Inhibition of NFkappa B activation with antioxidants is correlated with reduced cytokine transcription in PTC

Gopala K. Rangan, Yiping Wang, Yuet-Ching Tay, and David C. H. Harris

Department of Renal Medicine, University of Sydney at Westmead Hospital, Westmead, Sydney, Australia 2145


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We recently reported that inhibition of the transcription factor nuclear factor-kappa B (NFkappa 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 NFkappa B activation and cytokine transcription in primary cultured rat proximal tubular epithelial cells (PTC) stimulated with lipopolysaccharide. Antioxidant-mediated inhibition of NFkappa B activation (PDTC, 20-100 µM; NAC, 100 mM; and quercetin, 50 µM) diminished the induction of both pro- [interleukin (IL)-1beta , tumor necrosis factor-alpha , monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha , and MIP-2] and anti-inflammatory (IL-10, transforming growth factor-beta 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 NFkappa B activation in PTC at subtoxic and physiologically relevant concentrations. Furthermore, antioxidant-mediated inhibition of NFkappa B is correlated with the nonselective reduction of cytokine transcription in activated tubular cells. These data might explain the protective effects of PDTC-mediated NFkappa B inhibition in tubulointerstitial disease in vivo.

N-acetylcysteine; pyrrolidinedithiocarbamate; quercetin; primary culture; osteopontin; proximal tubular cells; nuclear factor-kappa B


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NFkappa 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, NFkappa B is known to regulate the production of other cytokines [such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha ], chemokines [such as macrophage inflammatory protein (MIP)-1alpha and MIP-2], and many early response genes (2).

The antioxidants pyrrolidinedithiocarbamate (PDTC), N-acetylcysteine (NAC), and quercetin inhibit NFkappa B 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 NFkappa B by PDTC reduced the expression of inducible nitric oxide synthase (iNOS) and RANTES, respectively (1, 52). NAC also suppressed NFkappa B activation in LLC-PK1 cells after stimulation with cysteine S-conjugates (35). However, in activated rabbit alveolar macrophages, PDTC increased TNF-alpha despite inhibition of NFkappa B (6), and in an adenocarcinoma cell line, NAC paradoxically increased NFkappa B (11). Together, these data suggest that antioxidants are cell specific in their ability to inhibit NFkappa 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)-beta 1 (3). In vitro, in human monocytes, PDTC reduced lipopolysaccharide (LPS)-induced IL-10 secretion and increased TNF-alpha production (23). In contrast, in vivo, in rats with LPS-induced endotoxemia, PDTC increased in vivo plasma levels of IL-10 but suppressed TNF-alpha , IL-12, MIP-1alpha , and nitric oxide production and had no effect on IL-1alpha , IL-6, and interferon-gamma induction (31). In another study, diethyldithiocarbamate (a related dithiocarbamate) did not affect the increase in transcription of TGF-beta 1 in the postischemic myocardium of rats but reduced IL-1beta , IL-6, TNF-alpha , and iNOS together with NFkappa B DNA-binding activity (7). The differential modulation of pro- and anti-inflammatory cytokine transcription by antioxidant-mediated NFkappa B inhibition may explain the cytoprotective effects of dithiocarbamates in both models (7, 31).

Recently, we reported that PDTC reduced renal cortical NFkappa B 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 NFkappa B (p50) in this model (unpublished observations). Therefore, we hypothesized that antioxidants (NAC, PDTC, and quercetin) could differ in their ability to 1) suppress NFkappa B activation in PTC and/or 2) selectively modulate cytokine transcription in PTC, such that proinflammatory cytokines (IL-1beta , TNF-alpha , and osteopontin) and chemokines (MCP-1, MIP-1alpha , and MIP-2) are reduced, in preference to those with anti-inflammatory properties (IL-10 and TGF-beta 1). The following studies were designed to test these hypotheses.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 × 10-8 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 NFkappa B 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 NFkappa 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.

The antioxidants were dissolved in media, filter sterilized, and prepared immediately before the experiment. The pH of NAC in the medium solution was 2.4, and this was corrected to pH 7.4 with 6 M sterile sodium hydroxide. The concentrations of NAC (5-100 mM), PDTC (5-100 µM), quercetin (2-50 µM), and the other agents were determined from previous reports (1, 11, 17, 30, 35, 41, 42, 46, 50, 52) and pilot studies.

