Twenty male Kunming mice (KM) (6–8 weeks) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (China). DN model was established in mice by streptozotocin (STZ, Sigma, Darmstadt, Germany) induction. The mice were subject to a fasting period of 4~6 h and then injected with 50 mg/kg of STZ intraperitoneally daily for five days. The control group was treated with the same volume of citrate buffer. The DN model was determined to be successfully established when blood glucose levels increased to ≥16.7 mmol/L. Subsequently, all mice were euthanized under halothane anesthesia at the end of the experiments. All animal experiments complied with ARRIVE guidelines. This study was approved by the Hunan SJA Laboratory Animal Co., Ltd. (China) (ethics approval number IACUC-SJA2021019-4).

Human bone marrow MSCs were purchased from Pricella (Wuhan, China). MSCs were maintained in Dulbecco’s modified Eagle medium (DMEM)/F-12 medium (Sigma, Darmstadt, Germany) containing 1% penicillin-streptomycin (Beyotime, Shanghai, China). Then, MSCs were grown in a serum-free medium for 2 days. Cell supernatants of MSCs were collected, and cell debris was removed by centrifugation at 3000 g for 15 min. The supernatants were mixed with exosome extraction reagent (System Biosciences, Palo Alto, USA) in a ratio of cell supernatant:ExoQuick TC = 5:1, and the mixture was fully mixed and left overnight at 4°C. The mixture was centrifuged with 1500 g for 30 min to separate the exosomes from the cell culture. For exosome characterization, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) analysis were performed following the protocol in a previous study (Jiang et al., 2021). Exosomal markers, including CD63, HSP70, and calnexin, were determined by western blotting.

The extracted exosomes were stained with Dil dye solution (AmyJet Scientific Inc., Wuhan, China). Stained exosomes (20 μg) were added to the cell culture medium and incubated with MPC5, and the fluorescence status was checked under a fluorescence microscope (AE31E, Motic, Baltimore, USA) and photographed and recorded.

Mouse Podocyte Clone-5 (MPC5, Tongpai, Shanghai, China) was cultured in a DMEM medium (D5796, Sigma, Darmstadt, Germany) (containing 10% fetal bovine serum + 1% double resistance). MPC5 was treated with 0, 10, 20, and 30 mM glucose for 48 h. MPC5 was cultured with 30 mmol/L glucose for 0, 12, 24, 36, and 48 h. MPC5 treated with 30 mM glucose medium for 48 h was considered the high glucose group (HG). MPC5 cultured in DMEM containing 5.5 mM glucose was considered the control group (NC). Mannitol-treated MPC5 acted as an osmotic pressure control group (MA).

Lipofectamine 3000 (Invitrogen, Carlsbad, USA) was used for transient transfection. MiR-30a-5p mimics, a NLRP3 overexpression plasmid, and their corresponding negative controls were used for transfection in the HG-induced MPC5 group. The experiments are grouped as follows: NC group, HG group, HG + mimics NC group, HG + miR-30a-5p mimics group, and the HG group, HG + mimics NC + oe-NC group, HG + miR-30a-5p mimics group, HG + oe-NLRP3 group, HG + miR-30a-5p mimics + oe-NLRP3 group.

The cells were grouped into: Control group (MPC5 was cultivated with normal glucose), HG group (HG-induced MPC5), MSC-EXOs group (the exosomes that were isolated from MSCs were used to treat HG-induced MPC5), MSC-EXOs mimics NC group (the exosomes separated from MSCs were transfected with NC mimics and used to treat HG-induced MPC5), MSC-EXOs miR-30a-5p mimics group (the exosomes separated from MSCs were transfected with miR-30a-5p mimics and used to treat HG-induced MPC5).

Total RNA was extracted from renal tissues, MPC5, and exosomes using Trizol reagent (Invitrogen, Carlsbad, USA). RNA quality was assessed using a NanoDrop2000 spectrophotometer (Thermo Fisher Science, Waltham, USA). The RNA was transcribed into cDNA using an mRNA reverse transcription Kit (CW2569, CWBIO, Beijing, China). Primers designed for RT-qPCR are mentioned in Table 1. RT-qPCR was carried out at 95°C for 10 min, 95°C for 15 s, and 60°C for 30 s. The 2^−ΔΔCt method was used for analysis, and β-actin and U6 were used as internal references to calculate the relative expression of the target gene.

