Chemicals and reagents
The primary antibodies used in our study, anti-α-SMA (ab5694), anti-Fibronectin (ab2413), anti-CD34 (ab81289), and anti-CD44 (ab189524) were obtained from Abcam (Cambridge, UK). Anti-E-cadherin (20874-1-AP), anti-SOD1 (10269-1-AP), anti-Catalase (21260-1-AP), anti-cleaved caspase-3 (19677-1-AP), and anti-PARP1(13371-1-AP) were obtained from Proteintech (Wuhan, China). Anti-RhoA (sc-418), anti-ROCK1 (sc-17794), and anti-MFG-E8 (sc-271574) were purchased from Santa Cruz Technology (California, USA). Anti-p-MYPT1 (4563T) was purchased from Cell Signaling Technology (Massachusetts, USA). Anti-CD11b (201807), anti-CD90 (206105) were purchased from Biolegend (California, USA). GAPDH (AG019) and Reactive Oxygen Species Assay Kit were obtained from Beyotime (Shanghai, China). Y-27632 (S104921) was purchased from Selleck (Texas, USA). Recombinant Human TGF-β1 (100-21) was purchased from Peprotech (New Jersey, USA). Mesenchymal Stem Cell Adipogenic Differentiation Medium (RASMX-90031), Mesenchymal Stem Cell Chondrogenic Differentiation Medium (RASMX-90041), and Mesenchymal Stem Cell Osteogenic Differentiation Medium (RASTA-90021) were purchased from Cyagen (California, USA). RhoA Activation Assay Combo Biochem Kit (BK030) was purchased from Cytoskeleton (California, USA).
Animals
Eight-week-old male Sprague-Dawley (SD) rats (weighing 180–220 g) were used for this study. The animal experiments were approved by the Ethics Committee of Laboratory Animal Science, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine (SCMCIACUC-K2019025). All rats were housed in a specific pathogen free (SPF) animal room under standard conditions with free access to water and food.
Animal experiments
Thirty male SD rats were randomly divided into six groups (n=6 in each group): the Sham group, the Sham + EVs group, the UUO group, the UUO + EVs group, the UUO + EVsCtrl group, and the UUO + EVsshMFGE8 group. Briefly, the rats were anesthetized with 60 mg/kg pentobarbital and kept on a heated surface to maintain a body temperature of approximately 36.5°C. In the UUO group, the left ureter was exposed via abdominal incision and then ligated to the ureteral-pelvic junction with 5-0 silk sutures. In the Sham group, the left ureter was physically exposed but not ligated. The rats in the Sham and UUO groups received 0.5 mg/kg phosphate-buffered saline (PBS) vehicle intravenously. For the Sham + EVs group and the UUO + EVs group, BMSC-EVs were injected via the tail vein a day after the procedure at a dose of 0.5 mg/kg. For the UUO + EVsCtrl group, 0.5 mg/kg EVsCtrl was administered intravenously. For the UUO + EVsshMFGE8 group, 0.5 mg/kg EVsshMFGE8 was administered intravenously. All rats were sacrificed by pentobarbital anesthesia on postoperative day 14, and the kidneys were collected for further investigation.
BMSC isolation and culturing
BMSCs were isolated from the bone marrow of 4-week-old SD rat. The cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C incubator with 5% CO2. The marrow was plated in T75 culture flasks, and nonadherent hematopoietic cells were removed with PBS 3 days later, followed by the addition of fresh culture medium; the medium was subsequently changed every 2–3 days. When the BMSCs reached 80%–90% confluence, they were digested with 0.25% trypsin. The cells were passaged three times prior to verification of their identity. Flow cytometry was used to screen the cells for CD44, CD90, CD11b and CD34, known markers for BMSCs. The cells were also tested for their ability to be differentiated into adipocytes, osteocytes, and chondrocytes. BMSCs at passages 3–5 were used for further experiments.
Lentiviral vector construction and transduction
To down-regulate MFG-E8 expression in BMSC-EVs, BMSCs were divided into two groups: BMSCs transfected with null lentivirus vector (BMSCsCtrl) and BMSCs transfected with a lentiviral vector (final concentration, 100 nM) designed to knock down MFG-E8 expression (BMSCsshMFGE8). Lipofectamine 2000 was used to perform the transfections. The medium was removed 24 h after transfection and replaced with fresh medium containing 10% FBS. BMSC-EVs were purified from BMSCsCtrl or BMSCsshMFGE8 as described below.
