Intercalated cell H+/OH– transporter expression is reduced in Slc26a4 null mice
http://www.100md.com
《美国生理学杂志》
1Department of Medicine, Emory University, Atlanta, Georgia
2Department of Medicine, University of Florida College of Medicine, Gainesville, Florida
5The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom
6Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland
7The Water and Salt Research Center, Aarhus University, Aarhus, Denmark
3Division of Nephrology, University California School of Medicine, Los Angeles, California
4Nephrology and Hypertension Section, North Florida/South Georgia Veterans Health System, Gainesville, Georgia
ABSTRACT
Slc26a4 (Pds) encodes pendrin, a Cl–/HCO3– exchanger expressed in the apical region of type B and non-A, non-B cells, which mediates secretion of OH– equivalents. Thus genetic disruption of Slc26a4 leads to systemic alkalosis in some treatment models. However, humans and mice with genetic disruption of Slc26a4 have normal acid-base balance under basal conditions. Thus we asked: 1) Is net acid excretion altered in Slc26a4 (–/–) mice under basal conditions 2) In the absence of pendrin-mediated OH– secretion, are increases in intracellular and systemic pH minimized through changes in intercalated cell subtype abundance or intercalated cell H+/OH– transporter expression To answer these questions, net acid excretion and H+/OH– transporter expression were examined in Slc26a4 (–/–) and Slc26a4 (+/+) mice using balance studies, immunolocalization, and immunoblotting. Excretion of ammonium, titratable acid, and citrate were the same in Slc26a4 null and wild-type mice. However, urinary pH and PCO2 were much lower in Slc26a4 null relative to wild-type mice due to reduced urinary buffering of secreted H+ by HCO3–. Abundance of non-A, but not type A intercalated cells, was reduced within the cortical collecting ducts of Slc26a4 null mice. Moreover, kidneys from Slc26a4 null mice had reduced H+-ATPase, NBC3 and RhBG total protein expression, particularly within type B and non-A, non-B intercalated cells, although RhCG protein expression was unchanged. Reduced intercalated cell H+/OH– transporter expression is observed in Slc26a4 null mice, which likely attenuates the rise in intracellular and systemic pH expected with genetic disruption of Slc26a4.
NBC3; AE1; acid-base balance
INTERCALATED CELLS ARE PRESENT in the renal collecting duct, distal convoluted tubule, and connecting tubules (CNT) and respond to changes in systemic acid-base balance by appropriately regulating net acid excretion (1, 26).1 Thus intercalated cells are an important component of the kidney's robust ability to finely regulate net acid secretion, thereby contributing to the maintenance of normal arterial pH. Whether an intercalated cell subtype secretes net H+ or OH– equivalents corresponds to the polarity of the H+-ATPase as well as anion exchangers within the cell (1, 3, 26). Type A intercalated cells express the H+-ATPase on the apical plasma membrane which functions in series with the Cl–/HCO3– exchanger AE1 (Slc4a1), expressed on the basolateral plasma membrane to mediate net H+ secretion (26). These transporters are upregulated during metabolic acidosis (32), which augments secretion of H+ equivalents, normalizing arterial pH. Type B intercalated cells mediate secretion of OH– equivalents through apical Cl–/HCO3– exchange, which functions in series with H+-ATPase-mediated H+ efflux across the basolateral plasma membrane (1, 6, 13, 26). Apical anion exchange is upregulated in tandem in models of metabolic alkalosis, which augments secretion of OH– equivalents, attenuating the metabolic alkalosis (16, 26, 31). The function of non-A, non-B intercalated cells is unknown (13).
Apical Cl–/HCO3– exchange of the type B intercalated cell is critical not only in excretion of OH– equivalents during metabolic alkalosis but also in the regulation of intracellular pH (pHi). Emmons and Kurtz (8) observed that when apical Cl–/HCO3– exchange of the type B cell is paralyzed through luminal Cl– removal, pHi increased 0.3–0.5 pH units. Thus the alkalinization expected in the absence of apical anion exchange might lead to changes in cellular function (20), unless attenuated through other H+/OH– transport mechanisms. Apical anion exchange and pendrin are both Cl–/HCO3– exchangers (27–29), both are regulated in tandem (10, 31), both localize to the apical regions of type B and non-A, non-B intercalated cells (8, 15, 25, 36), and both have similar substrate specificity (25, 28, 29, 37). Thus the apical anion exchanger is thought to be the gene product of Slc26a4.
Mutations in Slc26a4 (Pds) result in Pendred syndrome, an autosomal recessive disorder characterized by goiter and deafness (23). While Slc26a4 is highly expressed in the kidney (15, 25, 36), under basal conditions arterial pH does not change either in humans or in mice with genetic disruption of Slc26a4/SLC26A4 (25, 31). However, under conditions that upregulate pendrin in normal mice, such as with administration of aldosterone analogs (31) or with dietary NaCl restriction (37), Slc26a4 (–/–) mice have a more pronounced metabolic alkalosis than wild-type animals, consistent with their reduced capacity to secrete OH– equivalents. The lack of a renal phenotype in persons with Pendred syndrome or in Slc26a4 null mice under basal conditions may reflect the low levels of pendrin expression observed in the apical plasma membrane in type B intercalated cells under this treatment condition. If so, genetic disruption of Slc26a4 should have little effect on net acid excretion and arterial pH under basal conditions. Alternatively, Slc26a4 may be very important under basal conditions, but powerful compensatory mechanisms within the kidney make up for the absence of Slc26a4-mediated net OH– secretion, thereby minimizing changes in systemic and intracellular pH.
We therefore asked the following questions: 1) Is net acid excretion the same in Slc26a4 (–/–) and Slc26a4 (+/+) mice under basal conditions 2) Do type A intercalated cells appropriately regulate net acid secretion in Slc26a4 (–/–) mice under basal conditions, through decreased cell number or though downregulation of H+-ATPase or NH3/NH4+ transporter expression and 3) In mice lacking Slc26a4-mediated net OH– secretion, are transporters that mediate uptake of OH– equivalents, such as NBC3, downregulated in type B and non-A, non-B intercalated cells
METHODS
Animals. Slc26a4 (–/–) mice (25 g) developed by Everett et al. (9) were bred in parallel with coisogenic wild-type mice (129S6/SvEv Tac, Taconic Farms, Germantown, NY). For 7 days, age- and sex-matched Slc26a4 (–/–) and Slc26a4 (+/+) mice were pair-fed a balanced rodent diet prepared as a gel (53881300, Zeigler Brothers, Gardeners, PA) supplemented with NaCl (0.8 meq/day NaCl) (31). Mice were placed in individual metabolic cages for 2 days, urine was collected at 4°C under oil for 24 h before death. Urine total ammonia and titratable acid were measured as reported previously (37). Urinary citrate was measured using a kit (Boehringer Mannheim/R-Biopharm, Darmstadt, Germany). Urinary PCO2 and pH were measured with an ABL5 (Radiometer America, Westlake, OH) and/or a pH-sensitive electrode (MI 410, Microelectrodes, Londonderry, NH) from samples obtained by bladder puncture of mice under anesthesia with isoflurane (31, 37). Urinary HCO3– concentration was calculated from the Henderson-Hasselbalch equation using the measured PCO2 and pH, assuming an of 0.0309 and correcting the first dissociation constant of carbonic acid with the method of Hastings and Sendroy (5, 11). Kidney tissue from Sprague-Dawley rats fed a normal diet was fixed in situ and processed as described previously (34). The Institutional Animal Care and Use Committee at Emory approved all animal treatment protocols.
Antibodies. Rabbit polyclonal antibodies that recognize amino acids 766–780 of the human pendrin protein sequence were employed (25). Protein expression of AE1 (2), AQP2 (4), NBC3 (19), RhBG (33), RhCG (33), and H+-ATPase (14, 22) was detected using polyclonal antibodies reported previously.
Immunohistochemistry. In situ fixation of mouse kidneys was performed as described previously (36). For paraffin embedding, tissues were dehydrated in a series of graded ethyl alcohol followed by xylene and then embedded in paraffin. The sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 30 min at room temperature. To reveal antigens, sections were incubated in 1 mM Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated in a microwave oven for 10 min. Nonspecific binding of IgG was prevented by incubating the sections in 50 mM NH4Cl/PBS for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After sections were rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin, labeling was visualized with horseradish peroxidase-conjugated secondary antibody (1:200, DAKO), followed by incubation with 3,3'-diaminobenzidine (DAB; brown stain). Sections were washed with distilled water, dehydrated with graded ethanol and xylene, mounted in Eukitt, and examined by light microscopy. In parallel with the paraffin embedding described above, kidney tissue was processed for preembedding peroxidase staining using the H+-ATPase antibody and embedded in the Epon mixture described previously (13). Tissue was cut into 1-μm sections, stained with hematoxylin, and examined by light microscopy.
