HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by THÉVENOD, F. Articles by FRIEDMANN, J. M. Search for Related Content PubMed PubMed Citation Articles by THÉVENOD, F. Articles by FRIEDMANN, J. M.

Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na⁺/K⁺-ATPase through proteasomal and endo-/lysosomal proteolytic pathways

II. Department of Physiology, University of Saarland, D-66421 Homburg, Germany

¹Correspondence: II. Physiologisches Institut, Universität des Saarlandes, Medizinische Fakultät, D-66421 Homburg/Saar, Germany. E-mail: frank.thevenod{at}med-rz.uni-sb.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The mechanisms of cadmium (Cd) -dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl₂ (5 µM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24–48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4±1.6%; Cd: 8.1±1.9%), but not necrosis (controls: 0.5 ± 0.3%; Cd: 1.4± 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (1) of Na⁺/K⁺-ATPase, a long-lived membrane protein (t_1/2>48 h) that drives reabsorption of ions and nutrients through Na⁺-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na⁺/K⁺-ATPase 1-subunit, as determined by immunoblotting, by 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 µM) or lactacystin (10 µM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH₄Cl (30 mM), significantly reduced the decrease of Na⁺/K⁺-ATPase 1-subunit induced by Cd, and in combination abolished the effect of Cd on Na⁺/K⁺-ATPase. Immunofluorescence labeling of Na⁺/K⁺-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na⁺/K⁺-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na⁺/K⁺-ATPase may contribute to the `Fanconi syndrome'-like Na⁺-dependent transport defects associated with Cd-nephrotoxicity.—Thévenod, F., Friedmann, J. M. Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na⁺/K⁺-ATPase through proteasomal and endo-/lysosomal proteolytic pathways.

Key Words: nephrotoxicity • heavy metals • oxygen radicals • Fanconi syndrome • ubiquitin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CADMIUM (Cd),² is a well-known environmental hazard with a potent nephrotoxic action. The proximal tubule of the mammalian kidney is a major target of chronic Cd-induced toxicity (1) . Cd-induced nephrotoxicity is thought to be mediated through the Cd-metallothionein (CdMT) complex, which is synthesized in the liver, released into the circulation, and taken up by renal tubule cells. In the kidney, CdMT is degraded in lysosomal compartments, and the released Cd stimulates the intracellular synthesis of heavy-metal detoxifying peptides, such as MT. Renal damage is believed to occur once the Cd concentration in the kidney cortex reaches or exceeds the binding capacity of heavy metal binding proteins (2) . Still, the mechanisms underlying Cd nephrotoxicity remain poorly understood, both at the cellular level and in vivo (3) . Clinically, Cd nephropathy resembles acquired Fanconi's syndrome, i.e., massive polyuria, glucosuria, aminoaciduria, proteinuria, calciuria and phosphaturia (1) , which are caused by a number of reabsorptive and secretory transport defects, mainly of Na⁺-dependent transporters (4) .

One of these transporters is the Na⁺/K⁺-ATPase, an integral membrane protein that directly couples the hydrolysis of ATP to the vectorial transport of Na⁺ and K⁺ across the plasma membrane. This in turn produces the electrochemical gradient of Na⁺ and K⁺, which is the primary source of energy for the active transport of various inorganic ions and small organic molecules (5) . In polarized transport epithelia, as in the proximal tubule, Na⁺/K⁺-ATPase is located at the basolateral plasma membrane, where it carries out vectorial Na⁺ transport and thereby provides the gradient for transepithelial fluxes of various ions, nutrients, metabolites and xenobiotics via Na⁺-dependent transporters (6) .

Na⁺/K⁺-ATPase consists of a catalytic subunit () with an M_r of 100,000 and a smaller glycoprotein subunit (ß) with an M_r of ~55,000 (7 , 8) . The catalytic subunit of Na⁺/K⁺-ATPase belongs to a stable class of membrane proteins (half-life t_1/224–48 h) (9) . Damage to this enzyme will therefore have a major impact on basic cellular functions and lead to secondary dysfunction of other Na⁺-dependent transporters, even if these transporters remain undamaged or are able to turn over rapidly.

In eukaryotic cells, the rates of intracellular protein degradation appear to be controlled differentially for individual proteins (10) . Relatively long-lived proteins with half-lives of more than 20 h coexist with extremely short-lived proteins (t_1/21 min), whose rapid turnover is part of the machinery regulating cellular function. In addition, damaged or otherwise abnormal proteins are also degraded in vivo. For instance, reactive oxygen species resulting from oxidative stress promote structural changes or misfolding of cellular proteins (11) ; to overcome the potentially toxic accumulation of oxidatively modified proteins, cells increase the rates of proteolysis of these abnormal proteins (12) .

Two major pathways are available for selective protein degradation: the lysosomal proteases and the proteasome complex (13) . Proteins that enter cells from the outside via endocytosis or phagocytosis are cleaved within lysosomes; lysosomes also play a role in the degradation of long-lived proteins and transmembrane proteins (14 15 16) . The main turnover of short-lived proteins, cytosolic proteins, and proteins originating from the endoplasmic reticulum is conducted by the proteasome complex (17 18 19) . In this pathway, proteins are marked for rapid degradation by conjugation with multiple molecules of ubiquitin, which leads to their rapid hydrolysis by the 26S proteolytic complex. The proteolytic core of this structure is the 20S (700 kDa) proteasome, which contains multiple peptidase activities that function together in proteolysis (20 , 21) . Abnormal proteins are thus marked for rapid disposal (13 , 22 , 23) .

Previous in vitro studies on purified Na⁺/K⁺-ATPase indicated that oxidants may inactivate enzyme function (24 , 25) as well as induce a higher susceptibility to degradation by intracellular proteinases, such as lysosomal cathepsin D or calpains (26) . We therefore hypothesized that Cd-induced nephrotoxicity might be caused by oxidative stress and increased proteolytic degradation of oxidatively modified Na⁺/K⁺-ATPase. In the present study, we used a rat kidney proximal tubule cell line as an in vitro model of Cd-induced nephrotoxicity to investigate the effect of Cd on the production of reactive oxygen species and protein stability of the 1-subunit of Na⁺/K⁺-ATPase and to identify putative cellular mechanisms underlying the proteolytic degradation of damaged Na⁺/K⁺-ATPase proteins in proximal tubule cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials
The following reagents were obtained from the listed sources and used at the concentrations indicated in the text. Stock solutions of lactacystin (Calbiochem, Bad Soden, Germany) and N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-leucinal (MG-132; Biomol, Hamburg, Germany) were made by solubilization in dimethyl sulfoxide (DMSO). Rat tail collagen type I (Sigma, Deisenhofen, Germany) was dissolved in 100 mM acetic acid. Horseradish peroxidase-conjugated sheep-anti-mouse and donkey-anti-rabbit immunoglobulin G (IgG) and enhanced chemiluminescence reagents were purchased from Amersham-Buchler (Braunschweig, Germany). Donkey anti-mouse IgG coupled to indocarbocyanin (CY3) was obtained from Dianova (Hamburg, Germany). Nonfat dry milk and prestained protein standards were from Bio-Rad (Munich, Germany). Polyvinylidene difluoride (PVDF) membranes were from NEN-Dupont (Bad Homburg, Germany). The thiol antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate, and the DNA dyes ethidium bromide (EB) and Hoechst 33342 were from Sigma. All other substances were from commercial sources and of analytical grade.

