2.1 Isolation, culture, and characterization of mouse bone marrow-derived EPC
EPCs were isolated from mouse bone marrow according to the previous study. Bone marrow medulla was centrifuged by Density Gradient with a Human peripheral blood lymphocyte isolation fluid (Ficoll Plus 1.077, Solarbio, P4350, Beijing China) to obtain the bone marrow Mononuclear Cells (MNCs). MNCs were plated in fibronectin (Solarbio, F8180, Beijing, China) coated cell culture bottle with a ventilation filter and maintained in Endothelial Growth Medium-2 (EGM-2, Lonza, CC-4176, USA) with 10% fetal bovine serum (FBS, EPHRAIM, 26-500-FBS China) in a cell incubator with 37℃ and 5% CO2. The medium was replaced every 48h. Cell colonies those appeared about the first 7days later, these cells had a fusiform morphology and were defined as the early EPC. After 28 days of culture, cells emerged abilities of proliferation and tube formation, had a pebble-like compact arrangement as endothelial cells (EC), and were called endothelial colony-forming cell (ECFC), outgrowth endothelial cells (OECs), or late-EPC.
There is no consensus about EPC cell typing now, but the most recognized characterizations of EPC are CD34, CD133, and VEGFR2 positive and Human Dil-Acetylated Low-Density Lipoprotein (DiI-Ac-LDL) and FITC-UEA-I double-positive. The cells cultured about 28 days were plated on sterilized coverslips (Beyotime FCGF18, shanghai, China) and cultured in the medium with 5µg/ml Human DiI-Ac-LDL (Maokangbio, MP6013, Shanghai, China) at 37℃ for 4h, 4% paraformaldehyde (PFA) fixed 30min, 10µg/ml FITC-UEA-I (Maokangbio, MP6038, Shanghai, China) incubated for 1h at room temperature, washed by PBS for three times and then the fluorescence images were captured by a fluorescence microscope (Carl Zeiss,Axio Scope A1, Germany). The fixed cells on coverslips were blocked with 5% bovine serum albumin (BSA Solarbio, A8010, Beijing, China) for 1.5h The primary antibodies, rabbit anti-CD34 (1:100, Wanleibio, WL02529, Shenyang, China), rabbit anti-CD133 (1:100, Proteintech, 66666-1-Ig, Wuhan China) and rabbit anti-VEGFR2 (1:100, Wanleibio, WL02294, Shenyang, China) incubated overnight at 4℃༌the second antibody (Cy3-conjugated Goat Anti-rabbit IgG H+L༌1:100༌Proteintech, Wuhan China SA00009-2) incubated for 2h at room temperature in the dark. After being washed by PBS, the images were captured by a fluorescence microscope (Carl Zeiss, Axio Scope A1, Germany). The mouse aorta vascular endothelial cells (MAVECs) as a negative control.
Adult male BALB/c nude mice, weighing 20-25g, were obtained from Gempharmatech, Nanjing, China.
High-fat diet (HFD, 10% saccharose, 10% lard, 10% sugar, 5% egg yolk powder, 0.5% cholesterol, 64.5% basal chow) and a low dose of streptozotocin (STZ) to develop diabetes according to the method described previously. Briefly, after 4 weeks of feeding with HFD, mice were administered STZ (65mg/kg body weight, pH 4.5) or citrate buffer (vehicle) by intraperitoneal (i.p.) injections once a day for 5 days. After one week, the mice fasted for 12h for fasting blood glucose (FBG) testing. Plasma glucose concentration was detected using a commercial glucometer (GA-3, Sinocare, Changsha, China). Fasted mice with blood glucose levels higher than 11.1mmol/L were considered as diabetic mice. 24h urinary albumin quantitation and plasma and urinary creatinine quantification and urinary albumin-to-creatinine ratio were used to evaluate renal function in diabetic mice. Urinary albumin and creatinine were detected by a commercial kit purchased from Jiancheng bioengineering institute (C035-2-1 and C011-2-1 Nanjing, China)
2.3 HE and PAS staining of the glomerulus
Mouse kidney was perfused by PBS and fixed in 4% PFA, dehydrated and embedded in paraffin. Paraffin sections were cut at 5µm. After deparaffinization and rehydration, glomerulus sections were dyed by hematoxylin solution (hematoxylin 1g, sodium iodate 0.2g, aluminum potassium sulfate, 50g, citric acid 1g, chloral hydrate 50g, distilled water added to 1 liter) for 10 minutes, alcoholic eosin (2.5g eosin, 500ml distilled water, hydrochloric acid 10ml, filter product dissolved in 1000ml 95% alcohol, double diluted before use) for 30 seconds respectively.
