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DOI 10.1002/pmic.201300371
Proteomics 2014, 14, 1922–1932
REVIEW
Urinary extracellular microvesicles: Isolation methods and prospects for urinary proteome Danqi Wang and Wei Sun Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, P. R. China
Extracellular microvesicles (EVs) are membranous vesicles, which are released from diverse cells. These EVs have also been found in a wide range of body fluids. The cargo of EVs, including proteins, lipids, carbohydrates, and nucleic acids, can be stably preserved in EVs. Researchers have found that EVs can mediate intercellular communication by shuttling the cargo components. Therefore, EVs can be used for the identification of disease-specific biomarkers. As one class of EVs, urinary exosomes can reflect the status of the renal system. Moreover, urinary exosome analysis can minimize the interference of high abundant proteins in the whole urine sample. Therefore, urinary exosomes have gained much attention in recent years. In this review, we present a comprehensive summary of urinary exosome studies in recent years, including collection, storage, and isolation methods. The normal and disease proteomic analyses of urinary exosomes are also presented. Thus, this review may provide a valuable reference for future research.
Received: August 22, 2013 Revised: May 7, 2014 Accepted: June 18, 2014
Keywords: Biomarkers / Biomedicine / MS / Urinary exosomes
1
Introduction
Extracellular microvesicles (EVs) which are spherical bilayered proteolipids vesicles, can be secreted by a variety of cells with a mean diameter of 20–1000 nm [1–3]. In recent decades, EVs have gained significant attention as they play important roles in intercellular communication, pathogenesis, drug and gene vector delivery, and are possible reservoirs of biomarkers [4]. Urinary EVs have attracted increasing research interest due to their relationship with kidney physiology and disease [5]. This review aims to summarize the progress in urinary EVs isolation methods and proteomic analysis as a reference for future research. Correspondence: Dr. Wei Sun, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, P. R. China E-mail: [email protected] Fax: +86-010-69156943 Abbreviations: ALIX, ALG-2 interacting protein X; AQP2, aquaporin-2; CD10, neprilysin; CP, ceruloplasmin; ELVs, exosome-like vesicles; EVs, extracellular microvesicles; HMW, high molecular weight; IgAN, IgA nephropathy; MVBs, multivesicular bodies; NSCLC, nonsmall cell lung cancer; RCC, renal cell carcinoma; TBMN, thin basement membrane nephropathy; THP, Tamm–Horsfall protein
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2
Classification of EVs
EVs can be classified into three main classes: Exosomes, which were first named by Trams et al. [6] in the early 1980s, can be secreted by various cells and found in a wide range of body fluids. In their study, exosomes were defined as small (50–250 nm) vesicles [6] that pellet at 100 000 × g [2,4,7]. Ectosomes (also known as microvesicles) are larger EVs that sediment at 10 000 × g and are thought to be large membranous vesicles (50–1000 nm) [2,4,7]. Apoptotic blebs (50–5000 nm), released from fragmented apoptotic cells, are by-products of programmed cell death [4, 7, 8]. In addition, other secreted vesicles, such as exosome-like vesicles (ELVs; 20–50nm) and membrane particles (50–80nm), are also sorted into EVs [2]. It should be noted that many published studies either used a crude preparation of exosomes with contaminants, including other sources of EVs and protein aggregates, or did not restrict the population of vesicles being studied, therefore, we have used the term EVs in this review [4, 7, 9].
3
Characteristics of EVs
First, EVs are secreted by many kinds of cells, such as B cells, T cells, erythroleukemia cells, and other tumor cells www.proteomics-journal.com
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[10]. They have also been reported in various body fluids such as urine, blood, amniotic fluid, and malignant ascites [11]. Second, EVs contain proteins, lipids, carbohydrates, and nucleic acids (mitochondrial DNA, mRNA, micro-RNA, rRNA, and tRNA), and have the capacity to transmit these species to neighboring cells; thus, they may be a novel mechanism of cell-to-cell signaling [1, 7, 12–14]. Third, exosomes create a stable environment for their associated components [15–17]. Kalra et al. [15] reported that plasma exosomes were stable for up to 90 days when stored at −80⬚C, and Sokolova et al. [16] demonstrated that size and integrity of exosomes in cell line remain unchanged within 4 days when stored at −20⬚C. Fourth, EVs are enriched with membrane proteins and other cytosolic proteins, some of which are attractive sources of disease-specific biomarker targets [10]. Exosome markers have been found in colorectal cancer cell exosomes [18], in plasma exosomes from melanoma patients [19], colon tumor cell line LIM1215 exosomes [11], and glioblastoma microvesicles [20].
4
Formation of urinary exosomes
Pisitkun et al. [21] first reported urinary exosomes in 2004. They found that the water channel aquaporin-2 (AQP2) and other apical plasma membrane proteins from the renal tubule segment could be measured in small and low-density membrane vesicles from the urine. They hypothesized that these apical plasma membrane proteins were secreted through the process of exosome formation. This hypothesis was proved by a number of observations [21]. First, under immunoelectron microscopy, they found the vesicles were oriented with their cytoplasmic side inward and therefore likely formed by multivesicular bodies (MVBs) fusing with the plasma membrane. Second, typical transmission electron microscopy demonstrated that the vesicles were small (35–40 nm) and relatively uniform in size [21]. Third, proteomic analysis showed that the endosomal sorting complex required for the transport (ESCRT)-I protein complex, the ESCRT-III protein complex, ALG-2 interacting protein X (ALIX), and vacuolar proteinsorting 4 were required for both the recruitment of endosomederived cargo proteins and the formation of MVBs (late endosomes). Overall, this evidence indicated that intraluminal vesicles (known as “exosomes”) were released into the urine by fusion of MVBs with the plasma membrane [14, 21–23]. The excretion process is shown in Fig. 1. In addition to the above mechanism of exosome formation, Yang and Gould [7] and Shen et al. [24] reported that plasma membrane budding may be another mechanism of exosome formation. Briefly, the cargo molecules may be trafficked to the site of outward vesicle budding and enriched in initial vesicles. Then, the cargo-containing vesicles separate from the plasma membrane and are released into the extracellular milieu. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Characteristics of urinary EVs
First, urine proteome have numerous high abundant proteins filtered from plasma and secreted by renal tubular cells, such as albumin and Tamm–Horsfall protein (THP, also known as uromodulin) [5, 10]. These high abundant proteins may complicate the detection of low abundant proteins especially in proteinuria [10]. The isolation of urinary exosomes could minimize these high abundant proteins. Second, in normal urine samples, urinary exosomes account for 3% of total urine proteins, and their components include membrane proteins and cytosolic proteins that can reflect changes in corresponding organs [5, 10]. Third, urinary exosomes are derived from epithelial cells lining the urinary tract [21], thus, could act as a reservoir for kidney disease biomarkers [10, 14, 25]. Fourth, besides mRNA and miRNA, urinary exosomes also contain preeminent 18s and 28s rRNA that have little or no presence in exosomes isolated from cultured cell lines [13, 26], serum, and saliva [26]. Matignon et al. [27] demonstrated that the combination of urinary cell mRNAs (CD3, CD105, CD14, CD46) and 18s rRNA could be used as a noninvasive test to distinguish T cell mediated rejection from acute antibodymediated rejection in patients with acute dysfunction of the kidney allograft. As for the nucleic acids in urinary exosomes, the potential medical value should be tested.
6
Collection of urinary EVs
Recent in-depth studies have reported on the collection time of urine samples in proteome analysis. Mid-stream of random-catch urine or the second morning urine has been recommended from the tentative standard protocol (http://www.hkupp.org/). For urinary EVs analysis, in 2006, Zhou et al. [28] compared first and second urine samples from healthy volunteers. Four specific exosome-associated markers were chosen for Western blot analysis, and no significant differences were found between the recovery of first and second urinary exosome proteins. This indicated minimal degradation in the urinary tract and bladder; therefore, first and second urine could be used for exosome isolation [28]. It is worth noting that, to date, there is only one report on the collection time for urinary exosomes. Therefore, more work is needed to define whether the collection time determined by Zhou et al. [28] is a standardized method. Moreover, new evidence showed that exosomes were related to posttranscriptional regulation in the circadian system [29, 30]. It is unknown whether the exosome components are affected by the circadian system and this requires further study.
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Storage of urinary EVs
Previous reports have presented different opinions on whether protease inhibitors should be added in urine. In www.proteomics-journal.com
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Figure 1. Exosomes are released from renal epithelial cells (a recapitulation previously provided by Pisitkun et al. [21]). First, apical membrane proteins enter cells through endocytosis pathways. Second, endosomes orient to and fuse with MVBs using monoubiquitination signals. Subsequently, the apical membrane proteins invaginate into MVBs and become internal vesicles. Last, the outer membrane of MVBs fuses with the plasma membrane of renal epithelial cells and secrets exosomes into the urinary space. Ub: ubiquitin; AP: adaptor protein; ESCRT: endosomal sorting complex required for transport; ALIX: ALG-2 interacting protein X.