Cell culture
Immortalized cells (WKPT-0293 Cl.2) of the S1 segment of the proximal tubule of normotensive Wistar-Kyoto rats (RPTC) were cultured as described earlier (27 , 28) . Briefly, cells were maintained in renal tubular epithelium medium composed of Dulbecco's modified Eagle's medium: F-12 [nutrient mixture F-12 (Ham)] 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 at a density of 5 x 10⁴/ml on rat tail collagen type I (125 µg/ml 100 mM acetic acid) -coated flasks, passaged at 80% confluency, and split 1:10 twice a week. For inhibitor studies, cells were pretreated for 2 h with inhibitors of the proteasome (lactacystin, MG-132), of lysosomal proteases (chloroquine, NH₄Cl) or N-acetylcysteine before addition of CdCl₂ for the times indicated. Medium, inhibitors, and CdCl₂ were replaced every 24 h during the stress period.

Fluorescence measurements
Chambers containing cells grown on glass coverslips were placed on an inverted microscope (IMT2-F, Olympus) with a x20, 0.85 objective (Fluor 20 or 40, Nikon). A variable monochromatic light source (Till Photonics, Munich, Germany) was coupled to the microscope with a light guide. Images were recorded with an integrating slow scan camera (Theta System, Munich, Germany) and digitized to 8 bits/pixel (29) . Camera and scanner were controlled by a TILLvisION v3.02 software, which was also used to analyze the stored images. For evaluation of individual cells, regions of interest were defined on an image and the fluorescence intensities were expressed as mean gray values per 200 ms exposure time per area. Comparable areas were selected in different experiments. The excitation wavelength used for measurements of rhodamine 123 fluorescence was 485 nm. Emitted light was collected with the appropriate dichroic mirrors.

Determination of reactive oxygen species (ROS) with DHRh123
Cells grown on collagen-coated coverslips were incubated for 4–8 h in Tyrode solution containing 5 µM CdCl₂ or 5 µM CdCl₂ + 15 mM N-acetylcysteine in the presence of dihydrorhodamine 123 (DHRh123) (2 µM) (Molecular Probes, Eugene, Oreg.). DHRh123 was dissolved in DMSO. During the cellular production of ROS, the intracellular DHRh123 was irreversibly converted to the green fluorescent compound rhodamine 123 (Rh123⁺) (500–540 nm). Rh123⁺, being membrane impermeable, accumulates in the cells. Images were taken before addition of 5 µM CdCl₂ and after washing out the incubation solution twice with Tyrode solution.

Detection of apoptosis and necrosis by fluorescence microscopy
4 x 10⁴ cells were seeded onto 35 mm tissue culture dishes and grown for 3 days before experiments began. Cells were stained intravitally with the DNA dyes Hoechst 33342 (H-33342; 2 µg/ml) and ethidium bromide (5 µg/ml), as described by Sun et al. (30) with minor modifications. Under UV epi-illumination (_ex: 330–380 nm; _em: >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 lost their plasma membrane integrity (i.e., underwent 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 (200x magnification) were counted per dish. Cell proliferation was estimated semiquantitatively by counting cell numbers and mitotic figures.

Antibodies
The monoclonal antibody to the 1-subunit of Na⁺/K⁺-ATPase (purified rabbit kidney Na⁺/K⁺-ATPase) was obtained from Upstate Biotechnology (Hamburg, Germany). The rabbit polyclonal antiserum to ubiquitin was from Sigma.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting
After washing three times with phosphate-buffered saline (PBS), WKPT-0293 Cl.2 cells were scraped off the culture flasks with a rubber policeman, centrifuged, and the cell pellet was resuspended in 100 µl of a buffer containing (in mM) mannitol 280, HEPES 10, EDTA 5, MgSO₄ 0.1, Pefabloc 0.2, and 10 µg/ml leupeptin (pH 7.0 adjusted with Tris). Cells were solubilized by addition of 50 µl of 3x concentrated SDS sample buffer and sonicated for 30 s on ice. After centrifugation at 12,000 x g for 5 min, the protein content of the supernatant was determined by the Lowry method. After incubation of samples at 37°C for 30 min, 50 µg of membrane protein was loaded onto each lane of the gel. Electrophoresis and blotting procedures were performed essentially as described earlier (28 , 31) . Proteins were separated by SDS-PAGE on 7.5% acrylamide Laemmli minigels and transferred onto PVDF membranes overnight. The efficiency of protein transfer was monitored with prestained protein standards. Blots were blocked with 3% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 6 h and incubated overnight at 4°C with primary antibodies against Na⁺/K⁺-ATPase (0.5 µg/ml) or ubiquitin (1:100). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:6,000 dilution) for 60 min at 4°C, blots were developed in enhanced chemiluminescence reagents and signals were visualized on X-ray films. X-ray films were scanned with a single pass flat-bed scanner (Linotype-Hell, Eschborn, Germany) and processed for documentation using a JASC Paint Shop Pro 4.1 software (Jameln, Germany). Signals from different experiments were scanned and the intensity (optical density) of the chemiluminescence signals was quantified on a Bioprofil computer assisted imaging and scanning system (Vilber-Lourmat; Marne La Vallée, France).

