ACTA SCIENTIFIC MEDICAL SCIENCES (ISSN: 2582-0931) Volume 3 Issue 11 November 2019 Comparison of Different Methods to Isolate Urinary Extracellular Vesicles Useful as Possible Kidney Damage Biomarkers Andrea Carraro 1 *, Susanna Negrisolo 1 , Diana Marzenta 1 , Michele Grassi 2 , Anna Maria Tolomeo 2 , Piera De Gaspari 3 , Maurizio Muraca 2 , Giorgio Perilongo 2 and Luisa Murer 1 1 Laboratory of Immunopathology and Molecular Biology of the Kidney of Pediatric Nephrology, Dialysis and Transplant Unit, Department of Women’s and Children’s Health, University-Hospital of Padua, Italy 2 Department of Women's and Children's Health, Hospital-University of Padua, Italy 3 Neuroimmunology Laboratory, IRP Città Della Speranza, Padua, Italy *Corresponding Author: Andrea Carraro, Laboratory of Immunopathology and Molecular Biology of the Kidney of Pediatric Nephrology, Dialysis and Transplant Unit, Department of Women’s and Children’s Health, University-Hospital of Padua, Italy. Research Article Received: October 09, 2019; Published: October 22, 2019 Abstract Keywords: Urinary Extracellular Vesicles; Biomarkers Isolation; Purification; Tunable Resistive Pulse Sensing Technology Introduction Extracellular vesicles are lipid membrane-bound nanoparticles released from cells in different biological fluids. Particularly, uri- nary extracellular vesicles (UEVs) arising from the nephron cells, could be helpful as novel biomarkers for kidney allograft injury. This comparative study aims to identify the most efficient UEVs isolation method to detect novel kidney biomarkers. UEVs were isolated from urine samples using four commercial kits and purified by Izon qEV single SEC. The UEVs isolated by different methods showed high variability in both concentration and size besides a remarkable decrease between pre and post purified samples. Based on these results the most efficient and cheap method to isolate UEVs is the Izon qEV single SEC column. Methods Summary: Four UEVs isolation techniques and Izon SEC purification were compared. Urine samples were collected and processed to discharge cellular debris. UEVs were isolated by Norgen, Exo Quick, QIAGEN, AMICON and Izon SEC methods. All sam- ples were characterized in size and concentration by qNano instrument before and after protein purification. The protein concen- tration was evaluated by Pierce TM BCA assay. Based on samples concentration and protein contamination we identified the most efficient method to isolate extracellular vesicles in urine. Recently, the clinical and scientific interest in the study of ex- tracellular vesicles (EVs) has been increased due to their potential role as noninvasive biomarkers of pathophysiological health status in different medical areas. This small membrane bound vesicles are secreted from MVs-multivesicular body (exosome) in the ex- tracellular space, or directly released from the cell through plasma membrane fusion and exocytosis (microvesicles) [1]. Their iden- tity and characterization is not well defined, although there are many studies that focus on their lipidomic [2,3], proteomic [4,5], and transcriptomic profiling [6,7], to exploit their possible role as biomarkers in different human pathologies. These nanoparticles are involved in intercellular communica- tion [8] and in many different biological processes such as: regu- lation of immune response, tumor proliferation, inflammatory response and antigen presentation [9,10]. EVs are present in vari- ous type of body fluids (e.g. blood, serum, saliva, milk and urine) [11] and their concentration, cellular origin, composition, function is body health-(state) dependent [10]. Particularly, EVs content is useful to understand in which physiological regulation process they are involved. Many studies have shown that EVs carry differ- ent type of cargo, such as micro RNA (miRNA) and proteins. miRNA are short single-strand RNA molecules that are involved in several biological processes including cell death, proliferation, apoptosis, fat metabolism and oncogenesis [12]. Thus, measuring the concen- tration, cellular origin and content of EVs in body fluids has also a Abbreviation EVS: Extracellular Vesicles; UEVS: Urinary Extracellular Vesicles; MVS: Multivesicular; Mirna: Micro RNA; SEC: Size Exclusion Col- umn; TRPS: Tunable Resistive Pulse Sensing Technology; PSD: Par- ticle Size Distribution DOI: 10.31080/ASMS.2019.03.0446 Citation: Andrea Carraro., et al. “Comparison of Different Methods to Isolate Urinary Extracellular Vesicles Useful as Possible Kidney Damage Biomarkers". Acta Scientific Medical Sciences 2.11 (2019): 99-104.