Cadmium-metallothionein nephrotoxicity in the rat: transient calcuria and proteinuria

After a s.c. injection of 0.4 mg Cd/kg as cadmium-metallothionein (CdMT) in rats, a marked increase in urinary protein concentration appeared at 16-40 h. There was a peak of urinary Cd content during the first 4 h after the treatment. Urinary Ca was increased at 8 h after the CdMT injection and returned to normal level at 32 h. Luminal and basolateral renal membrane vesicles were isolated from both control group and CdMT (0.4 mg Cd/kg) group at 24 h after the injection. Calcium uptake and binding of both fractions were decreased in the group treated with CdMT. Cd, Zn and MT concentrations in the kidney cortex were increased, but Ca concentration was not significantly changed. Since injected CdMT is probably only partly reabsorbed by tubular cells at the dose level of 0.4 mg Cd/kg as CdMT, excessive plasma CdMT is rapidly excreted in urine, explaining the increased Cd excretion during the first few hours observed in the present experiment. Decreased Ca binding in the luminal membranes as observed in vitro could be one of the mechanisms of production of calcuria if occurring in vivo. Another possible explanation of calcuria is that Cd ions released from CdMT into the cytoplasm of the tubular cell, may exert ionic interference with Ca transport across the luminal membranes and produce decreased Ca reabsorption. It is known that a disturbance of Ca metabolism could influence the membrane stability and such a change may contribute to explaining the proteinuria characteristic of CdMT nephrotoxicity. The reversibility of the proteinuria observed after a single dose of CdMT may be related to the induction of metallothionein synthesis in the renal cells.     

Dependence of cadmium-metallothionein nephrotoxicity on glutathione.

Acute cadmium-metallothionein (CdMT) injection is frequently used as a model to study the mechanism of chronic Cd-induced nephrotoxicity. The purpose of this study was to investigate the relationship between glutathione (GSH) status and the ability of CdMT, either administered as a bolus dose or infused over a 24-h period by an osmotic minipump, to cause nephrotoxicity. GSH levels were modulated by pretreatment with either buthionine sulfoximine (BSO) or GSH. BSO enhanced while GSH suppressed acute CdMT nephrotoxicity. An infused dose of CdMT (150 microg Cd/kg) that was well tolerated when delivered over a 24-h period became nephrotoxic when GSH synthesis was inhibited by BSO. With depletion of GSH, as little as 0.4 microg Cd/g renal cortex was sufficient to cause nephrotoxicity after an acute dose of CdMT. While BSO had no effect on renal Cd accumulation, pretreatment with GSH reduced renal cortical Cd accumulation by 36%. CdMT nephrotoxicity was enhanced by depleting renal GSH, but without increasing renal Cd accumulation, which suggests that intracellular GSH is directly involved in protection against CdMT nephrotoxicity. Reduced Cd accumulation in the renal cortex following GSH pretreatment suggests an additional extracellular mechanism of GSH protection. It is concluded that GSH status is an important determinant of CdMT nephrotoxicity, with low GSH levels enhancing and high GSH levels reducing its toxicity, and that the mechanism appears to involve both intracellular and extracellular sites.     

镉诱发大鼠肾重吸收钙障碍的机理(英文)

给雄性Wistar大鼠sc不同剂量的镉金属硫蛋白(CdMT),结果表明镉接触组尿镉、尿钙和尿蛋白浓度都高于对照组,并与镉剂量间存在剂量效应关系,而血清游离钙浓度无变化;镉接触组肾皮质钠泵和钙泵活性低于对照组,体外试验结果也显示镉能抑制钙泵活性,谷胱甘肽(GSH)和半胱氨酸对这种抑制有保护作用}镉接触组肾皮质GSH含量低于对照组,而丙二醛(MDA)含量则高于对照组,高剂量组肾皮质cAMP/cGMP比值低于对照组。肾皮质钙泵活性、cAMP/cGMP比值、GSH含量三者都与尿钙浓度间呈负相关,MDA含量则与尿钙呈正相关,揭示镉引起的钙尿症是由肾重吸收钙障碍造成的,可能与镉引起的肾脏钠泵、钙泵、环核苷酸、GSH和脂质过氧化等改变有关。     