Double immunolabeling with H+-ATPase and AE1, AQP2 and AE1. AE1 immunostaining was performed as described above and then labeled for either AQP2 or the H+-ATPase. Sections that labeled AE1, as visualized by DAB staining, were washed with distilled water and incubated in 0.5% H2O2 in absolute methanol for 30 min at room temperature to quench the remaining peroxidase activity. Sections were washed with distilled water and incubated in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin followed by overnight incubation at 4°C with primary antibodies against AQP2 or H+-ATPase. Sections were rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, 0.2% gelatin, and labeling was visualized with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:200, DAKO). For detection of immunoreactivity, Vector SG (Vector Laboratories) was used as the chromogen to produce a blue gray label, which is easily distinguishable from the brown staining produced by DAB, which labeled AE1.
Electron microscopy and morphometric analysis. Kidneys were preserved in vivo and processed for immunogold localization as described previously (36). Electron microscopy was performed as described previously (31, 36) on ultrathin sections. Type B and non-A, non-B intercalated cell subtypes were identified based on morphological characteristics established previously (31, 36). Cell profile area was quantified in type B intercalated cells from each group (12, 31). In each animal 5 cells of each intercalated cell subtype were selected at random and photographed at a primary magnification of x5,000 and examined at a final magnification of approximately x18,200. The exact magnification was determined using a calibration grid with 2,160 lines/mm.
Immunoblot analysis. Immunoblots of kidney lysates from Slc26a4 (–/–) and Slc26a4 (+/+) mice were done as reported previously (14). The cortex, outer medulla, and inner medulla were dissected and homogenized in dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 μM leupeptin, 1 mM PMSF). Protein samples were dissolved in Laemmli buffer and resolved by SDS-PAGE. The proteins were electrophoretically transferred onto nitrocellulose membranes and probed with antibodies to the H+-ATPase, RhBG, or RhCG. Immunolabeling was detected with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Upstate Biotechnology, Lake Placid, NY) by using an enhanced chemiluminescence system (Amersham International).
Statistical analysis. For data without normal distribution or equal variance, a Mann-Whitney rank sum test was used. In all other studies, comparisons were made between two groups using an unpaired Student’s t-test. P < 0.05 indicates statistical significance. Data are displayed ± SE.
RESULTS
Slc26a4 null mice have decreased urinary pH and PCO2, although NH4+ excretion, titratable acid, and citrate excretion are unchanged. On a normal, NaCl-replete diet, serum chemistries and arterial pH are the same in wild-type and Slc26a4 null mice (31, 37). Moreover, under this treatment condition urinary ammonium excretion, titratable acid, and citrate excretion are also the same in mutant and wild-type mice (Table 1). However, urinary pH was much lower in Slc26a4 (–/–) than in wild-type mice, possibly from reduced urinary buffering. With reduced base secretion, less H+ is titrated, which lowers the concentration of the dissociation products of carbonic acid, which are CO2 and H2O. To determine whether HCO3 buffering is reduced, urinary PCO2 was measured (Table 1). As shown, urinary PCO2 was lower in Slc26a4 null than in wild-type mice. Therefore, under basal conditions and at steady state, although arterial pH is the same in wild-type and Slc26a4 null mice, Slc26a4 null mice have a lower urinary pH and urinary PCO2, which reflects their reduced capacity to secrete HCO3–/OH–(25).
H+-ATPase immunolabel is decreased in Slc26a4 (–/–) mice. Cortical sections from Slc26a4 (+/+) mice showed strong pendrin immunolabeling in the apical region of intercalated cells of the CNT and collecting ducts (CCD) of wild-type mice (Fig. 1A). No pendrin immunolabel was observed in the cortex of Slc26a4 (–/–) mice (Fig. 1B).
We asked whether expression of other intercalated cell H+/OH– transporters is altered in Slc26a4 null mice. Figure 2 shows that total H+-ATPase immunolabel intensity appears decreased in both the cortex and medulla of Slc26a4 (–/–) mice. In wild-type mice, H+-ATPase labeling was clearly observed either in the apical regions (, Fig. 2B) or diffusely distributed within a subset of cells (arrows, Fig. 2B), consistent with the expected polarity of the H+-ATPase within the various intercalated cell subtypes (13). In the cortex of Slc26a4 (–/–) mice, apical H+-ATPase labeling was also readily detected (, Fig. 2E). However, immunolabel was barely detectable in intercalated cells of Slc26a4 null mice, which express the H+-ATPase in a diffuse pattern (arrows, Fig. 2E).
To compare H+-ATPase immunolabel in the different intercalated cell subtypes, double labeling experiments were performed using AE1 as a marker of type A intercalated cells (Fig. 3). In wild-type mice, strong H+-ATPase labeling was observed in the apical regions of AE1-positive (type A) cells (, Fig. 3, A and B). AE1-negative cells (type B and non-A, non-B intercalated cells) also showed strong H+-ATPase labeling in a diffuse pattern (arrows, Fig. 3, A and B). In Slc26A4 (–/–) mice, strong apical H+-ATPase labeling was observed in AE1-positive cells (, Fig. 3, C and D), similar to observations made in wild-type mice. However, AE1-negative intercalated cells (type B or non-A, non-B intercalated cells) from these mutant mice showed extremely weak H+-ATPase labeling (arrows, Fig. 3, C and D). Moreover, by immunogold cytochemistry, H+-ATPase immunolabel appeared weaker in type B intercalated cells of Slc26a4 null than in wild-type mice (Fig. 4).
Figure 2 shows that H+-ATPase expression might be lower in the medulla of Slc26a4 null than in wild-type mice. Thus H+-ATPase expression was quantified in the cortex, outer medulla, and inner medulla from Slc26A4 (+/+) and Slc26A4 (–/–) mice (Fig. 5). The anti-H+-ATPase antibody detected a protein by immunoblot that migrates at 56 kDa, the expected mobility of the B1 subunit of H+-ATPase. Band density was reduced in all regions of the kidney from Slc26A4 (–/–) mice relative to wild-type. However, these differences were most striking in the cortex, where all intercalated cell subtypes are expressed (1, 13). Differences were the least striking within the inner medulla, where type A intercalated cells are present but where type B and non-A, non-B intercalated cells are absent (21). We conclude that H+-ATPase expression is reduced in kidneys from Slc26a4 (–/–) mice. However, the reduced H+-ATPase expression observed in Slc26a4 null mice is much more apparent within type B and non-A, non-B cells than within type A intercalated cells.
Decreased immunolabeling of NBC3 in the cortex of Slc26A4 (–/–) mice. Because H+-ATPase immunoreactivity is reduced in non-A intercalated cells (i.e., type B and non-A, non-B cells) of Slc26a4 null mice, further studies investigated whether expression of other H+/OH– transporters, such as the Na+/HCO3– cotransporter, NBC3, is changed within type B and non-A, non-B intercalated cells of Slc26a4 null mice. In rat kidney, NBC3 localizes to the apical plasma membrane of type A intercalated cells and the basolateral plasma membrane and cytoplasmic vesicles of type B intercalated cells (19). However, the localization of NBC3 has not been determined in non-A, non-B intercalated cells. Figure 6 shows that in rat non-A, non-B intercalated cells, NBC3 localizes to the apical plasma membrane and subapical cytoplasmic vesicles (arrows).
NBC3 labeling within the cortex and the initial portion of the inner medulla of Slc26a4 (+/+) and Slc26a4 (–/–) mice is shown in Fig. 7. Abundant NBC3-positive cells were detected in the cortex of wild-type mice. Strong apical NBC3 labeling was observed in both the apical regions (, Fig. 7, A–C) and in the cytoplasm and basal regions (arrows, Fig. 7B) of wild-type mice. In Slc26A4 (–/–) mice, NBC3 labeling was readily observed in the apical regions of a subset of cells (, Fig. 7, E and F), whereas labeling was weak in cells expressing NBC3 in the cytoplasm and in the basolateral regions (arrows, Fig. 7E). Double immunolabeling of NBC3 and AE1 (Fig. 8) in Slc26a4 null mice showed that cells strongly expressing NBC3 are AE1-positive, type A intercalated cells (, Fig. 8, C and D), whereas NBC3 labeling is very weak in cells that are AE1 negative (arrows, Fig. 8, C and D).
