Sample Criteria
Combined with the United Nations World Health Organization (WHO) age classification standards, the age groups were classified A(0-6 years old), B(7-14 years old), C(15-30 years old), D(31-44 years old), E(45-59 years old), F(60-79 years old), and G(≥80 years old), and each group was assigned 30 people (including 15 males and 15 females). Altogether, 210 people were recruited, and they were all of Han nationality. The physical examination illustrated that all participants had no hypertension, diabetes, cardiovascular or cerebrovascular diseases, or other diseases, and no abnormal liver or kidney function was identified. They had no history of taking medicines in the past month. With the consent of the participants, 100 mL of clean mid-morning urine under normal living conditions was obtained from each of them. The sample population information is shown in Table 1.
Table 1 Basic information of experimental subjects
|
Group
|
Sex
|
Age
|
Total Protein Concentration
|
Healthy (210 person)
|
A(0-6Y)
|
Male(15 person)
|
4.53±0.88
|
0.53±0.07
|
Female(15 person)
|
4.73±1.00
|
0.45±0.08
|
Total
|
4.63±0.95
|
0.49±0.09
|
B(7-14Y)
|
Male(15 person)
|
9.40±1.62
|
0.43±0.10
|
Female(15 person)
|
8.33±1.53
|
0.47±0.06
|
Total
|
8.87±1.67
|
0.45±0.09
|
C(15-30Y)
|
Male(15 person)
|
25.67±2.47
|
0.41±0.13
|
Female(15 person)
|
27.60±1.25
|
0.40±0.17
|
Total
|
26.63±2.18
|
0.40±0.15
|
D(31-44Y)
|
Male(15 person)
|
37.07±3.36
|
0.43±0.10
|
Female(15 person)
|
35.67±3.38
|
0.50±0.14
|
Total
|
36.37±3.44
|
0.47±0.13
|
E(45-59Y)
|
Male(15 person)
|
52.53±3.59
|
0.47±0.12
|
Female(15 person)
|
51.00±3.35
|
0.46±0.10
|
Total
|
51.77±3.56
|
0.47±0.11
|
F(60-79Y)
|
Male(15 person)
|
69.67±5.25
|
0.44±0.15
|
Female(15 person)
|
69.40±4.74
|
0.45±0.19
|
Total
|
69.53±5.00
|
0.44±0.17
|
G(≥80Y)
|
Male(15 person)
|
83.73±2.91
|
0.48±0.14
|
Female(15 person)
|
83.07±7.54
|
0.42±0.12
|
Total
|
83.40±5.72
|
0.45±0.13
|
Isolation and Preservation of Urinary Exosomes
Regarding the standard operating procedures of HKUPP and EuroKUP (http://www.eurokup.org), the obtained urine samples were immediately placed in a refrigerator at 4 °C, and the urine was processed within 2 hours. First, a urine sample was processed at 1500 g×10 minutes at 4 °C and then 5000 g×30 minutes to remove the cells. Second, sterilization and cell debris were filtered with a 0.22 μm filter (Millipore, SLGVR33RB). Third, the urine was concentrated through an ultrafiltration tube (Millipore, UFC910024). Finally, the size exclusion method (Izon Science Ltd, qEV10/35 nm) was used to extract the exosomes in the concentrated urine samples, which had been resuspended in PBS and stored directly at -80 °C.
Identification of Urinary Exosomes
TEM
Five microliters of urine exosome samples were dropped on the copper mesh. After incubation for 5 minutes at room temperature, the excess liquid was absorbed with absorbent paper from one side. Then, a drop of 2% uranyl acetate was put on the sample and incubated at room temperature for 1 minute, and then, the surface liquid from one side was absorbed by the absorbent paper. After a 20-minute drying process, the morphology of the urine exosomes was observed under a microscope (Tecnai G2 Spirit BioTwin, FEI).
NTA
The sample was diluted with prechilled 1×PBS to a suitable concentration and directly used for NTA (ZetaVIEW S/N 17-310, PARTICLE METRIX) detection (ZetaView 8.04.02) of the urine exosomal particle size.
