Localization of the ammonium transporter proteins RhBG and RhCG in mouse kidney

Jill W. Verlander1, R. Tyler Miller2, Amy E. Frank1, Ines E. Royaux3, Young-Hee Kim1, and I. David Weiner1,4

1 University of Florida College of Medicine, and 4 North Florida/South Georgia Veterans Health System, Gainesville, Florida 32610; 2 Case Western Reserve University and Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106; and 3 Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ammonia is both produced and transported by renal epithelial cells, and it regulates renal ion transport. Recent studies have identified a family of putative ammonium transporters; mRNA for two members of this family, Rh B-glycoprotein (RhBG) and Rh C-glycoprotein (RhCG), is expressed in the kidney. The purpose of this study was to determine the cellular location of RhBG and RhCG protein in the mouse kidney. We generated RhBG- and RhCG-specific anti-peptide antibodies. Immunoblot analysis confirmed that both proteins were expressed in the mouse kidney. RhBG localization with immunohistochemistry revealed discrete basolateral labeling in the connecting segment (CNT) and in the majority of initial collecting tubule (ICT) and cortical collecting duct (CCD) cells. In the outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD) only a subpopulation of cells exhibited basolateral immunoreactivity. Colocalization of RhBG with carbonic anhydrase II, the thiazide-sensitive transporter, and the anion exchangers AE1 and pendrin demonstrated RhBG immunoreactivity in all CNT cells and all CCD and ICT principal cells. In the ICT and CCD, basolateral RhBG immunoreactivity is also present in A-type intercalated cells but not in pendrin-positive CCD intercalated cells. In the OMCD and IMCD, only intercalated cells exhibit RhBG immunoreactivity. Immunoreactivity for a second putative ammonium transporter, RhCG, was present in the apical region of cells with almost the same distribution as RhBG. However, RhCG immunoreactivity was present in all CCD cells, and it was present in outer stripe OMCD principal cells, in addition to OMCD and IMCD intercalated cells. Thus the majority of RhBG and RhCG protein expression is present in the same epithelial cell types in the CNT and collecting duct but with opposite polarity. These findings suggest that RhBG and RhCG may play important and cell-specific roles in ammonium transport and signaling in these regions of the kidney.

Rh B-glycoprotein; Rh C-glycoprotein; collecting duct; intercalated cell; principal cell; immunohistochemistry; connecting segment


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AMMONIUM TRANSPORT AND SIGNALING are important for the renal regulation of electrolyte homeostasis.1 Briefly, ammonium is produced in the proximal tubule, preferentially secreted into the luminal fluid, reabsorbed via specific membrane proteins in the medullary thick ascending limb of the loop of Henle, and then secreted into the luminal fluid by the collecting duct (4, 18). Production and excretion of ammonium result in production of equimolar amounts of new bicarbonate molecules, which is important for replenishing base consumed by endogenous and exogenous acid production (4, 18). In addition to being a metabolic product and transported molecule, ammonia also regulates collecting duct sodium, potassium, and acid-base transport (6, 7, 9, 11, 40). Accordingly, understanding ammonium transport mechanisms and the mechanisms through which ammonia might alter collecting duct ion transport are important.

A novel ammonium (NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) transporter family of proteins was recently identified in yeast and plants (29, 32) and is present in species throughout nature, including mammals (13, 14, 27). These proteins are glycosylated, integral membrane proteins that possess 12 predicted membrane-spanning segments. When expressed in heterologous expression systems, they transport ammonium and its analog, methylammonium (13, 27). They can also function, at least in yeast, as ammonia sensors, altering cellular function in response to changes in extracellular ammonia (24, 25).

Three mammalian members of this family, Rh A-glycoprotein (RhAG) (26), Rh B-glycoprotein (RhBG) (23), and Rh C-glycoprotein (RhCG) (22, 26), have been identified. They exhibit substantial homology to each other and to the erythrocyte Rh factor, a protein well recognized as an antigen in transfusion medicine but whose function in erythrocyte membranes is not known (12, 14). RhAG protein is expressed in the erythrocyte membrane, where it may contribute to erythrocyte ammonium transport (21). RhBG mRNA is expressed in liver, kidney, and skin (23), and RhCG mRNA is expressed in kidney, testes, and brain (22). Thus both RhBG and RhCG mRNA are expressed in organs where ammonium transport is a critical function.

The purpose of the present studies was to determine whether RhBG and RhCG proteins are expressed in the mammalian kidney and, if so, to identify their specific cellular locations by using light microscopic immunolocalization techniques. Anti-peptide antibodies to cytoplasmic regions of the COOH terminus of mouse RhBG and mouse RhCG were generated and shown to be specific to these proteins. These antibodies were then used to detect the specific cellular locations of RhBG and RhCG in the normal mouse kidney.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies

Synthetic peptides corresponding to a hydrophilic cytoplasmic region near the COOH terminus of RhBG and RhCG were synthesized, purified, and coupled to keyhole limpit hemocyanin through a cysteine link by using standard techniques (Interdisciplinary Center for Biotechnology Research, University of Florida College of Medicine, Gainesville, FL). The sequences used were TETQRPLRGGESDTRA for RhBG and EEVNTVYIPEDLAHK for RhCG. Rabbit polyclonal antibodies were then generated by using standard techniques (Cocalico Biologicals, Reamstown, PA). A mouse monoclonal anti-peptide antibody directed against RhBG was generated by using the same peptide sequence and standard techniques (Interdisciplinary Center for Biotechnology Research).

For immunohistochemical localization of carbonic anhydrase II (CA II), we used a polyclonal antibody raised in rabbit against mouse erythrocyte CA II. This antibody has been used in previous studies to identify mouse intercalated cells (36). These antibodies were a gift from Dr. Paul Linser (University of Florida Department of Anatomy and Cell Biology and Whitney Marine Laboratory, Gainesville, FL), have been characterized previously (16, 20), and have been used previously to identify mouse intercalated cells (36). Separate rabbit anti-pendrin antibodies were kindly supplied by Dr. Søren Nielsen (University of Aarhus, Aarhus, Denmark) and by Dr. Ines Royaux (National Institutes of Health, Bethesda, MD) and were used at a dilution of 1:1,000 unless otherwise detailed. Both have been characterized in detail previously (17, 34, 41). Rabbit anti-thiazide-sensitive transporter (TSC) antibodies were supplied by Dr. Stephen Hebert (Yale University), have been characterized previously (33), and were used at a dilution of 1:1,000. Antibodies against AE1 were kindly provided by Dr. Philip S. Low (Purdue University, West Lafayette, IN), have been characterized previously (36, 37), and were used at a dilution of 1:400.