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 kappa B binding site of the kappa -immunoglobulin light-chain gene (5'-AGT TGA GGG GAC TTT CCC AGG-3'; Promega, Madison, WI) was end-labeled with [gamma -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 NFkappa 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 -20°C. Two or four percent of the diluted cDNA was amplified by PCR with the use of primers specific for rat GAPDH or cytokine cDNA, respectively (Table 1). The PCR reactions were performed in a final volume of 50 µl and contained cDNA, 0.2 mM dNTP, 1.5 mM MgCl2, 0.4 µM each of upstream and downstream primers, 1.5 U thermostable DNA polymerase (red hot Taq DNA polymerase; Advanced Biotechnologies, Surrey, UK), 75 mM Tris · HCl, 20 mM (NH4)2SO4, 0.01% (vol/vol) Tween 20, 1.5 mM MgCl2 (Advanced Biotechnologies, Surrey, UK) and DW. A two-step cycling program was used and consisted of the following: initial template melting step at 94°C for 3 min, denature at 94°C for 30 s, annealing and extension at 60-68°C (depending on the primer pair; see Table 1) for 1 min and 30 s, and final extension at 72°C for 5 min.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Sequences of upstream and downstream rat primers for RT-PCR analysis

The PCR products (20% of the amplified product) were analyzed by agarose gel electrophoresis (1.6% in 1× TAE buffer) and visualized by ethidium bromide staining (0.5 µg/ml) under ultraviolet light. The gels were photographed with a camera, using positive/negative film (type 665; Polaroid, Cambridge, MA). The negatives were scanned by a laser densitometer (Molecular Dynamics, Sunnyvale, CA) with the use of image analysis software (ImageQuaNT, version 4.1, 1994; Molecular Dynamics). A box was drawn around the bands, and the volume density (integrated intensity of all the pixels in the area, excluding the background) was calculated with the use of the edge operator algorithm. To control for variation in RNA quality and RT efficiency between different samples, cytokine volume density was divided by GAPDH volume density and expressed in arbitrary units.

The semiquantitative method of RT-PCR was validated in preliminary experiments. First, the optimal PCR conditions that yielded a single band on agarose gel electrophoresis were determined for each primer. Second, to determine whether the method was semiquantitative, serial dilutions of total RNA extracted from LPS and vehicle-treated PTC underwent RT-PCR amplifications with the use of each of the primer pairs (Table 1), as described previously (10, 21). Third, experiments were performed to determine the optimal number of PCR cycles for each primer pair (that yielded PCR products in the linear phase of amplification). Finally, to ensure that results were consistent, PCR reactions were performed at least twice, and each contained cDNA samples from all experiments. Only one of these reactions was included for final densitometric analysis, and the selection was arbitrary. All reactions included a negative PCR control (cDNA replaced with DW).

Sequences of rat cytokines and GAPDH were retrieved from the Australian National Genomic Information Service (GenBank database), and primer pairs for each were designed with oligonucleotide software (Oligo Primer Analysis, version 5.0; National Biosciences, Plymouth, MN). The sequences of the upstream and downstream primers are shown in Table 1. The primers were intron spanning and synthesized by Life Technologies (Mulgrave, Australia). The identity of the amplified product was confirmed by determination of the molecular size on agarose gel electrophoresis with DNA molecular markers (Promega, Sydney, Australia). In addition, the amplified PCR products were purified (plasmid purification kit; Qiagen, Clifton Hill, Victoria, Australia), and the sequence of the base pairs was determined by an automated DNA sequencing system (Applied Biosystems 373A) utilizing fluorescent dye-labeled dideoxynucleotides (performed by Mark Wheeler, DNA Sequencing Facility, Westmead Institutes of Health Research, Westmead, Sydney, Australia) and compared with the known sequences of rat cytokines and GAPDH (Table 1).

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of NAC, PDTC, and quercetin on NFkappa B activation. In control cells, virtually no NFkappa 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 NFkappa 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 NFkappa 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 NFkappa B activation than that needed for PDTC and quercetin. LPS-induced NFkappa 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 NFkappa B activation in control cells (data not shown).


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of antioxidants [N-acetylcysteine (NAC), pyrrolidinedithiocarbamate (PDTC), and quercetin] on activation of nuclear factor-kappa B (NFkappa B) by lipopolysaccharide (LPS) in rat proximal tubular epithelial cells (PTC) in primary culture. Confluent PTC were treated with vehicle (control, lane 1), LPS (5 µg/ml, lane 2), LPS+NAC (5, 20, and 100 mM; lanes 3-5), LPS+PDTC (5, 20, and 100 µM; lanes 6-8), or LPS+quercetin (10 and 50 µM, lanes 9-10). After 8 h, nuclear protein was extracted and an electrophoretic mobility shift assay (EMSA) was performed, using a consensus 32P-labeled NFkappa B binding oligonucleotide. Results are representative of 2 independent experiments performed in duplicate. Specificity of EMSA was determined in competition experiments in which nuclear extracts from LPS-stimulated PTC were also incubated with a 100-fold excess of unlabeled consensus NFkappa B oligonucleotide (UL, lane 11) or a 100-fold excess of an irrelevant labeled oligonucleotide consensus for activator protein-1 (AP-1, lane 12). * Faster migrating band cannot be depleted or retarded by antisera reactive against NFkappa B subunits in supershift analysis (49) and could represent specific binding by a non-NFkappa B transcription factor.

