Animal experimental design

Thirty male SD rats (8 weeks old,180–220 g) were purchased from the Experimental Animal Center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The rats were acclimatized to a 12 h light/dark cycle in a specific pathogen-free animal house for 1 week before the start of the experiments. The rats were divided into six groups with 5 rats each group: the control group: normal feeding without any treatments; the GAM group: intraperitoneal injection of GAM (glyoxylic acid monohydrate) at 80 mg/kg/day for 9 days; the EXO(C) group: renal subcapsular injection of EXO(C) at 40 mg/day every week for 2 weeks; the EXO(S) group: renal subcapsular injection of EXO(S) at 40 mg/day every week for 2 weeks; renal subcapsular injection of normal saline (NS) was conducted in these two groups on the other side of the rat kidney as the sham group. For the EXO(C) + GAM group, pre-renal subcapsular injection with EXO(C) was performed every week for 2 weeks, followed by intraperitoneal injection of GAM at 80 mg/kg/day for 9 days; for the EXO(S) + GAM group, prerenal subcapsular injection with EXO(S) was performed every week for 2 weeks, followed by intraperitoneal injection of GAM at 80 mg/kg/day for 9 days; renal subcapsular injection of NS was conducted in these two groups on the other side of the rat kidney as the sham group. All procedures were performed following the Animal Management Rules of the Ministry of Health of the People’s Republic of China. The operations were approved by the Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (IACUC Number:2746). All rats were euthanised after the above treatments. The rat kidney was ground up with a tissue grinder. Then, the tissue was centrifuged at 3000 rpm for 10 min, and the supernatant was stored for further exploration. The rest kidney tissues were used for histological examination and Von Kossa staining.

Cell culture and treatment

The HK2 cell line was purchased from Procell (Wuhan, China) and cultured in RPMI 1640 medium (HyClone, USA) containing 10% foetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), which had been depleted of exosomes before being added to the medium. The cells were exposed to CaOx for 24 h at a concentration of 2 mM, and then, the supernatant was collected for isolation of exosomes. Exosomes derived from HK2 cells without CaOx crystal stimulation were defined as EXO(C), Exosomes derived from HK2 cells stimulated with CaOx crystals were defined as EXO(S). EXO(C) and EXO(S) were added to HK2 cells and cocultured for 48 hours.

Exosome isolation

Exosomes derived from cells were isolated through multistep centrifugation. First, HK2 cells cultured in RPMI 1640 containing exosomes depleted of FBS were centrifuged at 300 g for 15 minutes to eliminate the dead cells and debris. Then, the samples were centrifuged at 2000 g for 30 minutes and 10,000 g for 30 minutes. Each supernatant was filtered in a 100-kDa MWCO ultrafiltration centrifuge tube (Amicon-Ultra, Millipore, USA). Then, the supernatant was centrifuged at 4 °C and 120,000 g for 90 minutes, the supernatant was discarded, and the pellet was resuspended in PBS and centrifuged at 4 °C, 120000 g for 90 minutes again to collect exosomes.

In vivo fluorescence imaging

Briefly,40 μg of exosomes labelled with PKH26 (UR52302, Umibio, China) were locally injected into the rat kidneys (via methods described previously in the animal design section). Exosome accumulation and dynamics were tracked by in vivo fluorescence imaging on Day 1, 3, 7, 10and 14 via a Lago/Lago X Imager (Lago/Lago, Spectral Instruments Imaging, USA).

Exosome labelling with Dil and uptake study

The purified exosomes were labelled with a Dil red fluorescent labelling kit (Biodee, Beijing) according to the manufacturer’s instructions. Then, the labelled exosomes were cocultured with HK2 cells. Observations were conducted at 0, 6, and 12 hours after incubation with a laser confocal microscope (SP8, Leica, Germany).

Transmission Electron microscopy (TEM) and nanoparticle tracking analysis (NTA)

The procedures for conducting transmission electron microscopy and nanoparticle tracking analysis were the same as those described in our previous study [13].