Membrane Protein Preparation

Normal BALB/c mice were obtained from Harlan Sprague Dawley (Indianapolis, IN) and were maintained on a normal mouse diet and ad libitum water intake until the day of study. The animals were anesthetized intraperitoneally with pentobarbital sodium (50 mg/kg body wt), and the kidneys were rinsed by in vivo cardiac perfusion with PBS (pH 7.4), rapidly removed, and stored frozen at -70°C until used. Tissues were then homogenized with a Tissue Tearor homogenizer (BioSpec Products) and then diluted in buffer B (250 mM sucrose, 10 mM Tris buffer, and 1 mM EDTA, pH 7.4) containing PMSF. The sample was then centrifuged at 1,000 g for 10 min at 4°C. The supernatant was removed and centrifuged at 10,000 g for 20 min at 4°C. The supernatant was decanted, and the 10,000 g centrifugation was repeated twice more. The collected supernatants were then centrifuged at 100,000 g for 1 h at 4°C. The pellet was resuspended in 500 µl buffer B and gently homogenized in a Dounce homogenizer. An aliquot was obtained for protein determination by using a Lowry assay, and the remainder was stored frozen at -70°C until used.

Immunoblotting Procedure

Five micrograms of the renal membrane protein were electrophoresed on 10% PAGE ReadyGel (Bio-Rad, Hercules, CA). Gels were then transferred electrophoretically to nitrocellulose membranes, blocked with 5 g/dl nonfat dry milk, and incubated for 1 h with primary antibody diluted 1:1,000 in Blotto buffer (50 mM Tris, 150 mM NaCl, 5 mM Na2EDTA, and 0.05% Tween-20, pH 7.5) with 5 g/dl nonfat dry milk. After washing, membranes were exposed to secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase; Promega, Madison, WI) at a dilution of 1:5,000. Sites of antibody-antigen reaction were visualized by using enhanced chemiluminescence (SuperSignal West Pico Substrate, Pierce, Rockford, IL) and a Kodak Image Station 440CF digital imaging system.

Tissue Preparation for Immunohistochemical Localization of RhBG and RhCG

Female BALB/c mice weighing 17-20 g (n = 6) were anesthetized with pentobarbital sodium (10-30 mg/kg ip). The kidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4) followed by periodate-lysine-2% paraformaldehyde (PLP) (30) and then cut transversely into several 2- to 4-mm-thick slices and immersed overnight at 4°C in the same fixative. Samples of kidney from each animal were embedded in polyester wax (polyethylene glycol 400 distearate, Polysciences, Warrington, PA), and 5-µm-thick sections were cut and mounted on gelatin-coated glass slides.

Immunohistochemistry

Immunolocalization of RhBG and RhCG was accomplished by using immunoperoxidase procedures and a commercially available kit (Vectastain Elite, Vector Laboratories, Burlingame, CA). The sections were dewaxed in ethanol and rehydrated, rinsed in PBS, treated for 15 min with 5% normal goat serum (Vector Laboratories) in PBS, and then incubated at 4°C overnight with either the anti-RhBG or anti-RhCG antibody, diluted 1:10,000 in PBS. The sections were then washed in PBS, and endogenous peroxidase activity was blocked by incubating the sections in 0.3% H2O2 for 30 min. The sections were washed in PBS and incubated for 30 min with biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories) diluted 1:200 in PBS and again washed with PBS. The sections were treated for 30 min with the avidin-biotin complex reagent, rinsed with PBS, and then exposed to diaminobenzidine. The sections were washed in distilled water, and in some experiments the sections were counterstained with hematoxylin. The sections were then dehydrated with xylene, mounted with Permount (Fisher Scientific, Fair Lawn, NJ), and observed by light microscopy.

In some experiments localizing RhCG, the methods described above were modified by inclusion of an antigen retrieval step. In these experiments, after the slides were dewaxed in ethanol and rehydrated, they were microwaved at medium power in 0.1 M sodium citrate and 0.1 M citric acid, pH 6.0, for 10 min. The slides were then rinsed in PBS before blocking with normal goat serum and incubation with the primary antibody at a 1:8,000 dilution.

Colocalization of RhBG and RhCG with CA II Immunoreactivity

Colocalization was accomplished by using sequential immunoperoxidase procedures and Vectastain Elite. Five-micrometer sections were dewaxed in ethanol, rehydrated, and then rinsed in PBS. Endogenous peroxidase activity was blocked by incubation of the sections in 0.3% H2O2 for 30 min. The sections were rinsed in PBS, treated for 20 min with 5% goat serum in PBS, and then incubated at 4°C overnight with either the anti-RhBG or the anti-RhCG antibody diluted 1:10,000 in PBS. The sections were washed in PBS for 1 min, in 0.1% SDS in PBS for 10 min, and then again in PBS for 1 min. The sections were then incubated for 30 min with the biotinylated goat anti-rabbit IgG secondary antibody diluted 1:200 in PBS and then washed with PBS. The sections were treated for 30 min with the avidin-biotin complex reagent, rinsed with PBS, and then exposed to diaminobenzidine. The sections were washed in glass-distilled water and then in PBS and incubated in 0.3% H2O2 for 30 min. The sections were again washed in PBS and incubated for 20 min with 5% normal serum in PBS. The sections were treated for 60 min with the anti-CA II antibody diluted 1:1,600 or 1:2,000 in PBS, washed in PBS, and incubated with the biotinylated anti-rabbit secondary antibody. The sections were washed with PBS, incubated with the avidin-biotin complex reagent, and washed with PBS. For detection of CA II immunoreactivity, Vector SG (Vector Laboratories) was used as the chromogen to produce a blue label. This label was easily distinguishable from the brown label produced by the diaminobenzidine used for detection of RhBG or RhCG immunoreactivity. The sections were washed with glass-distilled water, dehydrated with xylene, mounted with Permount, and observed by light microscopy.

