ToxSci Advance Access originally published online on March 21, 2006
Toxicological Sciences 2006 91(2):493-500; doi:10.1093/toxsci/kfj168
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Inducible Nitric Oxide Synthase and Apoptosis in Murine Proximal Tubule Epithelial Cells
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 West Markham Street, # 611 Little Rock, AR 72205. Fax: (501) 686-5521. E-mail: prmayeux{at}uams.edu.
Received December 15, 2005; accepted March 2, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since inducible nitric oxide synthase (iNOS) and proximal tubule injury are known to be critical determinants of lipopolysaccharide (LPS)-induced renal failure, the role of nitric oxide (NO) in proximal tubule cell apoptosis was examined. An 18-h treatment with a combination of LPS (5 µg/ml) and interferon-
(IFN-, 100 units/ml) synergistically induced iNOS and produced a 20-fold increase in NO generation in the TKPTS murine proximal tubule cell line. NO generation by LPS + IFN- was blocked by a specific iNOS blocker, L-N6-(1-iminoethyl)-lysine (L-NIL, 1mM). To assess the role of iNOS-derived NO in proximal tubule cell apoptosis, annexin V– and propidium iodide–labeled cells were analyzed by flow cytometry. Neither the induction of iNOS nor its inhibition produced significant apoptotic cell death in TKPTS cells. Two exogenous NO donors were used to examine the role of NO more directly in proximal tubule apoptosis. Although both sodium nitroprusside (SNP), an iron-containing, nitrosonium cation donor, and S-nitroso-N-acetylpenicillamine (SNAP), a noniron-containing, NO generator, produced a concentration-dependent increase in NO generation, only SNP increased apoptotic cell death in TKPTS cells (5.9 ± 0.7% in control cells vs. 21.6 ± 3.8% in SNP [500µM]-treated cells; n = 4–9; p < 0.01). SNP-mediated tubule cell apoptosis was not dependent on the activation of caspases or p53 but was possibly related to the generation of reactive oxygen species by SNP. Thus, in TKPTS cells induction of iNOS and generation of NO by LPS does not lead to tubular epithelial cell death. Key Words: iNOS; nitric oxide; apoptosis; proximal tubule; sodium nitroprusside; S-nitroso-N-acetylpenicillamine.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acute renal failure (ARF) in experimental sepsis is a result of lipopolysaccharide (LPS)-induced pathologic alterations accompanied by changes in renal function. Renal pathologic changes include morphologic changes predominantly in the proximal tubules including the appearance of apoptotic nuclei, proximal tubule vacuolization, loss of brush border, and mild tubule degeneration (Guo et al., 2004
; Tiwari et al., 2005a). Consequently, proximal tubule function is also compromised (Kang et al., 1995).
Sepsis produces a systemic inflammatory state that is associated with an increase in the expression of proinflammatory genes, cytokines, and inducible nitric oxide synthase (iNOS) (De Vriese, 2003). ARF associated with sepsis appears to be strongly influenced by the unregulated production of nitric oxide (NO) through the cytokine-mediated upregulation of iNOS in various cell types, including kidney tubule epithelial cells (Schrier and Wang, 2004). This makes NO an attractive target for intervention in LPS-induced renal failure (Guo et al., 2004; Tiwari et al., 2005a). However, the role NO may play in the development of renal failure is not well understood.
Tubule epithelial cell function and blood flow are important regulators of glomerular filtration rate (GFR) (Welch et al., 2000). NO can directly act on the vasculature and cause systemic vasodilatation and decrease GFR (Schrier and Wang, 2004). However, excessive, unregulated NO production can also lead to the formation of reactive nitrogen species (RNS) that can oxidize lipids, damage DNA, and cause protein modifications of important cellular proteins and enzymes (Dedon and Tannenbaum, 2004). This could lead to NO-dependent tubule epithelial injury and apoptosis. The ability of NO or RNS to alter mitochondrial function and damage proteins may result in NO-mediated apoptosis or necrosis depending on the amount of NO and RNS generated (Lin et al., 1998; Meij et al., 2004). In general, low "physiological" levels of NO depress caspase activity through nitrosylation of caspase enzymes (Mannick et al., 2001; Rossig et al., 1999) via cGMP and inhibit apoptosis (Brune et al., 1993; Nicotera and Melino, 2004). In contrast, high levels of NO or prolonged synthesis of NO, as occurs following induction of iNOS, can promote apoptosis by mitochondrial damage, stabilization of the tumor suppressor p53, or direct DNA damage (Thomas et al., 2004). If oxidative damage is too great and ATP levels fall below critical levels, necrosis is more likely to result (Brune, 2003; Thomas et al., 2004). Thus, the regulatory role of NO in the process of apoptosis is directed by the source and concentration of NO. Furthermore, the cellular consequences of elevated NO generation are difficult to predict because they also appear to be cell-type specific (Kim et al., 2001) and dependent on the redox status of the cell and the expression of survival proteins (Kim et al., 1997, 2000).
