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Characterization of PKD Protein-Positive Exosome-Like Vesicles

Marie C. Hogan^*, Luca Manganelli^*,, John R. Woollard^*, Anatoliy I. Masyuk, Tatyana V. Masyuk, Rachaneekorn Tammachote^*, Bing Q. Huang, Alexey A. Leontovich, Thomas G. Beito^||, Benjamin J. Madden^¶, M. Cristine Charlesworth^¶, Vicente E. Torres^*, Nicholas F. LaRusso, Peter C. Harris^* and Christopher J. Ward^*

^* Division of Nephrology and Hypertension, Center for Basic Research in Digestive Diseases, Bioinformatics Core Facility, Division of Biomedical Informatics, ^|| Mayo Ab Core, and ^¶ Mayo Proteomics Core, Mayo Clinic, Rochester, Minnesota; and Department of Nephrology, University of Modena and Reggio Emilia, Modena, Italy

Correspondence: Dr. Christopher J. Ward, Division of Nephrology & Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: 507-266-3050; Fax: 507-266-9315; E-mail: ward.christopher{at}mayo.edu

Received for publication June 2, 2008. Accepted for publication September 5, 2008.

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1^del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

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Copyright © 2009 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673