Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis

Saskia Huber1, Esther Asan1, Thomas Jöns2, Christiane Kerscher1, Bernd Püschel1, and Detlev Drenckhahn1

1 Institute of Anatomy, Julius-Maximilians-University of Würzburg, 97070 Würzburg; and 2 Institute of Anatomy, Humboldt-Universität Berlin-Charité, 10115 Berlin, Germany


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By enzyme-linked in situ hybridization (ISH), direct evidence is provided that acid-secreting intercalated cells (type A IC) of both the cortical and medullary collecting ducts of the rat kidney selectively express the mRNA of the kidney splice variant of anion exchanger 1 (kAE1) and no detectable levels of the erythrocyte AE1 (eAE1) mRNA. Using single-cell quantification by microphotometry of ISH enzyme reaction, medullary type A IC were found to contain twofold higher kAE1 mRNA levels compared with cortical type A IC. These differences correspond to the higher intensity of immunostaining in medullary versus cortical type A IC. Chronic changes of acid-base status induced by addition of NH4Cl (acidosis) or NaHCO3 (alkalosis) to the drinking water resulted in up to 35% changes of kAE1 mRNA levels in both cortical and medullary type A IC. These experiments provide direct evidence at the cellular level of kAE1 expression in type A IC and show moderate capacity of type A IC to respond to changes of acid-base status by modulation of kAE1 mRNA levels.

enzyme-linked in situ hybridization; microphotometry; spleen; erythroblasts


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THE KIDNEY PLAYS an essential role in the control of the body's acid-base balance by reabsorbing or reclaiming the filtered HCO-3, and, in addition, by secreting protons or HCO-3 in the forming urine. The final urinary acidification occurs in the connecting segment and the cortical and medullary collecting duct (CD) and is accomplished by the mitochondria-rich intercalated cells (IC; see Refs. 18 and 28). The acid-secreting IC (type A IC) contain an apically located vacuolar type of H+-ATPase that pumps protons into the tubular lumen (9). Cytosolic alkalization is prevented by a basolateral release mechanism for HCO-3 that is mediated by a variant of the erythrocyte HCO-3/Cl- anion exchanger (eAE1; see Ref. 15). The functional importance of this anion exchanger in regulation of systemic acid-base balance is reflected by cases of primary distal renal tubular acidosis caused by mutations of the AE1 gene (10, 29).

Kidney AE1 (kAE1) lacks exons 1-3 of AE1 and contains a unique sequence at the 5'-end of the mRNA (exon 3A) that results from an alternative transcription initiation site within intron 3 of the AE1 gene (17, 19). kAE1 mRNA occurs in a major long (>= 90%) and a minor short splice variant (<= 10%), the latter lacking nucleotides (nt) 37-377 of the longer form (19). Because antibodies specific for the NH2-terminal portion of eAE1 (that is absent in kAE1) do not stain type A IC, it is generally concluded that only kAE1 is expressed in these cells (20, 33). However, positive proof for kAE1 expression in type A IC has not been given yet. Moreover, the location of a minor transcript possibly indicating transcription of the eAE1 mRNA in rodent kidney (3, 8, 22, 30) is not known.

The cortical CD of mammalian kidney is supplied with a second type of IC (type B IC) that contains the vacuolar type of H+-ATPase at the basolateral membrane domain and a still unknown type of HCO-3/Cl- anion exchanger at the apical membrane (4, 27). The proton-secreting activity of both types of IC appears to be regulated mainly by recycling of H+-ATPase between the plasma membrane and cytoplasmic storage vesicles. In acidosis, increasing amounts of the H+-ATPase are inserted in the apical membrane of type A IC, whereas in type B IC the H+-ATPase is endocytosed from the basolateral membrane under these conditions. Alkalosis induces a reverse mechanism (6, 26). No change in the amount of H+-ATPase protein or mRNA was detected under chronic metabolic perturbation (6).

