Fig. 3
Conditional knockout of the HS polymerizing enzyme, Ext1, in endothelial cells leads to reduced eGlx and compromised microvascular barrier function. a Schematic of experimental timeline. Age and sex matched littermate controls (LMCs) or Ext1 endothelial specific conditional knockout mice (Ext1(ECKO)) were given Doxycycline water for three weeks to induce knock-out. At three weeks, fluorescein angiographies were performed for solute flux measurements and urine analysis experiments conducted. Eye and kidney tissue was collected and analysed after three weeks of Doxycycline treatment. b Representative images of lectin-stained retinal tissue from LMC and Ext1(ECKO) mice. Lectin staining in green, R18 cell membrane stain in red, and DAPI in blue. Inset images show green signal on luminal side of the vessel, indicating eGlx staining. c Confocal fluorescence profile peak-to-peak measurements in capillaries were taken for a minimum of three vessels per animal. Mouse averages shown (n = 6, *P < 0.05, unpaired t-test). d Solute flux measured in LMC (n = 8 mice) and Ext1(ECKO) (n = 5 mice) after three weeks of doxycycline treatment (**P < 0.01, unpaired t-test). e Representative glomerular filtration barrier TEM images of litter mate control (LMC) and Ext1 endothelial specific conditional knockout mice (Ext1(ECKO)) showing podocyte (P), podocyte slit diaphragm (SD), endothelial cell (E), basement membrane (BM), podocyte Glx (open arrow heads), and eGlx (solid arrow heads). f Quantification of TEM images measuring eGlx depth (n = 6 mice,**P < 0.01, unpaired t-test). g End point urine albumin creatinine ratios (uACR) for LMC (n = 10) and Ext1(ECKO) (n = 7) mice (not significant, P = 0.24, unpaired t-test). h Fold change uACR from pre and post L-lysine (Lys) treatment in LMC (n = 12) and Ext1(ECKO) (n = 9) mice (*P < 0.05, unpaired t-test). i Glomerular albumin permeability (Ps’alb) measured for LMC (n = 6) and Ext1(ECKO) (n = 5 mice). Number of glomeruli analysed shown on graph and in parentheses. Stats performed on mouse number (***P < 0.001, unpaired t-test).