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Superoxide from NADPH oxidase as second messenger for the expression of osteopontin and monocyte chemoattractant protein‐1 in renal epithelial cells exposed to calcium oxalate crystals
Author(s) -
Umekawa Tohru,
Tsuji Hidenori,
Uemura Hirotsugu,
Khan Saeed R.
Publication year - 2009
Publication title -
bju international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.773
H-Index - 148
eISSN - 1464-410X
pISSN - 1464-4096
DOI - 10.1111/j.1464-410x.2009.08374.x
Subject(s) - osteopontin , nadph oxidase , superoxide , reactive oxygen species , chemistry , monocyte , microbiology and biotechnology , biochemistry , biology , enzyme , endocrinology , immunology
OBJECTIVE To test the hypothesis that exposure of a renal epithelial cell line, NRK52E, to calcium oxalate monohydrate crystals (COM) would up‐regulate NADPH oxidase subunit p47 phox , enhance superoxide production and increase monocyte chemoattractant protein‐1 (MCP‐1) and osteopontin mRNA levels. MATERIALS AND METHODS Confluent cultures of NRK52E cells were exposed to COM (66.7 µg/cm 2 ) with or with no pretreatment with diphenileneiodium chloride (DPI, 10 × 10 −6 m ) an inhibitor for NADPH oxidase, under serum‐free conditions. The conditioned medium was collected and total cellular RNA isolated from the cells, and subjected to enzyme‐linked immunosorbent assay and real‐time polymerase chain reaction (PCR). Production of reactive oxygen species (ROS) was estimated by dihydroethidium (DHE) staining using a fluorescence microscope. Immunohistochemistry and real‐time PCR were used to analyse p47 phox in NRK52E cells. RESULTS In COM treated NRK52E cells there was enhanced expression of p47 phox and production of superoxide. COM‐induced production of MCP‐1 and osteopontin was significantly reduced after treatment with DPI. CONCLUSIONS While the generation of a lot of ROS might play a major role in tissue injury or death, the regulated generation of low concentration of ROS, possibly by NADPH oxidase, may represent a second messenger system for generation of COM‐induced MCP‐1 and osteopontin production in the renal tubules.