Cell Culture

HK-2 human proximal tubular epithelial cells (ATCC, Rockville, MD) were grown on RPMI 1640 (GIBCO, Grand Island, NY), 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL sodium selenite, and 5 ng/mL hydrocortisone in 5% CO₂ at 37°C as previously described [23]. For experiments cells were rested in serum-free media for 24 h prior to sample collection.

Clinical Characteristics of Patients

Second morning void urine samples were obtained from human healthy volunteers or stable CKD patients who donated urine samples. All the study “OPG in exosome-like vesicles from human cultured tubular cells and urine”, and the human samples used, has been approved by the IIS-Fundacion Jimenez Diaz Biobank Local Ethics Biobank. Informed consent were signed by all the patients involved in the study, and all clinical investigation were conducted according to the principles expressed in the Declaration of Helsinki. Clinical characteristics are summarized in Table 1. The eGFR was estimated from serum creatinine using the Modification of Diet in Renal Disease (MDRD) Study Group equation: eGFR (ml/min/1.73 m2) = 186×(SCr)−1.154×(age in years)−0.203×(0.742, if patient is female)×(1.212, if patient ethnicity is black) [24]. Healthy controls were 3 males and one female, 61±10-year-old, non-diabetics, with serum creatinine <1.2 mg/dl and albuminuria <30 mg/g creatinine. A cocktail of protease inhibitors (2 mM AEBSF, 0.3 µM aprotinin, 130 µM bestatin, 1 mM EDTA, 14 µM E-64, 1 µM leupeptin, Sigma-Aldrich) were added to 100 ml urine and samples were frozen at −80°C until microvesicle extraction.

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Table 1. Clinical characteristics.

https://doi.org/10.1371/journal.pone.0072387.t001

Microvesicle Isolation

Microvesicles were obtained by serial ultracentrifugation at 4°C. In each centrifugation the pellet was discarded and the supernatant used in the next step. The first steps eliminate dead cells and large cell debris [25]. For cell culture supernatants, we normalized the values to 1 ml of culture medium conditioned per million cells. The yield was 1251±349.95 ng of exosomal protein/million cells. The isolation starts with a 300×g (cells and cell debris removal) centrifugation for 10 minutes; 1,100×g for 10 minutes twice; 6,000×g for 10 minutes; 17,000×g for 30 minutes and 200,000×g for 70 minutes. This last step precipitates small particles corresponding to exosome-like vesicles secreted into the culture medium. This pellet was washed with a large volume of PBS to avoid contamination with soluble proteins and then centrifuged again for 70 minutes at 200,000×g. The final pellet obtained was resuspended in various solvents (PBS, lysis buffer), depending on the purpose of the extraction. For urine samples we followed a modified protocol, optimized for urine. After thawing the 50–100 ml samples, an aliquot was removed and whole urine was centrifuged at 17,000×g for 30 minutes. The pellet was resuspended in isolation buffer (200 mM sucrose, 10 mM triethanolamine) and dithiothreitol (DTT) was added (200 mg/ml) to disrupt Tamm-Horsfall protein network that entraps exosome-like vesicles, thus increasing isolation efficiency [4], [26]. After adding DTT, the solution was centrifuged again at 17,000×g for 10 minutes, the pellet containing precipitated Tamm-Horsfall protein was discarded and the supernatant containing exosome-like vesicles was mixed with the first centrifugation supernatant kept at 4°C. The newly reconstituted samples were centrifuged at 200,000×g for 70 minutes at 4°C. The pellet obtained was resuspended in PBS or lysis buffer as required. Exosomal protein was assessed by the Bradford assay. All centrifugations were performed following described standard procedures [27], using a 70 Ti fixed angle rotor with 26 ml polycarbonate tubes. Conversion from rpms to RFCs (Relative Centrifugal Force; ǵs) were made automatically by the centrifuge.

