ARTICLE |
Correspondence to: Marcel Tappaz, INSERM U 433, Faculté de Médecine RTH Laennec, Rue Guillaume Paradin, F 69372 Lyon, Cedex 08, France. E-mail : tappaz@lyon151.inserm.fr
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Summary |
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Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of the pathway that forms taurine, a putative osmolyte in the kidney, which was previously localized in various segments of the nephron. Although CSD is known to be expressed in whole kidney extracts, no information on CSD mRNA regional expression and histological localization is yet available. Western blotting and Northern blotting were performed in four dissected regions of the kidney using an antiserum against recombinant CSD and a [32P]-dCTP-labeled CSD cDNA probe, respectively. In situ hybridization was carried out using a [35S]-CTP-labeled CSD RNA probe. A single protein (53 kD) and a single mRNA (2.5 kb) were detected, both of which appeared to be most enriched in the outer stripe of the outer medulla. In situ hybridization of CSD mRNA showed strong labeling of the thick tubules in the outer stripe of the outer medulla and in cortical medullary rays that corresponded to the proximal straight tubules. The significance of this restricted expression of CSD is discussed in relationship to the data previously reported on the location of taurine and the location of the taurine transporter along the nephron. (J Histochem Cytochem 48:14611468, 2000)
Key Words: Taurine, cysteine sulfinate, decarboxylase, kidney, nephron, proximal straight tubule
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Introduction |
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Cysteine sulfinate decarboxylase (CSD) is the rate-limiting step of the so-called cysteine sulfinate pathway that leads to the synthesis of taurine from cysteine (
The cellular localization of taurine in the kidney was investigated by immunocytochemistry. The most comprehensive study (
Taurine is taken up through a high-affinity sodium-dependent transporter. A taurine transporter has been cloned from a rat brain cDNA library and characterized by expression in mammalian cells (
Taurine shows a broader distribution in the kidney than that of its transporter. This discrepancy may reflect the contribution of local synthesis of taurine in some segments of the nephron. In contrast to the taurine transporter, the expression and regulation of which have been well studied (
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Materials and Methods |
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Animals and Tissue Preparation
Male adult SpragueDawley rats (200250 g body weight) were used (Iffa Credo; Lyon, France). Animals were maintained under controlled temperature, humidity, and light/dark cycle for an adaptation period of 2 days. Commercial food (Souffirat; Jenthon, France) and tapwater were supplied ad libitum. The animals were anesthetized with chloral hydrate (400 mg/kg body weight) and sacrificed by decapitation. Kidneys were rapidly removed. A sagittal section about 1 mm thick was cut with a razor blade and the different regions of the kidney [superficial cortex (called "cortex" in the text below), outer stripe of the outer medulla, inner stripe of the outer medulla and inner medulla] were dissected out under a microscope and immediately frozen in liquid nitrogen. For in situ hybridization the kidneys were rapidly removed and frozen in isopentane at -70C.
CSD Antiserum
A recombinant fusion protein glutathione-S-transferaseCSD (GSTCSD) was produced, isolated by affinity chromatography on glutathioneagarose beads, and cleaved by thrombin protease as previously reported (
Nucleotide Probes
The DNA probe used in Northern blotting was the 1.9-kb CSD cDNA cloned previously from a rat liver cDNA library (
Western Blotting
The tissues, whole kidney and dissected kidney regions, were homogenized in water (10% w/v) with a polytron (Polyblock; Strasbourg, France) and ultracentrifuged at 100,000 x g for 1 hr at 4C. The proteins of the supernatant were precipitated with 5% TCA for 90 min at 4C. The pellet was solubilized in 8 M urea. Proteins (20 µg/well) were separated by electrophoresis on a 10% acrylamide/SDS gel and electrophoretically transferred to a nitrocellulose membrane (Schlei-cher & Schuell; Dassel, Germany) in an electroblotting apparatus (BioRad Laboratories; Hercules, CA). The blots were incubated for 45 min in PBS containing 1% fat-free dry milk and 0.1% Triton X-100. They were further incubated overnight with rabbit CSD antiserum diluted 1:10,000 in the same solution. The blots were washed in PBS containing 0.1% Triton X-100 and then incubated with anti-rabbit secondary antibodies conjugated to peroxidase (Boehringer; Mannheim, Germany) diluted 1:1000 for 2 hr. Bands were revealed using a chemiluminescence kit according to the supplier's protocol (Pierce; Rockford, IL).
