Journal of Histochemistry and Cytochemistry, Vol. 45, 875-882, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Immunohistochemical Localization of Arginine and Citrulline in Rat Renal Tissue

Eiko Aokia and Ikuo K. Takeuchia
a Department of Embryology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan

Correspondence to: Eiko Aoki, Dept. of Embryology, Inst. for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-03, Japan.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Biochemical studies have revealed considerable amounts of free amino acids in the kidney (Dantzler and Silbernagl 1988 ; Blazer-Yost et al. 1979 ). The cellular distribution of some of these amino acids, including glutamate, aspartate, taurine, and GABA, was determined by immunohistochemical methods using antibodies against these amino acids (Ma et al. 1994 ; Trachtman et al. 1993 ; Parducz et al. 1992). The patterns of distribution of each amino acid were different from one another and were helpful in understanding their possible functional roles in the kidney. Arginine in kidney is synthesized largely from citrulline by means of the urea cycle (Dhanakoti et al. 1990 , Dhanakoti et al. 1992 ; Featherson et al. 1973 ). It is believed that nitric oxide (NO) and citrulline are produced from arginine in the kidney and that NO affects renal blood flow, glomerular filtration, and tubule function (Bachmann and Mundel 1994 ). However, it is unclear which cells of the kidney contain arginine and/or citrulline. We raised antibodies against L-arginine and L-citrulline and studied the localization of these amino acids in rat kidney by immunocytochemical techniques.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Schmechel et al. 1980 ). After perfusion, kidneys were removed and immersed in the same fixative for 4-5 hr. Then they were rinsed several times with 50 mM Tris-HCl buffer, pH 7.4, dehydrated with a graded alcohol series, and embedded in paraffin. Seven-µm-thick sections were mounted on albumin-coated slides.

Antibodies
The preparation and specificity of anti-L-arginine and anti-L-citrulline antibodies were the same as reported previously (Aoki et al. 1991 ). In brief, L-arginine and L-citrulline were coupled with guinea pig serum albumin and rabbit serum albumin, respectively, via glutaraldehyde. Each amino acid conjugate was emulsified with an equal volume of complete Freund's adjuvant and repeatedly injected intracutaneously into multiple sites on the backs of guinea pigs or rabbits. Each antibody was purified by affinity chromatography with the respective amino acid immobilized on formyl cellulofine (Seikagaku Kogyo; Tokyo, Japan). Specificities of the purified antibodies were examined by a dot immunobinding assay (Hawkes et al. 1982 ). Reactivity of the antibodies was studied against L-citrulline-, L-arginine-, L-ornithine-, L-aspartate-, L-glutamate-, GABA-, L-glycine-, L-taurine-, L-leucine-, L-isoleucine-, L-valine-, and ß-alanine-albumin complexes produced as described previously (Aoki et al. 1991 ). The purified anti-arginine and anti-citrulline antibodies were found to be specific to the L-arginine- and L-citrulline-albumin complex, respectively (Figure 1).



View larger version (23K):
[in this window]
[in a new window]
 
Figure 1. Dot immunobinding assay of anti-arginine and anti-citrulline antibodies. Specificity of the antibodies was studied by a dot immunobinding assay on a nitrocellulose membrane. Rabbit or guinea pig serum albumin conjugates of various amino acids, including L-arginine (Arg), L-citrulline (Cit), L-ornithine (Orn), ß-alanine (ß-Ala), L-glutamate (Glu), L-aspartate (Asp), taurine (Tau), GABA, L-glycine (Gly), L-valine (Val), L-leucine (Leu), and L-isoleucine (Iso-leu) were applied to a nitrocellulose membrane and incubated with an affinity-purified antibody at a dilution of 1:1000. Antigen-antibody reactions were visualized by the peroxidase-anti-peroxidase method. Purified antibody gave a strong immunostaining only on the spot of the specific amino acid against which the antiserum had been raised (arrows). (Left) Arginine-specific antibody; (Right) citrulline-specific antibody.

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 (Sternberger 1974 ). 3,3'-Diaminobenzidine tetrahydrochloride (Dojindo Chemical Institute; Kumamoto, Japan) was used as chromogen, which had been freshly prepared as a solution of 20 mg in 100 ml Tris buffer (50 mM, pH 7.6) that contained 0.01% H2O2. Control sections incubated with non-immune serum showed no positive staining.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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).



View larger version (109K):
[in this window]
[in a new window]
 
Figure 2. Photomicrographs showing the distribution of L-arginine immunoreactivity. (A) Overview of L-arginine distribution in different kidney zones. The outer stripe (OS) of the outer medulla is intensely labeled. C, cortex; OS, outer stripe of the outer medulla; IS, inner stripe of the outer medulla, IM; inner medulla. Bar = 100 µm. (B) Higher magnification of the cortex. Reactivity is mainly confined to epithelial cells of collecting ducts (arrowheads). Proximal and distal tubules and glomerulus (G) are not stained. Bar = 50 µm. (C) Higher magnification of the outer stripe. Almost all cells of the proximal tubule (arrows) and collecting duct (arrowheads) are positive staining. Bar = 50 µm. (D) In the inner medulla, epithelial cells of the collecting duct (arrowheads) display positive staining, whereas cells of the thin segments of Henle's loop are negative. Bar = 50 µm.

