Fig. 3

Glycocalyx shedding and the NF-κB signaling pathway in the four groups of mice. Representative images of immunofluorescence staining and corresponding scores were used to analyze the expression of heparan sulfate (A and B) and syndecan-1 (D and E) in the glomeruli of the four groups (× 630, Scale bar = 10 μm, n = 6 per group, glomeruli were outlined with dotted lines, five randomly glomeruli were selected per mouse). ELISA was used to measure the heparan sulfate level (C) and syndecan-1 (F) level in the serum of mice (n = 8 per group). G Electron micrographs were mcaptured to observe the glycocalyx more intuitively (scale bar = 200 nm). Representative electron micrographs of the glomerular capillary wall were shown. The measurements were carried out on 3 capillary loops per glomerulus and 5 glomeruli were used per mouse. Red labels indicate endothelial glycocalyx (eGLX),and glomerular basement membrane (GBM). Quantification of eGLX depth (H), percentage endothelium with GLX coverage (I) and foot processes (J)(n = 3 per group). The protein levels of p-IKKβ, IKKβ, p-IκBα, IκBα, p-NF-κBp65, and NF-κBp65 in the glomeruli of mice were determined by western blotting (K and L) (n = 3). β-actin was used as an internal reference control. Immunofluorescence staining (M) and E-selectin positive area (N) were used to assess activation of E-selectin(× 630, Scale bar = 10 μm, n = 6 per group). Results are expressed as mean ± SEM; ^***P < 0.001 vs. Cxcr2^L/Lgroup; ^##P < 0.01, and ^###P < 0.001 vs. DKD-Cxcr2^L/L group