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

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

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

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

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

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

Baseline characteristics of patients with FSGS who received a renal transplant and presented recurrence of proteinuria.

<p>All patients were treated with plasmapheresis and had their plasmapheresis eluates processed for partial purification of Permeability Factor (PF). P_alb activity reported in the Table is referred to that measured in the partially purified fraction prepared from plasmapheresis eluates following a procedure based on protein A Sepharose and differential precipitation in ammonium sulphate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188045#sec002" target="_blank">Methods</a> for details).</p

sCD40L concentrations in serum samples from children and adult patients with idiopathic nephrotic syndrome and from healthy subjects.

<p>(A) Serum sCD40L levels in healthy controls and in patients with idiopathic nephrotic syndrome (NS), subdivided according to clinical features (<i>i</i>.<i>e</i>. age at onset and response to steroids). Baseline clinical characteristic of the patients with idiopathic nephrotic syndrome are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188045#pone.0188045.t002" target="_blank">Table 2</a>. Children who presented proteinuria under the 1^st year of age underwent biopsy and had a diagnostic molecular approach for several genes implicated in NS; they were classified as congenital NS (cNS). (B) Serum sCD40L levels in healthy controls and in patients with steroid-resistant idiopathic nephrotic syndrome according to the levels of proteinuria (< or > 0.5 g/day). (C) Serum sCD40L levels in patients with nephrotic syndrome and a biopsy-proven diagnosis of FSGS or of iMN. Patients with FSGS were further subdivided according to their age in < or > 18 years. Patients with iMN were always older than 40 years.All patients had eGFR >60 ml/min at the time of serum sampling for sCD40L measurement.</p

Permeability activity of albumin (P_alb) induced by sCD40L in isolated rat glomeruli.

<p>P_alb was determined after the incubation of rat glomeruli for 30 min with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer). A significant increase in glomerular permeability is expressed by values of P_alb greater than 0.5 (black bar). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to glomerulus stimulation, whereas the CD40L-muCD8 fusion protein (50 ng/ml) was added to the glomeruli 30 minutes before adding hr-sCD40L (dashed bars) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188045#sec002" target="_blank">Methods</a> for details). At least five animals were studied per each experimental group. *<i>P</i> < 0.05 and **<i>P</i> < 0.01 versus unstimulated controls; ^#<i>P</i> < 0.01 versus rh-sCD40L.</p