JASN Express. Published on December 3, 2008 as doi: 10.1681/ASN.2008040406 BASIC RESEARCH www.jasn.org Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes Patricia A. Gonzales,*† Trairak Pisitkun,* Jason D. Hoffert,* Dmitry Tchapyjnikov,* ʈ Robert A. Star,‡ Robert Kleta,§ ¶ Nam Sun Wang,† and Mark A. Knepper* *Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, ‡Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, §Section of Human ʈ Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, and Office of Rare Diseases, Office of the Director, National Institutes of Health, Bethesda, and †Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland; and ¶London Epithelial Group, Centre for Nephrology, University College London, London, United Kingdom ABSTRACT Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phospho- proteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. J Am Soc Nephrol ●●: –, 2009. doi: 10.1681/ASN.2008040406 Urinary exosomes are small extracellular vesicles In this study, we thoroughly expanded the (Ͻ100 nm in diameter) that originate from the in- known proteome of human urinary exosomes by ternal vesicles of multivesicular bodies (MVB) in using a highly sensitive LC-MS/MS system, im- renal epithelial cells, including glomerular podo- proved software for identification of peptide ions cytes, renal tubule cells, and the cells lining the uri- and a more elaborate data analysis strategy than in nary drainage system.1 Exosomes are released into our previous study. In addition, we used a neutral the urine when the outer membrane of the MVB loss scanning approach4 to investigate the phos- fuses with the apical plasma membrane of the epi- phoproteome of human urinary exosomes. The thelial cell. study identified 1412 proteins including 14 phos- Exosomes can be recovered from the urine by differential centrifugation as a low-density mem- Received April 21, 2008. Accepted July 30, 2008. brane fraction. Exosome isolation can result in Published online ahead of print. Publication date available at marked enrichment of low-abundance urinary pro- www.jasn.org. teins that have potential pathophysiologic signifi- Correspondence: Dr. Mark A. Knepper, 10 Center Drive, MSC- cance. As a consequence, we and others have been 1603, National Institutes of Health, Bethesda, MD 20892-1603. working to define optimal conditions for their iso- Phone: 301-496-3064; Fax: 301-402-1443; E-mail: knep@helix. lation and purification as a prelude to their use in nih.gov biomarker discovery studies.1–3 Copyright ᮊ ●●●● by the American Society of Nephrology J Am Soc Nephrol ●●: –, 2009 ISSN : 1046-6673/●●00- 1 BASIC RESEARCH www.jasn.org phoproteins in human urinary exosomes. Overall, there are port into intracellular organelles.6 These proteins also mediate 177 proteins that are associated with diseases as judged by their proton transport across the apical plasma membrane of type A presence on the Online Mendelian Inheritance in Man intercalated cells and across the basolateral plasma membrane (OMIM) database, 34 of which are known to be associated with of type B intercalated cells.7 The B1 subunit is selectively ex- renal diseases. The potential clinical usefulness of urinary exo- pressed in intercalated cells, and its detection in urinary exo- somes was demonstrated using the well-defined renal tubu- somes establish that intercalated cells secrete exosomes as do lopathy, Bartter syndrome type I, as an example. The rich in- other types of epithelial cells lining the renal tubule. These formation from the proteomic analysis also provides further proteins constitute 78% of the subunits of the V0 and V1 do- ϩ insight into the biogenesis of urinary exosomes. mains of the vacuolar H -ATPase.8 An example of the utility of exosome analysis is shown in Fig- ure 1, describing immunoblotting in patients with Bartter syn- RESULTS drome type I, associated with mutations in the SLC12A1 gene, which encodes for the NKCC2 sodium-potassium-chloride co- Large-Scale Proteomic Profiling of Human Urinary transporter protein.9 The NKCC2 protein was found in the pro- Exosomes teome of the human urinary exosomes as shown in Table 1. Urine In this study, we carried out proteomic profiling of a low- samples were obtained from two patients (patients 1 and 2) with density membrane fraction from human urine consisting clinical phenotypes consistent with Bartter syndrome type I (Fig- chiefly of exosomes, using a highly sensitive LC-MS/MS sys- ure 1A).10 The clinical diagnosis for the patients with Bartter syn- tem, based on an ion trap mass spectrometer (LTQ; Thermo- drome type I was confirmed by the ultrasound images showing Finnigan; Thermo Electron, San Jose, CA). We unambiguously deposits of calcium in the kidney also known as nephrocalcino- identified 1132 proteins including 205 proteins seen in our sis9,10 (Figure 1B) and other typical laboratory findings. The two previous study and 927 proteins not seen in our previous study urinary exosome samples obtained from the patients with Bartter of human urinary exosomes.1 The full list (ambiguous and syndrome type I were analyzed by immunoblotting for the pres- unambiguous identifications) contains 1412 proteins and can ence of the NKCC2 protein (Figure 1C). Compared with the re- be viewed in Supplemental Table 1, and the list of proteins that spective control samples, patients 1 and 2 showed an absence of were unambiguously identified in both studies can be viewed the NKCC2 protein bands, expected at 160 kD for monomeric at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. The ex- NKCC2 and 320 kD for dimeric NKCC2. In addition, the samples panded list of exosomal proteins includes 177 proteins that are (patients 1 and 2) were probed for the thiazide-sensitive co-trans- disease related, on the basis of their presence in the OMIM porter (NCC) protein to ensure that urinary exosomes were suc- database (Table 1). cessfully isolated and loaded properly. Strong NCC bands were Predictably, a large number of proteins that were identified obtained in samples from both patients with Bartter syndrome were integral membrane proteins involved in solute and water type I and control samples. transport (Table 2). As seen in our previous study,1 these pro- teins predominantly represent apical transporters present in Phosphoproteomic Analysis of Human Urinary every renal tubule segment, including the proximal tubule (so- Exosomes dium-hydrogen exchanger 3, sodium-glucose co-transporter 1 Protein phosphorylation is a key element of most cell regula- and 2, and aquaporin-1 [AQP1]), the thick ascending limb tory processes. Recently, technical approaches that allow phos- (sodium-potassium-chloride co-transporter 2 [NKCC2]), the phoproteomic profiling on a large scale have been intro- distal convoluted tubule (thiazide-sensitive Na-Cl co-trans- duced.4,11–13 We used neutral loss scanning with high- porter [NCC]), and connecting tubule/collecting duct (AQP2, stringency target-decoy analysis to identify phosphorylation rhesus blood group C glycoprotein [RhCG, an ammonia chan- sites present in exosomal proteins from human urine samples. nel], B1 subunit of vacuolar Hϩ-ATPase, and pendrin). Note Nineteen phosphorylation sites corresponding to 14 phos- that both polycystin-1 and polycystin-2 were detected in hu- phoproteins were identified (Table 5). These included both man urinary exosomes. newly identified phosphorylation sites and sites that had been Exosomes derive from MVB and are delivered to the urine previously identified. Two orphan G-protein–coupled recep- when the outer membranes of MVB fuse with the apical plasma tors are included in the former group, viz. GPRC5B and membrane. Interestingly, 22 of the proteins identified in this GPRC5C. In GPRC5B, we identified one new phosphorylation study are recognized as components of the apparatus respon- site, T389, and, in GPRC5C, we identified three new phosphor- sible for the formation of MVB (Table 3). These 22 proteins ylation sites, T435, S395, and Y426. These proteins are also account for approximately 75% of the proteins that constitute known as retinoic acid–induced gene 2 (GPRC5B) and retinoic the ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III complexes acid–induced gene 3 (GPRC5C). involved in multivesicular body formation.5 A new phosphorylation site was also identified in the In