TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
117,242-248 (1992)
Accumulation and Degradation of the Protein Moiety of CadmiumMetallothionein (CdMT) in the Mouse Kidney’ CATHERINEDORIAN,~ Departments
VINCENT H. GATTONE II, ANDCURTIS
of Pharmacology, Toxicology University of Kansas Medical
& Therapeutics. Center. Kansas
D.KLAASSEN~
and Anatomy and Ceil Biology, City, Kansas 66160
Received May 5, 1992; accepted August 10, 1992
Evidence of kidney damage after Cd exposure was obAccumulation and Degradation of the Protein Moiety of Cad- served as early as 1948 by Friberg and was confirmed by mium-Metallothionein (CdMT) in the Mouse Kidney. DORIAN, other investigators in the following years (Axelsson et al., C.,GATTONEII,V. H., ANDKLAASSEN,~. D.( 1992). Toxicof. 1968; Friberg et al., 1974; Nordberg et al., 1975). Factors Appl. Pharmacol. 117,242-248. such as length of exposure (chronic or acute), route of exOf major concern in Cd toxicity is its ability to produce renal posure, and nutritional status (calcium, vitamins, etc.) can damage after chronic exposure in humans and experimental an- affect the outcome of the uptake, the severity of the toxicity. imals. Renal injury affects predominantly the proximal tubules and the initial site of toxicity (Nomiyama et al., 1975). and more specifically the first segments of these tubules. Similar Acute exposure to inorganic Cd produces hepatotoxicity toxic effects to the kidneys are observed after administration of (Dudley et al., 1984) but with chronic administration, the cadmium bound to metallothionein (CdMT). Therefore, CdMT target organ of toxicity changes from liver to kidney (Friberg wasused in this study as a model to understand the mechanism(s) of Cd nephrotoxicity. It has heen recently demonstrated that Cd et al., 1974). In the liver, Cd is stored bound to metallothiis a nonenzymatic, inducfrom CdMT was preferentially taken up by the proximal con- onein (CdMT). Metallothionein voluted tubules. Therefore, the purpose of these studies was to ible, low-molecular-weight protein which is thought to play determine if the organic portion of the complex was also accu- a role in the metabolism and detoxication of a number of mulated in these tubules. [‘%]CdMT prepared from rat liver was essential and nonessential metals. This protein has a rapid administered intravenously to mice at a nonnephrotoxic dose turnover but the liver has a very high capacity to synthesize (0.1 mg Cd/kg). The radioactivity in the kidney showed maxi- MT. Therefore, Cd is efficiently trapped in the liver as CdMT mum level (80% of the dose) 15 min after the injection. This complex. Metallothionein may play a protective role in Cd preferential renal uptake was also observed after administration exposure by sequestering the toxic metal ion and forming a of various doses of [35S]CdMT. In contrast to the earlier observed nontoxic intracellular metal-protein complex (Cherian et persistency of io9Cd in the kidney after “‘%dMT administration, al., 1976). Metallothionein may be involved in the tolerance 35Sdisappeared rapidly (with a half-life of approximately 2 hr), to Cd. Pretreatment by metals, such as Cd and Zn, which and 24 hr after injection of [35S]CdMT, there was very little 35S induce MT, reduces Cd-induced lethality (Yoshikawa, 1970; left in the kidneys. These observations indicate that the protein Leber and Miya, 1976; Probst et al., 1977; Goering and portion of CdMT is rapidly degraded after renal uptake of CdMT and the released Cd is retained in the kidney. Within the kidney, Klaassen, 1983, 1984a) and hepatotoxicity (Goering and 35Sdistributed mainly to the cortex. Light microscopic autora- Klaassen, 1984a,b; Suzuki et al., 1990). diography showed that [35S]CdMT preferentially distributed to With prolonged exposure, CdMT is released from the liver the proximal convoluted tubule (Sl and S2), which is the site into the bloodstream, probably due to minor hepatic injury of nephrotoxicity. Within the Sl and S2 segments, a greater (Dudley et al., 1985). Following glomerular filtration, this distribution of 3sSto the apical portion of the cells was observed low-molecular-weight complex (MW about 6500) binds to after administration of both a nonnephrotoxic (0. I mg Cd/kg) the brush border of the proximal tubular cells (Foulkes, 1978) and a nephrotoxic (0.3 mg Cd/kg) dose. lo9Cd administered as lo9CdMT also distributed to the apical portion of the Sl and S2 and is taken up by these cells. The toxicity of Cd is primarily cells. Therefore, both the organic (35S) and inorganic (‘@‘Cd) confined to S 1 and S2 (convoluted) segments of the proximal portions of CdMT are rapidly and efficiently taken up by the Sl tubules. In these segments, morphological changes (vacuolar degeneration and/or swelling) can be observed. Morphologand S2 cells of the proximal tubules, the site of nephrotoxicity. These observations support the concept that CdMT is readily ical and functional alterations produced by chronic exposure taken up by the proximal tubular cells as a complex, and then its protein portion is rapidly degraded to release Cd that binds permanently to intracellular sites and produces nephrotoxicity. ’ Supported by NIH Grant ES-01 142. 0 1992 Academic
2 Supported by a fellowship from Merck Sharp & Dohme. ’ To whom all correspondence should be addressed.
Press, Inc.
