Establishment and grouping of the animal model
Female NZB/WF1 and ICR mice (specific pathogen free, SPF) aged 16 weeks were acquired from Jackson Laboratory by the iBio Logistics Co., Ltd. (Beijing, China) and the Zhejiang Academy of Traditional Chinese Medicine, respectively, and were housed in accordance with relevant guidelines. All procedures involving mice were approved by the Animal Ethics Committee of Zhejiang Academy of Traditional Chinese Medicine (approval number: ZATCM-2019045) and were carried out in accordance with the ARRIVE guidelines.
The SR lupus mouse (SRLM) model was established by the intraperitoneal injection of low-dose Meyhylprednisolone(MP, Pfizer, New York, USA) for eight weeks (0.8 mg·kg-1·day-1) according to our previous study. Compared with untreated mice, the criterion for the successful construction of the SRLM model was that the expression of P-gp increased and the accumulation of Rh-123 decreased in the splenic lymphocytes of LN mice (SLCsL)(Supplementary Fig. 1). ICR mice were used as a control group (normal saline, 8 weeks). NZB/WF1 mice were randomly divided into the LN group (normal saline, 8 weeks), SR group (0.8 mg·kg-1·day-1 MP, 8 weeks), SR with PNS (Guangxi Wuzhou Pharmaceutical Co., Ltd, Guangxi, China) (Supplementary Fig. 2) group (0.8 mg·kg-1·day-1 MP, 100 mg·kg-1·day-1 PNS, 8 weeks) and SR with P-gp inhibitor Tariquidar (Tariq, Selleckchem, TX, USA) group (0.8 mg·kg-1·day-1 MP, 8 mg·kg-1·day-1 Tariq, 8 weeks).
Preparation of splenic lymphocytes
After the early intervention, five groups of mice were euthanized through CO2 asphyxiation using slow displacement of chamber air with compressed CO2 (25%/min), and their splenic lymphocytes were extracted, namely, the control group (SLCsC), LN group (SLCsL), SR group (SLCsL/S), SR with PNS group (SLCsL/S+PNS), and SR with Tariq group (SLCsL/S+Tariq). Lymphocytes were isolated and cultured as previously described.
Preparation of GECs
The GECs of ICR and NZB/WF1 mice were separated as previously described and cultured in endothelial cell medium supplemented with 10% FBS at 37 °C with 5% CO2.
Cell co-culture assay
SLCsC and SLCsL, SLCsL/S, SLCsL/S+Tariq(5 × 105/well) were seeded into the upper chamber of a co-culture system and GECs(3 × 105/well) from ICR and NZB/WF1 mice were placed in the lower chamber and incubated for 48h. Co-cultured cells were separated using Transwell permeable supports(0.4 μm) in poly carbonate membrane(Costar, Cambridge, MA, USA).
Isolation of exosomes
Exosomes were isolated by ultracentrifugation as described previously. In brief, the culture supernatants of cells were collected and sequentially centrifuged at 300 g for 10 min, 2000 g for 15 min, and 10,000 g for 30 min to remove cell debris and large vesicles, and then, the cleared sample was further ultracentrifuged for 2 h at 100,000 g to pellet the exosomes. The extracted exosomes were allocated and stored at -80 °C or used for downstream experiments.
Transmission electron microscopy (TEM)
The morphology of exosomes was observed by TEM. After absorbing onto Formvarcarbon-coated copper mesh grids, exosomes were negatively stained with aqueous phosphotungstic acid for 1-2 min. After air dry, samples were visualized with a Hitachi 7100 transmission electron microscope.
Nanoparticle-tracking analysis (NTA)
We measured the exosome particle size and concentration using nanoparticle tracking analysis (NTA) at VivaCellBiosceinces with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). Isolated exosome samples were appropriately diluted using 1X PBS buffer (Biological Industries, Israel) to measure the particle size and concentration. NTA measurement was recorded and analyzed at 11 positions. The ZetaView system was calibrated using 110 nm polystyrene particles. Temperature was maintained around 23°C and 30°C.
Western blot analysis
Exosomes were collected, and total protein was extracted and quantified using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Next, the immunoreactive proteins were detected using the SimonTM machine (ProteinSimple, San Jose, USA). The primary and secondary antibodies used were the following: rabbit anti-CD63 antibody(Abcam, USA), anti-TSG101 antibody(Abcam), anti-Calnexin antibody(Abcam), anti-P-gp antibody(Abcam) and anti-β-actin antibody(Cell Signaling Technology, USA), anti-rabbit IgG(ProteinSimple). Quantization of detected proteins and image preparation were performed with Compass Software (ProteinSimple).
Exosomes were labeled with the green lipophilic fluorescent dye PKH67 (Sigma–Aldrich, St. Louis, MO) according to the manufacturer’s instructions. After coculture with PKH67-stained exosomes for 24 h, GECs were stained with DAPI (Abcam) for the nucleus and phalloidin (Abcam) for the intracellular cytoskeleton F-actin according to the instruction manual and observed under a confocal laser microscope.
GECs were trypsinized and washed twice in 1×PBS. After resuspension in 100 μl of 1×PBS, FITC-conjugated antibodies against P-gp (Abcam) and Rh-123 (Sigma) or their respective isotype controls were stained for 30 min at 4 °C. Following washing twice in 1×PBS, labeled cells were measured by flow cytometry (BD Biosciences) and analyzed with FlowJo software (Tree Star).
