Fig 2: Validation of the relationship between SERPINA3 and DN. (A) Kaplan–Meier curve of SERPINA3 provided by dbPKD and the corresponding ROC curve. (B) Correlation between the SERPINA3 expression level and Log2GFR (GFR, glomerular filtration rate). (C) Correlation between median centered log2 (SERPINA3 expression value) and serum creatinine levels. Both GFR and serum creatinine level data were obtained from the database Nephroseq. (D) Comparison of the concentration of SERPINA3 proteins in serum samples between patients with DN and healthy controls. (E) Comparison of the concentration of SERPINA3 proteins in urine samples between patients with DN and healthy controls. (F) Correlation between the SERPINA3 protein concentration and urinary protein/creatinine levels. (G) The relative mRNA expression level of SERPINA3 in the HK-2 cells treated/untreated with high glucose levels was measured using qPCR. (H) SERPINA3 immunohistochemical staining in kidney tubular sections from patients with DN and healthy controls. Scale bars = 25 µm. (I) Semi-quantitative analyses of the immunohistochemically stained SERPINA3-positive tubule area. **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, no significance.

Fig 3: Correlation between SERPINA3 expression levels and chymase activity in mast cells. (A) Images of immunohistochemical staining for chymase activity in kidney tubular tissue samples from patients with DN and healthy controls. Scale bars = 100 µm. (B) Semi-quantitative analyses of the chymase activity area. (C) Observation of mast cell degranulation in the renal tubular tissue of patients with DN and healthy controls by toluidine blue staining. Red circles indicate cells in a degranulated state, while blue circles indicate cells in a resting state. Scale bars = 250 µm (images above) and scale bars = 50 µm (images below). Microscopes were used at magnifications of 100× and 500×, respectively. (D) Histogram of the number of activated and resting mast cells in the DN and control groups. (E) Proportion of mast cell degranulation observed in patients with DN and healthy controls determined by toluidine blue staining. Data shown are the mean ± SEM (n = 4). (F) Colocalization of SERPINA3 (green) and chymase (red) in tubule tissue. *P < 0.05 and ****P < 0.0001.

Fig 4: (A) The expression distribution of risk score IRGs in the short-survival time group and the long-survival time group. The asterisks represent levels of significance *p < 0.05, ns: not statistically significant. (B) Western blot analysis of four differentially expressed IRGs in the signature model. The protein expression level of TNC in the short survival group was significantly upregulated, and the protein expression level of SSTR2 was significantly downregulated. There was no significant difference in the expression of SERPINA3 and TNFRSF19 between the two groups. (C) High expression of SSTR2 was negatively related to tumor invasion, EMT and metastasis. (D) High expression of TNV was positively related to tumor metastasis and hypoxia.