0041-008X/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
242
RENAL DISTRIBUTION
to Cd have been described, but the mechanism(s) of these nephrotoxic effects is (are) not clearly understood. One important finding in the study of Cd-induced nephrotoxicity is that renal damage induced by Cd bound to thionein (CdMT) is similar to that caused by chronic exposure to CdC& (Nordberg et al., 1975; Suzuki et al., 1979; Cherian and Nordberg, 1980). Therefore, CdMT was used as a probe to assessthe mechanisms of chronic Cd toxicity on the kidney. Two hypotheses have been proposed to explain the development of this nephrotoxicity. Several investigators (Webb and Etienne, 1977; Fowler and Nordberg, 1978; Squibb et al., 1979; Cain and Holt, 1983) have suggested that renal damage is caused by Cd ions which are released intracellularly by the lysosomal degradation of the reabsorbed complex. This “lysosomal theory” is based on studies showing that the protein is degraded when it is reabsorbed in the kidney. In support of this hypothesis, it has been shown by subcellular fractionation that Cd is concentrated in the lysosomes 30 min after CdMT administration (Squibb et al., 1979; Sato and Nagai, 1986). Cd is then redistributed to the cytosol where it is bound to highmolecular-weight proteins. Although the kidney is also able to synthesize MT, the rate of synthesis is slower than that in the liver (Sendelbach and Klaassen, 1988). Within 24 to 72 hr after administration, Cd again binds to thionein in the cytosol (Sato and Nagai, 1986). Renal injury according to this hypothesis is due to the Cd cation and not to the intact complex CdMT. In contrast, Cherian et al. ( 1976) have suggested that the process of pinocytosis during the uptake of injected CdMT produces membrane changes which are the cause of the toxicity. Therefore, CdMT is a nephrotoxin. In support of this hypothesis, Cd administered as CdMT does not accumulate in lysosomal subfractions (Cherian, 1978). Some degradation of CdMT occurs, followed by synthesis of new thionein, hence, Cd is bound to thionein at all times. Consequently, the role of the lysosomal system in renal CdMT toxicity by this hypothesis is minimal. While these hypotheses on how CdMT produces its nephrotoxicity are intriguing, much data is yet to be collected to support or refute them. A considerable amount of work has been performed on the distribution of Cd after CdMT administration, but little is available on the distribution of the organic portion of the molecule (Bremner et al., 1978; Cain and Holt, 1983; Andersen et al., 1987). We have recently demonstrated that the Cd in CdMT is rapidly and efficiently taken up by the Sl and S2 segments of the proximal tubule cells, the site of Cd nephrotoxicity (Dorian et al., 1992), and that practically no Cd is eliminated from these cells within a week. Therefore, the purpose of the present study was to quantify the uptake of the peptide portion of the CdMT complex by the S 1 and S2 cells of the proximal tubules. The fate of the protein portion of CdMT was studied by using [35S]CdMT and by light microscopic autoradiography, a
243
OF [35S]CdMT
technique previously used to study Cd nephrotoxicity itatively. MATERIALS
qual-
AND METHODS
Preparation of [‘5qCdMT. Male Sprague-Dawley rats (350-400 g) (Sasco, Omaha, NE) were injected subcutaneously with CdC12 (3 mg Cd/ kg/day) for 4 days. Each day, 4 hr after CdCl* injection, [35S]cysteine(Amersham Co., Arlington Heights, IL) was administered ip (50 pCi/animal for 3 days and 100 &i/animal on the fourth day) to each rat. [3SS]CdMT was purified as described previously (Dorian et al., 1992). Following separation of the two isoforms (MT-I and MT-II), the two fractions were dried by lyophilization and resolubilized in 5-10 ml of ultrapure water. The protein concentration was determined by ultraviolet spectroscopy (Waddell, 1956) with a DU-8 Spectrophotometer (Beckman Instruments, Inc., Arlington Heights, IL). Cd and Zn contents were analyzed with a Perkin-Elmer Model 2380 flame atomic absorption spectrophotometer (Notwalk, CT). The CdMT solutions were then stored in aliquots at -20°C. Because of its purity and abundance. [35S]CdMT II was used in all the experiments. Injection of CdMT for the dose-response study. [-“S]CdMT (I .4 &i/ nmole MT) was diluted with isotonic saline and used for injection. Male Swiss mice, weighing 25-30 g (Harlan, Indianapolis, IN) and kept in the same conditions as rats, were used throughout the experiments. [35S]CdMT was injected via the tail vein at dosages of 50 to 1000 pg Cd/kg. Kidneys and liver were removed 15 min after the injection. Injection of CdMT for the time-response study. [35S]CdMT (1.4 pCi/ nmole MT) was administered via the tail vein at 0.1 mg Cd/kg (a nonnephrotoxic dose: Maitani et al., 1988). Kidneys and liver were removed at 5, 15, 30, 45, and 60 min, as well as 2, 4. 8, and 24 hr after the injection. Determination of [“.5JCdMT in tissues. Kidney and liver were digested in Protosol (DuPont NEN Research Products, Boston. MA). Approximately 100 mg of tissue was minced with scissors, put in I ml of solution. and digested at 50°C for 3 hr in open glassvials. After cooling to room temperature and addition of 15 ml of scintillation liquid (Econo-safe, Research Products International Corp., Mount Prospect, IL), the concentration of [“S]CdMT in kidney and liver samples was determined by quantifying the radioactivity in a liquid scintillation analyzer (Packard Model 2000 CA, Downers Grove, IL). Tissue [35S]CdMT concentrations are expressed in nmoles of MT equivalents per gram of tissue. Light microscopic autoradiography study. Mice were anesthetized I5 min after administration of [35S]CdMT with sodium pentobarbital(30 mg/ kg ip) (Anthony Products Co., Arcadia, CA). An intracardiac perfusion was performed with a 60-ml syringe equipped with a blunt needle (Monoject 200, 2 1 X 1 in.). Following perfusion with saline, the left kidney was ligated and removed, digested in Protosol, and the concentration of 35Sdetermined. The remainder of the mice was perfused with fixative (2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4) and the right kidney was removed and used for autoradiography. Preparation of the tissue samples for the light microscopic autoradiography was done as previously described (Dorian et al., 1992). The exposure time of the specimens was 12 weeks. The slides were developed (developer D-19 and fixer, Kodak Lab, Rochester, NY), stained, and photographed under a light microscope (objective 40X). Cellular and subcellular areas in different parts of the nephron were determined on 15 randomly chosen tubules per nephron segment per mouse with a Bioquant Image Analysis system (R&M Biometrics, Inc., Nashville, TN). The silver grains were then counted manually. MT concentrations are expressed as density of grains per cellular area (cm2). Background grain density was determined from mice treated with unlabeled CdMT. The values were subtracted from the tubule grain density prior to analysis of the data. Statistics. Statistical comparisons were based on analysis of variance followed by Duncan’s multiple range test at the 5% level of significance.
244
DORIAN, GATTONE,
5’
AND KLAASSEN
15’ 30’ 4S 60’ Zhr4hrBhr24hr
ZJ-
FE
Liver
Liver
1 -L-0’
I
I
5’
15’
30’
45’
60’
2 hrs
4 hrs
8 hrs
24 hrs
FIG. 1. Time-dependent accumulation of [%]MT equivalents in kidney and liver after a single iv injection of a nonnephrotoxic dose (0.1 mg Cd/ kg) of [%]CdMT II to mice. Symbols represent the mean + SE of five mice. The inset represents the results on a logarithmic scale.
RESULTS
Figure 1 depicts the distribution of 35S at various times after administration of a nonnephrotoxic dose (0.1 mg Cd/ kg) of [35S]CdMT. The 35S was preferentially taken up by the kidneys (approximately 80% of the dose) and very little of it was found in the liver. The concentration of 35Sin the kidney was 10 to 12 times higher than that in the liver during the first hour following the injection. The concentration of 35Sin the kidney s increased rapidly after the injection. The maximum concentration was reached 15 min after [35S]CdMT administration. However, 35S concentration in the kidney decreased rapidly. The half-life of [35S]thionein was about 2 hr. Within 24 hr after [35S]CdMT administration, the concentration of “S in the kidney and the liver was nearly the same (0.6 nmol MT/g tissue in the kidney versus 0.45 nmol MT/g tissue in the liver). Fifteen minutes after administration of [35S]CdMT, the concentration of “S in kidneys increased as the dosage of CdMT increased from 0.05 to 0.4 mg Cd/kg, but remained relatively constant with further increase in dosage (Fig. 2). The plateau in renal “S concentration obtained with dosages larger than 0.4 mg Cd/kg suggests a saturation of the reabsorption process. In contrast, low and relatively constant concentrations of [35S]MT were found in liver (about 0.1 nmol MT/g tissue). A dose-response study (unpublished data) using ‘@‘CdMT prepared from rat liver (Dorian et al., 1992) was done at the same dosages as for [35S]CdMT. Figure 3 shows a comparison of the renal CdMT concentrations after administration of either lmCdMT or [35S]CdMT at various dosages. It is known that 1 mol of thionein can bind up to 7 mol of cadmium. As the MT in the solution used in these studies was almost saturated with Cd (6.7 mol Cd/mol MT), it was possible to estimate MT concentration based on Cd concentration. In
[35 S I-CdMT
(mg Cd/kg)
FIG. 2. Dosedependent accumulation of [“S]MT equivalents in kidney and liver 15 min after a single injection of various doses of [?S]CdMT II to mice. Symbols represent mean f SE of five mice.