Although these findings show that the plasmalemmal amount of the H+-ATPase is predominantly controlled at a posttranslational level, regulation of the surface deposition of kAE1 is less well understood. In rabbit cortical CD, AE1 immunoreactivity is predominantly located in vesicular structures of type A IC, and AE1 immunostain is shifted to the plasma membrane under conditions of chronic metabolic acidosis (NH4Cl gavage, 12 days; see Ref. 33). In type A IC of the human CD, a fraction of AE1 immunolabel is associated with intracellular vesicular structures (14). In type A IC of the rat kidney cortical CD, vesicular concentration of AE1 immunolabel was not observed at the light microscope level. However, elevated levels of basolateral AE1-immunostaining intensity were observed in response to acute metabolic acidosis (6 h, NH4Cl gavage), whereas substantially reduced levels of staining were found in acute metabolic alkalosis (6 h, NaHCO3 gavage; see Ref. 26). The acid-base response of medullary type A IC was not included in the study by Sabolic et al. (26). Northern blot analysis indicated two- to threefold elevation of AE1 mRNA in both cortex and medulla of rat kidneys in response to chronic respiratory acidosis (5 days of hypercapnia; see Ref. 30). In rabbits, RT-PCR indicated four- to fivefold higher levels of AE1 mRNA in dissociated cortical CD cells in animals subjected to subacute metabolic acidosis (16-20 h, NH4Cl gavage) compared with animals subjected to metabolic alkalosis (NaHCO3 gavage; see Ref. 17).

These results indicate that, in addition to possible posttranslational mechanisms, kAE1 may be regulated at a transcriptional level. However, a potential problem of all studies dealing with mRNA quantification in kidney homogenates is that the cellular source of the mRNA measured cannot be exactly determined. Although most studies agree that kAE1 is only present in type A IC, AE1 protein has been reported to occur also in other cell types of rabbit and rat kidneys (1, 2, 16, 17). Therefore, the possibility has to be considered that there may be other cells besides type A IC expressing low levels of kAE1 and/or eAE1 that might contribute to total AE1 mRNA measured in homogenates and thus render the recognition of kAE1 mRNA regulation in acid-base perturbations difficult. Moreover, the possibility of differential contributions of cortical versus medullary type A IC to renal kAE1 regulation has been largely neglected in previous studies.

In the present study, we present evidence by in situ hybridization (ISH) using specific oligonucleotide probes for kAE1 mRNA (covering a portion of exon 3A) and for eAE1 (covering a portion of exon 2) that, in the rat kidney, type A IC selectively express kAE1 in both cortex and medulla, whereas eAE1 mRNA is not detectable. Using single-cell quantification of AE1 mRNA content by microphotometry of enzyme-linked ISH, we document twofold higher kAE1 mRNA signals in medullary type A IC compared with cortical type A IC. Only a small but significant increase of kAE1 mRNA in both cortical and medullary type A IC was observed in acidosis compared with alkalosis.


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Animal Experiments

The studies were carried out on 3- to 4-mo-old male Wistar rats, weighing 200-250 g. All rats had a standard diet (food intake was restricted to 60-90 g/day) and access to drink ad libitum. For investigations of the distribution of different AE1 mRNA splice variants in the kidney, untreated rats were used. Chronic metabolic acidosis was induced by NH4Cl (75 mmol/l), and metabolic alkalosis was induced by NaHCO3 (150 mmol/l) added to the drinking water for 12 days. Control animals were maintained on ad libitum tap water. For the last 12 h before the experiments, the rats were starved. Beginning on day 5, urine pH was monitored daily. On the day of killing, arterial blood was taken for analysis of acid-base parameters (ABL 330; Radiometer, Wittich, Germany).

Tissue Preparation

For blood analyses, mRNA extraction, immunocytochemistry, and ISH/immunocytochemistry double labeling, rats were killed by cervical dislocation, followed by immediate opening of the thoracic cavity. A blood sample was taken from the left cardiac ventricle, and the animals were perfused with 50 ml PBS (137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.3). For ISH only, rats were decapitated in ether anesthesia. Kidneys and spleens were rapidly removed. For mRNA extraction, kidneys were immediately frozen in liquid nitrogen. For immunostaining and ISH, kidneys were cut in 5-mm-thick slices through their midportions (level of the papilla), frozen in liquid nitrogen-cooled isopentane, and stored at -80°C until needed. For immunostaining of semithin (1 µm) sections, small pieces (a few mm2) of frozen tissue were freeze-dried and embedded in Epon (13).

