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.
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 microg/cm(2)) with or with no pretreatment with diphenileneiodium chloride (DPI, 10 x 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.
Umekawa
Tohru
T
Department of Urology, Kinki University School of Medicine, Osaka, Japan.
Tsuji
Hidenori
H
Uemura
Hirotsugu
H
Khan
Saeed R
SR
D000818:Animals; D002129:Calcium Oxalate / Q000494:pharmacology; D002460:Cell Line; D018932:Chemokine CCL2 / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D006801:Humans; D007150:Immunohistochemistry; D007669:Kidney Calculi / Q000209:etiology / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D053495:Osteopontin / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015290:Second Messenger Systems / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology
D018932:Chemokine CCL2; D012333:RNA, Messenger; D053495:Osteopontin; D013481:Superoxides; D002129:Calcium Oxalate; D019255:NADPH Oxidases