Department of Renal Medicine, University of Sydney at Westmead Hospital, Westmead, Sydney, Australia 2145
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ABSTRACT |
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We recently reported that inhibition of the transcription factor
nuclear factor-B (NF
B) with pyrrolidinedithiocarbamate (PDTC)
reduced interstitial monocyte infiltration in rats with proteinuric
tubulointerstitial disease, whereas
N-acetylcysteine (NAC) was not
effective. Here we investigate the effects of antioxidants (PDTC, NAC,
and quercetin) on NF
B activation and cytokine transcription in
primary cultured rat proximal tubular epithelial cells (PTC) stimulated
with lipopolysaccharide. Antioxidant-mediated inhibition of NF
B
activation (PDTC, 20-100 µM; NAC, 100 mM; and quercetin, 50 µM) diminished the induction of both pro- [interleukin
(IL)-1
, tumor necrosis factor-
, monocyte chemoattractant
protein-1, macrophage inflammatory protein (MIP)-1
, and MIP-2]
and anti-inflammatory (IL-10, transforming growth factor-
1) cytokine
transcription in PTC (RT-PCR analysis). PDTC and quercetin did not
affect PTC viability, but NAC (100 mM) caused a threefold increase in
lactate dehydrogenase leakage (P < 0.001). We conclude that NAC is unable to suppress NF
B activation in
PTC at subtoxic and physiologically relevant concentrations.
Furthermore, antioxidant-mediated inhibition of NF
B is correlated
with the nonselective reduction of cytokine transcription in activated
tubular cells. These data might explain the protective effects of
PDTC-mediated NF
B inhibition in tubulointerstitial disease in vivo.
N-acetylcysteine; pyrrolidinedithiocarbamate; quercetin; primary culture; osteopontin; proximal tubular cells; nuclear factor-B
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INTRODUCTION |
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THE ACCUMULATION OF macrophages within the interstitial space of the renal cortex plays a pathogenic role in the development of tubular injury and interstitial fibrosis in progressive chronic renal diseases (47). Proximal tubular epithelial cells (PTC) are thought to mediate the interstitial macrophage infiltration because of their anatomic proximity and ability to produce chemotactic cytokines and other proinflammatory mediators (36).
In PTC, the ubiquitous proinflammatory transcription factor nuclear
factor-B (NF
B) has a pivotal role in the regulation of chemokines
[cytokine-induced neutrophil chemoattractant (CINC), regulated on
activation normal T cell expressed and secreted (RANTES), and monocyte
chemoattractant protein (MCP)-1] (33, 49, 52) and adhesion
molecules [intercellular adhesion molecule (ICAM)-1 and vascular
cellular adhesion molecule-1] (32). In other
cell types, NF
B is known to regulate the production of other
cytokines [such as interleukin (IL)-1
and tumor necrosis
factor (TNF)-
], chemokines [such as macrophage
inflammatory protein (MIP)-1
and MIP-2], and many early
response genes (2).
The antioxidants pyrrolidinedithiocarbamate (PDTC),
N-acetylcysteine (NAC), and quercetin
inhibit NFB activation in a wide variety of cells, possibly by
suppressing the production of intracellular reactive oxygen species (2,
41, 42, 46). In murine and porcine
(LLC-PK1) PTC lines, inhibition
of NF
B by PDTC reduced the expression of inducible nitric oxide
synthase (iNOS) and RANTES, respectively (1, 52). NAC also suppressed
NF
B activation in LLC-PK1 cells
after stimulation with cysteine
S-conjugates (35). However, in
activated rabbit alveolar macrophages, PDTC increased TNF-
despite
inhibition of NF
B (6), and in an adenocarcinoma cell line, NAC
paradoxically increased NF
B (11). Together, these data suggest that
antioxidants are cell specific in their ability to inhibit NF
B and
in the cytokines that are modulated.