Recently, ARF in murine models of endotoxemia was shown to be dependent on caspase activation and subsequently increased renal apoptosis (Guo et al., 2004; Tiwari et al., 2005a). In addition, we have shown that iNOS-derived NO is a critical determinant of LPS-induced renal failure in the mouse (Tiwari et al., 2005a) and rat (Zhang et al., 2000). Although iNOS, renal apoptosis, and proximal tubule dysfunction appear to be critical determinants of LPS-induced ARF, the role of NO in proximal tubule cell apoptosis has not been well examined. This study was undertaken to examine the effect of overproduction of NO and its role in apoptosis in murine proximal tubule epithelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LPS (Escherichia coli serotype O55:B5), S-nitroso-N-acetylpenicillamine (SNAP), deferoxamine (DFX), and all other chemicals (unless otherwise noted) were purchased from Sigma-Aldrich (St Louis, MO). Sodium nitroprusside (SNP), mouse recombinant interferon-
(IFN-), pifithrin-
, and Z-VAD-fmk were purchased from Calbiochem (San Diego, CA). L-N6-(1-iminoethyl) lysine (L-NIL) was purchased from Alexis Biochemicals (San Diego, CA). 5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate (DCF-DA) was purchased from Invitrogen Corp. (Carlsbad, CA). Apoptosis detection kit was purchased from Biovision Inc. (Mountain View, CA).
Culture of TKPTS cells.
The TKPTS cell line is a mouse proximal tubule cell line developed by Dr Elsa Bello-Reuss (Ernest and Bello-Reuss, 1995) and was a gift from Dr Elsa Bello-Reuss. This cell line has previously been used to study drug transport in proximal tubules (Ernest and Bello-Reuss, 1995), receptor signaling in proximal tubules (Tiwari et al., 2005b), and proximal tubule injury (Arany et al., 2004, 2005; Di Mari et al., 1999). Cells were grown at 37°C under 5% CO2 in 50% Dulbecco's modified Eagle's medium and 50% Ham's F-12 medium containing penicillin (100 U/ml) and streptomycin (100 µg/ml) and supplemented with insulin (50 µU/ml) and 5% fetal bovine serum. Experiments were performed on cells from passages 36 to 48.
Treatment of TKPTS cells.
The concentrations of LPS and IFN- used to induce iNOS were based on reports by others (Poljakovic et al., 2002) and preliminary studies. TKPTS cells were grown in 25-cm2 flasks, 6- or 12-well plates to a density of 80% confluence. Cells were washed once with phosphate-buffered saline (PBS) and incubated with the following treatments for 18 h: PBS (control), LPS, LPS + IFN-, IFN- alone, LPS + IFN- + L-NIL, SNP, or SNP with various inhibitors diluted in culture medium. For iNOS induction time course experiments, cells were incubated with PBS (control) or LPS + IFN- for 0, 18, 24, and 48 h. Since SNAP has an aqueous half-life of about 5 h (Ignarro et al., 1981), cells were treated with SNAP at time 0, 5, 10, and 15 h. At the end of the treatment, culture media supernatants were collected for nitrite measurements. Adherent cells were lysed in RIPA buffer (pH 7.8) containing 1mM EGTA, 1mM MgCl2, 150mM NaCl, 1mM Na3VO4, 50mM Tris (pH 7.8), 1% NP40, 0.5% sodium deoxylate, 0.1% SDS, and a protease inhibitor cocktail (aprotinin, leupeptin, pepstatin A). Total protein concentrations of the cell extracts were determined by a bicinchoninic acid protein assay (Pierce, Rockford, IL).
iNOS Western blot analysis.
