Regulation of the apical Clminus /HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger pendrin in rat cortical collecting duct in metabolic acidosis

Snezana Petrovic1,2, Zhaohui Wang1, Liyun Ma1, and Manoocher Soleimani1,2

1 Department of Medicine, University of Cincinnati, and 2 Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio 45267-0485


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ABSTRACT
INTRODUCTION
METHODS
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Pendrin is an apical Cl-/OH-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in beta -intercalated cells (beta -ICs) of rat and mouse cortical collecting duct (CCD). However, little is known about its regulation in acid-base disorders. Here, we examined the regulation of pendrin in metabolic acidosis, a condition known to decrease HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in CCD. Rats were subjected to NH4Cl loading for 4 days, which resulted in metabolic acidosis. Apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -ICs was determined as amplitude and rate of intracellular pH change when Cl was removed in isolated, microperfused CCDs. Intracellular pH was measured by single-cell digital ratiometric imaging using fluorescent pH-sensitive dye 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein-AM. Pendrin mRNA expression in kidney cortex was examined by Northern blot hybridizations. Expression of pendrin protein was assessed by indirect immunofluorescence. Microperfused CCDs isolated from acidotic rats demonstrated ~60% reduction in apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -ICs (P < 0.001 vs. control). Northern blot hybridizations indicated that the mRNA expression of pendrin in kidney cortex decreased by 68% in acidotic animals (P < 0.02 vs. control). Immunofluorescence labeling demonstrated significant reduction in pendrin expression in CCDs of acidotic rats. We conclude that metabolic acidosis decreases the activity of the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in beta -ICs of the rat CCD by reducing the expression of pendrin. Adaptive downregulation of pendrin in metabolic acidosis indicates the important role of this exchanger in acid-base regulation in the CCD.

kidney; intercalated cells


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ONE-THIRD OF THE KIDNEY CORTICAL collecting duct (CCD) cell population comprises intercalated cells, which are responsible for the final adjustments of acid-base balance (31). In response to acid or alkali loading, CCD reabsorbs or secretes more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, respectively. This is accomplished through the coordinated activity of two types of intercalated cells: alpha -intercalated cells (alpha -ICs), which are modeled to secrete acid through an apical H+-ATPase and basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>exchanger, and beta -intercalated cells (beta -ICs), which are modeled to secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> through an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger and a basolateral H+-ATPase. Immunocytochemical experiments identified H+-ATPase in both cell types as vacuolar-type ATPase and the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger of alpha -ICs as anion exchanger 1 (AE1) (reviewed in Ref. 31). However, the molecular identity of the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger of beta -ICs has long remained unknown.

Recent molecular studies have identified a large, highly conserved family of membrane proteins (designated as SLC26A), many of which have been shown to transport anions. Three closely related members of this family are downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and PAT1 (CFEX or SLC26A6) (14, 17, 19, 20, 44). All three transporters mediate Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (22, 36, 44). DRA is expressed on the apical membranes of colonocytes, whereas PAT1 or CFEX is expressed on the apical membranes of kidney proximal tubule and duodenum (19, 22, 27, 44). Pendrin mRNA expression is detected in proximal tubule and CCD (36). However, immunocytochemical studies localize pendrin only to the apical membranes of a subpopulation of CCD cells, which also express H+-ATPase on their basolateral membranes (28, 35, 36). In addition, CCDs of pendrin-deficient mice failed to secret HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in response to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> loading (28). Taken together, these studies are consistent with pendrin functioning as an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in beta -ICs of CCD (28, 35, 36).

Metabolic acidosis decreases the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in rabbit beta -ICs (25, 30, 32, 33, 40). However, little is known about the regulation of the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in beta -ICs of either control or acidotic rats. Furthermore, no study has examined the molecular adaptation of beta -IC apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in acid-base disorders. In the present study, we sought to correlate the kidney expression of pendrin with the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in single beta -ICs in rats subjected to acid loading.


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Animals. Metabolic acidosis was generated according to established protocols (1, 9). Female Sprague-Dawley rats, 100-150 g, were given 280 mM NH4Cl in their drinking water for 4 days. Serum HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was 14 ± 1.2 mM in rats on NH4Cl, consistent with metabolic acidosis vs. 24 ± 1.5 mM in control (P < 0.03, n = 4 for each). Both groups were allowed free access to water and food.

