Nuclear Factor {kappa}B Activity Determines the Sensitivity of Kidney Epithelial Cells to Apoptosis: Implications for Mercury-Induced Renal Failure

Francisco J. Dieguez-Acuña*, William W. Polk{dagger}, Maureen E. Ellis{dagger}, P. Lynne Simmonds{dagger}, John V. Kushleika{dagger} and James S. Woods{dagger},1

* Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, and {dagger} Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105

Received June 11, 2004; accepted July 23, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear factor kappa B (NF-{kappa}B) is a thiol-dependent transcriptional factor that promotes cell survival and protects cells from apoptotic stimuli. Numerous studies have demonstrated increased sensitivity to apoptosis associated with inhibition of NF-{kappa}B activation in various cell types. We have previously demonstrated that mercuric ion (Hg2+), one of the strongest thiol-binding agents known, impairs NF-{kappa}B activation and DNA binding at low µM concentrations in kidney epithelial cells. In the present studies we investigated the hypothesis that inhibition of NF-{kappa}B activation by Hg2+ and other selective NF-{kappa}B inhibitors would increase the sensitivity of kidney epithelial (NRK52E) cells to apoptogenic agents to which these cells are normally resistant. Fewer than 10% of untreated cells in culture were found to be apoptotic when evaluated by DNA fragmentation (TUNEL) assay. Treatment of cells with Hg2+ in concentrations up to 5 µM or with tumor necrosis factor-{alpha} (TNF) (300 units/ml) did not significantly increase the proportion of apoptotic cells, compared with untreated controls. However, when TNF was given following Hg2+ pretreatment (0.5 to 5 µM for 30 min), the proportion of cells undergoing apoptosis increased by 2- to 6-fold over that seen in untreated controls. Kidney cells pretreated with specific NF-{kappa}B inhibitors (Bay11-7082 or SN50) prior to TNF also showed a significant increase in apoptosis. Increased sensitivity to apoptotic cell death following these treatments was significantly attenuated in cells transfected with a p65 expression vector. In studies in vivo, rats pretreated by intraperitoneal injection with Hg2+ (0.75 mg/kg) 18 h prior to administration of bacterial lipopolysaccharide (LPS) (10 mg/kg) displayed impaired NF-{kappa}B activation and an increased mitochondrial cytochrome c release in kidney cortical cells. These findings are consistent with the view that prevention of NF-{kappa}B activity in vitro or in vivo enhances the sensitivity of kidney cells to apoptotic stimuli to which these cells are otherwise resistant. Since apoptosis is known to play a seminal role in the pathogenesis of renal failure caused by toxicant injury to tubular cells, the present findings suggest that inhibition of NF-{kappa}B activity may define a molecular mechanism underlying the pathogenesis of Hg2+ toxicity in kidney cells.

Key Words: nuclear factor kappa B; kidney epithelial cells; renal failure; Hg2+ toxicity; apoptosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear factor kappa B (NF-{kappa}B) is a mammalian transcriptional activator known to be involved in the inducible expression of a variety of genes, particularly those involved in cellular proliferative, survival, and antiapoptotic processes (Chen et al., 1999Go; Kretz-Remy et al., 1996Go; Sha et al., 1995Go). Depending on the cell type, NF-{kappa}B is activated by a wide range of stimuli including viruses, bacterial lipopolysaccharide (LPS), cytokines such as tumor necrosis factor-{alpha} (TNF), and growth factors. Many of the steps in the signal transduction pathway leading to NF-{kappa}B activation are thiol-dependent and require reduced cysteine moieties at critical steps of activation and DNA binding (DiDonato et al., 1996Go; Matthews et al., 1992Go).

Numerous agents are known to trigger apoptosis in kidney cells. Among the physiological activators of particular interest are members of the tumor necrosis factor family, notably, TNF per se (Ortiz, 2000aGo; Ortiz et al., 1995Go). Renal epithelial cells possess surface receptors for TNF (TNFR1) engagement of which by exogenous TNF triggers apoptosis. Moreover, TNF is synthesized intrinsically by tubular epithelial cells as well as other renal cell types, and participates in cellular physiology (Ortiz et al., 1995Go). Additionally, TNF is known to activate a wide array of cellular signaling pathways that result in divergent biological responses, including activation of NF-{kappa}B (Luster et al., 1999Go). Under basal conditions, tubular cells are quite resistant to TNF-induced apoptosis, as would be expected by its constitutive expression in these cells. By contrast, sensitivity to apoptotic cell death induced by TNF has been reported to be increased by the presence of other cytokines and nephrotoxicants (Ortiz, 2000bGo; Ortiz et al., 2000aGo,bGo), suggesting a cooperative role of exogenous agents that accumulate in tubular cells at low concentrations in the pathophysiology of acute and chronic diseases. This prospect assumes particular importance in light of emerging evidence suggesting that apoptosis, rather than necrosis or other cell death paradigms, may play a seminal role in the pathogenesis of those forms of renal failure in which tubular epithelial cells are the primary target of toxicant injury (Amore and Coppo, 2000Go; Lieberthal and Levine, 1996Go; Rana et al., 2001Go), as occurs in the case of mercury exposure.

Mercuric ion (Hg2+) is a potent nephrotoxicant with principal effects directed toward proximal tubular epithelial cells of the S3 segment (pars recta) (Gritzka and Trump, 1968Go). Previously, we reported (Dieguez-Acuña et al., 2001Go; Woods et al., 2002Go) that Hg2+, within a low concentration range that does not predispose to necrotic cell death, nonetheless specifically impairs thiol-dependent signal transduction processes that are involved in activation of NF-{kappa}B and that these effects may increase the susceptibility of kidney cells to the cytotoxic effects of other endogenous or exogenous agents such as TNF or LPS, respectively. However, the mechanisms and functional consequences of these effects remain to be demonstrated.