Northern Blotting
Total RNA was extracted from whole kidney or main kidney regions by the RNA B method (Bioprobe Systems; Montreuil-sous-bois, France). After alcohol precipitation, the pellet was air-dried and solubilized in RNase-free water. Twenty µg of total RNA was run on a 1.2% agarose/0.2 M formaldehyde gel and transferred to a nitrocellulose membrane (Schleicher & Schuell). Prehybridization was performed at 42C for 3 hr in a mixture made of 50% deionized formamide in 5 x SSPE (50 mM, pH 7.4 sodium phosphate buffer containing 0.75 M NaCl and 5 mM EDTA), 5 x Denhardt's solution, 0.05% SDS, and 100 mg/ml denatured salmon sperm DNA. Hybridization was carried out at 42C overnight in the same mixture to which the [32P]-dCTP-labeled random-primed cDNA probe was added (about 3.106 cpm/ml). The membrane was washed at 42C as follows : (a) once in 2 x SSPE containing 0.1% SDS for 15 min; (b) once in 0.5 x SSPE containing 0.1% SDS for 15 min; and (c) twice in 0.1 x SSPE containing 0.1% SDS for 20 min. The membrane was exposed to Reflection film (NEN Life Science Products; Boston, MA) for autoradiography with an intensifying screen at -80C overnight. The membranes were treated with hot water containing 0.1% SDS to remove the CSD cDNA probe. They were then hybridized with a [32P]-dCTP-labeled random-primed 1-kb cDNA probe of a housekeeping gene (GAPDH) (Clontech; Heidelberg, Germany).
In Situ Hybridization
Cryostat sections (16 µm) were collected on silanated RNase-free slides. They were dipped in acetone for 5 min at 4C and air-dried. Fixation was performed with 4% paraformaldehyde in 100 mM phosphate buffer (PB), pH 7.4, for 15 min at 4C. The sections were acetylated in 0.1 M triethanolamine-HCl buffer (pH 8) containing 0.25% acetic anhydride for 10 min at room temperature and then treated with 1 x SSC containing 50% formamide for 10 min at 55C. After alcohol dehydration and air-drying, each slide was hybridized overnight at 52C in 50 µl of hybrization solution made of 20 mM Tris-HCl, pH 6.8, containing 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 1 mM Na2HPO4, 5 mM EDTA, 1 x Denhardt's solution, 0.01 M DTT, 0.5 mg/ml yeast tRNA, and the labeled probe (about 3.106cpm). The sections were rinsed in 1 x SSC containing 50% formamide for 30 min at 55C, 2 x SSC for 10 min at room temperature, and then incubated with 20 µg/ml RNase A in 0.01 M Tris-HCl, pH 7.5, containing 0.5 M NaCl and 5 mM EDTA for 20 min at 37C. They were washed twice in 1 x SSC containing 50% formamide for 1 hr at 55C, rinsed with 0.1 x SSC for 10 min at 55C, dehydrated in alcohol, and air-dried. Slides were initially exposed to X-ray films for 23 days to provide an indication of the intensity of the hybridization signal. They were then coated with Ilford Nuclear Research emulsion (Ilford, UK) diluted 1:1 with water. Sections were exposed for 23 weeks at 4C in sealed dark boxes. They were developed in Kodak D19, fixed in Ilford Hypam, and washed abundantly. They were counterstained with toluidine blue for histological identification.
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Results |
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By Western blotting, the CSD antiserum labeled a single band of 53 kD in the soluble fraction of homogenates of each of the four main dissected regions of the rat kidney (Fig 1A). The molecular weight of this labeled protein subunit is identical to that of the recombinant CSD produced in bacteria. The labeling of this 53-kD band was most intense in the outer stripe. By Northern blotting, the CSD cDNA probe recognized a band of 2.5 kb in the total kidney, in the outer stripe, and in the inner stripe (Fig 1B). It was not detectable in the inner medulla and was barely visible in the cortex. In the latter extract, the signal appeared underestimated because of some degradation of the RNA revealed by the reduced level of GAPDH mRNA. The CSD mRNA was much more overexpressed in the outer stripe.
