1 Unit of Molecular Toxicology, Institute for Medical Research and Occupational Health, 10001 Zagreb, Croatia; and 2 Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nephrotoxicity in humans and
experimental animals due to chronic exposure to cadmium (Cd) is
manifested by defects in the reabsorptive and secretory functions of
proximal tubules (PT). The main symptoms of Cd nephrotoxicity,
including polyuria, phosphaturia, aminoaciduria, glucosuria, and
proteinuria, suggest that various brush-border membrane (BBM)
transporters are the main targets of Cd. Specific transporters may be
either directly inhibited by Cd or lost from the BBM after Cd
treatment, or both. We have recently proposed that Cd may impair the
vesicle-dependent recycling of BBM transporters by inhibiting vacuolar
H+-ATPase (V-ATPase) activity and endocytosis in PT cells
(Herak-Kramberger CM, Sabolic I, and Brown D. Kidney Int 53:
1713-1726, 1998). The mechanism underlying the Cd effect was
further explored in an in vivo model of experimental Cd nephrotoxicity
induced by Cd-metallothionein (Cd-MT; 0.4 mg Cd/kg body mass; a single
dose sc) in rats. The time-dependent redistribution of various BBM
transporters was examined in this model by fluorescence and
gold-labeling immunocytochemistry on tissue sections and by
immunoblotting of isolated renal cortical BBM. In PT cells of
Cd-MT-treated rats, we observed 1) shortening and loss of
microvilli; 2) time-dependent loss of megalin, V-ATPase, aquaporin-1 (AQP1), and type 3 Na+/H+ exchanger
(NHE3) from the BBM; 3) redistribution of these transporters into vesicles that were randomly scattered throughout the cell cytoplasm; and 4) redistribution of NHE3, but not megalin,
into the basolateral plasma membrane. The internalization of BBM
transporters was accompanied by fragmentation and loss of microtubules
and by an increased abundance of -tubulin monomers in PT cells.
Transporter redistribution was detectable as early as 1 h after
Cd-MT treatment and increased in magnitude over the next 12 h. We
conclude that the early mechanism of Cd toxicity in PT cells may
include a colchicine-like depolymerization of microtubules and impaired
vesicle-dependent recycling of various BBM proteins. These processes
may lead to a time-dependent loss of cell membrane components,
resulting in reabsorptive and secretory defects that occur in
Cd-induced nephrotoxicity.
cytoskeleton; heavy metals; immunocytochemistry; kidney; microtubules; nephrotoxicity
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NEPHROPATHY IN HUMANS AND experimental animals due to chronic exposure to cadmium (Cd) is manifested by reabsorptive and secretory dysfunction of renal tubules. The principal symptoms of tubular damage (proteinuria, phosphaturia, glucosuria, aminoaciduria, and hyperosmolar polyuria) indicate that Cd preferentially targets various transporters in the proximal tubule brush-border membrane (BBM) (3, 5, 30, 34, 35, 37). Several Na+-dependent BBM transporters, such as those for phosphate (NaPi-2a) (4, 5, 30), glucose (37), and amino acids (34), were inhibited by inorganic Cd in isolated BBM vesicles and in experimental animals treated with CdCl2 for 2-3 wk. The inhibition in vitro may be due to a direct interaction of Cd with the transporters (4, 5, 34, 36, 37, 58) and/or a loss of BBM vesicle integrity (29). However, tubular dysfunction in vivo may result from the loss of BBM functional capacity due to 1) shortening and loss of microvilli (19, 28); 2) loss of specific BBM transporters (28, 30); 3) direct inhibition of BBM transporters by Cd (4, 5, 28, 34, 36, 37, 50, 58); 4) indirect inhibition of BBM transporters caused by oxidative stress and peroxidation of membrane lipids (60); and 5) a combination of these phenomena.
How Cd reaches and damages proximal tubule cells is not entirely clear. While acute exposure of experimental animals to a high dose of CdCl2 has little nephrotoxic effect, nephrotoxicity develops with either repeated parenteral small daily doses of CdCl2 or after prolonged oral ingestion of this substance. In the liver, either treatment may stimulate the synthesis of a cystein-rich metal-binding protein, metallothionein (MT), to which Cd binds and forms a Cd-MT complex (6-7 kDa) that may be released into the circulation (21). However, Cd-MT in the blood may also originate from food, after absorption of an intact molecule in the gastrointestinal tract (15, 16) or after binding of ingested inorganic Cd to MT in gastrointestinal tract cells and release of this complex into the circulation (33). The circulating Cd-MT may be filtered by the glomeruli, endocytosed in proximal tubule cells, and degraded in lysosomes (20). The released Cd may then stimulate production of MT in proximal tubule cells (23, 59) and target various cellular structures and functions (28, 30, 31, 41, 55, 60, 67).
The possibility that Cd-MT mediates Cd action in the kidney has been used to develop short-term animal models of Cd nephrotoxicity. Animals treated with a single dose of Cd-MT developed full-blown structural (16, 22, 61, 62) and functional (22, 32, 63, 64) damage of proximal tubules after only 6-24 h. Similar tubule damage in long-term animal models exposed to CdCl2 required weeks or months to manifest. The damage induced by a single or, in some studies, repeated Cd-MT injections qualitatively resembled those achieved by long-term treatment with CdCl2 (23, 38, 47, 70), although, for unknown reasons, they occurred at much lower levels of tissue Cd than after treatment with inorganic Cd (21, 31, 48).
The intracellular events in proximal tubule cells that lead to structural and functional damage in Cd-MT nephrotoxicity have not been established. In proximal tubule cells of rats treated with CdCl2 for 2 wk we recently showed 1) greatly diminished expression of NaPi-2a and the vacuolar H+-ATPase (V-ATPase), 2) impaired endocytosis of a fluorescent marker, FITC-dextran, and 3) deranged and partially depolymerized actin and microtubule cytoskeleton (28, 30, 55). Because V-ATPase-mediated acidification of intracellular organelles (42) and the cytoskeleton (6, 11, 14) play a pivotal role in the trafficking, targeting, and recycling of many plasma membrane proteins, we assumed that Cd may acutely interfere with these pathways and thereby affect the structural and functional polarity of proximal tubule cells. The experiments in this report were designed to examine the early intracellular effects of Cd-MT in proximal tubules that may contribute to the development of Cd nephrotoxicity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and treatment. Two-month-old male Wistar rats (body mass, 180-200 g) from the breeding colony at the Institute in Zagreb were used. Animals were bred and maintained according to the Guide for Care and Use of Laboratory Animals (Washington, DC: Academy Press, 1996). Before and during experiments, animals had free access to standard laboratory food and tap water. The studies were approved by the Institutional Ethics Committee.
