URINARY EXOSOMES AND PROTEOMICS Pyong-Gon Moon,1,2 Sungyong You,3 Jeong-Eun Lee,1,2 Daehee Hwang,3 and Moon-Chang Baek1,2* 1 Department of Molecular Medicine 2 Cell and Matrix Biology Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422, Republic of Korea 3 School of Interdisciplinary Bioscience and Bioengineering & Department of Chemical Eng., POSTECH, Pohang 790-784, Republic of Korea Received 21 October 2009; revised 23 July 2010; accepted 23 July 2010 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.20319 A number of highly abundant proteins in urine have been identified through proteomics approaches, and some have been considered as disease-biomarker candidates. These molecules might be clinically useful in diagnosis of various diseases. However, none has proven to be specifically indicative of perturbations of cellular processes in cells associated with urogenital diseases. Exosomes could be released into urine which flows through the kidney, ureter, bladder and urethra, with a process of filtration and reabsorption. Urinary exosomes have been recently suggested as alternative materials that offer new opportunities to identify useful biomarkers, because these exosomes secreted from epithelial cells lining the urinary track might reflect the cellular processes associated with the pathogenesis of diseases in their donor cells. Proteomic analysis of such urinary exosomes assists the search of urinary biomarkers reflecting pathogenesis of various diseases and also helps understanding the function of urinary exosomes in urinary systems. Thus, it has been recently suggested that urinary exosomes are one of the most valuable targets for biomarker development and to understand pathophysiology of relevant diseases. # 2011 Wiley Periodicals, Inc. Mass Spec Rev Keywords: urinary exosomes; urogenital diseases; biomarkers; proteomics I. INTRODUCTION The attempts to identify disease biomarkers, for diagnostics and prognostics, increase tremendously during the last 10 years as proteomics technology has been developed and optimized (Azad et al., 2006; Qian et al., 2006; Geho et al., 2007; Tao & Lazarev, 2007). Urine could be easily used for clinical as well as basic research because urine might be obtained in a simple, non-invasive, sufficient and stable fashion compared to other clinical samples (Theodorescu et al., 2006; Decramer et al., 2008; Caubet et al., 2010). Urine contains proteins, peptides, and metabolites derived from kidney as well as blood (Decramer et al., 2008). Therefore, urine is applicable to study Contract grant sponsor: Korean Ministry of Education, Science and Technology; Contract grant sponsor: Regional Technology Innovation Program of the Ministry of Knowledge Economy; Contract grant number: RTI04-01-01. *Correspondence to: Moon-Chang Baek, Department of Molecular Medicine, School of Medicine, Kyungpook National University, 101 Dongin-dong 2 Ga, Jung-gu, Daegu 700-422, Republic of Korea. E-mail: mcbaek@knu.ac.kr Mass Spectrometry Reviews # 2011 by Wiley Periodicals, Inc. with regards to urogenital diseases and non-urogenital diseases from associated organs in the body (Thongboonkerd, 2008). The advent of the proteomics era has enormously affected the methodology to study proteins, and has accelerated the ability to acquire information on proteins for basic and clinical research areas. The number of urine proteins identified during the past 5 years exceeded by 10-fold that of urine proteins found previously, and facilitated the search for urinary biomarkers (Spahr et al., 2001; Pieper et al., 2004; Adachi et al., 2006; Wang et al., 2006; Moon et al., 2008; Zurbig & Mischak, 2008). Various mass spectrometry techniques including 2-D PAGE-MS, surface-enhanced laser desorption/ionization timeof-flight mass spectrometry (SELDI-TOF MS), LC-MS/MS and capillary electrophoresis mass spectrometry (CE-MS), have been applied to analyze urine proteome. SELDI-TOF MS (Cadieux et al., 2004; Khurana et al., 2006; Poon, 2007), and CE-MS (Mischak et al., 2004; Weissinger et al., 2004; Haubitz et al., 2005; Wittke et al., 2005; Decramer et al., 2006) have been used for profiling approaches, and LC-MS/MS (Castagna et al., 2005; Sun et al., 2005; Adachi et al., 2006; Ru et al., 2006; Wang et al., 2006; Pieper, 2008; Smalley et al., 2008) and 2-D PAGE-MS (Thongboonkerd, Klein, & Arthur, 2003; Saito et al., 2005; Thongboonkerd, 2007) have been used for biomarker discovery. The advantages and disadvantages of each MS-based proteomics techniques have been reviewed in detail elsewhere (Fliser et al., 2007; Decramer et al., 2008). For these methods, usually the whole urine has been used. One of the challenges for biomarker identification is to reduce the dynamic range of protein concentrations in samples. To overcome this difficulty, an immunosubtraction method such as albumin depletion, affinity chromatography method such as lectin (Yang & Hancock, 2004, 2005; Wang et al., 2006; Moon et al., 2008) and immobilized metal-ion chromatography (IMAC) columns (Belew et al., 1987; Hoffert et al., 2006; Villen & Gygi, 2008), and ligand library beads method (Righetti et al., 2006) have been introduced, and the identification efficiency of low-abundance proteins has been improved. However, it is still difficult to select good biomarker candidates for the next validation step among proteins with differentially expressed proteins. Urinary exosomes are microparticles identified recently in urine (Pisitkun, Shen, & Knepper, 2004). Proteomic research of these urinary exosomes has become one of the newer trends in the field of urine-biomarker discovery. Various mammalian cells such as reticulocytes (Pan & Johnstone, 1983; Johnstone et al., 1987), mast cells (Skokos et al., 2001a, 2003; Valadi & MOON ET AL. et al., 2007), B lymphocytes cell lines (Escola et al., 1998; Dukers et al., 2000; Wubbolts et al., 2003), and human dendritic cells (DCs) (Thery et al., 1999, 2001; Kim et al., 2005) secrete exosomes by physiological signals. Many studies related to exosomes have been reported, and the recent high increase in exosome studies might be related to the growth of proteomics research (Fig. 1). In 2004, the Knepper group first identified exosomes in human urine, and demonstrated the potential to use the urinary exosomes as a starting sample for biomarker discovery in urine (Pisitkun, Shen, & Knepper, 2004). Subsequently, the number of studies of urinary exosomes has gradually increased as shown by other exosomes studies (Fig. 1). To secrete the exosomes, an endocytic pathway and the fusion of multivesicular bodies (MVBs) with the plasma membrane are involved (Fevrier & Raposo, 2004). Intra-lumenal vesicles (ILVs) in MVB lumen are released into the extracellular environment. The released small particles (40–100 nm) were called exosomes. The exosomes obtain cytosolic portions, including proteins and various RNAs, at the point when endosomes are fused into MVBs, and also contain membrane proteins and lipids from original cells (Lin et al., 2005; Fang et al., 2007; Lakkaraju & RodriguezBoulan, 2008). There are several reports that urinary exosomes could be excreted into urine via exocytosis from kidney cells by a similar mechanism to other exosomes (Pisitkun, Shen, & Knepper, 2004; Zhou et al., 2006b; Keller et al., 2007; Lakkaraju & Rodriguez-Boulan, 2008; Gonzales et al., 2009). Only 3% of the total urinary protein in urine is contained in urinary exosomes, which excludes other high-abundance proteins such as albumin (Hoorn et al., 2005). In addition, the urinary exosomes might contain many clues regarding the state of organs through the urinary track (Gonzales, Pisitkun, & Knepper, 2009). Therefore, urinary exosomes could be important sources to study urogenital diseases, especially human diseases without proper animal models. Many researchers have focused their current efforts on biomarkers that are biologically relevant to the pathophysiology of diseases as reviewed recently (Knepper, 2009; Gonzales, Pisitkun, & Knepper, 2009). Therefore, urinary exosomes could be good target materials to develop biomarkers in clinical samples due to their attractive characteristics, including origin from cells lining the urinary track, low complexity of