Detection of Na^+/K⁺-ATPase by indirect immunofluorescence
WKPT-0293 Cl.2 cells grown on glass coverslips were fixed in 4% paraformaldehyde/PBS for 30 min at room temperature. All subsequent steps were also carried out at room temperature. Cells were rinsed 3 x 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 anti-Na⁺/K⁺-ATPase antibody (5 µg/ml) on Parafilm 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) for 30 min. After three more rinses for 5 min in PBS, coverslips were mounted in a medium consisting of 50% Tris^.HCl (pH 8.0), 50% glycerol, and 4% n-propyl gallate as an antiquenching agent. They were examined using an Olympus BX50F microscope equipped with a 40x Olympus UPlanFI objective and a narrow band green fluorescence exciter filter ( 530–550 nm). Images were recorded with a 3CCD color video camera (Sony DXC-950) and digitized to 8 bits/pixel using a software (`µ-Slicer') developed by Professor B. Lindemann (II. Department of Physiology, University of Saarland, Homburg/Saar, Germany). Digitized images were processed for documentation using an Adobe Photoshop D1–4.0 software (Herzogenaurach, Germany). The brightness and contrast of the images used in this study were enhanced by 20% and 40%, respectively.

Statistics
All experiments were repeated at least three times with different batches of cell preparations, and representative data or means ± SD are shown. Statistical analysis was carried out with the Statgraphics program using unpaired Student's t test. Results with levels of P < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cadmium induces the production of ROS in proximal tubule cells (WKPT 0293 Cl.2)
Cd is not a Fenton metal and therefore cannot generate reactive oxygen species by itself (32) . However, Cd does elevate lipid peroxidation in tissues soon after exposure (see ref 33 for review). Moreover, free radical scavengers and antioxidants protect against Cd toxicity, suggesting that Cd does increase radical formation, though indirectly (33) . To measure Cd-induced ROS formation, immortalized cells (WKPT 0293 Cl.2 from the S1 segment of rat proximal tubule) were loaded with DHRh123 (2 µM) and incubated with 5 µM CdCl₂ for various time periods. The dye was washed out from the medium before analysis of fluorescence images. Though ROS may be generated at earlier time points, significant increases of Rh123⁺ fluorescence were not detected earlier than 4–8 h after addition of CdCl₂ (Fig. 1 ). To further investigate the hypothesis of Cd-induced ROS formation, two different thiol antioxidants, N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), were tested on the potential of Cd to induce ROS production: NAC can raise intracellular glutathione levels and thereby protect cells from the effects of ROS (34) . In addition, the SH-group of the agent can react directly with radicals. PDTC (35) is also a potent chelator of various metals, including Cd (36) . Both NAC (15 mM) (Fig. 1B ) and PDTC (0.1 mM) (data not shown), when incubated together with Cd for 8 h, prevented the increase of Rh123⁺ fluorescence induced by Cd alone.

View larger version (36K): [in this window] [in a new window]   Figure 1. Effect of CdCl2 on the production of reactive oxygen species in rat proximal tubule cells. A) Transmission light and fluorescence micrographs of cultured rat proximal tubule cells (WKPT-0293 Cl.2) incubated with the probe for reactive oxygen species (ROS), dihydrorhodamine 123 (DHRh123; 2 µM), in the absence or presence of 5 µM CdCl2 for 8 h. B) Statistical analysis of 3 different experiments also showing the effect of the thiol-antioxidant N-acetylcysteine (NAC; 15 mM) on the production of ROS. NAC prevents conversion of nonfluorescent DHRh123 to green fluorescent Rh123+ by 5 µM CdCl2. Means ± SD. *P 0.01 using unpaired Student's t test.

Cadmium induces apoptosis but not necrosis in proximal tubule cells (WKPT 0293 Cl.2)
Incubation of cells with micromolar Cd concentrations for up to 72 h raises the legitimate question of whether under the experimental conditions selected in this study, proximal tubule cells are still viable and able to activate their protein synthesizing and modifying machineries. Cd-induced proteinuria, glucosuria, calciuria, and aminoaciduria are thought to result from proximal tubule cell necrosis (1) . But Cd is also known to induce apoptosis in renal tubule cells (3 , 37) when used at low concentrations that do not lead to rapid metabolic collapse and necrosis. By subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent ethidium bromide, apoptotic cells, whose plasma membrane integrity is still intact, could easily be distinguished from cells that underwent necrosis and therefore can be labeled by the plasma membrane impermeable charged dye ethidium bromide (30) . Incubation of proximal tubule (PT) cells with 10 µM CdCl₂ for 20 h induced typical features of apoptosis such as chromatin condensation, fragmentation, and the appearance of apoptotic bodies (see Fig. 2 A). The basal rate of apoptosis under control conditions of 2.4 ± 1.6% was significantly increased by Cd treatment (8.1±1.9%; P0.001). Increased apoptosis was also observed after incubation of PT cells with 10 µM CdCl₂ for 72 h (controls: 2.0±0.4%; Cd: 4.0±0.6%; P0.001), though the percentage of apoptotic cells was smaller compared to Cd exposure for 20 h (Fig. 2B ). In contrast, the percentage of necrotic cells was not increased by Cd exposure either at 20 h (controls: 0.5±0.3%; Cd: 1.4±2.5%) or 72 h (controls: 0.2±0.1%; Cd: 0.3±0.3%). These data clearly show that at the concentrations of Cd selected in this study, the rates of cell death (apoptosis+necrosis) were significantly increased, but never exceeded 10%. Moreover, Cd did not affect cell proliferation, as estimated by comparison of cell numbers and percentage of mitotic figures (data not shown).

View larger version (67K): [in this window] [in a new window]   Figure 2. Effect of CdCl2 on the rates of apoptosis and necrosis in rat proximal tubule cells. A) Cells were stained with 5 µg/ml ethidium bromide and 2 µg/ml Hoechst 33342, as described in Materials and Methods, and photographed under phase contrast and fluorescence microscopy. The control photograph shows vital cells stained blue by Hoechst 33342. With CdCl2 (10 µM), typical features of apoptosis can be seen: fragmentation (arrow) and condensation (arrowheads) (bar =30 µM). B) Statistical analysis of 6–12 different experiments analyzed as described in Materials and Methods. The rate of apoptosis is significantly increased by incubation with 10 µM CdCl2 for 20 or 72 h. The rate of necrosis is not affected by CdCl2 treatment. Means ± SD. *P 0.001 using unpaired Student's t test.

Cd induces a time-dependent increase of ubiquitin-protein conjugates in WKPT 0293 Cl.2 cells
Cd-dependent formation of ROS may provoke oxidative breaks or misfolding of proteins, which may then be targeted for degradation by the ubiquitin-proteasome proteolytic pathway. Therefore, we examined whether Cd incubation of proximal tubule cells leads to an accumulation of ubiquitinated proteins. As shown in Fig. 3 A, B, the levels of ubiquitin conjugates increased ~twofold after 24 h and ~sixfold after 72 h of Cd incubation. Coincubation with 15 mM NAC, which prevents the generation of ROS (see Fig. 1B ), strongly reduced the ubiquitination levels of cellular proteins (Fig. 3) .

