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

Clinical characteristics.

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

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

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

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

Psychologické a sociální aspekty umírání a smrti

<p>Data are expressed as mean ± SEM. *p<0.05 vs. CONTROL; ^#p<0.05 vs. ALDO</p

Quantitative analyses of protein levels measured by Western blot for ColI (A), FN (B), CTGF (C) and MMP-2(D) in control (CONTROL), spironolactone treated animals (SPIRO), aldosterone+salt-treated animals (ALDO) and aldosterone+salt plus spironolactone treated animals (ALDO+SPIRO).

<p>Data are expressed as mean ± SEM. *p<0.05 vs. CONTROL; ^#p<0.05 vs. ALDO</p

(A) Representative images showing Sirius red staining, Masson trichrome and Hematoxilin/Eosin staining in control+PBS liposome (Control+Lipo), aldosterone+PBS liposome (Aldo+Lipo), control+clodronate liposome (Control+Clodr), or aldosterone+clodronate liposome (Aldo+Clodr) treated animals. Kidney collagen content (B), quantitative analyses of Col I and Fibronectin (FN) protein (C-D) and mRNA expression (E) in treated rats.

<p>Data are expressed as mean ± SEM. *p<0.05 vs Control+Lipo; #p<0.05 vs. Aldo+Lipo.</p