Expression of Metallothionein Isoform 3 (MT-3) Determines the Choice between Apoptotic or Necrotic Cell Death in Cd+2-Exposed Human Proximal Tubule Cells
Seema Somji*,
Scott H. Garrett*,
Mary Ann Sens*,
Volkan Gurel* and
Donald A. Sens
,1
* Department of Pathology and
Department of Surgery, School of Medicine and Health Sciences, University of North Dakota, 501 N. Columbia Road, Grand Forks, North Dakota 58202-9037
Received February 9, 2004;
accepted April 22, 2004
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ABSTRACT
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This laboratory has shown that the third isoform of metallothionein (MT-3) is expressed in the human kidney in situ, including the cells of the proximal tubule. A subsequent analysis of MT-3 expression in cell cultures derived from the human proximal tubule (HPT) demonstrated that mortal HPT cells expressed MT-3, while the HPV-immortalized HK-2 cells had no expression of MT-3. In the present study, the effect of MT-3 expression on Cd+2-induced cytotoxicity was determined by stable transfection of the MT-3 coding sequence into the HK-2 cell line. The results demonstrated that HK-2 cells stably transfected with MT-3 were more sensitive to the cytotoxic effects of Cd+2. Furthermore, this increase in Cd+2-induced cytotoxicity was correlated to an alteration in the mechanism of cell death, being changed from an apoptotic mechanism in cells not expressing the MT-3 gene to a necrotic mechanism in cells expressing the MT-3 gene. The present study provides evidence that MT-3 could play a role in controlling the choice between apoptosis and necrosis in multiple epithelial cell types of the human kidney.
Key Words: cadmium; proximal tubule; nephrotoxicity; metallothionein; apoptosis; necrosis; kidney.
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INTRODUCTION
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Numerous studies in both animals and humans have shown that the kidney, and the proximal tubule in particular, are the organ and cell type critically affected by chronic exposure to Cd+2 (Andrews, 2000
; Bernard et al., 1976
; Bosco et al., 1984
; Gonich et al., 1980
). Studies employing transgenic mice deficient in the expression of the first and second isoforms (MT-1 and MT-2) of the metallothionein protein family members have confirmed the many studies suggesting that MT plays an important protective role against the chronic nephrotoxicity elicited by Cd+2 exposure (Liu et al., 1998
, 2000
). Recently, this laboratory has shown that an additional MT family member, the third isoform (MT-3), is also expressed in the human kidney in situ, including the cells of the proximal tubule (Garrett et al., 1999
). A subsequent analysis of MT-3 expression in cell cultures of mortal human proximal tubule (HPT) cells (Detrisac et al., 1984
) and immortalized HK-2 cells (Ryan et al., 1994
) derived from the human proximal tubule demonstrated that the mortal HPT cells expressed MT-3, while the HPV-immortalized HK-2 cells had no expression of MT-3 mRNA or protein (Kim et al., 2002
). This difference in MT-3 expression was used to demonstrate that the expression of MT-3 had a role in the maintenance of proximal tubule active ion transport. Specifically, it was shown that HK-2 cells stably transfected to overexpress the MT-3 gene regained the capacity expected of a proximal tubule cell for vectorial active transport as evidenced by the reestablishment of dome formation by the MT-3 transfected HK-2 cells (Kim et al., 2002
). To date, there has been no examination of the potential role that MT-3 expression might have in mediating Cd+2-induced damage to the proximal tubule.
This lack of examination is presumably due to the fact that, in contrast to the MT-1 and MT-2 isoforms which have been extensively studied for many decades, the MT-3 isoform was isolated relatively recently in 1992 and assigned as a brain-specific MT family member (Palmiter et al., 1992
). The MT-3 protein, which was originally called growth inhibitory factor (GIF), has been shown to retain all the characteristic features of the traditional MTs, including transition metal binding, and was subsequently renamed MT-3 (Tsuji et al., 1992
; Uchida et al., 1991
). Structurally, the MT-3 isoform possesses seven additional amino acids that are not present in any other member of the MT gene family, a six amino acid C-terminal sequence and a Thr in the N-terminal region (Palmiter et al., 1992
; Tsuji et al., 1992
; Uchida et al., 1991
). Functionally, MT-3 has been shown to possess a neuronal cell growth inhibitory activity that is not duplicated by the other human MT classes (Amoureux et al., 1995
; Uchida et al., 1991
). This nonduplication of function occurs despite a 6369% homology in amino acid sequence among MT-3 and the other human MT isoforms (Sewell et al., 1995
). The goal of the present study was to employ cultures of HPT and HK-2 cells to determine if MT-3 expression might mediate Cd+2-induced damage to the proximal tubule.
