Aristolochic acid I (Sigma-Aldrich, St. Louis, MO) was dissolved as previously described and used for cell culture studies at a concentration of 10µM (Ren et al. 2020). β-actin, NOX-4, kidney injury molecule-1 (KIM-1) primary antibodies and goat anti-rabbit or anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) secondary antibodies were purchased from Bioss (Beijing, China). TSG101, CD63, ARF6, Cleaved caspased-3, LRG1 antibody was obtained from Abcam (Cambridge, UK). Human recombinant LRG1 were purchased from Abcam (Cambridge, UK). Cr and BUN assay kit was purchased from Njjcbio (Nanjing, China). Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Bestbio (Shanghai, China). Cytochalasin D was obtained from APExBIO (USA). PKH67 was obtained from Sigma (St. Louis, MO).
C57BL/6 mice supplied by the Experimental Animal Center of Anhui Medical University were used to establish the AAN model. All the animal experiments were performed in accordance with the Regulations of the Experimental Animal Administration issued by the State Committee of Science and Technology of China. Efforts were made to minimize the number of animals used and their suffering. Animals were maintained in accordance with the guides of the Center for Developmental Biology, Anhui Medical University for the Care and Use of Laboratory. Animals and all experiments used protocols approved by the institutions’ subcommittees on animal care. Mice were injected with AAI (5 mg/kg, i.p.; Sigma-Aldrich, St. Louis, MO) every other day for 12 days. Kidney tissues and blood samples were obtained at 3, 9, 15, 21 and 27 days (n = 6 mouse per group). Control mice were treated with the same dosage of saline via intraperitoneal injection.
4.3 Cell Lines and culture.
HK-2 and mTEC, kindly provided by Prof. Huiyao Lan, were cultured in DME/F-12 (HyClone, Logan, UT, USA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Merck Millipore, Darmstadt, Germany) at 37°C in a humidified incubator under 5% CO2. THP-1 cells were cultured in RPMI-1640 (HyClone, Logan, UT, USA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Merck Millipore, Darmstadt, Germany) at 37°C in a humidified incubator under 5% CO2. For all experiments, THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (5 ng/mL).
4.4 Primary cell isolation and culture.
Bone marrow-derived macrophages (BMDM) were isolated from wild-type mice as previously described (Thery et al. 2006). Briefly, bone marrow was flushed from dissected mouse tibia and femur with sterile PBS. Cells were resuspended and plated in RPMI-1640 (Gibco, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and M-CSF (10 ng/mL) conditioned media. BMDM were used for experiments after a differentiation period of 7 days. For real-time quantitative PCR analysis, 3 × 105 cells were plated per well into 12-well plates. The next day, BMDM were treated with EV in RPMI 1640 medium for 8h before RNA isolation.
Extracellular vesicle isolation. Renal primary epithelial cell and macrophage derived EV isolated from T2D mice were collected over a 24 h time period in EV-depleted complete media, which was prepared by overnight centrifugation at 100,000 × g at 4°C. Unless stated otherwise, EV was isolated from cell culture medium by differential ultracentrifugation using a modified version of a protocol by Thery et al (Thery, Amigorena 2006). Collected medium was depleted of cells and cell debris by consecutive, low-speed centrifugations (2,000 × g for 15 min and 16,000 × g for 20 min). The supernatants obtained were carefully collected and centrifuged for 90 min at 100,000 × g at 4°C. Pellets from this centrifugation step were washed in PBS, pooled, and centrifuged again for 90 min at 100,000 × g at 4°C. The obtained pellets were resuspended in lysis buffer (see below), PBS solution or RPMI-1640 medium, depending on subsequent experiments. EV solutions intended for cell treatment were sterile filtered through 0.22 µm syringe filter. Resuspended EV were either used for subsequent analysis or aliquoted and stored at -80°C. For isolation of EV derived from primary cells, a commercially available kit from Invitrogen (Carlsbad, CA) was utilized.
4.5 Electron microscopy.
Isolated EV were fixed in 2% paraformaldehyde in 0.1 M phosphate buffer overnight at 4°C. The samples were then placed on Formvar-carbon-coated grid and air dried for 20 min. After being rinsed with PBS, grids were transferred to 1% glutaraldehyde for 5 min and washed with distilled water. The grids were first contrasted with uranyl-oxalate solution and then contrasted and embedded in a mixture of 4% uranylacetate and 2% methylcellulose (1:9 ratio). The grids were air dried and visualized with a JEOL 1400 electron microscope (JEOL USA, Peabody, MA) at 80 kV. For immunogold staining, grids were block with 10% FBS for 20 min followed by overnight incubation at 4°C with primary anti-TRAIL and anti-LRG1 antibody diluted 1:20 in blocking solution overnight. Next, grids were incubated with secondary antibody for 1 h. In negative control samples, primary antibody was omitted. Samples were then labeled with protein A-10-nm gold for 1 h. The grids were contrasted and embedded with a mixture of 4% uranyl acetate and 2% methylcellulose (1:9 ratio) and observed as described above.
