Fig. 2
Enzymatic removal of HS results in reduced eGlx and loss of barrier function in the retina and glomerulus. a Representative TEM images of retinal vessels from mice perfused with inactive or active heparinase III and then Alcian Blue to visualise eGlx and glutaraldehyde for EM preparation. Basement membrane (BM) and retinal endothelial cell (E) indicated. Arrowheads point to eGlx on endothelial cell. b Glycocalyx depth measured. Mouse averages shown (n = 6 mice (inactive) and n = 7 mice (active), unpaired t-test indicated, *P < 0.05). c Representative images of lectin-stained retina sections from mice treated with inactive or active heparinase III. Lectin (FITC-LEL) staining in green, R18 cell membrane stain in red, and DAPI in blue. Inset images show green signal on the luminal side of the vessel, indicating eGlx staining. d Example of confocal fluorescence peak-to-peak (P-P) measurement for measurements of eGlx depth using LEL and cell membrane (R18) staining. e Confocal fluorescence profile peak-to-peak measurements in capillaries were taken for a minimum of three vessels per animal. Mouse averages shown (n = 6 mice/group, *P < 0.05, unpaired t-test indicated). f Image of mouse retina perfused with sodium fluorescein angiography. Within the selected ROI, the area in main vessel (V1) and in the tissue next to vessel (V2) measured over time to calculate solute flux. g Solute flux after treatment with inactive or active enzyme. (n = 6 mice/group, *P < 0.05, unpaired t-test indicated) h Representative glomerular filtration barrier TEM images of glomerular capillary in inactive and active heparinase III treated mice showing podocyte (P), podocyte slit diaphragm (SD), endothelial cell (E), basement membrane (BM), podocyte Glx (open arrowhead), and eGlx (solid arrowhead). i Quantification of EM images measuring eGlx depth (n = 5 mice/group,*P < 0.05, unpaired t-test). j Glomerular albumin permeability (Ps’alb) measured for both groups. Glomeruli analysed shown on graph and in parentheses. Stats performed on mouse number (n = 5 mice/group,*P < 0.05, unpaired t-test).