Less studied are the effects of antioxidants on the expression of
cytokines with macrophage deactivating properties, such as IL-10 and
transforming growth factor (TGF)-1 (3). In vitro, in human
monocytes, PDTC reduced lipopolysaccharide (LPS)-induced IL-10
secretion and increased TNF-
production (23). In contrast, in vivo,
in rats with LPS-induced endotoxemia, PDTC increased in vivo plasma
levels of IL-10 but suppressed TNF-
, IL-12, MIP-1
, and nitric
oxide production and had no effect on IL-1
, IL-6, and interferon-
induction (31). In another study, diethyldithiocarbamate (a related
dithiocarbamate) did not affect the increase in transcription of
TGF-
1 in the postischemic myocardium of rats but reduced IL-1
, IL-6, TNF-
, and iNOS together with NF
B DNA-binding activity (7).
The differential modulation of pro- and anti-inflammatory cytokine
transcription by antioxidant-mediated NF
B inhibition may explain the
cytoprotective effects of dithiocarbamates in both models (7,
31).
Recently, we reported that PDTC reduced renal cortical NFB
activation, tubular injury, and interstitial monocyte infiltration in
rats with doxorubicin-induced chronic glomerular disease (39). In
contrast, NAC had no significant effect on these parameters. By
immunohistochemical staining, tubular cells are the predominant cellular source of activated NF
B (p50) in this model (unpublished observations). Therefore, we hypothesized that
antioxidants (NAC, PDTC, and quercetin) could differ in their ability
to 1) suppress NF
B activation in
PTC and/or 2) selectively modulate
cytokine transcription in PTC, such that proinflammatory cytokines
(IL-1
, TNF-
, and osteopontin) and chemokines (MCP-1, MIP-1
,
and MIP-2) are reduced, in preference to those with anti-inflammatory
properties (IL-10 and TGF-
1). The following studies were designed to
test these hypotheses.
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METHODS |
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Isolation and primary culture of rat
PTC.
PTC were isolated and cultured from normal male Wistar rats with the
use of isopycnic centrifugation, as previously described (8, 48, 49).
The cells were grown on plastic tissue culture dishes (coated with rat
tail collagen) in DMEM supplemented with epidermal growth factor (10 ng/ml), insulin (5 µg/ml), transferrin (5 mg/ml), and hydrocortisone
(5 × 108 M) in a 5%
CO2 atmosphere at 37°C. The
medium was supplemented with 5% FCS for the first 24 h, after which
point it was replaced with serum-free media. The cells formed dome
structures in culture and were positive for alkaline phosphatase
staining, confirming their PTC origin, as described previously (8, 48,
49). Experiments were commenced when cells reached confluence,
~4-5 days after isolation and initial plating.
Experimental protocol.
LPS (Escherichia coli, serotype
026:B6; Sigma-Aldrich, Sydney, Australia) was used as a stimulant of
NFB activation in PTC (1, 49). Confluent cultures of PTC were
incubated with LPS (5 µg/ml) or vehicle, with or without the
antioxidants (NAC, PDTC, or quercetin), and harvested after 8 h for
analysis of NF
B activation, cytokine gene transcription, and cell
viability (48, 49). The antioxidants were added 1 h before LPS and were
continued until the end of the experiment. In some
experiments, catalase (1,000 units/ml; human erythrocyte source),
desferrioxamine (DFO; 200-800 µM), and hydrogen peroxide
(H2O2;
200 µM) were added 1 h before the addition of NAC without LPS.
Preparation of nuclear protein
extracts.
Nuclear proteins were extracted from PTC with the use of methods
described by Dignam et al. (12) with minor modification (49). Approximately 1 × 108 cells were washed in PBS and
then scraped from culture plates and transferred to microcentrifuge
tubes. The cells were resuspended in buffer
A (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM
MgCl2 · 6H2O, and 0.1 mM EDTA) containing a cocktail of protease inhibitors [0.5 mM dithiothreitol (DTT), 0.1 mM pepstatin A, 1 µM
phenylmethylsulfonyl fluoride (PMSF), 0.05 µg/ml leupeptin, and 0.01 mM aprotinin] and lysed by 10 even strokes of a glass-Teflon
homogenizer. Successful release of nuclei was checked by phase-contrast
microscopy. The mixture was centrifuged for 6 min at 6,000 rpm at
4°C, and the resultant pellet was resuspended in
buffer C [20 mM HEPES, pH 7.9, 25% (vol/vol) glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 0.01 mM KCl, and 300 mM NaCl] and incubated on ice for
30 min. After the addition of buffer D
(20 mM HEPES, 19% glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 1 mM PMSF),
the mixture was centrifuged for 10 min at 13,000 rpm at 4°C. The
supernatant (containing nuclear protein extract) was removed and placed
in a separate tube. The protein concentration was determined by the Bradford method (Bio-Rad) (5) and stored in diluted aliquots (3 µg/µl) at 70°C.