2006, Zhou et al. [28] adopted Western blot analysis of Na–K– Cl cotransporter from normal urine samples with and without protease inhibitors, and found that the signal in samples with protease inhibitors was higher than the one without protease inhibitors. However, in 2009, Mitchell et al. [31] incubated LNCaP (a prostate cancer cell line commonly used in the field of oncology) cell exosomes with fresh urine samples from patients with advanced prostate cancer in the presence or absence of protease inhibitors. Western blot analysis of several exosomal associated markers showed that urinary exosomes could resist endogenous proteolytic activity for at least 18 h at 37⬚C [31]. In 2012, Musante et al. [32] collected the first morning urine from nonsmoking healthy volunteers. The specimens were processed within 3 h without protease inhibitors. From Western blot analysis of six urinary vesicular markers, the pattern did not show a specific degradation suggesting that the addition of protease inhibitors may be unnecessary [32]. In terms of storage procedures, in 2006, Zhou et al. [31] used pooled normal urine samples at different storage temperatures (4⬚C within 1 h, −20⬚C and -80⬚C for 1 week). Through Western blot analysis of four exosome markers, they found that freezing at −20⬚C caused a major loss of urinary exosome proteins compared to samples stored at 4⬚C and processed within 1 h, whereas freezing at −80⬚C preserved 86% of exosome-associated proteins. In 2008, Cheruvanky et al. [33], using human urine sample from healthy volunteers, made a comparison of three different storage conditions: 4⬚C for 1 h, −80⬚C for 1 week, and 4⬚C for 24 h and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
then stored at −80⬚C for 1 week. Four urinary exosome markers were analyzed by Western blot analysis and the results showed that the recoveries from urine samples placed on wet ice at 4⬚C within 1 h were not striking distinct from the latter two conditions, and protein patterns were similar in all three cases. In 2013, Jacquillet et al. [34] proposed that for shorter storage times, a temperature of 4⬚C or −20⬚C may suffice, but for longer storage periods (ࣙ1 month), a temperature of −70⬚C or −80⬚C is optimal.
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Isolation of urinary EVs
The isolation of urinary EVs is an essential step for subsequent proteomic analysis. Thus, we summarize present EVs isolation methods and the advantages and limitations of each method are listed in Table 1.
8.1 Two-step ultracentrifugation In 2004, Pistikun et al. [21] first published the two-step differential centrifugation method. The protocol contains five steps: Step 1, normal urine samples were centrifuged at 17 000 × g to remove large membrane fragments, cells, and other debris. Step 2, the supernatant from Step 1 was then centrifuged at 200 000 × g to acquire the low-density pellets (crude exosomes). Step 3, resuspend the pellets using isolation solution. Step 4, to deplete the high abundant THP, a reducing
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Proteomics 2014, 14, 1922–1932 Table 1. Advantages and limitations of different isolation methods
Number
Isolation method
Advantages
Limitations
References
1
Ultracentrifugation
Sucrose density gradient
Time-consuming Expensive equipment Labor-intensive Crude preparation Not preferable for large vesicles with similar sedimentation velocities
[21, 32, 34–37]
2
3
Immunoaffinity
Maintaining the exosome structure Isolation of all vesicular particles Avoid high abundant proteins Divide EVs into different populations Derive specific exosomes
[15, 17, 60]
4
Ultrafiltration
Lower limit of samples Faster, easier
5
UC-SEC
6
Exoquick-TCTM
Avoid high abundant proteins High quantities of EVs protein Simple, fast, highly scalable
Nonspecific binding Cleanup procedure may be rarely 100% efficient Retain contaminating proteins Low quantities of EVs protein More time consuming Labor-intensive Incomplete recovery of exosomes Low quantities of EVs protein
[15, 17, 36, 39]
[33, 35, 36]
[34, 35]
[17, 35]
UC-SEC: size-exclusion chromatography following ultracentrifugation; Exoquick-TCTM : exosome precipitation solution (System Biosciences).
agent (DTT) was added to the suspension. Step 5, repeat ultracentrifugation step to obtain pure exosomes (Fig. 2). The two-step ultracentrifugation method can obtain pure exosome proteins [35], and maintain the exosome structure. However, ultracentrifugation has limitations as it is time-consuming, labor-intensive, and has high equipment costs [36]. In 2012, Musante et al. [32] used urine from healthy volunteers to identify whether ultracentrifugation can isolate all vesicles in urine samples. In their workflow, ammonium sulfate (97%, w/v saturation) was added to the supernatant from Step 2. The precipitate was subjected to Step 1 and Step 2 again. Six vesicle markers were analyzed by Western blot analysis, and they found that an additional 40% of urinary vesicle proteins were still retained in the supernatant of the first Step 2 spin, which indicated ultracentrifugation was less effective and might result in the loss of a large number of important vesicles. In 2010, Fern´andez-Llama et al. [37] isolated exosomes from normal urine samples with ultracentrifugation [21] and used immunoblotting to analyze four exosome markers. They found that urinary exosomes were also present in the pellet of Step 1, which might be due to the entrapment by THP. They modified ultracentrifugation by adding DTT to the resuspended pellet of Step 1 and subsequently repeated the Step 1 spin. Using immunoblotting analysis, exosome markers (ALIX and tumor susceptibility gene [TSG] 101) were markedly diminished in the Step 1 pellets with additional DTT and the urinary exosome signal was more enhanced in the Step 2 pellets. In addition, they optimized DTT concentrations, temperatures, incubation times, and recommended
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Two-step ultracentrifugation procedure for the isolation of urinary exosomes. Step 1, the 17 000 × g centrifugation: removes large membrane fragments, cells, and other debris. Step 2, the 200 000 × g centrifugation: the supernatant from Step 1 is centrifuged to acquire low-density pellets (crude exosomes). Step 3, isolation solution (250 mM sucrose/10 mM triethanolamine/0.5 mM PMSF/1 mM leupeptin) is used to resuspend the pellet. Step 4, the reducing agent (DTT) is added to the suspension to deplete the high abundant THP. Step 5, ultracentrifugation step mentioned in Step 2 might be repeated more times to sediment the low-density membrane vesicles (pure exosomes).
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200 mg/mL of DTT at 37⬚C for 5–10 min for the enrichment of urinary exosomes [37].
8.2 Sucrose density gradient In 2012, Raj et al. [38] used a double-cushion sucrose/D2 O centrifugation method to obtain more homogeneous exosomes from normal urine specimens. Briefly, the pellets from Step 2 were resuspended in 20 mM Tris, and the supernatant was ultracentrifuged in a solution containing 1 M and 2 M sucrose. Through Western blot analysis of seven exosome markers, all of them were upregulated in the 1 M sucrose fraction compared with ultracentrifugation and single cushion method [38]. In 2013, Hogan and coworkers [39] modified ultracentrifugation by recentrifuging pellets from Step 2 in continuous 5–30% sucrose/D2 O at 275 000 × g. ELVs can be divided into three fractions depending on their origins, the collecting ducts, the proximal tubule, and glomeruli. Western blot analysis showed that THP was driven into the pellet.
8.3 Ultrafiltration Due to the time-consuming and labor-intensive limitations of ultracentrifugation [21, 35], in 2008, Cheruvanky et al. [33] developed a nanomembrane ultrafiltration method to enrich urinary exosomes from healthy volunteers and patients with focal segmental glomerulosclerosis. First, the supernatant from Step 1 was transferred to a polyethersulfone nanomembrane concentrator (100 kDa molecular weight cut off) and centrifuged at 3000 × g for 10–30 min to collect the urinary exosomes. Second, the retentate was removed from the concentrator. Adhering proteins were removed using an equal volume of preheated 1× solubilizing buffer (2× Laemmli buffer with 60 mg/mL DTT) and incubated for 10 min at room temperature. Exosomes were found in the retentate and adhering fraction. This nanomembrane ultrafiltration approach has a lower limit of urine sample volume and is faster and easier [40]. However, the high abundance of proteins in urine may obstruct the nanomembrane, thus reducing the isolation efficiency [40]. In 2010, Merchant et al. [36] proposed a microfiltration approach using a hydrophilized PVDF membrane (0.1 m hydrophilized polyvinylidene difluoride (VVLP) filter, Millipore, Bedford, MA, USA). This commercially available membrane has a low protein binding property compared with the nanofiltration membrane used by Cheruvanky et al. [33]. Using Western blot analysis of exosome markers (neprilysin [CD10], Na+/H+ exchanger 3 and AQP2), the microfiltration method had an equivalent efficiency of exosome recovery to that of ultracentrifugation [21] and ultrafiltration [33]. This VVLP isolation method has the advantages of low carryover of urine protein and low cost (less than $1000 per setup) [36]. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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8.4 Ultracentrifugation followed by SEC In 2010, Rood et al. [40] used an ultracentrifugation followed by SEC column (BioSep-SEC-S4000, Phenomenex, Torrance, CA, USA) to increase the yield of microvesicles from nephrotic urine. Briefly, the pellet from Step 2 resuspended in PBS was loaded on a SEC column to obtain a high molecular weight (HMW) and lower molecular weight fractions. Both fractions were centrifuged at 3000 × g using a 10 kDa filter and the proteins adhering to the nanomembrane stripped by adding 1% octyl -glucopyranoside as microvesicular proteins. Through Western blot analysis, both CD10 and AQP2 (exosome markers) could be detected in the HMW fraction, whereas the lower molecular weight fraction had no signals. Besides, the recovery of CD10 and AQP2 in the HMW fraction was shown to be higher than fractions obtained from ultracentrifugation and ultrafiltration [40].
