Development of urinary exosomes as biomarkers of drug-induced kidney injury

Aims

This project aims to develop the use of urinary exosomes as a non-invasive approach to assessing renal toxicity.

Background

Renal toxicity is estimated to account for around 2% of drug attrition in preclinical development and 19% in phase III clinical trials. Improving the identification of kidney toxicity and to ensuring better translation of biomarkers has the potential to reduce and refine the use of animals.

Damage to the kidneys as a result of drug toxicity is assessed as part of preclinical studies in animals. They tend not to be standalone studies unless a specific toxicity has been identified which requires further investigation. Assessment typically focuses on circulating biomarkers of kidney damage such as serum creatinine levels and post-mortem tissue analysis, however, these provide little information on the cellular site of injury or the underlying pathophysiology.

The use of urinary exosomes provides an opportunity to non-invasively investigate renal toxicity. This has a number of 3Rs advantages; early signs of renal toxicity should allow decisions to be made to terminate studies before animals suffer, and longitudinal studies in the same animal should be possible allowing animals to be used as their own control. Importantly, improved biomarkers which translate to the clinic should allow drugs to be dropped earlier in the pipeline before the extensive animal testing required to support late stage clinical trials.

Research details and methods

Exosomes are nanoparticle-sized vesicles which transport a range of molecules including microRNAs. They are derived from derived from a range of cells and released into blood and urine. This research aims to investigate whether rat urinary exosomes can be used as a non-invasive marker of renal toxicity.

Urinary exosomes have been shown to capsulate microRNA originating from the glomerulus and nephron. Recent evidence suggests that microRNAs are mediators of drug toxicity and therefore may provide potential biomarkers of cell injury. The research will explore the utility and reproducibility of urinary exosomes for analysis of kidney toxicity. It will employ nanoparticle tracking analysis technology and antibody labelling to identify exosome concentration and cellular origin. 

Related Content:

The kidney is often affected by drug toxicity, this being a frequent reason for promising drugs to fail to reach humans. Rodent models are an essential part of preclinical testing for kidney toxicity. Current circulating biomarkers such as serum creatinine are slow to report injury and do not provide information regarding the cellular site of injury or the underlying pathophysiological process. Therefore, post-mortem analysis of kidney tissue is essential which prevents repeated measurements on the same animal. This cross-sectional design is sub-optimal: important information concerning the temporal progression of disease can be lost and the total number of animals required for any given study is high. Furthermore, tissue biomarkers have limited translational utility since renal biopsies usually cannot be performed in phase I clinical trials. Our objective is to refine the approach for drug toxicity studies by developing urinary exosomes (a specific type of cell-derived nanoparticle) as a reservoir for non-invasive sampling of kidney tubular cell microRNA (miRNA). This approach is designed for longitudinal studies: the ability to make repeated measurements as injury progresses promises to reduce the total number of animals required yet provide more informative and translatable data. Exosomes are vesicles formed within cells that are released into biological fluids such as urine. Urinary exosomes encapsulate miRNA originating from cells of the kidney's glomerulus and each region of the nephron. MiRNA are promising new biomarkers of cell injury because organ-specific miRNA species are released from cells following injury and they are stable in a range of biofluids including urine. The first aim of this studentship is to characterize the rat urinary exosome population and determine the effect of nephrotoxic AKI on exosome excretion. We will use a new technology, nanoparticle tracking analysis that can directly measure urinary exosome concentration and, in combination with antibody labelling, identify the exosomal cell of origin. Our hypothesis is that exosomes will be released by injured tubular cells and create a toxicity signal in the urine. Our second aim is to determine if changes in kidney tissue miRNA are faithfully reflected in exosomes and, therefore, whether exosomes could replace the need for post-mortem tissue and allow non-invasive, repeat miRNA sampling. Our third aim is to explore the biological function of exosomes as inter-cellular signals in a kidney cell model. Exosomes shuttle protein, mRNA and miRNA  between cells. This can change the proteome, and therefore function, of the recipient cell either directly by protein transfer, or indirectly via translation of exosomal mRNA or miRNA interference of target proteins. With exosomes released into the urine along the entire renal tubule, the capacity to traffic downstream protein or RNA species and thereby influence cell physiology is a novel mechanism for signalling within the kidney.

McCrae JC, Sharkey N, Webb DJ, Vliegenthart AD, Dear JW (2016).  Ethanol consumption produces a small increase in circulating miR-122 in healthy individuals.  Clinical Toxicology 54(1):53-5. doi: 10.3109/15563650.2015.1112015

Vliegenthart AD, Shaffer JM, Clarke JI, Peeters LE, Caporali A, Bateman DN, Wood DM, Dargan PI, Craig DG, Moore JK, Thompson AI, Henderson NC, Webb DJ, Sharkey J, Antoine DJ, Park BK, Bailey MA, Lader E, Simpson KJ, Dear JW (2015). Comprehensive microRNA profiling in acetaminophen toxicity identifies novel circulating biomarkers for human liver and kidney injury. Scientific Reports 5:15501. doi: 10.1038/srep15501

Liga A, Vliegenthart A D B, Oosthuyzen W, Dear J W, Kersaudy-Kerhoas M (2015).  Exosome isolation: a microfluidic road-map. Lab on a Chip 15(11):2388-94. doi: 10.1039/c5lc00240k

Vliegenthart AD, Antoine DJ, Dear JW (2015).  Target biomarker profile for the clinical management of paracetamol overdose.  British Journal of Clinical Pharmacology 80(3):351-362. doi: 10.1111/bcp.12699

Vliegenthart AD, Starkey Lewis P, Tucker CS, Del Pozo J, Rider S, Antoine DJ, Dubost V, Westphal M, Moulin P, Bailey MA, Moggs JG, Goldring CE, Park BK, Dear JW (2014).  Retro-orbital blood acquisition facilitates circulating microRNA measurement in zebrafish with paracetamol hepatotoxicity.  Zebrafish 11(3):219-26. doi: 10.1089/zeb.2013.0912

Vliegenthart AD, Tucker CS, Del Pozo J, Dear JW (2014).  Zebrafish as model organisms for studying drug-induced liver injury.  British Journal of Clinical Pharmacology 78(6):1217-27. doi: 10.1111/bcp.12408

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PhD Studentship

Status:

Closed

Principal investigator

Dr James Dear

Institution

University of Edinburgh

Co-Investigator

Dr Matthew Bailey
Professor David John Webb

Grant reference number

NC/K001485/1

Award date:

Oct 2013 - Sep 2016

Grant amount

£90,000