Ethics statement and clinical sample collection

This study was approved by the ethics committee of the Shanghai East Hospital and was conducted as per the Declaration of Helsinki. All participating patients submitted their written informed consent prior to the investigation. Human kidney biopsy tissues of patients (n = 63) with DN were obtained from the Shanghai East Hospital between August 2017 and January 2018, and normal kidney tissues from nephrectomies performed for renal hamartoma (n = 10) served as the control. All diabetic donors had type 2 diabetes. Diabetes status was defined as hemoglobin A1c ≥ 6.5%. Inclusion criteria required that: (i) donors be between 17 and 80 years of age, (ii) diabetic status be confirmed as noted above, and (iii) the postmortem time be within 24 h from cross-clamping in the operating room. The only exception with respect to hemoglobin A1c testing was if a donor had been prescribed and/or using glycemic agent(s), including but not limited to metformin and insulin, in which case repeated hemoglobin A1c testing was not mandatory. Exclusion criteria included active infection; malignancy; severe glomerulosclerosis, as determined in frozen operating room biopsy material; history of renal replacement therapy (hemodialysis or peritoneal dialysis at any time); and any known genetic renal condition, such as polycystic kidney disease. Tissue was placed into RNALater and manually microdissected at 4 °C for glomerular and tubular compartment. Glomeruli that readily released from the capsule and corresponding tubuli compartment were collected and placed into cold RNAeasy lysis buffer solution (RNeasy Mini Kit; Qiagen, Shanghai, China; 74106). All demographic data and anthropometric parameters were obtained at the time of enrollment.

Immunohistochemistry

Immunohistochemical (IHC) staining was performed to validate WTAP protein expression following the standard protocol using the anti-WTAP antibody (ab195380; Abcam; 1:100 dilution), followed by incubation of the secondary antibody (D-3004; Shanghai Long Island Biotech Co., Ltd.). IHC evaluation was done on the basis of the percentage of positively stained cells (graded on a scale of 0–4: 0, < 5%; 1, 5–25%; 2, 25–50%; 3, 50–75%; 4, > 75%) and the intensity of staining (graded on a scale of 0–3: 0, negative; 1, weak; 2, moderate; 3, strong), which ranged from 0 to 12. Two pathologists who were blinded to the patients’ clinical and pathological characteristics conducted the immunohistochemical evaluation. The 63 patients were subsequently divided into two groups: low expression (IHC score < 4) and high expression (IHC score ≥ 4).

Cell culture

Human renal tubular epithelial HK-2 cells and mouse kidney epithelial cell line (TCMK-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA; 10564029) with 10% fetal bovine serum (Gibco; 16000044) and 100 U/mL penicillin and streptomycin (Solarbio, Beijing, China; P1400-100). HK-2 cells were treated with 5.5 mM normal glucose (NG) and 30 mM high glucose (HG) for 48 h. The NG group was additionally treated with 24.5 mM mannitol to establish the control osmolarity.

Adenovirus production

The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The shRNA sequence was cloned into the pShuttle-H1 adenovirus plasmids (shWTAP). Otherwise, the human WTAP coding sequence was synthesized and cloned into pShuttle-CMV adenovirus shuttle plasmids (Agilent, Beijing, China; 240007). The blank pAdEasy-1 adenovirus skeleton plasmid (Addgene Headquarters, Watertown, MA, USA; #16400) and pShuttle-H1-shWTAP or pShuttle-CMV-WTAP were co-transfected into HEK 293T cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) to produce a high-titer adenovirus vector. After 48 h transfection, recombined adenovirus plasmids were collected and transduced into HK-2 or TCMK-1 cells. Cells that were transduced with pShuttle-H1-nonspecific scramble shRNA plasmid (shNC) or blank pShuttle-CMV plasmid (vector) were used as the negative control.

Transfection

HK-2 cells were transfected at 60–75% confluence with siRNA targeting IGF2BP1, IGF2BP2, IGF2BP3, or NLRP3 using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s protocol. Scramble siRNA (siNC) for IGF2BP1, IGF2BP2, IGF2BP3, or NLRP3 were utilized as negative controls. The interfering RNA sequences are listed in Additional file 1: Table S1.

Cell Counting Kit-8

HK-2 cells (3 × 103 cells per well) were cultured in 96-well plates and incubated at 37 °C overnight. After 0, 12, 24, and 48 h treatment, 10 μL of the Cell Counting Kit-8 (CCK-8; Signalway Antibody LLC, College Park, MD, USA; CP002) solution was added into each well and incubated for an extra 1 h. Cell viability was subsequently determined using a microplate reader (PERLONG MEDICAL, Beijing, China; DNM-9602) at OD450 nm.