Immunocytochemistry

AE1 antibody. An affinity-purified rabbit polyclonal antibody raised against the cytoplasmic domain of rat eAE1 (reAE1) was used for immunolocalization of AE1 (15). Cryostat sections (5 µm) were cut at -20°C, mounted on Superfrost microscopic slides (Menzel, Braunschweig, Germany), and dried at room temperature (RT). Semithin Epon sections were etched and processed for immunostaining as described (13). Unless otherwise stated, the following steps were carried out at RT in PBS containing 1% ovalbumin (Sigma, Deisenhofen, Germany). After preincubation for 1 h, the sections were incubated with affinity-purified AE1 antibody diluted 1:30 overnight at 4°C. For control sections, the antibody was omitted from the incubation. After being washed (5 × 5 min in PBS), the sections were incubated for 30 min with tetramethylrhodamine isothiocyanate- or cyanine conjugate 3 (Cy3)-labeled goat anti-rabbit IgG (Biotrend, Cologne, Germany) diluted 1:100 or 1:600, respectively. Finally, the sections were thoroughly washed and mounted in 60% glycerine/PBS containing 1.5% n-propyl gallate as antifadant.

ISH

Probes. The following oligonucleotide probes were custom-synthesized and covalently labeled with one molecule of alkaline phosphatase (AP) per oligonucleotide (DNA Technology, Aarhus, Denmark). Additionally, for competition controls (see Controls for ISH), unlabeled oligonucleotides were provided by DNA Technology. The probe sequences were as follows: 1) human AE1 (hAE1) probe (for all AE1 mRNA splice variants), 5'-CTG-ACA-ATC-AGC-GTG-GTG-ATC-TGA-GAC-TCC-AGG, complementary to nt 2193-2151 of the human eAE1 mRNA (22) and 96% homologous (one base exchange in position 30, C for A) to a probe complementary to the corresponding sequence of the rat eAE1 (reAE1) mRNA (nt 1990 to 1949; see Ref. 22); 2) rat AE1 (rAE1) probe (for all AE1 mRNA splice variants), 5'-TGA-GCG-CAT-CGG-TGA-TGT-CAC-TCA-GGT-AGT-AAG, complementary to nt 1019-996 of the rat kAE1 (rkAE1) mRNA (22); 3) rkAE1 probe (for the long kAE1 mRNA splice variant only), 5'-CTA-CCG-TAC-TAC-TTT-CAG-GGA-GCC-TCA-GGA-AGC, complementary to nt -199 to -157 of the rkAE1 mRNA (22), this sequence is transcribed from exon 3A of the AE1 gene and is thus not present in the eAE1 mRNA; and 4) reAE1 probe (for the eAE1 mRNA splice variant only), 5'-GCT-GTC-CTC-GGT-GTC-CAG-TTG-TCC-TCT-GCT-CTC, complementary to nt 149-116 of the reAE1 mRNA. This sequence is transcribed from exon 2 of the AE1 gene and is thus not present in the kidney-specific kAE1 mRNA splice variant (22).

For all probes, homology comparisons were carried out with sequences registered in the GCG and EMBL GenBanks. None of the probe sequences had significant homologies with registered sequences for other proteins.

ISH protocol. ISH was carried out on unfixed kidney sections from 19 control (3 of which were untreated), 15 alkalotic, and 15 acidotic rats according to the method of Dågerlind et al. (12) as modified by Asan and Kugler (5). Briefly, 12-µm-thick cryostat sections were mounted on Superfrost slides (see AE1 antibody), thawed to RT, and immediately covered with hybridization solution containing 6 fmol/µl of one or several probes, 50% deionized formamide, 4× standard saline citrate (SSC), 10% dextran sulfate, 1× Denhardt's solution (Sigma), and 500 µg/ml salmon testes DNA (Sigma). Hybridization was carried out at 37°C for 16-20 h and was stopped by rinsing the sections with 1× SSC at 55°C. Posthybridization washes were with 1× SSC for 4 × 15 min at 55°C. After cooling to RT, the sections were washed in 0.1 M Tris · HCl and 0.15 M NaCl (pH 7.5) for 30 min and in 0.1 M Tris · HCl, 0.1 M NaCl, and 0.05 M MgCl2 (pH 9.5) for 10 min. AP development was carried out in the last buffer supplemented with 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate (Boehringer, Mannheim, Germany) and 0.4 mM tetranitro blue tetrazolium chloride (Serva, Heidelberg, Germany) at 25°C. The medium was filtered immediately before use.

