1Department of Physiology, University of Saarland, D-66421 Homburg; 2Department of Neuroanatomy, University of Göttingen, D-37075 Göttingen; and 3Department of Physiology & Pathophysiology, University of Witten/Herdecke, D-58448 Witten, Germany
Submitted 27 May 2003 ; accepted in final form 21 July 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
trafficking; endocytosis; necrosis; chloroquine; LY-294002
When free Cd2+ enters the cytosol of PT cells, reactive oxygen species (ROS) are generated, partly as a consequence of Cd2+-induced displacement of endogenous redox active metals (Fe2+, Cu2+) (29) and subsequent damage to critical organelles (e.g., mitochondria) (36). ROS resulting from oxidative stress promote structural changes or misfolding of cellular proteins including vital membrane transporters, such as the Na+-K+-ATPase (38). To overcome the toxic accumulation of oxidatively modified proteins, cells increase the rates of proteolysis of abnormal transporters, either by endocytosis and subsequent degradation by lysosomal proteases, or proteolysis via the ubiquitin-proteasome complex (26). If this plethora of ROS-mediated stress events is not sufficiently balanced by repair processes, affected cells may be induced to undergo cell death via apoptosis or necrosis. We have shown that low micromolar Cd2+ concentrations induce apoptosis, but not necrosis, of PT cells by a process involving ROS (38, 39).
Apical endocytosis of Cd2+ complexed to metallothionein (MT) represents the major form in which Cd2+ is delivered to the kidneys in vivo (28). This is because enterally absorbed Cd2+ is initially taken up into the liver. Once taken up by cells, Cd2+ binds to small molecules (such as amino acids) or peptides, such as glutathione (GSH), which contain a sulfhydryl group and protect the intracellular milieu from oxidative damage, or to proteins including MTs. At least four different MTs are known. MT-1 and MT-2, which are found expressed in epithelial cells, are small proteins of 6 kDa that are distinguished by their high metal content (9). MTs contain numerous thiol groups due to their high cysteine content, which provide the basis for high-affinity binding of many transition metals. MTs are highly inducible and accumulate intracellularly in response to a variety of stimulants, such as Cd2+ (22). Because free cytosolic Cd2+ is sequestered by GSH or by intracellularly induced MT, toxicity may occur if an imbalance between these protective factors and the cellular Cd2+ load occurs. Some Cd2+ bound to MT is also released from damaged cells into the plasma, from where CdMT complexes are easily filtered through the glomerulus and translocated from the primary urinary filtrate into renal PT cells (reviewed in Ref. 22). It has been suggested that once taken up by endo/lysosomes, Cd2+ may be freed from MT and subsequently transported into the cytoplasmic compartment, where the free Cd2+ ion may trigger apoptosis and/or induce intracellular de novo MT synthesis. However, in vivo as well as in vitro studies have yielded conflicting results as to the nephrotoxic potency of cadmiummetallothionein (CdMT) (16, 18, 20, 22, 24, 31).
The cellular processes underlying in vivo cadmium nephrotoxicity as well as the uptake pathways for Cd2+ and CdMT in PT cells are poorly understood. Thus the present study was designed to determine the role of the endo/lysosomal uptake pathway for cell viability and MT levels in cultured immortalized rat PT cells exposed to CdCl2 or Cd7MT-1.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The following reagents were obtained from the listed sources and used at the concentrations indicated in the text. Stock solutions of LY-294002 (Calbiochem, Bad Soden, Germany) were made by solubilization in dimethyl sulfoxide (DMSO). Polyvinylidene difluoride (PVDF) membranes were from NEN-Dupont (Bad Homburg, Germany). Enhanced chemiluminescence reagents and 109CdCl2 with a specific activity of 50-1,000 µCi/µg Cd2+ were purchased from Amersham Pharmacia Biotech Europe (Freiburg, Germany). Chelex 100, chloroquine diphosphate, and metallothionein 1 (MT-1 from rabbit liver) were purchased from Sigma (Deisenhofen, Germany). Rat tail collagen type I (Sigma) was dissolved in 100 mM acetic acid. Hoechst 33342 (dissolved in H2O) was from Calbiochem. Ethidium bromide (Sigma) was dissolved in 150 mM HEPES, pH 7.4. All other substances were from commercial sources and of analytic grade.
Antibodies. The mouse monoclonal antibody (E9) against horse metallothionein (MT-1 and MT-2) was obtained from DAKO Diagnostika (Hamburg, Germany). The mouse antilysosome-associated membrane protein 1 (LAMP1) monoclonal antibody (MAb) (Bioquote Limited, York, UK) was used for the detection of late endosomal and lysosomal membrane fractions. The rabbit polyclonal antibody against Rab5a (S-19) was from Santa Cruz Biotechnology (Santa Cruz, CA) and served as a marker of early endosomal vesicles. Donkey anti-mouse IgG coupled to indocarbocyanin (Cy3) was obtained from Dianova (Hamburg, Germany). Horseradish-peroxidase conjugated sheep anti-mouse or donkey anti-rabbit IgG were purchased from Amersham-Buchler (Braunschweig, Germany) and were used as secondary antibodies for immunofluorescence and immunoblotting, respectively.