ELISA

OPG was quantified by ELISA (Biomedica Gruppe) following the manufacturer instructions. Proteins from the following origins were assayed: tubular epithelial cells, whole human urine, tubular cell culture supernatant, tubular cell derived exosome-like vesicle and urinary exosome-like vesicles. Optical density (OD) was read in all wells on a plate reader using 450 nm wavelength (correction wavelength 630 nm). We construct the standard curve from the OD values of an OPG standard with a range 0 to 20 pmol/l and obtained the sample concentration from this standard curve. The detection limit of the assay is 0.07 pmol/l.

Transmission Electron Microscopy

The pellet containing exosome-like vesicles was resuspended in 2% paraformaldehyde/PBS, and 5 µl were plated on Formvar/carbon-coated grids (Ted Pella Inc. CA, USA). After adsorption, grids were washed several times in PBS and transferred to 1.5% glutaraldehyde for 5 minutes. After another series of washes, grids were transferred to a uranyl-oxalate solution for 5 minutes and to methylcellulose-uranyl-oxalate for 10 minutes. Grids were dried and observed in a transmission electron microscope at 80 kV.

Flow Cytometry

Because of their small size, exosomes can only be analyzed in a flow cytometer after linkage to larger particles of known size. Exosome-like vesicles were adsorbed to solid 3.9 mm latex microspheres composed of aldehyde-free sulfate surfactants (Interfacial Dynamics, USA). Microspheres/exosomes were incubated with anti-CD63-PE antibody (Becton-Dickinson) and analyzed on a FACScalibur flow cytometer (Becton-Dickinson).

Western Blot

Western blots were performed as previously described [28]. Membranes were incubated overnight at 4°C with anti-OPG antibody (1∶500, Acris Antibodies GmbH, Germany), anti-TRAIL (1∶1,000, R&D systems), anti-ALIX (1∶1000; Santa Cruz Biotechnology, CA, USA), anti-TSG101 (1∶1000; AbCam, Boston, MA, USA), anti-CD63 (1∶2000; Millipore, MA, USA), anti-Calnexin (Calbiochem), anti-Tweak (1∶500, Santa Cruz Biotechnology), anti-Fibronectin (1∶1000, Millipore, MA, USA), anti-C3 (kind gift from Dr. Pastor-Vargas) and anti-TGFβ-iH3 (1∶1000, Santa Cruz Biotechnology, CA, USA) followed by incubation with horseradish peroxidase-conjugated secondary antibody (1∶2,000, Amersham, Aylesbury, UK). The use of a different anti-OPG antibody (Santa Cruz) yielded results similar to those shown in the figures. Blots were developed with the enhanced chemiluminescence method (ECL) following the manufacturer’s instructions (Amersham). All assays were performed under reducing conditions (adding β-mercaptoethanol as reducing agent into the loading buffer) except for CD63. Autoradiographs were scanned using the GS-800 Calibrated Densitometer (Quantity One, Bio-Rad).

Sample Preparation for LC-MS/MS

Exosomal protein extract (50 µg) from tubular epithelial cell supernantants was concentrated and desalted. In a first attempt electrophoresis was performed until the sample passed through the stacking gel and reached the running gel. At that moment, electrophoresis was stopped and the gel was stained using colloidal Coomassie (Fermentas). The piece of stained gel in which the sample was concentrated was excised and digested. In this way, all proteins were concentrated in a unique band, eliminating sample contaminants [29]. To further increase the number of identified proteins, full electrophoresis was additionally performed and the stained gel pieces were excised. Digestion was performed according to Schevchenko et al [30] with minor modifications: the gel piece was incubated with 10 mM DTT in 50 mM ammonium bicarbonate (99% purity; Scharlau) for 30 min at 56°C and after reduction, alkylated with 55 mM iodoacetamide (Sigma Aldrich) in 50 mM ammonium bicarbonate for 20 min at RT. Gel plugs were washed with 50 mM ammonium bicarbonate in 50% methanol (gradient, HPLC grade, Scharlau), rinsed in acetonitrile (gradient, HPLC grade, Scharlau) and dried in a Speedvac. Dry gel pieces were then embedded in sequencing grade modified porcine trypsin (Promega, Madison, WI, USA) at a final concentration of 20 ng/µL in 20 mM ammonium bicarbonate. After digestion at 37°C overnight, peptides were extracted with 60% acetonitrile (ACN) in 0.1% formic acid (99.5% purity; Sigma Aldrich) and the sample were resuspended in 98% water with 2% formic acid (FA) and 2% ACN.

