ARTICLE |
Correspondence to: Nobuteru Usuda, Dept. of Anatomy and Cell Biology, Shinshu U. School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan..
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Summary |
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We studied the developmental changes in the localization of peroxisome-specific enzymes in rat kidney tissues from embryonic Day 16 to postnatal Week 10 by immunoblot analysis and immunohistochemistry, using antibodies for the peroxisomal enzymes catalase, D-amino acid oxidase, L--hydroxyacid oxidase (isozyme B), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein. Peroxisomal enzymes were detected in the neonatal kidney by immunoblot analysis and their amount increased with kidney development. By light microscopic immunohistochemistry, they were first localized in a few proximal tubules in the juxtamedullary cortex of 18-day embryos. The distribution of proximal tubules positive for them expanded towards the superficial cortex with development. The full thickness of the cortex became positive for the staining by 14 days after birth. Peroxisomes could be detected by electron microscopy in structurally immature proximal tubules in 18-day embryos. Their size increased and the ultrastructure of subcompartments became clear with continuing development of proximal tubules. These results show that peroxisomal enzymes appear in the immature proximal tubules in the kidney of embryos and that the ultrastructure of the peroxisomes and localization of the peroxisomal enzymes develop along with the maturation of proximal tubules and kidney tissues. (J Histochem Cytochem 46:11611173, 1998)
Key Words: peroxisome, peroxisomal enzymes, kidney, development, immunohistochemistry
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Introduction |
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The peroxisome is a cell organelle widely distributed in various organs of vertebrates and predominantly located in the liver and the kidney. The peroxisome is defined as an organelle containing H2O2-reducing catalase and at least one H2O2-producing oxidase, such as D-amino acid oxidase and L--hydroxyacid oxidase (
-hydroxyacid oxidase (HOX-B), acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein or multifunctional protein 1 (BP), and 3-ketoacyl-CoA thiolase are localized in the electron-dense peripheral matrix (
-hydroxyacids to the corresponding
-keto acids forming H2O2 (
The fine structure of peroxisomes during ontogenesis has been studied in various organs, including the kidney (
As a first step towards a basis for understanding the ontogenic appearance of peroxisomes and to create a bridge between the structural maturation of peroxisomes and the appearance of peroxisomal enzymes, this study attempted to clarify the changes in the localization of peroxisome-specific enzymes in developing kidney. We performed immunoblot analysis and light and electron microscopic immunohistochemistry using monospecific antibodies for four peroxisomal enzymes, i.e., catalase, DAOX, HOX-B, and BP. We demonstrated that the appearance of the various peroxisomal enzymes is temporally parallel and that the appearance of peroxisomes and their maturation correspond to the period of differentiation of nephrons. The subcompartments were shown to be formed along with the distinct localization of peroxisomal enzymes.
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Materials and Methods |
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Animals and Tissues
Kidney tissues from Wistar rats from various ages were examined: embryonic Days 16, 18, and 20, postnatal Days 1, 4, 7, 14, and 28, and postnatal Week 10. Five embryos irrespective of gender in each prenatal group and five male rats in each postnatal group (45 animals in total) were used. Animals were sacrificed under deep anesthesia with diethylether according to Shinshu University School of Medicine Animal Care Committee recommendations. Small pieces of kidney tissue from each animal were immersed in fixatives immediately for light and electron microscopic immunohistochemistry. To obtain identical fixation conditions, all tissues were fixed by immersion because the perfusion technique was not possible for embryos or small animals. Other pieces of tissue without fixation were kept frozen at -30C until the immunoblot analysis was conducted.
Preparation of Antibodies
Catalase and BP were purified from rat livers and DAOX and HOX-B were purified from rat kidneys, as described previously: catalase (
Immunoblot Analysis
The kidney tissues were homogenized in 10 volumes of 10 mM potassium phosphate, pH 7.5/150 mM NaCl/1% Triton X-100 with a teflon homogenizer. The solubilized preparations were heated at 100C for 5 min in sample buffer solution containing 1% SDS. They were subjected to SDS-polyacrylamide slab gel electrophoresis and immunoblot analysis was performed. Electrophoresis was conducted with a small slab gel (6 x 7 cm). After transfer to nitrocellulose membranes, they were stained as follows: 3% bovine serum albumin/150 mM sodium phosphate, pH 7.4; staining with each primary antibody, anti-rat catalase, DAOX, HOX-B, and BP; goat anti-rabbit IgG antibody conjugated with horseradish peroxidase. Visualization was done with the reaction using 4-chloro-1-naphthol.