8.5 Exoquick-TC In 2012, Alvarez et al. [35] tried a commercial exosome precipitation reagent, Exoquick-TC (System Biosciences, Mountain View, CA, USA) to isolate exosomes from normal urine specimens. First, the sample was centrifuged at 3000 × g to remove cells and cell debris. Then, an appropriate volume of ExoQuick-TC solution was added to the urine and incubated. A 1500 × g centrifugation was used to acquire crude exosomes. Western blot analysis of two exosome markers ALIX and TSG101 from Exoquick-TC method showed weak signals compared with ultracentrifugation, suggesting that the purity of exosome proteins from this method were lower than those from the ultracentrifugation method. However, for ascites exosomes, Taylor et al. [17] reported that the ExoQuick-TC yielded a higher purity and quantity of protein compared with ultracentrifugation. This method is simple, fast, and requires less laboratory equipment.
8.6 Immunoaffinity purification Several studies reported an immunoaffinity method to purify exosomes. This method relies on magnetic beads coated with antibodies directed against a specific exosomal membrane surface protein [15]. In 2011, Taylor et al. [17] isolated tumorsecreted exosomes from ascites of patients with stage III serous adenocarcinoma of the ovary. Anti-EpCAM coupled to the microbeads was mixed with ascites specimens and incubated at room temperature. After washing the beads/exosome complex with TBS, Trizol was used to extract exosome proteins for proteomic analysis. Following Western blot analysis, the use of immunoaffinity purification yielded a higher concentration of exosome protein than the ultracentrifugation method. In 2013, Kalra et al. [15] successfully used the same method to isolate exosomes from human plasma. It should be noted that before LC-MS/MS analysis, the eluted exosomes www.proteomics-journal.com
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Table 2. Summary of high-throughput human urinary EVs proteome datasets of healthy volunteer studies, including data published on EVpedia (http://evpedia.info) and one article published in 2013
Author
Year
Isolation methods
Proteomic analysis strategies
Pisitkun et al.
2004
Ultracentrifugation
Gonzales et al.
2009
Ultracentrifugation
Hogan et al.
2009
Sucrose/D2 O density
Wang et al.
2012
Ultracentrifugation
Raj et al.
2012
Sucrose/D2 O density
Zubiri et al.
2013
Ultracentrifugation combined with THP, IgG, and albumin depletion
1DE-1DLC-MS/MS (LCQ Deca XP Plus) 1DE-1DLC-MS/MS (LTQ linear ion trap) 1DLC-MS/MS (LTQ-Orbitrap linear ion trap) 2D-LC-MS/MS (LTQ linear ion trap and LTQ Velos linear ion trap) 1DE-1DLC-MS/MS (QSTAR Elite) 2DE-LC-MS/MS (LTQ-Orbitrap XL and LTQ linear ion trap)
should be thoroughly washed. However, the cleanup using protein extracting reagent is rarely 100% efficient, thus there may be potential interference on subsequent MS analysis. Moreover, the cleanup procedure might result in loss of exosome proteins.
9
Proteomic analysis of urinary EVs
Many groups have to urinary EVs proteomic analysis. We summarize these reports in Table 2 (normal exosome analyses) and Table 3 (disease differential analyses). For normal EVs analysis, in 2004, Pisitkun et al. [21] first used ultracentrifugation to isolate urinary exosomes. By in-gel digestion and 1D LC-MS/MS analysis, total 295 urinary exosome proteins were identified, 14 were associated with kidney diseases and seven with hypertension [21]. In 2009, they combined ultracentrifugation [21] and in-gel trypsin digestion. Forty sliced peptide samples were analyzed by 1DLC-MS/MS. One thousand one hundred thirty-two proteins were unambiguously identified including 927 new proteins. In addition, they extended the urinary exosome proteomic analysis to phosphoproteomic profiling, which yielded 19 phosphorylation sites corresponding to 14 phosphoproteins [41]. In the same year, Hogan et al. [42] isolated urinary ELVs from normal volunteers using density ultracentrifugation on a 5–30% continuous sucrose gradient with D2O and purified the fraction enriched in polycystin-1, fibrocystin, and polycystin-2. Using 1D LC-MS/MS analysis, a total of 552 proteins were identified (232 unique proteins not yet in urinary proteomic databases). In 2012, Wang et al. [14] adopted the ultracentrifugation method [21] to isolate urinary exosomes. By combining trifluoroethanol solution-phase digestion with Multidimensional Protein Identification Technology, they produced the largest urinary exosome proteome dataset so far. A total of
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Number of identified proteins
References
294
[21]
1314
[41]
552
[42]
3280
[14]
378
[38]
526
[43]
3280 proteins were identified, 1788 of which were not reported in previous studies [14]. In addition, they found that more than 1000 proteins can be detected from just a 25 mL urine sample using their approach [14]. In 2012, Raj et al. [38] used double-cushion sucrose/D2 O centrifugation to separate urinary vesicles from healthy volunteers. The fractions were subjected to in-gel digestion. Using 1D LC-MS/MS analysis, 378 unique urinary exosome proteins were identified. In 2013, Zubiri et al. [43] combined a serial ultracentrifugation with three major proteins (THP, IgG, and albumin) depletion to get intact exosomes. Following 2DE and LC-MS/MS analysis, 526 proteins were identified including 230 new proteins in urinary exosomes. As a noninvasive source of sample in proximity to the affected tissues, urine is arguably the ideal body fluid for the identification of biomarkers for urological diseases [44, 45]. (The summary of disease associated urinary exosome proteins can be seen in Table 4.) In 2011, Moon et al. [46] used ultracentrifugation [21] to isolate urinary exosomes from patients with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN). Following 1D LC-MS/MS and label-free quantification (peptide intensity), 31 proteins were upregulated in IgAN group and 52 proteins were upregulated in TBMN nephropathy group, respectively. Four markers, including ceruloplasmin (CP), aminopeptidase, vasorin precursor, and ␣-1-antitrypsin were validated by Western blot analysis. Among the four markers, CP had the highest AUC value (0.993) from ROC curves, which could effectively distinguish IgAN from TBMN. In 2014, Zubiri et al. [47] combined ultracentrifugaR tion with high abundant protein depletion (ProteoPrep Immunoaffinity Albumin & IgG Depletion Kit, SigmaAldrich) to isolate urinary exosomes from patients with diabetic nephropathy and normal controls. Exosome proteins were further analyzed by 2DE-LC-MS/MS. Using a spectra count quantitative method, 25 proteins were
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2008
2011
2011
2012
2013
2014
Smalley et al.
Li et al.
Moon et al.
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Chen et al.
Raimondo et al.
Zubiri et al.
Diabetic nephropathy
Renal cell carcinoma
Bladder cancer
Early IgAN and TBMN
NSCLC
Bladder cancer
Sample sources
Ultracentrifugation combined with Albumin and IgG depletion
Ultracentrifugation
Ultracentrifugation
Sucrose density gradient
Ultracentrifugation
Ultracentrifugation
Isolation methods
2DE-LC-MS/MS (LTQ-Orbitrap XL)
2DLC-MS/MS (LTQ-Orbitrap linear ion trap) 1DLC-MS/MS (MaXis hybrid UHR-QTof system)
1DLC-MS/MS (Waters, Q-Tof premier)
1DLC-MS/MS (LTQ-FT linear ion trap) 1DLC-chip-MS/MS (MSD Trap)
Proteomic analysis strategies
Label-free (spectra count)
Label-free (spectra count)
Isotopic dimethyl labeling
Label-free (peptide intensity)
Label-free (spectra count) Label-free (peptide intensity)
Quantitative method
Western blot (MMP-9, CP, PODXL, DKK4, CAIX, AQP1, EMMPRIN, CD10, dipeptidase 1, syntenin-1) SRM (AMBP, MLL3, VDAC1)
Western blot (EPS8L2, Mucin 4) Western blot and immunohistochemistry (LRG1) Western blot (CP, aminopeptidase, vasorin precursor, ␣-1-antitrypsin) MRM and ELISA (TACSTD2)
Verification method
25
219
107
83
15
9
Number of differentially expressed proteins
[47]
[48]
[50]
[46]
[53]
[49]
References
D. Wang and W. Sun
AMBP: ␣-1-microglobulin/bikunin precursor; AQP1, aquaporin-1; CAIX, carbonic anhydrase IX; CD10: neprilysin; CP: ceruloplasmin; DKK4, dickkopf-related protein 4; EMMPRIN: extracellular matrix metalloproteinase inducer; EPS8L2: epidermal growth factor receptor kinase substrate 8-like protein 2; LRG1: leucine-rich ␣-2-glycoprotein; MLL3: mixed-lineage leukemia 3; MMP-9: matrix metalloproteinase 9; PODXL, podocalyxin, TACSTD2: tumor-associated calcium-signal transducer 2; VDAC1: voltage-dependent anion-selective channel protein 1.