Isolation and identification of BMSC-EVs
BMSC-EVs were isolated and identified as described previously. Briefly, BMSCs at 80%–90% confluency were rinsed with PBS and cultured for 48 h in Mesen Gro MSC medium. The conditioned medium was then collected and centrifuged at 300 x g for 10 min followed by another round of centrifugation at 2000 x g for 10 min at 4 °C to remove cellular debris and dead cells. Then, the supernatant was ultra-centrifuged at 100,000 x g for 70 min at 4 °C to obtain a pellet containing the EVs, which was rinsed in 200 μl of PBS and ultracentrifuged again at 100,000 x g for another 70 min at 4 °C. The protein content of the BMSC-EVs was quantitated using a BCA Protein Assay Kit. Western blot and transmission electron microscopy (TEM) were used to examine the quality and morphology of the BMSC-EVs, and qNano was used to assess their size. The purified BMSC-EVs were stored at -80°C for later experiments.
HK-2 cell culturing and treatment
Human renal proximal tubular epithelial (HK-2) cells (XY Biotechnology, Guangzhou, China) were cultured in DMEM/F12 with 10% fetal bovine serum supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a 5% CO2/95% air atmosphere with nearly 100% relative humidity. To generate renal fibrosis cell models, the cells were incubated with TGF-β1 at a final concentration of 10 ng/ml for 72 h. After this pretreatment step, the HK-2 cells were then incubated with or without 30 μg/ml BMSC-EVs in the presence of TGF-β1. To inhibit ROCK, Y-27632 (10 μM) was added to cells treated with TGF-β1 for the final 24 h of incubation.
Renal morphology analysis
For histological examination, the kidneys were isolated, fixed in 4% formaldehyde overnight and embedded in paraffin. Hematoxylin and eosin (HE) staining was performed according to standard methods. Renal interstitial lesions were characterized by the degree of changes to the glomerulus and tubules. At least 10 randomly chosen non-overlapping fields of view at a magnification of 200 x were observed and recorded for each section. Masson’s trichrome staining was used to assess the degree of renal interstitial fibrosis based on the amount of collagen deposition observed at 200 x magnification. An optical microscope equipped with image analysis software was applied to analyze images of the renal interstitium. The total area occupied by fibrotic lesions was calculated for arbitrarily chosen fields of view and expressed as the percentage of fibrotic area relative to the entire image.
Renal immunohistochemical analysis
For immunohistochemical (IHC) staining, paraffin-embedded kidney sections were rehydrated and incubated in 10 mM citrate with 0.05% Tween for antigen retrieval. After blocking, the sections were incubated with primary antibody at 4°C overnight, followed by incubation with a horseradish peroxidase–conjugated secondary antibody for 1 h at 37°C. All images were recorded using Image-Pro Plus 6.0.
RhoA activation assay
The RhoA activation assay was performed using a GTP pulldown assay kit according to the manufacturer’s instructions. After cells were lysed, 300–500 μg of total protein was added to 10 μL RBD-binding beads, then incubated at 4°C on a rotator for 1 h, washed three times with PBS, and resuspended in Laemmli buffer. Finally, the protein samples were heated at 100 °C for 10 min and stored at -20 °C.
Western blot
Total protein was extracted by suspending kidney tissues in ice-cold RIPA buffer containing a protease inhibitor cocktail, followed by homogenization and subsequent centrifuged at 12000 x g for 20 min, after which the pellets were discarded and the protein concentration was quantified using t a bicinchoninic acid (BCA) assay method. Next, a 4 x volume of loading buffer was added each to supernatant for Western blot. Equal amounts of each protein sample (40 μg/lane) were loaded onto a 8%–12% SOD1ium dodecyl sulfate-polyacrylamide gel, electrophoresed for 2 h and then transferred to poly-vinylidene fluoride membrane. The membranes were blocked for 1.5 h at room temperature with 5% nonfat milk followed by incubation at 4 °C overnight with primary antibodies against the following proteins: α-SMA, E-cadherin, Fibronectin, Catalase, SOD1, cleaved caspase-3, PARP1, and GAPDH. The membranes were then incubated with an HRP-conjugated goat anti-mouse secondary antibody or with rabbit lgG. The protein bands were detected by enhanced chemiluminescence (ECL) and imaged. Semi-quantitative analysis was performed by measuring band intensities for three experiments using ImageJ software.