Colocalization of RhBG with TSC, RhCG, AE1, and Pendrin

Immunofluorescent localization and confocal laser scanning microscopy were used in some studies to colocalize RhBG with RhCG, TSC, AE1, or pendrin. In these studies, we used our mouse monoclonal RhBG antibody; the other primary antibodies were rabbit polyclonal antibodies, thereby facilitating separate identification of their localization by using species-specific secondary antibodies. Briefly, the sections were dewaxed with ethanol, rehydrated, rinsed in PBS, and then treated for 30 min with 50 mM NH4Cl. They were then rinsed in PBS, blocked for 20 min with 5% normal goat serum (Vector Laboratories) in PBS, and incubated at 4°C overnight with primary antibody, or antibodies, diluted in PBS. The sections were then washed in PBS and incubated for 30 min with a fluorescently tagged species-specific secondary antibody [either FITC-labeled goat anti-rabbit IgG, 1:50 dilution (Sigma), or AF488-labeled goat anti-rabbit IgG, 1:100 dilution, and tetramethylrhodamine isothiocyanate-labeled goat anti-mouse IgG, 1:50 dilution (Sigma)] diluted in PBS. The sections were then rinsed with PBS and mounted by using Fluoromount (Southern Biotechnology Associates, Birmingham, AL). We then visualized the tissue by using either an Axiovert 100 M laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) with LSM 510 Software, version 2.8 (Carl Zeiss), or an MRC-1024 laser scanning confocal microscope and LaserSharp software (Bio-Rad Laboratories).

Colocalization of RhBG with Pendrin

We performed double labeling both on wax-embedded mouse kidney and on 1-µm sections embedded in Epon 812, as summarized below.

Double labeling on wax-embedded mouse kidney. Slices of PLP-fixed kidney tissue were dehydrated and embedded in polyester wax, and sections were cut and mounted on gelatin-coated glass slides. The sections were dewaxed with xylene and ethanol, and after rinsing in tap water, sections were treated with methanolic H2O2 for 30 min. Before incubation with the primary antibody, the sections were permeabilized by incubation for 15 min in 0.5% Triton X-100 in PBS, subsequently blocked with normal goat serum, diluted 1:10 in PBS, for 1 h, and then incubated overnight at 4°C with rabbit antiserum against pendrin (kindly provided by Dr. Søren Nielsen) diluted 1:3,000 in PBS. The sections were rinsed in PBS and incubated for 2 h in peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Sections were then incubated with the peroxidase-substrate solution, a mixture of 0.05% 3,3'-diaminobenzidine and 0.01% H2O2, for 5 min at room temperature. After being rinsed with Tris · HCl buffer, the sections were treated with the same protocol for the second antiserum, polyclonal antibodies directed against RhBG, diluted 1:1,000 in PBS, except for the use of a Vector SG kit (Vector Laboratories) as the chromogen to produce a gray-blue label, which is easily distinguished from the brown label produced by 3,3'-diaminobenzidine in the first immunolocalization procedure for pendrin. The sections were rinsed in tap water and examined with light microscopy.

Double labeling on 1-µm Epon 812-embedded sections. Sections of PLP-fixed tissue were cut transversely on a Vibratome at a thickness of 50 µm and processed for immunohistochemistry by using an indirect immunoperoxidase method as described in detail in a previous study (17). All sections were washed with 50 mM NH4Cl in PBS three times for 15 min. Before incubation with the primary antibodies, all tissue sections were incubated for 3 h with PBS containing 1% bovine serum albumin, 0.05% saponin, and 0.2% gelatin (solution A). The tissue sections were then incubated overnight at 4°C with rabbit antiserum against pendrin, 1:3,000 dilution, in PBS plus 1% bovine serum albumin (solution B). After several washes with solution A, the tissue sections were incubated for 2 h in peroxidase-conjugated goat anti-rabbit Fab fragment (Jackson ImmunoResearch Laboratories), diluted 1:100 in solution B. The tissues were then rinsed, first in solution A and subsequently in 0.05 M Tris (hydroxymethyl) aminomethane (Tris) buffer, pH 7.6. For the detection of horseradish peroxidase, the sections were incubated in 0.1% 3,3'-diaminobenzidine in 0.05 M Tris buffer for 5 min, after which H2O2 was added to a final concentration of 0.01% and the incubation was continued for 10 min. After washing with 0.05 M Tris buffer three times, the sections were dehydrated in a graded series of ethanol and embedded in Epon 812. From the flat-embedded 50-µm-thick sections, areas from the cortex were excised and glued onto empty blocks of Epon 812, and consecutive 1.5-µm sections were cut for postembedding immunolabeling. The sections were treated for 5 min with a saturated solution of sodium hydroxide to remove the resin. After three brief rinses in absolute ethanol, the sections were hydrated with graded ethanol and rinsed in tap water. The sections were rinsed with PBS, incubated in normal goat serum for 30 min, and subsequently incubated overnight with anti-RhBG antibody (1:3,000) at 4°C. The sections were rinsed with PES and incubated for 2 h in peroxidase-conjugated donkey anti-rabbit IgG, Fab fragment, and washed again with PBS. For detection of RhBG antibody, Vector SG was used as the chromogen, and the sections were washed with distilled water, dehydrated with graded ethanol and xylene, mounted in Canada balsam, and examined by light microscopy.

