Collection of gene expression dataset

We downloaded a dataset of glomerular transcriptional profiles of DN patients from the Gene Expression Omnibus Database (www.ncbi.nlm.nih.gov/geo/). The dataset (GSE96804) includes transcriptomes of glomerular tissues extracted from the kidneys of DN patients and control glomerular tissues from healthy living donors, including 42 patients with DN and 19 controls. We screened out the data related to IL-33 and sST2 gene expression.

Animals

We obtained male C57BL/6J mice from Beijing Vital River Laboratory Animal Technology Co. Ltd. Mice aged 6 to 8 weeks and weighing 18 ~ 20 g. Animals were cared for and used in accordance with the Guidelines for Care and Use of Laboratory Animals. The Local Committee on Animal Care Use and Protection of Hebei Medical University approved the experiments.

Experimental design and animal model

The experimental mice were maintained at a room temperature of about 22℃, humidity of about 45%, and a circadian rhythm of 12 h of light/12 h of darkness, and acclimatized feeding for 1 week [22, 23].

Forty-eight mice were randomly assigned to the DN (n = 36) and control (n = 12) groups. All mice were fasted for 12 h. The DN mouse model was established by intraperitoneal injection of streptozotocin (0.1% STZ, 100 mg/kg) on 3 consecutive days [24]. The modeling was established in 30 min with an injection of STZ (STZ was dissolved in 0.1 mol/L of citrate buffer (pH 4.5) before use) into the abdominal cavity. The control group received an equivalent volume of PBS by intraperitoneal injection. After STZ injections were administered over 72 h, fasting blood glucose (FBG) levels were measured after the mice had fasted for 12 h, and the animals were considered diabetic when the FBG level was ≥ 11.1 mmol/L. Urine samples were collected after two weeks. Urinary creatinine excretion (UCE) and random urine protein (UPR) were measured, and the urine protein creatinine ratio (UPC) was calculated. All the values were significantly increased, suggesting the successful establishment of the DN model. Thirty-six mice in which the model was successfully established were randomly divided into three groups, namely, the STZ group, the STZ + IL-33 group, and the STZ + IL-33 + sST2 group. The STZ + IL-33 group was given 14 g/kg intraperitoneally of IL-33 and STZ + IL-33 + sST2 group was given 16 g/kg intraperitoneally of sST2 every day for six consecutive days.

Sample collection

All experimental mice were euthanized after anesthesia. The specific measures of euthanasia were as follows: all experimental mice were intraperitoneally injected with pentobarbital sodium (150 mg/kg).Urine samples and blood samples were collected and centrifuged at 3500 r for 15 min. The supernatants were stored at -80 °C. The blood was collected by extirpating the eyeball, and the samples were centrifuged at 2000 r for 10 min to obtain serum, and then stored at -80 °C. Kidneys were harvested after euthanasia, and we fixed one kidney with 4% paraformaldehyde for histological analysis and stored the other at -80 °C until use.

Renal function analysis

Biochemical indices, including blood urea nitrogen (BUN), UPR, and UCE, were measured in the serum and urine samples using commercial kits (IDEXX Laboratories, Westbrook, ME, USA), and the results reflected the degree of kidney damage.

Cell culture

Mouse glomerular endothelial cells (MGECs) were cultured in RPMI 1640 medium (HyClone) containing 10% FBS (HyClone), 100 U/mL penicillin, and 100 µg/mL streptomycin placed in a 5% CO2 incubator at 37 °C. The cells were first treated with IL-33 (10 ng/ml or 100 ng/ml, Sino Biological, 10,368-HNAE) or sST2 (Sino Biological, 10,105-H08H) for 24 h and then treated with 30 mmol/l glucose (high glucose; HG) for 24 h.

Quantitative real-time PCR analysis

We extracted total RNA from tissues using a Promega total RNA isolation kit (Promega, Madison, WI) and reverse transcribed it with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA). A Bio-Rad iQ5 real-time PCR detection system with SuperReal PreMix (SYBR Green) (TIANGEN, Beijing, China) was then used to perform quantitative real-time PCR. The ∆∆CT method was used to determine the relative changes in gene expression normalized to mouse GAPDH gene expression. The primers used are listed in Table 1.

Western blotting

MGECs were homogenized and lysed with RIPA lysis buffer (Beyotime, Shanghai, China). We quantified total proteins using a BCA protein assay kit (Beyotime, Shanghai, China), separated by SDS‒PAGEand then transferred to polyvinylidene difluoride membranes. The blots were incubated with primary antibodies against the following target proteins: GRP78 (1:1000; ABclonal), Caspase-12 (1:1000; ABclonal), CHOP (1:1000; ABclonal), PERK (1:1000; Affinity), p-PERK (1:1000; Affinity), IRE1α (1:1000; Affinity), and p-IRE1α (1:1000; Affinity). The membranes were then washed with PBST (3 × 10 min) before incubation with horseradish peroxidase-conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (1:5000) in 5% milk–PBST for 1 h. After another PBST (3 × 10 min) wash, the blots were incubated in the chemiluminescent substrate for 1 min and finally exposed to X-ray film (ChemiDoc™XRS+, Bio-Rad).

Flow cytometry

An Annexin V-FITC/PI Apoptosis Detection kit (Vazyme, Nanjing, China) was used to analyze apoptosis in MGECs cultured in six-well plates. Following treatment with IL33 or sST2 for 24 h, the cells were treated with HG for 48 h. The treated cells were collected, washed with PBS (1000 r for 5 min), and resuspended in 100 µl of binding buffer. Five microliters of Annexin FITC and 5 µl of PI were added successively, and the samples were incubated in the dark at room temperature for 10 and 15 min, respectively. All the samples were analyzed using a cytometer (CytoFLEX S, Beckman Coulter).

Immunohistochemistry

Histological examination of the kidneys was conducted after the kidneys were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and cut into 5-mm sections. By incubating the sections with 3% bovine serum albumin in PBS, nonspecific binding was blocked, and endogenous peroxidase was quenched by 3% hydrogen peroxide. The sections were immuno-stained with anti-CHOP and anti-caspase 3 primary antibodies, followed by secondary antibodies (R&D, MN, USA), and the pathological changes were observed under an ordinary light microscope.

Double immunofluorescence staining

Sections were treated with EDTA antigenic repair buffer (pH 9.0) for antigenic repair, and to quench autofluorescence, added 3% BSA, and incubated for 30 min at room temperature; then stained with an anti-rat CD31 primary antibody, an anti-rabbit IL-33 primary antibody overnight at 4 °C. Anti-rat Alexa Fluor 488 and anti-rabbit CY3 secondary antibodies were used. The cells were incubated at room temperature in the dark for 50 min, and the nuclei were stained with DAPI in the dark for 10 min at room temperature. Anti-fluorescence quenching tablets were sealed and the samples were observed and images were collected by confocal microscopy.

Statistical analysis

All the data were analyzed using IBM SPSS software. The values are presented as the mean ± SEM. Student’s t-test was used to analyze experiments with two groups. Statistical analyses were conducted using a one-way ANOVA followed by Tukey’s multiple comparison test to determine the significant differences when more than two groups were involved. A P value less than 0.05 was considered significant.