Year
Author
Table 3. Summary of high-throughput human urinary EVs proteome datasets of patients with kidney disease, renal cell carcinoma, bladder cancer, and nonsmall cell lung cancer, including data published on EVpedia (http://evpedia.info) and two articles published in 2013 and 2014
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Table 4. Proteins identified in urinary exosomes, which associated with kidney disease, bladder cancer, hypertension, and endosome diseases Disease Kidney disease Hyperuricemic nephropathy medullary cystic disease 2 Renal aminoglycoside accumulation and nephrotoxicity Normocalaiuric renal hypomagnesemia Membranous glomerulonephritis Farber disease Gitelman syndrome Renal tubule dysgenesis, diabetic nephropathy Bartter syndrome
Autosomal dominant polycystic kidney disease Renal cell carcinoma
Antenatal Bartter syndrome type 1 Autosomal dominant and autosomal recessive nephrogenic diabetes insipidus Fechtner syndrome and Epstein syndrome Medullary cystic kidney disease 2 and familial juvenile hyperuricemic nephropathy Autosomal recessive steroid-resistant nephrotic syndrome Autosomal recessive pseudohypoaldosteronism type 1 Family renal hypomagnesemia Proximal renal tubular acidosis Liddle syndrome Autosomal dominant polycystic kidney disease type 1 Autosomal recessive syndrome of osteopetrosis with renal tubular acidosis 2,8-Dihydroxyadenine urolithiasis Early podocyte injury Diabetic nephropathy Bladder cancer NSCLC Hypertension
Endosomal diseases
Protein name
References
THP
[25]
Gp330 precursor EGF MME neprilysin PHP NaCl electroneutral thiazide-sensitive cotransporter Angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 Solute carrier family 12 member 1 (bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2) Polycystin-1 and polycystin-2 AQP1, CAIX, CD10, CP, dipeptidase 1, DKK4, EMMPRIN, MMP-9, PODXL, Syntenin-1 Sodium potassium chloride cotransporter-2 AQP2
[42] [48]
[10, 21]
Nonmuscle myosin heavy chain IIA THP
Podocin Epithelial sodium channel ␣, , ␥ FXYD domain-containing ion transport regulator-2 Carbonic anhydrase IV Epithelial sodium channel , ␥ Polycystin-1 Carbonic anhydrase II
Adenine phosphoribosyltransferase WT-1 AMBP, MLL3, VDAC1 EPS8L2, Mucin 4, TACSTD2 Leucine-rich ␣-2-glycoprotein Aminopeptidase A, aminopeptidase N, aminopeptidase P, angiotensin I converting enzyme isoform 1, CD10, dimethylarginine dimethylaminohydrolase 1, NAD Beta-galactosidase precursor Cerebroside sulfate activator protein protective protein for beta-galactosidase
[61, 62] [47] [49, 50] [53] [10, 21]
[25]
AMBP: ␣-1-microglobulin/bikunin precursor; AQP1: aquaporin-1; AQP2: aquaporin-2; CAIX: carbonic anhydrase IX; CD10: neprilysin; CP: ceruloplasmin; DKK4: dickkopf-related protein 4; EGF: proepidermal growth factor precursor; EMMPRIN: extracellular matrix metalloproteinase inducer; EPS8L2: epidermal growth factor receptor kinase substrate 8-like protein 2; MLL3: mixed-lineage leukemia 3; MMP-9: matrix metalloproteinase 9; NAD: 15-hydroxyprostaglandin dehydrogenase; NSCLC: nonsmall cell lung cancer; PHP: putative heart protein; PODXL: podocalyxin; TACSTD2: tumor-associated calcium-signal transducer 2; VDAC1: voltage-dependent anion-selective channel protein 1; WT-1: Wilm’s tumor 1. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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significantly changed in diabetic nephropathy. Three markers (␣-1-microglobulin/bikunin precursor, isoform 1 of histonelysine N-methyltransferase mixed-lineage leukemia 3, and voltage-dependent anion-selective channel protein 1) were further confirmed by SRM technology. In 2013, Raimondo et al. [48] isolated urinary exosomes from renal cell carcinoma (RCC) patients by ultracentrifugation [21]. Apart from the THP band, other bands were in-gel digested and further injected into 1D LC-MS/MS. Using the spectral count quantitative method, 72 were detected only in the RCC group, whereas 147 were present only in the control group. A panel of ten markers was subjected to Western blot analysis. The AUC results showed that matrix metalloproteinase 9, CP, podocalyxin, dickkopf-related protein 4, and carbonic anhydrase IX were significantly upregulated in RCC patients, while aquaporin-1, extracellular matrix metalloproteinase inducer, CD10, dipeptidase 1, and syntenin-1 were downregulated. In 2008, Smalley et al. [49] highlighted bladder cancer associated proteins in urinary microparticles. The samples were random urine from bladder cancer individuals. After ultracentrifugation [21], the proteins in microparticles were analyzed by 1D LC-MS/MS. Following label-free quantification based on spectra count, nine proteins were differentially expressed between bladder cancer and control groups. Eight proteins were found to be specifically enriched in the urinary microparticles from the bladder cancer samples and one protein was enriched in the healthy control samples. Through Western blot analysis, two markers (epidermal growth factor receptor kinase substrate 8 like protein 2 and mucin 4) were both upregulated in bladder cancer patients versus healthy controls [49]. In 2012, Chen et al. [50] used the ultracentrifugation method [21, 49, 51] to isolate urinary microparticles from bladder cancer patients and hernia patients (control group). They applied isotopic dimethyl labeling and the 2D LC-MS/MS approach and found 107 differentially expressed proteins. Using MRM quantification, 24 proteins were verified to be significantly changed with AUC values ranging from 0.702 to 0.896. Tumor-associated calcium-signal transducer 2 was further validated by ELISA as a novel urine marker for early bladder cancer detection and diagnosis. In addition to urological diseases, lung cancer was also investigated by urinary exosome proteome analysis. Early researchers adopted serum or pleural fluid as the preferred specimen in the detection, prognosis, and follow-up of lung cancer [52]. In 2011, Li et al. [53] proposed that changes in urinary exosome protein composition could reflect the status of the urinary and circulatory systems, thus urinary exosomes had potential in identifying nonsmall cell lung cancers (NSCLC) related biomarkers for early clinical diagnosis. Urinary exosomes from NSCLC patients were isolated by ultracentrifugation [21]. Urinary exosome proteins in 35– 45 kDa bands of a 1D gel (proteins in this band range were highly expressed in NSCLC samples) were in-gel digested. By 1D LC-chip-MS/MS and peptide intensity quantification,
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11 proteins were only identified in NSCLC samples, whereas four proteins only detected in control group. Leucine-rich ␣-2glycoprotein was further validated as an upregulated NSCLC biomarker in urine exosomes by Western blot analysis and in lung tissue by immunohistochemistry [53].
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Conclusion
In this review, we comprehensively summarize urinary EVs isolation methods, however, there is a dispute regarding whether these methods should be widely adopted for clinical use, as each isolation protocol has advantages and limitations (Table 1). More work will be necessary to establish standard methods for hospital laboratories to increase the quantity and quality of urinary EVs for sensitive downstream proteomic analysis [34]. In 2008, Mann et al. [54] defined “precision proteomics” a term derived from major improvements in MS that have greatly improved resolution and mass accuracy. Great technology has advanced from 2DE [55] and SELDI [56] to LCMS/MS [57]. Up until now, the LTQ-Orbitrap has made enormous progress for shotgun proteomics. Continued improvements in sequencing speed, high mass accuracy, and resolving power have been combined in routine proteomic analysis [25,54]. This has led to the identification of a large number of proteins in urinary EVs proteome. Recently, more researchers have performed urinary EVs proteomics; thus the creation of a dataset achieved by high-resolution MS would be more desirable for future research. Intra- and interindividual variabilities exist in urinary proteomics [57, 58]. In different populations, food, stress, exercise, physiological conditions, environmental factors, and genetic background vary between individuals [58, 59]. There are differences in urinary protein profiles across individuals and between different days of specimen collection [58]. The level of variation in the proteome of urinary EVs is worth examining for future clinical applications. The authors are grateful to Xiaoqiang Tang for critical reading of this manuscript and great suggestions in all sincerity, and benefit assistance from Lili Zou for collection of literatures. This work was supported by grants from National Natural Science Foundation (No. 30970650), a Foundation for the Author of National Excellent Doctoral Dissertation of P.R. China (No. 2007B64). The authors have declared no conflict of interest.
