Urinary Exosomes Enrichment, Purification, And The Internal Proteome Profile Analysis

BackgroundIn 1987, Johnstone and his team found a kind of 60nm vesicles in the goat reticulocyte culture supernatant after ultracentrifuge, during his research on the mature process of reticulocyte. He named these nanoscale small vesicles as "exosomes". Exosomes, the cup-shaped vesicles under the electron microscope, is composed of lipid billayer, transmembrane proteins, mRNAs, microRNAs. It’s density is between 1.13ug/ml and 1.19g/ml. The following researches revealed that exosomes can be released by most cell types in the extracellular space, such as epithelial cells, fibroblast cells, mast cells, antigen presenting cells, tumor cells, hepatic cells, intestinal epithelial cells, plastocyte, etc. Moreover, exosomes present in vivo in many body fluids, including blood, urine, breast milk, amniotic fluid, sperm, saliva, synovial fluid, hydrothorax, ascites, bronchial alveolar lavage, and so on. The releasing of exosomes to the extracellular space is not just for removing the waste protein in the cells, exosomes also play a pivotal role in cell-to-cell communication. It could participate in a variety of physiological and pathological process by mediating biological information.Exosomes from different cell origins share common groups of proteins that are involved in exosome biogenesis, and thus reflect many common biological functions. In addition, cell-type specific proteins are present that may reflect the origin and biological functions of the parent cells.Currently, the researches on exosomes are not only focus on it’s function, but also practical application, exploring the technologies associate with disease diagnosis and treatment. Exosomes have the ability to transport proteins, mRNAs, and microRNAs and to modulate immune reaction, angiogenesis, cell proliferation, and tumor cell invasion, thus represent an ideal resource for biomarker identification which is aimed to early diagnosis of disease. By for now, some diseases associted biomarkers have been identified by studies on exosomes in blood, urine, saliva and other body fluids, which bring new hope to individual treatment of diseases at early stage.Exosome vesicles was first description in the urinary solid phase in 2004 by Pisitkun. Normal urine consist large number of exosomes, which are released from every renal epithelial cell of the the urinary system(inclding kidney, bladder and prostate, etc.). Urinary exosomes, like other kinds of exosomes, contains lots of information of the original cells. Thus, their study opens the possibility of discovering physical function and pathological changes of special cells. More than 1000 proteins have been identified from the isolated urinary exosomes with proteomic technologies. Comparing to other kinds of body fluids, urine has unique advantage of large volume and noninvasion. Urinary exosomes represent source of biological material for finding biomarkers of early renal and urogential disorder, for exploring target point of disease treatment and monitoring, and for the studies on disease pathogenesis. Recent studies have shown the changes of urinary exosomes proteome profile could be associated with special kidney injury. Urinary exosomes, which cargo biological information, could shuttle between different kidney cells and change the proteome profile and function of receptor cells. This represent a kind of signal-transmitting mechanism between cells of nephron.Combining with exosomes isolation method and advanced proteomic technologies, some urinary exosomes biomarkers associated with kidney structure and function injury have been identified in latest years. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinaryexosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunteers or steroid-sensitive nephrotic syndrome (SSNS) patients. Urinary exosomal WT-1 was also significantly decreased in patients in remission for either FSGS or SSNS or following steroid treatment in six SSNS subjects. We conclude that urinary exosomal WT-1 is a promising noninvasive biomarker with apparent podocyte specificity that can detect early progression and treatment-induced regression of podocyte injury in FSGS or SSNS. Recently (in 2014), Zubiri I et al. evaluated different protocols for urinary exosome isolation. The optimal methodology was then applied to investigate the proteome of human urinary exosomes in DN and controls using spectral counting LC-MS/MS analysis. A panel of 3 proteins (AMBP, MLL3, and VDAC1) is differentially present in urinary exosomes from DN patients. These proteins also could be the potential DN biomarkers. Beside above researches, there were other reports about urinary exosomes on acute kidney injury, polycystic kidney, renal fibrosis, bladder cancer and so on.Urinary exosomes bring us great prospect of reseaches on urinary system diseases, especially the kidney diseases. But the associated studies are still on the preliminary stage, numbers of challenges need be solved.First of all, we are facing the problem of urinary exosomes isolation, enrichment and purification, as there is no gold standard method has been builded untill now. There are two exosomes