Conditionally immortalized mouse podocytes (BLUEFBIO Biotechnology Company, Shanghai, China, ATCC number BFN60700330) were cultured in RPMI 1640 medium. Cells were grown in a 5% CO₂ humidified atmosphere at 33 °C and medium containing 100 U/ml IFNγ and 10% fetal bovine serum (FBS). Then podocytes were exposed to 37°C without IFNγ for 10 days to induce differentiation. The differentiated podocytes were grown in serum-free RPMI 1640 medium for 24 h prior to the experiment, which followed by treatment with 0.1% DMSO (solvent control DMSO) or glucose (HG, 40 mM, G7021, Sigma) or quercetin (5280343, Sigma, St. Louis, MO, United States) at a concentration of 10 μmol/L (Q10), 20 μmol/L (Q20), 40 μmol/L (Q40) or 1 μmol/L AG1478 (2934816, EMD Millipore, DE) for 24 h.

Eight-week-old genetically diabetic C57BL/KSJ db/db mice and their age-matched nondiabetic C57/KSJ db/m littermates (used as control animals) were obtained from the Model Animal Research Center of Nanjing University, following the Guiding Principles for Care and Use of Laboratory Animals of Xuzhou Medical University. All mice used in experiments were male. The mice were housed in an animal facility conditioned with 12–12 h light-dark cycles and allowed free access to normal food and water. After acclimatization for 8 weeks, the mice were randomly divided into five groups with at least six mice in each group. The average initial body weight of each group was not significantly different (p > 0.05). These treatment groups were designated as db/m group (control group), db/db group (diabetes model group), QL group (low-dose quercetin-treated group, 50 mg kg⁻¹), QM group (medium-dose quercetin-treated group, 100 mg kg⁻¹), and QH group (high-dose quercetin-treated group, 150 mg kg⁻¹). Quercetin was dissolved in 0.5% carboxy methyl cellulose (CMC-Na) according to the expected dose of the treatment groups and was given to the mice via intragastric administration, whereas the mice of the db/m group and db/db group were given 0.5% CMC-Na through the same administration method for 8 weeks. After administration for 8 weeks, the mice were sacrificed and part of the kidney tissue was fixed in 4% paraformaldehyde, while the remaining tissue was stored at –80°C for biochemical analysis. Animal experiments were conducted in accordance to the principles provided by the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Experiments were conducted with approval of the Animal Ethics Committee of Xuzhou Medical University, which also conformed the Guidelines for Ethical Conduct in the Care and Use of Animals.

Network pharmacology was carried out to identify the interactions between compounds and network target proteins. The putative quercetin targets were screened from ETCM (http://www.tcmip.cn/ETCM/), SymMap (http://www.symmap.org/), TCM-MESH (http://mesh.tcm.microbioinformatics.org/), TCMSP (https://old.tcmsp-e.com/tcmsp.php) four databases. Similarly, information on DN-associated target genes were gathered from the following databases: DrugBank (https://www.drugbank.ca/), OMIM (https://www.omim.org/), DisGeNet (http://www.disgenet.org/), Proteomics, then the possible targets of quercetin against DN were screened through the overlap analysis between putative targets of quercetin and known DN-associated targets. The potential target gene in quercetin was mapped to the disease target gene by using the ImageGP platform, and a Venn diagram was drawn to show results. In addition, the protein-protein interaction (PPI) network analysis was constructed to determine the potential targets and inherent pathways for investigating the actions of drugs by STRING (https://string-db.org/). Then, 12 topology analysis algorithms of Cytoscape 3.2.1 were used to screen out the key targets in PPI network. Screening of significant therapeutic targets was based on the high throughput reverse molecular docking (Gao et al., 2016; Sheng et al., 2017), hub results and the tissue distribution. The database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/) was used to analyze the Gene Ontology (GO) function and KEGG pathway enrichment of significant therapeutic targets. Finally, the relationships between these significantly enriched pathways and DN were further validated by literature reports.

Fasting blood glucose (FBG) was measured with a glucose assay Kit (Jiancheng Bioengineering Institute, Nanjing, China). Urinary albumin and the creatinine levels were detected by ELISA kits (Lanpai Biotechnology, Shanghai, China). The UACR (mg/g) was computed as urinary albumin/urinary creatinine. Blood urea nitrogen (BUN) was measured with ELISA kits. The ELISA kits were purchased from Lanpai Biotechnology (Shanghai, China). Results are expressed as the mean ± SEM. These biochemical indices were measured for estimating the progression of DN.

Tissue sections of 4-μm thickness were prepared from paraffin-embedded kidney tissue. The sections were stained with periodic acid Schiff (PAS) (Liu et al., 2018), periodic acid-silver metheramine (PASM), Masson, and Sirius red after deparaffinization. Staining was conducted to assess kidney morphology, the glomerular basement membrane, glycogen deposition, and collagen accumulation. These kits were purchased from Solarbio, Beijing, China. Photographs were taken randomly and blindly under a microscope (OLYMPUS, Tokyo, Japan). Quantification of staining was performed using Image Pro Plus 6.0 and was expressed as the positive region. Representative views were shown.

Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay (Roche, United States) is used to detect the nuclear DNA fragmentation of tissue cells in the early process of apoptosis. Sections were dewaxed and rehydrated according to standard protocols. After deparaffinization, sections were incubated with proteinase K working solution and prepared TUNEL reaction mixture for 15 min at 37°C. Sections were counterstained with DAPI (Beyotime Institute of Biotechnology, Nantong, China). The number of TUNEL positive cells per DAPI positive cells was used for quantitation of apoptotic cell.

Hoechst 33342 is a blue fluorescent dye that can penetrate cell membranes and is less toxic to cells. It was often used to detect apoptosis and the staining was observed by fluorescence microscope. The Annexin-V-FITC Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, United States, catalog no. 556547) was used to detect apoptosis by flow cytometry. Cells were exposed to various conditions of treatment for 24 h, and they were harvested and processed according to the manufacturer’s instructions. Cells were considered viable if FITC Annexin V and PI staining were all negative; early apoptotic if FITC Annexin V staining was positive with negative PI staining; and late apoptotic or already dead if both FITC Annexin V and PI staining were positive (Li et al., 2017; Yao et al., 2020).

The fixed kidney section of a 4 μm thickness (Leica Company, Germany, RM2235) were deparaffinized in xylene 3333 rehydrated in a graded series of alcohols. Subsequently the sections were placed in 3% H₂O₂ for 10 min to eliminate endogenous peroxidase activity. After pepsin antigen retrieval for 30 min, the sections were washed with PBS 3 times for 3 min each time and then blocked with 2% BSA for 0.5 h at room temperature. The sections were incubated with mouse anti-synaptopodin antibody (1:200, santa cruz biotechnology, sc-515842) at 37°C for 2 h or 4°C overnight. The sections were stained using a polymer HRP detection system (ZSGB-BIO, Beijing, China) and visualized with a DAB detection kit (Vector Laboratories Inc., Burlingame, CA, United States). After conventional dewatering and neutral balsam mounting, photographs were blindly taken at random felds under an Olympus BX43F fuorescence microscope (OLYMPUS, Japan) (Chen et al., 2019).

Differentiated podocytes were fixed with cold methanol at −20°C for 20 min and permeabilized with 0.1% Triton X-100/PBS. After they were washed three times with cold PBS. The cells were blocked with 2% bovine serum albumin (BSA) for 1 h at room temperature and incubated with mouse anti-synaptopodin antibody (1:200, santa cruz biotechnology, sc-515842) or rabbit anti-cleaved caspase three antibody (1:400 Cell Signaling Technology, 9661) at 37°C for 2 h or 4°C overnight. Then, the cells were washed three times with PBS and incubated with a secondary antibody conjugated to DyLight 488 or DyLight 594 (Earthox, Millbrae, CA, United States) at 37°C for 1 h, respectively. Nuclei were counterstained with DAPI. The coverslips were mounted onto glass slides, and the images were viewed with an Olympus BX43F fluorescence microscope (OLYMPUS, Japan).

Protein analysis was performed on mouse renal cortex tissues (Ji et al., 2017) and cultured podocytes as described previously. EGFR, p-EGFR and cleaved caspase three antibodies were purchased from Cell Signaling (Beverly, MA, United States). ERK1/2, p-ERK1/2, Bcl2, Bax and synaptopodin antibodies were purchased from Abcam (Cambridge, United Kingdom). β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

All data were presented as the means ± SEM. Statistical analysis was performed using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, United States). Statistical differences were determined using analysis of variance followed by Dunnett’s test (Exp. versus Con.) using one trial analysis. A significant difference was defined as p < 0.05 compared with the control.

The renal function levels of the experimental mice were examined after treatment with quercetin for 8 weeks. BUN and UACR levels are important indicators of renal function. The levels of BUN and UACR in diabetic mice treated with quercetin were significantly decreased, compared with those in the diabetic mice. In parallel, the FBG levels of the diabetic mice were obviously retrieved. During the period of the study, quercetin effectively prevented the progression of albuminuria as shown in Table 1. Collectively, these results indicated quercetin exerts protective effects on renal function.

To investigate the protection of quercetin toward DN, a series of staining methods was used in this study. PASM staining is an essential adjunct to the evaluation of change in the GBM. The GBM was markedly thickened in the diabetic group compared with the normal group when observed by PASM stain. Glycogen and collagen are components of the extracellular matrix. PAS, masson and sirius redstaining showed that glycogen and collagen deposition was enhanced in the diabetic mice renal cortex compared with control group, however, administration of quercetin in medium-dose and high-dose effectively reversed these levels (Figures 1A–E). We quantified expression levels of a podocyte marker protein, nephrin, indicated much less nephrin expression in diabetic mice by immunofluorescence (Figure 1F), immunohistochemistry (Figures 1G,H) and western blotting analysis (Figure 1I,J). However, treatment with quercetin significantly prevented reduction of nephrin. These results indicated that quercetin protects against glomerular injury in diabetic mice.

