ARTICLE |
Correspondence to: Reiji Semba, Dept. of Anatomy, Mie U. School of Medicine, Tsu, Mie 514, Japan.
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Summary |
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Recent studies suggest that carbon monoxide (CO), which is formed by the enzyme heme oxygenase (HO) during the conversion of heme to biliverdin, shares some of the chemical and biological properties of nitric oxide (NO) and may play roles similar to those of NO. Heme oxygenase activity in the kidney has been reported for many years, and there are some reports on the expression of mRNA for two HO isozymes (HO-1 and HO-2) and cellular localization of HO-1 protein. However, cellular localization of HO-2 protein in the kidney under normal conditions has not been reported. In the present study we examined the expression and distribution of HO-2 mRNA and HO-2 protein in rat kidney using RNA protection assay and light and electron immunocytochemistry. RNA protection assay confirmed constitutive expression of HO-2 transcript in rat kidney. HO-2 immunoreactivity was selectively found in epithelial cells of the thick ascending limb and distal convoluted tubule, connecting tubule cells, and principal cells of the collecting duct. These results suggest that HO-2 is synthesized in the kidney and that HO-2 in the epithelial cells of renal tubules may serve as a source for CO generation under normal conditions. (J Histochem Cytochem 46:249256, 1998)
Key Words: carbon monoxide, RNA protection assay, Western blot analysis, immunocytochemistry, distal tubule, collecting duct
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Introduction |
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Nitric OXIDE (NO) and carbon monoxide (CO) have been considered as toxic gases without physiological function. However, the discovery of NO as a metabolic product has opened a rapidly expanding area of research. It is proposed that CO may function in a fashion similar to that of NO (
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Materials and Methods |
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Animals
Adult Wistar rats bred in our laboratory and kept under standardized animal room and food conditions were used.
Preparation of RNA Probe
Total cytoplasmic RNA was extracted from rat kidney and brain using the CLONsep total RNA isolation kit (Clontech; Palo Alto, CA). RNA concentration was determined by absorbance at 260 nm. Rat HO-2 cDNA was generated from rat kidney by using RT-PCR with the two oligonucleotide primers P1 (5'-TGAAAACTGGGAGGAGCAGG-3'), homologous to rat HO-2 cDNA nucleotides +543 to +562, and P2 (5'-AACAGGTAGAACTGGGTCCC-3'), complementary to rat HO-2 cDNA nucleotides +755 to +736 (
RNA probes were generated from the restriction enzyme-linearized plasmids containing the 198 BP of rat HO-2 cDNA insert according to the manufacturer's instruction with [-32P]-UTP (ICN Biomedicals; Costa Mesa, CA). The resultant anti-sense RNA probe was approximately 300 nucleotides long, containing 198 nucleotides attributed to rat HO plus 95 nucleotides of vector-derived ends. Therefore, hybridization between the cRNA probe and rat mRNA yielded the protection of a 198-nucleotide fragment.
RNA Protection Assay
According to a previous method (
Antibodies
Rabbit polyclonal antibodies raised against the synthesized N-terminal fragment of rat testis HO-2 were used. The preparation and specificity of the antibodies have been previously described in detail (
Western Blot Analysis
Fresh kidneys and brains were homogenized in 50 mM Tris-HCl buffer, pH 8.0. Protein concentration was determined with a protein assay kit (Bio-Rad; Richmond, CA). Crude homogenates (50 µg) were loaded, separated on a 12% SDS-PAGE gel, and transferred onto a nitrocellulose membrane. The membrane was immunostained using the HO-2 antibodies and the peroxidaseanti-peroxidase complex (PAP) method.
Fixation
Rats were anesthetized with an IP injection of sodium pentobarbital and were perfused transcardially with Schmechel's fixative (
Light Microscopic Immunocytochemistry
One hundred-µm-thick sections of kidney were cut on a vibratome. Endogenous peroxidase activity and nonspecific staining were blocked by incubation with 0.3% hydrogen peroxide and 10% normal goat serum for 30 min. Then the sections were incubated with HO-2 antibodies at a concentration of 0.87 µg/ml in PBS overnight at room temperature. After washing with PBS, sections were immunostained by the PAP method and visualized with 3,3'-diaminobenzidine tetrahydrochloride as chromogen. Sections were postfixed in 1.0% osmium tetroxide for 1 hr, dehydrated, and embedded in Epon resin. Semithin sections cut at 3-µm thickness were examined under a light microscope. Preimmune rabbit serum and preabsorbed antibodies, prepared by passing through the peptide-bound column, were used for control immunostainings.
