Targeting ANGPTL3 and IL‐33/ST2 Ameliorates Diabetic Kidney Disease by Reducing Lipotoxicity, Alleviating Inflammation and Inhibiting Fibrosis

FIGURE 1. Expression of ANGPTL3 and IL‐33 in murine DKD models and patients with DKD. (A) Gene expression of ANGPTL3 and IL‐33 in control mice (n = 5) and DKD mice (n = 5). (B) GSEA of pathways related to negative regulation of ANGPTL3 and IL‐33. (C) Il33 mRNA expression in kidney tissue and serum IL‐33 levels in control mice (n = 8) and DKD mice (n = 8). (D) IL‐33 gene expression in human controls (n = 9) and patients with DKD (n = 21). (E) Serum IL‐33 levels in human controls (n = 20) and patients with DKD (n = 20). (F) Correlation of IL‐33 with serum CRE and serum BUN in patients with DKD. Data in (A, C, D, E) are presented as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant. Statistical significance was assessed by one‐way ANOVA with Tukey's multiple‐comparisons test.

FIGURE 2. Expression, purification, and characterization of FD03‐sST2. (A) Schematic illustration of protein design, generation, and expression. (B) SDS‐PAGE of purified proteins under reducing and non‐reducing conditions. (C) SEC‐HPLC profiles of purified proteins (RT, retention time). (D) Particle size distribution of the proteins. (E) Thermal stability assessment of the proteins.

FIGURE 3. Binding affinity and bioactivity of FD03‐sST2. (A–C) Surface plasmon resonance analysis of FD03 and FD03‐sST2 binding to murine half‐length ANGPTL3 (A), human full‐length ANGPTL3 (B), and of sST2‐Fc and FD03‐sST2 to murine IL‐33 (C). (D) FD03‐sST2 exhibited ANGPTL3‐binding affinity comparable to FD03 and IL‐33‐binding affinity similar to sST2‐Fc. (E) FD03 and FD03‐sST2 counteracted ANGPTL3‐mediated LPL inhibition in a dose‐dependent manner, as determined by fluorescence intensity of a lipase substrate. (F) sST2‐Fc and FD03‐sST2 suppressed IL‐33‐stimulated luciferase reporter activity, measured by fluorescence intensity.

FIGURE 4. Reno protective effects of FD03‐sST2 in db/db mice: functional improvement and histopathological amelioration. (A) Experimental timeline and dosing protocol for protein administration. (B) Temporal profile of body weight changes (n = 8). (C) Longitudinal ACR measurements throughout the 6‐week intervention (n = 8). (D) ACR, serum BUN, and 12 h urine volume after 6 weeks of protein treatment in db/db mice (n = 8). (E) Representative photomicrographs of renal histology: H&E and PAS trichrome staining (scale bar = 20 µm). (F) Quantification of the glomerular mesangial index (n = 13). Data in (B, C, D, F) are presented as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant. Statistical significance was assessed by one‐way ANOVA with Tukey's multiple‐comparisons test.

FIGURE 5. FD03‐sST2 alleviated hepatic and renal steatosis in db/db mice. (A) GLU, serum TC, serum TG, and LDL‐C after 6 weeks of protein administration (n = 8). (B) KEGG enrichment of lipid metabolism‐related genes significantly downregulated in FD03‐sST2‐treated mice. (C) Oil Red O staining of neutral lipids (NLs) in kidney and liver sections (scale bar = 200 µm). (D) Quantification of Oil Red O‐positive staining in kidney (n = 8) and liver (n = 8). (E) OPLS‐DA score plots from metabolomics analyses in BEH negative‐ion mode and C18 positive‐ion mode (n = 6). (F) Heat map of significantly altered glyceride‐related metabolites among db/m, db/db, and FD03‐sST2‐treated mice. Data in (A, D) are presented as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant. Statistical significance was assessed by one‐way ANOVA with Tukey's multiple‐comparisons test.

FIGURE 6. Modulation of the NF‐κB/NLRP3 inflammasome pathway by FD03‐sST2 in db/db mice. (A) Heat map of significantly altered inflammation‐related metabolites among db/m, db/db, and FD03‐sST2‐treated mice. (B, C) Western blot analysis of p65 and IκBα in renal lysates post‐treatment; GAPDH was used as an internal loading reference (n = 6). (D) KEGG enrichment of inflammation‐related genes significantly downregulated in FD03‐sST2‐treated mice. (E) Representative immunohistochemical images of NLRP3 in renal sections at 100× and 400× magnification; scale bars: 200 µm (100×) and 50 µm (400×). (F) Quantification of NLRP3 staining in kidney sections (n = 9). (G) Renal Nlrp3 mRNA expression after protein administration (n = 8). (H) Serum IL‐1β levels and renal Il1b mRNA expression after protein administration (n = 8). (I, J) Western blot analysis of caspase‐1 and its cleaved form in renal lysates following treatment (n = 6). Data in (C, F, G, H, J) are presented as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant. Statistical significance was assessed by one‐way ANOVA with Tukey's multiple‐comparisons test.

FIGURE 7. FD03‐sST2 mitigates renal fibrosis in db/db mice through suppression of ILC2‐derived fibrosis‐related factors (A) Representative images of kidney sections: Masson's trichrome (scale bar = 20 µm) and immunohistochemical staining ofα‐SMA and fibronectin (scale bar = 20 µm). (B) Quantitative analysis of collagen deposition based on Masson‐stained sections (n = 14). (C) Quantitative assessment of renal α‐SMA (n = 8) and fibronectin (n = 8) levels following treatment. (D) Representative immunofluorescence micrographs of db/db mouse kidneys (n = 8) showing DAPI (blue), IL‐33 (green), and GATA3 (red). Magnification: 100× and 400×; scale bars: 200 µm (100×) and 100 µm (400×). (E) KEGG enrichment of fibrosis‐related genes significantly downregulated in FD03‐sST2‐treated mice. (F) Transcript levels of TGF‐β, fibronectin, α‐SMA, and collagen I in cultured ILC2s (n = 6). Data in (B, C, F) are presented as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant. Statistical significance was assessed by one‐way ANOVA with Tukey's multiple‐comparisons test.

FIGURE 8. Proposed model of DKD progression and the role of FD03‐sST2 in improving dysregulated lipid metabolism, alleviating inflammation, and inhibiting fibrosis.