BMMSCs collection and culture
Femurs and tibias of 6-week-old C57BL/6 female mice were collected, washed with phosphate buffered solution (PBS) containing 2% fetal bovine serum (FBS), and inoculated in DMEM medium containing 2% FBS and penicillin/streptomycin. The adherent cells were removed 48 h later. After the adherent cells reached 90% fusion, the adherent cells were treated with trypsin . Mice were raised in the Animal Center of Zhengzhou University. All animal procedures met the standards of the Institutional Animal Care and Use Committee of Zhengzhou University People's Hospital.
BMMSCs exosomes (BMMSC-Ex) collection
BMMSCs cells were cultured in depleted exosomal medium (cells were centrifuged at 100000 g), the supernatant was collected and centrifuged at 300 g for 10 min, 3000 g for 10 min, 20,000 g for 30 min, and 120,000 g for 70 min. BMMSC-Ex were obtained after resuspended in PBS .
Transmission electron microscopy (TEM)
TEM was conducted to observe the morphology of BMMSC-Ex using a Tecnai™ transmission electron microscope (FEI, USA). Before photography, BMMSC-Ex was suspended in 2.5% glutaraldehyde and loaded on the copper grids .
Nanoparticles Tracking Analysis (NTA)
The size of BMMSC-Ex was determined by NTA with Nanosight NS300 (Malvern, UK). Exosomes were resuspended by PBS, diluted, and injected into the sample chamber. The NTA software was used to capture and analyze the data.
Proteins of BMMSC-Ex or CD4+ T cells were collected with RIPA Lysis Buffer (Beyotime, Shanghai), and the samples were quantified by Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The samples were administrated with SDS-PAGE followed by transferred to the polyvinylidene difluoride membrane (Millipore, Germany). The membrane was blocked and then incubated with primary antibodies against CD9 (ab92726, 1:2000), TSG101 (ab125011, 1:1000), CD63 (ab217345, 1:1000), SIRT1 (ab12193, 1:2000). Calnexin (ab22595, 1:1000) and β-actin (ab8227, 1:1000) overnight at 4°C. The membranes were then incubated with Goat Anti-Rabbit IgG H&L (HRP) for 2 h. Finally, the blots were observed with BeyoECL Moon (Beyotime Biotechnology, Shanghai) .
Mice were grouped into isograft control (BALB/c to BALB/c, n=7), allograft (C57BL/6 to BALB/c, n=7), allograft + PBS (C57BL/6 to BALB/c, n=7), allograft + BMMSC-Ex (C57BL/6 to BALB/c, n=7). For the model of isogenic kidney transplantation, BALB/c mice were performed as donors and recipients. For the model of allograft kidney transplantation, C57BL/6 mice were acted as donors and BALB/c mice were the recipients.
Mice were anaesthetized with pentobarbital sodium and ether, and subcutaneously injected with butorphanol (1 mg/kg) for analgesia. In donor mice, a median abdominal incision was made, and the left kidney was used as the donor kidney, the left kidney and blood vessels were exposed. The abdominal aorta and the inferior vena cava were dissociated, ligated, and blocked. After perfusion, the abdominal aorta, inferior vena cava and ureter were moved, and the left kidney and its associated blood vessels and ureter were stored in 4°C high osmotic citron salt adenine solution. In recipient mice, a median abdominal incision was made, and the left kidney was resected, the donor kidney was placed into the left lower abdomen. Then, 2 mL (50 U/mL) of 4°C heparin saline was injected into the right peritoneum for systemic heparinized. The transplanted blood vessel was first anastomosed to the recipient aorta and then to the inferior vena cava. After the anastomosis, the blood supply was restored and the ureter was implanted into the bladder. The cold ischemia time was 60 min and the hot ischemia time was 20 min.
For allograft + PBS group, mice were infused with PBS through caudal vein 1 day before and 1 day after kidney transplantation. For allograft + BMMSC-Ex group, mice were infused with BMMSC-Ex (10 μg, suspended in PBS) through caudal vein 1 day before and 1 day after kidney transplantation. All animal procedures met the standards of the Institutional Animal Care and Use Committee of Zhengzhou University People's Hospital .
HE (hematoxylin-eosin) staining
Six days after transplantation, the kidney tissues were collected, fixed with 10% formaldehyde, dehydrated with different concentrations of alcohol gradient, embedded in paraffin, and cut into 5 μm slices. Tissues were then dewaxed with xylene, rehydrated with gradient alcohol and stained with HE (Beyotime Biotechnology, Shanghai). After gradient dehydration and transparency, the kidney tissues were sealed with neutral gum and observed under a light microscope (Olympus BX51, Japan).
