Changes in subcellular distribution of the ammonia transporter, Rhcg ...

Am J Physiol Renal Physiol 290: F1443–F1452, 2006. First published January 24, 2006; doi:10.1152/ajprenal.00459.2005.

Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis Ramanathan M. Seshadri,1 Janet D. Klein,2 Tekla Smith,2 Jeff M. Sands,2 Mary E. Handlogten,1 Jill W. Verlander,1 and I. David Weiner1,3 3

Nephrology and Hypertension Section, North Florida/South Georgia Veterans Health System, and Division of Nephrology, Hypertension, and Transplantation, University of Florida College of Medicine, Gainesville, Florida; and 2Renal Division, Emory University, Atlanta, Georgia 1

Submitted 21 November 2005; accepted in final form 19 January 2006

Seshadri, Ramanathan M., Janet D. Klein, Tekla Smith, Jeff M. Sands, Mary E. Handlogten, Jill W. Verlander, and I. David Weiner. Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis. Am J Physiol Renal Physiol 290: F1443–F1452, 2006. First published January 24, 2006; doi:10.1152/ajprenal.00459.2005.—The primary mechanism by which the kidneys mediate net acid excretion is through ammonia metabolism. In the current study, we examined whether chronic metabolic acidosis, which increases ammonia metabolism, alters the cell-specific and/or the subcellular expression of the ammonia transporter family member, Rhcg, in the outer medullary collecting duct in the inner stripe (OMCDi). Chronic metabolic acidosis was induced in normal SD rats by HCl ingestion for 7 days; controls were pair-fed. The subcellular distribution of Rhcg was determined using immunogold electron microscopy and morphometric analyses. In intercalated cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane. Intracellular Rhcg decreased significantly, and basolateral Rhcg was unchanged. Because apical plasma membrane length increased in parallel with apical Rhcg immunolabel, apical plasma membrane Rhcg density was unchanged. In principal cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane while decreasing the intracellular proportion. In contrast to the intercalated cell, chronic metabolic acidosis did not significantly alter apical boundary length; accordingly, apical plasma membrane Rhcg density increased. In addition, basolateral Rhcg immunolabel increased in response to chronic metabolic acidosis. These results indicate that in the rat OMCDi 1) chronic metabolic acidosis increases apical plasma membrane Rhcg in both the intercalated cell and principal cell where it may contribute to enhanced apical ammonia secretion; 2) increased apical plasma membrane Rhcg results from both increased total protein and changes in the subcellular distribution of Rhcg; 3) the mechanism of Rhcg subcellular redistribution differs in intercalated and principal cells; and 4) Rhcg may contribute to regulated basolateral ammonia transport in the principal cell. intercalated cell; principal cell; immunogold; morphometry RENAL AMMONIA METABOLISM IS the primary mechanism of acidbase homeostasis (4, 9, 10). Ammonia1 is produced by the proximal tubule in association with equimolar bicarbonate generation and is secreted preferentially into the luminal fluid. The majority of luminal ammonia is reabsorbed by the thick

Address for reprint requests and other correspondence: I. D. Weiner, Univ. of Florida College of Medicine, PO Box 100224, Gainesville, FL 32610-0224 (e-mail: [email protected]). 1 The term ammonia is used to refer to the combination of the two molecular species, NH3 and NH⫹ 4 . When referring specifically to the molecular species NH3, we specifically state “NH3,” and when referring to NH⫹ 4 we specifically state “NH⫹ 4 .” http://www.ajprenal.org

ascending limb of the loop of Henle into the renal interstitium before finally being secreted by the collecting duct into the luminal fluid. Under normal conditions, 70 – 80% of total urinary ammonia is secreted by the collecting duct, and almost the entire increase in net acid excretion in response to chronic metabolic acidosis is due to increased ammonia excretion (10, 28). Importantly, this increased renal ammonia excretion is associated with substantial increases in collecting duct ammonia secretion (10, 28). Accordingly, understanding the regulation of collecting duct ammonia transport is important. A recently identified family of ammonia transporter proteins may mediate important roles in renal ammonia transport (23, 36). The nonerythroid Rh glycoprotein, Rh C glycoprotein (Rhcg), transports ammonia (1, 20, 43) and is expressed in the renal distal convoluted tubule, connecting segment, initial collecting tubule, and collecting duct (5, 29, 32). Moreover, in vitro studies using cultured mouse collecting duct cells that express Rhcg suggest that the majority of both apical and basolateral ammonia transport occurs via ammonia-specific transport mechanisms, and not via nonionic NH3 diffusion (12, 13). Thus it is likely that changes in Rhcg-mediated ammonia transport may be important in the collecting duct response to chronic metabolic acidosis. Transepithelial ion transport can be regulated through a variety of mechanisms. One potential mechanism is a quantitative change in transporter expression. In fact, we recently demonstrated that chronic metabolic acidosis increases Rhcg protein expression in the outer and inner medulla (29). When changes in protein expression in the heterogeneous collecting duct are being examined, it is important to assess separately the contribution of its two epithelial cell types, the intercalated and the principal cells. Although these two cell types are generally thought to mediate different transport functions (11), some (29, 32), but not all (5), studies show that both cell types in both the rat and mouse outer medullary collecting duct in the inner stripe (OMCDi) express Rhcg, suggesting that both may contribute to regulated ammonia secretion. A second regulatory mechanism can be a change in the membrane location of ion-transporting proteins. Our recent observation that the rat kidney exhibits basolateral, in addition to apical, Rhcg immunoreactivity (29) raises the possibility that Rhcg may contribute to regulated ammonia transport across both the basolateral and the apical plasma membranes. Finally, a change in the subcellular distribution of a protein is an important mechanism 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. F1443