Controls for ISH

Competition controls (34) were performed with hybridization solutions containing either of the oligonucleotide probes 1-4 or a mixture of two or three probes for probes 1-3, supplemented with the appropriate unlabeled oligonucleotides at a 100-fold excess. All controls showed no specific labeling.

Combined ISH/Immunocytochemistry

After ISH and AP development, sections were washed in PBS for 3 × 10 min and were preincubated in PBS-1% Triton X-100 (Sigma) and 5% normal goat serum (NGS; Sigma) for 1.5-2 h. Thereafter, the sections were incubated with AE1 antibody diluted 1:10 in PBS-0.5% Triton X-100-2% NGS for 72 h at 4°C. Subsequently, the sections were washed in PBS for 6 × 10 min, incubated in Cy3-labeled goat anti-rabbit IgG, diluted 1:600 in incubation buffer overnight at 4°C, washed again for 6 × 10 min in PBS, and mounted as described (see AE1 antibody).

Two series of controls were carried out for the double-labeling experiments. In the first series, competition control sections were subjected to immunocytochemistry. In these sections, the "normal" immunocytochemical labeling pattern and no ISH signal was observed. In the second series, sections hybridized with the labeled oligonucleotides (without competing unlabeled probes) were subjected to the immunocytochemical incubations omitting the primary antiserum. In these sections, a normal ISH pattern but no immunofluorescence signal was found.

Quantitative Measurements

A computer-controlled Leitz MPV-3 scanning microscope photometer (Leitz, Wetzlar, Germany; for details see Refs. 23 and 24) was used for quantitative measurements. The microphotometer settings were as has been described previously (5), with a measuring wavelength of 557 nm and a final magnification of ×40. The measuring square of 100 µm2 was resolved into 400 measuring steps, and the results were integrated to give a mean optical density (MOD). The measuring square was positioned in such a way to include the cell nucleus and the AP-reactive ring around it, and MODs of ISH-reactive cells were calculated relative to those of unreactive CD cells measured in the same section (5).

Quantitative analyses were carried out after ISH using a mixture of the hAE1 and the rAE1 mRNA probes. Measurements for the determination of the time course of AP development (5) were done on individual reactive cells from both the medulla and outer cortex (connecting segments and cortical CDs were not differentiated) in kidney sections of two untreated, two control, two acidotic, and two alkalotic rats. For comparative quantitative measurements in different metabolic conditions, at least 10 reactive cells in randomly chosen well-reacted areas of the medulla and of the cortex in 3 kidney sections from each of 5 control, 10 alkalotic, and 10 acidotic rats were evaluated.

Statistics

MODs measured in individual reactive cells were grouped according to their metabolic state (control, acidotic, and alkalotic) and their localization (outer medulla or cortex). Because preliminary experiments had indicated that the data did not display a strictly normal nor log normal distribution, comparisons between different groups (e.g., medullary cells in acidotic vs. in control kidneys) were carried out with Mann-Whitney U-tests. Overall significance of <0.05 was asserted using Bonferroni correction as modified by Holm (7, 19) at P < 0.0083 for the most significant difference and at P < 0.01 for the second highest and so on.


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Specificity of ISH Probes

For specificity tests, spleen and kidney sections of the rat were processed in parallel. Spleen sections incubated with the rAE1 probe (detects both eAE1 and kAE1 mRNA) and the reAE1 probe (specific for eAE1 mRNA) showed strong staining of cell groups in the red pulp, presumably representing erythroblastic colonies (31). No staining was observed in spleen sections hybridized with the rkAE1 probe (specific for kAE1 mRNA), indicating that this probe indeed does not detect eAE1 mRNA (Fig. 1).


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Fig. 1.   Serial cryostat sections of the rat spleen processed for in situ hybridization (ISH) using alkaline phosphatase (AP)-linked oligonucleotide probes for rat anion exchanger 1 (rAE1) mRNA. Note strong labeling of cells in the red pulp (r) [but not the white pulp (w)] applying non-isoform-specific rAE1 and rat erythrocyte-specific AE1 (reAE1) probes. No signal is seen with the rat kidney-specific (rkAE1) probe.