Periodic Acid-Schiff stain (PAS) of glomeruli was performed using the PAS stain kit (Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China). After deparaffinization and rehydration, glomerulus sections were immersed in periodic acid for 5min, washed by running water and distilled water respectively, then in Schiff reagent for 10min in the dark. After being washed with running water for 10min, the nuclear was stained by the hematoxylin solution. The sections were washed with running water until the nuclear turning blue.
The sections were dehydrated by the gradient of ethanol, and graphs were captured by microscope (Carl Zeiss, Axio Scope A1, Germany) in the light field mode.
2.4 Western Blot Analysis and co-immunoprecipitation
Western blot analysis was performed as described previously. Briefly, cell total protein and membrane protein samples were extracted using Membrane and Cytosol Protein Extraction Kit (Beyotime, P0033, Shanghai, China), nuclear protein samples were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027, Shanghai, China). Protein concentration was quantified using a BCA Protein Assay Kit (Keygentec, KGP903, Nanjing, China). According to the quantification, protein samples were adjusted to equal with 5×loading buffer (Beyotime, P0015, Shanghai, China), and samples were boiled for 5 minutes at 95°C. Equal amounts of cell lysate protein were resolved by SDS-PAGE using 5% (w/v) stacking and 8%-15% (w/v) separating polyacrylamide gels, and then transferred to 0.45µm polyvinylidene difluoride membrane (PVDF, Millipore, USA), which were then blocked for 1.5 hours in 5% (w/v) non-fat milk diluted in Tris-buffered saline (TBST, 100 mM Tris-HCL, pH 7.4) with 0.01% (v/v) Tween-20, and incubated with the primary antibodies overnight at 4°C. The primary antibodies used were rabbit anti-TXNIP (1:500, Beyotime AF8277, Shanghai, China), rabbit anti-NLRP3 (1:1000, Abways Technology, Inc., CY5651, Shanghai, China), rabbit anti-p20 (1:500, Abways Technology, Inc., AY0406 Shanghai, China), rabbit anti-Ki67 (1:500, Wanleibio, WL01384a, Shenyang, China), rabbit anti-IL-1β (1:1000,Wanleibio, WL00891, Shenyang, China), rabbit anti- Histone H2A.X (1:500, Wanleibio, WL00616a, Shenyang, China), rabbit anti-CD31 (1:500, affinity, P16284, Changzhou, China), rabbit anti-VWF (1:1000, affinity, AF3000, Changzhou, China), rabbit anti-pan-cadherin (1:1000, Wanleibio, WL03295, Shenyang, China), rabbit anti-α-tubulin (1:4000, Abways, AB0048, Shanghai, China)༌rabbit anti-Histone H3 (1:1000, Wanleibio, WL0984a, Shenyang, China). After being washed for 3 times, the membranes were incubated with goat anti-rabbit IgG (1:5000, Abways Technology, Inc., Shanghai, China) for 1.5 h. The blot was detected by an automatic chemiluminescence/fluorescence image analysis system (5200 Multi, Tanon, Shanghai, China) with electrochemiluminescence (ECL, Tanon, Shanghai, China). Densitometric analysis of the images was performed with Image J software (NIH, Littleton, CO, USA).
2×107 treated cells were lysed and extracted total protein using RIPA Lysis Buffer (Beyotime, P0013B, Shanghai, China). 1µg Rabbit IgG (Bioworld technology, co, Ltd, Nanjing, China) and 20µl Protein A+G Agarose (Beyotime, P2055, Shanghai, China) were added to each sample to remove the nonspecific binding. The primary rabbit anti-catalase antibody (Abways, CY6783, Shanghai, China) was added and combined with catalase, and samples were shocked slowly overnight at 4℃. The immunoprecipitation was performed using 30µl Protein A+G Agarose each sample for 2h, 4℃. After 1000g centrifugation, the supernatant was discarded, the precipitate was suspended with 1×SDS loading buffer (Beyotime, P0015A, Shanghai, China), and boiled at 100℃ for 3min. Samples were separated by SDS-PAGE gel, and CAT-p20 conjunction was detected by rabbit anti-p20 antibody (Wanleibio, WL02996a, Shenyang, China). As same as these steps, the p20-CAT conjunction was detected by rabbit anti-catalase antibody (Abways, CY6783, Shanghai, China).