NH4+/NH3 transporter expression is reduced in kidneys from Slc26a4 (–/–) mice. RhBG and RhCG are NH4+/NH3 transporters expressed along the CNT and collecting duct (33). Because these transporters may participate in the process of ammonium secretion (38), limiting their expression might reduce net acid secretion, thereby attenuating a metabolic alkalosis. Thus RhBG and RhCG expression was determined in kidneys from wild-type and Slc26a4 null mice (Fig. 9). As shown, although RhCG expression was the same in kidneys from wild-type and mutant mice, RhBG expression was markedly reduced in kidneys from Slc26a4 null mice.
Type A intercalated cell expression is maintained in Slc26a4 (–/–) mice. Because type A intercalated cells mediate net H+ secretion, we asked whether net acid excretion is maintained in the absence of Slc26a4-mediated OH– secretion by decreased type A intercalated cell numbers. To answer this question, cells were divided into three groups: 1) AQP2-positive, AE1-negative cells (principal cells, arrowheads); 2) AE1-positive, AQP2-negative cells (type A intercalated cells, ); and 3) AE1-negative, AQP2-negative cells (type B and non-A, non-B intercalated cells, arrows). These cell types were quantified per high-power field (Fig. 10, Table 2). In the CNT, initial portion of the collecting duct, and the outer medullary collecting duct, the number of principal and type A intercalated cells per high-power field was similar in Slc26a4 (–/–) and Slc26a4 (+/+) mice, although non-A intercalated cell number (type B and non-A, non-B) was reduced by 20% in the CCD of Slc26a4 null mice relative to wild-type. However, reduced cell counts per field could reflect either reduced total cell number per kidney or smaller cell size. Because type B cells are much more prevalent than non-A, non-B intercalated cells within the mouse CCD (30), it is more likely that type B, rather than non-A, non-B intercalated cell number is reduced in the CCD of Slc26a4 null mice relative to wild-type. Thus type B intercalated cell size within the CCD was measured. In random sections of type B intercalated cells from wild-type mice, cell profile area was 6.84 ± 0.27 x 10–5 mm2 (n = 3 mice) but 4.62 ± 0.27 x 10–5 mm2 (n = 4 mice) in cells from Slc26a4 null mice (P < 0.05). Thus the reduced non-A intercalated cell number observed by light microscopy in the CCD of these Slc26a4 (–/–) mice reflects, in part, the smaller type B cell size (30).
Morphological characteristics of type B intercalated cells from Slc26a4 null mice. Because type B cell size and H+/OH– transporters expression are markedly reduced in kidneys of Slc26a4 null mice, the morphological characteristics of this cell type were studied (Fig. 4). As shown, type B cells of Slc26a4 null mice have a less prominent apical vesicle-free band and have more prominent apical plasma membrane microprojections compared with wild-type mice. Relative to wild-type mice, type B cells of Slc26a4 null mice also have markedly fewer cytoplasmic vesicles, and less complex basolateral plasma membrane infoldings.
DISCUSSION
Previous studies by our laboratory have shown that the Cl–/HCO3– exchanger pendrin mediates secretion of OH–/HCO3– into the luminal fluid of the CCD (25). Thus the purpose of this study was to explore how mice maintain intracellular and systemic pH under basal conditions in the absence of Slc26a4-mediated OH– secretion. We observed that H+-ATPase expression is reduced in the cortex as well and in the outer and the inner medulla. Because the H+-ATPase is expressed most highly within type A intercalated cells of the inner medulla, H+-ATPase expression is reduced in type A intercalated cells, at least within the inner medullary collecting duct. However, the most striking finding of this study was the marked downregulation of transporters that mediate net OH– uptake/or H+ efflux within type B and non-A, non-B intercalated cells. The marked downregulation of the H+-ATPase and NBC3 expression observed in these cell types should decrease net OH– uptake or decreased net H+ efflux by these cells, thereby attenuating the rise in pHi expected in the absence of Slc26a4-mediated OH– secretion.
NBC3 and the H+-ATPase are closely associated proteins (24), which coimmunoprecipitate and colocalize within intercalated cells. The present study and previous studies have shown that the H+-ATPase and NBC3 localize to the apical plasma membrane and subapical intracellular vesicles in type A and non-A, non-B intercalated cells (19, 24, 30), whereas in the type B intercalated cell they localize to the basolateral plasma membrane and to cytoplasmic vesicles (19, 24, 30). Moreover, NBC3 and the H+-ATPase are regulated in parallel in treatment models such as metabolic acidosis (18). Although the H+-ATPase is a critical component of net H+ secretion along the collecting duct, NBC3 plays only a minor role in transepithelial transport of H+ equivalents (39). Instead, NBC3 is thought to regulate pHi (39). Yip et al. (39) observed that in type A intercalated cells, pHi recovery following an acid load is sensitive to Na+ and EIPA present in the luminal fluid, which reflects NBC3-mediated OH– uptake (39). Thus downregulation of NBC3 and the H+-ATPase within non-A, non-B and type B intercalated cells of Slc26A4 (–/–) mice should limit net OH– uptake or net H+ efflux by these cells (7), thereby attenuating the intracellular alkalization expected in the absence of Slc26a4-mediated OH– efflux (25).
Because type A intercalated cells secrete H+ through the H+-ATPase, which localizes to the apical plasma membrane, we explored whether the metabolic alkalosis expected in the absence of Slc26a4-mediated OH– secretion is attenuated by reducing type A intercalated cell number or through reduced H+-ATPase protein expression. We observed that Slc26a4 null mice do not maintain normal arterial pH by reducing type A intercalated cell number. However, H+-ATPase protein expression is reduced in the medulla of Slc26a4 null mice, which should reduce secretion of net H+ equivalents and thus minimize the metabolic alkalosis expected in the absence of Slc26a4-mediated OH– secretion.
Despite the reduced H+-ATPase expression observed along the collecting duct, urinary pH was markedly reduced in Slc26a4 null mice relative to wild-type. Thus we explored the possibility that the fall in urinary pH occurred from reduced urinary buffering, rather than increased H+ secretion. Secreted HCO3– titrates secreted H+ to form carbonic acid, which dissociates into H2O and CO2. Thus the lower urinary PCO2 observed is consistent with the reduced capacity of Slc26a4 null mice to secrete HCO3–. However, we cannot exclude the possibility that the lower PCO2 observed in urine of Slc26a4 null mice reflects, at least in part, increased renal CO2 absorption or decreased renal carbonic anhydrase activity (5).
Reduced secretion of protonated nonbicarbonate buffers should attenuate the alkalosis expected in Slc26a4 null mice. Excretion of ammonium is the major regulatable component of net acid excretion (35). Because urinary pH was much lower in Slc26a4 null than in wild-type mice, the driving force for NH3 secretion into the luminal fluid should be markedly increased. Thus greater urinary excretion of NH4+ is expected in Slc26a4 null than in wild-type mice (35). However, this was not observed. Several possible mechanisms might explain why NH4+ excretion was unchanged in Slc26a4 null mice despite their extremely low urinary pH, including reduced ammonium production by the proximal tubule (17). Alternatively, reduced transfer of NH3/NH4+ from the interstitium to the collecting duct lumen could increase renal NH4+ exit through the renal vein while decreasing renal NH4+ exit through excretion in the urine. We observed a marked reduction in expression of the NH3/NH4+ transporter RhBG, which is expressed along the basolateral region of all cells within the CNT and within principal cells and type A intercalated cells of the CCD and medullary collecting duct (33). However, within the medullary collecting duct, RhBG is most highly expressed in type A intercalated cells (33). RhBG is thought to contribute to transepithelial ammonium secretion in the CNT and collecting duct, although this has not been proven experimentally (38). If this protein participates in the process of NH4+/NH3 secretion along the collecting duct, by reducing its expression in Slc26a4 null mice, ammonium secretion should be limited, thereby reducing net acid secretion and attenuating the expected metabolic alkalosis. NH4+ not excreted in the urine should exit the kidney through the renal vein, stimulating hepatic ureagenesis, which consumes NH4+ and HCO3–. By stimulating NH4+ exit through the renal vein while reducing NH4+ excretion into the final urine, arterial pH could be kept within the normal range, despite a very low urinary pH (17). In some treatment models, blood urea nitrogen is higher in Slc26a4 null mice than in wild-type mice (31, 37), which may reflect increased ureagenesis in the mutant mice.
