Animal experiment

All experimental protocols on animals were approved by the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology (Approval No. 2663). Experiments were carried out with 6-8-week-old male C57BL/6 mice (SPF Biotechnology, Beijing, China). All animals were cultivated in a specific pathogen-free environmental animal room at Tongji Medical College, Huazhong University of Science and Technology. All surgical operations were performed after anesthesia was induced using 1% pentobarbital.

For LPS-AKI model, the mice were divided into three groups (n = 8 per group): phosphate-buffered saline (PBS) group, LPS-12 h group (LPS 20 mg/kg for 12 h), and LPS-16 h group (20 mg/kg for16 h). The mice were anesthetized, and blood was collected. Creatinine and urea nitrogen levels were measured in the blood, and the kidney tissue was collected for further analysis.

For experiment in ASM knock out mice, the mice were divided into four groups (n = 6 per group): PBS group in ASM+/+ mice, PBS group in ASM+/− mice, LPS (20 mg/kg for16 h) in ASM+/+ mice, and LPS (20 mg/kg for16 h) in ASM+/− mice. The mice were anesthetized, and blood was collected. Creatinine and urea nitrogen levels were measured in the blood, and the kidney tissue was collected for further analysis.

For in vivo experiments on EXOs injection, the mice were divided into three groups (n = 3 per group): PBS group, control-RAW264.7-EXO injection group (40 µg) and LPS-RAW264.7-EXO injection group (40 µg). EXOs were injected into mice through tail vein. After 24 h, blood was collected under anesthesia to detect creatinine and urea nitrogen, and kidney tissue was collected for subsequent analysis.

Cell culture

Rat glomerular endothelial cells (RGECs) were a gift from Shandong University; they were cultured in Dulbecco’s Modified Eagle Medium F-12 (DMEM-F12) with 1% penicillin streptomycin (Gibco, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS; Science Cell, USA). RAW264. 7 cells were purchased from American Type Culture Collection and culture in DMEM medium with 1% penicillin streptomycin (Gibco) and 10% FBS (Science cell).

For the use of the ASM inhibitor amitriptyline, pretreatment with amitriptyline (20 µM) 0.5 h followed by other stimuli (LPS, or EXOs). The RGECs was knocked down ASM with siRNA (sequence: GCTACCGTTTACCAAAT) and then stimulated with EXO. The siRNA was transfected into RGECs with Lippo 2000 (thermo, USA), and the solution was changed 4 h after transfection.

Isolation and characterization of EXOs

RAW264.7 cells were cultured in DMEM containing EXO-free FBS (ultracentrifugation at 120,000 ×g for 12 h) before LPS stimulation. RAW264.7 cells were cultured to 80% confluence, and then the control group was not given any treatment. The LPS group was stimulated with 1 µg/mL LPS (#L2880; Sigma, St Loui, MO, USA) for 24 h, and the supernatant was collected and subjected to a series of gradient centrifugation to extract the EXOs.

For the extraction of renal EXOs, 20 mg of renal cortex was digested with collagenase at 37 °C for 120 min. The samples were then subjected to EXOs extraction. The gradient centrifugation included 300 ×g for 10 min and 2000 ×g for 10 min, followed by 10000×g for 30 min. The supernatants were then centrifuged at 120000 ×g for 70 min. The pellets were washed once with PBS, centrifuged again at 120000×g for 70 min and resuspended in PBS (Type 70 Ti rotor; Beckman Coulter Optima, USA) .

The morphology of RAW264.7-EXO was examined using a transmission electron microscope (Hitachi HT770, Tokyo, Japan). Nanoparticle tracking analysis (NTA) was performed by Zetaview, PMX 110 (Particle Metrix, Meerbusch, Germany), and the protein levels was quantified using the BCA Protein Assay Kit (Aspen, Wuhan, China) following the manufacturer’s instructions.

Macrophage-derived EXO labeling and uptake assay

RAW264.7-EXOs were incubated with PKH26 red fluorescent dye (Sigma) for 3 min, and the reaction was stopped with Dilute C. Subsequently, centrifugation was performed to obtain PKH26-labeledEXOs, and the cells were seeded in a 35 mm confocal culture dish and treated with 5 µg/mL PKH26-labeled EXOs. After culturing for 12 h, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. The cytoskeleton with phalloidin (Sigma), and the nuclei with 4′,6-diamidino-2-phenylindole (Aspen Biotech, Shanghai, China) for 10 min. The internalization of EXOs was observed under a confocal microscope.

RAW264.7-EXO were purified and stained with DIR. After dyeing for approximately 30 min, the dye was stopped with PBS, and the excess dye was removed by centrifugation at 120000 ×g for 70 min. DIR-labeled EXOs were injected into C57BL/6 mice via the tail vein. After 24 h, the distribution locations of DIR-labeled-EXOs in mice were observed using an in vivo fluorescence imaging system. After the preliminary observation, the mice were anesthetized, dissected and the kidneys were removed and observed directly using the in vivo fluorescence imaging system.

Cell proliferation

Cell Counting Kit-8 (CCK8; Boster, China) was used to determine cell viability. RGECs were planted in 96-well plates according to the instructions. RAW264.7-EXO (5 µg/mL) were used to stimulate RGECs. After 24 h, the medium was changed and 10 μL CCK-8 mixed solution was added. After 1 h, the absorbance was measured at a wavelength of 450 nm.

Western blotting analysis

Cells, kidney tissues, and purified EXOs were lysed in radioimmunoprecipitation assay lysis buffer containing protease inhibitors, and protein concentration was determined by BCA assay (aspen). The protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (Servicebio, China) and transferred to PVDF membrane (Millipore, USA). The membrane was blocked with 5% BSA (Biosharp, Hefei, China) for 1 h, and then the primary antibody was applied and incubated overnight at 4 ℃. The other reagents are listed here: anti- cluster of differentiation (CD) 9, anti-CD63, anti- interleukin (IL)-1β, anti- nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3), anti-adaptor apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), anti- vascular cell adhesion molecule-1 (VCAM-1), anti-ASM, anti-neutrophil gelatinase-associated lipocalin (NGAL) antibodies, all purchased from Abcam (USA); anti-caspase-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-GAPDH (Proteintech, Wuhan, China). Anti-mouse secondary antibody or anti-rabbit secondary antibodies (Proteintech) were used for detection in the BioSpectrum 600 imaging system (UVP, CA, USA).

Histological analysis

After harvesting kidneys from male C57 BL/6 mice, they were fixed with 4% paraformaldehyde, dehydrated using a series of gradient ethanol, embedded in paraffin, and cut into 6 μm thick longitudinal sections. Sections were stained with hematoxylin and eosin, and histopathological changes were observed under a microscope.

Statistical analysis

All data are expressed as mean ± SEM. The t-test was used to compare the differences between the two groups, and one-way or two-way analysis of variance was used for more than two groups. Statistical significance was set at P < 0.05. Statistical analysis was performed using GraphPad Prism 8.0 software (version 8.0; GraphPad Software, Inc., San Diego, CA, USA).