Nephrol Dial Transplant (2005) 20: 870–878
doi:10.1093/ndt/gfh750
Advance Access publication 8 March 2005
Original Article
Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate
monohydrate and brushite crystal-induced upregulation of MCP-1
in NRK 52E cells
Tohru Umekawa1, Karen Byer2, Hirotsugu Uemura1 and Saeed R. Khan2
1
Department of Urology, Kinki University, School of Medicine, Osaka, Japan and 2Department of Pathology
and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, USA
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
Correspondence and offprint requests to: Saeed R. Khan,
Department of Pathology and Laboratory Medicine, University
of Florida College of Medicine, Box 100275, Gainesville, FL
32610-0275, USA. Email:
[email protected]fl.edu
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
Introduction
Stone formation involves interactions between renal
epithelial cells and ions such as oxalate (Ox) and
crystals such as calcium phosphate and calcium
oxalate. The interactions trigger a cycle of pathological changes [1], leading to up- or downregulation of
specific genes and activation of various inflammatory
factors. We have shown recently that Ox, as well as
calcium oxalate, and the brushite (Br) form of calcium
phosphate crystals upregulate the expression and
production of monocyte chemoattractant protein-1
(MCP-1) by NRK52E, a rat renal epithelial cell line
in culture [2,3]. The results also indicated the possibility of free radical involvement in the upregulation
since catalase treatment reduced MCP-1 expression and
production. MCP-1 is a chemokine with potent chemoattractant activity towards monocytes/macrophages
[4] and has been proposed as a possible mediator
of the inflammatory response to crystal deposition.
It is already known that exposure to high levels of
Ox and calcium oxalate crystals can induce oxidative
stress [5,6] as shown by: (i) an increase in free radical
generation; (ii) increased lipid peroxidation; (iii) a
decrease in cellular anti-oxidant status; and (iv) an
increase in phospholipase-A2 (PLA2)-induced release of
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Crystal-induced expression of MCP-1 by renal epithelial cells
arachidonic acid [7]. Sustained exposure to high levels
of Ox and/or calcium oxalate crystals injures the cells
[1]. Sublethal doses promote DNA synthesis and
cellular proliferation [8], induce a variety of genes [5]
including immediate early genes (eg. c-myc and egr-1),
osteopontin, bikunin and clusterin, and promote
redistribution of phosphatidylserine to the membrane
surfaces [9].
The sources of reactive oxygen species (ROS) in Oxand calcium oxalate crystal-induced alterations remain
unclear. Even though mitochondria have been shown
to be a source of free radicals [10], the possibility of
other sites and sources has not been ruled out. Recent
studies have provided evidence that NADPH oxidase
is a major source of ROS in the kidneys [11] and is
involved in the upregulation of MCP-1 [12]. Therefore,
the specific aim of this study was to investigate
NADPH oxidase as a source of free radicals during
Ox- and crystal-induced changes in the kidneys and
Ox- and crystal-induced upregulation of MCP-1. We
investigated whether diphenyleneiodonium (DPI)
chloride, a selective NADPH oxidase inhibitor, would
reduce the production of ROS, lipid peroxidation and
cell injury, and affect the expression and production
of MCP-1 by NRK52E rat renal tubular epithelial cells
exposed to Ox, or crystals of calcium oxalate and Br.
We hypothesized that DPI would inhibit NADPH
oxidase activity reducing free radical production and
MCP-1 upregulation. ROS were determined as H2O2.
Products of lipid peroxidation were determined as
8-isoprostane (8-IP). Cell injury was determined by
release of lactate dehydrogenase (LDH). We have
recently shown that DPI treatment causes a reduction
in Ox- and calcium oxalate crystal-induced LDH
release by the renal epithelial cells in culture, indicating
the possible involvement of NADPH oxidase [13].
Materials and methods
Cell culture
A normal rat kidney epithelial-derived cell line, NRK52E,
was obtained from American Type Culture Collection
(CRL-1571; Manasses, VA). Cells were maintained as
continuously growing monolayers in 75 cm2 Falcon T-flasks
(Fisher, Atlanta, GA) in culture in a 1:1 ratio of Dulbecco’s
modified essential medium nutrient mixture and F-12
(DMEM/F-12, Gibco BRL, Grand Island, NY) containing
4% fetal calf serum, 15 mmol/l. HEPES, 20 mmol/l. sodium
bicarbonate, 0.5 mmol/l sodium pyruvate, 17.5 mmol/l
glucose, streptomycin and penicillin at 37 C in a 5% CO2
air atmosphere incubator. Under these conditions, cells
achieved confluence. They were washed with serum- and
sodium pyruvate-free DMEM/F-12 medium. Then the cells
were exposed to calcium oxalate monohydrate (COM:
66.7 mg/ml, generously provided by Dr Y. Nakagawa,
University of Chicago) or calcium phosphate (Br: 66.7 mg/ml,
Sigma, St Louis, MO) crystals. Some were exposed to oxalate
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