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REDISTRIBUTION OF RHCG IN RESPONSE TO METABOLIC ACIDOSIS

used to regulate transepithelial transport. Our recent observation that chronic metabolic acidosis induces a sharper and more discrete apical Rhcg immunoreactivity in OMCD and IMCD intercalated cells (29) raises the possibility that either increased apical membrane Rhcg or redistribution from subapical vesicles to the apical plasma membrane regulates ammonia transport. The purpose of the current study was to determine the effect of chronic metabolic acidosis on the expression of Rhcg in the OMCD intercalated and principal cell, with particular emphasis on its cell-specific abundance, on the amount of apical and basolateral plasma membrane immunolabel, and on its subcellular distribution in these two epithelial cell types. To do so, we used a chronic metabolic acidosis model which we have used previously (17, 29) and quantified Rhcg expression at the ultrastructural level in OMCDi intercalated and principal cells using immunogold electron microscopy and morphometric analysis. METHODS

Antibodies. Affinity-purified antibodies to rodent Rhcg were generated in our laboratory and have been characterized previously (14, 29, 32, 38). Animals. Normal male Sprague-Dawley rats, weighing ⬃250 g, were obtained from Charles River Laboratories. Metabolic acidosis was induced as described previously (17, 29). Briefly, acidosis rat chow was prepared by adding 1 liter of 0.8 M HCl solution to 1 kg rat chow. A control rat diet was made by mixing 1 liter of H2O to 1 kg of rat chow. The weight of food eaten by chronic metabolic acidosis rats was recorded every 24 h, and control rats were provided food on a pair-fed basis. Although plasma electrolytes were not obtained in this study, this model is identical to that used in studies we reported recently, and results in chronic metabolic acidosis, increased renal ammonia excretion, and no significant change in either urinary urea excretion or plasma potassium (17, 29). On the day of the experiment, rats were anesthetized and the kidneys were preserved by retrograde aortic perfusion at 170 mmHg with 1% glutaraldehyde, 3% polyvinylpyrrolidone in Tyrode buffer made with 12 g NaCl/l, pH 7.4, then immersed in the same fixative for 3–5 h at room temperature. The tissue was rinsed in Tyrode buffer made with 12 g NaCl/l, and samples from the inner stripe of the outer medulla were immersed in 0.1 M NH4Cl for 1 h at 4°C. The tissue samples were then dehydrated in a graded series of alcohols and processed and embedded in Lowicryl K4M (Electron Microscopy Sciences, Ft. Washington, PA). Lowicryl polymerization was carried out under ultraviolet light for 24 h at ⫺20°C and then for 48 h at room temperature. Samples containing well-preserved collecting ducts were selected after light microscopic examination of 0.5-␮m-thick sections stained with toluidine blue. Ultrathin sections of these were mounted on Formvar/carbon-coated nickel grids for immunogold cytochemistry. All animal interventions were reviewed and approved by the Emory University Institutional Animal Care and Use Committee. Immunogold labeling. Briefly, the immunogold labeling procedure was performed by exposure of the ultrathin tissue sections to the primary antibody and then to a goat anti-rabbit IgG secondary antibody conjugated to 0.8-nm colloidal gold particles (AuroProbe EM GAR G10, Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Unless noted otherwise, all steps were done by floating the grids on droplets of solution at room temperature. The following solutions were used: an incubation solution, 0.2% acetylated BSA (Aurion BSA-c, Electron Microscopy Sciences) and 10 mM NaN3, in PBS, pH 7.4; and a blocking solution, 5% BSA, 0.1% cold-water fish-skin gelatin, and 5% normal goat serum, in PBS. The sections were exposed to 0.1 M NH4Cl for 1 h, rinsed with PBS, treated with AJP-Renal Physiol • VOL

the blocking solution for 30 min, washed with the incubation solution, and then incubated in a humidified chamber overnight at 4°C with the affinity-purified primary antibody diluted in the incubation solution. The sections were washed and exposed for 1.5 h to the secondary antibody diluted in the incubation solution. The sections were washed with the incubation buffer, washed with PBS, postfixed with 1.25% glutaraldehyde in PBS, washed with PBS, and counterstained with saturated uranyl acetate. Each group of sections subjected to the immunogold procedure included a control section that was exposed to the incubation buffer in place of the primary antibody. Electron microscopy. Ultrathin sections were examined using a Zeiss EM10A transmission electron microscope by an observer blinded to the experimental conditions. The OMCDi was identified by its characteristic heterogeneous epithelial cell population, which included principal cells and intercalated cells. For morphometric analyses, a minimum of five intercalated cells and five principal cells in the OMCDi in each animal (3 acidosis mice, 5 control mice) was selected randomly and photographed at a primary magnification of ⫻4,800, recorded on Kodak SO-163 Electron Microscopy film. Individual photomicrographs were examined at a final magnification of ⬃⫻18,880; the exact magnification was calculated using a calibration grid with 2,160 lines/mm. Morphometric data were collected by point and intersection counting using the Merz curvilinear test grid with a distance of 20 mm between the points, corresponding to 1.059 ␮m (d). Raw data from the individual images were pooled to produce a single value for each parameter in each animal for statistical analysis. The boundary length of the apical and basolateral plasma membranes, cytoplasmic and cell profile area, and surface density were calculated using standard morphometric formulas (35). The number of gold particles along the apical and basolateral plasma membranes and the number of gold particles over the cytoplasm, including cytoplasmic vesicles, were counted manually in the same images. Boundary length (B) was calculated from the equation B ⫽ I ⫻ d, where I represents the number of intersections between the test line and the plasma membrane. B was expressed in micrometers. Apical plasma membrane surface density (SV) was calculated using the formula SV ⫽ 2 ⫻ I/L, where I is the number of intersections between the test line and the plasma membrane, and L is the length of the test line over the cell profile (35). Using the Merz grid, L ⫽ P ⫻ ␲/2 ⫻ d, where P represents the number of points over the cell. SV was expressed as square millimeters per cubic millimeters. Gold particles that were touching the apical or basolateral plasma membranes and those over the cytoplasm, including cytoplasmic vesicle membranes, were counted and were related to the respective plasma membrane boundary length (gold particles/␮m of plasma membrane boundary length). In addition, the ratio of gold particles associated with the apical or basolateral plasma membrane to total gold particles per cell was determined for each cell type. Statistics. Results are presented as means ⫾ SE. Statistical analyses were performed using Student’s unpaired t-test, and P ⬍ 0.05 was taken as statistically significant. RESULTS

