Differential Induction of Podocyte Heat Shock Proteins by Prolonged Single and Combination Toxic Metal Exposure
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《毒物学科学杂志》
Pediatric Nephrology Division, Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109
ABSTRACT
Cadmium, mercury, and arsenite are among the most abundant toxic metals (TM) in our environment, and chronic TM exposure leads to injury to the kidney's glomerular filtration barrier. The small heat shock protein hsp25, highly expressed in glomerular podocytes, is induced during development of experimental nephrotic syndrome, and hsp25 overexpression can protect cultured podocytes from injury. Because little is known about the effect of multiple TM on podocytes, we measured the response of cultured podocytes to prolonged exposures to single and multiple TM. Podocyte viability declined by approximately 50% after 3 days of treatment with 20 μM cadmium, mercury, or arsenite, and 40 μM of any of these metals was lethal. The toxicity of equimolar concentrations of two or all three metals in combination was significantly altered compared to individual metal treatments. Single TM treatments induced only modest increases in the amounts of hsp25, B-crystallin, and inducible hsp70. Toxic metal combinations induced greater stress protein accumulation, especially arsenite + cadmium or arsenite + cadmium + mercury treatments, the TM mixtures with the lowest toxicity. All TM treatments caused a rapid and sustained increase in hsp25 phosphorylation. The intracellular accumulation of cadmium was greater and that of mercury was less in cells treated with TM combinations than in cells treated with a single TM. Our results showed that multiple TM effects on podocyte viability were neither additive nor synergistic and that induction of heat shock proteins correlated with increased resistance to TM injury, suggesting that induction of small heat shock proteins may play an important role in preventing TM-induced podocyte injury.
Key Words: cadmium; arsenite; mercury; Hsp25; Hsp70; B-crystallin.
INTRODUCTION
Environmental or occupational exposure to toxic metals is well known to induce chronic renal disease, and cadmium, mercury, and arsenite (a metalloid) are among the most environmentally abundant nephrotoxic metals (Fowler, 1993). Although the renal toxicological effects are dependent on the dosage, duration, and type of metal exposure (Madden and Fowler, 2000), the kidney's proximal tubules are known to be especially sensitive to injury, primarily because of their characteristically high reabsorptive activity (Robertson 2000). Importantly, toxic metal–induced glomerular injury, although far less well characterized than renal tubular injury, has also been reported (Enestrom and Hultman, 1984; Hoedemaeker et al., 1988).
Despite the extensive range of debilitating diseases caused by toxic metals, relatively little is known about the specific cellular and molecular mechanisms involved in metal toxicity. A variety of molecular factors likely play major roles in regulating cellular toxicity, including metal-binding proteins, inclusion bodies, and cell-specific receptor-like proteins. In addition, although most studies to date have examined exposure only to individual toxic metals, it is likely that chronic environmental toxic metal exposure typically includes sequential or simultaneous exposure to multiple toxic metals. Such environmental exposures (as opposed to occupational exposures typically involving acute or chronic exposure to a single toxic metal) highlight the potential importance of altered toxicity from interactions between the common environmental toxic metals or their cellular mechanisms of action. These poorly characterized interactions may result in additive, synergistic, or antagonistic effects on the overall toxicity compared to the effects of individual metals.
One important reaction to toxic metal exposure is induction of a cellular stress response, including increased expression of the major cytosolic stress protein hsp70i (Ait-Aissa et al., 2000; Bonham et al., 2003; Hernadez-Pando et al., 1995) and increased expression and phosphorylation of the small heat shock protein hsp25 (hsp25 is the murine or rat isoform; hsp27, the human isoform: the combined hsp25/27 will be used subsequently when referring in general to this protein), a known regulator of actin polymerization (Bonham et al., 2003; Radloff et al., 1998; Somji et al., 1999a; Wu and Welsh, 1996). Although this stress response to toxic injury has been best characterized in renal tubular cells (Bonham et al., 2003; Liu et al., 1996; Somji et al., 1999a), we have previously reported analogous induction of hsp25 expression and phosphorylation in renal podocytes in the kidney's glomerular filtration barrier in response to another toxin, puromycin aminonucleoside (Smoyer et al., 1996). Subsequent studies further demonstrated that hsp25 had the ability to regulate the podocyte response to injury (Smoyer and Ransom, 2002). In addition, few studies have addressed the long-term effects of toxic metals on cytotoxicity and stress protein accumulation (Bonham et al., 2003; Croute et al., 2000; Rossi et al., 2002; Somji et al., 1999a, 1999b). From these findings, as well as the poorly characterized glomerular injury induced by toxic metals, we hypothesized (1) that toxic metal exposure would alter small heat shock protein expression and phosphorylation in glomerular podocytes, (2) that exposure to combinations of toxic metals would result in altered toxicity as compared to individual metal exposure alone, and (3) that this altered toxicity may be a result of increased heat shock protein expression in cells treated with multiple toxic metals. To begin to test these hypotheses, we designed a series of studies to analyze in detail the stress response and intracellular toxic metal accumulation of cultured podocytes after prolonged exposure to individual and various combinations of the common environmental toxic metals, cadmium, mercury, and arsenite.
MATERIALS AND METHODS
Cell culture and toxic metal treatments. A conditionally immortalized mouse podocyte clonal cell line (MPC-5) isolated from the "Immortomouse" was used in these studies, and was the kind gift of Dr. Peter Mundel. Bulk culture of podocytes was carried out in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco-BRL, Rockville, MD), and 10 U/ml mouse -interferon (Sigma, St. Louis, MO) at 33°C with 100% relative humidity and 5% CO2 atmosphere. Proliferating podocytes were detached from the substrate with trypsin, seeded into culture dishes at 1000 cells/well in 96-well plates for MTT assays or at 30,000 cells/well in 6-well plates for all other experiments, and then induced to differentiate by transfer to 37°C and culture in the above medium without -interferon for 10–14 days. These culture conditions repress the promoter-driven expression and inactivate the thermosensitive protein product of a transgene encoding a variant of the SV40 T-antigen that results in conditional immortalization of this cell line.
Differentiated podocytes were treated with toxic metals by replacement of the culture medium with medium containing vehicle alone or single or multiple toxic metal salts. Medium containing toxic metals was prepared by addition of 1000x solutions of metal salts (HgCl2, CdCl2, or NaAsO2, all 95% A.C.S. grade, Sigma-Aldrich, St. Louis, MO) to medium immediately prior to use. Treatments consisted of individual metals at 0, 10, 20, or 40 μM concentrations, and dual and triple metal treatments at the same total metal concentrations but consisting of equimolar concentrations of each constituent metal summing to the total metal concentrations (e.g., 20 μM dual metal treatments consisted of 10 μM of each metal, and 20 μM triple metal treatments consisted of 6.7 μM of each metal). Podocytes were cultured in this medium for various times up to 3 days and then analyzed as described below.
Cell survival assays. Podocyte viability was measured using MTT reduction as previously described (Mosmann, 1983) after 3 days of toxic metal treatment. Wells used for the negative controls were not seeded with cells. After treatment medium containing metal salts was removed and replaced with 200 μl/well of fresh medium, and then 50 μl/well of a 50 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) solution was added and the cells cultured for 4 h. The MTT-media solution was then removed, and 200 μl/well of DMSO (dimethyl sulfoxide; Sigma, St. Louis, MO) was added, followed by the addition of 25 μl/well glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). Finally, the absorbance of each well was immediately read at 570 nm in a spectrophotometric plate reader (SpectraMAX 250, Molecular Devices, Sunnyvale, CA).
Metal uptake assays. After 3 days of toxic metal treatments, podocytes were washed with PBS, detached from the substrate with trypsin, and collected by centrifugation. The cell pellets were weighed to determine fresh weight, and analysis of metal accumulation in podocytes was performed using Environmental Protection Agency (EPA) methods detailed in the EPA document SW-846, "Test Methods for Evaluating Solid Waste" by the University of Michigan Occupational Safety and Environmental Health Environmental Labs. All data are reported in terms of grams cell material analyzed (μg toxic metal/g cellular fresh weight).