In serial kidney sections, the hAE1, rAE1, and rkAE1 probes labeled scattered IC-like epithelial cells of both cortical and medullary ducts. All three probes displayed similar labeling patterns (Fig. 2). When hAE1 and rAE1 probes (which detect all AE1 mRNA splice variants) were used together, the signal intensity per labeled cell was considerably increased (Fig. 2). Therefore, and to ensure that both the long and the short splice variant of rkAE mRNA (which is not detected by the rkAE1 oligo probe) were quantified, this probe combination was used for quantitative studies. Addition of the rkAE1 probe to the mixture led to a further signal intensification, but with a higher background staining. That the ISH-reactive cells are type A IC was demonstrated by ISH for either rAE1, rkAE1, or a combination of both probes followed by immunofluorescent labeling for AE1. Immunofluorescence of cells ISH-reactive for rAE1 and rkAE1 was preferentially located over the basolateral membrane (Fig. 3). These findings demonstrate that all three probes detect mRNA in the same cell type and most likely hybridize with the same species of mRNA. Occasionally, particularly in very strongly ISH-reactive cells, immunostaining was not detectable. On the other hand, especially in sections reacted with only one AE1 probe, few immunoreactive cells were observed that did not display ISH signal.


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Fig. 2.   Serial cryostat sections of an individual medullary ray of the inner kidney cortex processed for ISH using non-isoform-specific probes for human AE1 (hAE1) and rAE1 as well as rkAE1. Note similar labeling patterns for scattered cells in a collecting duct (CD)-like tubule. Strongest reactivity is observed by combination of hAE1 and rAE1 probes.



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Fig. 3.   Section of the outer medulla processed for ISH for rAE1 and rkAE1 followed by immunofluorescence labeling for AE1 (a-AE1). Note ISH signals for rAE1 and rkAE1 in AE1-immunoreactive cells, thereby identified as type A intercalated cells.

ISH of kidney sections with the reAE1 probe did not result in any labeling (not shown). Because, with the use of this probe, erythroblastic cells were easily detectable in spleen sections treated in the same experiment (Fig. 1), this finding indicates that the eAE1 mRNA splice variant is absent from rat kidney or, if present, represents only a negligible constituent of the AE1 mRNA species in this organ, not detectable by ISH.

Physiological Data

Blood acid-base parameters were determined in six control, nine acidotic, and six alkalotic animals. The values (mean ± SE) for the blood pH were 7.32 ± 0.02 (control), 6.95 ± 0.03 (acidosis), and 7.40 ± 0.01 (alkalosis); for plasma HCO-3, the values (mM) were 22.9 ± 0.31 (control), 10.49 ± 0.43 (acidosis), and 33.5 ± 0.35 (alkalosis). The urine pH was 6.93 ± 0.13 (control), 5.36 ± 0.21 (acidosis), and 9.37 ± 0.24 (alkalosis).

Immunocytochemistry

Regardless of the metabolic state, AE1 immunoreactivity was localized in basolateral membranes of individual cells of medullary and cortical ducts (Fig. 4). Immunostaining of cross sections through entire kidneys at the level of the midportion (tip of the papilla) revealed clearly stronger staining intensities of medullary than of cortical type A IC (Fig. 5). This difference was most pronounced in alkalosis and less obvious in acidosis. Under no condition were differences seen in the immunoreactivity between type A IC of the medulla. On the other hand, clear-cut differences in staining intensities were observed between cortical type A IC in acidotic, alkalotic, and control kidneys. In cortical IC, immunostaining was clearly weaker in alkalotic compared with acidotic and control kidneys but did not completely disappear.


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Fig. 4.   Semithin section of outer kidney cortex of control, acidotic, and alkalotic rats immunostained for AE1. Note significantly weaker immunostaining intensity in cortical type A intercalated cells of the alkalotic rat compared with the control and acidotic rat.



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Fig. 5.   Areas of the cortex and medulla photographed from individual 5-µm-thick cryostat sections of the entire midportion of kidneys processed for immunostaining with AE1 antibody. Blood has been removed by vascular perfusion. Note weaker immunoreactivity of type A intercalated cells in the cortex (arrowheads) compared with type A intercalated cells of the outer and inner medulla. In alkalosis, immunostaining of cortical type A intercalated cells is barely visible at this magnification.

