Albuminuria is not associated with elevated urinary vesicle concentration but can confound nanoparticle tracking analysis

Abstract

There was no increase in urinary vesicle concentration in patients with albuminuria. Furthermore, these results demonstrate the need for cautious interpretation of nanoparticle tracking analysis of vesicle concentration in biological fluids containing protein and for sophisticated preparative methods in vesicle purification from urine.

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs

“…We confirmed a wide variety (or heterogeneity) of different types of vesicles of different sizes (40-500 nm) and morphology (round, oval/flattened) with complex architecture made of a set of vesicles enclosed inside some larger ones independent of the presence of filament of uromodulin. This is consistent with Cryo-TEM analysis of EVs enriched from other biofluids, such as plasma 30,31 , synovial fluid 32 , ejaculate 33 and urine 34 .…”

“…However, similar discrepancies were observed in other studies on different biofluids 32,33 . www.nature.com/scientificreports www.nature.com/scientificreports/ We can explain this discrepancy in count numbers between TRPS and NTA on the basis that NTA is more sensitive to detect small particles made also of soluble proteins such as albumin, which can scatter light 34 . For example, the particle counts in the SN21 TCPE , P21 TCEP and SN21 TCEP is still significant which might be due to the fact that THP can scatter and be detected as a particle like albumin.…”

The Authors Reply

“…Importantly, we confirmed the results by additional experiments with the EXO-CET Exosome Quantitation Kit (System Biosciences, CA), which measured the esterase activity known to be within exosomes. The finding of Gleadle et al that albumin can form exosome-like particles is important and interesting, 4 and confirms the importance of combining techniques for characterizing urinary extracellular vesicles, rather than a single approach like nanoparticle tracking analysis.…”

New insight into the role of extracellular vesicles in kidney disease

“…A recent study report that ultracentrifugation followed by size exclusion chromatography (UC‐SEC) yielded the most homogenous population of exosomes and less non‐vesicle co‐precipitated proteins from urine . The optimal method for EV purification is important especially for urine samples from proteinuric nephropathy patients . Besides, large cohort of validation study is necessary for biomarker development and clinic translation.…”

“…84 The optimal method for EV purification is important especially for urine samples from proteinuric nephropathy patients. 85 Besides, large cohort of validation study is necessary for biomarker development and clinic translation. Meanwhile, to determine the cell origin of urinary exosomes and the relevance of candidate biomarker to the development of kidney disease are the critical issues that deserve in-depth consideration.…”