The specificity of the NFkappa B EMSA was demonstrated in competition experiments. Incubation of nuclear extracts from LPS-treated cells with a 100-fold excess of unlabeled NFkappa B probe abolished the retarded band (Fig. 1, lane 11), whereas a 100-fold excess of an irrelevant oligonucleotide (consensus for the transcription factor, activator protein-1) had no effect (Fig. 1, lane 12). In a separate study (49), we showed that the retarded band is composed of p50/65, p50/c-Rel, and p50/50 heterodimers in activated PTC, as determined by supershift analysis with the use of antisera specific for rat NFkappa B subunits. A faster migrating constitutively expressed band was also abrogated by excess unlabeled NFkappa B probe (Fig. 1). This band is not depleted or retarded by antisera reactive against NFkappa B subunits (49) and could represent specific binding by a non-NFkappa B transcription factor.

Effect of NAC, PDTC, and quercetin on cytokine transcription in PTC. To assess the effect of antioxidant-mediated NFkappa B 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).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Validation of method to semiquantitate cytokine gene transcription in rat PTC by RT-PCR analysis (10, 21). Total RNA was extracted from PTC stimulated with LPS (5 µg/ml) for 8 h, and RT-PCR was performed with primers specific for rat monocyte chemoattractant protein (MCP)-1. PCR products were visualized by electrophoresis on an ethidium bromide-stained (0.5 µg/ml) 1.6% agarose gel and photographed under ultraviolet light illumination with uniform exposure times. Volume density of band was quantitated by densitometry of negative film. Positive and negative film are shown at top, and numerical values for volume density (in arbitrary units) are plotted on graphs at bottom. A: serial dilutions of total RNA (62.5-4,000 ng) were reverse transcribed and amplified by PCR for 32 cycles. B: determination of optimal cycle number for PCR amplification with the use of 1 µg of total RNA. C: total RNA (1 µg) from a sample (L1) was divided into 8 portions and underwent RT and PCR amplification (32 cycles) in separate tubes to determine intra-assay coefficient of variation (standard deviation divided by mean: L1 = 7.03/171.60 = 4.1%).

The housekeeping gene GAPDH was constitutively expressed in all experimental groups (Fig. 3). In resting PTC, there was weak expression of IL-1beta mRNA, which increased 29-fold after exposure to LPS (5 µg/ml) for 8 h (P < 0.001; Figs. 3 and 4A). NAC, PDTC, and quercetin alone had no significant effect on IL-1beta transcription in unstimulated cells (P = 0.14, by 1-way ANOVA). Exposure of PTC to 100 mM NAC 1 h before LPS reduced IL-1beta induction almost to the level of control cells (P = 0.006 compared with LPS alone). Lower doses of NAC (5 and 20 mM) partially increased IL-1beta transcription (P = 0.01 and 0.04, respectively) in LPS-stimulated cells. Similarly, PDTC (20 and 100 µM) dramatically reduced IL-1beta induction in LPS-treated PTC (P = 0.005 and 0.003, respectively). Likewise, quercetin (50 µM only) also reduced IL-1beta induction (P = 0.003). Concentrations of PDTC (5 µM) and quercetin (10 µM) that did not inhibit NFkappa B activation had no effect on the increase in IL-1beta in LPS-stimulated PTC (Figs. 3 and 4A).


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of NAC, PDTC, and quercetin on LPS-induced cytokine transcription in rat PTC. RT-PCR was performed, using specific primers for GAPDH, proinflammatory cytokines [interleukin (IL)-1beta , tumor necrosis factor (TNF)-alpha , and osteopontin], chemokines [MCP-1, macrophage inflammatory protein (MIP)-1alpha , and MIP-2], and anti-inflammatory cytokines [IL-10 and transforming growth factor (TGF)-beta 1]. PCR products were analyzed by 1.6% agarose gel electrophoresis stained with ethidium bromide. Lanes 1-2, control; lanes 3-4, LPS; lanes 5-7, NAC+LPS; lane 8, NAC+vehicle; lanes 9-11, PDTC+LPS; lane 12, PDTC+vehicle; lanes 13-15, quercetin+LPS; lane 16, quercetin+vehicle. Molecular size (in bp) of PCR products is shown at right. PCR reactions not containing cDNA or RT reactions not containing RNA or reverse transcriptase produced no bands (data not shown). Results are representative of 3 separate experiments.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of NAC, PDTC, and quercetin on transcription of proinflammatory cytokines in rat PTC. A: IL-1beta . B: TNF-alpha . C: osteopontin. Individual bars are ratios of mean volume density of cytokine divided by mean volume density of GAPDH, determined by densitometry. Results are from 3 separate experiments (n = 6 for control, n = 9 for other groups). * P < 0.05 compared with control. # P < 0.05 compared with LPS groups.

