Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome

Abstract

Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome.

Extracellular Vesicles in Renal Diseases

“…39,43,44 These different techniques have varying recovery rates of EVs. 42,44 Lower recovery rates of EVs have been especially a challenge in patients with heavy or nephrotic-range proteinuria. Changes of urinary concentration represent another challenge for accurate analysis of uEVs.…”

“…TammHorsfall protein (THP), the most abundant protein in urine, is capable of entrapping uEVs. 41 Several studies have, therefore, looked at various methods to isolate and enrich uEVs, which include ultracentrifugation with and without size exclusion chromatography (at 100,000-200,0003g for at least 1 h), ultrafiltration, 42 precipitation, and immunoisolation. 39,43,44 These different techniques have varying recovery rates of EVs.…”

Quantitative body fluid proteomics in medicine — A focus on minimal invasiveness

“…More than 35000 proteins were found in the extracellular vesicles (http://www.microvesicles.org/) and using quantitative proteomics techniques 32 potential biomarkers for bladder and prostate cancer, kidney diseases, deep vein thrombosis and brain tumors were identified in body fluids. 10 of the potential biomarkers were validated using antibody-based methods (Table 9) [208][209][210][211][212][213][214][215]. [90] J. Wiesner, A. Vilcinskas, Antimicrobial peptides: the ancient arm of the human immune [100] D. Alexiou, A. Anagnostopoulos, C. Papadatos, Total free amino acids, ammonia, and protein in the sweat of children., The American Journal of Clinical Nutrition.…”

Does RBC Storage Age Effect Inflammation, Immune Function and Susceptibility to Transfusion Associated Microchimerism in Critically Ill Patients? Adverse Effects of RBC Storage in Critically Ill Patients

“…More recently, researchers have shown that EVs can be manipulated to deliver tailored thera peutic cargo to specific targets within the body (37,38). A number of methods have been used to analyze EVs, including scanning electron microscopy (39,40), transmission electron microscopy (TEM) (41), atomic force microscopy and dynamic light scattering (42 45), and western blotting (46,47). Clinically, flow cytometry (FCM) is the most com monly used method for analyzing EVs in blood (5,48 50); however, accurate characterization of EVs remains challeng ing.…”