THE ROLE OF GLOMERULAR ENDTHELIAL CELLS IN THE PATHOGENESIS OF GLOMERULONEPHRITIS.

Research Project

Project/Area Number	04670573
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Pediatrics
Research Institution	HIROSAKI UNIVERSITY
Principal Investigator	KAKIZAKI Yoshiki HIROSAKI UINIVERSITY SGHOOL OF MEDICINE,LECTURER, 医学部, 講師 (40160973)
Co-Investigator(Kenkyū-buntansha)	WAGA Shinobu HIROSAKI UNIVERSITY SCHOOL OF MEDICINE,ASSIST.PROF., 医学部, 助教授 (10167744)
Project Period (FY)	1992 – 1994
Project Status	Completed (Fiscal Year 1994)
Budget Amount *help	¥2,100,000 (Direct Cost: ¥2,100,000) Fiscal Year 1994: ¥400,000 (Direct Cost: ¥400,000) Fiscal Year 1993: ¥900,000 (Direct Cost: ¥900,000) Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
Keywords	Glomerulonephritis / Monocyte / Macrophage / Glomerular endothelial cell / Monocyte chemoattractant protein-1 / Protein kinase / gene expression / MCP-1 / 転写因子NFκB / 単球遊走因子 / メサンギウム細胞 / 細胞外基質成分 / 接着分子 / 単球
Research Abstract	To explore the role of glomerular endothelial cells (GEN) in the pathogenesis of glomerulonephritis, the in vitro production of monocyte chemoattractant protein-1 (MCP-1) by bovine GEN was determined by chemotaxis assay, Northern blot analysis, and immunocytochemistry. Monocyte chemotactic activity of GEN-conditioned media was detectable by a chemotaxis assay using human peripheral blood monocytes. Exposure to human recombinant interleukin-1beta (IL-1beta) and phorbol myristate acetate (PMA) significantly increased the chemotactic activity of GEN-conditioned media. A checcurboard analysis showed that the response of monocytes to GEN-conditioned media was truly chemotactic. Immunoadsorption with a monoclonal antibody to human MCP-1 reduced the chemotactic activity of GEN-conditioned media by 85%. Northern blot analysis revealed that MCP-1 mRNA was constitutively expressed by GEN and that IL-1beta and tumor necrosis factor-alpha (TNF-alpha) increased MCP-1 mRNA levels in a dose- and time … More -dependent manner. PMA also induced an increase in MCP-1 mRNA levels that was suppressed by the protein kinase C inhibitors staurosporine and H-7, whereas dibutyryl cyclic AMP and forskolin had minimal effects, indicating involvement of protein kinase C.However, MCP-1 mRNA expression induced by IL-1beta and TNF-alpha was suppressed by the tyrosine kinase inhibitor genistein, not by staurosporine, H-7, or the protein kinase A inhibitor H-89, suggesting importance of the tyrosine kinase activation on the cytokine-induced MCP-1 gene expression. Dexamethasone had a small inhibitory effect on constitutive and PMA-induced MCP-1 mRNA expression, but no effect on the induction by TNF-alpha.By immunoperoxidase staining with an anti-MCP-l monoclonal antibody, MCP-1 protein was detected in untreated GEN and increased by exposure to PMA,correspondeng to mRNA expression. These results demonstrate the production of MCP-1 by GEN at gene and protein levels as well as bioactivity, and suggest that GEN may participate in the development of glomerulonephritis through the production of MCP-1. Less