Abstract
Introduction
Pre-analytical processing significantly affects tissue metabolomes. Since most frozen kidney samples are stored after embedding, standardization of cryoprotective medium removal before metabolomics is essential.
Objectives
We used rodent and human kidney samples to develop an easy and robust pre-analytical procedure compatible with 1H-nuclear magnetic resonance (NMR)-based metabolomics.
Methods
In mice, renal ischemia was induced for 30 min, followed by 48-h reperfusion (I/R, n = 6). Right kidneys were transversally cut in two fragments, and snap-frozen in liquid nitrogen (LN2) or in Optimal Cutting Temperature ® (OCT) fixative. In man, double kidney biopsies were simultaneously obtained before transplantation (n = 15), and snap-frozen in LN2 or OCT.
Results
1H-NMR spectrum of pure OCT highlighted two major peaks, i.e. from 3.4 to 4.2 ppm (47.2%) and from 1.2 to 2.2 ppm (42.5%). 1H-NMR spectra of mouse OCT kidneys were biased at 3.7. By contrast, 1H-NMR analyses of mouse OCT kidneys iteratively rinsed in saline significantly discriminated sham versus I/R groups, with Q² at 0.695 (to be compared with Q² at 0.866 for LN2 sham vs. I/R kidneys). Discriminant metabolites were analogous in both OCT and LN2 kidneys, with a correlation coefficient of 0.83. In man, iteratively rinsing OCT kidneys in saline eliminated the spectral 3.7-peak, thereby making metabolomes of OCT kidneys interpretable and similar to LN2 samples, with a correlation coefficient of 0.73.
Conclusion
NMR metabolomics using OCT-frozen kidney samples is valuable in mouse and man, following standardized OCT removal. This may help use residual biobanked human tissues to better understand renal pathophysiology.
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Acknowledgements
FJ is a Fellow of the Fonds National de la Recherche Scientifique (FNRS), and received support from the University of Liège (Fonds Spéciaux à la Recherche, Fonds Léon Fredericq) and the FNRS (Research Credit #3309). PDT is Senior Research Associate of the Fonds National de la Recherche Scientifique (FNRS).
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The authors have no conflict of interest to disclose.
Ethical approval
Our experimental mouse model was approved by the Institutional Animal Care and Use Committee of the University of Liège in Liège, Belgium (approval number: #1335). Our protocol using human kidney samples was approved by the Ethics Committee of the Cliniques Universitaires Saint-Luc (Université catholique de Louvain) in Brussels, Belgium (approval number; #2016/08MAR/086).
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Leenders, J., Buemi, A., Mourad, M. et al. Nuclear magnetic resonance-based metabolomics of OCT-embedded frozen kidney samples in mouse and man following standardized pre-analytics. Metabolomics 13, 94 (2017). https://doi.org/10.1007/s11306-017-1232-9
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DOI: https://doi.org/10.1007/s11306-017-1232-9