Batch effect exerts a bigger influence on the rat urinary metabolome and gut microbiota than uraemia: a cautionary tale

Randall et al. Microbiome (2019) 7:127 https://doi.org/10.1186/s40168-019-0738-y SHORT REPORT Open Access Batch effect exerts a bigger influence on the rat urinary metabolome and gut microbiota than uraemia: a cautionary tale David William Randall1*, Julius Kieswich1, Jonathan Swann2, Kieran McCafferty1, Christoph Thiemermann1, Michael Curtis3, Lesley Hoyles4 and Muhammed Magdi Yaqoob1 Abstract Background: Rodent models are invaluable for studying biological processes in the context of whole organisms. The reproducibility of such research is based on an assumption of metabolic similarity between experimental animals, controlled for by breeding and housing strategies that minimise genetic and environmental variation. Here, we set out to demonstrate the effect of experimental uraemia on the rat urinary metabolome and gut microbiome but found instead that the effect of vendor shipment batch was larger in both areas than that of uraemia. Results: Twenty four Wistar rats obtained from the same commercial supplier in two separate shipment batches underwent either subtotal nephrectomy or sham procedures. All animals undergoing subtotal nephrectomy developed an expected uraemic phenotype. The urinary metabolome was studied using 1H-NMR spectroscopy and found to vary significantly between animals from different batches, with substantial differences in concentrations of a broad range of substances including lactate, acetate, glucose, amino acids, amines and benzoate derivatives. In animals from one batch, there was a complete absence of the microbiome-associated urinary metabolite hippurate, which was present in significant concentrations in animals from the other batch. These differences were so prominent that we would have drawn quite different conclusions about the effect of uraemia on urinary phenotype depending on which batch of animals we had used. Corresponding differences were seen in the gut microbiota between animals in different batches when assessed by the sequencing of 16S rRNA gene amplicons, with higher alpha diversity and different distributions of Proteobacteria subtaxa and short-chain fatty acid producing bacteria in the second batch compared to the first. Whilst we also demonstrated differences in both the urinary metabolome and gut microbiota associated with uraemia, these effects were smaller in size than those associated with shipment batch. Conclusions: These results challenge the assumption that experimental animals obtained from the same supplier are metabolically comparable, and provide metabolomic evidence that batch-to-batch variations in the microbiome of experimental animals are significant confounders in an experimental study. We discuss strategies for reducing such variability and the need for transparency in research publications about the supply of experimental animals. Keywords: Microbiome, Urinary metabolome, 1H-NMR spectroscopy, Uraemia, Batch effect, Hippurate, Lactate, Acetate * Correspondence: 1 Centre for Translational Medicine and Therapeutics, William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London EC1M 8BQ, UK Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Randall et al. Microbiome (2019) 7:127 Background The lack of reproducibility in pre-clinical animal research remains a major challenge in experimental biology [1] and is at least partially explained by variation between animal microbiomes [2]. Animal research has been based on the assumption that whilst experimental animals in different facilities may have differences at species level between their gut microbiota [3], at a population level, in healthy laboratory animals on identical diets, these diverse collections of microorganisms achieve a shared set of basic metabolic functions—an assumption supported by evidence of significant functional redundancy within gut microbial communities [4]. A number of toxic molecules that accumulate in renal failure have been shown to be produced by bacterial metabolism of dietary protein in the large intestine [5, 6], leading to an interest in the gut microbiome as a potential therapeutic target to reduce the cardiovascular morbidity of patients with chronic kidney disease [7]. Based on an assumption of metabolic similarity between experimental animals, we sought to investigate this ‘gut-kidney axis’ in a rodent model of uraemia, by demonstrating the effect of experimental uraemia on the urinary metabolome and gut microbiota of rats, purchased from the same supplier in two separate shipment batches for logistical reasons. We actually found that the effect of shipment batch had a larger effect in both areas than uraemia and that conclusions drawn about the effect of uraemia on gut-derived metabolites would have been radically different depending on the batch of animals used. Results We obtained 24 wild-type outbred Wistar International Genetic Standard (IGS) rats in two shipment batches, 3 weeks apart, from the same supplier (Charles Rivers, Kent, UK). Fourteen were rendered uraemic by undergoing a two-stage subtotal (five-sixth) nephrectomy (eight from batch 1, six from batch 2), whilst 10 underwent sham procedures (six from batch 1, four from batch 2, Fig. 1a). There were no differences in animal husbandry or diet between batches. At the time of sacrifice 8 weeks later, the urinary metabolome was assessed by untargeted proton nuclear magnetic resonance (1H-NMR) spectroscopy, and composition of the gut microbiota was assessed by sequencing 16S rRNA gene amplicons. All animals undergoing subtotal nephrectomy developed an expected uraemic phenotype, including elevations in serum urea and creatinine, weight loss, and polyuria compared to sham-operated controls, and there were no gross phenotypic differences between animals from different batches (Fig. 1b–f; Additional file 1). Principal component analysis (PCA) of normalised and aligned urinary NMR spectral profiles identified that Page 2 of 10 shipment batch was responsible for the largest source of variance in the biochemical data, seen chiefly in principal component 1, which accounted for 38% of variance. Surgical treatment accounted for a smaller but nonetheless definite source of variance, with these differences being seen chiefly in the second principal component, which accounted for 17.7% of total variance (Fig. 2a). Sep (...truncated)