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DOI 10.1002/pmic.201300371
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REVIEW
Urinary extracellular microvesicles: Isolation methods and prospects for urinary proteome Danqi Wang and Wei Sun Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, P. R. China
Extracellular microvesicles (EVs) are membranous vesicles, which are released from diverse cells. These EVs have also been found in a wide range of body fluids. The cargo of EVs, including proteins, lipids, carbohydrates, and nucleic acids, can be stably preserved in EVs. Researchers have found that EVs can mediate intercellular communication by shuttling the cargo components. Therefore, EVs can be used for the identification of disease-specific biomarkers. As one class of EVs, urinary exosomes can reflect the status of the renal system. Moreover, urinary exosome analysis can minimize the interference of high abundant proteins in the whole urine sample. Therefore, urinary exosomes have gained much attention in recent years. In this review, we present a comprehensive summary of urinary exosome studies in recent years, including collection, storage, and isolation methods. The normal and disease proteomic analyses of urinary exosomes are also presented. Thus, this review may provide a valuable reference for future research.
Received: August 22, 2013 Revised: May 7, 2014 Accepted: June 18, 2014
Keywords: Biomarkers / Biomedicine / MS / Urinary exosomes
1
Introduction
Extracellular microvesicles (EVs) which are spherical bilayered proteolipids vesicles, can be secreted by a variety of cells with a mean diameter of 20–1000 nm [1–3]. In recent decades, EVs have gained significant attention as they play important roles in intercellular communication, pathogenesis, drug and gene vector delivery, and are possible reservoirs of biomarkers [4]. Urinary EVs have attracted increasing research interest due to their relationship with kidney physiology and disease [5]. This review aims to summarize the progress in urinary EVs isolation methods and proteomic analysis as a reference for future research. Correspondence: Dr. Wei Sun, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, P. R. China E-mail: [email protected] Fax: +86-010-69156943 Abbreviations: ALIX, ALG-2 interacting protein X; AQP2, aquaporin-2; CD10, neprilysin; CP, ceruloplasmin; ELVs, exosome-like vesicles; EVs, extracellular microvesicles; HMW, high molecular weight; IgAN, IgA nephropathy; MVBs, multivesicular bodies; NSCLC, nonsmall cell lung cancer; RCC, renal cell carcinoma; TBMN, thin basement membrane nephropathy; THP, Tamm–Horsfall protein
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2
Classification of EVs
EVs can be classified into three main classes: Exosomes, which were first named by Trams et al. [6] in the early 1980s, can be secreted by various cells and found in a wide range of body fluids. In their study, exosomes were defined as small (50–250 nm) vesicles [6] that pellet at 100 000 × g [2,4,7]. Ectosomes (also known as microvesicles) are larger EVs that sediment at 10 000 × g and are thought to be large membranous vesicles (50–1000 nm) [2,4,7]. Apoptotic blebs (50–5000 nm), released from fragmented apoptotic cells, are by-products of programmed cell death [4, 7, 8]. In addition, other secreted vesicles, such as exosome-like vesicles (ELVs; 20–50nm) and membrane particles (50–80nm), are also sorted into EVs [2]. It should be noted that many published studies either used a crude preparation of exosomes with contaminants, including other sources of EVs and protein aggregates, or did not restrict the population of vesicles being studied, therefore, we have used the term EVs in this review [4, 7, 9].
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Characteristics of EVs
First, EVs are secreted by many kinds of cells, such as B cells, T cells, erythroleukemia cells, and other tumor cells www.proteomics-journal.com
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[10]. They have also been reported in various body fluids such as urine, blood, amniotic fluid, and malignant ascites [11]. Second, EVs contain proteins, lipids, carbohydrates, and nucleic acids (mitochondrial DNA, mRNA, micro-RNA, rRNA, and tRNA), and have the capacity to transmit these species to neighboring cells; thus, they may be a novel mechanism of cell-to-cell signaling [1, 7, 12–14]. Third, exosomes create a stable environment for their associated components [15–17]. Kalra et al. [15] reported that plasma exosomes were stable for up to 90 days when stored at −80⬚C, and Sokolova et al. [16] demonstrated that size and integrity of exosomes in cell line remain unchanged within 4 days when stored at −20⬚C. Fourth, EVs are enriched with membrane proteins and other cytosolic proteins, some of which are attractive sources of disease-specific biomarker targets [10]. Exosome markers have been found in colorectal cancer cell exosomes [18], in plasma exosomes from melanoma patients [19], colon tumor cell line LIM1215 exosomes [11], and glioblastoma microvesicles [20].
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Formation of urinary exosomes
Pisitkun et al. [21] first reported urinary exosomes in 2004. They found that the water channel aquaporin-2 (AQP2) and other apical plasma membrane proteins from the renal tubule segment could be measured in small and low-density membrane vesicles from the urine. They hypothesized that these apical plasma membrane proteins were secreted through the process of exosome formation. This hypothesis was proved by a number of observations [21]. First, under immunoelectron microscopy, they found the vesicles were oriented with their cytoplasmic side inward and therefore likely formed by multivesicular bodies (MVBs) fusing with the plasma membrane. Second, typical transmission electron microscopy demonstrated that the vesicles were small (35–40 nm) and relatively uniform in size [21]. Third, proteomic analysis showed that the endosomal sorting complex required for the transport (ESCRT)-I protein complex, the ESCRT-III protein complex, ALG-2 interacting protein X (ALIX), and vacuolar proteinsorting 4 were required for both the recruitment of endosomederived cargo proteins and the formation of MVBs (late endosomes). Overall, this evidence indicated that intraluminal vesicles (known as “exosomes”) were released into the urine by fusion of MVBs with the plasma membrane [14, 21–23]. The excretion process is shown in Fig. 1. In addition to the above mechanism of exosome formation, Yang and Gould [7] and Shen et al. [24] reported that plasma membrane budding may be another mechanism of exosome formation. Briefly, the cargo molecules may be trafficked to the site of outward vesicle budding and enriched in initial vesicles. Then, the cargo-containing vesicles separate from the plasma membrane and are released into the extracellular milieu. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Characteristics of urinary EVs
First, urine proteome have numerous high abundant proteins filtered from plasma and secreted by renal tubular cells, such as albumin and Tamm–Horsfall protein (THP, also known as uromodulin) [5, 10]. These high abundant proteins may complicate the detection of low abundant proteins especially in proteinuria [10]. The isolation of urinary exosomes could minimize these high abundant proteins. Second, in normal urine samples, urinary exosomes account for 3% of total urine proteins, and their components include membrane proteins and cytosolic proteins that can reflect changes in corresponding organs [5, 10]. Third, urinary exosomes are derived from epithelial cells lining the urinary tract [21], thus, could act as a reservoir for kidney disease biomarkers [10, 14, 25]. Fourth, besides mRNA and miRNA, urinary exosomes also contain preeminent 18s and 28s rRNA that have little or no presence in exosomes isolated from cultured cell lines [13, 26], serum, and saliva [26]. Matignon et al. [27] demonstrated that the combination of urinary cell mRNAs (CD3, CD105, CD14, CD46) and 18s rRNA could be used as a noninvasive test to distinguish T cell mediated rejection from acute antibodymediated rejection in patients with acute dysfunction of the kidney allograft. As for the nucleic acids in urinary exosomes, the potential medical value should be tested.
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Collection of urinary EVs
Recent in-depth studies have reported on the collection time of urine samples in proteome analysis. Mid-stream of random-catch urine or the second morning urine has been recommended from the tentative standard protocol (http://www.hkupp.org/). For urinary EVs analysis, in 2006, Zhou et al. [28] compared first and second urine samples from healthy volunteers. Four specific exosome-associated markers were chosen for Western blot analysis, and no significant differences were found between the recovery of first and second urinary exosome proteins. This indicated minimal degradation in the urinary tract and bladder; therefore, first and second urine could be used for exosome isolation [28]. It is worth noting that, to date, there is only one report on the collection time for urinary exosomes. Therefore, more work is needed to define whether the collection time determined by Zhou et al. [28] is a standardized method. Moreover, new evidence showed that exosomes were related to posttranscriptional regulation in the circadian system [29, 30]. It is unknown whether the exosome components are affected by the circadian system and this requires further study.
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Storage of urinary EVs
Previous reports have presented different opinions on whether protease inhibitors should be added in urine. In www.proteomics-journal.com
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Figure 1. Exosomes are released from renal epithelial cells (a recapitulation previously provided by Pisitkun et al. [21]). First, apical membrane proteins enter cells through endocytosis pathways. Second, endosomes orient to and fuse with MVBs using monoubiquitination signals. Subsequently, the apical membrane proteins invaginate into MVBs and become internal vesicles. Last, the outer membrane of MVBs fuses with the plasma membrane of renal epithelial cells and secrets exosomes into the urinary space. Ub: ubiquitin; AP: adaptor protein; ESCRT: endosomal sorting complex required for transport; ALIX: ALG-2 interacting protein X.