View larger version (40K): [in this window] [in a new window]   Figure 3. Effect of N-acetylcysteine on CdCl2-induced ubiquitin-conjugation of cellular proteins in rat proximal tubule cells. A) Immunoblots showing ubiquitinated proteins (brackets) in lysates of WKPT-0293 Cl.2 cells (50 µg of protein/lane). Cells were incubated for 1–72 h without, with 5 µM CdCl2, or with CdCl2 (5 µM) + N-acetylcysteine (NAC; 15 mM). B) Quantitative analysis of immunoblots shown in panel A. Changes in the levels of ubiquitinated proteins detected in cells treated for different time periods without or with 5 µM CdCl2 or CdCl2 (5 µM) + N-acetylcysteine (NAC; 15 mM) are shown. Ubiquitination levels with no added CdCl2 (0 h) were set to 100%. Data are means ± SD of three identical experiments for each condition tested. Quantification of the immunoblots was by image analysis as described in Materials and Methods.

Cd enhances the degradation of Na⁺/K⁺-ATPase in WKPT 0293 Cl.2 cells
The 1-isoform of Na⁺/K⁺-ATPase is expressed in WKPT 0293 Cl.2 cells (Fig. 4 and Fig. 6 ). After addition to the culture medium of 10 µg/ml cycloheximide, which blocks protein synthesis, protein levels of the 1-subunit of Na⁺/K⁺-ATPase did not change significantly over 72 h (Fig. 4A ) and were similar to those of control cells without cycloheximide (not shown), confirming that Na⁺/K⁺-ATPase belongs to the class of long-lived proteins (9) . In contrast, in cells that had been incubated for up to 72 h with 5 µM CdCl₂, the 1-subunit of the Na⁺/K⁺-ATPase decreased to ~25% of the control level after 72 h incubation (Figs. 4 B, C). This indicates that Cd has induced proteolytic degradation of the 1-subunit. When the scavenger of ROS, NAC (15 mM), was coincubated with Cd, it largely prevented the decrease of 1-subunit expression observed with Cd alone, thus underscoring the role of ROS in Cd-induced degradation of Na⁺/K⁺-ATPase.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effect of CdCl2 on protein expression of the 1-subunit of Na+/K+-ATPase in rat proximal tubule cells. A) Protein levels of the 1-subunit of Na+/K+-ATPase after addition of the inhibitor of protein synthesis, cycloheximide (10 µg/ml), during a period of 72 h. A single protein band of ~100 kDa was labeled by the antibody to the 1-subunit of Na+/K+-ATPase. One out of four similar experiments. B) Immunoblots showing the effect of 5 µM CdCl2 or 5 µM CdCl2 plus 15 mM N-acetylcysteine (NAC) on the expression of the 1-subunit of Na+/K+-ATPase in rat proximal tubule cells during a period of 72 h. C) Statistical analysis of three similar experiments to that shown in panel B. Expression (optical density) of the 1-subunit in the absence of CdCl2 (0 h) was set to 100%. Data are means ± SD for each condition tested. Quantification of the immunoblot was by image analysis as described in Materials and Methods. An asterisk identifies the values that are significantly different from cells incubated with CdCl2, with P 0.05 using unpaired Student's t test.

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of the proteasomal inhibitor lactacystin and of the lysosomotropic drug chloroquine on protein expression of the 1-subunit of Na+/K+-ATPase in Cd-treated rat proximal tubule cells. A) Immunoblots showing protein levels of the 1-subunit of Na+/K+-ATPase in lysates of WKPT-0293 Cl.2 cells (50 µg of protein/lane). Cells were coincubated for up to 72 h with 5 µM CdCl2 and either 10 µM lactacystin, 0.2 mM chloroquine, or a combination of both drugs. B) Statistical analysis of three similar experiments to that shown in panel A. Changes in the expression levels of the 1-subunit of Na+/K+-ATPase detected in cells incubated for different periods with 5 µM CdCl2 in the absence or presence of inhibitors of the proteasome and/or lysosomes. Protein levels (optical density) of the 1-subunit in the absence of CdCl2 (0 h) were set to 100%. Data are means ± SD for each condition tested. Quantification of the immunoblot was by image analysis as described in Materials and Methods. *Values significantly different from cells incubated with CdCl2 alone, with P 0.05 using unpaired Student's t test.

Degradation of Na⁺/K⁺-ATPase is sensitive to proteasomal and endo/lysosomal inhibitors
Degradation of ubiquitinated proteins is carried out by either the proteasome (usually degradation of cytosolic or misfolded proteins in the endoplasmic reticulum, but also oxidatively damaged proteins) (15 , 20 21 22 , 25 , 26) or the endo-/lysosomal system in the case of several transmembrane proteins (17 18 19) . In the past few years, various agents have been identified as useful tools for the study of the proteasome (26) . Several peptide aldehydes, including MG-132, reversibly inhibit the peptidase activities of the 20S proteasome in vitro and in intact cells. However, the peptide aldehydes also inhibit the function of the lysosomal cysteine proteinases (e.g., cathepsin B, H, and L) and the Ca²⁺-activated proteinases (23 , 26) . In contrast, a highly specific, irreversible inhibitor of the 20S proteasome, lactacystin (38) , has not been found to inhibit any other known protease. When the potent proteasome inhibitors lactacystin (10 µM) or MG-132 (20 µM) were added during Cd stress, the levels of ubiquitin-conjugated cellular proteins were strongly and significantly enhanced at all time points 6 h (compare Fig. 3 and Fig. 5 and data not shown), whereas degradation of Na⁺/K⁺-ATPase was strongly reduced (Fig. 6 and Table 1 ). This suggests that the proteasome is responsible for both degradation of the bulk of Cd stress-induced ubiquitin-conjugated proteins and is involved in Cd-dependent proteolysis of Na⁺/K⁺-ATPase. Chloroquine (0.2 mM) or NH₄Cl (30 mM)—weak bases that dissipate the late endo-/lysosomal acidic pH, thereby inhibiting lysosomal protease activity in these compartments—also led to a significant inhibition of Na⁺/K⁺-ATPase degradation (see Fig. 6 and Table 1 ). However, though chloroquine moderately increased the levels of ubiquitin-conjugated proteins induced by Cd in some experiments (compare Fig. 3A and Fig. 5A ), the levels of ubiquitinated cellular proteins in Cd-treated or Cd plus chloroquine-treated cells were not statistically different (compare Fig. 3B and Fg. 5B). This suggests that though the majority of ubiquitinated proteins is not degraded by the endo-/lysosomal pathway, oxidatively damaged Na⁺/K⁺-ATPase molecules are also degraded by endo-/lysosomes. When both, chloroquine (0.2 mM) and lactacystin (10 µM) were coincubated with Cd, degradation of the Na⁺/K⁺-ATPase was abolished (Fig. 6) .