Protein from renal tissues and MPC5 was extracted using RIPA lysis buffer (Beyotime Shanghai, China) containing phosphatase inhibitor (P1260, Applygen, Beijing, China) and protease inhibitor (583794, Gentihold, Beijing, China). The protein sample was mixed with the loading buffer and heated at 95°C to 100°C for 5 min. The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. For blocking the membrane, 5% skim milk was used for 1 h at room temperature. Primary antibodies, anti-NLRP3 (1:1000, ab214185, Abcam, Cambridge, UK), anti-GSDMD-N (1:1000, ab215203, Abcam, Cambridge, UK), anti-caspase-1 (1:1000, 22915-1-AP, Proteintech, Rosemont, USA), anti-IL-1beta (1:2000, #12703, CST, Danvers, USA), anti-IL-18 (1:1000, 10663-1-Ap, Proteintech, Rosemont, USA), and anti-β-actin (1:5000, 66009-1-Ig, Proteintech, Rosemont, USA) were added to the membranes and kept overnight at 4°C. Secondary antibodies were added to react with the membranes for 2 h. The membranes were incubated with SuperECL Plus (Advansta, San Jose, USA) for 5 min and then visualized on a chemiluminescence imaging system (Chemiscope 6100, Qinxiang, Guangzhou, China).

Renal tissues were paraffin-embedded and sectioned. The sections were heated at 60°C for 12 h. The sections were dehydrated in xylene and ethanol and then microwave heated by immersing them in ethylenediamine tetraacetate buffer for 24 min. The sections were placed in sodium borohydride solution for 30 min. The sections were soaked in 75% ethanol solution for 15 s~1 min and placed in Sudan black staining solution for 15 min. The sections were incubated with 5% bovine serum albumin (BSA) for 60 min, followed by primary antibody (1:50, Nephrin, ab216341; GSDMD-N, ab219800, Abcam, Cambridge, UK) at 4°C overnight. The sections were then incubated with secondary antibodies at 37°C for 60~90 min. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at 37°C for 10~20 min. The sections were sealed with buffered glycerol and observed under a fluorescence microscope.

MPC5 on glass slides was fixed with 4% paraformaldehyde for 30 min. Triton (0.3%) was added to the glass slides and permeabilized for 30 min. BSA (5%) was added to the glass slides and incubated for 30 min. The slides were then incubated with primary antibody GSDMD-N (1:50, ab215203, Abcam, Cambridge, UK) and caspase-1 (1:50, 22915-1-AP, Proteintech, Rosemont, USA) at 4°C, overnight. Then, the cell slides were treated with secondary antibodies in a dark room for 1 h. Slides were treated with DAPI for 10 min. Next, the cells were sealed with buffered glycerol and observed under a fluorescence microscope.

ELISA kits were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China) to measure the protein levels of tumor necrosis factor (TNF)-alpha (CSB-E04741m), IL-1beta (CSB-E08054m), and IL-18 (CSB-E04609m) in serum and cell supernatant. Samples and standards were processed according to the manufacturer’s instructions.

The cell viability of MPC5 was evaluated by CCK-8 assay (NU679, Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer’s protocol. The cells were seeded in 96-well plates at 5 × 103 cells/well density. CCK8 solution (10 μL) was added to each well. After incubation at 37°C, 5% CO₂ for 4 h, the absorbance was measured at 450 nm using a Bio-Tek microplate.

A caspase-1 (active) staining assay kit (ab219935, Abcam, Cambridge, UK) was used to detect the pyroptosis of MPC5. 5 × 105 MPC5 were digested by trypsin and then incubated with FAM-YVAD-FMK solution for 1 h. The cells were treated with propidium iodide staining buffer for 10 min. The flow cytometer (A00-1-1102, Beckman, Brea, USA) was used to detect the fluorescence intensity.

The TargetScan software was used to predict that NLRP3 interacts with miR-30a-5p, and this was verified using the dual-luciferase reporter system. Briefly, NLRP3-wt and miRNA-NC or miR-30a-5p mimics, NLRP3-mut and miRNA-NC or miR-30a-5p-mimics were transfected into 293A cells using the Lipofectamine 3000 Kit (Invitrogen, Carlsbad, USA). The luciferase activity was detected by a dual-luciferase reporter assay kit (E1910, Promega, Madison, USA).

All experiments were carried out three times independently. The data were statistically analyzed by GraphPad Prism 8 software (GraphPad software, La Jolla, USA). The statistical significance between the two groups was determined by an unpaired t-test. The statistical significance of the difference between three or more groups was determined by One-way ANOVA. p < 0.05 was regarded as statistically significant, and all data were expressed as mean ± SD.

DN mice models were established by STZ injection. Compared with the control mice, the miR-30a-5p level was down-regulated in DN mice, and the NLRP3 level was up-regulated (Figs. 1A–1B). Western blotting also verified that the protein levels of caspase-1, GSDMD-N, IL-1beta, and IL-18 were considerably augmented in DN mice than in control mice (Fig. 1B). The immunofluorescence results showed enhanced expression of GSDMD-N in the DN model (Fig. 1C). Nephrin expression was decreased, while TNF-alpha, IL-18, and IL-1beta levels in the serum of DN mice increased compared to those in control mice (Fig. 1D). These results indicated that the down-regulation of miR-30a-5p expression might be related to increased pyroptosis and inflammation in DN.