2006, Zhou et al. [28] adopted Western blot analysis of Na–K– Cl cotransporter from normal urine samples with and without protease inhibitors, and found that the signal in samples with protease inhibitors was higher than the one without protease inhibitors. However, in 2009, Mitchell et al. [31] incubated LNCaP (a prostate cancer cell line commonly used in the field of oncology) cell exosomes with fresh urine samples from patients with advanced prostate cancer in the presence or absence of protease inhibitors. Western blot analysis of several exosomal associated markers showed that urinary exosomes could resist endogenous proteolytic activity for at least 18 h at 37⬚C [31]. In 2012, Musante et al. [32] collected the first morning urine from nonsmoking healthy volunteers. The specimens were processed within 3 h without protease inhibitors. From Western blot analysis of six urinary vesicular markers, the pattern did not show a specific degradation suggesting that the addition of protease inhibitors may be unnecessary [32]. In terms of storage procedures, in 2006, Zhou et al. [31] used pooled normal urine samples at different storage temperatures (4⬚C within 1 h, −20⬚C and -80⬚C for 1 week). Through Western blot analysis of four exosome markers, they found that freezing at −20⬚C caused a major loss of urinary exosome proteins compared to samples stored at 4⬚C and processed within 1 h, whereas freezing at −80⬚C preserved 86% of exosome-associated proteins. In 2008, Cheruvanky et al. [33], using human urine sample from healthy volunteers, made a comparison of three different storage conditions: 4⬚C for 1 h, −80⬚C for 1 week, and 4⬚C for 24 h and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
then stored at −80⬚C for 1 week. Four urinary exosome markers were analyzed by Western blot analysis and the results showed that the recoveries from urine samples placed on wet ice at 4⬚C within 1 h were not striking distinct from the latter two conditions, and protein patterns were similar in all three cases. In 2013, Jacquillet et al. [34] proposed that for shorter storage times, a temperature of 4⬚C or −20⬚C may suffice, but for longer storage periods (ࣙ1 month), a temperature of −70⬚C or −80⬚C is optimal.
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Isolation of urinary EVs
The isolation of urinary EVs is an essential step for subsequent proteomic analysis. Thus, we summarize present EVs isolation methods and the advantages and limitations of each method are listed in Table 1.
8.1 Two-step ultracentrifugation In 2004, Pistikun et al. [21] first published the two-step differential centrifugation method. The protocol contains five steps: Step 1, normal urine samples were centrifuged at 17 000 × g to remove large membrane fragments, cells, and other debris. Step 2, the supernatant from Step 1 was then centrifuged at 200 000 × g to acquire the low-density pellets (crude exosomes). Step 3, resuspend the pellets using isolation solution. Step 4, to deplete the high abundant THP, a reducing
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Proteomics 2014, 14, 1922–1932 Table 1. Advantages and limitations of different isolation methods
Number
Isolation method
Advantages
Limitations
References
1
Ultracentrifugation
Sucrose density gradient
Time-consuming Expensive equipment Labor-intensive Crude preparation Not preferable for large vesicles with similar sedimentation velocities
[21, 32, 34–37]
2
3
Immunoaffinity
Maintaining the exosome structure Isolation of all vesicular particles Avoid high abundant proteins Divide EVs into different populations Derive specific exosomes
[15, 17, 60]
4
Ultrafiltration
Lower limit of samples Faster, easier
5
UC-SEC
6
Exoquick-TCTM
Avoid high abundant proteins High quantities of EVs protein Simple, fast, highly scalable
Nonspecific binding Cleanup procedure may be rarely 100% efficient Retain contaminating proteins Low quantities of EVs protein More time consuming Labor-intensive Incomplete recovery of exosomes Low quantities of EVs protein
[15, 17, 36, 39]
[33, 35, 36]
[34, 35]
[17, 35]
UC-SEC: size-exclusion chromatography following ultracentrifugation; Exoquick-TCTM : exosome precipitation solution (System Biosciences).
agent (DTT) was added to the suspension. Step 5, repeat ultracentrifugation step to obtain pure exosomes (Fig. 2). The two-step ultracentrifugation method can obtain pure exosome proteins [35], and maintain the exosome structure. However, ultracentrifugation has limitations as it is time-consuming, labor-intensive, and has high equipment costs [36]. In 2012, Musante et al. [32] used urine from healthy volunteers to identify whether ultracentrifugation can isolate all vesicles in urine samples. In their workflow, ammonium sulfate (97%, w/v saturation) was added to the supernatant from Step 2. The precipitate was subjected to Step 1 and Step 2 again. Six vesicle markers were analyzed by Western blot analysis, and they found that an additional 40% of urinary vesicle proteins were still retained in the supernatant of the first Step 2 spin, which indicated ultracentrifugation was less effective and might result in the loss of a large number of important vesicles. In 2010, Fern´andez-Llama et al. [37] isolated exosomes from normal urine samples with ultracentrifugation [21] and used immunoblotting to analyze four exosome markers. They found that urinary exosomes were also present in the pellet of Step 1, which might be due to the entrapment by THP. They modified ultracentrifugation by adding DTT to the resuspended pellet of Step 1 and subsequently repeated the Step 1 spin. Using immunoblotting analysis, exosome markers (ALIX and tumor susceptibility gene [TSG] 101) were markedly diminished in the Step 1 pellets with additional DTT and the urinary exosome signal was more enhanced in the Step 2 pellets. In addition, they optimized DTT concentrations, temperatures, incubation times, and recommended
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Two-step ultracentrifugation procedure for the isolation of urinary exosomes. Step 1, the 17 000 × g centrifugation: removes large membrane fragments, cells, and other debris. Step 2, the 200 000 × g centrifugation: the supernatant from Step 1 is centrifuged to acquire low-density pellets (crude exosomes). Step 3, isolation solution (250 mM sucrose/10 mM triethanolamine/0.5 mM PMSF/1 mM leupeptin) is used to resuspend the pellet. Step 4, the reducing agent (DTT) is added to the suspension to deplete the high abundant THP. Step 5, ultracentrifugation step mentioned in Step 2 might be repeated more times to sediment the low-density membrane vesicles (pure exosomes).
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200 mg/mL of DTT at 37⬚C for 5–10 min for the enrichment of urinary exosomes [37].
8.2 Sucrose density gradient In 2012, Raj et al. [38] used a double-cushion sucrose/D2 O centrifugation method to obtain more homogeneous exosomes from normal urine specimens. Briefly, the pellets from Step 2 were resuspended in 20 mM Tris, and the supernatant was ultracentrifuged in a solution containing 1 M and 2 M sucrose. Through Western blot analysis of seven exosome markers, all of them were upregulated in the 1 M sucrose fraction compared with ultracentrifugation and single cushion method [38]. In 2013, Hogan and coworkers [39] modified ultracentrifugation by recentrifuging pellets from Step 2 in continuous 5–30% sucrose/D2 O at 275 000 × g. ELVs can be divided into three fractions depending on their origins, the collecting ducts, the proximal tubule, and glomeruli. Western blot analysis showed that THP was driven into the pellet.
8.3 Ultrafiltration Due to the time-consuming and labor-intensive limitations of ultracentrifugation [21, 35], in 2008, Cheruvanky et al. [33] developed a nanomembrane ultrafiltration method to enrich urinary exosomes from healthy volunteers and patients with focal segmental glomerulosclerosis. First, the supernatant from Step 1 was transferred to a polyethersulfone nanomembrane concentrator (100 kDa molecular weight cut off) and centrifuged at 3000 × g for 10–30 min to collect the urinary exosomes. Second, the retentate was removed from the concentrator. Adhering proteins were removed using an equal volume of preheated 1× solubilizing buffer (2× Laemmli buffer with 60 mg/mL DTT) and incubated for 10 min at room temperature. Exosomes were found in the retentate and adhering fraction. This nanomembrane ultrafiltration approach has a lower limit of urine sample volume and is faster and easier [40]. However, the high abundance of proteins in urine may obstruct the nanomembrane, thus reducing the isolation efficiency [40]. In 2010, Merchant et al. [36] proposed a microfiltration approach using a hydrophilized PVDF membrane (0.1 m hydrophilized polyvinylidene difluoride (VVLP) filter, Millipore, Bedford, MA, USA). This commercially available membrane has a low protein binding property compared with the nanofiltration membrane used by Cheruvanky et al. [33]. Using Western blot analysis of exosome markers (neprilysin [CD10], Na+/H+ exchanger 3 and AQP2), the microfiltration method had an equivalent efficiency of exosome recovery to that of ultracentrifugation [21] and ultrafiltration [33]. This VVLP isolation method has the advantages of low carryover of urine protein and low cost (less than $1000 per setup) [36]. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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8.4 Ultracentrifugation followed by SEC In 2010, Rood et al. [40] used an ultracentrifugation followed by SEC column (BioSep-SEC-S4000, Phenomenex, Torrance, CA, USA) to increase the yield of microvesicles from nephrotic urine. Briefly, the pellet from Step 2 resuspended in PBS was loaded on a SEC column to obtain a high molecular weight (HMW) and lower molecular weight fractions. Both fractions were centrifuged at 3000 × g using a 10 kDa filter and the proteins adhering to the nanomembrane stripped by adding 1% octyl -glucopyranoside as microvesicular proteins. Through Western blot analysis, both CD10 and AQP2 (exosome markers) could be detected in the HMW fraction, whereas the lower molecular weight fraction had no signals. Besides, the recovery of CD10 and AQP2 in the HMW fraction was shown to be higher than fractions obtained from ultracentrifugation and ultrafiltration [40].
