Isolation and characterization of hucMSCs
All experiment protocols were approved by the medical ethics committee of Jiangsu University (2012258). Fresh umbilical cords were obtained from consenting mothers, and processed within 6 h. HucMSCs were isolated as previously described . All cords were cultured in low glucose Dulbecco’s modified Eagle’s medium (LG-DMEM) containing 10% fetal bovine serum (FBS; Shanghai ExCell Biology, China) and 1% penicillin and streptomycin at 37°C with 5% CO2. Medium was replaced every 3 days after initial plating. When confluence reached 80%, the cells were passaged into new flasks for further expansion. As control cells, human lung fibroblasts (HFL-1; Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences) were cultured with minimal essential medium alpha (MEM-a) containing 15% FBS.
To detect the typical markers of different passages of hucMSCs, flow cytometric analysis was performed using the following fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated antibodies: CD13, CD29, CD44, CD90, CD105, human leukocyte antigen (HLA)-I, CD34, CD45, and HLA-DR (Becton Dickinson, San Jose, CA, USA). Mouse PE-IgG1 and FITC-IgG1 isotypic immunoglobulins were used as isotype controls.
To determine the multidirectional differentiation potential of hucMSCs, the cells of passage 2 were seeded into six-well plates at 1 × 105 cells per well. The next day, the medium was changed to osteogenic medium (0.1 μM dexamethasone, 10 μM β-glycerophosphate, 50 μM ascorbate-phosphate) and adipogenic medium (Cyagen, Guangzhou, China). The medium was replaced every 3 days. After 2 weeks, osteogenic differentiation of hucMSCs was examined by neutrophil alkaline phosphatase (NAP) staining according to the manufacturer’s protocol (SUNBIO, Shanghai, China). At 3 weeks later, adipocytes were stained with Oil-Red-O staining.
Isolation and characterization of exosomes derived from hucMSCs and HFL-1
HucMSCs and HFL-1 were cultured in serum-free medium. After 48 h, cell culture media were collected and density gradient centrifugations performed. Exosomes were isolated as previously described . Cell supernatants were centrifuged at several times, and then passed through a 0.22-μm filter. Final exosomes were obtained and stored at −70°C. The morphology of the collected exosomes was observed by transmission electron microscopy (FEI Tecnai 12, Philips, The Netherlands). The CD9 (Bioworld, Louis Park, MN, USA), CD63 (Bioworld), and CD81 (Epitomics, Burlingame, USA) molecules, frequently located on the surface of exosomes, were analyzed by western blotting. As described above, after hucMSCs were cultured in media for 48 h, the conditioned media (hucMSC-CM) was collected for the following experiment in vivo. Exosome-depleted hucMSC-conditioned media (non-hucMSC-ex) was also gathered for use in the rat model of AKI.
Rat model of AKI
Adult female Sprague–Dawley rats (weighing 220 ± 20 g) were purchased from the Animal Centre of Chinese Academy of Sciences (Shanghai, China), and housed in a specific pathogen-free animal facility under constant temperature and humidity, and with a 12 h/12 h light/dark cycle with sufficient qualified food and water. All protocols and surgical procedures were approved by the Institutional Animal Care Committee of Jiangsu University.
To evaluate whether the exosomes derived from hucMSCs could repair AKI but not other non-MSC cells or MSC-secreted other cells except MSC, animals were divided into six groups of six rats each, treated as follows. (1) Normal group (no cisplatin treatment). (2) Phosphate-buffered saline (PBS) group: intraperitoneal injection of a single dose of 6 mg/kg cisplatin. Cisplatin (Dezhou Pharmaceutical, Shandong, China) was dissolved in 0.9% saline. After 24 h, both kidneys in one rat received a renal capsule injection of PBS. (3) hucMSC-ex group: 24 h after cisplatin administration, both kidneys in one rat received a renal capsule injection of 200 μg exosomes from hucMSCs. (4) hucMSC-CM group: after cisplatin treatment for 24 h, rats received a renal capsule injection of equal volumes of hucMSC-CM with hucMSC-ex. (5) Non-hucMSC-ex group: after 24 h of cisplatin administration, rats were given a renal capsule injection of exosome-depleted hucMSC-conditioned media. (6) HFL-1-ex group: 24 h after cisplatin treatment, rats received a renal capsule injection of 200 μg exosomes from HFL-1 to both kidneys.