Mercury accumulation was measured according to SW-846, Method 7471A, "Mercury in Solid or Semisolid Waste (Manual Cold-Vapor Technique)." Briefly, samples were digested with sulfuric acid, nitric acid, potassium permanganate, and potassium persulfate at 95°C for 2 h. Excess potassium permanganate was removed by the addition of sodium chloride-hydroxylamine sulfate. Immediately prior to analysis, stannous chloride was added, and the sample was analyzed by atomic absorption (AA), with a Leeman Labs Hydra AA Automated Mercury Analyzer (Leeman Labs, Hudson, NH). The limits of detection for mercury were 0.6 ng/sample, equivalent to approximately 0.12 μg Hg per gram cellular fresh weight in our approximately 5 mg samples.
Cadmium and arsenic accumulation was measured according to SW-846 Method 3050B, "Acid Digestion of Sediments, Sludges, and Soils," followed by SW-846 Method 6020, "Inductively Coupled Plasma-Mass Spectrometry" (ICP-MS). Briefly, samples were digested with nitric acid followed by hydrogen peroxide, then heated at 95°C for 2 h and analyzed by ICP-MS with an Agilent Technologies 4500 ICP-MS (Agilent Technologies, Palo Alto, CA). The limits of detection for arsenite and cadmium were 0.04 and 0.02 μg/sample, equivalent to approximately 8 μg As and 4 μg Cd per gram cellular fresh weight in our 5 mg samples.
Western blotting analyses. Proteins were extracted from treated podocyte cultures, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or iso-electric focusing (IEF), transferred to PVDF membranes, and analyzed by quantitative Western blotting as previously described (Smoyer et al., 2000), with the exception that cells were extracted into sample buffers (200 μL/well) without reducing agents (SDS-PAGE buffer: 2% SDS, 62.5 mM Tris-Cl pH 6.8, 10% glycerol; IEF buffer: 8.5 M urea, 2% [wt/vol] NP-40, 67 mM -D-glycerophosphate, 50 mM sodium fluoride, 5 mM pyrophosphate, 1 mM ethylenediaminetetra-acetic acid, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 50 μg/ml each of leupeptin, pepstatin, and aprotinin), an aliquot of extracts was removed for protein assay (see below), reducing agents were added (2-mercaptoethanol to 2% [vol/vol] for SDS-PAGE, DTT to 5 mM for IEF), and samples prepared in SDS-PAGE sample buffer were incubated at 100°C for 5 min. Briefly, IEF separation of hsp25 phospho-isoforms was performed using urea buffer protein extracts containing similar amounts of hsp25 (as determined by prior analysis of SDS-PAGE sample buffer extracts by quantitative Western blotting) separated by slab-gel IEF using the Bio-Rad Model 111 Mini-IEF Cell essentially according to the manufacturer's instructions except that the support film was omitted to permit subsequent transfer of proteins to polyvinylidene difluoride (PVDF) membranes. Proteins were focused at 100 V for 15 min, 200 V for 15 min, and finally at 450 V for 1 to 2 h. After focusing, proteins were transferred to PVDF membranes by semi-dry transfer for 30 min at 75 mA.
Western blotting was performed as previously described (Smoyer et al., 2000). The primary antibodies used were 1:5000 rabbit anti-murine hsp25 polyclonal, rabbit anti-murine B-crystallin polyclonal, or mouse anti-hsp70i monoclonal (StressGen, Victoria, BC, Canada), and secondary antibody solutions consisted of 1:10,000 goat anti-rabbit or goat anti-murine IgG horseradish-peroxidase conjugates (Jackson ImmunoResearch, West Grove, PA). Antibody binding was visualized with the ECL chemiluminescence system (Amersham, Arlington Heights, IL) as detected using the Bio-Rad Chemidoc system (Bio-Rad, Hercules, CA). Densitometric analyses of captured images were performed using version 4.1.0.026 of the Bio-Rad Quantity One software. The absolute amounts of specific protein in each band were measured by linear regression analysis using six different amounts of standard proteins (hsp25, B-crystallin, or hsp70i [StressGen]) on each blot to generate standard curves of protein content versus densitometric units (mean density value times area).
Protein assays. The total protein concentration in protein extracts was measured prior to SDS-PAGE or IEF separation by the bicinchoninic acid (BCA) microassay according to the manufacturer's protocol (Pierce Biotechnology, Rockford, IL). Equal volumes of SDS-PAGE or IEF sample buffers without reducing agents were added to albumin protein standards to normalize for the effects of buffer components on the assay results.
Statistics. Statistical analyses of results were performed using Statview software (version 4.57) (Abacus Concepts, Berkeley, CA) to compare results using unpaired, two-tailed t-tests. Comparisons with P values <0.05 were considered significant, and P values <0.01 were noted separately.
RESULTS
Podocyte Viability after Toxic Metal Treatments
The viability of podocyte cultures was measured by the MTT assay, and the results shown in Figure 1A demonstrate that individual cadmium, mercury, and arsenite treatments all caused dose-dependent decreases in podocyte viability after 3 days of treatment. Treatment with the lowest concentration (10 μM) of any of the toxic metals had only modest effects on podocyte viability, whereas 20 μM treatments significantly decreased podocyte viability by 20–50%, and 40 μM of any single metal was completely lethal. However, arsenite treatments were the most injurious to podocytes, resulting in a significantly greater decrease in podocyte viability after 10 μM treatment than cells treated with cadmium, and a significantly greater decrease in viability after 20 μM treatment than cells treated with either cadmium or mercury.
The MTT assay results shown in Figure 1B demonstrate that two- and three-metal cadmium, mercury, and arsenite combination treatments induce disparate effects on podocyte viability. Combination metal treatments consisted of equimolar concentrations of each toxic metal summing to the indicated total toxic metal concentration (i.e., a 10 μM cadmium + mercury treatment contained 5 μM cadmium and 5 μM mercury). When compared to single-metal treatments of the same total metal concentration, equimolar combinations of mercury + cadmium or mercury + arsenite at both 10 μM and 20 μM were more toxic than treatment with the same concentration of a single metal constituent of the mixture. However, the combination of cadmium + arsenite, in contrast to combinations containing mercury, was significantly less toxic to podocytes than either cadmium or arsenite alone at both 20 μM and 40 μM, and, most notably, the toxicity of 20 μM cadmium + arsenite was less than 20 μM of arsenite alone. Moreover, none of the 40 μM combination-metal treatments were completely lethal to podocytes, in marked contrast to the uniform lethality of 40 μM single-metal treatments.
The differential effects of combinations containing mercury versus the cadmium + arsenite combination were especially apparent in comparisons of 20 μM combination versus single-metal treatments (Fig. 1A, 1B). Treatment with a combination of 10 μM each of cadmium + mercury resulted in a 54.7% decrease in podocyte viability compared to controls, but treatment with 20 μM of cadmium or mercury alone only decreased viability by 31.2% and 38.3%, respectively. Similarly, the viability of podocytes treated with 20 μM arsenite alone decreased by 59.9%, but it decreased by 74.4% in cells treated with 10 μM each of arsenite and mercury. In addition, the decrease in podocyte viability after treatment with 20 μM combinations containing mercury was also considerably greater than the sum of the decreases in viability caused by 10 μM of each constituent metal. The decreases in viability compared to controls induced by 10 μM cadmium (10.5%) or mercury (17.4%) summed to approximately half the decrease induced by a 20 μM combination of both metals (54.7%). Similarly, the decreases induced by 10 μM mercury or arsenite (26.3%) summed to approximately half the decrease induced by a 20 μM combination of both metals (74.4%). In marked contrast to the higher toxicity of mercury-containing combinations compared to single-metal treatments, podocyte viability only decreased by 18.1% compared to controls after 3 days of treatment with a 20 μM total concentration of a combination of cadmium + arsenite, a value significantly lower than that obtained with 20 μM treatments of either cadmium (31.2%) or arsenite (59.9%) alone, and only approximately half that of the sum of the decreases caused by 10 μM of each metal alone (36.8%). In addition, the decrease in podocyte viability from treatment with a combination of 20 μM cadmium and 20 μM arsenite (59.8%) was actually less than the decrease in viability induced by 20 μM of arsenite alone (59.9%).