Isolation of CCDs and in vitro microperfusion. Rats were killed by intraperitoneal injection of pentobarbital sodium (100 mg/kg of body wt). Kidneys were quickly removed and placed in ice-cold dissection medium (solution 1; Table 1). Thin coronal slices (~1 mm) were cut and transferred to the dissection chamber. CCDs were obtained by freehand dissection. Dissected tubules were quickly transferred to the 1.5-ml temperature-controlled specimen chamber mounted on an inverted Zeiss Axiovert S-100 microscope (Carl Zeiss, Thornwood, NY). Tubules were perfused by using concentric glass pipettes according to the method of Burg and colleagues (7, 8) with modifications (40) at 5-cm water pressure. Solutions used to perfuse and bath the tubules are listed in Table 1. Solutions were delivered to the specimen chamber in tubing impermeable to CO2 and O2 (Cole Palmer, Chicago, IL) by a peristaltic pump (Peristar, WPI, Sarasota, FL) at a rate of 1 ml/min. Fluid in the chamber was constantly superfused with 95% O2-5% CO2 to minimize gas loss and help keep the pH of the bath fluid constant. Chamber pH was frequently checked on a pH meter (model B213, Horiba). Initially, tubules were perfused with fast green dye (Sigma, St. Louis, MO) to identify the damaged cells, because damaged cells take up the dye. Tubules were carefully inspected and discarded if damaged cells were found (41).

                              
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Table 1.   Solutions utilized in microperfusion experiments

Intracellular pH measurement in intercalated cells. After 15-20 min equilibration in solution 2, the tubule was perfused with 5 µM 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein AM (BCPCF-AM) for 5 min. Only intercalated, but not principal, cells take up the pH-sensitive dye when perfused from the luminal side (45). BCPCF-AM is a close analog of BCECF-AM, with improved spectral characteristics (a higher absorption at isosbestic point yields a better signal-to-noise ratio) (16). In our preliminary experiments, we noticed that BCPCF-AM was better retained in rat intercalated cells. Fluorescent measurements were done with the Zeiss Axiovert S-100 inverted microscope equipped with Attofluor RatioVision digital imaging system (Attofluor, Rockville, MD). An Achroplan ×40/0.8 water objective with 3.6-mm working distance was used. Excitation wavelengths were recorded at 488 and 440 nm, and emission was measured at 520 nm. Attofluor RatioVision software allowed for "regions of interest" to be applied to individual cells so that multiple cells in a single tubule were simultaneously examined. Generally, three to seven cells were examined per tubule. Only one tubule per animal was examined. Digitized images were analyzed by using Attograph software (Attofluor). Intracellular calibration was performed by using the high-K+-nigericin method (24, 30, 33, 37, 45). A three-point calibration curve was used to convert the recorded ratios into intracellular pH (pHi) values. pH clamp calibration values (7.5, 7.0, and 6.5) were recorded from each cell that was selected for experimental measurements in every tubule at the end of the experiment.

Apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -ICs was assessed as the rate of pHi change (calculated as a linear tangent to the initial pH change) as well as amplitude of pHi response when the luminal perfusate (solution 2; Table 1) was switched to a Cl--free solution (solution 3; Table 1). This maneuver causes cell alkalinization in beta -ICs via reversal of the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, whereas pHi of alpha -ICs remains unchanged. When pHi stabilization occurred in Cl--free medium, the luminal perfusate was switched back to Cl--containing solution, resulting in recovery of pHi to baseline levels via Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (24, 30, 32, 33, 45, 46).

In separate maneuvers, bath solution was switched from Cl--containing (solution 2; Table 1) to a Cl--free solution (solution 3; Table 1). Under this protocol, alpha -ICs alkalinize whereas beta -ICs acidify (24, 30, 32, 33, 45, 46). The alkalinization of alpha -ICs in response to the removal of bath Cl- is due to the reversal of basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. The acidification of beta -ICs in response to bath Cl- removal is due to the stimulation of apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, as intracellular Cl- exits the cell via basolateral Cl- conductance. This increases the inward gradient for luminal Cl- and therefore stimulates the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (46).