Cd-MT (from rabbit liver, 7% metal as Cd; Sigma, St. Louis, MO) was dissolved in 0.9% NaCl and injected subcutaneously in a single dose of 0.4 mg Cd/kg body mass. The animals were killed at various times (hours) thereafter. As shown previously (32), 8-16 h later, the above-mentioned treatment induced urinary symptoms of nephrotoxicity that included proteinuria and calciuria. Control animals received an equivalent amount (0.2-0.3 ml) of vehicle 12 h before death. In preliminary experiments, by checking the tissue content of Cd and the immunocytochemical distribution of various antigens, we showed that these parameters in untreated rats and in rats treated with vehicle 6 or 12 h before death were similar (data not shown). In one experiment, to compare effects of inorganic Cd, rats were injected subcutaneously with a solution of CdCl2 (in water) in a single dose of 0.4 mg Cd/kg body mass, and the animals were killed at various time points (hours) thereafter.Determination of tissue cadmium. Rats were decapitated at various time points (hours) after treatment with Cd-MT, and the kidney cortex was weighed and dry ashed at 450°C for 24 h. Ashed samples were dissolved in 2% nitric acid, and the Cd concentration was measured by atomic absorption spectrometry (Varian-AA375; flame mode) using an appropriate standard solution (999 ± 2 mg Cd/l; 19777.0500, Merck, Darmstadt, Germany) as a reference.
Antibodies.
Primary antibodies included polyclonal (rabbit immune serum) and
monoclonal (1H2) anti-megalin holoprotein antibodies (1, 6,
26); polyclonal antibodies (whole rabbit immune serum and an
affinity-purified chicken antibody) against the COOH-terminal sequence
of the 31-kDa ("E") V-ATPase subunit (6, 27, 54); polyclonal anti-aquaporin-1 (AQP1) antibodies (native rabbit immune serum or an antibody purified by immunoadsorption onto Immobilon strips
containing AQP1 protein) (57); and monoclonal antibodies (cell culture media) 19F5 and 2B9 against a type 3 N+/H+ exchanger (NHE3) fusion protein (8,
56). A commercial monoclonal antibody (affinity purified) to
-tubulin (T-9026, Sigma) was used to label microtubules
(6). The reason for the use of some polyclonal antibodies
in the form of either immune serum or affinity-purified IgG was
indicated in our previous publication (27): whereas the
specificity of labeling with both forms was the same, the immune sera
and the affinity-purified antibodies gave stronger labeling in
immunoblotting and immunocytochemical studies, respectively.
Tissue fixation and immunocytochemistry. Rats were anesthetized with Nembutal (65 mg/kg body mass ip) and then perfused via the left cardiac ventricle, first with PBS [(in mM) 140 NaCl, 4 KCl, 2 KH2PO4, pH 7.4] at 37°C for 2-3 min to remove circulating blood and then with 180 ml PLP fixative (2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate) for 5 min (43). Kidneys were removed, decapsulated, sagitally sliced, and kept overnight in the same fixative at 4°C, followed by extensive washing in PBS and storage in PBS containing 0.02% NaN3 at 4°C until further use.
To cut 4-µm frozen sections, tissue slices were infiltrated with 30% sucrose (in PBS) overnight, frozen in liquid nitrogen, and sectioned in a Leica CM 1850 cryostat (Leica Instruments, Nussloch, Germany). Sections were collected on Superfrost/Plus Microscope slides (Fisher Scientific, Pittsburgh, PA) and rehydrated in PBS for 10 min. Sections for V-ATPase and NHE3 staining were pretreated for 5 min with 1% SDS (in PBS) to expose cryptic antigenic sites (12); SDS was removed by extensive washing with PBS. Sections for megalin, AQP1, and tubulin staining were used without SDS pretreatment. Nonspecific binding of antibodies was reduced by incubating sections with 1% bovine serum albumin (in PBS) for 15 min before application of antibodies against megalin (immune serum diluted 1:800 with PBS); V-ATPase (affinity-purified chicken antibody, 1:20); AQP1 (affinity purified, used undiluted); NHE3 (cell culture medium 19F5, 1:20); andImmunogold electron microscopy. The immunogold-labeling of ultrathin frozen tissue sections was performed according to the method of Tokuyasu (65). The kidney cortex was infiltrated with 2.3 M sucrose overnight, frozen in liquid nitrogen, and 70- to 80-nm-thick sections were cut on a Leica ultracryomicrotome (EMFCS Ultracut UCT) and mounted on Formvar-coated nickel grids. The grids with sections were processed at room temperature in a humid chamber using the following steps: rehydration with PBS containing 20 mM glycine for 10 min followed by PBS alone (2 × 5 min); blocking with 1% BSA in PBS (BSA/PBS) for 15 min; incubation with 1H2 (1:500) or anti-NHE3 antibody (19F5; 1:20) for 30 min; washing with BSA/PBS for 5 min and PBS alone (3 × 5 min); incubation with gold-conjugated goat anti-mouse IgG antibody (10-nm gold particles, Electron Microscopy Sciences, Fort Washington, PA; diluted 1:5) for 60 min; washing with PBS (4 × 5 min); fixation with 1% glutaraldehyde (in water) for 5 min; washing in water (3 × 5 min); staining with a mixture (8:5:1) of 3% methylcellulose-water-3% uranyl acetate for 10 min; drying; and viewing on a Philips CM10 electron microscope.
Preparation of tissue homogenates and isolation of BBM vesicles.
Animals were killed by decapitation. The kidneys were removed and
decapsulated, and cortical tissue slices (~0.5 mm in depth from the
kidney surface) were cut, collected in chilled buffer (300 mM mannitol,
12 mM HEPES/Tris, pH 7.4), which contained the protease inhibitors
phenylmethylsulfonyl fluoride (1 mM), benzamidine (0.1 mM), and
antipain (0.1 µg/ml), and homogenized in a Polytron homogenizer
(Kinematica, Littau, Lucerne, Switzerland; setting 5) for 60 s.