ISH

The number and the distribution of ISH-reactive type A IC were similar in all metabolic states, and no differences in the morphology or the intracellular labeling pattern were observed (Fig. 6). ISH reaction product was mainly confined to a rim around the nucleus, often with some deposition over the nucleus (Figs. 3 and 6). Medullary type A IC were generally more intensely reactive than cortical ones. The highest density of reactive type A IC was found in the outer medullary CD. Cortical type A IC yielded the impression of somewhat lighter labeling in alkalotic than in acidotic kidneys (Fig. 6). Variations in labeling intensities between medullary type A IC of the different groups were not discernible. As in the control, staining of medullary type A IC was stronger than in cortical ones both in alkalotic and in acidotic states.


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Fig. 6.   ISH of sections of the rat kidney using combination of hAE1 and rAE1 probes at low magnification (a) and higher magnification (b-d). Images shown from the cortex and medulla (b-d) were taken from individual cross sections of the kidney, such as that in a. Note somewhat weaker reactivity of cortical vs. medullary type A intercalated cells. In alkalosis, cortical type A intercalated cells react less intensely than in acidotic and control conditions. Significant differences in ISH reactivity between medullary type A intercalated cells are not obvious under different acid-base conditions. OM, outer medulla; IM, inner medulla.

To ensure that, for quantitative evaluations, MODs in type A IC were measured at a time point where ISH reaction product accumulation was in its linear phase and could thus be regarded as a direct measure for AE1 mRNA levels, time course measurements were done on AE1 mRNA-reactive cells in medulla and cortex of untreated rat kidneys (Fig. 7A). Even though the increase in the more strongly reactive cells of the medulla was steeper, it was linear for both groups of cells for at least 12-13 h. Because measurements on 10 medullary and 10 cortical cells in three sections from each of two acidotic, two alkalotic, and two control rat kidneys also showed linearity in this time range regardless of the absolute staining intensity, quantitative measurements were generally carried out after 10 h of development.


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Fig. 7.   Microphotometric quantification of enzyme (AP)-linked ISH in type A intercalated cells [IC; mean optical density (MOD) ± SE] of outer cortical collecting duct and connecting segment (CCD) and outer medullary collecting duct (OMCD). A: time course of the increase of the MOD in 20 individual cortical and 20 individual medullary AE1 mRNA reactive cells (mean values ± SE) in kidney sections from two untreated rats. Regression analysis was carried out for the MOD values up to 16 h and shows that both in the strongly reacting medullary and in the less strongly reactive cortical type A IC there is a linear increase over at least 12-13 h. B: in control, acidosis, and alkalosis the optical density of ISH reaction product in type A IC of OMCD is ~2-fold higher than in CCD. Note small but significant capacity of type A IC to adapt kAE1 mRNA levels to disturbances of acid-base status.

As in normal rats, the mean MOD values (Fig. 7B) were significantly higher in medullary than in outer cortical type A IC in both acidosis and alkalosis (relation between MOD of cells in the medulla and in the cortex was in controls 1.93, in acidosis 1.94, and in alkalosis 1.99). Comparison of MOD values between reactive cells in the medulla showed a conspicuous increase in acidosis (~27%) and a slight decrease in alkalosis (~9%) compared with controls (controls: 0.276 ± 0.027; acidosis: 0.350 ± 0.06; alkalosis: 0.254 ± 0.041). Accordingly, MOD values in reactive type A IC in the kidney cortex were increased in acidotic (~20%) and decreased in alkalotic kidneys (~15%) compared with controls (control: 0.151 ± 0.014; acidosis: 0.19 ± 0.036; alkalosis: 0.129 ± 0.026). Differences between the MOD values in acidosis and in alkalosis were significant both in the medullary and in the cortical AE1 mRNA-reactive cells (P = 0.0022 and P = 0.00361, respectively).


    DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
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kAE1 Is Expressed in Type A IC

A number of years ago, the presence of AE1 protein in the basolateral membrane of type A IC of the rat was documented using an antibody specific for sequence portions shared by all AE1 variants (15). Later, indirect evidence for the presence of a kidney-specific AE1 splice variant (kAE1) expressed in type A IC of the human kidney was obtained by the observation that antibodies directed against an epitope located close to the NH2-terminus of AE1 and lacking in the kAE1 sequence did not stain type A IC (20). Further evidence for predominant kAE1 expression in the rat kidney came from the observation that kAE1 mRNA is much more abundant in the kidney than AE1 mRNA (8, 22, 30).