Controls

For controls in each of the immunolocalization procedures, preimmune serum diluted at the same concentration as the primary antibody in PBS or PBS only was substituted for the primary antibody. In other experiments, the polyclonal RhBG and RhCG antibodies were incubated overnight at 4°C with an excess of the antigenic peptide, and the antigen/antibody mixture was substituted for the primary antibody. For colocalization procedures, in each experiment controls included substitution of buffer only for the first primary antibody, the second primary antibody, and both primary antibodies.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Immunoblot Analyses

Immunoblot analyses of membrane protein isolated from mouse kidney by using both the anti-RhBG and the anti-RhCG antibodies demonstrated specific immunoreactive proteins at the appropriate molecular masses. The anti-RhBG polyclonal and monoclonal antibodies recognized a native protein of ~52 kDa (Fig. 1, A and B, respectively), consistent with the reported molecular mass of 50-55 kDa for RhBG (23). The monoclonal anti-RhBG antibodies also recognized a faint band at ~40 kDa. This may represent the nonglycosylated form of RhBG (23). Importantly, all immunoreactivity was blocked by coincubation with the immunizing peptide with both the polyclonal and the monoclonal RhBG antibodies. The anti-RhCG antibodies recognized a protein of ~58 kDa (Fig. 1C), consistent with the reported molecular mass of ~58 kDa for RhCG (22). An additional band at ~46 kDa was observed; coincubation with the immunizing peptide did not alter recognition of this band, suggesting that it represents nonspecific cross-reactivity. With all three antibodies, the broad band seen on the immunoblot is consistent with the known glycosylation of these proteins (22, 23). Control immunoblots in which the primary antibody was substituted with the respective preimmune sera were negative (not shown). These results identify that both RhBG and RhCG protein are expressed in normal mouse kidney.


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Fig. 1.   A: polyclonal anti-Rh B-glycoprotein (RhBG) antibodies. An immunoblot utilizing 5 µg of a mouse renal membrane vesicle preparation is probed with rabbit polyclonal anti-RhBG antibodies. B: monoclonal anti-RhBG antibody. An immunoblot utilizing 5 µg of a mouse renal membrane vesicle preparation is probed with a mouse monoclonal anti-RhBG antibody. C: polyclonal anti-Rh C-glycoprotein (RhCG) antibodies. An immunoblot utilizing 5 µg of a mouse renal membrane vesicle is probed with rabbit polyclonal anti-RhCG antibody. Arrows, appropriate band.

Immunolocalization of RhBG

Intense basolateral RhBG immunoreactivity was observed in epithelial cells in the connecting segment (CNT) and in the collecting duct throughout the cortex and outer medulla and extending into the inner medulla (cortex, Figs. 2 and 3; medulla, Fig. 4). Proximal tubules and thick ascending limbs of Henle's loop were negative. Parallel studies with a second polyclonal anti-RhBG antiserum and with a monoclonal anti-RhBG antibody yielded identical results (not shown). In sections in which the primary antibody was substituted with either preimmune serum or buffer only, no immunoreactivity was observed (Fig. 2f). Similarly, when we incubated the primary antibody with the immunizing peptide, we observed no immunoreactivity (Fig. 5, A and B).


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Fig. 2.   Light micrographs of mouse kidney cortex labeled for RhBG by using immunohistochemistry; blue is due to hematoxylin counterstain. The majority of cells in the cortical collecting ducts (CCDs) and connecting segments (CNTs) throughout the cortex exhibit intense basolateral immunostaining for RhBG. Proximal tubules are negative and glomeruli are faintly stained. a and b: low-magnification images of the renal cortex. a: continuous intense basolateral immunolabeling in a branched profile of the CNT (marked c in the lumen). A transition from a strongly labeled CNT to the distal convoluted tubule (DCT), where only sporadic cells are strongly labeled, is also evident and is illustrated at higher magnification in c (*). b: heterogeneous RhBG localization observed in the CCD (*). Although the great majority of cells in the CCD exhibited strong basolateral immunostaining for RhBG, occasional cells are negative. These are illustrated at higher magnification in d (arrows). e: similar labeling in the initial collecting duct. The majority of cells were strongly positive for basolateral RhBG, whereas occasional cells were negative (arrow). Rare cells were observed that appeared to have weak immunostaining in the apical region of the cell (arrowhead). f: low-magnification image of negative control, in which preimmune serum was substituted for the primary antibody.



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Fig. 3.   Light micrographs of mouse kidney cortex labeled for both RhBG (brown) and carbonic anhydrase II (CA II; blue); hematoxylin counterstain was omitted. a: multiple CNTs with continuous intense basolateral immunolabel for RhBG, which labels both intercalated cells, identified by intense label for CA II, and CNT cells, which are negative for CA II. One profile that contains a transition from the CNT to the DCT is illustrated (*), demonstrating the decrease in intensity of RhBG immunolabel in the majority of cells in the DCT and strong RhBG label in intercalated cells. A portion of the CNT (c) is illustrated at higher magnification in c. Although the majority of intercalated cells in the CNT had discrete basolateral label only (arrows), rare cells were observed that appeared to have immunolabel apical to the cell nucleus (arrowhead). b: heterogeneous RhBG immunolabeling observed in the CCD, colocalized with CA II. Although CA II is present in both intercalated cells and principal cells in the mouse CCD, cells that are intensely labeled for CA II can be identified as intercalated cells. Some cells in the CCD that had not only the characteristic shape of intercalated cells but also intense label for CA II, were negative for RhBG (arrowhead, and illustrated at higher magnification in d). Other intercalated cells (arrow, and illustrated at higher magnification in e) had basolateral RhBG immunostaining that was more intense than that in the principal cells.



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Fig. 4.   Light micrographs of mouse renal medulla immunolabeled for RhBG and either counterstained with hematoxylin (a, b, and d) or colabeled for CA II (c). In the outer stripe of the outer medullary collecting duct (OMCDo) (A) and the inner stripe of the OMCD (OMCDi) (b), only a subpopulation of cells have intense basolateral immunolabel for RhBG. The majority of cells in these segments were negative for RhBG. c: colocalization of RhBG and CA II in the OMCDo, demonstrating that the RhBG-positive cells in the OMCD are also CA II positive and thus are intercalated cells; blue indicates CA II immunoreactivity. d: immunolocalization of RhBG in the inner medulla demonstrated strong basolateral labeling in a small minority of cells in the inner medullary collecting duct (arrows), which is consistent with the distribution of intercalated cells in this segment.