In the present studies we tested the hypothesis that attenuation of NF-{kappa}B activation increases the sensitivity of kidney epithelial cells to the apoptosis-inducing effects of TNF, an endogenous proapoptotic cytokine to which kidney cells are normally resistant. In addition to Hg2+, SN50, a cell membrane-permeable peptide carrying the nuclear localization signal of the NF-{kappa}B p50 subunit (Pampfer et al., 2000Go), and Bay 11-7082, a specific inhibitor of cytokine-inducible I{kappa}B{alpha} phosphorylation (Pampfer et al., 2000Go), were employed as specific inhibitors of NF-{kappa}B activation. We demonstrate that attenuation of NF-{kappa}B activity dramatically increases the proapoptotic effects of TNF or LPS in kidney cells in vitro or in vivo, suggesting a potential mechanism underlying the pathogenesis of renal tubular injury during mercury exposure.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures and treatments. NRK52E cell stocks (ATCC CRL 1571) were acquired from the American Type Culture Collection (Manassas, VA) and were propagated as previously described (Dieguez-Acuña et al., 2001Go). Chemical treatments were performed when cells covered 70–90% of the flask surface. Treatments of TNF (Calbiochem, Human recombinant, E. Coli, Cat. # 654205, 1 ng = 110 WHO IU) and LPS (Sigma, E. coli serotype 026:B6, Cat. # L8274, 1 µg ≥ 10 endotoxin units) were made from stock solutions maintained at –70°C. Bay 11-7082 was obtained from Sigma (St. Louis, MO). SN50 was purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). All other drugs and chemicals were obtained from standard commercial sources and were of the highest purity available.

Electrophoretic mobility shift assays (EMSAs) and autoradioaugraphy. Nuclear and cytoplasmic extracts were prepared as previously described (Woods et al., 1999Go). Following preparation, a 4 µl aliquot of each extract was isolated for protein determination by the bicinchoninic acid assay (Pierce, Rockford, IL). The remaining portions were employed in EMSAs or other analyses as described below. For binding reactions, an oligonucleotide containing the sense {kappa}B sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3'), obtained as an annealed probe from IDT (Coralville, IA), was end-labeled with [{gamma}-P32]dATP (DuPont, Wilmington, DE) using T4 kinase (Boehringer Mannheim, Indianapolis, IN). The radiolabeled probe was separated from free nucleotide using a NucTrap push column (Stratagene, La Jolla, CA). Following electrophoresis in 0.5X TBE running buffer (44.5 mM Tris base, 44.5 mM boric acid, and 1 mM EDTA) at 4°C for 1.5–2 h at 90 V constant O/C, the gels were dried and exposed to Kodak X-OMAT AR X-ray film with intensifying screens for up to 48 h. Autoradiograms were analyzed using a BioRad Gel Doc 1000 and the BioRad Molecular Analyst version 2.1.1 software from BioRad Laboratories (Hercules, CA).

Western blot analysis. Western analyses were performed as published (Woods et al., 1999Go). Briefly, 10–30 µg of protein was mixed with 5X sample buffer and resolved with SDS-PAGE using 4–20% Tris-glycine pre-cast gels and then transferred onto PVDF paper (or nitrocellulose) using the NOVEX published protocol. The blots were visualized using ECL, as described by the manufacturer. When appropriate, selected blots were stripped with ChemiStrip (Chemicon International, Inc., Temecula, CA) and reblotted.

Transient transfections and reporter gene assays. Plasmids containing a construct composed of a 4X tandem repeat of the NF-{kappa}B promoter response element inserted upstream of the coding region of a firefly (Photinus pyralis) luciferase gene in pGL2-basic [p4x-NF-{kappa}B-luc], as well as the control vector [pCDNA 3] (Scatena et al., 1998Go), were generously provided by Dr. Nelson Fausto, Department of Pathology, University of Washington. The plasmids were propagated via Subcloning Efficiency DH5{alpha} Competent Cells (Life Technologies, Grand Island, NY), extracted with Endofree Plasmid Maxi Kit (Qiagen, Chatsworth, CA), and evaluated with a 0.5% agarose-tris buffer gel, eluted, and stored in TE buffer at 4°C. Cells were seeded into 6-, 12- or 24-well plates and grown to approximately 60% confluence. Using the Effectene Transfection Reagent kit manufacturer's protocol (Qiagen, Valencia, CA), cotransfection complexes were formed with the NF-{kappa}B luciferase reporter plasmid and with a commercially available constitutively active renilla reinformis luciferase reference plasmid pRL-CMV (Renilla reniformis, Promega, Madison, WI) in a ratio of 1000:1 experimental reporter:reference reporter. The latter was employed to adjust for well-to-well variation in cell number and transfection efficiency. The cotransfection mixture was introduced to the cell cultures for 24 h followed by refreshment of the culture medium. Cells were used in induction experiments at about 80% confluency 24–60 h following cotransfection. Experimental cultures were harvested and assayed according to product instructions with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) and the Lumat LB9507 luminometer (EG&G Berthold, Bundorra, Australia). Luminescence values were normalized and expressed as small, whole number transforms of the ratio of the firefly signal:Renilla reference signal. Vectors expressing mouse p65/relA (CMV-p65) and deletion mutant (p65dC) (CMV-p65 lacking the transactivation domain) (Ruben et al., 1992Go) were generously provided by Dr. Nancy Rice, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD, and were transiently transfected as described above.