Intercalated cell: qualitative observations. Qualitative observation of the OMCDi intercalated cell showed that Rhcg immunolabel was present in the apical plasma membrane, in subapical vesicles, and in the basolateral plasma membrane (Fig. 1). Under control conditions, apical plasma membrane immunolabel was moderately abundant, whereas numerous gold particles were present in the apical cytoplasm. The majority of the cytoplasmic label was clearly associated with vesicles and tubulovesicles. Rhcg immunoreactivity in the basal and lateral cellular regions was evident and was associated with the basolateral plasma membrane, consistent with our previous observation using light microscopy (29). Rhcg was

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Numerous differences were observed in response to chronic metabolic acidosis. Apical plasma membrane microprojections were markedly increased in length and number, and Rhcg immunolabel along the apical plasma membrane was abundant and appeared greater than under control conditions (Fig. 3). In addition, apical cytoplasmic vesicles and associated Rhcg immunolabel were sparse compared with control intercalated cells. No qualitative differences were apparent in basolateral plasma membrane complexity or immunolabel (Figs. 1C vs. 3C). Intercalated cell: quantitative measurements. Quantifying the number of gold particles in individual cell profiles demonstrated that chronic metabolic acidosis increased total cellular Rhcg immunolabel significantly (control, 174.4 ⫾ 13.0 vs. acidosis, 237.7 ⫾ 20.2 gold particles/cell profile, P ⬍ 0.05). This observation is consistent with chronic metabolic acidosis increasing Rhcg protein expression in the outer medulla, as we recently showed (29). Figure 4 summarizes these results. Chronic metabolic acidosis increased apical plasma membrane Rhcg immunolabel significantly (control, 34.7 ⫾ 3.1 vs. acidosis, 136.6 ⫾ 10.0 gold particles/cell profile, P ⬍ 0.0001, Fig. 5A). Because the relative increase exceeded the relative increase in total cellular Rhcg, the proportion of total cellular Rhcg present in the apical plasma membrane increased significantly (control, 20 ⫾ 2% vs. acidosis, 58 ⫾ 2%, P ⬍ 0.0002, Fig. 5B). Simultaneously chronic metabolic acidosis decreased significantly both cytoplasmic Rhcg immunolabel (control

Fig. 1. Transmission electron micrographs of Rhc glycoprotein (Rhcg) immunolabel in a representative inner stripe of the outer medullary collecting duct (OMCDi) intercalated cell in control kidney. A: entire cell. B: apical region. C: basal region. OMCDi intercalated cells in control animals typically exhibited few apical plasma membrane microprojections and numerous apical cytoplasmic vesicles (A and B). In the apical region, Rhcg immunolabel was predominantly associated with cytoplasmic vesicles (arrowheads, B); immunolabel in the apical plasma membrane was moderate (arrows, B). Strong Rhcg immunolabel was also present in the basolateral plasma membrane (arrows, C). Magnification: ⫻9,000 (A) and ⫻22,000 (B and C).

not observed in the adjacent medullary thick ascending limb of the loop of Henle (Fig. 2), consistent with previous observations in both the rat and mouse kidney using the same antibody (29, 32) and with studies from other laboratories using other anti-Rhcg antibodies (5, 27). AJP-Renal Physiol • VOL

Fig. 2. Rhcg immunolabel in thick ascending limb of the loop of Henle (TAL). A: low-magnification transmission electron micrograph showing Rhcg immunolabel in an OMCDi intercalated cell (IC) and in an adjacent medullary TAL (magnification: ⫻8,000). B: high-magnification image. Arrows denote Rhcg immunolabel. Rhcg immunolabel is observed only in the intercalated cell and not in the adjacent TAL (magnification: ⫻13,000).

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Fig. 4. Effect of chronic metabolic acidosis on OMCDi cell-specific Rhcg expression. Total cellular Rhcg expression is shown as the total number of gold particles (Au) per cell profile. Total Rhcg expression was the sum of apical plasma membrane, intracellular and basolateral plasma membrane expression and was determined using immunogold electron microscopy. Intracellular and basolateral plasma membrane expression was quantified in OMCDi intercalated and principal cells under control conditions and in response to chronic metabolic acidosis using immunogold electron microscopy. Under basal conditions, intercalated cell Rhcg expression exceeded principal cell Rhcg expression. Chronic metabolic acidosis increased total cellular Rhcg expression in both intercalated cells and principal cells. Results are from 25 cells of each type in control animal (5/animal) and 15 cells of each type in chronic metabolic acidosis animals (5/animal).

Fig. 3. Transmission electron micrographs of Rhcg immunolabel in representative OMCDi intercalated cell in a chronic metabolic acidosis kidney. A: entire cell. B: apical region. C: basal region. Compared with control conditions (Fig. 1), the apical plasma membrane microprojections in OMCDi intercalated cells were markedly increased in length and number, and the abundance of apical cytoplasmic vesicles was greatly reduced (A and B). Rhcg immunolabel was redistributed; apical plasma membrane Rhcg immunolabel (arrows, B) was markedly increased, and label in apical cytoplasmic vesicles (arrowheads, B) was greatly reduced compared with intercalated cells under control conditions. Strong RhCG immunolabel was associated with the basolateral plasma membrane (arrows, C), but there were no appreciable differences in the basolateral plasma membrane complexity or immunolabel compared with control intercalated cells. Magnification: ⫻9,000 (A) and ⫻22,000 (B and C).