View larger version (38K): [in this window] [in a new window]   Figure 5. Effects of the proteasomal inhibitor lactacystin and of the lysosomotropic drug chloroquine on ubiquitin-conjugation of proteins from Cd-exposed rat proximal tubule cells. A) Immunoblots showing ubiquitin-conjugated proteins (brackets) in lysates of WKPT-0293 Cl.2 cells (50 µg of protein/lane). Cells were coincubated for up to 72 h with 5 µM CdCl2 and either 10 µM lactacystin or 0.2 mM chloroquine. B) Quantitative analysis of immunoblots shown in panel A. Data are means ± SD of four similar experiments for each condition tested. Shown are the changes in the levels of ubiquitinated proteins detected in cells treated for different periods of time with 5 µM CdCl2 + 10 µM lactacystin or 5 µM CdCl2 + 0.2 mM chloroquine. The values are shown as percentage of condition without drugs. Quantification of the immunoblot was as described in Materials and Methods.

View this table: [in this window] [in a new window]   Table 1. Effects of proteasomal and lysosomal inhibitors on CdCl2-induced degradation of the 1-subunit of Na+/K+-ATPasea

Cd-induced decrease of Na⁺/K⁺-ATPase expression in the plasma membrane of WKPT 0293 Cl.2 cells involves retrieval of the enzyme into intracellular compartments
To characterize further Cd-induced damage of the 1-subunit of Na⁺/K⁺-ATPase of PT cells and the cellular degradation pathways involved, cells were incubated with Cd or with Cd plus proteasomal and endo-/lysosomal inhibitors for 48 h, as described in Figs. 3 and 5 , and immunofluorescence labeling of the 1-subunit of Na⁺/K⁺-ATPase of PT cells was carried out in situ. As shown in the immunofluorescent images of Fig. 7 , immunolabeling of Na⁺/K⁺-ATPase was found at the plasma membrane of control WKPT 0293 Cl.2 cells. The expression of Na⁺/K⁺-ATPase in the plasma membrane was strongly reduced in most cells that had been exposed to Cd for 48 h, both in the absence (Fig. 7B ) and presence of lactacystin plus chloroquine (Fig. 7C ), compared to control cells (Fig. 7A ). However, Cd-exposed cells that had been coincubated with chloroquine and lactacystin developed a different structure, clearly showing a round shape and a distinct accumulation of immunoreactive material in intracellular vesicular structures. This indicates that after Cd exposure, damaged Na⁺/K⁺-ATPase molecules are endocytosed and routed toward endo-/lysosomal degradation.

View larger version (30K): [in this window] [in a new window]   Figure 7. Localization of the 1-subunit of Na+/K+-ATPase by indirect immunofluorescence in controls (A) and in Cd-exposed PT cells without (B) or with lactacystin and chloroquine (C). WKPT-0293 Cl.2 cells grown on glass coverslips were studied at subconfluency. Cells were treated for 48 h without or with CdCl2 (5 µM), lactacystin (10 µM), and chloroquine (0.2 mM). Magnification = 400x.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The cellular processes underlying Cd nephrotoxicity are poorly understood: Cd reacts with thiol groups and may substitute for zinc in critical metabolic processes (39) , but it also causes DNA strand breaks, lipid peroxidation, and generation of oxidatively modified proteins (33 , 40 , 41) . Cd, not being a Fenton metal, does not appear to generate free radicals by itself (33) , but it has been shown to produce hydroxyl radicals in the presence of metallothioneins, containing Fenton metals (32) . This suggests that Cd-mediated production of ROS takes place as a consequence of Cd-induced displacement of endogenous redox active metals (Fe, Cu) and subsequent damage to critical organelles (e.g., mitochondria) or is due to a decrease of endogenous radical scavengers, such as glutathione and/or protein sulfhydryls (33) . What, then, is the mechanism underlying the damaging effect of Cd on renal Na⁺-dependent membrane transporters? Though transfection and in vitro studies with isolated renal brush-border membrane vesicles have shown a direct inhibitory effect of Cd on the functional activity of various Na⁺-dependent membrane transporters with an IC₅₀ of ~50–300 µM (42 , 43) as well as ROS-mediated inhibition of Na⁺/K⁺-ATPase function within hours (24) (see also Fig. 1 ), the present data rather demonstrate that low micromolar concentrations of Cd may already decrease Na⁺-dependent transport in proximal tubule cells by increasing the proteolysis of Na⁺/K⁺-ATPase through the proteasome system and lysosomal proteases.

A major factor determining protein stability is the amino-terminal residue (`N-end rule') (44) . We may speculate that subsequent to Cd-induced oxidative damage, those Na⁺/K⁺-ATPase protein molecules may be targeted to selective degradation by the proteasome whose amino-terminal residues have been cleaved so as to expose a destabilizing residue at the amino terminus (45) . The participation of the proteasome in the degradation of Cd-induced, potentially toxic oxidatively modified proteins, as implied in the present study for Na⁺/K⁺-ATPase, is supported by the observation in yeasts that mutants in the proteasome are Cd hypersensitive (46) .

Cd-induced degradation of Na⁺/K⁺-ATPase was reduced by the inhibitors of the lysosomal pathway chloroquine (0.2 mM) and NH₄Cl (30 mM) (Fig. 6 and Table 1 ), suggesting that in Cd-exposed cells, degradation of oxidatively modified Na⁺/K⁺-ATPase molecules also occurs through the lysosomal pathway. Lysosomal degradation of a mammalian plasma membrane transporter has recently been demonstrated for parathyroid hormone-induced degradation of a renal Na⁺-dependent phosphate transporter type II (47) . In contrast, the cystic fibrosis conductance regulator, a Cl^- channel involved in epithelial salt secretion, is mostly degraded intracellularly by the ubiquitin-proteasome system (48 , 49) . The aldosterone-inducible, amiloride-sensitive epithelial Na⁺ channel (ENaC), however, is degraded by both ubiquitin-proteasomal and endo-/lysosomal proteolytic pathways (50) , and the same applies to connexin43-containing gap junction proteins (51) . These plasma membrane proteins all have a rapid physiological turnover (t_1/21–3 h), as expected for transporters involved in the regulation of ion and water homeostasis. This differs from the present study, however, which shows oxidative stress-induced degradation of a long-lived protein, the 1-subunit of Na⁺/K⁺-ATPase, by both the proteasome and the lysosome.