The combination treatments containing equimolar amounts of all three metals resulted in changes in podocyte viability that were generally not significantly different from single-metal treatments at 10 μM and 20 μM (Fig. 1B), but again the 40 μM combination treatment was significantly less toxic than the 40 μM single-metal treatments that were completely lethal after 3 days of treatment. Together, these findings provide evidence for both synergistic and inhibitory effects of toxic metal combinations on podocyte toxicity.
Accumulation of Toxic Metals in Cultured Podocytes
The quantity of each toxic metal present in cells after 3 days of selected treatments was measured (Table 1) by inductively coupled plasma mass spectrometry (ICP-MS) analysis for cadmium and arsenic and by cold vapor atomic absorption (CVAA) analysis for mercury. Podocytes treated with cadmium or mercury salts accumulated detectable amounts of these metals, but the amount of arsenic present in our samples was below the limits of detection (<8 μg arsenic/g fresh weight).
The amount of cadmium detected in podocytes treated with 20 μM of the triple-metal combination containing 6.7 μM cadmium chloride (27 μg cadmium/g fresh weight) was roughly proportional to the amount that accumulated in podocytes treated with the 40 μM triple-metal combination containing 13.4 μM cadmium chloride (45 μg/g). However, cells treated with the triple-metal combination accumulated greater amounts of cadmium than the cells treated with cadmium alone. Specifically, cells treated with 20 μM cadmium chloride accumulated 37 μg cadmium/g cellular fresh weight, whereas cells treated with the 20 μM or 40 μM of the triple-metal combination, which contained only 6.7 or 13.4 μM cadmium, accumulated 27 μg and 45 μg cadmium/g cellular fresh weight, respectively, suggesting that cadmium cellular accumulation is enhanced in combination-metal treatments.
The amount of mercury that accumulated in podocytes treated with 20 μM mercury for 3 days was less that 25% of the amount of cadmium that accumulated in podocytes treated with 20 μM cadmium. In addition, although the accumulation was proportional between the concentrations of mercury present in the 20 μM mercury (9 μg Hg/g fresh weight) versus the 20 μM triple-metal combination containing 6.7 μM mercury ( 3 μg/g), the accumulation of mercury was less than 2 μg/g in cells treated with 13.4 μM of mercury in a 40 μM triple-metal combination, suggesting that mercury cellular accumulation is inhibited in combination-metal treatments.
For comparison, 1 g of a 20 μM aqueous solution of cadmium chloride or mercuric chloride would contain, respectively, 2.2 or 4.0 μg of metal. Our results demonstrate that cadmium accumulation in podocytes was much greater (15–37x) than would be expected from simple diffusion of the metal in the medium into cells, whereas the amount of mercury in cells varied between 0.6x and 2.3x the amount expected if the HgCl2 concentration equilibrated between the treatment medium and the cell.
Hsp70i Accumulation in Podocytes in Response to Toxic Metal Treatment
The quantity of the inducible form of hsp70 (hsp70i) in cultured podocytes was measured by quantitative Western blotting after 3 days of treatment (Fig. 2), and, as expected, there was no detectable accumulation of the stress-induced hsp70i in control podocytes treated with vehicle alone. However, there was also no detectable hsp70i accumulation in cultures treated with 10 μM of any single toxic metal or two-metal combination, nor did treatment with 20 μM of cadmium + mercury or arsenite + mercury induce hsp70i accumulation. All of the 20 μM single-metal treatments induced detectable, if modest, accumulation of hsp70i in podocytes, as did 20 μM cadmium + arsenite, 40 μM arsenite + mercury, and both the 10 μM and 20 μM triple-metal combination. However, the greatest accumulation of hsp70i occurred in cells treated with 40 μM of cadmium + mercury, cadmium + arsenite, and the triple-metal combination, with levels 2–4x greater than other treatments that induced detectable hsp70i accumulation. Podocytes treated with 40 μM of any single metal for 3 days did not survive (see Fig. 1), precluding analysis of changes in hsp70i protein expression.
The greatest accumulation of podocyte hsp70i occurred in cells treated with cadmium or mixtures containing cadmium. The accumulation of hsp70i in podocytes treated with 40 μM cadmium + mercury was almost 3x greater than would be expected if the accumulation of hsp70i were additive (i.e., comparing the sum of the quantity of hsp70i induced by separate 20 μM cadmium and 20 μM mercury treatments to the accumulation in cells treated with 20 μM of both metals together), and more than 4x greater than would be expected in cells treated with cadmium + arsenite. In contrast, the hsp70i accumulation in cells treated with 40 μM mercury + arsenite was almost exactly the sum of the accumulation in cells treated with 20 μM of each metal individually. Accumulation of hsp70i in podocytes treated with the triple-metal combination was also almost 4x greater than would be expected if the accumulation induced by 20 μM cadmium, mercury, or arsenite alone were additive, despite the fact that the 40 μM triple-metal combination treatment contained only 13.4 μM of each metal. These results demonstrate that the accumulation of hsp70i in podocytes is greatest after treatments containing cadmium, and the accumulation in combination treatments containing cadmium is not additive.
B-Crystallin and Hsp25 Accumulation in Podocytes in Response to Toxic Metal Treatment
The amounts of the closely related small heat shock proteins, hsp25 (Fig. 3) and B-crystallin (Fig. 4), in cultured podocytes were measured by quantitative Western blotting after 3 days of treatment. The changes in the amounts of these proteins in response to treatment with individual toxic metals were modest (maximum increase of 1.6x control level) and only significantly increased after treatment with 10 μM cadmium or arsenite. The 20 μM and 40 μM combination treatments containing cadmium + arsenite induced greater increases in the amount of the small heat shock proteins in podocytes (2.5x control hsp25 and B-crystallin amounts in cells treated with 40 μM combinations). Treatment with the triple-metal combination was the only other combination treatment that consistently induced accumulation of both small heat shock proteins, particularly the accumulation of hsp25. As observed for hsp70i, the accumulation of hsp25 or B-crystallin in cells treated with cadmium + arsenite was greater than the sum of the changes in the accumulation of these proteins induced by equimolar treatment with the individual metals alone. In contrast to our hsp70i results, higher concentrations of toxic metals did not generally induce greater accumulation of small heat shock proteins. These findings provide evidence suggesting that, while the small stress proteins are comparatively less induced than hsp70i in podocytes by toxic metal exposure, the induction of all three of these major cytosolic stress proteins appears to be enhanced in the presence of toxic metal combinations compared to equimolar concentrations of individual toxic metals.
Hsp25 Phosphorylation in Podocytes in Response to Toxic Metal Treatment
The relative amount of hsp25 phosphorylation (expressed as the percent mono- and di-phosphorylated hsp25 of the total phosphorylated + unphosphorylated hsp25) was measured by quantitative Western blotting of IEF slab gel separations of hsp25 phospho-isoforms (Table 2). We found that almost all toxic metal treatments tested induced rapid, dramatic, and persistent changes in hsp25 phosphorylation. All single and combination metal treatments induced a rapid (within 45 min) increase in hsp25 phosphorylation, with the percentage of hsp25 containing at least one phosphorylated serine residue, increasing from less than 50% to nearly or as much as 100%. Importantly, in all cases the increased hsp25 phosphorylation compared to controls persisted for the entire course of the treatments. These findings demonstrate that hsp25 phosphorylation is strongly, similarly, and persistently induced in sublethally injured podocytes exposed to cadmium, mercury, and arsenic both individually and in various combinations.
