ARTICLE |
Correspondence to: Masaaki Kurasaki, Dept. of Environmental Medicine and Informatics, Graduate School of Environmental Earth Science, Hokkaido Univ., Kita 10 Nishi 5, Kita-Ku, Sapporo 060, Japan.
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Summary |
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Menkes disease is an X-linked disorder of copper metabolism. Excess amounts of copper in the kidney of Macular mice, a model for this disease, were found as copper-metallothionein (Cu-MT) from kidney of the mice. Histochemical studies of Cu-MT based on its autofluorescent emission properties showed that the protein was predominant in the proximal convoluted tubule (PCT) cells of the cortex. PCT cells are known to be the primary site of the nephrotoxicity caused by heavy metals. MT mRNA was also observed in the cortex, indicating that the protein was biosynthesized in this region. On the basis of these results, we suggest that biosynthesis and degradation of Cu-MT occur repeatedly in the PCT cells of the cortex. We also compared the histochemical localization of Cu-MT in Macular mice and Long-Evans cinnamon rats, a model for Wilson's disease. The significance of this comparison is discussed. (J Histochem Cytochem 45:1493-1501, 1997)
Key Words: copper, metallothionein, Menkes disease, Macular mouse, kidney, in situ blotting, autofluorescence
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Introduction |
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Menkes kinky hair disease (
Recently, the gene encoding a P-type, cation-transporting ATPase was shown to be responsible for the decrease in hepatic copper content in Menkes patients (
Copper is an essential trace element that requires a delicate cellular balance between necessary and toxic concentrations. Metallothionein (MT), a low molecular weight heavy metal binding protein, plays an important role in copper homeostasis and detoxification (
Hepatic copper accumulation is observed in some other disorders, such as Wilson's disease (
To gain insights into the developmental mechanism of the abnormal copper metabolism, we investigated the histochemical distribution of Cu-MT in the kidney of Macular mice by a method based on the autofluorescent emission properties of thiolate clusters in the protein (
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Materials and Methods |
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Materials
Original breeding pairs of the Macular mouse strain were kindly supplied by Dr. M. Nishimura. The C3H male mice used as a control, weighing 20-25 g, were bought from Japan SLC (Hamamatsu, Japan). Both strains of mice were fed MF-4 standard pellets ad libitum (Oriental Yeast; Tokyo, Japan), containing 1.5 mg of copper/100 g, and water, and were housed under conditions of constant temperature (22 ± 1C) and humidity (50 ± 10%). Sephadex G-75, DEAE-Sephadex A-25, Octyl-Sepharose CL-4B, and ProRPC HR5/2 were obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden). Other chemicals were reagent grade. Nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoryl phosphate (BCIP) were obtained from Sigma (St Louis, MO). Zeta probe membrane and nylon membrane were from Bio-Rad (Richmond, CA) and Pall Ultrafine Filtration (Glen Cove, NY), respectively. The DIG DNA labeling and detection kit was bought from Boehringer Mannheim (Mannheim, Germany).
Copper Therapy of Macular Mice
Copper was administered to the mice by SC injections in the mid-dorsal region of 10 µl of copper chloride solution (10 µg of copper/g bw in 0.9% NaCl) on the morning of postnatal Days 7 and 10. Injections were made with a 10-µl Terumo microsyringe MT-10 (Tokyo, Japan).
Purification of Copper Binding Protein from Kidneys of Macular Mice
Copper binding proteins from 7.5 g of the kidneys were purified by a simple method that we previously described (
Further purification of the low molecular weight copper-containing fractions was carried out on a column of DEAE-Sephadex A-25 (2.1 x 4 cm) with a linear gradient. The main copper-containing peak was immediately subjected to a column (1.1 x 3 cm) of Octyl-Sepharose CL-4B. The pass-through fractions were gathered.