Phosphate excretion represents the major component of titratable acid. Because phosphate excretion was the same in Slc26a4 null and wild-type mice (37), greater urinary acidification should increase protonation of HPO42– to H2PO4–, thus increasing titratable acid. Although titratable acid excretion was approximately twice as great in Slc26a4 (–/–) mice as in wild-type mice, the results did not achieve statistical significance. Interestingly, titratable acid is greater in Slc26a4 null than wild-type mice under treatment conditions that increase Slc26a4 expression in normal mice, such as with dietary NaCl restriction (37).
The present study shows that Slc26a4 null mice regulate net acid excretion through adaptations that occur within intercalated cells, resulting in cell-specific decreases in the expression of many H+/OH– transport mechanisms. Compensatory mechanisms that may occur in other cell types will require further study. In conclusion, H+-ATPase and NBC3 expression are differentially regulated in intercalated cell subtypes in Slc26a4 (–/–) mice. The decrease in H+/OH– transporter expression observed within intercalated cells is probably a compensatory mechanism that attenuates the increases in net acid excretion and pHi expected in the absence of Slc26a4-mediated OH– secretion.
ACKNOWLEDGMENTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52935 (to S. M. Wall).
FOOTNOTES
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.
1 Net acid secretion is equivalent to urinary excretion of NH4+ + titratable acid (TA) – urinary HCO3–.
REFERENCES
Alper SL, Natale J, Gluck S, Lodish HF, and Brown D. Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase. Proc Natl Acad Sci USA 86: 5429–5433, 1989.
Blomqvist SR, Vidarsson H, Fitzgerald S, Johansson BR, Ollerstam A, Brown R, Persson AEG, Bergstrom G, and Enerback S. Distal renal tubular acidosis in mice that lack the forkhead transcription factor Foxi1. J Clin Invest 113: 1560–1570, 2004.
Brown D, Hirsch S, and Gluck S. Localization of a proton-pumping ATPase in rat kidney. J Clin Invest 82: 2114–2126, 1988.
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.
DuBose TD Jr. Hydrogen ion secretion by the collecting duct as a determinant of the urine to blood PCO2 gradient in alkaline urine. J Clin Invest 69: 145–156, 1982.
Emmons C. New subtypes of rabbit CCD intercalated cells as functionally defined by anion exchange and H+-ATPase activity (Abstract). J Am Soc Nephrol 4: 838, 1993.
Emmons C. H+ extrusion in mouse cortical collecting duct (CCD) intercalated cells (Abstract). J Am Soc Nephrol 8: 5A, 1997.
Emmons C and Kurtz I. Functional characterization of three intercalated cell subtypes in the rabbit outer cortical collecting duct. J Clin Invest 93: 417–423, 1994.
Everett LA, Belyantseva IA, Noben-Trauth K, Cantos R, Chen A, Thakkar SI, Hoogstraten-Miller SL, Kachar B, Wu DK, and Green ED. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome. Hum Mol Genet 10: 153–161, 2001.
Frische S, Kwon TH, Frkir J, Madsen KM, and Nielsen S. Regulated expression of pendrin in rat kidney in response to chronic NH4Cl or NaHCO3 loading. Am J Physiol Renal Physiol 284: F584–F593, 2003.
Hastings AB and Sendroy J Jr. The effect of variation in ionic strength on the apparent first and second dissociation constants of carbonic acid. J Biol Chem 65: 445–455, 1925.
Henger A, Tutt P, Riesen WF, Hulter HN, and Krapf R. Acid-base and endocrine effects of aldosterone and angiotensin II inhibition in metabolic acidosis in human patients. J Lab Clin Med 136: 379–389, 2000.
Kim J, Kim YH, Cha JH, Tisher CC, and Madsen KM. Intercalated cell subtypes in connecting tubule and cortical collecting duct of rat and mouse. J Am Soc Nephrol 10: 1–12, 1999.
Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frkir J, and Nielsen S. Altered expression of renal acid-base transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol 285: F1244–F1257, 2003.
Kim YH, Kwon TH, Frische S, Kim J, Tisher CC, Madsen KM, and Nielsen S. Immunocytochemical localization of pendrin in intercalated cell subtypes in rat and mouse kidney. Am J Physiol Renal Physiol 283: F744–F754, 2002.
Knepper MA, Good DW, and Burg MB. Mechanism of ammonia secretion by cortical collecting ducts of rabbits. Am J Physiol Renal Fluid Electrolyte Physiol 247: F729–F738, 1984.
Kurtz I, Dass PD, and Cramer S. The importance of renal ammonia metabolism to whole body acid-base balance: a reanalysis of the pathophysiology of renal tubular acidosis. Miner Electrolyte Metab 16: 331–340, 1990.
Kwon TH, Fulton C, Wang W, Kurtz I, Frkir J, Aalkjr C, and Nielsen S. Chronic metabolic acidosis upregulates rat kidney Na+-HCO3– cotransporters NBCn1 and NBC3 but not NBC1. Am J Physiol Renal Physiol 282: F341–F351, 2002.
Kwon TH, Pushkin A, Abuladze N, Nielsen S, and Kurtz I. Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney. Am J Physiol Renal Physiol 278: F327–F336, 2000.
Lagadic-Gossmann D, Huc L, and Lecureur V. Alterations of intracellular pH homeostasis in apoptosis: origins and roles. Cell Death Differ 11: 953–961, 2004.
Madsen KM and Tisher CC. Structural-functional relationships along the distal nephron. Am J Physiol Renal Fluid Electrolyte Physiol 250: F1–F15, 1986.
Nelson RD, Guo XL, Masood K, Brown D, Kalkbrenner M, and Gluck S. Selectively amplified expression of an isoform of the vacuolar H+-ATPase 56-kilodalton subunit in renal intercalated cells. Proc Natl Acad Sci USA 89: 3541–3545, 1992.
Pendred V. Deaf-mutism and goitre. Lancet 2: 532, 1896.
Pushkin A, Yip KP, Clark I, Abuladze N, Kwon TH, Tsuruoka S, Schwartz GJ, Nielsen S, and Kurtz I. NBC3 expression in rabbit collecting duct: colocalization with vacuolar H+-ATPase. Am J Physiol Renal Physiol 277: F974–F981, 1999.
Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, and Green ED. Pendrin, encoded by the pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci USA 98: 4221–4226, 2001.
Schuster VL. Function and regulation of collecting duct intercalated cells. Annu Rev Physiol 55: 267–288, 1993.
Scott DA and Karniski LP. Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange. Am J Physiol Cell Physiol 278: C207–C211, 2000.
Scott DA, Wang R, Kreman TM, Sheffield VC, and Karniski LP. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nature Genetics 21: 440–443, 1999.
Soleimani M, Greeley T, Petrovic S, Wang Z, Amlal H, Kopp P, and Burnham CE. Pendrin: an apical Cl–/OH–/HCO3– exchanger in the kidney cortex. Am J Physiol Renal Physiol 280: F356–F364, 2001.
Teng-ummnuay P, Verlander JW, Yuan W, Tisher CC, and Madsen KM. Identification of distinct subpopulations of intercalated cells in the mouse collecting duct. J Am Soc Nephrol 7: 260–274, 1996.
Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett LA, Green ED, and Wall SM. Deoxycorticosterone upregulates Pds (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003.
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.
Verlander JW, Miller RT, Frank AE, Royaux IE, Kim YH, and Weiner ID. Localization of the ammonium transporter proteins RhBg and RhCg in mouse kidney. Am J Physiol Renal Physiol 284: F323–F337, 2003.
Verlander JW, Tran TM, Zhang L, Kaplan MR, and Hebert SC. Estradiol enhances thiazide-sensitive NaCl cotransporter density in the apical plasma membrane of the distal convoluted tubule in ovariectomized rats. J Clin Invest 101: 1661–1669, 1998.
Wall SM. Mechanisms of NH4+ and NH3 transport during hypokalemia. Acta Physiol Scand 179: 325–330, 2003.
Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, and Verlander JW. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284: F229–F241, 2003.
Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, and Verlander JW. NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl– conservation. Hypertension 44: 1–6, 2004.
Weiner ID. The Rh gene family and renal ammonium transport. Curr Opin Nephrol Hypertens 13: 533–540, 2004.