DISCUSSION
Because glomerular injury induced by environmental toxic metals has not been well characterized, the present study was designed to analyze in detail the stress response and intracellular toxic metal accumulation of cultured podocytes after exposure to individual and combinations of the common environmental toxic metals, cadmium, mercury, and arsenite. Our results demonstrate that prolonged exposure to each of these individual toxic metals induced similar dose-dependent podocyte cytotoxicity, despite markedly different intracellular accumulations of the metals. However, exposure to combinations of these metals resulted in significantly disparate cytotoxicity compared to equimolar concentrations of individual toxic metals. Combinations associated with decreased toxicity correlated broadly with greater increases the cytosolic stress proteins, hsp25, B-crystallin, and hsp70i compared to equimolar treatment with individual toxic metals. Despite these differences, all individual and combination toxic metal treatments resulted in a similar strong and sustained induction of hsp25 phosphorylation. These findings provide clear evidence of dose-related toxicity to podocytes after prolonged individual exposure to common environmental toxic metals (despite marked differences in their cellular uptake) and dramatic differential effects of combinations of these metals on both podocyte viability and heat shock protein accumulation.
The most notable evidence of a differential effect on cultured podocyte toxicity between single-metal and combination-metal treatment, that 40 μM cadmium + arsenite (20 μM cadmium + 20 μM arsenite) treatment resulted in toxicity that was not significantly greater than treatment with 20 μM of arsenite alone (Fig. 1), was in striking contrast to findings in mice treated with cadmium and arsenite together, a treatment that resulted in significantly greater toxicity to murine renal tissues (including glomerular swelling, an indication of glomerular injury) than either metal alone (Liu et al., 2000). However, the toxicity of cadmium was decreased in mice treated sequentially with arsenite and then cadmium, although cadmium pre-treatment had no effect on subsequent arsenite toxicity (Hochadel and Waalkes, 1997).
The accumulation of both cadmium and mercury by podocytes was affected by the presence of other toxic metals in the treatment solution (Table 1). Inhibition of mercury uptake in the renal cortex has previously been observed in rats exposed to both metals (Zalups and Barfuss, 2002), though another study found decreased renal toxicity but not decreased mercury uptake in rat pups exposed to both cadmium and mercury (Peixoto et al., 2003), and an older study described cadmium-induced enhanced uptake of mercury, increases in its incorporation into renal metallothionein, and protection from mercury nephrotoxicity (Webb and Magos, 1976). In contrast to our uptake results showing an increase in cadmium uptake in combination-metal treatments, the majority of cadmium uptake in cultured renal cortical cells was previously found to be by diffusion (Shaikh et al., 1995), and two studies using cultured cortical cells found mercury could modestly (10%) antagonize cadmium uptake (Endo and Shaikh, 1993; Shaikh et al., 1995). This pattern of mercury antagonism of cadmium uptake has also been observed in rat hepatocytes (Blazka and Shaikh, 1992; Gerson and Shaikh, 1984) and in human intestinal cells, where mercury uptake was found to occur by nonspecific passive diffusion whereas cadmium uptake likely involved a thiol-mediated reaction (Aduayom et al., 2003). These findings (summarized in Table 3) and our results suggest that both the mechanism of cadmium uptake and the effect of other toxic metals on cadmium uptake by podocytes differ significantly from other described cultured cells. However, the differences observed between podocytes and cultured cortical cells may be a result of the variety of treatment conditions (serum-free or serum-containing culture medium, metal concentrations, single or repeated additions) used in different studies. In addition, our study was performed using conditionally immortalized, differentiated podocytes that do not proliferate appreciably in culture.
We observed only relatively modest changes in the amounts of the heat shock proteins hsp25, B-crystallin, or hsp70i in response to 3-day treatments with the individual toxic metals cadmium, mercury, or arsenite. Although induction of heat shock protein expression immediately (6–12 h) after treatment with these metals has been well described (reviewed in Del Razo et al., 2001; Pinot et al., 2000; Sarafian et al., 1996), few studies have examined heat shock protein expression after prolonged (>24 h) exposure or exposure to combinations of metals. Previous studies also found no significant changes in the expression of hsp27 and hsp70i in cultured kidney epithelial cells in response to long-term (12 h–6 days) exposure to cadmium (Somji et al., 1999a, 1999b), although other studies have discovered accumulation of hsp70i in cultured lung cells in response to long-term cadmium exposure (Croute et al., 2000), of hsp70i but not hsp27 in cultured bladder urothelium cells in response to long-term arsenite exposure (Rossi et al., 2002), and of hsp25/27 and hsp70i but not B-crystallin in two separate renal epithelial cell lines in response to long-term cadmium exposure (Bonham et al., 2003). It is difficult to draw any firm conclusions from the relative paucity of data concerning heat shock protein induction caused by long-term exposure to toxic metals, especially in light of the differences in treatment protocols. For example, culture medium containing cadmium was added to cultured cells either every day (Bonham et al., 2003) or every 3 days (Somji et al., 1999a, 1999b) in the studies of the effects of long-term exposure of cultured renal epithelial cells to cadmium, making it difficult to directly compare the results of these studies to our work using a single addition of toxic metal.
The results of our measurements of the accumulation of heat shock proteins in response to combinations of toxic metals showed a correlation between their accumulation and a decrease in toxic injury to podocytes, especially combinations of toxic metals containing cadmium and arsenite. These results are supported by a previous study that found that cadmium and arsenite induced an increase in hsp70i in human and rat kidney cell lines that occurred at lower concentrations than the increase caused by either metal alone (Madden et al., 2002), and a correlation between a decrease in the heat shock response and the onset of cell injury and death. These results together suggest that the decreased toxicity of toxic metal mixtures containing cadmium and arsenite are a result of the increased expression of one or more of the heat shock proteins in podocytes. Heat shock proteins have previously been shown to confer protection against injury caused by metals in a variety of systems. For example, a direct correlation was found between the expression of hsp25 in embryonic stem cells transformed with hsp25 sense and antisense expression vectors and their resistance to injury by cadmium, mercury, or arsenite (Wu and Welsh, 1996). The expression of hsp27 and B-crystallin together conferred greater protection than the expression of either of these proteins alone (Fu and Liang, 2003), a finding that may be explained by co-localization, because fluorescence resonance energy transfer (FRET), and two-hybrid studies have shown that hsp25/27 interacts directly with B-crystallin (Fu and Liang, 2003; Klemenz et al., 1993; Liu and Welsh, 1999), an association that is thought to stabilize B-crystallin (Bova et al., 2000).
We observed a rapid (within 45 min) and sustained (3 days) increase in the phosphorylation of hsp25 in response to all single and combination toxic metal treatments. The induction of hsp27 phosphorylation in cultured cells by arsenite, cadmium, and lead treatment has been well documented (Landry et al., 1992; Leal et al., 2002), but to our knowledge our work shows the first published description of the induction of hsp25 phosphorylation by mercury. Interestingly, mercury induced almost complete conversion of unphosphorylated to mono- or di-phosphorylated hsp25, an effect greater than that induced by an equal concentration of cadmium and roughly equivalent to the hsp25 phosphorylation induced by arsenite. That effect also persisted longer than treatments with the other toxic metals (Table 2). The hsp25 phosphorylation induced by combinations of toxic metals was approximately intermediate between the values induced by the constituents of the mixtures, suggesting that toxic metals induce hsp25 phosphorylation via similar mechanisms. There did not appear to be any correlation between treatments that induced more hsp25 phosphorylation and podocyte survival or heat shock protein accumulation.
In conclusion, we have demonstrated that treatments with combinations of toxic metals caused disparate effects on podocyte viability, heat shock protein accumulation, and accumulation of toxic metals in cells, but not on hsp25 phosphorylation. We also described the novel finding that mercuric chloride is a strong inducer of hsp25 phosphorylation, and we found that the combination treatments that induced the smallest decreases in podocyte viability induced the greatest increases in the accumulation of the heat shock proteins hsp70i, hsp25, and B-crystallin. Our results suggest the stress response, characterized by an increase in the expression of heat shock proteins, may be vital in the podocyte's ability to protect against and recover from toxic metal injury, especially from exposure to combinations of the toxic metals cadmium and arsenite.
ACKNOWLEDGMENTS
Portions of this work were supported by a Program Project grant (P01 ES11188–01) from the National Institute of Environmental Health Sciences (W.E.S.: Subproject Principal Investigator).