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MATERIALS AND METHODS
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Cell culture, stable transfection, RT-PCR, MT protein determination, and cell viability. Stock cultures of HPT and HK-2 cells for use in experimental protocols were grown using serum-free conditions as previously described by this laboratory (Detrisac et al., 1984
; Kim et al., 2002
). The procedures for the stable transfection of the HK-2 cells with the MT-3 coding sequence has been detailed previously (Kim et al., 2002
). An identical procedure was used to stably transfect the HK-2 cells with the coding sequence of the MT-1E isoform gene. The primers and conditions used to determine the expression of MT-1, MT-2, and MT-3 isoform-specific mRNAs have been detailed previously (Garrett et al., 1998a
,b
; Kim et al., 2002
). The levels of the MT-1/2 and MT-3 proteins were determined using an antibody-based method that determines the combined expression of the MT-1 and MT-2 proteins and the specific expression of the MT-3 protein (Garrett et al., 1998a
; Kim et al., 2002
). Cell viability of confluent monolayers, as an indicator of cytotoxicity of Cd+2, was determined by measuring the capacity of the cells to reduce MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan (Rossi et al., 2002
).
Visualization of DAPI-stained cells. The effect of metal treatment on cell viability and the number of fragmented nuclei was determined by the visualization and counting of 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei as described previously by this laboratory (Garrett et al., 1998a
). At the indicated time points, wells containing the monolayers were rinsed with phosphate-buffered saline (PBS), fixed for 15 min in 70% ethanol, rehydrated with 1 ml PBS, and stained with 10 µl DAPI (10 µg/ml in distilled water). For each time point, a minimum of 20 fields per well and three wells per data point were examined. The nuclear counts as well as the number of fragmented nuclei were determined for each field. The percentage of fragmented nuclei were determined for each well and the results presented as the mean ± SEM for the triplicate wells.
DNA laddering. The procedure described by Borner and coworkers (1995)
was used to extract DNA from the cells. At each time point, adherent and detached cells were collected and combined from each well, centrifuged, and the pellet resuspended in lysis buffer consisting of 5 mM Tris (pH 7.4), 5 mM EDTA, and 0.5% Triton X-100 and incubated for 2 h at 4°C. The cell lysate was centrifuged and the supernatant was incubated with 200 µg/ml proteinase K for 1 h at 50°C. The DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v) and precipitated overnight with absolute ethanol in the presence of 20 µg glycogen. The DNA pellet was washed twice with 70% alcohol, air dried, and dissolved in Tris-EDTA buffer. After treatment with ribonuclease A for 1 h at 50°C, the DNA was loaded onto a 2% (w/v) agarose gel containing ethidium bromide.
Determination of LDH release. The release of lactate dehydrogenase (LDH) from cells was determined by the Cyto Tox 96 assay kit (Promega). Briefly, 50 µl of the cell culture supernatant was transferred to a 96 well enzymatic assay plate. Reconstituted substrate mix (50 µl) was added to each sample and the enzymatic reaction was allowed to proceed for 30 min at room temperature in the dark. The assay was stopped by adding 50 µl of the stop solution (1 M acetic acid) and the plate was read at 490 nm using an ELISA plate reader.
Caspase-3 assay. The caspase-3 activity of the cells was determined using the protocol provided with the caspase-3 assay kit from R&D Systems. Briefly, the cells were collected by centrifugation and the pellet was lysed by the addition of the cell lysis buffer as provided in the kit. The cell lysate was incubated on ice for 10 min and centrifuged. The supernatant was transferred to a new tube and the protein content was determined using the BCA protein assay.