LC-MS/MS and Database Searching

The LC/MSMS system consisted of a TEMPO nano LC system (Applied Biosystems) combined with a nano LC Autosampler. LC-MS/MS analysis was performed on an AB/MDS Sciex 4000 Q TRAP System with NanoSprayII Source (Applied Biosystems). Two replicate injections (4.5 µL) were made for each sample using mobile phase A (2% ACN/98% water, 0.1% FA) with a flow rate of 10 µL/min for 5 min. Peptides were loaded onto a µ-Precolumn Cartridge (Acclaim Pep Map 100 C18, 5 µm, 100 Å; 300 µm i.d. X 5 mm, LC Packings) to preconcentrate and desalt samples. RPLC was achieved on a C18 column (Onyx Monolithic C18 150×0.1 mm, Phenomenex) using mobile phase A (2% ACN/98% water, 0.1% FA) and mobile phase B (98% ACN/2% water, 0.1% FA). Peptides were eluted at a flow rate of 300 nL/min by the following gradient: initial conditions 5% B, increased to 40% B over 40 min, 40 to 95% B for 1 min and then 95% B for 4 min, returning next to initial conditions (5% B) in 2 min and maintaining them for 14 more minutes.

The TEMPO nano LC system and 4000 QTRAP were both controlled by Analyst Software v.1.5.1. Analyst software creates wiff format files including all the spectra data. Wiff files were batch-processed by ProteinPilot™ Software 2.0.1 (Applied Biosystems/MDS Sciex) which automatically generated peak lists that were searched against the Swissprot database version 2011_01 using MASCOT version 2.2 (Matrix Science) [31], with 0.8 Da precursor and MS/MS fragment tolerance, allowing 1 missed cleavage and setting carbamidomethyl cysteines and methionine oxidation as modifications.

All the MS and MS/MS data were achieved in positive ion mode with an ion spray voltage of 2800 V, a declustering potential of 80 V and a nanoflow interface temperature of 150°C. Nitrogen was applied as both curtain and collision gas, setting source gas 1 and curtain gas to 20 psi. An Information Dependent Acquisition (IDA) method was programmed, with a full scan Enhanced MS (EMS) experiment at 4000 amu/s for ion profiling, followed by an enhanced resolution (ER) MS experiment at 250 amu/s. ER experiment allowed charge state recognition, further used by the IDA criteria to select precursor ions and to estimate the collision energy to fragment them. This IDA criteria was set to select the 8 doubled, tripled or quadrupled charged most intense ions from 400–1200 m/z that exceed 100,000 counts for fragmentation in the LINAC collision cell, excluding isotopes within a 4.0 amu window and with a mass tolerance of 1000,000 mmu. These 8 ions were submitted to 8 independent Enhanced Product Ion (EPI) MS/MS experiments at 4,000 amu/s with Dynamic Fill Time (DFT). The total number of MS and MS/MS experiments per cycle was 10 (1 EMS, 1 ER and 8 EPI), resulting in a total cycle time of 5.0058 s.