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).




View larger version (206K):
[in this window]
[in a new window]
 
Figure 3. Immunohistochemical demonstration of L-citrulline in the rat kidney. (A) Overview of L-citrulline immunoreactivity in cortical and medullary zones. C, cortex; OS, outer stripe of the outer medulla; IS, inner stripe of the outer medulla, IM; inner medulla; pw, pelvic wall. Bar = 200 µm. (B-D) Photomicrographs of the cortical labyrinth, demonstrating immunoreactivity in podocytes of the glomerulus (G), endothelial cells near the vascular pole (arrows), and the collecting ducts (arrowheads). Many epithelial cells of collecting ducts are intensely stained, whereas some cells are weakly stained. Proximal and distal tubules are not stained. Bars = 50 µm. (E) In the outer stripe of the outer medulla, epithelial cells of collecting ducts (arrowheads) and proximal tubules (arrows) exhibit positive staining; collecting duct cells exhibit particularly intense staining. Bar = 50 µm. (F) Distribution pattern in the inner stripe of the outer medulla is similar to that of the outer stripe. Bar = 50 µm. (G) In the inner medulla, epithelial cells of the collecting ducts (arrowheads) display intense staining. Bar = 50 µm.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Immunohistochemical localization of some neutral and acidic amino acids (GABA, taurine, aspartate, and glutamate) were studied in the rat kidney (Ma et al. 1994 ; Trachtman et al. 1993 ; Parducz et al. 1992), and none of the distribution patterns of these amino acids was identical to any other. Therefore, it was of interest to determine how arginine and citrulline, basic amino acids, are distributed in rat kidney. Arginine and citrulline were found to be highly concentrated in the proximal tubules, in which none of the four amino acids studied previously had been proved. Consequently, the distribution of the individual amino acids examined to date appears to be specific for each amino acid.

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 (Levillain et al. 1990 , Levillain et al. 1993 ). By immunohistochemical and in situ hybridization studies, arginine-synthesizing enzymes and their mRNAs were also shown to be localized in proximal tubules (Morris et al. 1991 ). The localization of arginine immunoreactivity in proximal tubules, as revealed in the present study, is in good agreement with these reports. Arginine synthesized in the kidney is largely released into the renal blood flow and a portion of it is used within the kidney (Levillain et al. 1990 ). Dantzler and Silbernagl 1988 suggested that arginine is concentrated and recycled in the medulla. In the epithelium of the collecting duct, several amino acids were accumulated and their physiological significance in increasing the cellular osmotic pressure by acting as organic osmolytes has been proposed (Trachtman et al. 1988 ). Therefore, arginine in the collecting duct may be accumulated from the bloodstream or tubule fluid and may contribute to increasing the cellular osmotic pressure. Arginine in the collecting duct may also be used as the substrate for an enzyme that catabolizes argi-nine, NO synthase, as suggested by several reports (Markewitz et al. 1993 ; Stoos et al. 1992 ; Terada et al. 1992 ).

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 (Levillain et al. 1993 ). Therefore, the localization of these citrulline-synthesizing enzymes is similar to that of the citrulline immunoreactivity revealed in the present study. Citrulline is also produced as the co-product of NO formation, and NO plays a role in activating guanyl-ate cyclase. It has been shown that NO regulates solute transport in the cortical and inner medullary collecting duct (Stoos et al. 1992 ), and mRNAs for NO synthase and guanylate cyclase are expressed in both cortical and medullary collecting ducts (Markewitz et al. 1993 ; Terada et al. 1992 ). These reports are consistent with our results showing that citrulline immunoreactivity is present in collecting ducts.

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 (Fawcett 1994 ). Therefore, the immunopositive cells are considered to be the principal cells.

Immunohistochemical demonstration of free amino acids was first achieved by Storm-Mathisen et al. 1983 . Since that study, many amino acids have been successfully localized in various tissues by many laboratories (Ma et al. 1994 ; Storm-Mathisen and Ottersen 1990 ; Aoki et al. 1987 , Aoki et al. 1988 ). In those studies, free amino acids in the tissue were fixed on tissue proteins by glutaraldehyde contained in the fixative and the antiserum against each amino acid was raised by immunizing animals with amino acid-glutaraldehyde-serum albumin complex. Antibody thus raised was reactive with the respective amino acid conjugated with glutaraldehyde but was not reactive to amino acid contained in the peptide chains of the serum albumin or other proteins. This was also the case in the present study, and although guinea pig serum albumin contains a lot of arginine in its peptide chain (in bovine serum albumin, 23 arginine residues are contained within 585 amino acids), a dot of the serum albumin was not immunostained with our anti-arginine antibody. Consequently, the antibody against arginine was considered to be reactive with a special form of arginine that is free in vivo and is immobilized by glutaraldehyde on proteins.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Aoki E, Semba R, Kato K, Kashiwamata S (1987) Purification of specific antibody against aspartate and immunocytochemical localization of aspartergic neurons in the rat brain. Neuroscience 21:755-765[Medline]