Electrophoretic mobility shift assay.
Double-stranded oligonucleotide consensus for the B binding site of
the
-immunoglobulin light-chain gene (5'-AGT TGA
GGG GAC TTT CCC
AGG-3'; Promega, Madison, WI) was end-labeled with [
-32P]ATP (Amersham
Life Science, Sydney, Australia) with the use of T4 kinase (Promega,
Sydney, Australia). Unincorporated label was removed with a G-50
Sephadex spin column. The binding reaction was performed for 30 min at
room temperature and contained 5 µg of nuclear protein, 2 µl of
binding buffer (5 mM MgCl2, 50 mM Tris · HCl, 250 mM NaCl, 20% glycerol, 2.5 mM EDTA,
2.5 mM DTT, and 0.25 mg/ml poly[dI-dC]), 1 µl of
32P-labeled NF
B probe (5,000 counts/min, Cerenkov counting), and distilled water (DW) to a total
volume of 10 µl. The DNA-protein complexes were resolved by
electrophoresis on a 10 × 12-cm, 7% polyacrylamide gel (1×
TBE buffer; TBE is 89 mM Tris base + 89 mM boric acid + 2 mM
EDTA). The gel was run at 10 V/cm for 60 min and then
dried onto filter paper under vacuum with a gel dryer (80°C for 1 h). Autoradiographs were prepared by exposing the dried gel to X-ray
film (Hyperfilm HP film; Amersham Life Science) with an intensifying
screen for 3-6 h at
70°C (49).
RT-PCR. Total RNA was extracted from cell monolayers with the use of a one-step phenol-guanidinium isothiocyanate procedure based on the method of Chomczynski and Sacchi (9), using RNAzol B (Teltest, Friendswood, TX). The total RNA concentration was determined by absorbance at 260 nm with the use of a spectrophotometer (Beckman DU-68; Beckman Instruments, Fullerton, CA).
First-strand complementary DNA (cDNA) synthesis was performed in a 20-µl reaction containing 1 µg of total RNA, 50 U murine leukemia virus reverse transcriptase, 20 U RNase inhibitor, 2.5 µM oligo(dT)16, 2 µl of 10× PCR buffer II (100 mM Tris · HCl and 500 mM KCl), 1 µl of 25 mM MgCl2 (Perkin-Elmer, Melbourne, Australia), 1 mM dNTP and diethyl pyrocarbonate (DEPC)-treated water. The reaction was performed at 25°C for 10 min, 42°C for 90 min, and 99°C for 5 min with the use of a thermocycler (PTC-100; MJ Research, Boston, MA). Two negative controls were included with all reverse transcription reactions (total RNA replaced with DEPC-treated water, and reverse transcriptase replaced with DEPC-treated water containing RNA). The resultant cDNA was diluted to 100 µl with DW and stored at
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Assessment of PTC viability. The effect of NAC, PDTC, and quercetin on PTC viability was assessed biochemically by measuring the cellular leakage of the cytosolic enzyme lactate dehydrogenase (LDH) (26). This assay is a sensitive and early marker of nonspecific injury that can precede evidence of irreversible cellular damage (18). The assay has previously been used in our laboratory to assess PTC damage (8). LDH activity in cell culture supernatants was measured at 37°C as the amount of pyruvate consumed [by continuously measuring the decrease in absorbance at 339 nm with a spectrophotometer (Cary 2300; Varian Techtron, Victoria, Australia)] because of oxidation of NADH in 0.05 M NaPO4 (pH 7.4) (26). Total cellular LDH was determined after cells were lysed with 1% (vol/vol) Triton X-100 for 30 min. The supernatant LDH activity divided by the total cellular LDH activity, expressed as a percentage, was defined as the amount of LDH leakage (8).