Six days after transplantation, the kidney tissues were collected, fixed with 4% paraformaldehyde, incubated with 0.3% H2O2 for 30 min, blocked with Tris-buffered saline containing 10% normal serum and 1% Bull Serum Albumin for 2 h, and incubated overnight with Anti-CD4 antibody (ab183685, 1:200) at 4°C. The next day, sections were washed and incubated with biotin-labeled secondary antibody at room temperature for 1 h, following stained with 3,3'-diaminobenzidine for 10 min. Finally, the images were observed under a microscope (Zeiss, Germany) 
Serum creatinine (SCr) levels
Blood samples were taken from mice and centrifuged at 1500 g for 15 min. SCr levels were examined using an Indiko automatic biochemical analyzer (Thermo Fisher Scientific, USA) .
ELISA (enzyme-linked immunosorbent assay)
Levels of IL-2, IL-17, and IFN-γ were examined by ELISA assay referring to its instructions. The ELISA kits for IFN-γ, IL-2, and IL-17 were all purchased from Invitrogen™ (Thermo Fisher Scientific, USA) .
Six days after transplantation, the recipient mice were sacrificed, and spleen and kidney samples were collected and prepared into single-cell suspension. Cells were incubated with anti-CD4-APC antibody and anti-Foxp3-PE antibody following the instructions (eBiosciences). Data were collected and analyzed with flow cytometry (FACSCalibur, BD Biosciences, USA) .
The LV, LV-DANCR, LV-shRNA, LV-shDANCR were cloned into a lentiviral vector and transferred into 293T cells. After that, the obtained virus particles were used to infect BMMSCs or BMMSC-Ex.
For the BMMSCs-LV-Ex group, BMMSCs-LV-DANCR-Ex group, BMMSCs-LV-shRNA-Ex group, and BMMSCs-LV-shDANCR-Ex group, the LV, LV-DANCR, LV-shRNA, or LV-shDANCR was transfected into BMMSCs followed by the collection of BMMSC-Ex.
CD4+ T cell isolation
CD4+ T cells were isolated from spleens of healthy C57BL/6 mice using Dynabeads™ Untouched™ Mouse CD4 Cells Kit (Thermo Fisher Scientific, USA) referring to the instructions. CD4+ T cells were in the supernatant. Then cultured with different concentrations of BMMSC-Ex (10, 50, 100 ng/mL) for 2 days .
RNA was isolated from BMMSCs, BMMSC-Ex, CD4 + T cells, spleen and kidney tissues using Trizol (Beyotime Biotechnology, Shanghai). After reverse transcription, DANCR levels were determined using SYBR qPCR Mix (Takara, Japan) on a Real-time fluorescence quantitative PCR instrument (Applied Biosystems, USA). GAPDH was used as an internal reference and the intergroup multiples were calculated by the 2-ΔΔCt method.
DANCR: (F: 5′-CGTCTCTTACGTCTGCGGAA-3′
GAPDH: (F: 5′-TGCACCACCAACTGCTTAG-3′
RNA pull-down assay
RNA pull-down assay was performed according to the instructions of the Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA). CD4+ T cells were lysed using RIP buffer containing protease inhibitor and RNase inhibitor. The biotin-labeled lncRNA DANCR (Bio-DANCR) or control (Bio-NC) was captured with beads and then transfected into CD4+ T cell lysate. Western blot was carried out to examine the protein levels of SIRT1.
RNA immunoprecipitation (RIP) assay
RIP assay was performed according to the instructions of Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Germany). CD4+ T cells were lysed using RIP buffer containing protease inhibitor and RNase inhibitor. The CD4+ T cell lysate was incubated with magnetic beads coated with anti-SIRT1 (ab32441, 1:30). Normal Rabbit IgG was used as the negative control. After washing and purification, qRT-PCR was carried out to examine DANCR levels.
The FLAG-SMURF2, LV-DANCR (or LV) constructs were transfected into CD4+ T cells and treated with 10 μM MG132 (proteasome inhibitor) for 4 h. Cells were then lysed with RIP Lysis Buffer and incubated with magnetic beads coated with anti-FLAG (SMURF2, Cell Signaling Technology, 1:50). Western blot was carried out to examine the protein levels of SIRT1.
The HA-Ub, FLAG-SMURF2, LV-DANCR (or LV) constructs were transfected into CD4+ T cells and treated with 10 μM MG132 for 4 h. Cells were then lysed with RIP Lysis Buffer and incubated with magnetic beads coated with anti-HA-Ub (ab3341, 1:1000). Western blot was carried out to examine the protein levels of SIRT1.
Analyses were performed with GraphPad Prism 8.01. The difference was assessed by Student's t-test or one-way ANOVA followed by the LSD post hoc test. P<0.05 was considered statistically significant .