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RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.

Brown AC, Hallouane D, Mawby WJ, Karet FE, Saleem MA, Howie AJ, Toye AM - Am. J. Physiol. Renal Physiol. (2009)

Bottom Line: A proportion of RhCG staining labeled kAE1-positive cells, confirming that RhCG is localized to the alpha-IC cells.The same antibodies, however, could detect RhBG in rat tissue.We conclude that under normal conditions, RhCG is the major putative ammonia transporter expressed in the human kidney and RhBG is not expressed at detectable levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medical Sciences, Univ. Walk, Bristol BS8 1TD, UK.

ABSTRACT
Rhesus glycoprotein homologs RhAG, RhBG, and RhCG comprise a recently identified branch of the Mep/Amt ammonia transporter family. Animal studies have shown that RhBG and RhCG are present in the kidney distal tubules. Studies in mouse and rat tissue suggest a basolateral localization for RhBG in cells of the distal tubules including the alpha-intercalated cells (alpha-IC), but no localization of RhBG has been reported in human tissue. To date RhCG localization has been described as exclusively apical plasma membrane in mouse and rat kidney, or apical and basolateral in humans, and some mouse and rat tissue studies. We raised novel antibodies to RhBG and RhCG to examine their localization in the human kidney. Madin-Darby canine kidney (MDCKI) cell lines stably expressing human green fluorescent protein-tagged RhBG or RhCG and human tissue lysates were used to demonstrate the specificity of these antibodies for detecting RhBG and RhCG. Using immunoperoxidase staining and antigen liberation techniques, both apical and basolateral RhCG localization was observed in the majority of the cells of the distal convoluted tubule and IC of the connecting tubule and collecting duct. Confocal microscopic imaging of normal human kidney cryosections showed that RhCG staining was predominantly localized to the apical membrane in these cells with some basolateral and intracellular staining evident. A proportion of RhCG staining labeled kAE1-positive cells, confirming that RhCG is localized to the alpha-IC cells. Surprisingly, no RhBG protein was detectable in the human kidney by Western blot analysis of tissue lysates, or by immunohistochemistry or confocal microscopy of tissue sections. The same antibodies, however, could detect RhBG in rat tissue. We conclude that under normal conditions, RhCG is the major putative ammonia transporter expressed in the human kidney and RhBG is not expressed at detectable levels.

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Immunoperoxidase staining of a human kidney with rabbit αRhCG-CT1 antibody and various markers of parts of the tubule. A: control section of cortex stained by αRhCG-CT1 preincubated with the immunizing peptide. B: low-magnification view of the cortex (c) and outer medulla (m). Glomeruli and proximal tubules are not stained. Only some parts of tubules are detected, and the staining appears mostly continuous in the cortex and intermittent in the medulla. The arrows indicate parts of tubules that show staining for RhCG-CT1 but not for uromodulin in an adjacent section (C) or vice versa. RhCG is therefore not expressed in the thick limb of the loop of Henle. D: high-magnification image of the cortex to show that in some tubules every cell expresses RhCG, and the arrows indicate that the apical membrane is most strongly stained, although there is also staining of the basolateral membrane and to a lesser extent the cytoplasm. E: high-magnification image of the inner medulla to show that only some cells in collecting ducts (cd) are stained, mainly on the basolateral membrane (arrowed). In F there is a group of tubules expressing RhCG, and G shows that the same tubules in an adjacent section express the sodium-chloride cotransporter on their apical membrane (arrows). This confirms expression of RhCG in the distal convoluted tubule. In H, RhCG-CT1 immunostaining is seen in cortical tubules not only continuously as in C and F, but also discontinuously (arrows). Adjacent sections show that the discontinuous part expresses the amiloride-sensitive epithelial sodium channel (I) and that both parts express 11β-hydroxysteroid dehydrogenase type 2 (J). This shows that the continuous staining of the distal convoluted tubule changes to staining only of intercalated cells in the connecting tubule. In K, a connecting tubule has RhCG-CT1 staining only in intercalated cells, while in an adjacent section principal cells express aquaporin-2 on their apical membrane (arrows; L).
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f5: Immunoperoxidase staining of a human kidney with rabbit αRhCG-CT1 antibody and various markers of parts of the tubule. A: control section of cortex stained by αRhCG-CT1 preincubated with the immunizing peptide. B: low-magnification view of the cortex (c) and outer medulla (m). Glomeruli and proximal tubules are not stained. Only some parts of tubules are detected, and the staining appears mostly continuous in the cortex and intermittent in the medulla. The arrows indicate parts of tubules that show staining for RhCG-CT1 but not for uromodulin in an adjacent section (C) or vice versa. RhCG is therefore not expressed in the thick limb of the loop of Henle. D: high-magnification image of the cortex to show that in some tubules every cell expresses RhCG, and the arrows indicate that the apical membrane is most strongly stained, although there is also staining of the basolateral membrane and to a lesser extent the cytoplasm. E: high-magnification image of the inner medulla to show that only some cells in collecting ducts (cd) are stained, mainly on the basolateral membrane (arrowed). In F there is a group of tubules expressing RhCG, and G shows that the same tubules in an adjacent section express the sodium-chloride cotransporter on their apical membrane (arrows). This confirms expression of RhCG in the distal convoluted tubule. In H, RhCG-CT1 immunostaining is seen in cortical tubules not only continuously as in C and F, but also discontinuously (arrows). Adjacent sections show that the discontinuous part expresses the amiloride-sensitive epithelial sodium channel (I) and that both parts express 11β-hydroxysteroid dehydrogenase type 2 (J). This shows that the continuous staining of the distal convoluted tubule changes to staining only of intercalated cells in the connecting tubule. In K, a connecting tubule has RhCG-CT1 staining only in intercalated cells, while in an adjacent section principal cells express aquaporin-2 on their apical membrane (arrows; L).

