| BackgroundThe Nobel Prize in Physiology or Medicine held on Nov, 2013, was awarded jointly to James E. Rothman (American), Randy W. Schekman (American) and Thomas C. Sudhof (German) "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells". Randy Schekman discovered a set of genes required for vesicle traffic. James Rothman unrivalled protein machinery that allows vesicles to fuse with their targets to permit transfer of cargo. Thomas Sudhof revealed how signals instruct vesicles to release their cargo with precision. The award of Nobel Prize in Physiology or Medicine definitely will put forward the research of vesicles. Exosomes, as a kind of vesicles, are getting more and more attraction.In 1981, Trams and his team was the first group which observed the vesicles about 40 nm in diameter under electron microscopy, when they were doing cell cultures from various normal and neoplastic cell lines exfoliated vesicles with 5'-nucleotidase activity which reflected the ecto-enzyme activity of the parent monolayer culture. Exosomes are 40-100nm diameter membrane vesicles of endocytic origin that are released by most types of cells. There are prteins, DNA,RNA, lipids and others in exosomes. Firstly, endocytic origin visicle develeps into early endsomes. Secondly, exosomes biogenesis begins with formation of intraluminal vesicles (ILVs) through inward budding of endosomes to form multivesicular bodies (MVBs) with the help of endosomal sorting complex required for transport (ESCRT). Then, the MVBs either are transported to lysomes for degradation or fuse with the outer cell membrane to release their cargo of ILVs (now exosomes) to the extracellular environment.Exosomes may perform their roles of cell-cell interaction in local position or play roles in distal site through the circulation of blood. Depending on their origin,dendritic cell-drived exosomes can modulate immune-regulatory processes. HIV may exploit a natural host cell function, exosomes biogenesis, to aid to escape from cell.Tumor cell-drived exosomes may transfer proteins,mRNA and miRNA,set up tumor escape mechanisms, aid to angiogenesis, cell proliferation and the survival of cells.Exosomes also mediate regenerative or degenerative processes amongst others.Urinary exosomes are derived from renal epithelial cells, and released into urine lumin. Usually urinary exosomes contain several renal dysfunction or structure injury biomarkers. Recent researches found that there are biomarkers of renal epithelium extending from the glomerular podocytes (e.g., podocinhe, podocalyxin and WT1),biomarkers of the proximal tubule (e.g.,megalin,cubilin,APN,AQP1,type IV carbonic anhydrase and ?-glutamyltransferase), biomarkers of the thick ascending limb of Henle loop (e.g., THP, CD9 and type 2 Na-K-2C1 cotransporter), biomarkers of the distal convoluted tubules (e.g., NCC), and the biomarkers of the collecting duct(e.g., AQP2, mucin-1 and Rh type C glycoprotein). Furthermore, highly abundant proteins of the transitional epithelium of the urinary bladder, uroplakin-1 and -2, were identified. Thus, urinary exosomes analysis can potentially provide insight into the physiological or pathophysiological processes in every epithelial cell type facing the urinary space.Urine, as a source of non-invasive biomarkers, has got more and more attention from scientist. In 2009, the first exosomes data base was established. It included almost all the recent updates of exosomes researches. However, it still need to be timely update every year, because exosomes remain to be a new interesting field. In 2011, The International Society for Extracellular Vesicles (ISEV, about Microvesicles, Exosomes, Ectosomes and other Extracellular Vesicles) pronounced to be established. It will be a milestone of the history of exosomes researches. There were a couple of international conferences about urinary exosomes hosted every year.Some researches of urinary exosomes had been applied in renal and urogenital disorders, such as chronic kidney disease, carcinoma of urinary bladder and prostatic cancer, etc,. Also some published researches about acute myocardial infarction and non-small cell lung cancer.As it is known to everyone, urine, clear, amber-colored fluid formed by the kidney may carries metabolic wastes out of the body. Analysis of the urine is important in detecting diseases of the urogenital organs, as well as disorders of other body systems. However, Tamm-Horsfall protein is the most contamination during of urinary exosomes enrichment. Tamm-Horsfall protein polymers can entrap exosomes,bring difficulties to urinary exosomes recovery. Currently, many labs had establish their standard protocol of exosomes recovery, but most of them remains to be validate,since there was no authoritative, unified and acceptable urinary exosomes enrichment standard operating procedure.Generally speaking, there are two methods of urinary exosomes recovery:ultracentrifuge and nanomembrane concentrator with advantages and disadvantages.They both are a bit complicated, according to the literatures, and take time for operation. Sometimes they need to be equipped with expensive