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Fig. 5.   Prevention of RhBG and RhCG immunolabeling by coincubation with immunizing peptide. Mouse kidney sections were examined for RhBG immunoreactivity by using either primary antibody alone (A) or after coincubation with the immunizing peptide (B). Coincubation with the immunizing peptide completely blocked immunolabeling. Similarly, mouse kidneys were examined for RhCG immunoreactivity by using primary antibody alone (C) or after coincubation with the immunizing peptide (D). Coincubation with the immunizing peptide completed blocked RhCG immunolabeling.

In the CNT, nearly all cells exhibited strong basolateral RhBG immunoreactivity (Fig. 2a). Rarely, cells were observed that had diffuse apical label in addition to basolateral label (Fig. 3c). Occasionally, tubule profiles were seen that appeared to include transitions from the CNT to the distal convoluted tubule (DCT) (Fig. 2, a and c). In these, the basolateral label for RhBG disappeared entirely in the DCT.

In the initial collecting tubule (ICT) and in the CCD, most, but not all, cells expressed basolateral RhBG immunoreactivity. The great majority of cells in the ICT and CCD exhibited strong basolateral immunoreactivity; within this group, a small population had more intense immunoreactivity (Fig. 2, b, d, and e). The cells with more intense basolateral immunoreactivity often exhibited a profile that bulged into the tubule lumen, suggesting that they were intercalated cells rather than principal cells. A small minority of cells had no detectable RhBG immunoreactivity (Fig. 2, d and e). Rarely, cells that had no detectable basolateral label appeared to have weak apical immunoreactivity (Fig. 2e).

In the medulla, basolateral RhBG immunoreactivity was present only in a subpopulation of cells in the collecting duct; no other structures in the medulla were positive for RhBG (Fig. 4). In both the outer and the inner stripe of the outer medullary collecting duct (OMCDo and OMCDi, respectively), the RhBG-positive cells represented a minority of the total cells. The incidence of positive cells in the inner medullary collecting duct (IMCD) was less than in the OMCD and diminished in the more distal portions of the IMCD, until they disappeared entirely at the papillary tip. In both the OMCD and the IMCD, RhBG-positive cells frequently had a rounded outline and bulging apical surface. Throughout the medullary collecting duct, the distribution of the positive cells and their morphology suggested they were intercalated cells.

Colocalization of RhBG and TSC

The DCT is a distinct portion of the nephron. To determine whether RhBG is expressed in the DCT, we examined immunofluorescent colocalization of RhBG with TSC, the thiazide-sensitive Na-Cl cotransporter. TSC is expressed solely in the apical region of DCT cells (33). Using immunofluorescence, cells that expressed apical TSC did not express RhBG, and cells that expressed basolateral RhBG did not express apical TSC (Fig. 6). We observed several tubules with abrupt transitions from TSC-positive to RhBG-positive cells (Fig. 6B). These results show that RhBG immunoreactivity is not identifiable in the DCT by using immunofluorescence microscopy.


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Fig. 6.   Colocalization of thiazide-sensitive Na-Cl cotransporter (TSC) and RhBG by using immunofluorescent labeling and confocal laser scanning microscopy. TSC immunoreactivity (green) labels the apical membrane of DCT cells. Tubule cells that expressed apical TSC did not express RhBG immunoreactivity (red), and those with RhBG immunoreactivity did not express TSC immunoreactivity (A). Occasionally, tubule segments with abrupt transition from TSC-positive cells to RhBG-positive cells were observed (B), identifying a transition from the DCT to the CNT. These studies, as do all confocal microscopy colocalization studies in this report, utilize a mouse monoclonal anti-RhBG antibody.

Colocalization of RhBG with CA II

The absence of basolateral RhBG immunoreactivity in a population of ICT and CCD cells suggested that the expression of RhBG protein is cell type specific. To begin identifying the types of cells that were positive or negative for basolateral immunoreactivity, we colocalized RhBG with CA II. CA II is highly expressed by mouse CCD intercalated cells, although low expression levels are also detectable in mouse CCD principal cells (3).

In the CNT, essentially all cells exhibited basolateral RhBG immunoreactivity. Colocalization with CA II clearly demonstrated that the RhBG-positive cells included both CNT cells, or principal cells, which are CA II negative, and intercalated cells, which are strongly CA II positive (Fig. 3, a and c). Furthermore, the rare cells that appeared to have diffuse apical RhBG labeling in addition to the basolateral label were CA II positive and thus were intercalated cells (Fig. 3c).

In the CCD, the distinction between intercalated cells and principal cells was less clear, because mouse CCD principal cells express CA II but at a much lower level than intercalated cells (3). However, cells that were strongly positive for CA II were easily identifiable, and within this group there were cells that were intensely positive for basolateral RhBG and other cells that were negative for RhBG (Fig. 2, b, d, and e). The majority of CCD cells were weakly positive for CA II; thus they were principal cells and expressed basolateral RhBG immunoreactivity that was less intense than that observed in the RhBG-positive intercalated cells. Thus all CCD principal cells and a subset of intercalated cells exhibit basolateral RhBG immunoreactivity.

In the medullary collecting duct, CA II immunoreactivity is present only in intercalated cells. In the OMCD, all CA II-positive cells exhibited intense basolateral RhBG immunoreactivity (Fig. 4c). Furthermore, CA II-negative cells did not express identifiable RhBG immunoreactivity. The results of CA II and RhBG colocalization in the IMCD were similar to that in the OMCD. All CA II-positive cells were also RhBG positive. Thus basolateral RhBG immunoreactivity is present in OMCD and IMCD intercalated cells and not in OMCD principal cells or in IMCD cells.

Colocalization of RhBG with AE1

To identify the specific intercalated cell subtypes that exhibit RhBG immunoreactivity, we first colocalized RhBG with AE1. AE1 is present in the basolateral plasma membrane of the intercalated cells in the medulla and the type A intercalated cells in the cortex (1, 36, 38).

In RhBG and AE1 colocalization studies, CCD, OMCD, and IMCD cells with basolateral AE1 immunoreactivity coexpressed basolateral RhBG immunoreactivity (Fig. 7). OMCD and IMCD cells that were AE1 negative were also RhBG negative. Thus CCD A-type intercalated cells and intercalated cells in the OMCD and IMCD express basolateral RhBG immunoreactivity.


