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

Tohru Umekawa, Hidenori Tsuji, Hirotsugu Uemura, Saeed R. Khan

Research output: Contribution to journalArticlepeer-review

Abstract

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 p47phox, 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 g/cm2) 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 p47phox in NRK52E cells. RESULTS In COM treated NRK52E cells there was enhanced expression of p47phox 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.

Original languageEnglish (US)
Pages (from-to)115-120
Number of pages6
JournalBJU International
Volume104
Issue number1
DOIs
StatePublished - Jul 2009
Externally publishedYes

Keywords

  • Calcium oxalate
  • Monocyte chemoattractant protein-1
  • Nephrolithiasis
  • Osteopontin
  • Reactive oxygen species
  • Second messenger

ASJC Scopus subject areas

  • Urology

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