (RRVs) are of increasing relevance to kidney disease and transplant outcomes. It is currently understood that the presence of RRVs in donors negatively impacts kidney allograft survival in an autosomal recessive pattern of inheritance. Less well known is the interplay between ischemia and alternative allograft preservation methods, such as normothermic machine perfusion (NMP), on APOL1 gene expression. To investigate this, we examined the effects of APOL1 RRVs on APOL1 gene expression in ischemic donor kidneys and compared the differences in cytokine and APOL1 expression patterns between the alternative preservation methods, static cold storage (CS) and NMP. Methods: Non-utilized deceased donor kidney pairs from donors of African ancestry were procured from Mid-America Transplant after being deemed unsuitable for kidney transplant. Samples were collected from each donor kidney pair and DNA was extracted for APOL1 genotyping. APOL1 RRVs G1 (rs73885319) (rs60910145) and G2 (rs71785313) were identified by Sanger sequencing. From each pair, one kidney underwent 6 h NMP (n = 3) and the contralateral kidney 6 h of CS (n = 3) following the initial CS. Renal perfusion and biochemical, and histologic parameters were recorded. NMP was directly compared with CS using paired donor kidneys using NMP with allogeneic red blood cells, followed by assessment of perfusion, biochemical, and histologic parameters, in addition to gene expression. Results: Donor genotyping identified kidney pairs as heterozygous for the G1 RRV (G1/G0), homozygous for the G0 allele (G0/G0), and homozygous for the G2 RRV (G2/G2), respectively. All kidneys were successfully reperfused, with mRNA transcript levels of APOL1-related genes subsequently measured. Significant differences in APOL1 gene expression were observed among all three groups of kidneys. In paired kidneys from baseline to hour 6 of NMP, mRNA expression varied significantly between G1/G0 and G2/G2 homozygous pairs (p = 0.002) as well as between the G0/G0 and G2/G2 pairs (p = 0.002). APOL1 expression shifted by a significantly higher-fold change of 2.4 under NMP conditions in the G2/G2 genotype (p < 0.001). The inflammatory cytokine marker IFN-γ was also significantly upregulated in the G2/G2 genotype kidney, in both CS and NMP groups (p = 0.001). Other related genes such as KIM-1 were upregulated by a change of 3.9-fold in the NMP group for the G2/G2 kidney. Conclusion: Donor kidney pairs with the high-risk APOL1 genotype, especially G2/G2, show increased APOL1 expression and inflammation, particularly under NMP conditions. NMP enables detection of genotype-specific molecular changes in an ischemic reperfusion injury model, supporting its potential to improve donor kidney assessment before transplantation.

Intestinal transplantation (IT) is the only treatment for patients with intestinal failure who cannot tolerate parenteral nutrition. However, IT often faces high rejection rates, partly due to damage from the currently practiced preservation method, static cold storage (CS). Furthermore, CS is limited to approximately 8 hour duration to enhance chances of success. Normothermic machine perfusion (NMP) preserves organs under physiological conditions, has improved outcomes in other transplants, and may benefit IT. This study aimed to evaluate the effects of NMP versus CS on reducing epithelial injury in porcine IT and to test the possibility of prolonging storage times via NMP. We hypothesized that NMP limits tissue damage, enhances epithelial recovery, and promotes graft stability for prolonged periods compared to CS. Small intestines from 18 Yorkshire-cross pigs were preserved using either CS (n=9) or NMP (n=9) and transplanted. In initial experiments, jejunal full-thickness biopsies were collected at intestinal procurement (T0), post-storage (T6), after 1-hour post-transplant reperfusion (T1RP), and 48 hours after transplantation (T48). In follow-up experimentation, the intestine was continuously perfused for increasing durations up to 12 hours. Histological injury was assessed using the Chiu-Park grade and, for those transplanted, a graft rejection scoring system including subepithelial bleb or Gruënhagen’s space formation, epithelial surface erosion/denuded villi, and columnar attenuation. Shotgun proteomics were employed to evaluate wound healing protein expression following NMP. NMP significantly reduced subepithelial bleb grade (NMP: 1.0 ± 0.7 vs. CS: 2.2 ± 0.4, p = 0.0125) at T1RP. Moreover, at T6, jejunal columnar attenuation grade was elevated (NMP: 1.4 +/- 1.342 vs. CS: 0 +/- 0, p = 0.0479), suggesting increased epithelial flattening and barrier restitution with NMP compared to CS. Compared to control tissue, CS incurred significantly higher Chiu/Park score, subepithelial bleb grade, and denuded villi grade at T1RP. In contrast, NMP preserved epithelial integrity up to 12 hours of storage (Chiu/Park 1.2 +/- 0.8367). Proteomic analyses revealed a 7-log2 fold increase in wound healing proteins such as Filamin A and Filamin binding LIM protein 1 in NMP-stored grafts (p &lt; 0.05). NMP reduces epithelial injury and promotes recovery, in part through increased expression of filamin proteins, even following prolonged durations of storage. Increased durations of storage would significantly expand the pool of intestines available to patients in need. Overall, these findings demonstrate the potential of this novel allograft storage method to improve transplantation outcomes and thus the quality of life for many patients. U.S. Department of Defense PR181265; NIH K01OD010199 SERCA, NIH 5T32OD011130-15; 1R01Al182590-01 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Normothermic machine perfusion (NMP) of donor kidneys provides the opportunity for improved graft preservation and objective pre-transplant ex-vivo organ assessment. Currently, a multitude of perfusion solutions exist for renal NMP. This study aimed to evaluate four different perfusion solutions side-by-side and determine the influence of different perfusate compositions on measured renal perfusion parameters. Porcine kidneys and blood were obtained from a slaughterhouse. Kidneys underwent NMP at 37°C for 7 hours, with 4 different perfusion solutions (n = 5 per group). Group 1 consisted of red blood cells (RBCs) and a perfusion solution based on Williams’ Medium E. Group 2 consisted of RBCs, albumin and a balanced electrolyte composition. Group 3 contained RBCs and a medium based on a British clinical NMP solution. Group 4 contained RBCs and a medium used in 24-hour perfusion experiments. NMP flow patterns for solutions 1 and 2 were similar, solutions 3 and 4 showed lower but more stable flow rates. Thiobarbituric acid reactive substances were significantly higher in solution 1 and 4 compared to the other groups. Levels of injury marker N-acetyl-β-D glucosaminidase were significantly lower in solution 2 in comparison with solution 3 and 4. This study illustrates that the perfusate composition during NMP significantly impacts the measured perfusion and injury parameters and thus affects the interpretation of potential viability markers. Further research is required to investigate the individual influences of principal perfusate components to determine the most optimal conditions during NMP and eventually develop universal organ assessment criteria.