Cell debris was removed from the total (crude) homogenate by
centrifugation at 2,500 g for 15 min. The pellet was
discarded, and the supernatant (referred to below as the
"homogenate") was used in immunoblotting experiments to detect
-tubulin and in vesicle purification experiments to isolate BBM by
the Mg/EGTA precipitation method (7). The final membrane
preparation was dissolved in a low-ionic-strength buffer (150 mM
mannitol, 6 mM HEPES/Tris, pH 7.4) and stored in liquid nitrogen until
further use. Protein was determined by Bradford assay using bovine
serum albumin as a standard (10).
SDS-PAGE and immunoblotting.
Proteins from the homogenate (to test -tubulin) or from isolated BBM
(to test all other antigens) were denatured in sample buffer (1% SDS,
12% vol/vol glycerol, 30 mM Tris/HCl, pH 6.8) without (to detect
megalin and V-ATPase) or with 5%
-mercaptoethanol (to detect all
other antigens) at either 37°C for 30 min (to assay AQP1), 65°C for
15 min (to detect megalin, V-ATPase, and NHE3), or 95°C for 5 min (to
detect
-tubulin). Proteins (6 µg/lane for AQP1, 20 µg/lane for
megalin, 40 µg/lane for V-ATPase and NHE3, 50 µg/lane for
-tubulin) were separated through either 4-10% gradient (for
megalin) or 12% linear (for all other antigens) SDS-polyacrylamide
minigels (SDS-PAGE) and transferred to Immobilon membranes (Millipore,
Bedford, MA). Each membrane was briefly stained with Coomassie
brilliant blue to check the efficiency of the transfer, destained,
blocked in blotting buffer (5% nonfat dry milk, 0.15 M NaCl, 1%
Triton X-100, 20 mM Tris/HCl, pH 7.4), and incubated at 4°C overnight
(12-14 h) in the same buffer that contained an antibody to either
megalin (rabbit immune serum, diluted 1:1,000); V-ATPase (rabbit immune
serum, 1:500); AQP1 (rabbit immune serum, 1:6,000); NHE3 (cell culture
medium 2B9, 1:10); or
-tubulin (1:500). After extensive washing in
antibody-free blotting buffer, the membrane was incubated for 1 h
with the same buffer that contained either GARAP or GAMAP (0.1 µg/ml), washed, and stained for alkaline phosphatase activity with
the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium method.
The density of specific protein bands was scanned (Ultroscan XL Laser
Densitometer, LKB, Bromma, Sweden), and the integrated surface of each
scan was expressed in arbitrary units relative to the surface of the
densest band (=100 arbitrary units) in samples from control animals.
Presentation of the data. The immunofluorescence and immunogold-labeling data represent findings from three rats in the control and each experimental group. The figures were prepared from color slides or black-and-white negatives that were scanned using a Polaroid SprintScan 35 Plus scanner or an Epson Perfection 1640 SU with an Epson EU-33 transparency adaptor. Scans were imported into Adobe Photoshop 4.0 software, appropriately processed and labeled, and printed on an Epson Stylus Color printer. The numerical data (means ± SE) were statistically evaluated either by two-tailed t-test or by ANOVA/MANOVA, followed by Duncan's multiple-range test at 5% level of difference. The evaluation was performed using Statistica 5.0 for Windows (release 1995; www.Statsoft.com).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of Cd-MT treatment on Cd content of renal cortical tissue.
Cd concentration in wet and dry renal cortex was determined in control
rats (injected with saline 12 h before death; time 0)
and in rats injected with Cd-MT and killed 3, 6, or 12 h later. As
shown in Table 1, 3 h after Cd-MT
injection, the wet tissue Cd concentration reached 40 µg/g; i.e., it
increased 1,000-fold compared with the amount in control animals. The
accumulated Cd remained unchanged at 6 h and slightly decreased
(12%) at 12 h after Cd-MT injection. However, the dry tissue Cd,
which increased 1,067-fold 3 h after Cd-MT injection, increased
further by 37% (1,462-fold compared with that in the controls) at
6 h, indicating an increased abundance of tissue water (edema) at
this time point. At 12 h after Cd-MT treatment, the dry tissue Cd
was 30% lower than at 6 h. Thus the injection protocol used in
these experiments was successful in raising the tissue Cd levels
considerably, as reported previously (16).
|
Distribution of BBM transporters in proximal tubules after Cd-MT
treatment.
Megalin, an apical membrane protein in proximal tubule cells, is a
receptor involved in the reabsorption of several filtered proteins
(18). Treatment of rats with Cd-MT induced in most (but
not in all) tubules a major, time-dependent intracellular redistribution of megalin (Fig. 1,
A-H). In cells
of proximal convoluted tubules in control animals, in accordance with
previous observations (6, 14, 26), the anti-megalin
antibody strongly stained the BBM and apical membrane coated pits, with
little staining of the rest of the cytoplasm (Fig. 1A).
Already at 1 h after Cd-MT treatment, the apical staining became
weaker and thinner in most tubules, and numerous granule-like
protrusions of staining (which we have termed "granulation") were
observed projecting from the apex of the cell into the cytoplasm (Fig.
1, B and C). Two hours after the injection of
Cd-MT, the intensity of the apical staining was further diminished, and
numerous small intracellular vesicles were seen in the subapical domain
of many cells (Fig. 1D). This process continued with time,
resulting in progressively decreased apical staining and an increased
abundance of labeled intracellular vesicles 3 (Fig. 1E), 4 (Fig. 1F), 6 (Fig. 1G), and 12 h (Fig. 1H) after Cd-MT injection. In addition to this cytoplasmic
accumulation of megalin, many tubules exhibited various signs of
necrosis 6 and 12 h after Cd-MT treatment, including cell edema,
pycnotic nuclei, damaged apical surface, loss of epithelium, and
presence of cell debris in the tubule lumen. This typical structural
damage for Cd nephrotoxicity is clearly seen in Fig. 1H and
was previously observed in animals treated with Cd-MT (16, 22,
61, 62) or subchronically intoxicated with CdCl2
(19, 23, 28).
|
|
Relocation of BBM transporters into the BLM: immunogold labeling.