A rabbit polyclonal anti-iNOS antibody (Upstate Cell Signaling Solutions, Lake Placid, NY) was used to detect iNOS protein. Cell lysates (30 µg protein per lane) were separated by 10% SDS-PAGE and then transferred to nitrocellulose membrane using an electroblotting transfer apparatus. Nitrocellulose membranes were incubated in 5% nonfat milk blocking buffer for 90 min at room temperature. Following this, membranes were washed and incubated with rabbit polyclonal anti-iNOS antibody (1:2000 dilution in 5% nonfat milk) overnight at 4°C with agitation. Subsequently, membranes were washed and incubated with anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:5000 dilution in 5% nonfat milk) for 90 min at room temperature. Finally, membranes were washed and developed using Supersignal West Pico Chemiluminescent detection kit (Pierce) as described by the manufacturer.
Measurement of nitrite in culture media.
Culture media (190 µl) were deproteinated by incubation with 10 µl of 30% ZnSO4 at room temperature for 15 min. Samples were then centrifuged at 2000 x g for 5 min. Nitrite levels were estimated in supernatants using the Griess reagent. Griess reagent was made of equal volumes of 1% sulfanilamide and 0.1% N-(1-naphthyl)-ethylenediamine in water and 0.5 N HCl. Supernatants and Griess reagent were mixed in a ratio of 1:1 and incubated at room temperature for 15 min, and absorbance was read at 543 nm. Results were compared against a NaNO2 standard curve (0.25–10 µM), and nitrite concentration was calculated. Nitrite concentrations were then normalized to protein as assayed by BCA protein assay and expressed as nmol nitrite/mg protein. In a preliminary study, deproteinated supernatants containing nitrite from cells treated with LPS + IFN- were incubated for 18 h with cadmium beads to reduce any nitrate to nitrite prior to nitrite determination (Tiwari et al., 2005a). No measurable nitrate was detected. Therefore, subsequent studies used only nitrite determination to estimate NO generation.
Annexin V– and propidium iodide–labeling assay.
Subconfluent monolayers of cells grown in 25-cm2 flasks were treated as indicated above for 18 h. Cells were then washed once with binding buffer and labeled with annexin V and propidium iodide according to the manufacturer's instructions. Briefly, cells were incubated in dark with propidium iodide in apoptosis binding buffer for 15 min with constant agitation on ice. Following this, cells were washed once with binding buffer on ice for 10 min. Cells were then labeled with annexin V diluted in apoptosis binding buffer for 10 min at room temperature. Finally, cells were washed again with binding buffer for 10 min and removed with a rubber policeman. The collected cells were then analyzed using a flow cytometer. Ten thousand events were analyzed for each sample.
Dichlorofluorescein fluorescence assay.
Reactive oxygen species (ROS) generation was estimated using dichlorofluorescein (DCF). TKPTS cells were grown in 6-well plates and treated as indicated above for 18 h. Upon washing the cells with PBS, cells were incubated with 10µM DCF-DA for 30 min at 37°C. Adherent cells were then treated with 0.25% trypsin-EDTA for 6 min and neutralized with media. Cell suspension was then centrifuged at 800 x g for 4 min. The cell pellet was then resuspended in PBS and analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA) using excitation and emission wavelengths of 488 and 535 nm, respectively (Eligini et al., 2005).
Data analysis.
Data were analyzed using Prism 4.0 for Mac (GraphPad Software Inc., San Diego, CA). Data are presented as mean ± SEM unless otherwise noted. Data with three or more groups were analyzed using ANOVA followed by Newman-Keuls posttest. A p value < 0.05 was considered significant. Each "n" represents a unique flask or well of cells from passages 36 to 48.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of iNOS in TKPTS Cells
Western blot analysis of treated TKPTS cells revealed that a combination of LPS and IFN-
produced a synergistic induction of iNOS protein at 18 h (Fig. 1). NO in aqueous solution produces nitrite predominantly (Ignarro et al., 1993). Preliminary studies revealed that induction of iNOS resulted in measurable nitrite but not nitrate generation in cell culture supernatants. Consequently, nitrite levels in the cell culture supernatants were used as a marker of NO generation. Similar to what was observed for protein induction, the combination of LPS (5 µg/ml) and IFN- (100 units/ml) produced a synergistic increase in nitrite generation (Fig. 2A). The rise in nitrite levels seen with LPS and IFN- was completely blocked by the addition of a specific iNOS blocker, L-NIL (1mM) (Fig. 2A). These data suggest that LPS and IFN- synergistically lead to generation of NO through the induction of iNOS.