enrichment approaches are widely adopted now: one is based on the ultracentrifuge, the other is based on ultrafiltration with nanomembrane. But both have disadvantages. Another barrier to exosomes enrichment and purification is the knotty problem of Tamm-Horsfall protein excretion. The kidney excretes large amounts of Tamm-Horsfall protein(THP) every day. This protein is a member of the zona pellucida protein family and tends to form vast fibrillar aggregates in urine. These aggregates can entrain the exosomes and prevent their efficient isolation and purification by centrifugation methods, which could affect the results of exosomal proteomic analysis. According to the literature survey, no study has reported that Tamm-Horsfall protein could be completely elimilated from the outside of the urinary exosomes. Another barrier for urinary exosomes research is lacking normalizing variables, which might restrain the clinical application in the future. Ideally, we would like to know the absolute rate of excretion of each protein and compare excretion rates among patients and controls. Simple measurement of the concentration of individual proteins in urine is of limited use because variation in water excretion would be the main determinant of the concentration of a given biomarker candidate. Normalization by urinary creatinine concentration is a traditional substitute as a normalizing variable. For example the concentration of urine albumin is usually normalized by urine creatinine. But it’s not precise as creatinine excretion rate varies widely among humans, potentially masking true changes in the excretion of individual protein markers. Alternatively, the excretion of specific exosomal proteins can be normalized by the excretion of exosomal markers such as Alix or TSG101 that are found in all exosomes. Unfortunately, the measurement of TSG101 and Alix are complicated and time consuming. So it’s necessary to explore a new normalizing variable that is applicable.During last two years, our team has created a new approach, named "hydrostatic dialysis", for urinary exosomes enrichment. This method has the following advantages:no expensive equipment is need; low cost; large amount of urine could be proceed; the soluble proteins are effectively removed; could eliminate other factors that might affect the subsequent biological analysis(the PH of different urine samples will be the same after hydrostatic dialysis); minimize the loss of exosomes; could easily combing with the other two exosomes enrichment methods, which are based on ultracentrifuge and ultrafiltration, respectively. Before the wide popularization and application of the "hydrostatic dialysis" method, we should validate it’s reproducibility and effectiveness with urine samples from Chinese patients with different degree of kidney disorder. In addition, based on our exosomes enrichment method, we will examine the candidate normalizing variable for urinary exosomes. Meanwhile, we will carry out reduction, alkylation and trypsin digestion reaction for urinary exosomes purification(completely elimilate the disturbing protein outside the exosomes). Then, we will conduct Mass Spectrometry to detect the proteome profile inside the urinary exosomes.Methods1. Urine samples selecting and collection The urine samples for "hydrostatic dialysis" method validation were selected from the Chinese biobank. All the samples from individuals of normal group, prediabetes group, diabetes without proteinuria group, diabetes with microalbuminuria group, diabetes with macroalbuminuria group were stored in-80℃ ultra-low temperature freezer before experiment.The urine samples for exosomes normalization, purification and Mass Spectrometry were collected from the healthy volunteers in Dublin City University. All the participants signed the information consent, which had been approved by the Ethics Committee of National Centre of Sensor Research of Dublin City University before enrollment. First morning urine were collected from these participants. Samples were sent to laboratory in 2 hours without protein inhibitors or preservative.2. Urinary exosomes enrichment with "hydrostatic dialysis" methodAt first, urine was performed 2,000g centrifuge at room temprature for 30 min, in order to exclude the cells, cell debris, and some Tamm-Horsfall protein. Then self-design instrument for hydrostatic dialysis was used for exosomes recovery. After checking the collection of the system, the urine was introduced to the 1000KD membrane. When samples was concentrated to 6-8ml,200ml of millQ water was added to rinse the 1000KD membrane. Then, we collet the final 6-8ml conentrated retain solution inside the 1000KD dialysis membrane(↑1000KDa fraction).3. Urinary exosomes identificationWe observed if there were vesicles in ↑1000KDa fraction after hydrostatic dialysis under the transmission electrom microscopy, and the shape and size of the urinary vesicles. In addition, we checked the existence of urinary exosomes biomarker TSG101 by western blot.4. Exploring the normalizing variable for urinary exosomesWe explored the possiblity of applicating Tamm-Horsfall protein and albumin as the normalizing variable for urinary exosomes. After conducting