ARTICLE |
Correspondence to: Eiko Aoki, Dept. of Embryology, Inst. for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-03, Japan.
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Summary |
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Using antibodies highly specific for L-arginine and L-citrulline, we localized these amino acids in rat kidney with immunohistochemical methods. Highest levels of arginine immunoreactivity were observed in epithelial cells of proximal tubules in the outer stripe of the outer medulla and the collecting ducts in the cortex. Staining intensity of proximal convoluted tubules in the outer stripe decreased from the inner side to the outer side. In the inner medulla, collecting ducts were labeled with moderate intensity. Staining within the cortex was apparent only with collecting ducts. Citrulline immunoreactivity was localized in the epithelial cells of collecting ducts both in the cortex and medulla. Immunoreactivity was also found in glomerular podocytes and in the epithelial cells of proximal convoluted tubules in the outer medulla. These localizations were different from those of other amino acids previously reported. The precise cellular distribution of arginine and citrulline in rat kidney was determined for the first time by an immunohistochemical method in the present study. (J Histochem Cytochem 45:875-881, 1997)
Key Words: amino acid, arginine, citrulline, kidney, immunohistochemistry, renal tubules
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Introduction |
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Biochemical studies have revealed considerable amounts of free amino acids in the kidney (
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Materials and Methods |
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Animals
Experiments were performed using 2-month-old male rats of the Sprague-Dawley strain that were bred in our laboratory. They were maintained in an animal room at a temperature of 24 ± 2C with a relative humidity of 55 ± 10% and a light/dark cycle of 12 hr. Rats were allowed free access to a commercial diet (NMF; Oriental Yeast, Tokyo, Japan) and tapwater.
Tissue Preparation
For the immunohistochemical demonstration of L-arginine and L-citrulline, the rats were perfused via the heart with a mixture of 1% GAL, 4% paraformaldehyde, 0.2% picric acid, and 2% sucrose in 0.1 M sodium acetate buffer, pH 6.0 (
Antibodies
The preparation and specificity of anti-L-arginine and anti-L-citrulline antibodies were the same as reported previously (
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Immunohistochemical Processing
Sections were deparaffinized in xylene, dehydrated through a graded alcohol series, and washed twice in 50 mM Tris-HCl buffer containing 500 mM NaCl, pH 7.6. Sections were incubated with the anti-arginine (0.5 µg/ml) or anti-citrulline (0.2 µg/ml) antibody, left overnight at room temperature, and followed by an immunohistochemical procedure using the peroxidase-anti-peroxidase method (
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Results |
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L-Arginine Immunoreactivity
Low-power magnification of the section demonstrated that L-arginine immunoreactivity is predominantly located in the outer stripe of the outer medulla (Figure 2A). Higher magnification revealed it to be localized in the cells of the proximal tubules and the collecting ducts (Figure 2C). Staining intensity of proximal convoluted tubules in the outer stripe of the outer medulla decreased from the inner side to the outer side (Figure 2A and Figure 2C). Staining within the cortex was apparent exclusively in collecting ducts. Immunopositive cells of these collecting ducts exhibited a characteristic mosaic-like pattern, with some of the epithelial cells being immunoreactive for arginine whereas other cells were immunonegative (Figure 2B). Glomeruli and proximal and distal tubules in the cortex were not stained. Interestingly, proximal straight tubules and collecting ducts were not stained in the inner stripe of the outer medulla (Figure 2A). In the inner medulla, collecting ducts were labeled with moderate intensity (Figure 2D).
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L-Citrulline Immunoreactivity
L-citrulline immunoreactivity was present in both the cortex and the medulla of the kidney (Figure 3A). In the cortex, the immunoreactivity was clearly localized in podocytes of the glomeruli, endothelial cells near the vascular pole, and endothelial cells of the collecting ducts (Figure 3B-D). Immunostaining of the collecting ducts was more intense in about half of the cells than in the other half. Cells of proximal and distal tubules were not stained. In the outer and inner stripes of the outer medulla, immunoreactivity was observed in the medullary ray, epithelial cells of proximal convoluted and straight tubules, and collecting ducts (Figure 3E and Figure 3F). Staining intensity in the collecting ducts was greater than that of proximal tubules. In the inner medulla, all cells of collecting ducts were strongly labeled (Figure 3G). Cells of the pelvic wall were also labeled intensely (Figure 3A).
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Discussion |
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Immunohistochemical localization of some neutral and acidic amino acids (GABA, taurine, aspartate, and glutamate) were studied in the rat kidney (
The kidney synthesizes a considerably large portion of endogenous arginine, and the proximal convoluted tubules have been suggested as the predominant site for arginine synthesis (
Citrulline immunoreactivity was localized in cortical and medullary collecting duct cells, proximal tubules in outer medulla, and glomerular podocytes. Arginase and ornithine aminotransferase, enzymes of the urea cycle that catabolize arginine to citrulline, were localized in the cortex and outer medulla (
In the collecting ducts, about half of the epithelial cells in the cortex and most cells in the inner medulla were immunoreactive to both anti-arginine and anti-citrulline antibodies. Two types of cells, principal cells and intercalated cells, are known in the collecting ducts, and the latter cells gradually decrease from about 35% of the cell population in the ducts of the outer medulla to 10% in the inner medulla (
Immunohistochemical demonstration of free amino acids was first achieved by
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