Methods
Cell culture. Immortalized cells (WKPT-0293 Cl.2) of the S1 segment of the PT of normotensive Wistar-Kyoto rats were cultured as described earlier (38, 39). Briefly, cells were maintained in renal tubular epithelium medium composed of Dulbecco's modified Eagle's medium (DMEM)/F-12 [nutrient mixture F-12 (Ham's); 1:1] and supplemented with 15 mM HEPES, 1.2 mg/ml NaHCO3, 5 µg/ml insulin, 5 µg/ml transferrin, 10 ng/ml epidermal growth factor, 4 µg/ml dexamethasone, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 5% fetal calf serum. Cells were plated on 25-cm2 culture flasks, passaged at 80% confluence, and split 1:10 once a week. Drugs and solvents were applied 1 h before addition of CdCl2, MT-1, or Cd7MT-1 for the times indicated. Media ± drugs/solvents were replaced every 48 h.
109Cd2+ kinetic transport and inhibitor studies. Experiments were performed on cells seeded into six-well plates. The cell number per well (3-5 x 105 cells) was adjusted to obtain cell confluence within 48 h after seeding. 109Cd2+ and 109Cd2+-MT-1 uptake into PT cells was performed according to the method described by Templeton (37) with some modifications. All uptake measurements were performed in culture medium. The concentration of CdCl2 was adjusted to 10 µM from a 1,000-fold concentrated stock solution and labeled with 109CdCl2 to give a final activity of 0.5 µCi/ml. MT-1 was saturated with 109CdCl2 as described below. At particular time intervals, the labeling solution was removed from duplicate wells and the monolayers were washed three times with 1 ml of ice-cold Hanks' balanced salt solution (HBSS) containing 2 mM EGTA and solubilized in 1 M NaOH overnight. The 109CdCl2 content in the solubilized cell layer was determined using a Cobra II Auto-Gamma counter (Packard Instrument, Meriden, CT) with the upper and lower energy limits of detection adjusted to 30 and 15 keV, respectively, yielding a counting efficiency of 68%.
The lysosomotropic weak base chloroquine, which buffers acidic intracellular organelles (11), was applied to cultured cells for 40 h together with 109CdCl2 or 109Cd7MT-1 and affected neither medium pH nor basal rates of apoptosis or necrosis at the tested concentration of 0.1 mM. The same applied to LY-294002 (5 µM), a selective inhibitor of phosphatidylinositol 3-kinases (PI 3-kinase), which interferes with trafficking between endosomes and lysosomes (4, 10, 34) (higher concentrations of LY-294002 induced apoptosis of cultured cells). In control experiments, cells were exposed to 0.1% DMSO exactly as described for experimental drugs. These protocols were optimized such that application of the drugs did not induce apoptosis.
Fractionation of homogenate from PT cells by continuous sucrose gradient centrifugation. Cell monolayers of WKPT-0293 Cl.2 cells from four to six culture flasks were washed three times with HBSS, scraped off the culture dishes with a rubber policeman, pelleted by centrifugation at 14, 000 g for 5 min, and resuspended in homogenization buffer. Homogenization was performed in a cell disruption chamber (Parr Instrument, Moline, IL) by nitrogen pressure cavitation (1,000 lb./in.2 for 10 min at 4°C) in 0.5 ml of ice-cold homogenizing buffer containing (in mM) 280 mannitol, 10 HEPES, 10 KCl, 1 MgCl2, adjusted to pH 7.0, and a protease inhibitor cocktail (10 µM leupeptin, 2 mM benzamidine, and 0.1 mM Pefabloc SC). The homogenate was centrifuged at 50 g for 5 min, and the supernatant was collected. The pellet containing unbroken cells was resuspended in 0.5 ml of the same buffer and homogenized once more. After centrifugation at 50 g for 5 min, both supernatants were combined.
A continuous 0.25-2.0 M sucrose gradient was prepared in a total volume of 10 ml by mixing 0.25 M sucrose, 10 mM HEPES, and 1 mM EDTA (pH 7.0 with Tris) with 2.0 M sucrose, 10 mM HEPES, and 1 mM EDTA (pH 7.0 with Tris) in a linear gradient maker. The cell homogenate was loaded on top of the gradient and centrifuged at 25,000 g for 90 min, using a swing-out Beckman SW 41 rotor in a Beckman Optima L-70K ultracentrifuge (Beckman Coulter, High Wycombe, UK). The gradient was unloaded from the top, and ten 1-ml fractions were thus collected. Membrane pellets were obtained by centrifugation at 150,000 g for 90 min in a Beckman Optima MAX tabletop ultracentrifuge, using a TLA-55 fixed-angle rotor, and protein concentration was determined according to Bradford (3). Samples were processed for immunoblotting.
Immunoblotting. Electrophoresis and blotting procedures were performed essentially as described earlier (38, 39). Membrane proteins were separated by SDS-PAGE on 9% acrylamide Laemmli minigels and transferred onto PVDF membranes. After blocking with Tris-buffered saline containing 0.1% Tween 20 and 3% nonfat dry milk for 8 h, the membranes were incubated at 4°C with anti-LAMP1 (1:500) or anti-Rab5a (1:400) overnight, followed by horseradish peroxidase-conjugated secondary antibodies (1:10,000) for 1 h. The blots were developed in enhanced chemiluminescence reagents (ECL+; Amersham-Buchler), and signals were visualized on X-ray films. For documentation and quantification of LAMP1 and Rab5a expression, X-ray films from different experiments were scanned with a Fuji Film LAS-1000plus system hardware and stored using LAS-1000 Pro Image Reader software (Fuji Photo Film, Tokyo, Japan). The intensity (optical density) of the chemiluminescence signals was quantified by using AIDA Image Analyzer software (Raytest Isotopenmessgeräte, Straubenhardt, Germany).