Fig. 3, we plotted CdMT concentrations as determined from 35S measured after [35S]CdMT administration, and CdMT concentrations as calculated from ‘@‘Cd concentration measured after ‘09CdMT administration. There is a good correlation between the concentration measured and the concentration calculated. Figure 3 indicates that regardless of the position of the radioactive label (on the metal or on the protein moiety), similar renal CdMT concentrations were observed after administration of the complex. An increase in renal MT concentration occurred in a dose-dependent manner; above the dosage of 0.5 mg Cd/kg, reabsorption of MT by the kidney appears to be saturated (Fig. 3). The distribution of 35Sadministered as [35S]CdMT to different parts of the nephron was determined after injection of a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose. The areas examined were glomerulus (without Bowman’s capsule), S 1 segment and convoluted portion
0.2
0.4
CdMT
0.6
0.6
1.0
(mg Cd/kg)
FIG. 3. Dose-dependent accumulation of CdMT II in kidney after administration of lssCdMT and [35S]CdMT, expressed in nmol of MT equivalents/g tissue. Symbols represent the mean & SE of five mice.
RENAL DISTRIBUTION
245
OF [35S]CdMT
FIG. 4. Autoradiography of kidney from [‘%]CdMT-treated mouse. Brightfield (a) and darkfield (b) micrographs of renal cortex showing autoradiographic labeling (arrows, silver grains are black dots in a and white dots in b) of the proximal tubules. The grains are mainly over the brush border (apical region) of the cells. 400X.
of the S2 segment of the proximal tubules (termed hereon S 1 and S2) and distal convoluted tubules (DCT) in the cortex, as well as the straight proximal tubules (S3) and distal straight tubules (DST) in the outer stripe of the outer medulla. A typical autoradiograph is shown in Fig. 4. Fifteen minutes after administration of [35S]CdMT, areas of high-grain density were observed in the cortex and were in contrast to the lower density observed in the outer medulla. In the cortex, the highest concentration of [35S]MT was observed in the proximal convoluted tubules (Sl and S2 segments) (Fig. 5). The grain density in these first segments of the proximal tubules was approximately two times higher than that in the proximal straight (S3) segments. The grain density in the glomeruli was low and it was even lower in the distal tubules. This pattern of grain distribution was very similar after administration of either a nonnephrotoxic or a nephrotoxic dose of lo9Cd as ‘09CdMT (Dorian et al., 1992). Thus, after administration of both [35S]CdMT and lo9CdMT, the highest level of radioactivity is found in the Sl and S2 segments of the proximal tubules. As the proximal tubules are the primary site of Cd toxicity (Friberg et al., 1974) we also determined the distribution of [35S]CdMT in the apical (brush border) and basal (cytoplasm) portions of the proximal tubules (Fig. 6). The apical portion, at the luminal side of the cell, includes the brush border or supranuclear region. The portion of the cell designated as basal contains the midregions and the basal regions. After administration of both nonnephrotoxic and nephrotoxic doses of [35S]CdMT, three to five times higher concentrations of 35Swere observed in the apical than in the basal portion of the proximal convoluted tubules (S 1 and S2). In the proximal straight tubules (S3), a preferential apical distribution
of 35Swas noted only after administration dose (0.3 mg Cd/kg).
of the nephrotoxic
DISCUSSION The renal proximal tubules are the most vulnerable sites of the kidney to chronic cadmium intoxication in humans and in experimental animals. Renal dysfunction (glycosuria,
0.1
[ %+CdMT
0.3
(mg Cd/kg)
FIG. 5. Distribution of [35S]MT equivalents, as quantified by light microscopic autoradiography, in different parts of the nephron, I5 min after a single iv injection of a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic dose (0.3 mg Cd/kg) of [3SS]CdMT. Sl and S2 indicate the proximal convoluted tubules, S3 the proximal straight tubules, DCT the distal convoluted tubules, and DST the distal straight tubules. Bars represent mean + SE of five mice. The asterisk indicates that [35S]MT concentration is significantly higher in the Sl and S2 segments of the proximal tubules than in the other tubules.
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DGRIAN, GATTONE,
AND KLAASSEN
CdMT preferentially distributes to the cortex (Nordberg and Nordberg, 1975; Murakami et al., 1983; Dorian et al., 1992). A similar preferential distribution to the cortex has been observed after [3H]CdMT administration (Andersen et al., 1987). In the present work, within the kidney, 35Safter administration of [35S]CdMT attains a concentration three times higher in the cortex than in the outer medulla. Cd administered as CdMT in the nephron distributes mainly to the first segments of the proximal tubules (Dorian et al., 1992). It is well known that CdMT selectively alters proximal convoluted tubule cells while glomeruli and distal tubule cells consistently show no changes (Cherian et al., 1976; Nordberg et al., 1975; Webb and Etienne, 1977; Mu0.1 0.3 rakami et al., 1983). Proximal convoluted tubules are con[ 3%]-CdMT (mg Cd/kg) sidered to be the primary site of Cd nephrotoxicity. Little FIG. 6. Distribution of [35S]MT equivalents, as quantified by light miwas known about the tubular distribution of the organic porcroscopic autoradiography, in apical (brush border) and basal (cytoplasm) tion of CdMT prior to this study. The quantification of 35S, portions of the proximal tubules in the cortex and the outer medulla 15 by light microscopic autoradiography, revealed that 35Sconmin after a single iv injection of a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose of [‘%]CdMT. Bars represent the mean + centrated predominantly in the convoluted (S 1 and S2) segSE of five mice. The asterisk indicates that [35S]MT concentration is signif- ments of the proximal tubules, after administration of either icantly higher in the apical than in the basal portion of the proximal tubules. a nonnephrotoxic (0.1 mg Cd/kg) or a nephrotoxic (0.3 mg Cd/kg) dose of [35S]CdMT. The S3 segment of the proximal tubules as well as glomeruli and distal tubules accumulated low-molecular-weight proteinuria) and morphological 35S to a much lesser extent. At nonnephrotoxic dose, the changes (swelling, vacuolization, necrosis) are consistently presence of MT in these tubules may be due to a saturation observed (Axelsson et al., 1968; Friberg et al., 1974) after of the reabsorption process occurring in the convoluted long-term exposure to Cd. These functional and pathological proximal tubules. At nephrotoxic doses, a significant increase changes observed after chronic exposure to Cd can be re- in MT concentration was noted. This increase may result produced by an acute administration of CdMT, a low-mofrom a decrease in reabsorption by the damaged Sl and S2 lecular-weight protein with a high affinity for metals (Nordsegments. The finding that ‘09Cd- as well as 35S-labeled MT berg et al., 1975; Cherian et al., 1976; Webb, 1979). Inforare found in the highest concentration in the proximal conmation is available on the renal distribution and uptake of voluted tubules (Sl and S2), suggests that CdMT is reabCd after CdMT