OPG Analysis by Selected Reaction Monitoring (SRM) in a QQQ-LC/MS

SRM was performed to specifically identify OPG in exosome-like vesicles. Exosome-like vesicles were lysed in 7 M urea, 2 M thiourea and 4% Chaps and proteins were precipitated in acetone and solved in 8 M urea, reduced with 10 mM DTT (Sigma Aldrich) for 30 min at 37°C and alkylated for 20 min at room temperature with 55 mM iodoacetamide (Sigma Aldrich). Proteins were then digested in 50 mM ammonium bicarbonate (pH 8.5), with sequencing grade modified bovine trypsin (Merck) at a final concentration of 1∶50 (trypsin:protein). After overnight digestion at 37°C, 1 µl trifluoroacetic acid (Merck) was added to stop the reaction and tryptic peptide solutions were cleaned with C18 spin columns (Protea Biosciences) according to the manufacturer’s instructions. Tryptic digests were dried in a Speedvac and resuspended in mobile phase A (5% acetonitrile (Merck), 0.1% formic acid (Fluka)). Samples were analyzed in Selected Reaction Monitoring (SRM) mode using a 6460 Triple Quadrupole LC/MS/MS on-line connected to an nLC-ChipCube interface (Agilent Technologies) and 1200 Series LC Modules (Agilent Technologies) provided with a pre-cooled nLC autosampler.

The following is the FASTA sequence for OPG, where the 4 measured peptides are in bold and underlined:

MNNLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKWKT

VCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCLK

HRSCPPGFGVVQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQKGNAT

HDNICSGNSESTQKCGIDVTLCEEAFFRFAVPTKFTPNWLSVLVDNLPGTKVNAESVERI

KRQHSSQEQTFQLLKLWKHQNKDQDIVKKIIQDIDLCENSVQRHIGHANLTFEQLRSLME

SLPGKKVGAEDIEKTIKACKPSDQILKLLSLWRIKNGDQDTLKGLMHALKHSKTYHFPKT

VTQSLKKTIRFLHSFTMYKLYQKLFLEMIGNQVQSVKISCL

Peptide separation was carried out onto a ProtID chip with 43×0.075-mm analytical column and 40 nL enrichment column (Agilent Technologies). Samples were injected at 4 µL/min and separation took place at 0.8 µL/min in a continuous acetonitrile gradient: 5–30% B at 0.5 min, 30–99% B at 1 min and 99% B at 3.5 min (phase B: 100% acetonitrile, 0.1% formic acid). Mass Hunter Software (v4.0) (Agilent Technologies) controlled the system. The mass spectrometer was operated in positive ion mode with capillary voltage of 1950 V, 325°C source gas temperature and 5 L/min source gas flow. Fragmentor was set to 130 V, dwell time to 250 ms, delta EMV to 200 V and collision energy was optimized for each SRM transition. SRM methodology allows specific detection of OPG proteotypic peptides. Theoretical SRM transitions were designed using Skyline (v.1.1.0.2905) [32] and manually inspected. OPG specificity was confirmed by protein blast and prototypic peptides were selected for SRM analysis.

OPG Immunohistochemistry

The site of OPG protein expression in the kidney cortex was localized by immunohistochemistry in 4 nephrectomy specimens from non-diabetic patients obtained from the IIS-Fundacion Jimenez Diaz biobank: A sample with preserved kidney histology from a 76-year-old male suffering from kidney cancer with serum creatinine 0.9 mg/dl and estimated glomerular filtration rate (eGFR) >60 ml/min; another kidney from a 74-year-old male had histological features of CKD secondary to ureteral obstruction by urothelial cancer, with serum creatinine 1.6 mg/dl and eGFR 45 ml/min. In addition 10 renal cysts from 2 autosomal dominant polycystic kidney disease (ADPKD) patients on dialysis, male and female, 54- and 62-year-old were studied. Paraffin-embedded sections 5 µm thick were processed as described [23] and antigen retrieval was performed at pH 9 (Dako). The primary antibody was polyclonal rabbit anti-human OPG (1∶500; Acris) and secondary antibody was HRP-conjugated. Sections were counterstained with Carazzìs hematoxylin. Negative controls included incubation with a non-specific immunoglobulin of the same isotype as the primary antibody.

Database Mining

In order to put the results in context and further define the potential clinical and biological implications the sub-set of proteins compiled in Table 2 was additionally analyzed by the following databases, Kidney & Urinary Pathway Knowledge Base [33], Nephromine [34] and STRING 9.0 software [35], consisting of a database of known and predicted protein interactions, including direct (physical) and indirect (functional) associations derived from genomics, high-throughput experiments, coexpression and previous knowledge. The highest confidence (0.900) filter was applied.

