Volume 86, Issue 5, Pages 932-942 (November 2014) Modulation of heparan sulfate in the glomerular endothelial glycocalyx decreases leukocyte influx during experimental glomerulonephritis  Angelique LWMM Rops, Markus A. Loeven, Jasper J. van Gemst, Iris Eversen, Xander M. Van Wijk, Henry B. Dijkman, Toin H. van Kuppevelt, Jo H.M. Berden, Ton J. Rabelink, Jeffrey D. Esko, Johan van der Vlag  Kidney International  Volume 86, Issue 5, Pages 932-942 (November 2014) DOI: 10.1038/ki.2014.115 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Ndst1 deficiency leads to a decreased glomerular expression of the specific HS domain recognized by AO4B08. (a) Immunofluorescence staining and (b) semiquantitative analysis of the glomerular expression of the inflammatory heparan sulfate (HS) domains recognized by the anti-HS single-chain variable fragment (scFv) antibodies AO4B08, EW3D10, and EW4G2 and of the highly sulfated HS domain recognized by the anti-HS antibody HS4C3 revealed a decreased expression of the HS domain defined by AO4B08 (HS domain with N-, 2-O, and 6-O sulfation, and C5 epimerization) in N-deacetylase-N-sulfotransferase-1 (Ndst1)-deficient mice compared with wild-type (WT) mice. Staining intensities are expressed as means±s.e.m. from four Ndst1f/f TEKCre− WT and four Ndst1f/f TEKCre+ (Ndst1f/f) mice in arbitrary units (a.u.). (a) Magnification × 400. (b) *P<0.001 vs. WT mice. Kidney International 2014 86, 932-942DOI: (10.1038/ki.2014.115) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Ndst1 deficiency leads to minor segmental podocyte foot process effacement and focal thickening and irregularities in the glomerular basement membrane (GBM). The glomerular filtration barrier of four wild-type (WT) and four N-deacetylase-N-sulfotransferase-1 (Ndst1)-deficient mice, 14–18 weeks old, was analyzed by transmission electron microscopy. WT glomeruli showed the typical pedicel arrangement of podocytes (arrow), whereas in some Ndst1-deficient glomeruli segmental podocyte foot process effacement (arrow) and the presence of thrombocytes (star) were observed. Furthermore, Ndst1-deficient glomeruli showed focal thickening and irregularities in the GBM and some endothelial activation (arrowhead). Magnification × 5000. Kidney International 2014 86, 932-942DOI: (10.1038/ki.2014.115) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Ndst1 deficiency significantly decreases glomerular granulocyte and macrophage influx during anti-GBM nephritis. (a) Glomerular polymorphonuclear granulocyte (PMN) and (b) macrophage influx, analyzed by immunofluorescence staining, peaked at 2h and 1 day, respectively, after rabbit anti-GBM IgG administration in both wild-type (WT) and N-deacetylase-N-sulfotransferase-1 (Ndst1)-deficient mice. Ndst1-deficient mice showed a significantly reduced glomerular PMN and macrophage influx during the acute heterologous phase. (c) Glomerular CD4+ T-cell influx was lower in Ndst1-deficient mice compared with WT mice after 1 day but was not significantly different compared with that in untreated controls. In the autologous phase, glomerular CD4+ T-cell and (d) CD8+ T-cell influx in both WT and Ndst1-deficient mice was significantly higher compared with that in both untreated controls. Results are expressed as means±s.e.m. from four control WT and four control Ndst1-deficient mice and five WT and five Ndst1-deficient mice with induced anti-GBM nephritis. **P<0.001 vs. anti-GBM-injected WT mice. *P<0.05 vs. anti-GBM-injected WT mice. ^P<0.05 vs. control. Kidney International 2014 86, 932-942DOI: (10.1038/ki.2014.115) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Ndst1-deficient mice show better renal function during anti-GBM nephritis. (a) The albumin/creatinine ratio (mg/mg) (ACR) was significantly higher in wild-type (WT) mice compared with untreated controls after 1 day during anti-GBM nephritis. N-deacetylase-N-sulfotransferase-1 (Ndst1)-deficient