Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosomelike vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.This work was supported by grants from the Instituto de Salud Carlos III (ISCIIIRETIC REDINREN RD06/0016, RD12/0021, PI11/01854, PI10/00072 PI09/ 00641 and PS09/00447); Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD-2378); Sociedad Española de NefrologÍa; European Network (HEALTH F2-2008-200647); DIALOK European project LSHB-CT-2007-036644; Fundacion Lilly and IRSIN/FRIAT to JE; Programa Intensificación Actividad Investigadora (ISCIII/ Agencia Laín-Entralgo/CM) to AO; Instituto de Salud Carlos III (FIS PI11/01401, CP09/00229); and Fundación Conchita Rábago de Jiménez DÍaz to GAL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

<div><p>Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.</p></div

Proximal tubular cell exosomes contain OPG.

<p><b>A)</b> 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>B)</b> OPG is observed in HK-2-derived exosomes when reducing, denaturizing conditions are applied. Each lane contains 10 µg exosomal protein. <b>C)</b> OPG expression in HK-2-derived exosomes detected by ELISA. Results expressed as pg/µg of total protein. Mean+SEM of 3 independent experiments. <b>D)</b> 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 (<u>YLHYDEETSHQLL</u>) 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.</p

OPG immunohistochemistry in human kidneys.

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

Exosomes from human urine contain OPG.

<p><b>A)</b> 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>B)</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.</p

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

<p>Microvesicles from tubular cell conditioned serum-free media present exosomal features. <b>A)</b> 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>B)</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. <b>C)</b> Standard exosome markers were present in exosomes secreted by HK2 cells (Western blot). Each lane contains 6 µg of exosomal proteins. <b>D)</b> 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.</p

Clinical characteristics.

<p>Summary data expressed as mean±SD.</p><p>sCr: serum creatinine, eGFR: estimated glomerular filtration rate, UProt: urinary protein.</p

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

<p>STRING map of predicted associations for the sub-set of proteins compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072387#pone-0072387-t002" target="_blank">Table 2</a>. 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.</p

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

<p>Exocarta: Presence (Y) or absence (N) in the exosomal database Exocarta, accessed December 28, 2012. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072387#pone.0072387-Mathivanan1" target="_blank">[37]</a>. In addition several keratins were identified (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072387#pone.0072387.s005" target="_blank">Table S1</a>).</p><p>Conf.: confidence of the identification. ECM: extracellular matrix.</p