Cell culture and treatment

Human renal tubular epithelial cell (HK-2) was provided by Shanghai Mingjin Biotechnology Co., Ltd (Shanghai, China). The cells were maintained in the endothelial culture medium supplemented with 5.6 mmol/L glucose and 10% fetal bovine serum (FBS; Gibco). In order to induce the DKD model, HK-2 cells were treated with 30 mmol/L d-glucose for 48 h, named high glucose (HG) group. In addition, HK-2 cells were treated with 5.6 mmol/L d-glucose in the normal glucose (NG) group, while cells cultured in 25 mM mannitol were used as the osmotic control (M group).

Cell transfection

FTO over-expressing vector (oe-FTO), NLRP3 over-expressing vector (oe-NLRP3) and empty vector (oe-NC) were provided by GenePharma (Shanghai, China). In brief, pUC57-FTO and pLVX-mCMV-ZsGreen-IRES-Puro were digested by EcoRI and SpeI, and the products were connected by T4 DNA Ligase. Then, an Agarose Gel Extraction Kit was used to retrieve fragments. JM109, which was transfected with the ligation product, was inoculated and amplificated, and the sequencing primer CMV-F was used to verify the bacterial fluid. rLV-FTO/NLRP3 was obtained in 293T cells co-transfected with recombinant plasmid or control plasmid using Lipofectamine 3000 (Invitrogen, USA). HK-2 cells were transfected with rLV-FTO/NLRP3 on the basis of MOI = 20. Two days after transfection, lentivirus-infected HK-2 cells were cultured normally.

Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR)

The total RNA was extracted with Trizol Kits (Beyotime, Shanghai, China), and the total RNA was reverse transcribed into cDNA according to the instructions of the PrimeScript RT reagent kit (Takara, Japan). The synthetic cDNA was used as the templates for the RT-qPCR reaction with a Fast SYBRGREEN PCR kit (Takara), and in a ABIPRISM 7300 RT-PCR system (Applied Biosystems). The amplification program was set as follows: initial denaturation, 95℃ for 15 min; denaturation, 95℃ for 15 s, 45 cycles; annealing, 55℃ for 15 s; extension, 72℃ for 30 s. In this experiment, β-actin was used as the internal control of genes. Relative quantification method (2−ΔΔCt method) was used to calculate the relative levels of related genes. The primer sequences were as follows: FTO, 5ʹ-GCTGCTTATTTCGGGACCTG-3ʹ and 5ʹ-AGCCTGGATTACCAATGAGGA-3ʹ; NLRP3, 5ʹ-CCACAAGATCGTGAGAAAACCC-3ʹ and 5ʹ-CGGTCCTATGTGCTCGTCA-3ʹ; CASP1, 5ʹ-TTTCCGCAAGGTTCGATTTTCA-3ʹ and 5ʹ-GGCATCTGCGCTCTACCATC-3ʹ; ASC, 5ʹ-CCCAAGCAAGTCAAGCGACA-3ʹ and 5ʹ-AAGCCGCTGAAGTTGAGCC-3ʹ; GSDMD, 5ʹ-GGACAGGCAAAGATCGCAG-3ʹ and 5ʹ-CACTCAGCGAGTACACATTCATT-3ʹ; and GAPDH, 5ʹ-GTGTTCCTACCCCCAATGTGT-3ʹ and 5ʹ-ATTGTCATACCAGGAAATGAGCTT-3ʹ.

RNA stability detection

For the determination of mRNA stability of NLRP3, the cells were treated with Actinomycin D for 2, 4, 6, 8 h. After the treatment of different times, the mRNA levels of NLRP3 were detected using RT-qPCR as mentioned above.