PTC viability was also assessed morphologically by transmission electron microscopy. Confluent cell monolayers exposed for 8 h to vehicle, NAC (100 mM), PDTC (100 µM), or quercetin (50 µM) were immersion fixed in Karnovsky's buffer for 1 h at room temperature. The cells were gently removed from culture plates with a cell scraper and resuspended in MOPS buffer overnight at 4°C. They were then encapsulated in BSA and postfixed in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 3 h. After being rinsed in distilled water, they were dehydrated in a graded ethanol series (50, 70, 95, and 100% ethanol in 0.1% NaCl for 10 min each, followed by 100% acetone for 10 min, repeated once) and embedded in Spurr's epoxy resin. The blocks were polymerized at 70°C for 14 h. Semithin sections were cut with a microtome (Reikhardt-Jung Ultracut), stained with methylene blue, and examined by light microscopy. Ultrathin sections were stained with 2% uranyl acetate and Reynold's lead citrate and then examined with an electron microscope (Philips CM 10).Statistical analysis. All data points are means of values obtained from two to three separate experiments, each containing duplicate or triplicate samples (culture dishes), as specified in RESULTS. Statistical analyses were performed with JMP statistical software (SAS Institute, Cary, NC). The Shapiro-Wilk test was used to determine whether the experimental groups were parametric or nonparametric in distribution. The Kruskal-Wallis one-way nonparametric ANOVA was used to analyze differences among three or more groups. Post hoc tests to compare the differences between two groups were performed with the Mann-Whitney U test. Data are presented as means ± SE. P < 0.05 was considered significant.
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RESULTS |
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Effect of NAC, PDTC, and quercetin on
NFB activation.
In control cells, virtually no NF
B proteins could be detected by
electrophoretic mobility shift assay (EMSA) in nuclear extracts of PTC
(Fig. 1, lane
1). After 8 h, LPS (5 µg/ml) increased NF
B DNA
binding activity in nuclear extracts of PTC (Fig. 1,
lane 2). The addition of NAC, PDTC,
or quercetin to the media 1 h before LPS completely prevented
LPS-induced activation of NF
B in a dose-dependent manner (Fig.1,
lanes 3-10). However, at least
a 500-fold higher concentration of NAC was required to suppress NF
B
activation than that needed for PDTC and quercetin. LPS-induced NF
B
activation was not affected by antioxidants at low concentrations (NAC
5-20 mM, PDTC 5 µM, and quercetin 10 µM). The incubation of
PTC with NAC (100 mM), PDTC (100 µM), or quercetin (50 µM) alone
for 8 h did not induce NF
B activation in control cells (data not
shown).
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Effect of NAC, PDTC, and quercetin on cytokine
transcription in PTC.
To assess the effect of antioxidant-mediated NFB inhibition on the
transcription of multiple cytokines, a semiquantitative method of
RT-PCR was used as described previously (10, 21). To validate the
method, varying amounts of total RNA from LPS-stimulated PTC were
amplified for 32 cycles with primers specific for rat MCP-1 cDNA (48)
(Fig.
2A). A
curvilinear relationship exists between the quantity of template RNA
and the volume density of the amplified product when plotted
semilogarithmically (Fig. 2A). The
same relationship exists at 30 cycles of amplification for MCP-1 (data
not shown). Similar results were obtained with the use of primer pairs
for the other cytokines and GAPDH (with 1 µg of total RNA within the
linear section of the curve) but at cycle numbers different to that
used for MCP-1 (Table 1). The cycle number chosen for each of the
primer pairs was within the linear phase of PCR amplification (Fig.
2B and data not shown). The
intra-assay coefficient of variation of RT-PCR amplification followed
by agarose gel electrophoresis and densitometry was <10% (Fig.
2C). Negative PCR and RT controls
that accompanied the PCR reactions produced no bands on agarose gel
electrophoresis (data not shown).
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Effect of LPS, NAC, PDTC, and quercetin on PTC
viability.