proteins, and content of cytosolic and membrane proteins of donor cells (Hoorn et al., 2005), even if exosomal proteins are much harder to be isolated compared to soluble urine proteins. This review presents the characteristics of urinary exosomes, methodologies to study urinary exosomes, and proteomics studies on various urinary exosomes. II. THE CHARACTERISTICS OF URINARY EXOSOMES A. Kidney Function and Urine The kidneys are reddish, bean-shaped, paired organs, which play a major role in the production of urine. In a human, an adult kidney is 10–12 cm long, 5–7 cm wide, and 3 cm thick with a mass of 115–170 g (Boron & Boulpaep, 2005; Tortora & Grabowski, 2003). The left kidney is slightly larger than the right. The concave surface, called the renal hilum is the point through which the ureter emerges from the kidney along with blood vessels, lymphatic vessels, and nerves. The kidney has two major structures; the renal cortex at the outer region, and the renal medullar at the inner region. Nephrons, the urine-producing functional structures of the kidney, span the cortex and medullar (Tortora & Grabowski, 2003; Boron & Boulpaep, 2005). Urine is produced by the urinary system that is composed of two kidneys, two ureters, one urinary bladder, and one urethra. The kidneys are the major part of the urinary system, and the other parts are primarily for passageways and storage of urine. Furthermore, the kidneys play important roles in the regulation of ionic composition, pH, volume, pressure, and glucose level in blood. Each nephron, the functional unit of FIGURE 1. The relationship between exosomes research and proteomics. The high increase of publications related to exosomes research ( ) might be related to the growth of proteomics research ( ). Study on ) started in 2004, and the number of publications has increased annually since urinary exosomes ( then. The number of publications was calculated based on PubMed searches with key words ‘‘exosomes,’’ ‘‘urine þ exosomes,’’ or ‘‘proteomics,’’ respectively. 2 Mass Spectrometry Reviews DOI 10.1002/mas URINARY EXOSOMES & the kidneys, consists of two parts: a renal corpuscle, where bold plasma is filtered, and a renal tubule into which the filtered fluid passes. The two components of a renal corpuscle are the glomerulus and the glomerular (Bowman’s) capsule, a double-walled epithelial cup that surrounds the glomerular capillaries. Blood plasma is filtered in the glomerular capsule, and the filtered fluid passes into the renal tubule, which has three main sections. In the order that fluid passes through them, first the renal tubule consists of a (1) proximal convoluted tubule, (2) loop of Henle (nephron loop), and (3) distal convoluted tubule. The distal convoluted tubules of several nephrons empty into a single collecting duct. Second, collecting ducts unite and converge into several hundred large papillary ducts, which drain into the minor calyces. Third, the collecting ducts and papillary ducts extend from the renal cortex through the renal medulla to the renal pelvis. Thus, one kidney has about 1 million nephrons, but a much smaller number of collecting ducts and even fewer papillary ducts. About 170–179 L of water is reabsorbed into the bloodstream, and the actual volume of urine produced is only 1– 2 L. The urine is composed of water, proteins, glucose, amino acids, urea, and ions such as sodium, potassium, calcium, chloride, bicarbonate, and phosphate (Tortora & Grabowski, 2003; Boron & Boulpaep, 2005) (Fig. 2A). In healthy individuals, urinary proteins consist of kidney-originated proteins (70%) (Christensen & Birn, 2001) and plasma-derived proteins (30%) (Thongboonkerd et al., 2002; Pieper et al., 2004; Thongboonkerd & Malasit, 2005) (Fig. 2B). Therefore, urine is an ideal biofluid for biomarker discovery of kidney diseases as well as non-kidney diseases (Thongboonkerd, 2008). Urine protein is divided two parts, soluble proteins, and solidphase elements. The solid-phase elements can be divided into sediment precipitated with low-speed centrifugation and lowdensity exosomes precipitated with ultracentrifugation. The proteins that are present in urine are a major area of investigation for proteomics researchers. In normal urine, half of the proteins are soluble proteins (49%), and the remaining 48% are sediment precipitated with low-speed centrifugation and exosomes (3%) (Serafini-Cessi, Malagolini, & Cavallone, 2003; Hoorn et al., 2005; Zhou et al., 2006b; Pisitkun, Johnstone, & Knepper, 2006) (Fig. 2C). The exosomes are prepared with high-speed centrifugation, and the size of the low-density vesicle is less than 100 nm, which originates from multivesicular bodies (MVBs) in cells (Pisitkun, Shen, & Knepper, 2004). B. Characteristics of Exosomes 1. Exosome Excretion It has been reported that human body fluids such as urine (Pisitkun, Shen, & Knepper, 2004; Keller et al., 2007; Gonzales et al., 2009), plasma (Looze et al., 2009), physiological pregnancy-associated sera (Sabapatha, Gercel-Taylor, & Taylor, 2006), bronchial lavage fluid (Admyre et al., 2003), synovial fluid (Skriner et al., 2006), saliva (Castle, Huang, & Castle, 2002; Kapsogeorgou et al., 2005; Gonzalez-Begne et al., 2009), breast milk (Admyre et al., 2007), and malignant pleural effusion (Andre et al., 2002; Bard et al., 2004) contain exosomes. In addition, many groups have reported that several cell types, including B cells (Escola et al., 1998; Dukers et al., 2000; Wubbolts et al., 2003), dendritic cells (Thery et al., 1999, Mass Spectrometry Reviews DOI 10.1002/mas FIGURE 2. The composition of urine and urine proteins in human. The volume of urine excreted a day is about 1–2 L. A: The ratio of the urine composition is; water (97.1%), urea (1.94%), ions (0.8%), creatinine (0.1%), uric acid (0.05%), and protein (0.01%). B: Urine proteins used for proteomics are classified into two parts based on their origin; kidney and urinary tract (70%), filtered plasma (30%). C: Urine proteins are also divided two parts based on their solubility; Soluble (49%) and solid-phase elements (51%). The solid-phase elements can be also divided into sediment (48%) precipitated with low-speed centrifugation and exosomes (3%) precipitated with ultracentrifugation. 2001), mast cells (Skokos et al., 2001a, 2003; Valadi et al., 2007), T cells (Blanchard et al., 2002; Zakharova, Svetlova, & Fomina, 2007), platelets (Heijnen et al., 1999), neuronal cells (Andrews & Chakrabarti, 2005; Faure et al., 2006; Smalheiser, 3 & MOON ET AL. 2007), intestinal epithelial cells (van Niel et al., 2001; Hundorfean et al., 2007), retinal pigment epithelium (McKechnie, Copland, & Braun, 2003; Wang et al., 2009), Schwann cells (Beswetherick, Lane, & Allt, 1992), and tumor cells (Segura, Amigorena, & Thery, 2005; Taylor & Gercel-Taylor, 2005, 2008; Hao et al., 2006; Choi et al., 2007), tumor cell lines (Andre et al., 2002), and sperm (Sullivan et al., 2005) also secrete exosomes in vitro. A urinary exosome was first identified by the Knepper group in 2004 (Pisitkun, Shen, & Knepper, 2004). It was predicted that some exosomes originated from epithelial cell types that face the urinary space, because specific proteins that correspond to various epithelial cell types are identified as follows; podocin and podocalyxin (glomerular podocytes), megalin, cubilin, APSN, AQP-1, type IV carbonic anhydrase, gamma-glutamyltransferase (proximal tubule), Tamm–Horsfall protein (THP), CD9, the type 2 Na-K-2Cl cotransporter (thick ascending limb of Henle), NCC (distal convoluted tubule), AQP-2, mucin-1, the RH type C glycoprotein (collecting duct), and uroplakin-1 and -2 (Urinary bladder) (Pisitkun, Shen, & Knepper, 2004; Hoorn et al., 2005). 