ISOLATION AND CHARACTERIZATION OF PIG URINARY EXOSOMES FOR BIOMARKER DISCOVERY

In recent years exosome research pubblications are rapidly expanding (1). These small vesicles (30‐100 nm) of endocytic origin are thought to participate in cell—cell communication and protein and RNA delivery. A wide range of cells have been shown to release exosomes, but they have also been detected in several biological fluids, including plasma, urine, saliva and breast milk. In particular urinary exosomes have been proposed as starting material to detect protein biomarkers of renal dysfunction and structural injury or overall to shed much insight on the health status of the kidney (2, 3, 4). We aimed to validate methods for exosome isolation from urine in a large animal model, the pig one. The swine shares with the man anatomical and physiological characteristics that make it preferred species as a pre‐clinical model, in particular for kidney functions, surgical approaches and in the view to obtain a significant amount of biological specimens (urine and blood samples, tissue for renal biopsy) compared to other animals. Exosomes were purified by differential ultracentrifugation, identified by electron microscopy and described in morphology, shape, size and distribution using atomic force microscopy (AFM). Validation methods include Western blot with pan‐exosome markers and urinary specific exosome antibodies. Exosome protein content was analyzed through nanospray liquid chromatography‐tandem mass spectrometry (LM‐MS/MS). The total exosomal RNA purificated was evaluated using gel electrophoresis and a Bioanalyzer. We found that the vesicles displayed a typical exosome‐like size and morphology as analyzed by electron microscopy and AFM. Western blot and mass spectrometry further confirmed the presence of several exosome‐associated molecules. Exosome RNA profile detected is typical of these vesicles, lacking rRNA subunits which are prominent in analysis of cellular RNA. Characterization of isolated exosomes indicates that the described isolation methods are suitable for the subsequent RNA and protein profiling.(1) Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell‐to‐cell communication. Kidney Int. 2010 Nov;78(9):838‐48. (2) Fernández‐Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA. Tamm‐Horsfall protein and urinary exosome isolation. Kidney Int. 2010 Apr;77(8):736‐42. (3) Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13368‐73. (4) Lässer C, Eldh M, Lötvall J. Isolation and characterization of RNA‐containing exosomes. J Vis Exp. 2012 Jan 9;(59):e3037.