| Autor: |
Beltrami C; Department of Nephrology, Wales Kidney Research Unit, School of Medicine, College of Biomedical and Life Sciences, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. c.beltrami@bristol.ac.uk., Clayton A; Section of Oncology and Palliative Medicine, Institute of Cancer and Genetics, School of Medicine, College of Biomedical and Life Sciences, Velindre Hospital, Heath Park, Cardiff CF14 2TL, UK. claytona@cf.ac.uk., Newbury LJ; Department of Nephrology, Wales Kidney Research Unit, School of Medicine, College of Biomedical and Life Sciences, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. newburyl@cf.ac.uk., Corish P; BBI Group, The Courtyard, Ty Glas Avenue, Cardiff CF14 5DX, UK. PeterCorish@the-bbigroup.com., Jenkins RH; Department of Nephrology, Wales Kidney Research Unit, School of Medicine, College of Biomedical and Life Sciences, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. jenkinsrh2@cf.ac.uk., Phillips AO; Department of Nephrology, Wales Kidney Research Unit, School of Medicine, College of Biomedical and Life Sciences, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. phillipsao@cf.ac.uk., Fraser DJ; Department of Nephrology, Wales Kidney Research Unit, School of Medicine, College of Biomedical and Life Sciences, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. fraserdj@cf.ac.uk., Bowen T; Department of Nephrology, Wales Kidney Research Unit, School of Medicine, College of Biomedical and Life Sciences, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. bowent@cf.ac.uk. |
| Abstrakt: |
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. |