Fifty µl of the cell lysate was transferred to a 96 well flat bottom micro plate. Fifty microliters of 2X reaction buffer and 5 µl of caspase-3 colorimetric substrate (DEVD-pNA) was added to each well. Enzyme activity was determined by enzymatic cleavage of the tetrapeptide substrate DEVD-pNA and the release of the chromophore p-nitroanilide, which was measured spectrophotometrically at a wavelength of 405 nm. The results are reported as absorbance units per mg total cell protein.
Statistical analysis. All experiments were performed in triplicate and the results are expressed as the SEM. Statistical analyses were performed using Systat software using separate variance t-tests, ANOVA with Tukey post-hoc testing. Unless otherwise stated, the level of significance was 0.05.
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RESULTS
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Basal Expression of MT-1/2 and MT-3 in HPT and HK-2 Cells
The basal expression of the MT-1, MT-2, and MT-3 isoform specific genes has been detailed previously for HPT cells, parental HK-2 cells, HK-2 cells stably transfected with the MT-3 coding sequence, and HK-2 cells stably transfected with the blank vector control (Garrett et al., 1998a
,b
; Kim et al., 2002
). The basal levels of expression of MT-1/2 and MT-3 proteins were confirmed in the present study for each of these cultures (Table 1). The stable transfection of the HK-2 cells with the MT-1E coding sequence was accomplished using methodology identical to that for the prior transfection with MT-3, but the results have not been detailed previously. Briefly, the coding sequence of the MT-1E gene was obtained from human proximal tubule cell RNA by RT-PCR, blunt end ligated into the EcoR V site of pcDNA3.1/Hygro(+), and linearized by Fsp I prior to transfection of the HK-2 cells. The HK-2 cells were transfected with the MT-1E plasmid construct in the sense direction. Following selection, five clones of MT-1E transfectants were selected for further characterization. It was demonstrated that each of the five HK-2 clones overexpressed MT-1E mRNA when compared to cells containing the blank pcDNA3.1 vector (Fig. 1A). The MT-1E mRNA was first detected from each of the five overexpressing clones between 23 and 25 cycles of PCR and from the blank vector controls at between 30 and 33 cycles of PCR, both at 500 ng total RNA inputs (data not shown). It was also demonstrated that the transfection protocol had no effect on the expression on the glyceraldehyde 3-phosphate dehydrogenase housekeeping gene (Fig. 1B). The five HK-2 MT-1E stable transfectants expressed MT-1/2 protein over a range of 7.5 to 9.6 ng MT-1/2 protein/µg total protein, and the clone expressing the intermediate value in this overall range was used in the present study (Table 1).

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FIG. 1. MT-1E and g3pdh expression in transfected HK-2 cells. HK-2 cells were transfected with the blank vector or the vector containing the MT-1E coding sequence. Expression of mRNA was confirmed by RT-PCR using MT-1E and glyceraldehyde 3-phosphate dehydrogenase (g3pdh) specific primers on total RNA from cultured cells. Shown in (A) are PCR products at 35 cycles for five blank vector controls and at 25 cycles for the five MT-1E transfected clones; (B) the g3pdh products at 28 cycles for five blank vector controls and five MT-1E expressing clones.