Aoki E, Semba R, Keino H, Kato K, Kashiwamata S (1988) Glycine-like immunoreactivity in the rat auditory pathway. Brain Res 442:63-71[Medline]

Aoki E, Semba R, Mikoshiba K, Kashiwamata S (1991) Predominant localization in glial cells of free L-arginine. Immunocytochemical evidence. Brain Res 547:190-192[Medline]

Bachmann S, Mundel P (1994) Physiology and cell biology update. Nitric oxide in the kidney: synthesis, localization, and function. Am J Kidney Dis 24:112-129[Medline]

Blazer-Yost B, Reynold R, Segal S (1979) Amino acid content of rat renal cortex and the response to in vitro incubation. Am J Physiol 236:F398-F404[Medline]

Dantzler WH, Silbernagl S (1988) Amino acid transport by juxtamedullary nephrons: distal reabsorption and recycling. Am J Physiol 255:F397-F407[Abstract/Free Full Text]

Dhanakoti SN, Brosnan JT, Herzberg GR, Brosman ME (1992) Cellular and subcellular localization of enzymes of arginine metabolism in rat kidney. Biochem J 282:369-375[Medline]

Dhanakoti SN, Brosnan JT, Herzberg GR, Brosman ME (1990) Renal arginine synthesis: studies in vitro and in vivo. Am J Physiol 259:E437-E442[Abstract/Free Full Text]

Fawcett DW (1994) The urinary system. In Bloom DW, Fawcett W, eds. A Textbook of Histology. 12th ed. New York, Chapman & Hall, 728-767

Featherson WR, Rogers R, Freedland RA (1973) Relative importance of kidney and liver in synthesis of arginine by the rat. Am J Physiol 224:127-129[Medline]

Hawkes R, Niday E, Gordon J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119:142-147[Medline]

Levillain O, Hus-Citharel A, Morel F, Bankir L (1993) Arginine synthesis in mouse and rabbit nephron: localization and functional significance. Am J Physiol 264:F1038-F1045[Abstract/Free Full Text]

Levillain O, Hus-Citharel A, Morel F, Bankir L (1990) Localization of arginine synthesis along rat nephron. Am J Physiol 259:F916-F923[Abstract/Free Full Text]

Ma N, Aoki E, Semba R (1994) An immunohistochemical study of aspartate, glutamate, and taurine in rat kidney. J Histochem Cytochem 42:621-626[Abstract/Free Full Text]

Markewitz BA, Michael JR, Kohan DE (1993) Cytokine-induced expression of a nitric oxide synthase in rat renal tubule cells. J Clin Invest 91:2138-2143[Medline]

Morris SM JR, Sweeney WE JR, Kepka DM, O'Brien WE, Avner ED (1991) Localization of arginine biosynthetic enzymes in renal proximal tubules and abundance of mRNA during development. Pediatr Res 29:151-154[Medline]

Párducz Á Dobo E, Wolff JR, Petrusz P, Erdö SL (1992) GABA- immunoreactive structures in rat kidney. J Histochem Cytochem 40:675-680[Abstract/Free Full Text]

Schmechel DE, Brightman MW, Marangos PJ (1980) Neurons switch from non-neuronal enolase to neuron-specific enolase during differentiation. Brain Res 190:195-214[Medline]

Sternberger LA (1974) The unlabeled antibody enzyme method. In Sternberger LA, ed. Immunocytochemistry. Englewood Cliffs, NJ, Prentice-Hall, 129-171

Stoos BA, Carretero OA, Farhy RD, Scicli G, Garvin JL (1992) Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells. J Clin Invest 89:761-765[Medline]

Storm-Mathisen J, Leknes AK, Bore AT, Vaaland JL, Edminson P, Haug FMS, Ottersen OP (1983) First visualization of glutamate and GABA in neurons by immunocytochemistry. Nature 301:517-520[Medline]

Storm-Mathisen J, Ottersen OP (1990) Antibodies and fixatives for the immunocytochemical localization of glycine. In Ottersen OP, Storm-Mathisen J, eds. Glycine Neurotransmission. Chichester, John Wiley & Sons, 281-301

Terada Y, Tomita K, Nonoguchi H, Marumo F (1992) Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segments. J Clin Invest 90:659-665[Medline]

Trachtman H, Barbour R, Sturman JA, Finberg L (1988) Taurine and osmoregulation: taurine is a cerebral osmoprotective molecule in chronic hypernatremic dehydration. Pediatr Res 23:35-39[Abstract]

Trachtman H, Lu P, Sturman JA (1993) Immunohistochemical localization of taurine in rat renal tissue: studies in experimental disease states. J Histochem Cytochem 41:1209-1216[Abstract/Free Full Text]