Blood samples were collected from the rats via the tail vein every day and centrifuged at 900 g for 15 minutes. Then the serum was separated and stored at −70°C until assayed. Rats were killed at day 5 after administration of cisplatin; both kidneys were immediately excised and cut into two coronal sections each. Two pieces of kidney were fixed in 4% paraformaldehyde at room temperature, the others were stored at −70°C. The blood urea nitrogen (BUN) and creatinine (Cr) levels were determined by a Biochemistry Autoanalyzer (Olympus, Tokyo, Japan).
Location of hucMSC-ex in AKI kidney
To investigate whether exosomes could incorporate into tubular epithelial cells, immunofluorescence was used. First, we incubated exosomes and CM-Dil dye (Molecular Probes, Eugene, OR, USA) together for 30 minutes at 37°C. The CM-Dil dye-labeled exosomes were injected into the AKI kidneys via the renal capsule, with unlabeled exosomes used as a control. After 24 h, fresh kidney tissues were removed for frozen sectioning. Then, frozen sections were blocked using 5% bovine serum albumin (BSA) for 20 minutes. The sections were then incubated with cytokeratin 19 antibody (Bioworld) at 37°C for 1 h. After washing three times, green fluorescent anti-rabbit antibody was used as secondary antibody. Finally, Hoechst 33342 dye (Sigma Saint Louis, USA) was added to the slices. Cytokeratin 19 antibody was used for staining the cytoplasm of tubular epithelial cells and Hoechst 33342 dye was used for nuclei staining.
In vitro experiments
NRK-52E cells were purchased from Cell Bank, and maintained in DMEM containing 10% newborn calf serum (NBS; Gibco, Grand Island, USA) at 37°C with 5% CO2. For in vitro treatments, NRK-52E cells were seeded in six-well plates at 1 × 105 cells per well. At approximately 70% confluence, the control and cisplatin groups were grown with or without 5 μM cisplatin for 6 h, then the control and cisplatin groups were changed to fresh medium. In the other two groups, after NRK-52E cells were treated with 5 μM cisplatin for 6 h the culture solutions were changed to 1 mL fresh medium with 160 μg/mL exosomes derived from hucMSCs or HFL-1, respectively. After 24 h, cells were fixed in 4% paraformaldehyde for histologic staining or were collected for protein extraction, and cell suspensions were collected to detect glutathione (GSH) and malondialdehyde (MDA). In order to determine whether hucMSC-ex promote cell proliferation through activation of the extracellular-signal-regulated kinase (ERK)1/2 pathway, cisplatin-treated NRK-52E cells were cultured in fresh medium containing 160 μg/mL hucMSC-ex and 15 μM U0126 (Promega, Wisconsin, USA); 24 h later, cells were collected for protein detection.
To detect the injury of kidney tubules, the kidneys were fixed in 4% paraformaldehyde (pH 7.4) gradually dehydrated, embedded in paraffin, cut into 4-μM sections and stained with H&E stain.
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay
Tissue slices underwent deparaffination and dehydration, then renal tubular cell apoptosis was measured by the TUNEL assay using an in situ cell apoptosis detection kit (Boster, Wuhan, China) according to the manufacturer’s instructions.