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Expression of MT-3 in HK-2 Cells Increases the Susceptibility to Cd+2-Induced Cell Death
The first goal of this study was to determine if the overexpression of MT-3 protein would protect the HK-2 cells from Cd+2-induced cytotoxicity. For this determination, confluent cultures of parental HK-2 cells, HK-2 cells transfected with the MT-3 gene, and HK-2 cells transfected with the blank vector were exposed to increasing concentrations of Cd+2 for a 16-day time course (Figs. 2A2E). Instead of MT-3 expression conferring protection to the HK-2 cells, the results of this determination clearly showed that HK-2 cells expressing the MT-3 gene were more sensitive to the cytotoxic effects of Cd+2 than the HK-2 cells containing the blank vector control. The parental cell line, HK-2, showed a similar resistance to Cd+2 as that of the cells transfected with the blank vector (data not shown). A baseline was established where exposure of the cells to 0.9 µM Cd+2 had no effect on cell viability over the 16-day growth period for the MT-3 transfectants or the blank vector transfectants (Fig. 2A). Increasing the exposure of the cells to 2.25 µM Cd+2 reduced the viability of the HK-2 MT-3 transfectants by day 7 of exposure while having no effect on the HK-2 cells transfected with the blank vector (Fig. 2B). An additional increase in Cd+2 concentration to 4.5 µM resulted in a further reduction in the viability of the HK-2 cells transfected with the MT-3 gene, as noted by a loss of viability by day 4 of exposure, while having no effect on the viability of the HK-2 cells transfected with the blank vector (Fig. 2C). This trend continued when the cells were exposed to 6.75 or 9 µM Cd+2 (Figs. 2D and 2E). There was no loss in viability of the HK-2 cells transfected with the blank vector control following exposure to 9 µM Cd+2. Further sequential increases in Cd+2 concentration to 13.5 and 18 µM resulted in a total loss of cell viability for all the cell lines following four days and one day of exposure, respectively (Figs. 2F and 2G).

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FIG. 2. Effect of cadmium on the viability of HK-2 transfected cells. Confluent cultures of HK-2 cells transfected with the 3.1 blank vector control, HK-2 cells transfected with the MT-1E coding sequence, and HK-2 transfected with the MT-3 coding sequence were exposed to (A) 0.9, (B) 2.25, (C) 4.5, (D) 6.75, (E) 9.0, (F) 13.5, and (G) 18.0 µM cadmium for 16 days. Cell viability was determined by measuring the capacity of the cells to reduce MTT to formazan. Viability is expresses as percent of control. *Significant difference (p < 0.05) compared to control.
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The Increased Sensitivity of MT-3 Transfected HK-2 Cells to Cd+2 Is Specific for Expression of the MT-3 Isoform
The second goal was to determine if the effect of MT-3 expression on Cd+2-induced cell death of the HK-2 cells was specific for the MT-3 isoform. Evidence for specificity of effect was obtained using HK-2 cells stably transfected with the MT-1E coding sequence in an identical analysis of cell viability as described above for the MT-3 transfected HK-2 cells. The results of this determination showed that when the MT-1E stable transfectants were exposed to increasing concentrations of Cd+2 for a 16-day period of exposure, they had the same sensitivity to Cd+2 as the HK-2 parental cells or the HK-2 cells containing the blank vector control (Figs. 2A2E). Unlike the MT-3 transfectants, the MT-1E transfected HK-2 cells were generally not more susceptible to Cd+2-induced cytotoxicity. Increased susceptibility of MT-1E transfected cells was shown only at single time points at the two highest levels of Cd+2 exposure, and these were highly cytotoxic levels of Cd+2 that produced 100% cell death following 24 and 96 h of exposure, respectively (Figs. 2F and 2G).
MT-3 Expression Inhibits Cd+2-Induced Apoptosis in HK-2 Cells
The next goal was to determine the contribution of apoptosis and necrosis in Cd+2-induced cell death of HK-2 cells and if this was altered by MT-3 expression. In a preliminary examination, confluent cultures of the HK-2 cells (blank vector control transfectant, the MT-3 transfectant, and the MT-1E transfectant) were exposed to 13.5 µM Cd+2 and monitored by light microscopy until the first sign of toxicity was noted as indicated by rounding of the cells. The cells were then treated with the nuclear stain DAPI and examined by fluorescent microscopy for the presence of nuclear fragmentation. Nuclear fragmentation was taken as a positive marker for apoptosis. This preliminary examination suggested that apoptosis was the major mechanism of cell death in parental HK-2 cells exposed to Cd+2 as noted by the frequent profiles of nuclear fragmentation when compared to control cells (Figs. 3A and 3B). In contrast, an identical examination of the HK-2 cells transfected with the MT-3 gene disclosed only rare instances of fragmented nuclei (Fig. 3C). That the reduction in fragmented nuclei was specific for the MT-3 gene was suggested by the finding that the frequency of fragmented nuclei was similar between the Cd+2-treated HK-2 cells carrying the blank vector and those with the MT-1E coding sequence (Figs. 3B and 3D). Thus, these results suggest that Cd+2-induced cell death occurs by apoptosis in HK-2 cells and that MT-3 transfection alters this process.