Measures of apoptosis. Apoptotic cell death was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method using the fluorescein-based TUNEL cell death detection kit (Boehringer Mannheim, Indianapolis, IN). For this assay, cells were grown on Lab-Teck II chamber slides (Nalge Nunc International, Naperville, IL) to 50–80% confluence prior to treatments. Cells were then fixed in 100% acetone at –20°C. Following incubation with TUNEL reaction mixture, cells were incubated with anti-fluorescein antibody (Converter-POD) containing Fab fragments from sheep, conjugated with horse-radish peroxidase (POD). Fragmentation was visualized using POD substrate (DAB, metal-enhanced substrate set) (Roche Molecular Biochemicals, Mannheim, Germany). The percent apoptotic cells was calculated using an RT (real time) slider spot camera (Diagnostic Instruments Inc., Sterling Heights, MI) and NIH Image software (Universal Imaging Corp., Downingtown, PA).

Cytochrome c determination. Cytochrome c in mitochondrial and cytosolic fractions was evaluated by Western analysis following fractionation of cells using the ApoAlert Fractionation kit obtained from BD Biosciences Clontech (Palo Alto, CA). Cytochrome c was detected by immunoblotting the mitochondrial or cytosolic fraction with the monoclonal antibody 7H8.2C12 (1:2000) (Pharmingen, San Diego, CA), as described by Bossy-Wetzel and Green (2000)Go.

Statistical analyses. Analysis of differences between treatment groups was determined using a paired, one-tailed t-test. The level of significance was chosen at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NF-{kappa}B Activation in Kidney Cells Is Attenuated by Hg2+
We have previously described LPS-mediated activation of NF-{kappa}B in NRK52E cells, characterized by specific activation of the p50p65 heterodimer (Woods et al., 1999Go). Unlike in many other cell types, this effect is mediated through a redox-insensitive signaling transduction pathway that is not modulated by antioxidants, prooxidants, or modulation of endogenous GSH levels. Nonetheless, LPS-mediated activation of NF-{kappa}B in kidney cells was found to be substantially attenuated by pretreatment with Hg2+ at low µM concentrations. Although a potential prooxidant (Lund et al., 1993Go), Hg2+ did not by itself activate NF-{kappa}B or affect NF-{kappa}B expression in kidney cells for up to 4 h after treatment. Subsequent studies showed that Hg2+ attenuates LPS-mediated NF-{kappa}B activation by targeting thiol-dependent pathways involved in various steps of NF-{kappa}B activation and DNA-binding (Dieguez-Acuña et al., 2001Go). Here, we demonstrate comparable effects of Hg2+ on the activation of NF-{kappa}B by the proapoptotic regulatory cytokine, TNF. As in the case with LPS, TNF (300 units/ml, 2.1 ng/ml) administration to NRK52E cells resulted in peak activation of NF-{kappa}B at 30 min. As shown in Figure 1, this effect was significantly attenuated by Hg2+ pretreatment. Significant concentration-related inhibition of TNF-mediated NF-{kappa}B activation to 73, 56, and 45% of control was observed following pretreatments with Hg2+ at 0.5, 2, or 5 µM, respectively. The kinetics of Hg2+-mediated inhibition of NF-{kappa}B activation by TNF were similar to those previously observed with LPS (Dieguez-Acuña et al., 2001Go).



View larger version (31K):
[in this window]
[in a new window]
 
FIG. 1. Effects of Hg2+ on TNF-mediated NF-{kappa}B activation in kidney cells. (A) EMSA showing the concentration-response effect of Hg2+ on NF-{kappa}B activation when administered to cells 30 min prior to TNF. TNF was administered at 300 U/ml of culture medium 30 min prior to preparation of nuclear extracts. Arrow indicates location of the TNF-induced p50p65 complex. (B) Binding signals (optical densities in arbitrary units) are presented as a percentage of that for TNF alone (0 mM Hg2+). NT = control cells treated with vehicle only. *Significantly different from TNF-treated value (p < 0.05).

 
Hg2+ Diminishes NF-{kappa}B-Dependent Transcriptional Activity
To evaluate the capacity of Hg2+ to impair TNF-mediated NF-{kappa}B transcriptional activity, NRK52E cells were transiently transfected with a luciferase reporter plasmid driven by 4 {kappa}B tandem repeats inserted upstream of the coding region of a firefly luciferase gene. Luciferase activity was increased approximately 4-fold in cells after treatment with TNF alone. Similar to findings previously reported with respect to LPS (Dieguez-Acuña et al., 2001Go), TNF-induced NF-{kappa}B-dependent luciferase activity was decreased by 20 and 50% in cells pretreated with 2 or 5 µM Hg2+, respectively, for 30 min prior to TNF (not shown). Inhibition of transcriptional activity was not a general feature of Hg2+ toxicity, as no effect was observed with regard to the reference plasmid. Moreover, Hg2+ pretreatments did not affect cell viability as measured by lactate dehydrogenase (LDH) release from kidney cells (not shown). These results suggest, therefore, that the reduction in NF-{kappa}B-mediated transcriptional activity is attributable to attenuation of NF-{kappa}B activation and DNA binding targeted by Hg2+ in kidney cells.

