Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine
2013; Elsevier BV; Volume: 85; Issue: 5 Linguagem: Inglês
10.1038/ki.2013.422
ISSN1523-1755
AutoresMarie C. Hogan, Kenneth L. Johnson, Roman M. Zenka, M. Cristine Charlesworth, Benjamin Madden, Doug W. Mahoney, Ann L. Oberg, Bing Huang, Alexey A. Leontovich, Lisa Nesbitt, Jason L. Bakeberg, Daniel McCormick, H. Robert Bergen, Christopher J. Ward,
Tópico(s)Complement system in diseases
ResumoUrinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm–Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)–enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm–Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)–enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. Urinary microparticles known as exosome-like vesicles (ELVs) are derived from two distinct cellular sources: the multivesicular body (true exosomes) and apical cell membrane (membrane vesicles).1Keller S. Sanderson M.P. Stoeck A. et al.Exosomes: from biogenesis and secretion to biological function.Immunol Lett. 2006; 107: 102-108Crossref PubMed Scopus (670) Google Scholar, 2Johnstone R.M. Adam M. Hammond J.R. et al.Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes).J Biol Chem. 1987; 262: 9412-9420Abstract Full Text PDF PubMed Google Scholar, 3Johnstone R.M. Revisiting the road to the discovery of exosomes.Blood Cells Molecules Diseases. 2005; 34: 214-219Crossref PubMed Scopus (125) Google Scholar, 4Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1623) Google Scholar, 5Pisitkun T. Johnstone R. Knepper M.A. Discovery of urinary biomarkers.Mol Cell Proteomics. 2006; 5: 1760-1771Crossref PubMed Scopus (342) Google Scholar, 6Knepper M.A. Pisitkun T. Exosomes in urine: who would have thought...?.Kidney Int. 2007; 72: 1043-1045Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar They are shed from the entire genitourinary epithelium (kidney, urothelium, prostate, and bladder), and an important sub-population appears to originate from the glomerulus. These could be a rich source of biomarkers permitting noninvasive assessment of glomerular health.5Pisitkun T. Johnstone R. Knepper M.A. Discovery of urinary biomarkers.Mol Cell Proteomics. 2006; 5: 1760-1771Crossref PubMed Scopus (342) Google Scholar Bulk ELV subfractionation has not been possible owing to large amounts of the Tamm–Horsfall protein (THP) in urine, as under physiological conditions THP oligomerizes into long double-helical strings.7Bayer M.E. An electron microscope examination of urinary mucoprotein and its interaction with influenza virus.J Cell Biol. 1964; 21: 265-274Crossref PubMed Scopus (21) Google Scholar In centrifugation-based protocols, it tends to precipitate under high 'g' forming a gel that traps and sequesters ELVs.8Fernandez-Llama P, Khositseth S, Gonzales PA et al. Tamm-Horsfall protein and urinary exosome isolation. Kidney Int 77: 736–742.Google Scholar A variety of techniques have been used to reduce the amount of THP in ELV preparations, the most important being the use of dithiothreitol to reduce the disulfide bonds in the THP ZP (Zona pellucida) domains, abolishing its ability to oligomerize, but even under these conditions4Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1623) Google Scholar significant amounts of reduced THP still precipitate upon ultracentrifugation, and it dominates the proteomic landscape of urine ELVs especially in the range 80–110kDa.4Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1623) Google Scholar,8Fernandez-Llama P, Khositseth S, Gonzales PA et al. Tamm-Horsfall protein and urinary exosome isolation. Kidney Int 77: 736–742.Google Scholar Another approach is to generate an exclusion list of THP MS1 peptide masses, which can be used to exclude THP peptides from further analysis in the MS2 dimension (which generates sequence data).9Hiemstra T.F. Charles P.D. Hester S.S. et al.Uromodulin exclusion list improves urinary exosomal protein identification.J Biomol Tech. 2011; 22: 136-145PubMed Google Scholar However, this runs the risk of excluding peptides from non-THP proteins, although it tends to uncover more peptides than it loses.9Hiemstra T.F. Charles P.D. Hester S.S. et al.Uromodulin exclusion list improves urinary exosomal protein identification.J Biomol Tech. 2011; 22: 136-145PubMed Google Scholar Our D2O 5–30% sucrose gradient method pellets the vast majority of the THP to the bottom of the ultracentrifuge tube (see Figure 2b and d in Hogan et al10Hogan M.C. Manganelli L. Woollard J.R. et al.Characterization of PKD protein-positive exosome-like vesicles.J Am Soc Nephrol. 