1 Department of Urology, Kinki University, School of Medicine, Osaka, Japan and 2 Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, USA
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: Khan{at}pathology.ufl.edu
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Abstract |
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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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Introduction |
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The sources of reactive oxygen species (ROS) in Ox- and 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].
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Materials and methods |
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Reverse transcriptase for real-time PCR
The transcription of MCP-1 mRNA in NRK52E was determined using real-time polymerase chain reaction (PCR). Total RNA was isolated from treated and untreated cells (1 x 106 cells) using TRIZOL reagent (Gibco-BRL, Grand Island, NY) according to the manufacturer's protocol. A 2 µg aliquot of total RNA was reverse transcribed to cDNA. In brief, 50 µl reactions contained 3 µl of 100 mM MgCl, 1.25 µl of RNase inhibitor, 5 µl of 10x PCR buffer, 10 µl of 10 mM dNTP mix, 1.3 µl of oligo(dT) and 1.5 µl of reverse transcriptase (all the above materials come from Gibco-BRL, Grand Island, NY). This mixture was incubated for 60 min at 37°C, and then heating the reaction mixture to 94°C for 5 min stopped the reaction.
Primers for real-time PCR
Primers were designed using Primer Express software (PE Applied Biosystems, Foster City, CA), according to the cDNA sequences of rat MCP-1 (accession no. M57441) and rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (accession no. NM_017008) as follows: 5'-CAGATCTCTCTTCCTCCACCACTAT-3' (forward primer, bases 1741) and 5'-CAGGCAGCAACTGTGAACAAC-3' (reverse primer, bases 6989) for MCP-1; 5'-TGCCAAGTATGATGACATCAAGAA-3' (forward primer, bases 780803) and 5'-AGCCCAGGATGCCCTTTAGT-3' (reverse primer, bases 831850) for GAPDH.
Real-time quantitative PCR
PCR product was monitored directly by measuring the increase in fluorescence of dye (SYBR GREEN, PE Applied Biosystems) bound to the amplified double-stranded DNA (dsDNA). The parameter of threshold cycle (CT) was defined as the fractional cycle number at which fluorescence exceeds a threshold level. The comparative CT method quantifies the amount of mRNA relative to that of a reference sample, termed the calibrator, for comparison of the expression level of every unknown sample. The calibrator sample was analysed on every assay plate. To normalize the relative amount of MCP-1 mRNA, GAPDH was chosen as an internal reference. The changes in expression are given by unknown samples of interest.
All PCRs were performed using an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). For each PCR run, a master mix was prepared: 1x SYBR PCR buffer, 3 mM MgCl2, 200 µM dATP, dCTP and dGTP, 400 µM dUTP, 300 µM primer set for MCP-1 and GAPDH and 1.25 U of AmpliTaq Gold DNA polymerase. A 5 µl aliquot of diluted (1:20) cDNA was added to 45 µl of the PCR master mix. After an initial 10 min denaturation at 95°C, the thermal cycling comprised 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min.
MCP-1 measurements by ELISA
Cells were maintained as continuously growing monolayers in 25 cm2 Falcon T-flasks (Becton Dickinson Labware, Franklin Lakes, NJ) and cultured in the same conditions as for mRNA isolation, described above. Under these conditions, cells achieved confluence, and they were washed with serum- and sodium pyruvate-free DMEM/F-12 medium and then the cells were exposed to COM, Br or Ox in the same medium (10 ml) for 24 h. The content of MCP-1 in the culture supernatants were determined by ELISA using an ELISA kit for rat MCP-1 (BIOSOURCE, Camarillo, CA) according to the manufacturer's instructions repeated four times. Protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL). Data are presented a means±SD in which values was determined in four different samples.
Lactate dehydrogenase
Since catalase appeared to prevent membrane peroxidation, we further determined whether or not catalase could prevent LDH release on COM, Br or Ox exposure. The 24 h incubated culture media from the control and experimental wells were recovered and centrifuged to remove crystals and cellular debris. LDH activity from the media, at all time periods, was determined with a commercial kit (Proteins International, Inc., Rochester Hills, MI) by a microtitre assay method. All determinations were against appropriate reagent blanks.