administration, but very little is known about sorbed by the proximal tubule cells as a complex and that the fate of the protein moiety after administration of this the site of CdMT uptake corresponds to the site of nephrocomplex. Therefore, CdMT labeled on the protein with toxicity. [35S]cysteine was used in these experiments to study its disThe distribution of 35Sin the apical and basal portions of tribution in the nephron. the cells was also compared in the convoluted and the straight The distribution of Cd in the body has been extensively proximal tubules. Fifteen minutes after injection of a nonstudied. Its distribution differs markedly depending on the nephrotoxic or a nephrotoxic dose of CdMT, a higher conchemical form of Cd. After administration of CdCl*, the centration of 35Swas observed in the apical than in the basal highest concentration of Cd is found in the liver. In contrast, portion of the Sl and S2 segments of the proximal tubules. Cd after CdMT administration is predominantly taken up In the straight (S3) segment of the proximal tubules, a difby the kidney (Cherian and Shaikh, 1975; Nordberg and ference in distribution between apical and basal was noted Nordberg, 1975; Tanaka et al., 1975; Webb and Etienne, only after administration of the nephrotoxic dose. The dis1977; Squibb et al., 1979; Suzuki et al., 1979; Dorian et al., tribution of Cd administered as CdMT was previously quan1992). Several studies have shown that much of 35S-, 14C-, tified in the apical and basal portions of the proximal tubules or 3H-labeled metallothionein is also concentrated in the (Dorian et al., 1992). Cd was evenly distributed throughout kidney (Cherian and Shaikh, 1975; Webb and Etienne, 1977; the cell at a nonnephrotoxic dose, but concentrated mainly Cain and Holt, 1983; Andersen et al., 1987). In the present in the apical portion of the proximal convoluted cells, 24 hr study, the distribution of [35S]CdMT purified from rat liver after injection of a nephrotoxic dose. Thus, lo9Cd and 35S was followed after intravenous administration of a non- from ‘09CdMT and [35S]CdMT, respectively, are not only accumulated in the same Sl and S2 cells, but they are also nephrotoxic dose (0.1 mg Cd/kg). The 35Safter [35S]CdMT administration was also predominantly accumulated by the localized similarly inside the cells. This further supports the kidney. conclusion that the whole CdMT complex is taken up from n
Apical
Sl & 52
l
RENAL DISTRIBUTION
the glomerular filtrate by the proximal convoluted tubular cells. Predominant apical localization of CdMT within the Sl and S2 cells appears to be due to uptake of the complex at the luminal side of these cells. It has been demonstrated that CdMT is freely filtered at the glomerulus and reabsorbed by the brush border of the proximal tubules by a system that appears to be specific for anionic proteins (Foulkes, 1978), and CdMT does not react with basolateral cell membranes in vivo (Foulkes and Blanck, 1990). The renal uptake of Cd is very rapid (maximum concentration reached 30 min after injection) after administration of ‘09CdMT (Tanaka et al., 1975; Dorian et al., 1992) or after administration of 13H]CdMT (maximum concentration reached 10 min after injection) (Andersen et al., 1987). In the present study, we also found that the renal uptake of “S is extremely fast and the maximum concentration is reached 15 min after administration of [35S]CdMT. Once in the kidney, ‘09Cd concentration remains unchanged for up to 7 days after administration (Tanaka et al., 1975; Suzuki et al., 1979; Dorian et al., 1992). In contrast, the half-life of the injected [35S]CdMT may be only a few hours (Cherian et al., 1975; Bremner et al., 1978). The results from the time-course study (Fig. 1) also show that [“S]CdMT is rapidly degraded and the renal half-life of the 35S-labeled Cd-thionein is approximately 2 hr. The half-life of the protein moiety of injected CdMT is, therefore, much shorter than that found for endogenous renal CdMT, which is approximately 5 days (Cain and Holt, 1979). The difference between rates of disappearance of the protein moiety of endogenous and exogenous CdMT suggests that CdMT reabsorbed by the kidney cells is much more rapidly degraded than CdMT synthesized within the cell itself. In summary, our studies on the renal distribution of radioactivity after administration of lo9CdMT and [35S]CdMT collectively indicate that CdMT is taken up by the cells of the proximal tubules as an intact complex of Cd and MT. Moreover, after internalization, the protein moiety of CdMT is rapidly degraded and the released Cd is retained inside the cells. Preferential uptake of CdMT by the proximal convoluted tubular cells and the subsequent intracellular release of the toxic Cd ions may explain the fact that these cells represent the targets of the Cd-induced nephrotoxicity. REFERENCES Andersen, K. J., Haga, H. J., and Dobrota, M. (1987). Lysosomes of the renal cortex: Heterogeneity and role in protein handling. Kidney Znt. 31, 886-897. Axelsson, B., Dahlgren, S. E., and Piscator, M. (1968). Renal lesions in the rabbit after long-term exposure to cadmium. Arch. Environ. Health 17, 24-28. Bremner, I., Hoekstra, W. G., Davies, N. T., and Young, B. W. (1978). Metabolism of 35S-labelled copper-, zinc- and cadmium-thionein in the rat. Chem. Biol. Interact. 23, 355-367.
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Dorian, C., Gattone, V. H., and Klaassen, C. D. (1992). Renal cadmium deposition and injury as a result of accumulation of cadmium-metallothionein (CdMT) by the proximal convoluted tubules - a light microscopic autoradiography study with imCdMT. Toxicol. Appl. Pharmacol. 114, 173-181. Dudley, R. E., Gamma], L. M., and Klaassen, C. D. (1985). Cadmiuminduced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol. 77,4 14-426. Dudley, R. E., Svoboda, D. J., and Klaassen, C. D. (1984). Time course of cadmium-induced ultrastructural changes in rat liver. Toxicol. Appl. Pharmacol. 76, 150-l 60. Foulkes, E. C. (1978). Renal tubular transport of cadmium-metallotbionein. Toxicol. Appl. Pharmacol. 45, 505-S 12. Foulkes, E. C., and Blanck, S. (1990). Acute cadmium uptake by rabbit kidneys: Mechanism and effects. Toxicol. Appl. Pharmacol. 102, 464473.
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Nordberg, G. F., Goyer, R., and Nordberg, M. (1975). Comparative toxicity of cadmium-metallothionein and cadmium chloride on mouse kidney. Arch. Pathol. 99, 192-197. Nordberg, M., and Nordberg, G. F. (1975). Distribution of metallothioneinbound cadmium and cadmium chloride in mice: Preliminary studies. Environ. Health Perspect. 12, 103-108. Probst, G. S., Bousquet, W. F., and Miya, T. S. ( 1977). Correlation of hepatic metallothionein concentrations with acute cadmium toxicity in mice. Toxicol. Appl. Pharmacol. I, 37-44. Sato, M., and Nagai, Y. (1986). Cadmium in rat kidney subcellular particles after injection of cadmium-metallothionein. J. Toxicol. Sci. 11, 29-39. Sendelbach, L. E., and Klaassen, C. D. (1988). Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmiummetallothionein. Toxicol. Appl. Pharmacol. 92, 95-102. Squibb, K. S., Ridlington, J. W., Carmichael, N. G., and Fowler, B. A. (1979). Early cellular effects of circulating cadmium-thionein in kidney proximal tubules. Environ. Health Perspect. 28, 287-296.