Measurement of MT content by indirect immunofluorescence labeling and quantitative morphometry. The WKPT-0293 Cl.2 cells grown on glass coverslips were fixed in 4% paraformaldehyde/PBS for 30 min at room temperature (RT) (38). All subsequent steps were also carried out at RT. Cells were rinsed three times for 5 min in PBS and permeabilized by incubation for 5 min in PBS containing 0.1% Triton X-100. After three rinses in PBS, coverslips were inverted on 100-µl drops of a mouse MAb against a highly conserved peptide sequence (PNCSC) from MT-1 and MT-2 (E9; 1:300) and incubated for 1 h in a moist chamber. After three more rinses in PBS for 5 min, coverslips were incubated on drops of donkey anti-mouse IgG coupled to indocarbocyanin (Cy3; 1:600) for 30 min in the dark. After three more rinses for 5 min in PBS, coverslips were mounted with 2:1 Vectashield/Tris · HCl (pH 8.0) (Vector Laboratories, Peterborough, UK). The cells were examined with an Olympus BX50F microscope equipped with x40/0.75 and x20/0.5 Olympus UPlanFI objectives, a dichroic NB filter block, and a narrow-band green fluorescence exciter filter (wavelength 530-550 nm). Images were recorded with a three-charge-coupled device color video camera (Sony DXC-950P) and digitized to 8 bits/pixel using software (µ-Slicer) developed by Prof. B. Lindemann (Department of Physiology, University of the Saarland, Homburg/Saar, Germany) (38).
Digitized images (x400 magnification) were processed for documentation and a semiquantitative morphometric analysis using Adobe Photoshop Dl-4.01 software (Herzogenaurach, Germany). First, a level adjustment was performed by increasing the gamma value (slope of the curve) from 1.0 to 3.0 to raise the midtones without altering the range of brightness values (full range). Thus, by increasing the brightness of the midtones, weakly fluorescing images (e.g., controls) can be detected and analyzed. Images were then converted from a three-channel color model (RGB) to the grayscale mode. To minimize a bias due to cell proliferation (particularly in experiments in which time courses were analyzed), which occurs independently from the specific treatments applied, a fixed number of cells was selected for each image analyzed. To avoid a further bias caused by variations of fluorescence intensities, cells were selected for analysis that were neither too dark nor too bright, i.e., with a representative
or
average
level of brightness. Images were analyzed twice by blind assignment to three different investigators. A freehand selection of five different cells was carried out with a mask, and a pixel histogram of selected cells was performed. The fluorescence intensity of an individual cell, which reflects MT expression, was estimated from the mean grayscale value, and a mean value was calculated from the values of five individual cells. For data analysis, cells from control and experimental conditions from an individual experiment were matched to account for variations in the labeling protocol and cell culture conditions.
Cd2+ toxicity and inhibitor studies. Cells were exposed to given concentrations of CdCl2 (10 µM), Cd7MT-1 (1.4 µM MT-1 containing 7 Cd2+ ions per molecule of MT-1), or MT-1 (1.4 µM) for time periods varying between 4 and 72 h. At all incubation periods tested, CdCl2 (10 µM) did not affect cell proliferation, which has previously been assessed by comparison of cell numbers and percentages of mitotic figures (39). To prepare Cd7-reconstituted MT-1, apo-MT-1 was prepared by acidification of commercially available rabbit MT-1 in 0.1 N HCl, followed by filtration on a Centricon-3 (Amicon, Stonehouse, UK) equilibrated with 0.01 N HCl (30). Apo-MT-1 (1 mM) was reconstituted with 10-fold Cd2+ ions per mole of apo-MT-1 protein (Kd 10-20-10-25 M). The unbound metals were removed with a 2:1 excess of Chelex 100. Inhibitors were applied as described for 109Cd2+ transport studies. Chloroquine (0.1 mM) or LY-294002 (5 µM) were applied to cultured cells for various time periods in controls or together with CdCl2 or Cd7MT-1.
Detection of apoptosis and necrosis by fluorescence microscopy. Cells (3 x 105) cells were seeded into 35-mm tissue culture dishes and grown for 24 h before experiments were started. For detection of apoptosis and necrosis, cells were stained intravitally with the DNA dyes Hoechst 33342 (H-33342; 2 µg/ml) for 20 min and ethidium bromide (EB; 5 µg/ml) for 10 min, as previously described (39). Under ultra-violet (UV) epi-illumination (excitation wavelength 330-380 nm; emission wavelength >430 nm), necrotic cells fluoresce pink due to EB, whereas normal and apoptotic cells emit blue fluorescence due to H-33342. EB stains nuclei from cells that have lost the integrity of their plasma membrane, i.e., have undergone necrosis. In contrast, the lipophilic DNA dye H-33342 freely enters viable as well as apoptotic cells. Apoptotic cells can be distinguished from viable cells by their nuclear morphology with nuclear condensation and fragmentation as well as by the higher intensity of blue fluorescence of the nuclei. After washing out of the dyes, cells were examined under a UV/VIS fluorescence microscope (IMT-2 Olympus). Cells from five microscopic fields (x200 magnification) were counted per dish.