2. Mechanism of Exosome Excretion The excretion pathway of exosomes in apical membranes is shown at Figure 3. The mechanism of exosomes excretion could be usually described as three steps. First, the incorporation of proteins into multivesicular body (MVB) begins when plasmamembrane proteins are monoubiquitinated. This monoubiquitination is different from polyubiquitination that targets proteins to the proteasome (Polo et al., 2002; Shekhtman & Cowburn, 2002). Proteomic data of urinary exosomes showed that they contain many ubiquitinated proteins, including E2 and E3 components related to the ubiquitination pathway. Second, these ubiquitinated proteins are endocytosed, follow the endosomal pathway (Pisitkun, Shen, & Knepper, 2004), and finally fuse into MVBs (Futter & White, 2007). In this stage, internal vesicles could take up proteins, mRNAs, and micro-RNAs of cytoplasm of the cell. Some proteins identified from the urinary exosome include members of the endosomal-sorting complex required for transport (ESCRT) Complex, ATPase complexes, and accessory proteins involved in MVB formation (Pisitkun, Shen, & Knepper, 2004). Endocytosed proteins are sorted within intralumenal vesicles (ILVs) via ESCRT component lipids and/ or tetraspanines-enriched microdomains (Gruenberg, 2003; van Niel et al., 2006). ESCRT consist of several components. Sorting is initiated by recognition of mono-ubiquitinated cargo proteins by TAM, epidermal growth factor receptor substrate 15 (Eps15) and hepatocyte growth factor-regulated kinase substrate (Hrs), a component of ESCRT-0. Hrs also recruits tumor susceptibility gene 101 (Tsg101) of ESCRT I complex, which binds to ESCRT II. ESCRT-II moves to the plasma membrane, and clusters at the ubiquitinated cargo. Ubiquitin of cargo proteins are removed by de-ubiquitylating enzyme recruited by ESCRT-III before cargo proteins enter into ILVs. Vps4 with ATPase activity play a role in disassembling all ESCRTs from membranes (Babst, 2005). Alix identified from urine are class E Vps proteins that act as a linker between various ESCRT complexes (Morelli et al., 2004). As an alternative pathway, ESCRT-independent mechanisms to sort proteins into ILVs within MVBs have been identified recently that are regulated by ceramide-mediated budding of exosome vesicles into multivesicular endosomes (Trajkovic et al., 2008). Third, exosomes are secreted into the urinary space by fusion of MVBs into the corresponding epithelial membrane. The mechanisms of exosome release are similar to those involved in the fusion of secretory lysosomes with the plasma membrane, but the mechanism was not defined (Stinchcombe, Bossi, & Griffiths, 2004; Yu, Harris, & Levine, 2006; Proux-Gillardeaux et al., 2007; Charette & Cosson, 2008). It has been reported that the major factors for exosomes release are calcium ionophores (Jaiswal, Andrews, & Simon, 2002; Khvotchev et al., 2003; Savina et al., 2003), decrease of membrane cholesterol (Lebrand et al., 2002; Stoeck et al., 2006; Llorente, van Deurs, & Sandvig, 2007; Chen et al., 2008), inhibition of cholesterol biosynthesis, overexpression of citron kinases, and Rab11 (Savina, Vidal, & Colombo, 2002) or RhoA effector (Perret et al., 2005; Rodriguez-Boulan, Kreitzer, & Musch, 2005; Chen et al., 2008). Recent imaging techniques, such as stimulated-emission depletion (STED) and stochastic optical reconstruction microscopy (SORM) (Willig et al., 2006; Bates et al., 2007), increased spatial resolutions of 20–40 nm to assist the visual- FIGURE 3. The excretion pathway of exosomes in apical membranes. The mechanism of exosomes excretion could be usually described as three steps. First, plasma membrane-containing membrane proteins are internalized by a signal such as ubiquitination. Second, the internal vesicles are fused into multivesicular body (MVB) outer membrane. In this stage, internal vesicles could take up proteins, mRNAs, and micro-RNAs of cytoplasm of the cell. MVBs contain intralumenal vesicles (ILVs). Third, vesicles, or exosomes, are secreted to the extracellular region by fusion of MVBs into plasma membrane. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com] 4 Mass Spectrometry Reviews DOI 10.1002/mas URINARY EXOSOMES ization of the real-time trafficking of exosomes in cells, and finally it will provide the opportunity to better understand the biogenesis of various vesicles, including exosomes in living cells. 3. Characteristics of Exosomes There are other membrane vesicles, called apoptotic blebs, microparticles (MPs) (Diamant et al., 2004; Lynch & Ludlam, 2007), and microvesicles (MVs) (Majno & Joris, 1995; Aupeix et al., 1997; MacKenzie et al., 2001) in the extracellular environment. These apoptotic blebs, MPs, and MVs show heterogeneous populations of membrane vesicles with variable size of ranges 100–1,000 nm, compared to exosomes, which showed homogeneous populations of membrane vesicles in size (40–100 nm) (Mears et al., 2004; Pisitkun, Shen, & Knepper, 2004; Xiu et al., 2007; Simpson, Jensen, & Lim, 2008). These heterogeneous membrane vesicles are shed directly from the plasma membrane of stimulated cells. In the case of MPs, different agents can induce release of phenotypically distinguishable particles (Baj-Krzyworzeka et al., 2002). These membrane vesicles have the same membrane topology; cytosolic side of the lipid bilayer inside the vesicle, the luminal part of the membrane exposed (Hugel et al., 2005). Exosomes contain protein, lipid (Laulagnier et al., 2004; Subra et al., 2007), and RNA (Ratajczak et al., 2006; Lotvall & Valadi, 2007; Smalheiser, 2007; Valadi et al., 2007; Belting & Wittrup, 2008; Rabinowits et al., 2009). The exosome composition varies depending on the cell type of origin (Schorey & Bhatnagar, 2008). Nevertheless, all exosomes contain a number of common protein components (van Niel et al., 2006). The cytosolic proteins present on exosomes include Rabs, annexins, as well as several adhesion molecules. Exosomes also contain heat-shock proteins (Gastpar et al., 2005), and also carry some cell-specific proteins like MHC II (Raposo et al., 1997; Denzer et al., 2000) and CD86 present only on exosomes isolated from antigen-presenting cells (APCs) (Raposo et al., 1996; Segura, Amigorena, & Thery, 2005) and MFG-E8/lactadherin present on exosomes from immature DCs (Veron et al., 2005) and adipocytes (Aoki et al., 2007). Exosomes are also enriched in proteins that participate in vesicle formation and trafficking like the lysobisphosphatidic acid-binding protein, Alix. Other proteins detected on exosomes are the metabolic enzymes. Consistent with their origin, exosomes typically do not contain endoplasmic reticulum, mitochondria, or nuclear proteins. The lipid compositions of exosomes are characteristic of the cell origin. The analysis of lipid compositions have been performed with exosomes derived from guinea pig reticulocytes (Vidal et al., 1989), rat mast cells (Laulagnier et al., 2004), human B cell (Wubbolts et al., 2003), and human dendritic cells (Laulagnier et al., 2004). Lipids, such as lyos(bis)phosphatidic acid (LBPA) are enriched in internal membranes of MVBs (Kobayashi et al., 1998), and play a key role in ILV formation (Chu, Witte, & Qi, 2005) to indicate the important roles of lipids in exosome biogenesis. Also, exosomes derived from mast cells and dendritic cells showed different phospholipid composition compared to the parent cells. Especially, a high sphingomyelin level is shown in exosomes derived from parent cells such as mast cells, dendritic cells, and B-cells (Wubbolts et al., 2003). In addition, a nonasymmetrical distribution of phosphatidylethanolamine and a Mass Spectrometry Reviews DOI 10.1002/mas & rapid flip–flop of lipids between the two membranes are observed (Laulagnier et al., 2004). Furthermore, although most lipids, including phosphatidylcholine, phosphatikylserine, phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin, are also present on exosomes isolated from other cell types, the ratios of these lipids vary. The presence of lipid-raft domains on the exosomes has been investigated (de Gassart et al., 2003; Kobayashi et al., 2007). Various typical raft components, glycolipids, Src tyrosine kinases (Lyn), GPI-anchored proteins (AChE), stomatin, and flotillin-1, were associated in a detergent-resistant membrane on the secreted exosomes (de Gassart et al., 2003; Echarri, Muriel, & Del Pozo, 2007). Recently, biologically active exosomal mRNAs were identified in a mouse mast cell line, a human mast cell line, and primary bone marrow-derived mouse mast cells (Valadi et al., 2007). With microarray and microRNA chip analysis, 1,300 mRNAs and 120 micro RNAs were detected in mast cells, many of which were exosome-specific, and were not detected