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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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Novel RhBG and RhCG antibodies are specific to GFP-RhBG or GFP-RhCG when analyzed by Western blotting. MDCKI cells stably expressing GFP-tagged RhBG or RhCG were treated with sodium butyrate to increase protein expression, lysed, and immunoblotted with polyclonal antibodies to GFP (A), αRhCG-CT1 (B), αRhCG-CT2 (C), αRhBG-CT1 (D), αRhBG-NT (E), and αRhBG-CT (F). All RhBG and RhCG antibodies bind specifically to the Rh glycoprotein against which they were raised.
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f2: Novel RhBG and RhCG antibodies are specific to GFP-RhBG or GFP-RhCG when analyzed by Western blotting. MDCKI cells stably expressing GFP-tagged RhBG or RhCG were treated with sodium butyrate to increase protein expression, lysed, and immunoblotted with polyclonal antibodies to GFP (A), αRhCG-CT1 (B), αRhCG-CT2 (C), αRhBG-CT1 (D), αRhBG-NT (E), and αRhBG-CT (F). All RhBG and RhCG antibodies bind specifically to the Rh glycoprotein against which they were raised.

Mentions: We confirmed the specificity of these antibodies to human RhCG and RhBG proteins by Western blotting experiments using lysates from MDCKI GFP-RhCG and GFP-RhBG cell lines (Fig. 2). Figure 2A shows using an anti-GFP antibody that GFP-RhCG and GFP-RhBG were expressed at the correct size (∼80 kDa) in MDCKI cells. GFP-RhBG cells also exhibited a smaller band, which is likely to be a differently glycosylated form of the protein. Figure 2, B and C, demonstrates that the RhCG antibodies bound specifically to GFP-RhCG with no cross-reactivity with GFP-RhBG. For RhBG, all antibodies, αRhBG-CT1, αRhBG-CT, and αRhBG-NT, specifically detected the GFP-RhBG protein and did not cross-react with RhCG (Fig. 2, D–F). This validation of the antibodies raised to RhCG and RhBG is important as it demonstrates that all antibodies used in these studies detect the correct protein and are specific to either human RhCG or RhBG.


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)

Novel RhBG and RhCG antibodies are specific to GFP-RhBG or GFP-RhCG when analyzed by Western blotting. MDCKI cells stably expressing GFP-tagged RhBG or RhCG were treated with sodium butyrate to increase protein expression, lysed, and immunoblotted with polyclonal antibodies to GFP (A), αRhCG-CT1 (B), αRhCG-CT2 (C), αRhBG-CT1 (D), αRhBG-NT (E), and αRhBG-CT (F). All RhBG and RhCG antibodies bind specifically to the Rh glycoprotein against which they were raised.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Novel RhBG and RhCG antibodies are specific to GFP-RhBG or GFP-RhCG when analyzed by Western blotting. MDCKI cells stably expressing GFP-tagged RhBG or RhCG were treated with sodium butyrate to increase protein expression, lysed, and immunoblotted with polyclonal antibodies to GFP (A), αRhCG-CT1 (B), αRhCG-CT2 (C), αRhBG-CT1 (D), αRhBG-NT (E), and αRhBG-CT (F). All RhBG and RhCG antibodies bind specifically to the Rh glycoprotein against which they were raised.
Mentions: We confirmed the specificity of these antibodies to human RhCG and RhBG proteins by Western blotting experiments using lysates from MDCKI GFP-RhCG and GFP-RhBG cell lines (Fig. 2). Figure 2A shows using an anti-GFP antibody that GFP-RhCG and GFP-RhBG were expressed at the correct size (∼80 kDa) in MDCKI cells. GFP-RhBG cells also exhibited a smaller band, which is likely to be a differently glycosylated form of the protein. Figure 2, B and C, demonstrates that the RhCG antibodies bound specifically to GFP-RhCG with no cross-reactivity with GFP-RhBG. For RhBG, all antibodies, αRhBG-CT1, αRhBG-CT, and αRhBG-NT, specifically detected the GFP-RhBG protein and did not cross-react with RhCG (Fig. 2, D–F). This validation of the antibodies raised to RhCG and RhBG is important as it demonstrates that all antibodies used in these studies detect the correct protein and are specific to either human RhCG or RhBG.

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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