AND KLAASSEN Suzuki, C. A. M., Ohta, H., Albores, A., Koropatnick, J., and Cherian, M. G. (1990). Induction of metallothionein synthesis by zinc in cadmium pretreated rats. Toxicology 63, 273-284. Suzuki, K. T., Takenaka, S., and Kubota, K. (1979). Fate and comparative toxicity of metallothioneins with differing Cd/Zn ratios in rat kidney. Arch. Environ. Contam. Toxicol. 8, 85-95. Tanaka, K., Sueda, K., Onosaka, S., and Okahara, K. (1975). Fate of ‘@‘Cdlabelled metallothionein in rats. Toxicol. Appl. Pharmacol. 33, 258-266. Waddell, W. J. (1956). A simple ultraviolet spectrophotometric method for the determination of protein. J. Lab. Clin. Med. 48, 3 1 l-3 14. Webb, M., and Etienne, A. T. (1977). Studies on the toxicity and metabolism of cadmium-thionein. Biochem. Pharmacol. 26,25-30. Webb, M. (1979). The metallothioneins. In The Chemistry, Biochemistry and Biology of Cadmium (M. Webb, Ed.). Elsevier, Amsterdam. Yoshikawa, H. (1970). Preventive effect of pretreatment with low dose of metals on the acute toxicity of metals in mice. Ind. Health 8, 184- 19 1.
AND
APPLIED
PHARMACOLOGY
117,242-248 (1992)
Accumulation and Degradation of the Protein Moiety of CadmiumMetallothionein (CdMT) in the Mouse Kidney’ CATHERINEDORIAN,~ Departments
VINCENT H. GATTONE II, ANDCURTIS
of Pharmacology, Toxicology University of Kansas Medical
& Therapeutics. Center. Kansas
D.KLAASSEN~
and Anatomy and Ceil Biology, City, Kansas 66160
Received May 5, 1992; accepted August 10, 1992
Evidence of kidney damage after Cd exposure was obAccumulation and Degradation of the Protein Moiety of Cad- served as early as 1948 by Friberg and was confirmed by mium-Metallothionein (CdMT) in the Mouse Kidney. DORIAN, other investigators in the following years (Axelsson et al., C.,GATTONEII,V. H., ANDKLAASSEN,~. D.( 1992). Toxicof. 1968; Friberg et al., 1974; Nordberg et al., 1975). Factors Appl. Pharmacol. 117,242-248. such as length of exposure (chronic or acute), route of exOf major concern in Cd toxicity is its ability to produce renal posure, and nutritional status (calcium, vitamins, etc.) can damage after chronic exposure in humans and experimental an- affect the outcome of the uptake, the severity of the toxicity. imals. Renal injury affects predominantly the proximal tubules and the initial site of toxicity (Nomiyama et al., 1975). and more specifically the first segments of these tubules. Similar Acute exposure to inorganic Cd produces hepatotoxicity toxic effects to the kidneys are observed after administration of (Dudley et al., 1984) but with chronic administration, the cadmium bound to metallothionein (CdMT). Therefore, CdMT target organ of toxicity changes from liver to kidney (Friberg wasused in this study as a model to understand the mechanism(s) of Cd nephrotoxicity. It has heen recently demonstrated that Cd et al., 1974). In the liver, Cd is stored bound to metallothiis a nonenzymatic, inducfrom CdMT was preferentially taken up by the proximal con- onein (CdMT). Metallothionein voluted tubules. Therefore, the purpose of these studies was to ible, low-molecular-weight protein which is thought to play determine if the organic portion of the complex was also accu- a role in the metabolism and detoxication of a number of mulated in these tubules. [‘%]CdMT prepared from rat liver was essential and nonessential metals. This protein has a rapid administered intravenously to mice at a nonnephrotoxic dose turnover but the liver has a very high capacity to synthesize (0.1 mg Cd/kg). The radioactivity in the kidney showed maxi- MT. Therefore, Cd is efficiently trapped in the liver as CdMT mum level (80% of the dose) 15 min after the injection. This complex. Metallothionein may play a protective role in Cd preferential renal uptake was also observed after administration exposure by sequestering the toxic metal ion and forming a of various doses of [35S]CdMT. In contrast to the earlier observed nontoxic intracellular metal-protein complex (Cherian et persistency of io9Cd in the kidney after “‘%dMT administration, al., 1976). Metallothionein may be involved in the tolerance 35Sdisappeared rapidly (with a half-life of approximately 2 hr), to Cd. Pretreatment by metals, such as Cd and Zn, which and 24 hr after injection of [35S]CdMT, there was very little 35S induce MT, reduces Cd-induced lethality (Yoshikawa, 1970; left in the kidneys. These observations indicate that the protein Leber and Miya, 1976; Probst et al., 1977; Goering and portion of CdMT is rapidly degraded after renal uptake of CdMT and the released Cd is retained in the kidney. Within the kidney, Klaassen, 1983, 1984a) and hepatotoxicity (Goering and 35Sdistributed mainly to the cortex. Light microscopic autora- Klaassen, 1984a,b; Suzuki et al., 1990). diography showed that [35S]CdMT preferentially distributed to With prolonged exposure, CdMT is released from the liver the proximal convoluted tubule (Sl and S2), which is the site into the bloodstream, probably due to minor hepatic injury of nephrotoxicity. Within the Sl and S2 segments, a greater (Dudley et al., 1985). Following glomerular filtration, this distribution of 3sSto the apical portion of the cells was observed low-molecular-weight complex (MW about 6500) binds to after administration of both a nonnephrotoxic (0. I mg Cd/kg) the brush border of the proximal tubular cells (Foulkes, 1978) and a nephrotoxic (0.3 mg Cd/kg) dose. lo9Cd administered as lo9CdMT also distributed to the apical portion of the Sl and S2 and is taken up by these cells. The toxicity of Cd is primarily cells. Therefore, both the organic (35S) and inorganic (‘@‘Cd) confined to S 1 and S2 (convoluted) segments of the proximal portions of CdMT are rapidly and efficiently taken up by the Sl tubules. In these segments, morphological changes (vacuolar degeneration and/or swelling) can be observed. Morphologand S2 cells of the proximal tubules, the site of nephrotoxicity. These observations support the concept that CdMT is readily ical and functional alterations produced by chronic exposure taken up by the proximal tubular cells as a complex, and then its protein portion is rapidly degraded to release Cd that binds permanently to intracellular sites and produces nephrotoxicity. ’ Supported by NIH Grant ES-01 142. 0 1992 Academic
2 Supported by a fellowship from Merck Sharp & Dohme. ’ To whom all correspondence should be addressed.
Press, Inc.