RNA isolation and Northern blot hybridization. Total cellular RNA was extracted from cortex by the method of Chomczynski and Sacchi (10), quantitated spectrophotometrically, and stored at -80°C. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes (MSI). Membranes were cross-linked by ultraviolet light and baked for 1 h. Hybridization was performed according to Church and Gilbert (11). A pendrin-specific cDNA probe (36) was labeled with [32P]deoxynucleotides by using the Rad-Prime DNA labeling kit (GIBCO-BRL). The membranes were washed, blotted dry, exposed to a PhosphorImager cassette at room temperature for 24-72 h, and read by the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

For pendrin hybridization, a PCR fragment encoding nucleotides 1473-1961 was generated from rat kidney using oligonucleotide primers 5'-CAT TCT GGG GCT GGA CCT C and 5'-CCT TCG GGA CAT TCA CTT TCA C that were designed on the basis of rat pendrin cDNA (GenBank accession no. AF-167412).

Nephron segment RT-PCR. CCDs were dissected as single-nephron segments from freshly killed normal or acidotic rat kidneys at 4-6°C as described (36). The dissection media comprises 140 mM NaCl, 2.5 mM K2HPO4, 2 mM CaCl2, 1.2 mM MgSO4, 5.5 mM D-glucose, 1 mM Na citrate, 4 mM Na lactate, and 6 mM L-alanine, pH 7.4, and bubbled with 100% O2. Tubule lengths were ~0.5-0.7 mm for both control and acidotic animals. For each RT-PCR, two nephron segments (CCDs) from each rat were pooled in a small volume (5-10 µl) of ice-cold PBS. The tubules were centrifuged at 12,000 g for 1 min at room temperature, and the PBS was removed and replaced with 10 µl of a tubule lysis solution consisting of 0.9% Triton X-100, 5 mM DTT, and 1 U/µl rRNasin (Promega). After 5 min on ice, the tubules were gently agitated by tapping the tube, and 1 µl (0.5 µg) oligo(dT) primer, 1 µl H2O, 4 µl 5× reverse transcription buffer, 2 µl DTT (0.1 M), and 1 µl dNTPs (10 mM each) were added. The reaction was equilibrated to 42°C for 2 min, and 1 µl SuperScript II RT (Life Technologies) was added, mixed, and incubated for 1 h at 42°C. After reverse transcription, 30 µl of TE (10 mM Tris · Cl and 1 mM EDTA, pH 8.0) were added, and the combined mixture was heated to 95°C for 5 min and placed on ice.

The following oligonucleotide primers (5'-CAT TCT GGG GCT GGA CCT C and 5'-CCT TCG GGA CAT TCA CTT TCA C) were designed on the basis of rat pendrin cDNA (GenBank accession no. AF-167412) and used for nephron segment RT-PCR. These primers should amplify a PCR fragment of 488 bp. Amplification of the pendrin cDNA by the PCR was performed by using parameters previously established with rat CCD (36). Briefly, each PCR contained 10 µl cDNA, 5 µl 10× PCR buffer (with 20 mM MgCl2), 1 µl 10 mM dNTPs, 10 pmol/primer, and 2.5 µl Taq DNA polymerase in a final volume of 50 µl. Cycling parameters were 95°C for 45 s, 47°C for 45 s, and 72°C for 2 min. The expression of beta -actin was examined in each sample, and pendrin/beta -actin mRNA ratios were calculated in control and acidosis and compared. Four separate CCD segment samples from two control and two acidotic rats (2 samples/rat) were isolated and examined.

Immunocytochemistry: antibodies. For pendrin, polyclonal antibodies were raised in two rabbits against a synthetic peptide corresponding to amino acids 734-752 (CKSREGQDSLLETVARIRDC). The sequence of the synthetic peptide used for antibody generation was identical for rat, mouse, and human pendrin. Antibodies were purified with cystein-affinity columns. This antibody is highly specific and labels the apical membranes of a subset of CCD cells (35).

For aquaporin 2 (AQP2), peptide-derived polyclonal antibodies specific to the AQP2 water channel were raised in our laboratory as described (2). The rat AQP2 peptide has the following sequence: NH2-CEVRRRQSVELHSPQSLPRG- SKA-COOH, which corresponds to amino acid residues 250-271 of the COOH-terminal tail of the vasopressin-regulated AQP2 water channel. This antibody is highly specific and has been successfully used to examine the regulation of AQP2 in pathophysiological disorders (2).

