Two gene expression datasets (GSE30529 and GSE86300) were obtained from the GEO database, a public functional genomics data repository of high throughput gene expression data.¹ The GSE30529 dataset contained 10 human diabetic kidney disease renal tissue samples and 12 control tissue samples. The GSE86300 dataset contained 5 db/db mouse renal tissue samples and 5 control tissue samples.

Eight-week-old db/db mice obtained from Junke Biological Company (Nanjing, China) were randomly divided into two groups. For repertaxin experiments (db/db + repertaxin, n = 6), the mice in the db/db + repertaxin group were treated with repertaxin (20 mg/kg/day) by intragastric administration once every 2 days for 12 weeks. At the age of 20 weeks, the mice were euthanized and kidney, urine and serum samples were harvested for subsequent experimental measurements. To generate HFD + STZ-induced DN mice, 8-week-old C57BL/6 mice were fed a high-fat diet for 4 weeks and then treated with streptozotocin (STZ) (Sigma-Aldrich) in citrate buffer, pH 4.5 (50 mg/kg) for five consecutive days by intraperitoneal injection. After 2 days, the blood glucose level was higher than 16.7 mmol/L, indicating successful modeling of diabetes. After continued feeding for 12 weeks, the mice were euthanized, and kidney and serum samples were harvested for the subsequent experimental measurements. All mice were housed in quiet rooms at 22–26°C with a 12-h light-dark cycle. All animal experiments were conducted in accordance with the relevant institutional guidelines of the Animal Experimentation Ethics Committee of Second Xiangya Hospital of Central South University.

Diabetic nephropathy patients (n = 6) were diagnosed by renal biopsy and cohorts with non-diabetic renal diseases, such as a minor glomerular lesion (GML) (n = 6), were used as the control group. The Institutional Human Experimentation Ethics Committee, Second Xiangya Hospital, Central South University approved and supervised all experiments described above.

Hematoxylin-eosin (H&E) and Masson trichrome staining were used to assess kidney damage in 4-μm-thick paraffin-embedded kidney sections from patients with renal biopsy or DN mice. The degree of tubulointerstitial injury was evaluated by a semiquantitative scoring system as described previously (Yang et al., 2019).

To observe CXCL1/CXCR2 expression and inflammatory infiltration, IHC staining was performed on 4-μm-thick paraffin-embedded kidney sections with a Servicebio immunohistochemical kit (G1212-200T). After deparaffinization, rehydration, antigen repair, and blocking, paraffin sections of renal tissues were incubated with CXCL1 (Proteintech, 1:200), CXCR2 (Proteintech, 1:100), and F4/80 (Servicebio, 1:1,000) for 12 h in 4°C, The tissues were incubated with HRP-conjugated secondary antibody (Abcam, 1:200) for 30min at 37°C. Then, a DAB kit was used for development, followed by observation under a light microscope.

According to the manufacturer’s instructions (16), terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining was used to evaluate apoptotic cells in renal tissues from different groups (Yang et al., 2019).

The human proximal tubular epithelial cell line HK-2 was cultured with DMEM/F12 containing 10% FBS in a 37°C incubator containing 5% CO₂. After overnight culture with DMEM/F12 without FBS, the HK-2 cells were pretreated with repertaxin (10 μM) 30 min in advance before high-glucose (HG) treatment. After an additional 24 h of culture, the cells were collected for subsequent experiments.

Total RNA was extracted from kidneys and HK-2 cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and quantified by a NanoDrop 2,000 (Thermo Fisher Scientific, Madison, WI, United States). A PrimeScript reagent kit with gDNA Eraser (Takara) was used to synthesize first-strand cDNA. Real-time PCRs were performed with SYBR GreenER qPCR SuperMix (Thermo Fisher Scientific) in a 7300 Real-Time PCR System (Applied Biosystems, CA).

Protein from mice and HK-2 cells was collected by radioimmunoprecipitation assay (RIPA) buffer with protease or phosphatase inhibitor, and the protein was quantified by a BCA Protein Assay Kit (Beyotime Biotechnology, China). Then, 5 × SDS loading buffer was added, followed by boiling for 10 min. Equal amounts of proteins were used for western blot analysis.

SPSS 13.0 software was used for statistical analysis of the experimental data. The results are presented as the mean ± SE. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis was used to compare differences among the groups. Statistical significance was indicated at a P-value less than 0.05.

