INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

SYSTEMIC AND TISSUE-SPECIFIC HCO homeostasis is an essential physiological function. The kidney cortical collecting duct (CCD) plays an important role in systemic acid-base homeostasis (22, 23, 30). HCO secretion and absorption by this segment of the nephron is mediated by an intricate array of luminal and basolateral H⁺/HCO transporters residing in selective cells and selective membranes. The CCD is a heterogeneous epithelium consisting of three main cell types: -, -, and -intercalated cells (3, 22, 31, 37, 39). The -intercalated cells are characterized by expression of a vacuolar type H⁺-ATPase pump in the luminal membrane and a Cl/HCO exchanger in the basolateral membrane (3, 11, 37, 39). Immunocytochemical and molecular evidence suggests that at least part of the Cl/HCO exchange in the basolateral membrane of these cells is mediated by a splice variant of anion exchanger isoform 1 (AE1) of the SLC4 family of anion exchanger (2). This arrangement allows -intercalated cells to mediate HCO absorption (2, 11, 22, 39). The -intercalated cells display the opposite polarity in terms of expression of the H⁺/HCO transporters. The -intercalated cells express a Cl/HCO exchange activity in the luminal membrane (8, 9, 39). Accordingly, these cells mediate the bulk of HCO secretion by the CCD (22, 23, 30). The -intercalated cells appear to express Cl/HCO exchange activity in both membranes (9). The exact role of these cells in acid-base homeostasis is not well understood.

An unresolved and intriguing question is the identity of the protein(s) responsible for Cl/HCO exchange activity in the luminal membrane of the -intercalated cells. This activity is unique in that it is resistant to inhibition by DIDS, the classic and defining inhibitor of the SLC4 family, AE1-AE3 (2, 12). Very recently, two studies identified members of two families of anion exchangers as the possible proteins (29, 38). The first is a new member of the AE family, named AE4 (38). AE4 was cloned from isolated rabbit -intercalated cells and was shown to function as a Na⁺-independent Cl/HCO exchanger, although it has higher homology to members of the Na⁺-HCO cotransporter (NBC) family (27, 33) than to the SLC4 family. Important features of AE4 reported in the study of Tsuganezawa et al. (38) are the resistance to DIDS when expressed in Xenopus oocytes and the colocalization of AE4 and peanut lectin in the luminal membrane of the rabbit CCD. Peanut lectin is a marker of the rabbit -intercalated cells. These studies were interpreted to suggest that AE4 is the protein responsible for HCO secretion by -intercalated cells.

A different view emerged from examination of HCO secretion in the PDS^/ mouse CCD (29). The PDS gene codes for pendrin, a protein mutated in Pendred syndrome (10). Pendrin belongs to a new family of proteins named SLC26 (20). Members of this family, including pendrin (34), were shown to function as Cl/HCO exchangers (24). In the mouse and human kidney, pendrin is expressed exclusively in the luminal membrane of the CCD (18, 29). Most notably, deletion of the PDS gene eliminated net HCO secretion by isolated, perfused CCD. In fact, the perfused CCD from PDS^/ mice absorbed, rather than secreted, HCO under the same condition that CCD from wild-type mice secreted HCO (29). These findings provide strong evidence that pendrin mediates most, if not all, of the HCO secretion by the CCD, at least in the mouse.

The findings with the PDS^/ mouse (29) raise the question of the role of AE4 in acid-base transport by the kidney and other cells. An organ that absorbs HCO at the resting state and secretes copious amounts of HCO at the stimulated state is the salivary duct (21). Therefore, it was of interest to determine the localization of AE4 in submandibular gland (SMG) duct cell. We independently cloned AE4 from a rat kidney library at the time that the report on the rabbit AE4 appeared. Here we report the functional properties and localization of the AE4 cloned from the rat kidney. Although we also find that AE4 functions as a Na⁺-independent Cl/HCO exchanger and is expressed mostly in the CCD, our findings reveal three fundamental differences from those reported in the rabbit CCD (38). We find that AE4 activity is completely inhibited by DIDS, and AE4 is expressed in the rat and rabbit -intercalated cells. Membrane localization of AE4 proved to be species specific. In the rat, AE4 is expressed in the basolateral membrane, whereas in the rabbit it was found in the luminal and lateral membrane. Importantly, the localization of AE4 was not influenced by the metabolic status of the animals. We conclude that in the rat AE4 is likely to function in HCO absorption by -intercalated cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cloning of AE4. A human expressed sequence tag (EST) clone (GenBank accession no. AI183992 from testis) was identified by a BLAST search using the NBC family as queries. The EST clone was purchased from Research Genetics (Huntsville, AL) and sequenced to confirm its identity as a new member of the NBC family. A rat kidney cDNA library (14) was screened using the EST clone as probe, and eight clones were obtained. All the clones were sequenced and found to derive from the same gene. The longest clone (3.2 kb) was chosen for further analysis.