Hg2+ Pretreatment Increases TNF-Mediated Apoptosis of Kidney Epithelial Cells
Using a fluorescein-based TUNEL cell death detection kit, we showed that treatments of kidney cells with Hg2+ at 0.5, 2, or 5 µM for 22 h did not result in a significant increase in the percentage of apoptotic cells when compared to untreated controls (Figs. 2A–2D). In contrast, when cells were pretreated with Hg2+ at 0.5, 2, or 5 µM for 30 min prior to TNF administration, a significant (p < 0.05) increase in the percentage of TUNEL positive cells was observed. The greatest increase in TNF-induced apoptosis was seen after pretreatment of cells with 2 µM Hg2+, where the amount of apoptosis increased from 8.4% (2 µM Hg2+ alone) to 43.5% (Figure 2C vs. 2F and Figure 2G). Further evidence of increased apoptosis was also obtained when evaluated by mitochondrial cytochrome c (Cyt-C) release, using mitochondrial and cytoplasmic fractions of cells treated with Hg2+ (2 µM) followed by TNF. As shown in Figure 3, no change in the levels of mitochondrial Cyt-C was observed following treatment of kidney cells with either TNF or 2 µM Hg2+ alone. However, when cells were pretreated with Hg2+ followed by TNF, the level of mitochondrial Cyt-C decreased to less than 20% of that of seen in untreated (NT) cells 4 h after TNF administration. A corresponding increase in cytochrome c was found in cytoplasmic extracts of the same samples (Figs. 4A and 4B).



View larger version (42K):
[in this window]
[in a new window]
 
FIG. 2. Hg2+ pretreatment increases the apoptogenic effect of TNF. (A–F) Representative images from kidney cells pretreated with Hg2+ as indicated 30 min prior to TNF (300 U/ml) for 22 h. Six images for each treatment, from two replicate groups, were taken using a 20X optical lens and analyzed for apoptosis. Representative TUNEL positive cells are indicated by arrows. (G) Percent apoptotic kidney cells measured by TUNEL assay. Six images for each treatment, from two replicate groups, were analyzed to determine the percent of TUNEL positive cells for the indicated treatments. *Significantly different from Hg2+ alone (p < 0.05).

 


View larger version (30K):
[in this window]
[in a new window]
 
FIG. 3. Effects of Hg2+ on TNF-mediated cytochrome c release from mitochondrial extracts of kidney cells. (A) Western blot showing release of cytochrome c (Cyt-C) from mitochondria of kidney cells treated with Hg2+ (2.5 µM) for 30 min followed by TNF (300 U/ml) for 4 h. Cox-4, a mitochondrial protein not released during apoptosis was used as a loading control. (B) The binding signal (optical density) of the Cyt-C released from the mitochondria was standardized to 100 percent of untreated (NT) kidney cells. *Significantly different from Hg2+ alone (p < 0.05).

 


View larger version (32K):
[in this window]
[in a new window]
 
FIG. 4. Effects of Hg2+ pretreatment on TNF-mediated mitochondrial cytochrome c release to cytoplasmic fraction in kidney cells. (A) Western blot showing an increase in cytochrome c (Cyt-C) in cytoplasmic fraction of kidney cells treated with Hg2+ (2.5 µM) for 30 min followed by TNF (300 U/ml) for 4 h. (B) The binding signal (optical density) of the Cyt-C released to the cytoplasm was standardized to 100% of untreated (NT) kidney cells. *Significantly different from Hg2+ alone (p < 0.05).

 
Bay 11-7082 Prevents NF-{kappa}B Activation in Kidney Cells and Increases TNF-Mediated Apoptosis
To demonstrate that the increased sensitivity of kidney cells to TNF-induced apoptosis following Hg2+ treatment was attributable specifically to inhibition of NF-{kappa}B activation, we sought to show comparable increased sensitivity using specific NF-{kappa}B inhibitors. Treatment of cells with Bay11-7082 (Bay 11), a specific inhibitor of I{kappa}B{alpha} phosphorylation, resulted in a dose-related inhibition of TNF-mediated NF-{kappa}B activation, when evaluated by EMSA. As shown in Figure 5A, NF-{kappa}B-DNA binding was blocked by 42 and 60% at Bay 11 concentrations of 2.5 and 5 µM, respectively, and completely inhibited at 10 µM. Treatment with Bay 11 also elicited a concentration-dependent inhibition of TNF-mediated NF-{kappa}B-dependent luciferase activity at 2.5 and 5 µM (Fig. 5B), suggesting inhibition of NF-{kappa}B-mediated transcriptional activity at comparable concentrations of Hg2+.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 5. Effects of Bay 11-7082 on TNF-mediated NF-{kappa}B activation and transcriptional activity in kidney cells. (A) EMSA showing a concentration-dependent effect of Bay 11 on NF-{kappa}B activation when administered to cells 1 h prior to TNF. TNF was administered at 300 U/ml of culture medium 30 min prior to preparation of nuclear extracts. Binding signals (optical densities in arbitrary units) are presented as a percentage of that for TNF alone. Gel density analysis shows mean of at least three replicate treatments. *Significantly different from control (0 µM Bay 11) value (p < 0.05). (B) Cells transiently transfected with the 4xNF-{kappa}B-luciferase reporter plasmid show a concentration-dependent inhibition in NF-{kappa}B transcriptional activity by Bay 11-7082. *Significantly different from NT (p < 0.05). **Significantly different from TNF-treated value (p < 0.05).

 
To support the hypothesis that prevention of NF-{kappa}B activation sensitizes kidney cells to the proapoptotic effects of TNF, we measured the effect of Bay 11 pretreatment of TNF-mediated apoptosis in kidney cells. As shown in Figures 6A–6C, treatment of cells with TNF or Bay 11 (2.5 µM) alone did not promote a significant increase in the number of TUNEL positive cells, compared with untreated (NT). In contrast, the proportion of apoptotic cells increased from 8.4 to 28.5% after treatment with 2.5 µM Bay 11 followed by TNF (Fig. 6D). The proportion of TUNEL-positive cells observed after TNF treatment increased to 54.2% when the pretreatment concentration of Bay 11 was doubled from 2.5 to 5 µM (not shown), consistent with the concentration-related inhibition of NF-{kappa}B-DNA binding observed under the same treatment conditions.