in the cytoplasm of the donor cells. The exosomal mRNA of mouse donor cells was transferred to human recipient cells, and new mouse proteins were synthesized in human cells. In addition to normal cells, tumor cells such as glioblastoma tumor cells also release exosomes that contain mRNAs, micro-RNAs and, angiogenic proteins (Skog et al., 2008). The mRNAs in exosomes from tumor cells also could be delivered to normal recipient cells in the tumor environment and were translated. The tumor-specific mRNAs and micro-RNAs could be detected in serum exosomes of the tumor patients; those data suggest that the exosomes in serum could be used for diagnosis and therapeutic decision for tumor patients (Rabinowits et al., 2009). For urinary exosomes, recently it has been reported that mRNA was extracted from prostate cancer-derived urinary exosomes by a general RNA-isolation method (Nilsson et al., 2009). In that study, two known prostate-cancer biomarkers, PCA-3 and TMPRSS2: ERG, were detected in exosomes isolated from the urine of patients. This result suggested that urinary exosomes also contain mRNAs as exosomes secreted from other cells, even if many studies on urinary exosomal RNAs, including micro-RNAs, have not been reported so far. It has been reported that exosomes secreted by tumor cell lines induced tumor invasion (Hao et al., 2006; Segura, Amigorena, & Thery, 2005). Tumor-derived exosomes mediated immunosuppression during tumor-infiltrating lymphocytes processes, and exosomes secreted from tumor-cell lines played a role as a strong angiogenic factor in vascular development around the tumor (Kim et al., 2002; Koga et al., 2005). It has been reported that the supernatants of primary cortical cultures contain exosomes and GluR2/3 subunits of AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor, integral membrane proteins, are secreted into the exosome fraction (Menager et al., 2007). Exosomes secreted from oligodendrocytes also contains myelin proteins (Trajkovic et al., 2008). Exosomes regulate the production and turnover of myelin-membrane proteins, and also affect the progression of neurodegenerative diseases (Vella et al., 2008). Retroviruses such as HIV-1 accumulate in MVBs in human macrophages, and escape with the intracellular machinery of MVBs for budding at plasma membranes (Fevrier & Raposo, 2004). The HIV particles have similar components to exosomes (Loomis et al., 2006). Exosomes can also transport 5 & MOON ET AL. prions (Fevrier et al., 2004), which could cause neurodegenerative disorders in humans and animals. III. METHODOLOGY TO STUDY URINARY EXOSOMES Various studies with proteomics and other biochemical approaches on exosomes from various sources (Mathivanan & Simpson, 2009) are shown in Table 1. The preparation and digestion method for urinary exosomes are described in detail below. A. Preparation of the Exosomes There are many reports to prepare exosomes from various cells and tissues. Usually, differential centrifugation, a combination of differential centrifugation with gradient centrifugation, ultraTABLE 1. PUBMED ID 10878338 9685355 12519789 19109410 1 6302 7 29 17641064 14582906 15940614 1 7956 14 3 16446100 10545503 11390481 19367702 11487543 18452139 12147373 14975938 11231627 111 4566 2 11306949 126 2 655 8 6 filtration, and gel filtration, are used. To isolate exosomes from a culture supernatant, cells and debris were removed with lowspeed centrifugation with 500–3,000g for 15 min. In this case, to eliminate large-cell debris, filtration with 0.22 mm filters or 0.1 mm filters are used. To further purify exosomes, a linear sucrose gradient (2.0–0.25 M sucrose) has been used. Recently, an affinity capture method with magnetic beads has been introduced. With a culture supernatant of prostate cell lines (Jansen et al., 2009), breast adenocarcinoma cell lines (Koga et al., 2005), and ascites of ovarian cancer patient (Clayton et al., 2001), tumor exosomes are collected with antibody-coated magnetic beads that have affinity to tumor-specific protein (KeryerBibens et al., 2006). In a study by Lamparski et al. (2002), ultrafiltration (500 kDa membrane) and ultracentrifugation with a 30% sucrose/deuterium oxide (98%) cushion (density, 1.210 g/cm3) were used; that method was useful to miniscale process clinical studies with high yield. Current studies of various exosomes, including urinary exosome SAMPLES B cell lymphoblastoid cells B cells B cells Brain tumor Breast adenocarcinoma Breast milk Bronchoalveolar lavage fluid Colorectal cancer cells Colorectal cancer cells Cortical neurones Dendritic cells Dendritic cells Hepatocytes Intestinal epithelial cells Keratinocytes Malignant pleural effusions Malignant pleural effusions Mammary adenocarcinoma Mast cells Mast cells Mast cells METHODS REFERENCES Western blotting Duke rs et a l ., 2 0 00 Western blotting/ Immunoelectron Microscopy Mass spectrometry/ Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting Western blotting/ FACS Mass spectrometry/ Western blotting/ Immunoelectron Microscopy/ FACS Escola et al., 1998 Wubbolts et al., 2003 Graner et al., 2009 Koga et al., 2005 Admyre et al., 2007 FACS/ Immunoelectron Microscopy Admyre et al., 2003 Western blotting/ Immunoelectron Microscopy/ FACS Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting Mass spectrometry/ Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting Western blotting Huber et al., 2005 Choi et al., 2007 Faure et al., 2006 Thery et al., 1999 Thery et al., 2001 Conde-Vancells et al., 2008 van Niel et al., 2001 Chavez-Munoz et al., 2008 Western blotting A n dr e e t a l . , 2 0 0 2 Mass spectrometry/ Western blotting Bard et al., 2004 Western blotting/ Immunoelectron Microscopy Wolfers et al., 2001 Skokos et al., 2001b Skokos et al., 2001a Skokos et al., 2003 17486113 Mast cells 15478216 15111327 16081791 Melanoma cells Mesothelioma cells Microglia 18309083 Oligodendrocytes 15908444 Plasma ELISA Western blotting Western blotting Mass spectrometry/ Western blotting/ Microarray/ miRCURY LNA Array Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting Mass spectrometry/ Western blotting/ Immunoelectron Microscopy Western blotting/ FACS/ Immunoelectron Microscopy 170766 7 9 Plasma Western blotting 19028452 Plasma 10572093 630 7529 3597417 15210972 18520029 185 89 210 Platelets Reticulocytes Reticulocytes Rov epithelial cells Saliva Serum 17133577 Synovial fluid 11907077 T cells 19190083 Tracheobronchial cells 153 26 28 9 1 7 700 6 4 0 1905 6867 19158352 Uri n e U ri n e Urine Ur ine Mass spectrometry/ Western blotting/ Immunoelectron Microscopy Western blotting/ FACS/ Immunoelectron Microscopy Antibody based methods Enzyme assay/ Antibody based methods Western blotting/ Immunoelectron Microscopy Mass spectrometry Western blotting/microRNA Array Immunoelectron Microscopy/ Mass spectrometry/ Western blotting Western blotting/ Immunoelectron Microscopy Mass spectrometry/ Western blotting/ Immunoelectron Microscopy/ FACS Mass spectrometry/ Western blotting Western blotting/ FACS Mass spectrometry Mass spectrometry Valadi et al., 2007 Mears et al., 2004 Hegmans et al., 2004 Potolicchio et al., 2005 Trajkovic et al., 2008 Caby et al., 2005 Sabapatha, Gercel-Taylor & Taylor 2006 Looze et al., 2009 Heijnen et al., 1999 Pan & Johnstone, 1983 Johnstone et al., 1987 Fevrier et al., 2004 Ogawa et al., 2008 Taylor & Gercel-Taylor, 2008 Skriner et al., 2006 Blanchard et al., 2002 Kesimer et al., 2009 Pisitkun, Shen & Knepper, 2004 Keller et al., 2007 Gonzales et al., 2009 Hogan et al., 2009 Mass Spectrometry Reviews DOI 10.1002/mas URINARY EXOSOMES For the stability of urinary exosomes, addition of protease inhibitor to urine sample increased the signal of Na–K–Cl cotransporter isoform 2 in Western blotting experiment to suggest that protease inhibitor prevented the protein from degradation (Zhou et al., 2006b). For storage and recovery of urinary exosomes, three different temperatures, 808C, 208C, and 48C, have been compared for periods of either 1 week or 7 months. Storage at 208C reduced the exosome recovery compared to fresh urine. Storage at 808C with extensive vortexing after thawing increased exosome recovery, even in cases where samples had been frozen for 7 months. The first