Statistics. All experiments were repeated at least three times with different batches of cell preparations. For kinetic studies, each time point was assayed in duplicate. Representative data or means ± SD or SE of at least three different preparations are shown. Uptake and efflux data were curve fitted using the Sigma Plot 8.0 spreadsheet program. Statistical analysis was carried out with the SPSS 11.0 program. Unpaired Student's t-test was applied when two groups were compared. For more than two groups, statistical differences were compared using a one-way ANOVA and Bonferroni post hoc test for multiple comparisons, assuming equality of variance with Levene's test. Results with levels of P 0.05 were considered to be statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The kinetics of 109Cd2+ uptake by confluent PT cells incubated in cell culture medium with 10 µM CdCl2 and 0.5 µCi 109Cd2+/ml are shown in Fig. 1. It has been previously shown that transport-mediated uptake of Cd2+ is nearly completely inhibited at 4°C (37). To differentiate between membrane-bound Cd2+ and transport-mediated Cd2+ uptake into PT cells, experiments were carried out at 4 and 37°C, respectively. Cellular 109CdCl2 increased linearly over time at 4°C with 141.3 ± 38.6 pmol 109Cd2+/mg protein (n = 4) measured after 40 h of incubation (data not shown), possibly reflecting a time-dependent saturation of binding sites at the surface of the confluent monolayer. At 37°C, after 3.5 h of incubation, cells rapidly took up Cd2+ (365.4 ± 15.7 pmol/mg protein; n = 4), with a further uptake to near saturation to 1,568.8 ± 255.8 pmol/mg protein (n = 4) at 40 h. With 109Cd7MT-1(1.4 µM MT-1 saturated with 10 µM Cd2+; see Methods), the number of binding sites determined by incubation at 4°C was similar to that for 109CdCl2 (data not shown). In contrast, 109Cd7MT-1 uptake was significantly reduced compared with that for 109CdCl2. Uptake was about one-third the 109CdCl2 uptake at 3.5 h. At 40 h, 109Cd7MT-1 uptake was still significantly lower than that observed with 109CdCl2.
|
The lysosomotropic drug chloroquine is a weak base that buffers acidic compartments and thereby prevents MT-1 degradation by acid proteases, such as cathepsins (7, 25). LY-294002 is a selective inhibitor of PI 3-kinases and interferes with vesicular transport to lysosomes (4, 10, 34). In PT cells exposed for 40 h with 109Cd7MT-1, 109Cd2+ content was significantly reduced with 0.1 mM chloroquine or 5 µM LY-294002 (Fig. 2). In contrast, when cells were incubated with 109CdCl2, 109Cd2+ uptake was not affected by chloroquine or LY-294002. This indicates that 109Cd7MT-1 is taken up via endo/lysosomal pathways.
|
Subcellular Distribution of 109Cd7MT-1 in PT Cells
PT cells were exposed for 24 h with 109Cd7MT-1 and homogenized, and subcellular membrane vesicles fractions were separated by ultracentrifugation of the homogenate layered on top of a continuous sucrose gradient ranging from 0.25 M (fraction 1) to 2.0 M (fraction 10) (Fig. 3A). Immunoblotting of the membrane fractions with an antibody against the marker of early endosomal vesicles, Rab5a (5), showed an enrichment of Rab5a-associated vesicles in fractions 3-5, with a sharp peak in fraction 4 (Fig. 3, A and B). LAMP1, a marker of late endosomes and lysosomes (6), was more broadly distributed between fractions 3 and 8, with a peak in fractions 5 and 6 (Fig. 3, A and B). 109Cd2+ accumulated in fractions 4-7, with a maximum in fractions 5-7, which correlates best with the distribution of LAMP1 and partly overlaps with the distribution of Rab5a (Fig. 3B).
|
MT Immunoreactivity of Rat PT Cells Exposed to CdCl2 or Cd7MT-1
Another means of monitoring uptake of Cd7MT-1 is to determine the content of MT-1 in PT cells by quantitative morphometry following immunofluorescence labeling with a MAb that recognizes MT-1 and MT-2 (E9). Thus the antibody detects not only exogenously applied end endocytosed MT-1 or Cd7MT-1 but also endogenously expressed MT-1 and MT-2, which may be induced by cytosolic heavy metals such as Cd2+. In PT cells exposed to either 1.4 µM MT-1 or Cd7MT-1 (Fig. 4), MT immunoreactivity increased rapidly at 4 h and then slowed down at 20 and 48 h, suggesting cumulative intracellular uptake of Cd7MT-1/MT-1 via endocytosis. In cells exposed to 10 µM CdCl2, MT immunore-activity was not significantly different from background at 4 h but was followed by a large and significant increase of MT expression after 20 h, which remained at this level after 48 h and could represent Cd2+-induced MT-1 and MT-2 (Fig. 4).
|
Time Course of Apoptosis in Rat PT Cells exposed to CdCl2 or Cd7MT-1
Compared with controls, CdCl2 significantly increased apoptosis at 4 h (controls: 3.0 ± 0.3%; CdCl2: 5.0 ± 0.5%; P < 0.01, n = 6-7) (Fig. 5). The maximal increase of apoptosis was observed at 24 h and decreased again at 72 h, which confirms previous observations (39). Application of Cd7MT-1 for up to 12 h did not significantly alter rates of apoptosis compared with controls. Exposure times of 36 h clearly increased apoptosis, and rates of apoptosis reached the maximal levels obtained with CdCl2 at 48 and 72 h. With equimolar concentrations of MT-1, rates of apoptosis were, however, not significantly different from that for controls (Fig. 5), indicating that the protein moiety of Cd7MT-1 does not contribute to the development of apoptosis. With CdCl2, MT-1 and Cd7MT-1 rates of necrosis were not significantly different from that for controls (data not shown), similar to previously published reports (39).
