Volume 59, Issue 6, Pages 2134-2145 (June 2001) Electroporation-mediated PDGF receptor-IgG chimera gene transfer ameliorates experimental glomerulonephritis  Hiroyuki Nakamura, Yoshitaka Isaka, Michiko Tsujie, Yoshitaka Akagi, Tetsuo Sudo, Noriko Ohno, Enyu Imai, Masatsugu Hori  Kidney International  Volume 59, Issue 6, Pages 2134-2145 (June 2001) DOI: 10.1046/j.1523-1755.2001.00728.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Immunoglobulin heavy chain (IgGHc; A), human β–platelet-derived growth factor receptor (βPDGFR; B), and platelet-derived growth factor receptor fused with IgG-Fc (PDGFR/Fc) chimeric inhibitor molecule (C). Consequent to ligand binding, signals are transduced into target cells (B). In contrast, the PDGFR/Fc chimeric molecule can bind to PDGF ligand and thereby block the signal transduction of PDGF-BB (C). PDGFR cDNA encoding extracellular domain was recombined in frame with the Fc portion of the human IgGFc at the hinge region. The BamH I restriction site is underlined (D). Abbreviations are: VH, variable region; CH, constant region; H, hinge region; EC, extracellular domain of βPDGFR; TK, tyrosine kinase domain; and TM, transmembrane domain. Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Effect of PDGFR/Fc chimera on tyrosine phosphorylation of βPDGFR. The level of tyrosine phosphorylation of the βPDGFR was determined in human mesangial cells. Cells were incubated with PDGF-BB (10 ng/mL) with or without the PDGFR/Fc chimeric molecule (10 μg/mL) for 10 minutes. Cells were solubilized, and βPDGFR was isolated by immunoprecipitation with anti-human βPDGFR antibody-conjugated immunobeads. Immunobead complexes were extensively washed, separated on SDS-PAGE, and transferred to nitrocellulose membranes. The level of phosphotyrosine within βPDGFR was determined by immunoblot analysis with antiphosphotyrosine antibody. Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Effect of chimeric molecule on cell proliferation. (A) The effect of the chimeric PDGFR/Fc molecule on PDGF-BB-induced cell proliferation was evaluated by colony-forming efficiency assay. NIH3T3 cells were seeded at a density of 1000 cells per 35mm dish and stimulated with PDGF-BB (30 ng/mL) and/or PDGFR/Fc (10 μg/mL). Two weeks later, the number of colonies with a diameter of larger than 50 μm was counted. (B) The effect of the chimeric PDGFR/Fc molecule on PDGF-BB–induced thymidine incorporation was evaluated. Rat mesangial cells were growth arrested with 0.4% FCS for 72 hours and stimulated by PDGF-BB (10 ng/mL) with PDGFR/Fc (100 ng/mL) or CD4/Fc (100 ng/mL) for 24 hours. [3H]-thymidine incorporation was determined in final eight hours of stimulation. The values are expressed as mean ± SD (N = 4) and are representative of two independent experiments. *P < 0.001 vs. the other groups. Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 Effect of chimeric gene transfer on glomerular cell proliferation. Representative immunohistochemical photomicrographs of glomeruli stained for PCNA on day 5 in normal rats (A), untreated nephritic rats (B), and PDGFR/Fc gene-transfected nephritic rats (C). Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 Representative photomicrographs of glomeruli stained for PAS on day 7 in normal rats (A), untreated nephritic rats (B), and PDGFR/Fc gene-transfected nephritic rats (C). Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 Quantitative analysis of the effect on glomerular cell proliferation. The number of PCNA-positive cells per glomerulus (A) and the total cell number (B) in normal rats (), untreated nephritic rats (▪), and PDGFR gene-transfected nephritic rats (□). The values are expressed as mean ± SD. *P < 0.001 vs. the other groups. Each group contained six animals. Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Semiquantitation of the pathological effects of chimeric PDGFR/Fc gene transfection. On day 7, the severity of the glomerular damage of untreated nephritic rats (▪) and nephritic rats with PDGFR/Fc gene transfection (□) was evaluated by ECM accumulation. The values are expressed as mean ± SD. *P < 0.001 vs. the other groups. Each group contained six animals. Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 8 Effect of chimeric gene transfer on phenotypic alteration. Representative immunohistochemical photomicrographs of glomeruli stained for α-smooth muscle actin on day 5 in normal rats (A), untreated nephritic rats (B), and PDGFR/Fc gene-transfected nephritic rats (C). Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 9 Northern blot analysis of glomerular α-SMA, TGF-β1, and type I collagen mRNA in nephritic rats treated with PDGFR/Fc transfection. The glomeruli were obtained from age-matched normal control rats (lane 1), nephritic rats treated with PDGFR/Fc gene transfer (lane 2), and untreated nephritic rats (lane 3). Kidney International 2001 59, 2134-2145DOI: (10.1046/j.1523-1755.2001.00728.x) Copyright © 2001 International Society of Nephrology Terms and Conditions