Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells.
BACKGROUND
Our earlier studies have demonstrated upregulation of monocyte chemoattractant protein-1 (MCP-1) in NRK52E rat renal epithelial cells by exposure to oxalate (Ox) ions and crystals of calcium oxalate monohydrate (COM) or the brushite (Br) form of calcium phosphate. The upregulation was mediated by reactive oxygen species (ROS). This study was performed to investigate whether NADPH oxidase is involved in ROS production.


METHODS
Confluent cultures of NRK52E cells were exposed to Ox ions or COM and Br crystals. They were exposed for 1, 3, 6, 12, 24 and 48 h for isolation of MCP-1 mRNA and 24 h for enzyme-linked immunosorbent assay (ELISA) to determine the secretion of protein into the culture medium. We also investigated the effect of free radical scavenger, catalase, and the NADPH oxidase inhibitor diphenyleneiodium (DPI) chloride, on the Ox- and crystal-induced expression of MCP-1 mRNA and protein. The transcription of MCP-1 mRNA in the cells was determined using real-time polymerase chain reaction. Hydrogen peroxide and 8-isoprostane were measured to investigate the involvement of ROS.


RESULTS
Exposure of NRK52E cells to Ox ions as well as the crystals resulted in increased expression of MCP-1 mRNA and production of the chemoattractant. Treatment with catalase reduced the Ox- and crystal-induced expression of both MCP-1 mRNA and protein. DPI reduced the crystal-induced gene expression and protein production but not Ox-induced gene expression and protein production.


CONCLUSIONS
Exposure to Ox ions, and COM and Br crystals stimulates a ROS-mediated increase in MCP-1 mRNA expression and protein production. Reduction in ROS production, lipid peroxidation, low-density lipoprotein release, and inducible MCP-1 gene and protein in the presence of DPI indicates an involvement of NADPH oxidase in the production of ROS.
Umekawa
Tohru
T
Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Box 100275, Gainesville, FL 32610-0275, USA.
Byer
Karen
K
Uemura
Hirotsugu
H
Khan
Saeed R
SR
D000818:Animals; D002129:Calcium Oxalate / Q000494:pharmacology*; D002130:Calcium Phosphates / Q000494:pharmacology*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D015227:Lipid Peroxidation; D019255:NADPH Oxidases / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology*; D010070:Oxalates / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017382:Reactive Oxygen Species; D015854:Up-Regulation
D002130:Calcium Phosphates; C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D009861:Onium Compounds; D010070:Oxalates; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D002129:Calcium Oxalate; C007517:diphenyleneiodonium; D007770:L-Lactate Dehydrogenase; D019255:NADPH Oxidases; C494366:calcium phosphate, dibasic, dihydrate