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Table 2. Proteins identified in tubular epithelial cell-derived exosomes by SDS-PAGE and LC-MS/MS analysis.

https://doi.org/10.1371/journal.pone.0072387.t002

Statistical Analysis

Results are expressed as mean ± standard error of mean. Differences between groups were assessed by the non-parametric test Mann-Whitney test and p<0.05 was considered significant. All the analysis was conducted using the SPSS statistical software (version 11.0).

Proximal Tubular Epithelial Cells Release Microvesicles with Exosomal Features

Human proximal tubular HK2 cells constitutively release microvesicles with exosomal features when cultured in serum-free medium for 24 hours (Figure 1). Microvesicles were isolated following a standard differential centrifugation exosome purification protocol. Electron microscopy of the pellet obtained in the last centrifugation showed that tubular epithelial cells secrete microvesicles into the culture media. These vesicles were 50–100 nm in diameter, consistent with previous descriptions of exosomes (Figure 1A). To further characterize the purified vesicles as exosomes, we studied common exosomal proteins using Western blot and flow cytometry (Figure 1B–D). Pellets from the different centrifugation steps were assayed by Western blot to assess the exosomal tetraspanin CD63 (Figure 1C). Cells and dead cells/debris express CD63. CD63 expression decreases through the isolation process and is enriched in the final exosome-like vesicle pellet. Microvesicles secreted by tubular cells were also positive for Alix and TSG101 but calnexin was absent, indicating absence of cytoplasmic contamination (Figure 1C). Flow cytometry confirmed the presence of CD63 in exosomal vesicles from proximal tubular cells (Figure 1D). These features indicate that human tubular epithelial cells secrete exosome-like vesicles.

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Figure 1. Characterization of exosomal-like vesicles isolated by serial centrifugation from cultured human proximal tubular epithelial cells.

Microvesicles from tubular cell conditioned serum-free media present exosomal features. A) Transmission electron microscopy shows vesicles have a diameter 50–100 nm, consistent with exosomes. i) Scale bar = 200 nm, ii) Scale bar = 100 nm, iii & iv) Scale bar = 50 nm. Arrows point to exosomes. B) Representative Western blot for the exosome marker CD63. Each lane contains 20 µg protein obtained from the pellet of the following sequential centrifugation steps: P1 1,100×g, P2 1,100×g, P3 6,000×g, P4 17,000×g, and P5 100,000×g supernatant. Exo: exosomes present in the 200,000×g pellet. C) Standard exosome markers were present in exosomes secreted by HK2 cells (Western blot). Each lane contains 6 µg of exosomal proteins. D) CD63 expression assessed by latex bead flow cytometry. Black peak: control beads. White peak: beads+PE–conjugated anti-CD63. Grey peak: beads+PE–conjugated anti-CD63+5 µg exosomes. FLH3: fluorescence intensity.

https://doi.org/10.1371/journal.pone.0072387.g001

Tubular Epithelial Cell-derived Exosome-like Vesicles Contain OPG but not TRAIL nor TWEAK

We have recently shown that cultured human tubular cells and tubular cells from human CKD biopsies express TRAIL, and that TRAIL promotes tubular cell death [16]. TRAIL is present in T cell-derived exosomes [36] and membrane bound-TRAIL is more lethal than soluble TRAIL. For these reasons we assessed the presence of TRAIL in exosomes secreted by human proximal tubular epithelial cells. However tubular epithelial cell-derived exosome-like vesicles did not contain TRAIL nor TWEAK, another TNF superfamily cytokine expressed by tubular cells (Figure 2A). Interestingly, anti-OPG antibodies identified a band of the expected size in Western blots run under reducing conditions (Figure 2B). A second, different anti-OPG antibody yielded similar results (not shown). The presence of OPG in exosomes secreted by tubular epithelial cells was confirmed using a specific ELISA assay for OPG (Figure 2C). We further confirmed the presence of OPG in tubular epithelial cell-derived exosome-like vesicles by selected reaction monitoring (SRM) in a QQQ-LC-MS/MS (Agilent Technologies). The aim of the SRM was solely to confirm specifically and unequivocally the presence of OPG in those samples, confirming WB data. In particular, two different transitions of the same precursor peptide could be detected co-eluting in time (Figure 2D). Three additional peptides were measured with several transitions per peptide (Figure S1).