Finally, analytical and preparative chromatography was carried out on a reverse-phase FPLC column, ProRPC HR5/2, attached to the fast protein liquid chromatography (FPLC) equipment from Pharmacia LKB Biotechnology. Fractions were collected and measured for absorbance at 220 nm with a Beckman spectrophotometer model DU-50 and for copper content with a Hitachi flameless atomic absorption spectrophotometer model 170-70. The isolated protein was lyophilized. All steps were performed at 4C.
Amino Acid Analysis
The proteins were oxidized with performic acid at 4C for 16 hr and dried with a Yamato rotary evaporator model RE-40 (Tokyo, Japan) at 40C. Amino acid analysis after hydrolysis at 110C with 6 N HCl for 24 hr was performed on a Hitachi amino acid analyzer model 305.
Histochemical Observation of Luminescence from Cu-MT
All procedures were carried out according to the methods of
The fluorescent emissions of Cu-MT were visualized and photographed by using an Olympus BX-50-FLA (Tokyo, Japan) biological microscope system with U-MWU filter cube (DM-400 dichroic mirror, 330-385-nm excitation filter, 530-nm barrier filter) (Tokyo, Japan) with a 100-W mercury lamp as a UV light source. The Cu-MT on the Zeta probe nylon membrane was visualized by illumination with a UV light (365 nm).
Northern Hybridization
To examine the genomic expression of Cu-MT in the kidney, the distribution of mRNA encoding MT was observed according to the method of
Histochemical Study with Acid Phosphatase
The histochemical distribution of acid phosphatase was investigated by the method of Gossrau et al. (1978), using BCIP as a substrate. The incubation with 0.5 mM BCIP, 0.5 mM NBT, and 45 mM dimethylformamide in 40 mM CH3COOH and 100 mM CH3COONa was carried out at 25C for 60 min.
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Results |
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Treatment of Macular Mice
Because some investigators have reported that a single dose of copper at 7th days after birth permitted the mutant mice to survive, we injected copper (10µg of copper/kg bw) into the mice at Days 7 and 10 after birth and compared their survival rates (Table 1). We found that mutant mice injected twice with copper had higher longterm survival rates than mice that received a single injection. The significant difference of the survival rates between two groups of mice receiving one or two injections was observed (p<0.005) by 2 study. Other studies used single injections (
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Purification of Low Molecular Weight Copper Binding Protein from Kidneys of Macular Mice
Figure 1A and Figure 1B show representative elution patterns of Sephadex G-75 chromatography of the kidney extracts from Macular mice and control mice, respectively. No peak corresponding to the low molecular weight protein (Peak A) in the chromatogram of Macular mice can be detected in the chromatogram of the control mice. The Kd value of Peak A is 0.76, which agreed well with that of MT from the liver of rats reported by
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The final purification step of the protein was carried out by FPLC on a reverse-phase ProRPC column (Figure 1D). One copper-containing peak was eluted at a retention time of 8.5 min, the same as the retention time of Cu-MT from the livers of copper-injected rats (
Amino Acid Composition
Table 2 lists the amino acid composition of the copper binding protein obtained by FPLC. Typically for mammalian MTs, the protein showed a high cysteine content and completely lacked aromatic amino acids and histidine. The composition agreed with that deduced from the cDNA sequence of MT (
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Overall Localization of Cu-MT
We examined the overall histochemical localization of Cu-MT in the kidney of Macular mice by direct autofluorescent observation (
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Expression of MT mRNA
The expression of MT mRNA was investigated to determine whether Cu-MT in the cortex was biosynthesized in the kidney or transported from other tissues (Figure 2C and Figure 2D). A high level of mRNA was expressed throughout the renal cortex. The expression of MT mRNA was not detected in the renal medulla and pelvis of the mutant mice. The kidneys of the control mice did not exhibit expression of MT mRNA. Both MT mRNA and its transcripts were detected in the renal cortex of Macular mice, indicating that biosynthesis of MT occurred in this region.