Hydrogen peroxide (H2O2)
H2O2 was measured in cell culture media using the Amplex Red Hydrogen/Peroxidase Assay kit (Molecular Probes, A-22188). A standard curve for H2O2 was prepared at 101.25 µM. Samples, standards and blank were aliquoted to a 96-well fluorescent ready plate. A working solution of 100 µM Amplex red reagent and 0.2 U/ml horseradish peroxidase was prepared and added to all the samples, standards and blank. The plate was protected from light and incubated for 30 min at room temperature. The fluorescence emission was measured at 590 nm excitation and 560 nm emission [16].
8-Isoprostane (8-IP)
To assess lipid peroxidation, 8-IP was measured, using an 8-IP ELISA kit (Cayman Chemical, cat. # 516351). This assay is based on competition between 8-IP and an 8-IP acetylcholinesterase (AchE) conjugate for a limited number of 8-IP-specific rabbit antiserum-binding sites. Samples, standard, 8-IPsupplied with the kit at dilutions of 5003.9 pg/ml controls (total activity, non-specific binding and maximum binding)and blank were aliquoted to designated wells on the 96-well plate. 8-IP AchE tracer and 8-IP antiserum were added to all wells according to the protocol. The plate was incubated for 18 h at room temperature. After incubation, the plate was washed five times with washing buffer (supplied with the kit). Tracer was added to the total activity wells and Ellman's reagent added to all wells. The plate was incubated in the dark at room temperature with gentle shaking for 7590 min. Absorbance was read at 405 nm using a Bio-Rad 3550 microplate reader [16].
Experiment with free radical scavenger and NADPH oxidase inhibitor
To evaluate the protective effect of free radical scavenger (catalase: 2000 U/ml, Sigma) or NADPH oxidase inhibitor (DPI: 10 µM, Sigma), NRK52E cells were exposed to COM, Br or Ox. In the case of catalase, cells were pre-treated for 24 h and co-treated for 6 h for mRNA and 24 h for ELISA and LDH analysis. In the case of DPI, cells were pre-treated for 90 min. Both pre-treatments were followed by replacement of media and DPI or catalase. According to the manufacturer's specifications, DPI solutions remain stable for at least 12 h. We prepared fresh DPI solutions immediately before use. Concentrations of DPI and catalase and duration of exposure were selected based on results of earlier studies. In previous studies, DPI has been used at concentrations of 5100 µM and for up to 48 h [17].
Statistical analysis
We used one-way analysis of variance to test for overall differences among the groups, followed by Fisher's modified least significant difference to compare the separate groups (StatView, SAS Institute Inc, NC). P-values <0.05 denoted the presence of a statistically significant difference. Real-time PCR, ELISA and LDH assay were done on four different samples.
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Results |
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Production of MCP-1 protein by NRK52E cells
There was marked upregulation of MCP-1 protein synthesis by the NRK52E cells after COM, Br and Ox exposure (Figure 4A). The amount of MCP-1 protein production was higher after an exposure to COM or Br crystals than after exposure to the Ox only. Catalase treatment significantly reduced the MCP-1 protein synthesis by NRK52E cells on COM, Br or Ox exposure. The protein synthesis was, however, still higher by cells exposed to COM, Br or Ox than by the unexposed control cells. DPI treatment significantly inhibited the production of MCP-1 synthesis on COM and Br exposure, but not on Ox exposure.
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Production of the reactive oxygen species, H2O2 and the product of lipid peroxidation 8-IP
Exposure to Ox as well as COM and Br crystals resulted in significant increases in the production of H2O2 (Figure 5A). Pre-treatment with DPI was associated with a significant reduction in the Ox-, COM- or Br-induced production of H2O2. However, the presence of DPI did not completely abolish the production of H2O2, and cells exposed to various challenges produced similar amounts of H2O2.