ultracentrifuge.Therefore, we determine to search for the simple and effective method of urinary exosomes recovery in order to provide proof for clinical researches.Diabetes has been a global public health problem, especially in China.According to the 5th version of updates coming from the International Diabetes Federation in 2012, there were 92,300,000 people suffered from diabetes in age 20-79 in China; ranking the No.1, the rest are India and America. The most important is that a half of diabetes don't know that they have diabetes. Some of the people with the impaired fasting glucose and/or impaired glucose tolerance were called as prediabetes since they haven't reached the criteria of diabetes diagnosis. Before presenting obvious clinical symptoms, prediabetes may be got complications, even if it was not observed. According to the survey of 1999-2006 in America, incidence of prediabetic nephropathy had been reached to 17.1%.Therefore, we conducted the research of comparison of the urinary exosomes of prediabetes, diabetes with normal proteinuria, diabetic nephropathy with microalbuminuria, diabetic nephropathy with macroproteinuria to normal controls,based on the method of 1000kDa nanomemrane concentrator we had established for urinary exosomes recovery, in order to search for the earlier protein biomarkers of diabetes and diabetic nephropathy.AimTo search potential protein biomarkers of diabetes and diabetic nephropathy in disease progression of different period, including of prediabetes, diabetes with normal proteinuria, diabetic nephropathy with microalbuminuria and diabetic nephropathy with macroproteinuria.Methods1 Optimize two methods of urinary exosomes recovery protocolTwo methods were established in our pilot study, one is ultracentrifuge method,and another is nanomembrane concentrator method.Approach 1: ultracentrifuge method. Firstly,urine was performed 2,000g centrifuge, room temperature, half an hour, in order to exclude the cell fragments,bacteria and other substance out. Secondly, 3.5kDa membrane was used to dialysis,three times ultra-pure water change, overnight in the cold room. After dialysis, speed vacuum system was used to concentrate urine to 10% or original volume. Then 18,000g centrifuge was performed, room temperature, half an hour, in order to remove contaminated protein. Finally, 200,000g ultracentrifuge, 4?, two hours, then collet pellet and suspend with ultra-pure water.Approach 2: nanomembrane concentrator method. Firstly, urine was performed 2,000g centrifuge, room temprature, half an hour, in order to exclude the cell fragments, bacteria and other substance out. Secondly, self-made 1,000kDa nanomembranse concentrator instrument was used to concentrate samples after check the function by normal urine concentrator. When samples were left about 6-8ml in the process of urine concentrator, add 200ml of ultra-pure water in order to wash the nanomembrane and filter the yellow pigments of urine. Finally, collet the 4-5 ml concentrated urine. After nonamembrane concentrator of urine, perform ultracentrifuge, 40,000g, 4?, 1 hours. Then collect the pellet and suspend with ultra-pure water.2 Optimize method of urine collectionCollect healthy volunteers'urine,and separate it into two groups: 15ml group and 50ml group. 15ml group was separated into two sub groups: add proteinase inhibitor group and no proteinase inhibitor group. The same as 50ml group, separated into add proteinase inhibitor group and no proteinase inhibitor group. Each group has 8 participants. Nanomembrane concentrator method was used to enrich urine exosomes. In order to compare the difference between each group, Bradford coomassie brilliant blue protein assay was used to quantify exosomes.3 Disposing of morning urine coming from patients of diabetes and diabetic nephropathy during different period of the diasease.75 community residents were included in our study from epidemiological survey in Zhuhai, including of 15 participants in each group of normal controls, prediabetes,diabetes mellitus with normal proteinuria, diabetic nephropathy with microalbuminuria, and diabetic nephropathy with macroproteinuria. Nanomembrane concentrator method was used to enrich exosomes from different groups.4 Identification of exosomesBradford coomassie brilliant blue was used to quantify exosomes protein assay.Transmission electron microscope (TEM) was used to observe the morphology of exosomoes and nanoparticle diameter. Nanoparticle tracking analysis (NTA) was used to observe the Brownian motion, morphology, nanoparticles diameter, and intensity of exosomse. Colloidal gels were stained by coomassie brilliant blue to observe the protein pattern. TSG101 was blotted, as a biomarker of biogenesis of exosomes.5 Application of proteomics analysis techniques in diabetes researchDifferential gel electrophoresis (DIGE) was used to isolate protein from different groups, Typhoon image scanner was used to scan the fluorescent gel. DeCyder 5.0 software (GE Healthcare) was used for 2-D DIGE analysis according to the manufacturer's recommendation. The DeCyder differential in-gel analysis (DIA)module