Immunofluorescense. Animals were killed with an overdose of pentobarbital sodium and perfused through the left ventricle of the heart with 200 ml of 0.9% saline followed by cold 500 ml of 4% paraformaldehyde in 0.1 M sodium-phosphate buffer (pH 7.4). Kidneys were removed, cut in tissue blocks, and left in the same fixative solution overnight at 4°C.

For cryosections, tissue blocks were removed from the fixative solution and soaked in 30% sucrose overnight. The tissue was frozen on dry ice, and 5-µm sections were cut with a cryostat and stored at -80°C until use. For staining, cryosections were washed twice in 0.01 M PBS (pH 7.4) and blocked with 10% goat serum-0.3% Triton X-100-PBS solution for 45-60 min. Primary pendrin antibody was diluted 1:40 in 1% BSA-0.3% Triton X-100-PBS solution and applied to sections overnight at room temperature. Primary AQP2 antibody was diluted 1:20 in 1% BSA-0.3% Triton X-100-PBS solution and applied to sections overnight at room temperature. Sections treated with either primary antibody were rinsed twice in 0.01 M PBS for 10 min and then incubated with a secondary antibody for 2 h at room temperature. Rhodamine TRITC (Jackson Immunoresearch Laboratories, West Grove, PA)-conjugated goat-anti-rabbit IgGs was used as secondary antibody for pendrin (1:200 dilution). In some sections we used green fluorescent secondary, Oregon green-conjugated goat-anti-rabbit IgGs (Molecular Probes, Eugene, OR) at a dilution of 1:150, because we noticed that there was less background with the green fluorescent secondary dye. Sections were then washed four times, air dried, and mounted in Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). Sections were examined, and images were acquired on the Nikon PCM 2000 laser confocal scanning microscope as 0.5-1 µm "optical sections" of the stained cell membrane; a ×20 objective and ×60 oil-immersion objective were used. The 543.5-nm single-line output of the HeNe laser was used for the red dye excitation, and the standard red channel long-pass 565-nm filter was used as an emission filter. A standard argon laser 488-nm line and 515/30-nm emission filter were used for the green- emitting dye. Instrument settings, black level, gain, and integration time, which is pixel dwell time, were kept the same when sections from control and acid-loaded animals were compared. In addition, all sections from control and acidotic animals were processed the same day and with the same dilutions of primary and secondary antibodies. More than 20 sections from four separate animals were examined in each group of control or acidosis.

Materials. [32P]dCTP was purchased from New England Nuclear (Boston, MA). The RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF-AM and BCPCF-AM were from Molecular Probes. Nitrocellulose filters and and all other chemicals were purchased from Sigma. Nigericin was dissolved in ethanol as 10 mM stock and diluted 1:1,000 for the final concentration of 10 µM.

Statistics. Results are expressed as means ± SE. Statistical significance between experimental groups was determined by Student's t-test, as required. Significance was asserted if P < 0.05. Comparison of the number of intercalated cell types in control and acidotic animals was analyzed by chi 2-test as computed on SAS software (version 8, SAS Institute, Cary, NY).


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mRNA expression of pendrin in the kidneys of control and acidotic rats. In the next series of experiments, we sought to examine the effect of metabolic acidosis on the expression of pendrin in rat kidney. Rats were made acidotic by addition of NH4Cl to their drinking water (see METHODS). Animals were killed, and kidney RNA and sections were utilized for expression studies. Figure 1A is a representative Northern blot hybridization experiment and demonstrates that the mRNA expression of pendrin in the kidney cortex is decreased in metabolic acidosis. The results of four samples from separate animals, (summarized in Fig. 1B) indicate that the expression of pendrin is decreased by 68% in acidotic rats (n = 4, P < 0.02 vs. control rats).