It is not known how the proteasomal and endo-/lysosomal proteolytic pathways interact temporally and spatially. The quantitative immunoblot analyses of the present study indicate that both degradation pathways equally contribute to Cd-induced proteolysis of Na⁺/K⁺-ATPase (see Fig. 6 and Table 1 ). However, several experiments failed to demonstrate ubiquitination of immunoprecipitated 1-subunits of Na⁺/K⁺-ATPase (data not shown). Nevertheless, polyubiquitination of Na⁺/K⁺-ATPase 1- and 2-subunits has recently been demonstrated in transfected COS-7 cells, suggesting that polyubiquitination and proteasomal degradation are involved in regulating the number of Na⁺/K⁺-ATPase molecules, at least under certain conditions (52) .

In conclusion, the present data show that low micromolar Cd concentrations directly or indirectly increase the production of reactive oxygen intermediates. This oxidative stress decreases the stability of the 1-subunit of the Na⁺/K⁺-ATPase, a long-lived protein, which is then removed from the cells by the proteasome complex and the lysosomal pathway (Fig. 8 ). An increased degradation of oxidatively modified plasma membrane Na⁺/K⁺-ATPase (and possibly of other Na⁺-dependent transporters) by both proteolytic pathways could therefore contribute to the dysfunction of Na-dependent transport associated with Cd nephrotoxicity.

View larger version (16K): [in this window] [in a new window]   Figure 8. Model of the proteolytic pathways involved in the degradation of oxidatively modified Na+/K+-ATPase in cadmium (Cd2+) -incubated proximal tubule cells. For further details, see Discussion. Ub = ubiquitin; E1/E2/E3 = ubiquitin-conjugating enzyme complex; = inhibition.

	ACKNOWLEDGMENTS

 
We would like to thank Dr. I. A. Hauser (Department of Nephrology, J. W.-Goethe University, Frankfurt, Germany) for critical comments on the manuscript, and Drs. E. Roussa and A. Katsen (Department of Anatomy, University of Saarland) for introducing us to the techniques of indirect immunofluorescence and apoptosis assay, respectively. The WKPT-0293 Cl.2 rat renal proximal tubule cell line was generously provided by Dr. U. Hopfer (Department of Physiology and Biophysics, CWRU, Cleveland, Ohio). Work in the author's laboratory is supported by DFG grant Th 345/6–1.

	FOOTNOTES

 
² Abbreviations: Cd, cadmium; DHRh123, dihydrorhodamine 123; DMSO, dimethyl sulfoxide; EB, ethidium bromide; IgG, immunoglobulin G; MG-132, N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-leucinal; MT, metallothionein; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; PDTC, pyrrolidene dithiocarbamate; PT, proximal tubule; PVDF, polyvinylidene difluoride; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WK, Wistar-Kyoto.

