Analysis of Perfusates During Hypothermic Machine Perfusion by NMR Spectroscopy

Bon, D.; Billault, C.; Thuillier, Raphaël; Hébrard, William; Boildieu, N.; Celhay, O.; Irani, Jacques; François, Sabine; Hauet, Thierry

doi:10.1097/tp.0000000000000046

Cited by 32 publications

(38 citation statements)

References 36 publications

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“…The experimental conditions in these experiments were standardised as much as possible to reduce inter-experimental variability and designed to replicate clinical conditions. Whilst previous studies have repeatedly demonstrated the viability of porcine kidneys after similar periods of HMP [ 23 , 33 ], this study does not seek to corroborate this and as such we are unable to correlate de novo metabolism with functional outcome. Given the findings from earlier studies [ 6 ], we hypothesise that glycolytic activity during perfusion may correlate with post-transplant function and propose a human observational study to clarify this, which is possible due to the non-radioactive nature of the 13 C isotope used in this study.…”

Section: Discussionmentioning

confidence: 71%

“…This paper provides further evidence that a significant amount of metabolism occurs during HMP and that NMR methods are useful for identifying these [ 6 , 7 , 23 ]. However, unlike 1 H NMR reports, this paper provides unequivocal evidence that de novo metabolism occurs in these organs during HMP with the glucose present in the perfusate used as a substrate.…”

Section: Discussionmentioning

confidence: 96%

“…Furthermore, our 2D database of splitting patterns and chemical shift parameters for different metabolites is evolving and other labelled metabolites may be identified once these have been further clarified. Whilst we feel the 2D NMR methods in this paper relay unparalleled amounts of metabolic information, they are both time consuming and resource intensive and would not be suitable for real-time analysis to inform clinical decisions, as proposed for 1D 1 H NMR studies [ 23 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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BackgroundThe aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies (13C-enriched glucose) to detect ex vivo de novo metabolism in the perfusion fluid and cortical tissue of porcine kidneys during hypothermic machine perfusion (HMP).MethodsPorcine 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 13C ([U-13C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D 1H and 2D 1H,13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing 13C in both perfusate and tissue samples.ResultsThere was significant enrichment of 13C 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.ConclusionsThis 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 13C-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.

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Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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“…Within transplantation, porcine models have been studied extensively and normothermic perfusion is a good example of how this has translated into clinical practice [20] . On a functional level, analysis using 1 H-NMR spectroscopy of perfusate from autotransplanted pig kidneys has demonstrated that metabolite concentrations do correspond to graft outcome indicating that there is a strong correlation between pre-transplant metabolism and graft function [21] .…”

Section: Introductionmentioning

confidence: 99%

Metabolomic Perfusate Analysis during Kidney Machine Perfusion: The Pig Provides an Appropriate Model for Human Studies

Nath¹,

Guy

Smith

et al. 2014

PLoS ONE

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IntroductionHypothermic machine perfusion offers great promise in kidney transplantation and experimental studies are needed to establish the optimal conditions for this to occur. Pig kidneys are considered to be a good model for this purpose and share many properties with human organs. However it is not established whether the metabolism of pig kidneys in such hypothermic hypoxic conditions is comparable to human organs.MethodsStandard criteria human (n = 12) and porcine (n = 10) kidneys underwent HMP using the LifePort Kidney Transporter 1.0 (Organ Recovery Systems) using KPS-1 solution. Perfusate was sampled at 45 minutes and 4 hours of perfusion and metabolomic analysis performed using 1-D 1H-NMR spectroscopy.ResultsThere was no inter-species difference in the number of metabolites identified. Of the 30 metabolites analysed, 16 (53.3%) were present in comparable concentrations in the pig and human kidney perfusates. The rate of change of concentration for 3-Hydroxybutyrate was greater for human kidneys (p<0.001). For the other 29 metabolites (96.7%), there was no difference in the rate of change of concentration between pig and human samples.ConclusionsWhilst there are some differences between pig and human kidneys during HMP they appear to be metabolically similar and the pig seems to be a valid model for human studies.

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“…36 In analysis of perfusate biomarkers during the randomized Eurotransplant trial, Moers et al 37 determined that GST, N-acetyl-beta-D-glucosaminidase, and heart-type fatty acid binding protein were all significantly associated with DGF, but not with primary nonfunction or graft survival. In a preclinical model, Bon et al 38 performed metabolomics analysis using nuclear magnetic resonance spectroscopy to determine that changes in the concentration of lactate, choline, and several amino acids exhibited a greater increase in kidneys with poorer outcomes than in kidneys that fared better. Thus it appears that perfusate analysis has the potential to predict DGF that may lead to alterations in posttransplant management; however, perfusate analysis has not yet been shown appropriate for making renal discard decisions.…”

Section: Prediction Of Graft Quality With Mpmentioning

confidence: 99%

Machine perfusion for improving outcomes following renal transplant: current perspectives

Franklin¹,

Cannon²

2016

TRRM

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There is a disparity between the number of kidneys available for transplantation and the number of patients awaiting an organ while on dialysis. The current kidney waiting list in the US contains more than 100,000 patients. This need has led to the inclusion of older donors with worsening renal function, as well as greater utilization of kidneys from non-heartbeating (donation after cardiac death) donors. Coinciding with this trend has been a growing interest in technology to improve the function of these more marginal organs, the most important of which currently is machine perfusion (MP) of donated kidneys after procurement. While this technology has no standard guidelines currently for comprehensive use, there are many studies that demonstrate higher organ yield and function after a period of MP. Particularly with the older donor and during donation after cardiac death cases, MP may offer some significant benefits. This manuscript reviews all of the current literature regarding MP and its role in renal transplantation. We will discuss both the experience in Europe and the US using machine perfusion for donated kidneys.

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Analysis of Perfusates During Hypothermic Machine Perfusion by NMR Spectroscopy

Abstract: Multivariate analysis of lesion biomarkers during kidney perfusion using NMR data could be an interesting tool to assess graft quality, particularly because analyses times (2 hr total) are compatible with clinical application.

Cited by 32 publications

References 36 publications

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

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

Metabolomic Perfusate Analysis during Kidney Machine Perfusion: The Pig Provides an Appropriate Model for Human Studies

Machine perfusion for improving outcomes following renal transplant: current perspectives

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