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Figure 2. Proximal tubular cell exosomes contain OPG.

A) Exosomes from HK-2 conditioned serum-free cell culture media. Representative Western blots of TRAIL, TWEAK and exosome markers. Each lane contains 5 µg exosomal protein. B) OPG is observed in HK-2-derived exosomes when reducing, denaturizing conditions are applied. Each lane contains 10 µg exosomal protein. C) OPG expression in HK-2-derived exosomes detected by ELISA. Results expressed as pg/µg of total protein. Mean+SEM of 3 independent experiments. D) OPG analysis by selected reaction monitoring (SRM) in a LC-(QQQ)-MS/MS showing two different transitions corresponding to the same precursor peptide which coelute in time. The mass and charge of the precursor and its fragments are shown. A single peptide (YLHYDEETSHQLL) and a single precursor were measured under two different charge state (1025.9624+2 and 684.3107+3), each of them with its own fragment (1230.5783+ and 1138.4687+, respectively), thus yielding two different peaks or transitions.

https://doi.org/10.1371/journal.pone.0072387.g002

Additional Proteins Present in Proximal Tubular Epithelial Cell-derived Exosome-like Vesicles

Additional proteins present in exosome-like vesicles secreted by proximal tubular epithelial cells were identified by proteomic profiling. Exosomal protein extracts were concentrated and desalted by SDS-PAGE and identified by LC-MS/MS (Table S1). Twenty-one proteins were identified with a confidence ≥90%. Neither canonical exosomal markers nor OPG were identified by LC-MS/MS (Table 2). Most of the identified proteins had already been described in exosome-like vesicles as assessed by Exocarta [37]. However, this is the first time that vitamin D binding protein is described in exosome-like vesicles. Extracellular matrix proteins and, specifically, basement membrane proteins were overrepresented in tubular cell exosome-like vesicles. In this regard, nidogen-1 and agrin were only recently identified in exosome-like vesicles [37] and join type IV collagen, laminin, and fibulin-1 as basement membrane matrix proteins present in exosome-like vesicles. The presence of Fibronectin, C3 and TGFβ-ih3 was confirmed by Western blot (Figures S2 and S3). A search of the database STRING disclosed relationships between OPG and proteins identified in tubular cell-derived exosomal-like vesicles by LC-MS/MS (Figure 3).

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Figure 3. Relationships between OPG and proteins identified in tubular cell-derived exosomal-like vesicles by LC-MS/MS as evidenced by the STRING database.

STRING map of predicted associations for the sub-set of proteins compiled in Table 2. The network nodes are proteins and the edges represent the predicted functional associations based on evidence which are represented by different color lines: green line, neighborhood in the genome; blue line, co-occurrence across genomes; purple line, experimental evidence; yellow line, text mining evidence; light blue line, database evidence; black line, co-expression evidence. The highest confidence filter was applied. NID1: Nidogen 1; COL4A1/2: Colagen 4 A1/2; THBS1: Thrombospondin-1; FBLN1: Fibulin 1, FN1: Fibronectin, TNFRSF11B: Osteoprotegerin; LGALS3BP: Galectin-3 Binding Protein, TGFBI: TGF-β-induced protein ig-h3, GC: Vitamin D-binding protein; C3: Complement C3.

https://doi.org/10.1371/journal.pone.0072387.g003

Exosome-like Vesicles are Present in Human Urine and Contain OPG

The next step was to assess whether OPG was present in exosome-like vesicles from human urine samples. First we confirmed by electron microscopy that the isolation protocol applied to urine resulted in microvesicles with exosomal features in size and shape (Figure 4A). We confirmed the presence in these vesicles of the exosomal markers ALIX, CD63 and TSG101 (Figure 4B). As it was the case for exosome-like vesicles derived from human proximal tubular cells, human urine exosome-like vesicles contained OPG as assessed by Western blot (Figure 4B) and ELISA (3528±1994 pg OPG/µ g exosomal protein, not shown).