Western Blot
Urine exosome samples were lysed with RIPA strong lysis buffer containing PMSF. Then, the sample with the determined protein concentration was loaded on a 15% sodium dodecyl sulfate- polyacrylamide gel. Next, 65 V to 100 V was used for electrophoresis, and then the membrane was transferred with a rapid transfer system (Bio-Rad, TURBO). Furthermore, a 5% blocking solution with TBST and skim milk powder was made and placed on a shaker at room temperature for 2 hours or at 4 °C overnight. Anti-CD9(Abcam, ab236630), Anti-CD63 (Abcam, ab271286), Anti-CD81 (Thermo, MA1-10290), Anti-RAB8A (Abcam, ab188574), Anti-RAB8B (Abcam, ab222017), Anti-Plexin B2/MM1 (Abcam, ab193355), Anti-Semaphorin 5A (Bioss, bs-20430R), Anti-Junctional adhesion molecule A (Abcam, ab269948) and Anti-STUB1/CHIP (ab134064) antibodies were then diluted at a ratio of 1:1000, followed by incubation in a refrigerator at 4 °C. After washing the membrane, a 1:2000 diluted secondary antibody was incubated for 2 hours at room temperature. Then, the membrane was rewashed with TBST and poured with chemiluminescent HRP solution (Millipore, WBKLS0500) for exposure.
Enzyme-linked immunosorbent assay
Took the standard solution to make a standard curve, added 100 μL sample to be tested to the sample well, sealed the plate with a sealing film, and incubated at 37 °C for 90 minutes. Next, discard the solution in the plate, washing the plate two times with wash buffer. Added 100 μL of biotin-labeled RAB8A (FineTest, EH1615) and RAB8B (FineTest, EH11620) antibody working solution into the above wells, covered the plate, and incubated at 37°C for 60 minutes. Remove the cover and washing the plate three times with wash buffer. Added 100ul of HRP-streptavidin conjugate (SABC)working solution into each well, cover the plate, and incubate at 37°C for 30 minutes. After washing, added 90μL TMB substrate and 50μL stop solution into each well. The OD value was measured at 450 nm and calculated the concentration according to the standard curve.
Immunofluorescence
The suspension containing EVs was added to a shaker box, centrifuged at 1500 r/min×7 min, permeabilized with 0.3% Triton X-100 for 10 min, and then blocked with 2% BSA (prepared in PBS) at room temperature for 30 min. The specific primary antibody (prepared in 2% BSA, dilution ratio 1:2000) and fluorescein-labeled mouse secondary antibody (Abcam, ab150077) were incubated in a humid box at 37 °C for 30 minutes and observed under an upright fluorescent microscope (EUROStar III Plus).
LC-MS/MS
Protein Isolation
The samples were mixed evenly, and then the urea particles were added to obtain a mixed sample suspension with a final urea concentration of 8 M. The sample was shaken until the urea particles were fully dissolved. Then, the sample was concentrated in an ultrafiltration tube. Ten microliters were maintained for Bradford protein quantification, and the remaining sample aliquots were kept at -80 °C.
Enzymatic Desalination
Then, 100 μg of sample protein was added to 10 mM DTT and reduced to 56 °C for 1 hour. Then, it was alkylated with iodoacetamide for 1 hour in a dark room at room temperature. The protein was digested with trypsin at a ratio of 1:50 and 37 °C overnight, desalted with a C18 cartridge to remove high urea, and dried by vacuum centrifugation.
Library construction and mass spectrometry data acquisition in DIA mode
A Q Exactive HF-X mass spectrometer and Nanospray Flex™ (NSI) ion source were employed. The ion spray voltage was 2.4 kV, and the temperature of the ion transfer tube was 275 °C. Data-dependent acquisition mode was performed, with a scanning range of 350-1500 m/z to construct the library. Mass spectrometry data were obtained by data-independent acquisition (DIA) mode, with a spectrum scanning range of m/z 350-1500.
Statistics and Analysis
Bioinformatics Analysis
This experiment employed the Homo sapiens database. Data analysis and visualization of DDA and DIA data were performed using the Proteome Discoverer 2.4 (PD 2.4, Thermo) platform, Biognosys Spectronaut 13 version, and R statistical framework. The original DDA-MS file was analyzed with PD software (version 2.4). The peak list was also searched against the protein database. Clusters of Orthologous Groups (COG) were performed for homology classification. The protein database was used for Gene Ontology (GO) to perform the statistical analysis of cell components, molecular functions, and biological processes (CC). The identified proteins data from each group of samples were uploaded to the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg) website to obtain and analyze all the map results from all channels.
Statistical Analysis
All experiments were performed three times. All values are expressed as the mean± standard deviation. Here, we took a 1.5-fold change as a cutoff for differentially expressed proteins, and P<0.05 was considered statistically significant. For the Enzyme-linked immunosorbent assay, tests were completed by R3.6.3 software. Due to the Shapiro-Wilk normality test, some groups did not meet the normal distribution. Therefore, to ensure the uniformity of all tests, the Kruskal-Wallis test was used for the different analyses for the simultaneous comparison of multiple groups. For pairwise comparisons, Wilcoxon rank-sum test was used for different analyses.