Yip KP, Tsuruoka S, Schwartz GJ, and Kurtz I. Apical H+/base transporters mediating bicarbonate absorption and pHi regulation in the OMCD. Am J Physiol Renal Physiol 283: F1098–F1104, 2002.(Young-Hee Kim, Jill W. Verlander, Sharon)
2Department of Medicine, University of Florida College of Medicine, Gainesville, Florida
5The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom
6Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland
7The Water and Salt Research Center, Aarhus University, Aarhus, Denmark
3Division of Nephrology, University California School of Medicine, Los Angeles, California
4Nephrology and Hypertension Section, North Florida/South Georgia Veterans Health System, Gainesville, Georgia
ABSTRACT
Slc26a4 (Pds) encodes pendrin, a Cl–/HCO3– exchanger expressed in the apical region of type B and non-A, non-B cells, which mediates secretion of OH– equivalents. Thus genetic disruption of Slc26a4 leads to systemic alkalosis in some treatment models. However, humans and mice with genetic disruption of Slc26a4 have normal acid-base balance under basal conditions. Thus we asked: 1) Is net acid excretion altered in Slc26a4 (–/–) mice under basal conditions 2) In the absence of pendrin-mediated OH– secretion, are increases in intracellular and systemic pH minimized through changes in intercalated cell subtype abundance or intercalated cell H+/OH– transporter expression To answer these questions, net acid excretion and H+/OH– transporter expression were examined in Slc26a4 (–/–) and Slc26a4 (+/+) mice using balance studies, immunolocalization, and immunoblotting. Excretion of ammonium, titratable acid, and citrate were the same in Slc26a4 null and wild-type mice. However, urinary pH and PCO2 were much lower in Slc26a4 null relative to wild-type mice due to reduced urinary buffering of secreted H+ by HCO3–. Abundance of non-A, but not type A intercalated cells, was reduced within the cortical collecting ducts of Slc26a4 null mice. Moreover, kidneys from Slc26a4 null mice had reduced H+-ATPase, NBC3 and RhBG total protein expression, particularly within type B and non-A, non-B intercalated cells, although RhCG protein expression was unchanged. Reduced intercalated cell H+/OH– transporter expression is observed in Slc26a4 null mice, which likely attenuates the rise in intracellular and systemic pH expected with genetic disruption of Slc26a4.
NBC3; AE1; acid-base balance
INTERCALATED CELLS ARE PRESENT in the renal collecting duct, distal convoluted tubule, and connecting tubules (CNT) and respond to changes in systemic acid-base balance by appropriately regulating net acid excretion (1, 26).1 Thus intercalated cells are an important component of the kidney's robust ability to finely regulate net acid secretion, thereby contributing to the maintenance of normal arterial pH. Whether an intercalated cell subtype secretes net H+ or OH– equivalents corresponds to the polarity of the H+-ATPase as well as anion exchangers within the cell (1, 3, 26). Type A intercalated cells express the H+-ATPase on the apical plasma membrane which functions in series with the Cl–/HCO3– exchanger AE1 (Slc4a1), expressed on the basolateral plasma membrane to mediate net H+ secretion (26). These transporters are upregulated during metabolic acidosis (32), which augments secretion of H+ equivalents, normalizing arterial pH. Type B intercalated cells mediate secretion of OH– equivalents through apical Cl–/HCO3– exchange, which functions in series with H+-ATPase-mediated H+ efflux across the basolateral plasma membrane (1, 6, 13, 26). Apical anion exchange is upregulated in tandem in models of metabolic alkalosis, which augments secretion of OH– equivalents, attenuating the metabolic alkalosis (16, 26, 31). The function of non-A, non-B intercalated cells is unknown (13).
Apical Cl–/HCO3– exchange of the type B intercalated cell is critical not only in excretion of OH– equivalents during metabolic alkalosis but also in the regulation of intracellular pH (pHi). Emmons and Kurtz (8) observed that when apical Cl–/HCO3– exchange of the type B cell is paralyzed through luminal Cl– removal, pHi increased 0.3–0.5 pH units. Thus the alkalinization expected in the absence of apical anion exchange might lead to changes in cellular function (20), unless attenuated through other H+/OH– transport mechanisms. Apical anion exchange and pendrin are both Cl–/HCO3– exchangers (27–29), both are regulated in tandem (10, 31), both localize to the apical regions of type B and non-A, non-B intercalated cells (8, 15, 25, 36), and both have similar substrate specificity (25, 28, 29, 37). Thus the apical anion exchanger is thought to be the gene product of Slc26a4.
Mutations in Slc26a4 (Pds) result in Pendred syndrome, an autosomal recessive disorder characterized by goiter and deafness (23). While Slc26a4 is highly expressed in the kidney (15, 25, 36), under basal conditions arterial pH does not change either in humans or in mice with genetic disruption of Slc26a4/SLC26A4 (25, 31). However, under conditions that upregulate pendrin in normal mice, such as with administration of aldosterone analogs (31) or with dietary NaCl restriction (37), Slc26a4 (–/–) mice have a more pronounced metabolic alkalosis than wild-type animals, consistent with their reduced capacity to secrete OH– equivalents. The lack of a renal phenotype in persons with Pendred syndrome or in Slc26a4 null mice under basal conditions may reflect the low levels of pendrin expression observed in the apical plasma membrane in type B intercalated cells under this treatment condition. If so, genetic disruption of Slc26a4 should have little effect on net acid excretion and arterial pH under basal conditions. Alternatively, Slc26a4 may be very important under basal conditions, but powerful compensatory mechanisms within the kidney make up for the absence of Slc26a4-mediated net OH– secretion, thereby minimizing changes in systemic and intracellular pH.
We therefore asked the following questions: 1) Is net acid excretion the same in Slc26a4 (–/–) and Slc26a4 (+/+) mice under basal conditions 2) Do type A intercalated cells appropriately regulate net acid secretion in Slc26a4 (–/–) mice under basal conditions, through decreased cell number or though downregulation of H+-ATPase or NH3/NH4+ transporter expression and 3) In mice lacking Slc26a4-mediated net OH– secretion, are transporters that mediate uptake of OH– equivalents, such as NBC3, downregulated in type B and non-A, non-B intercalated cells
METHODS
Animals. Slc26a4 (–/–) mice (25 g) developed by Everett et al. (9) were bred in parallel with coisogenic wild-type mice (129S6/SvEv Tac, Taconic Farms, Germantown, NY). For 7 days, age- and sex-matched Slc26a4 (–/–) and Slc26a4 (+/+) mice were pair-fed a balanced rodent diet prepared as a gel (53881300, Zeigler Brothers, Gardeners, PA) supplemented with NaCl (0.8 meq/day NaCl) (31). Mice were placed in individual metabolic cages for 2 days, urine was collected at 4°C under oil for 24 h before death. Urine total ammonia and titratable acid were measured as reported previously (37). Urinary citrate was measured using a kit (Boehringer Mannheim/R-Biopharm, Darmstadt, Germany). Urinary PCO2 and pH were measured with an ABL5 (Radiometer America, Westlake, OH) and/or a pH-sensitive electrode (MI 410, Microelectrodes, Londonderry, NH) from samples obtained by bladder puncture of mice under anesthesia with isoflurane (31, 37). Urinary HCO3– concentration was calculated from the Henderson-Hasselbalch equation using the measured PCO2 and pH, assuming an of 0.0309 and correcting the first dissociation constant of carbonic acid with the method of Hastings and Sendroy (5, 11). Kidney tissue from Sprague-Dawley rats fed a normal diet was fixed in situ and processed as described previously (34). The Institutional Animal Care and Use Committee at Emory approved all animal treatment protocols.
Antibodies. Rabbit polyclonal antibodies that recognize amino acids 766–780 of the human pendrin protein sequence were employed (25). Protein expression of AE1 (2), AQP2 (4), NBC3 (19), RhBG (33), RhCG (33), and H+-ATPase (14, 22) was detected using polyclonal antibodies reported previously.
Immunohistochemistry. In situ fixation of mouse kidneys was performed as described previously (36). For paraffin embedding, tissues were dehydrated in a series of graded ethyl alcohol followed by xylene and then embedded in paraffin. The sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 30 min at room temperature. To reveal antigens, sections were incubated in 1 mM Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated in a microwave oven for 10 min. Nonspecific binding of IgG was prevented by incubating the sections in 50 mM NH4Cl/PBS for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After sections were rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin, labeling was visualized with horseradish peroxidase-conjugated secondary antibody (1:200, DAKO), followed by incubation with 3,3'-diaminobenzidine (DAB; brown stain). Sections were washed with distilled water, dehydrated with graded ethanol and xylene, mounted in Eukitt, and examined by light microscopy. In parallel with the paraffin embedding described above, kidney tissue was processed for preembedding peroxidase staining using the H+-ATPase antibody and embedded in the Epon mixture described previously (13). Tissue was cut into 1-μm sections, stained with hematoxylin, and examined by light microscopy.
