Metabolic differences between cold stored and machine perfused porcine kidneys: A 1H NMR based study.

Hypothermic machine perfusion (HMP) and static cold storage (SCS) are the two methods used to preserve deceased donor kidneys prior to transplant. This study seeks to characterise the metabolic profile of HMP and SCS porcine kidneys in a cardiac death donor model. Twenty kidneys were cold flushed and stored for two hours following retrieval. Paired kidneys then underwent 24 h of HMP or SCS or served as time zero controls. Metabolite quantification in both storage fluid and kidney tissue was performed using one dimensional 1H NMR spectroscopy. For each metabolite, the net gain for each storage modality was determined by comparing the total amount in each closed system (i.e. total amount in storage fluid and kidney combined) compared with controls. 26 metabolites were included for analysis. Total system metabolite quantities following HMP or SCS were greater for 14 compared with controls (all p < 0.05). In addition to metabolic differences with control kidneys, the net metabolic gain during HMP was greater than SCS for 8 metabolites (all p < 0.05). These included metabolites related to central metabolism (lactate, glutamate, aspartate, fumarate and acetate). The metabolic environments of both perfusion fluid and the kidney tissue are strikingly different between SCS and HMP systems in this animal model. The total amount of central metabolites such as lactate and glutamate observed in the HMP kidney system suggests a greater degree of de novo metabolic activity than in the SCS system. Maintenance of central metabolic pathways may contribute to the clinical benefits of HMP.

(13)C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys.

BACKGROUND: The aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies ((13)C-enriched glucose) to detect ex vivo de novo metabolism in the perfusion fluid and cortical tissue of porcine kidneys during hypothermic machine perfusion (HMP). METHODS: Porcine kidneys (n = 6) were subjected to 24 h of HMP using the Organ Recovery Systems LifePort Kidney perfusion device. Glucose, uniformly enriched with the stable isotope (13)C ([U-(13)C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D (1)H and 2D (1)H,(13)C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing (13)C in both perfusate and tissue samples. RESULTS: There was significant enrichment of (13)C in a number of central metabolites present in both the perfusate and tissue extracts and was most pronounced for lactate and alanine. The total amount of enriched lactate (per sample) in perfusion fluid increased during HMP (31.1 ± 12.2 nmol at 6 h vs 93.4 ± 25.6 nmol at 24 h p < 0.01). The total amount of enriched alanine increased in a similar fashion (1.73 ± 0.89 nmol at 6 h vs 6.80 ± 2.56 nmol at 24 h p < 0.05). In addition, small amounts of enriched acetate and glutamic acid were evident in some samples. CONCLUSIONS: This study conclusively demonstrates that de novo metabolism occurs during HMP and highlights active metabolic pathways in this hypothermic, hypoxic environment. Whilst the majority of the (13)C-enriched glucose is metabolised into glycolytic endpoint metabolites such as lactate, the presence of non-glycolytic pathway derivatives suggests that metabolism during HMP is more complex than previously thought. Isotopic labelled ex vivo organ perfusion studies using 2D NMR are feasible and informative.