Microscopic Observation of Fluorescence from Cu-MT
To determine the detailed localization of Cu-MT in the kidney, microscopic observation was carried out. In the Macular mice, yellow fluorescence was observed exclusively in the proximal convoluted tubules (PCT) of the cortex (Figure 2E). The renal proximal tubules are the most sensitive sites in kidneys exposed to large doses of heavy metals (
To clarify the subcellular distribution of Cu-MT in the PCT cells, the mechanism of the renal damage caused by copper was investigated. Strong emission signals of spherical granules (Figure 2G and Figure 2H) indicated that Cu-MT was concentrated in certain organelles, such as the lysosomes. Emissions from Cu-MT appeared also in the cytoplasm (Figure 2G) and in some nuclei of the PCT cells (Figure 2H). It has been reported that MT bound heavy metals present in nuclei (
In the quenching test of the blot, the fluorescent emission disappeared after soaking in 1 mM HgCl2 (data not shown) as we described previously (
Localization of Acid Phosphatase
To confirm that Cu-MT exists mainly in the lysosomes, we investigated the histochemical localization of acid phosphatase in the PCT cells. This enzyme is recognized as a histochemical marker for lysosomes (
Microscopic Observation of Fluorescence from Cu-MT in LEC Rat Kidney
LEC rats are a model for Wilson's disease. Copper and Cu-MT were abnormally accumulated in LEC kidneys. To compare the localization of the fluorescence from Cu-MT in Macular mouse kidney with that in LEC rat kidney, microscopic observation of the fluorescence from Cu-MT was carried out. Orange autofluorescence from Cu-MT was detected in the outer stripe of outer medulla and yellow signals from Cu-MT were observed in the PCT cells of the renal cortex of LEC rat (Figure 2K) as previously described (
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Discussion |
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Menkes disease is a disorder of copper metabolism (Menkes 1962). Accumulation of abnormal amounts of copper in the kidney and intestinal wall of patients and in mouse models of disease is a significant feature. This study showed that excess amounts of copper were present as Cu-MT in the kidney of Macular mice. The properties of Cu-MT autofluorescence enabled us to observe the histochemical localization of the protein directly, as previously described (
There have been few reports on the direct identification of Cu-MT from the tissues of Menkes patients or from model animals for Menkes disease. Many investigators have believed that the low molecular weight protein associated with Menkes disease is Cu-MT because of its features, e. g., the metal (
Microscopic studies of the detailed localization of Cu-MT showed that it is dominant in the PCT cells of the cortex in Macular mice (Figure 2E). It has been reported that after copper administration Cu-MT accumulates mainly in the renal PCT cells (
The subcellular distribution of Cu-MT in the PCT cells of Macular mice showed a large concentration in lysosomes (Figure 2E). This result coincides with the evidence of
Some studies (
Whereas orange fluorescent emissions were observed in the S3 segment in the kidney of LEC rats (Figure 2K), yellow fluorescence appeared in the PCT cells of Macular mice. Emissions of a model complex of Cu-MT changed from orange to yellow according to a decrease in the molar ratio of copper to thiol (
LEC rats are an inbred strain. These animals exhibit an abnormal accumulation of copper in tissues and die because of the hepatotoxicity induced by copper accumulation; (
The apparent discrepancy in copper-transporting systems between Menkes disease and Wilson's disease may be due to the candidate genes for these conditions. Two types of P-type cation-transporting ATPases encoded by an X chromosome-linked gene have been reported to be concerned with copper metabolism: one is a copper-efflux pump (ATP7A) (
We have identified Cu-MT in the kidney of Macular mice, and we suggest that Cu-MT is biosynthesized in the renal cortex. Localization of Cu-MT in the kidney of these mice has been also confirmed. These findings, including the lysosomal cycle postulated here, give some clues to future studies on the mechanism of the abnormal copper accumulation in Menkes disease.
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Acknowledgments |
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We are grateful to Dr M. Nishimura, Institute for Experimental Animals, Hamamatsu University of Medicine, Hamamatsu, Japan, who provided us with a breeding pair of Macular mice, and to Prof Yutaka Kojima, Graduate School of Environmental Earth Science, Hokkaido University, for suggestions and discussion during this study.
Received for publication December 9, 1996; accepted May 29, 1997.
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