Light Microscopic Immunohistochemistry
Small pieces of kidney tissue were fixed in Methacarn solution (methanolchloroformacetic acid 6:3:1) for 48 hr at room temperature and embedded in paraffin (
Electron Microscopic Immunohistochemistry
Kidney tissues from seven groups were examined: embryonic Day 18, superficial and juxtamedullary cortex at postnatal Days 7, 14, and 28. Tissue processing and immunostaining were done basically as reported previously (
Classification of Developmental Stages
The presence of the immunoreaction was observed in relation to the developmental maturation stage of kidney tissues. For light microscopic observation, classification of developmental stages of the nephron was done according to the description by
Quantitative Analysis
The size of peroxisomes was determined in the kidney tissues stained for catalase of the seven groups described above. The diameters or the long axes of the peroxisomes in circular or elongate profile were measured on the electron micrographs of the proximal tubules in each group. The developmental stage and part of the proximal tubule evaluated were as follows: stage III at embryonic Day 18, Stage III and early Stage IV in the superficial cortex at postnatal Day 7, convoluted part at Stage IV or later in the juxtamedullary cortex at postnatal Day 7 and in the superficial and juxtamedullary cortex at postnatal Day 14, and the almost mature convoluted part in the superficial and juxtamedullary cortex at postnatal Day 28. The labeling density, i.e., the number of gold particles per unit area of peroxisomes, for each enzyme was estimated in the proximal tubules at three developmental stages: Stage III at embryonic Day 18, early Stage IV in the superficial cortex at postnatal Day 7, and the almost mature convoluted part in the juxtamedullary cortex at postnatal Day 28. Cell organelles immunopositive for each enzyme were regarded as peroxisomes. Their area and the number of gold particles on them were measured with an image analyzer, LUZEX-III (Nireco; Tokyo, Japan). Fifty peroxisomes photomicrographed at x30,000 from each group were evaluated. Statistical analysis was carried out by analysis of variance (ANOVA), and the differences between respective groups were judged to be significant at p<0.05.
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Results and Discussion |
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The course of the appearance of peroxisomal enzymes was analyzed by immunoblot analysis and by light and electron microscopic immunohistochemistry. Methacarn solution was used as fixative for light microscopic immunohistochemistry. The excellence of Carnoy's fixative for light microscopic visualization of peroxisomal enzymes in paraffin sections was reported by
Figure 1 shows the results of the immunoblot analyses performed to demonstrate the presence of four peroxisome-specific enzymes: catalase, DAOX, HOX-B, and BP (Figure 1AD) in the developing kidney tissues at various ages. Each antibody gave a single signal with rat kidney homogenates at the same mobility as the purified enzyme. All of them were undetectable at prenatal Days 16 and 18. Each was weakly detectable in the kidney homogenate at postnatal Day 1. The signal became obvious at postnatal Day 7, and the size of the signal increased at postnatal Day 14, along with development. The time courses of the appearance of each enzyme seemed to be generally parallel.