Resistance to acute nephrotoxicity induced by cadmium-metallothionein dependence on pretreatment with cadmium chloride

Three groups of rats (B-D) were given various daily doses of CdCl2 (0.5-2 mg Cd/kg) continuously or in intervals during time periods of 1-8 weeks. Another group of animals (A) were kept untreated. At the end of the period, selected subgroups of groups A-D were given a single subcutaneous injection of 109Cd-metallothionein (109CdMT) 0.05 or 0.4 mg Cd/kg ("challenge dose"). Subsequently, urinary creatinine, protein, Cd, 109Cd and MT and kidney cortex Cd, 109Cd and MT were determined. In group A (no long term pretreatment), an increased proteinuria was observed after the rats had received the lower of the challenge doses of 109CdMT, and an even greater increase after the higher challenge dose of 109CdMT. No such increase appeared in group B, C and D (repeatedly pretreated with CdCl2) at either of the challenge doses. Higher metallothionein concentrations in kidney cortex observed in the pretreated groups constitute a plausible explanation of the protective effects of pretreatment against the development of increased proteinuria after challenge dosing. It is likely that increasing Cd concentrations, gradually accumulating in the renal cortex (22-226 micrograms/g wet wt.) as a result of the pretreatment, served to induce the synthesis of metallothionein in the renal cortical cells, thus making them resistant to the challenge from 109CdMT.     

Protection by Zinc-Metallothionein (ZnMT) against Cadmium-Metallothionein-Induced Nephrotoxicity

In contrast to inorganic Cd, acute iv administration of Cd boundto metallothionein (CdMT) concentrates in renal tissue. Thisuptake of CdMT produces functional and morphological changesin kidneys, similar to those observed after chronic exposureto inorganic Cd. In order to examine the importance of the metalcomponent of MT in the renal uptake of MT, the renal concentrationof ³⁵S after administration of [³⁵S]ZnMT and [³⁵S]CdMT was compared.Renal uptake of ³⁵S from both CdMT and ZnMT was very rapid,with peak concentrations observed 15–30 min after administration.³⁵S in kidneys increased in a dose-dependent manner after administrationof various doses of [³⁵S]ZnMT, up to 1.3 µmole MT/kg;however, higher doses did not further increase renal ³⁵S concentrations.A similar saturation of ³⁵S reabsorption was observed for therenal uptake of [³⁵S]CdMT. CdMT produced renal injury with dosesas low as 0.26 µmol MT/kg (0.2 mg Cd/kg). In contrast,with a dose of ZnMT as high as 5.12 µmol MT/kg (2 mg Zn/kg),no histopathological changes were observed. Therefore, ZnMTappears to be nontoxic even though ZnMT delivers more MT tothe kidney than does CdMT. Because ZnMT and CdMT are apparentlyhandled by the same renal transport mechanism, the effects ofZnMT on ¹⁰⁹CdMT renal uptake and nephrotoxicity were determined.One group of mice was given a nephrotoxic dose of ¹⁰⁹CdMT (0.51µmol MT/kg containing 0.4 mg Cd/kg, iv), and the othergroup received an equimolar dose of unlabeled ZnMT 1 min before¹⁰⁹CdMT administration. Renal function was evaluated by measuringurinary glucose and protein excretion, as well as histopathology.Marked renal toxicity was observed 24 hr after ¹⁰⁹CdMT administration.In contrast, renal function appeared normal in mice receivingZnMT before ¹⁰⁹CdMT. However, a similar concentration of ¹⁰⁹Cdwas found in kidneys of both groups. The present results demonstratethat ZnMT is not only nontoxic to the kidney at a dose as highas 5 µmol MT/kg, but can also protect against the nephrotoxiceffect of CdMT without decreasing renal Cd concentration.     