western blot, we examined the fluorescence signal of Tamm-Horsfall protein with TSG101, and albumin with TSG101 in the same membrane respectively. We calculated the band intensity of Tamm-Horsfall protein, albumin and TSG101. Statistic analysis was performed by using SPSS 13 software. The correlation between THP and TSG101 band intensity, albumin and TSG101 band intensity from the 10 samples were analyzed with Spearman’s rank correlation coefficient test. P< 0.05 was considered significant.5. Eliminating the interfering proteins outside the exosomes with reduction and alkylation reactionThe polymer struction of Tamm-Horsfall protein consists lots of disulfide band. After using the reduction and alkylation agents to break the disulfide band, we analysed the effect of reaction on intefering proteins outside the exosomes(especially the degradation and remove of Tamm-Horsfall protein) and the exosomes itself. This results could provide guidence for urinary exosomes purification.6. Eliminating the interfering proteins outside the exosomes with trypsin digestion and exosomes purificationAfter reduction, alkylaion and trypsin digestion for ↑1000KDa fraction after"hydrostatic dialysis". The interfering proteins outside the exosomes (especially the Tamm-Horsfall protein) were degradated into peptides, which was removed by 30KD molecular weight cutoff ultrafiltration device. The degradation and elimination of Tamm-Horsfall protein outside exosomes and the affection to exosomes itself were observed by coomassie blue staining of SDS-PAGE gel, western blot and transmission electron microscope.7. Internal proteome profile analysis of urinary exosomes based on the exosomes purificationThe urinary exosomes were purified from samples after "hydrostatic dialysis". Sodium deoxycholate was used for breaking the membrane of exosomes, then the materials inside exosomes were exposed. After reduction, alkylation and trypsin digestion again, the internal proteome profile was analyzed by mass spectrometry.Results1. The validation of new urinary exosomes enrichment methodWe effectively enriched the exosomes by means of "hydrostatic dialysis" method in the urine of patients with different degree of kidney injury. It showed good reproducibility and stablity. There was a heterogeneous population of vesicles, main of which ranging in size from 40 to 100nm in diameter. We also observed small size vesicles around 20-30nm in diameter, and the biggest size vesicle could be 400nm in diameter. The fluorescence signals of TSG101, the biomarker of exosomes, were positive in all groups. It also indicate the exosomes during the enrichment procedure are intact.2. The normalizing variable for urinary exosomesWe calculated the band intensity of fluorescence signals with Quantity-one analysis software from the Odyssey infrared scanned pictures. The Spearmans rank correlation analysis showed that the correlation between Tamm-Horsfall protein and TSG101 was significant, r=0.842, p=0.002. This means there is positive correlationship between these two items.3. Eliminate the interfering proteins outside the exosomes with reduction and alkylation reactionThe results of coomassie blue staining of SDS-PAGE gel and western blot indicated that the Tamm-Horsfall protein outside the exosomes was partially degradated. We can hardly completely eliminate the interference of Tamm-Horsfall protein to urinary exosomes with two kinds of reduction and alkylation agents.4. Eliminate the interfering proteins outside the exosomes with Trypsin digestion and exosomes purificationThe results from coomassie blue staining of SDS-PAGE gel and western blot indicated that the Tamm-Horsfall protein outside the exosomes were fully degradated after reduction, alkylaion and trypsin digestion. The peptides after digestion could be removed by 30KD molecular weight cutoff ultrafiltration device. With the above process, urinary exosomes could be purified.5. Internal proteome profile analysis of urinary exosomes based on the exosomes purificationBased on the purification approach mentioned above, the internal materials of exosomes was received MS/MS analysis.942 proteins inside urinary exosomes were identified, and their cellular component, molecular function and biological process were also analyzed.Conclusions1. The "hydrostatic dialysis" method invented by our team could effectively enrich urinary exosomes, and it has good reproducibility and stablity. This method are applicable in different laboratories, and urine from different population and various pathologic conditions. It is worth wide popularization and application.2. Tamm-Horsfall protein could be the candidate normalizing variable for urinary exosomes associated measurement.3. Urinay exsomes could be purified with reduction, alkylation and trypsin digestion reaction combining with ultrafiltration. The internal proteome profile could be acquired after urinary exosomes purification.4. Our present study establish a series of urinary exosomes research protocols, including urine collection, exosomes isolation, enrichment and purification, and corresponding proteomic analysis. These protocols could provide a guideline for studies on disease biomarkers in urinary exosomes.