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Fig. 1.   A: expression of pendrin mRNA (PDS) in the kidneys of control and acidotic rats. Representative Northern blot hybridizations indicate the downregulation of pendrin mRNA in kidney cortices of acidotic rats. The results of 4 separate samples from 4 separate animals indicate that the expression of pendrin is decreased by 68% in acidotic rats (P < 0.02 vs. control rats). RNA was loaded 30 µg/each lane. The expression of 28S rRNA is shown as a constitutive control. B: summary of results. Pendrin mRNA/28S rRNA ratios (n = 4 for each bar). C: nephron segment RT-PCR with primers specific for pendrin. A representative ethidium bromide staining of agarose gel (left and right) demonstrates a PCR product of expected size (488 bp) for rat pendrin in the cortical collecting duct (CCD) of control and acidotic rats. The expression of beta -actin mRNA is shown as control in the same nephron segments. No PCR product was observed in the negative (no RT) reactions (not shown). For statistical analysis, pendrin/beta -actin mRNA ratios from 4 different CCD samples from normal and acidotic rats (2 animals/each group) were acquired and analyzed by ANOVA. The expression of pendrin decreased by 62% in kidneys of rats with acidosis (P < 0.05). The identity of PCR fragments was verified by DNA sequencing.

Pendrin mRNA is detected in both proximal tubule and CCD in normal kidney (36). To assess the CCD-specific regulation, we examined the mRNA expression of pendrin in acidosis by nephron segment RT-PCR. Single CCDs were isolated from kidneys of control and acid-loaded rats as before (36) and subjected to RT-PCR. Figure 1C shows a representative ethidium bromide gel image from semiquantitative nephron segment RT-PCR experiments and demonstrates that pendrin mRNA is decreased in acidosis. beta -Actin mRNA expression was measured in the same samples and is shown as control. The expression of pendrin, as assessed by semiquantitative pendrin/beta -actin mRNA ratio, decreased by 62% in CCDs of acidotic rats (P < 0.05, n = 4, separate CCD samples from 2 separate rats/each group). These results indicate that the reduction in the cortical expression of pendrin in acidosis (Fig. 1A) is in part due to downregulation in CCD.

Immunofluorescent staining of pendrin in the kidneys of control and acidotic rats. In the next series of experiments, the effect of acidosis on pendrin abundance was examined by indirect immunofluorescence. In kidney sections from control rats, immunofluorescent staining with the purified polyclonal pendrin antibody (Fig. 2A) shows apical labeling in a subpopulation of CCD cells. Specificity of the staining is demonstrated in Fig. 2B. As indicated, labeling was completely prevented by preadsorption of the immune sera with the synthetic peptide. The limited expression of pendrin in CCD is in agreement with recent reports on the expression of pendrin in the kidney (28, 35).


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Fig. 2.   Specificity of pendrin immunofluorescent labeling. A: immunocytochemical staining of rat kidney with pendrin polyclonal purified antibody. As indicated, the antibody labeled the apical membranes in a subpopulation of CCD cells. B: labeling was completely prevented by preadsorption of the antibody with the synthetic peptide.

As shown at a higher magnification, the apical staining in CCDs decreased in acidotic rats vs. control animals (Fig. 3A, bottom and top, respectively). Because of a high background with the secondary red fluorescent dye, we repeated the experiments with a green fluorescent dye to tag the pendrin antibody. Figure 3B represents an experiment that was performed with a green fluorescent dye and shows decreased intensity of pendrin immunostaining in kidneys of rats with acidosis vs. control animals (Fig. 3B, bottom and top, respectively). A broader view compares pendrin expression in control and acidotic animals (Fig. 3C, top and bottom, respectively).


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Fig. 3.   Pendrin expression in metabolic acidosis. A: pendrin immunofluorescent staining with the use of a red fluorescent secondary dye. As indicated, the apical labeling with pendrin antibody in CCDs decreased in acidotic rats (bottom) compared with control animals (top). PT, proximal tubule. B: pendrin immunofluorescent staining with the use of a green fluorescent secondary dye. As indicated, the apical labeling with pendrin antibody in CCDs decreased in acidotic rats (bottom) compared with control animals (top). C: pendrin immunofluorescent staining (low magnification) of kidney sections in control (top) and acidotic animals (bottom). When images from the kidney sections of control and acid-loaded rats were compared, instrument settings (camera gain, black level, and integration time) were kept the same at both magnifications. All sections were fixed and stained the same day and examined with the same concentration of primary and secondary antibodies. Arrows, typical apical staining pattern of pendrin in a subpopulation of CCD cells. Magnification, ×600.

