Materials
Mannitol (SM8120) was obtained from Solarbio. RPMI-1640 (SH30809.01B), DMEM/F12 medium (SH30023.01B), and BSA (SH30574.03) were purchased from Hyclone. Collagenases I (17100-017), II (17101-015), IV (17104-019), and 0.25% Trypsin (15050065) were purchased from Gibco. PI (25535-16-4), RNase A (9001-99-4), dexamethasone (D4902), β-glycerophosphoric acid (G9422), ascorbic acid (A7562), insulin (I5500), indomethacin (I7378), and 3 isobutyl 1 methylxanthine (IBMX; I7018) were from Sigma. 1% Triton x-100 was obtained from SBJBio Life Sciences (SBJ-1141).
Animal treatment
Eight-week-old C57BL/KsJ db/m (control mice, n = 10) and C57BL/KsJ db/db (spontaneous diabetes mice, n = 20) male mice were obtained from Cavens Lab Animal (SCX, 2016-0010, Changzhou, China). All animal care procedures were approved by Laboratory Animals of Zhejiang Provincial People’s Hospital. The mice were housed three to five per cage in a room under controlled light (12 h/day) and temperature (22 ± 2 °C) conditions with free access to food and water. At 12 weeks old, periodic acid-Schiff (PAS) staining was performed to for histopathology investigation to verify disease phenotype of DN according to previous study. At 13 weeks old, mice were treated with tail intravenous injection of PBS or ADSCs-Exo for additional 12 weeks.
Cell isolation, culture, and treatment
Eight-week-old C57BL/KsJ db/m male mice was anesthetized using pentobarbital (200 mg/kg i.p.), and subcutaneous adipose tissue in groin was harvested. ADSCs were isolated by utilizing collagenase digestion method. Briefly, adipose tissue was minced and washed with PBS buffer twice followed by centrifugation at 1200 rpm for 10 min. The supernatant was removed, and the mixed collagenases were added into the precipitate. After digestion for 40 min at 37 °C, complete medium was added to stop the reaction and the digested mixture was filtered through 40-mm cell strainer and centrifuged at 1500 rpm for 8 min. The precipitate was then resuspended in RPMI-1640 complete medium and cultured at 37 °C in a humidified incubator in 5% CO2. In miRNA-486 inhibitor assay, ADSC cells were transfected with miRNA-486 inhibitor and negative control in ADSCs for 48 h. Then, ADSCs-Exo were isolated for the next experiments. The sequences of miRNA-486 inhibitor and negative control were as follows: AUCCUGUACUGAGCUGCCCCG and CAG UAC UUU UGU GUA GUA CAA.
Mouse podocyte MPC5 cells were purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (3111C0001CCC000230). MPC5 cells were cultured in DMEM medium containing with 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere containing 5% CO2. For CCK8 assay, MPC5 cells were treated with 5.5 mM d-glucose (NG), 5.5 mM d-glucose+ 24.5 mM mannitol (MA), 30 mM d-glucose (HG), and combination of HG and concentration gradient ADSCs-Exo for 24 h, 48 h, 72 h, and 96 h. For apoptosis and autophagy experiments, MPC5 cells were treated with NG, MA, HG, and combination of HG and ADSCs-Exo for 48 h.
Osteogenic and adipogenic differentiation and authentication of ADSCs
ADSCs in passage 3 were subjected to stem cell induction and appraisal system for examining differentiation ability of ADSCs. For the ability of osteogenic differentiation, 1 × 105 cells were seeded on coverslip in six-well plate. After incubation for 24 h, cells were induced with osteogenic medium containing 10% FBS, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid in high-glucose DMEM medium. Normal high-glucose DMEM was served as the control group. At day 14, the induced cells were harvested for adipogenic assessments by utilizing alkaline phosphatase (ALP) detection kit (A059-2, Jiangcheng biotechnology, Nanjing, China) and Alizarin Red Staining. The ability of ALP was according to the manufacturer’s instruction. ALP activity was measured at the wavelength of 520 nm, and activity relative to control was calculated after normalization to the total protein content. Alizarin Red Staining was another employed method for detecting mineralization after osteogenic differentiation. In brief, the induced ADSCs on the glass were washed with PBS for three times and fixed with 4% paraformaldehyde for 15 min. Next, the glass was stained with 0.2% Alizarin Red for 20 min. After washing with PBS, staining results were observed and taken photographs under optical microscope. The positive cells presented as nacarat.
For adipogenic differentiation of ADSCs, cells in passage 3 were incubated with adipogenic medium containing 10% FBS, 1 μM dexamethasone, 60 μM indomethacin, 500 nM IBMX, and 10 μg/m insulin in high-glucose DMEM medium. Oil Red O staining was used for quantitatively adipogenic assessments as per previous study. Briefly, cells were washed with PBS for twice and fixed using 4% paraformaldehyde for 5 min. Then, cells were washed again twice and stained in propylene glycol for 5 min, Oil Red O solution for 15 min at 60 °C. After Oil Red O solution was removed, cells were differentiated using 85% propylene glycol for 5 min. Next, cells were stained with hematoxylin for 30s. After washing again, results were observed and taken with optical microscopes.