|
Effect of Endo/Lysosomal Inhibitors on MT Immunoreactivity in Rat PT Cells Exposed to CdCl2 or Cd7MT-1
Basal MT levels were similar in controls and in cells treated with the endo/lysosomal inhibitors chloroquine (0.1 mM) or LY-294002 (5 µM) for 48 h, respectively, suggesting that the drugs do not affect basal MT expression in PT cells (data not shown). PT cells were also exposed to CdCl2 or Cd7MT-1 for 48 h, a period that demonstrated significant increase of MT levels with both conditions tested (see Fig. 4B), without or with coincubation with the drugs (Fig. 6). When cells were exposed to chloroquine or LY-294002, MT immunoreactivity induced by exposure to 10 µM CdCl2 for 48 h did not statistically differ from that measured in cells with no drug exposure. These values were, however, significantly different from the basal levels of MT found in cells with no Cd2+ exposure. After application of 1.4 µM Cd7MT-1 for 48 h, coincubation with chloroquine or LY-294002 significantly reduced MT immunoreactivity (both conditions: P < 0.01 compared with condition without drug). In line with these observations, coincubation of PT cells exposed to 109Cd7MT-1 with 0.1 mM chloroquine or 5 µM LY-294002 strongly reduced 109Cd2+ distribution in all membrane vesicle fractions investigated (see Fig. 3B).
|
Effect of Endo/Lysosomal Inhibitors on Apoptosis in Rat PT Cells Exposed to CdCl2 or Cd7MT-1
Inhibitors were tested in cells exposed to CdCl2 or Cd7MT-1 for 48 h because of the similar rates of apoptosis (see Fig. 5). As shown in Fig. 7, the increase of apoptosis induced by CdCl2 or Cd7MT-1 was statistically significant. Chloroquine (0.1 mM) did not affect basal levels of apoptosis, but it virtually abolished apoptosis induced by Cd7MT-1, whereas apoptosis induced by CdCl2 was not affected. With LY-294002 (5 µM), similar results were observed (Fig. 7).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apical uptake of free Cd2+. At the apical side, in vivo perfusion and studies with isolated PT segments or cell lines indicate that Cd2+ is avidly taken up at the luminal membrane in all three segments (S1, S2, and S3), but most rapidly in S1 segments (for review, see Refs. 14, 40, 41). In WKPT-0293 Cl.2 cells, Cd2+ uptake consisted of two components, similar to previous descriptions in LLC-PK1 cells (37). A temperature-insensitive component was likely caused by surface binding of 109Cd2+ to cell membranes, and the temperature-sensitive part represented intracellular transport. Significant 109Cd2+ uptake was observed at a time point as early as 3.5 h of exposure, increased linearly over a period of up to 24 h, and saturated at 40 h (Fig. 1). Uptake of free 109Cd2+ was not affected by chloroquine or LY-294002 (Fig. 2), which indicates that endo/lysosomal acidification and/or trafficking is not involved in the uptake process. It has been suggested that Cd2+ may cross the apical membrane through Ca2+ channels, but this evidence is derived from data in hepatocytes obtained with the use of inhibitors of L-type Ca2+ channels. In renal cells, the lack of effect of Ca2+ channel blockers argues against a significant contribution of Ca2+ channels in the renal transport and accumulation of Cd2+ (37) (Thévenod F, unpublished observations). Although in vitro studies suggest that Cd2+ may be transported into PT cells via Zn2+ and/or Cu2+ transporters (15), the identity of putative transport systems for free Cd2+ at the apical and basolateral plasma membrane of PT cells remains to be determined.
Apical uptake of Cd7MT-1 complex. Apical uptake of 109Cd7MT-1 was delayed compared with that of 109CdCl2 (Fig. 1), suggesting that the uptake of Cd7MT-1 by PT cells might involve several time-consuming steps. Current models assume that Cd7MT is taken up at the apical membrane of PT cells, particularly in the S1 and S2 segments (12), the site of Cd2+ nephrotoxicity, by what is thought to be receptor-mediated endocytosis (RME) (reviewed in Ref. 22) and then sorted to the lysosomal compartment. In a manner similar to PT reabsorption of other low-molecular-weight proteins (for review, see Ref. 8), uptake of Cd7MT is probably triggered by binding to the promiscuous multiligand receptor proteins megalin and cubilin, followed by endocytosis of the megalin-Cd7MT complex. Subsequent steps may involve pH-dependent release of megalin from its ligand in endosomes, recycling of megalin to the plasma membrane, and fusion of the Cd7MT-containing late endosomes with lysosomes. In the lysosomes, the MT moiety may be degraded by acidic proteases, which frees Cd2+ for transport into the cytoplasmic compartment. This model, for which evidence is still lacking, is nevertheless supported by data demonstrating that acute uptake of Cd7MT by the kidney in rats is blocked by low-molecular-weight proteins, including 2-microglobulin (2), a ligand that binds megalin (8). Application of the weak bases NH4Cl and chloroquine to dissipate the intraorganellar acidic pH, or use of the PI 3-kinase inhibitor LY-294002, which prevents endosomal receptor recycling and interferes with vesicular transport to lysosomes, strongly inhibited 109Cd7MT-1 uptake (Fig. 2), providing supporting evidence that apical uptake of Cd7MT takes place through RME. The colocalization of markers of the endo/lysosomal pathway (LAMP1, Rab5a) with 109Cd7MT-1 accumulation into membrane vesicles and the inhibition of 109Cd7MT-1 uptake by chloroquine and LY-294002 demonstrates that Cd7MT is transported into endosomes and lysosomes (Fig. 3).
