Increased expression of heparanase in overt diabetic nephropathy M.J. van den Hoven, A.L. Rops, M.A. Bakker, J. Aten, N. Rutjes, P. Roestenberg, R. Goldschmeding, E. Zcharia, I. Vlodavsky, J. van der Vlag, J.H. Berden  Kidney International  Volume 70, Issue 12, Pages 2100-2108 (December 2006) DOI: 10.1038/sj.ki.5001985 Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 1 Expression of HS, HPSE, and agrin in human DNP. Immunofluorescence double stainings of (a and c) agrin (red) with HS (green) and (b and d) HS (green) with HPSE (red) in kidney sections from (c and d) patient with overt DNP and (a and b) a control. (a) Co-staining of agrin with HS revealed a linear co-localization (yellow) along the GBM in controls, (c) whereas reduction of glomerular HS was observed in patients with DNP. Decrease of glomerular HS expression in diabetic patients was associated with an upregulation of HPSE in the glomeruli of (d) diabetic patients compared to (b) controls. Original magnification × 63. Kidney International 2006 70, 2100-2108DOI: (10.1038/sj.ki.5001985) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 2 Quantitative analysis of HS and HPSE expression in human DNP. Staining of HS and HPSE was scored by three independent observers on a scale between 0 and 10 for HS linearity or on a scale between 0 and 5 for HPSE intensity in arbitrary units (a.u.). Points represent mean scores from each individual glomerulus or tubular photograph and horizontal lines represent median values of these scores. HS expression was significantly reduced both in (a) glomeruli and (c) tubuli, whereas expression of HPSE was increased in both (b) glomeruli and (d) tubuli of patients with overt DNP compared to the controls. There was an inverse correlation between (e) glomerular HS and proteinuria and between (f) glomerular HS and glomerular HPSE expression. ***P<0.001. Kidney International 2006 70, 2100-2108DOI: (10.1038/sj.ki.5001985) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 3 Localization of HPSE in human DNP. Immunofluorescence double staining of HPSE with glomerular markers for the GBM (agrin), podocytes (synaptopodin), or mesangial cells (perlecan) in a patient with overt DNP. HPSE expression co-localizes with both (a) agrin and (b) synaptopodin, representing the extracellular and intracellular HPSE expression, respectively. (c) However, HPSE is not expressed by mesangial cells. (d) Tubular HPSE is mainly expressed in proximal tubuli, determined by co-staining with anti-P-glycoprotein. Original magnification × 63. Kidney International 2006 70, 2100-2108DOI: (10.1038/sj.ki.5001985) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 4 HS and HPSE expression in rats with STZ-induced diabetes. (a) Immunofluorescence staining of the increased HPSE expression and decreased HS expression in kidneys of diabetic rats. (b) Western blot analysis was used to confirm an increased HPSE protein expression in renal cortex of rats with STZ-induced diabetes compared to controls. (c) HS expression was decreased, however, no statistical significance could be observed. (d) Quantification revealed that both HPSE protein and HPSE mRNA expression were upregulated in glomeruli of diabetic rats compared to controls. Horizontal lines represent median values. *P<0.05. a.u. arbitrary units; rHPSE, recombinant heparanase. Kidney International 2006 70, 2100-2108DOI: (10.1038/sj.ki.5001985) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 5 HS and HPSE expression in mice with STZ-induced diabetes. (a) Renal cortex of mice with STZ-induced diabetes contained a significantly higher amount of HPSE mRNA compared to controls. (b) HPSE protein expression was significantly increased and (c) HS expression was decreased, although not significant. Sections were coded randomly and staining was evaluated by two independent observers on a scale of 0–10 for HS linearity and on a scale of 0–5 for HPSE staining intensity. Horizontal lines represent median values. (d) HS expression inversely correlated with HPSE mRNA expression. **P<0.01, *P<0.05. a.u.; arbitrary units. Kidney International 2006 70, 2100-2108DOI: (10.1038/sj.ki.5001985) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 6 Glomerular expression of HPSE, HS, and agrin in mice with transgenic HPSE overexpression and after treatment with active HPSE. (a) HPSE expression is increased in the HPSEtg mouse kidney compared to the control. (a) HS expression was markedly reduced in the kidney of mice with transgenic overexpression of HPSE, whereas agrin core protein expression did not differ. (b) A significant increase in the urinary albumin excretion was observed in HPSEtg mice compared to controls. (c) After exposure of kidney sections to recombinant active HPSE, HS staining almost completely disappeared, whereas it did not affect agrin expression. Original magnification × 100. *P<0.05. Kidney International 2006 70, 2100-2108DOI: (10.1038/sj.ki.5001985) Copyright © 2006 International Society of Nephrology Terms and Conditions