View larger version (57K):
[in this window]
[in a new window]
 
FIG. 6. Effects of Bay 11-7082 on TNF-mediated apoptosis. (A–D) Representative images from kidney cells pretreated with Bay 11 as indicated 1 h prior to TNF treatment (300 U/ml) for 22 h. Twelve images for each treatment, from three replicate groups, were taken using a 20X optical lens and analyzed for apoptosis. Representative TUNEL positive cells are indicated by arrows.

 
Further evidence of increased sensitivity to TNF-mediated apoptosis in cells pretreated with Bay 11 was obtained by analysis of cytochrome c levels in mitochondrial and cytosolic extracts prepared from cells treated with either TNF or Bay 11 (2.5 µM) alone or with Bay 11 followed by TNF. As shown in Figure 7, a 70% decrease in the levels of mitochondrial Cyt-C content was observed following treatment of cells with both Bay 11 and TNF, compared to untreated (NT) controls. A corresponding increase in cytoplasmic Cyt-C levels was observed (not shown). In contrast, no changes in either mitochondrial or cytosolic Cyt-C levels were observed following treatment with either TNF or Bay 11 alone.



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 7. Effects of Bay 11-7082 on TNF-mediated cytochrome c release from mitochondrial extracts of kidney cells. (A) Western blot showing release of cytochrome c (Cyt-C) from mitochondria of kidney cells treated with Bay11-7082 (2.5 µM) for 1 h prior to TNF (300 U/ml) for 4 h. Cox-4, a mitochondrial protein not released during apoptosis was used as a loading control. (B) The binding signal (optical density) of the Cyt-C released from the mitochondria was standardized to 100% of untreated (NT) kidney cells. *Significantly different from Bay11 alone (p < 0.05).

 
SN50 Prevents NF-{kappa}B Activation in Kidney Cells and Increases TNF-Mediated Apoptosis
Findings similar to those observed with Hg2+ and Bay 11 were obtained when kidney epithelial cells were pretreated with the NF-{kappa}B nuclear translocation-inhibitor peptide, SN50, prior to TNF administration. As shown in Figure 8A, lanes 1–7, and 8B, pretreatment with SN50 (50–100 µg/ml) decreased NF-{kappa}B-DNA binding intensity in a concentration-related manner when added to cells 15 min prior to TNF. In contrast, pretreatment with an inactive SN50 mutant control (SN50M) resulted in no inhibition of TNF-mediated NF-{kappa}B activation (Figure 8A lanes 8, 9, and 8B). Moreover, pretreatment with SN50 dramatically increased the apoptosis-inducing potential of TNF in kidney cells. As shown in Figure 9, the percentage of TUNEL-positive cells increased from 8% to 14 and 56% following pretreatment with 50 or 100 µg/ml SN50, respectively. In contrast, the percentage of apoptotic cells treated with TNF and SN50 alone, or with SN50M followed by TNF, did not differ from that in untreated cells.



View larger version (30K):
[in this window]
[in a new window]
 
FIG. 8. Effects of SN50 on TNF-mediated NF-{kappa}B activation in kidney cells. (A) EMSA showing a concentration-dependent effect of SN50 on NF-{kappa}B activation when administered to cells 15 min prior to TNF (lanes 4–7). TNF was administered at 300 U/ml of culture medium 30 min prior to preparation of nuclear extracts. An inactive mutant form of the SN50 peptide (SN50 M) had no effect on NF-{kappa}B activation (lanes 8 and 9). (B) Binding signals (optical densities in arbitrary units) are presented as a percentage of that for TNF alone (0 µg/ml SN50). Gel density analysis shows mean of at least two replicate treatments. NT = control cells treated with vehicle only. *Significantly different from TNF alone (0 µg/ml SN50) (p < 0.05).

 


View larger version (11K):
[in this window]
[in a new window]
 
FIG. 9. Effects of SN50 on TNF-mediated apoptosis. Ten representative images from three replicate samples were analyzed for apoptotic cells. Kidney cells were pretreated with SN50 15 minutes prior to TNF (300 U/ml) for 22 h. *Significantly different from NT (untreated) control (p < 0.05).

 
Constitutive Expression of p65 Attenuates Sensitivity to Apoptosis
To further demonstrate the functional role of NF-{kappa}B in mediating the sensitivity of kidney cells to TNF-induced apoptosis, we determined whether constitutive expression of p65 would attenuate or prevent this effect. Constitutive expression of p65 was achieved by cotransfection of NRK52E cells with vectors expressing mouse p65/relA (CMV-p65) or deletion mutant (p65dC), as described in Materials and Methods, along with the NF-{kappa}B-luciferase expression vector. Co-transfection with the p65 expression vector resulted in a 12-fold increase in luciferase activity compared with cells co-transfected with the p65 mutant control vector (not shown). Whereas 41% of cells transfected with the deletion mutant (controls) displayed condensed and fragmented nuclei 24 h after Hg2+ (20 µM) followed by TNF (300 U/ml) treatment, fewer than 15% of the p65-expressing cells appeared apoptotic after comparable treatment (not shown).