Quantitative real-time PCR
Total RNA was isolated from kidney tissues using TRIzol according to the standard protocol. Reverse transcription was carried out to synthesize complementary DNA (cDNA) from 500 ng total RNA using 5 x primescript RT Master Mix 2 μl (TAKARA) in a 10-μl reaction volume. The quantitative real-time PCR reaction was performed using qPCR Master Mix, (TAKARA, Japan), primers (designed and synthesized by Shanghai Sangon Biological Co, Ltd), and RNase-Free ddH2O in a 10-μl reaction volume. An Applied Biosystems® 7500 Fast Real-time PCR System was used to run the following program: 95 °C for 30 seconds, 40 cycles of 95 °C for 5 seconds and 60 °C for 34 seconds, followed by 95 °C for 15 seconds, 60°C for 1 min and 95 °C for 15 seconds. The threshold cycle (Ct) was recorded by the instrument’s software (7500), and fold changes in mRNA expression were calculated according to the comparative Ct method (2-△△CT). Results were normalized to mRNA expression in the kidney tissues of rats from the Sham group. The rat primers used for RT-PCR were as follows: α-SMA, forward 5’-CAGGGAGTGATGGTTGGAAT -3’ and reverse 5’-GGTGATGATGCCGTGTTCTA-3’;E-cadherin, forward 5’-CTCAGTGTTTGCTCGGCGTTTGC-3’ and reverse 5’-GCTCTGGGTTGGATTCAGAG-3’; Fibronectin, forward 5’-GATTCTTCTGGCGTCTGCAC-3’ and reverse 5’-GCCCCGGAACATGAGGATAG-3’; IL-1β, forward 5’-AGCAGCTTTCGACAGTGAGG-3’ and reverse 5’-CTCCACGGGCAAGACATAGG-3’; IL-6, forward 5’—3’ and reverse 5’—3’; TNF-α, forward 5’-CAACCAGGCCATCAGCAACAACAT-3’ and reverse 5’-TCTGTGGGTTGTTCACCTCGAACT-3’; IL-10, forward 5’-GGACTTTAAGGGTTACTTGGG-3’ and reverse 5’-AGAAATCGATGACAGCGTCG-3’; GAPDH, forward 5’-TGACTCTACCCACGGCAAGTTCAA-3’ and reverse 5’-ACGACATACTCAGCACCAGCATCA-3’.
Measurement of oxidative stress
Oxidative stress in kidney tissues was determined by Western blot analysis of SOD1 and Catalase expression. Oxidative stress in HK-2 cells was assessed using the oxidation-sensitive probe DCFH-DA, as previously described. After culturing under various conditions, the ROS levels in HK-2 cells from each group were assessed using the ROS detection kit. The cells were cultured with DCFH-DA for 20 min at 37 °C. After washing with PBS three times, the ROS levels were observed using a fluorescent microscope.
Analysis of apoptosis
Apoptosis in kidney tissues and HK-2 cells was assessed using different three methods. First, apoptosis in kidney tissues was determined by Western blot analysis of cleaved caspase-3 and PARP expression. Second, a TUNEL assay was performed using an in situ Cell Death Detection kit according to the manufacturer’s instruction (Roche Applied Science, Indianapolis, IN) to examine apoptosis in kidney tissues. The number of apoptotic cells was counted under a fluorescent microscope at 200 x magnification. At least 10 areas around the proximal tubules from sections from different rats from each group were counted and averaged. Third, HK-2 cell apoptosis was detected by flow cytometry using an Annexin V-FITC/PI Apoptosis Detection Kit. The cells were collected and re-suspended at 1 x 106 in 300 μl of binding buffer. Then, 10 μl PI solution and 5 μl Annexin V-FITC solution were added, and the cells were incubated for 5 min. A total of 10,000 events were collected, and the proportion of apoptotic cells was analyzed by flow cytometry.
Statistical analysis
Data are presented as the mean ± standard deviation (SD). Significant differences were examined by one-way analysis of variance with Bonferroni correction; p <0.05 was deemed to be statistically significant. All statistical analyses were performed using SPSS version 20.0 statistical software.