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Light Microscopic Immunohistochemistry
Figure 2 Figure 3 Figure 4 illustrate the light microscopic immunohistochemical localization of four peroxisome-specific enzymes in the developing kidney tissues, shown as a brown deposit of DAB reaction product. Figure 2 shows developing kidney tissues stained for catalase, indicating the tissue area positive for catalase. Figure 3 consists of high-power magnification pictures of the same specimens, showing the nephron positive for catalase. Immunostaining for catalase was negative in the kidney cortex at embryonic Day 16, in which immature nephrons at the vesicle and S-shaped body stages were identified (Figure 2A). Kidney cortex tissue at embryonic Day 18 consisted of developing nephrons from the vesicle to maturing glomerulus stages (Figure 2B and Figure 3A). The positive staining was localized in a small number of proximal tubules near the juxtamedullary renal corpuscles at the developing capillary loop or maturing glomerulus stage, but the nephrons at the vesicle and S-shaped body stages in the outer zone of the cortex were negative for staining (Figure 3A). The number of immunopositive nephrons at the developing capillary loop and maturing glomerulus stages increased in the juxtamedullary cortex at embryonic Day 20 (Figure 2C). The tissues of the cortex matured along with kidney development, in which the outermost zone of the cortex remained immature and its thickness gradually decreased relative to the entire thickness of the cortex (Figure 2DF). The distribution of proximal tubules with positive staining expanded towards the surface of the kidney. The staining was more intense in the proximal tubules located in the juxtamedullary cortex and outer medulla than in those of the superficial cortex. The outermost zone of the cortex, negative for staining, consisted of nephrons at the vesicle and S-shaped body stages at postnatal Days 1 and 4 (Figure 2D, Figure 2E, and Figure 3B). The immunopositive nephrons in the inner zone of the cortex belong to the developing capillary loop and maturing glomerulus stages in the same periods, in which convoluted and straight parts of the proximal tubules gradually came to be distinguishable (Figure 2D, Figure 2E, and Figure 3B). At postnatal Day 7, the outermost zone of the cortex became weakly positive for the staining where nephrons at the developing capillary loop stage were predominant, and the proximal convoluted and straight tubules were easily distinguished in the superficial cortex (Figure 2F and Figure 3C). The positive staining in the proximal tubules was observed in the full thickness of the cortex and the outer stripe of the outer medulla at postnatal Day 14 (Figure 2G and Figure 3D). The kidney tissues at postnatal Day 28 (Figure 2H) and Week 10 (Figure 2I), whose cortices were positively stained in the full thickness, exhibited a characteristic staining pattern, with the straight part or S3 segment of the proximal tubules in the outer stripe of the outer medulla and medullary rays being most intense. This staining pattern for catalase has been reported in mature kidney tissues in previous studies employing the DAB reaction and in situ hybridization (
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Figure 4 illustrates the localization of DAOX (Figure 4A and Figure 4B), HOX-B (Figure 4C and Figure 4D), and BP (Figure 4E and Figure 4F) in the developing kidney. Because the changes in localization of each enzyme were generally similar to those of catalase, findings in representative age groups are presented: embryonic Day 18 and postnatal Day 4. Immunostaining for each enzyme was negative in the kidney cortex at embryonic Day 16. Positive staining for each enzyme was found for the first time in a small number of proximal tubules around the renal corpuscles at the developing capillary loop and maturing glomerulus stages at embryonic Day 18 (Figure 4A, Figure 4C, and Figure 4E). The distribution of the proximal tubules positive for each enzyme was observed to expand towards the superficial cortex progressively during kidney development (Figure 4B, Figure 4D, and Figure 4F). The outermost zone of the cortex became weakly positive for each enzyme at postnatal Day 7 and the proximal tubules just below the renal capsule became clearly positive for each enzyme by postnatal Day 14. More intense staining for DAOX and BP, but less evident for HOX-B, in the proximal straight tubules was observed at postnatal Day 28 and Week 10, as previously reported for DAOX (
Electron Microscopic Immunohistochemistry
Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 show the electron microscopic immunohistochemical localization of peroxisomal enzymes in developing kidney tissues at the representative ages of embryonic Day 18, and postnatal Days 7, 14, and 28. Figure 5 illustrates the localization of catalase in developing proximal tubule epithelial cells at embryonic Day 18 (Figure 5A), postnatal Day 7 (Figure 5B and Figure 5C), and postnatal Day 14 (Figure 5D) at low-power magnification. Staining was negative in the nephrons at Stages I and II in the kidney tissues at embryonic Day 18 as far as we observed (data not shown). Positive staining was observed in small particles, peroxisomes, in the proximal tubule epithelial cells in the juxtamedullary cortex at embryonic Day 18 (Figure 5A) and in the superficial cortex at postnatal Day 7 (Figure 5B), where weak positive staining for catalase was observed by light microscopic observation. Because some of these proximal tubules did not have dense microvilli forming the typical brush border, they can be categorized as Stage III (Figure 5A and Figure 5B). Peroxisomes were also observed in more differentiated proximal tubules in the juxtamedullary cortex at postnatal Day 7 (Figure 5C) and superficial cortex at postnatal Day 14 (Figure 5D). These proximal tubules possess dense microvilli or brush borders and are categorized as stage IV or later. The size of peroxisomes in the proximal convoluted tubules appeared to be larger in the juxtamedullary cortex at postnatal Day 7 than in the superficial cortex at postnatal Day 14 (Figure 5C and Figure 5D).