Isolation of exosome
Passage 3 ADSCs were seeded into 6-cm dish and incubated in a 37 °C incubator containing 5% CO2. When grown to 70–80% confluence, cells were cultured with fresh medium containing with exosome-free FBS for another 24 h which was obtained by ultracentrifugation at 100,000g for 18 h followed by filtration through a 0.22-μm filter. After 24 h of incubation, the conditioned medium (CM) was collected and exosome was isolated by utilizing exosome extraction kit (Wako pure Chemicals Industry, 293-77601) according to the manufacturer’s protocol. Briefly, 30 ml CM were centrifuged at 300×g for 5 min, 1200×g for 20 min, and then 10,000×g for 30 min at 4 °C to remove cells, debris, and large extracellular vesicles (EVs). Next, the CM were concentrated to 1 mL by using Amicon Ultra-15 Ultracel-100 K (Merck KGaA, Darmstadt, Germany, UFC910024). Then concentrated CM were incubated with Streptavidin Magnetic Beads (60 mg) accompanied by additional 1 μg of biotinylated mouse Tim4-Fc, 350 μL Exosome Capture Immobilizing Buffer, and 50 μL Exosome Binding Enhancer overnight at 4 °C. The next day, the beads were washed with washing buffer for three times and the bound EVs were eluted by utilizing Exosome Elution Buffer.
Transmission electron microscopy (TEM)
Exosome morphology was analyzed using TEM analysis kit (E1610, Weihui Biotechnology, Peking, China). Firstly, exosome was placed on parafilm membrane as a 10 μL drop of exosome suspension and the EM membrane covered with formvar carbon was put up on the suspension for 10 min to adsorb exosome as much as possible. After washing with wash buffer, EM containing exosome was observed by transmission electron microscopy (JEOL, JSM-IT300LV) and images were taken using an electron sensitive Olympus KeenView CCD camera.
Nanoparticle tracking analysis (NTA)
Exosome was diluted to a volume of 1 mL in TPM to analysis. Size and concentration of exosome were determined through Nanosight Tracking Analysis by utilizing ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) according to previous protocol [19].
Urine protein, serum creatinine (Scr), and blood urea nitrogen (BUN) measurement
Urine was collected after 12 weeks of exosome treatment and measured using BCA kit. In brief, mixture of BCA working solution and urine was co-incubated at 37 °C for 30 min. The OD value was tested at the wavelength of 562 nm. In the end of exosome treatment, blood plasma was collected by abdominal aortic method and stand for 2 h at room temperature. After centrifugation at 3500 rpm for 5 min, Scr and BUN levels were measured through detection kits (Scr: C011-1, Jiangcheng Bio, Nanjing; BUN: C013-2, Jiangcheng, Nanjing), respectively.
Plasmid construction and cell transfection
For luciferase reporter plasmids, Smad1-3′UTR-wt (wild type) and Smad1-3′UTR-mut (mutant) fragments were inserted into pYr-MirTarget vector. For Smad1-overexpressed plasmid construction, mus Smad1 open reading frame fragment was inserted into pcDNA3.1 vector. Next, recombinant plasmids were transferred into DH5α coli cells and screened by clone assay. PCR amplification primers were as follows: Smad1-3′UTR-wt, forward: GGTTCTTTTCCAACGCTATT, reverse: CACTTCAGAAAGACTATCAG; Smad1-3′UTR-mut, forward: TTTGTTTGTTTTTAATGAAGACGTTAATCGTTATGACATGCATAG, reverse: ATAACGATTAACGTCTTCATTAAAAACAAACAAAAAACCCATTCA. After sequencing, the reporter plasmids were extracted by utilizing plasmid extraction kit.
Dual luciferase reporter assay
HEK-293 T cells were seeded on 24-well plate and incubated for 24 h. Then, cells were transfected with Smad1-3′UTR reporter and miR-486 mimic NC/miR-486 mimic plasmids for another 48 h. Culture medium was removed, and cells were washed with PBS for twice. Cells were lysed through adding PLA buffer, and luciferase activity was measured by utilizing dual luciferase reporter assay system (E1910, Promega). OD value was observed through microplate reader.
Immunofluorescence (IF)
Cells were fixed in 4% paraformaldehyde for 30 min followed by washing for three times using PBS. Cells were treated with 0.1% Triton X-100 for 15 min and blocked using 5% BSA (Hyclone, SH30574.03) for 1 h. For renal tissue, specimen was fixed in 4% paraformaldehyde for 24 h or more. Through the production of paraffin sections and antigen retrieval, tissue sections were blocked using 5% BSA for 1 h. Then, cells and sections were incubated using primary against CD29 (sc-9970, 1:200, Santa Cruz), CD34 (bs-0646R, 1:500, bioss), CD44 (bs-4916R, 1:400, bioss), CD45 (bs-0522R, 1:300, bioss), CD90 (bs-0778R, 1:500, bioss), nephrin (sc-377246, 1:100, Santa Cruz), LC3 (12135-1-AP, 1:200, Proteintech) at 4 °C overnight. After incubation with secondary antibodies at 37 °C for 1 h, samples or cells were stained with DAPI for 5 min and laser confocal microscopy was adopted for observation and photo taking.
