Role of receptor for advanced glycation end-products and signalling events in advanced glycation end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytes

• Published in: Nephrology, dialysis, transplantation dialysis, transplantation, 2006-02, Vol.21 (2), p.299-313
• Main Authors: Gu, Leyi, Hagiwara, Shinji, Fan, Qiuling, Tanimoto, Mitsuo, Kobata, Mami, Yamashita, Michifumi, Nishitani, Tomohito, Gohda, Tomohito, Ni, Zhaohui, Qian, Jiaqi, Horikoshi, Satoshi, Tomino, Yasuhiko
• Format: Article
• Language: English
• Subjects: AGE; Animals; Cell Differentiation; Cells, Cultured; Chemokine CCL2 - biosynthesis; ERK; MCP-1; Membrane Proteins - physiology; Mice; podocyte; Podocytes - cytology; Podocytes - metabolism; RAGE; Receptor for Advanced Glycation End Products; Receptors, Immunologic - physiology; ROS
• ISSN: 0931-0509; 1460-2385
• DOI: 10.1093/ndt/gfi210

Background. Upregulation of local monocyte chemoattractant protein-1 (MCP-1) production is involved in glomerular damage through macrophage recruitment and activation in diabetic nephropathy. Treatment of db/db mice with soluble receptor for advanced glycation end-products (RAGE) prevented recruitment of macrophages to the glomeruli and reduced albuminuria, suggesting that binding of ligands and RAGE may be involved in MCP-1 expression. Therefore, we investigated the role of advanced glycation end-products (AGEs) in MCP-1 production by podocytes and signalling events after RAGE activation. Methods. MCP-1 gene and protein expression were examined by using reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay in differentiated mouse podocytes. Dichlorofluorescein-sensitive intracellular reactive oxygen species (ROS) generation was measured by confocal microscopy. RAGE, phosphorylation of mitogen-activated protein kinases, nuclear factor (NF)-κB, c-Jun and Sp1 were studied using western blotting and immunocytochemistry. Results. Both differentiated and undifferentiated podocytes expressed RAGE. MCP-1 was induced by AGEs and carboxymethyllysine (CML) in a time-dependent and dose-dependent manner in differentiated podocytes. Neutralizing antibody for RAGE suppressed AGE- and CML-induced MCP-1 production. AGEs and CML rapidly generated intracellular ROS in podocytes. Blocking of ROS by using N-acetyl-l-cysteine abolished CML and H2O2-induced MCP-1 expression. Phosphorylated extracellular signal-regulated kinase (ERK) was found in podocytes incubated with CML and was prevented by N-acetyl-l-cysteine or 7′-amino 4 [trifluoromethyl]. PD98059, an inhibitor of ERK, partially prevented CML-induced MCP-1 gene expression. NF-κB and Sp1 were translocated into the nucleus after podocytes were incubated with CML for 60 min. Parthenolide and mithramycin A, inhibitors of NF-κB and Sp1, respectively, abolished CML-induced MCP-1 gene expression in a dose-dependent manner. Conclusions. These results suggest that AGEs and CML induce MCP-1 expression in podocytes through activation of RAGE and generation of intracellular ROS. NF-κB and Sp1 regulate MCP-1 gene transcription.