By using an oligonucleotide probe specific for the kidney splice variant of AE1 mRNA (rkAE1 probe), the present study provides the first positive in situ evidence for the selective expression of this mRNA species in rat kidney type A IC. The similarity of the hybridization patterns for oligonucleotide probes detecting all variants of AE1 mRNA (including the short kAE1 mRNA variant; hAE1 and rAE1 probes) and of the rkAE1 probe, and the fact that application of mixtures of the three probes resulted in increased signal intensities, indicated that all three probes recognized mRNA in the same cells (34). Combination of ISH with immunostaining confirmed that AE1 mRNA-expressing cells are type A IC.

The observation that a minor portion of duct cells that are positive for kAE1 mRNA were not immunoreactive for AE1 and vice versa could be taken to indicate that kAE1 mRNA is transcribed in additional cell types as has been suggested previously (e.g., see Ref. 2) and that some cells have the capacity to synthesize AE1-like protein but do not transcribe the kAE1 or eAE1 mRNA. However, we assume that this partial discrepancy between ISH and antibody reactivity is due to technical reasons. Thus immunofluorescence signal was generally lower in sections subjected to the ISH procedure than in nonpretreated sections. Also, most cells negative for AE1 immunostaining displayed particularly strong ISH reactivity, suggesting that high concentrations of the formazan enzyme reaction product may cover antigenic binding sites. On the other hand, cells positive for AE1 immunofluorescence but lacking ISH signal may be sectioned in such a way that the amount of mRNA still present in the sectioned cells is too low to be detectable.

eAE1 mRNA Is not Detectable in the Rat Kidney

Because the detection of a minor non-kAE1 transcript in kidney homogenates (8, 22, 30) indicated expression of another splice variant somewhere in the kidney, the question of whether type A IC cells express only kAE1 or both the kAE1 and eAE1 splice variant remained to be answered. We carried out ISH with an oligonucleotide probe directed against a sequence present only in the eAE1 and lacking in kAE1 (reAE1 probe). The probe failed to give an ISH signal in rat kidney sections processed in parallel with spleen sections, where the probe showed intense labeling of presumed erythroblastic cell groups. This finding suggests that eAE1 is not expressed in rat kidney and strongly suggests that the kAE1 mRNA splice variants are the only AE1 form expressed in rat type A IC. This observation confirms earlier studies that could not detect immunostaining in the rat kidney with eAE1-specific antibodies (20, 33). Because in the cited studies the kidneys analyzed were not perfused before mRNA isolation, the minor transcripts detected in Northern blots may have represented a contamination of kidney homogenates with reticulocytes (8, 21, 30). In the following descriptions of AE1 expression in the rat kidney, therefore, we will use the term kAE1 for describing the occurrence and alterations in AE1 expression in normal and metabolically disturbed situations.

Quantitation of kAE1 mRNA Levels

To our knowledge, the present study is the first to apply this method of single-cell ISH quantitation to kidney tissue. The use of probes detecting both kAE1 mRNA splice variants (22) ensured that quantitative measurements represented differences in single cell levels of all kidney-specific AE1 mRNA species. Whether differences in the relative contribution of the different splice variants to the entire kAE1 mRNA levels exist in different locations or after metabolic disturbances remains to be studied.

Cortical and Medullary Type A CD Cells Express Different Levels of kAE1

Quantitative ISH revealed approximately twofold lower amounts of kAE1 mRNA in cortical compared with medullary type A IC. These differences on the mRNA level correspond to an overall lower intensity of immunostaining and ISH of cortical versus medullary type A IC (Figs. 5 and 6). The lower mRNA expression and protein content of kAE1 in cortical type A IC indicate that the capacity of the type A IC in the cortical CD to reabsorb HCO-3 and to secrete equivalent amounts of H+ in the forming urine is significantly lower than the capacity of type A IC of the medullary CD.