2009; 20: 278-288Crossref PubMed Scopus (239) Google Scholar), with ELV subfractions banding at different specific densities.10Hogan M.C. Manganelli L. Woollard J.R. et al.Characterization of PKD protein-positive exosome-like vesicles.J Am Soc Nephrol. 2009; 20: 278-288Crossref PubMed Scopus (239) Google Scholar In prior studies, we identified ELV subfractions enriched for specific kidney disease proteins, including a fraction containing the major polycystic kidney disease proteins polycystin 1, 2 and fibrocystin (all thought to be of tubular origin), which permitted examination of the post-translational processing of these proteins and provided the first in vivo evidence of polycystin 1 G protein coupled receptor proteolytic site domain cleavage.10Hogan M.C. Manganelli L. Woollard J.R. et al.Characterization of PKD protein-positive exosome-like vesicles.J Am Soc Nephrol. 2009; 20: 278-288Crossref PubMed Scopus (239) Google Scholar, 11Arac D. Boucard A.A. Bolliger M.F. et al.A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (272) Google Scholar, 12Bakeberg J.L. Tammachote R. Woollard J.R. et al.Epitope-tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin.J Am Soc Nephrol. 2011; 22: 2266-2277Crossref PubMed Scopus (51) Google Scholar Along with others, we have also identified podocin and several other glomerular disease proteins in ELVs.4Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1623) Google Scholar,13Gonzales P.A. Pisitkun T. Hoffert J.D. et al.Large-scale proteomics and phosphoproteomics of urinary exosomes.J Am Soc Nephrol. 2009; 20: 363-379Crossref PubMed Scopus (556) Google Scholar, 14Zhou H. Yuen P.S. Pisitkun T. et al.Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.Kidney Int. 2006; 69: 1471-1476Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 15Wang Z. Hill S. Luther J.M. et al.Proteomic analysis of urine exosomes by multidimensional protein identification technology (MudPIT).Proteomics. 2012; 12: 329-338Crossref PubMed Scopus (140) Google Scholar, 16Raj D.A.A. Fiume I. Capasso G. et al.A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes.Kidney Int. 2012; 81: 1263-1272Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar A comprehensive analysis of the shed glomerular membrane vesicle (GMV) proteome together with the measurement of intra-individual variability is required before attempts are made to study the GMV proteome in preparation for biomarker discovery studies. By using D2O 5–30% sucrose gradient density centrifugation, we have now focused our isolation method to study the GMV subfraction. Antibodies to podocalyxin and podocin, regarded as major podocyte surface antigens (the visceral epithelial cells of Bowman's capsule), were used to study their morphology.17Kershaw D.B. Beck S.G. Wharram B.L. et al.Molecular cloning and characterization of human podocalyxin-like protein. Orthologous relationship to rabbit PCLP1 and rat podocalyxin.J Biol Chem. 1997; 272: 15708-15714Crossref PubMed Scopus (121) Google Scholar,18Mundel P. Shankland S.J. Podocyte biology and response to injury.J Am Soc Nephrol. 2002; 13: 3005-3015Crossref PubMed Scopus (579) Google Scholar We performed a comprehensive proteomic analysis of this subfraction enriched in GMVs (apical membrane vesicles) and then assessed the overlap of our GMV proteome with the published glomerular tissue and urine exosome proteomes.19Miyamoto M. Yoshida Y. Taguchi I. et al.In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry.J Proteome Res. 2007; 6: 3680-3690Crossref PubMed Scopus (52) Google Scholar We studied the post-translational processing of a number of known glomerular disease proteins for the first time in vivo and provide these data in a searchable database. Urine was collected from seven healthy volunteers, four male and three female volunteers, aged 17–34 years (mean age 27 years, average albumin/creatinine ratio 1.63mg/g (0–5.76mg/g) (IRB #09–003355), normal random urine albumin excretion <17mg/g