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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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RhBG is not detectable by immunoperoxidase stain on human renal cortex, renal medulla, or liver sections but is detectable on immunoperoxidase-stained rat kidney. Human renal cortex and medulla sections were stained with αRhBG-CT (A and B) and αRhBG-CT1 (C and D) at a high concentration (1:50). There is no reactivity to RhBG using αRhBG-CT in any region of the cortex (A) or medulla (B) and only a faint cytoplasmic staining in thick ascending limbs using αRhBG-CT1 (C and D). Preincubation of αRhBG-CT1 with immunizing peptide did remove this antibody reactivity (E). Rat renal cortex immunostained with αRhBG-CT showed basolateral RhBG staining in the distal tubules (F). Human liver sections were first stained with αRhCG-CT1 antibody (G). A few scattered hepatocytes show strong membrane staining (G), which is removed following preincubation of αRhCG-CT1 antibody with immunizing peptide (H). Liver immunostained with αRhBG-CT1 showed no reactivity in any cell type (I).
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f7: RhBG is not detectable by immunoperoxidase stain on human renal cortex, renal medulla, or liver sections but is detectable on immunoperoxidase-stained rat kidney. Human renal cortex and medulla sections were stained with αRhBG-CT (A and B) and αRhBG-CT1 (C and D) at a high concentration (1:50). There is no reactivity to RhBG using αRhBG-CT in any region of the cortex (A) or medulla (B) and only a faint cytoplasmic staining in thick ascending limbs using αRhBG-CT1 (C and D). Preincubation of αRhBG-CT1 with immunizing peptide did remove this antibody reactivity (E). Rat renal cortex immunostained with αRhBG-CT showed basolateral RhBG staining in the distal tubules (F). Human liver sections were first stained with αRhCG-CT1 antibody (G). A few scattered hepatocytes show strong membrane staining (G), which is removed following preincubation of αRhCG-CT1 antibody with immunizing peptide (H). Liver immunostained with αRhBG-CT1 showed no reactivity in any cell type (I).

Mentions: To confirm the result observed by Western blot analysis, the anti-RhBG antibodies were also used for immunoperoxidase staining and immunofluorescence experiments on human kidney sections. In previous studies in mice and rats, RhBG has been shown to be localized in cells of the DCT, CNT, and CD and to costain cells expressing RhCG or kAE1 (39). Importantly, immunoperoxidase staining for RhBG under a range of antibody dilutions and antigen-retrieval techniques showed no detectable RhBG in the human kidney or liver. αRhBG-CT1 at high concentrations, especially at 1:50, gave a slight cytoplasmic stain in the cells of the TAL that was competed by peptide (Fig. 7, C and D), but no membrane staining was detected, and there was no reactivity at dilutions comparable to those at which the RhCG antibodies reacted. Even at such high antibody concentrations, αRhBG-CT revealed no detectable reactivity (Fig. 7, A and B). Since both the RhBG COOH-terminal antibodies used here detected a protein in rat tissue lysate (Fig. 6), we also assessed the ability of the antibodies to stain rat kidney sections. Visualization of the stained rat sections revealed strong plasma membrane reactivity of the RhBG-CT antibodies in the DCT, CNT, and CD (Fig. 7F), a result consistent with previously published RhBG localization in animal studies (30, 33, 39).


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)

RhBG is not detectable by immunoperoxidase stain on human renal cortex, renal medulla, or liver sections but is detectable on immunoperoxidase-stained rat kidney. Human renal cortex and medulla sections were stained with αRhBG-CT (A and B) and αRhBG-CT1 (C and D) at a high concentration (1:50). There is no reactivity to RhBG using αRhBG-CT in any region of the cortex (A) or medulla (B) and only a faint cytoplasmic staining in thick ascending limbs using αRhBG-CT1 (C and D). Preincubation of αRhBG-CT1 with immunizing peptide did remove this antibody reactivity (E). Rat renal cortex immunostained with αRhBG-CT showed basolateral RhBG staining in the distal tubules (F). Human liver sections were first stained with αRhCG-CT1 antibody (G). A few scattered hepatocytes show strong membrane staining (G), which is removed following preincubation of αRhCG-CT1 antibody with immunizing peptide (H). Liver immunostained with αRhBG-CT1 showed no reactivity in any cell type (I).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2692438&req=5

f7: RhBG is not detectable by immunoperoxidase stain on human renal cortex, renal medulla, or liver sections but is detectable on immunoperoxidase-stained rat kidney. Human renal cortex and medulla sections were stained with αRhBG-CT (A and B) and αRhBG-CT1 (C and D) at a high concentration (1:50). There is no reactivity to RhBG using αRhBG-CT in any region of the cortex (A) or medulla (B) and only a faint cytoplasmic staining in thick ascending limbs using αRhBG-CT1 (C and D). Preincubation of αRhBG-CT1 with immunizing peptide did remove this antibody reactivity (E). Rat renal cortex immunostained with αRhBG-CT showed basolateral RhBG staining in the distal tubules (F). Human liver sections were first stained with αRhCG-CT1 antibody (G). A few scattered hepatocytes show strong membrane staining (G), which is removed following preincubation of αRhCG-CT1 antibody with immunizing peptide (H). Liver immunostained with αRhBG-CT1 showed no reactivity in any cell type (I).
Mentions: To confirm the result observed by Western blot analysis, the anti-RhBG antibodies were also used for immunoperoxidase staining and immunofluorescence experiments on human kidney sections. In previous studies in mice and rats, RhBG has been shown to be localized in cells of the DCT, CNT, and CD and to costain cells expressing RhCG or kAE1 (39). Importantly, immunoperoxidase staining for RhBG under a range of antibody dilutions and antigen-retrieval techniques showed no detectable RhBG in the human kidney or liver. αRhBG-CT1 at high concentrations, especially at 1:50, gave a slight cytoplasmic stain in the cells of the TAL that was competed by peptide (Fig. 7, C and D), but no membrane staining was detected, and there was no reactivity at dilutions comparable to those at which the RhCG antibodies reacted. Even at such high antibody concentrations, αRhBG-CT revealed no detectable reactivity (Fig. 7, A and B). Since both the RhBG COOH-terminal antibodies used here detected a protein in rat tissue lysate (Fig. 6), we also assessed the ability of the antibodies to stain rat kidney sections. Visualization of the stained rat sections revealed strong plasma membrane reactivity of the RhBG-CT antibodies in the DCT, CNT, and CD (Fig. 7F), a result consistent with previously published RhBG localization in animal studies (30, 33, 39).

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.

Show MeSH
Related in: MedlinePlus