Abstract
Chronic kidney disease is a progressive, incurable condition that involves a gradual loss of kidney function. While there are no non-invasive biomarkers available to determine whether individuals are susceptible to developing chronic kidney disease, small RNAs within urinary exosomes have recently emerged as a potential candidate to use for assessing renal function. Ultracentrifugation is the gold standard for urinary exosome isolation. However, extravesicular small RNA contamination can occur when isolating exosomes from biological fluids using ultracentrifugation, which may lead to misidentifying the presence of certain small RNA species in human urinary exosomes. Therefore, we characterized human urinary exosomal preparations isolated by ultracentrifugation alone, or via ultracentrifugation followed by size exclusion chromatography (SEC) column-purification. Using nanoparticle tracking analysis, we identified SEC fractions containing robust amounts of exosome-sized particles, that we further characterized using immunoblotting. When compared to exosomal preparations isolated by ultracentrifugation only, SEC fractionated exosomal preparations showed higher levels of the exosome-positive marker CD81. Moreover, while the exosome-negative marker calnexin was undetectable in SEC fractionated exosomal preparations, we did observe calnexin detection in the exosomal preparations isolated by ultracentrifugation alone, which implies contamination in these preparations. Lastly, we imaged SEC fractionated exosomal preparations using transmission electron microscopy to confirm these preparations contained human urinary exosomes. Our results indicate that combining ultracentrifugation and SEC column-purification exosome isolation strategies is a powerful approach for collecting contaminant-free human urinary exosomes and should be considered when exosomes devoid of contamination are needed for downstream applications.
Highlights
Fractions 6 and 7 demonstrated a similar diameter size pattern and overall particle number profile when compared to human urinary exosomal preparations isolated via ultracentrifugation only, while we observed a relatively inferior particle number and diameter size in fraction 8 when this fraction was compared to all the other preparations (Figure 1A–E and Supplementary Figure S1A–E)
We wanted to test whether human urinary exosomes which were collected via SECtocolumn-purification post-ultracentrifugation provide preparations that are
We wanted test whether human urinary exosomes which were colcomparable to human urinary exosomes which were isolated using ultracentrifugation lected via size exclusion chromatography (SEC) column-purification post-ultracentrifugation provide preparations that are only
Summary
CKD is thought to afflict millions of Americans annually and many individuals are unaware that they have this condition [2]. There are no feasible treatments for CKD [1,2]. There are many patients afflicted with CKD who are deemed ineligible for kidney transplantation [4,5]. The predominant treatment for CKD once the kidneys have failed is dialysis [6]. Dialysis is both burdensome and exhausting on patients, is expensive, and needs to be performed as a regular, life-long therapy [7,8]. Life expectancy and quality-of-life are drastically decreased in dialysis patients, too [9,10,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