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ABSTRACT
Cadmium, mercury, and arsenite are among the most abundant toxic metals (TM) in our environment, and chronic TM exposure leads to injury to the kidney's glomerular filtration barrier. The small heat shock protein hsp25, highly expressed in glomerular podocytes, is induced during development of experimental nephrotic syndrome, and hsp25 overexpression can protect cultured podocytes from injury. Because little is known about the effect of multiple TM on podocytes, we measured the response of cultured podocytes to prolonged exposures to single and multiple TM. Podocyte viability declined by approximately 50% after 3 days of treatment with 20 μM cadmium, mercury, or arsenite, and 40 μM of any of these metals was lethal. The toxicity of equimolar concentrations of two or all three metals in combination was significantly altered compared to individual metal treatments. Single TM treatments induced only modest increases in the amounts of hsp25, B-crystallin, and inducible hsp70. Toxic metal combinations induced greater stress protein accumulation, especially arsenite + cadmium or arsenite + cadmium + mercury treatments, the TM mixtures with the lowest toxicity. All TM treatments caused a rapid and sustained increase in hsp25 phosphorylation. The intracellular accumulation of cadmium was greater and that of mercury was less in cells treated with TM combinations than in cells treated with a single TM. Our results showed that multiple TM effects on podocyte viability were neither additive nor synergistic and that induction of heat shock proteins correlated with increased resistance to TM injury, suggesting that induction of small heat shock proteins may play an important role in preventing TM-induced podocyte injury.
Key Words: cadmium; arsenite; mercury; Hsp25; Hsp70; B-crystallin.
INTRODUCTION
Environmental or occupational exposure to toxic metals is well known to induce chronic renal disease, and cadmium, mercury, and arsenite (a metalloid) are among the most environmentally abundant nephrotoxic metals (Fowler, 1993). Although the renal toxicological effects are dependent on the dosage, duration, and type of metal exposure (Madden and Fowler, 2000), the kidney's proximal tubules are known to be especially sensitive to injury, primarily because of their characteristically high reabsorptive activity (Robertson 2000). Importantly, toxic metal–induced glomerular injury, although far less well characterized than renal tubular injury, has also been reported (Enestrom and Hultman, 1984; Hoedemaeker et al., 1988).
Despite the extensive range of debilitating diseases caused by toxic metals, relatively little is known about the specific cellular and molecular mechanisms involved in metal toxicity. A variety of molecular factors likely play major roles in regulating cellular toxicity, including metal-binding proteins, inclusion bodies, and cell-specific receptor-like proteins. In addition, although most studies to date have examined exposure only to individual toxic metals, it is likely that chronic environmental toxic metal exposure typically includes sequential or simultaneous exposure to multiple toxic metals. Such environmental exposures (as opposed to occupational exposures typically involving acute or chronic exposure to a single toxic metal) highlight the potential importance of altered toxicity from interactions between the common environmental toxic metals or their cellular mechanisms of action. These poorly characterized interactions may result in additive, synergistic, or antagonistic effects on the overall toxicity compared to the effects of individual metals.
One important reaction to toxic metal exposure is induction of a cellular stress response, including increased expression of the major cytosolic stress protein hsp70i (Ait-Aissa et al., 2000; Bonham et al., 2003; Hernadez-Pando et al., 1995) and increased expression and phosphorylation of the small heat shock protein hsp25 (hsp25 is the murine or rat isoform; hsp27, the human isoform: the combined hsp25/27 will be used subsequently when referring in general to this protein), a known regulator of actin polymerization (Bonham et al., 2003; Radloff et al., 1998; Somji et al., 1999a; Wu and Welsh, 1996). Although this stress response to toxic injury has been best characterized in renal tubular cells (Bonham et al., 2003; Liu et al., 1996; Somji et al., 1999a), we have previously reported analogous induction of hsp25 expression and phosphorylation in renal podocytes in the kidney's glomerular filtration barrier in response to another toxin, puromycin aminonucleoside (Smoyer et al., 1996). Subsequent studies further demonstrated that hsp25 had the ability to regulate the podocyte response to injury (Smoyer and Ransom, 2002). In addition, few studies have addressed the long-term effects of toxic metals on cytotoxicity and stress protein accumulation (Bonham et al., 2003; Croute et al., 2000; Rossi et al., 2002; Somji et al., 1999a, 1999b). From these findings, as well as the poorly characterized glomerular injury induced by toxic metals, we hypothesized (1) that toxic metal exposure would alter small heat shock protein expression and phosphorylation in glomerular podocytes, (2) that exposure to combinations of toxic metals would result in altered toxicity as compared to individual metal exposure alone, and (3) that this altered toxicity may be a result of increased heat shock protein expression in cells treated with multiple toxic metals. To begin to test these hypotheses, we designed a series of studies to analyze in detail the stress response and intracellular toxic metal accumulation of cultured podocytes after prolonged exposure to individual and various combinations of the common environmental toxic metals, cadmium, mercury, and arsenite.
MATERIALS AND METHODS
Cell culture and toxic metal treatments. A conditionally immortalized mouse podocyte clonal cell line (MPC-5) isolated from the "Immortomouse" was used in these studies, and was the kind gift of Dr. Peter Mundel. Bulk culture of podocytes was carried out in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco-BRL, Rockville, MD), and 10 U/ml mouse -interferon (Sigma, St. Louis, MO) at 33°C with 100% relative humidity and 5% CO2 atmosphere. Proliferating podocytes were detached from the substrate with trypsin, seeded into culture dishes at 1000 cells/well in 96-well plates for MTT assays or at 30,000 cells/well in 6-well plates for all other experiments, and then induced to differentiate by transfer to 37°C and culture in the above medium without -interferon for 10–14 days. These culture conditions repress the promoter-driven expression and inactivate the thermosensitive protein product of a transgene encoding a variant of the SV40 T-antigen that results in conditional immortalization of this cell line.
Differentiated podocytes were treated with toxic metals by replacement of the culture medium with medium containing vehicle alone or single or multiple toxic metal salts. Medium containing toxic metals was prepared by addition of 1000x solutions of metal salts (HgCl2, CdCl2, or NaAsO2, all 95% A.C.S. grade, Sigma-Aldrich, St. Louis, MO) to medium immediately prior to use. Treatments consisted of individual metals at 0, 10, 20, or 40 μM concentrations, and dual and triple metal treatments at the same total metal concentrations but consisting of equimolar concentrations of each constituent metal summing to the total metal concentrations (e.g., 20 μM dual metal treatments consisted of 10 μM of each metal, and 20 μM triple metal treatments consisted of 6.7 μM of each metal). Podocytes were cultured in this medium for various times up to 3 days and then analyzed as described below.
Cell survival assays. Podocyte viability was measured using MTT reduction as previously described (Mosmann, 1983) after 3 days of toxic metal treatment. Wells used for the negative controls were not seeded with cells. After treatment medium containing metal salts was removed and replaced with 200 μl/well of fresh medium, and then 50 μl/well of a 50 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) solution was added and the cells cultured for 4 h. The MTT-media solution was then removed, and 200 μl/well of DMSO (dimethyl sulfoxide; Sigma, St. Louis, MO) was added, followed by the addition of 25 μl/well glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). Finally, the absorbance of each well was immediately read at 570 nm in a spectrophotometric plate reader (SpectraMAX 250, Molecular Devices, Sunnyvale, CA).
Metal uptake assays. After 3 days of toxic metal treatments, podocytes were washed with PBS, detached from the substrate with trypsin, and collected by centrifugation. The cell pellets were weighed to determine fresh weight, and analysis of metal accumulation in podocytes was performed using Environmental Protection Agency (EPA) methods detailed in the EPA document SW-846, "Test Methods for Evaluating Solid Waste" by the University of Michigan Occupational Safety and Environmental Health Environmental Labs. All data are reported in terms of grams cell material analyzed (μg toxic metal/g cellular fresh weight).
