Clinical samples

This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the Medical Ethics Committee of Xiangya Hospital Central South University (ethical approval number: 202,106,247). A total of 30 patients (19 male and 11 female) with sepsis-associated AKI, aged 24 ~ 57 years (average age of 39.3 ± 7.8 years), were recruited consecutively from our hospital. The control group in our hospital consisted of 30 healthy volunteers (16 men and 14 women) aged 23 ~ 56 years (average age of 37.4 ± 8.2 years). There was no significant difference between patients with AKI and healthy volunteers with regard to sex and age. AKI cases were identified according to the Kidney Disease Improving Global Outcomes criteria and defined as any of the following symptoms: urine volume ≤ 0.5 mL/kg/h for 6 h; increase in serum creatinine (SCr) to ≥ 50% baseline within 7 days; or increase in SCr by more than 26.5 µmol/L within 48 h. Sepsis is the systemic response to infection, manifested by two or more of the following conditions as a result of infection: (1) body temperature < 36 °C or > 38.5 °C; (2) respiratory rate > 20 breaths/min or PaCO2 < 32 mmHg; (3) heart rate > 90 beats/min; and (4) white blood cell count < 4,000/mm3 or > 12,000/mm3, or > 10% immature band forms. The exclusion criteria were as follows: (1) patients with end-stage renal disease; (2) patients with a history of kidney transplantation; (3) patients who were diagnosed with cancer; (4) patients with acquired immunodeficiency syndrome; and (5) patients who received high-dose steroid therapy. All patients provided written informed consent, and venous blood tests were performed on all patients at the time of diagnosis.

Isolation and identification of AMSCs [29]

Human AMSCs from Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China) were soaked in MesenCult™-ACF medium (STEMCELL Technologies, CA) supplemented with 2 mM L-glutamine (Thermo Fisher Scientific, Wilmington, DE, USA) and 1% antibiotic antibacterial agent (Thermo Fisher Scientific). The phenotypes of the 3rd to 6th generation of AMSCs were evaluated by flow cytometry analysis (BD Accuri®C6 flow cytometry) after incubation with antibodies against CD29, CD31, CD44, CD45, CD73, CD90, CD105, and HLA-DR (Biolegend, CA, USA). IgG1 served as a negative control. Adipogenic differentiation medium (Invitrogen, Carlsbad, CA, USA), osteogenic differentiation medium (Invitrogen), and chondrogenic differentiation medium (Invitrogen) were used to induce the differentiation of AMSCs. After 14 or 21 days of induction, the cells were stained with Oil Red O dye (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), Alizarin Red S (Sigma-Aldrich), and Alcian Blue (Sigma-Aldrich).

Extraction of exosomes by differential centrifugation [30]

After 24 h of cell transfection, the culture medium was replaced with exosome-free fetal bovine serum (FBS)-supplemented medium for 48 h of incubation at 37 °C with 5% CO2. Exosomes were harvested by differential centrifugation: the supernatant was obtained, followed by low-speed centrifugation (300 × g for 10 min, 2,000 × g for 10 min) and ultracentrifugation (10,000 × g for 30 min, 100,000 × g for 70 min) at 4 °C. Cell precipitates were resuspended in PBS and then subjected to ultracentrifugation (100,000 × g for 70 min). Then, cell precipitates were resuspended in 200 µL PBS prior to the detection of protein concentration using the bicinchoninic acid (BCA) method or storage at -80 °C for later use. Quantitative analysis of exosomes was performed using the BCA protein assay kit (Beyotime Biotech, Shanghai, China) in accordance with the manufacturer’s instructions.

Observation of the morphology of exosomes by transmission electron microscope (TEM) [31]

Exosome suspensions (10 µL) were diluted with PBS and added to a 2 mm formvar-coated copper grid for 1 min of incubation at room temperature. After removing excess liquid with filter paper, exosomes were negatively stained with 3% (w/v) sodium phosphotungstate solution (Sigma-Aldrich) for 5 min. Then, exosomes were washed with PBS, dried naturally in the air prior to observation, and photographed using a TEM (H-8100, Hitachi, Tokyo, Japan) at 80 ~ 120 kV (Scare bar = 0.2 μm).

Nanosight tracking analysis [32]

Exosomes were diluted 1:100, and 1 mL exosome solution was added to a quartz colorimetric utensil. The particle size was determined using a dynamic light-scattering instrument (Nano S90, Marvin, UK).

Fluorescence labeling of exosomes [33]

AMSCs were labeled with 1.25 µM lipophilic carbocyanine DiLC16 (3) for 10 min at 37 °C, followed by washing and resuspension in fresh culture medium for 48 h. Exosomes labeled by fluorescence were added into HK-2 cells for incubation of 6 h and the cells were fixed in formaldehyde and sealed. Images were captured using a fluorescence microscope.