Immunohistochemistry was used for detection of proliferating cell nuclear antigen (PCNA) and the renal oxidative stress product 8-hydroxy-2′-deoxyguanosine (8-OHdG) in vivo and in vitro. The kidney tissue slices underwent deparaffination and dehydration, and were then immersed in 30% hydrogen peroxide for 10 minutes to block endogenous peroxidase and antigen retrieval for 10 minutes in 0.01 M citrate buffer (pH 6.0) in turn. Then, sections were incubated with PCNA antibody (Bioworld) and 8-OHdG antibody (10 μg/mL, Japan Institute for Control of Aging, Shizuoka, Japan) at 37°C for 1 h. Sections were washed with PBS three times and incubated with biotinylated goat anti-rabbit or anti-mouse IgG (Bostar, Wuhan, China) at 37°C for 20 minutes followed by streptavidin-biotin complex (SABC) for 20 minutes. The antibody binding sites in the tissue slices were visualized with 3,3′-diaminobenzidine (DAB), and counterstained with hematoxylin. In vitro, the NRK-52E cells treated in different ways were fixed in 4% paraformaldehyde for 30 minutes. After washing three times with PBS, cells were incubated with 8-OHdG antibody at 37°C for 1 h, and incubated with a secondary antibody for 20 minutes. Cells were visualized with diaminobenzidine substrate and counterstained with hematoxylin. The morphological tissue and cell sections were evaluated by high-power light microscopy examination (Nikon, Tokyo, Japan).
Mitochondrial membrane potential assay
A mitochondrial membrane potential assay kit with JC-1 staining solution (Beyotime, Nantong, China) was used to detect early apoptosis. After NRK-52E cells were treated in six well plates as described above, the medium was changed for 1 mL fresh medium with 1 mL JC-1 (5 μg/mL) and cells incubated at 37°C for 20 minutes then washed twice with JC-1 staining solution (1 μg/mL). Images were obtained by fluorescent microscopy and analyzed for green and red fluorescence. Mitochondrial membrane potential depolarization was expressed by an increase in the green/red fluorescence intensity ratio.
Measurement of GSH and MDA
In vivo, frozen kidney tissues were thawed, weighed and grinded in homogenate medium (pH 7.4, 0.01 mol/L Tris–HCl, 0.0001 mol/L ethylenediaminetetra-acetic acid (EDTA)-Na2, 0.01 mol/L sucrose, 0.8% NaCl solution). Prepared 10% homogenate was centrifuged at 400 g, 10 minutes. In vitro, cells were collected and ground in PBS. The total protein concentration from kidney tissues or cultured cells was determined using the BCA assay kit (Pierce, USA). Commercial assay kits used to determine GSH and MDA were purchased from Jiancheng Bioengineering Institute (Nanjing, China). All the procedures were performed according to the manufacturer’s instructions.
Western blotting analysis
Kidney tissues and cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1 mM ethylene glycol tetra-acetic acid (EGTA), 0.1% SDS, 1 mM NaF, 1 mM Na3VO4, 1 mg/mL aprotinin, and 1 mg/mL leupeptin in 10 mM Tris, pH 7.4) containing 1 mM phenylmethanesulfonyl fluoride (PMSF). The protein concentration of each sample was determined using the BCA assay kit. A total of 150 μg protein were electrophoresed on 12% SDS-polyacrylamide gels, which were transferred to polyvinylidene fluoride membranes, blocked with 5% skim milk for 1 h and then blotted against primary antibodies at 4°C overnight. Primary antibodies were as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Kangchen Bio-tech, Shanghai, China), B cell lymphoma 2 (Bcl-2) protein (Santa Cruz, Minneapolis, USA), Bcl-2-associated X protein (Bax) (Bioworld), p-ERK (Santa Cruz), ERK (Santa Cruz), phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) (Santa Cruz) and p38MAPK (Santa Cruz), caspase 3 (Bioworld). After this, the membrane was washed three times with Tris-buffered saline/Tween (TBST) and incubated in goat anti-rabbit or mouse antibodies (Bioworld) for 1 h at 37°C. Western blotting detection was performed using Luminata™ crescendo western horseradish peroxidase (HRP) substrate (Millipore, Billerica, MA, USA).
All data were shown as means ± standard deviation (SD). The statistically significant differences between groups were assessed by analysis of variance (ANOVA) with two-way classification, or ANOVA with Student-Newman-Keuls multicomparison test using Prism software (GraphPad, San Diego, USA). A P value of <0.05 was considered significant.