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93.6 ⫾ 8.8 vs. acidosis, 35.5 ⫾ 2.1 gold particles/cell profile, P ⬍ 0.005, Fig. 5A) and the proportion of total Rhcg present in the cytoplasmic compartment (control, 53 ⫾ 2% vs. acidosis, 15 ⫾ 0.4%, P ⬍ 0.000005, Fig. 5B). The increase in apical plasma membrane Rhcg expression was accompanied by a parallel increase in both apical plasma membrane boundary length (control, 19.4 ⫾ 3.0 vs. acidosis, 54.2 ⫾ 6.4 ␮m, P ⬍ 0.002, Fig. 5C) and surface density (control, 460 ⫾ 28 vs. acidosis, 1,024 ⫾ 84 mm2/mm3, P ⬍ 0.0005). These findings are consistent with a previous report using a different model of chronic metabolic acidosis (19). Because the increase in apical plasma membrane boundary length was of the same magnitude as the increase in apical Rhcg immunolabel, apical plasma membrane Rhcg immunolabel density did not change significantly [control, 1,896 ⫾ 204 vs. acidosis, 2,605 ⫾ 415 ⫻ 103 gold particles/␮m, P ⫽ not significant (NS), Fig. 5D]. We reported recently in studies using light microscopy that OMCDi intercalated cells express basolateral Rhcg immunoreactivity in addition to apical immunoreactivity (29). The current study confirms that observation and extends it by demonstrating, at the ultrastructural level, that the basolateral immunoreactivity observed by light microscopy represents basolateral plasma membrane Rhcg expression, and not cytoplasmic expression in the basal region of the cell. Quantitatively, basolateral Rhcg expression exceeded apical Rhcg expression under control conditions (basolateral, 46.1 ⫾ 4.3 vs. apical, 34.7 ⫾ 3.1 gold particles/cell profile, P ⬍ 0.05, Fig. 5A). In response to chronic metabolic acidosis, there was a tendency, which did not reach statistical significance, for an increase in basolateral plasma membrane Rhcg immunolabel (control, 46.1 ⫾ 4.3 vs. acidosis, 65.6 ⫾ 8.9 gold particles/cell profile, P ⫽ 0.066, Fig. 5A) but not for a change in the

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Fig. 5. Quantitative assessment of the OMCDi intercalated cell response to chronic metabolic acidosis. A: Rhcg immunolabel quantified as the mean number of gold particles (Au) per cell profile in the apical plasma membrane, cytoplasm, and the basolateral plasma membrane under control conditions and in response to chronic metabolic acidosis. Under control conditions, basolateral plasma membrane Rhcg immunolabel exceeds apical plasma membrane Rhcg immunolabel. Chronic metabolic acidosis increases OMCDi intercalated cell apical plasma membrane Rhcg expression, decreases intracellular Rhcg expression, and does not alter basolateral plasma membrane expression. B: relative proportion of total cellular Rhcg immunolabel present in the apical plasma membrane, intracellular compartment, and the basolateral plasma membrane. The relative proportion was calculated for each cell examined as the compartment-specific expression divided by the total expression in that cell. Chronic metabolic acidosis increases the relative percentage of total OMCDi intercalated cell Rhcg expressed in the apical plasma membrane, decreases the relative intracellular expression, and does not alter the relative basolateral plasma membrane expression. C: intercalated cell apical and basolateral plasma membrane boundary length under control and chronic metabolic acidosis conditions (␮m/cell profile). Chronic metabolic acidosis increases apical plasma membrane boundary length significantly, consistent with increased apical plasma membrane microprojections demonstrated in Fig. 3, but does not alter basolateral plasma membrane boundary length significantly. D: density of Rhcg in the apical and basolateral plasma membranes (Au particles ⫻ 103/␮m boundary length). Chronic metabolic acidosis does not significantly alter the density of Rhcg label per unit membrane length in either the apical or basolateral plasma membranes (P ⫽ not significant). Results are from 25 cells of each type in control animal (5/animal) and 15 cells of each type in chronic metabolic acidosis animals (5/animal).

proportion of total cellular Rhcg that was present in the basolateral plasma membrane (control, 26 ⫾ 1% vs. acidosis, 27 ⫾ 2%, P ⫽ NS, Fig. 5B). Similarly, neither basolateral plasma membrane boundary length (control, 23.6 ⫾ 3.6, vs. acidosis, 23.7 ⫾ 2.7 ␮m, P ⫽ NS, Fig. 5C) nor basolateral plasma membrane Rhcg density changed significantly (control, 2,047 ⫾ 247 vs. acidosis, 2,757 ⫾ 195 gold particles/mm, P ⫽ NS, Fig. 5D). Thus chronic metabolic acidosis did not substantially change basolateral Rhcg expression in OMCDi intercalated cells, either assessed as total basolateral Rhcg immunolabel, the proportion of total cellular Rhcg immunolabel, or basolateral plasma membrane Rhcg density. Principal cell: qualitative observations. Because some physiological studies indicate that the OMCDi principal cell can contribute to regulated acid-base transport (37, 39), we examined the effect of chronic metabolic acidosis on OMCDi principal cell Rhcg expression. Similar to the intercalated cell, principal cell Rhcg immunolabel was present in both the apical and basolateral plasma membranes, and it was scattered throughout the cytoplasm (Fig. 6). Immunolabel in both the apical and basolateral plasma membrane compartments and in the cytoplasm of the principal cell was less intense than in intercalated cells. In response to chronic metabolic acidosis, principal cell apical plasma membrane Rhcg immunolabel appeared to inAJP-Renal Physiol • VOL

crease, and there appeared to be a relative decrease in cytoplasmic immunolabel (Fig. 7, A and B). Basolateral plasma membrane Rhcg label appeared increased in intensity compared with control conditions (Fig. 7C). There were no observable differences in either apical or basolateral plasma membrane microprojections or complexity compared with principal cells from control kidneys. Principal cell: quantitative observations. Chronic metabolic acidosis increased principal cell total cellular Rhcg immunolabel significantly (control, 28.0 ⫾ 2.1 vs. acidosis, 67.5 ⫾ 4.6 gold particles/cell profile, P ⬍ 0.0002, Fig. 4). Thus the increased Rhcg protein expression in the outer medulla in response to chronic metabolic acidosis (29) is due to increased Rhcg expression in both the intercalated cell and the principal cell. Chronic metabolic acidosis increased principal cell apical plasma membrane Rhcg immunolabel significantly (control, 5.6 ⫾ 1.0 vs. acidosis, 21.3 ⫾ 0.4 gold particles/cell profile, P ⬍ 0.0001, Fig. 8A). This increase was greater than the increase in total cellular Rhcg immunolabel; thus chronic metabolic acidosis significantly increased the proportion of total cellular Rhcg present in the apical plasma membrane (control, 20 ⫾ 3% vs. acidosis, 32 ⫾ 2%, P ⬍ 0.05, Fig. 8B). Although intracellular Rhcg increased significantly (control, 11.4 ⫾ 1.4 vs. acidosis, 20.4 ⫾ 1.4, P ⬍ 0.01), the relative