Differential calcium transport disturbances in renal membrane vesicles after cadmium-metallothionein injection in rats

Cadmium-metallothionein (CdMT) induced calciuria may result from disturbed calcium (Ca) transport through the renal tubular epithelium. The present study aimed at defining time of onset and the degree of disturbed calcium transport. Kidneys were obtained from rats at 4, 12 and 24 h after a single injection of CdMT (dose 0.4 mg Cd/kg b.w.), and compared to saline injected controls. Rapid-filtration 45Ca-assays were performed on basolateral and luminal membrane vesicles, isolated from kidney cortex using a sequential ultracentrifugation procedure. Luminal 45Ca uptake was increased at 4 h and then declined to about 80% of controls, suggesting an early phase perturbation of Ca absorption. Basolateral 45Ca uptake was reduced to less than 50% of controls, starting already at 4 h while 45Ca binding was reduced at 8 h. This may reflect an inhibited basolateral Ca pump mechanism after the binding step. Since the Ca pump normally expels Ca from the cell, an accumulation of intracellular calcium was indicated. Metal analysis verified a four-fold increase of Ca in kidney cortex at 24 h. This suggests that Cd impact on tubular cells involves disturbances on cellular absorption as well as expulsion of Ca.     

Renal cadmium deposition and injury as a result of accumulation of cadmium-metallothionein (CdMT) by the proximal convoluted tubules--A light microscopic autoradiography study with 109CdMT.

Chronic, but not acute, exposure to inorganic Cd produces renal damage. However, a single injection of cadmium bound to metallothionein (CdMT) produces renal injury. It is hypothesized that an interorgan redistribution of Cd as CdMT is responsible for the chronic nephrotoxic effect of Cd. To better understand the mechanism(s) of CdMT-induced nephrotoxicity, the intrarenal distribution of 109CdMT was examined. 109CdMT isolated from rat liver was injected into mice at a nonnephrotoxic dose (0.1 mg Cd/kg, iv). The radioactivity in the kidney reached a maximum level (85% of the dose) as early as 30 min following administration and remained essentially constant for up to 7 days after injection. Within the kidney, 109Cd distributed almost entirely to the cortex. Light microscopic autoradiography of the kidney showed that, within the cortex, 109Cd distributed preferentially to the S1 and S2 segments of the proximal convoluted tubules. Within the S1 and S2 segments, the concentration of 109Cd in the basal and apical parts of the cells was similar to that after the nonnephrotoxic dose of CdMT, but after a nephrotoxic dose (0.3 mg Cd/kg) the radioactivity distributed preferentially to the apical portion of the cells. In contrast, light microscopic autoradiography studies with 109CdCl2 revealed that 109Cd was more evenly distributed throughout the proximal tubules. Moreover, after administration of a large dose of inorganic Cd (3 mg Cd/kg), a similar concentration of Cd was found in the convoluted and straight proximal tubules. These data support the hypothesis that CdMT-induced nephrotoxicity might be due, at least in part, to its preferential uptake of CdMT into the S1 and S2 segments of the proximal tubules, the site of Cd-induced nephrotoxicity.     

Accumulation and degradation of the protein moiety of cadmium-metallothionein (CdMT) in the mouse kidney.