mice showed a lower trend in albuminuria at this time point (P=0.06 vs. WT mice). (b) Plasma creatinine concentration (mg/dl) and (c) blood urea nitrogen (BUN) levels (mg/dl) were significantly lower in Ndst1-deficient mice than in WT mice at 4 days. Plasma creatinine concentrations of WT and Ndst1-deficient mice after 2h and 1 day were similar to that of untreated control WT and Ndst1-deficient mice. Results are expressed as means±s.e.m. from four control WT and four control Ndst1-deficient mice and five WT and five Ndst1-deficient mice with induced anti-GBM nephritis. ^P<0.05 vs. control. *P<0.05 vs. anti-GBM-injected WT mice. Kidney International 2014 86, 932-942DOI: (10.1038/ki.2014.115) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Ndst1-deficient mice show less glomerular injury during anti-GBM nephritis. (a) Representative pictures of renal histology and (b) semiquantitative analysis of the percentage of glomeruli with hyalinosis. At least 50 glomeruli per mouse were analyzed for the presence of hyalinosis. Wild-type (WT) mice showed significantly more glomerular injury compared with N-deacetylase-N-sulfotransferase-1 (Ndst1)-deficient mice at 1 and 4 days during anti-GBM nephritis. Results are expressed as means±s.e.m. from five WT and five Ndst1-deficient mice. *P<0.05 vs. anti-GBM-injected WT mice. Kidney International 2014 86, 932-942DOI: (10.1038/ki.2014.115) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Ndst1 deficiency significantly reduces the expression of inflammatory heparan sulfate (HS) domains during antiglomerular basement membrane (anti-GBM) nephritis. (a) Immunofluorescence staining and (b) semiquantitative analysis of the glomerular expression of the inflammatory HS domains recognized by anti-HS single-chain variable fragment (scFv) antibodies AO4B08, EW3D10, and EW4G2 and of the highly sulfated HS domain recognized by the anti-HS scFv antibody HS4C3 showed decreased glomerular expression of the inflammatory HS domains at 2h during anti-GBM nephritis in N-deacetylase-N-sulfotransferase-1 (Ndst1)-deficient mice compared with wild-type (WT) mice. Staining intensities are expressed as means±s.e.m. from five Ndst1f/f TEKCre− WT and five Ndst1f/f TEKCre+ (Ndst1f/f) mice in arbitrary units (a.u.). (a) Magnification × 400. (b) ^P<0.05 vs. control. *P<0.01 vs. anti-GBM-injected WT mice. Kidney International 2014 86, 932-942DOI: (10.1038/ki.2014.115) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Ndst1 silencing in glomerular endothelial cells leads to decreased adhesion of granulocytes, chemokines and L-selectin in vitro. (a) Adhesion of wild-type (WT) and N-deacetylase-N-sulfotransferase-1 (Ndst1)-deficient granulocytes to a monolayer of Ndst1- or scrambled siRNA (25nM)–transfected, and tumor necrosis factor (TNF)-α-activated, mouse glomerular endothelial cells (mGEnC-1) was measured. Adhesion of granulocytes to tumor necrosis factor (TNF)-α-activated mGEnC-1 that were transfected with scrambled small interfering RNA (siRNA) was set at 100%. (b–d) Glomerular endothelial expression of specific heparan sulfate (HS) domains defined by the anti-HS antibodies AO4B08, EW3D10, EW4G2, and HS4C3 and binding of the chemokines CXCL1, MCP1, and MIP-2α and of recombinant L-selectin to Ndst1-silenced mGEnC-1 were measured with enzyme-linked immunosorbent assay (ELISA). Binding of anti-HS antibodies, chemokines, and L-selectin to control mGEnC-1 was set at 100%. (e) Inhibitory capacity of L-selectin on adhesion of Ndst1-deficient granulocytes to a monolayer of Ndst1-silenced mGEnC-1. Percentage adhesion of Ndst1-deficient granulocytes after blocking with an isotype control is set at 100%. Results are shown as means±s.e.m. from three independent experiments (n=5). *P<0.01 vs. control. ^P<0.05 vs. control. Kidney International 2014 86, 932-942DOI: (10.1038/ki.2014.115) Copyright © 2014 International Society of Nephrology Terms and Conditions