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Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 are high-power magnification electron micrographs of peroxisomes stained for four peroxisome-specific enzymes, showing the intra-cell organellar localization. Figure 6 Figure 7 Figure 8 show the immunohistochemical localization of the peroxisomal enzymes catalase (Figure 6A, Figure 7A, and Figure 8A), DAOX (Figure 6B, Figure 7B, and Figure 8B) HOX-B (Figure 6C, Figure 7C, and Figure 8C), and BP (Figure 6D, Figure 7D, and Figure 8D) in the juxtamedullary cortex at embryonic Day 18 (Figure 6), and in the superficial (Figure 7) and juxtamedullary (Figure 8) cortices at postnatal Day 7. Positive staining for each enzyme was observed in peroxisomes in the juxtamedullary cortex at embryonic Day 18 (Figure 6). The peroxisomes in the proximal tubules at Stage III were small, in diameter, approximately 0.10.3 µm (mean 0.202 µm) (Table 1) and can be categorized as microperoxisomes. The substructures such as the electron-dense peripheral matrix and central electron-lucent matrix were not clearly observed and the four enzymes were distributed diffusely over the matrix in most peroxisomes [ Figure 6A, Figure 6B (left), 6C, and 6D]. However, in some peroxisomes the localization of DAOX was confined to the central area [ Figure 6B (right)], showing the presence of the central electron-lucent matrix. In the superficial cortex at postnatal Day 7, positive staining for each enzyme was detected in peroxisomes in the proximal tubules at Stage III (Figure 7) and those at early Stage IV (data not shown). The ultrastructure of small peroxisomes in the proximal tubules at Stage III, approximately 0.10.3 µm (mean 0.199 µm) in diameter (Table 1), was similar to that seen in the juxtamedullary cortex at embryonic Day 18, i.e., some did not have substructures and others did (Figure 7B). The peroxisomes in the proximal tubules at early Stage IV were larger than at Stage III, approximately 0.10.5 µm (mean 0.277 µm) in diameter (Table 1). In the juxtamedullary cortex at postnatal Day 7, peroxisomes of various sizes were observed in the proximal tubules that were more differentiated than at Stage IV, approximately 0.10.6 µm (mean 0.329 µm) in diameter in the convoluted part (Table 1). Four enzymes were localized preferentially in the appropriate subcompartments of large peroxisomes, as described previously (
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The peroxisome is defined as a cell organelle containing at least one H2O2-producing oxidase and H2O2-reducing catalase (
The appearance of peroxisome-specific enzymes or peroxisomes with progressing development is consistent with previous reports dealing with the mammalian kidney. By biochemical and morphometric analyses, the enzyme activity of catalase was shown to be present in the kidney tissues of mouse embryos and to increase during postnatal development, correlating with the increase in the number of peroxisomes in the proximal tubules (
Our observations and that on the regenerating kidney tissues indicate that the fine structure of peroxisomes might be a good marker for the differentiation of nephrons together with microvilli, apical endocytic vacuoles, and other structures in the proximal tubules. In other words, the presence of structurally or enzymologically discernible peroxisomes can be useful in identifying the nephrons at Stage III or at more differentiated stages.
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by the Occupational Health Promotion Foundation.
We thank Dr Takashi Hashimoto for kindly providing the purified HOX-B and BP proteins and their antibodies. We appreciate the skillful technical assistance of Ms Naoko Ogiwara.
Received for publication September 29, 1997; accepted May 21, 1998.
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