TNF-alpha mRNA was not detectable in control cells but was strongly induced after stimulation with LPS (P = 0.003; Figs. 3 and 4B). Incubation of PTC with antioxidants alone had no effect on the basal transcription of TNF-alpha . The induction of TNF-alpha by LPS was completely abolished by 100 mM NAC (P = 0.004) and 20 and 100 µM PDTC (P = 0.004 and 0.002, respectively) and was partially reduced by 50 µM quercetin (P = 0.012). Lower doses of the antioxidants were not effective, and, in cells treated with 20 mM NAC and LPS, TNF-alpha was increased slightly (P = 0.035; Fig. 4B).

The cytokine osteopontin is chemotactic for macrophages and may play an important role in the pathogenesis of tubulointerstitial disease in chronic glomerular disease, but it is not known to be regulated by NFkappa B (2, 40). Osteopontin mRNA was constitutively expressed in resting PTC (Figs. 3 and 4C). The expression of osteopontin was not altered by LPS (P = 0.14), antioxidants alone (P = 0.18, by 1-way ANOVA), or a combination of the antioxidants and LPS (P = 0.24, by 1-way ANOVA) compared with the control group (Fig. 4C).

LPS significantly increased the transcription of the chemokines MCP-1 (9-fold), MIP-1alpha (not detectable in control cells), and MIP-2 (43-fold) (all P < 0.001 compared with unstimulated PTC; Figs. 3 and 5, A-C). Prophylactic treatment with any of the three antioxidants at NFkappa B inhibitory concentrations only prevented the increase in MCP-1, MIP-1alpha , and MIP-2 (all P < 0.05 compared with LPS alone; Figs. 3 and 5A).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of NAC, PDTC, and quercetin on transcription of chemokines in rat PTC. A: MCP-1. B: MIP-1alpha . C: MIP-2. Results are from 3 separate experiments (n = 6 for control, n = 9 for other groups). * P < 0.05 compared with control. # P < 0.05 compared with LPS groups.

LPS caused a twofold increase in the transcription of TGF-beta 1 in PTC, but this did not reach statistical significance (P = 0.08; Figs. 3 and 6A). Antioxidants alone had no significant effect on the basal expression of TGF-beta 1 mRNA (P = 0.09, by 1-way ANOVA). In LPS-treated cells, both 20 and 100 mM NAC reduced the transcription of TGF-beta 1 (P = 0.023 and 0.009, respectively). In contrast to NAC, PDTC and quercetin suppressed TGF-beta 1 only at concentrations that reduced NFkappa B activation (PDTC, 20 and 100 µM, P = 0.035 and 0.027, respectively; quercetin, 50 µM, P = 0.018) (Fig. 6A).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of NAC, PDTC, and quercetin on transcription of anti-inflammatory cytokines in rat PTC. A: TGF-beta 1. B: IL-10. Results are from 3 separate experiments (n = 6 for control, n = 9 for other groups). * P < 0.05 compared with control. # P < 0.05 compared with LPS groups.

IL-10 mRNA was not detectable by RT-PCR in resting PTC or those exposed to the antioxidants (Figs. 3 and 6B), whereas it was strongly induced by LPS (P < 0.001). The induction of LPS was reduced by prophylactic treatment with 100 mM NAC (P = 0.005) and 20 and 100 µM PDTC (P < 0.005 for both). Quercetin reduced IL-10 induction at all concentrations, including those that did not inhibit LPS-induced NFkappa B activation (P = 0.003, 0.04, and 0.023 for 2, 10, and 50 µM, respectively; Fig. 6B).

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 NFkappa B inhibition can unmask TNF-alpha -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).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of vehicle (control, C), LPS, PDTC (P), quercetin (Q), and NAC on LDH leakage in cultured proximal tubule cells. Results are from 3 separate experiments (n = 6 for control, n = 9 for other groups). * P < 0.05 compared with control group.