and second morning urine, after normalizing with urine creatinine, showed similar exosome recovery; those data suggested low protein degradation in the bladder/urinary tract. For biomarker identification, the normalization step after obtaining biofluid from each animal or person is important (Zhou et al., 2006a,b). Normalization with urine flow rate is the best method. However, normalization with urine creatinine is practical for urinary exosome with spot urine. Briefly, the following is the method used to prepare urinary exosomes (Pisitkun, Shen, & Knepper, 2004). Protease inhibitor (1.67 mL of 100 mM NaN3/2.5 mL of 10 mM PMSF/50 mL of 1 mM Leupeptin per 50 mL urine) was used to treat urine immediately after collection. To eliminate debris such as cell-shedding membrane proteins, the urine was centrifuged at 17,000g for 15 min at 48C. The supernatant from the first centrifugation was ultracentrifuged again at 200,000g for 1 hr at 48C, and the pellet was suspended in fresh buffer (250 mM sucrose/10 mM triethanolamine/0.5 mM PMSF/ 1 mM leupeptin) and transferred to 1.5 mL of polypropylene tube. To remove THP, the most-abundant protein in urine, the solution was incubated with dithiothreithol for 2 min at 958C. Finally, the solution was re-ultracentrifuged, and the pellet was used in the subsequent study. As an alternative method, the ultrafiltration method with a 100 kDa MWCO filter can be used to prepare exosomes. In this method, filter containing urine is centrifuged at 3,000g for 10–30 min at room temperature, and the upper phase is used for urinary exosome capture. Whereas this method is rapid, it led to a low yield of urinary exosomes compared to Pisitkun and coworkers (Pisitkun, Shen, & Knepper, 2004; Cheruvanky et al., 2007). B. Digestion Methods To digest urinary exosomes, in-gel trypsin digestion has been used for gel slices from 1-D or 2-D SDS–PAGE. It is expected that a general in-solution method would not work well because the exosome is a small-vesicle structure that contains cytosolic proteins and membrane proteins. Therefore, it will increase the digestion efficiency of urinary exosomes by denaturation with detergent of membrane proteins on exosomes. As a similar method of in-gel trypsin digestion, Tube-Gel digestion (Lu & Zhu, 2005) and gel-assisted digestion (Han et al., 2008) methods were introduced. These two methods used detergent to denature membrane proteins in polyacrylamide gel without electrophoresis. For Tube-Gel digestion, protein, acrylamide, ammonium persulfate, and TEMED were mixed, and this solution was solidified in a small glass tube for 30 min at room temperature. The gel was cut into small pieces, and the steps that follow are similar to the in-gel Mass Spectrometry Reviews DOI 10.1002/mas & trypsin digestion method. This method increased the number of proteins and the coverage of proteins. Gel-assisted digestion is a modified method of Tube-Gel digestion. This digestion contains urea, EDTA, SDS, and polyacrylamide. The number of protein identified is three-times more in this gel-assisted digestion method than in with Tube-Gel digestion. In any event, these Tube-Gel and gel-assisted digestion methods are used to efficiently increase the number of proteins in several studies: for HeLa (Han et al., 2008) and prostate-cancer cell line (Lu & Zhu, 2005), mouse kidney (Han et al., 2008), and mouse-brain proteome study (Yu et al., 2007). Another approach to efficiently digest membrane protein is the chemical-digestion method. Membrane proteins contain transmembrane domains, which are difficult to identify with proteomics analysis. Therefore, it is necessary to eliminate membrane lipids for efficient digestion to identify membrane proteins with shotgun proteomics. Some methods to extract membrane proteins for a proteomics study were compared with human cell membrane proteins (Speers & Wu, 2007). Methods with acid-labile surfactant, urea, and organic solvent have been compared (Zhang et al., 2007). Finally, a total of 729 proteins were identified, and among them 44–50% were membrane proteins; that result suggested the necessity of lipid solubilization for an efficient identification of membrane proteins (Zhang et al., 2007). One of the methods to isolate and digest membrane proteins is to use CNBr in formic acid at room temperature (Washburn, Wolters, & Yates, 2001; Samyn, Sergeant, & Van Beeumen, 2006). CNBr digests the C-terminus of Methionine (M) at 48C or at room temperature. Also, formic acid can digest at either or both sides of aspartate (D) residues at high temperature (Li et al., 2001; Swatkoski et al., 2007; Zhang & Basile, 2007; Swatkoski et al., 2008). Because these chemical digestion methods produce too-large peptides for nano-LC MS/MS analysis, they need an additional protease treatment, such as with trypsin. IV. PROTEOMICS APPROACHES WITH URINARY EXOSOMES Several proteomic studies on urinary exosomes have been performed to identify biomarkers predicative of urinary track diseases. Pisitkun, Shen, and Knepper (2004) and Pisitkun, Johnstone, and Knepper (2006) first showed that the urine exosomes can enhance the detectability of relatively low-abundant proteins that have potential pathophysiological significance by isolating the exosomes (<100 nm) from urine samples collected from normal human subjects with differential centrifugation. In these studies, 295 proteins contained in these urinary exosomes were measured with LC-MS/MS analysis. These 295 proteins were suggested to be originated from epithelia that face the urine space throughout the urinary system. Also, a majority of these proteins were involved in (1) membrane trafficking, mostly in the endosomal pathway, as well as in (2) the membrane proteins (cargo proteins, transporters, channels, and peripheral-membrane proteins), and GTP-binding proteins (Rab, ARF, Rho, and Ral families). In addition to aquaporin-2, a urinary biomarker exploited in clinical studies of various waterbalance disorders, this study proposed several exosomal proteins that can be used as potential biomarkers. These exosomal proteins associate with kidney and systemic diseases, including autosomal dominant polycystic kidney disease (Polucystin-1), 7 & MOON ET AL. Gitelman syndrome (SLC12A3, a NaCl cotrasporter), Batter syndrome (Sodium potassium chloride cotransporter-2), autosomal recessive syndrome of osteopetrosis with renal tubular acidosis (Carbonic anhydrase 2), and familial renal hypomagnesemia (FXYD domain-containing ion transport regulator-2). Zhou et al. (2006a) from the same group also provided 27 proteins, whose abundances were increased (18 proteins) or decreased (9 proteins) between the exosomes isolated from urine samples of rats 8 hr after cisplatin injection to induce an acute kidney injury (AKI) with 2D-DIGE followed by MALDI-TOF-TOF analysis for the spots that showed differences in the two conditions. In this study, Fetuin-A was suggested as a novel urinary exosomal biomarker to detect acute kidney injury. Recently, Gonzales et al. (2009) from the same group further provided the comprehensive proteome and phosphoproteome profiles of urinary exosomes with LC-MS/ MS analyses. These profiles include 1,132 urinary exosomal proteins, among which 177 proteins are associated with various diseases according to the OMIM database, as well as 14 urinary exosomal phosphoproteins that correspond to 19 phosphorylation sites. In addition, the bladder cells, uroepithelial cells that line the bladder lumen, directly contact the urine space. Bladder cancer is the fourth most common cause of cancer in men in the United States (Jemal et al., 2006). Cytoscopy is the only method to diagnose the disease even if it is invasive with poor sensitivity. Therefore, it needs to identify biomarkers instead of this cytoscopy to diagnose and monitor this disease. Recently, the urinary exosomes originated from the bladder-cancer cells have been used to identify biomarkers for early diagnosis of bladder cancer (Smalley et al., 2008). They performed LC-MS/MS analysis on the urinary exosomes collected from five healthy individuals and four bladder cancer patients; eight proteins had elevated abundances in the urinary exosomes from the bladder-cancer patients. Among