Of major concern in Cd toxicity is its ability to produce renal damage after chronic exposure in humans and experimental animals. Renal injury affects predominantly the proximal tubules and more specifically the first segments of these tubules. Similar toxic effects to the kidneys are observed after administration of cadmium bound to metallothionein (CdMT). Therefore, CdMT was used in this study as a model to understand the mechanism(s) of Cd nephrotoxicity. It has been recently demonstrated that Cd from CdMT was preferentially taken up by the proximal convoluted tubules. Therefore, the purpose of these studies was to determine if the organic portion of the complex was also accumulated in these tubules. [35S]CdMT prepared from rat liver was administered intravenously to mice at a nonnephrotoxic dose (0.1 mg Cd/kg). The radioactivity in the kidney showed maximum level (80% of the dose) 15 min after the injection. This preferential renal uptake was also observed after administration of various doses of [35S]CdMT. In contrast to the earlier observed persistency of 109Cd in the kidney after 109CdMT administration, 35S disappeared rapidly (with a half-life of approximately 2 hr), and 24 hr after injection of [35S]CdMT, there was very little 35S left in the kidneys. These observations indicate that the protein portion of CdMT is rapidly degraded after renal uptake of CdMT and the released Cd is retained in the kidney. Within the kidney, 35S distributed mainly to the cortex. Light microscopic autoradiography showed that [35S]CdMT preferentially distributed to the proximal convoluted tubule (S1 and S2), which is the site of nephrotoxicity. Within the S1 and S2 segments, a greater distribution of 35S to the apical portion of the cells was observed after administration of both a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose. 109Cd administered as 109CdMT also distributed to the apical portion of the S1 and S2 cells. Therefore, both the organic (35S) and inorganic (109Cd) portions of CdMT are rapidly and efficiently taken up by the S1 and S2 cells of the proximal tubules, the site of nephrotoxicity. These observations support the concept that CdMT is readily 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.     

Induction of metallothionein by cadmium-metallothionein in rat liver: a proposed mechanism.

Distribution of Cd to various organs following iv administration of CdCl2 (3.5 mg Cd/kg) resulted in more than 43% of total tissue Cd accumulating in the liver. In contrast, after CdMT administration (0.5 mg Cd/kg), only 1% of the Cd was found in liver. Rats administered CdCl2 (1.0 mg Cd/kg) had hepatic MT values 30-fold greater than controls and a hepatic Cd concentration of 17 micrograms/g. In comparison, rats treated with CdMT (0.4 mg Cd/kg) had hepatic MT concentrations 7-fold greater than controls and a hepatic Cd concentration of 0.80 micrograms/g. However, when hepatic MT levels were normalized to tissue Cd concentrations, induction of MT by CdMT was 5-fold greater than by CdCl2. Northern and slot-blot analyses of mRNA showed that both CdCl2 and CdMT coordinately increased MT mRNA. These data suggest that both CdMT and CdCl2 increase hepatic MT by similar mechanisms. A dose-response increase in MT produced by CdCl2 indicated a biphasic response, with low doses producing relatively more hepatic MT than higher doses. In addition, the amount of MT produced per unit Cd after CdMT treatment was similar to those observed after low doses of CdCl2 in the dose-response experiment. These data provide strong evidence to support the conclusion that the apparent potency of CdMT observed here and in previous studies is most likely due to the small amount of Cd distributed to the liver, which is relatively more effective in inducing MT than are higher concentrations.     

Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmium-metallothionein

Acute exposure to Cd produces liver injury, whereas chronic exposure results in kidney injury. Tolerance to the hepatotoxicity is observed during chronic exposure to Cd due to the induction of metallothionein (MT). The nephrotoxicity produced by chronic Cd exposure purportedly results from renal uptake of Cd-metallothionein (CdMT) synthesized in liver. The change in target organ from liver to kidney might be due to a lower amount of MT synthesized in the kidney in response to CdMT. Therefore, the purpose of the present study was to quantitate hepatic and renal MT induced by CdCl2 and CdMT. MT levels in mice were quantitated using the Cd-heme assay 24 hr after administration of CdCl2 (0.5-3.0 mg Cd/kg) and CdMT (0.1-0.5 mg Cd/kg). In both liver and kidney, MT reached higher levels following administration of CdCl2 (220 and 60 micrograms/g, respectively) than of CdMT (25 and 35 micrograms/g, respectively), probably because higher dosages of CdCl2 than CdMT are tolerated. CdMT produced 19 and 3 micrograms MT/micrograms Cd in liver and kidney, respectively, while CdCl2 produced 11 and 6 micrograms MT/micrograms Cd, respectively. In conclusion, induction of MT occurs in both the liver and kidney after administration of CdCl2 and CdMT. However, the kidney is less responsive than the liver to the induction of MT by both forms of Cd, which may contribute to making the kidney the target organ of toxicity during chronic Cd exposure.     

Cd-metallothionein nephrotoxicity in inbred strains of mice.