We used high glucose conditions to treat MPC5 and simulate DN and then measured miR-30a-5p and NLRP3 mRNA levels in MPC5 exposed to different glucose concentrations (0, 10, 20, and 30 mM). The level of miR-30a-5p in podocytes decreased in a dose-dependent manner (Fig. 2A). In contrast, the level of NLRP3 increased. In MPC5 exposed to 30 mM glucose (0, 12, 24, 36, and 48 h), miR-30a-5p expression decreased, while that of NLRP3 increased in a time-dependent manner (Fig. 2B). The comparison revealed that MPC5 treated with NC or mannitol (MA), miR-30a-5p expression in MPC5 treated with HG was down-regulated, and NLRP3 was up-regulated (Fig. 2C). The above results further verified that miR-30a-5p and inflammatory corpuscle NLRP3 have potential roles in DN.

To study the effect of miR-30a-5p in HG-induced MPC5 injury, we transfected HG-induced MPC5 with miR-30a-5p mimics. RT-qPCR result confirmed the efficiency of miR-30a-5p up-regulation (Fig. 3A). CCK 8 results verified that cell viability decreased when exposed to HG but this was reversed by the transfection of miR-30a-5p mimics (Fig. 3B). Flow cytometry demonstrated that up-regulation of miR-30a-5p reduced HG-induced podocyte pyroptosis (Fig. 3C). Immunofluorescence analysis showed that miR-30a-5p mimics reversed HG-induced up-regulation of caspase-1 and GSDMD-N (Fig. 3D). TNF-alpha, IL-18, and IL-1beta levels were higher in the supernatant of MPC5 treated with HG compared with the NC group, and miR-30a-5p overexpression could significantly reduce the secretion of these inflammatory factors (Fig. 3E). The above results show that miR-30a-5p could modulate the pyroptosis and inflammation of MPC5 in DN.

Next, we verified the target relationship between miR-30a-5p and the inflammasome NLRP3. Bioinformatic analysis predicted the binding sites of miR-30a-5p and NLRP3 (Fig. 4A). In the luciferase assay, the luciferase activity of MPC5 transfected with miR-30a-5p mimics in the NLRP3-wt group was considerably lower than that of MPC5 cells transfected with mimics-NC. Meanwhile, the luciferase activity of MPC5 transfected with miR-30a-5p mimics or NC mimics in the NLRP3-mut group did not differ significantly (Fig. 4B). The expression of NLRP3 in HG-induced podocytes was decreased by the transfection of miR-30a-5p mimics (Figs. 4C–4D). The data indicate that NLRP3 may target and inhibit the expression of miR-30a-5p in MPC5.

Next, we studied the effects of overexpression of NLRP3 and miR-30a-5p on podocyte damage. NLRP3 was overexpressed in MPC5, and RT-qPCR was used to verify the overexpression efficiency (Fig. 5A). After transfection of HG-induced MPC5 with miR-30a-5p mimics, the cell viability increased, and the pyroptosis rate decreased as compared to those in the NC-mimics group (Figs. 5B–5C). After the transfection of oe-NLRP3, the above results were reversed (Figs. 5B–5C). Compared to the NC-mimics group, NLRP3, and caspase-1 were significantly descended in MPC5 transfected with miR-30a-5p mimics. Oe-NLRP3 transfection increased the expression levels of these proteins (Fig. 5D). Immunofluorescence results verified that the changing trend of caspase-1 was similar to the above results (Fig. 5E). The levels of TNF-alpha, IL-18, and IL-1beta in the cell supernatant of the oe-NLRP3 group and the miR-30a-5p mimics+oe-NLRP3 group were significantly higher than in the miR-30a-5p mimics group (Fig. 5F). The above results suggested that NLRP3 overexpression could reverse the effects of the miR-30a-5p on HG-induced MPC5.

We extracted exosomes from MSCs. TEM and NTA assays revealed that the exosomes were cup-shaped, and their diameters ranged from 100 to 200 nm (Figs. 6A and 6B). The expression of CD63, HSP70, and calnexin in exosomes was examined by western blot analysis. CD63 and HSP70 were enriched in exosomes, while calnexin was expressed at lower levels (Fig. 6C). MiR-30a-5p was highly expressed in exosomes (Fig. 6D). The exosome uptake assay showed that the loaded exosome miR-30a-5p could be taken up by the podocytes (Fig. 6E).

To investigate the effect of exosomes on podocytes, we treated podocytes with exosomes obtained from MSCs and transfected with miR-30a-5p mimics. RT-qPCR results showed a good overexpression efficiency of miR-30a-5p (Fig. 7A). The flow cytometry results showed that exosome intervention inhibited HG-induced pyroptosis of podocytes (Fig. 7B). Further detection of pyroptosis-related proteins showed that exosome treatment inhibited NLRP3, GSDMD-N, and caspase-1 expression in HG-induced cells (Fig. 7C). In addition, the secretion of inflammatory factors in HG-induced podocytes was reduced by exosome intervention (Fig. 7D). Overall, the exosomes released by miR-30a-5p mimics-treated MSCs had a stronger reversal effect as compared to MSC exosomes alone.