these eight proteins, five proteins are associated with EGFR pathway, whereas the other three proteins are alpha subunit of GsGTP-binding protein, resistin, and retinoic acid-induced protein 3. As an another study for bladder cancer, they presented proteomic analysis of exosomes, with high quality purification, from cultured HT1376 bladder cancer cells and identified 353 proteins with new 72 proteins not reported previously (Welton et al., FIGURE 4. Proteome profiles identified from the urinary exosomes from healthy individuals. A: The Venn diagram shows the overlaps between the urinary exosomal proteins found in two independent studies: Pisitkun et al. and Gonzales et al. (right), and Smalley et al. (left). Among a total of 1,224 proteins, 283 proteins were shared in the two data sets. B: The subcellular localization of the 283 shared proteins according to GO cellular components (GOCCs). C: The GO biological processes (GOBPs) in which the 283 shared proteins are involved. The number inside the parenthesis indicates the number of proteins that belong to the corresponding GO annotation out of the 283 shared proteins. D: The lists of proteins that belong to several GOBPs (C) that are likely to be closely associated with functions of urinary organs. 8 Mass Spectrometry Reviews DOI 10.1002/mas URINARY EXOSOMES in press). Among them, 18 proteins were confirmed as exosomal proteins by a combination of Western blotting, flotation on linear sucrose gradients and flow cytometry (Welton et al., in press). Some proteins such as CD36, CD44, 5T4, Basigin and CD73, detected from exosomes of cultured HT1376 bladder cancer cells were confirmed positive on urinary exosomes from a bladder cancer patient (Welton et al., in press). In addition to kidney and bladder, prostate is a small gland in men that is about the size and shape of a walnut (Weiss, Kaplan, & Fair, 2004). It lies at the base of the urinary TABLE 2. & bladder and surrounds the urethra which carries urine from the bladder and out of the body. Prostate cancer occurs when cells in the gland are damaged and start multiplying out of control and it is the second most common cause of cancer death in UK men (Kommu, Edwards, & Eeles, 2004; Mohammed et al., 2007). Until now, researchers have used levels of proteins, like prostate specific antigen (PSA), produced by cancer cells to try to spot the aggressive tumors (Cadeddu et al., 1993). Recently, to identify novel markers for prostate cancer with a new approach, proteomics analysis with serum Urogenital disease-related proteins identified from human urinary exosomes Diseases OMIM Proteins References DIABETES INSIPIDUS 125800 aquaporin 2 a,b FECHTNER SYNDROME 153640 myosin, heavy polypeptide 9, non-muscle a EPSTEIN SYNDROME 153650 myosin, heavy polypeptide 9, non-muscle a HYPERURICEMIC NEPHROPATHY 162000 CYSTINURIA uromodulin precursor a,b uromodulin precursor b 220100 solute carrier family 3, member 1 a RENAL GLUCOSURIA 233100 solute carrier family 5 (sodium/glucose cotransporter), member 2 a,b OSTEOPETROSIS WITH RENAL TUBULAR ACIDOSIS 259730 carbonic anhydrase II a,b Disease description CAUSE; ◦The inability of the renal collecting ducts to absorb water in response to arginine vasopressin ◦type I (X-linked recessive form) : Mutation in the gene encoding the vasopressin V2 receptor gene ◦type II (autosomal form) : Mutation in the AQP2 gene SYMPTOM; ◦Polyuria in kidneys ◦Lower urinary tract dilatation in bladder CAUSE; ◦Allelic mutations in the gene encoding nonmuscle myosin heavy chain-9 SYMPTOM; ◦Nephritis ◦End stage renal disease (20-40 years)(28% of patients) in kidneys CAUSE; ◦Mutation in the myosin heavy chain-9 nonmuscle gene SYMPTOM; ◦Nephritis ◦End stage renal disease (33% of patients) ◦Hypertension in kidneys CAUSE; ◦Mutation in the gene encoding uromodulin SYMPTOM; ◦Nephropathy ◦Renal failure CAUSE; ◦Mutation in the SLC3A1 amino acid transporter gene ◦Mutation in the SLC7A9 gene SYMPTOM; ◦ Nephrolithiasis CAUSE; ◦Mutation in the sodium/glucose cotransporter SGLT2 encoded by the SLC5A2 gene ◦Mutation in the SLC16A12 gene SYMPTOM; ◦Enuresis nocturna ◦Polyuria CAUSE; ◦Mutation in the gene encoding carbonic anhydrase II SYMPTOM; ◦Renal tubular acidosis CAUSE; ◦Mutation in the thiazide-sensitive Na-Cl GITELMAN SYNDROME 263800 fibrocystin isoform 2 a RENAL TUBULAR ACIDOSIS 267300 ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B1 a,b RENAL TUBULAR DYSGENESIS a,b angiotensin I converting enzyme isoform 1 precursor a,b pendrin a RENAL TUBULAR DYSGENESIS PENDRED SYNDROME ENLARGED VESTIBULAR AQUEDUCT 267430 274600 600791 pendrin a BARTTER SYNDROME 601678 sodium potassium chloride cotransporter 2 a,b RENAL TUBULAR ACIDOSIS 602722 ATPase, H+ transporting, lysosomal V0 subunit a4 a cotransporter SYMPTOM; ◦Polyuria ◦Renal potassium wasting ◦Renal magnesium wasting in kidneys CAUSE; ◦Mutation in the ATP6V1B1 (ATP6B1) gene SYMPTOM; ◦Kidney stones CAUSE; ◦Mutations in various genes(renin, angiotensinogen, angiotensin-converting enzyme, or angiotensin II receptor type 1) SYMPTOM; ◦Absence of differentiated proximal tubules, primitive renal tubules may exist ◦Thickening of renal arterial walls ◦Anuria CAUSE; ◦Mutation in the SLC26A4 gene ◦Heterozygous mutation in the FOXI1 gene CAUSE; ◦Mutation in the SLC26A4 gene ◦Heterozygous mutation in the FOXI1 gene CAUSE; ◦Mutation in the sodium-potassium-chloride cotransporter-2 gene SYMPTOM; ◦Renal salt wasting ◦Renal potassium wasting ◦Renal juxtaglomerular cell hypertrophy/hyperplasia ◦Polyuria ◦Nephrocalcinosis in kidneys CAUSE; ◦Mutation in the ATP6V0A4 (ATP6N1B) gene CAUSE; ◦Mutation in the gene encoding uromodulin MEDULLARY CYSTIC KIDNEY DISEASE 2 603860 uromodulin precursor a,b (UMOD; 191845) SYMPTOM; ◦Impaired renal function ◦Impaired renal creatinine clearance and renal uric acid clearance ◦Salt wasting and small kidneys ◦Tubulointerstitial nephritis and fibrosis ◦Interstitial inflammation ◦Disintegration of the tubular basement membrane ◦Progression to end stage renal failure in late adulthood (fifth to seventh decade) (Continued ) Mass Spectrometry Reviews DOI 10.1002/mas 9 & MOON ET AL. TABLE 2. (Continued ) RENAL TUBULAR ACIDOSIS AMYLOIDOSIS POLYCYSTIC KIDNEYS 604278 solute carrier family 4, sodium bicarbonate cotransporter, member 4 a 105200 lysozyme precursor fibrinogen, alpha polypeptide isoform alpha-E preproprotein apolipoprotein A-I preproprotein a,b protein kinase C and casein kinase substrate in neurons 2 a polycystin 1 isoform 1 precursor a uroplakin 3A precursor a,b a a,b CAUSE; ◦Mutation in the SLC4A4 gene SYMPTOM; ◦Proximal renal tubular acidosis ◦Renal bicarbonate wasting ◦Normal distal tubule acid excretion CAUSE; ◦Mutation in the apolipoprotein A1 gene, the fibrinogen alpha-chain gene, or the lysozyme gene. SYMPTOM; ◦Nephropathy with hematuria ◦Nephrotic syndrome and uremia CAUSE; ◦Mutations in any of several human disease 173900 loci. SYMPTOM; ◦Hypertension ◦Extrarenal cysts ◦Subarachnoid hemorrhage ◦Polycystic kidney ◦Renal failure CAUSE; ◦Mutations in the RET gene or in the RENAL ADYSPLASIA 191830 uroplakin IIIA gene SYMPTOM; ◦Renal adysplasia and renal agenesis ◦Renal dysplasia ◦Megalocystis CAUSE; ◦Contiguous gene deletion syndrome WILLIAMS-BEUREN SYNDROME 194050 eukaryotic translation initiation factor 4H isoform 1 a BARDET-BIEDL SYNDROME 209900 ADP-ribosylation factor-like 6 a resulting from the hemizygous deletion of several genes on chromosome 7q11.23 SYMPTOM; ◦Small kidneys ◦Solitary kidney and nephrocalcinosis ◦Renal insufficiency, renal artery stenosis in kidney ◦Vesicoureteral reflux in ureters ◦Bladder diverticula ◦Urethral stenosis ◦Recurrent urinary tract infections in bladder CAUSE; ◦Modification of CCDC28B gene ◦Mutations in MKS1 and MKS3 SYMPTOM; ◦Renal anomalies ◦Nephrogenic diabetes insipidus DONNAI-BARROW SYNDROME 222448 low density lipoprotein-related protein 2 a,b CAUSE; ◦Mutation in the LRP2 gene SYMPTOM; ◦Non-acidotic proximal tubulopathy in kidneys FRUCTOSE INTOLERANCE 229600 aldolase B, fructosebisphosphate b GM1-GANGLIOSIDOSIS, TYPE I 230500 galactosidase, beta 1 isoform a a,b ACTION MYOCLONUS-RENAL FAILURE SYNDROME 254900 scavenger receptor class B, member 2 a,b GLYCOGEN STORAGE DISEASE X 261670 bisphosphoglycerate mutase 2 b