Mercury accumulation was measured according to SW-846, Method 7471A, "Mercury in Solid or Semisolid Waste (Manual Cold-Vapor Technique)." Briefly, samples were digested with sulfuric acid, nitric acid, potassium permanganate, and potassium persulfate at 95°C for 2 h. Excess potassium permanganate was removed by the addition of sodium chloride-hydroxylamine sulfate. Immediately prior to analysis, stannous chloride was added, and the sample was analyzed by atomic absorption (AA), with a Leeman Labs Hydra AA Automated Mercury Analyzer (Leeman Labs, Hudson, NH). The limits of detection for mercury were 0.6 ng/sample, equivalent to approximately 0.12 μg Hg per gram cellular fresh weight in our approximately 5 mg samples.
Cadmium and arsenic accumulation was measured according to SW-846 Method 3050B, "Acid Digestion of Sediments, Sludges, and Soils," followed by SW-846 Method 6020, "Inductively Coupled Plasma-Mass Spectrometry" (ICP-MS). Briefly, samples were digested with nitric acid followed by hydrogen peroxide, then heated at 95°C for 2 h and analyzed by ICP-MS with an Agilent Technologies 4500 ICP-MS (Agilent Technologies, Palo Alto, CA). The limits of detection for arsenite and cadmium were 0.04 and 0.02 μg/sample, equivalent to approximately 8 μg As and 4 μg Cd per gram cellular fresh weight in our 5 mg samples.
Western blotting analyses. Proteins were extracted from treated podocyte cultures, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or iso-electric focusing (IEF), transferred to PVDF membranes, and analyzed by quantitative Western blotting as previously described (Smoyer et al., 2000), with the exception that cells were extracted into sample buffers (200 μL/well) without reducing agents (SDS-PAGE buffer: 2% SDS, 62.5 mM Tris-Cl pH 6.8, 10% glycerol; IEF buffer: 8.5 M urea, 2% [wt/vol] NP-40, 67 mM -D-glycerophosphate, 50 mM sodium fluoride, 5 mM pyrophosphate, 1 mM ethylenediaminetetra-acetic acid, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 50 μg/ml each of leupeptin, pepstatin, and aprotinin), an aliquot of extracts was removed for protein assay (see below), reducing agents were added (2-mercaptoethanol to 2% [vol/vol] for SDS-PAGE, DTT to 5 mM for IEF), and samples prepared in SDS-PAGE sample buffer were incubated at 100°C for 5 min. Briefly, IEF separation of hsp25 phospho-isoforms was performed using urea buffer protein extracts containing similar amounts of hsp25 (as determined by prior analysis of SDS-PAGE sample buffer extracts by quantitative Western blotting) separated by slab-gel IEF using the Bio-Rad Model 111 Mini-IEF Cell essentially according to the manufacturer's instructions except that the support film was omitted to permit subsequent transfer of proteins to polyvinylidene difluoride (PVDF) membranes. Proteins were focused at 100 V for 15 min, 200 V for 15 min, and finally at 450 V for 1 to 2 h. After focusing, proteins were transferred to PVDF membranes by semi-dry transfer for 30 min at 75 mA.
Western blotting was performed as previously described (Smoyer et al., 2000). The primary antibodies used were 1:5000 rabbit anti-murine hsp25 polyclonal, rabbit anti-murine B-crystallin polyclonal, or mouse anti-hsp70i monoclonal (StressGen, Victoria, BC, Canada), and secondary antibody solutions consisted of 1:10,000 goat anti-rabbit or goat anti-murine IgG horseradish-peroxidase conjugates (Jackson ImmunoResearch, West Grove, PA). Antibody binding was visualized with the ECL chemiluminescence system (Amersham, Arlington Heights, IL) as detected using the Bio-Rad Chemidoc system (Bio-Rad, Hercules, CA). Densitometric analyses of captured images were performed using version 4.1.0.026 of the Bio-Rad Quantity One software. The absolute amounts of specific protein in each band were measured by linear regression analysis using six different amounts of standard proteins (hsp25, B-crystallin, or hsp70i [StressGen]) on each blot to generate standard curves of protein content versus densitometric units (mean density value times area).
Protein assays. The total protein concentration in protein extracts was measured prior to SDS-PAGE or IEF separation by the bicinchoninic acid (BCA) microassay according to the manufacturer's protocol (Pierce Biotechnology, Rockford, IL). Equal volumes of SDS-PAGE or IEF sample buffers without reducing agents were added to albumin protein standards to normalize for the effects of buffer components on the assay results.
Statistics. Statistical analyses of results were performed using Statview software (version 4.57) (Abacus Concepts, Berkeley, CA) to compare results using unpaired, two-tailed t-tests. Comparisons with P values <0.05 were considered significant, and P values <0.01 were noted separately.
RESULTS
Podocyte Viability after Toxic Metal Treatments
The viability of podocyte cultures was measured by the MTT assay, and the results shown in Figure 1A demonstrate that individual cadmium, mercury, and arsenite treatments all caused dose-dependent decreases in podocyte viability after 3 days of treatment. Treatment with the lowest concentration (10 μM) of any of the toxic metals had only modest effects on podocyte viability, whereas 20 μM treatments significantly decreased podocyte viability by 20–50%, and 40 μM of any single metal was completely lethal. However, arsenite treatments were the most injurious to podocytes, resulting in a significantly greater decrease in podocyte viability after 10 μM treatment than cells treated with cadmium, and a significantly greater decrease in viability after 20 μM treatment than cells treated with either cadmium or mercury.
The MTT assay results shown in Figure 1B demonstrate that two- and three-metal cadmium, mercury, and arsenite combination treatments induce disparate effects on podocyte viability. Combination metal treatments consisted of equimolar concentrations of each toxic metal summing to the indicated total toxic metal concentration (i.e., a 10 μM cadmium + mercury treatment contained 5 μM cadmium and 5 μM mercury). When compared to single-metal treatments of the same total metal concentration, equimolar combinations of mercury + cadmium or mercury + arsenite at both 10 μM and 20 μM were more toxic than treatment with the same concentration of a single metal constituent of the mixture. However, the combination of cadmium + arsenite, in contrast to combinations containing mercury, was significantly less toxic to podocytes than either cadmium or arsenite alone at both 20 μM and 40 μM, and, most notably, the toxicity of 20 μM cadmium + arsenite was less than 20 μM of arsenite alone. Moreover, none of the 40 μM combination-metal treatments were completely lethal to podocytes, in marked contrast to the uniform lethality of 40 μM single-metal treatments.
The differential effects of combinations containing mercury versus the cadmium + arsenite combination were especially apparent in comparisons of 20 μM combination versus single-metal treatments (Fig. 1A, 1B). Treatment with a combination of 10 μM each of cadmium + mercury resulted in a 54.7% decrease in podocyte viability compared to controls, but treatment with 20 μM of cadmium or mercury alone only decreased viability by 31.2% and 38.3%, respectively. Similarly, the viability of podocytes treated with 20 μM arsenite alone decreased by 59.9%, but it decreased by 74.4% in cells treated with 10 μM each of arsenite and mercury. In addition, the decrease in podocyte viability after treatment with 20 μM combinations containing mercury was also considerably greater than the sum of the decreases in viability caused by 10 μM of each constituent metal. The decreases in viability compared to controls induced by 10 μM cadmium (10.5%) or mercury (17.4%) summed to approximately half the decrease induced by a 20 μM combination of both metals (54.7%). Similarly, the decreases induced by 10 μM mercury or arsenite (26.3%) summed to approximately half the decrease induced by a 20 μM combination of both metals (74.4%). In marked contrast to the higher toxicity of mercury-containing combinations compared to single-metal treatments, podocyte viability only decreased by 18.1% compared to controls after 3 days of treatment with a 20 μM total concentration of a combination of cadmium + arsenite, a value significantly lower than that obtained with 20 μM treatments of either cadmium (31.2%) or arsenite (59.9%) alone, and only approximately half that of the sum of the decreases caused by 10 μM of each metal alone (36.8%). In addition, the decrease in podocyte viability from treatment with a combination of 20 μM cadmium and 20 μM arsenite (59.8%) was actually less than the decrease in viability induced by 20 μM of arsenite alone (59.9%).