Double immunolabeling with H+-ATPase and AE1, AQP2 and AE1. AE1 immunostaining was performed as described above and then labeled for either AQP2 or the H+-ATPase. Sections that labeled AE1, as visualized by DAB staining, were washed with distilled water and incubated in 0.5% H2O2 in absolute methanol for 30 min at room temperature to quench the remaining peroxidase activity. Sections were washed with distilled water and incubated in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin followed by overnight incubation at 4°C with primary antibodies against AQP2 or H+-ATPase. Sections were rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, 0.2% gelatin, and labeling was visualized with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:200, DAKO). For detection of immunoreactivity, Vector SG (Vector Laboratories) was used as the chromogen to produce a blue gray label, which is easily distinguishable from the brown staining produced by DAB, which labeled AE1.
Electron microscopy and morphometric analysis. Kidneys were preserved in vivo and processed for immunogold localization as described previously (36). Electron microscopy was performed as described previously (31, 36) on ultrathin sections. Type B and non-A, non-B intercalated cell subtypes were identified based on morphological characteristics established previously (31, 36). Cell profile area was quantified in type B intercalated cells from each group (12, 31). In each animal 5 cells of each intercalated cell subtype were selected at random and photographed at a primary magnification of x5,000 and examined at a final magnification of approximately x18,200. The exact magnification was determined using a calibration grid with 2,160 lines/mm.
Immunoblot analysis. Immunoblots of kidney lysates from Slc26a4 (–/–) and Slc26a4 (+/+) mice were done as reported previously (14). The cortex, outer medulla, and inner medulla were dissected and homogenized in dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 μM leupeptin, 1 mM PMSF). Protein samples were dissolved in Laemmli buffer and resolved by SDS-PAGE. The proteins were electrophoretically transferred onto nitrocellulose membranes and probed with antibodies to the H+-ATPase, RhBG, or RhCG. Immunolabeling was detected with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Upstate Biotechnology, Lake Placid, NY) by using an enhanced chemiluminescence system (Amersham International).
Statistical analysis. For data without normal distribution or equal variance, a Mann-Whitney rank sum test was used. In all other studies, comparisons were made between two groups using an unpaired Student’s t-test. P < 0.05 indicates statistical significance. Data are displayed ± SE.
RESULTS
Slc26a4 null mice have decreased urinary pH and PCO2, although NH4+ excretion, titratable acid, and citrate excretion are unchanged. On a normal, NaCl-replete diet, serum chemistries and arterial pH are the same in wild-type and Slc26a4 null mice (31, 37). Moreover, under this treatment condition urinary ammonium excretion, titratable acid, and citrate excretion are also the same in mutant and wild-type mice (Table 1). However, urinary pH was much lower in Slc26a4 (–/–) than in wild-type mice, possibly from reduced urinary buffering. With reduced base secretion, less H+ is titrated, which lowers the concentration of the dissociation products of carbonic acid, which are CO2 and H2O. To determine whether HCO3 buffering is reduced, urinary PCO2 was measured (Table 1). As shown, urinary PCO2 was lower in Slc26a4 null than in wild-type mice. Therefore, under basal conditions and at steady state, although arterial pH is the same in wild-type and Slc26a4 null mice, Slc26a4 null mice have a lower urinary pH and urinary PCO2, which reflects their reduced capacity to secrete HCO3–/OH–(25).
H+-ATPase immunolabel is decreased in Slc26a4 (–/–) mice. Cortical sections from Slc26a4 (+/+) mice showed strong pendrin immunolabeling in the apical region of intercalated cells of the CNT and collecting ducts (CCD) of wild-type mice (Fig. 1A). No pendrin immunolabel was observed in the cortex of Slc26a4 (–/–) mice (Fig. 1B).
We asked whether expression of other intercalated cell H+/OH– transporters is altered in Slc26a4 null mice. Figure 2 shows that total H+-ATPase immunolabel intensity appears decreased in both the cortex and medulla of Slc26a4 (–/–) mice. In wild-type mice, H+-ATPase labeling was clearly observed either in the apical regions (, Fig. 2B) or diffusely distributed within a subset of cells (arrows, Fig. 2B), consistent with the expected polarity of the H+-ATPase within the various intercalated cell subtypes (13). In the cortex of Slc26a4 (–/–) mice, apical H+-ATPase labeling was also readily detected (, Fig. 2E). However, immunolabel was barely detectable in intercalated cells of Slc26a4 null mice, which express the H+-ATPase in a diffuse pattern (arrows, Fig. 2E).
To compare H+-ATPase immunolabel in the different intercalated cell subtypes, double labeling experiments were performed using AE1 as a marker of type A intercalated cells (Fig. 3). In wild-type mice, strong H+-ATPase labeling was observed in the apical regions of AE1-positive (type A) cells (, Fig. 3, A and B). AE1-negative cells (type B and non-A, non-B intercalated cells) also showed strong H+-ATPase labeling in a diffuse pattern (arrows, Fig. 3, A and B). In Slc26A4 (–/–) mice, strong apical H+-ATPase labeling was observed in AE1-positive cells (, Fig. 3, C and D), similar to observations made in wild-type mice. However, AE1-negative intercalated cells (type B or non-A, non-B intercalated cells) from these mutant mice showed extremely weak H+-ATPase labeling (arrows, Fig. 3, C and D). Moreover, by immunogold cytochemistry, H+-ATPase immunolabel appeared weaker in type B intercalated cells of Slc26a4 null than in wild-type mice (Fig. 4).
Figure 2 shows that H+-ATPase expression might be lower in the medulla of Slc26a4 null than in wild-type mice. Thus H+-ATPase expression was quantified in the cortex, outer medulla, and inner medulla from Slc26A4 (+/+) and Slc26A4 (–/–) mice (Fig. 5). The anti-H+-ATPase antibody detected a protein by immunoblot that migrates at 56 kDa, the expected mobility of the B1 subunit of H+-ATPase. Band density was reduced in all regions of the kidney from Slc26A4 (–/–) mice relative to wild-type. However, these differences were most striking in the cortex, where all intercalated cell subtypes are expressed (1, 13). Differences were the least striking within the inner medulla, where type A intercalated cells are present but where type B and non-A, non-B intercalated cells are absent (21). We conclude that H+-ATPase expression is reduced in kidneys from Slc26a4 (–/–) mice. However, the reduced H+-ATPase expression observed in Slc26a4 null mice is much more apparent within type B and non-A, non-B cells than within type A intercalated cells.
Decreased immunolabeling of NBC3 in the cortex of Slc26A4 (–/–) mice. Because H+-ATPase immunoreactivity is reduced in non-A intercalated cells (i.e., type B and non-A, non-B cells) of Slc26a4 null mice, further studies investigated whether expression of other H+/OH– transporters, such as the Na+/HCO3– cotransporter, NBC3, is changed within type B and non-A, non-B intercalated cells of Slc26a4 null mice. In rat kidney, NBC3 localizes to the apical plasma membrane of type A intercalated cells and the basolateral plasma membrane and cytoplasmic vesicles of type B intercalated cells (19). However, the localization of NBC3 has not been determined in non-A, non-B intercalated cells. Figure 6 shows that in rat non-A, non-B intercalated cells, NBC3 localizes to the apical plasma membrane and subapical cytoplasmic vesicles (arrows).
NBC3 labeling within the cortex and the initial portion of the inner medulla of Slc26a4 (+/+) and Slc26a4 (–/–) mice is shown in Fig. 7. Abundant NBC3-positive cells were detected in the cortex of wild-type mice. Strong apical NBC3 labeling was observed in both the apical regions (, Fig. 7, A–C) and in the cytoplasm and basal regions (arrows, Fig. 7B) of wild-type mice. In Slc26A4 (–/–) mice, NBC3 labeling was readily observed in the apical regions of a subset of cells (, Fig. 7, E and F), whereas labeling was weak in cells expressing NBC3 in the cytoplasm and in the basolateral regions (arrows, Fig. 7E). Double immunolabeling of NBC3 and AE1 (Fig. 8) in Slc26a4 null mice showed that cells strongly expressing NBC3 are AE1-positive, type A intercalated cells (, Fig. 8, C and D), whereas NBC3 labeling is very weak in cells that are AE1 negative (arrows, Fig. 8, C and D).
