Volume 66, Issue 4, Pages (October 2004)

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Volume 66, Issue 4, Pages 1471-1478 (October 2004) Regulation of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) expression in podocytes Kaustubh Datta, Jinping Li, S. Ananth Karumanchi, Enfeng Wang, Eric Rondeau, Debabrata Mukhopadhyay Kidney International Volume 66, Issue 4, Pages 1471-1478 (October 2004) DOI: 10.1111/j.1523-1755.2004.00910.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) mRNA level in podocytes in the presence of different matrices. Immortalized glomerular podocytes were grown on plastic, human extracellular matrix (ECM), type IV collagen, or on laminin for 48 hours. Real-time polymerase chain reaction (PCR) was performed using primers specific for VPF/VEGF-A with the total RNA isolated from the podocytes cultured on these different matrices. The data represented here are the mean of three independent results. Kidney International 2004 66, 1471-1478DOI: (10.1111/j.1523-1755.2004.00910.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) protein level in podocytes in the presence of different matrices. Sandwich enzyme-linked immunosorbent assay (ELISA) was performed using specific antibodies targeting VPF/VEGF-A using the conditioned media collected from the podocytes cultured on different matrices. The data presented are the mean of two independent experiments. Kidney International 2004 66, 1471-1478DOI: (10.1111/j.1523-1755.2004.00910.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 (A) Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) mRNA level after incubating the podocytes with antibodies against α3 and β1. Real-time polymerase chain reaction (PCR) was performed using primers specific for VPF/VEGF-A with the total RNA isolated from the podocytes incubated with antibodies against α3 and β1 and on different matrices. The data represented here are the mean of three independent results. (B) Adhesion of podocytes to laminin in the presence of antibodies against α3 and β1. Adhesion experiment to laminin was performed with podocytes in the presence of antibodies against integrin α3 and β1 or with mouse normal IgG which served as a control. Kidney International 2004 66, 1471-1478DOI: (10.1111/j.1523-1755.2004.00910.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) promoter activity in podocytes in the presence of different matrices. Plasmid DNA containing the full-length VPF/VEGF-A promoter (2.6 kb) fused with luciferase reporter gene was transiently transfected in podocytes cultured on different matrices. Total luciferase activities in the cells were measured by a chemiluminescent reader. The data represented here are the average of three independent results. Kidney International 2004 66, 1471-1478DOI: (10.1111/j.1523-1755.2004.00910.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Hypoxia-inducible factor (HIF)-1α and HIF-2α protein level is unchanged in podocytes cultured on extracellular matrices (ECMs). Western blots were performed with the nuclear extract of podocytes grown in the presence or absence of ECM. Antibodies against HIF-1α and HIF-2α protein were used for this purpose. Kidney International 2004 66, 1471-1478DOI: (10.1111/j.1523-1755.2004.00910.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Association between hypoxia-inducible factor (HIF) and p300 increases in podocytes in the presence of extracellular matrix (ECM) and laminin. Nuclear extracts were prepared from podocytes in the presence or absence of ECM. (A) Immunoprecipitation of the nuclear extracts with anti p300 antibody followed by Western blot with anti-HIF-1α antibody. (B) Immunoprecipitation experiment with anti-p300 antibody followed by Western blot with anti-HIF-2α antibody. (C) Nuclear extracts were prepared from podocytes in the presence or absence of laminin. Immunoprecipitation of the nuclear extracts with anti-p300 antibody followed by Western blot with anti-HIF-1α antibody. Kidney International 2004 66, 1471-1478DOI: (10.1111/j.1523-1755.2004.00910.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 7 Classical protein kinase C (PKC) regulates the association between hypoxia-inducible factor (HIF) and p300 in podocytes. Nuclear extracts were prepared from podocytes in the presence or absence of laminin. Upper panel represents the immunoprecipitation of the nuclear extracts with ant- p300 antibody followed by Western blot with anti-HIF-1α antibody. Lower panel represents the immunoprecipitation experiment with anti-p300 antibody followed by Western blot with the same antibody. Kidney International 2004 66, 1471-1478DOI: (10.1111/j.1523-1755.2004.00910.x) Copyright © 2004 International Society of Nephrology Terms and Conditions