Intravascular space (plasma) was labeled with iv-injected albumin–Alexa Fluor 594 (red). (A) Representative image and calculation of endothelial surface FITC-WGA signal width on linear intensity profiles as an index of glycocalyx thickness. FITC-WGA fluorescence decreases as it meets the plasma (luminal end) and the endothelial cell (abluminal end). FITC-WGA signal width was measured as the distance between half maximal intensities as shown. Glycocalyx measurements relied on the selection of capillary segments where clear plasma and FITC-WGA peak signals could be seen. Only clear longitudinal or cross-sectioned (round) capillary profiles and the level at one-half the capillary depth (deduced from the Z-stack) were analyzed to ensure the glycocalyx was measured perpendicular to the endothelial membrane. (B) Summary of FITC-WGA signal width in various mouse groups as shown. (C–G) Representative images of FITC-WGA lectin labeling and intensity before and 1 hour after H treatment. (H) Summary and statistical analysis of FITC-WGA fluorescence intensity (ratio of glomerular labeling/proximal tubule autofluorescence). (B–H) Mice were 4–6 weeks old unless stated otherwise. Scale bar: 10 μm. n = 8 glomeruli from n = 4 mice each group. Values are expressed as means ± SEM, *P < 0.005, **P < 0.001, based on 1-way ANOVA followed by Tukey’s multiple-comparisons test (B–H). GC, glomerular capillary; ESL, endothelial surface layer; NOD, nonobese diabetic; H, hyaluronidase.