0041-008X/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
242
RENAL DISTRIBUTION
to Cd have been described, but the mechanism(s) of these nephrotoxic effects is (are) not clearly understood. One important finding in the study of Cd-induced nephrotoxicity is that renal damage induced by Cd bound to thionein (CdMT) is similar to that caused by chronic exposure to CdC& (Nordberg et al., 1975; Suzuki et al., 1979; Cherian and Nordberg, 1980). Therefore, CdMT was used as a probe to assessthe mechanisms of chronic Cd toxicity on the kidney. Two hypotheses have been proposed to explain the development of this nephrotoxicity. Several investigators (Webb and Etienne, 1977; Fowler and Nordberg, 1978; Squibb et al., 1979; Cain and Holt, 1983) have suggested that renal damage is caused by Cd ions which are released intracellularly by the lysosomal degradation of the reabsorbed complex. This “lysosomal theory” is based on studies showing that the protein is degraded when it is reabsorbed in the kidney. In support of this hypothesis, it has been shown by subcellular fractionation that Cd is concentrated in the lysosomes 30 min after CdMT administration (Squibb et al., 1979; Sato and Nagai, 1986). Cd is then redistributed to the cytosol where it is bound to highmolecular-weight proteins. Although the kidney is also able to synthesize MT, the rate of synthesis is slower than that in the liver (Sendelbach and Klaassen, 1988). Within 24 to 72 hr after administration, Cd again binds to thionein in the cytosol (Sato and Nagai, 1986). Renal injury according to this hypothesis is due to the Cd cation and not to the intact complex CdMT. In contrast, Cherian et al. ( 1976) have suggested that the process of pinocytosis during the uptake of injected CdMT produces membrane changes which are the cause of the toxicity. Therefore, CdMT is a nephrotoxin. In support of this hypothesis, Cd administered as CdMT does not accumulate in lysosomal subfractions (Cherian, 1978). Some degradation of CdMT occurs, followed by synthesis of new thionein, hence, Cd is bound to thionein at all times. Consequently, the role of the lysosomal system in renal CdMT toxicity by this hypothesis is minimal. While these hypotheses on how CdMT produces its nephrotoxicity are intriguing, much data is yet to be collected to support or refute them. A considerable amount of work has been performed on the distribution of Cd after CdMT administration, but little is available on the distribution of the organic portion of the molecule (Bremner et al., 1978; Cain and Holt, 1983; Andersen et al., 1987). We have recently demonstrated that the Cd in CdMT is rapidly and efficiently taken up by the Sl and S2 segments of the proximal tubule cells, the site of Cd nephrotoxicity (Dorian et al., 1992), and that practically no Cd is eliminated from these cells within a week. Therefore, the purpose of the present study was to quantify the uptake of the peptide portion of the CdMT complex by the S 1 and S2 cells of the proximal tubules. The fate of the protein portion of CdMT was studied by using [35S]CdMT and by light microscopic autoradiography, a
243
OF [35S]CdMT
technique previously used to study Cd nephrotoxicity itatively. MATERIALS
qual-
AND METHODS
Preparation of [‘5qCdMT. Male Sprague-Dawley rats (350-400 g) (Sasco, Omaha, NE) were injected subcutaneously with CdC12 (3 mg Cd/ kg/day) for 4 days. Each day, 4 hr after CdCl* injection, [35S]cysteine(Amersham Co., Arlington Heights, IL) was administered ip (50 pCi/animal for 3 days and 100 &i/animal on the fourth day) to each rat. [3SS]CdMT was purified as described previously (Dorian et al., 1992). Following separation of the two isoforms (MT-I and MT-II), the two fractions were dried by lyophilization and resolubilized in 5-10 ml of ultrapure water. The protein concentration was determined by ultraviolet spectroscopy (Waddell, 1956) with a DU-8 Spectrophotometer (Beckman Instruments, Inc., Arlington Heights, IL). Cd and Zn contents were analyzed with a Perkin-Elmer Model 2380 flame atomic absorption spectrophotometer (Notwalk, CT). The CdMT solutions were then stored in aliquots at -20°C. Because of its purity and abundance. [35S]CdMT II was used in all the experiments. Injection of CdMT for the dose-response study. [-“S]CdMT (I .4 &i/ nmole MT) was diluted with isotonic saline and used for injection. Male Swiss mice, weighing 25-30 g (Harlan, Indianapolis, IN) and kept in the same conditions as rats, were used throughout the experiments. [35S]CdMT was injected via the tail vein at dosages of 50 to 1000 pg Cd/kg. Kidneys and liver were removed 15 min after the injection. Injection of CdMT for the time-response study. [35S]CdMT (1.4 pCi/ nmole MT) was administered via the tail vein at 0.1 mg Cd/kg (a nonnephrotoxic dose: Maitani et al., 1988). Kidneys and liver were removed at 5, 15, 30, 45, and 60 min, as well as 2, 4. 8, and 24 hr after the injection. Determination of [“.5JCdMT in tissues. Kidney and liver were digested in Protosol (DuPont NEN Research Products, Boston. MA). Approximately 100 mg of tissue was minced with scissors, put in I ml of solution. and digested at 50°C for 3 hr in open glassvials. After cooling to room temperature and addition of 15 ml of scintillation liquid (Econo-safe, Research Products International Corp., Mount Prospect, IL), the concentration of [“S]CdMT in kidney and liver samples was determined by quantifying the radioactivity in a liquid scintillation analyzer (Packard Model 2000 CA, Downers Grove, IL). Tissue [35S]CdMT concentrations are expressed in nmoles of MT equivalents per gram of tissue. Light microscopic autoradiography study. Mice were anesthetized I5 min after administration of [35S]CdMT with sodium pentobarbital(30 mg/ kg ip) (Anthony Products Co., Arcadia, CA). An intracardiac perfusion was performed with a 60-ml syringe equipped with a blunt needle (Monoject 200, 2 1 X 1 in.). Following perfusion with saline, the left kidney was ligated and removed, digested in Protosol, and the concentration of 35Sdetermined. The remainder of the mice was perfused with fixative (2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4) and the right kidney was removed and used for autoradiography. Preparation of the tissue samples for the light microscopic autoradiography was done as previously described (Dorian et al., 1992). The exposure time of the specimens was 12 weeks. The slides were developed (developer D-19 and fixer, Kodak Lab, Rochester, NY), stained, and photographed under a light microscope (objective 40X). Cellular and subcellular areas in different parts of the nephron were determined on 15 randomly chosen tubules per nephron segment per mouse with a Bioquant Image Analysis system (R&M Biometrics, Inc., Nashville, TN). The silver grains were then counted manually. MT concentrations are expressed as density of grains per cellular area (cm2). Background grain density was determined from mice treated with unlabeled CdMT. The values were subtracted from the tubule grain density prior to analysis of the data. Statistics. Statistical comparisons were based on analysis of variance followed by Duncan’s multiple range test at the 5% level of significance.
244
DORIAN, GATTONE,
5’
AND KLAASSEN
15’ 30’ 4S 60’ Zhr4hrBhr24hr
ZJ-
FE
Liver
Liver
1 -L-0’
I
I
5’
15’
30’
45’
60’
2 hrs
4 hrs
8 hrs
24 hrs
FIG. 1. Time-dependent accumulation of [%]MT equivalents in kidney and liver after a single iv injection of a nonnephrotoxic dose (0.1 mg Cd/ kg) of [%]CdMT II to mice. Symbols represent the mean + SE of five mice. The inset represents the results on a logarithmic scale.