Cell culture and cell transfection [34]

The human renal proximal tubular epithelial cell line HK-2 (National Collection of Authenticated Cell Cultures, Shanghai, China) was immersed in Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY, USA) containing 10% FBS, 1% penicillin, and 1% streptomycin for cell culture in an incubator at 37 °C and 5% CO2. For the construction of sepsis cellular models, HK-2 cells were induced by 1 µg/mL lipopolysaccharide (LPS, Sigma-Aldrich) for 24 h. To investigate the effect of autophagy on sepsis, HK-2 cells were treated with autophagy activator rapamycin (Rapa, 10 nM, Sigma-Aldrich) or autophagy inhibitor 3-Methyladenine (3-MA, 10 µM, Sigma-Aldrich) for 30 min before LPS treatment. For incubation with exosomes (20 µg), HK-2 cells were treated with LPS (1 µg/mL) for 24 h before being treated by AMSC-Exo for 24 h.

The LPS-induced cells were placed into plates and incubated for 16 h at 37 °C with 5% CO2 before cell transfection. Next, cells were transfected with miR-342-5p mimic, miR-342-5p inhibitor, miR-67 mimic, miR-67 inhibitor (50 nM), pcDNA3.1-TLR9, si-TLR9 (2 µg), or their negative controls (GenePharma, Shanghai, China) using the LipoFiterTM transfection reagent (Hanbio Biotech, Shanghai, China) following the manufacturer’s instructions. Three replicates were established, and the following experiments were carried out 24 h after transfection.

Lentiviral Transfection

The AMSCs (1 × 106 cells) were immersed in 10 mL MesenCultTM-ACF medium overnight. Then, AMSCs were transfected with LV-miR-342-5p (lentiviral vector overexpressing miR-342-5p) and LV-miR-67 (lentiviral vector overexpressing cel-miR-67) (MOI = 10.0). Cel-miR-67 was chosen as the control because it was demonstrated to have no binding sites with mRNAs in humans. Twenty-four hours after transfection, AMSCs were collected for qRT-PCR analysis.

Animals [34]

All experiments involving animals were performed according to the protocol approved by the Animal Experiment Ethics Committee of Central South University (ethical approval number: 202,106,247). Six-to-eight-week-old male C57BL/6J mice (n = 36) were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. All mice were bred and maintained under specific pathogen-free conditions at the humidity of 40 ~ 60% for 1 week with a 12 h light-dark cycle. Water and standard laboratory food were provided ad libitum.

The sepsis-associated AKI mouse models (the CLP group, n = 6) were established by cecal ligation and puncture (CLP). Briefly, mice were anesthetized with ketamine (100 mg, Ketavet®)/xylazine (200 mg/kg, Rompun®) after being weighed. A 1 ~ 1.5 cm incision was made in the middle of the lower abdomen, and the cecum was carefully separated. The distal 1/3 of the cecum was ligated with a 4 − 0 silk ligature. The end of the cecum was punctured twice with a 25-gauge sterile needle, and a small amount of feces was gently squeezed out to ensure that the punctures were unblocked. The cecum was then put back into the abdomen, and the incision was sutured. Mice in the sham group (n = 6) underwent surgery without ligation and puncture. Mice in the CLP + AMSC-Exo-67 group (n = 6) or the CLP + AMSC-Exo-342 group (n = 6) were injected with 100 µg AMSC-Exo-67 or AMSC-Exo-342 by tail vein 4 h after CLP. In the CLP + Rapa group (n = 6), 4 mg/kg Rapa (Sigma-Aldrich) was intraperitoneally injected into mice 6 h before surgery, and 200 µL normal saline was injected into mice via the tail vein 4 h after surgery. Mice in the CLP + 3-MA + AMSC-Exo-342 group were administered 20 mg/kg 3-MA by intraperitoneal injection 6 h before operation and injected with 100 µg AMSC-Exo-342 by tail vein 4 h after CLP. Kidney tissues and blood samples from each group were obtained 72 h later.

Quantitative Reverse transcription-polymerase Chain Reaction (qRT-PCR)

Total RNA from cells or tissues was extracted according to the instructions included with TRIzol reagent (Invitrogen), and the concentration and quality of total RNA were determined. After adjustment to an appropriate concentration, total RNA was reverse transcribed using a reverse transcription kit (TaKaRa, Tokyo, Japan). Random primers were applied as reverse transcription primers, and the operation was performed according to the instructions included with the reverse transcription kit and random primers. Gene expression was assessed using a LightCycler 480 fluorescence quantitative PCR instrument (Roche, Indianapolis, IN, USA). The reaction conditions were consistent with the instructions for the fluorescent quantitative PCR kit (SYBR Green Mix, Roche Diagnostics, Indianapolis, IN, USA). The reaction conditions were initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 20 s. Three independent experiments were run in parallel. The quantification of mRNA was normalized to that of GAPDH, whereas that of miRNAs was normalized to U6. All primers used are listed in Table 1.