NF-{kappa}B Activation in Kidney Cells Is Attenuated by Hg2+ in Vivo
Further studies were performed in order to ascertain the functional relevance of the findings derived from cell culture studies to events in vivo. For these assessments, male Sprague Dawley rats (four/group) were injected ip with physiologic saline (0.9% NaCl) or with a nontoxic dose (0.75 mg/kg) of Hg2+ as HgCl2 dissolved in physiologic saline 18 h prior to a second dose of saline or LPS (10 mg/kg, ip). Animals were subsequently sacrificed 2 h after LPS treatment, and kidneys were removed. Gel shifts assays were performed using nuclear extracts prepared from slices of kidney cortex. Figure 10A shows EMSA results performed on nuclear extracts prepared from renal cortical samples from animals treated with either Hg2+, LPS (2 h), or Hg2+ followed by LPS. Similar to findings from cultured cells, LPS treatment significantly increased NF-{kappa}B-DNA binding activity (lanes 6, 7), and Hg2+ pretreatment significantly inhibited this effect (lanes 7, 8). In contrast, NF-{kappa}B-DNA binding activity from kidneys of rats treated with Hg2+ alone (lanes 3, 4) did not differ from that seen in kidneys of untreated (NT) rats. Similarly, analysis of cytoplasmic extracts prepared from kidneys of animals exposed to LPS for 2 h showed a slight increase in Cyt-C content. In contrast, animals treated with both Hg2+ and LPS showed a more than additive increase in the levels of Cyt-C (Fig. 11), consistent with findings derived from studies with NRK52E cells.



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 10. Effects of Hg2+ on LPS-mediated NF-kB activation in vivo. Nuclear extracts of rat kidney cortex were harvested from male rats (four/group) treated with Hg2+ (0.75 mg/kg, IP) alone or for 18 h prior to LPS (10 mg/kg, ip) for 2 h. (A) EMSA showing inhibition of LPS-mediated NF-{kappa}B activation by Hg2+ (lanes 7–8). Arrow indicates location of the LPS-induced p50p65 complex. (B) Binding signals (optical densities in arbitrary units) are presented as a percentage of that for LPS alone. NT = saline treated only. *Significantly different from LPS-treated value (p < 0.05).

 


View larger version (32K):
[in this window]
[in a new window]
 
FIG. 11. Effects of Hg2+ on LPS-mediated cytochrome c release from mitochondrial to cytoplasmic fraction of rat kidney in vivo. (A) Western blot showing an increase in cytochrome c (Cyt-C) in cytoplasmic fractions of kidney from rats (4/group) treated with Hg2+ (0.75 mg/kg, IP) for 18 h followed by LPS (10 mg/kg, ip) for 2 h. (B) The binding signal (optical density) of the Cyt-C released to the cytoplasm was standardized to 100% of saline-treated (NT). *Significantly different from NT (p < 0.05). **Significantly different from Hg2+ alone (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kidney tubular cell injury is a primary event in the pathogenesis of mercury (Hg2+)-induced renal failure. Of central importance to understanding the toxicity of Hg2+ is the identification of the specific and most sensitive steps in cellular processes that are involved in Hg2+-mediated cell injury and cell death. Previous studies (Fowler and Woods, 1977Go; Lund et al., 1993Go; Southard et al., 1974Go; Weinberg et al., 1982aGo,bGo) have focused largely on disruption of mitochondrial bioenergetics as a principal event underlying Hg2+ toxicity. In contrast, few studies have sought to define the specific molecular mechanisms through which Hg2+ initiates toxicity via alteration of signal transduction pathways that regulate cellular proliferation and survival. The present studies demonstrate that Hg2+ inhibits activation of NF-{kappa}B and that this effect increases the sensitivity of kidney cells to the apoptosis-inducing effects of other toxicants to which kidney cells are otherwise resistant.

The functional consequences of impairment of NF-{kappa}B activation and DNA binding by Hg2+ as pertains to increased sensitivity of kidney cells to apoptogenic potential of other agents is supported by the present morphologic and biochemical indications of apoptosis observed in cells treated with specific inhibitors of NF-{kappa}B activation (Bay 11 and SN50). Numerous studies have demonstrated an important role for NF-{kappa}B in mediating resistance to apoptosis in various cell types, including kidney epithelial cells (Guijarro and Egido, 2001Go; Ortiz, 2000aGo,bGo; Zoja et al., 1998Go). This effect is associated with increased expression of NF-{kappa}B-regulated antiapoptotic gene products (Ortiz, 2000aGo). Several gene products that may play a role in blocking apoptosis and whose expression is regulated by NF-{kappa}B have been identified, including various members of the IAP family of apoptosis inhibitors as well as the antiapoptotic Bcl-2 family members (Aggarwal, 2000Go; Rana et al., 2001Go). The antiapoptotic effect of NF-{kappa}B is unlikely to be restricted to any one specific apoptotic agent in light of the wide variety of diverse stimuli that activate NF-{kappa}B in kidney epithelial cells (Amoah Apraku et al., 1995Go; Woods et al., 1999Go). Notably, inhibition of NF-{kappa}B has been shown to diminish the activity of a number of renal survival factors including insulin and platelet-derived growth factor (Rana et al., 2001Go). Thus, NF-{kappa}B may be a potent antiapoptotic response to a ubiquitous array of apoptotic triggers in kidney cells. The finding that NF-{kappa}B is a sensitive molecular target for Hg2+ suggests a possible mechanism by which mercury initiates toxicity in kidney by enhancing the apoptotic potential of endogenous cytokines and other toxicants through impaired NF-{kappa}B activation.

The biological relevance of the findings observed in the present cell culture studies is supported by the comparable observations obtained in vivo from rats treated with bacterial lipopolysaccharide (LPS) subsequent to Hg2+ administration. The level and duration of Hg2+ treatment employed in the present studies (0.75 mg/kg) is well below the lowest dose of Hg2+ (1.5 mg/kg) previously demonstrated (Woods, 1989Go) to elicit evidence of nephrotoxicity in this strain of rat. Nonetheless, we observed here a significant decrease in NF-{kappa}B activation as measured in nuclear extracts of kidney cortical cells from rats treated with Hg2+ prior to LPS administration, as compared with that from rats treated with LPS alone. The association of this effect with cytochrome c release from mitochondria isolated from the same cells further supports a mechanistic association of impaired NF-{kappa}B activity with increased sensitivity to apoptosis in kidney cells in vivo.