RESULTS
Figure 1 depicts the distribution of 35S at various times after administration of a nonnephrotoxic dose (0.1 mg Cd/ kg) of [35S]CdMT. The 35S was preferentially taken up by the kidneys (approximately 80% of the dose) and very little of it was found in the liver. The concentration of 35Sin the kidney was 10 to 12 times higher than that in the liver during the first hour following the injection. The concentration of 35Sin the kidney s increased rapidly after the injection. The maximum concentration was reached 15 min after [35S]CdMT administration. However, 35S concentration in the kidney decreased rapidly. The half-life of [35S]thionein was about 2 hr. Within 24 hr after [35S]CdMT administration, the concentration of “S in the kidney and the liver was nearly the same (0.6 nmol MT/g tissue in the kidney versus 0.45 nmol MT/g tissue in the liver). Fifteen minutes after administration of [35S]CdMT, the concentration of “S in kidneys increased as the dosage of CdMT increased from 0.05 to 0.4 mg Cd/kg, but remained relatively constant with further increase in dosage (Fig. 2). The plateau in renal “S concentration obtained with dosages larger than 0.4 mg Cd/kg suggests a saturation of the reabsorption process. In contrast, low and relatively constant concentrations of [35S]MT were found in liver (about 0.1 nmol MT/g tissue). A dose-response study (unpublished data) using ‘@‘CdMT prepared from rat liver (Dorian et al., 1992) was done at the same dosages as for [35S]CdMT. Figure 3 shows a comparison of the renal CdMT concentrations after administration of either lmCdMT or [35S]CdMT at various dosages. It is known that 1 mol of thionein can bind up to 7 mol of cadmium. As the MT in the solution used in these studies was almost saturated with Cd (6.7 mol Cd/mol MT), it was possible to estimate MT concentration based on Cd concentration. In
[35 S I-CdMT
(mg Cd/kg)
FIG. 2. Dosedependent accumulation of [“S]MT equivalents in kidney and liver 15 min after a single injection of various doses of [?S]CdMT II to mice. Symbols represent mean f SE of five mice.
Fig. 3, we plotted CdMT concentrations as determined from 35S measured after [35S]CdMT administration, and CdMT concentrations as calculated from ‘@‘Cd concentration measured after ‘09CdMT administration. There is a good correlation between the concentration measured and the concentration calculated. Figure 3 indicates that regardless of the position of the radioactive label (on the metal or on the protein moiety), similar renal CdMT concentrations were observed after administration of the complex. An increase in renal MT concentration occurred in a dose-dependent manner; above the dosage of 0.5 mg Cd/kg, reabsorption of MT by the kidney appears to be saturated (Fig. 3). The distribution of 35Sadministered as [35S]CdMT to different parts of the nephron was determined after injection of a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose. The areas examined were glomerulus (without Bowman’s capsule), S 1 segment and convoluted portion
0.2
0.4
CdMT
0.6
0.6
1.0
(mg Cd/kg)
FIG. 3. Dose-dependent accumulation of CdMT II in kidney after administration of lssCdMT and [35S]CdMT, expressed in nmol of MT equivalents/g tissue. Symbols represent the mean & SE of five mice.
RENAL DISTRIBUTION
245
OF [35S]CdMT
FIG. 4. Autoradiography of kidney from [‘%]CdMT-treated mouse. Brightfield (a) and darkfield (b) micrographs of renal cortex showing autoradiographic labeling (arrows, silver grains are black dots in a and white dots in b) of the proximal tubules. The grains are mainly over the brush border (apical region) of the cells. 400X.
of the S2 segment of the proximal tubules (termed hereon S 1 and S2) and distal convoluted tubules (DCT) in the cortex, as well as the straight proximal tubules (S3) and distal straight tubules (DST) in the outer stripe of the outer medulla. A typical autoradiograph is shown in Fig. 4. Fifteen minutes after administration of [35S]CdMT, areas of high-grain density were observed in the cortex and were in contrast to the lower density observed in the outer medulla. In the cortex, the highest concentration of [35S]MT was observed in the proximal convoluted tubules (Sl and S2 segments) (Fig. 5). The grain density in these first segments of the proximal tubules was approximately two times higher than that in the proximal straight (S3) segments. The grain density in the glomeruli was low and it was even lower in the distal tubules. This pattern of grain distribution was very similar after administration of either a nonnephrotoxic or a nephrotoxic dose of lo9Cd as ‘09CdMT (Dorian et al., 1992). Thus, after administration of both [35S]CdMT and lo9CdMT, the highest level of radioactivity is found in the Sl and S2 segments of the proximal tubules. As the proximal tubules are the primary site of Cd toxicity (Friberg et al., 1974) we also determined the distribution of [35S]CdMT in the apical (brush border) and basal (cytoplasm) portions of the proximal tubules (Fig. 6). The apical portion, at the luminal side of the cell, includes the brush border or supranuclear region. The portion of the cell designated as basal contains the midregions and the basal regions. After administration of both nonnephrotoxic and nephrotoxic doses of [35S]CdMT, three to five times higher concentrations of 35Swere observed in the apical than in the basal portion of the proximal convoluted tubules (S 1 and S2). In the proximal straight tubules (S3), a preferential apical distribution
of 35Swas noted only after administration dose (0.3 mg Cd/kg).
of the nephrotoxic
DISCUSSION The renal proximal tubules are the most vulnerable sites of the kidney to chronic cadmium intoxication in humans and in experimental animals. Renal dysfunction (glycosuria,
0.1
[ %+CdMT
0.3
(mg Cd/kg)
FIG. 5. Distribution of [35S]MT equivalents, as quantified by light microscopic autoradiography, in different parts of the nephron, I5 min after a single iv injection of a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic dose (0.3 mg Cd/kg) of [3SS]CdMT. Sl and S2 indicate the proximal convoluted tubules, S3 the proximal straight tubules, DCT the distal convoluted tubules, and DST the distal straight tubules. Bars represent mean + SE of five mice. The asterisk indicates that [35S]MT concentration is significantly higher in the Sl and S2 segments of the proximal tubules than in the other tubules.