Measurement of reactive oxygen species (ROS) by dihydroethidium (DHE)

Intracellular ROS production was detected via a Reactive Oxygen Species Assay Kit (Beyotime, S00335). Cells were cultured in a 6-well plate and stimulated by EXO(C) and EXO(S), digested these cells with 0.25% trypsin without EDTA and collected after termination of digestion, washed the cells once with PBS, and set aside. Following the instructions of the ROS detection kit, ROS were detected by flow cytometry (Beckman, CytoFLEX, USA). ROS in renal tissues were measured with DHE fluorescence. Kidney tissues of different groups were ground into homogenate and used for detection of ROS production following the instructions of the ROS detection kit. Then, the fluorescence of DCF was detected via multimode microplate reader (Tecan,M200 PRO).

Measurement of CAT,SOD and MDA

MDA represents products of lipid peroxidation, and SOD and CAT are key antioxidative biomarkers. The MDA level and SOD and CAT activity were detected by chemiluminescence methods via MDA, CAT, and SOD detection kits (NanJing，JianCheng Bioengineering Institute, China) were used, and detection was conducted following the manufacturer’s instructions. The OD values of MDA were 532 nm, and those of CAT were 405 nm. The OD values at 550 nm were measured for SOD.

Western blot analysis

Western blotting was performed following the method we used in our previous study [13]. The antibodies included anti-rabbit BMP2 (1:2000, BOSTER, China), anti-rabbit OPN (1:2000, SANTA CRUZ, USA), anti-rabbit OCN (1:2000, SANTA CRUZ, USA), anti-rabbit β-actin antibodies (1:2000, BOSTER, China), anti-mouse TSG101 (1:2000, Abcam, USA), anti-mouse CD9 (1:2000, Abcam, USA), anti-mouse Alix (1:2000, Abcam, USA),anti-mouse Calnexin (1:2000, Abcam, USA), and anti-rabbit and mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody (1:5000, BOSTER, China). All WB quantification was performed using three independent WBs.

RT-qPCR

Total RNA was extracted from cells with different treatments via TRIzol reagent (Ambion, USA), and reverse transcription reactions were performed with a complementary DNA Synthesis Kit (Yeasen, China). RT-qPCR was conducted via SYBR Green Master Mix (Hieff qPCR SYBR Green Master Mix (High Rox Plus)) on the ABI Prism 7300 system (Thermo Fisher Scientific, USA). The 2 − ΔΔCt method was used to analyse the mRNA expression levels. The primer sequences used in this study are listed in Supplementary Table 1 (Stable1).

Von Kossa staining

The renal tissues of rats with different treatments were collected and fixed in 10% paraformaldehyde, dehydrated, embedded, sliced and washed them with ddH2O for 1 min. Crystal depositions of these renal tissues were then analysed by Von Kossa staining following the manufacturer’s instructions (HEPENGBIO VON KOSSA Kit, China).

Immunofluorescence (IF) assays and immunohistochemical staining (IHC)

Cells with different treatments were cultured in 6-well plates with slides preset in the wells. The slides were washed for 3 times in PBS,fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5%Triton X-100 (PBS preparation) for 20 min(room temperature) and blocked with 5% BSA for 30 min. The cells were incubated with OPN and OCN antibodies (1:100) overnight (4 °C), and then incubated with FITC-conjugated goat anti-rabbit IgG (1:100) for 1 h (25 °C). DAPI (4′,6-diamidino-2-phenylindole) was coincubated with cells for 5 min, and then, the fluorescent signals were observed via a BX53 microscope (Olympus, Tokyo, Japan). Immunohistochemical staining was used to detect tissue proteins. Antibodies against BMP2, OPN and OCN (1:100) were coincubated with these section preparations. Images were then observed with a BX53 microscope (Olympus, Tokyo, Japan).

Statistical analysis

The results are reported as the mean of at least three independent experiments. GraphPad Prism 5 (GraphPad Software, Inc., CA, USA) and SPSS24.0 (SPSS Statistics Inc., Chicago, USA) were used. The statistical methods were selected as we previous described [6]. A * p < 0.05 was considered statistically significant.