SIMPSON-GOLABI-BEHMEL SYNDROME, TYPE 2 300209 oral-facial-digital syndrome 1 protein b PHOSPHOGLYCERATE KINASE 1 DEFICIENCY 300653 phosphoglycerate kinase 1 b OROFACIODIGITAL SYNDROME I 311200 oral-facial-digital syndrome 1 protein b CAUSE; ◦Mutation in the gene encoding aldolase B SYMPTOM; ◦Proximal tubular acidosis in renal CAUSE; ◦Mutation in the gene encoding betagalactosidase-1 SYMPTOM; ◦Glomerular epithelial cytoplasmic vacuolization in kidneys CAUSE; ◦Mutations in the SCARB2 gene SYMPTOM; ◦Renal failure ◦Focal segmental glomerulosclerosis ◦Collapsing glomerulopathy in kidneys CAUSE; ◦Mutation in the gene encoding muscle phosphoglycerate mutase SYMPTOM; ◦Renal failure CAUSE; ◦Mutation in the CXORF5 gene in 1 affected family SYMPTOM; ◦Multicystic kidneys CAUSE; ◦Mutation in the PGK1 gene SYMPTOM; ◦Renal failure with myoglobinuria CAUSE; ◦Mutations in the CXORF5 gene SYMPTOM; ◦Adult onset polycystic kidney (50%) in kidneys (Continued ) from mice grafted with human prostate cancer xenografts were processed and identified 44 tumor-derived proteins (Jansen et al., 2009). Several proteins such as CD9 and proteasome b1 (PSMZB1) were identified in the exosome fraction isolated from the prostate cancer PC346C cell line (Jansen et al., 2009). Also, they showed that these exosomes contained RNAs, including the gene fusion TMPRSS2-ERG product. Furthermore, the RNAs of TMPRSS2-ERG and PCA-3 were detected in urinary exosomes from prostate cancer patients (Nilsson et al., 2009). This suggested that cancer-derived urinary exosomes offer possibilities for the identification of novel biomarkers for prostate cancer. 10 Until now, proteome profiles identified from the urinary exosomes from healthy individuals are compared and analyzed in this study (Fig. 4). Figure 4A shows the relationships between 1,081 proteins detected from Pisitkun, Shen, and Knepper (2004) and Gonzales et al. (2009) and 426 proteins identified from Smalley et al. (2008). Two hundred eighty three proteins (23.1%) are shared in the data sets. Figure 4B shows that a majority of the shared proteins are localized in vesicle-related organelles (e.g., cytoplasmic and membrane-bound vesicles, early and late endosome, lysosome, and ER-Golgi intermediate compartment), plasma membrane and extracellular region. Figure 4C shows that the shared proteins are involved in the Mass Spectrometry Reviews DOI 10.1002/mas URINARY EXOSOMES & TABLE 2. (Continued ) CAUSE; ◦Homozygous deletion on chromosome HYPOTONIA-CYSTINURIA SYNDROME 606407 GLOMERULOCYSTIC KIDNEY DISEASE WITH HYPERURICEMIA AND ISOSTHENURIA 609886 solute carrier family 3, member 1 a uromodulin precursor a,b uromodulin precursor b 2p21 that disrupts the SLC3A1 and PREPL genes SYMPTOM; ◦Nephrolithiasis in kidney ◦Bladder cystine calculi in bladder CAUSE; ◦Mutation in the UMOD gene SYMPTOM; ◦Cystic dilatation of the Bowman space ◦The initial proximal convoluted tubule CAUSE; ◦Heterozygous mutation in the APOE gene SYMPTOM; ◦Proteinuria LIPOPROTEIN GLOMERULOPATHY 611771 apolipoprotein E precursor a,b NEPHROLITHIASIS/OSTEOPOROSIS, HYPOPHOSPHATEMIC, 2 612287 solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 1 a,b 612624 angiotensin I converting enzyme isoform 2 precursor angiotensin I converting enzyme isoform 1 precursor MICROVASCULAR COMPLICATIONS OF DIABETES, SUSCEPTIBILITY TO, 3 a,b a,b 607832 CD2-associated protein a,b HYPEROXALURIA, PRIMARY, TYPE II 260000 glyoxylate reductase/hydroxypyruvate reductase a 2,8-DIHYDROXYADENINE UROLITHIASIS 145500 angiotensin I converting enzyme isoform 2 precursor angiotensin I converting enzyme isoform 1 precursor membrane alanine aminopeptidase precursor dimethylarginine dimethylaminohydrolase 1 glutamyl aminopeptidase (aminopeptidase A) hydroxyprostaglandin dehydrogenase 15-(NAD) membrane metalloendopeptidase CAUSE; ◦Mutation in the SLC9A3R1 gene SYMPTOM; ◦Nephrolithiasis ◦Renal phosphate wasting in kidneys FOCAL SEGMENTAL GLOMERULOSCLEROSIS 3 HYPERTENSION ◦Progressive kidney failure ◦Distinctive lipoprotein thrombi in glomerular capillaries CAUSE; ◦Variation in the gene encoding angiotensin I-converting enzyme (ACE; 106180) on chromosome 17q23 Haploinsufficiency for CD2-associated protein (CD2AP; 604241) increases the risk of focal segmental glomerulosclerosis (FSGS). Focal segmental glomerulosclerosis (FSGS) is a histopathologic finding in several renal disorders characterized by proteinuria and progressive decline in renal function. CAUSE; ◦Mutation in the glyoxylate reductase/hydroxypyruvate reductase gene SYMPTOM; ◦Calcium oxalate urolithiasis a,b a,b a,b Variations in many genes contribute to essential hypertension. Examination of the biochemical processes that effect blood pressure homeostasis should elucidate some of the interactive physiologic regulators that malfunction in persons with elevated pressure and show whether single genes of large effect are important in some. a a,b a a,b adenine phosphoribosyltransferase isoform a a adenine phosphoribosyltransferase isoform b a 102600 PROXIMAL RENAL TUBULAR ACIDOSIS 114760 carbonic anhydrase IV precursor a,b RENAL CELL CARCINOMA 118955 clathrin heavy chain 1 a Adenine phosphoribosyltransferase catalyzes the formation of AMP from adenine and phosphoribosylpyrophosphate. It can act as a salvage enzyme for recycling of adenine into nucleic acids. Complete or partial deficiency of APRT can lead to accumulation of the insoluble purine 2,8dihydroxyadenine (DHA). Renal failure in kidney, and urolithiasis in ureters are symptom of 2,8dihydroxyadenine urolithiasis. Carbonic anhydrases (CAs) are a family of zinc metalloenzymes. For background information on the CA family. CA IV is a glycosylphosphatidylinositolanchored membrane isozyme expressed on the luminal surfaces of pulmonary (and certain other) capillaries and on the luminal surface of proximal renal tubules. CA IV has ancient evolutionary status among CA isozymes. It is functionally important in CO2 and bicarbonate transport and has a possible role in inherited renal abnormalities of bicarbonate transport. Clathrin is a major protein component of the cytoplasmic face of intracellular organelles, called coated vesicles and coated pits. These specialized organelles are involved in the intracellular trafficking of receptors and endocytosis of a variety of macromolecules. Clathrin molecules have a triskelion structure composed of 3 noncovalently bound heavy chains (CLTC) and 3 light chains. (Continued ) cellular processes including vesicle-related processes (e.g., secretion, vesicle-mediated transport, membrane fusion, actin cytoskeleton organization, and biogenesis), small GTPasemediated signal transduction, regulation of body-fluid levels (vasodilation), inflammatory response, cell motility, and blood vessel development. Figure 4D summarizes a list of the proteins that belong to several cellular processes that might reflect physiology of the urinary organs. Six proteins (KNG1, SERPINA1, SERPING1, ENPEP, ANPEP, and MASP2) are involved in regulation of body fluid levels. KNG1, a bradykinin upstream regulator, has been known to regulate the Mass Spectrometry Reviews DOI 10.1002/mas permeability of glomerular basement membrane (GBM) to various molecules (Iarovaia, 2001; Pike et al., 2005). The protein and chemical transporters (ABCB1, AQP2, SLC2A5, SLC12A1, and SLC12A3) in the urinary exosomes might provide the state of the filtration system in the proximal tubule. In particular, AQP2, an apical plasma-membrane protein, has been suggested as an indicator of water-balance in the filtration system (Funayama et al., 2004). These proteomic studies have generated comprehensive proteome profiles of the urinary exosomes from healthy individuals and patients with kidney disease and bladder 11 & MOON ET AL. TABLE 2. (Continued ) NEPHROPATHIC CYSTINOSIS 219800 cystinosis, nephropathic isoform 1 a HYPOMAGNESEMIA 2, RENAL 154020 FXYD domain-containing ion transport regulator 2 isoform 1 a podocin a AUTOSOMAL RECESSIVE STEROIDRESISTANT NEPHROTIC SYNDROME 600995 CAUSE; ◦Misrouting of the Na+,K(+)-ATPase gamma subunit CAUSE; ◦Mutations in the NPHS2 gene(podocin) SYMPTOM; ◦Massive proteinuria ◦Hypoalbuminemia, hyperlipidemia and edema ◦Minimal glomerular changes. CAUSE; ◦Mutation in the