To assess whether the doses of LPS or those of the antioxidants
affected cell viability, the effect of LPS, NAC, PDTC, and quercetin on
LDH leakage was determined. LDH leakage was similar to that in control
cells after an 8-h exposure to LPS (5 µg/ml), PDTC (100 µM), or
quercetin (50 µM) alone (Fig. 7). In
contrast, NAC (100 mM) increased LDH leakage up to threefold. The
increase in LDH leakage was dose dependent, starting at 20 mM
(P = 0.004) and peaking at 100 mM
(P < 0.001). Because NFB
inhibition can unmask TNF-
-induced cytotoxicity in
LLC-PK1 cells (51), LDH leakage
was also assessed in LPS-stimulated PTC treated with each of the
antioxidants. LDH leakage in LPS-treated PTC exposed to either PDTC
(100 µM) or quercetin (50 µM) was similar to that in control cells.
In PTC treated with both LPS and NAC (100 mM), LDH leakage was
increased to the same levels as those in unstimulated cells (data not
shown).
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Effect of
H2O2,
DFO, and catalase on NAC-induced LDH leakage.
NAC can undergo metal-catalyzed autooxidation, resulting in the
formation of superoxide anion and
H2O2.
H2O2
can then cause cellular damage directly or lead to the formation of
· OH via the Haber-Weiss and Fenton reactions
(17, 30, 45). Therefore, we investigated the effect of catalase and DFO
on NAC-induced LDH leakage. Exposure of PTC to catalase (1,000 U/ml) or
DFO (200 µM) alone did not induce LDH leakage (Fig. 8). This dose of
catalase has previously been reported to attenuate thiol-induced
autooxidation in other cell types (30), and we have previously shown
that 200 µM DFO can prevent iron-induced toxicity in PTC (8).
Treatment of PTC with either DFO or catalase 1 h before NAC did not
affect NAC-induced LDH leakage (Fig. 9). A
higher dose of DFO (800 µM) was also not effective (14.2 ± 2.7%;
P value not significant compared with
NAC, 100 mM alone). A higher dose of catalase alone (5,000 U/ml)
increased LDH leakage in PTC compared with control cells (4.1 ± 0.2 vs. 2.9 ± 0.2% in control cells;
P < 0.05). Also, neither catalase
(1,000 U/ml) nor DFO (200 µM) was able to prevent LDH leakage induced
by a lower dose of NAC (20 mM) (data not shown).
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DISCUSSION |
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This study has investigated the effects of three structurally diverse
antioxidants (NAC, PDTC, and quercetin) on NFB activation, cytokine
transcription, and cell viability in rat PTC in primary culture.
Similar studies (1, 33, 35, 50, 52) were performed in cell lines, and
the differential effects of antioxidant-mediated NF
B inhibition on
cytokine transcription were not known. The results of the present paper
show that both PDTC and quercetin are potent suppressors of NF
B
activation, whereas NAC is ineffective at subtoxic concentrations.
Furthermore, and contrary to our original hypothesis,
antioxidant-mediated inhibition of NF
B was associated with the
reduction of both pro- (IL-1
, TNF-
, MCP-1, MIP-1
, and MIP-2)
and anti-inflammatory (TGF-
1 and IL-10) cytokine transcription.
Quercetin is a prototypical polyphenolic plant flavonoid that has
potent antioxidant and anti-inflammatory effects (20, 24, 41, 44). On
the basis of the ability of quercetin to suppress NFB activation in
nonrenal cells (41), we predicted that it would have similar effects in
PTC. In vitro, quercetin prevented cisplatin-induced cellular injury in
LLC-PK1 cells (20). In vivo, in
rats with acute renal ischemia, the prophylactic administration of quercetin prevented tubular injury and the upregulation of chemokines (MCP-1 and RANTES) in the renal cortex (44). Data from the
present study suggest that the mechanism of these effects could, at
least in part, involve the suppression of NF
B activation and the
reduction of cytokine and chemokine transcription in PTC. Further
studies are needed to determine whether the continuous administration
of quercetin could attenuate the chronic upregulation of NF
B in
renal tubulointerstitial disease in vivo (29, 39).