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Histological localization of CSD and CSD mRNA was then performed. Although the antiserum we produced gave a reliable detection of a single protein on Western blots of kidney extracts, no satisfactory immunolabeling could be achieved on cryostat sections of paraformaldehyde-fixed tissue with this antiserum. Apparently this antiserum recognized the native form of the protein very poorly. This may be due to the fact that the antiserum was produced using as antigen the denatured form of the recombinant protein isolated after SDS-PAGE. Histological localization of the CSD mRNA along the nephron was then achieved through in situ hybridization. On film autoradiography of sections incubated with the antisense riboprobe, strong labeling was observed in the outer stripe, from which characteristic labeled indentations penetrated the deeper part of the cortex (Fig 2A). In the inner medulla no labeling was detectable, but a very faint darkening of the film could be distinguished over the inner stripe. No labeling was obtained when the tissue sections were incubated with the sense probe (Fig 2B). On emulsion-dipped tissue sections conterstained with toluidine blue, an intense signal was observed in the tubules located in the outer stripe, which abruptly stopped at the limit between the outer stripe and the inner stripe (Fig 3A and Fig 3B). A strong labeling was present over the tubules of the cortical medullary rays. No labeling was detected in the inner medulla. The glomeruli were devoid of labeling. Faint but detectable labeling was visible over the structures of the inner medulla and the cortical labyrinths between the medullary rays. At higher magnification, the strong labeling in the outer stripe was localized over many slightly sinuous, thick-walled tubules, around 30 µm in diameter, whereas thinner tubules (<10 µm in diameter) were not labeled (Fig 3C and Fig 3D).
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Discussion |
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In this study, we provide new information on the regional distribution of CSD and CSD mRNA and on the histological localization of CSD mRNA in the kidney. By Western blotting only one protein band appeared labeled in soluble protein extracts from the dissected regions of the kidney. This protein has the same molecular weight (53 kD) as the recombinant CSD. By Northern blotting only one mRNA band appeared labeled with the CSD cDNA probe. This mRNA has the same size (2.5 kb) as that found in liver or brain RNA extracts (
In agreement with the regional distribution of the CSD mRNA, in situ hybridization with an antisense riboprobe showed very strong labeling in the tubules located in the outer stripe of the outer medulla and the cortical medullary rays, and only faint labeling in the inner stripe of the outer medulla and the cortical labyrinths. The lack of any labeling in the inner medulla indicates that the inner medullary collecting ducts, as well as the thin limbs of the long-looped nephrons, do not significantly express CSD. The strong labeling in the tubules of the outer stripe of the outer medulla might be located in some of the tubules present at this level, i.e., the proximal straight tubules, the distal straight tubules, and the collecting ducts. If the labeling were associated with the latter two, it would not be restricted to the outer stripe but should also extend into the inner stripe according to the anatomic organization of the nephrons (
Our results thus indicate that taurine formation through the sulfinate pathway in which CSD is held as the rate limiting step (
The main conclusions of our investigation are that CSD expression occurs primarily in the part of the nephron, i.e., the proximal straight tubules, in which taurine is concentrated (
Taurine was reported to be highly concentrated in the collecting ducts throughout the renal cortex and the renal medulla (
In conclusion, in this report we have provided evidence for a restricted localization of CSD mRNA in the cortical and medullary proximal straight tubules. Accordingly, a significant synthesis of taurine through the cysteine sulfinate pathway occurs only in this restricted portion of the nephron, where a taurine transporter was reported to be also highly expressed. Because taurine was reported to have a broader distribution, notably in the collecting ducts, where CSD or taurine transporter are not expressed, other synthesis pathways and/or transporters may be involved, which remain to be identified and precisely localized in the kidney.
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Footnotes |
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1 This work was presented as a poster and in abstract form at the International Taurine Symposium, Siena, Italy, August 48, 1999, and at the 6th International Congress on Amino-Acids, Bonn, Germany, August 37, 1999.
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Acknowledgments |
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Marc Bitoun is the recipient of a DRET fellowship DGA.
We thank Didier Decimo (INSERM U 412 ENS, Lyon) for his help in setting up the in situ hybridization and Jean Louis Borach (INSERM U 94, Bron) for the photographic work.
Received for publication November 11, 1999; accepted May 10, 2000.
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