Detection of cell pyroptosis

The cells were stained using FLICA 660-YVAD-FMK (FLICA 660 in vitro Active Caspase-1 Detection Kit, ImmunoChemistry Technologies, Bloomington, MN, USA; #9122) according to manufacturer’s instructions and with propidium iodide (PI; Invitrogen, Waltham, MA, USA; P3566) to mark cells with membrane pores. Cells were stained with 60× FLICA 660 and incubated for 1 h at 37 °C and protected from light. After which, the cells were washed three times with 1× Cellular Wash Buffer and stained with 3 μM PI for 15 min at 25 °C. The percentage of pyroptotic cells was then determined using a CytoFLEX flow cytometer (Beckman Coulter Cytoflex S, Krefeld, Germany). The output images from the cell pyroptosis assay included four fields, of which the field with active caspase-1+PI+ represents pyroptotic cells.

Lactate dehydrogenase release assay

Pyroptosis was also evaluated by assaying the lactate dehydrogenase (LDH) released into the supernatants. A lactate dehydrogenase assay kit (Nanjing Jiancheng Bioengineering Institute, China; A020-2) was used to detect LDH.

Real-time qPCR

RNA samples from human kidney tissues and HK-2 cells were extracted using TRIzol (Invitrogen; 15596026), and cDNA was generated using the Toyobo reverse transcription reagent kit (Takara, Japan; RR047A). Real-time qPCR (RT-qPCR) was performed using an ABI 7500 fast machine (Applied Biosystems, Foster City, CA, USA) with the SYBR Premix EX Taq kit (Takara; RR420A). The primer pair sequences are listed in Additional file 1: Table S2. Gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in either tissues or HK-2 cells. Relative quantification was determined using the 2−ΔΔCT method.

Western blot

Total protein samples from mouse kidney tissues and HK-2 cells were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer (JRDUN Biotechnology Co. Ltd, Shanghai, China; BYL40825). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was conducted to isolate protein samples, after which, the proteins were electrotransferred onto nitrocellulose membranes followed by blocking with 5% skim milk for 2 h at room temperature. The blots were incubated with primary antibodies against WTAP (Cell Signaling Technology, MA, USA; #56501), NLRP3 (Abcam; ab263899), caspase-1 (Abcam; ab207802), GSDMD (Affinity; AF4012), IGF2BP1 (Abcam; ab82968), IGF2BP2 (Abcam; ab129071), IGF2BP3 (Abcam; ab177477), H3K27ac (Abcam; ab4729), and GAPDH (Cell Signaling Technology; #5174) at 4 °C overnight. Incubation with goat anti-rabbit IgG (Beyotime, Shanghai, China; A0208) was subsequently performed for 1 h at room temperature. The western blotting bands were determined using ImageJ software.

ELISA

The release of IL-1β (KAC1211; BMS6002TEN), IL-18 (KHC0181; KMC0181), TNF-α (KHC3014C; BMS607-3TEN), and IL-6 (BMS213-2; KMC0062 all from Invitrogen) in HK-2 cell supernatant and mouse serum was analyzed using ELISA kits according to the manufacturer’s instructions. The OD450 nm of each sample was measured using a microplate reader.

m6A content analysis

TRIzol reagent was used to extract total RNA. Poly(A)+ RNA was purified using GenElute mRNA Miniprep Kit (Sigma, Louis, MO, USA; MRN10). m6A content was assayed using the m6A RNA Methylation Assay Kit (Abcam, ab185912). Briefly, 80 µL of binding solution and 200 ng of sample RNA were added into each designated well, then incubated at 37 °C for 90 min for RNA binding. Each well was washed three times with wash buffer. Fifty microliters of the diluted capture antibody was added to each well, then incubated at room temperature for 60 min. Each well was incubated with detection antibody and enhancer solution at room temperature for 30 min subsequently. Finally, the wells were incubated with developer solution in the dark for 1–10 min at 25 °C. Reaction was stopped with stop solution and determined using a microplate reader at 450 nm wavelength within 2–10 min.

