Cell Culture and HG Treatment. The conditionally immortalized mouse podocyte cell line MPC5 was provided by BeNa Biotechnology (Beijing, China). The mouse podocytes were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 10 U/ml interferon-γ at 33°C for cell proliferation. To induce differentiation, the medium was replaced with fresh medium without interferon-γ and cells were cultivated at 37°C for 14 days. To induce the HG injury of podocytes, MPC5 cells were placed in medium containing 30 mM glucose and cultivated for 48 h. Cells maintained in medium containing normal glucose (NG, 5.5 mM) were utilized as a control.
Real-Time Quantitative PCR (RT-qPCR). Total RNAs in cultured podocytes were extracted and purified using an RNApure Tissue & Cell Kit (Cowin Biosciences, Beijing, China). Total RNAs were reversely transcribed into cDNA using a HiFi-Script cDNA Synthesis Kit (Cowin Biosciences). FastSYBR Mixture (Cowin Biosciences) was adopted to amplify cDNA by RT-qPCR with appropriate primers. The generated data by RT-qPCR was assessed via 2−ΔΔCt method, and relative gene expression was obtained using GAPDH for normalization.
Western Blotting. Cultured podocytes were collected and lysed into lysis buffer (Solarbio, Beijing, China) supplemented with phosphatase inhibitors, proteinase inhibitors and phenylmethanesulfonyl fluoride. After being lysed, the lysates were centrifuged and the supernatants were collected. Protein concentration in the supernatants was quantified via a BCA Protein Assay Kit (Tiangen Biotech, Beijing, China). The same amounts of proteins were placed into each lane of sodium dodecyl sulfate (SDS) polyacrylamide gels, followed by being separated via electrophoresis. Then, proteins were transferred to Polyvinylidene Fluoride membranes via an electro-transfer method using Bio-Rad Trans-Blot apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Polyvinylidene fluoride membranes were immersed into 5% skim milk for blocking prior to incubation with primary antibodies. The membranes were hybridized with matched secondary antibody, followed by incubation with enhanced chemiluminescent reagents (Solarbio) for the visualization of protein bands. The primary antibodies against TRIM32 (Abcam, Cambridge, UK), Akt (Proteintech Group, Wuhan, China), phospho-Akt (Ser473) (Proteintech Group), GSK-3β (Proteintech Group), phopho-GSK-3β (Ser9) (Proteintech Group), Nrf2 (Proteintech Group), GAPDH (Proteintech Group) and Lamin B1 (Proteintech Group).
Cell Transfection. The siRNAs targeting TRIM32 or Nrf2 were synthesized via Genepharma (Shanghai, China). The transfection of siRNAs into podocytes was implemented via using TransIntro EL Transfection Reagent (Transgen, Beijing, China) according to the protocol provided by the manufacturer. The down-regulation of target genes was confirmed by RT-qPCR or western blotting after 48 h transfection.
Cell Viability Assay. MPC5 podocytes were cultured in a 96-well plate and transfected with indicated siRNAs when they reached ~ 70% confluence. After transfection for 48 h, the medium was replaced with fresh medium harboring HG, and podocytes were cultivated for a further 48 h. Then, cell counting kit-8 (CCK-8) reagents (Solarbio, Beijing, China) were added to each well to determine the viability of podocytes. The absorbance of each well at 450 nm was measured via a microplate reader (BioTeke, Beijing, China).
Terminal Deoxynucleotidyl Transferase dUTP Nick end Labeling (TUNEL) Assay. TUNEL assay was carried out using the TransDetect In Situ Fluorescein TUNEL Cell Apoptosis Detection Kit (Transgen, Beijing, China) following the manufacturer’s protocol. In brief, podocytes were fixed by formaldehyde fixing solution at the time of detection. Then, 0.1% Triton X-100 solution was adopted to permeabilize the podocytes. Afterwards, podocytes were incubated with TdT reagent and Labeling Solution at 37°C for 1 h, protected from light. After being stained with DAPI, cells were visualized via a fluorescence microscope.
Annexin V-FITC/PI Apoptosis Assay. Annexin V‐FITC/PI apoptosis assay was performed via flow cytometry analysis using an Annexin V‐FITC/PI Apoptosis Kit (Solarbio, Beijing, China). Briefly, cultured podocytes were dissociated by trypsin digestion and washed with ice-cold phosphate buffer saline (PBS). Podocytes were collected and re-suspended into Binding Buffer, followed by adding Annexin V‐FITC/PI Solution. After being cultivated for 15 min at room temperature protected from light, cells were assessed via the FACScan flow cytometry system.
Detection of ROS Generation. The intracellular levels of ROS was evaluated via 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) which can be oxidized into the fluorescent DCFH. Generally, at the time of detection, the old medium was discarded and fresh medium supplemented with 10 µM DCFH-DA (Beyotime, Shanghai, China) was added to cells. After being cultivated for 30 min at 37°C, cells were washed with PBS and then analyzed via the FACScan flow cytometry system to quantify the fluorescence intensity.
Measurement of Malondialdehyde (MDA) and Superoxide Dismutase (SOD) Activity. The contents of MDA and SOD in podocytes were measured by Lipid Peroxidation MDA Assay Kit (Beyotime, Shanghai, China) and Total Superoxide Dismutase Assay Kit (Beyotime), respectively, according to the protocols of the manufacturer.
Enzyme-Linked Immuno-Sorbent Assay (ELISA). The levels of pro-inflammatory cytokine levels, including interleukin (IL)-6, the tumor necrosis factor-α (TNF-α), and IL-1β in the supernatants of cultured podocytes were quantified using ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocol.
Luciferase Activity Assays. Luciferase reporter vector pARE (Beyotime, Shanghai, China), which contains an ARE binding site, was adopted to measure the transcriptional activity of Nrf2. The luciferase reporter vector pNF-κB (Beyotime) was utilized to detect the transcriptional activity of NF-κB. TRIM32 siRNAs and corresponding luciferase reporter vectors were co-transfected into MPC5 podocytes and cultivated for 48 h prior to HG stimulation. Then, cells were collected and lysed to measure luciferase activity using a Luciferase Reporter Gene Assay Kit (Beyotime).
Statistical Analysis. Experimental results were expressed as mean ± standard deviation. Statistical analysis and graphing were implemented using GraphPad Prism 8. Student’s t test was adopted for the two-group comparison. When there were three or more groups, comparisons were performed using one-way analysis of variance (ANOVA). Differences were considered statistically significant when p < 0.05.