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iNOS-Derived NO Does Not Induce Apoptosis in TKPTS Cells
To examine the role of iNOS-derived NO in proximal tubule cell apoptosis, TKPTS cells treated with LPS (5 µg/ml) and IFN-
(100 units/ml) for 18 h were labeled with annexin V and propidium iodide and analyzed by flow cytometry. No increase in either apoptotic or necrotic cell death was observed with LPS and IFN- treatment (4.7 ± 0.9% cells stained positive for annexin following the 18-h treatment with LPS + IFN- compared to 5.1 ± 0.9% control cells; n = 3–8; p > 0.05). A 10-fold higher concentration of LPS (50 µg/ml) + IFN- was also tested and also failed to increase cell death (6.5 ± 1.1%). Neither the induction of iNOS nor its inhibition by L-NIL produced marked apoptosis in TKPTS cells (Fig. 2B).
Time Course of iNOS Induction
TKPTS cells were treated with LPS (5 µg/ml) and IFN- (100 units/ml) for 0, 18, 24, and 48 h and examined for iNOS induction. Nitrite levels showed a time-dependent increase, indicating progressively increased cumulative NO generation from iNOS (Fig. 3A).
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Effect of Time on iNOS-Mediated Apoptosis
Since no LPS-induced apoptosis was observed at 18 h in these cells, longer incubations of cells with a combination of LPS 5 µg/ml and IFN-
(100 units/ml) were carried out to examine the effect of time on apoptotic events in TKPTS cells. Incubation of cells for 24 or 48 h did not result in significant cell death compared to control cells (n = 3–10; p > 0.05), indicating that even longer duration of treatment did not result in iNOS-derived NO-mediated cell death in TKPTS cells (Fig. 3B). To examine the possibility of substrate limitation, 10mM L-arginine was included in the incubation mixture along with LPS (5 µg/ml) and IFN- (100 units/ml). This treatment did not result in any significant increase in NO generation or apoptosis compared to LPS + IFN-–treated cells, indicating that there was no substrate limitation for iNOS to generate NO.
NO Production by SNAP
The ability of SNAP to generate NO was assessed by nitrite levels in the supernatants (Fig. 4A). SNAP increased NO generation in a concentration-dependent manner as evidenced by elevated nitrite levels (p < 0.01 compared to control for the 50 and 500µM SNAP treatments).
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SNAP Does Not Produce Apoptosis in TKPTS Cells
Flow cytometry analysis of annexin- and propidium iodide–labeled cells revealed that SNAP did not produce apoptosis even at the highest concentrations used in the study (n = 4–10; p > 0.05). Less than 10% of the cells were apoptotic at the end of the 18-h SNAP treatment (Fig. 4B).
NO Production by SNP
To further examine the role of NO in apoptosis in these proximal tubule cells, an iron-containing NO donor, SNP, was used. Addition of SNP to culture media for 18 h increased NO generation as assessed by nitrite measurement in supernatants. SNP increased NO generation in a concentration-dependent manner in TKPTS cells as evidenced by a progressive increase in nitrite levels in culture supernatants (Fig. 5A). Statistically significant NO generation occurred with a 500µM concentration of SNP (0.76 ± 0.08 nmol/mg in control cells vs. 31.48 ± 3.52 nmol/mg in SNP [500µM]-treated cells; n = 5–6; p < 0.001).
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SNP Produces Apoptosis in TKPTS Cells
SNP treatment of TKPTS cells for 18 h resulted in increased apoptosis of cells. SNP produced changes in cell morphology that were consistent with apoptosis and confirmed by a predominant increase in annexin V–labeled cells (Fig. 5B). Apoptotic cell death increased from 5.9 ± 0.7% in control cells to 21.6 ± 3.8% in SNP (500µM)-treated cells (n = 5–6; p < 0.001). However, no significant increase in propidium iodide–labeled cells was observed with SNP treatment (0.7 ± 0.3% in control cells vs. 2.8 ± 1% in cells treated with SNP [500µM]; n = 5–12; p > 0.05). Total cell death with SNP treatment was also significantly increased in TKPTS cells (23 ± 2.1%; p < 0.001 compared to control).
