Aberrant Heparan Sulfate Profile in the Human Diabetic Kidney Offers New Clues for Therapeutic Glycomimetics  Tessa J.M. Wijnhoven, MSc, Joost F.M. Lensen, MSc, Angelique L.W.M.M. Rops, MSc, Johan van der Vlag, PhD, Svein O. Kolset, MD, PhD, Hans-Jacob Bangstad, MD, PhD, Per Pfeffer, MD, PhD, Mabel J.W. van den Hoven, MSc, Jo H.M. Berden, MD, PhD, Lambert P.W.J. van den Heuvel, PhD, Toin H. van Kuppevelt, PhD  American Journal of Kidney Diseases  Volume 48, Issue 2, Pages 250-261 (August 2006) DOI: 10.1053/j.ajkd.2006.05.003 Copyright © 2006 National Kidney Foundation, Inc. Terms and Conditions

Fig 1 Immunofluorescence staining of renal cryosections of control subjects versus microalbuminuric patients with type 1 diabetes with the anti-HS antibodies (A-D) LKIV69, (E, F) HS4E4, (G, H) AO4B08, and (I, J) MPB49 (irrelevant antibody, negative control). Patients showed increased staining of renal tubules with LKIV69. Other antibodies stained similarly in control subjects and patients with diabetes. Abbreviation: G, glomerulus. (Bar: 50 μm; magnification is identical for each photograph.) American Journal of Kidney Diseases 2006 48, 250-261DOI: (10.1053/j.ajkd.2006.05.003) Copyright © 2006 National Kidney Foundation, Inc. Terms and Conditions

Fig 2 Double-immunostaining of renal cryosections of control subjects versus microalbuminuric patients with type 1 diabetes with the anti-HS antibody LKIV69 and anti-aquaporin-1 antibody. In contrast to control subjects, proximal tubules of patients were highly immunoreactive for the HS structure recognized by LKIV69. Abbreviation: G, glomerulus. (Bar: 50 μm; magnification is identical for each photograph.) American Journal of Kidney Diseases 2006 48, 250-261DOI: (10.1053/j.ajkd.2006.05.003) Copyright © 2006 National Kidney Foundation, Inc. Terms and Conditions

Fig 3 Immunofluorescence staining of renal cryosections of control subjects versus microalbuminuric patients with type 1 diabetes with the anti-HS antibodies (A-D) HS4C3, (E, F) EW3D10, and (G, H) EW4G2. No aberrant staining was observed in patients, except for HS4C3, which increased in the mesangium of some patients, especially the patient who had developed macroalbuminuria after an 8-year follow-up (arrow). (Bar: 50 μm; magnification is identical for each photograph.) American Journal of Kidney Diseases 2006 48, 250-261DOI: (10.1053/j.ajkd.2006.05.003) Copyright © 2006 National Kidney Foundation, Inc. Terms and Conditions

Fig 4 Analysis of chemical structures defined by the anti-HS antibodies LKIV69 and HS4C3 by means of (A) ELISA and (B) immunohistochemistry. (A) Reactivity of antibodies with immobilized test molecules relative to unmodified heparin. Values expressed as mean ± SEM (n = 4). Both N- and 2-O-sulfates are essential for LKIV69 binding, whereas N- and 6-O-sulfates are essential for HS4C3 binding. (B) Immunofluorescence staining of wild-type (K1) and 2-O-sulfotransferase–deficient (psF-17) CHO cell lines with anti-HS antibody LKIV69. Note the lack of staining of the mutant cell line. (Bar: 50 μm; magnification is identical for each photograph.) American Journal of Kidney Diseases 2006 48, 250-261DOI: (10.1053/j.ajkd.2006.05.003) Copyright © 2006 National Kidney Foundation, Inc. Terms and Conditions

Fig 5 Competition of FGF-2 with the anti-HS antibody LKIV69 for HS by means of (A) ELISA and (B) immunohistochemistry. (A) Reactivity of LKIV69 with immobilized HS in the presence of increasing amounts of FGF-2. Values expressed as mean ± SEM (n = 4). (B) Immunofluorescence staining of renal cryosections of microalbuminuric patients with type 1 diabetes with LKIV69 in the absence or presence of 1 μg of FGF-2/mL. FGF-2 decreased binding of LKIV69 to HS, whereas staining for FGF-2 increased. Abbreviation: G, glomerulus. (Bar: 50 μm; magnification is identical for each photograph.) American Journal of Kidney Diseases 2006 48, 250-261DOI: (10.1053/j.ajkd.2006.05.003) Copyright © 2006 National Kidney Foundation, Inc. Terms and Conditions

Fig 6 Reactivity of the anti-HS antibodies LKIV69 and HS4C3 with GAG-based drugs by means of (A) ELISA and (B) immunohistochemistry. (A) Reactivity of antibodies with immobilized GAG-based drugs relative to unmodified heparin. Values expressed as mean ± SEM (n = 7). Reactivity of LKIV69 was highest for heparin and sulodexide. HS4C3 binds to all GAG-based drugs. (B) Immunofluorescence staining of renal cryosections of microalbuminuric patients with type 1 diabetes with LKIV69 in the absence or presence of 100 μg of sulodexide/mL. Note decreased staining in the presence of sulodexide. Abbreviation: G, glomerulus. (Bar: 50 μm; magnification is identical for each photograph.) American Journal of Kidney Diseases 2006 48, 250-261DOI: (10.1053/j.ajkd.2006.05.003) Copyright © 2006 National Kidney Foundation, Inc. Terms and Conditions