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Fig. 7.   Colocalization of AE1 and RhBG in the CCD. Basolateral AE1 immunoreactivity (green) identifies type A intercalated cells (A) and is shown with white arrows. A single erythrocyte in a peritubular capillary (red arrow) is also identified. Basolateral RhBG immunoreactivity is present in the majority of CCD cells (B) but is absent in occasional cells (arrowhead). Colocalization of AE1 with RhBG (C, yellow) identifies that all CCD type A cells express strong basolateral RhBG immunoreactivity. Cells without identifiable basolateral RhBG immunoreactivity do not express basolateral AE1 immunoreactivity.

Colocalization of RhBG with Pendrin

Pendrin is a recently identified apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger expressed in the apical membrane of both the B-type intercalated cell (B cell) and the non-A-non-B intercalated cell (17, 41). We next examined whether pendrin-positive intercalated cells express RhBG immunoreactivity.

The colocalization of RhBG with pendrin differed in the CCD, CNT, and ICT. In the CCD, cells that expressed apical pendrin immunoreactivity did not express basolateral RhBG immunoreactivity (Fig. 8, A and B). Moreover, CCD cells that lacked basolateral RhBG immunoreactivity expressed apical pendrin immunoreactivity. Occasionally, there appeared to be colocalization of apical RhBG immunoreactivity with apical pendrin immunoreactivity. In contrast, pendrin-positive cells in the CNT almost always expressed basolateral RhBG immunoreactivity. The ICT appeared to have both pendrin-positive, RhBG-positive cells and pendrin-positive, RhBG-negative cells. Identical results were obtained with the two different pendrin antibodies. Thus, in general, pendrin-positive CCD cells do not express identifiable basolateral RhBG immunoreactivity, CNT pendrin-positive cells do express basolateral RhBG immunoreactivity, and ICT pendrin-positive cells are both RhBG positive and RhBG negative.


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Fig. 8.   Colocalization of pendrin and RhBG in the CCD and CNT. CCD cells with apical pendrin immunoreactivity (A, green; B, brown) do not express identifiable basolateral RhBG immunoreactivity (A, red; B, blue). In the CNT, almost all cells with apical pendrin immunoreactivity express basolateral RhBG immunoreactivity (C and D). A and C utilize the monoclonal anti-RhBG antibody described in this study and an anti-pendrin antibody previously characterized (34, 41). B and D utilize the polyclonal anti-RhBG antibody described in this study and an anti-pendrin antibody supplied by Dr. Søren Nielsen (University of Aarhus, Aarhus, Denmark) and also previously characterized (17). Identical results were obtained with the 2 different sets of anti-RhBG and anti-pendrin antibodies.

Localization of RhCG

Next, we examined the localization of RhCG protein, a second putative ammonium transporter expressed in the kidney. RhCG immunoreactivity was present in epithelial cells in CNT and collecting duct segments throughout the cortex, outer medulla, and inner medulla (Fig. 9). Coincubation of the primary antibody with the immunizing peptide completely prevented immunoreactivity (Fig. 5, C and D). However, in contrast to RhBG, RhCG immunoreactivity was apical rather than primarily basolateral. Identical results were obtained by using a second anti-RhCG antibody developed in our laboratory (data not shown). The labeling pattern was similar in sections subjected to antigen retrieval and those where antigen retrieval was omitted. However, the sections subjected to antigen retrieval exhibited more distinct labeling.


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Fig. 9.   Light micrographs of mouse kidney labeled for RhCG by using immunohistochemistry after antigen retrieval. The segmental distribution of RhCG was similar to that of RhBG in that it was present in the CNT and in collecting ducts throughout the kidney. Proximal tubules and distal tubules were negative. However, in contrast to RhBG, the cellular distribution of RhCG immunolabel was apical rather than basolateral. a: low-magnification image of RhCG immunolabel in mouse kidney cortex illustrating the intense apical localization in the CNT (c) and CCD (*). b: higher magnification of CNT in a. The apical label in the CNT was intense in most cells, although some cells appeared to have broader apical labeling (arrows), whereas other cells had weaker apical labeling (arrowhead). c: higher magnification image of CCD illustrated in a. In the CCD, a minority of cells exhibited broad, intense apical immunolabel for RhCG (arrows), whereas the remainder of cells had narrower and weaker apical immunolabel (arrowheads). d: outer stripe of mouse outer medulla. Apical immunolabel was present in virtually all cells in the mouse OMCDo. However, as in the CCD, heterogeneity in the intensity of label was present, with a minority of cells exhibiting intense, broad apical immunoreactivity (arrows) and the majority of cells labeled with only a thin apical band (arrowheads). e: inner stripe of mouse outer medulla. Intense apical immunolabel was present in a minority of cells in the OMCDi (arrows), whereas the majority of cells in this segment were negative. f: inner medulla. Only occasional cells exhibited apical immunolabel (arrow). g: low-magnification image of negative control, in which preimmune serum was substituted for the primary antibody.

In the cortex, virtually all cells in the CNT, ICT, and CCD exhibited apical labeling (Fig. 9, a-c). The apical labeling of cells in the CNT was more intense and homogeneous than in the CCD (compare Fig. 9, b and c). In the CCD, the majority of cells exhibited weak apical immunolabeling, whereas a small population of cells had intense apical immunolabeling (Fig. 9c). The intensely labeled cells frequently had a rounded, bulging apical surface, suggesting that they were intercalated cells. RhCG immunoreactivity was not observed in proximal tubule cells or thick ascending limb cells, even in experiments in which higher concentrations of the primary antibody were used.

The OMCDo expressed RhCG immunoreactivity that was apical and present in virtually all cells (Fig. 9d). The pattern was similar to that observed in the CCD, with the majority of cells having weak apical labeling and a minority exhibiting intense apical labeling. However, in the OMCDi, the majority of cells were negative for the RhCG immunolabel, although a minority had intense apical labeling (Fig. 9e). In the IMCD, only a small minority of cells exhibited apical immunolabeling (Fig. 9f).