creatinine (males) and 60ml/min42ml/min/SAaMeasured by 24-h urine creatinine clearance.60ml/minTreatmentLosartan 100mg/dFurosemide 40mg/dayCyBorD protocolbCyBorD protocol: 150mg/m2 of cyclophosphamide po once weekly (dose reduced for renal function and age), 20mg of dexamethasone po once weekly, 3mg (1.5mg/m2) of subcutaneous bortezomib once weekly. Each cycle consisted of four treatments, usually 3–6 cycles. cycle 1Amlodipine 5mg/dayBumetanide 0.5mg qodMetoprolol succinate 25mg/dayLisinopril/HCTZ 30–37.5mg/dayLisinopril 40mg/dayMetoprolol succinate 25mg/dayProteins identified325831974189Abbreviations: eGFR, estimated glomerular filtration rate; SA, corrected to body surface area.a Measured by 24-h urine creatinine clearance.b CyBorD protocol: 150mg/m2 of cyclophosphamide po once weekly (dose reduced for renal function and age), 20mg of dexamethasone po once weekly, 3mg (1.5mg/m2) of subcutaneous bortezomib once weekly. Each cycle consisted of four treatments, usually 3–6 cycles. Open table in a new tab Abbreviations: eGFR, estimated glomerular filtration rate; SA, corrected to body surface area. We obtained 270ml of fresh urine from each of seven individuals and centrifuged the samples at low g-force to remove cells and debris, and then ultracentrifuged them at 150,000g for 1h to pellet a mixture of THP and ELVs, termed 'crude exosomes'. THP was further removed from the ELV fractions by centrifugation on a 5–30% sucrose gradient in D2O (200,000g × 24h). We found three distinct bands of visible ELVs scattering light when the 5–30% sucrose D2O gradient was illuminated along its long axis and which we could reproducibly and accurately collect (Figure 1a).20Hogan M. In-Depth Proteomic Analysis of Podocin-Rich Exosomes in Human Urine. JASN, Denver, CO, USA2010Google Scholar We designated the three bands as A, B, and C: A being the lightest, RI η=1.3436 s.d.±0.00124, B the band of intermediate density (PKD-ELVs), RI η=1.3539 s.d.±0.000831, and C the highest density (GMVs), RI η=1.3625 s.d.±0.000911. On transmission electron microscopy of the purified ELV fractions, fraction A was characterized by the presence of large ∼200-nm ELVs, which have a classical 'punched-out soccer ball' appearance (median diameter 93.0nm (interquartile range (IQR) 75.3–128.8nm) with a right skewed distribution), fraction B contained mainly classical ELVs (median diameter 79.4nm (IQR 54.6–103.9nm), and GMVs (membrane particles) were most abundant in fraction C (median diameter 72.1nm (IQR 50.2–93.8nm), left skew). In fraction C, there were smaller membrane fragments that lacked the distinct appearance of classical ELVs, stained poorly with uranyl acetate, and were podocin/podocalyxin positive (termed GMVs). GMVs accounted for 23.3% of all particulate content in fraction B and 44.7% of particulate content in the fraction C grids surveyed, with the remainder in each being 'classical' ELVs that did not stain for podocin/podocalyxin (Figures 1c and 2a). The median classical exosome diameter was 91.4nm (IQR 76.9–109.2nm) compared with a GMV diameter of 45.8nm (IQR 35.9–56.1nm; P-value<2e-16, Wilcoxon test) in fraction C (Figure 2a and b). Using Kolmogorov–Smirnov permutation testing, we also observed a clear shift indicating a difference in the diameters of two particle types (P<0.0001; Supplementary Figure S1 online). Download .jpg (.02 MB) Help with files Supplementary Figure 1 These differences were also reflected in western blots of the three fractions, with fraction B being enriched for polycystin-1 more than fraction A or C and fraction C being podocalyxin positive with only weak detection of polycystin-1 (Figure 3a). Thus, although this method isolates a heterogenous population of vesicles (albeit a not completely pure GMV fraction), it permits a first-time assessment of their morphology and peptide-level proteomic data at depth not previously accessible (Supplementary Table S2A online). Virtually all THP migrated to the pellet at the bottom of the ultracentrifuge tube, leaving the banded ELV fractions clear of THP. This was seen when we analyzed ELV fraction C by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Fraction C protein ran as a multitude of bands, whereas the starting material 'crude exosomes' was dominated by a large band of