The basolateral localization of some apical transporters in
Cd-MT-treated rats, e.g., NHE3, was studied in more detail in rats that
had been treated with Cd-MT 12 h earlier, e.g., when the
redistribution of megalin, V-ATPase, AQP1, and NHE3, in proximal tubules was extensive (Fig. 3). In
the case of megalin, the basolateral domain of most tubules was
unstained, but some tubules exhibited a weak staining at their
basolateral pole (Fig. 3A). This basolateral megalin may be
confined to intracellular vesicles located in the vicinity of the BLM,
as previously described in colchicine-treated rats (26).
The V-ATPase was also strongly internalized, but the basolateral pole
of the proximal tubule remained unstained (Fig. 3B). On the
other hand, in many tubules with extensive loss and internalization of
AQP1, basolateral AQP1 was also barely detectable, whereas the tubules
showing less extensive endocytosis of apical AQP1 still exhibited a
substantial basolateral staining of this antigen (Fig. 3C).
In contrast, a clear and sharp staining of NHE3 in the BLM was always
found in proximal tubules after 12 h of Cd-MT treatment, in
parallel with a reduced intensity of apical staining (Fig.
3D).
|
|
|
Immunoblotting of isolated BBM. Renal cortical BBM from control and Cd-MT-treated rats were immunoblotted with the same battery of antibodies used for immunocytochemical studies. The final membrane preparations from all animal groups were similarly enriched in LAP activity [enrichment factor was 9.6 ± 0.83 (n = 4), 10.2 ± 0.83 (n = 4), 10.5 ± 0.89 (n = 4), and 11.9 ± 0.35 (n = 3) in BBM from control rats and from rats treated with Cd-MT 3, 6, and 12 h earlier, respectively] and were also contaminated to a similar extent with thrombomodulin and Na+-K+-ATPase, markers for endothelial cell plasma membranes (52) and proximal tubule cell BLM, respectively (data not shown), indicating that the membranes isolated from the different groups of rats were similar in origin. However, the yield of BBM, expressed as milligrams of BBM protein obtained per gram of cortical tissue, showed a steady decline, being 84, 73, and 66% of that in control rats 3, 6, and 12 h after Cd-MT treatment, respectively (Table 1).
Representative immunoblots, obtained with two independent BBM preparations from each animal group, are shown in Fig. 6, and the densitometric values of the specific protein bands, pooled from two separate experiments, are listed in Table 1. The abundance of megalin (molecular mass: ~520 kDa) and the 31-kDa V-ATPase subunit in isolated BBM steadily diminished 3, 6, and 12 h after Cd-MT treatment (Fig. 6, A and B, respectively; Table 1). This finding was in accordance with the time-dependent reduction in apical immunofluorescence observed with the respective antibodies and its redistribution into intracellular vesicles. However, the abundance of AQP1 and NHE3 in isolated BBM exhibited no consistent decline (Fig. 6, C and D, respectively; Table 1), which is in apparent contrast to the marked reduction in these antigens that was found by immunofluorescence microscopy (cf. Fig. 2, C and D and E-F, respectively). However, the quantitative immunogold-labeling data described above show that the amount of NHE3 gold label per micrometer length of BBM is not decreased after Cd-MT treatment and even appears greater than in control tissues. Thus the observed decrease in apical NHE3 staining detected by immunofluorescence microscopy (Fig. 2F) is due to a general reduction in size and number of the brush-border microvilli and not to a decrease in concentration of the protein in the apical plasma membrane after Cd-MT treatment. In contrast, the concentration of megalin in the apical membrane does decrease after Cd-MT treatment, resulting in a parallel reduction in both immunocytochemical staining and immunoblotting.
|
Microtubules.
The time-dependent internalization of various apical transporters
and the finding of NHE3 in the BLM in proximal tubules of Cd-MT-injected rats described above is remarkably similar to the effect
on these transporters of the microtubule-depolymerizing agent
colchicine in rats (6, 13, 26, 56). Therefore, the effect
of Cd-MT treatment on microtubule organization in proximal tubules was
examined. In accordance with previous findings from our laboratory
(2, 6, 56), apical-to-basolateral bundles of microtubules
were abundant in proximal tubule cells of control rats (Fig.
7A). However, already 1 h
after Cd-MT injection there was a substantial loss of overall staining
intensity as well as a shortening and fragmentation of microtubules in
most cortical proximal tubules (Fig. 7B). The loss of
staining was more marked after 2 h (Fig. 7C) and was
nearly complete 3-4 h after Cd-MT injection (Fig. 7D).
At 6 and 12 h, a partial reestablishment of the microtubule
network was apparent in some cells, albeit in a somewhat abnormal
pattern (Fig. 7, E and F, respectively). However,
despite the loss of polymerized microtubules, the abundance of
-tubulin in cortical homogenates from Cd-MT-treated rats increased 82, 110, and 198% above control values, 3, 6, and 12 h after
Cd-MT injection, respectively (Fig. 6E, Table 1).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To gain greater insight into the mechanism of Cd nephrotoxicity in
mammals, the acute effects of Cd-MT treatment in rats on the
distribution of several BBM transporters and microtubules in proximal
tubules were studied. In proximal tubule cells of Cd-MT-treated rats,
we observed 1) shortening and loss of microvilli; 2) time-dependent loss of megalin, V-ATPase, AQP1, and
NHE3 from the BBM; 3) redistribution of these transporters
into randomly scattered cytoplasmic vesicles; 4)
redistribution of NHE3, but not megalin, into the BLM; and
5) time-dependent fragmentation and loss of microtubules,
accompanied by an increased abundance of -tubulin monomers.