After exosomes intervention, GECs were washed twice in 1×PBS，and then incubated with 3.0×10-8M 3H-labeled dexamethasone for 20 minutes at 37 °C. The intracellular dexamethasone concentration was counted with a scintillation counter, according to previously study.
GECs exosomal miRNA sequencing
The total RNA was extracted using the Total RNA Purification Kit (LC Sciences, Houston, USA), according to the manufacturer’s protocol. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Then, the first strand cDNA was synthesized and PCR amplification was performed according to protocol of TruSeq Small RNA Sample Prep Kits(Illumina, San Diego, USA),and Samll RNAs were used for library preparation. Finally, we performed the single-end sequencing (1x50 bp) on an Illumina Hiseq2500 at the LC-BIO (Hangzhou, China) following the vendor’s recommended protocol.
To determine the role of SLCsL/S exosomes and PNS in the SR and inflammatory injury of GECs in vivo, 16-weeks-old female ICR and NZB/WF1 mice were injected via the tail vein with exosomes from lymphocytes. ICR mice were used as a control group: SLCsC-exo group (n=6, SLCsC-derived exosomes treatment). NZB/WF1 mice were randomly divided into four groups: SLCsL-exo group (n=6, SLCsL-derived exosomes treatment), SLCsL/S-exo group (n=6, SLCsL/S-derived exosomes treatment), SLCsL/S+PNS-exo group (n=6, SLCsL/S+PNS-derived exosomes treatment), and SLCsL/S+Tariq-exo group (n=6, SLCsL/S+Tariq-derived exosomes treatment). All the groups were injected five times every week for three consecutive weeks and then the serum, urine, and renal cortex samples were harvested for further analysis.
Enzyme-linked immunosorbent assay (ELISA)
GECs supernatant and serum samples were centrifuged at 3500×g for 10 min at 4 °C. The concentrations of IL-1β, IL-6, MCP-1, VCAM-1, ANA and anti-dsDNA were detected by using ELISA kits according to the manufacturer’s instructions.
Real-time PCR analysis
The total RNA of exosomes, GECs and tissues was isolated by using E.Z.N.A total RNA kit (Omega Biotech Inc., Norcross, GA, USA) according to the manufacturer’s protocol and converted to cDNA with the PrimeScriptTM RT reagent kit (Takara, Dalian, China). Quantitative real-time PCR was performed using SYBR-green mix (TaKaRa) and run on a 7500 Real-Time PCR System (Applied Biosystems). The relative expression levels of mRNA and miRNA were normalized against β-actin and U6, analyzed using the 2−ΔCt method. IL-1β forward primer: 5’-TCGCAGCAGCACATCAACAAGAG-3’; reverse primer: 5’-AGGTCCACGGGAAAGACACAGG-3’. IL-6 forward primer: 5’-CTTCTTGGGACTGATGCTGGTGAC-3’; reverse primer: 5’-AGGTCTGTTGGGAGTGGTATCCTC-3’. MCP-1 forward primer: 5’-CCACTCACCTGCTGCTACTCATTC-3’; reverse primer: 5’-CTTCTTTGGGACACCTGCTGCTG-3’. VCAM-1 forward primer: 5’-GAGGGTGGTGCTGTGACAATGAC-3’; reverse primer: 5’-GGGTGGCATTTCCTGAGAGAAGC-3’. MDR1 forward primer: 5’-TGATCGCTCACCGCCTGTCC-3’; reverse primer: 5’-GTGCCGTGCTCCTTGACCTTG-3’. β-actin forward primer: 5’-TATGCTCTCCCTCACGCCATCC-3’; reverse primer: 5’-GTCACGCACGATTTCCCTCTCAG-3’. miR-125b-5p forward primer: 5’-TGAGACCCTAACTTGTGAGTCGTATC-3’; reverse primer: 5’-CAGTGCAGGGTCCGAGGTAT-3’. miR-128-3p forward primer: 5’-GCGGTCTCTTTGTCGTATCCA-3’; reverse primer: 5’-CAGTGCAGGGTCCGAGGTA-3’. U6 forward primer: 5’-CTCGCTTCGGCAGCACA -3’; reverse primer: 5’-AACGCTTCACGAATTTGCGT-3’.
Hematoxylin and eosin (HE) staining and Immunohistochemical analysis (IHC)
To determine renal histopathology, 3-μm-thick paraffin sections of the kidney were stained with hematoxylin and eosin (HE). Renal histology was assessed blindly by an experienced pathologist. Renal histopathological changes were quantitated as described previously.
After deparaffinization and rehydration, paraffin sections were blocked with Dual Endogenous Enzyme Block (DAKO, Glostrup, Denmark) for IHC. The primary antibody was rabbit anti-P-gp (or IL-1β, IL-6, MCP-1, VCAM-1) IgG (Abcam). The secondary antibody was polymer-horseradish peroxidase-labeled goat anti-rabbit IgG (Abcam). Finally, sections were incubated with 3, 3′-diaminobenzine-chromogen substrate (DAKO) and counterstained with hematoxylin. The stains were scored by a renal pathologist blinded to the mice grouping.
Serum and urine analyses
The levels of 24 h urinary protein (24 h Upro), serum creatinine (Scr) and nitrogen concentration (BUN) were assessed using commercially available kits (Jian Cheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s directions.
The analysis process was performed by SPSS 22.0 software (SPSS Inc., Chicago, IL, USA), and measurement data consistent with a normal distribution are expressed as the mean ± standard deviation. Student's t-test and One-way ANOVA were used to compare differences between two or more groups. A value of P<0.05 indicated statistically significant differences.