title: 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.
abstract: 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)-kappaB, 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 H(2)O(2)-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-kappaB and Sp1 were translocated into the nucleus after podocytes were incubated with CML for 60 min. Parthenolide and mithramycin A, inhibitors of NF-kappaB 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-kappaB and Sp1 regulate MCP-1 gene transcription.
authors.affiliation: Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Leyi
authors.initials: L
authors.lastname: Gu
authors.forename: Shinji
authors.initials: S
authors.lastname: Hagiwara
authors.forename: Qiuling
authors.initials: Q
authors.lastname: Fan
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Tanimoto
authors.forename: Mami
authors.initials: M
authors.lastname: Kobata
authors.forename: Michifumi
authors.initials: M
authors.lastname: Yamashita
authors.forename: Tomohito
authors.initials: T
authors.lastname: Nishitani
authors.forename: Tomohito
authors.initials: T
authors.lastname: Gohda
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Ni
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Qian
authors.forename: Satoshi
authors.initials: S
authors.lastname: Horikoshi
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Tomino
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis*; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D050199:Podocytes / Q000166:cytology / Q000378:metabolism*; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000502:physiology*
chemical_list: D018932:Chemokine CCL2; D008565:Membrane Proteins; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic