Utilizing pathophysiological concepts of ischemia-reperfusion injury to design renoprotective strategies and therapeutic interventions for normothermic ex vivo kidney perfusion.

Normothermic machine perfusion (NMP) has emerged as a promising tool for the preservation, viability assessment, and repair of deceased-donor kidneys prior to transplantation. These kidneys inevitably experience a period of ischemia during donation, which leads to ischemia-reperfusion injury when NMP is subsequently commenced. Ischemia-reperfusion injury has a major impact on the renal vasculature, metabolism, oxygenation, electrolyte balance, and acid-base homeostasis. With an increased understanding of the underlying pathophysiological mechanisms, renoprotective strategies and therapeutic interventions can be devised to minimize additional injury during normothermic reperfusion, ensure the safe implementation of NMP, and improve kidney quality. This review discusses the pathophysiological alterations in the vasculature, metabolism, oxygenation, electrolyte balance, and acid-base homeostasis of deceased-donor kidneys and delineates renoprotective strategies and therapeutic interventions to mitigate renal injury and improve kidney quality during NMP.

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.

Ex-Vivo Kidney Perfusion With Hemoglobin-Based Oxygen Carriers, Red Blood Cells, or No Oxygen Carrier.

INTRODUCTION: Normothermic machine perfusion (NMP) of donor kidneys provides the opportunity to assess and improve organ viability prior to transplantation. This study explored the necessity of an oxygen carrier during NMP and whether the hemoglobin-based oxygen carrier (HBOC-201) is a suitable alternative to red blood cells (RBCs). METHODS: Porcine kidneys were perfused with a perfusion solution containing either no-oxygen carrier, RBCs, or HBOC-201 for 360 min at 37°C. RESULTS: Renal flow and resistance did not differ significantly between groups. NMP without an oxygen carrier showed lower oxygen consumption with higher lactate and aspartate aminotransferase levels, indicating that the use of an oxygen carrier is necessary for NMP. Cumulative urine production and creatinine clearance in the RBC group were significantly higher than in the HBOC-201 group. Oxygen consumption, injury markers, and histology did not differ significantly between these two groups. However, methemoglobin levels increased to 45% after 360 min in the HBOC-201 group. CONCLUSIONS: We conclude that HBOC-201 could be used as an alternative for RBCs, but accumulating methemoglobin levels during our perfusions indicated that HBOC-201 is probably less suitable for prolonged NMP. Perfusion with RBCs, compared to HBOC-201, resulted in more favorable renal function during NMP.

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.

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.

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.

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

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

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.

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.

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.

Prolonged ex-vivo normothermic kidney perfusion: The impact of perfusate composition.

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-ß-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.

Improved Normothermic Machine Perfusion After Short Oxygenated Hypothermic Machine Perfusion of Ischemically Injured Porcine Kidneys.

BACKGROUND: In an era where global kidney shortage has pushed the field of transplantation towards using more marginal donors, modified kidney preservation techniques are currently being reviewed. Some techniques require further optimization before implementation in full scale transplantation studies. Using a porcine donation after circulatory death kidney model, we investigated whether initial kidney hemodynamics improved during normothermic machine perfusion if this was preceded by a short period of oxygenated hypothermic machine perfusion (oxHMP) rather than static cold storage (SCS). METHODS: Kidneys subjected to 75 minutes of warm ischemia were randomly assigned to either SCS (n = 4) or SCS + oxHMP (n = 4), with a total cold storage time of 240 minutes. Cold preservation was followed by 120 minutes of normothermic machine perfusion with continuous measurement of hemodynamic parameters and renal function. RESULTS: oxHMP preserved kidneys maintained significantly lower renal resistance throughout the normothermic machine perfusion period compared to SCS kidneys (P < 0.001), reaching lowest levels at 60 minutes with means of 0.71 ± 0.35 mm Hg/mL/min/100 g (SCS) and 0.45 ± 0.15 mm Hg/mL/min/100 g (oxHMP). Accordingly, the oxHMP group had a higher mean renal blood flow versus SCS kidneys (P < 0.001). oxHMP kidneys had higher oxygen consumption during normothermic machine perfusion compared to SCS preserved kidneys (P < 0.001). Creatinine clearance remained similar between groups (P = 0.665). CONCLUSIONS: Preceding oxHMP significantly improved initial normothermic machine perfusion hemodynamics and increased total oxygen consumption. With the long period of warm ischemia, immediate kidney function was not observed, reflected by the findings of low creatinine clearance in both groups.

