A novel fusion protein consisting of anti-ANGPTL3 antibody and interleukin-22 ameliorates diabetic nephropathy in mice

Figures

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  Figure 1. Expression and purification of anti-ANGPTL3/IL22 fusion protein. (A) Schematic representation of the preparation and purification of anti-ANGPTL3/IL22 fusion protein. Mouse IL22 was fused to the C-terminus of the anti-ANGPTL3 heavy chain via a flexible linker (linker sequence: GGGGSGGGGSGGGGS). (B) Schematic representation of the preparation and purification of anti-ANGPTL3. (C) Schematic representation of the preparation and purification of mIL22Fc (Mouse IL22 and mouse IgG2a Fc fusion protein). (D) SDS-PAGE analysis of anti-ANGPTL3 (lane 1), anti-ANGPTL3/IL22 (lane 2) and mIL22Fc (lane 3) under reducing condition (anti-ANGPTL3: approximately 150 KD, IL22Fc: approximately 90 KD, anti-ANGPTL3/IL22: approximately 190 KD). (E) SEC-HPLC analysis of anti-ANGPTL3 and anti-ANGPTL3/IL22 fusion protein (RT, retention time). (F) Thermal stability analysis of anti-ANGPTL3, anti-ANGPTL3/IL22 and mIL22Fc.

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  Figure 2. Affinity of anti-ANGPTL3 and anti-ANGPTL3/IL22 for ANGPTL3 and the validation of in vitro activity. (A) Affinity of anti-ANGPTL3 for mouse (S17-T455) and human (S17-E460) ANGPTL3 measured by surface plasmon resonance (SPR). (B) Affinity of anti-ANGPTL3/IL22 for mouse and human ANGPTL3 measured by SPR. (C) Affinity summaries of anti-ANGPTL3 antibody and anti-ANGPTL3/IL22 fusion protein suggested that the functional integrity of anti-ANGPTL3 was preserved. (D) Mouse podocytes (MPC5) were first incubated with anti-ANGPTL3, mIL22Fc, anti-ANGPTL3/IL22 and FGDyk for 1 hour, followed by the addition of high concentration glucose (30 mmol/L) for 6 hours. Then, the cells were stained with AP5 and Hoechst 33342 to assess intracellular exposed PSI epitopes. (Scale bar: 20 μm). (E) Statistical analysis of AP5 levels by immunofluorescence staining (n = 4). (F) After mouse proximal tubular epithelial cells (mPTC) were incubated overnight, 10 nM anti-ANGPTL3/IL22-induced phosphorylation of STAT3 (p-STAT3) was analyzed by Western blot at different times. (G) Densitometric values of p-STAT3 expression after 2 h, 4 h and 6h. (n = 4). (H) Western blot analysis of p-STAT3 induced by 10 nM mIL-22Fc…

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  Figure 3. Efficacy in improving renal function of anti-ANGPTL3/IL22 fusion protein in DN mice. (A) The tendency of ACR and serum BUN during eight weeks of drug administration. (B) Quantitative analysis of body weight trends. * and # respectively represent the Student’s t-test of db/db group and anti-ANGPTL3/IL22 treatment compared with db/m group at twelve week. $ and & respectively represent the Student’s t-test of anti-ANGPTL3 and mIL22Fc treatment compared with anti-ANGPTL3/IL22 group. (C) Renal index (kidney weight/body weight) was measured to reveal the degree of kidney injury. (D-G) Trends of ACR, serum BUN, 24-hour urine volume and urine protein in db/db mice after 8 weeks of treatment with anti-ANGPTL3/IL22. (H-J) Measurements of blood glucose, serum triglyceride and serum T-CHO after the 8 weeks of anti-ANGPTL3/IL22 administration. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. & P < 0.05; && P < 0.01. $ P < 0.05; $$ P< 0.01.### P < 0.001.

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  Figure 4. Treatment with anti-ANGPTL3/IL22 attenuated histological damage in DN mice (A) Representative images of PAS staining of kidney tissue from db/db mice. (Scale bar: 100 μm, 200 ×; 50 μm, 400 ×). (B) Expressions of nephrin and WT1 were detected by immunohistochemical staining. (Scale bar: 20 μm and 50 μm) (C) Quantification of WT1-positive cells in the glomerulus. (D) Statistical analysis of nephrin-positive areas in the kidney. (E, F) Representative electron microscopic images in db/db mice and quantitative analysis of glomerular basement membrane (GBM) thickness after the treatment of anti-ANGPTL3/IL22. (Scale bar: 10 μm) (n = 5-7). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Figure 5. Anti-ANGPTL3/IL22 fusion protein reduced renal fibrosis in db/db mice. (A) Representative Masson staining images of mouse kidney tissues after 8 weeks of anti-ANGPTL3/IL22 treatment. (B) Measurements of Masson-positive staining. (Scale bar: 100 μm, 200 ×; 50 μm, 400 ×). (C) The levels of vimentin in the kidney sections were measured by immunofluorescence. (Scale bar: 50 μm). (D) Statistical assessment of vimentin expression in the kidney sections. (n = 5). (E) The levels of α-SMA in the kidney sections were measured by immunofluorescence. (Scale bar: 50 μm). (F) Quantitative analysis of α-SMA expression (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001 ; ****P<0.0001.

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  Figure 6. Anti-ANGPTL3/IL22 fusion protein attenuated the inflammatory response in db/db mice. (A-C) Measurements of serum inflammatory factors including TNF-α, IL-1β and IL-6 in db/db mice after treatment with anti-ANGPTL3/IL22. (D) Representative immunohistochemical images of NF-κB p65 in the kidney sections. (Scale bar: 20μm). (E) Representative immunohistochemical images of NLRP3 inflammasome in the kidney sections. (Scale bar: 50 μm). (F) Quantitative analysis of NF-κB. (n = 3). (G) Quantitative analysis of NLRP3 inflammasome (n = 5). *P < 0.05; **P < 0.01; ****P< 0.0001.

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  Figure 7. Transcriptomic analysis of db/db mice treated with anti-ANGPTL3/IL22. (A) Differential expression of genes related to inflammation, podocyte injury and metabolism in db/m mice compared to db/db mice were shown in volcano plots (log2 fold change ≥ 1 or ≤ -1 in the horizontal coordinate and p value ≤ 0.05 in the vertical coordinate). (B) Differential expression of genes related to inflammation, podocyte injury and metabolism (log2 fold change ≥ 1 or ≤ -1 in the horizontal coordinate and p value ≤ 0.05 in the vertical coordinate) in db/db compared to anti-ANGPTL3/IL22 treatment mice. (C) Cluster analysis of differentially expressed genes including inflammation-related, metabolism-related and podocyte damage process-related genes between db/m and db/db mice. (D) Cluster analysis of differentially expressed genes including inflammation-related, metabolism-related and podocyte damage process-related genes between anti-ANGPTL3/IL22 treated mice and db/db mice. (E) KEGG analysis of genes significantly up-regulated in db/db mice. (F) KEGG analysis of genes obviously down-regulated in anti-ANGPTL3/IL22 administration mice.