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principal cell response of increased apical Rhcg due to increased apical plasma membrane Rhcg density contrasts with the intercalated cell response of increased apical plasma membrane Rhcg without a change in apical plasma membrane Rhcg density. Rhcg was also present in the rat OMCDi principal cell basolateral plasma membrane under both control and chronic metabolic acidosis conditions. Under control conditions, basolateral plasma membrane Rhcg immunolabel was low but exceeded apical Rhcg (basolateral, 11.0 ⫾ 0.9 vs. apical, 5.6 ⫾ 1.0 gold particles/cell profile, P ⬍ 0.02, Fig. 8A). In response to chronic metabolic acidosis, basolateral plasma membrane Rhcg expression increased significantly (control, 11.0 ⫾ 0.9

Fig. 6. Transmission electron micrographs of Rhcg immunolabel in representative OMCDi principal cell in control kidney. A: entire cell. B: apical region. C: basal region. In control rats, OMCDi principal cells exhibited few, short apical microprojections (A and B). Rhcg immunolabel was present in principal cells in both the apical and basolateral plasma membranes, but the abundance of label was low compared with intercalated cells (arrows in B and C, respectively; compare with Fig. 1, B and C). Sparse immunolabel was also present throughout the cytoplasm; these particles were occasionally associated with distinct membrane vesicles (arrowheads). Magnification: ⫻9,000 (A) and ⫻22,000 (B and C).

increase was less than the increase in total cellular Rhcg. As a result, the proportion of total cellular Rhcg in the intracellular compartment decreased significantly in response to chronic metabolic acidosis (control, 40 ⫾ 4% vs. acidosis, 30 ⫾ 0.4%, P ⬍ 0.05, Fig. 8B). Furthermore, the relative decrease in cytoplasmic immunolabel was quantitatively similar to the relative increase in apical Rhcg immunolabel. In contrast to the intercalated cell, chronic metabolic acidosis did not alter principal cell apical plasma membrane boundary length significantly (control, 11.4 ⫾ 1.0 vs. acidosis, 12.4 ⫾ 1.1 ␮m, P ⫽ NS, Fig. 8C). Because apical Rhcg label increased significantly, apical plasma membrane Rhcg density increased significantly (control, 489 ⫾ 75 vs. acidosis, 1,754 ⫾ 161 ⫻ 103 gold particles/␮m, P ⬍ 0.0002, Fig. 8D). Thus the AJP-Renal Physiol • VOL

Fig. 7. Transmission electron micrographs of Rhcg immunolabel in representative OMCDi principal cell in chronic metabolic acidosis kidney. A: entire cell. B: apical region. C: basal region. In response to chronic metabolic acidosis, there was increased apical plasma membrane Rhcg label in principal cells (arrows, B; compare with Fig. 6B) but no apparent change in apical plasma membrane complexity (A and B). However, apical Rhcg immunolabel in individual principal cells remained much less than that in intercalated cells in chronic metabolic acidosis (compare with Fig. 1B). Chronic metabolic acidosis also increased basolateral plasma membrane Rhcg immunolabel compared with control principal cells (arrows, C; compare with Fig. 6, C). Magnification: ⫻9,000 (A) and ⫻22,000 (B and C).

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Fig. 8. Quantitative assessment of OMCDi principal cell response to chronic metabolic acidosis. A: morphometric analysis of Rhcg immunolabel in the apical plasma membrane, cytoplasm, and the basolateral plasma membrane in response to chronic metabolic acidosis (Au particles/cell profile). Under control conditions, basolateral plasma membrane Rhcg immunolabel exceeds apical plasma membrane Rhcg immunolabel. Chronic metabolic acidosis increases total Rhcg expression in each of the above-mentioned compartments significantly. B: percentage of total cellular Rhcg immunolabel in each of the above-mentioned compartments. The relative proportion was calculated for each cell examined as the compartment-specific expression divided by the total expression in that cell. Chronic metabolic acidosis increases the proportion of total cellular Rhcg in the OMCDi principal cell apical plasma membrane, decreases it in the intracellular compartment, and does not alter the proportion in the basolateral plasma membrane. C: quantifies apical and basolateral plasma membrane boundary length under control and chronic metabolic acidosis conditions (␮m/cell profile). Chronic metabolic acidosis results in a significant increase in principal cell apical plasma membrane boundary length. D: density of Rhcg in the apical and basolateral plasma membranes (Au particles ⫻ 103/␮m boundary length). Chronic metabolic acidosis increases significantly the density of Rhcg label present in both the apical and the basolateral plasma membranes of the OMCDi principal cell. Results are from 25 cells of each type in control animal (5/animal) and 15 cells of each type in chronic metabolic acidosis animals (5/animal).

vs. acidosis, 25.6 ⫾ 3.0 gold particles/cell, P ⬍ 0.002, Fig. 8A). The increase in basolateral Rhcg expression paralleled the increase in total cellular Rhcg expression, resulting in no change in the proportion of total cellular Rhcg in the basolateral plasma membrane (control, 40 ⫾ 3% vs. acidosis, 38 ⫾ 2% P ⫽ NS, Fig. 8B). Similar to the intercalated cell, principal cell basolateral plasma membrane boundary length did not change (control, 15.7 ⫾ 1.0, vs. acidosis, 17.7 ⫾ 2.0 ␮m, P ⫽ NS, Fig. 8C). Thus metabolic acidosis increased basolateral plasma membrane Rhcg density significantly (control, 726 ⫾ 95 vs. acidosis, 1,476 ⫾ 189 ⫻ 103 gold particles/␮m, P ⬍ 0.01, Fig. 8D). DISCUSSION

