(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.

Jay Nath,Sam Ebbs,Daniel A Tennant,Sefa W Canbilen,Andrew R Ready,Christian Ludwig,Tom Smith,Alex Hollis

doi:10.1186/s13737-016-0037-0

Abstract

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.

Highlights

The 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)
Using a one-dimensional (1D) 1H nuclear magnetic resonance (NMR) metabolomic approach, we have previously identified a panel of metabolites within the perfusion fluid during HMP that are predictive of post-transplant graft function [6]
An understanding of the active metabolic pathways during HMP could potentially facilitate the development of a more nutritionally supportive perfusion fluid enabling the metabolic optimisation of these valuable organs prior to transplantation. In this donation after cardiac death (DCD) large animal model of kidney transplant preservation, enrichment of 13C in a number of metabolites was observed in both perfusate solution and kidney cortex tissue

Summary

Introduction

The 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). There is good evidence to support the use of hypothermic machine perfusion (HMP) in clinical practice [1,2,3], there are surprisingly few reports detailing renal metabolism during this process This is likely to reflect the widely held belief that metabolism is deleterious during organ preservation and should be minimised by both hypothermia and keeping cold ischaemia times (CIT) as short as possible. We propose performing isotopic 13C-labelled glucose studies

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