Transmission electron microscopy was undertaken to determine the clinical significance of the increased LDH leakage in NAC-treated cells. Cells exposed to vehicle alone had normal PTC morphology with numerous mitochondria and abundant microvilli (Fig. 8). Very occasionally, cells in the vehicle-treated group had evidence of cellular damage and necrosis. The ultrastructure of PTC exposed to NAC (100 mM), PDTC (100 µM), or quercetin (50 µM) for 8 h was the same as that of control cells (data not shown).


View larger version (110K):
[in this window]
[in a new window]
 
Fig. 8.   Transmission electron microscopy of PTC grown in culture for 5 days and exposed to vehicle for 8 h. Cell has numerous mitochondria and abundant microvilli (inset; magnification = ×16,800). Cells exposed to NAC (100 mM) alone for 8 h were similar in ultrastructural appearance to vehicle-treated group (data not shown).

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).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of catalase (1,000 U/ml), desferrioxamine (DFO, 200 µM), and hydrogen peroxide (H2O2, 200 µM) on NAC-induced LDH leakage. Results are from 2 separate experiments (n = 6/group). * P < 0.05 compared with control. # P < 0.05 compared with NAC alone.

Due to the fact that catalase may not penetrate cellular membranes easily because of its molecular size, we sought to determine the effect of exogenous peroxide on PTC. Exogenous H2O2 can accelerate thiol oxidation and potentially worsen cellular cytotoxicity induced by NAC (30). The addition of H2O2 (200 µM) alone at doses that increase intracellular peroxide levels to those achieved after treatment with NAC (11) did not increase LDH leakage in PTC (Fig. 9). However, paradoxically, the addition of H2O2 with NAC (100 mM) in PTC reduced NAC-induced LDH leakage (P = 0.046). This effect was not inhibited by the addition of catalase (1,000 U/ml) (Fig. 9).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study has investigated the effects of three structurally diverse antioxidants (NAC, PDTC, and quercetin) on NFkappa B 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 NFkappa B inhibition on cytokine transcription were not known. The results of the present paper show that both PDTC and quercetin are potent suppressors of NFkappa B activation, whereas NAC is ineffective at subtoxic concentrations. Furthermore, and contrary to our original hypothesis, antioxidant-mediated inhibition of NFkappa B was associated with the reduction of both pro- (IL-1beta , TNF-alpha , MCP-1, MIP-1alpha , and MIP-2) and anti-inflammatory (TGF-beta 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 NFkappa B 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 NFkappa 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 NFkappa 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 NFkappa B activation in tubular epithelial cells. The reasons why NAC is a less potent inhibitor of NFkappa 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 NFkappa 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 NFkappa 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 NFkappa B 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 NFkappa B in tubular epithelial cells (50). Paradoxically, the prooxidant and metal-chelating properties of PDTC could be involved in its ability to inhibit NFkappa 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 NFkappa B inhibition. Similarly, the suppression of protein tyrosine kinases and protein kinase C could play an important role in quercetin-induced NFkappa B inhibition (24).

Having demonstrated that NAC, PDTC, and quercetin suppressed NFkappa B activation in PTC, we next examined their effects on cytokine gene transcription. The promoter regions of several proinflammatory cytokine genes contain binding sites for NFkappa 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 NFkappa B (49). Nevertheless, it cannot be assumed that NFkappa B plays an essential role in the transcription of these cytokines (1, 6). For example, in PTC stimulated with LPS, NFkappa B was necessary but not sufficient for induction of the iNOS gene (1). In the present study, activation of NFkappa B was associated with the upregulation of IL-1beta , TNF-alpha , MCP-1, MIP-1alpha , and MIP-2 transcription. In contrast, the latter were suppressed by prophylactic treatment with the antioxidants, but only at NFkappa B inhibitory concentrations. These data suggest that NFkappa 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 NFkappa B (2, 40). Consistent with this hypothesis, the modulation of NFkappa 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 NFkappa B inhibition in vivo (39).

Activation of NFkappa B with LPS in PTC was correlated with an increase in IL-10 and TGF-beta 1 transcription, cytokines with macrophage-deactivating properties (3). Whereas the posttranslational activation of the latent form of TGF-beta 1 is regulated by NFkappa B (25), neither IL-10 nor TGF-beta 1 transcriptions are known to be directly controlled by NFkappa B in rats (2). An unexpected result of this study, therefore, was that antioxidant-mediated inhibition of NFkappa B was associated with reduced IL-10 and TGF-beta 1 transcription. These data suggest that in rats, the transcriptional control of IL-10 and TGF-beta 1 could directly or indirectly involve NFkappa B. In support of this possibility, the mouse IL-10 gene was found to have three NFkappa B-like binding sites (20). In addition, in monocytes, the induction of IL-10 by LPS is mediated by the autocrine effects of TNF-alpha (23), and, as is shown in the present study, the latter is correlated with NFkappa B activation.