246
DGRIAN, GATTONE,
AND KLAASSEN
CdMT preferentially distributes to the cortex (Nordberg and Nordberg, 1975; Murakami et al., 1983; Dorian et al., 1992). A similar preferential distribution to the cortex has been observed after [3H]CdMT administration (Andersen et al., 1987). In the present work, within the kidney, 35Safter administration of [35S]CdMT attains a concentration three times higher in the cortex than in the outer medulla. Cd administered as CdMT in the nephron distributes mainly to the first segments of the proximal tubules (Dorian et al., 1992). It is well known that CdMT selectively alters proximal convoluted tubule cells while glomeruli and distal tubule cells consistently show no changes (Cherian et al., 1976; Nordberg et al., 1975; Webb and Etienne, 1977; Mu0.1 0.3 rakami et al., 1983). Proximal convoluted tubules are con[ 3%]-CdMT (mg Cd/kg) sidered to be the primary site of Cd nephrotoxicity. Little FIG. 6. Distribution of [35S]MT equivalents, as quantified by light miwas known about the tubular distribution of the organic porcroscopic autoradiography, in apical (brush border) and basal (cytoplasm) tion of CdMT prior to this study. The quantification of 35S, portions of the proximal tubules in the cortex and the outer medulla 15 by light microscopic autoradiography, revealed that 35Sconmin after a single iv injection of a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose of [‘%]CdMT. Bars represent the mean + centrated predominantly in the convoluted (S 1 and S2) segSE of five mice. The asterisk indicates that [35S]MT concentration is signif- ments of the proximal tubules, after administration of either icantly higher in the apical than in the basal portion of the proximal tubules. a nonnephrotoxic (0.1 mg Cd/kg) or a nephrotoxic (0.3 mg Cd/kg) dose of [35S]CdMT. The S3 segment of the proximal tubules as well as glomeruli and distal tubules accumulated low-molecular-weight proteinuria) and morphological 35S to a much lesser extent. At nonnephrotoxic dose, the changes (swelling, vacuolization, necrosis) are consistently presence of MT in these tubules may be due to a saturation observed (Axelsson et al., 1968; Friberg et al., 1974) after of the reabsorption process occurring in the convoluted long-term exposure to Cd. These functional and pathological proximal tubules. At nephrotoxic doses, a significant increase changes observed after chronic exposure to Cd can be re- in MT concentration was noted. This increase may result produced by an acute administration of CdMT, a low-mofrom a decrease in reabsorption by the damaged Sl and S2 lecular-weight protein with a high affinity for metals (Nordsegments. The finding that ‘09Cd- as well as 35S-labeled MT berg et al., 1975; Cherian et al., 1976; Webb, 1979). Inforare found in the highest concentration in the proximal conmation is available on the renal distribution and uptake of voluted tubules (Sl and S2), suggests that CdMT is reabCd after CdMT administration, but very little is known about sorbed by the proximal tubule cells as a complex and that the fate of the protein moiety after administration of this the site of CdMT uptake corresponds to the site of nephrocomplex. Therefore, CdMT labeled on the protein with toxicity. [35S]cysteine was used in these experiments to study its disThe distribution of 35Sin the apical and basal portions of tribution in the nephron. the cells was also compared in the convoluted and the straight The distribution of Cd in the body has been extensively proximal tubules. Fifteen minutes after injection of a nonstudied. Its distribution differs markedly depending on the nephrotoxic or a nephrotoxic dose of CdMT, a higher conchemical form of Cd. After administration of CdCl*, the centration of 35Swas observed in the apical than in the basal highest concentration of Cd is found in the liver. In contrast, portion of the Sl and S2 segments of the proximal tubules. Cd after CdMT administration is predominantly taken up In the straight (S3) segment of the proximal tubules, a difby the kidney (Cherian and Shaikh, 1975; Nordberg and ference in distribution between apical and basal was noted Nordberg, 1975; Tanaka et al., 1975; Webb and Etienne, only after administration of the nephrotoxic dose. The dis1977; Squibb et al., 1979; Suzuki et al., 1979; Dorian et al., tribution of Cd administered as CdMT was previously quan1992). Several studies have shown that much of 35S-, 14C-, tified in the apical and basal portions of the proximal tubules or 3H-labeled metallothionein is also concentrated in the (Dorian et al., 1992). Cd was evenly distributed throughout kidney (Cherian and Shaikh, 1975; Webb and Etienne, 1977; the cell at a nonnephrotoxic dose, but concentrated mainly Cain and Holt, 1983; Andersen et al., 1987). In the present in the apical portion of the proximal convoluted cells, 24 hr study, the distribution of [35S]CdMT purified from rat liver after injection of a nephrotoxic dose. Thus, lo9Cd and 35S was followed after intravenous administration of a non- from ‘09CdMT and [35S]CdMT, respectively, are not only accumulated in the same Sl and S2 cells, but they are also nephrotoxic dose (0.1 mg Cd/kg). The 35Safter [35S]CdMT administration was also predominantly accumulated by the localized similarly inside the cells. This further supports the kidney. conclusion that the whole CdMT complex is taken up from n
Apical
Sl & 52
l
RENAL DISTRIBUTION
the glomerular filtrate by the proximal convoluted tubular cells. Predominant apical localization of CdMT within the Sl and S2 cells appears to be due to uptake of the complex at the luminal side of these cells. It has been demonstrated that CdMT is freely filtered at the glomerulus and reabsorbed by the brush border of the proximal tubules by a system that appears to be specific for anionic proteins (Foulkes, 1978), and CdMT does not react with basolateral cell membranes in vivo (Foulkes and Blanck, 1990). The renal uptake of Cd is very rapid (maximum concentration reached 30 min after injection) after administration of ‘09CdMT (Tanaka et al., 1975; Dorian et al., 1992) or after administration of 13H]CdMT (maximum concentration reached 10 min after injection) (Andersen et al., 1987). In the present study, we also found that the renal uptake of “S is extremely fast and the maximum concentration is reached 15 min after administration of [35S]CdMT. Once in the kidney, ‘09Cd concentration remains unchanged for up to 7 days after administration (Tanaka et al., 1975; Suzuki et al., 1979; Dorian et al., 1992). In contrast, the half-life of the injected [35S]CdMT may be only a few hours (Cherian et al., 1975; Bremner et al., 1978). The results from the time-course study (Fig. 1) also show that [“S]CdMT is rapidly degraded and the renal half-life of the 35S-labeled Cd-thionein is approximately 2 hr. The half-life of the protein moiety of injected CdMT is, therefore, much shorter than that found for endogenous renal CdMT, which is approximately 5 days (Cain and Holt, 1979). The difference between rates of disappearance of the protein moiety of endogenous and exogenous CdMT suggests that CdMT reabsorbed by the kidney cells is much more rapidly degraded than CdMT synthesized within the cell itself. In summary, our studies on the renal distribution of radioactivity after administration of lo9CdMT and [35S]CdMT collectively indicate that CdMT is taken up by the cells of the proximal tubules as an intact complex of Cd and MT. Moreover, after internalization, the protein moiety of CdMT is rapidly degraded and the released Cd is retained inside the cells. Preferential uptake of CdMT by the proximal convoluted tubular cells and the subsequent intracellular release of the toxic Cd ions may explain the fact that these cells represent the targets of the Cd-induced nephrotoxicity. REFERENCES Andersen, K. J., Haga, H. J., and Dobrota, M. (1987). Lysosomes of the renal cortex: Heterogeneity and role in protein handling. Kidney Znt. 31, 886-897. Axelsson, B., Dahlgren, S. E., and Piscator, M. (1968). Renal lesions in the rabbit after long-term exposure to cadmium. Arch. Environ. Health 17, 24-28. Bremner, I., Hoekstra, W. G., Davies, N. T., and Young, B. W. (1978). Metabolism of 35S-labelled copper-, zinc- and cadmium-thionein in the rat. Chem. Biol. Interact. 23, 355-367.
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