Received for publication January 13, 1999. Revised for publication April 16, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Piscator, M. (1986) The nephropathy of chronic cadmium poisoning. In Cadmium. Handbook of Experimental Pharmacology (Foulkes, E. C., ed) Vol. 80, pp. 194, Springer, New York
2. Klaassen, C. D., Liu, J. (1997) Role of metallothionein in cadmium-induced hepatotoxicity and nephrotoxicity. Drug. Metab. Rev. 29,79-102[Medline]
3. Liu, J., Habeebu, S. S., Liu, Y., Klaassen, C. D. (1998) 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[Medline]
4. Kim, Y. K., Choi, J. K., Kim, J. S., Park, Y. S. (1988) Changes in renal function in cadmium-intoxicated rats. Pharmacol. Toxicol. 63,342-350[Medline]
5. Jorgensen, P. L. (1980) Sodium and potassium ion pump in kidney tubules. Physiol. Rev. 60,864-917[Free Full Text]
6. Ullrich, K. J. (1979) Sugar, amino acid, and Na⁺ cotransport in the proximal tubule. Annu. Rev. Physiol. 41,181-195[Medline]
7. Shull, G. E., Schwartz, A., Lingrel, J. B. (1985) Amino-acid sequence of the catalytic subunit of the (Na⁺ + K⁺) ATPase deduced from a complementary DNA. Nature (London) 316,691-695[Medline]
8. Fambrough, D. M., Lemas, M. V., Hamrick, M., Emerick, M., Renaud, K. J., Inman, E. M., Hwang, B., Takeyasu, K. (1994) Analysis of subunit assembly of the Na-K-ATPase. Am. J. Physiol. 266,C579-C589[Abstract/Free Full Text]
9. Chambers, S. K., Gilmore-Hebert, M., Kacinski, B. M., Benz, E. J., Jr (1992) Changes in Na, K-ATPase gene expression during granulocytic differentiation of HL60 cells. Blood 80,1559-1564[Abstract/Free Full Text]
10. Hershko, A., Ciechanover, A. (1982) Mechanisms of intracellular protein breakdown. Annu. Rev. Biochem. 51,335-364[Medline]
11. Stadtman, E. R. (1992) Protein oxidation and aging. Science 257,1220-1224[Abstract/Free Full Text]
12. Grune, T., Reinheckel, T., Davies, K. J. (1997) Degradation of oxidized proteins in mammalian cells. FASEB J 11,526-534[Abstract]
13. Olson, T. S., Dice, J. F. (1989) Regulation of protein degradation rates in eukaryotes. Curr. Opin. Cell Biol. 1,1194-1200[Medline]
14. Hare, J. F. (1990) Mechanisms of membrane protein turnover. Biochim. Biophys. Acta 1031,71-90[Medline]
15. Mellman, I. (1996) Endocytosis and molecular sorting. Annu. Rev. Cell. Dev. Biol. 12,575-625[Medline]
16. Hicke, L. (1997) Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins. FASEB J 11,1215-1226[Abstract]
17. Goldberg, A. L., Rock, K. L. (1992) Proteolysis, proteasomes and antigen presentation. Nature (London) 357,375-379[Medline]
18. Ciechanover, A. (1994) The ubiquitin-proteasome proteolytic pathway. Cell 79,13-21[Medline]
19. Hochstrasser, M. (1996) Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30,405-439[Medline]
20. Coux, O., Tanaka, K., Goldberg, A. L. (1996) Structure and functions of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65,801-847[Medline]
21. Pickart, C. M. (1997) Targeting of substrates to the 26S proteasome. FASEB J 11,1055-1066[Abstract]
22. Etlinger, J. D., Goldberg, A. L. (1977) A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc. Natl. Acad. Sci. USA 74,54-58[Abstract/Free Full Text]
23. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., Goldberg, A. L. (1994) Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78,761-771[Medline]
24. Huang, W. H., Wang, Y., Askari, A., Zolotarjova, N., Ganjeizadeh, M. (1994) Different sensitivities of the Na⁺/K⁽⁺⁾-ATPase isoforms to oxidants. Biochim. Biophys. Acta 1190,108-114[Medline]
25. Gonzalez-Flecha, B., Evelson, P., Ridge, K., Sznajder, J. I. (1996) Hydrogen peroxide increases Na⁺/K⁽⁺⁾-ATPase function in alveolar type II cells. Biochim. Biophys. Acta 1290,46-52[Medline]
26. Zolotarjova, N., Ho, C., Mellgren, R. L., Askari, A., Huang, W. H. (1994) Different sensitivities of native and oxidized forms of Na⁺/K⁽⁺⁾-ATPase to intracellular proteinases. Biochim. Biophys. Acta 1192,125-131[Medline]
27. Woost, P. G., Orosz, D. E., Jin, W., Frisa, P. S., Jacobberger, J. W., Douglas, J. G., Hopfer, U. (1996) Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int 50,125-134[Medline]
28. Hauser, I. A., Koziolek, M., Hopfer, U., Thévenod, F. (1998) Therapeutic concentrations of cyclosporine A, but not FK506, increase P-glycoprotein expression in endothelial and renal tubule cells. Kidney Int 54,1139-1149[Medline]
29. Messler, P., Harz, H., Uhl, R. (1996) Instrumentation for multiwavelength excitation imaging. J. Neurosci. Methods 69,137-147[Medline]
30. Sun, X. M., Snowden, R. T., Skilleter, D. N., Dinsdale, D., Ormerod, M. G., Cohen, G. M. (1992) A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes. Anal. Biochem. 204,351-356[Medline]
31. Thévenod, F., Hildebrandt, J. P., Striessnig, J., DeJonge, H. R., Schnulz, I. (1996) Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a 80-kDa mdr1a gene product. J. Biol. Chem. 271,3300-3305[Abstract/Free Full Text]
32. O'Brien, P., Salacinski, H. J. (1998) Evidence that the reactions of cadmium in the presence of metallothionein can produce hydroxyl radicals. Arch. Toxicol. 72,690-700[Medline]
33. Stohs, S. J., Bagchi, D. (1995) Oxidative mechanisms in the toxicity of metal ions. Free Rad. Biol. Med. 18,321-336[Medline]
34. Aruoma, O. I., Halliwell, B., Hoey, B. M., Butler, J. (1989) The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Rad. Biol. Med. 6,593-597[Medline]
35. Schreck, R., Rieber, P., Baeuerle, P. A. (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J 10,2247-2258[Medline]
36. Shimada, H., Kamenosono, T., Kawagoe, M., Funakoshi, T., Kiyozumi, M., Takadate, A., Kojima, S. (1991) Mobilization of renal and hepatic cadmium by dithiocarbamates in rats. J. Pharmacobiodyn. 14,555-560[Medline]
37. Hamada, T., Tanimoto, A., Iwai, S., Fujiwara, H., Sasaguri, Y. (1994) Cytopathological changes induced by cadmium-exposure in canine proximal tubular cells: a cytochemical and ultrastructural study. Nephron 68,104-111[Medline]
38. Fenteany, G., Standaert, R. F., Lane, W. S., Choi, S., Corey, E. J., Schreiber, S. L. (1995) Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268,726-731[Abstract/Free Full Text]
39. Tanaka, M., Yanagi, M., Shirota, K., Une, Y., Nomura, Y., Masaoka, T., Akahori, F. (1995) Effect of cadmium in the zinc deficient rat. Vet. Hum. Toxicol. 37,203-208[Medline]
40. Dally, H., Hartwig, A. (1997) Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 18,1021-1026[Abstract/Free Full Text]
41. Berlett, B. S., Stadtman, E. R. (1997) Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272,20313-20316[Free Full Text]
42. Kinne, R. K., Schütz, H., Kinne-Saffran, E. (1995) The effect of cadmium chloride in vitro on sodium-glutamate cotransport in brush border membrane vesicles isolated from rabbit kidney. Toxicol. Appl. Pharmacol. 135,216-221[Medline]
43. Wagner, C. A., Waldegger, S., Osswald, H., Biber, J., Murer, H., Busch, A. E., Lang, F. (1996) Heavy metals inhibit Pi-induced currents through human brush-border NaPi-3 cotransporter in Xenopus oocytes. Am. J. Physiol. 271,F926-F930[Abstract/Free Full Text]
44. Bachmair, A., Finley, D., Varshavsky, A. (1986) In vivo half-life of a protein is a function of its amino-terminal residue. Science 234,179-186[Abstract/Free Full Text]
45. Rechsteiner, M. (1991) Natural substrates of the ubiquitin proteolytic pathway. Cell 66,615-618[Medline]
46. Jungmann, J., Reins, H.-A., Schobert, C., Jentsch, S. (1993) Resistance to cadmium mediated by ubiquitin-dependent proteolysis. Nature (London) 361,369-371[Medline]
47. Pfister, M. F., Ruf, I., Stange, G., Ziegler, U., Lederer, E., Biber, J., Murer, H. (1998) Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter. Proc. Natl. Acad. Sci. USA 95,1909-1914[Abstract/Free Full Text]
48. Ward, C. L., Omura, S., Kopito, R. R. (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83,121-127[Medline]
49. Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., Riordan, J. R. (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83,129-135[Medline]
50. Staub, O., Gautschi, I., Ishikawa, T., Breitschopf, K., Ciechanover, A., Schild, L., Rotin, D. (1997) Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO J 16,6325-6336[Medline]
51. Laing, J. G., Tadros, P. N., Westphale, E. M., Beyer, E. C. (1997) Degradation of connexin43 gap junctions involves both the proteasome and the lysosome. Exp. Cell Res. 236,482-492[Medline]
52. Coppi, M. V., Guidotti, G. (1997) Ubiquitination of Na,K-ATPase 1 and 2 subunits. FEBS Lett 405,281-284[Medline]

This article has been cited by other articles:

				P. Kolman, A. Pica, N. Carvou, A. Boyde, S. Cockcroft, A. Loesch, A. Pizzey, M. Simeoni, G. Capasso, and R. J. Unwin Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1227 - F1237. [Abstract] [Full Text] [PDF]

