MPC5 mouse podocytes were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. LM22B-10 (ERK1/2 agonist, 5 mM) (13) and p79350 (p38 agonist, 50 µM) (14) were purchased from MedChemExpress. Podocytes were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). The podocytes were divided into normal glucose (NG, 5 mM glucose), mannitol control (MA, 5 mM glucose + 25 mM mannitol), HG (30 mM glucose) and transfection groups. The podocytes were cultured in an incubator with 5% CO₂ at 37˚C.

The podocytes were plated into a six-well plate at a density of 6x10⁵ cells/well and incubated at 37˚C for 24 h. The podocytes were subsequently transfected with 2 µg/ml pGPU6 short hairpin RNA vector targeting ADAM10 (shRNA-ADAM10) and scrambled shRNA-negative control (NC) (Shanghai GenePharma Co., Ltd.) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h at 37˚C. 24 h after the end of transfection, the cells were utilized in subsequent experiments. Target sequences for shRNA-ADAM10 and shRNA-NC were 5'-GCAAAGATGATAAGGAATTAT-3' and 5'-GACAAGATGATAAGGATATAT-3', respectively.

Total RNA was extracted from podocytes in a 6-well plate at a density of 2x10⁵ cells/well using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the ReverTra Ace™ qPCR RT kit (cat. no. FSQ-101; Toyobo Life Science) according to the manufacturer's instructions. qPCR was performed using the SYBR-Green qPCR Master Mix fit (MedChemExpress) using 2 µl cDNA as a template. The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 2 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 1 min. The RNA expression levels of ADAM10 were quantified using the 2^-∆∆Cq method (15) and normalized to the internal reference gene GAPDH. The following primer pairs were used for qPCR: ADAM10 forward, 5'-TCATCAAGACTCGTGGTGGC-3' and reverse, 5'-GCATGCTTCTCTGGATGTGC-3'; GAPDH forward, 5'-GGGTCCCAGCTTAGGTTCATC-3' and reverse, 5'-TACGGCCAAATCCGTTCACA-3'.

Total protein was extracted from the podocytes (2x10⁶) using RIPA lysis (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA assay kit (Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated by SDS-PAGE on a 10% gel (Bio-Rad Laboratories, Inc.). The separated proteins were transferred onto PVDF membranes (MilliporeSigma) and blocked with 5% skimmed milk at room temperature for 1 h. The membranes were incubated overnight at 4˚C with the following primary antibodies: Anti-ADAM10 (1:1,000; cat. no. ab124695; Abcam), anti-cyclooxygenase (COX)-2 (1:1,000; cat. no. ab179800; Abcam), anti-inducible nitric oxide synthase (iNOS; 1:1,000; cat. no. ab178945; Abcam), anti-Bax (1:1,000; cat. no. ab53154; Abcam), anti-Bcl-2 (1:1,000; cat. no. ab59348; Abcam), anti-cleaved caspase-3 (1:1,000; cat. no. PA5-114687; Thermo Fisher Scientific, Inc.), anti-cleaved caspase-9 (1:1,000; cat. no. ABP50009; AmyJet Scientific, Inc.), anti-phosphorylated (p-)ERK1/2 (1:1,000; cat. no. ab176640; Abcam), anti-ERK1/2 (1:1,000; cat. no. ab17942; Abcam), anti-p-p38 (1:1,000; cat. no. ab4822; Abcam), anti-p38 (1:2,000; cat. no. ab170099; Abcam), anti-p-JNK (1:5,000; cat. no. ab124956; Abcam), anti-JNK (1:500; cat. no. ab208035; Abcam), anti-NLR family pyrin domain containing 3 (NLRP3; 1:1,000; cat. no. ab263899; Abcam), anti-cleaved caspase-1 (1:1,000; cat. no. sc-22166; Santa Cruz Biotechnology, Inc.), anti-apoptosis-associated speck-like protein containing a CARD (ASC; 1:1,000; cat. no. ARG41743; Arigo Biolaboratories Corp.), anti-caspase-1 (1:1,000; cat. no. 3019-100; AmyJet Scientific, Inc.), anti-cleaved N-terminal gasdermin-D (GSDMD-N; 1:1,000; cat. no. DF13758; Affinity Biosciences, Ltd.), anti-podocin (1:1,000; cat. no. 113216; NovoPro Bioscience, Inc.), anti-CD2-associated protein (CD2AP; 1:1,000; cat. no. DF2298; Affinity Biosciences, Ltd.), anti-nephrin (1:2,000; cat. no. DF7501; Affinity Biosciences, Ltd.) and anti-GAPDH (1:2,000; cat. no. MA1-16757; Thermo Fisher Scientific, Inc.). Following primary antibody incubation, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. ab6721; Abcam) for 1 h at room temperature. The membranes were visualized using Hypersensitive ECL kit (Hanbio Biotechnology Co., Ltd.). Blots were quantified using ImageJ v1.52 software (National Institutes of Health).

The podocytes seeded into a 24-well plate (5x10⁴ cells/well) were centrifuged at 500 x g for 5 min at 4˚C and supernatant was collected. The levels of TNF-α (cat. no. ZC-39024), IL-6 (cat. no. ZC-37988), IL-1β (cat. no. ZC-37974) and IL-18 (cat. no. ZC-37973; all ZCIBIO Technology Co., Ltd.) in the podocytes were measured using ELISA kits, according to the manufacturer's instructions. A microplate reader (Molecular Devices, LLC) was used to measure the optical density at 450 nm.