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FIG. 3. DAPI staining of HK-2 cells. HK-2 parental and transfected cells were exposed to 13.5 µM cadmium and visualized by light microscopy. At the first sign of cell toxicity (rounding of cells), the cells were fixed and stained with the nuclear dye, DAPI. Nuclear morphology was visualized by fluorescent microscopy. Arrows indicate fragmented nuclei. (A) HK-2 parental cells without treatment. (B) HK-2 parental cells after cadmium treatment. (C) HK-2 cells transfected with the MT-3 coding sequence after cadmium treatment. (D) HK-2 cells transfected with the MT-1E coding sequence after cadmium treatment.
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The above preliminary results were confirmed by four additional studies. The first was an analysis of DNA laddering from the three HK-2 transfectants as a function of Cd+2 exposure. In this analysis, confluent cultures of the respective HK-2 transfectants were treated with 0, 4.5, 6.75, 9.0, 13.5, and 18.0 µM Cd+2 for 24 h and the extent of DNA laddering determined by agarose gel electrophoresis (Figs. 4A, 4B, and 4C). The times and concentrations were chosen from the viability studies in Figure 2 and represent the following cell toxicities: 4.5 µMno loss of cell viability for any of the HK-2 transfectants; 6.75 µMno loss of cell viability for blank vector or MT-1E transfectants, but a 20% loss of cell viability for the MT-3 transfectant; 9.0 µM30% loss of viability for blank vector and MT-1E transfectant and 55% loss of viability for the MT-3 transfectant; 13.5 µM30 to 40% loss of viability for blank vector and MT-1E transfectants and 60% loss of viability for the MT-3 transfectant; and 18.0 µM40% loss of viability for blank vector and MT-1E transfectants and 65% loss of viability for the MT-3 transfectant. The results demonstrated that Cd+2-induced cell death for HK-2 cells transfected with the blank vector or MT-1E coding sequence have a qualitative correlation with DNA laddering, a marker of apoptotic cell death. The absence of laddering noted at 9 µM Cd+2 for MT-1E transfected cells is a result of experimental variation that occurred due to performance of the experiments at independent points in time. In contrast, HK-2 cells transfected with the MT-3 coding sequence demonstrated no evidence of DNA laddering at identical concentrations of Cd+2; even though these concentrations produced even greater losses in cell viability.

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FIG. 4. DNA laddering for control and cadmium treated cells. The control and transfected cells were exposed to Cd+2 for 24 h and the DNA extracted and analyzed by agarose gel electrophoresis. (A) HK-2 cells transfected with the blank vector control. (B) HK-2 cells transfected with the MT-1E coding sequence. (C) HK-2 cells transfected with the MT-3 coding sequence. In each panel, lane 1 is the commercial DNA size ladder, lane 2 the untreated sample, and lanes 37 DNA samples from cells exposed to 4.5, 6.75, 9.0, 13.5, or 18 µM cadmium.
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A second study determined the activation of caspase-3 by the three HK-2 transfectants as a function of Cd+2 exposure. In this analysis, confluent cultures of the respective transfectants were exposed to 9.0, 13.5, or 18 µM Cd+2 for 6, 8, and 12 h (Figs. 5A, 5B, and 5C). The results demonstrated that caspase-3 was activated when the HK-2 cells carrying the blank vector control or the MT-1E coding sequence were exposed to the three Cd+2 concentrations. In contrast, there was no activation of caspase-3 when the HK-2 cells expressing the MT-3 construct were exposed to identical Cd+2 concentrations. A third study expanded the initial studies assessing nuclear fragmentation by DAPI fluorescence to include quantification of the amount of nuclear fragmentation over a time course of Cd+2 exposure. In this analysis, confluent cultures of the respective transfectants were exposed to 0, 4.5, 6.75, 9.0, 13.5, or 18 µM Cd+2 and the percentage of fragmented nuclei determined at 12, 18, 24, 36, and 48 h (Figs. 6A, 6B, and 6C). The results demonstrated that nuclear fragmentation induced by Cd+2 exposure was elevated for the HK-2 cells carrying the blank vector control or the MT-1E coding sequence when compared to the HK-2 cells expressing the MT-3 construct.