Mentions: To investigate the localization of RhCG in the human kidney, we conducted immunoperoxidase staining using both αRhCG-CT1 and αRhCG-CT2 antibodies on paraffin-embedded human kidneys. The distribution of staining using either RhCG-CT1 or RhCG-CT2 antibodies was similar and is shown in Fig. 5 for αRhCG-CT1, with comparisons to markers of identified parts of the nephron. The immunoreactivity of each antibody was removed by preincubation with their respective immunizing peptide (Fig. 5A).


RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.

Brown AC, Hallouane D, Mawby WJ, Karet FE, Saleem MA, Howie AJ, Toye AM - Am. J. Physiol. Renal Physiol. (2009)

Immunoperoxidase staining of a human kidney with rabbit αRhCG-CT1 antibody and various markers of parts of the tubule. A: control section of cortex stained by αRhCG-CT1 preincubated with the immunizing peptide. B: low-magnification view of the cortex (c) and outer medulla (m). Glomeruli and proximal tubules are not stained. Only some parts of tubules are detected, and the staining appears mostly continuous in the cortex and intermittent in the medulla. The arrows indicate parts of tubules that show staining for RhCG-CT1 but not for uromodulin in an adjacent section (C) or vice versa. RhCG is therefore not expressed in the thick limb of the loop of Henle. D: high-magnification image of the cortex to show that in some tubules every cell expresses RhCG, and the arrows indicate that the apical membrane is most strongly stained, although there is also staining of the basolateral membrane and to a lesser extent the cytoplasm. E: high-magnification image of the inner medulla to show that only some cells in collecting ducts (cd) are stained, mainly on the basolateral membrane (arrowed). In F there is a group of tubules expressing RhCG, and G shows that the same tubules in an adjacent section express the sodium-chloride cotransporter on their apical membrane (arrows). This confirms expression of RhCG in the distal convoluted tubule. In H, RhCG-CT1 immunostaining is seen in cortical tubules not only continuously as in C and F, but also discontinuously (arrows). Adjacent sections show that the discontinuous part expresses the amiloride-sensitive epithelial sodium channel (I) and that both parts express 11β-hydroxysteroid dehydrogenase type 2 (J). This shows that the continuous staining of the distal convoluted tubule changes to staining only of intercalated cells in the connecting tubule. In K, a connecting tubule has RhCG-CT1 staining only in intercalated cells, while in an adjacent section principal cells express aquaporin-2 on their apical membrane (arrows; L).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: Immunoperoxidase staining of a human kidney with rabbit αRhCG-CT1 antibody and various markers of parts of the tubule. A: control section of cortex stained by αRhCG-CT1 preincubated with the immunizing peptide. B: low-magnification view of the cortex (c) and outer medulla (m). Glomeruli and proximal tubules are not stained. Only some parts of tubules are detected, and the staining appears mostly continuous in the cortex and intermittent in the medulla. The arrows indicate parts of tubules that show staining for RhCG-CT1 but not for uromodulin in an adjacent section (C) or vice versa. RhCG is therefore not expressed in the thick limb of the loop of Henle. D: high-magnification image of the cortex to show that in some tubules every cell expresses RhCG, and the arrows indicate that the apical membrane is most strongly stained, although there is also staining of the basolateral membrane and to a lesser extent the cytoplasm. E: high-magnification image of the inner medulla to show that only some cells in collecting ducts (cd) are stained, mainly on the basolateral membrane (arrowed). In F there is a