was used for pairwise comparisons of each sample with the internal standard in each gel. The differential protein spots (|ratio|>1.2, p <0.05) which were altered consistently in all fifteen protein spot maps were selected for further identification.Protein identification was carried out by the ABI 4800 Proteomic Analyzer MALDI-TOF-MS mass spectrometry. Mass spectrometry spectra were searched in the Swiss Prot database for the protein identifications using Global Proteome Server Explorer software. Bioinformatics analysis and literature searching were used to identify interesting protein.6 Statistical analysisSPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All of these data from 15ml and 50ml morning urine groups with and without protein inhibitor was shown as mean ± SD (u g/ml),The factorial design analysis of variance was used for analysis. Sex, age, fasting blood glucose, ACR, SCr, BUN of residents in our study were included in statistical analysis. One-way Anova was used for continuous data analysis with LSD methods for equal variance, and Dunnett's T3 method and Welch correction for equal variances not assumed. ?2 test was used for numerous data analysis. P<0.05 was considered as significant difference.Results1. Comparison with ultracentrifuge method, nanomembrane concentrator method have these advantages, such as little times of centrifugation, simple method of dialysis for cleaning up, concentrate samples quickly, saving time in all the protocol, general laboratory can manage, may dispose large volume of urine one go.2. Adding or no proteinase inhibitor has little influence on quantification of urinary exosomes protein assay by Bradford coomassie brilliant blue protein assay, there was no significant difference (F=0.828, P>0.05). However, 50ml of urine yield more exosomes than 15ml,there were significant difference (F=58.661, P<0.001).There were significant difference in the age (F=11.950, P<0.001), fasting blood glucose (F=28.950,P<0.001), ACR (F=161.456,P<0.001),SCr (F=3.111,P<0.05) , BUN (F=5.612, P<0.05), male (?2=32.000, P<0.001) , and female(?2=43.000, P<0.001) of residents in our study.3. Exosomes exhibit cup shape, or ellipse under TEM, equally distribution, and 30-100nm diameter in nanoparticles. NTA analysis shows the same distribution,same morphology and Brownian motion activity, the mean nanoparticles diameter is 55-1 10nm. Bradford coomassie brilliant blue protein assay show that protein concentration in normal controls is 15.4?g/ml, in prediabetes is 26.56?g/ml, in diabetes with normal proteinuria is 51.55?g/ml, in diabetic nephropathy with microalbuminuria is 76.62?g/ml,in diabetic nephropathy with macroproteinuria is 89.14?g/ml. Protein pattern is clear in each group of colloidal gel stained with coomassie brilliant blue. Exosomes biogenesis biomarker of TSG101 is present in each gel blotted by anti-TSG101 antibody.4. 22 differential proteins were identified by combination of 2D-DIGE and MALDI-TOF/TOF mass spectrometry. Bioinformatics analysis show that these proteins' biological function involved in catalytic activity, binding activity,structural activity, enzyme regulator activity,mRNA processing activity,transcription regulator activity; these proteins' biological process involved in cell killing process, chronic inflammatory response, immune response, signal pathway, development process, extracellular matrix organization, hemopoiesis,signal transduction process, differentiation process, transport process, biological process, and metabolic process. Four interesting protein of MASP2, CALB1,Protein S100 A8, and Protein S100 A9 were selected by bioinformatics analysis and literature searching.Conclusions1. Nanomembrane concentrator method was determined to enrich urinary exosomes for proteomics analysis.2. 50ml morning urine adding proteinase inhibitor or not both can recover more exosomes than 15ml morning urine, and have little effect on enrichment of urinary exosomes. 50ml morning urine is better for proteomics analysis.3. Secreted exosomes protein quantification vary ar different stage of diabetes and diabetic nephropathy, it increase with the development of disease progression,therefore exosomes may change the cargo of diabetes, and involve in the pathogenesis and progression of disease.4. MASP2, CALB1, Protein S100 A8 and Protein S100 A9 may involve in variation of different stage of diabetes and diabetic nephropathy, and may be potential biomarkers of disease progression.Innovation pointWe didn't find same report about proteomic analysis of diabetic disease in different stage urinary exosomes by DIGE and MALDI-TOF/TOF mass spectrometry,after research of bibliographic retrieval domestic and overseas. Our research objects coming from community residents of epidemiological survey let our results more reliable. Hunting for differential protein by DIGE method has the advantage of good biological repeat and much better sensitivity. This is the first time report of MASP2,CALB1, Protein S100 A8 and Protein S1000 A9 in urinary exosomes vary significantly in diabetes of different stage. |
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