Electron Microscopic Immunocytochemistry
One hundred-µm-thick vibratome sections were treated with Lugol's iodine solution for 10 min, rinsed with 2.5% sodium thiosulfate, then incubated with 10% normal goat serum for 10 min. Sections were incubated with HO-2 antibodies overnight at 4C, washed with PBS, and left in 3-nm gold-labeled goat anti-rabbit IgG (Funakoshi; Tokyo, Japan) for 3 hr. Then sections were developed with the silver enhancement kit (Amersham; Poole, UK) for 510 min and osmicated in 1.0% osmium tetroxide for 1 hr. Areas of interest were selected, dehydrated, and embedded in Epon resin. Seventy-nm-thick ultrathin sections were cut, counterstained in 4% uranyl acetate and lead acetate, and examined with an electron microscope H-800 (Hitachi; Tokyo, Japan). Control sections were made by omission of HO-2 antibodies.
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Results |
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The constitutive expression of HO-2 mRNA was examined in rat kidney by RNA protection assay. As shown in Figure 1, a protected band of 198 nucleotides (lower arrowhead) was detected in the RNA samples of rat kidney and brain, a positive control, by using the anti-sense probe for the hybridization. However, no corresponding band was detected in yeast RNA or with sense probe. The relative abundance of HO-2 transcripts in the kidney was smaller than that in the brain.
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The constitutive expression of HO-2 protein was examined in rat kidney by Western blot analysis. Immunoblottings from rat kidney and brain homogenates revealed a single clear protein band with a molecular mass of 36 kD in both lanes, with greater abundance in the brain (Figure 2).
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Distribution of HO-2 protein in rat kidney under normal conditions was examined by light and electron microscopic immunocytochemistry using HO-2 antibodies. In light microscopic immunocytochemistry, positive staining for HO-2 was obtained in both the renal cortex and the medulla. In the renal cortex, on the basis of cell morphology and cell composition, immunoreactive segments were identified as the distal convoluted tubule, the connecting tubule, and the cortical collecting duct, as follows. The distal convoluted tubule is composed of homogeneous epithelial cells which have extensive basolateral membrane interdigitations (
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Electron microscopic immunocytochemistry further defined HO-2 immunoreactivity in the specific cells of the renal tubules. Figure 5 shows HO-2 immunogoldsilver particles which were predominantly located in epithelial cells of the distal convoluted tubule (Figure 5A), the connecting tubule cells (Figure 5B), and the principal cells (Figure 5C). Particles were not found in the intercalated cells of the connecting tubule and the cortical collecting duct. Particles for HO-2 immunoreactivity were observed throughout the cytoplasm and were concentrated in the apical region of the stained cells. Control sections were made by omission of HO-2 antibodies. In these sections, positive staining was absent from the above-mentioned epithelial cells. However some particles were found predominantly on the surfaces of some interstitial cells (Figure 5D). At high magnification (Figure 6A and Figure 6B), most of the silver-enhanced gold particles were associated with the endoplasmic reticulum and the nuclear outer membrane. In contrast, mitochondria, lysosomes, and Golgi complex were not labeled.
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Discussion |
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The RNA protection assay revealed a fairly high level of HO-2 transcript expression in rat kidney. Constitutive expression of HO-2 mRNA in rat kidney is consistent with the results of Northern blot analyses (
Degradation of heme takes place largely in tissues of the reticuloendothelial system, such as the spleen, by heme oxygenase-1. The brain is an organ usually considered not to play a significant role in degradation of heme. However, HO-2 mRNA and protein are abundant in the brain (
HO-2 immunoreactivity was found in the thick ascending limb, distal convoluted tubule, connecting tubule, and collecting duct. In the thick ascending limb and the distal convoluted tubule, all epithelial cells were immunoreactive, whereas in the connecting tubule and the collecting duct, immunonegative epithelial cells were found scattered among immunopositive epithelial cells. Those immunonegative cells were considered to be intercalated cells, based on the knowledge that the incidence of these cells is about 40% in the connecting tubule and the cortical collect duct and about 10% in the inner medullary collecting duct (
To our knowledge, the present study has revealed the precise intracellular localization of HO-2 protein in rat kidney for the first time. Although further work is necessary to clarify the physiological role of HO-2, our present results suggest that HO-2 is synthesized in the kidney and that HO-2 in the epithelial cells of the renal tubules may serve as a source for CO generation under normal conditions.
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Literature Cited |
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