RNA immunoprecipitation assays

RNA immunoprecipitation (RIP) assays were carried out using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Billerica, MA, USA; 17-701) following the manufacturer’s protocol. The RNA–protein complexes were conjugated with anti-m6A (Abcam, ab208577), anti-IGF2BP1 (ab184305), or anti-IgG antibody (ab172730) at 4 °C for 1 h. Once incubation was complete, agarose beads and 50 µL of protein A/G were added and cells were incubated for a further 60 min at 4 °C. Subsequently, the precipitated beads were washed with RIP-wash buffer for 10 min at 4 °C and then RIP-lysis buffer for 5 min at 4 °C. The RNA in the immunoprecipitated complex and the RNA in the previously saved input fraction were released by incubating cells at 65 °C for 2 h with 200 mM NaCl and 20 µg proteinase K, which reversed the cross-linking. The coprecipitated RNAs were purified using phenol:chloroform:isoamyl alcohol and subjected to RT-qPCR.

mRNA stability measurements

HK-2 cells were treated with 0.2 mM actinomycin D (GlpBio, Montclair, CA, USA; GC16866) for 30 min and were designated as the 0 h samples. The 2, 4, and 6 h samples were then collected for the extraction of total mRNA. cDNA synthesis by reverse transcriptase was subsequently performed using an oligo(dT) primer. The quantitated mRNA levels were determined by RT-qPCR.

Chromatin immunoprecipitation (ChIP)

ChIP analysis was performed as previously described [31]. Briefly, cells with 10 μM C646 treatment were cross-linked in 1% formaldehyde, and the DNA was sonicated into a size range of 200–1000 base pairs using a Bioruptor Sonicator (Diagenode) for five cycles of 3 s on/3s off. The extracts were precleared in BSA-blocked protein A/G beads and incubated with anti-H3K27ac (Abcam; ab4729) or control IgG (Santa Cruz Biotechnology; sc-2027) overnight at 4 °C. After being washed, the DNA was eluted and reverse-cross-linked overnight at 65 °C; then, it was purified and confirmed by RT-qPCR (WTAP primers sequences: F, 5′-TTTCCACTCCCACCAGGAAAG-3′ and R, 5′-TAAGACTGCCATCTGGACCG-3′).

Reporter gene assays

The NLRP3 3′UTR or 5′UTR sequence was cloned into the pGl3 vector (Promega, Madison, WI, USA). HK-2 cells were treated with 30 mM HG and transduced with WTAP shRNA or overexpression vector [cotransfected with either the pGl3-NLRP3 3′UTR or 5′UTR luciferase reporter plasmid and the pRL-TK vector (Promega) expressing the Renilla luciferase] using Lipofectamine 2000 (Invitrogen). Otherwise, H3 wild-type or K27R-mutant HK-2 cells, generated as previously described [32], were transfected with the pRL-TK vector and the pGL3-basic plasmid containing WTAP promoter sequence in the presence of 10 μM C646, a histone acetyltransferase p300 inhibitor, following the Lipofectamine 2000 (Invitrogen) transfection protocol. A dual-luciferase assay was conducted on the basis of the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

Animals

A total of 20 C57BL/KsJ diabetic mice (db/db) (male; 8 weeks old) were housed under a 12 h:12 h light–dark cycle (7:00 am, on; 7:00 pm, off) (relative humidity: 55–60%; temperature: 22 °C ± 1 °C) and fed ad libitum with standard chow and water before being sacrificed. Another six C57BL/KsJ nondiabetic mice (db/m) (male; 8 weeks old) were used as control. The db/db mice exhibited obesity, hyperinsulinemia, and hyperglycemia as previously reported [33] and were randomly allocated into two groups, namely db/db + shNC group (n = 10) and db/db + shWTAP group (n = 10), wherein either control shNC or shWTAP were injected via the tail vein. A total of 12 mice (n = 6 per group) were successfully modeled for adenovirus injection via tail vein, with a success rate of 60.0%. Four weeks after injection, serum and urine samples were collected and mice were anesthetized by inhalation with 3% isoflurane, sacrificed by cervical dislocation, and kidney tissues were collected for H&E and Masson’s Trichrome staining as described in a previous study [34]. The serum creatinine, serum blood urea nitrogen (BUN), and urine protein levels were analyzed using a creatinine assay kit (C011-1-1), urea assay kit (C013-2-1), and urine protein test kit (C035-2-1; all from Nanjing Jiancheng Bioengineering Institute) as per the manufacturer’s instructions, respectively. Primary tubular segments were obtained from the cortex of mouse kidneys in each group and the primary tubular epithelial cells were cultured in DMEM/F-12 (Invitrogen) medium as previously described [35]. All procedures were authorized by the Institutional Animal Care and Use Committee (IACUC) and the ethics committee of of Shanghai Rat@Mouse Biotech Co., Ltd., China.

Data analysis

All data in this study were processed using GraphPad Prism 8.4.2 and expressed as mean ± standard deviation (SD). We also used the Shapiro–Wilk normality test to evaluate the normal distribution of the collected data. When data showed normal distribution, two-sided unpaired Student’s t-test and one-way analysis of variance Tukey’s post-hoc test were adopted. P-value < 0.05 was considered statistically significant.