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NDT Advance Access published online on March 8, 2005
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfh750
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
Received July 19, 2004
Accepted January 14, 2005

Original Articles

Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells

Tohru Umekawa 1, Karen Byer 2, Hirotsugu Uemura 1, and Saeed R. Khan 2*

1 Department of Urology, Kinki University, School of Medicine, Osaka, Japan
2 Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, USA

* To whom correspondence should be addressed.
Saeed R. Khan, E-mail: Khan{at}pathology.ufl.edu



  Abstract

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.

Keywords: calcium oxalate; calcium phosphate; kidney stones; MCP-1; NADPH oxidase; reactive oxygen species.
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