group of tubules expressing RhCG, and G shows that the same tubules in an adjacent section express the sodium-chloride cotransporter on their apical membrane (arrows). This confirms expression of RhCG in the distal convoluted tubule. In H, RhCG-CT1 immunostaining is seen in cortical tubules not only continuously as in C and F, but also discontinuously (arrows). Adjacent sections show that the discontinuous part expresses the amiloride-sensitive epithelial sodium channel (I) and that both parts express 11β-hydroxysteroid dehydrogenase type 2 (J). This shows that the continuous staining of the distal convoluted tubule changes to staining only of intercalated cells in the connecting tubule. In K, a connecting tubule has RhCG-CT1 staining only in intercalated cells, while in an adjacent section principal cells express aquaporin-2 on their apical membrane (arrows; L).
Mentions: To investigate the localization of RhCG in the human kidney, we conducted immunoperoxidase staining using both αRhCG-CT1 and αRhCG-CT2 antibodies on paraffin-embedded human kidneys. The distribution of staining using either RhCG-CT1 or RhCG-CT2 antibodies was similar and is shown in Fig. 5 for αRhCG-CT1, with comparisons to markers of identified parts of the nephron. The immunoreactivity of each antibody was removed by preincubation with their respective immunizing peptide (Fig. 5A).

Bottom Line: A proportion of RhCG staining labeled kAE1-positive cells, confirming that RhCG is localized to the alpha-IC cells.The same antibodies, however, could detect RhBG in rat tissue.We conclude that under normal conditions, RhCG is the major putative ammonia transporter expressed in the human kidney and RhBG is not expressed at detectable levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medical Sciences, Univ. Walk, Bristol BS8 1TD, UK.

ABSTRACT
Rhesus glycoprotein homologs RhAG, RhBG, and RhCG comprise a recently identified branch of the Mep/Amt ammonia transporter family. Animal studies have shown that RhBG and RhCG are present in the kidney distal tubules. Studies in mouse and rat tissue suggest a basolateral localization for RhBG in cells of the distal tubules including the alpha-intercalated cells (alpha-IC), but no localization of RhBG has been reported in human tissue. To date RhCG localization has been described as exclusively apical plasma membrane in mouse and rat kidney, or apical and basolateral in humans, and some mouse and rat tissue studies. We raised novel antibodies to RhBG and RhCG to examine their localization in the human kidney. Madin-Darby canine kidney (MDCKI) cell lines stably expressing human green fluorescent protein-tagged RhBG or RhCG and human tissue lysates were used to demonstrate the specificity of these antibodies for detecting RhBG and RhCG. Using immunoperoxidase staining and antigen liberation techniques, both apical and basolateral RhCG localization was observed in the majority of the cells of the distal convoluted tubule and IC of the connecting tubule and collecting duct. Confocal microscopic imaging of normal human kidney cryosections showed that RhCG staining was predominantly localized to the apical membrane in these cells with some basolateral and intracellular staining evident. A proportion of RhCG staining labeled kAE1-positive cells, confirming that RhCG is localized to the alpha-IC cells. Surprisingly, no RhBG protein was detectable in the human kidney by Western blot analysis of tissue lysates, or by immunohistochemistry or confocal microscopy of tissue sections. The same antibodies, however, could detect RhBG in rat tissue. We conclude that under normal conditions, RhCG is the major putative ammonia transporter expressed in the human kidney and RhBG is not expressed at detectable levels.

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