Genetic differences in the acute hepatic and testicular toxicity of Cd occur among different strains of mice. However, it is not known whether genetic variation to the renal damage caused by Cd-metallothionein (CdMT) exists. Therefore, male mice of the C3H/HeJ, C57/Bl10, CBA/CA, and DBA/2J strains, previously shown to differ in hepatic and testicular injury due to Cd, were treated with CdMT at dosages of 0.2, 0.4, 0.8, and 1.6 mg/kg (sc). For all strains of mice, tissue accumulation of Cd occurred predominantly in kidney, which had two to three times as much Cd as liver, while testes had no measurable amounts of Cd. Hepatic and renal metallothionein (MT) concentrations were increased with increasing dosage of CdMT, and no differences between strains were demonstrated. Urinary glucose was increased significantly at the three highest dosages of CdMT, with no differences between strains. At each dose level, light microscopic manifestations of CdMT nephropathy did not differ between strains. In summary, all CdMT-treated strains of mice responded similarly with respect to all measured renal parameters (accumulation of Cd and MT and nephrotoxicity). Unlike the strain differences in hepatic and testicular injury from Cd in these strains of mice, CdMT nephrotoxicity shows no such genetic variation.     

Renal tubular transport of cadmium-metallothionein

Immediately following arterial injection of labeled cadmium-metallothionein (CdMT) into rabbits, all of the Cd isotope excreted in the urine is contained in the CdMT fraction; the label can be used, therefore, for analysis of the renal handling of CdMT. No evidence was found for tubular secretion of injected CdMT. At high transient plasma concentrations following bolus injection of 1 mg of CdMT, fractional recovery in urine approached that of inulin. This suggests that CdMT is freely filterable; in addition, its reabsorption appears completely saturable, not only partially as described previously. Of several proteins tested, only myoglobin interfered with CdMT reabsorption. Reabsorption was also depressed following Cd exposure, at a time when renal cortical concentrations of Cd exceeded 200 μg/g wet weight, although PAH and creatinine clearances remained normal. This represents another instance of the relatively specific nephrotoxicity of Cd. The conclusion is reemphasized that for quantitative reasons tubular rejection of filtered CdMT is unlikely to play a role in Cd excretion by rabbits.     

Distribution of cadmium chloride and cadmium-metallothionein to liver parenchymal, Kupffer, and endothelial cells: their relative ability to express metallothionein.

Acute exposure to cadmium (Cd) salts results in liver toxicity, while administration of cadmium-metallothionein (CdMT) iv, causes renal damage. When CdMT is administered iv there is a rapid accumulation of Cd in the proximal tubule cells of the kidney. In comparison, only small amounts of Cd accumulate in the liver following administration of CdMT. Thus, in order to better understand the regulation of MT as well as the toxicity of Cd, the present study has examined the ability of each of the three primary liver cells, parenchymal (PC), Kupffer (KC), and endothelial (EC), to accrue Cd after administration of either inorganic or organic forms of Cd. In addition, the relative ability of each cell type to express metallothionein (MT) mRNA and protein was examined. Following CdCl2 (3.5 mg Cd/kg) treatment, Cd concentrations increased to about the same degree in PC and KC, but EC had about 2-fold more than PC. After administration of CdCl2 (1.0 mg Cd/kg) each cell responded to the presence of Cd by increasing intracellular MT mRNA and protein. However, PC showed the greatest response, with a 30-fold increase in mRNA and a 21-fold increase in protein. Interestingly, KC and EC possessed intracellular Cd concentrations equal to or greater than that of PC, but contained less MT than would have been expected on the basis of their intracellular Cd concentrations. Thus, KC had a 7-fold increase in MT mRNA and a 2-fold increase in protein, while EC increased mRNA 3-fold and protein 2-fold over control values. In contrast, following CdMT (0.5 mg Cd/kg) administration, only low levels of Cd were detected, with similar concentrations in each cell type. After administration of CdMT (0.4 mg Cd/kg), PC again showed the greatest response, with a 3-fold increase in mRNA and a 6-fold increase in MT protein. Only slight changes were observed in KC and EC. In conclusion, the present study has shown the following: (1) Endogenous levels of MT in KC and EC are higher than those in PC. (2) Cd is readily accumulated by all three cell types, when administered as CdCl2, but not when given as CdMT. (3) PC, KC, and EC are capable of responding to intracellular Cd by increasing MT.     