The major finding was that Cd-MT induced a rapid, time-dependent internalization of several apical membrane transporters. The transporters initially accumulated in subapical invaginations (granulation pattern), followed by their continued loss (by endocytosis) from the BBM and relocation into vesicles that were randomly scattered throughout the cytoplasm. This process was already apparent 1 h after Cd-MT treatment and increased in magnitude over the next 12 h. The time dependence of antigen redistribution was similar whether the Cd-MT was injected subcutaneously or intravenously, showing that the phenomenon was not limited by the rate of Cd-MT resorption and transport to the kidney. Rather, the rate-limiting step may be endocytosis and intracellular processing of filtered Cd-MT by proximal tubules and/or the rate of antigen internalization via endocytosis. However, when the effects of Cd in the form of Cd-MT and CdCl2 were compared, the inorganic Cd caused the initial step of granulation, but no subsequent intracellular antigen redistribution was apparent. This finding agrees with previous observations in rats and mice, in which inorganic Cd in doses of up to 3 mg/kg body mass did not induce significant functional and morphological damage to proximal convoluted tubules within 24 h after treatment (16, 21, 22). These data, therefore, support the concept that organic Cd (Cd-MT) is primarily nephrotoxic whereas inorganic Cd (CdCl2) may be primarily hepatotoxic (16, 21). Nevertheless, the key player in nephrotoxicity seems to be Cd; rats treated with ZnMT showed no functional and structural damage to proximal tubules (61). In (sub)chronic Cd nephrotoxicity, after endocytosis and lysosomal degradation of filtered Cd-MT (20), the liberated Cd may stimulate the production of MT in proximal tubule cells (23, 55, 59), which may bind Cd and prevent immediate toxicity. This may be why, in experimental animals treated with small doses of CdCl2 for weeks or months, Cd in the kidney cortex has to reach a "critical" concentration of ~200 µg/g wet mass to overcome the binding capacity of newly synthesized MT before the unbound Cd becomes nephrotoxic (24, 53, 55). However, the data in this report (although representing more acute conditions) show that tissue Cd in the renal cortex reached only ~40 µg/g wet mass 3 h after Cd-MT injection, and yet there was a marked internalization of BBM transporters. At shorter time points, when the processes of granulation (1 h) and endocytosis (2 h) had already begun, the tissue Cd concentration was probably even lower. This indicates that Cd may act as a nephrotoxin immediately after its release from lysosomes, at much lower intracellular levels than previously thought, supporting the conclusions of other studies (16, 31).
Although the intracellular events that trigger Cd-MT-induced
endocytosis of BBM transporters are not known, our data suggest that
depolymerization of microtubules may be an important event in this
process. Fragmentation and loss of microtubules in proximal tubule
cells were already extensive 1 h after Cd-MT injection, while the
internalization of megalin was still at the initial granulation stage
at this time point. This suggests that depolymerization of microtubules
may precede the process of endocytosis of BBM transporters. As the loss
of microtubules became more extensive over the next 2-3 h, the
internalization of megalin was also more extensive. At 6 and 12 h
after Cd-MT injection, patches of microtubules in a state of apparent
repolymerization were detected. At the same time, the significantly
increased amount of -tubulin in tissue homogenates, which in its
nonpolymerized state cannot be readily visualized by
immunocytochemistry, indicates that Cd-MT either stimulated synthesis
or inhibited degradation of
-tubulin, or both. The patches of
repolymerized but disorganized microtubules may indicate the start of
regenerative processes in proximal tubule cells that take place
6-12 h after a single Cd-MT injection. These rapid regenerative
processes may be specific for acute Cd-MT toxicity, as in this study,
but may be slower in animals treated with multiple, short-interval
Cd-MT treatments, e.g., in conditions that may be more relevant for
chronic Cd intoxication (23, 38, 47, 70). However, we
recently reported a similar limited loss and/or derangement of
microtubules, actin filaments, and villin in proximal convoluted
tubules of rats treated with inorganic Cd for 2 wk (56),
suggesting that similar phenomena occur in (sub)chronic Cd
nephrotoxicity. In the present report, other cytoskeletal components were not examined, but our data indicate that the effect of Cd on
microtubules might be an early event that may trigger other cellular
responses, beginning with inhibition of the vesicle-mediated recycling
of BBM transporters. The process continues with a loss of BBM
components and microvilli, leading ultimately to more general structural and functional damage. However, a direct connection between depolymerization of microtubules and internalization of BBM
transporters in Cd-MT-treated rats, as suggested by our data, remains
to be established in future experiments.
As shown in our previous studies (6, 13, 26, 56), colchicine-induced loss of microtubules in proximal convoluted tubule cells led to a similar internalization of several BBM transporters, including megalin, V-ATPase, AQP1, and NHE3. However, only NHE3 was also relocated into the BLM after microtubule disruption (56). These findings were reproduced in this study after Cd-MT injection. Normally an apical protein, NHE3 was detected in the BLM 6 and 12 h after Cd-MT treatment, suggesting that Cd-MT had a colchicine-like effect on vesicle trafficking, resulting in a partial loss of cell polarity. This supports our recent hypothesis that the intracellular recycling of NHE3 in proximal tubules may include microtubule-independent targeting of newly synthesized NHE3 to the BLM and subsequent microtubule-dependent internalization and transcytosis to the apical membrane (56). After depolymerization of microtubules by colchicine (56) or Cd-MT (this study), NHE3 may slowly accumulate in the BLM due to disrupted internalization and deficient transcytosis. This would explain why the accumulation of NHE3 in the BLM was more pronounced at 12 h than at 6 h after Cd-MT treatment. Our gold-labeling data also support this contention. Whereas the BBM in the affected tubules was nearly depleted of megalin 12 h after Cd-MT exposure, the amount of gold-labeled NHE3 in this membrane domain was actually greater than in control tissues. This indicates the presence of (at least) two pools of BBM proteins that respond differently to Cd-MT exposure and microtubule disruption. Some proteins, such as megalin (and probably the V-ATPase), are rapidly internalized under these conditions and are removed from the BBM in a time-dependent manner (indicated by the decreasing density of the corresponding protein bands in immunoblots). Other proteins, such as NHE3 (and probably AQP1), are internalized more slowly and may even become more concentrated in the BBM as the total amount of BBM decreases over time. By Western blotting, significant differences in the apical abundance of these antigens that would correspond more closely to the immunocytochemical observations were probably partially masked by a contribution from the BBM of proximal tubules in which Cd-MT-induced protein redistribution was less marked or absent.