4.6 Nanoparticle tracking analysis.
Concentration and size distribution of isolated EV were assessed by nanoparticle tracking analysis (NTA) using NanoSight NS300 instrumentation (NanoSight, Amesbury, UK). EV samples were diluted with PBS at a range of concentrations between 4 × 108 and 8 × 108 particles per milliliter in a total volume of 1 milliliter. Each sample was continuously run through a flow-cell top-plate set up to 23.3°C using a syringe pump at a rate of 25 µL/min. At least three videos of 30 seconds documenting Brownian motion of nanoparticles were recorded and at least 1000 of completed tracks were analyzed by NanoSight software (NTA 2.3.5).
4.7 Cell EV uptake.
THP-1-derived purified EV were labeled with a green fluorescent dye PKH67 (Sigma) and washed by a 100,000 × g spin. Differentiated HK-2 cells were then incubated with these EV (1010/mL) for 1 h (with or without 10 µ Mcytochalasin D pre-Hirsova et al. treatment) and their cellular internalization was observed using an LSM880 confocal microscope (Carl Zeiss, Jena, Germany).
4.8 Flow Cytometry.
The extent of the programmed cell death was detected by flow cytometry (CytoFlex, Beckman Coulter, USA) using AV-FITC/PI apoptosis detection kit (Bestbio, Shanghai, China). To evaluate the apoptosis level of HK-2, the attached and supernatant cells were stained with 5 µl of Annexin V-FITC and10 µl PI in the dark, detected by flow cytometry, and analyzed using CytExpert 2.1 software (CytExpert, Beckman Coulter, USA).
4.9 Transient transfection.
A small interfering RNA (siRNA) was used to silence TGFβR1 in THP-1 cells. Cells transfected with scramble siRNA were used as control. Cells were grown in 60-mm dishes and transiently transfected with Smart Pool siRNA (5 nM, Hanbio) using LipoFiter 3.0 (Hanbio). Experiments were performed 48 h after transfection.
4.10 Immunofluorescence Staining.
Sections were blocked with 10% bovine serum albumin (BSA) solution to avoid non-specific staining. Sections were incubated with rabbit polyclonal primary antibodies against KIM-1 (1:500). Sections were incubated overnight at 4◦C, followed by incubation with anti-rabbit Cy-3 (1:200)-conjugated secondary antibodies and nuclei staining with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China). Stained sections were examined with an inverted fluorescence microscope (Carl Zeiss Axio Vert.A1, Jena, Germany).
Renal tissues of mice were fixed in 4% paraformaldehyde for 24 h immediately following killing, processed for histological examination according to a conventional method, and stained with PAS, H&E and F4/80. The slides were scored in a blinded manner and de-identified.
4.12 TUNEL Assay.
Renal cell apoptosis was examined by TUNEL assay using the One step TUNEL Apoptosis Assay Kit from Beyotime Biotechnology (Beyotime, Jiangsu, China). Briefly, cells were fixed with 4% paraformaldehyde in PBS and then exposed to the TUNEL reaction mixture containing TM red–labeled dUTP. Finally, samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). TUNEL-positive nuclei were identified by fluorescence microscopy.
4.13 Western Blot.
Whole extracts were separated by 10 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidenedifluoride membrane, which were incubated with primary antibodies against LRG1/ NOX-4/ KIM-1/ TSG101/ ARF6/ CD63/ cleaved-caspased-3/ β-actin (1:500; Santa Cruz biotechnology, Dallas, TX, USA). The membranes were then washed in TBS-Tween 20 and incubated with secondary antibodies correspondingly. After extensive washing in TBS-Tween 20, protein bands were visualized with ECL chemiluminescent kit (ECL-plus; Thermo Fisher Scientific, Pittsburgh, PA, USA).
4.14 Real-Time Reverse Transcriptase-PCR.
Total RNA was collected from kidney tissues, THP-1 and BMDM cells using TRIzol reagents (Invitrogen). First-strand cDNA was synthesized using Thermoscript RT-PCR synthesis kit (Fermentas, Pittsburgh, PA, USA) according to the manufacturer’s instructions. Real-time quantitative PCR analyses for mRNA were performed using Thermoscript RT-qPCR kits in an ABI Prizm step-one plus real-time PCR System (Applied Biosystems, Foster City, CA, USA). The products were used as templates for amplification using the SYBR Green PCR amplification reagent (Qiagen, Valencia, CA, USA) and gene-specific primers. Relative expression levels were calculated according to the standard 2−ΔΔCt method (Schmittgen and Livak 2008). The forward and reverse primers used for PCR were as listed in Supporting Information Tab S1.
4.15 Statistical Analysis.
Data are expressed as the means ± SEM and represent at least three independent experiments. Differences between two groups were compared using the two-tailed Student’s t-test. Differences between multiple groups were compared using one-way analysis of variance followed by Student’s t-test. Differences were considered significant at P < 0.05. All analyses were performed using GraphPad Prism 5.0 software (San Diego, CA).