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【摘要】 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.
【关键词】 intercalated cell principal cell immunogold morphometry
RENAL AMMONIA METABOLISM IS the primary mechanism of acid-base homeostasis ( 4, 9, 10 ). Ammonia 1 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 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 NH 3 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 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 H 2 O 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 NH 4 Cl 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-µm-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 NaN 3, 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 NH 4 Cl for 1 h, rinsed with PBS, treated with 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 x 4,800, recorded on Kodak SO-163 Electron Microscopy film. Individual photomicrographs were examined at a final magnification of x 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 µm ( 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 x 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 ( S V ) was calculated using the formula S V = 2 x 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 x /2 x d, where P represents the number of points over the cell. S V 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/µm 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 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 ).
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: x 9,000 ( A ) and x 22,000 ( B and C ).
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: x 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: x 13,000).
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. 1 C vs. 3 C ).
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: x 9,000 ( A ) and x 22,000 ( B and C ).
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.
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).
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. 5 A ). 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. 5 B ). Simultaneously chronic metabolic acidosis decreased significantly both cytoplasmic Rhcg immunolabel (control 93.6 ± 8.8 vs. acidosis, 35.5 ± 2.1 gold particles/cell profile, P < 0.005, Fig. 5 A ) and the proportion of total Rhcg present in the cytoplasmic compartment (control, 53 ± 2% vs. acidosis, 15 ± 0.4%, P < 0.000005, Fig. 5 B ).
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 (µm/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 x 10 3 /µm 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).
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 µm, P < 0.002, Fig. 5 C ) and surface density (control, 460 ± 28 vs. acidosis, 1,024 ± 84 mm 2 /mm 3, 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 x 10 3 gold particles/µm, P = not significant (NS), Fig. 5 D ].
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. 5 A ). 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. 5 A ) but not for a change in the proportion of total cellular Rhcg that was present in the basolateral plasma membrane (control, 26 ± 1% vs. acidosis, 27 ± 2%, P = NS, Fig. 5 B ). Similarly, neither basolateral plasma membrane boundary length (control, 23.6 ± 3.6, vs. acidosis, 23.7 ± 2.7 µm, P = NS, Fig. 5 C ) nor basolateral plasma membrane Rhcg density changed significantly (control, 2,047 ± 247 vs. acidosis, 2,757 ± 195 gold particles/mm, P = NS, Fig. 5 D ). 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.
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: x 9,000 ( A ) and x 22,000 ( B and C ).
In response to chronic metabolic acidosis, principal cell apical plasma membrane Rhcg immunolabel appeared to increase, 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. 7 C ). There were no observable differences in either apical or basolateral plasma membrane microprojections or complexity compared with principal cells from control kidneys.
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. 6 B ) 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. 1 B ). Chronic metabolic acidosis also increased basolateral plasma membrane Rhcg immunolabel compared with control principal cells (arrows, C; compare with Fig. 6, C ). Magnification: x 9,000 ( A ) and x 22,000 ( B and C ).
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. 8 A ). 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. 8 B ). Although intracellular Rhcg increased significantly (control, 11.4 ± 1.4 vs. acidosis, 20.4 ± 1.4, P < 0.01), the relative 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. 8 B ). Furthermore, the relative decrease in cytoplasmic immunolabel was quantitatively similar to the relative increase in apical Rhcg immunolabel.
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 (µm/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 x 10 3 /µm 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).
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 µm, P = NS, Fig. 8 C ). Because apical Rhcg label increased significantly, apical plasma membrane Rhcg density increased significantly (control, 489 ± 75 vs. acidosis, 1,754 ± 161 x 10 3 gold particles/µm, P < 0.0002, Fig. 8 D ). Thus the 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. 8 A ). In response to chronic metabolic acidosis, basolateral plasma membrane Rhcg expression increased significantly (control, 11.0 ± 0.9 vs. acidosis, 25.6 ± 3.0 gold particles/cell, P < 0.002, Fig. 8 A ). 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. 8 B ). 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 µm, P = NS, Fig. 8 C ). Thus metabolic acidosis increased basolateral plasma membrane Rhcg density significantly (control, 726 ± 95 vs. acidosis, 1,476 ± 189 x 10 3 gold particles/µm, P < 0.01, Fig. 8 D ).
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 subcellular 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. Previous studies have shown that OMCD ammonia secretion involves net NH 3 transport ( 6, 7 ), a finding compatible with transporter-mediated NH 4 + /H + exchange or NH 3 transport, or with nonionic NH 3 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 NH 3 diffusion, that mediates net transmembrane NH 3 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 NH 3 concentrations exist. Several studies have shown that Rhcg, along with the related ammonia transporter family members, Rhag and Rhbg, mediate NH 3 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 NH 3 transport ( 1 ). The reason for these differing findings is not clear at the present time. Finally, facilitated NH 3 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 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 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.
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.
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.
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作者单位：3 Nephrology and Hypertension Section, North Florida/South Georgia Veterans Health System, and 1 Division of Nephrology, Hypertension, and Transplantation, University of Florida College of Medicine, Gainesville, Florida; and 2 Renal Division, Emory University, Atlanta, Georgia