Colocalization of RhCG with RhBG

The convoluted tubule cells in which RhCG is expressed could be the same cells in which RhBG is expressed, i.e., the CNT, or it could be the DCT or both the DCT and the CNT. To differentiate these possibilities, we examined colocalization of RhBG and RhCG immunoreactivity. In all cases, convoluted tubule cells either expressed apical RhCG and basolateral RhBG or expressed neither (Fig. 10). Thus RhCG is expressed in the same convoluted tubule cells as RhBG, i.e., CNT cells, and not in DCT cells.


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Fig. 10.   Colocalization of RhBG and RhCG. CNT cells with apical RhCG immunoreactivity (green) expressed basolateral RhBG immunoreactivity (red) and vice versa.

Colocalization of RhCG and CA II

In the CNT, colocalization of RhCG with CA II demonstrated that essentially all CA II-negative cells, that is, CNT cells, expressed apical RhCG immunoreactivity (Fig. 11, a and c). The majority of CA II-positive cells expressed intense RhCG apical immunoreactivity that was much greater than that observed in CA II-negative cells. However, a few CA II-positive cells exhibited only faint or no immunoreactivity (Fig. 11c).


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Fig. 11.   Light micrographs of mouse kidney labeled for both RhCG (brown) and CA II (blue). a and b: low-magnification images of the renal cortex illustrating immunolocalization in the CNT and CCD, respectively. c: higher magnification image of the CNT in A. CNT cells (arrowheads), which are negative for CA II, exhibit apical immunolabel for RhCG, as do the majority of intercalated cells (black arrows), which are identified by CA II immunolabel. Occasional intercalated cells (gray arrow) exhibited only faint label for RhCG. d: higher magnification of the CCD illustrated in b, demonstrating that intercalated cells that were intensely labeled for CA II exhibited strong apical RhCG immunolabel (arrows). Principal cells (arrowheads) also had apical RhCG immunoreactivity, but the label was less prominent. e and f: outer stripe of the outer medulla. e: low-magnification image demonstrating labeling in the OMCDo. f: higher magnification image demonstrating that in the OMCDo, both intercalated cells, which were CA II positive (arrows), and principal cells (arrowheads) had apical RhCG immunoreactivity, although the label was more prominent in intercalated cells. g: inner stripe of the outer medulla. h: inner medulla. In the OMCDi and the inner medullary collecting duct, only intercalated cells were RhCG positive (arrows).

In the CCD, although the distinction between principal cells and intercalated cells was not as clear as in the CNT, cells identified as intercalated cells by strong CA II immunoreactivity exhibited intense apical RhCG immunoreactivity (Fig. 11, b and d). The majority of cells had weaker CA II immunoreactivity and a thin apical band of RhCG immunoreactivity. Thus principal cells exhibited less abundant apical RhCG compared with intercalated cells.

Although RhCG immunoreactivity was present in all cells in the OMCDo, the immunolabel was more intense in CA II-positive cells than in CA II-negative cells (Fig. 11, e and f). Thus the OMCDo intercalated cell appears to contain more apical RhCG than the OMCDo principal cell.

In the OMCDi and the IMCD, RhCG immunoreactivity was present only in CA II-positive cells (Fig. 11, g and h). Thus in these segments, RhCG is present in intercalated cells and is not detectable in the OMCDi principal cell or in the IMCD cell.

Thus apical RhCG immunoreactivity is present in virtually all cells in the CNT, ICT, CCD, and OMCDo and in intercalated cells in the OMCDi and IMCD. In the cortical segments and the OMCDo, intercalated cells were more intensely labeled than principal cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies are the first to determine the specific cellular location of the putative ammonium transporter family proteins RhBG and RhCG in the mouse kidney. Our findings demonstrate that the putative ammonium transporters RhBG and RhCG are widely distributed throughout the collecting duct and CNT. However, the intensity of the immunoreactivity and the prevalence of positive cells are markedly greater in the CNT and the CCD than in the medullary collecting duct segments. We find that in the CCD and CNT virtually all epithelial cell subtypes express both basolateral RhBG and apical RhCG, including CNT cells, principal cells, and type A and non-A-non-B intercalated cells but, interestingly, not type B intercalated cells.

In medullary collecting duct segments, the coexistence of basolateral RhBG and apical RhCG persists in the intercalated cells, which are similar in function to type A intercalated cells in the CCD. However, expression of these transporters is largely absent in the majority cell types, principal cells, and IMCD cells and is limited to weak apical RhCG immunoreactivity in OMCDo principal cells.

The coexistence and polarized expression of these transporters in multiple epithelial cell subtypes in collecting duct segments and the marked axial heterogeneity with respect to both the intensity of immunoreactivity and the prevalence of labeled cells, particularly among principal cells, suggest that RhBG and RhCG mediate specific and important roles in the kidney.

The cellular localization of RhBG protein in the present study adds to the reported localization of RhBG. In situ hybridization studies suggested that renal convoluted tubules express RhBG mRNA (23). Whether these were proximal convoluted tubules, DCTs, or CNTs was not determined. The present studies identify no RhBG immunoreactivity in proximal convoluted tubule cells, even in experiments in which we used higher concentrations of the RhBG antibody than was necessary to produce intense immunoreactivity in the CNT and collecting duct. Thus our findings of intense RhBG immunoreactivity in the CNT but not in proximal tubules or DCT suggest that the convoluted tubules that were positive for RhBG mRNA in the in situ hybridization studies (23) represent the CNT. Our observation that RhBG immunoreactivity is substantially less in the CCD than in the CNT suggests that RhBG protein expression is less in the CCD than in the CNT. Thus CCD RhBG mRNA expression may also be lower, potentially below the level of detection by using in situ hybridization. This might explain the failure to detect RhBG mRNA expression by in situ hybridization in a previous study (23).