How could Cd-MT cause depolymerization of microtubules in the proximal convoluted tubule? Being highly reactive with thiol groups, Cd released from Cd-MT could directly chelate essential SH groups in tubulin and block polymerization of tubulin monomers (25, 40, 45, 46). In addition, in a model of experimental Cd-MT nephrotoxicity identical to that used in the present study, a strongly reduced uptake of 45Ca into isolated rat renal cortical BBM and BLM vesicles 4-24 h after Cd-MT injection was reported (39). This was associated with accumulation of Ca in cortical tissue (39) and with calciuria (38). Furthermore, in various nonrenal cells and/or organelles, Cd was found to 1) mobilize Ca2+ from intracellular stores via inositol lipid hydrolysis (9); 2) inhibit Ca2+-ATPase activity (68, 69); 3) inhibit Na+/Ca2+ exchange (66); and 4) compete with Ca2+ in activating calmodulin associated with microtubules (51). At least some of these processes may also take place in proximal tubule cells and elevate the intracellular concentration of Ca2+, which is a powerful inducer of microtubule disassembly (25, 49, 51).
Because microtubules are involved in vesicle trafficking and
maintaining epithelial cell polarity (14),
depolymerization of microtubules may have deleterious consequences with
regard to the structure and function of proximal tubule cells. Together with a functional V-ATPase in various organelles, microtubules are
required for the continuous recycling of various domain-specific membrane proteins (14). Recent data have demonstrated that
vesicle acidification is necessary for the selective recruitment of
various vesicle coat proteins, including Arf6, ARNO, and -coat
protein from the cell cytosol (Ref. 42 and references
therein), a process that may play an important role in intracellular
vesicle trafficking and receptor-mediated endocytosis. We have shown
previously that inorganic Cd inhibits V-ATPase activity and impairs
acidification in various organelles of the vacuolar system in proximal
tubule cells (28). This process may also contribute to the
inhibitory effect of Cd on the recycling of BBM transporters. With
time, the diminished expression of many apical proteins in the proximal tubule cell BBM (Refs. 28, 30, and
53 and the present study) and the impaired endocytosis of
proteins from the ultrafiltrate (28, 30) may contribute to
the loss of proximal tubule structure and function that is associated
with Cd-induced nephrotoxicity.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Eva Hersak, Djurdja Breski, and Mary McKee for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by Grants 00220101 (I. Sabolic), 0022111 (C. M. Herak-Kramberger), and 0022011 (I. Sabolic) from the Croatian Ministry of Science and Technology, by National Institutes of Health Fogarty International Research Collaborative Award 1-R03-TW-01057-01 (I. Sabolic and D. Brown) and Grant DK-42956 (D. Brown). The electron microscopy studies were performed in a Core facility that is partially supported by National Institutes of Health Grants DK-57521 and Grant DK-43351.
Address for reprint requests and other correspondence: I. Sabolic, Unit of Molecular Toxicology, Institute for Medical Research and Occupational Health, Ksaverska cesta 2, PO Box 291, HR-10001 Zagreb, Croatia (E-mail: sabolic{at}imi.hr).
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.
July 30, 2002;10.1152/ajprenal.00006.2002
Received 15 February 2002; accepted in final form 25 July 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abbate, M,
Bachinsky D,
Zheng G,
Stamenkovic I,
McLaughlin M,
Niles JL,
McCluskey RT,
and
Brown D.
Location of gp330/2-m receptor-associated protein (
2-MRAP) and its binding sites in kidney: distribution of endogenous
2-MRAP is modified by tissue processing.
Eur J Cell Biol
61:
139-149,
1993[ISI][Medline].
2.
Abbate, M,
Bonventre J,
and
Brown D.
The microtubule network of renal epithelial cells is disrupted by ischemia and reperfusion.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F971-F978,
1994
3.
Adams, RG,
Harrison JF,
and
Scott P.
Cadmium-induced proteinuria, impaired renal function, and osteomalacia in alkaline battery workers.
QJM
38:
425-443,
1969[Medline].
4.
Ahn, W,
Kim YK,
Kim KR,
and
Park YS.
Cadmium binding and sodium-dependent solute transport in renal brush-border membrane vesicles.
Toxicol Appl Pharmacol
154:
212-218,
1999[ISI][Medline].
5.
Ahn, DW,
and
Park YS.
Transport of inorganic phosphate in renal cortical brush-border membrane vesicles of cadmium-intoxicated rats.
Toxicol Appl Pharmacol
133:
239-243,
1995[ISI][Medline].
6.
Baus, M,
Medjugorac-Popovski M,
Brown D,
and
Sabolic I.
In colchicine-treated rats, cellular distribution of AQP-1 in convoluted and straight proximal tubule segments is differently affected.
Pflügers Arch
439:
321-330,
2000[ISI][Medline].
7.
Biber, J,
Stieger B,
Haase W,
and
Murer H.
A high yield preparation for rat kidney brush-border membranes; different behavior of lysosomal markers.
Biochim Biophys Acta
647:
169-176,
1981[ISI][Medline].
8.
Biemesderfer, D,
Rutherford PA,
Nagy T,
Pizzonia JH,
Abu-Alfa AK,
and
Aronson PS.
Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal kidney.
Am J Physiol Renal Physiol
273:
F289-F299,
1997
9.
Bingham-Smith, J,
Dwyer SD,
and
Smith L.
Cadmium evokes inositol polyphosphate formation and calcium mobilization.
J Biol Chem
264:
7115-7118,
1989
10.
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].
11.
Brown, D,
Lee R,
and
Bonventre JV.
Redistribution of villin to proximal tubule basolateral membranes after ischemia and reperfusion.
Am J Physiol Renal Physiol
273:
F1003-F1012,
1997
12.
Brown, D,
Lydon J,
McLaughlin M,
Stuart-Tilley A,
Tyszkowski R,
and
Alper S.
Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).
Histochem Cell Biol
105:
261-267,
1996[ISI][Medline].
13.
Brown, D,
Sabolic I,
and
Gluck S.
Colchicine-induced redistribution of proton pumps in the proximal tubule.
Kidney Int
40, Suppl33:
S79-S83,
1991[ISI].
14.
Brown, D,
and
Stow JL.
Protein trafficking and polarity in kidney epithelium: from cell biology to physiology.
Physiol Rev
76:
245-297,
1998
15.
Cherian, GM.
Metabolism of orally administered cadmium-metallothionein in mice.
Environ Health Perspect
28:
127-130,
1979[ISI][Medline].
16.
Cherian, MG,
Goyer RA,
and
Delaquerriere-Richardson L.
