Mouse Experiments
All animal experiments were approved by the Animal Ethics Committee of China Medical University (CMU2019277). Wild type (WT) and NR4A1-targeted mutant (No: 006187; NR4A1-/-) mice with a C57BL/6J background were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). NR4A1-targeted mutant mice were produced with a neomycin cassette introduced to exon 2 of the mouse Nur77 to block the transcription of both the DNA binding domain (DBD) and ligand-binding (LBD) domain38. All mice were housed in a temperature and climate-controlled barrier system (23 ± 2°C and 45–60% relative humidity, 12 h cycle of light and darkness), and fed regular rodent chow. Naturally aging mice were divided into 4 groups for analysis (5-, 8-, 15-, 18-month-old). Young serum (YS) and old serum (OS) were extracted from 5- and 18-month-old mice, respectively. The number of surviving mice for WT (n=10) and NR4A1-/- (n=10) was recorded every month for 24 months. Then the mice were divided into four groups for analysis: 5-month-old mice (WT-5 MO; n=6) and (NR4A1-/--15 MO; n=6) groups; 15-month-old mice (WT-15 MO; n=6) and (NR4A1-/--15 MO; n=6) groups. Urine samples were collected by placing each mouse in an individual metabolic cage (Tecniplast, Gazzada, Italy) for 24 h. Oral glucose tolerance test (OGTT) was performed at 0, 30, 60, 90, 120 min using an Accu-Chek meter (Roche; MO, USA). Fasting blood glucose (FBG), total cholesterol (CHOL), triglyceride (TAG), creatine (Cr) and urea nitrogen (BUN) were determined as per the test kit manufacturer’s instructions (Jiancheng, Nanjing, China).
Histological Staining for Tissues and Analysis
For H&E, Sirius red, Masson’s trichrome and Periodic acid–Schiff (PAS) staining, tissues were isolated and fixed overnight at 4°C, and treated with 30% sucrose for cryoprotection. Sections of kidney were dewaxed with xylene and a series of descending ethanol gradients. Then sections were stained with Mayer’s H&E (Jiancheng) or Sirius Red F3B and a saturated aqueous solution of picric acid (Solarbio, beijing, China), Masson (Jiancheng) or PAS kit (Solarbio), and observed with a Nikon Eclipse Ni-U microscope. For immunohistochemistry (IHC), paraffin sections were stained with anti-Nephrin (Abcam, Cambridge, UK), assessed by light microscopy and quantified using ImageJ (Wayne Rasband, US National Institutes of Health, Bethesda, MD, USA). For β-GAL, TUNEL and immunofluorescence staining, frozen tissue slices were stained with a β-GAL kit (Beyotime, Biotechnology, Shanghai, China), TUNEL kit (Beyotime), or anti-53BP1 (NOVUS, Abingdon, UK), respectively, photographed using an Olympus IX-71 fluorescence microscope and quantified using ImageJ. For DHE staining, 24 h before killing, mice received a 200 μL intravenous injection of dihydroethidium kit (Sigma-Aldrich, St Louis, MI, USA) at 25 mg/kg, and frozen tissue slices were photographed using an Olympus IX-71 fluorescence microscope and quantified using ImageJ.
Transmission Electron Microscopy (TEM)
For electron microscopy, sample handling and detection were performed by Wuhan servicebio technology. Tissues were collected and fixed with 2.5% glutaraldehyde at 4°C. Sections were washed with PBS and fixed in 1% osmium tetroxide at room temperature for 2 h. Specimens were then dehydrated using a series of ascending ethanol gradients and 100% acetone in series. After dehydration, the sections were embedded in Pon 812 resin overnight at 37°C using acetone as a transitional solvent. The ultra-thin sections were stained with 2% saturated uranyl acetate and lead citrate. The glomerular Basement Membrane (GBM) thickness, foot process width and the number of foot processes per μm of GBM, TEM images were analyzed using ImageJ.
Glutathione Peroxidase (GSH-PX) and Superoxide dismutase (SOD) detection
Tissues were washed with PBS and homogenized with cold Tris–HCl buffer (pH 7.4) to obtain 10% homogenate. GSH-PX and SOD in serum and kidney tissues were measured kinetically according to the method of the corresponding enzymatic assays (Jiancheng), and quantified using an automatic analyzer.
RNA-Sequencing Analysis
RNA sequencing data were downloaded and referenced to the Gene Expression Omnibus under accession number (GSE54714, GSE6591, GSE8150, GSE25905). A log fold change (log2FC) > 1.5 and an adjusted p-value < 0.05 were set as the thresholds for the identification of differentially expressed genes. Bioinformatic analyses using the Metascape pathway analysis39 and Ingenuity Pathways Analysis40 were carried out to determine molecular functions and upstream signaling pathways.
