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

Tohru Umekawa, Karen Byer, Hirotsugu Uemura, Saeed R. Khan

Research output: Contribution to journalArticle

Abstract

Background. Our earlier studies have demonstrated upregulation of monocyte chemoattractant protein-1 (MCP-1) in NRK52F 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 24h 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.

Original languageEnglish (US)
Pages (from-to)870-878
Number of pages9
JournalNephrology Dialysis Transplantation
Volume20
Issue number5
DOIs
StatePublished - May 2005
Externally publishedYes

Fingerprint

Calcium Oxalate
Oxalates
Chemokine CCL2
Up-Regulation
Ions
Reactive Oxygen Species
Messenger RNA
NADPH Oxidase
Proteins
8-epi-prostaglandin F2alpha
Catalase
Gene Expression
Free Radical Scavengers
dibasic calcium phosphate dihydrate
Chemotactic Factors
LDL Lipoproteins
Hydrogen Peroxide
Lipid Peroxidation
Culture Media
Real-Time Polymerase Chain Reaction

Keywords

  • Calcium oxalate
  • Calcium phosphate
  • Kidney stones
  • MCP-1
  • NADPH oxidase
  • Reactive oxygen species

ASJC Scopus subject areas

  • Nephrology
  • Transplantation

Cite this

Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells. / Umekawa, Tohru; Byer, Karen; Uemura, Hirotsugu; Khan, Saeed R.

In: Nephrology Dialysis Transplantation, Vol. 20, No. 5, 05.2005, p. 870-878.

Research output: Contribution to journalArticle

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title = "Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells",
abstract = "Background. Our earlier studies have demonstrated upregulation of monocyte chemoattractant protein-1 (MCP-1) in NRK52F 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 24h 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",
author = "Tohru Umekawa and Karen Byer and Hirotsugu Uemura and Khan, {Saeed R.}",
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T1 - Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells

AU - Umekawa, Tohru

AU - Byer, Karen

AU - Uemura, Hirotsugu

AU - Khan, Saeed R.

PY - 2005/5

Y1 - 2005/5

N2 - Background. Our earlier studies have demonstrated upregulation of monocyte chemoattractant protein-1 (MCP-1) in NRK52F 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 24h 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.

AB - Background. Our earlier studies have demonstrated upregulation of monocyte chemoattractant protein-1 (MCP-1) in NRK52F 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 24h 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.

KW - Calcium oxalate

KW - Calcium phosphate

KW - Kidney stones

KW - MCP-1

KW - NADPH oxidase

KW - Reactive oxygen species

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