The study was approved by the ethical committee of Jiangsu University (2014280).
Fresh human umbilical cords were obtained from consenting mothers in the affiliated hospital of Jiangsu University. HucMSCs were isolated and identified as previously described  and cultured in serum-free Dulbecco’s modified Eagle’s medium (DMEM, Gibco, CA) with 10% fetal bovine serum (FBS, Excell), penicillin and streptomycin (Gibco). Platelet-rich plasma (PRP) was provided by the central blood bank of Zhenjiang city, Jiangsu, China. The primary concentration was 1.2×1012 platelets/mL. In the following experiments, hucMSCs treated with 1×108 platelets/mL PRP 12 h, and then the stimulator (PRP medium) was remove, MSCs were cultured in DMEM for 24 hours and collected cells as PRP-MSCs, MSCs: hucMSCs treated with DMEM as the control. Rat renal tubular epithelial cell lines (NRK-52E) were purchased from Cell Bank (Chinese Academy of Sciences, Shanghai, China) and maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% FBS at 37℃ with 5% CO2. The cells at passage 3 were used for the following studies.
Osteogenic and adipogenic differentiation in vitro
MSCs and PRP-MSCs were seeded in 35-mm plates in an osteogenic differentiation medium (0.1 mM dexamethasone, 10 mM β-glycerophosphate, and 50 mg/L ascorbic acid) or adipogenic differentiation medium (Cyagen Biosciences, USA) for 2 weeks, according to the manufacturer’s instructions. After the induction, the osteogenic and adipogenic potential was evaluated through alkaline phosphatase, alizarin red and oil red O staining, respectively.
Isolation and Characterization of PRP-MSC-Ex
The supernatant of MSCs stimulated by PRP 12 hours was collected, to a certain volume then exosomes were extracted and purified . The protein concentration, as the quantification of exosomes, was determined by using a BCA protein assay kit. The morphology of exosomes was observed by using transmission electron microscopy (FEITecnai 12, Philips, Netherlands). The size of exosomes was analyzed the NanoSight LM10 system (nanosight tracking analysis, UK). For in vivo, to investigate the roles of PRP-MSCs-Ex in AKI model, MSCs-Ex and PRP-MSCs-Ex (10mg/kg) were treatment for AKI model via tail vein injected. CM-DiR (sigma) labeled exosomes were incubated at 37 ℃ for 30 minutes, and the bio-distribution of exosomes was detected via IVIS Lumina system (Perkin Elmer, USA) in vivo.
Glycerin-induced model of AKI
Adult male Sprague-Dawley rats weighing 180-200g (provided by the experimental animal center, JiangSu University, Permit Number: 2014280) were selected, the in vivo experiments were conducted following the regulatory standards. All the experimental rats were reared in the environment of room temperature of about 26 ℃, and the room should be kept clean, ventilated and quiet. After feeding for one week, the experimental rats were divided into four groups according to the random number table: control group, glycerin group, MSCs group, PRP-MSCs group, 10 rats in each group. The AKI rat model was established as described previously . As previously described, AKI was performed in rat by intramuscular injection of 50% solution of hypertonic glycerin (10mL/kg, Sigma) into inferior hindlimbs. On day 1 post injury, rat received a tail vein injection of 1×106 MSCs and 1×106 PRP-MSCs in 200 mL saline and the same volume of saline as control. All animals were sacrificed at Day 3 after glycerin injection. Renal function (serum BUN, Creatinine), histopathological structure changes and tubular cells apoptosis were evaluated.
Renal tissues or NRK-52E cells were lysed in a radioimmunoprecipitation assay buffer containing proteinase inhibitors. Protein samples were separated by SDS-polyacrylamide gel electrophoresis, transferred to the polyvinylidene difluoride membrane (Millipore), blocked in 5% skim milk, and incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Invitrogen).
Primary antibodies used in research were as following: Bax (1:200, Bioworld, USA), Actived-caspase3 (1:500, CST, USA), and β-actin (1:800, CST, USA). The secondary antibodies were HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies (1:1500, CWBIO, China).
TUNEL and immunohistochemistry staining
For histologic analysis, the kidneys were prepared by perfusion of the rat through the left ventricle and slides of the kidney were prepared, fixed in 4% paraformaldehyde, embedded in paraffin and then cut into sections. We detected apoptosis cells by employing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining according to the manufacturer’s protocol (Vazyme, USA). The sections were stained with hematoxylin and eosin (HE) staining, and the histological changes of renal tissues were observed under a microscope (DP73; Olympus, Tokyo, Japan). After inactivating endogenous enzymes by 10% H2O2 with 30 minutes, the slices of kidney tissues and cells were incubated with actived-caspase3 antibody (1:100, CST, USA) overnight at 4°C, then incubated with biotinylated sheep anti-rabbit IgG. The signal was observed by DAB staining and hematoxylin counterstaining under microscope (DP73; Olympus, Tokyo, Japan).
MSCs and PRP-MSCs cells slide were placed in 4% paraformaldehyde at 4°C for 12 h. Then permeabilized with PBS solution containing 0.15% Triton X-100 for 30min and incubated with 5% bovine serum albumin (BSA) for 1h to block non-specific antibody binding. Primary antibody AKT (1:100, CST), Rab27 (1:100, CST), CD63 (exosomes marker) (1:50, CST) were incubated overnight, followed by incubation with Cy3-labeled (Red) anti-rabbit IgG (1: 200, invitrogen), FITC-labeled (Green) anti-rabbit IgG secondary antibody (1: 200, invitrogen) at 37°C for 30 min. The nuclei were counterstained with Hoechst 33342 (1:200; Sigma). The slides were visualized with Confocal microscope (DeltaVision Elite, GE, USA).
All data were shown as mean ± SD. Statistical analysis between groups was performed by GraphPad Prism 5.0 software (San Diego, USA). Statistical differences between two groups were determined by two-tailed paired Student’s t-test. In multiple groups (>2) were determined by one-way analysis, and variance followed by ANOVA Tukey’s post tests. *P <0.05 was considered statistically significant.