Fig. 4

Molecular changes in the glomerulus of Wt and Hif1α ^+/− mice. a Representative confocal microscopy images of 8 μm sections of the glomerulus stained with anti-VEGFA antibody (green) show the highest expression of VEGFA in diabetic Hif1α ^+/− kidneys. The area of the glomerulus is outlined by the yellow dashed line. Arrows indicate VEGFA expression (sharp green) in the glomerulus. Negative control is without primary antibody. Images are stacked Z-plane sections; nuclei are counterstained with Hoechst 33,342 (blue); scale bar 20 μm. b Delineated VEGFA⁺ area by ImageJ. c Staining of mature and fully functional podocytes with anti-WT1 antibody (red) shows the largest loss of podocytes in the diabetic Hif1α ^+/− renal cortex; nuclei are counterstained with Hoechst 33,342 (blue); scale bar 50 μm. d Relative quantification of VEGFA expression was determined as a percentage of VEGFA⁺ area per glomerular area. e Quantification of podocyte density as a number of WT1⁺ cells per glomerular area. The values represent means ± SEM (n = 10 glomerulus/3 sections/3 mice/group). Two-way ANOVA detected statistical significance of diabetes effect (P < 0.0001) followed by post hoc pairwise comparison tests *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: vascular endothelial growth factor (VEGFA), Wilms tumor 1 (WT1)