* Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, and Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105
Received June 11, 2004; accepted July 23, 2004
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ABSTRACT |
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Key Words: nuclear factor kappa B; kidney epithelial cells; renal failure; Hg2+ toxicity; apoptosis.
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INTRODUCTION |
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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, 2000a; Ortiz et al., 1995
). 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., 1995
). Additionally, TNF is known to activate a wide array of cellular signaling pathways that result in divergent biological responses, including activation of NF-
B (Luster et al., 1999
). 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, 2000b
; Ortiz et al., 2000a
,b
), 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, 2000
; Lieberthal and Levine, 1996
; Rana et al., 2001
), 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, 1968). Previously, we reported (Dieguez-Acuña et al., 2001
; Woods et al., 2002
) 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-
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-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-
B p50 subunit (Pampfer et al., 2000
), and Bay 11-7082, a specific inhibitor of cytokine-inducible I
B
phosphorylation (Pampfer et al., 2000
), were employed as specific inhibitors of NF-
B activation. We demonstrate that attenuation of NF-
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.
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MATERIALS AND METHODS |
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Electrophoretic mobility shift assays (EMSAs) and autoradioaugraphy. Nuclear and cytoplasmic extracts were prepared as previously described (Woods et al., 1999). 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
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 [
-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 1.52 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., 1999). Briefly, 1030 µg of protein was mixed with 5X sample buffer and resolved with SDS-PAGE using 420% 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-B promoter response element inserted upstream of the coding region of a firefly (Photinus pyralis) luciferase gene in pGL2-basic [p4x-NF-
B-luc], as well as the control vector [pCDNA 3] (Scatena et al., 1998
), were generously provided by Dr. Nelson Fausto, Department of Pathology, University of Washington. The plasmids were propagated via Subcloning Efficiency DH5
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-
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 2460 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., 1992
) 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 5080% 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).
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.
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RESULTS |
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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. 2A2D). 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).
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NF-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-B-DNA binding activity (lanes 6, 7), and Hg2+ pretreatment significantly inhibited this effect (lanes 7, 8). In contrast, NF-
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.
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DISCUSSION |
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The functional consequences of impairment of NF-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-
B activation (Bay 11 and SN50). Numerous studies have demonstrated an important role for NF-
B in mediating resistance to apoptosis in various cell types, including kidney epithelial cells (Guijarro and Egido, 2001
; Ortiz, 2000a
,b
; Zoja et al., 1998
). This effect is associated with increased expression of NF-
B-regulated antiapoptotic gene products (Ortiz, 2000a
). Several gene products that may play a role in blocking apoptosis and whose expression is regulated by NF-
B have been identified, including various members of the IAP family of apoptosis inhibitors as well as the antiapoptotic Bcl-2 family members (Aggarwal, 2000
; Rana et al., 2001
). The antiapoptotic effect of NF-
B is unlikely to be restricted to any one specific apoptotic agent in light of the wide variety of diverse stimuli that activate NF-
B in kidney epithelial cells (Amoah Apraku et al., 1995
; Woods et al., 1999
). Notably, inhibition of NF-
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., 2001
). Thus, NF-
B may be a potent antiapoptotic response to a ubiquitous array of apoptotic triggers in kidney cells. The finding that NF-
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-
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, 1989) to elicit evidence of nephrotoxicity in this strain of rat. Nonetheless, we observed here a significant decrease in NF-
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-
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-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-
B has been described to play a key regulatory role in the survival and prevention of apoptosis in various cell types (Bhakar et al., 2002
; Jarosinski et al., 2001
; Koulich et al., 2001
). In this respect, NF-
B deficiency in hippocampal neurons is associated with learning and memory deficits (Kassed et al., 2002
), events also associated with and exacerbated by prolonged elemental mercury exposure (Danielsson et al., 1993
). Similarly, NF-
B expression promotes survival and prevents apoptosis of cerebellar neurons (Koulich et al., 2001
), whereas inhibition of NF-
B activation is reported to induce apoptosis of cerebellar granular cells (Piccoli et al., 2001
), both selective targets of organic mercurials, particularly, methyl mercury (Chang and Hartmann, 1972
). These observations provide a potential mechanistic basis for the exacerbation of neurologic toxicity associated with impaired NF-
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-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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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