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FIG. 5. Caspase-3 activation for control and cadmium treated cells. Caspase-3 activation after exposure to 9, 13.5, or 18 µM cadmium for 6, 8, and 12 h for (A) HK-2 cells transfected with the blank vector control, (B) HK-2 cells transfected with MT-1E, and (C) HK-2 cells transfected with MT-3. Caspase-3 activity was determined by the release of the chromophore, p-nitroanilide, which was quantitated spectrophotometrically at a wavelength of 405 nm. *Significant difference (p < 0.05) compared to control.
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FIG. 6. Quantification of nuclear fragmentation for control and cadmium treated cells. The percentage of apoptotic nuclei was determined after exposure to 4.5, 6.75, 9, 13.5, and 18 µM cadmium for 12, 18, 24, 36, and 48 h for (A) HK-2 cells transfected with the blank vector control, (B) HK-2 cells transfected with MT-1E, and (C) HK-2 cells transfected with MT-3. The number of fragmented nuclei was determined by fluorescent microscopy of the DAPI stained cells. Results are expressed as percentage of apoptotic nuclei. *Significant difference (p < 0.05) compared to control.
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Lastly, the release of LDH from the cell was assessed for the HK-2 cells transfected with the blank vector control and the MT-3 coding sequence as a function of Cd+2 exposure. In this analysis, confluent cultures of the HK-2 blank vector control and the MT-3 transfectant were exposed to 0, 4.5, 6.75, 9.0, 13.5, or 18 µM Cd+2 and the release of LDH into the growth medium determined 48 h after Cd+2 addition to the cells (Fig. 7). The results demonstrated that LDH release was highly elevated for the HK-2 cells transfected with the MT-3 coding sequence at Cd+2 exposure levels eliciting marked cell cytotoxicity. In contrast, LDH release remained near control values for HK-2 cells carrying the blank vector control under identical conditions of treatment.

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FIG. 7. Release of LDH from control and cadmium treated cells. Confluent cultures of HK-2 cells containing the blank vector control or the MT-3 coding sequence were exposed to 4.5, 6.75, 9, 13.5, or 18 µM cadmium for 48 h. The release of LDH by the cells was quantified spectrophotometrically at a wavelength of 490 nm. The results are expressed as percentage of total LDH release. Total LDH release was obtained by treating control cells with Triton-X100. *Significant difference (p < 0.05) compared to control.
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DISCUSSION
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The results of this study show that immortalized human proximal tubule cells stably transfected to express the MT-3 gene are more sensitive to the cytotoxic effects of Cd+2 than blank vector controls not expressing this gene. The finding that MT-3 expression can regulate the choice between apoptosis and necrosis in a renal cell culture model exposed to Cd+2 provides evidence that MT-3 may function in a similar manner in the in situ human kidney. Previous studies from this laboratory employing immunohistochemical localization with an MT-3 specific antibody demonstrated that MT-3 was widely and variably expressed in the epithelial elements of the human kidney (Garrett et al., 1999
). In the glomerulus, cytoplasmic staining of moderate intensity was demonstrated in the parietal epithelial cells of Bowman's capsule and in visceral epithelial cells of the glomerular tuft. The cells of the proximal tubules also exhibited moderate cytoplasmic MT-3 reactivity. The distal tubules showed strong cytoplasmic staining for MT-3, particularly in the medullary rays. In the medulla, MT-3 staining was the most variable, with weak to moderate staining in the medullary collecting ducts and a general absence of staining in the thin loops of Henle and in the transitional epithelium of the renal pelvis. Thus, the present study provides evidence that MT-3 could play a role in controlling the choice between apoptosis and necrosis in multiple epithelial cell types of the human kidney.