ROS Generation by SNP and LPS
The ability of SNP and LPS to cause the generation of ROS was investigated using the fluorescent probe DCF and flow cytometry (Fig. 6). Four treatment groups were studied at 18 h: control, SNP at 50µM, SNP at 500µM, LPS (5 µg/ml) + IFN- (100 units/ml), and LPS + IFN- + L-NIL (1mM). Of the groups studied, only SNP at 500µM produced a significant (approximately sevenfold) elevation in DCF intracellular fluorescence, indicating the generation of ROS (p < 0.05 compared to control; n = 4).
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Mechanism of SNP-Mediated Apoptosis
The mechanism of apoptosis produced by SNP was investigated further. SNP-mediated apoptotic cell death was not reversed by treatment with Z-VAD (20µM, a broad-spectrum caspase inhibitor) or pifithrin-
(30µM, a p53 inhibitor). These agents have been shown to be effective at these concentrations in other tubular epithelial cell lines (Murphy et al., 2004; Seth et al., 2005). Z-VAD treatment resulted in 16.5 ± 1.2% annexin-positive cells (p > 0.05 compared to SNP 500µM) and 8.8 ± 1.5% propidium iodide–positive cells (p < 0.001 compared to SNP 500µM), indicating a slight shift from apoptotic cell death to necrotic cell death (Fig. 7B). However, SNP-induced total cell death was unchanged with Z-VAD treatment (25.0 ± 1.2%; p > 0.05). DFX (500µM, an iron chelator) completely prevented both SNP-mediated apoptosis (8.4 ± 1.6%; p > 0.05 compared to control) and total cell death (9.6 ± 1.1%; p > 0.05 compared to control) (Fig. 7A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the vascular effects of NO in LPS-induced ARF have been well examined (Boffa and Arendshorst, 2005
; Millar and Theimermann, 1997; Schrier and Wang, 2004), emerging evidence indicates that the role of NO in renal apoptosis could also impact renal function in a major way (Guo et al., 2004; Tiwari et al., 2005a). Cytokine production is a hallmark of human sepsis (Simmons et al., 2004) and murine models of LPS-induced renal failure (Guo et al., 2004; Tiwari et al., 2005a; Wang et al., 2005). LPS and/or cytokines can induce iNOS and apoptosis in various kidney cell types, such as human proximal tubules (Bussolati et al., 2002), cultured human proximal tubule cells (Jo et al., 2002), cultured bovine glomerular endothelial cells (Messmer et al., 1999), and cultured rat mesangial cells (Trachtman et al., 2000). The murine model of sepsis is a frequently used model to study sepsis-induced ARF (Tiwari et al., 2005a). Therefore, studies were undertaken to examine the potential for iNOS-derived NO to induce apoptosis in the murine proximal tubules using the murine proximal tubule epithelial cell line, TKPTS.
Our results indicate that LPS and IFN- act synergistically to induce iNOS in TKPTS cells. Pharmacologic inhibition of iNOS with a specific blocker, L-NIL, resulted in complete blockade of LPS + IFN-–induced NO production, indicating that the rise in NO generation seen with LPS + IFN- was due to induction of active iNOS protein. A combination of LPS and cytokines, particularly IFN-, has been shown to synergistically induce iNOS in various cell types, such as macrophages (Chan and Riches, 2001; Hecker et al., 1996), vascular smooth muscle cells (Wileman et al., 1995), renal epithelial cells (Amoah-Apraku et al., 1995), cardiomyocytes (Kinugawa et al., 1997), and chondrocytes (Shiraishi et al., 1997). Although the combination of LPS and IFN- significantly enhanced NO generation in TKPTS cells, no cell death was observed with the treatment. This is in contrast to a report in the HK-2 human proximal tubule cell line showing that LPS or IFN- stimulated apoptosis (Jo et al., 2002). Differences in the response to LPS or IFN- could be due to species or cell line differences. Although NO-mediated apoptosis has been shown to occur in various cell types (Brune et al., 1998), the degree and type of cytotoxicity is dependent on source and concentration of NO, cell type (Kim et al., 2001), redox status of the cell (Kim et al., 2000), and the expression of survival proteins (Kim et al., 1997). While we observed no cell death, LPS and NO have been shown to cause proximal tubule dysfunction even in the absence of overt cell death (Fissell et al., 2002; Kang et al., 1995; Nakamura et al., 2004), and NO-mediated dysfunction cannot be ruled out in these proximal tubular epithelial cells.