The current study is the first to quantify cell-specific and subcellular changes in the expression of any ammonia transporter family member in response to a physiological stimulus in either mammalian or nonmammalian tissues. Several important observations result from these studies. First, both the intercalated cell and the principal cell express Rhcg, and chronic metabolic acidosis increases expression in both cell types. Second, Rhcg is present in the apical and the basolateral plasma membranes and in intracellular sites, and the amount of Rhcg in these different compartments is regulated in response to chronic metabolic acidosis. Thus changes in the subcellular distribution of Rhcg may be an important mechanism regulating ammonia transport. However, the regulation of the subcelAJP-Renal Physiol • VOL

lular distribution of Rhcg may differ in these two cell types. Third, substantial basolateral plasma membrane Rhcg is present in both the intercalated cell and the principal cell, and it is increased in response to chronic metabolic acidosis in the principal cell. Thus chronic metabolic acidosis induces both cell-specific changes in OMCDi Rhcg expression and changes in Rhcg’s subcellular location. Ammonia transport is essential for all organisms. The ammonia transporter family includes the Mep family in yeast, Amt family in bacteria and plants, and Rh glycoprotein homologs in higher organisms (21, 23, 33, 36). Previous studies in yeast, plants, and rat kidney have demonstrated increased ammonia transporter family expression in response to conditions associated with increased ammonia transport (8, 22, 34). However, no previous study has examined the cell-specific response in the renal collecting duct or the role of altered subcellular distribution as a possible mechanism regulating protein-mediated ammonia transport. The current study, by demonstrating changes in the subcellular distribution of Rhcg in the rat kidney in response to chronic metabolic acidosis, identifies a previously unrecognized mode of regulation of ammonia transport. Chronic metabolic acidosis induces significant increases in apical plasma membrane Rhcg immunolabel in both the intercalated cell and the principal cell, and these changes are likely to be important in the increased collecting duct ammonia secretion that occurs in response to chronic metabolic acidosis.

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Previous studies have shown that OMCD ammonia secretion involves net NH3 transport (6, 7), a finding compatible with ⫹ transporter-mediated NH⫹ exchange or NH3 transport, or 4 /H with nonionic NH3 diffusion. In vitro studies using cultured mouse collecting duct cells showed that the primary mechanism of both apical and basolateral ammonia transport was a transporter-mediated mechanism, not nonionic NH3 diffusion, that mediates net transmembrane NH3 transport (12, 13). Diffusive transport was a minor component of both apical and basolateral ammonia transport in these in vitro studies but may have a role in transepithelial ammonia secretion, particularly in the inner medulla where high interstitial NH3 concentrations exist. Several studies have shown that Rhcg, along with the related ammonia transporter family members, Rhag and Rhbg, mediate NH3 transport (20, 40, 41, 43). However, some studies suggest Rhcg either mediates only electrogenic NH⫹ 4 transport (24) or that it mediates both electrogenic NH⫹ 4 and electroneutral NH3 transport (1). The reason for these differing findings is not clear at the present time. Finally, facilitated NH3 transport is consistent with transport characteristics of the bacterial ammonia transporter family member AmtB (16, 42). Taken as a whole, these findings strongly suggest that collecting duct ammonia secretion involves transporter-mediated ammonia transport involving Rhcg and that the increased ammonia secretion that occurs in response to chronic metabolic acidosis is due, at least in part, to increased plasma membrane Rhcg expression in both the intercalated cell and the principal cell. Regulation of the subcellular distribution of Rhcg appears to be an important mechanism regulating apical plasma membrane Rhcg expression. Our demonstration that chronic metabolic acidosis increases the proportion of total cellular Rhcg that is present in the apical plasma membrane while simultaneously decreasing the proportion of total cellular Rhcg present in the intracellular compartment suggests that there is redistribution of Rhcg in response to chronic metabolic acidosis. In the intercalated cell, in particular, this appears to be the predominant component of the increased apical plasma membrane Rhcg response to chronic metabolic acidosis. Moreover, the redistribution appears to be membrane specific, as there was no evidence of redistribution to the basolateral plasma membrane, even in the principal cell where there was increased basolateral Rhcg expression in response to chronic metabolic acidosis. The patterns of changes in apical plasma membrane expression appear to differ in the intercalated cell and the principal cell. The intercalated cell responded to chronic metabolic acidosis with increased apical plasma membrane boundary length and no change in the density of Rhcg immunolabel. This contrasts with the principal cell, in which plasma membrane boundary length did not change but plasma membrane Rhcg density increased significantly. These differing patterns are similar to those observed previously using different stimuli and different proteins. For example, the rat intercalated cell responds to respiratory and metabolic acidosis with increased apical plasma membrane boundary length but no change in H⫹-ATPase density (18, 30). Conversely, the rat principal cell responds to vasopressin with increased apical AQP2 density, but not apical boundary length (25). Further evidence that the intercalated and principal cell differ in their response to chronic metabolic acidosis is shown by comparing the relative roles of increased total cellular Rhcg AJP-Renal Physiol • VOL

expression and altered subcellular distribution in the determination of the increase in apical plasma membrane Rhcg expression. In the intercalated cell, apical plasma membrane Rhcg expression increased about fourfold, whereas total cellular Rhcg expression increased only minimally and was accompanied by a substantial decrease in cytoplasmic Rhcg. Conversely, in the principal cell the relative increase in apical plasma membrane Rhcg was similar to the increase in total cellular Rhcg. Thus the pattern of regulation of Rhcg appears to differ in the intercalated and principal cell. In addition to changes in the subcellular distribution of Rhcg in the OMCDi in response to chronic metabolic acidosis, the current studies demonstrate that there is increased Rhcg protein expression by both the intercalated cell and the principal cell. This dual regulatory mechanism is similar to the effect of vasopressin on AQP2 expression and subcellular distribution in collecting duct principal cells, where there is both increased apical plasma membrane targeting and, in response to chronic increases in vasopressin, increased AQP2 protein expression (3, 25, 26). More importantly, changes in both subcellular distribution and protein expression enable synergistic regulation of apical plasma membrane Rhcg expression. The present ultrastructural studies make several important new observations regarding basolateral Rhcg expression. First, the current studies confirm at the ultrastructural level that the basolateral Rhcg immunoreactivity previously reported (29) represents expression in the basolateral plasma membrane and that it does not represent cytoplasmic expression in the basal region of the cell. Quantitatively, basolateral plasma membrane Rhcg expression exceeded apical plasma membrane expression under control conditions in both the intercalated and principal cell, suggesting that Rhcg may play an important role in basolateral ammonia transport under basal conditions in the rat kidney. Second, increased principal cell basolateral expression in response to chronic metabolic acidosis suggests basolateral Rhcg may contribute to the increased ammonia secretion. Because the proportion of total cellular Rhcg in the basolateral plasma membrane did not change with chronic metabolic acidosis, whereas relative apical plasma membrane expression increased, the mechanisms regulating the subcellular distribution of Rhcg in apical and basolateral plasma membranes may differ. It is also important to note that we have reported that renal cells in the rat connecting segment and cortical and outer medullary ducts that express apical Rhcg also express basolateral Rhcg immunoreactivity (29). Thus it is possible that basolateral plasma membrane Rhcg, in species such as the rat, which expresses basolateral RhCG, may contribute to transepithelial ammonia secretion in regions other than the OMCDi and may mediate an important role in renal ammonia metabolism. Interestingly, genetic ablation of Rhbg in the mouse does not alter renal ammonia metabolism (2). This, combined with the apparent absence of basolateral Rhcg in the mouse kidney (32), suggests that neither basolateral Rhbg nor Rhcg is necessary for transepithelial ammonia secretion by the mouse kidney. The mechanisms underlying these differences between the mouse and the rat kidney are not well understood at present. Increasing evidence shows that the OMCDi principal cell plays an important role in acid-base homeostasis. In previous studies, we have shown that the rabbit OMCDi principal cell has apical proton secretion, that mineralocorticoids increase