The podocytes were seeded into a 24-well plate at a density of 5x10⁵ cells/well. After reaching 70-80% confluency, the podocytes were fixed with 4% paraformaldehyde for 30 min and treated with permeable fluid for a further 5 min, both at room temperature. TUNEL staining was performed at 37˚C for 1 h using the TUNEL assay kit (cat. no. C1086; Beyotime Institute of Biotechnology), according to the manufacturer's protocol. 1 mg/ml DAPI (Beyotime Institute of Biotechnology) was used to counterstain the nuclei for 10 min at room temperature. The results were observed in five random fields of view under a fluorescence microscope (magnification, x200; Olympus Corporation).

Graph Pad Prism 8.0 software (GraphPad Software; Dotmatics) was utilized to analyze all the experimental data. Data are presented as the mean ± standard deviation (n=3). One-way ANOVA followed by Tukey's post hoc test was applied to compare differences between groups. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of ADAM10 were determined using RT-qPCR and western blot analysis (Fig. 1A and B). ADAM10 expression was significantly upregulated in the HG group compared with that in the NG group, while it was downregulated in the ADAM10 knockdown group compared with that in the HG + sh-NC group. The HG + sh-ADAM10-1 group showed a greater decrease in ADAM10-1 mRNA and protein levels; therefore, this group of podocytes was used as the ADAM10-1 knockdown group in subsequent experiments. The levels of inflammatory factors in podocytes were measured using ELISA kits (Fig. 1C). The levels of TNF-α, IL-1β, IL-6 and IL-18 in the HG group were upregulated compared with those in the NG group, whereas the levels of these cytokines were decreased in the HG + sh-ADAM10 group compared with those in the HG + sh-NC group. In addition, protein expression levels of iNOS and COX-2 were semi-quantified using western blot analysis (Fig. 1D). Expression levels of iNOS and COX-2 were increased in the HG group compared with those in the NG group and decreased in the HG + sh-ADAM10 group compared with those in the HG + sh-NC group. These results suggested that ADAM10 knockdown inhibited HG-induced podocyte inflammation.

Moreover, levels of the podocyte markers podocin, CD2AP and nephrin significantly decreased following HG treatment compared with those in the NG group, whereas ADAM10 knockdown increased the levels of these protein levels compared with those in the HG + sh-NC group (Fig. 2A). Podocyte apoptosis levels in each group were evaluated using the TUNEL assay (Fig. 2B and C). The apoptotic podocytes in the HG group emitted more fluorescence than the NG group, whereas the HG + sh-ADAM10 group did not emit as much fluorescence. Subsequently, expression levels of proteins related to apoptosis were assessed using western blot analysis (Fig. 2D). The protein levels of Bax, cleaved caspase-3 and cleaved caspase-9 were increased in the HG group, while ADAM10 knockdown counteracted the effect of HG induction. However, the expression of Bcl-2 was decreased in the HG group, whereas it increased following ADAM10 knockdown. These results indicated that HG induced the apoptosis of podocytes, whereas ADAM10 knockdown attenuated apoptosis.

The expression levels of MAPK-associated proteins were assessed using western blot analysis (Fig. 3A). The expression levels of p-ERK, p-p38 and p-JNK were increased in the HG group compared with those in the NG group. ADAM10 knockdown decreased these expression levels compared with those in the HG + sh-NC group. Furthermore, the expression levels of pyroptosis-associated proteins were assessed using western blot analysis (Fig. 3B). The expression levels of NLRP3, cleaved caspase-1, ASC and GSDMD-N were increased in the HG group, whereas ADAM10 knockdown attenuated this effect. These results indicated that ADAM10 blocked the MAPK signaling pathway and suppressed pyroptosis.

Podocytes were pretreated with LM22B-10 or p79350 to investigate the role of the MAPK signaling pathway in the regulatory effects of ADAM10. Subsequently, levels of inflammatory factors and inflammation-related proteins in each group of podocytes were measured using ELISA kits and western blot analysis, respectively (Fig. 4A and B). The levels of inflammatory factors and expression of COX-2 and iNOS in podocytes pretreated with agonists increased, attenuating the inhibitory effects of ADAM10 knockdown on podocyte inflammation. The levels of podocyte markers decreased in response to LM22B-10 or p79350 pretreatment compared with those in the HG + sh-ADAM10 group (Fig. 4C). The apoptosis of podocytes in each group was assessed using TUNEL assay and western blot analysis (Fig. 4D-F). TUNEL assay demonstrated that the apoptotic rates of podocytes pretreated with agonist increased compared with those in the HG + sh-ADAM10 group, exhibiting a higher fluorescence intensity. The protein levels of Bax, cleaved caspase-3 and cleaved caspase-9 were increased in the pretreatment groups, whereas the expression of Bcl-2 decreased. The addition of the agonists also attenuated the inhibitory effects of AMAD10 knockdown on podocyte apoptosis. Finally, the effects of agonists on the expression levels of pyroptosis-associated proteins were evaluated using western blot analysis (Fig. 5). The levels of NLRP3, cleaved caspase-1, ASC and GSDMD-N were increased in the pretreatment groups as a consequence of the activation of the pathway. These results suggested that activation of the MAPK signaling pathway attenuated the suppressive effects of AMAD10 knockdown on podocyte inflammation, apoptosis and pyroptosis.