However, the inhibition of TGF-beta 1 and IL-10 transcription by NAC and quercetin, respectively, also occurred at non-NFkappa 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 NFkappa B in anti-inflammatory cytokine gene transcription in PTC.

Because the concentration of NAC required to inhibit NFkappa B 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 NFkappa 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 NFkappa B 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 NFkappa 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 NFkappa B suppression, as demonstrated in endotoxin-induced acute lung injury (45).

In conclusion, we have demonstrated that PDTC and quercetin potently suppress NFkappa B activation in PTC. In contrast to what has been shown in other cell types, NAC was not able to suppress NFkappa B activation in PTC at subtoxic and physiologically relevant concentrations. Furthermore, antioxidant-mediated inhibition of NFkappa 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 NFkappa B activation in vivo (39).


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amoah-Apraku, B., L. J. Chandler, J. K. Harrison, S. S. Tang, J. R. Ingelfinger, and N. J. Guzman. NF-kappa B and transcriptional control of renal epithelial-inducible nitric oxide synthase. Kidney Int. 48: 674-682, 1995[Medline].

2.   Baeuerle, P., and V. R. Baichwal. NF-kappa B as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv. Immunol. 65: 111-137, 1997[Medline].

3.   Bogdan, C., and C. Nathan. Modulation of macrophage function by transforming growth factor beta , interleukin-4 and interleukin-10. Ann. NY Acad. Sci. 685: 713-739, 1993[Abstract].

4.   Borgstrom, L., B. Kagedal, and O. Paulsen. Pharmacokinetics of N-acetylcysteine in man. Eur. J. Clin. Pharmacol. 31: 217-222, 1986[Medline].

5.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

6.   Bulger, E. M., I. Garcia, and R. V. Maier. Dithiocarbamates enhance tumour necrosis factor-alpha production by rabbit alveolar macrophages, despite inhibition of NF-kappa B. Shock 9: 397-405, 1998[Medline].

7.   Chandrasekar, B., J. E. Streitman, J. T. Colston, and G. L. Freeman. Inhibition of nuclear factor-kappa B attenuates proinflammatory cytokine and inducible nitric-oxide synthase expression in postischemic myocardium. Biochim. Biophys. Acta 1406: 91-106, 1998[Medline].

8.   Chen, L., B. H. Zhang, and D. C. H. Harris. Evidence suggesting that nitric oxide mediates iron-induced toxicity in cultured proximal tubule cells. Am. J. Physiol. 274 (Renal Physiol. 43): F18-F25, 1998[Abstract/Free Full Text].

9.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

10.   Cockfield, S. M., V. Ramassar, J. Noujaim, P. H. van der Meide, and P. F. Halloran. Regulation of IFN-gamma in vivo. IFN-gamma up-regulates expression of its mRNA in normal and lipopolysaccharide stimulated mice. J. Immunol. 150: 717-725, 1993[Abstract/Free Full Text].

11.   Das, K. C., Y. Lewis-Molock, and C. W. White. Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L588-L602, 1995[Abstract/Free Full Text].

12.   Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11: 1475-1489, 1983[Abstract].

13.   Driscoll, K. E., D. G. Hassenbein, B. W. Howard, R. J. Isfort, D. Cody, M. H. Tindal, M. Suchanek, and J. M. Carter. Cloning, expression and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen. J. Leukoc. Biol. 58: 359-364, 1995[Abstract].

14.   Estler, H. C., M. Grewe, R. Gaussling, M. Pavlovic, and K. Decker. Rat tumour necrosis factor-alpha . Transcription in rat Kupffer cells and in vitro post-translational processing based on a PCR-derived cDNA. Biol. Chem. 373: 271-281, 1992.

15.  Feeser, W., and B. D. Freimark. (1992). Nucleotide Sequence of Rat Pro-Interleukin-1beta mRNA (Accession Number M988200). [Online] Australian National Genomic Information Service (GenBank). (http: //www. angis. org. au). [1996].

16.   Goodman, R. E., J. Oblak, and R. G. Bell. Synthesis and characterization of rat interleukin-10 cDNA clones from the RNA of cultured OX8- OX22- thoracic duct T cells. Biochem. Biophys. Res. Commun. 189: 1-7, 1992[Medline].

17.   Held, K. D., and J. E. Biaglow. Mechanisms for the oxygen radical-mediated toxicity of various thiol-containing compounds in cultured mammalian cells. Radiat. Res. 139: 15-23, 1994[Medline].