THP that was no longer visible in the purified fraction C ELVs (Figure 3b compared with Figure 3c–e). This was also reflected in the proteomic analysis of gel slice D (70–90kDa, THP Mwt=85kDa) in fraction C where THP was no longer the most abundant protein (by either spectral counting or sequence coverage). We separated 30μg of ELV fraction B (PKD-ELVs) and fraction C (GMVs) material from each individual on a 4–12% SDS-PAGE gel and then sliced it into 10 horizontal gel band segments (A–J) for proteomic analysis by liquid chromatography–tandem mass spectrometry (Figure 3b), generating 140 individual samples. We also pooled equal protein amounts of the seven fraction C samples (GMV fraction) and ran this on both 10–14% and 5% SDS gels; each lane was then cut into 45 and 36 slices, respectively (Figures 3d and e). Each slice was analyzed by liquid chromatography–tandem mass spectrometry. Data from both liquid chromatography–tandem mass spectrometry experiments were searched by using a combination of Mascot, Sequest, and X!Tandem (Figure 3f).21Neubert H. Bonnert T.P. Rumpel K. et al.Label-free detection of differential protein expression by LC/MALDI mass spectrometry.J Proteome Res. 2008; 7: 2270-2279Crossref PubMed Scopus (90) Google Scholar,22Wiener M.C. Sachs J.R. Deyanova E.G. et al.Differential mass spectrometry: a label-free LC-MS method for finding significant differences in complex peptide and protein mixtures.Anal Chem. 2004; 76: 6085-6096Crossref PubMed Scopus (243) Google Scholar A total of 2190 proteins were identified by liquid chromatography–tandem mass spectrometry analysis from the ELVs from both sucrose gradient fractions B and C combining all methodologies (Figures 3f, 4a and c). A total of 1830 proteins were identified in the GMVs (fraction C), combining all methodologies (Figure 4b). The glomerular origin was confirmed by the presence of podocin in fraction C but not in fraction B (Figure 2c; Supplementary Table S1 online). We found that 1106 proteins were common to all seven individuals of fraction C (Supplementary Figure S2 online). We also found that 27% (599/2190) of proteins were present in the glomerular tissue proteome, implying that a specific subfraction of glomerular proteins is secreted (Figure 5a).19Miyamoto M. Yoshida Y. Taguchi I. et al.In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry.J Proteome Res. 2007; 6: 3680-3690Crossref PubMed Scopus (52) Google Scholar Download .doc (.06 MB) Help with doc files Supplementary Figure and Table Legends Download .jpg (.02 MB) Help with files Supplementary Figure 2Figure 5Venn diagrams of GMV proteome, glomerular tissue and other urine exosome proteomes. (a) Venn diagram comparison of our proteome with the Miyamoto glomerular tissue proteome. (b) Overlap of our data with the Wang et al. exosome proteome. (c) Comparison of human urine (NHLBI), Miyamoto, and Wang exosome proteomes with our data.View Large Image Figure ViewerDownload (PPT) We identified many hereditary glomerular disease proteins, the majority with greater peptide number than previously reported in other exosome studies (Table 2, Supplementary Tables S1 and S2 online).4Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1623) Google Scholar,13Gonzales P.A. Pisitkun T. Hoffert J.D. et al.Large-scale proteomics and phosphoproteomics of urinary exosomes.J Am Soc Nephrol. 2009; 20: 363-379Crossref PubMed Scopus (556) Google Scholar These included podocin, alpha-actinin-4, CD2-associated protein, myosin-9, myosin 1E, cdc42, CD151, Rho GDP-dissociation inhibitor 1, integrin alpha 3, and cubilin. Podocin (exclusively seen in gel slice F; ∼40–55kDa range across all seven controls) is reported to have two isoforms 42 and 34kDa, the latter characterized by the absence of amino acids 179–246 (Uniprot.org). The peptides identified were outside of that range, and therefore although the MS data could not distinguish which isoform was present, our molecular-weight data from the one-dimensional (1D)-SDS gel suggests that isoform 1 was the secreted GMV form (Supplementary Table S2 online). Several proteins mutated in atypical hemolytic uremic syndrome (complement C3, C4B and factor B; Table 2) were also present.23Manenti L. Vaglio A. Buzio C. 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