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both principal cell apical proton secretion rates, and that the relative increase in principal cell apical proton secretion exceeds that observed for the intercalated cell (37, 39). Thus the OMCDi principal cell has both apical ammonia and proton transport mechanisms, and both are regulated in response to physiological conditions. Even though principal cell apical proton secretory rates and apical Rhcg expression are less than in the intercalated cell, because the principal cell is the majority cell type in the OMCDi, it may mediate an important role in acid-base homeostasis. Quantitatively, the relative changes in Rhcg expression in the intercalated and principal cell were similar. Because there are no significant changes in the number of intercalated and principal cells in response the chronic metabolic acidosis (15, 31), it is likely that chronic metabolic acidosis increases transcellular ammonia secretion mediated by Rhcg in the intercalated cell and the principal cell by quantitatively similar amounts. In summary, the current study is the first to demonstrate changes in the subcellular distribution of the ammonia transporter family member, Rhcg, in response to chronic metabolic acidosis and to thereby identify a new mode of regulation of ammonia transport. Our data demonstrate enhanced Rhcg protein expression in the apical plasma membrane in the intercalated cell and the apical and basolateral plasma membranes in the principal cell in rat OMCDi and thus suggest that both cell types contribute to enhanced ammonia secretion in response to chronic metabolic acidosis. Finally, our data suggest that basolateral Rhcg, in addition to apical Rhcg, may mediate an important role in transepithelial ammonia transport. ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical support of Dr. Sharon Matthews, Wencui Zheng, and Karen Chamusco of the University of Florida College of Medicine Electron Microscopy Core Facility. The authors also thank Gina Cowsert for secretarial assistance. GRANTS These studies were supported by National Institutes of Health Grants DK-45788, NS-47624, DK-62081, and DK-63657 and by the Department of Veterans Affairs Merit Review Program. REFERENCES 1. Bakouh N, Benjelloun F, Hulin P, Brouillard F, Edelman A, CherifZahar B, and Planelles G. NH3 is involved in the NH⫹ 4 transport induced by the functional expression of the human Rh C glycoprotein. J Biol Chem 279: 15975–15983, 2004. 2. Chambrey R, Goossens D, Bourgeois S, Picard N, Bloch-Faure M, Leviel F, Geoffroy V, Cambillau M, Colin Y, Paillard M, Houillier P, Cartron JP, and Eladari D. Genetic ablation of Rhbg in mouse does not impair renal ammonium excretion. Am J Physiol Renal Physiol 289: F1281–F1290, 2005. 3. DiGiovanni SR, Nielsen S, Christensen EI, and Knepper MA. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc Natl Acad Sci USA 91: 8984 – 8988, 1994. 4. DuBose TD Jr, DW Good, LL Hamm, and SM Wall. Ammonium transport in the kidney: new physiological concepts and their clinical implications. J Am Soc Nephrol 1: 1193–1203, 1991. 5. Eladari D, Cheval L, Quentin F, Bertrand O, Mouro I, Cherif-Zahar B, Cartron JP, Paillard M, Doucet A, and Chambrey R. Expression of RhCG, a new putative NH3/NH⫹ 4 transporter, along the rat nephron. J Am Soc Nephrol 13: 1999 –2008, 2002. 6. Flessner MF, Wall SM, and Knepper MA. Permeabilities of rat collecting duct segments to NH3 and NH⫹ 4 . Am J Physiol Renal Fluid Electrolyte Physiol 260: F264 –F272, 1991. 7. Flessner MF, Wall SM, and Knepper MA. Ammonium and bicarbonate transport in rat outer medullary collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 262: F1–F7, 1992. AJP-Renal Physiol • VOL