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Discussion |
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That injury is most probably mediated by ROS is shown by the reduction in LDH release in the presence of the free radical scavenger catalase. In addition, DPI, an inhibitor of NADPH oxidase, reduced the Ox-, and crystal-induced production of H2O2 as well as 8-IP, a product of lipid peroxidation. Ox- or crystal-induced LDH release was only partially blocked by catalase or DPI treatment. Lipid peroxidation was also not completely prevented by either the DPI or catalase treatment. This indicates that exposure to Ox, and calcium oxalate or Br crystals produces many types of ROS and that NADPH oxidase is not their only source. Mitochondrial involvement in the production of ROS by renal epithelial cells on expsoure to Ox and calcium oxalate crystals has already been suggested [10].
ROS are influential molecules modulating a variety of cardiovascular and renal functions [11,18]. Superoxide (.), hydroxyl radical (.OH) and H2O2 are the most prominent ROS. Antioxidant enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase. Dismutation of .
by SOD produces H2O2, which is a precursor of the highly reactive .OH. H2O2 is more stable than other ROS such as the superoxide or hydroxyl radicals, can pass through the cell membrane and has been shown to cause lipid peroxidation, DNA damage and, in the end, cell death. H2O2 is converted to water and oxygen in the presence of catalase. NADPH oxidases are a major source of superoxide in vascular cells and monocytes [18]. When stimulated, NAD(P)H oxidases catalyse superoxide production by transferring a single electron from NADH or NADPH to molecular oxygen. The expression and activity of NADPH oxidase have also been detected in kidney cells such as proximal tubular epithelial cells, glomerular mesangial cells and cells of the thick ascending loops of Henle [11]. In response to growth factors, cytokines and other stimuli, they produce superoxide, which is metabolized to hydrogen peroxide [19].
Our results also indicate that DPI reduces the gene expression and production of MCP-1 by NRK52E cells exposed to Ox as well as CaOx or Br crystals. In addition, the production of ROS as determined by measuring the production of H2O2 and 8-IP, a product of lipid peroxidation, is also reduced. DPI is a selective inhibitor of NADPH oxidase and has been shown to reduce the production of ROS and associated injury. Interestingly, both DPI and catalase produced similar reductions in the expression of the MCP-1 gene and production of MCP-1 in response to an exposure to the CaOx and Br crystals. However, Ox-induced gene expression and protein production were not significantly reduced by DPI treatment, indicating differences in cellular response to Ox ions and calcific crystals. The Ox-induced increase in the production of H2O2 and 8-IP was, however, significantly inhibited by DPI treatment. In addition, crystals appear to act quicker than Ox with regard to the gene expression. These results suggest that the pathways triggered by the crystals and ionic Ox are complex and may be different. Ox can elicit a response from both inside and outside, while crystals which are externally applied interact at least initially with the cell surface membrane. Differences between responses to an ion and crystals may also be the result of dissimilarities in their interactions with the cells. The initial crystalcell interaction would most probably be mechanical. Studies of interactions between various cells and different types of disease-causing calcium phosphate crystals, collectively termed basic calcium phosphate, have indicated cell responses to be dependent upon, among other characteristics, crystallanity and crystal shape and size. Since the results of earlier studies have shown that exposure to non-calcium-containing uric acid crystals [3] and latex beads [20] does not trigger particularly significant production of macromolecules, it is conceivable that the response to the two calcium-containing crystals tested here is specific. Differences in cellular response to the two crystals were also noted. DPI did not significantly block the increase in Br-induced LDH release while it was reduced when cells were exposed to COM crystals in the presence of DPI, indicating the activation of different pathways by the two crystals.