Fifteen donor kidneys were subjected to HMP followed by 2 h of NMP before transplantation. NMP perfusate was collected at 3 time points (0, 1, 2 h) for the measurements of prorenin/renin, erythropoietin (EPO), and vitamin D, and urine samples were collected at 1 h and 2 h for urodilatin measurement. Fifteen HMP perfusate samples were collected for the same measurements. Kidneys on NMP secreted significantly more prorenin, renin, EPO, and active vitamin D than during HMP. EPO and vitamin D secretion remained stable during 2 h of NMP, whereas the prorenin release rate increased and renin release rate decreased after 1 h. Donation after brain death kidneys secreted more vitamin D and less EPO during NMP than donation after circulatory death kidneys. Twelve donor kidneys produced urine during NMP and released detectable levels of urodilatin. Kidneys exhibited a large variation in hormone release rates. No significant differences were found in hormone release capacity between delayed graft function (DGF) and non-DGF kidneys, and no significant correlations were found between hormone release rates and the duration of DGF or 1-mo posttransplant serum creatinine levels. Human transplant kidneys display endocrine activity during NMP. To explore whether correlations exist between hormone release rates and posttransplant kidney function, large numbers of kidneys are required.

Background/Objectives: The rising use of liver grafts from donation after circulatory death (DCD) has been enabled by advances in normothermic regional perfusion (NRP) and machine perfusion (MP) technologies. We aimed to identify predictive biomarkers in DCD grafts subjected to NRP, followed by randomization to either normothermic machine perfusion (NMP) or dual hypothermic oxygenated perfusion (D-HOPE). Methods: Among 57 DCD donors, 32 liver grafts were transplanted, and recipients were monitored for one week post-transplant. Biomarkers linked with oxidative stress, hepatic injury, mitochondrial dysfunction, inflammation, regeneration, and autophagy were measured during NRP, end-ischemic MP, and one week post-transplant. Results: Arginase-1 (ARG-1) levels were consistently higher in discarded grafts and in recipients who later developed early allograft dysfunction (EAD). Specifically, ARG-1 levels at the end of MP correlated with markers of hepatic injury. Receiver operating characteristic analysis indicated that ARG-1 at the end of MP had a good predictive accuracy for EAD (AUC = 0.713; p = 0.02). Lipid peroxidation (TBARS) elevated at the start of NRP, declined over time, with higher levels in D-HOPE than in NMP, suggesting a more oxidative environment in D-HOPE. Metabolites like flavin mononucleotide (FMN) and NADH exhibited significant disparities between perfusion types, due to differences in perfusate compositions. Inflammatory biomarkers rose during NRP and NMP but normalized post-transplantation. Regenerative markers, including osteopontin and hepatocyte growth factor, increased during NRP and NMP and normalized post-transplant. Conclusions: ARG-1 demonstrates strong potential as an early biomarker for assessing liver graft viability during perfusion, supporting timely and effective decision-making in transplantation.