Chemicals and reagents
PI3K inhibitor LY294002, P38 MAPK inhibitor SB203580, N-acetylcysteine (NAC), and d-glucose were obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from GIBCO Invitrogen (Carlsbad, CA, USA). DMEM-F12 was purchased from Corning (Steuben County, NY, USA). The Nrf2 activator TBHQ and Caspase-1 inhibitor VX-765 were obtained from MedChem Express (New Jersey, USA). Reverse transcription system and qPCR Master Mix were purchased from Vazyme (New Jersey, USA). Antibodies against phospho-AKT (473), phospho-AKT (308), Phospho-Smad3 and Phospho-P38MAPK (p-P38MAPK) were obtained from Cell Signaling Technology (Danvers, MA, USA). Nrf2, HO-1, TGF-β1, Caspase-1, Caspase-1-p10, FN, α-SMA and 𝛽-actin were purchased from Proteintech (Chicago, USA). GSDMD and GSDMD-N were obtained from Abcam (Cambridge, UK).
Cell culture and transfection
The human proximal tubular cell line (HK−2) was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in DMEM-F12 (3:1) medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in 95% air and 5% CO2 atmosphere at 37°C. The cells were stimulated with a normal concentration of D-glucose medium (NG, 5.5 mmol/L) and high D-glucose medium (HG, 30 mmol/L). The Nrf2 activator TBHQ, Caspase-1 inhibitor VX-765, PI3K inhibitor LY294002, P38 MAPK inhibitor SB203580, and N-acetylcysteine (NAC) were preincubated for 2 h.
siRNA-Nrf2 oligo and siRNA negative control (NC) were purchased from GenePharma (Shanghai, China). Cells were seeded in 6-well plates and transfected with 20 𝜇M siRNA when they were 60-70%. All transfections were performed using LipofectamineTM 3000 reagent (Invitrogen, USA) according to the manufacturer’s protocols. Cells were harvested at 48 h transfection to evaluate the silencing efficiency by Western blot.
Western blot
Total protein from cultured HK-2 cells was treated with RIPA lysis buffer (Best-Bio, Shanghai, China), separated by SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Massachusetts, USA). The bands were blocked with Tris-buffered saline Tween-20 (TBST) containing 5% nonfat milk for 2 h at room temperature before incubation with primary antibodies at 4°C overnight. The following day, the membranes were washed with TBST and incubated with HRP-peroxidase-conjugated secondary antibodies (Solarbio, Beijing, China). Images were obtained using the Odyssey Fc system (LI-COR, USA).
RNA extraction and quantitative real-time PCR
Total RNA from HK-2 cells was extracted using TRIzol reagent (Thermo Fisher, Carlsbad, CA, USA) following the manufacturer’s instructions. Next, cDNA was synthesized using the SuperScript Reverse Transcription Kit (Thermo Fisher, Carlsbad, USA). The mRNA expression levels were measured by real-time PCR using SYBR Green Master Mix (Vazyme, Nanjing, China). 𝛽-actin was used as an internal control for Nrf2, GSDMD, Caspase-1, α-SMA, and FN. The primers were listed as follows:
Nrf2 (forward: 5′-TCAGCG ACGGAAAGAGTATGA-3′ and reverse: 5′-CCACTGGTTTCTGACTGGATGT-3′), GSDMD (forward: 5′-CTAGCTAAGGCTCTGGAGACAA-3′ and reverse: 5′-GATTCTTTTCATCCCAGCAGTC-3′), Caspase-1 (forward: 5′-AGAGGATTTCTTAACGGATGCA-3′ and reverse: 5′-TCACAAGACCAGGCATATTCTT-3′), α-SMA (forward: 5′-TGCACAAACGAGGGGAATATAT-3′ and reverse: 5′-CGTTCCTTGTAGCCAATAAAGG-3′), FN (forward: 5′-TGGACCAAATCTCCACGGTCT-3′ and reverse: 5′-CTCTCCCCAGGTGATGCCTT-3′), and β-actin (forward: 5′-TGACGTGGACATCCGCAAAG-3′ and reverse: 5′-CTGGAAGGTGGACAGCGAGG-3′). The mRNA expression was analyzed using the classic 2-ΔΔCt method.
Immunofluorescence staining
HK-2 cells were seeded onto a six well plate with slides. After treatment, the cells were fixed with 4% paraformaldehyde and then washed with PBS. Next, 0.3% Triton X-100 (prepared with PBS) was added, and the solution was drilled for 10 min. Samples were blocked with goat serum for 30 min at 37°C. The cells were then incubated with anti-GSDMD (1:100), anti-α-SMA (1:100), and anti-Caspase-1 antibody (1:100) antibodies at 4°C overnight. The next day, the slices were removed for PBS cleaning and incubated with FITC-conjugated secondary antibodies (1:200) for 2 h at 37°C. Subsequently, the nuclei were stained with DAPI. Images were obtained and observed using a confocal microscope (Leica, Germany).
Detection of intracellular and mitochondrial ROS analysis
The generation of intracellular and mitochondrial ROS was evaluated using a ROS assay kit (DCFH-DA, 10 μM, Beyotime, Shanghai, China) and Mito Sox Red (5 μM, Sigma, St. Louis, USA). After peptide treatment, the cells were incubated for 30 min at 37°C according to the manufacturer’s instructions, and images were acquired using a confocal microscope (Leica, Germany).
Statistical analyses
All data were analyzed by one-way analysis of variance or Student’s t-test using SPSS 26.0, and are shown as the mean ± SD. Statistical significance was set at P < 0.05.