Acidotic and Alkalotic States

The immunocytochemical observations indicate that the amount of kAE1 protein inserted in the basolateral membranes of cortical type A IC is regulated according to the chronic acid-base perturbation, being considerably higher in acidotic than in alkalotic animals. These results are in agreement with the findings by Sabolic et al. (26) in cortical type A IC after induction of acute acidosis and alkalosis. The optical impression of rather large differences in AE1 protein content of the basolateral membranes does not reflect the rather small changes observed in mRNA content. This indicates an additional posttranslational mechanism of regulation, perhaps via endocytotic extraction of the protein in alkalosis, as has been suggested for rabbits (32) and humans (14). The fact that, in the rat kidney, cytoplasmic AE1-immunoreactive vesicles are not observed in alkalosis does not necessarily argue against this supposition, since the amount of protein in these vesicles may be too low to be detectable by immunocytochemistry. In medullary type A IC, we did not observe obvious differences in either ISH or immunostaining intensities between control, acidosis, and alkalosis. However, immunofluorescence even in alkalotic kidneys is extremely strong so that differences in the range documented here may not be recognizable due to a nonlinearity of fluorescence signal at high labeling intensities. The quantitative ISH results showed that the changes in kAE1 mRNA content amounted to an increase of ~35% in acidosis compared with alkalosis in both the medullary and cortical type A IC. Although no systematic analyses were carried out, no obvious changes in the number of kAE1-positive cells in the different metabolic states were observed. Thus it can be concluded that the rat kidney type A IC possess the capacity to adapt to chronic metabolic perturbations by a limited increase or decrease in kAE1 mRNA levels, presumably followed by an increased or decreased synthesis of the protein. Differential insertion of the protein into or extraction from the basolateral membranes may add to this regulatory capacity.

The changes in AE1 mRNA expression documented here are much less conspicuous than has been described for isolated type A IC in rabbit cortical CD after subacute alkalosis and acidosis (13.8-fold increase as judged by RT-PCR; see Ref. 17). Beyond technical considerations (no control animals were included in that study), one explanation for these apparent differences could be that, because of their alkaline diet, rabbits excrete an alkaline urine (32), whereas the normal urine of control rats is acidic so that the CD HCO-3 reabsorption in rabbits requires a comparatively low capacity under control conditions. Under acid load, a more significant increase in proton secretion and, consequently, in HCO-3 reabsorption capacity would be necessary in rabbits compared with rats. The increase in content of kAE1 mRNA documented in metabolic acidosis in this study (~20-30%) is lower than the increase of the Northern blotting signals reported for AE1 mRNA after 5 days of hypercapnia in rats (2- to 3-fold; see Ref. 30). This could be due to the fact that, in respiratory acidosis, the kidney is the primary organ to regulate the body's acid-base balance. Nevertheless, both studies show that kAE1 of both the cortex and medulla takes part in the regulatory process. In this regulation, the medullary type A IC, with their documented higher expression of kAE1 mRNA, may play the most important role, a finding in agreement with the fact that the most obvious changes in the distribution of H+-ATPase in chronic acid loading occurred in the outer medullary CDs (6).

The present findings document for the first time positively the presence of the kidney-specific splice variant of AE1 mRNA in type A IC of rat kidney. Medullary CD type A IC express twofold higher levels of kAE1 mRNA than cortical type A IC. Also, the absence of any detectable amount of the erythroid splice variant (eAE1) in type A IC and of any other cell type of the rat kidney is confirmed. Chronic metabolic perturbation of the acid-base status leads to small but significant changes in the kAE1 mRNA levels in type A IC, indicating some degree of regulation of the anion exchange capacity on the transcriptional or the mRNA level. Recycling of H+-ATPase between cytoplasmic stores and the cell surface appears to be the predominant regulative mechanism to adapt to changing acid-base situations.


    ACKNOWLEDGEMENTS

We are grateful to Theresia Manger-Harasim for skillful technical assistance.


    FOOTNOTES

Saskia Huber and Esther Asan made equally strong contributions to this study and should be both considered as first authors.

This work was supported by grants of the Deutsche Forschungsgemeinschaft (SFB 176 and PU185/1-1).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. Drenckhahn, Institute of Anatomy, Julius-Maximilians-Univ., Koellikerstr. 6, D-97070 Würzburg, Germany (E-mail: anat015{at}mail.uni-wuerzburg.de).

Received 8 February 1999; accepted in final form 8 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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