NH4+/NH3 transporter expression is reduced in kidneys from Slc26a4 (–/–) mice. RhBG and RhCG are NH4+/NH3 transporters expressed along the CNT and collecting duct (33). Because these transporters may participate in the process of ammonium secretion (38), limiting their expression might reduce net acid secretion, thereby attenuating a metabolic alkalosis. Thus RhBG and RhCG expression was determined in kidneys from wild-type and Slc26a4 null mice (Fig. 9). As shown, although RhCG expression was the same in kidneys from wild-type and mutant mice, RhBG expression was markedly reduced in kidneys from Slc26a4 null mice.
Type A intercalated cell expression is maintained in Slc26a4 (–/–) mice. Because type A intercalated cells mediate net H+ secretion, we asked whether net acid excretion is maintained in the absence of Slc26a4-mediated OH– secretion by decreased type A intercalated cell numbers. To answer this question, cells were divided into three groups: 1) AQP2-positive, AE1-negative cells (principal cells, arrowheads); 2) AE1-positive, AQP2-negative cells (type A intercalated cells, ); and 3) AE1-negative, AQP2-negative cells (type B and non-A, non-B intercalated cells, arrows). These cell types were quantified per high-power field (Fig. 10, Table 2). In the CNT, initial portion of the collecting duct, and the outer medullary collecting duct, the number of principal and type A intercalated cells per high-power field was similar in Slc26a4 (–/–) and Slc26a4 (+/+) mice, although non-A intercalated cell number (type B and non-A, non-B) was reduced by 20% in the CCD of Slc26a4 null mice relative to wild-type. However, reduced cell counts per field could reflect either reduced total cell number per kidney or smaller cell size. Because type B cells are much more prevalent than non-A, non-B intercalated cells within the mouse CCD (30), it is more likely that type B, rather than non-A, non-B intercalated cell number is reduced in the CCD of Slc26a4 null mice relative to wild-type. Thus type B intercalated cell size within the CCD was measured. In random sections of type B intercalated cells from wild-type mice, cell profile area was 6.84 ± 0.27 x 10–5 mm2 (n = 3 mice) but 4.62 ± 0.27 x 10–5 mm2 (n = 4 mice) in cells from Slc26a4 null mice (P < 0.05). Thus the reduced non-A intercalated cell number observed by light microscopy in the CCD of these Slc26a4 (–/–) mice reflects, in part, the smaller type B cell size (30).
Morphological characteristics of type B intercalated cells from Slc26a4 null mice. Because type B cell size and H+/OH– transporters expression are markedly reduced in kidneys of Slc26a4 null mice, the morphological characteristics of this cell type were studied (Fig. 4). As shown, type B cells of Slc26a4 null mice have a less prominent apical vesicle-free band and have more prominent apical plasma membrane microprojections compared with wild-type mice. Relative to wild-type mice, type B cells of Slc26a4 null mice also have markedly fewer cytoplasmic vesicles, and less complex basolateral plasma membrane infoldings.
DISCUSSION
Previous studies by our laboratory have shown that the Cl–/HCO3– exchanger pendrin mediates secretion of OH–/HCO3– into the luminal fluid of the CCD (25). Thus the purpose of this study was to explore how mice maintain intracellular and systemic pH under basal conditions in the absence of Slc26a4-mediated OH– secretion. We observed that H+-ATPase expression is reduced in the cortex as well and in the outer and the inner medulla. Because the H+-ATPase is expressed most highly within type A intercalated cells of the inner medulla, H+-ATPase expression is reduced in type A intercalated cells, at least within the inner medullary collecting duct. However, the most striking finding of this study was the marked downregulation of transporters that mediate net OH– uptake/or H+ efflux within type B and non-A, non-B intercalated cells. The marked downregulation of the H+-ATPase and NBC3 expression observed in these cell types should decrease net OH– uptake or decreased net H+ efflux by these cells, thereby attenuating the rise in pHi expected in the absence of Slc26a4-mediated OH– secretion.
NBC3 and the H+-ATPase are closely associated proteins (24), which coimmunoprecipitate and colocalize within intercalated cells. The present study and previous studies have shown that the H+-ATPase and NBC3 localize to the apical plasma membrane and subapical intracellular vesicles in type A and non-A, non-B intercalated cells (19, 24, 30), whereas in the type B intercalated cell they localize to the basolateral plasma membrane and to cytoplasmic vesicles (19, 24, 30). Moreover, NBC3 and the H+-ATPase are regulated in parallel in treatment models such as metabolic acidosis (18). Although the H+-ATPase is a critical component of net H+ secretion along the collecting duct, NBC3 plays only a minor role in transepithelial transport of H+ equivalents (39). Instead, NBC3 is thought to regulate pHi (39). Yip et al. (39) observed that in type A intercalated cells, pHi recovery following an acid load is sensitive to Na+ and EIPA present in the luminal fluid, which reflects NBC3-mediated OH– uptake (39). Thus downregulation of NBC3 and the H+-ATPase within non-A, non-B and type B intercalated cells of Slc26A4 (–/–) mice should limit net OH– uptake or net H+ efflux by these cells (7), thereby attenuating the intracellular alkalization expected in the absence of Slc26a4-mediated OH– efflux (25).
Because type A intercalated cells secrete H+ through the H+-ATPase, which localizes to the apical plasma membrane, we explored whether the metabolic alkalosis expected in the absence of Slc26a4-mediated OH– secretion is attenuated by reducing type A intercalated cell number or through reduced H+-ATPase protein expression. We observed that Slc26a4 null mice do not maintain normal arterial pH by reducing type A intercalated cell number. However, H+-ATPase protein expression is reduced in the medulla of Slc26a4 null mice, which should reduce secretion of net H+ equivalents and thus minimize the metabolic alkalosis expected in the absence of Slc26a4-mediated OH– secretion.
Despite the reduced H+-ATPase expression observed along the collecting duct, urinary pH was markedly reduced in Slc26a4 null mice relative to wild-type. Thus we explored the possibility that the fall in urinary pH occurred from reduced urinary buffering, rather than increased H+ secretion. Secreted HCO3– titrates secreted H+ to form carbonic acid, which dissociates into H2O and CO2. Thus the lower urinary PCO2 observed is consistent with the reduced capacity of Slc26a4 null mice to secrete HCO3–. However, we cannot exclude the possibility that the lower PCO2 observed in urine of Slc26a4 null mice reflects, at least in part, increased renal CO2 absorption or decreased renal carbonic anhydrase activity (5).
Reduced secretion of protonated nonbicarbonate buffers should attenuate the alkalosis expected in Slc26a4 null mice. Excretion of ammonium is the major regulatable component of net acid excretion (35). Because urinary pH was much lower in Slc26a4 null than in wild-type mice, the driving force for NH3 secretion into the luminal fluid should be markedly increased. Thus greater urinary excretion of NH4+ is expected in Slc26a4 null than in wild-type mice (35). However, this was not observed. Several possible mechanisms might explain why NH4+ excretion was unchanged in Slc26a4 null mice despite their extremely low urinary pH, including reduced ammonium production by the proximal tubule (17). Alternatively, reduced transfer of NH3/NH4+ from the interstitium to the collecting duct lumen could increase renal NH4+ exit through the renal vein while decreasing renal NH4+ exit through excretion in the urine. We observed a marked reduction in expression of the NH3/NH4+ transporter RhBG, which is expressed along the basolateral region of all cells within the CNT and within principal cells and type A intercalated cells of the CCD and medullary collecting duct (33). However, within the medullary collecting duct, RhBG is most highly expressed in type A intercalated cells (33). RhBG is thought to contribute to transepithelial ammonium secretion in the CNT and collecting duct, although this has not been proven experimentally (38). If this protein participates in the process of NH4+/NH3 secretion along the collecting duct, by reducing its expression in Slc26a4 null mice, ammonium secretion should be limited, thereby reducing net acid secretion and attenuating the expected metabolic alkalosis. NH4+ not excreted in the urine should exit the kidney through the renal vein, stimulating hepatic ureagenesis, which consumes NH4+ and HCO3–. By stimulating NH4+ exit through the renal vein while reducing NH4+ excretion into the final urine, arterial pH could be kept within the normal range, despite a very low urinary pH (17). In some treatment models, blood urea nitrogen is higher in Slc26a4 null mice than in wild-type mice (31, 37), which may reflect increased ureagenesis in the mutant mice.