The findings derived from the current kidney cell model suggest a potential role for NF-{kappa}B in Hg-induced cytotoxicity in other tissues in which mercury or mercury compounds are preferentially accumulated. Of particular interest in this regard is the central nervous system (CNS), a principal target of elemental and organic mercurials, and in which NF-{kappa}B has been described to play a key regulatory role in the survival and prevention of apoptosis in various cell types (Bhakar et al., 2002Go; Jarosinski et al., 2001Go; Koulich et al., 2001Go). In this respect, NF-{kappa}B deficiency in hippocampal neurons is associated with learning and memory deficits (Kassed et al., 2002Go), events also associated with and exacerbated by prolonged elemental mercury exposure (Danielsson et al., 1993Go). Similarly, NF-{kappa}B expression promotes survival and prevents apoptosis of cerebellar neurons (Koulich et al., 2001Go), whereas inhibition of NF-{kappa}B activation is reported to induce apoptosis of cerebellar granular cells (Piccoli et al., 2001Go), both selective targets of organic mercurials, particularly, methyl mercury (Chang and Hartmann, 1972Go). These observations provide a potential mechanistic basis for the exacerbation of neurologic toxicity associated with impaired NF-{kappa}B-mediated survival processes by mercury in the CNS, similar to that observed in kidney epithelial cells. Studies to test these hypotheses are in progress.

In conclusion, the present studies demonstrate that inhibition of NF-{kappa}B activation and transcriptional activity by Hg2+ in kidney epithelial cells increases their sensitivity to the apoptosis-inducing effects of other agents to which these cells are otherwise resistant. Since apoptosis is considered to be an underlying mechanistic event in the pathogenesis of kidney failure associated with toxicant injury to tubular epithelial cells, the present findings suggest a mechanistic basis underlying the loss of kidney function associated with mercury exposure.


    ACKNOWLEDGMENTS
 
This work was supported by the University of Washington NIEHS-sponsored Center for Ecogenetics and Environmental Health Grant P30ES07033 and by the University of Washington NIEHS-sponsored Superfund Program Project Grant P42ES04696. F.J.D-A. and W.W.P. were supported by the NIEHS Environmental Pathology/Toxicology Traning Grant ES07032. Additional funding was provided by the Wallace Research Foundation.


    NOTES
 

1 To whom correspondence should be addressed at Department of Environmental and Occupational Health Sciences, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98105. Fax: (206) 528-3550. E-mail: jwoods{at}u.washington.edu.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aggarwal, B. B. (2000). Tumor necrosis factor receptor associated signaling molecules and their role in activation of apoptosis, JNK and NF-kappaB. Ann. Rheum. Dis. 59(Suppl. 1), 6–16.[CrossRef][ISI]

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

Amore, A., and Coppo, R. (2000). Role of apoptosis in pathogenesis and progression of renal diseases. Nephron 86, 99–104.[CrossRef][ISI][Medline]

Bhakar, A. L., Tannis, L.-L., Zeindler, C., Russo, M. P., Jobin, C., Park, D. S., MacPherson, S., and Barker, P. A. (2002). Constitutive nuclear factor-{kappa}B activity is required for central neuron survival. J. Neurosci. 22, 8466–8475.[Abstract/Free Full Text]

Bossy-Wetzel, E., and Green, D. R. (2000). Assays for cytochrome c release from mitochondria during apoptosis. Meth. Enzymol. 322, 235–243.[ISI][Medline]

Chang, L. W., and Hartmann, H. A. (1972). Electron microscopic histochemical study on the localization and distribution of mercury in the nervous system after mercury intoxication. Exptl. Neurol. 35, 122–137.[CrossRef][ISI][Medline]

Chen, F., Castrovanova, V., Shi, X., and Demers, L. M. (1999). New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor, in the initiation of diseases. Clin. Chem. 45, 7–17.[Abstract/Free Full Text]

Danielsson, B. R., Fredriksson, A., Dahlgren, L., Gardlund, A. T., Olsson, L., Denker, L., Archer, T. (1993). Behavioral effects of prenatal metallic mercury inhalation exposure in rats. Neurotoxicol. Teratol. 15, 391–396.[CrossRef][ISI][Medline]

DiDonato, J. A., Mercurio, F., Rossette, C., Wu-Li, J., Suyang, H., Ghosh, S., and Karin, M. (1996). Mapping of the inducible I-{kappa}B phosphorylation sites that signal its ubiquitination and degradation. Molec. Cell. Biol. 16, 1295–1304.[Abstract]

Dieguez-Acuña, F. J., Ellis, M. E., Kushleika, J., and Woods, J. S. (2001). Mercuric ion attenuates nuclear factor-kappaB activation and DNA binding in normal rat kidney epithelial cells: Implications for mercury-induced nephrotoxicity. Toxicol. Appl. Pharmacol. 173, 176–187.[CrossRef][ISI][Medline]

Fowler, B. A., and Woods, J. S. (1977). Ultrastructural and biochemical changes in renal mitochondria during chronic oral methyl mercury exposure: The relationship to renal function. Exptl. Molec. Pathol. 27, 403–412.[CrossRef]

Gritzka, T. L., and Trump, B. F. (1968). Renal tubular lesions caused by mercuric chloride. Electron microscopic observations: Degeneration of the pars recta. Amer. J. Pathol. 52, 1225–1277.[ISI][Medline]

Guijarro, C., and Egido, J. (2001). Transcription factor-kappa B (NF-kappa B) and renal disease. Kidney Int. 2001 59, 415–424.[CrossRef][ISI][Medline]