Consistent with recent data reported by Woods and colleagues (50), our
results showed that at least 100 mM NAC was required to suppress NFB
activation in tubular epithelial cells. The reasons why NAC is a less
potent inhibitor of NF
B in PTC than either PDTC or quercetin are not
certain. In a previous study (39), we found that NAC (150 mg/kg twice
daily ip injection for 14 days) was not able to attenuate
renal NF
B activation and tubulointerstitial injury in rats with
doxorubicin-induced nephrosis. The treatment regimen used in the latter
study may have resulted in a peak plasma concentration of NAC between 3 and 15 mM (4, 45). Higher doses were limited by systemic toxicity in
nephrotic rats (39). Although it is not possible to directly
extrapolate the findings of the present in vitro study, the results
suggest that the failure of NAC to inhibit renal NF
B activation in
vivo may have been because therapeutic concentrations were not attained
in the kidney cortex.
The mechanisms by which PDTC, quercetin, and NAC suppress NFB
activation in PTC are not known. Although all three agents are
antioxidants, recent evidence suggests that this property may not be
responsible for their ability to inhibit NF
B in tubular epithelial
cells (50). Paradoxically, the prooxidant and metal-chelating properties of PDTC could be involved in its ability to inhibit NF
B
(37). In this regard, PDTC appears to act catalytically with micromolar
amounts to cause the oxidation of several hundred molar equivalents of
intracellular glutathione (37). The latter may explain the steep
concentration gradient of PDTC-mediated NF
B inhibition. Similarly,
the suppression of protein tyrosine kinases and protein kinase C could
play an important role in quercetin-induced NF
B inhibition (24).
Having demonstrated that NAC, PDTC, and quercetin suppressed NFB
activation in PTC, we next examined their effects on cytokine gene
transcription. The promoter regions of several proinflammatory cytokine
genes contain binding sites for NF
B (2). We have recently shown that
the 5'-flanking end of the rat MCP-1 gene (derived from the
genomic DNA of PTC) contained at least two putative binding sites for
NF
B (49). Nevertheless, it cannot be assumed that NF
B plays an
essential role in the transcription of these cytokines (1, 6). For
example, in PTC stimulated with LPS, NF
B was necessary but not
sufficient for induction of the iNOS gene (1). In the present study,
activation of NF
B was associated with the upregulation of IL-1
,
TNF-
, MCP-1, MIP-1
, and MIP-2 transcription. In contrast, the
latter were suppressed by prophylactic treatment with the antioxidants,
but only at NF
B inhibitory concentrations. These data suggest that
NF
B is likely to have a positive role in the transcriptional
regulation of these cytokines in PTC, as demonstrated for CINC and
RANTES (35, 52).
In contrast to other proinflammatory cytokines and chemokines,
osteopontin is not known to be regulated by NFB (2, 40). Consistent
with this hypothesis, the modulation of NF
B DNA binding activity in
LPS-stimulated PTC treated with or without the antioxidants did not
affect the basal transcription of osteopontin. Our data also provide
direct confirmation of the results reported by Madsen et al. (22), who
showed that LPS did not increase osteopontin expression in PTC in vivo.
Because osteopontin is increased in experimental models of proteinuric
renal disease (40), including that induced by doxorubicin hydrochloride
(unpublished observation), its persistent expression may explain why
interstitial monocyte infiltration was only partially reduced by
PDTC-mediated NF
B inhibition in vivo (39).
Activation of NFB with LPS in PTC was correlated with an increase in
IL-10 and TGF-
1 transcription, cytokines with
macrophage-deactivating properties (3). Whereas the posttranslational
activation of the latent form of TGF-
1 is regulated by NF
B (25),
neither IL-10 nor TGF-
1 transcriptions are known to be directly
controlled by NF
B in rats (2). An unexpected result of this study,
therefore, was that antioxidant-mediated inhibition of NF
B was
associated with reduced IL-10 and TGF-
1 transcription. These data
suggest that in rats, the transcriptional control of IL-10 and TGF-
1 could directly or indirectly involve NF
B. In support of this possibility, the mouse IL-10 gene was found to have three NF
B-like binding sites (20). In addition, in monocytes, the induction of IL-10
by LPS is mediated by the autocrine effects of TNF-
(23), and, as is
shown in the present study, the latter is correlated with NF
B activation.