Previous in vivo as well as in vitro studies on isolated rat kidney cortex endosomes have suggested that nephrotoxicity of Cd2+ and Cd7MT is partly mediated by impaired endo/lysosomal trafficking and vesicle recycling (19, 33) in PT cells and is caused by inhibition of endo/lysosomal V-ATPase by cytosolic Cd2+ (19). This is in contrast to the results of the present study, which demonstrates that endocytosis is unaffected in cells exposed to 1.4 µM Cd7MT-1 (Figs. 1, 2, 3) and is actually a prerequisite for apoptosis of PT cells to develop (Fig. 7). First, it could be argued that possible differences may exist between the models used (in vivo animal experiments vs. cultured cells). In addition, because of the high affinity of free Cd2+ to cytosolic proteins, such as GSH or MT, it has to be assumed that free cytosolic Cd2+ concentrations in cultured cells exposed to 1.4 µM Cd7MT-1 do not exceed submicromolar concentrations, whereas in the study by Sabolic and coworkers (19), Cd2+ inhibited endosomal acidification with a Ki of 25-50 µM. Nevertheless, the low free cytosolic Cd2+ concentrations obtained in our cultured PT cells are sufficient to induce significant levels of apoptosis within 40 h (Fig. 5). Our experimental in vitro model actually adequately mimics long-term animal models of chronic Cd2+ toxicity, where proximal tubule damage requires weeks to months to develop. Thus it is worth emphasizing that the toxic effects of Cd2+ (and Fanconi syndrome-associated features) appear to depend on RME of Cd7MT and not on an inhibitory effect of cytosolic Cd2+ on endo/lysosomal function.
A crucial step in the cascade of events leading to cellular toxicity induced by Cd7MT should be the release of free Cd2+ from the lysosomes into the cytosol. How does this transmembrane movement of Cd2+ into the cytosol occur? It is thought that Cd2+ is transported out of the endo/lysosome into the cytosol by a carrier-mediated process. The current dogma is that because Cd2+ is a nonessential metal, it must be transported by proteins with native ligands of similar chemical characteristics. The most likely candidates are proteins that translocate the essential divalent cations Fe2+, Cu2+, or Zn2+: the divalent metal transporter DMT1 (NRAMP2) translocates divalent cations (such as Cu2+, Fe2+, Zn2+, and also Cd2+), is proton coupled, and has been localized in endo/lysosomes of epithelial cells (17). Although DMT1 is expressed in PT cells, its cellular localization has not yet been determined with certainty, and its role in Cd2+ toxicity of PT cells remains to be investigated.
Origin of MT in PT Cells Exposed to Cd2+ or Cd7MT-1
Less than 10% of the Cd2+ taken up at the luminal membrane of PT is subsequently transported across the basolateral membrane, indicating that most of the transported Cd2+ is sequestered within the epithelial cells (41). Once taken up into PT cells, Cd2+ binds to small molecules (such as amino acids) or peptides, such as GSH, which contain a sulfhydryl group and protect the intracellular milieu from oxidative damage, or to proteins, such as MT. MT is thought to have several functions, including essential element homeostasis and toxic metal detoxification (22).
The kinetics of MT immunoreactivity in CdCl2-and Cd7MT-1-exposed cells and the inhibitor studies provide some clues as to the origin of intracellular MT. At early time points (4 h), MT immunoreactivity of cells exposed to Cd7MT-1 was increased (though not significantly) compared with background levels (Fig. 4B), which argues against de novo synthesis of MT and favors an exogenous source of MT. Furthermore, inhibition of the endo/lysosomal pathway significantly decreased MT immunoreactivity in PTC exposed to Cd7MT-1 (Fig. 6) and prevented accumulation of 109Cd7MT-1 into endo/lysosomes (Fig. 3B), suggesting that a main source of MT in these cells is endocytosed extracellular Cd7MT-1 (though induction of endogenous MT-1/MT-2 via free cytosolic Cd2+ released from endo/lysosomes may also contribute to cellular MT levels). In contrast, in cells treated with CdCl2, MT immunoreactivity was not different from that in controls after 4 h, which is in agreement with previous studies in PT cell lines showing induction of MT synthesis not earlier than 3-6 h after exposure to micromolar concentrations of CdCl2 at the apical cell side and maximal induction 24-48 h after treatment (13, 32). At 24 h, the increase of cellular MT levels widely exceeded that observed in PT cells exposed to Cd7MT-1. Moreover, chloroquine and LY-294002 had no effect on MT expression of PT cells exposed to CdCl2 (Fig. 6). The data therefore indicate that the source of MT in CdCl2-treated PT cells is unrelated to endo/lysosomal uptake. Because intracellular MT exists in at least three forms, cytosolic apo-MT, nuclear MT, and lysosomal MT, each displaying different turnover rates (35), it is possible that MT induced by cytosolic Cd2+ uptake rather represents induction of cytosolic and/or nuclear MT (see review, Ref. 9). Cytosolic uptake of Cd2+ may increase MT expression as the result of chemical induction of the MT gene through metal responsive elements (1). It is interesting to note that MT was also found expressed in the nucleus (Fig. 4A). Its function there is not completely clear; MTs may protect DNA against oxidative damage. MT protein has been found localized in the nucleus during S phase. The mRNA encoding the MT-1 isoform has a perinuclear localization and is associated with the cytoskeleton; this targeting, due to signals within the 3'-untranslated region, facilitates nuclear localization of MT-1 during S phase. Furthermore, recent evidence suggests that the perinuclear localization of MT mRNA is important for the function of MT in a protective role against DNA damage and apoptosis induced by external stress (9).