Jarosinski, K. W., Whitney, L. W., and Massa, P. T. (2001). Specific deficiency in nuclear factor-{kappa}B activation in neurons of the central nervous system. Lab. Invest. 81, 1275–1288.[ISI][Medline]

Kassed, C. A., Willing, A. E., Garbuzova-Davis, S., Sanberg, P. R., and Pennybacker, K. R. (2002). Lack of NF-{kappa}B p50 exacerbates degeneration of hippocampal neurons after chemical exposure and impairs learning. Exptl. Neurol. 176, 277–288.[CrossRef][ISI][Medline]

Koulich, E., Nguyen, T., Johnson, K., Giardina, C. A., and D'Mello, S. R. (2001). NF-{kappa}B is involved in the survival of cerebellar granule neurons: Association of I{kappa}B phosphorylation with cell survival. J. Neurochem. 76, 1188–1198.[CrossRef][ISI][Medline]

Kretz-Remy, C., Mehlen, P., Mirault, P., and Arrigo, A. P. (1996). Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression. J. Cell. Biol. 133, 1083–1093.[Abstract]

Lieberthal, W., and Levine, J. S. (1996). Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury. Amer. J. Physiol. 271, F477–F488.[ISI][Medline]

Lund, B. O., Miller, D. M., and Woods, J. S. (1993). Studies on Hg(II)-induced H2O2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria. Biochem. Pharmacol. 45, 2017–2024.[CrossRef][ISI][Medline]

Luster, M. I., Simeonova, P. P., Gallucci, R., and Matheson, J. (1999). Tumor necrosis factor alpha and toxicology. Crit. Rev. Toxicol. 29, 491–511.[ISI][Medline]

Matthews, J. R., Wakasugi, N., Virelizier, J. L., Todoi, J., and Hay, R. T. (1992). Thioredoxin regulates the DNA binding activity of NF-{kappa}B by reduction of a disulfide bond involving cysteine 62. Nucleic Acids Res. 20, 3821–3830.[Abstract]

Ortiz, A. (2000a). Nephrology forum: Apoptotic regulatory proteins in renal injury. Kidney Int. 58, 467–485.[CrossRef][ISI][Medline]

Ortiz, A. (2000b). Renal cell loss through cell suicide. Kidney Int. 58, 2235–2236.[ISI][Medline]

Ortiz, A., Gonzalez-Cuadrado, S., Bustos, C., Alonso, J., Homez-Guerrero, C., Lopez-Armada, M. J., Gonzalez, E., Plaza, J. J., and Egido, J. (1995). Tumor necrosis factor and glomerular damage. J. Nephrol. 8, 27–34.[ISI]

Ortiz, A., Lorz, C., Catalan, M. P., Danoff, T. M., Yamasaki, Y., Egido, J., and Neilson, E. G. (2000a). Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure. Kidney Int. 57, 969–981.[CrossRef][ISI][Medline]

Ortiz, A., Lorz, C., Catalãn, M. P., Justo, P., and Egido, J. (2000b). Role and regulation of apoptotic cell death in the kidney. Y2K update. Front. Biosci. 2000 5, 735–749.

Pampfer, S., Cordi, S., Cikos, S., Pidry, B., Vanderhayden, I., and De Hertogh, R. (2000). Activation of nuclear factor {kappa}B and induction of apoptosis by tumor necrosis factor-{alpha} in the mouse uterine epithelial WEG-1 cell line. J. Reproduct. 63, 879–886.

Piccoli, P., Porcile, C., Stanzione, S., Bisaglia, M., Bajett, A., Bonavio, R., Florio, T., and Schettini, G. (2001). Inhibition of nuclear factor-{kappa}B activation induces apoptosis in cerebellar granular cells. J. Neurosci. Res. 66, 1064–1073.[CrossRef][ISI][Medline]

Rana, A., Sathyanarayana, P., and Lieberthal, W. (2001). Role of apoptosis of renal tubular cells in acute renal failure: Therapeutic implications. Apoptosis 6, 83–102.[CrossRef][ISI][Medline]

Ruben, S. M, Narayanan, R., Klement, J. F., Chen, C.-H., and Rosen, C. A. (1992). Functional characterization of the NF-{kappa}B transcriptional activator and an alternatively spliced derivative. Molec. Cell. Biol. 12, 444–454.[Abstract]

Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli, C. M. (1998). NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival. J. Cell Biol. 141, 1083–1093.[Abstract/Free Full Text]

Sha, W. C., Liou, H. C., Toumanen, E. I., and Baltimore, D. (1995). Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 80, 321–330.[ISI][Medline]

Southard, J., Nitisewojo, P. and Green, D. E. (1974). Mercurial toxicity and the perturbation of the mitochondrial control system. Fedn. Proc. 33, 2147–2153.

Weinberg, J. M., Harding, P. G., and Humes, H. D. (1982a). Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. I. Direct effects of in vitro mercuric chloride on renal mitochondrial function. J. Biol. Chem. 257, 60–67.[Abstract/Free Full Text]

Weinberg, J. M., Harding, P. G., and Humes, H. D. (1982b). Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. II. Functional alterations of renal cortical mitochondria isolated after mercuric chloride treatment. J. Biol. Chem. 257, 68–74.[Abstract/Free Full Text]

Woods, J. S. (1989). Mechanisms of metal-induced alteration of cellular heme metabolism. Comments Toxicol. 3, 3–25.

Woods, J. S., Dieguez-Acuña, F. J., Ellis, M. E., Kushlieka, J., and Simmonds, P. L. (2002). Attenuation of nuclear factor kappa B (NF-kappaB) promotes apoptosis of kidney epithelial cells: A potential mechanism of mercury-induced nephrotoxicity. Environ. Health Perspect. 110(Suppl. 5), 819–822.[ISI][Medline]

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

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