Immortalized differentiated murine podocytes (MPC5) were obtained from Dr. Peter Mundel (Mount Sinai School of Medicine, New York, USA) and maintained in RPMI-1640 (Gibco BRL, Grand Island, New Jersey , USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL) and 100 U/ml, interferon (IFN)-γ (PeproTech, Inc., Rocky Hill, New  Jersey , USA) at 33°C with 5% CO2 for proliferation. To induce differentiation, podocytes were cultivated without IFN-γ at 37 °C for 10 days. For subsequent experiments, differentiated podocytes were treated with 50 μM puromycin aminonucleoside (PAN) (MedChemExpress New Jersey ,USA) with or without AS-IV （MedChemExpress New Jersey ,USA）for 24 h.
Cell viability assay.
Cell viability was measured using a CCK-8 assay (Beyotime ShangHai，China).The podocytes were seeded in 96-well plate cultured for 24h, then incubated with different concentrations of AS-IV with or without PAN at 37˚C, 5% CO2 for 24 h. Then 10 µl of CCK-8 was added to each well, and the cells were incubated at 37˚C for 1 -1.5h The absorbance was detected at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Small Interfering RNA Transfection
non-targeting siRNA Pool (Control-si) or Mfn2-specific siRNA pool were ordered from GenePharma (Shanghai, China). Mfn2-siRNA and control-siRNA were transfected into MPC5 using Opti-MEMI Reduced Serum Medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Lipofectamine 3000 transfection kit ( Invitrogen New York, USA). The expression of Mfn2 was examined by western-blot.
Lentiviral carrying Mfn2 or empty vector (GenePharma ,Shanghai, China) were infected MPC5, then incubated for 12 h. Cells were then switched to normal culture medium. The expression of Mfn2 was examined by western blotting.
Western blot assay
Cells and kidney tissues were lysed with cell lysis buffer ( Beyotime ，Shanghai, China) on ice for 30 minutes and then centrifuged at 12000 × g for 10 minutes at 4 °C. The protein concentration was determined by BCA Protein Assay Reagent Kit (Beyotime, Shanghai, China). Protein was loaded in each lane of SDS-polyacrylamide electrophoresis gel. Then transferred to polyvinylidene difluoride membrane, which was then blocked with 5% bovine serum albumin for 1 h at room temperature and further incubated with a specific primary antibody and horseradish peroxidase-conjugated second antibodies. Results were Quantified with ImageJ (National Institutes of Health, Bethesda, MD, USA)
Anti-pink1, anti-parkin, anti-Mfn2, anti-LC3,anti-podocin and anti-gapdh were purchased from Proteintech ( No.23274-1-AP, No.14060-1-AP, No.12186-1-AP, No.18725-1-AP,No.10494-1-AP,No.20384-1-AP),Anti-Synaptopodin,anti-nephrin were ordered from Abcam (No.ab224491,No.58968.).Anti-P62 was order from Abclonal( No.A7758).
Measurement of Oxidative Stress
Hydrogen peroxide, indicative of ROS generation, were measured with DCFH-DA (Beyotime, Shanghai, China) After the treatment, podocytes were incubated with 20 μM DCFH-DA at 37°C in the dark for 30 min. ROS generation was detected by flow cytometry and observed by the fluorescence microscope (ZEISS, Germany).
Measurement of Mitochondrial Membrane Potential
Mitochondrial membrane potential (MMP) was determined using the lipophilic cationic dye JC-1 (Beyotime, Shanghai, China). Podocytes were loaded with JC-1 in the dark for 20 min and fluorescence was measured by flow cytometry and observed by the fluorescence microscope (ZEISS, Germany). The ratio of red to green (590/520) fluorescence reflects the MMP.
Apoptosis was assessed by flow cytometry after cell staining with Annexin V-FITC Apoptosis Detection Kit I (Beyotime, Shanghai, China). Briefly, Cells were washed with PBS and following which 1x106cells were suspended in 195 µl of binding buffer. the solution then 5 µl of Annexin V and 10 µl of propidium iodide (PI) were added; the mixture was gently vortexed and incubated for 20 min at room temperature in the dark. Subsequent flow cytometric analysis was performed within 1 h.
Establishment of the experimental model
BALB/c mice male 10-12w [Grade SPF II, Certificate Number SCXK (Jing) 2016–0006] weighing 18–20 g were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai China). All mice were housed with drinking water and standard chow ad libitum, and were randomly divided into control group (n = 8), Adriamycin( ADR) group (n = 8 ), and ADR with AS-IV treatment group (n =8). FSGS was induced by giving a single dose of ADR (10.4mg/kg, MedChemExpress, USA) intravenous injection via tail vein, and normal saline was administered in control group. One week after adriamycin injection, mice in AS-IV treatment group started to received AS-IV (25 mg/kg, MedChemExpress, USA) by gavage daily for 8 weeks, and the other two groups of mice also received same volume of saline by gavage. By the end of the nine-week protocol, mice were euthanatized, and the renal tissues were fixed for further research. All animal procedures were performed according to the National Institutes of Health guidelines (Guide for the Care and Use of the Laboratory Animals). The animal protocols were reviewed and approved by the ethics committee of the Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University.
Mice were anaesthetized and blood samples were collected from the orbital sinus. Blood was centrifuged and serum was isolated. Serum creatinine (Scr) and blood urea nitrogen (BUN) were analyzed using FUJI DRI-CHEM CRE and BUN slides in a FUJI DRI-CHEM 7000i biochemistry analyser (Fujifilm, Tokyo, Japan).
Quantitative collection of overall urine was performed using metabolic cages for 12h. Albumin and creatinine concentrations were measured using ELISA (Abcam)
Kidney tissues were fixed with 4% paraformaldehyde overnight and dehydrated with 30% sucrose for 24 hours. Finally, fixed tissues were embedded in OCT compound and cut into 5-μm sections at -20℃ for further staining. Cells were grown on glass covers slips, then fixed with 4% paraformaldehyde for further process. The tissue sections and cells were blocked with 5% bovine serum albumin for 1 h at room temperature and further incubated with a specific primary antibody and FITC and Cy3–labeled secondary antibody. Nuclei was stained with DAPI for 5 min. Images were then acquired by laser scanning confocal microscopy (Leica Microsystems)
For mitochondria staining in cultured podocytes, cells were grown on glass coverslips and mitochondrial morphology was visualized by labeling the cells. with 50 nmol/L MitoTracker Red CMXRos (Thermo Fisher Scientific, USA) in the dark for 30 min at 37°C.
Kidneys and cells were perfused and fixed with glutaraldehyde and then cut into 1mm3 tissue blocks for epoxy embedding. Transmission electron microscopy was performed by standard procedures.
GraphPad Prism and SPSS 19.0 software were used for the statistical analysis. Data were presented as mean values ± standard deviation (Mean ± SD) for independent experiments. For two groups, t-test was performed. Comparisons among more than two groups were assessed by one-way analysis of variance (ANOVA) followed. P<0.05 was considered statistically significant.