The finding that MT-3 expression influences the choice between apoptosis and necrosis in Cd+2 exposed proximal tubule cells also defines a difference in function between MT-3 and the closely related MT-1 and MT-2 family members. Studies have shown that the susceptibility to cadmium-induced apoptosis is dependent on the basal and induced expression of MT-1 and MT-2, with the level of MT-1 and MT-2 expression having a negative correlation with the rate of apoptosis (Kondo et al., 1997
; McAleer and Tuan, 2001
; Shimoda et al., 2001
). However, to the authors' knowledge no study has shown MT-1 and MT-2 expression level to influence the choice between apoptotic and necrotic mechanisms of cell death in a given cell line exposed to a toxicant. This nonduplication of function between MT-3 and the other MT family members as regards mechanism of cell death occurs despite a high degree of homology in amino acid sequence between MT-3 and the other human MT isoforms (Sewell et al., 1995
). There is compelling evidence that the small difference in sequence between MT-3 and the other MT isoforms can result in a difference in protein function. As detailed in the Introduction, MT-3 was discovered based on its neuronal cell growth inhibitory activity, a functional feature not shared by any other MT family member (Tsuji et al., 1992
; Uchida et al., 1991
). In the present study, additional evidence that MT-3 was specific in influencing the mechanism of cell death was obtained by showing that stable transfection of the HK-2 cells with the MT-1E isoform had no effect on the mechanism of cell death when the cells were exposed to Cd+2 under identical conditions.
The finding that MT-3 can regulate the choice between necrosis and apoptosis when the proximal tubule cell is exposed to Cd+2 may explain some of the variable features of cell death noted in in vivo studies of Cd+2-induced nephrotoxicity. Early studies of Cd+2 nephrotoxicity demonstrated that the mode of cell death by the metal was necrosis (Cherian et al., 1976
; Nordberg et al., 1975
; Squibb et al., 1984
). Later studies, once apoptosis was recognized as a mechanism of toxicant-induced cell death, demonstrated apoptosis to be a major mode of cell death in Cd+2-induced nephrotoxicity (Ishido et al., 1998
; Liu et al., 1998
, 1999
, 2000
). The most likely explanation for the difference in mechanism of cell death between these studies is that experimental conditions can be developed that can favor either mechanism of cell death. An overall examination of the literature detailing Cd+2-induced nephrotoxicity shows a large number of studies that employ a variety of experimental conditions. Two previous findings by this laboratory on MT-3 expression in HPT cells might explain how the level of MT-3 could influence the choice between apoptotic and necrotic cell death in these in vivo models of Cd+2-induced nephrotoxicity. The first of these was the finding that basal MT-3 expression is markedly reduced following subculture of the HPT cells and returns to presubculture values once cell confluency and contact inhibition of cell growth is reestablished (Kim et al., 2002
). This finding suggests that conditions that favor cell proliferation/regeneration would result in reduced levels of MT-3 expression and render a cell more prone to elimination by an apoptotic mechanism of cell death. A second observation was that exposure of confluent cultures of HPT cells to Cd+2 caused a transient induction of MT-3 levels on a time scale similar to that of the early response genes (Garrett et al., 2002
). This transient increase in MT-3 level might be expected to increase the cells' resistance to apoptotic cell death during an acute exposure of the kidney to Cd+2. Thus, extrapolation of these findings of MT-3 expression in the in vitro proximal tubule might explain why studies of Cd+2 nephrotoxicity in animal models have found differing predominant mechanisms of cell death based on the different conditions of Cd+2 exposure.
Overall, the present study provides the initial evidence that MT-3 can play a role in the decision of a cell to undergo apoptotic or necrotic cell death when exposed to a toxicant.
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ACKNOWLEDGMENTS
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The project described was supported by grant number R01 ES11333 from the National Institutes of Environmental Health Sciences (NIEHS), NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. Project support was also provided by the North Dakota Biomedical Research Infrastructure Network, NIH.
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NOTES
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1 To whom correspondence should be addressed. Fax: (701) 777-3108. E-mail: dsens{at}medicine.nodak.edu.
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