Cadmium-metallothionein-induced kidney dysfunction increases magnesium excretion in the rat

Urinary excretion of the major minerals, calcium (Ca), magnesium (Mg), sodium (Na), and potassium (K), as well as of protein and metallothionein, was studied following the injection of cadmium-metallothionein (CdMT) in rats. Animals were given vehicle (saline) and 0.4 and 0.8 mg Cd/kg body wt as CdMT. A marked, relatively early, and reversible increase in Mg excretion was seen. The increase was dose-related, indicating a close connection to the typical Cd-derived cellular damage in the renal tubular epithelium, including an early reversible Ca excretion and a late reversible protein excretion. The increase in Mg excretion was similar in magnitude to the one for Ca and much more prominent than that recorded for Na and K. The appearance of Mg and Ca excretion peaks at an early stage after CdMT injection makes it likely that this effect is an early event in the process of development of cellular damage and does not merely represent unspecific cellular damage giving rise to proteinuria.     

Metallothionein protects against the nephrotoxicity produced by chronic CdMT exposure.

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, metal-binding protein. Induction of MT has been proposed to be an important adaptive mechanism in decreasing Cd toxicity. MT has been shown to protect against CdCl2-induced lethality and hepatotoxicity; however, MT does not protect against acute CdMT-induced nephrotoxicity. This study was aimed at clarifying the role of metallothionein in chronic CdMT-induced renal injury. Wild type and MT-I/II knockout (MT-null) mice were therefore given sc injections of CdMT (25 and 100 microg Cd/kg) or saline daily, 6 times/week for 6 weeks, and renal injury was evaluated. Multiple injections of CdMT to wild-type mice resulted in renal Cd concentrations up to 120 microg/g kidney, along with a 100-fold increase in renal MT (450 microg/g kidney). In contrast, renal Cd concentration in MT-null mice administered multiple injections of CdMT reached a much lower level than in wild-type mice (<10 microg/g kidney). Although less Cd accumulated in their kidneys, MT-null mice were more susceptible than wild-type mice to CdMT-induced nephrotoxicity, as indicated by increased urinary excretion of protein and N-acetyl-beta-D-glucosaminidase, as well as by elevated blood urea nitrogen levels. At the higher daily dose of CdMT (100 microg Cd/kg), kidneys of MT-null mice were enlarged. Chronic CdMT administration eventually damaged the entire kidney, which included glomerular swelling, interstitial inflammation, edema, tubular cell degeneration, and atrophy. In contrast to a single injection of CdMT that produces proximal tubular necrosis, chronic injection of CdMT results in tubular cell apoptosis in both wild-type and MT-null mice. These data indicate that chronic CdMT administration produces similar renal injury to that observed after chronic CdCl2 administration, and that intracellular MT protects against nephrotoxicity produced by chronic CdMT administration.     

Renal cortical mitochondrial dysfunction upon cadmium metallothionein administration to Sprague-Dawley rats