Addition of either an iron-containing NO donor, SNP, or a noniron-containing NO-generating compound, SNAP, resulted in a concentration-dependent increase in nitrite levels, indicating the ability of these compounds to generate NO. Such NO-generating actions of these donors have been shown to occur upon metabolic activation (Kowaluk and Fung, 1990). Addition of SNP, but not SNAP, resulted in a concentration-dependent increase in apoptotic cell death in these cells. The morphologic features of SNP-treated cells exhibited typical apoptotic characteristics that were confirmed by predominant annexin V labeling of the cells. Treatment of TKPTS cells with SNAP did not result in significant cell death, a finding similar to what was observed with iNOS induction. Although it is generally believed that higher levels of NO are responsible for apoptosis, in our studies, NO did not cause apoptotic cell death in TKPTS cells even at relatively high concentrations. This is evident from the NO generation observed at 24 and 48 h and higher SNAP concentrations that produced NO levels comparable to SNP and yet produced no cell death.
Since SNP but not SNAP or LPS/IFN- caused apoptosis, the mechanism of SNP-mediated cell death was investigated further. SNP-mediated cell death has previously been shown to be dependent on mechanisms such as p53 accumulation (Messmer and Brune, 1996), caspases (Chae et al., 2004), iron mobilization (Desole et al., 1998), and oxidative stress (Rabkin and Kong, 2000). We observed that SNP produced increased ROS generation at a concentration that produced cell death. In contrast, LPS/IFN-–induced iNOS and NO generation, but no ROS generation was detected with this treatment. SNP-mediated cell death in proximal tubule cells was not found to be dependent on activation of p53 or caspases since neither pifithrin- nor Z-VAD blocked SNP-mediated apoptosis at concentrations that block cisplatin-induced activation of the apoptotic cascade (Seth et al., 2005). In TKPTS cells Z-VAD treatment shifted a small population of cells (approximately 5%) from the apoptotic to the necrotic category, although this did not significantly alter total SNP-mediated cell death. This finding is similar to that reported in human chondrocytes (Kuhn and Lotz, 2003) and retinal photoreceptor cells (Sanvicens et al., 2004) where ROS scavengers protected against SNP-induced apoptosis while Z-VAD did not. The iron chelator DFX prevented apoptotic and total cell death produced by SNP in TKPTS cells. Previous reports have suggested that iron mobilization from ferritin stores can produce oxidative stress that may lead to SNP-mediated apoptosis in other cell types (Desole et al., 1998; Reif and Simmons, 1990). For example, in PC12 cells, DFX also prevented SNP-induced apoptosis (Desole et al., 1998). Since DFX is known to be a peroxynitrite scavenger (Denicola et al., 1995) in addition to its ability to chelate iron, the mechanism by which DFX protected TKPTS cells needs further investigation.
One possible reason for the differential sensitivity of the NO donors and iNOS-derived NO could be the formation of different redox-activated species of NO. SNP is known to generate NO in the NO+ form (Stamler et al., 1992b). Both NO· and NO+ can regulate the activity of proteins in physiologic conditions, and iNOS can lead to the formation of both species under different redox intracellular conditions. However, because of the unique character and chemical properties of NO· and NO+, these species may react at different target sites on proteins (Stamler et al., 1992a,b).
In conclusion, our results indicate that in TKPTS murine proximal tubule cells, LPS and IFN- lead to a synergistic induction of iNOS and NO generation. However, iNOS induction alone does not appear to lead to apoptosis or necrosis. These data support our in vivo findings in a murine model of LPS-induced ARF (Tiwari et al., 2005a) that suggest that iNOS-induced renal vascular dysfunction may play a more critical role in tubular injury and the development of LPS-induced ARF than direct tubular epithelium cytotoxicity.
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ACKNOWLEDGMENTS |
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This research was supported by funds from the University of Arkansas for Medical Sciences Graduate Student Research Fund awarded to M.M. Tiwari, K.J. Messer was supported by an Institutional Summer Undergraduate Research Fellowship from the American Society for Pharmacology and Experimental Therapeutics.
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