Flow cytometry
ADSCs in passage 3 were digested by 0.25% trypsin, and cell suspension were filtered using 100-mesh filter. After 5 min of centrifugation, supernatant was removed and cells were resuspended with PBS followed by incubating with primary antibodies. Positive marker of ADSCs were including CD29 (1:500), CD44 (1:300), and CD90 (1:500). CD34 (1:300) and CD45 (1:300) were the negative marker of ADSCs. For exosome, the exosome suspension was used directly for purity identification by FACS according to previous study [20]. Positive marker of exosome, CD9 (bs-2489R, 1:100, Bioss), CD63 (GTX41877, 1:100, GeneTex), and CD81 (GTX41794, 1:100, GeneTex) were used for purity identification. In brief, exosomes were resuspended in 200 μL of PBS and aldehyde/sulfate beads (10 μL, Life Technologies) were added into exosome solution. After mixing for 15 min at room temperature by using a benchtop rotator, 600 μL PBS were added and incubated overnight at 4 °C. The next day, the pellets were resuspended in 40 μL of 2% BSA in PBS and stained with CD9, CD63, CD81, and their isotype control antibodies at RT for 30 min. After centrifugation at 12,000 rpm for 1 min, the pellets were resuspended in 20 μL BSA and stained with secondary antibodies. Eventually, the beads containing exosome were analyzed using the LSR Fortessa X-20 cell analyzer (BD Biosciences).
For apoptosis detection, cells were digested using 0.25% trypsin without EDTA and centrifuged at 1500 rpm for 5 min. The precipitate was resuspended and washed gently with PBS. Through 5 min of centrifugation at 1500 rpm, cells were suspended using 300 μL binding buffer. Cells were stained with 5 μL Annexin V-FITC in darkness for 5 min following with 10 μL PI staining for 10 min at room temperature. Then, cells apoptosis was measured by Flow cytometry (BD Biosciences, FACSCalibur).
CCK8 assay and Tunel staining
MPC5 cells treated with high-glucose or ADSCs-Exo were seeded on 96-well plate at a density of 3000 cells/well. After 24 h, 48 h, 72 h, and 96 h of incubation, 100 μL CCK8 was added into cells following by 2 h of incubation. OD value was detected through microplate reader (Thermo, Multiskan MK3) at the wavelength of 450 nm.
For tissue apoptosis measurement, tissue sections were assessed according to the manufacturer’s instruction (Biobox, BA27). In brief, the specimens were stand in TUNEL reaction solution containing TdT and Streptavidin-FITC to label DNA fragments. After washing, the labeled preparations were incubated using POD-conjugated Anti-FITC at 37 °C for 30 min in darkness. Then, slides were added with DAB reaction solution and stained with DAPI followed by a step of hydrochloric acid alcohol differentiation, ethanol dehydration, xylol incubation, and mounting with neutral resins. The resulting images were processed and analyzed using microscope.
RNA isolation and quantitative real-time polymerase chain reaction
Tissue and cell total RNA were extracted using TRIZOL reagent. One microgram RNA was used to perform reverse transcription by using First Strand cDNA Synthesis Kit (Fermentas, K1622) and polymerase chain reaction using SYBR Green qPCR kit (Thermo, K0222). Primers of miR-486 and internal control were as follows: miR486, TATAGCTCCTGTACTGAGCTGC; miR universal primer, CCAGTCTCAGGGTCCGAGGTATTC. U6 F, CTCGCTTCGGCAGCACA; U6 R, AACGCTTCACGAATTTGCGT.
Western blotting
Protein of renal tissue and MPC5 cells were processed SDS-PAGE according to the previous study [21]. All primary antibodies were included CD9 (1:1000, Bioss, bs-2489R), CD63(1:1000, GeneTex, GTX41877), CD81 (1:800, GeneTex, GTX41794), β-actin (1:1000, CST, 4970), Caspase3 (1:1000, CST, 9662), mTOR (1:1000, CST, 2983), p-mTOR (1:500, CST, 5536), LC3 (1:500, Proteintech, 12135-1-AP), p62 (1:500, Proteintech, 18420-1-AP), Beclin1 (1:500, CST, 3495) and Smad1 (1:1000, Affinity, AF6451).
Statistical analysis
Statistical analysis was processed by utilizing SPSS 21.0 software. Data were presented according to three independent experiments and appeared as mean ± standard error of the mean. Histogram and line chart were analyzed by GraphPad Prism 5.0 (GraphPad Software Inc., USA) using Student’s t test or One-way ANOVA analysis. p < 0.05 was regarded as significant difference.