Through the GEO database, we found that 628 and 340 genes were upregulated and 158 and 151 genes were downregulated, with significant changes in the GSE30529 and GSE86300 datasets, respectively (Figures 1A,B). Forty-six overlapping upregulated genes (Figure 1C) and 6 overlapping of downregulated genes (Figure 1D) were identified in the two datasets. KEGG pathway analysis revealed that these genes were primarily concentrated in the inflammatory pathway (Figure 1E). The PPI network of the 52 DEGs was constructed by the STRING database (Figure 1F).

To elucidate whether inflammation levels increase in DN states, we detected inflammatory markers in the renal tissues of mice by RT-PCR. As shown in Figures 2A–C, IL-6, IL-1β, and TNF-α mRNA expression was significantly increased in the kidneys of STZ-induced DN mice compared with the control mice. The results show an increased level of inflammation in the kidneys in the DN state. In addition, we investigated the potential role of CXCL1/CXCR2 in the development of DN. HE and Masson staining were used to evaluate pathological lesion changes in the kidneys of DN mice and patients. Exfoliated tubule cells, enlarged lumens, increased fibrosis and increased tubular interstitial damage scores were observed (Figures 2D,E), which were accompanied by increased expression of CXCL1/CXCR2 in DN mice compared to the controls (Figures 2F,G). Similarly, IHC staining also showed increased expression of CXCL1/CXCR2 in DN mice and patients (Figures 2H–K). The results implied that CXCL1/CXCR2 mediated inflammation may play a vital role in the occurrence and development of diabetic nephropathy.

To further explore the role of the CXCL1/CXCR2 axis in DN development, repertaxin (a previously reported CXCR2 inhibitor) was used to block the CXCL1/CXCR2 axis in db/db mice. Compared with that in the control group, the UACR level was increased in db/db mice and decreased after repertaxin treatment (Figure 3A). Similarly, the mRNA expression of inflammatory makers (IL-6, IL-1β, and TNF-α) (Figures 3B–D) and pathological renal injury (Figures 3E,F) were significantly increased in the kidneys of db/db mice compared with the control group mice, while repertaxin mitigated these adverse changes (Figures 3B–F). Similarly, IHC staining revealed that CXCL1 and CXCR2 expressions were increased in the kidney of db/db mice compared to the db/m group, and their expressions were notably decreased after treatment with repertaxin (Figures 3G,H). F4/80 and TUNEL staining showed increased inflammatory cell infiltration (Figures 3I,J) and apoptosis (Figures 3I,K) in the db/db mice, and repertaxin partially block this effect (Figures 3J,K). These data suggest that when renal tissue is exposed to HG stimulation, the CXCL1/CXCR2 axis is activated, resulting in renal inflammation, while inhibiting the CXCL1/CXCR2 axis with repertaxin may relieve renal inflammation and pathological damage in DN.

Previous studies have demonstrated that the NF-κB/NLRP3 inflammasome signaling pathway is activated in DN and accelerates renal damage (Yi et al., 2017; Tian et al., 2021), while Liu et al. (2020) also showed that NF-κB is downstream of the CXCL1-CXCR2 signaling pathway. Therefore, we detected the expression of CXCL1/CXCR2-NF-κB-NLRP3 in HK-2 cells with HG intervention. Consistent with the in vivo studies, CXCL1/CXCR2 expression was increased in the HG group compared with the LG group, while repertaxin inhibited their expression (Figures 4A,B). Next, we detected the expression of the components in the NF-κB/NLRP3 pathway. Consistent with the changes in CXCL1/CXCR2, the phosphorylation of NF-κB was notably increased in HK-2 cells with HG treatment compared with that in LG group, and when CXCL1/CXCR2 was inhibited, its phosphorylation decreased (Figures 4C,D). In addition, the expression of the NLRP3 inflammasome and other inflammatory factors was detected by RT-PCR. Treatment with HG resulted in an increase in NLRP3 (Figure 4E), IL-18 (Figure 4F), IL-1β (Figure 4G), TNF-α (Figure 4H), and IL-6 (Figure 4I), while suppression of CXCL1/CXCR2 mitigated these adverse changes. Finally, the fibrosis-associated proteins FN and α-SMA were detected by WB analysis. As shown in Figures 4J,K, inhibition of CXCL1/CXCR2 alleviated the increase in fibrosis caused by HG treatment.