The present studies demonstrate that a subpopulation of CCD intercalated cells do not express identifiable basolateral RhBG immunoreactivity. Colocalization of RhBG with AE1 demonstrates that the type A intercalated cell in the CCD, as well as intercalated cells throughout the medullary collecting duct, express basolateral RhBG. Colocalization with pendrin, an apical anion exchanger present in type B and non-A-non-B intercalated cells (17, 41), revealed two patterns of RhBG localization in pendrin-positive cells. In the CCD, pendrin-positive cells were negative for RhBG, whereas in the CNT pendrin-positive cells typically exhibited basolateral RhBG immunoreactivity. In the mouse CNT, non-A-non-B intercalated cells are prevalent and type B intercalated cells are rare (36). Thus the pendrin-positive, RhBG-positive cells observed in the CNT likely are non-A-non-B intercalated cells. In the CCD, the identity of non-A intercalated cells is not as clear. Some investigators have reported that in the CCD, type B intercalated cells occur frequently and non-A-non-B intercalated cells are rare or absent (36), whereas other studies have reported that non-A-non-B intercalated cells occur as frequently as type B intercalated cells (15). Thus the pendrin-positive, RhBG-negative cells observed in the CCD likely include type B intercalated cells. It is also possible that non-A-non-B intercalated cells in the CCD, unlike those in the CNT, do not express RhBG. Nevertheless, our findings indicate that the CNT non-A-non-B intercalated cell and type A cells throughout the medullary collecting duct express basolateral RhBG immunoreactivity, whereas the CCD type B cell does not express it at detectable levels.

The cellular localization of RhCG protein that we observed in this study is both consistent with and adds to a previous report examining RhCG mRNA expression in the mouse kidney (22). Using in situ mRNA hybridization, it was suggested that RhCG mRNA was expressed by collecting duct cells (22). The immunohistochemical findings in the present study are consistent with this observation and extend these findings in important ways. First, the present study establishes that RhCG protein is present in the CNT and the collecting duct. In addition, the present study demonstrates that RhCG is located at the apical membrane of mouse renal cells in which it is expressed. We have observed similar findings in the rat (Verlander and Weiner, unpublished observations).

Although our findings of polarized expression of RhBG and RhCG in many specific epithelial cells in the CNT and collecting duct suggest these proteins have functional importance, their potential roles in the kidney presently must be surmised from evidence regarding the functions of related proteins in other cell types. In 1994, separate independent groups reported the cloning and expression of ammonium transporters from the yeast Saccharomyces cerevisiae, Mep1 (29), and from plants, Amt1 (32). Both Mep1 and Amt1 encode proteins of ~54 kDa with 9-12 transmembrane domains that transport both ammonium (NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) and [14C]methylammonium, a radiolabeled ammonium analog (29, 32). Further studies have identified multiple additional members of the ammonium transporter family: Mep2 and Mep3 in yeast (28) and Amt1;2, Amt1;3 and Amt2 in plants (10, 35). In the last few years, members of the ammonium transporter family have been identified in essentially all organisms, from bacteria to the slime mold, Dicytostelium discoideum, birds, and mammals (2, 12, 13, 39).

A fundamental characteristic of yeast and plant members of this protein family is ammonium transport (26, 27, 29). RhAG, another mammalian member of this transport family (26), transports the ammonium analog methylammonium (42). Similarly, a preliminary report suggests that mouse RhCG, when expressed in the Xenopus laevis oocyte, mediates electrogenic acid loading in the presence, but not the absence, of extracellular ammonia, consistent with RhCG functioning as an electrogenic NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transporter (31). Thus it is possible that RhBG or RhCG, or both, mediate transepithelial ammonium transport by the CNT and collecting duct, which are critically important sites for total ammonia secretion (4, 18).

Another possible function of these proteins is to function as ammonia "sensors." In the mammalian collecting duct, ammonia regulates several aspects of collecting duct ion transport. For example, ammonia stimulates collecting duct net proton secretion (6, 9, 19, 40) and potassium reabsorption (11). Simultaneously, ammonia inhibits CCD unidirectional bicarbonate and potassium secretion and sodium reabsorption (7, 11). The mechanisms through which ammonia alters ion transport are not completely identified. However, the effects of ammonia on CCD acid-base transport can be dissociated from ammonia transport (7-9). Thus ammonia may act through a cellular sensor to regulate collecting duct ion transport. Evidence from other organisms suggests that ammonium transporters related to RhBG and RhCG can serve as ammonia sensors and regulate cell function. Specifically, in yeast, the ammonium transporter Mep2 regulates the cellular response to nutrient depletion through mechanisms that are independent of ammonium transport (25). Thus it is possible that RhBG or RhCG may function as such a sensor and may regulate function in any of the specific epithelial cell types where they are present.

In summary, these studies are the first to identify the cellular localization of the ammonium transporter family of proteins RhBG and RhCG in the mouse kidney. RhBG and RhCG protein exhibit axial heterogeneity and polarized expression in many specific epithelial cell types in the mouse CNT and collecting duct, consistent with an important role for these proteins in either ammonium transport or signaling throughout these segments.


    NOTE ADDED IN PROOF

A report describing a similar localization of RhCG in the rat kidney (5) was published while this manuscript was under review.


    ACKNOWLEDGEMENTS

The authors thank Gina Cowsert and Lee Ann Day for secretarial support and Melissa A. Lewis and Lauren DeWitt of the University of Florida College of Medicine Electron Microscopy Core Facility who performed the majority of the immunohistochemical experiments. We also thank Dr. Shen-Ling Xia of the North Florida/South Georgia Veterans Health System confocal microscopy core facility and Timothy Vaught of the University of Florida Brain Institute for assistance with the confocal microscopy.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45788 and funds from the Department of Veterans Affairs and the Florida Affiliate of the American Heart Association.

Address for reprint requests and other correspondence: I. D. Weiner, Div. of Nephrology, Hypertension and Transplantation, Univ. of Florida College of Medicine, PO Box 100224, Gainesville, FL 32610 (E-mail: WeineID{at}ufl.edu).

1 Ammonia exists in solution in the molecular forms of NH3 and NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. When the term "ammonia" is used in this report, we are referring to the combination of these two molecular forms. When referring to the molecular form NH3, we will specifically state NH3. When referring to the molecular form NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, we will either specifically state NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or we will use the term ammonium.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 8, 2002;10.1152/ajprenal.00050.2002

Received 6 February 2002; accepted in final form 2 October 2002.


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