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Figure 4. Exosomes from human urine contain OPG.

A) Transmission electron microscopy representative images showing microvesicles purified from human urine. i) Scale bar = 200 nm; ii) Scale bar = 50 nm; iii) Scale bar = 200 nm; iv)Scale bar = 50 nm. ii&iv) show amplified areas from i&iii). B) Western blot. Representative images. Urine: whole urine collected from healthy donors. Supernat.: Urine supernatant from the last exosome isolation step. 5 µg protein were loaded per well.

https://doi.org/10.1371/journal.pone.0072387.g004

OPG Expression is Increased in Exosome-like Vesicles from CKD Urine

An exploratory study in urine from healthy controls or CKD patients (Table 1) disclosed that urinary exosome-like vesicle protein content was higher in urine from CKD patients than in non-CKD controls (Figure 5A). OPG-containing exosome-like vesicles were detected in human urine samples, as assessed by Western blot, both in healthy volunteers and in CKD patients. Since in a preliminary analysis the highest OPG content corresponded to patients with ADPKD, further studies were performed in this population. OPG is higher in exosome-like vesicles obtained from ADPKD CKD patients than from healthy controls (Figure 5B). Increased levels were also found in diabetic nephropathy (n = 3, 5.87±0.28 Arbitrary Density Units, A.D.U.), IgA nephropathy (n = 1, 18.78 A.D.U) and CAKUT (Congenital anomalies of the kidney and urinary tract) (n = 1, 22.58 A.D.U)(Figure S4).

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Figure 5. OPG in human urinary exosomes.

A) Urinary exosomal protein content is higher in CKD patients (n = 14) than in healthy volunteers (n = 4), * p = 0.042. B) Representative Western blot. 5 µg urinary exosomal protein were loaded. All CKD samples in this blot correspond to ADPKD patients. C) Quantification of OPG levels as assessed by Western blot in ADPKD CKD patients (n = 9) and healthy volunteers (n = 4). Results presented as mean+SEM, * p = 0.0476. A.D.U.

https://doi.org/10.1371/journal.pone.0072387.g005

Increased OPG mRNA had been observed in the transcriptome from human DN biopsies [16]. However results from urinary exosome-like vesicles suggested that other nephropathies might be associated to increased kidney OPG. A search of the Nephromine database of published kidney transcriptome datasets disclosed that OPG mRNA was increased in the tubulointerstitium of a different DN data set (fold-change vs control: 2.4, p = 4.83E-4) as well as in IgA nephropathy (fold-change vs control: 1.7, p = 2.07E-4) [34], [38], [39], [40]. Furthermore renal cortex OPG gene expression correlated with chronicity index quartile (correlation: 0.497, p = 3.60E-5) in aging humans [34], [40]. OPG immunohistochemistry of human kidneys showed that OPG is mainly expressed in the basolateral aspect of proximal tubules in control kidneys (Figure 6). In CKD whole tubular cells are intensely stained in the cortex. A similar pattern of intense whole cell staining is observed in cyst epithelial lining in ADPKD.

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Figure 6. OPG immunohistochemistry in human kidneys.

Control, CKD and autosomal dominant polycystic kidney disease kidneys samples were studied. For ADPKD a cortical cyst wall is shown. A) OPG mainly stained the basolateral aspect of proximal tubules in control kidneys (arrowheads). B) In CKD whole tubular cells are intensely stained in the cortex (arrows). C) A similar pattern of intense whole cell staining is observed in cyst epithelial lining (arrows). Original magnification ×200.

https://doi.org/10.1371/journal.pone.0072387.g006