In the next series of experiments, we examined the expression of an unrelated transporter in CCD in rats subjected to NH4Cl loading to determine the specificity of pendrin regulation in acidosis. Accordingly, the expression of AQP2 was examined by immunofluorescent labeling in rats with acidosis using AQP2-specific antibodies raised in our laboratory (2). Consistent with published reports, AQP2 staining (Fig. 4A) is observed on the apical membrane of the majority of CCD cells (23). Contrary to pendrin, AQP2 labeling does not decrease in kidneys of acidotic animals (Fig. 4B). Taken together, these experiments indicate that metabolic acidosis decreases the expression of pendrin in rat CCD.


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Fig. 4.   Immunofluorescent staining of AQP2. A: representative immunofluorescent staining of kidney AQP2 in normal rats. B: representative immunofluorescent staining of kidney AQP2 in acidotic rats. Results demonstrate that AQP2 labeling is not decreased in acidosis. Sections were made from the same animals that were used for pendrin immunolabeling. Magnification, ×600.

Cl-/HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> exchanger activity in intercalated cells of control and acidotic animals. The results of the above studies demonstrated decreased expression of pendrin in CCD in metabolic acidosis. To correlate these results with functional studies, the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -ICs was examined in control and acidotic animals. Toward this end, the beta -ICs and alpha -ICs were first identified by their pHi response to luminal or basolateral Cl- removal in microperfused CCDs, according to the established criteria (see METHODS). Representative pHi tracings in beta -ICs in control and acidosis are shown in Fig. 5, A and B, respectively.


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Fig. 5.   Apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -intercalated cells (beta -IC) of control and acidotic animals. A: representative intracellular pH (pHi) tracing of an individual normal beta -IC demonstrating cell alkalinization when luminal Cl- was removed and cell acidification when bath Cl- was removed. B: representative pHi tracing of an individual beta -IC from an acidotic rat. C: rate of intracellular alkalinization when luminal Cl- was removed in beta -ICs in normal and acidotic rats. As shown, apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity is significantly decreased in beta -ICs in acidosis. D: amplitude of pHi alkalinization when luminal Cl- was removed in beta -ICs in normal and acidotic rats. As shown, the magnitude of intracellular alkalinization in response to luminal Cl- removal is significantly diminished in beta -ICs in acidosis. E: rate of intracellular acidification when bath Cl- was removed in beta -ICs in normal and acidotic rats is significantly decreased in acidosis. F: amplitude of pHi acidification when bath Cl- was removed in beta -ICs in normal and acidotic rats. As shown, the magnitude of intracellular acidification in response to bath Cl- removal is significantly diminished in beta -ICs in acidosis.

In the control group, 15 of 44 cells that were labeled with BCPCF-AM in 10 CCDs alkalinized when luminal Cl- was removed at a rate of 0.16 ± 0.02 pH units/min (Fig. 5C). The cell pH increased from a baseline of 7.21 ± 0.01 to 7.39 ± 0.02 in response to luminal Cl- removal, with a change in pH (Delta pH) of 0.18 ± 0.02 (Fig. 5D). The same cells acidified when basolateral Cl- was removed at a rate of 0.12 ± 0.02 pH units/min (Fig. 5E). The cell pH decreased from a baseline of 7.20 ± 0.02 to 7.03 ± 0.03 in response to bath Cl- removal, with a Delta pHi of 0.16 ± 0.02 (Fig. 5F). These cells were therefore considered beta -ICs. There seemed to be fewer beta -ICs in CCDs from acidotic animals (9 beta -ICs of 41 intercalated cells) compared with control CCDs (15 beta -ICs of 45 intercalated cells). However, the difference did not reach statistical significance as assessed by chi 2-test (P = 0.24).

In acidotic rats, 9 of 41 cells that were labeled with BCPCF-AM in nine CCDs alkalinized when luminal Cl- was removed (and hence were identified as beta -ICs) at a rate of 0.10 ± 0.01 pH units/min (vs. 0.16 ± 0.02 in normal rats, P < 0.05, n = 9) (Fig. 5C). The cell pH increased from a baseline of 7.26 ± 0.01 to 7.32 ± 0.02 in response to luminal Cl- removal, with a Delta pHi of 0.06 ± 0.02 (P < 0.01 vs. 0.18 ± 0.02 in control animals) (Fig. 5D). Baseline pH was not statistically different from control animals (7.26 ± 0.01 in acidotic vs. 7.21 ± 0.01 in control animals, P > 0.05). The rate of acidification when basolateral Cl- was removed was 0.04 ± 0.003 pH units/min (P < 0.05 vs. 0.12 ± 0.02 in control animals) (Fig. 5E). The cell pH decreased from 7.26 ± 0.04 to 7.17 ± 0.04, with a Delta pHi of 0.07 ± 0.02 (P < 0.01 vs. 0.16 ± 0.02 in control) (Fig. 5F).