A bolus dose of cadmium metallothionein (CdMT) produces renal proximal tubular dysfunction because it accumulates in the tubular epithelial cells and undergoes rapid degradation, releasing Cd. Morphologically, mitochondria appear to be the target organelle. The present study examined changes in renal cortical mitochondrial function following CdMT administration and investigated whether some of these effects could be ascribed to Cd2+ accumulation in the mitochondria. Sprague-Dawley rats were injected ip with 0.3 mg Cd as CdMT/kg and the animals were sacrificed after 6, 8, or 12 h. Two- to threefold increases in urinary protein excretion and LDH activity were evident at 8 h, with marked elevations (11- and 29-fold) thereafter. Renal cortical mitochondria were swollen and rounded at 12 h. The mitochondrial Cd level was 399 pmol/mg protein at 6 h and did not change significantly during the next 6 h; however, mitochondrial respiratory function declined with time. At 12 h, state 3 oxygen consumption, respiratory control ratio (RCR), and ADP:O (P/O) ratio were 48, 49, and 76% of control values, respectively, indicating inhibition of electron transfer and oxidative phosphorylation. The direct effect of Cd on mitochondrial function was examined by incubating mitochondria from untreated rats with 0.1-2 microM CdCl2. Rapid uptake of Cd resulted in concentration-dependent effects on respiration. After 1 min of incubation with 2 microM Cd, the mitochondria contained 262 microgCd/mg protein and state 3 respiration and RCR values were 75 and 33% of control levels, respectively. Thus, renal proximal tubular cell damage following a bolus dose of CdMT involves perturbations in mitochondrial respiration, brought on by the accumulation of Cd.     

Influence of cadmium-metallothionein pretreatment on tolerance of rat kidney cortical cells to cadmium toxicity in vitro and in vivo

Kidney cells were isolated from rats pretreated by daily subcutaneous doses of cadmium metallothionein (CdMT: 0.05-0.2 mg Cd/kg X 5) and from non-pretreated rats. Upon exposure to CdCl2 in vitro (0-200 micrograms Cd/ml), a concentration dependent decrease in viability was observed in the non-pretreated cells, while no such decrease occurred in the pretreated cells indicating that these cells were more resistant to the toxic action of cadmium. There was a higher in vitro uptake of Cd+2 and an increased metallothionein (MT) concentration in the pretreated cells (compared to non-pretreated cells). Subcellular distribution studies revealed that Cd was mainly recovered in the "cytosol" fraction. The higher total cadmium uptake in pretreated cells corresponded to an increase of Cd in "cytosol" and "nuclear" fractions. This observation may be explained by MT-binding of Cd in the cells and is in accordance with a possible protective effect of induced MT in the pretreated cells. In order to assess whether pretreatment-induced tolerance to cadmium toxicity--indicated by the cellular studies--could also be observed in vivo, some whole animal experiments were also performed. A dose-related proteinuria was observed in non-pretreated rats after a single subcutaneous administration of 109Cd-MT at doses of 0.05 and 0.4 mg Cd/kg. Urinary total Cd, 109Cd and MT was also increased in a dose-related fashion. Cadmium concentrations in kidney were dose related and reached 19 micrograms/g wet weight. In contrast, in animals repeatedly pretreated with CdMT according to 1), no proteinuria was observed after administration of the same single doses of 109CdMT. Total Cd. 109Cd and particularly MT-concentrations in urine were lower in such pretreated animals than in in non-pretreated ones in spite of the accumulation of higher tissue concentrations of total Cd (up to 80 micrograms/g). The pretreatment was thus shown to prevent some of the acute nephrotoxicity of CdMT, possibly by means of induction of MT synthesis.     

Urinary metallothionein and tissue metal levels of rats injected with cadmium, mercury, lead, copper or zinc

Since Cd exposure results in a dose dependent increase in metallothionein level in urine, the present investigation was conducted to examine whether exposure to other divalent cations would also cause an elevation in urinary metallothionein. Female Sprague-Dawley rats were injected subcutaneously with either saline, 5 mumol/kg/day of CdCl2, HgCl2, Pb(C2H3O2)2, CuSO4 or ZnCl2 for 5 days. Significant increases in hepatic Cu levels in rats treated with not only Cu, but also Zn, Cd, or Hg, and in hepatic Zn levels in rats treated with Zn or Cd were noted. Similarly, renal Cu and Zn levels were elevated significantly in all groups except the Pb-injected group. These increases in tissue metal levels were presumably due to induction of metallothionein. The urinary metallothionein level in control rats on day 0, determined by radioimmunoassay, was 0.85 +/- 0.17 mg/g creatinine. There was no significant change in urinary metallothionein level in rats given up to 5 injections of saline or Pb. Hg-injected rats showed 25-fold increase in urinary metallothionein after 5 injections, whereas Cd-injected rats had 9-fold increase. There were also 2- and 3-fold increases of urinary metallothionein by Cu and Zn treatments for 5 days, respectively. Thus, urinary metallothionein levels were elevated in response to Cd, Hg, Cu and Zn, but not Pb; Hg had the most profound effect at equimolar doses.     