TUNEL assay was used to detect apoptosis in glomeruli for observing the effect of quercetin on podocyte apoptosis under diabetic conditions. The number of apoptotic cells in glomeruli was noticeably increased from diabetic mice compared to the control group (Figure 2A, B. Further results showed that the pro-apoptotic proteins Bax and cleaved Caspase-3 were significantly up-regulated and the anti-apoptotic protein Bcl-2 was markedly down-regulated in diabetic mice (Figure 2C, D). Above changes were reversed by the administration of quercetin, which demonstrated that quercetin inhibits apoptosis in diabetic mice.

We obtained 161 drugs targets and 640 DN-related disease protein targets from respective databases. A total of 56 potential targets were gained based on the intersection of protein targets acting on quercetin and these are related to DN by using the ImageGP platform. The targets were exhibited in PPI and the key targets were used to screen out through 12 topology analysis algorithms of Cytoscape 3.2.1. The six key targets MPO, MMP9, MMP2, MMP3, EGFR, AKT1 were screened in this process, which suggested that these six targets probably served as significant therapeutic targets in DN. Then the tissue distribution of these targets indicating that EGFR, MMP2, and AKT1 were expressed in the kidney. GO analysis revealed that the functions of these potential targets are related to cell proliferation, differentiation and apoptosis. In the last step, we found that EGFR enriched the most pathways by KEGG pathway enrichment analysis. Therefore, EGFR was choosed as the target for quercetin intervention in DN, which synthesized all of the above analysis. All were shown in Figures 3, 4. In addition, we evaluated the interaction between quercetin and EGFR by molecular docking scores (Supplementary Figure S1). The result showed that the docking score was -8.396, indicating a better combination of quercetin with EGFR.

The expressions of total EGFR, phospho-EGFR and the downstream ERK in this pathway were examined to observe whether the EGFR signaling pathway can be activated in diabetic mice. Even though the raise in total EGFR and ERK expression did not reach statistical significance, the expression of phospho-EGFR and phospho-ERK markedly increased in diabetic mice compared with the normal group, and immunohistochemical results showed the same changes Figure 5A, C), suggesting EGFR was activated under diabetic conditions and quercetin could down-regulate the expressions of phospho-EGFR and phospho-ERK in diabetic mice (Figure 5B, D). Collectively, the data demonstrated that quercetin inhibited the EGFR pathway.

In vitro, we evaluated the role of EGFR signaling through examining the expressions of phospho-EGFR and total EGFR and the downstream ERK in cultured podocytes, which were treated with HG, quercetin (40 μM) and AG1478 (EGFR inhibitor). DMSO was used as a control group for drug solvents and played no effect on their expression. CCK8 assay was used to investigate the cytotoxicity of the quercetin in podocyte. The data demonstrated that 40 μM of quercetin treatment for 24 h did not generate any conspicuous cytotoxic effects, whereas 80 μM significantly declined cell viability (Figure 6B). Then we examined cell viability under different treatments to further evaluate whether quercetin protects podocyte against HG-induced apoptosis. High glucose inhibited cell viability, while treatment with quercetin raised cell viability dose dependently. Thus, doses of 10, 20 and 40 μM quercetin were used to examine its potential ability to prevent against DN in the following experiments. Western blotting results showed that the same changes in HG-induced podocyte as in vivo (Figure 6C), suggesting EGFR was activated under high glucose conditions, and quercetin could down-regulate the high expressions (Figure 6D). The differentiated podocytes were treated with HG for 24 h prominently increased EGFR phosphorylation, and pretreatment of the differentiated podocytes with the EGFR tyrosine kinase inhibitor (AG1478) and HG markedly prevented EGFR (Figure 6E, F, Supplementary Figure S2). So we used AG1478 to further explore the mechanism of quercetin on DN.

Podocyte apoptosis was detected by Annexin V-staining (Figure 7C, E), immunofluorescence and western blot analysis. We found that quercetin reversed HG-induced podocyte apoptosis through examining the expression of Bcl-2, Bax and cleaved caspase-3 (Figure 7A, B, G, H). To further investigate whether quercetin improved HG-induced podocyte apoptosis through the EGFR pathway, we used AG1478 to inhibit EGFR and then added quercetin to observe its effect on podocyte apoptosis. Compared to Q40 group, the improvement level of podocyte apoptosis was reduced in Q40 + AG1478 group (Figure 7D, F, I, J), indicating that quercetin supressed HG-induced podocyte apoptosis through the EGFR pathway.

We identified expression levels of synaptopodin and nephrin by immunofluorescence and western blotting analysis. As shown in Figures 8A–F, quercetin raised the low expression of synaptopodin and nephrin in HG-induced podocytes, but the upregulation effect was supressed in Q40 + AG1478 group (Figure 8G–L), verifying that quercetin inhibited HG-induced podocyte injury through the EGFR pathway.