Cell counting kit (CCK)-8 assay

CCK-8 detection kit (Shanghai Liji Biotechnology Co., Ltd., Shanghai, China) was used to detect the cell viability. 100 μL of cell suspension was seeded in a 96-well plate at a density of 5 × 103/well. On the 24th, 48th, and 72th h after seeding, 10 μL of CCK-8 solution was added to each well, and mixed gently without bubbles. After incubated in a 5% CO2 incubator at 37 ℃ for 4 h, the absorbance of each well at 450 nm was measured with a microplate reader.

Annexin V and propidium iodide (PI) double staining

Pyroptosis of HK-2 cells was detected by using an Annexin V-FITC Detection Kit (Beyotime) according to the manufacturer’s instructions. Briefly, HK-2 cells at the concentration of 2 × 105 were centrifuged at 300 × g for 5 min, and the supernatant was discarded. The cells were re-suspended with 500 μL diluted 1 × Annexin V Binding Buffer. The cell suspension was added with 5 μL Annexin V-FITC staining solution and 5 μL PI staining solution (50 μg/mL). Cells ere incubated at room temperature for 15 ∼ 20 min away from light, and then analyzed by flow cytometry (Becton Dickinson, Germany). HK-2 cells in Q1 (Annexinv-/PI+) are necrotic cells [17], and were deemed as positive pyroptosis cells.

Western blot

The cultured cells were collected and the supernatant was discarded. Then the cells were lysed with enhanced RIPA lysate (Beyotime), and the protein concentration was determined with BCA protein quantitative Kit (Beyotime). Proteins (20 μg) were separated with 10% SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked in 5% skimmed milk at room temperature for 1 h. Then the blots were treated with primary antibodies against (NLRP3, 27458-1-AP, Proteintech, China; ASC, ab307560, Abcam, USA; caspase-1, ab286125, Abcam, USA; GSDMD-N, EPR20829-408, Abcam, USA; IL-6, ab290735, Abcam, USA; caspase-3, ab32351, Abcam, USA) at 4℃ overnight. Next day, after washing, the membranes were treated with HRP labeled secondary antibody for 1 h. Thereafter, the membranes were visualized with ECL solution (Biomiga, USA) for 1 min at room temperature. ImageJ software was used to quantify the gray scale of each band. β-actin was used as the internal control.

m6A dot blot assay

Total RNA was extracted from the blood, cells and tissues by Trizol (Beyotime). The m6A content relative to the total mRNA level was measured by EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric) (AmyJet Scientific, Wuhan, China) according to the instructions. Briefly, 4 μg of total RNA was and purified using the RNeasy Mini Kit (Qiagen). Then the RNA was separated on 1.2% formaldehyde-agarose gels and transferred onto Hybond N + membranes (Amersham). Then the membranes were incubated with m6A antibody overnight and and then treated with horseradish peroxidase conjugate anti-rabbit immunoglobulin G. Finally, after washing, the blots were developed on PhosphorImager screens and quantited with ImageQuant.

Me-RIP qPCR experiment

Magna RIPTM RNA binding protein immunoprediction kit was purchased from Millipore (MA, USA) and the experiment was carried out according to the instructions. After the cells were treated with Rip lysate, anti-m6A antibody was used to incubate the cell lysate. Then the mixture was incubated with magnetic beads. After that, protease K was used to remove proteins. Finally, the RNA was purified and reverse transcribed into cDNA, and the expression of target genes was detected by RT-qPCR.

RNA-binding protein immunoprecipitation (RIP) experiment

The Rip Kit (Millipore, USA) was used to detect the binding of FTO protein with NLRP3 RNA. HK-2 cells were washed twice with cold PBS and collected. The cells were then resuspended with RIPA lysate (Beyotime) and centrifuged (14 000 r/min, 4 ℃) for 10 min to collect the supernatant. The experimental procedures were as follows: 50 μL of magnetic beads were washed and then resuspended in 100 μL of RIP Wash Buffer, and then incubated with 5 μg of anti-FTO or IgG. The magnetic bead-antibody complexes were washed and resuspended in 900 μL RIP Wash Buffer, and then incubated with 100 μL cell extract at 4℃ overnight. The sample was then placed on a magnetic holder to collect the magnetic bead-protein complexes. The complex pulled down were digested with proteinase K to extract RNA, and the expression level of NLRP3 was detected by RT-qPCR as mentioned above.