gene encoding fibrocystin. SYMPTOM; ◦Enlarged kidneys 601313 polycystin 1 isoform 1 precursor a POLYCYSTIC KIDNEY DISEASE, ADULT, TYPE II 173910 polycystin 2 a DEFECTIVE KIDNEY ACID SECRETION LEADING TO DISTAL RENAL TUBULAR ACIDOSIS 179800 solute carrier family 4, anion exchanger, member 1 a GLUCOSE/GALACTOSE MALABSORPTION 606824 solute carrier family 5 (sodium/glucose cotransporter), member 1 a 234500 solute carrier family 6, member 19 RENAL HYPOURICEMIA 220150 urate anion exchanger 1 isoform a a METACHROMATIC LEUKODYSTROPHY DUE TO SAPOSIN B DEFICIENCY 249900 prosaposin isoform a preproprotein a,b FRONTOTEMPORAL DEMENTIA, CHROMOSOME 3-LINKED 600795 prosaposin isoform c preproprotein a a ◦Renal failure, ◦Renal calculi (urate and calcium oxalate) in kidneys SYMPTOM; ◦Renal magnesium wasting in kidneys POLYCYSTIC KIDNEY DISEASE, ADULT, TYPE I HARTNUP DISORDER CAUSE; ◦Mutation in the gene encoding cystinosin SYMPTOM; ◦Renal tubular Fanconi syndrome a ◦Cystic kidneys and renal failure ◦Increased echogenicity of entire parenchyma ◦Loss of corticomedullary differentiation ◦Interstitial fibrosis in kidneys SYMPTOM; ◦Enlarged kidneys ◦Cystic kidneys acd renal failure ◦Increased echogenicity of entire parenchyma ◦Interstitial fibrosis in kidneys ◦Polycystic kidney CAUSE; ◦Mutation in the SLC4A1 gene SYMPTOM; ◦Nephrocalcinosis CAUSE; ◦Mutation in the intestinal sodium/glucose transporter CAUSE; ◦Mutations in the SLC6A19 gene SYMPTOM; ◦Pellagra-like light-sensitive rash ◦Cerebellar ataxia ◦Emotional instability and amino aciduria CAUSE; ◦Mutation in the SLC22A12 gene SYMPTOM; ◦Uric acid urolithiasis ◦Uric acid nephrolithiasis ◦Tubular defect in presecretory reabsorption of urate ◦Exercise-induced acute renal failure with acute tubular necrosis in kidneys CAUSE; ◦Mutation in the prosaposin gene SYMPTOM; ◦Incontinence in bladder CAUSE; ◦Mutation in the CHMP2B gene SYMPTOM; ◦Urinary incontinence in bladder Pisitkun, Shen, and Knepper (2004) and Gonzales et al. (2009). Smalley et al. (2008). b cancer, identified differentially expressed proteins (DEPs) from the detected proteome between healthy and diseased conditions, and additionally proposed some of these DEPs that can be validated with Western blotting (Zhou et al., 2006a) as potential biomarkers for the investigated diseases. Table 2 summarizes the association of all the proteins detected from the above studies with urinary track diseases. V. CONCLUSION In this review, we described the characteristics of urinary exosomes, and methods for proteomics approaches to urinary exosomes, and showed proteomic profiles of urinary exosomes that may reflect pathophysiology of urogenital diseases. Many exosomes were identified from various cells, tissues, and body fluids, and urinary exosomes have also been identified recently and added into the existing exosomes category. Exosomes secreted from various cells contain proteins and RNAs, and play important roles in cell–cell communications among exosomes-secreting cells and exosomes-accepting cells. However, studies on the mechanism related to communications among cells via urinary exosomes have been not reported. It might be 12 difficult to investigate the details of this mechanism because interactions with other cells during urine production could be limited. Even though natural functions of urinary exosomes remain to be studied, the urinary exosomes have many advantages as starting materials for biomarker identification. The identification of candidate biomarkers relevant to pathophysiology of specific diseases could be very promising to proceed to validation steps with a large-scale human study. Urinary exosomes might be a valuable target to identify biologically meaningful biomarkers, finally available in clinics with assistance of systems approaches based on proteomics and/or microarray data derived from urinary exosomes, ‘‘informative vesicles.’’ VI. ABBREVIATIONS 2-D PAGE APCs AQP CE-MS DCs DEPs 2 dimensional polyacrylamide gel electrophoresis antigen-presenting cells aquaporin capillary electrophoresis mass spectrometry dendritic cells differentially expressed proteins Mass Spectrometry Reviews DOI 10.1002/mas URINARY EXOSOMES Eps15 ESCRT GBM Hrs ILVs IMAC MHC MPs MVBs MVs MWCO NCC PMSF SELDI-TOF MS SORM STED THP Tsg101 Vps epidermal growth factor receptor substrate 15 endosomal sorting complex required for transport glomerular basement membraney hepatocyte growth factor-regulated kinase substrate intralumenal vesicles immobilized metal-ion chromatography major histocompatibility complex microparticles multivesicular bodies microvesicles molecular weight cut off thiazide-sensitive Na–Cl cotransporter phenylmethanesulphonylfluoride surface enhanced laser desorption/ ionization time-of-flight mass spectrometry stochastic optical reconstruction microscopy stimulated emission depletion Tamm–Horsfall protein tumor susceptibility gene 101 vacuolar protein-sorting ACKNOWLEDGMENTS This work was supported by the Grant of the Korean Ministry of Education, Science and Technology (The Regional Core Research Program/Anti-aging and Well-being Research Center) and supported by the grant No. RTI04-01-01 from the Regional Technology Innovation Program of the Ministry of Knowledge Economy (MKE). 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Capillary electrophoresis coupled to mass spectrometry for biomarker discovery and diagnosis of kidney diseases. Contrib Nephrol 160:107–126. Pyong-Gon Moon received his M.Med. in School of Medicine from Kyungpook National University, Republic of Korea in 2008. He is currently a Ph.D. candidate in biomarker discovery and proteomics group at School of Medicine of Kyungpook National University. His main research interests are clinical proteomics, mass spectrometry-based urinary proteomics, and urinary exosomes under supervision of Prof. Moon-Chang Baek. Sungyong You received his M.Sc. in Pharmacy from Seoul National University, Republic of Korea in 2005. He is currently a Ph.D. candidate at Pohang University of Science and Technology. His research involves developing systems medicine approaches for finding key regulators associated with underlying mechanisms of complex human diseases under supervision of Prof. Daehee Hwang. Jeong-Eun Lee obtains a master at School of Medicine of Kyungpook National University, Republic of Korea in 2009. She is currently a Ph.D. candidate in the medical science at Kyungpook National University. She is interested in defining the relationship between post-translational modification and signaling pathway using proteomic approach under supervision of Prof. Moon-Chang Baek. Daehee Hwang during the Ph.D. study in Department of Chemical Engineering at MIT, he had participated in several systems biology projects on mortor neuron diseases. In 2003, he pursued his postdoctoral career at the Institute for Systems Biology where he developed (1) data integration and proteomic data analysis tools, (2) a systems medicine approach for understanding complex human diseases. In 2006, he joined POSTECH in Korea as a junior faculty and is currently engaged in developing integrative genomic/proteomic data analysis pipelines and various systems biology frameworks for finding key regulators associated with underlying mechanisms in several complex human diseases. Moon-Chang Baek received his Ph.D. degree in Pharmacy from Seoul National University in 1997 and has held post-doctoral fellow positions in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School (1998–2002) as well as in the Department of Life Science at POSTECH (2002–2005). During the periods, he has studied on traditional proteomics using various mass spectrometries (MALDI-TOF, LCQ, and Q-TOF) and also developed a fusion technology, called Ligand Profiling and Identification (LPI) technology, briding between cell biology and proteomics. He has been serving as an assistant professor at School of Medicine of Kyungpook National University since 2005. He has currently focused on the identification of biomarker candidates for kidney diseases by developing various proteomics technologies including glycoproteomics, membrane proteomics as well as enrichment technologies for clinic specimen analysis. 18 Mass Spectrometry Reviews DOI 10.1002/mas