Apoptotic Processes in PT Cells Exposed to Cd2+ or Cd7MT-1
In cultured immortalized cells (WKPT-0293 Cl.2) from the S1 segment of the rat PT, we have previously demonstrated that application of CdCl2 (10 µM) induces apoptosis within 3-4 h, but not necrosis, by a mechanism involving production of ROS (39). We also observed a biphasic time dependence of Cd2+-induced apoptosis in rat PT cells exposed to 10 µM CdCl2: after rapid onset, Cd2+-dependent apoptosis decreased with prolonged Cd2+ application, which partly involved up-regulation of the multidrug transporter MDR-1 (39). Figure 6 shows confirmation of these observations by application of 10 µM CdCl2 to WKPT-0293 Cl.2 cells for up to 72 h.
It has been proposed that the Cd2+-MT protein complex is highly toxic to the kidney after renal tubular reabsorption and induces apoptosis of PT cells (21, 23, 24, 27). However, other in vivo studies and particularly in vitro studies with different kidney cell lines have been less conclusive: when HEK-293 cells were exposed to Cd2+-MT (10-59 µg/ml protein), apoptosis was observed after 48 h of exposure (18). In contrast, exposure of LLC-PK1 cells with up to 100 µM Cd2+-MT for 24 h failed to induce apoptosis, whereas 10 µM CdCl2 did induce apoptosis (20). The experiments of Fig. 5 reconcile this apparent discrepancy from the literature by demonstrating the critical importance of the time period of exposure with Cd7MT-1 in PT cell lines.
The delayed onset of apoptosis with Cd7MT-1 compared with the rapid induction of apoptosis with CdCl2, whereas no apoptosis was observed with MT-1, suggests that delivery of free Cd2+ into the cytosol is critical for development of apoptosis. It also indicates that toxicity induced by Cd7MT-1 might involve several time-demanding processes, as expected for Cd2+-MT uptake up at the apical membrane of PT cells by receptor-mediated endocytosis and sorting to the lysosomal compartment. When the effect of chloroquine and LY-294002, which interfere with endo/lysosomal trafficking and protein degradation, was tested on the rates of apoptosis induced by CdCl2 or Cd7MT-1, both drugs strongly reduced apoptosis induced by Cd7MT-1 but not by CdCl2 (Fig. 7). This finding suggests that induction of apoptosis by Cd7MT-1 in PT cells requires integrity of vesicular trafficking to lysosomes and adequate functional activity of lysosomal proteases. In contrast, both compounds did not affect cell death rates in cells exposed to CdCl2 (Fig. 7). Further studies on the cellular and molecular mechanisms involved in endocytosis and trafficking of the Cd7MT-1 complex in PT cells are required to provide a better understanding of Cd2+ (and other heavy metal) nephropathies.
![]() |
DISCLOSURES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bernard AM, Ouled Amor A, and Lauwerys RR. The effects of low doses of cadmium-metallothionein on the renal uptake of beta 2-microglobulin in rats. Toxicol Appl Pharmacol 87: 440-445, 1987.[ISI][Medline]
3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976.[ISI][Medline]
4. Brown WJ, De Wald DB, Emr SD, Plutner H, and Balch WE. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J Cell Biol 130: 781-796, 1995.[Abstract]
5. Bucci C, Wandinger-Ness A, Lutcke A, Chiariello M, Bruni CB, and Zerial M. Rab 5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells. Proc Natl Acad Sci USA 91: 5061-5065, 1994.[Abstract]
6. Chen JW, Murphy TL, Willingham MC, Pastan I, and August JT. Identification of two lysosomal membrane glycoproteins. J Cell Biol 101: 85-95, 1985.[Abstract]
7. Choudhuri S, McKim JM Jr, and Klaassen CD. Role of hepatic lysosomes in the degradation of metallothionein. Toxicol Appl Pharmacol 115: 64-71, 1992.[ISI][Medline]
8. Christensen EI and Birn H. Megalin and cubilin: multifunctional endocytic receptors. Nat Rev Mol Cell Biol 3: 256-266, 2002.[Medline]
9. Coyle P, Philcox JC, Carey LC, and Rofe AM. Metallothionein: the multipurpose protein. Cell Mol Life Sci 59: 627-647, 2002.[ISI][Medline]
10. Davidson HW. Wortmannin causes mistargeting of procathepsin D. Evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J Cell Biol 130: 797-805, 1995.[Abstract]
11. De Duve C, de Barsy T, Poole B, Trouet A, Tulkens P, and Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol 23: 2495-2531, 1974.[ISI][Medline]
12. Dorian C, Gattone VH 2nd, and Klaasen CD. Renal cadmium deposition and injury as a result of accumulation of cadmium-metallothionein (CdMT) by the proximal convoluted tubules: a light microscopic autoradiography study with 109CdMT. Toxicol Appl Pharmacol 114: 173-181, 1992.[ISI][Medline]
13. Felley-Bosco E and Diezi J. Cadmium uptake and induction of metallothionein synthesis in a renal epithelial cell line (LLCPK1). Arch Toxicol 65: 160-163, 1991.[ISI][Medline]
14. Friberg L, Elinder CG, Kjellstrom T, and Nordberg GF. Cadmium and Health: A Toxicological and Epidemiological Approach. Boca Raton, FL: CRC, 1986, vol. 1 and 2.
15. Gachot B and Poujeol P. Effects of cadmium and copper on zinc transport kinetics by isolated renal proximal cells. Biol Trace Elem Res 35: 93-103, 1992.[ISI][Medline]
16. Goyer RA and Cherian MG. Toxicology of Metals: Biochemical Aspects. Handbook of Experimental Pharmacology. New York, NY: Springer, 1995, vol. 15.
17. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388: 482-488, 1997.[ISI][Medline]
18. Hamada T, Sasaguri T, Tanimoto A, Arima N, Shimajiri S, Abe T, and Sasaguri Y. Apoptosis of human kidney 293 cells is promoted by polymerized cadmium-metallothionein. Biochem Biophys Res Commun 219: 829-834, 1996.[ISI][Medline]
19. Herak-Kramberger CM, Brown D, and Sabolic I. Cadmium inhibits vacuolar H+-ATPase and endocytosis in rat kidney cortex. Kidney Int 53: 1713-1726, 1998.[ISI][Medline]
20. Ishido M, Tohyama C, and Suzuki T. Cadmium-bound metallothionein induces apoptosis in rat kidneys, but not in cultured kidney LLC-PK1 cells. Life Sci 64: 797-804, 1999.[ISI][Medline]
21. Klaassen CD and Liu J. Role of metallothionein in cadmium-induced hepatotoxicity and nephrotoxicity. Drug Metab Rev 29: 79-102, 1997.[ISI][Medline]
22. Klaassen CD, Liu J, and Choudhuri S. Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 39: 267-294, 1999.[ISI][Medline]
23. Liu J, Habeebu SS, Liu Y, and Klaassen CD. Acute CdMT injection is not a good model to study chronic Cd nephropathy: comparison of chronic CdCl2 and CdMT exposure with acute CdMT injection in rats. Toxicol Appl Pharmacol 153: 48-58, 1998.[ISI][Medline]
24. Liu J, Liu Y, Habeebu SS, and Klaassen CD. Susceptibility of MT-null mice to chronic CdCl2-induced nephrotoxicity indicates that renal injury is not mediated by the CdMT complex. Toxicol Sci 46: 197-203, 1998.[Abstract]
25. McKim JM Jr, Choudhuri S, and Klaassen CD. In vitro degradation of apo-, zinc-, and cadmium-metallothionein by cathepsins B, C, and D. Toxicol Appl Pharmacol 116: 117-124, 1992.[ISI][Medline]
26. Mehlhase J and Grune T. Proteolytic response to oxidative stress in mammalian cells. Biol Chem 383: 559-567, 2002.[ISI][Medline]
27. Nordberg GF, Goyer R, and Nordberg M. Comparative toxicity of cadmiummetallothionein and cadmium chloride on mouse kidney. Arch Pathol 99: 192-197, 1975.[ISI][Medline]
28. Nordberg M, Jin T, and Nordberg GF. Cadmium, metallothionein and renal tubular toxicity. IARC Sci Publ 118: 293-297, 1992.[Medline]
29. O'Brien P and Salacinski HJ. Evidence that the reactions of cadmium in the presence of metallothionein can produce hydroxyl radicals. Arch Toxicol 72: 690-700, 1998.[ISI][Medline]
30. Oikawa S, Kurasaki M, Kojima Y, and Kawanishi S. Oxidative and nonoxidative mechanisms of site-specific DNA cleavage induced by copper-containing metallothioneins. Biochemistry 34: 8763-8770, 1995.[ISI][Medline]
31. Petering DH and Fowler BA. Roles of metallothionein and related proteins in metal metabolism and toxicity: problems and perspectives. Environ Health Perspect 65: 217-224, 1986.[ISI][Medline]
32. Rodilla V, Miles AT, Jenner W, and Hawksworth GM. Exposure of cultured human proximal tubular cells to cadmium, mercury, zinc and bismuth: toxicity and metallothionein induction. Chem Biol Interact 115: 71-83, 1998.[ISI][Medline]
33. Sabolic I, Ljubojevic M, Herak-Kramberger CM, and Brown D. Cd-MT causes endocytosis of brush-border transporters in rat renal proximal tubules. Am J Physiol Renal Physiol 283: F1389-F1402, 2002.
34. Simonsen A, Wurmser AE, Emr SD, and Stenmark H. The role of phosphoinositides in membrane transport. Curr Opin Cell Biol 13: 485-492, 2001.[ISI][Medline]
35. Steinebach OM and Wolterbeek BT. Metallothionein biodegradation in rat hepatoma cells: a compartmental analysis aided 35S-radiotracer study. Biochim Biophys Acta 1116: 155-165, 1992.[ISI][Medline]
36. Takebayashi S, Jimi S, Segawa M, and Kiyoshi Y. Cadmium induces osteomalacia mediated by proximal tubular atrophy and disturbances of phosphate reabsorption: A study of 11 autopsies. Pathol Res Pract 196: 653-663, 2000.[ISI][Medline]
37. Templeton DM. Cadmium uptake by cells of renal origin. J Biol Chem 265: 21764-21770, 1990.
38. Thévenod F and Friedmann JM. Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K+-ATPase through proteasomal and endo-/lysosomal proteolytic pathways. FASEB J 13: 1751-1761, 1999.
39. Thévenod F, Friedmann JM, Katsen AD, and Hauser IA. Upregulation of multidrug resistance P-glycoprotein via NFB activation protects kidney proximal tubule cells from cadmiumand reactive oxygen species-mediated apoptosis. J Biol Chem 275: 1887-1896, 2000.
40. Wedeen RP and De Broe ME. Heavy metals and the kidney. In: Oxford Textbook of Clinical Nephrology, edited by Davison AM. Oxford, UK: Oxford University Press, 1998, vol. 2, p. 1175-1189.
41. Zalups RK and Koropatnick J. Molecular Biology and Toxicology of Metals. London: Taylor & Francis, 2000.