Double luciferase reporter assay

The wild-type and mutant fragments of NLRP3 were constructed and inserted into the pmirGLO reporter vector using endonuclease sites Spe I and Hind III, named as NLRP3-WT and NLRP3-MUT, respectively. These reporter plasmids along with oe-nc or oe-FTO were co-transfected into HK-2 cells using the LipofectamineTM3000 reagent. After 48 h, the cells were collected and fully lysed. The firefly luciferase activity was finally measured and normalized to the renin luciferase activity.

Animal experiment

The experimental procedures followed the recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by Laboratory Animal Ethics Committee of the Beijing Medconor Biotechnology Co., LTD. (MDKN-2022-70). Five weeks old male C57bl/6 mice (20 ± 2 g) were divided into DKD model group and control group. The fasting blood glucose of all mice was measured before the experiment, which was required to be less than 7 mmol/L. The mice in DKD model group were fed with high-fat and high-glucose diet for 8 weeks. Then, 1 day after restoring the high-fat and high-glucose diet, the mice in the DKD model group were fasted for 16 h and injected with streptozotocin (STZ) at 50 mg/kg per day for 5 days. The fasting blood glucose was measured 2 weeks after the last injection. If the fasting blood glucose was greater than 16 mmol/L, the medium-term diabetes model was established successfully. The mice in the control group were received standard chow and injected with the same amount of normal saline. Subsequently, all DKD mice were randomly divided into lv-NC group and lv-FTO group. After DKD model establishment, the lentiviruses carrying empty vector (NC) or FTO over-expressing vector (FTO) (MOI = 50) were injected into caudal vein at the dose of 1 μg/g according to the weight of mice, once a week for 8 weeks.

H&E staining

Mice were sacrificed with 160 mg/kg pentobarbital sodium injection to cause respiratory arrest. The kidneys of the mice were collected and fixed in 4% paraformaldehyde. After the fixation, dehydration and paraffin embedding, the paraffin sections were cut into sections with a thickness of 2 μm. H&E staining kit were purchased from Shanghai GEFAN Biotechnolog Co., Ltd. (Shanghai, China). The sections were stained according to the instructions of the kits. The pathological changes of renal glomerulus, basement membrane and mesangial matrix were observed under the microscope. The renal tubule injury was evaluated as described previously [18].

Detection of renal tubule injury

The IL-1β, IL-18 and LDH levels in the kidneys of the mice and the HK-2 cells were tested by corresponding ELISA kits purchased from Jiancheng Bio (Nanjing, China). All operations shall be carried out according to the operation instructions of the kits. Urine samples were collected from the metabolic cage 2 days before sacrifice, and the urinary albumin level and urinary albumin excretion rate (mg/24 h) were measured according to the instructions of enzyme-linked immunosorbent assay (ELISA) kit (Ethos Biosciences). Femoral vein blood was collected with heparin anti-coagulation capillary tubes for measurement of blood urea nitrogen, serum creatinine and glomerular filtration rate using biochemical analyzer (Mindray). To measure urinary N-acetyl-beta-D-glucosidase (NAG) and retinol-binding protein-4 (RBP-4) levels, particle-enhanced turbidimetric immunoassay (PETIA) kits (Diazyme, USA) were implemented.

Statistical analysis

SPSS 22.0 software was applied for data analysis. The results were expressed by mean ± SD. The independent sample T-test was used for the comparison between the two groups, and the one-way ANOVA was used for the comparison between multiple groups. The m6A methylation sites of NLRP3 were predicted using the SRAMP database (http://www.cuilab.cn/sramp). P < 0.05 was statistically significant.