18.   Keilhoff, G., and G. Wolf. Comparison of double fluorescence staining and LDH-test for monitoring cell viability in vitro. Neuroreport 5: 129-132, 1993[Medline].

19.   Kim, J. M., C. I. Brannan, N. G. Copeland, N. A. Jenkins, T. A. Khan, and K. W. Moore. Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes. J. Immunol. 148: 3618-3623, 1992[Abstract/Free Full Text].

20.   Kuhlmann, M. K., E. Horsch, G. Burkhardt, M. Wagner, and H. Kohler. Reduction of cisplatin toxicity in cultured renal tubular cells by the bioflavonoid quercetin. Arch. Toxicol. 72: 536-540, 1998[Medline].

21.   Lee, Y.-J., S.-J. Shin, M.-S. Tan, T.-J. Hsieh, and J.-H. Tsai. Increased renal atrial natriuretic peptide synthesis in rats with deoxycorticosterone acetate-salt treatment. Am. J. Physiol. 271 (Renal Physiol. 40): F779-F789, 1996[Abstract/Free Full Text].

22.   Madsen, K. M., L. Zhang, A. R. Abu Shamat, S. Siegfried, and J. H. Cha. Ultrastructural localization of osteopontin in the kidney: induction by lipopolysaccharide. J. Am. Soc. Nephrol. 8: 1043-1053, 1997[Abstract].

23.   Meisel, C., K. Vogt, C. Platzer, F. Randow, C. Liebenthal, and H. D. Volk. Differential regulation of monocytic tumour necrosis factor-alpha and interleukin-10 expression. Eur. J. Immunol. 26: 1580-1586, 1996[Medline].

24.   Middleton, E. Effect of plant flavonoids on immune and inflammatory cell function. Adv. Exp. Med. Biol. 439: 175-182, 1998[Medline].

25.   Mirza, A., S.-L. Liu, E. Frizell, J. Zhu, S. Maddukuri, J. Martinez, P. Davies, R. Schwarting, P. Norton, and M. A. Zern. A role for tissue transglutaminase in hepatic injury and fibrogenesis, and its regulation by NFkappa B. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G281-G288, 1997[Abstract/Free Full Text].

26.   Mitchell, D. B., K. S. Santone, and D. Acosta. Evaluation of cytotoxicity in cultured cells by enzyme leakage. J. Tissue Cult. Methods 6: 113-116, 1980.

27.   Morrissey, J., L. Duan, and S. Klahr. N-acetylcysteine treatment alters nuclear transcription factor activity and monocyte infiltration during ureteral obstruction (Abstract). J. Am. Soc. Nephrol. 7: 1830A, 1996.

28.   Morrissey, J., T. Tolley, and S. Klahr. N-acetylcysteine treatment affects monocyte adhesion (Abstract). J. Am. Soc. Nephrol. 9: 482A, 1998.

29.   Morrissey, J. J., and S. Klahr. Enalapril decreases nuclear factor-kappa B activation in the kidney with ureteral obstruction. Kidney Int. 52: 926-933, 1997[Medline].

30.   Mottley, C., K. Toy, and R. P. Mason. Oxidation of thiol drugs and biochemicals by the lactoperoxidase/hydrogen peroxide system. Mol. Pharmacol. 31: 417-421, 1987[Abstract].

31.   Nemeth, Z. H., G. Hasko, and E. S. Vizi. Pyrrolidine dithiocarbamate augments IL-10, inhibits TNF-alpha , MIP-1alpha , IL-12 and nitric oxide production and protects from the lethal effect of endotoxin. Shock 10: 49-53, 1998[Medline].

32.   Oertli, B., B. Beck-Schimmer, X. Fan, and R. P. Wuthrich. Mechanisms of hyaluronan-induced up-regulation of ICAM-1 and VCAM-1 expression by murine kidney tubular epithelial cells: hyaluronan triggers cell adhesion molecule expression through a mechanism involving activation of nuclear factor-kappa B and activating protein-1. J. Immunol. 161: 3431-3437, 1998[Abstract/Free Full Text].

33.   Ohtsuka, T., A. Kubota, T. Hirano, K. Watanabe, H. Yoshida, M. Tsurufuji, Y. Iizuka, K. Konishi, and S. Tsurufuji. Glucorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family through impairment of NF-kappa B activation. J. Biol. Chem. 271: 1651-1659, 1996[Abstract/Free Full Text].

34.   Oldberg, A., A. Franzen, and D. Heinegard. Cloning and sequencing of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell binding sequence. Proc. Natl. Acad. Sci. USA 83: 8819-8823, 1986[Abstract].