Table 1 Primer sequence information Western blot [35]

Cells or tissues were lysed with RIPA lysis buffer (Beyotime) and centrifuged to obtain protein samples. GAPDH served as an internal reference. The protein concentration was determined using a BCA kit (Beyotime), and the corresponding volume of protein was mixed with loading buffer (Beyotime), followed by 3 min of denaturation in a boiling water bath. SDS-PAGE (10%) was performed in compliance with the directions for the SDS-PAGE preparation kit (Beyotime) and was then applied to separate proteins. Electrophoresis was conducted at 80 V, and then for 1 ~ 2 h at 120 V once bromophenol blue reached the separation gel. Then, the proteins were transferred onto membranes at 300 mA for 60 min in an ice bath. The membranes were rinsed for 1 ~ 2 min with washing solution, sealed in blocking solution at room temperature for 60 min, or inactivated overnight at 4℃. The membranes were incubated with primary antibodies against rabbit anti-human GAPDH (1:1000, Cell Signaling Technology [CST], Boston, USA), CD9 (1:500, Santa Cruz, Texas, USA), CD81 (1:500; Santa Cruz), CD63 (1:1000, Abcam, MA, USA), LC-3 (1:2000, Abcam), p62 (1:10000, Abcam), Beclin-1 (1:1000, CST), and TLR9 (1:1000, Abcam) at room temperature on a shaking table for 1 h. Following primary incubation, the membranes were washed with the washing solution for 3 × 10 min and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature. The membranes were washed for 3 × 10 min with washing solution and subjected to color development. A chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA) was used for these observations.

Dual-luciferase reporter assay [36, 37]

TargetScan (http://www.targetscan.org/mamm_31/) was used to predict the binding sites of miR-342-5p and TLR9. The mutated and wild-type sequences (mut-TLR9 and wt-TLR9) were designed and synthesized in accordance with the predicted results, and cloned into a luciferase reporter vector (pGL3-Basic, Promega, Madison, WI, USA). The vectors were co-transfected with miR-342-5p mimic or mimic-NC (30 nM, GenePharma) into HEK293T cells. After transfection, the dual-luciferase reporter assay kit (Promega) was used to measure the fluorescence intensity of cells in each group and to determine the binding of miR-342-5p to TLR9. Cells were grouped into the mimic + mut-TLR9 group, mimic + wt-TLR9 group, mimic NC + mut-TLR9 group, and mimic NC + wt-TLR9 group. Three replicates were used for this test.

Hematoxylin-eosin (H&E) staining [38]

Kidney tissues were fixed in 10% paraformaldehyde for 24 h and then subjected to gradient dehydration and paraffin embedding. H&E staining was performed after 4 μm thick paraffin sections were prepared.

Immunofluorescence [39]

Cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 1% Triton X-100 for 25 min at room temperature, and washed three times with PBS. After 45 min of inactivation in 5% bovine serum albumin, the cells were incubated with the primary antibody overnight at 4 °C, followed by 3 × 5 min of PBS wash. The cells were then exposed to a secondary antibody (DyLight® 650, goat anti-rabbit IgG) at room temperature for 2 h. The nuclei of cells were stained with DAPI (Beyotime) and visualized under a microscope (Nikon, Japan).

TEM [38]

HK-2 cells were starved with Earle’s Balanced Salt Solution for 30 min, washed with PBS, and centrifuged for 10 min. The cells were then fixed in glutaraldehyde for 2 ~ 4 h and washed with 0.1 mol/L PBS for 1 h at 4 °C. The washing liquid was changed 3 times during this process. Cells were immersed in osmium tetroxide fixative for 1 ~ 2 h at 0 ~ 4℃, followed by exposure to acetone (30%, 70%, and 90%, each for 10 min) and pure acetone (3 × 10 min). After being embedded in the embedding medium, the cells were polymerized in an oven at 37 °C for 12 h and 60 °C for 24 h. For examination via TEM (H-8100, Hitachi, Tokyo, Japan), sections of 60 nm were produced using a Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems).

Enzyme-linked Immunosorbent Assay (ELISA)

The serum of humans and mice and the supernatant of HK-2 cells were collected. ELISA kits were used to detect the levels of blood urea nitrogen (BUN) (YS02947B, Jiya biological and technology co., Ltd, Shanghai, China), SCr (FT-P9S3176X, Fantai biological and technology co., Ltd, Shanghai, China), tumor necrosis factor (TNF)-α (ab285312, ab285312, Abcam), interleukin (IL)-1β (ab214025, ab255730, Abcam), IL-6 (ab178013, ab222503, Abcam), and monocyte chemoattractant protein (MCP)-1 (ab179886, ab208979, Abcam) in the serum and cell supernatant.

Statistical analysis

Three sets of samples were used for each group in each experiment. Statistical analysis was conducted using GraphPad software (version 7.0). Data were expressed as mean ± standard deviation (SD). A t-test was employed for comparisons between two groups. Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA) and confirmed by Tukey’s multiple comparison test. P values were considered significant at P < 0.05.