				B. Medicherla and A. L. Goldberg Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins J. Cell Biol., August 25, 2008; 182(4): 663 - 673. [Abstract] [Full Text] [PDF]

				H Yokota and H Tonami Experimental studies on the bone metabolism of male rats chronically exposed to cadmium intoxication using dual-energy X-ray absorptiometry Toxicology and Industrial Health, April 1, 2008; 24(3): 161 - 170. [Abstract] [PDF]

				A. Brandes, O. Oehlke, A. Schumann, S. Heidrich, F. Thevenod, and E. Roussa Adaptive redistribution of NBCe1-A and NBCe1-B in rat kidney proximal tubule and striated ducts of salivary glands during acid-base disturbances Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2007; 293(6): R2400 - R2411. [Abstract] [Full Text] [PDF]

				W.-K. Lee, B. Torchalski, and F. Thevenod Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells Am J Physiol Cell Physiol, September 1, 2007; 293(3): C839 - C847. [Abstract] [Full Text] [PDF]

				M. Abouhamed, N. A. Wolff, W.-K. Lee, C. P. Smith, and F. Thevenod Knockdown of endosomal/lysosomal divalent metal transporter 1 by RNA interference prevents cadmium-metallothionein-1 cytotoxicity in renal proximal tubule cells Am J Physiol Renal Physiol, September 1, 2007; 293(3): F705 - F712. [Abstract] [Full Text] [PDF]

				W.-K. Lee, M. Abouhamed, and F. Thevenod Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells Am J Physiol Renal Physiol, October 1, 2006; 291(4): F823 - F832. [Abstract] [Full Text] [PDF]

				N. A. Wolff, M. Abouhamed, P. J. Verroust, and F. Thevenod Megalin-Dependent Internalization of Cadmium-Metallothionein and Cytotoxicity in Cultured Renal Proximal Tubule Cells J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 782 - 791. [Abstract] [Full Text] [PDF]

				W.-K. Lee and F. Thevenod A role for mitochondrial aquaporins in cellular life-and-death decisions? Am J Physiol Cell Physiol, August 1, 2006; 291(2): C195 - C202. [Abstract] [Full Text] [PDF]

				A. P. Comellas, L. A. Dada, E. Lecuona, L. M. Pesce, N. S. Chandel, N. Quesada, G. R. S. Budinger, G. J. Strous, A. Ciechanover, and J. I. Sznajder Hypoxia-Mediated Degradation of Na,K-ATPase via Mitochondrial Reactive Oxygen Species and the Ubiquitin-Conjugating System Circ. Res., May 26, 2006; 98(10): 1314 - 1322. [Abstract] [Full Text] [PDF]

				W.-K. Lee, M. Spielmann, U. Bork, and F. Thevenod Cd2+-induced swelling-contraction dynamics in isolated kidney cortex mitochondria: role of Ca2+ uniporter, K+ cycling, and protonmotive force Am J Physiol Cell Physiol, September 1, 2005; 289(3): C656 - C664. [Abstract] [Full Text] [PDF]

				S. Othumpangat, M. Kashon, and P. Joseph Eukaryotic Translation Initiation Factor 4E Is a Cellular Target for Toxicity and Death Due to Exposure to Cadmium Chloride J. Biol. Chem., July 1, 2005; 280(26): 25162 - 25169. [Abstract] [Full Text] [PDF]

				X. Xia, G. Wang, Y. Peng, M.-G. Tu, J. Jen, and H. Fang The Endogenous CXXC Motif Governs the Cadmium Sensitivity of the Renal Na+/Glucose Co-Transporter J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1257 - 1265. [Abstract] [Full Text] [PDF]

				W.-K. Lee, U. Bork, F. Gholamrezaei, and F. Thevenod Cd2+-induced cytochrome c release in apoptotic proximal tubule cells: role of mitochondrial permeability transition pore and Ca2+ uniporter Am J Physiol Renal Physiol, January 1, 2005; 288(1): F27 - F39. [Abstract] [Full Text] [PDF]

				C. A. Wagner, K. E. Finberg, S. Breton, V. Marshansky, D. Brown, and J. P. Geibel Renal Vacuolar H+-ATPase Physiol Rev, October 1, 2004; 84(4): 1263 - 1314. [Abstract] [Full Text] [PDF]

				C. J. C. Phillips, P. C. Chiy, and H. M. Omed The effects of cadmium in feed, and its amelioration with zinc, on element balances in sheep J Anim Sci, August 1, 2004; 82(8): 2489 - 2502. [Abstract] [Full Text] [PDF]

				C. Erfurt, E. Roussa, and F. Thevenod Apoptosis by Cd2+ or CdMT in proximal tubule cells: different uptake routes and permissive role of endo/lysosomal CdMT uptake Am J Physiol Cell Physiol, December 1, 2003; 285(6): C1367 - C1376. [Abstract] [Full Text] [PDF]

				D. Z. Ellis, J. Rabe, and K. J. Sweadner Global Loss of Na,K-ATPase and Its Nitric Oxide-Mediated Regulation in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis J. Neurosci., January 1, 2003; 23(1): 43 - 51. [Abstract] [Full Text] [PDF]

				R. A. Shanely, M. A. Zergeroglu, S. L. Lennon, T. Sugiura, T. Yimlamai, D. Enns, A. Belcastro, and S. K. Powers Mechanical Ventilation-induced Diaphragmatic Atrophy Is Associated with Oxidative Injury and Increased Proteolytic Activity Am. J. Respir. Crit. Care Med., November 15, 2002; 166(10): 1369 - 1374. [Abstract] [Full Text] [PDF]

				S. Abu-Hayyeh, M. Sian, K. G. Jones, A. Manuel, and J. T. Powell Cadmium Accumulation in Aortas of Smokers Arterioscler. Thromb. Vasc. Biol., May 1, 2001; 21(5): 863 - 867. [Abstract] [Full Text] [PDF]

				F. Thevenod, J. M. Friedmann, A. D. Katsen, and I. A. Hauser Up-regulation of Multidrug Resistance P-glycoprotein via Nuclear Factor-kappa B Activation Protects Kidney Proximal Tubule Cells from Cadmium- and Reactive Oxygen Species-induced Apoptosis J. Biol. Chem., January 21, 2000; 275(3): 1887 - 1896. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by THÉVENOD, F. Articles by FRIEDMANN, J. M. Search for Related Content PubMed PubMed Citation Articles by THÉVENOD, F. Articles by FRIEDMANN, J. M.