Cell Culture and Treatments
Mouse embryonic fibroblasts (MEFs) were derived from E14.5 embryos from NR4A1+/- C57BL/6J mice, and cultured in DMEM medium with 15% FBS (Gibco, Carlsbad, CA, USA), 100 units/mL penicillin, and 10 mg/mL streptomycin. Then they were treated with serum from aged mice (OS) or young mice (YS) as a normal control for 24 h. Human embryonic kidney (HEK) 293 cell lines were obtained from the American Type Culture Collection and cultured in DMEM medium with 10% FBS. H1299 cells are a p53-deficient cell line obtained from the American Type Culture Collection and cultured in 1640 medium with 10% FBS. For DNA damage-triggered senescence, 293 cells were treated with 500 μM H2O2 for 4 h. Transfection was performed using Lipo3000 (Thermo Fisher Scientific, Waltham, MA, USA).
Plasmids
Nur77 overexpression and shRNA lentiviruses were purchased from GeneChem (Shanghai, China). The HA-tagged SIRT1 WT or deacetylase-inactive mutant (363HY) plasmids were constructed and described previously41. CBP, p300, GCN5 and PCAF plasmids were constructed and described previously42. SIRT1, MDM2 and CBP siRNA were purchased from RiboBio. GFP-MDM2, Ub and Site-directed mutagenesis (WT, S20A, S20E) of Flag-p53 plasmids was performed using QuikChange XL (Stratagene) and confirmed by sequencing. SIRT1-/- and Chk2-/- Cas9 HEK-293 cells were constructed and provided by Wenyu Zhang43.
Co-immunoprecipitation (Co-IP) and western blot analysis
Co-IP and western blot analysis were performed as previously described44. The different primary antibodies used are indicated in Supplementary Table 1.
Real-Time PCR
RNA was isolated from podocytes using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Copy DNA was prepared using a PrimeScript™ RT reagent Kit (TaKaRa, Kusatsu, Japan) followed by quantitative Real-Time PCR using SYBR Green (TaKaRa). Relative quantitation was carried out using 2−ΔΔCT. RT-PCR primers are listed in Supplementary Table 2.
Protein degradation assays
For the determination of the half-life of proteins, cells were pretreated with 100 μM H2O2 for 6 h, then incubated with 100 μg/mL cycloheximide (CHX; APExBIO, Houston, TX, USA) for 0, 3, 6 and 9 h (SIRT1) or 0, 30 ,60 and 90 min (p53), followed by sample harvesting and western blot analysis. To determine whether SIRT1 expression is affected by proteasome-dependent proteolysis, cells were treated with 50 μM MG132 (Selleck Chemicals, Houston, TX, USA) for 4 h, with/without 20 μM chloroquine (CQ; Sigma-Aldrich) for 10 h. For ubiquitination assays, cells were co-transfected with HA-SIRT1, Ubiquitin (Ub) and GFP-MDM2 plasmids. After 30 h post-transfection, cells were harvested followed by co-immunoprecipitation (Co-IP) analysis.
Flow Cytometry
Apoptosis assessments were made based on allophycocyanin (APC) Annexin V and propidium iodide (PI) staining (Thermo Fisher). Cells were harvested followed by staining with APC Annexin V and PI as per the manufacturer’s instructions. ROS was determined by a fluorometric intracellular ROS Kit (Sigma-Aldrich) according to the manufacturer’s instructions. After stimulation, cells were then incubated in Hank's balanced salt solution containing 5 mM 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) for 1 h, and analyzed by flow cytometry using FACS caliber (Becton Dickinson, Franklin Lakes, NJ, USA).
Cell Counting Kit-8 (CCK8)
After transfection and stimulation, cell viability was counted using the CCK8 kit. The fluorescence intensity in each well was then measured at 450 nm.
Statistical analyses
Data (n > 6) are expressed as mean ± standard error (SE). Data (n ≤ 6) are expressed as mean ± standard deviation (SD). Unpaired two-tailed Student’s t-test and Mann-Whitney test were used for comparison between two groups. One-way ANOVA coupled with Tukey’s multiple comparison test or two-way ANOVA coupled with Sidak’s multiple comparisons tests were used for comparisons over two groups. p < 0.05 was considered significant. The number of repetitions for each experiment is indicated in the figure legends.
Data availability
RNA sequencing data were referenced to the Gene Expression Omnibus under accession number (GSE54714, GSE6591, GSE8150, GSE25905). All other data supporting the findings of this study are available from the corresponding author upon reasonable request.