Fig. 4
Treatment with the heparanase inhibitor OVS/HS-1638 in db/db mice prevents systemic eGlx damage and associated microvascular permeability changes. a Schematic of experimental timeline. Lean or db/db mice were treated with vehicle or OVZ/HS-1638 daily for 14-days by i.p. End-point urine was collected for analysis and eye and kidney tissue collected for analysis. b Retinas from lean and db/db mice treated with vehicle (db/db) or with OVZ/HS-1638 (db/db + HI) were stained with FITC-LEL (green), R18 cell membrane stain (red) and DAPI (blue). Inset images show green staining on luminal side of vessel, indicating eGlx staining. c Glycocalyx depth in lean and diabetic animals was measured using confocal fluorescence profile peak-to-peak (n = 5, mice per group, *P ≤ 0.05, One-way ANOVA with Tukey’s multiple comparison test). d Retinas from lean and db/db mice treated with vehicle (db/db) or with OVZ/HS-1638 (db/db + HI) were stained for albumin (red) and DAPI (blue). Arrows point to vessels filled with albumin and arrowheads point to extravascular albumin. Extravascular albumin staining found near Ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) in db/db retina. e Extravascular corrected total cell fluorescence (CTFEXV) in arbitrary units (a.u.) (n = 5 mice per group, **P ≤ 0.01, ***P ≤ 0.001, One-way ANOVA with Tukey’s multiple comparison test). f Average CTFEXV value for each mouse was plotted against corresponding eGlx depth for same animal (n = 15 total mice, **P ≤ 0.01, Pearson correlation analysis). g Representative TEM images of lean, diabetic (db/db) and diabetic mice treated with OVZ/HS-1638 (db/db + HI) mouse glomerular filtration barrier showing podocyte (P), podocyte slit diaphragm (SD), endothelial cell (E), basement membrane (BM), podocyte Glx (open arrow heads), and eGlx (solid arrow heads). h Quantification of TEM images measuring eGlx depth (n = 5 mice,*P < 0.05, Kruskal–Wallis test). i End point urine albumin creatinine ratio for lean (n = 5), db/db (n = 4), and db/db + HI (n = 4) (**P < 0.01, Kruskal–Wallis test). j Glomerular albumin permeability (Ps’alb) measured. Glomeruli analysed shown on graph and in parentheses. Stats performed on mouse number (n = 5, **P < 0.01, ***P < 0.001, One way ANOVA, Tukey’s multiple comparison test). (k,l) Correlation analysis. (k) Ps’alb vs eGlx depth (n = 7,**P < 0.01), (l) Ps’alb vs podocyte foot process width (n = 7, P > 0.05, not significant (ns)).