The combination treatments containing equimolar amounts of all three metals resulted in changes in podocyte viability that were generally not significantly different from single-metal treatments at 10 μM and 20 μM (Fig. 1B), but again the 40 μM combination treatment was significantly less toxic than the 40 μM single-metal treatments that were completely lethal after 3 days of treatment. Together, these findings provide evidence for both synergistic and inhibitory effects of toxic metal combinations on podocyte toxicity.
Accumulation of Toxic Metals in Cultured Podocytes
The quantity of each toxic metal present in cells after 3 days of selected treatments was measured (Table 1) by inductively coupled plasma mass spectrometry (ICP-MS) analysis for cadmium and arsenic and by cold vapor atomic absorption (CVAA) analysis for mercury. Podocytes treated with cadmium or mercury salts accumulated detectable amounts of these metals, but the amount of arsenic present in our samples was below the limits of detection (<8 μg arsenic/g fresh weight).
The amount of cadmium detected in podocytes treated with 20 μM of the triple-metal combination containing 6.7 μM cadmium chloride (27 μg cadmium/g fresh weight) was roughly proportional to the amount that accumulated in podocytes treated with the 40 μM triple-metal combination containing 13.4 μM cadmium chloride (45 μg/g). However, cells treated with the triple-metal combination accumulated greater amounts of cadmium than the cells treated with cadmium alone. Specifically, cells treated with 20 μM cadmium chloride accumulated 37 μg cadmium/g cellular fresh weight, whereas cells treated with the 20 μM or 40 μM of the triple-metal combination, which contained only 6.7 or 13.4 μM cadmium, accumulated 27 μg and 45 μg cadmium/g cellular fresh weight, respectively, suggesting that cadmium cellular accumulation is enhanced in combination-metal treatments.
The amount of mercury that accumulated in podocytes treated with 20 μM mercury for 3 days was less that 25% of the amount of cadmium that accumulated in podocytes treated with 20 μM cadmium. In addition, although the accumulation was proportional between the concentrations of mercury present in the 20 μM mercury (9 μg Hg/g fresh weight) versus the 20 μM triple-metal combination containing 6.7 μM mercury ( 3 μg/g), the accumulation of mercury was less than 2 μg/g in cells treated with 13.4 μM of mercury in a 40 μM triple-metal combination, suggesting that mercury cellular accumulation is inhibited in combination-metal treatments.
For comparison, 1 g of a 20 μM aqueous solution of cadmium chloride or mercuric chloride would contain, respectively, 2.2 or 4.0 μg of metal. Our results demonstrate that cadmium accumulation in podocytes was much greater (15–37x) than would be expected from simple diffusion of the metal in the medium into cells, whereas the amount of mercury in cells varied between 0.6x and 2.3x the amount expected if the HgCl2 concentration equilibrated between the treatment medium and the cell.
Hsp70i Accumulation in Podocytes in Response to Toxic Metal Treatment
The quantity of the inducible form of hsp70 (hsp70i) in cultured podocytes was measured by quantitative Western blotting after 3 days of treatment (Fig. 2), and, as expected, there was no detectable accumulation of the stress-induced hsp70i in control podocytes treated with vehicle alone. However, there was also no detectable hsp70i accumulation in cultures treated with 10 μM of any single toxic metal or two-metal combination, nor did treatment with 20 μM of cadmium + mercury or arsenite + mercury induce hsp70i accumulation. All of the 20 μM single-metal treatments induced detectable, if modest, accumulation of hsp70i in podocytes, as did 20 μM cadmium + arsenite, 40 μM arsenite + mercury, and both the 10 μM and 20 μM triple-metal combination. However, the greatest accumulation of hsp70i occurred in cells treated with 40 μM of cadmium + mercury, cadmium + arsenite, and the triple-metal combination, with levels 2–4x greater than other treatments that induced detectable hsp70i accumulation. Podocytes treated with 40 μM of any single metal for 3 days did not survive (see Fig. 1), precluding analysis of changes in hsp70i protein expression.
The greatest accumulation of podocyte hsp70i occurred in cells treated with cadmium or mixtures containing cadmium. The accumulation of hsp70i in podocytes treated with 40 μM cadmium + mercury was almost 3x greater than would be expected if the accumulation of hsp70i were additive (i.e., comparing the sum of the quantity of hsp70i induced by separate 20 μM cadmium and 20 μM mercury treatments to the accumulation in cells treated with 20 μM of both metals together), and more than 4x greater than would be expected in cells treated with cadmium + arsenite. In contrast, the hsp70i accumulation in cells treated with 40 μM mercury + arsenite was almost exactly the sum of the accumulation in cells treated with 20 μM of each metal individually. Accumulation of hsp70i in podocytes treated with the triple-metal combination was also almost 4x greater than would be expected if the accumulation induced by 20 μM cadmium, mercury, or arsenite alone were additive, despite the fact that the 40 μM triple-metal combination treatment contained only 13.4 μM of each metal. These results demonstrate that the accumulation of hsp70i in podocytes is greatest after treatments containing cadmium, and the accumulation in combination treatments containing cadmium is not additive.
B-Crystallin and Hsp25 Accumulation in Podocytes in Response to Toxic Metal Treatment
The amounts of the closely related small heat shock proteins, hsp25 (Fig. 3) and B-crystallin (Fig. 4), in cultured podocytes were measured by quantitative Western blotting after 3 days of treatment. The changes in the amounts of these proteins in response to treatment with individual toxic metals were modest (maximum increase of 1.6x control level) and only significantly increased after treatment with 10 μM cadmium or arsenite. The 20 μM and 40 μM combination treatments containing cadmium + arsenite induced greater increases in the amount of the small heat shock proteins in podocytes (2.5x control hsp25 and B-crystallin amounts in cells treated with 40 μM combinations). Treatment with the triple-metal combination was the only other combination treatment that consistently induced accumulation of both small heat shock proteins, particularly the accumulation of hsp25. As observed for hsp70i, the accumulation of hsp25 or B-crystallin in cells treated with cadmium + arsenite was greater than the sum of the changes in the accumulation of these proteins induced by equimolar treatment with the individual metals alone. In contrast to our hsp70i results, higher concentrations of toxic metals did not generally induce greater accumulation of small heat shock proteins. These findings provide evidence suggesting that, while the small stress proteins are comparatively less induced than hsp70i in podocytes by toxic metal exposure, the induction of all three of these major cytosolic stress proteins appears to be enhanced in the presence of toxic metal combinations compared to equimolar concentrations of individual toxic metals.
Hsp25 Phosphorylation in Podocytes in Response to Toxic Metal Treatment
The relative amount of hsp25 phosphorylation (expressed as the percent mono- and di-phosphorylated hsp25 of the total phosphorylated + unphosphorylated hsp25) was measured by quantitative Western blotting of IEF slab gel separations of hsp25 phospho-isoforms (Table 2). We found that almost all toxic metal treatments tested induced rapid, dramatic, and persistent changes in hsp25 phosphorylation. All single and combination metal treatments induced a rapid (within 45 min) increase in hsp25 phosphorylation, with the percentage of hsp25 containing at least one phosphorylated serine residue, increasing from less than 50% to nearly or as much as 100%. Importantly, in all cases the increased hsp25 phosphorylation compared to controls persisted for the entire course of the treatments. These findings demonstrate that hsp25 phosphorylation is strongly, similarly, and persistently induced in sublethally injured podocytes exposed to cadmium, mercury, and arsenic both individually and in various combinations.