Comparative renal toxicity of metallothioneins with different cadmium/zinc ratios in rats

The comparative renal toxicity of rats after injection of cadmium (Cd) and zinc (Zn)-metallothioneins (MTs) with different Cd/Zn ratios at the same dose of 200 micrograms MT-bound Cd/kg was studied. From determination of the urinary excretion of protein, aspartate aminotransferase (AST) and glucose, which are indices of Cd-induced renal damage, the extent of the renal toxicity of the MTs used here was in the order (1 Cd/0Zn)-MT = (2Cd/1Zn)-MT greater than (1Cd/2Zn)-MT greater than (1Cd/6Zn)-MT. The characterization of Cd, Zn and Cu in the urine after injection of MTs was examined using a Sephadex G-75 column. (1Cd/0Zn)-MT injection showed that Cd was present mainly in lower-molecular-weight fractions, with only small amounts of Cd in the MT fraction. Upon injection of other MTs, Cd was present mainly in the MT fraction and increased with decreasing Cd/Zn ratio. Zn was present mainly in lower-molecular-weight fractions and Cu mainly in the MT fraction, indicating the replacement of MT-bound Zn by Cu. The cumulative urinary excretion of Cd during 12 days after injection of MTs decreased with increasing Cd/Zn ratio. The Cd content of the kidney and liver increased with increasing Cd/Zn ratio. The results of this study indicate that in rats injected with MTs with different Cd/Zn ratios, the renal uptake of Cd increases with increasing Cd/Zn ratio, resulting in more severe renal damage.     

Aluminium Accumulation in Some Tissues of Rats with Compromised Kidney Function Induced by Cadmium-Metallothionein

Abstract: Two experiments (I and II) were performed to study aluminium accumulation in brain as well as in several other tissues in male Wistar rats. A single intraperitoneal injection of cadmium-metallothionein (CdMT, 0.1-0.4 mg Cd/kg b. wt.) was used to compromise kidney function 12 hr before the final aluminium injection in both experiments. In experiment I, rats were maintained on diets deficient (0.01%, w/w) in calcium (-Ca) or providing adequate (+ Ca) dietary calcium (0.9%) for 6 weeks. Among animals given a daily intraperitoneal dose of aluminium chloride (10.8 mg Al/kg per day) on 6 consecutive days there was a tendency towards higher aluminium level in brains of rats with compromised kidney function from CdMT (in -Ca rats: the geometric mean [G] = 288 versus 205 ng/g wet weight [w., wt.], P=0.07, and in + Ca rats: G=242 versus 164, P<0.05) as compared to animals given no CdMT. The results from experiment II (all rats were given aluminium 5.6 mg Al/kg 2 and 12 hr after CdMT injection) demonstrated a higher level of aluminium (G: 41 ng/g w. wt., P<0.05) in brains of rats with only slightly damaged kidney function (0.1 mg Cd/kg) than in those given no CdMT (G: 29 ng/g w. wt.). It was also observed that 1) calcium deficiency had a statistically significant effect (P<0.05) in increasing kidney retention of intraperitoneal aluminium (G: 327 ug/g w. wt.) as compared to rats with a normal calcium supply in the diet (G: 54 ug/g w. wt.); 2) when aluminium concentration in kidney was at and above 54 ug/g wet tissue, kidney damage was observed. The above results indicate that compromised kidney function including tubular damage induced by a low-dose of CdMT may play a crucial role in the accumulation of aluminium in brain and other tissues. Since tubular function decreases with age in human populations, these findings in rats may be of considerable importance if a similar phenomenon would occur in humans. Therefore, the possibility of increased aluminium retention in persons with low calcium and high aluminium intakes may need to be further investigated.