Cadmium-metallothionein-induced nephropathy.
Toxicol Appl Pharmacol
38:
399-408,
1976[ISI][Medline].
17.
Cherian, MG,
Goyer RA,
and
Valberg LS.
Gastrointestinal absorption of oral cadmium chloride and cadmium-metallothionein in mice.
J Toxicol Environ Health
4:
861-868,
1978[ISI][Medline].
18.
Christensen, EI,
and
Birn H.
Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule.
Am J Physiol Renal Physiol
280:
F562-F573,
2001
19.
Condron, RJ,
Schroen CJ,
and
Marshall AT.
Morphometric analysis of renal proximal tubules in cadmium-treated rats.
J Submicrosc Cytol Pathol
26:
51-58,
1994[ISI][Medline].
20.
Dorian, C,
Gattone VH, II,
and
Klaasen CD.
Accumulation and degradation of the protein moiety of cadmium-metallothionein (CdMT) in the mouse kidney.
Toxicol Appl Pharmacol
117:
242-248,
1992[ISI][Medline].
21.
Dorian, C,
Gattone VH, II,
and
Klaasen CD.
Discrepancy between the nephrotoxic potencies of cadmium-metallothionenin and cadmium chloride and the renal concentration of cadmium in the proximal convoluted tubules.
Toxicol Appl Pharmacol
130:
161-168,
1995[ISI][Medline].
22.
Dorian, C,
Gattone VH, II,
and
Klaasen CD.
Renal cadmium deposition and injury as a result of accumulation of cadmium-metallothionein (CdMT) by the proximal convoluted tubulesa light microscopic autoradiography study with 109CdMT.
Toxicol Appl Pharmacol
114:
173-181,
1992[ISI][Medline].
23.
Dudley, RE,
Gammal LM,
and
Klaasen CD.
Cadmium-induced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity.
Toxicol Appl Pharmacol
77:
414-426,
1985[ISI][Medline].
24.
Friberg, L,
Piscator M,
Nordberg GF,
and
Kjellstrom T.
Cadmium in the Environment (2nd ed.). Cleveland, OH: CRC, 1974.
25.
Gan, SD,
Fan MM,
and
He GP.
The role of microtubules in axoplasmic transport in vivo.
Brain Res
369:
75-82,
1986[ISI][Medline].
26.
Gutmann, EJ,
Niles JL,
McCluskey RT,
and
Brown D.
Colchicine-induced redistribution of an apical membrane glycoprotein (gp330) in proximal tubules.
Am J Pathol
257:
C397-C407,
1989.
27.
Herak-Kramberger, CM,
Breton S,
Brown D,
Kraus O,
and
Sabolic I.
Distribution of the vacuolar H+-ATPase along the rat and human male reproductive tract.
Biol Reprod
64:
1699-1707,
2001
28.
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].
29.
Herak-Kramberger, CM,
and
Sabolic I.
The integrity of renal cortical brush-border and basolateral membrane vesicles is damaged in vitro by nephrotoxic heavy metals.
Toxicology
156:
139-147,
2001[ISI][Medline].
30.
Herak-Kramberger, CM,
Spindler B,
Biber J,
Murer H,
and
Sabolic I.
Renal type II Na/Pi-cotransporter is strongly impaired whereas the Na/sulphate-cotransporter and aquaporin 1 are unchanged in cadmium-treated rats.
Pflügers Arch
432:
336-344,
1996[ISI][Medline].
31.
Järup, L,
Berglund M,
Elinder CG,
Nordberg G,
and
Vahter M.
Health effects of cadmium exposurea review of the literature and a risk estimate.
Scand J Work Environ Health
24, Suppl1:
1-52,
1998[ISI].
32.
Jin, T,
Leffler P,
and
Nordberg GF.
Cadmium-metallothionein nephrotoxicity in the rat: transient calciuria and proteinuria.
Toxicology
45:
307-317,
1987[ISI][Medline].
33.
Jonah, MM,
and
Bhattacharyya MH.
Early changes in the tissue distribution of cadmium after oral but not intravenous cadmium exposure.
Toxicology
58:
325-338,
1989[ISI][Medline].
34.
Kim, KR,
Lee HY,
Kim CK,
and
Park YS.
Alteration of renal amino acid transport system in cadmium-intoxicated rats.
Toxicol Appl Pharmacol
106:
102-111,
1990[ISI][Medline].
35.
Kim, YK,
Choi JK,
Kim JS,
and
Park YS.
Changes in renal function in cadmium-intoxicated rats.
Pharmacol Toxicol
63:
342-350,
1988[ISI][Medline].
36.
Kinne, RKH,
Schutz H,
and
Kinne-Saffran E.
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,
1995[ISI][Medline].
37.
Lee, HY,
Kim KR,
and
Park YS.
Transport kinetics of glucose and alanine in renal brush-border membrane vesicles of cadmium-intoxicated rabbits.
Pharmacol Toxicol
69:
390-395,
1991[ISI][Medline].
38.
Leffler, PE,
Jin T,
and
Nordberg GF.
Nephrotoxic impact of multiple short-interval cadmium-metallothionein injections in the rat.
Toxicology
112:
151-156,
1996[ISI][Medline].
39.
Leffler, PE,
Jin T,
and
Nordberg GF.
Differential calcium transport disturbances in renal membrane vesicles after cadmium-metallothionein injection in rats.
Toxicology
143:
227-234,
2000[ISI][Medline].
40.
Liliom, K,
Wagner G,
Pacz A,
Cascante M,
Kovacz J,
and
Ovadi J.
Organization-dependent effects of toxic bivalent ions on microtubule assembly and glycolysis.
Eur J Biochem
267:
1-9,
2000
41.
Lonnerholm, G,
and
Ridderstrale Y.
Intracellular distribution of carbonic anhydrase in the rat kidney.
Kidney Int
17:
162-174,
1980[ISI][Medline].
42.
Maranda, B,
Brown D,
Bourgoin S,
Casanova JE,
Vinay P,
Ausiello DA,
and
Marshansky V.
Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes.
J Biol Chem
276:
18540-18550,
2001
43.
McLean, IW,
and
Nakane PF.
Periodate-lysine paraformaldehyde fixative: a new fixative for immunoelectron microscopy.
J Histochem Cytochem
22:
1077-1083,
1974[ISI][Medline].