DISCUSSION
Because glomerular injury induced by environmental toxic metals has not been well characterized, the present study was designed to analyze in detail the stress response and intracellular toxic metal accumulation of cultured podocytes after exposure to individual and combinations of the common environmental toxic metals, cadmium, mercury, and arsenite. Our results demonstrate that prolonged exposure to each of these individual toxic metals induced similar dose-dependent podocyte cytotoxicity, despite markedly different intracellular accumulations of the metals. However, exposure to combinations of these metals resulted in significantly disparate cytotoxicity compared to equimolar concentrations of individual toxic metals. Combinations associated with decreased toxicity correlated broadly with greater increases the cytosolic stress proteins, hsp25, B-crystallin, and hsp70i compared to equimolar treatment with individual toxic metals. Despite these differences, all individual and combination toxic metal treatments resulted in a similar strong and sustained induction of hsp25 phosphorylation. These findings provide clear evidence of dose-related toxicity to podocytes after prolonged individual exposure to common environmental toxic metals (despite marked differences in their cellular uptake) and dramatic differential effects of combinations of these metals on both podocyte viability and heat shock protein accumulation.
The most notable evidence of a differential effect on cultured podocyte toxicity between single-metal and combination-metal treatment, that 40 μM cadmium + arsenite (20 μM cadmium + 20 μM arsenite) treatment resulted in toxicity that was not significantly greater than treatment with 20 μM of arsenite alone (Fig. 1), was in striking contrast to findings in mice treated with cadmium and arsenite together, a treatment that resulted in significantly greater toxicity to murine renal tissues (including glomerular swelling, an indication of glomerular injury) than either metal alone (Liu et al., 2000). However, the toxicity of cadmium was decreased in mice treated sequentially with arsenite and then cadmium, although cadmium pre-treatment had no effect on subsequent arsenite toxicity (Hochadel and Waalkes, 1997).
The accumulation of both cadmium and mercury by podocytes was affected by the presence of other toxic metals in the treatment solution (Table 1). Inhibition of mercury uptake in the renal cortex has previously been observed in rats exposed to both metals (Zalups and Barfuss, 2002), though another study found decreased renal toxicity but not decreased mercury uptake in rat pups exposed to both cadmium and mercury (Peixoto et al., 2003), and an older study described cadmium-induced enhanced uptake of mercury, increases in its incorporation into renal metallothionein, and protection from mercury nephrotoxicity (Webb and Magos, 1976). In contrast to our uptake results showing an increase in cadmium uptake in combination-metal treatments, the majority of cadmium uptake in cultured renal cortical cells was previously found to be by diffusion (Shaikh et al., 1995), and two studies using cultured cortical cells found mercury could modestly (10%) antagonize cadmium uptake (Endo and Shaikh, 1993; Shaikh et al., 1995). This pattern of mercury antagonism of cadmium uptake has also been observed in rat hepatocytes (Blazka and Shaikh, 1992; Gerson and Shaikh, 1984) and in human intestinal cells, where mercury uptake was found to occur by nonspecific passive diffusion whereas cadmium uptake likely involved a thiol-mediated reaction (Aduayom et al., 2003). These findings (summarized in Table 3) and our results suggest that both the mechanism of cadmium uptake and the effect of other toxic metals on cadmium uptake by podocytes differ significantly from other described cultured cells. However, the differences observed between podocytes and cultured cortical cells may be a result of the variety of treatment conditions (serum-free or serum-containing culture medium, metal concentrations, single or repeated additions) used in different studies. In addition, our study was performed using conditionally immortalized, differentiated podocytes that do not proliferate appreciably in culture.
We observed only relatively modest changes in the amounts of the heat shock proteins hsp25, B-crystallin, or hsp70i in response to 3-day treatments with the individual toxic metals cadmium, mercury, or arsenite. Although induction of heat shock protein expression immediately (6–12 h) after treatment with these metals has been well described (reviewed in Del Razo et al., 2001; Pinot et al., 2000; Sarafian et al., 1996), few studies have examined heat shock protein expression after prolonged (>24 h) exposure or exposure to combinations of metals. Previous studies also found no significant changes in the expression of hsp27 and hsp70i in cultured kidney epithelial cells in response to long-term (12 h–6 days) exposure to cadmium (Somji et al., 1999a, 1999b), although other studies have discovered accumulation of hsp70i in cultured lung cells in response to long-term cadmium exposure (Croute et al., 2000), of hsp70i but not hsp27 in cultured bladder urothelium cells in response to long-term arsenite exposure (Rossi et al., 2002), and of hsp25/27 and hsp70i but not B-crystallin in two separate renal epithelial cell lines in response to long-term cadmium exposure (Bonham et al., 2003). It is difficult to draw any firm conclusions from the relative paucity of data concerning heat shock protein induction caused by long-term exposure to toxic metals, especially in light of the differences in treatment protocols. For example, culture medium containing cadmium was added to cultured cells either every day (Bonham et al., 2003) or every 3 days (Somji et al., 1999a, 1999b) in the studies of the effects of long-term exposure of cultured renal epithelial cells to cadmium, making it difficult to directly compare the results of these studies to our work using a single addition of toxic metal.
The results of our measurements of the accumulation of heat shock proteins in response to combinations of toxic metals showed a correlation between their accumulation and a decrease in toxic injury to podocytes, especially combinations of toxic metals containing cadmium and arsenite. These results are supported by a previous study that found that cadmium and arsenite induced an increase in hsp70i in human and rat kidney cell lines that occurred at lower concentrations than the increase caused by either metal alone (Madden et al., 2002), and a correlation between a decrease in the heat shock response and the onset of cell injury and death. These results together suggest that the decreased toxicity of toxic metal mixtures containing cadmium and arsenite are a result of the increased expression of one or more of the heat shock proteins in podocytes. Heat shock proteins have previously been shown to confer protection against injury caused by metals in a variety of systems. For example, a direct correlation was found between the expression of hsp25 in embryonic stem cells transformed with hsp25 sense and antisense expression vectors and their resistance to injury by cadmium, mercury, or arsenite (Wu and Welsh, 1996). The expression of hsp27 and B-crystallin together conferred greater protection than the expression of either of these proteins alone (Fu and Liang, 2003), a finding that may be explained by co-localization, because fluorescence resonance energy transfer (FRET), and two-hybrid studies have shown that hsp25/27 interacts directly with B-crystallin (Fu and Liang, 2003; Klemenz et al., 1993; Liu and Welsh, 1999), an association that is thought to stabilize B-crystallin (Bova et al., 2000).
We observed a rapid (within 45 min) and sustained (3 days) increase in the phosphorylation of hsp25 in response to all single and combination toxic metal treatments. The induction of hsp27 phosphorylation in cultured cells by arsenite, cadmium, and lead treatment has been well documented (Landry et al., 1992; Leal et al., 2002), but to our knowledge our work shows the first published description of the induction of hsp25 phosphorylation by mercury. Interestingly, mercury induced almost complete conversion of unphosphorylated to mono- or di-phosphorylated hsp25, an effect greater than that induced by an equal concentration of cadmium and roughly equivalent to the hsp25 phosphorylation induced by arsenite. That effect also persisted longer than treatments with the other toxic metals (Table 2). The hsp25 phosphorylation induced by combinations of toxic metals was approximately intermediate between the values induced by the constituents of the mixtures, suggesting that toxic metals induce hsp25 phosphorylation via similar mechanisms. There did not appear to be any correlation between treatments that induced more hsp25 phosphorylation and podocyte survival or heat shock protein accumulation.
In conclusion, we have demonstrated that treatments with combinations of toxic metals caused disparate effects on podocyte viability, heat shock protein accumulation, and accumulation of toxic metals in cells, but not on hsp25 phosphorylation. We also described the novel finding that mercuric chloride is a strong inducer of hsp25 phosphorylation, and we found that the combination treatments that induced the smallest decreases in podocyte viability induced the greatest increases in the accumulation of the heat shock proteins hsp70i, hsp25, and B-crystallin. Our results suggest the stress response, characterized by an increase in the expression of heat shock proteins, may be vital in the podocyte's ability to protect against and recover from toxic metal injury, especially from exposure to combinations of the toxic metals cadmium and arsenite.
ACKNOWLEDGMENTS
Portions of this work were supported by a Program Project grant (P01 ES11188–01) from the National Institute of Environmental Health Sciences (W.E.S.: Subproject Principal Investigator).
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