In control animals, 24 of 44 cells that were labeled with BCPCF-AM in 10 CCDs alkalinized when basolateral Cl- was removed at a rate of 0.22 ± 0.02 pH units/min (Fig. 6C). These cells were therefore considered alpha -ICs. Representative pHi tracings in alpha -ICs in control and acidosis are shown in Fig. 6, A and B, respectively. The cell pH increased from a baseline of 7.22 ± 0.04 to 7.43 ± 0.02 in response to basolateral Cl- removal, with a Delta pH of 0.21 ± 0.02 (Fig. 6D). Luminal Cl- removal did not significantly change pHi of these cells (from a baseline pHi of 7.29 ± 0.01 to 7.28 ± 0.02, P > 0.05).


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Fig. 6.   Basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in alpha -intercalated cells (alpha -ICs) of control and acidotic animals. A: representative pHi tracing of an individual normal alpha -IC demonstrating no pHi changes when luminal Cl- was removed but cell alkalinization when bath Cl- was removed. B: representative pHi tracing of an individual alpha -IC from an acidotic rat. C: rate of intracellular alkalinization when bath Cl- was removed in alpha -ICs in normal and acidotic rats. As shown, basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity is significantly increased in alpha -ICs in acidosis. D: amplitude of pHi alkalinization when bath Cl- was removed in alpha -ICs in normal and acidotic rats. As shown, the magnitude of intracellular alkalinization in response to bath Cl- removal is significantly increased in alpha -ICs in acidosis.

In acidotic rats, 29 of 41 cells that were labeled with BCPCF in nine CCDs alkalinized when basolateral Cl- was removed (and hence were designated as alpha -ICs) at a rate of 0.28 ± 0.02 pH units/min (P < 0.03 vs. 0.21 ± 0.02 in control animals) (Fig. 6C). Cell pH increased from a baseline of 7.29 ± 0.01 to 7.53 ± 0.01 in response to bath Cl- removal, with a Delta pH of 0.24 ± 0.01 (P < 0.05 vs. 0.21 ± 0.02 in control) (Fig. 6D). Luminal Cl- removal did not change the cell pH of alpha -ICs (from a baseline of 7.29 ± 0.01 to 7.3 ± 0.02, P > 0.05). In addition to alpha -ICs and beta -ICs that express Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger on their basolateral and luminal membranes, respectively, a subtype of intercalated cells in rabbit kidney has been described by several investigators that expresses Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity on its apical and basolateral membrane (13, 47). We observed that 5 of 44 cells in control animals alkalinized when either luminal or basolateral Cl- was removed at the rate of 0.17 ± 0.03 or 0.22 ± 0.04 pH units/min, respectively. In acidotic animals, 3 of 41 cells alkalinized when either luminal or basolateral Cl- was removed at the rate of 0.19 ± 0.031 or 0.22 ± 0.04 pH units/min, respectively. Although there was no significant difference between the rate of apical or basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in these cells in acidosis vs. control (P > 0.05), no firm conclusion could be reached at this stage due to the low abundance of these cells. A representative pHi tracing of this cell type is shown in Fig. 7.


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Fig. 7.   A representative pHi tracing of an individual intercalated cell demonstrating cell alkalinization when either luminal or bath Cl- was removed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present experiments examine the regulation of pendrin in metabolic acidosis in rat kidney. Functional studies in microperfused cortical collecting tubules demonstrate that apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -ICs is decreased by ~60% in acid-loaded rats (Fig. 5). Northern blot hybridization experiments demonstrated that the mRNA expression of pendrin decreased in the kidneys of acidotic animals (Fig. 1). Similarly, immunohistochemical studies indicated decreased expression of pendrin protein in intercalated cells of acid-loaded rats (Figs. 2 and 3). In contrast to the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in beta -ICs, the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in alpha -ICs increased in acidosis (Fig. 6).

A Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger is located on the apical membrane of beta -ICs and mediates the secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> into the lumen of CCD. Contrary to beta -ICs, alpha -ICs express a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger on their basolateral membrane, which mediates the reabsorption of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in CCDs (31). This transporter is a truncated splice variant of red cell AE1 or band 3 (6). On the basis of immunohistochemical studies indicating a lack of AE1 staining on the apical membranes of CCD cells, as well as functional studies showing differences in Cl affinity and DIDS sensitivity, it was concluded that the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger of beta -ICs is distinct from AE1 (31).

Recent findings identified pendrin as an important candidate for an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in beta -ICs (36). This conclusion was on the basis of functional and molecular studies indicating that pendrin is an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in the kidney cortex, with abundant expression in rat CCD (36). This was supported by immunocytochemical studies demonstrating apical localization of pendrin in a subset of CCD cells in mouse, rat, and human kidney that were distinct from alpha -ICs and principal cells (28). In addition, pendrin null mice failed to secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in their CCDs when subjected to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> loading, indicating the important role of pendrin in adaptation of the mouse CCD to change in alkali load (28). These findings are consistent with pendrin functioning as an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger and mediating HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion into rat and mouse CCD. CCDs from control or acid-loaded rats absorb HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (4, 15). Immunocytochemical data in rat kidney show that adaptation to systemic acidosis results from decreased beta -ICs function, as concluded from changes in H+-ATPase- and AE1-labeling patterns. The present studies are the first to measure the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -ICs in rat kidney (Fig. 5). Furthermore, our findings demonstrating decreased apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in rat beta -ICs (Fig. 5) are in agreement with previous immunocytochemical data indicating adaptive regulation of H+-ATPase in rat kidney in acidosis (5, 29, 38). The reduction in pendrin mRNA in acidosis is specific, as judged by a lack of reduction in AQP-2 mRNA in acidosis (3). These latter results correlate very well with immunocytochemical labeling performed in the present studies (Fig. 4). The reduction in apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity correlates with a significant downregulation in pendrin expression (Figs. 1 and 2).

Rat kidney AE1 mRNA levels increased in response to both respiratory and metabolic acidosis (11a, 18). Immunocytochemical studies indicated adaptive upregulation of AE1 in basolateral membranes of alpha -ICs in acidotic rats (21, 29, 38, 43). Our results demonstrating increased basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in alpha -ICs in acidosis are in complete agreement with the above molecular and immunocytochemical studies.

Adaptation of rabbit CCD to both in vivo and in vitro metabolic acidosis has been studied in detail and shown to result mainly from the decreased activity of the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in beta -ICs, as well as the increased activity of the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger of alpha -ICs and reversal of the functional polarity of beta -ICs in CCDs incubated in low pH in vitro (26, 30, 32, 33). However, molecular identity of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger mediating these changes is presently unknown. Tsuganezawa et al. (39) have recently cloned the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger of rabbit beta -ICs and named it AE4. AE4 mRNA expression has also been shown in rat kidney (26), but preliminary immunocytochemical labeling from different groups has been conflicting with respect to its subcelullar localization in rat CCDs (12, 26). Further studies are necessary to address this issue.

Some studies in rabbit CCDs have shown both apical and basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in intercalated cells (13, 47). In addition, immunocytochemical studies have identified a group of intercalated cells referred to as non-A-non-B intercalated cells in rat and mouse CCDs (21, 38, 43). Interestingly, we have observed a small number of cells that have apical as well as basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity (Fig. 7), which may resemble non-A-non-B intercalated cells identified by immunocytochemical staining. However, because of the low abundance of these cells, it is hard to draw any firm conclusions with regard to their adaptation in acidosis.

In conclusion, mRNA expression and protein abundance of pendrin are downregulated in metabolic acidosis in the rat kidney, resulting in decreased apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in beta -ICs. Taken together, these results suggest that pendrin plays an important role in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and, as a result, in acid-base regulation in rat kidney.


    ACKNOWLEDGEMENTS

These studies were supported by a Merit Review grant from the Department of Veterans Affairs, National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52821 and DK-54430, a Cystic Fibrosis Foundation grant, and grants from Dialysis Clinic, Inc.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Soleimani, Division of Nephrology and Hypertension, Dept. of Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, MSB G259, Cincinnati, Ohio 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.edu).

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.

August 13, 2002;10.1152/ajprenal.00205.2002

Received 30 May 2002; accepted in final form 13 August 2002.


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