Phosphate excretion represents the major component of titratable acid. Because phosphate excretion was the same in Slc26a4 null and wild-type mice (37), greater urinary acidification should increase protonation of HPO42– to H2PO4–, thus increasing titratable acid. Although titratable acid excretion was approximately twice as great in Slc26a4 (–/–) mice as in wild-type mice, the results did not achieve statistical significance. Interestingly, titratable acid is greater in Slc26a4 null than wild-type mice under treatment conditions that increase Slc26a4 expression in normal mice, such as with dietary NaCl restriction (37).
The present study shows that Slc26a4 null mice regulate net acid excretion through adaptations that occur within intercalated cells, resulting in cell-specific decreases in the expression of many H+/OH– transport mechanisms. Compensatory mechanisms that may occur in other cell types will require further study. In conclusion, H+-ATPase and NBC3 expression are differentially regulated in intercalated cell subtypes in Slc26a4 (–/–) mice. The decrease in H+/OH– transporter expression observed within intercalated cells is probably a compensatory mechanism that attenuates the increases in net acid excretion and pHi expected in the absence of Slc26a4-mediated OH– secretion.
ACKNOWLEDGMENTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52935 (to S. M. Wall).
FOOTNOTES
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.
1 Net acid secretion is equivalent to urinary excretion of NH4+ + titratable acid (TA) – urinary HCO3–.
REFERENCES
Alper SL, Natale J, Gluck S, Lodish HF, and Brown D. Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase. Proc Natl Acad Sci USA 86: 5429–5433, 1989.
Blomqvist SR, Vidarsson H, Fitzgerald S, Johansson BR, Ollerstam A, Brown R, Persson AEG, Bergstrom G, and Enerback S. Distal renal tubular acidosis in mice that lack the forkhead transcription factor Foxi1. J Clin Invest 113: 1560–1570, 2004.
Brown D, Hirsch S, and Gluck S. Localization of a proton-pumping ATPase in rat kidney. J Clin Invest 82: 2114–2126, 1988.
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.
DuBose TD Jr. Hydrogen ion secretion by the collecting duct as a determinant of the urine to blood PCO2 gradient in alkaline urine. J Clin Invest 69: 145–156, 1982.
Emmons C. New subtypes of rabbit CCD intercalated cells as functionally defined by anion exchange and H+-ATPase activity (Abstract). J Am Soc Nephrol 4: 838, 1993.
Emmons C. H+ extrusion in mouse cortical collecting duct (CCD) intercalated cells (Abstract). J Am Soc Nephrol 8: 5A, 1997.
Emmons C and Kurtz I. Functional characterization of three intercalated cell subtypes in the rabbit outer cortical collecting duct. J Clin Invest 93: 417–423, 1994.
Everett LA, Belyantseva IA, Noben-Trauth K, Cantos R, Chen A, Thakkar SI, Hoogstraten-Miller SL, Kachar B, Wu DK, and Green ED. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome. Hum Mol Genet 10: 153–161, 2001.
Frische S, Kwon TH, Frkir J, Madsen KM, and Nielsen S. Regulated expression of pendrin in rat kidney in response to chronic NH4Cl or NaHCO3 loading. Am J Physiol Renal Physiol 284: F584–F593, 2003.
Hastings AB and Sendroy J Jr. The effect of variation in ionic strength on the apparent first and second dissociation constants of carbonic acid. J Biol Chem 65: 445–455, 1925.
Henger A, Tutt P, Riesen WF, Hulter HN, and Krapf R. Acid-base and endocrine effects of aldosterone and angiotensin II inhibition in metabolic acidosis in human patients. J Lab Clin Med 136: 379–389, 2000.
Kim J, Kim YH, Cha JH, Tisher CC, and Madsen KM. Intercalated cell subtypes in connecting tubule and cortical collecting duct of rat and mouse. J Am Soc Nephrol 10: 1–12, 1999.
Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frkir J, and Nielsen S. Altered expression of renal acid-base transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol 285: F1244–F1257, 2003.
Kim YH, Kwon TH, Frische S, Kim J, Tisher CC, Madsen KM, and Nielsen S. Immunocytochemical localization of pendrin in intercalated cell subtypes in rat and mouse kidney. Am J Physiol Renal Physiol 283: F744–F754, 2002.
Knepper MA, Good DW, and Burg MB. Mechanism of ammonia secretion by cortical collecting ducts of rabbits. Am J Physiol Renal Fluid Electrolyte Physiol 247: F729–F738, 1984.
Kurtz I, Dass PD, and Cramer S. The importance of renal ammonia metabolism to whole body acid-base balance: a reanalysis of the pathophysiology of renal tubular acidosis. Miner Electrolyte Metab 16: 331–340, 1990.
Kwon TH, Fulton C, Wang W, Kurtz I, Frkir J, Aalkjr C, and Nielsen S. Chronic metabolic acidosis upregulates rat kidney Na+-HCO3– cotransporters NBCn1 and NBC3 but not NBC1. Am J Physiol Renal Physiol 282: F341–F351, 2002.
Kwon TH, Pushkin A, Abuladze N, Nielsen S, and Kurtz I. Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney. Am J Physiol Renal Physiol 278: F327–F336, 2000.
Lagadic-Gossmann D, Huc L, and Lecureur V. Alterations of intracellular pH homeostasis in apoptosis: origins and roles. Cell Death Differ 11: 953–961, 2004.
Madsen KM and Tisher CC. Structural-functional relationships along the distal nephron. Am J Physiol Renal Fluid Electrolyte Physiol 250: F1–F15, 1986.
Nelson RD, Guo XL, Masood K, Brown D, Kalkbrenner M, and Gluck S. Selectively amplified expression of an isoform of the vacuolar H+-ATPase 56-kilodalton subunit in renal intercalated cells. Proc Natl Acad Sci USA 89: 3541–3545, 1992.
Pendred V. Deaf-mutism and goitre. Lancet 2: 532, 1896.
Pushkin A, Yip KP, Clark I, Abuladze N, Kwon TH, Tsuruoka S, Schwartz GJ, Nielsen S, and Kurtz I. NBC3 expression in rabbit collecting duct: colocalization with vacuolar H+-ATPase. Am J Physiol Renal Physiol 277: F974–F981, 1999.
Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, and Green ED. Pendrin, encoded by the pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci USA 98: 4221–4226, 2001.
Schuster VL. Function and regulation of collecting duct intercalated cells. Annu Rev Physiol 55: 267–288, 1993.
Scott DA and Karniski LP. Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange. Am J Physiol Cell Physiol 278: C207–C211, 2000.
Scott DA, Wang R, Kreman TM, Sheffield VC, and Karniski LP. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nature Genetics 21: 440–443, 1999.
Soleimani M, Greeley T, Petrovic S, Wang Z, Amlal H, Kopp P, and Burnham CE. Pendrin: an apical Cl–/OH–/HCO3– exchanger in the kidney cortex. Am J Physiol Renal Physiol 280: F356–F364, 2001.
Teng-ummnuay P, Verlander JW, Yuan W, Tisher CC, and Madsen KM. Identification of distinct subpopulations of intercalated cells in the mouse collecting duct. J Am Soc Nephrol 7: 260–274, 1996.
Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett LA, Green ED, and Wall SM. Deoxycorticosterone upregulates Pds (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003.
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.
Verlander JW, Miller RT, Frank AE, Royaux IE, Kim YH, and Weiner ID. Localization of the ammonium transporter proteins RhBg and RhCg in mouse kidney. Am J Physiol Renal Physiol 284: F323–F337, 2003.
Verlander JW, Tran TM, Zhang L, Kaplan MR, and Hebert SC. Estradiol enhances thiazide-sensitive NaCl cotransporter density in the apical plasma membrane of the distal convoluted tubule in ovariectomized rats. J Clin Invest 101: 1661–1669, 1998.
Wall SM. Mechanisms of NH4+ and NH3 transport during hypokalemia. Acta Physiol Scand 179: 325–330, 2003.
Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, and Verlander JW. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284: F229–F241, 2003.
Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, and Verlander JW. NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl– conservation. Hypertension 44: 1–6, 2004.
Weiner ID. The Rh gene family and renal ammonium transport. Curr Opin Nephrol Hypertens 13: 533–540, 2004.
Yip KP, Tsuruoka S, Schwartz GJ, and Kurtz I. Apical H+/base transporters mediating bicarbonate absorption and pHi regulation in the OMCD. Am J Physiol Renal Physiol 283: F1098–F1104, 2002.(Young-Hee Kim, Jill W. Verlander, Sharon)