Anti-ANGPTL3-FLD monoclonal antibody treatment ameliorates podocyte lesions through attenuating mitochondrial damage

Figures

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  Fig. 1. In ADR-induced nephropathy mice, anti-ANGPTL3-FLD monoclonal antibody (5E5F6) preserve podocytopathy. A Schematic figure of experimental design and 5E5F6 dosing plan. B Evaluation of proteinuria at 0 to 8th weeks. Figures show individual data of each animal and the mean ± SEM of the different groups at each time point (n = 6 per group). C The expression of Angptl3 in glomeruli of mice was detected by immunohistochemistry staining. D TUNEL staining in different groups. A total of 50 glomeruli per kidney were calculated. White arrow: Both TUNEL and WT1-positive staining cells. Original magnification 400×. E Representative immunohistochemistry images against podocyte marker Wilms tumor protein1 (WT1; brown stained nuclei) for control mice, ADR nephropathy mice, and 5E5F6 mAb-treated ADR nephropathy mice (ADR + mAb) Magnification ×400. Bars = 20 μm. F Representative images of light microscopy of kidney sections of control mice, ADR nephropathy mice (ADR), and 5E5F6 mAb-treated ADR nephropathy mice (ADR + mAb), stained with periodic acid–Schiff (PAS) in 8w. Original magnification: ×400, Bar = 20 μm. Arrows indicate tuft adhesion. G Average WT1-positive cell numbers per glomeruli, the…

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  Fig. 2. Characterization of the anti-ANGPTL3-FLD monoclonal antibody (5E5F6). A The ribbon structure of the human ANGPTL3-FLD and 5E5F6-Fab complex based on the computer-guided modeling and docking methods. B SPR sensorgram depicting the interaction of immobilized human ANGPTL3-FLD with 5E5F6. Rate constants (ka and kd) were determined by kinetic fitting of the sensorgrams using a one-to-one binding equation, and equilibrium dissociation constant (KD) values were determined by calculating kd/ka. The KD values of human ANGPTL3-FLD to 5E5F6 was 42.04 nM. C SPR sensorgram depicting the interaction of immobilized mouse ANGPTL3-FLD with 5E5F6. Rate constants (ka and kd) were determined by kinetic fitting of the sensorgrams using a one-to-one binding equation, and equilibrium dissociation constant (KD) values were determined by calculating kd/ka. The KD values of mouse ANGPTL3-FLD to 5E5F6 was 11.93 nM.

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  Fig. 3. Effect of 5E5F6 mAb on PAN-induced podocyte apoptosis. A Flow cytometry analysis of apoptosis in podocytes incubated with different media and quantification of these results (n = 3). B Cell viability of podocytes was analyzed using an MTT assay following different treatments (n = 3). C Western blot assay showed the expression of ANGPTL3 at different time points in human podocytes after PAN treatment. *p < 0.05, **p < 0.01 compared with the Ctrl group. D Relative expression of ANGPTL3, BAX, Bcl2, caspase 8, cleaved caspase 9 and cleaved caspase3 in different groups (n = 3). *p < 0.05, **p < 0.01 compared with the PAN group.

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  Fig. 4. 5E5F6 mAb reduced integrin β3 activation and ROS production in the podocyte. A The ribbon structure of the human ANGPTL3-FLD and integrin β3-b1 domain complex, human ANGPTL3-FLD and 5E5F6-Fab complex, human ANGPTL3-FLD, 5E5F6-Fab and integrin β3-b1 domain based on the computer-guided modeling and docking methods. B Analysis of the activity of 5E5F6 blocking the binding of ANGPTL3-FLD to Integrin αvβ3 by competitive ELISA assay, the EC50 is 1.57 ug/ml. C Immunofluorescence analysis of kidney tissue in different treatment groups. Kidney tissue were stained with AP5 antibody that recognizes the active form of integrin β3 and WT1 antibody to mark podocytes. Magnification in ×400. D Representative images showing AP5 staining in podocytes from different groups. E Fresh renal cortical tissues were obtained from the different treated mice and prepared as frozen cryostat sections for staining DCFH-DA (green), a marker of ROS. F Representative images of podocytes stained with DCFH-DA. G Average fluorescence intensity representing the level of integrin β3 activation detected. Data represent measurements of >50 cells and are plotted as means ± SD (n = 3). H Average fluorescence intensity…

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  Fig. 5. 5E5F6 mAb restored mitochondrial dysfunction in podocytes. A Representative TEM images of glomeruli showing mitochondrial morphology in podocytes of kidney sections, mitochondria swelling, vacuolization, and cristae fragmentation in podocytes were observed in ADR nephropathy mice. Arrows indicate representative mitochondria. Bar = 1 μm, original magnification 6800. B Representative TEM images showing mitochondrial morphology. Bar = 0.5 μm, original magnification 13,600. C The mitochondrial diameter in cultured podocytes with different treatments was quantified. D Representative images of podocytes stained with JC-1. E Mito-Tracker staining was used to assess the mitochondrial morphology in podocytes. Elongated mitochondria were observed in healthy untreated podocytes (arrows), shortened fragmented mitochondria were noted in podocytes injured by PAN. Treatment with 5E5F6 mAb restored the mitochondrial morphology. Scale bar 25 μm. F Representative images of podocytes stained with MitoSOX. G Relative mitochondrial DNA copy number (mtDNA:18 s rRNA) in podocytes. The results were normalized to the ratio of mtDNA: 18 s rRNA of healthy untreated podocytes. H JC-1 fluorescence…

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  Fig. 6. 5E5F6 mAb treatment mitigates Rac1 activation and restores mitochondrial function. A Representative western blot gel documents of Rac1-GTP, Rac1 in podocytes with different treatments. Relative expression of Rac1-GTP, Rac1 in different groups. B Representative western blot gel documents of NRF1, TFAM, MFN-1and DRP1 in podocytes with different treatments. Relative expression of NRF1, TFAM, MFN-1and DRP1 in different groups. *p < 0.05, **p < 0.01 compared with the PAN group. C Representative western blot gel documents of PINK1, Parkin, p62 in podocytes with different treatments. *p < 0.05, **p < 0.01 compared with the PAN group. D Immunofluorescence analysis of podocytes in different treatment groups. Cox IV: mitochondrial marker, LAMP1: lysosome marker. Quantification of the colocalization area of Cox IV and LAMP1. *p < 0.05, **p < 0.01 compared with the PAN group. Asterisk: lysosome accumulation. E Illustration of treatment with 5E5F6 mAb improves mitochondrial dysfunction in podocyte damage. In response to ADR or PAN exposure, ANGPTL3 is highly expressed in podocytes, with the activation of integrin αvβ3 and Rac1 GTPase, and subsequent generation of ROS. ROS-induced…