Normothermic machine perfusion of ischaemically damaged porcine kidneys with autologous, allogeneic porcine and human red blood cells.

In porcine kidney auto-transplant models, red blood cells (RBCs) are required for ex-vivo normothermic machine perfusion (NMP). As large quantities of RBCs are needed for NMP, utilising autologous RBCs would imply lethal exsanguination of the pig that is donor and recipient-to-be in the same experiment. The purpose of this study was to determine if an isolated porcine kidney can also be perfused with allogeneic porcine or human RBCs instead. Porcine kidneys, autologous and allogeneic blood were obtained from a local slaughterhouse. Human RBCs (O-pos), were provided by our transfusion laboratory. Warm ischaemia time was standardised at 20 minutes and subsequent hypothermic machine perfusion lasted 1.5-2.5 hours. Next, kidneys underwent NMP at 37°C during 7 hours with Williams' Medium E and washed, leukocyte depleted RBCs of either autologous, allogeneic, or human origin (n = 5 per group). During perfusion all kidneys were functional and produced urine. No macroscopic adverse reactions were observed. Creatinine clearance during NMP was significantly higher in the human RBC group in comparison with the allogeneic group (P = 0.049) but not compared to the autologous group. The concentration of albumin in the urine was significantly higher in the human RBC group (P <0.001) compared to the autologous and allogeneic RBC group. Injury marker aspartate aminotransferase was significantly higher in the human RBC group in comparison with the allogeneic group (P = 0.040) but not in comparison with the autologous group. Renal histology revealed glomerular and tubular damage in all groups. Signs of pathological hyperfiltration and microvascular injury were only observed in the human RBC group. In conclusion, perfusion of porcine kidneys with RBCs of different origin proved technically feasible. However, laboratory analysis and histology revealed more damage in the human RBC group compared to the other two groups. These results indicate that the use of allogeneic RBCs is preferable to human RBCs in a situation where autologous RBCs cannot be used for NMP.

Treating Ischemically Damaged Porcine Kidneys with Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stromal Cells During Ex Vivo Normothermic Machine Perfusion.

Pretransplant normothermic machine perfusion (NMP) of donor kidneys offers the unique opportunity to perform active interventions to an isolated renal graft before transplantation. There is increasing evidence that mesenchymal stromal cells (MSCs) could have a paracrine/endocrine regenerative effect on ischemia-reperfusion injury. The purpose of this study was to determine which cytokines are secreted by MSCs during NMP of a porcine kidney. Viable porcine kidneys and autologous whole blood were obtained from a slaughterhouse. Warm ischemia time was standardized at 20 min and subsequent hypothermic machine perfusion was performed during 2-3 h. Thereafter, kidneys were machine perfused at 37°C during 7 h. After 1 h of NMP, 0, 107 cultured human adipose tissue-derived MSCs, or 107 cultured bone marrow-derived MSCs were added (n = 5 per group). In a fourth experimental group, 7-h NMP was performed with 107 adipose tissue-derived MSCs, without a kidney in the circuit. Kidneys perfused with MSCs showed lower lactate dehydrogenase and neutrophil gelatinase-associated lipocalin levels in comparison with the control group. Also, elevated levels of human hepatocyte growth factor, interleukin (IL)-6, and IL-8 were found in the perfusate of the groups perfused with MSCs compared to the control groups. This study suggests that MSCs, in contact with an injured kidney during NMP, could lead to lower levels of injury markers and induce the release of immunomodulatory cytokines.