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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 detectable on Western blot of rat kidney lysate but not of healthy human kidney lysate. Fifty micrograms of each lysate were prepared as in Fig. 4. Two COOH-terminal antibodies, αRhBG-CT (A) and affinity-purified αRhBG-CT1 (B) bind a ∼54-kDa polypeptide in rat cortex tissue that can be specifically competed by preincubation of the antibodies with 1 mg/ml of immunizing peptide. Neither antibody detects an equivalent polypeptide in human tissue that can be specifically competed. The NH2-terminal RhBG antibody, αRhBG-NT, binds nonspecifically in both rat and human tissue with equivalent background bands detected in the presence or absence of immunizing peptide (C).
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f6: RhBG is detectable on Western blot of rat kidney lysate but not of healthy human kidney lysate. Fifty micrograms of each lysate were prepared as in Fig. 4. Two COOH-terminal antibodies, αRhBG-CT (A) and affinity-purified αRhBG-CT1 (B) bind a ∼54-kDa polypeptide in rat cortex tissue that can be specifically competed by preincubation of the antibodies with 1 mg/ml of immunizing peptide. Neither antibody detects an equivalent polypeptide in human tissue that can be specifically competed. The NH2-terminal RhBG antibody, αRhBG-NT, binds nonspecifically in both rat and human tissue with equivalent background bands detected in the presence or absence of immunizing peptide (C).

Mentions: We tested the RhBG antibodies (αRhBG-NT, αRhBG-CT, and αRhBG-CT1) to assess the protein levels of RhBG in human renal cortex lysate, whole kidney lysates, and rat renal cortex lysates by Western blot analysis. We found no detectable RhBG protein that could be competed by preincubation with immunizing peptide, despite using three independent human kidney tissue samples. We successfully detected RhCG, kAE1, and β-actin in the same lysates by Western blotting, suggesting that this lack of detection is not due to protein degradation (see Fig. 4). In comparison, both COOH-terminal antibodies (αRhBG-CT and αRhBG-CT1) detected a clear band in rat tissue of the correct molecular weight for RhBG that could be competed by preincubation of the antibody with specific immunizing peptide (Fig. 6, A and B). For αRhBG-CT, this result supports previously published work on rat lysate utilizing this antibody, and for αRhBG-CT1, this is consistent with a 70% sequence homology between the human and rat RhBG sequence in the region to which the antibody was raised (30). For the NH2-terminal antibody αRhBG-NT, no specific binding was observed in rat lysate, a result that is consistent with a complete lack of homology between rat and human RhBG in the NH2-terminal region (Fig. 6C).


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 detectable on Western blot of rat kidney lysate but not of healthy human kidney lysate. Fifty micrograms of each lysate were prepared as in Fig. 4. Two COOH-terminal antibodies, αRhBG-CT (A) and affinity-purified αRhBG-CT1 (B) bind a ∼54-kDa polypeptide in rat cortex tissue that can be specifically competed by preincubation of the antibodies with 1 mg/ml of immunizing peptide. Neither antibody detects an equivalent polypeptide in human tissue that can be specifically competed. The NH2-terminal RhBG antibody, αRhBG-NT, binds nonspecifically in both rat and human tissue with equivalent background bands detected in the presence or absence of immunizing peptide (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: RhBG is detectable on Western blot of rat kidney lysate but not of healthy human kidney lysate. Fifty micrograms of each lysate were prepared as in Fig. 4. Two COOH-terminal antibodies, αRhBG-CT (A) and affinity-purified αRhBG-CT1 (B) bind a ∼54-kDa polypeptide in rat cortex tissue that can be specifically competed by preincubation of the antibodies with 1 mg/ml of immunizing peptide. Neither antibody detects an equivalent polypeptide in human tissue that can be specifically competed. The NH2-terminal RhBG antibody, αRhBG-NT, binds nonspecifically in both rat and human tissue with equivalent background bands detected in the presence or absence of immunizing peptide (C).
Mentions: We tested the RhBG antibodies (αRhBG-NT, αRhBG-CT, and αRhBG-CT1) to assess the protein levels of RhBG in human renal cortex lysate, whole kidney lysates, and rat renal cortex lysates by Western blot analysis. We found no detectable RhBG protein that could be competed by preincubation with immunizing peptide, despite using three independent human kidney tissue samples. We successfully detected RhCG, kAE1, and β-actin in the same lysates by Western blotting, suggesting that this lack of detection is not due to protein degradation (see Fig. 4). In comparison, both COOH-terminal antibodies (αRhBG-CT and αRhBG-CT1) detected a clear band in rat tissue of the correct molecular weight for RhBG that could be competed by preincubation of the antibody with specific immunizing peptide (Fig. 6, A and B). For αRhBG-CT, this result supports previously published work on rat lysate utilizing this antibody, and for αRhBG-CT1, this is consistent with a 70% sequence homology between the human and rat RhBG sequence in the region to which the antibody was raised (30). For the NH2-terminal antibody αRhBG-NT, no specific binding was observed in rat lysate, a result that is consistent with a complete lack of homology between rat and human RhBG in the NH2-terminal region (Fig. 6C).

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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Related in: MedlinePlus