Biochemical and Physical Characterisation of Urinary Nanovesicles following CHAPS Treatment

<div><p>Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, <em>e.g.</em> by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.</p> </div

Proteases activity.

<p>Membrane-bound DPP IV (Panel A) and NEP (Panel C) peptidase activity profiles recorded in absence and presence of 10 mM DTT. Samples were dialysed at a MWCO of 300 kDa. DTT pellet 200,000 g (sample 1), DTT SN 200,000 g (sample 3), CHAPS pellet 200,000 g (sample 2) and CHAPS SN 200,000 g (sample 4) are represented. Columns compare DTT vs CHAPS after dialysis with a membrane of MWCO 300 kDa and in the presence of 5 mM DTT. Values represent mean ± SD of units of peptidase (UP) per milligram of protein per minute. Panel B represents the Coomassie gel and DDP immunodetection of the same samples. Ten µg of protein per fraction obtained in Method 1 were loaded on the gels after 300 kDa MWCO dialysis.</p

Protein identification comparisons.

<p>Venn diagram showing the distribution of the number of identified proteins presents in SN 200,000 g after CHAPS and DTT treatments. Protein identifications from the current study were compared to two other studies which were carried out using high-resolution mass spectrometers in gels on 200,000 g pellets after DTT treatment (Gonzales et al. 2008) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Gonzales1" target="_blank">[5]</a> and 200,000 g supernatants (Kentsis et al. 2009) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Kentsis1" target="_blank">[23]</a>.</p

TEM analysis.

<p>Transmission electron micrographs of <b>P18</b> (Panel A) and <b>P200</b> (Panel B) at 10,000× and 5,000× magnifications, respectively. High-magnification (50,000×) of CHAPS- (Panels C-F) and DTT-treated (Panels G-I) vesicle preparations are represented.</p

Protein identification in DTT and CHAPS supernatant.

<p>Partial list of proteins not previously reported in urinary exosomes and in 200,000 g supernatants.</p>a<p>Unique peptides on the total number of peptides.</p

Western blotting analysis.

<p>Rabbit anti-CD63, Rabbit anti-TSG101, rabbit anti-MGF-E8/lactadherin and rabbit anti-nephrin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Hara1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Khatua1" target="_blank">[50]</a>. Ten µg of protein of fractions obtained in Method 1 were loaded on the gels.</p

SDS-PAGE.

<p><b>Panel A:</b> Gel Acrylamide T 12% constant. Fifteen µg of protein per lane of crude preparation <b>Panel B:</b> Gel Acrylamide T 8% constant. Ten µg of protein per fraction obtained in Method 1. <b>Panel C:</b> Gel Acrylamide T 12% constant. Ten µg of protein per fraction obtained in Method 2.</p

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X‑114 Phase Partitioning

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm–Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a “hydrostatic filtration dialysis’’ appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC–MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs

Prevalence of CKD in 1534 subjects based on HW phenotype.

<p>Prevalence of CKD in 1534 subjects based on HW phenotype.</p

Association of the hypertriglyceridemic waist phenotype with CKD.

<p>a.Adjusted for age, sex.</p><p>b.Adjusted for age, sex, history of hypertension, history of coronary heart disease, history of stroke, history of malignancy, current smoker, current alcohol use, physical inactivity, educational status.</p><p>c. Adjusted for above + diabetes and hypertension.</p