2.5 Catalase activity detection
The activity of catalase was detected by in-gel catalase stain according to the previous study. Cells were lysed by RIPA lysis buffer (Beyotime, P0013B, Shanghai, China) with PMSF (Beyotime, ST506, Shanghai, China) on ice, and the extract was centrifuged for 10min at 4℃ and 10000g. The supernatant was mixed with non-reducing 4×SDS loading buffer (50mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 12.5 mM EDTA, 0.02% bromophenol blue) and electrophoresed on a 10% PAGE gel in electrophoresis buffer (3.025g tris, 14.4glycine dissolve in 1L deionized water). The gel was washed with deionized water three times and then immersed in 0.003% H2O2 (2µl 30% H2O2 added to 20 ml deionized water) for 10min and stained with a freshly staining solution (2% potassium ferricyanide and 2% ferric chloride) until the bright band appeared. After three times washed by running water, the gels were photographed by an automatic chemiluminescence/fluorescence image analysis system (5200 Multi, Tanon, Shanghai, China). The α-tubulin detected by western blot was used to normalize the total protein.
2.6 Immunofluorescence Analysis
EPCs (5×104 cells) were seeded on sterilized coverslips. After treatment, cells were washed three times by PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4) and fixed by 4% PFA for 30min. The cell membrane was perforated by 0.3% Triton X-100 (Biosharp, BS084, Hefei, China) for 15min and the cell was blocked with 5% bovine serum albumin (BSA, Solarbio, A8010, Beijing, China) for 1.5 hours. The primary antibodies, rabbit anti-NLRP3 (1:200, Abways Technology, Inc., CY5651, Shanghai, China) and rabbit anti-PYCARD (ASC, 1:100, Abways Technology, Inc., AY0406, Shanghai, China), were incubated for 5 hours, respectively. After triple washed by PBS, NLRP3 and ASC were labeled by the second fluorescent antibodies Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (1:100, Proteintech, SA00009-2, Wuhan, China) and Goat Anti-Rabbit IgG (H+L) Alexa Fluor 488 (1:100, Abways Technology, Inc., AB0141, Shanghai, China) for 2 hours, respectively.
Diabetic nude mice received Sodium tanshinone IIA sulfonate (STS, 15mg/kg/d Solarbio, ST8030, Beijing, China), a water-soluble substance obtained by sulfonation of tanshinone IIA injection for 7days and once Dil (CM-DiI, 0.5µg/µl, HB171031, Yeasen, Shanghai, China) labeled EPCs (1×106) transplantation via tail vein. After PBS and 4% PFA perfusion, the diabetic kidneys were cut into tissue blocks. Optimal cutting temperature compound (OCT, CellPath, SA00009-2, U.K.) embedded mouse kidney sections were cut at 6µm and permeabilized with 0.1% Triton X-100-PBS after washing the OCT. The sections were fixed and blocked as same as the EPCs procedure. The sections were incubated with the primary antibodies, rabbit anti-CD31 (1:100, affinity, P16284, Changzhou, China) and rabbit anti-catalase (1:100, Abways Technology, Inc., CY6783, Shanghai, China), anti-p21 (1:100, Wanleibio, WL0362 Shenyang, China) and rabbit anti-p20 (ASC, 1:100, Wanleibio, WL02996a, Shenyang, China), rabbit anti-VDUP1 (TXNIP, 1:100, sc-271237, Santa Cruz, USA), followed by Alexa Fluor 488. images were photographed by a fluorescent microscope (Carl Zeiss, Axio Scope A1, Germany), and the images of glomerulus were processed by ZEN blue 2.3 software (Carl Zeiss, Germany). The Pearson’s coefficient which represents col-localization was calculated by Image J software (NIH, Littleton, CO, USA).