In addition, exposure to calcium phosphate crystals activates both calcium-independent and -dependent signalling pathways. Addition of basic calcium phosphate crystals to the culture medium causes an increase in the intracellular calcium, some of which is suggested to be the result of intracellular dissolution of phagocytosed crystals [21,37,38]. Increased intracellular calcium activates a variety of calcium-dependent signals eventually leading to the upregulation of various transcription factors. Calcium oxalate crystals are also endocytosed by renal epithelial cells and undergo dissolution [1], but currently there are no records of calcium oxalate crystal exposure and endocytosis-induced changes in intra- or extracellular calcium. As we have shown here, cell response to crystals was faster than to the Ox ions, indicating that the changes investigated here did not result from crystal endocytosis and dissolution. There are conflicting reports on the effect of oxalate exposure on intra- and extracellular calcium in renal epithelial cells. We found that an addition of oxalate to the culture medium of renal epithelial cells causes a decrease in free intracellular calcium without any change in the extracellular calcium levels [22]. It was concluded that calcium accumulated at the intracellular sites such as mitochondria and endoplasmic reticulum. An earlier study reported that exposure to high oxalate does not cause any change in either the extracellular or intracellular calcium [23].
Even though DPI also inhibits other ROS-generating enzymes such as NADH oxidase, inducible nitric oxide synthetase and mitochondrial NADH dehydrogenase, it is generally understood that membrane-associated NADPH oxidase is the major DPI target. Our recent animal model studies have shown that calcium oxalate crystal deposition in the kidneys activates the reninangiotensin system [24]. Angiotensin II is implicated in causing oxidative stress by stimulating membrane-bound NADPH oxidase, which leads to increased generation of superoxide [19].
MCP-1 is responsive to NADPH oxidase activation with the potential involvement of ROS. Generation of ROS by NADPH oxidase increased the transcription rates for MCP-1 in the mesangial cells in response to tumour necrosis factor- (TNF-
) [12]. Inhibition of NADH/NADPH oxidase by DPI reduced the induction of MCP-1 by angiotensin II [25] and platelet-derived growth factor [26] in rat and human vascular smooth muscle cells (VSMCs), respectively. Induction of MCP-1 gene expression in rat VSMCs was inhibited by catalase and in human VSMCs by superoxide dismutase. TNF-
, interleukin (IL)-1 and interferon-
(IFN-
) stimulated the secretion of MCP-1 and IL-6 by human umbilical endothelial cells through ROS production via NADH oxidase [27]. This cytokine-induced secretion by the endothelial cells was completely blocked in the presence of Tiron and Tempol. The NADPH oxidase inhibitor DPI blocked the IL-1ß and TNF-
-induced upregulation of PLA2 at the mRNA as well as protein levels in rat mesangial cells by attenuating NF-
B binding [14]. Thrombin-induced generation of ROS in human VSMCs was mediated by the activation of the P22phox, subunit of the NADPH oxidase. ROS derived from this P22phox-containing NADPH oxidase contribute to the thrombin-induced MCP-1 gene expression [28]. NADPH oxidase-derived ROS such as superoxide and H2O2 mediate TNF-
-induced expression of MCP-1 in endothelial cells [27].
In summary, our results demonstrate that Ox-, and COM and Br crystal-induced upregulation of MCP-1 is significantly reduced by DPI at both the mRNA and protein levels in the rat renal epithelial cell line NRK52E. Ox- and crystal-induced production of H2O2 and lipid peroxidation is also signficantly reduced by DPI treatment. We conclude that oxalate and calcific crystal-induced MCP-1 upregulation is mediated in part by ROS produced at many sites including membrane-associated NADPH oxidase. Both experimental [1,5] and clinical studies [29] indicate an association between stone formation and development of oxidative stress and renal epithelial injury. In addition renal crystal deposits induce inflammation in which MCP-1 may play a significant role [2,3]. Based on the results of these studies, a case can be made for therapeutic utilization of antioxidants to control stone recurrence [30]. Since ROS can be produced through different pathways, requiring different reagents to counter oxidative stress, it is essential to determine specific pathways involved in Ox- and crystal-induced oxidative stress.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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