8.5 Exoquick-TC In 2012, Alvarez et al. [35] tried a commercial exosome precipitation reagent, Exoquick-TC (System Biosciences, Mountain View, CA, USA) to isolate exosomes from normal urine specimens. First, the sample was centrifuged at 3000 × g to remove cells and cell debris. Then, an appropriate volume of ExoQuick-TC solution was added to the urine and incubated. A 1500 × g centrifugation was used to acquire crude exosomes. Western blot analysis of two exosome markers ALIX and TSG101 from Exoquick-TC method showed weak signals compared with ultracentrifugation, suggesting that the purity of exosome proteins from this method were lower than those from the ultracentrifugation method. However, for ascites exosomes, Taylor et al. [17] reported that the ExoQuick-TC yielded a higher purity and quantity of protein compared with ultracentrifugation. This method is simple, fast, and requires less laboratory equipment.
8.6 Immunoaffinity purification Several studies reported an immunoaffinity method to purify exosomes. This method relies on magnetic beads coated with antibodies directed against a specific exosomal membrane surface protein [15]. In 2011, Taylor et al. [17] isolated tumorsecreted exosomes from ascites of patients with stage III serous adenocarcinoma of the ovary. Anti-EpCAM coupled to the microbeads was mixed with ascites specimens and incubated at room temperature. After washing the beads/exosome complex with TBS, Trizol was used to extract exosome proteins for proteomic analysis. Following Western blot analysis, the use of immunoaffinity purification yielded a higher concentration of exosome protein than the ultracentrifugation method. In 2013, Kalra et al. [15] successfully used the same method to isolate exosomes from human plasma. It should be noted that before LC-MS/MS analysis, the eluted exosomes www.proteomics-journal.com
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Table 2. Summary of high-throughput human urinary EVs proteome datasets of healthy volunteer studies, including data published on EVpedia (http://evpedia.info) and one article published in 2013
Author
Year
Isolation methods
Proteomic analysis strategies
Pisitkun et al.
2004
Ultracentrifugation
Gonzales et al.
2009
Ultracentrifugation
Hogan et al.
2009
Sucrose/D2 O density
Wang et al.
2012
Ultracentrifugation
Raj et al.
2012
Sucrose/D2 O density
Zubiri et al.
2013
Ultracentrifugation combined with THP, IgG, and albumin depletion
1DE-1DLC-MS/MS (LCQ Deca XP Plus) 1DE-1DLC-MS/MS (LTQ linear ion trap) 1DLC-MS/MS (LTQ-Orbitrap linear ion trap) 2D-LC-MS/MS (LTQ linear ion trap and LTQ Velos linear ion trap) 1DE-1DLC-MS/MS (QSTAR Elite) 2DE-LC-MS/MS (LTQ-Orbitrap XL and LTQ linear ion trap)
should be thoroughly washed. However, the cleanup using protein extracting reagent is rarely 100% efficient, thus there may be potential interference on subsequent MS analysis. Moreover, the cleanup procedure might result in loss of exosome proteins.
9
Proteomic analysis of urinary EVs
Many groups have to urinary EVs proteomic analysis. We summarize these reports in Table 2 (normal exosome analyses) and Table 3 (disease differential analyses). For normal EVs analysis, in 2004, Pisitkun et al. [21] first used ultracentrifugation to isolate urinary exosomes. By in-gel digestion and 1D LC-MS/MS analysis, total 295 urinary exosome proteins were identified, 14 were associated with kidney diseases and seven with hypertension [21]. In 2009, they combined ultracentrifugation [21] and in-gel trypsin digestion. Forty sliced peptide samples were analyzed by 1DLC-MS/MS. One thousand one hundred thirty-two proteins were unambiguously identified including 927 new proteins. In addition, they extended the urinary exosome proteomic analysis to phosphoproteomic profiling, which yielded 19 phosphorylation sites corresponding to 14 phosphoproteins [41]. In the same year, Hogan et al. [42] isolated urinary ELVs from normal volunteers using density ultracentrifugation on a 5–30% continuous sucrose gradient with D2O and purified the fraction enriched in polycystin-1, fibrocystin, and polycystin-2. Using 1D LC-MS/MS analysis, a total of 552 proteins were identified (232 unique proteins not yet in urinary proteomic databases). In 2012, Wang et al. [14] adopted the ultracentrifugation method [21] to isolate urinary exosomes. By combining trifluoroethanol solution-phase digestion with Multidimensional Protein Identification Technology, they produced the largest urinary exosome proteome dataset so far. A total of
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Number of identified proteins
References
294
[21]
1314
[41]
552
[42]
3280
[14]
378
[38]
526
[43]
3280 proteins were identified, 1788 of which were not reported in previous studies [14]. In addition, they found that more than 1000 proteins can be detected from just a 25 mL urine sample using their approach [14]. In 2012, Raj et al. [38] used double-cushion sucrose/D2 O centrifugation to separate urinary vesicles from healthy volunteers. The fractions were subjected to in-gel digestion. Using 1D LC-MS/MS analysis, 378 unique urinary exosome proteins were identified. In 2013, Zubiri et al. [43] combined a serial ultracentrifugation with three major proteins (THP, IgG, and albumin) depletion to get intact exosomes. Following 2DE and LC-MS/MS analysis, 526 proteins were identified including 230 new proteins in urinary exosomes. As a noninvasive source of sample in proximity to the affected tissues, urine is arguably the ideal body fluid for the identification of biomarkers for urological diseases [44, 45]. (The summary of disease associated urinary exosome proteins can be seen in Table 4.) In 2011, Moon et al. [46] used ultracentrifugation [21] to isolate urinary exosomes from patients with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN). Following 1D LC-MS/MS and label-free quantification (peptide intensity), 31 proteins were upregulated in IgAN group and 52 proteins were upregulated in TBMN nephropathy group, respectively. Four markers, including ceruloplasmin (CP), aminopeptidase, vasorin precursor, and ␣-1-antitrypsin were validated by Western blot analysis. Among the four markers, CP had the highest AUC value (0.993) from ROC curves, which could effectively distinguish IgAN from TBMN. In 2014, Zubiri et al. [47] combined ultracentrifugaR tion with high abundant protein depletion (ProteoPrep Immunoaffinity Albumin & IgG Depletion Kit, SigmaAldrich) to isolate urinary exosomes from patients with diabetic nephropathy and normal controls. Exosome proteins were further analyzed by 2DE-LC-MS/MS. Using a spectra count quantitative method, 25 proteins were
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2008
2011
2011
2012
2013
2014
Smalley et al.
Li et al.
Moon et al.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chen et al.
Raimondo et al.
Zubiri et al.
Diabetic nephropathy
Renal cell carcinoma
Bladder cancer
Early IgAN and TBMN
NSCLC
Bladder cancer
Sample sources
Ultracentrifugation combined with Albumin and IgG depletion
Ultracentrifugation
Ultracentrifugation
Sucrose density gradient
Ultracentrifugation
Ultracentrifugation
Isolation methods
2DE-LC-MS/MS (LTQ-Orbitrap XL)
2DLC-MS/MS (LTQ-Orbitrap linear ion trap) 1DLC-MS/MS (MaXis hybrid UHR-QTof system)
1DLC-MS/MS (Waters, Q-Tof premier)
1DLC-MS/MS (LTQ-FT linear ion trap) 1DLC-chip-MS/MS (MSD Trap)
Proteomic analysis strategies
Label-free (spectra count)
Label-free (spectra count)
Isotopic dimethyl labeling
Label-free (peptide intensity)
Label-free (spectra count) Label-free (peptide intensity)
Quantitative method
Western blot (MMP-9, CP, PODXL, DKK4, CAIX, AQP1, EMMPRIN, CD10, dipeptidase 1, syntenin-1) SRM (AMBP, MLL3, VDAC1)
Western blot (EPS8L2, Mucin 4) Western blot and immunohistochemistry (LRG1) Western blot (CP, aminopeptidase, vasorin precursor, ␣-1-antitrypsin) MRM and ELISA (TACSTD2)
Verification method
25
219
107
83
15
9
Number of differentially expressed proteins
[47]
[48]
[50]
[46]
[53]
[49]
References
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AMBP: ␣-1-microglobulin/bikunin precursor; AQP1, aquaporin-1; CAIX, carbonic anhydrase IX; CD10: neprilysin; CP: ceruloplasmin; DKK4, dickkopf-related protein 4; EMMPRIN: extracellular matrix metalloproteinase inducer; EPS8L2: epidermal growth factor receptor kinase substrate 8-like protein 2; LRG1: leucine-rich ␣-2-glycoprotein; MLL3: mixed-lineage leukemia 3; MMP-9: matrix metalloproteinase 9; PODXL, podocalyxin, TACSTD2: tumor-associated calcium-signal transducer 2; VDAC1: voltage-dependent anion-selective channel protein 1.