2.7 Catalase mutant and HEK293T cell transfection
Catalase (CAT) point mutants were performed by transfecting pcDNA3.1 plasmid which was purchased from GENEWIZ (Suzhou, China). By calculating the molecular weight of the CAT fragment, aspartic acid sites in D298, D307, and D226 were replaced with alanine, generated D298A, D307A, and D226A CAT plasmids, and the wild type CAT was also constructed into pcDNA3.1 plasmid. These mutant plasmids were delivered into EPCs by Lipofectamine™ 2000 Transfection Reagent (Invitrogen, 11668019, USA) and HEK293T cells (Fenghbio, Changsha, China) by Calcium Phosphate Cell Transfection Kit (Beyotime, C0508, Shanghai, China). EPCs were treated by LG, HG, HG with LPS+ATP and WEHD, HEK293T cells were transfected by NLRP3, ASC, and caspase-1 plasmids those purchased from Fenghbio (Changsha, China), and the NLRP3 inflammasome was activated by LPS+ATP treatment. The protein samples extracted from EPCs and HEK293T cells were detected by western blot and co-IP.
2.8 BrdU incorporation assay
BrdU incorporation assay was performed as described previously. EPCs were seeded on coverslips in six-well plates with LG and HG medium to 60–70% confluences and then incubated with serum-deprived LG and HG medium for 12 h. EPCs were labeled with BrdU (10 µΜ, Solarbio, Beijing, China) for 2h. After fixed by 4% PFA, perforated by 0.3% Triton X-100 and denatured by 2M HCl, EPCs were incubated with mouse anti-BrdU primary antibody (1:100, Proteintech™, Wuhan, China) overnight, followed by Cy3–conjugated Goat Anti-mouse IgG (H + L) (1:100, ProteintechTM, Wuhan, China), nuclei were labeled by DAPI. The immuno-fluorescence images were taken by a Fluorescence microscope (Carl Zeiss, scope A1, Germany). The percentage of BrdU positive cells to the total amount of cells was calculated by Image J software (NIH, Littleton, CO, USA).
2.9 AGEs detection
The abundance of AGEs in EPC supernatant and mice serum was detected using a commercial kit (ab273298, Abcam, USA). EPCs (3×103/ml) were seeded in 96 well plate, cultured by LG (Ctrl), HG (10, 20, 35mM) for 48h, and HG (35mM) for 24, 48, 72h respectively. Followed the procedure described in the specification, BSA as the negative control, BSA-AGE as the positive control, supernatant and serum samples were added into white flat-bottom 96-well plate (FCP968, Beyotime, Shanghai, China). Relative Fluorescence Units (RFU) were detected by a fluorescent microplate reader (Molecular Devices SpectraMax M4, USA) at Ex360nm/Em460nm. A standard curve was made using the BSA-AGE standard reagent (10mg/ml). The relative abundance of AGEs in samples was calculated according to the standard curve.
2.10 Hydrogen Peroxide detection
Hydrogen Peroxide content in EPC was detected using a commercial kit (Solarbio, BC3595, Beijing, China). 2×107 treated cells were collected in reagent Ⅰ, and the cell membrane was broken by intermittent ultrasonic, cell debris was centrifuged at 8000g for 10min, the supernatant was collected. According to specification, the absorbance at 415nm was detected by a microplate reader (Molecular Devices SpectraMax M4, USA). The sample hydrogen peroxide content was calculated according to the absorbance of the standard substance with known concentration.
2.11 EPC SA-β-gal staining
Accumulation of Senescence-Associated β-galactosidase (SA-β-gal) is one of the hallmaker of senescence. EPCs (1×105/ml) were seeded on sterilized coverslips, synchronized growth by FBS-free medium, and incubated by LG, HG for 48h; the NLRP3 inflammasome positive control was induced by LPS treatment for 4h and ATP treatment for 1h. EPCs were washed twice by precooled PBS and fixed by an immobilized reagent in SA-β-gal kit (C0602, Beyotime, Shanghai, China) for 15min. Staining solution (Formulated as per kit instructions, 1ml containing A, B, C and X-Gal solutions 10µl, 10µl, 930µl, and 50µl) was added and incubated the cells for 36h at 37℃, avoiding light. The coverslips were moisturized with glycerine PBS (1:1) solution, observed, and photographed by a microscope (Carl Zeiss, Germany, Axio Scope A1) in the light field. The images were processed using ZEN Blue 2.3 software (Carl Zeiss, Germany). Image J software was used to analyze the percentage of SA-β-gal positive area (green staining).
Statistical analysis was performed with IBM SPSS Statistics 20 software. Data are presented as means ± SE. Significant differences between and within multiple groups were examined using ANOVA for repeated measures, followed by Duncan's multiple range test. The Independent-Samples t-test was used to detect significant differences between the two groups. p< 0.05 was considered statistically significant.