Characterization of PKD Protein-Positive Exosome-Like Vesicles

Abstract

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. PC1ϩ 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.

Strategies Targeting cAMP Signaling in the Treatment of Polycystic Kidney Disease

“…[1][2][3] The three proteins are found at low levels in tubular epithelial cells, but are abundant in urinary exosomes. 4,5 Polycystin-1 resembles a receptor or adhesion protein and is also found at the plasma membrane and, possibly, in the endoplasmic reticulum. [6][7][8] Polycystin-1 interacts with the inositol 1,4,5-trisphosphate receptor (IP3R).…”

Human Urinary Exosomes as Innate Immune Effectors

“…12 Consistent with a renal tubular epithelial origin, renal tubular epithelial cells contain MVBs at the apical surface, and urine exosomes contain apical membrane proteins from every cell type along the nephron. 13,14 The array of functions ascribed to exosomes in other tissues has kindled recent interest in the functional significance of urinary exosomes. Hogan et al 13 suggested interaction of exosome-like vesicles with primary cilia of renal epithelial cells, and Street and coworkers 15 showed in vitro uptake of exosomes by a renal cortical collecting duct cell line, leading to speculation that exosomes may provide intrarenal proximal-to-distal transapical renal tubular epithelial signaling through RNA transfer.…”

“…13,14 The array of functions ascribed to exosomes in other tissues has kindled recent interest in the functional significance of urinary exosomes. Hogan et al 13 suggested interaction of exosome-like vesicles with primary cilia of renal epithelial cells, and Street and coworkers 15 showed in vitro uptake of exosomes by a renal cortical collecting duct cell line, leading to speculation that exosomes may provide intrarenal proximal-to-distal transapical renal tubular epithelial signaling through RNA transfer. However, most studies on urine exosomes to date have focused on biomarker discovery, resulting in the publication of several urine exosome protein compendia.…”

“…However, most studies on urine exosomes to date have focused on biomarker discovery, resulting in the publication of several urine exosome protein compendia. 12,13,[16][17][18][19][20][21] Published reports of the urinary exosomal proteome have limited value in illuminating the potential functions of urinary exosomes for several reasons. First, protein identification by mass spectrometry (MS) has, until recently, yielded results with unacceptably low reproducibility and high false-positive rates, 22,23 and previous reports are not free of these limitations.…”

Autosomal dominant polycystic kidney disease: genetics, epidemiology, and treatment

“…In addition, both of the polycystin proteins in their cleaved forms have been found in urinary exosome-like vehicles that interact with the primary cilium. 12 This has been suggested as a mechanism to explain the localization of PC1 at disparate cellular locations. However, despite significant progress toward understanding the pathophysiology of ADPKD, the precise function of the polycystins, and their role in cystogenesis is incompletely understood.…”