Year
Author
Table 3. Summary of high-throughput human urinary EVs proteome datasets of patients with kidney disease, renal cell carcinoma, bladder cancer, and nonsmall cell lung cancer, including data published on EVpedia (http://evpedia.info) and two articles published in 2013 and 2014
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Table 4. Proteins identified in urinary exosomes, which associated with kidney disease, bladder cancer, hypertension, and endosome diseases Disease Kidney disease Hyperuricemic nephropathy medullary cystic disease 2 Renal aminoglycoside accumulation and nephrotoxicity Normocalaiuric renal hypomagnesemia Membranous glomerulonephritis Farber disease Gitelman syndrome Renal tubule dysgenesis, diabetic nephropathy Bartter syndrome
Autosomal dominant polycystic kidney disease Renal cell carcinoma
Antenatal Bartter syndrome type 1 Autosomal dominant and autosomal recessive nephrogenic diabetes insipidus Fechtner syndrome and Epstein syndrome Medullary cystic kidney disease 2 and familial juvenile hyperuricemic nephropathy Autosomal recessive steroid-resistant nephrotic syndrome Autosomal recessive pseudohypoaldosteronism type 1 Family renal hypomagnesemia Proximal renal tubular acidosis Liddle syndrome Autosomal dominant polycystic kidney disease type 1 Autosomal recessive syndrome of osteopetrosis with renal tubular acidosis 2,8-Dihydroxyadenine urolithiasis Early podocyte injury Diabetic nephropathy Bladder cancer NSCLC Hypertension
Endosomal diseases
Protein name
References
THP
[25]
Gp330 precursor EGF MME neprilysin PHP NaCl electroneutral thiazide-sensitive cotransporter Angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 Solute carrier family 12 member 1 (bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2) Polycystin-1 and polycystin-2 AQP1, CAIX, CD10, CP, dipeptidase 1, DKK4, EMMPRIN, MMP-9, PODXL, Syntenin-1 Sodium potassium chloride cotransporter-2 AQP2
[42] [48]
[10, 21]
Nonmuscle myosin heavy chain IIA THP
Podocin Epithelial sodium channel ␣, , ␥ FXYD domain-containing ion transport regulator-2 Carbonic anhydrase IV Epithelial sodium channel , ␥ Polycystin-1 Carbonic anhydrase II
Adenine phosphoribosyltransferase WT-1 AMBP, MLL3, VDAC1 EPS8L2, Mucin 4, TACSTD2 Leucine-rich ␣-2-glycoprotein Aminopeptidase A, aminopeptidase N, aminopeptidase P, angiotensin I converting enzyme isoform 1, CD10, dimethylarginine dimethylaminohydrolase 1, NAD Beta-galactosidase precursor Cerebroside sulfate activator protein protective protein for beta-galactosidase
[61, 62] [47] [49, 50] [53] [10, 21]
[25]
AMBP: ␣-1-microglobulin/bikunin precursor; AQP1: aquaporin-1; AQP2: aquaporin-2; CAIX: carbonic anhydrase IX; CD10: neprilysin; CP: ceruloplasmin; DKK4: dickkopf-related protein 4; EGF: proepidermal growth factor precursor; EMMPRIN: extracellular matrix metalloproteinase inducer; EPS8L2: epidermal growth factor receptor kinase substrate 8-like protein 2; MLL3: mixed-lineage leukemia 3; MMP-9: matrix metalloproteinase 9; NAD: 15-hydroxyprostaglandin dehydrogenase; NSCLC: nonsmall cell lung cancer; PHP: putative heart protein; PODXL: podocalyxin; TACSTD2: tumor-associated calcium-signal transducer 2; VDAC1: voltage-dependent anion-selective channel protein 1; WT-1: Wilm’s tumor 1. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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significantly changed in diabetic nephropathy. Three markers (␣-1-microglobulin/bikunin precursor, isoform 1 of histonelysine N-methyltransferase mixed-lineage leukemia 3, and voltage-dependent anion-selective channel protein 1) were further confirmed by SRM technology. In 2013, Raimondo et al. [48] isolated urinary exosomes from renal cell carcinoma (RCC) patients by ultracentrifugation [21]. Apart from the THP band, other bands were in-gel digested and further injected into 1D LC-MS/MS. Using the spectral count quantitative method, 72 were detected only in the RCC group, whereas 147 were present only in the control group. A panel of ten markers was subjected to Western blot analysis. The AUC results showed that matrix metalloproteinase 9, CP, podocalyxin, dickkopf-related protein 4, and carbonic anhydrase IX were significantly upregulated in RCC patients, while aquaporin-1, extracellular matrix metalloproteinase inducer, CD10, dipeptidase 1, and syntenin-1 were downregulated. In 2008, Smalley et al. [49] highlighted bladder cancer associated proteins in urinary microparticles. The samples were random urine from bladder cancer individuals. After ultracentrifugation [21], the proteins in microparticles were analyzed by 1D LC-MS/MS. Following label-free quantification based on spectra count, nine proteins were differentially expressed between bladder cancer and control groups. Eight proteins were found to be specifically enriched in the urinary microparticles from the bladder cancer samples and one protein was enriched in the healthy control samples. Through Western blot analysis, two markers (epidermal growth factor receptor kinase substrate 8 like protein 2 and mucin 4) were both upregulated in bladder cancer patients versus healthy controls [49]. In 2012, Chen et al. [50] used the ultracentrifugation method [21, 49, 51] to isolate urinary microparticles from bladder cancer patients and hernia patients (control group). They applied isotopic dimethyl labeling and the 2D LC-MS/MS approach and found 107 differentially expressed proteins. Using MRM quantification, 24 proteins were verified to be significantly changed with AUC values ranging from 0.702 to 0.896. Tumor-associated calcium-signal transducer 2 was further validated by ELISA as a novel urine marker for early bladder cancer detection and diagnosis. In addition to urological diseases, lung cancer was also investigated by urinary exosome proteome analysis. Early researchers adopted serum or pleural fluid as the preferred specimen in the detection, prognosis, and follow-up of lung cancer [52]. In 2011, Li et al. [53] proposed that changes in urinary exosome protein composition could reflect the status of the urinary and circulatory systems, thus urinary exosomes had potential in identifying nonsmall cell lung cancers (NSCLC) related biomarkers for early clinical diagnosis. Urinary exosomes from NSCLC patients were isolated by ultracentrifugation [21]. Urinary exosome proteins in 35– 45 kDa bands of a 1D gel (proteins in this band range were highly expressed in NSCLC samples) were in-gel digested. By 1D LC-chip-MS/MS and peptide intensity quantification,
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11 proteins were only identified in NSCLC samples, whereas four proteins only detected in control group. Leucine-rich ␣-2glycoprotein was further validated as an upregulated NSCLC biomarker in urine exosomes by Western blot analysis and in lung tissue by immunohistochemistry [53].
10
Conclusion
In this review, we comprehensively summarize urinary EVs isolation methods, however, there is a dispute regarding whether these methods should be widely adopted for clinical use, as each isolation protocol has advantages and limitations (Table 1). More work will be necessary to establish standard methods for hospital laboratories to increase the quantity and quality of urinary EVs for sensitive downstream proteomic analysis [34]. In 2008, Mann et al. [54] defined “precision proteomics” a term derived from major improvements in MS that have greatly improved resolution and mass accuracy. Great technology has advanced from 2DE [55] and SELDI [56] to LCMS/MS [57]. Up until now, the LTQ-Orbitrap has made enormous progress for shotgun proteomics. Continued improvements in sequencing speed, high mass accuracy, and resolving power have been combined in routine proteomic analysis [25,54]. This has led to the identification of a large number of proteins in urinary EVs proteome. Recently, more researchers have performed urinary EVs proteomics; thus the creation of a dataset achieved by high-resolution MS would be more desirable for future research. Intra- and interindividual variabilities exist in urinary proteomics [57, 58]. In different populations, food, stress, exercise, physiological conditions, environmental factors, and genetic background vary between individuals [58, 59]. There are differences in urinary protein profiles across individuals and between different days of specimen collection [58]. The level of variation in the proteome of urinary EVs is worth examining for future clinical applications. The authors are grateful to Xiaoqiang Tang for critical reading of this manuscript and great suggestions in all sincerity, and benefit assistance from Lili Zou for collection of literatures. This work was supported by grants from National Natural Science Foundation (No. 30970650), a Foundation for the Author of National Excellent Doctoral Dissertation of P.R. China (No. 2007B64). The authors have declared no conflict of interest.
11
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