Marginal donor kidneys are more likely to develop ischemia-reperfusion injury (IRI), resulting in inferior long-term outcomes. Perfusion techniques are used to attenuate IRI and improve graft quality. However, machine perfusion is still in its infancy, and more research is required for optimal conditions and potential repairing therapies. Experimental machine perfusion using porcine kidneys is a great way to investigate transplant-related IRI, but these experiments are costly and time-consuming. Therefore, an intermediate model to study IRI would be of great value. We developed a precision-cut kidney slice (PCKS) model that resembles ischemia-reperfusion and provides opportunities for studying multiple interventions simultaneously. Porcine kidneys were procured from a local slaughterhouse, exposed to 30 min of warm ischemia, and cold preserved. Subsequently, PCKS were prepared and incubated under various conditions. Adenosine triphosphate (ATP) levels and histological tissue integrity were assessed for renal viability and injury. Slicing did not influence tissue viability, and PCKS remained viable up to 72 h incubation with significantly increased ATP levels. Hypothermic and normothermic incubation led to significantly higher ATP levels than baseline. William’s medium E supplemented with Ciprofloxacin (and Amphotericin-B) provided the most beneficial condition for incubation of porcine PCKS. The porcine PCKS model can be used for studying transplant IRI.

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 (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 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.

Diminishing ischemia-reperfusion injury (IRI) by improving kidney preservation techniques offers great beneficial value for kidney transplant recipients. Mitochondria play an important role in the pathogenesis of IRI and are therefore interesting targets for pharmacological interventions. Hypothermic machine perfusion (HMP), as a preservation strategy, offers the possibility to provide mitochondrial–targeted therapies. This study focuses on the addition of a mitochondrial protective agent SUL—138 during HMP and assesses its effect on kidney function and injury during normothermic reperfusion. In this case, 30 min of warm ischemia was applied to porcine slaughterhouse kidneys before 24 h of non–oxygenated HMP with or without the addition of SUL—138. Functional assessment was performed by 4 h normothermic autologous blood reperfusion. No differences in renal function or perfusion parameters were found between both groups. ATP levels were lower after 30 min of warm ischemia in the SUL–138 group (n.s, p = 0.067) but restored significantly during 24 h of HMP in combination with SUL—138. Aspartate aminotransferase (ASAT) levels were significantly lower for the SUL—138 group. SUL—138 does not influence renal function in this model. Restoration of ATP levels during 24 h of HMP with the addition of SUL in combination with lower ASAT levels could be an indication of improved mitochondrial function.

Background/Objectives: Kidneys from marginal donors are more susceptible to ischemia–reperfusion injury (IRI). To diminish the deleterious effects of IRI, mitochondria should be preserved optimally between donation and transplantation. It is unknown which macronutrients are imperative to support mitochondrial function during normothermic preservation. The aim of this study is to investigate the effect of different macronutrient compositions on mitochondrial function during prolonged normothermic incubation of precision-cut kidney slices. Methods: Both porcine (n = 8) and human (n = 5) kidneys were studied. After warm ischemia and cold preservation, precision-cut kidney slices (PCKS) were made and incubated in different incubation media under normothermic conditions. PCKS were incubated with all different combinations of glucose, glutamine and/or fatty acids. At zero, 24 and 48 h, mitochondrial function and metabolite levels were assessed. Results: After 48 h of incubation, ATP levels were significantly higher with glucose and glutamine or fatty acids and glutamine, compared to incubation without nutrients (p = 0.001 and p = 0.003, respectively). Also, mitochondrial oxygen fluxes were higher with all nutrients compared to no nutrients (ADP stimulated basal respiration p = 0.040; proton leak p = 0.001 and maximal respiration p = 0.039). Conclusions: The addition of multiple macronutrients during prolonged normothermic incubation increases the mitochondrial function of PCKS. These data pave the way for optimising the nutritional support for ex vivo perfusion of marginal kidneys.

Background: With increasing interest in renal normothermic machine perfusion (NMP), a deeper understanding of ex vivo renal physiology is essential to establish NMP as a robust pre‐transplant assessment platform. This study utilized magnetic resonance imaging (MRI) to compare in vivo renal physiology in healthy volunteers with ex vivo renal function of human donor kidneys during NMP. Methods: Multiparametric MRI maps assessing water diffusion, oxygenation, tissue characteristics, and perfusion were obtained from 11 healthy volunteers to define an in vivo renal reference frame. For ex vivo evaluation, 25 discarded human donor kidneys underwent 4 h of oxygenated hypothermic machine perfusion, followed by 6 h of NMP in an MRI‐compatible setup. The kidneys were assessed hourly using comparable MRI modalities as in vivo. Results: Ex vivo renal physiology was substantially different from in vivo physiology in terms of MRI‐based diffusion patterns, oxygenation, tissue characteristics, and tissue perfusion. Most MRI measurements did not correlate well with conventional parameters such as flow, renal function, and injury markers during NMP. Conclusions: Our findings highlight distinct differences between in vivo and ex vivo MRI‐based renal characteristics in a human cohort, suggesting that parameters beyond conventional in vivo functional markers may warrant consideration when evaluating organ viability during NMP.

Background: Optimizing organ preservation techniques is imperative in the face of donor kidney shortage and high waiting list mortality. Hypothermic machine perfusion (HMP) has emerged as an effective method to improve graft function post-transplantation, particularly for deceased donor kidneys, prone to ischemia reperfusion injury (IRI). The perfusion solution includes glucose to support kidney metabolism; however, its effect on mitochondrial function remains unclear. The present study investigated the effect of glucose supplementation during 24 h of oxygenated HMP on mitochondrial function in porcine kidneys. Methods: After 30 min of warm ischemia, porcine slaughterhouse kidneys were preserved for 24 h using HMP with one of the following three solutions: the standard HMP preservation solution, University of Wisconsin machine perfusion (UW-MP) solution, which contains glucose; the solution used for static cold storage, University of Wisconsin cold storage (UW-CS) solution, which lacks glucose; or the UW-CS supplemented with 10 mmol/L glucose. Tissue and perfusate samples were collected before, during, and after perfusion for further analysis. Results: ATP production, mitochondrial respiration, and oxidative stress markers were not significantly different between groups. Glucose was released into the perfusion solution even from kidneys without exogenous glucose supplementation in the perfusate. Conclusions: These results suggest that kidney mitochondrial respiration does not depend on the presence of glucose in the HMP perfusion solution at the start of perfusion, underscoring the need for further exploration of nutrient supplementation and mitochondrial function in organ preservation strategies.