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8. Gazzarrini S, Lejay L, Gojon A, Ninnemann O, Frommer WB, and von Wiren N. Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11: 937–948, 1999. 9. Good DW and Knepper MA. Ammonia transport in the mammalian kidney. Am J Physiol Renal Fluid Electrolyte Physiol 248: F459 –F471, 1985. 10. Hamm LL and Simon EE. Roles and mechanisms of urinary buffer excretion. Am J Physiol Renal Fluid Electrolyte Physiol 253: F595–F605, 1987. 11. Hamm LL, Weiner ID, and Vehaskari VM. Structural-functional characteristics of acid-base transport in the rabbit collecting duct. Semin Nephrol 11: 453– 464, 1991. 12. Handlogten ME, Hong SP, Westhoff CM, and Weiner ID. Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3). Am J Physiol Renal Physiol 287: F628 –F638, 2004. 13. Handlogten ME, Hong SP, Westhoff CM, and Weiner ID. Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3). Am J Physiol Renal Physiol 289: F347–F358, 2005. 14. Handlogten ME, Hong SP, Zhang L, Vander AW, Steinbaum ML, Campbell-Thompson M, and Weiner ID. Expression of the ammonia transporter proteins, Rh B glycoprotein and Rh C glycoprotein, in the intestinal tract. Am J Physiol Gastrointest Liver Physiol 288: G1036 – G1047, 2005. 15. Hansen GP, Tisher CC, and Robinson RR. Response of the collecting duct to disturbances of acid-base and potassium balance. Kidney Int 17: 326 –337, 1980. 16. Khademi S, O’Connell J III, Remis J, Robles-Colmenares Y, Miercke LJ, and Stroud RM. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science 305: 1587–1594, 2004. 17. Klein JD, Rouillard P, Roberts BR, and Sands JM. Acidosis mediates the upregulation of UT-A protein in livers from uremic rats. J Am Soc Nephrol 13: 581–587, 2002. 18. Madsen KM and Tisher CC. Cellular response to acute respiratory acidosis in rat medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 245: F670 –F679, 1983. 19. Madsen KM and Tisher CC. Response of intercalated cells of rat outer medullary collecting duct to chronic metabolic acidosis. Lab Invest 51: 268 –276, 1984. 20. Mak DD, Dang B, Weiner ID, Foskett JK, and Westhoff CM. Characterization of transport by the kidney Rh glycoproteins, RhBG and RhCG. Am J Physiol Renal Physiol 290: F297–F305, 2006. 21. Marini AM, Soussi-Boudekou S, Vissers S, and Andre B. A family of ammonium transporters in Saccharomyces cerevisiae. Mol Cell Biol 17: 4282– 4293, 1997. 22. Montesinos ML, Muro-Pastor AM, Herrero A, and Flores E. Ammonium/methylammonium permeases of a cyanobacterium. Identification and analysis of three nitrogen-regulated amt genes in synechocystis sp PCC 6803. J Biol Chem 273: 31463–31470, 1998. 23. Nakhoul NL and Hamm LL. Non-erythroid Rh glycoproteins: a putative new family of mammalian ammonium transporters. Pflu¨gers Arch 447: 807– 812, 2004. 24. Nakhoul NL, Palmer SA, Abdulnour-Nakhoul S, Hering-Smith K, and Hamm LL. Ammonium transport in oocytes expressing Rhcg (Abstract). FASEB J 16: A53, 2002. 25. Nielsen S, Chou C, Marples D, Christensen EI, Kishore BK, and Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad Sci USA 92: 1013–1017, 1995. 26. Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663– 11667, 1993. 27. Quentin F, Eladari D, Cheval L, Lopez C, Goossens D, Colin Y, Cartron JP, Paillard M, and Chambrey R. RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron. J Am Soc Nephrol 14: 545–554, 2003. 28. Sajo IM, Goldstein MB, Sonnenberg H, Stinebaugh BJ, Wilson DR, and Halperin ML. Sites of ammonia addition to tubular fluid in rats with chronic metabolic acidosis. Kidney Int 20: 353–358, 1982. 29. Seshadri RM, Klein JD, Kozlowski S, Sands JM, Kim YH, Handlogten ME, Verlander JW, and Weiner ID. Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis. Am J Physiol Renal Physiol 290: F397–F408, 2006.

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30. Verlander JW, Madsen KM, Cannon JK, and Tisher CC. Activation of acid-secreting intercalated cells in rabbit collecting duct with ammonium chloride loading. Am J Physiol Renal Fluid Electrolyte Physiol 266: F633–F645, 1994. 31. Verlander JW, Madsen KM, and Tisher CC. Axial distribution of band 3-positive intercalated cells in the collecting duct of control and ammonium chloride-loaded rabbits. Kidney Int 57: S137–S147, 1996. 32. Verlander JW, Miller RT, Frank AE, Royaux IE, Kim YH, and Weiner ID. Localization of the ammonium transporter proteins, Rh B glycoprotein and Rh C glycoprotein, in the mouse kidney. Am J Physiol Renal Physiol 284: F323–F337, 2003. 33. Von Wiren N, Gazzarrini S, Gojon A, and Frommer WB. The molecular physiology of ammonium uptake and retrieval. Curr Opin Plant Biol 3: 254 –261, 2000. 34. Von Wiren N, Lauter FR, Ninnemann O, Gillissen B, Walch-Liu P, Engels C, Jost W, and Frommer WB. Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato. Plant J 21: 167–175, 2000. 35. Weibel ER and Bolender RP. Stereological techniques for electron microscopic morphometry. In: Principles and Techniques of Electron Microscopy, edited by Hayat MA. New York: Van Nostrand Reinhold, 1973, p. 237–296. 36. Weiner ID. The Rh gene family and renal ammonium transport. Curr Opin Nephrol Hypertens 13: 533–540, 2004.

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37. Weiner ID, Frank AE, and Wingo CS. Apical proton secretion by the inner stripe of the outer medullary collecting duct. Am J Physiol Renal Physiol 276: F606 –F613, 1999. 38. Weiner ID, Miller RT, and Verlander JW. Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein in the mouse liver. Gastroenterology 124: 1432–1440, 2003. 39. Weiner ID, Wingo CS, and Hamm LL. Regulation of intracellular pH in two cell populations of the inner stripe of the rabbit outer medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 265: F406 – F415, 1993. 40. Westhoff CM, Ferreri-Jacobia M, Mak DD, and Foskett JK. Identification of the erythrocyte Rh-blood group glycoprotein as a mammalian ammonium transporter. J Biol Chem 277: 12499 –12502, 2002. 41. Westhoff CM, Siegel DL, Burd CG, and Foskett JK. Mechanism of genetic complementation of ammonium transport in yeast by human erythrocyte Rh-associated glycoprotein (RhAG). J Biol Chem 279: 17443– 17448, 2004. 42. Zheng L, Kostrewa D, Berneche S, Winkler FK, and Li XD. The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli. Proc Natl Acad Sci USA 101: 17090 –17095, 2004. 43. Zidi-Yahiaoui N, Mouro-Chanteloup I, D’Ambrosio AM, Lopez C, Gane P, Kim CVAN, Cartron JP, Colin Y, and Ripoche P. Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells. Biochem J 391: 33– 40, 2005.

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