35.   Otieno, M. A., and M. W. Anders. Cysteine S-conjugates activate transcription factor NF-kappa B in cultured renal epithelial cells. Am. J. Physiol. 273 (Renal Physiol. 42): F136-F143, 1997[Abstract/Free Full Text].

36.   Palmer, B. F. The renal tubule in the progression of chronic renal failure. J. Investig. Med. 45: 346-361, 1997[Medline].

37.   Pinkus, R., L. M. Weiner, and V. Daniel. Role of oxidants and antioxidants in the induction of AP-1, NF-kappa B and glutathione-S-transferase gene expression. J. Biol. Chem. 271: 13422-13429, 1996[Abstract/Free Full Text].

38.   Qian, S. W., P. Kondaiah, A. B. Roberts, and M. B. Sporn. cDNA cloning by PCR of rat transforming growth factor beta-1. Nucleic Acids Res. 18: 3059, 1990[Medline].

39.   Rangan, G. K., Y. Wang, Y.-C. Tay, and D. C. H. Harris. Inhibition of NF-kappa B reduces cortical tubulointerstitial injury in proteinuric rats. Kidney Int. 56: 115-134, 1999.

40.   Rovin, B. H., and L. T. Phan. Chemotactic factors and renal inflammation. Am. J. Kidney Dis. 31: 1065-1084, 1998[Medline].

41.   Sato, M., T. Miyazaki, F. Kambe, K. Maeda, and H. Seo. Quercetin, a bioflavonoid, inhibits the induction of interleukin 8 and monocyte chemoattractant protein-1 expression by tumour necrosis-alpha in cultured human synovial cells. J. Rheumatol. 24: 1680-1684, 1997[Medline].

42.   Schreck, R., B. Meier, D. N. Mannel, W. Droge, and P. A. Baeuerle. Dithiocarbamates as potent inhibitors of nuclear factor-kappa B activation in intact cells. J. Exp. Med. 175: 1181-1194, 1992[Abstract].

43.   Shanley, T. P., H. Schmal, H. P. Friedl, M. L. Jones, and P. A. Ward. Role of macrophage inflammatory protein-1alpha in acute lung injury in rats. J. Immunol. 154: 4793-4802, 1995[Abstract/Free Full Text].

44.   Shoskes, D. A. Effect of bioflavonoids quercetin and curcumin on ischemic renal injury. Transplantation 66: 147-152, 1998[Medline].

45.   Sproong, R. C., A. M. L. Winkelhuyzen-Janssen, C. J. M. Aarsman, J. F. L. M. van Oirschot, T. van der Bruggen, and B. S. van Asbeck. Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality. Am. J. Respir. Crit. Care Med. 157: 1283-1293, 1998[Abstract/Free Full Text].

46.   Staal, F. J. T., M. Roederer, L. A. Herzenberg, and L. A. Herzenberg. Intracellular thiols regulate activation of nuclear factor-kappa B and transcription of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 87: 9943-9947, 1990[Abstract].

47.   Van Goor, H., G. Ding, D. Kees-Folt, J. Grond, G. F. Schreiner, and J. R. Diamond. Macrophages and renal disease. Lab. Invest. 71: 456-464, 1994[Medline].

48.   Wang, Y., J. Chen, L. Chen, Y. C. Tay, G. K. Rangan, and D. C. H. Harris. Induction of monocyte chemoattractant protein-1 in proximal tubular cells by urinary protein. J. Am. Soc. Nephrol. 8: 1537-1545, 1997[Abstract].

49.   Wang, Y., G. K. Rangan, Y. C. Tay, Y. Wang, and D. C. H. Harris. Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor-kappa B in proximal tubule cells. J. Am. Soc. Nephrol. 10: 1204-1213, 1999[Abstract/Free Full Text].

50.   Woods, J. S., M. E. Ellis, F. J. Dieguez-Acuna, and J. Corral. Activation of NF-kappa B in normal rat kidney epithelial (NRK52E) cells is mediated via a redox-insensitive, calcium-dependent pathway. Toxicol. Appl. Pharmacol. 154: 219-227, 1999[Medline].

51.   Zhou, X., S. Doi, A. Iwagaki, and P. Hirszel. Inactivation of NF-kappa B potentiates TNFalpha -induced cytotoxicity in a renal proximal tubular cell line (Abstract). J. Am. Soc. Nephrol. 9: 449A, 1998.

52.   Zoja, C., R. Donadelli, S. Colleoni, M. Figliuzzi, S. Bonazzola, M. Morigi, and G. Remuzzi. Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation. Kidney Int. 53: 1608-1615, 1998[Medline].


Am J Physiol Renal Physiol 277(5):F779-F789
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society