Volume 61, Issue 1, Pages (January 2002)

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Volume 61, Issue 1, Pages 148-156 (January 2002) AGEs bind to mesothelial cells via RAGE and stimulate VCAM-1 expression Eric Boulanger, Marie-Paule Wautier, M.D., Ph.D., Jean-Luc Wautier, Bernadette Boval, Yves Panis, Nicolas Wernert, Pierre-Marie Danze, Philippe Dequiedt Kidney International Volume 61, Issue 1, Pages 148-156 (January 2002) DOI: 10.1046/j.1523-1755.2002.00115.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Receptor of advanced glycation end-product (RAGE) expression on human peritoneal mesothelial cells (HPMC) and human unbilical vein endothelial cells (HUVEC) obtained with anti-RAGE antibody. (A) Typical flow cytometry profiles. (B) Mean fluorescence intensity (MFI) in four sets of experiments, expressed in AUF. The whiskers denote the standard deviation (*P < 0.05). (C) Polymerase chain reaction (PCR) amplification of reverse transcripted (RT) RAGE mRNA from HPMC and HUVEC. RAGE mRNA from HPMC and HUVEC had a similar electrophoretic profile and was reproducible in different cell cultures (two sets of experiments). The arrow indicates the amplified region of 469bp located in the coding region of RAGE. Kidney International 2002 61, 148-156DOI: (10.1046/j.1523-1755.2002.00115.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Immunohistochemical staining of mesothelial cells with anti-RAGE antibody. (A) Histomorphology of normal human peritoneal mesothelial cells (59-year old male) stained with hematoxylin and eosin (H&E). (B) Diffuse intracytoplasmatic staining of mesothelial cells (arrow) is evident at high magnification after immunohistochemical staining with anti-RAGE antibody (×4500). Kidney International 2002 61, 148-156DOI: (10.1046/j.1523-1755.2002.00115.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Adhesion molecule expression on HPMC. Intercellular (ICAM-1; A) and vascular (VCAM-1; B) cell adhesion molecule expressions were studied under basal conditions or after stimulation (TNF-α 5 ng/mL, IL-1β 50 U/mL and CML-albumin 50 μg/mL). All results are expressed in cpm and presented as the mean ± SEM of two series of experiments for ICAM-1 and four series for VCAM-1, all carried out in quadruplicate (*P < 0.05; **P < 0.01; ***P < 0.001). (C) VCAM-1 expression after preincubation of HPMC with anti-RAGE IgG (100 μg/mL) or rR-RAGE (60 μg/mL). Kidney International 2002 61, 148-156DOI: (10.1046/j.1523-1755.2002.00115.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 Leukocyte adhesion. Leukocyte adhesion on HPMC was measured under basal conditions and after stimulation by CML-albumin (50 μg/mL) with or without preincubation of HPMC with rR-RAGE (60 μg/mL) or anti-RAGE IgG (100 μg/mL) for blocking AGE binding to mesothelial RAGE. Preincubation of leukocytes with rR-VCAM-1 (200 μg/mL) also was carried out to block leukocyte adhesion. The results of three different series of experiments are expressed in leukocytes/mm2 and presented as the mean ± SEM of 30 determinations (NS is non-significant; ***P < 0.001). Kidney International 2002 61, 148-156DOI: (10.1046/j.1523-1755.2002.00115.x) Copyright © 2002 International Society of Nephrology Terms and Conditions