However, the inhibition of TGF-1 and IL-10 transcription by NAC and
quercetin, respectively, also occurred at non-NF
B inhibitory concentrations (20 mM and 10 µM). Hence other signal-transducing factors specifically targeted by NAC and quercetin, such as protein tyrosine kinase (24) or activator protein-1 (6, 31, 37, 42), could also
be involved in the transcriptional regulation of these cytokines. For
these reasons, transfection studies and gene reporter assays are needed
to further define the role played by NF
B in anti-inflammatory
cytokine gene transcription in PTC.
Because the concentration of NAC required to inhibit NFB in PTC was
10-fold higher than that needed in other cell types (35, 45, 46), we
investigated its effect on PTC viability. With the use of a sensitive
marker of cell toxicity, our results clearly showed that NAC caused a
dose-dependent increase in LDH leakage that was not accompanied by
significant ultrastructural damage. The latter is not surprising,
because LDH leakage is a relatively early marker of cell damage (18).
Therefore, the injury induced by NAC during the time points of the
study was clinically mild and probably reversible. In contrast, neither
PDTC or quercetin caused biochemical or ultrastructural damage to PTC
at NF
B inhibitory concentrations.
Autooxidation and the generation of reactive oxygen intermediates (particularly peroxides and iron-generated hydroxyl radicals) are the most common explanation of thiol-mediated toxicity in cells (13, 18, 31, 45). However, our results do not support the involvement of peroxide in NAC-induced injury of PTC because 1) neither catalase nor DFO prevented NAC-induced LDH leakage, 2) concentrations of H2O2 that increase intracellular peroxide to levels similar to those induced by NAC (11) did not cause LDH leakage in PTC, and 3) H2O2 did not exacerbate NAC-induced LDH leakage and paradoxically had a mild but significant protective effect. The latter has been observed in other studies and is due to the reaction of peroxides with thiols (17). Alternative hypotheses to explain NAC-induced cytotoxicity include the generation of toxic cysteine metabolites (35), formation of nitric oxide intermediates (8) or copper-catalyzed hydroxyl radicals (17), or the induction of intracellular hypoxia by high-dose antioxidants (45).
Cell- and stimulus-specific effects may explain why NAC has been shown
to reduce NFB activation in vivo in rats with ureteric obstruction
(27) and in other nonrenal experimental models (2). For example,
monocytes isolated from normal rats treated with NAC have reduced
adhesion in vitro and increased NF
B DNA binding activity. The latter
is due to an increase in nontransactivating p52 homodimers (28).
Alternatively, the cytoprotective effects of NAC may involve mechanisms
other than NF
B suppression, as demonstrated in endotoxin-induced
acute lung injury (45).
In conclusion, we have demonstrated that PDTC and quercetin potently
suppress NFB activation in PTC. In contrast to what has been shown
in other cell types, NAC was not able to suppress NF
B activation in
PTC at subtoxic and physiologically relevant concentrations.
Furthermore, antioxidant-mediated inhibition of NF
B was associated
with the nonselective reduction of cytokine transcription in activated
tubular cells. Together, these data provide a possible explanation for
the protective effect of PDTC in chronic tubulointerstitial
inflammation and the failure of NAC to inhibit renal cortical NF
B
activation in vivo (39).
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ACKNOWLEDGEMENTS |
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We thank Ross Boadle, Levina Dear, Carol Robinson, and Gayle Avis (Dept. of Electron Microscopy, Westmead Institutes of Health Research) for assistance with electron microscopic techniques.
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FOOTNOTES |
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This work was supported by a project grant (no. 970721) from the National Health and Medical Research Council of Australia.
G. K. Rangan is a recipient of a medical scholarship from the Australian Kidney Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. K. Rangan, Dept. of Renal Medicine, Westmead Hospital, Westmead, Sydney, Australia 2145.
Received 11 January 1999; accepted in final form 30 June 1999.
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