Abstract
A pressing need for new chronic kidney disease (CKD) biomarkers persists. MicroRNAs (miRNAs) are emerging as a novel class of disease biomarkers in body fluids, but mechanisms conferring their stability in urine have not been fully elucidated. Here we investigated stabilization in human urine of ubiquitously expressed miR-16, and miR-192, which we have shown previously to be downregulated in renal fibrosis, by association with extracellular vesicles and with argonaute protein (AGO) 2. Endogenous urinary miR-16 was significantly more resistant to RNase-mediated degradation than exogenous, spiked-in, Caenorhabditis elegans cel-miR-39. We used our previously optimized high-resolution exosome isolation protocol with sucrose gradient ultracentrifugation to sub-fractionate the primary extracellular vesicle-rich urinary pellet. MiR-16 and miR-192 were enriched in exosomal sucrose gradient fractions, but were also detected in all other fractions. This suggested association of urinary miRNAs with other urinary extracellular vesicles and/or pellet components, complicating previous estimates of miRNA:exosome stoichiometry. Proteinase K digestion destabilized urinary miR-16 and we showed, for the first time, RNA-immunoprecipitation of urinary miR-16:AGO2 and miR-192:AGO2 complexes. Association with exosomes and AGO2 stabilized urinary miR-16 and miR-192, suggesting quantitative urinary miRNA analysis has the potential to identify novel, non-invasive CKD biomarkers.
Highlights
Chronic kidney disease (CKD) is a major challenge to global health with serious implications for health and economic output [1,2]
We demonstrated the stability of endogenous urinary miR-16 and identified two stabilization mechanisms for miR-16 and miR-192
Endogenous Urinary MiR-16 Is Resistant to RNase Digestion
Summary
Chronic kidney disease (CKD) is a major challenge to global health with serious implications for health and economic output [1,2]. The present diagnostic and prognostic test for intrinsic renal disease, biopsy, has a 3% risk of major complications. A potential end-point for numerous causal mechanisms, CKD is frequently accompanied by proteinuria. The predictive value of current non-invasive prognostic indicators such as urine protein quantification is limited since changes only become apparent following disease onset. There is, a pressing need for new CKD biomarkers. Powerful RNA detection techniques are available for analysis of body fluids, but endogenous
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