44.
Mellman, I,
Fuchs R,
and
Helenius A.
Acidification of the endocytic and exocytic pathways.
Ann Rev Biochem
55:
663-700,
1986[ISI][Medline].
45.
Mills, JW,
and
Ferm VH.
Effect of cadmium on F-actin and microtubules of Madin-Darby canine kidney cells.
Toxicol Appl Pharmacol
101:
245-254,
1989[ISI][Medline].
46.
Miura, K,
Inokawa M,
and
Imura N.
Effects of methylmercury and some metal ions on microtubule networks in mouse glioma cells and in vitro tubulin polymerization.
Toxicol Appl Pharmacol
73:
218-231,
1984[ISI][Medline].
47.
Nomiyama, K,
Nomiyama H,
and
Kameda N.
Plasma cadmium-metallothionein, a biological exposure index for cadmium-induced renal disfunction, based on the mechanism of its action.
Toxicology
129:
157-168,
1998[ISI][Medline].
48.
Nordberg, GF,
Goyer R,
and
Nordberg M.
Comparative toxicity of cadmium-metallothionenin and cadmium chloride on mouse kidney.
Arch Pathol
99:
192-197,
1975[ISI][Medline].
49.
O'Brien, ET,
Salmon ED,
and
Erickson HP.
How calcium causes microtubule depolymerization.
Cell Motil Cytoskel
36:
125-135,
1997[ISI][Medline].
50.
Park, K,
Kim KR,
Kim JY,
and
Park YS.
Effect of cadmium on Na-Pi cotransport kinetics in rabbit renal brush-border membrane vesicles.
Toxicol Appl Pharmacol
145:
255-259,
1997[ISI][Medline].
51.
Perrino, BA,
and
Chou IN.
Role of calmodulin in cadmium-induced microtubule disassembly.
Cell Biol Int Rep
10:
565-573,
1986[ISI][Medline].
52.
Sabolic, I,
Culic O,
and
Brown D.
Localization of ecto-ATPase in the rat kidney and isolated cortical vesicles.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F217-F228,
1992
53.
Sabolic, I,
Herak-Kramberger CM,
Blanua M,
and
Brown D.
Loss of brush-border proteins in cadmium-induced nephrotoxicity in rat.
Period Biol
102:
33-41,
2000[ISI].
54.
Sabolic, I,
Herak-Kramberger CH,
Breton S,
and
Brown D.
Na/K-ATPase in intercalated cells along the rat nephron revealed by antigen retrieval.
J Am Soc Nephrol
10:
913-922,
1999
55.
Sabolic, I,
Herak-Kramberger CM,
and
Brown D.
Subchronic cadmium treatment affects the abundance and arrangement of cytoskeletal proteins in rat renal proximal tubule cells.
Toxicology
165:
205-216,
2001[ISI][Medline].
56.
Sabolic, I,
Herak-Kramberger CM,
Ljubojevic M,
Biemesderfer D,
and
Brown D.
NHE3 and NHERF are targeted to the basolateral membrane in proximal tubules of colchicine-treated rats.
Kidney Int
61:
1351-1364,
2002[ISI][Medline].
57.
Sabolic, I,
Valenti G,
Verbavatz JM,
Van Hoek AN,
Verkman AS,
Ausiello DA,
and
Brown D.
Localization of the CHIP28 water channel in rat kidney.
Am J Physiol Cell Physiol
263:
C1225-C1233,
1992
58.
Sato, K,
Kusaka Y,
and
Okada K.
Direct effect of cadmium on citrate uptake by isolated rat renal brush border membrane vesicles.
Toxicol Lett
80:
161-165,
1995[ISI][Medline].
59.
Sendelbach, LE,
and
Klaasen CD.
Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmium-metallothionein.
Toxicol Appl Pharmacol
92:
95-102,
1988[ISI][Medline].
60.
Shaikh, ZA,
Vu TT,
and
Zaman K.
Oxidative stress as a mechanism of chronic cadmium-induced hepatotoxicity and renal toxicity and protection by antioxidants.
Toxicol Appl Pharmacol
154:
256-263,
1999[ISI][Medline].
61.
Squibb, KS,
Pritchard JB,
and
Fowler BA.
Cadmium-metallothonein nephropathy: relationship between ultrastructural/biochemical alterations and intracellular cadmium binding.
J Pharmacol Exp Ther
229:
311-321,
1984[Abstract].
62.
Squibb, KS,
Ridlington JW,
Carmichael NG,
and
Fowler BA.
Early cellular effects of circulating cadmium-thionein on kidney proximal tubules.
Environ Health Perspect
28:
287-296,
1979[ISI][Medline].
63.
Sugihira, N,
Sagai M,
and
Suzuki KT.
Renal damage induced by cadmium-metallothionenin: effects on biochemical indicators.
Toxicology
44:
1-11,
1987[ISI][Medline].
64.
Suzuki, CAM,
and
Cherian G.
Renal toxicity of cadmium-metallothionein and enzymuria in rats.
J Pharmacol Exp Ther
240:
314-319,
1987[Abstract].
65.
Tokuyasu, KT.
Immunocytochemistry on ultrathin frozen sections.
Histochem J
12:
381-403,
1980[ISI][Medline].
66.
Trosper, TL,
and
Philipson KD.
Effects of divalent and trivalent cations on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.
Biochim Biophys Acta
731:
63-68,
1983[ISI][Medline].
67.
Vallee, BL,
and
Ulmer DD.
Biochemical effects of mercury, cadmium, and lead.
Ann Rev Biochem
41:
91-128,
1972[ISI][Medline].
68.
Verbost, PM,
Senden MHMN,
and
VanOs CH.
Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium.
Biochim Biophys Acta
902:
247-252,
1987[ISI][Medline].
69.
Visser, GJ,
Peters PHJ,
and
Theuvenet APR
Cadmium ion is a non-competitive inhibitor of red cell Ca2+-ATPase activity.
Biochim Biophys Acta
1152:
26-34,
1993[ISI][Medline].
70.
Wang, XP,
Chan HM,
Goyer RA,
and
Cherian MG.
Nephrotoxicity of repeated injections of cadmium-metallothionein in rats.
Toxicol Appl Pharmacol
119:
11-16,
1993[ISI][Medline].