Northern blot and RT-PCR analyses. Total RNA was isolated from rat tissues with an RNeasy kit (Qiagen) according to the manufacturer's instructions. Each lane was loaded with 10 µg RNA, which was separated in a 0.8% agarose gel and transferred to nylon membranes. A multiple rat tissue Northern blot containing 2 µg of poly(A) RNA (Clontech) was also probed. The membranes were hybridized with randomly primed full-length AE4 cDNA labeled with [³²P]dCTP for 3 h at 68°C in an ExpressHyb hybridization buffer (Clontech). Subsequently, the membranes were washed at high stringency and developed by radiography. Nephron segments were microdissected from the rat kidney and used to prepare mRNA. The mRNA was reverse transcribed with random primers as previously described (25). The synthesized cDNA was used for 30 cycles of PCR (94°C for 1 min, 60°C for 1 min, 72°C for 2 min) with specific primers for AE4: sense AGGCTTCTCGTGATGAGG (nucleotides 512-564) and antisense strand AATCGCTGGGGTACCAGC (nucleotides 1155-1138) with an expected product size of 609 bp. The PCR products were electrophoresed on 2% agarose gels and transferred to nylon membranes, which were hybridized at high stringency. The PCR product obtained from the CCD was subcloned into a plasmid using a TA cloning kit (Invitrogen) and sequenced.

Western blot. Anti-AE4 antibodies were raised against two peptide sequences [antibody A against QPKAPEINISVN, amino acids (aa) 948-959; and antibody B against EEEKTIPENRPEPEH, aa 919-933], at or near the carboxy terminus of AE4. Multiple antigenic peptides (35) were synthesized to generate polyclonal antibodies specific for AE4 by immunizing rabbits. The antisera with the highest titer in an ELISA assay (1:64,000) were IgG fractionated by an affinity column (HiTrap Protein G, Pharmacia Biotech). The IgG fractions were used for Western blots and immunolocalization. For Western blot, membrane vesicles were prepared from the cortex and medulla of the rat kidney by differential centrifugation as before (15). The pellet was suspended in homogenization buffer, and 10 µg of protein were separated by SDS-PAGE. The primary antibodies were used at a dilution of 1:1,000 and the secondary antibody at a dilution of 1:1,000. To verify the specificity of the antibodies, membranes were prepared from HEK-293 cells transfected with green fluorescent protein (GFP) only or GFP and AE4 plasmids. After transfer, the membranes were probed with each of the anti-AE4 antibodies.

Immunostaining of AE4 expressed in HEK-293 and LLC-PK₁ cells. Both cell types were plated on glass coverslips and grown in DMEM-high glucose (HG) medium supplemented with 10% fetal calf serum. The cells were transfected with AE4 using the Lipofectamine reagent and grown to confluency to allow development of cell-cell contacts and, in the case of LLC-PK₁ cells, to allow establishment of cell polarity. Between 48 and 72 h posttransfection the cells were fixed with 4% formalin for 5 min, washed, and permeabilized by incubation in cold ethanol. The cells were stained with a 1:1,000 dilution of the anti-AE4 antibodies and detected by a 1:400 dilution of a secondary goat anti-rabbit antibody tagged with Alexa 488, as detailed below for rat kidney sections. The cells were imaged by confocal microscopy.

Immunohistochemistry of rat kidney and mouse kidney and SMG. When used, anti-H⁺ pump antibody was the monoclonal E11 raised against the 31-kDa subunit of the vacuolar H⁺-ATPase (gift from Dr. S. Gluck). Rat kidneys were fixed by retrograde perfusion via the aorta with 3% paraformaldehyde, in 0.1 M cacodylate buffer, pH 7.4. Mouse kidneys and SMG were removed into an OCT reagent, frozen in liquid N₂, and stored at 80°C until sectioning. Immunostaining of mouse kidney and SMG sections was exactly as described (21). When the effect of the metabolic status of the rats on the localization of AE4 was examined, sections from kidneys of control, acidotic, and alkalotic rats were processed in the same manner as mouse tissue (21). For other forms of immunofluorescence microscopy, rat kidney blocks containing all kidney zones were dehydrated and embedded in paraffin. For light- and laser confocal microscopy the paraffin-embedded tissue was cut at 2 µm on a microtome (Leica). The sections were dewaxed and rehydrated. To reveal antigens, sections were put in 1 mM Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and were heated using a microwave oven for 10 min. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH₄Cl for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections from all animals and tissues were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The sections were then rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 3 × 10 min. The sections for laser confocal microscopy were incubated in Alexa 488-conjugated goat anti-rabbit antibody (Molecular Probes) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100 for 60 min at room temperature. For double labeling, Alexa 546-conjugated goat anti-mouse antibody (Molecular Probes) was added as well. After rinsing with PBS for 3 × 10 min, the sections were mounted in glycerol supplemented with antifade reagent (N-propyl gallate). The microscopy was carried out using a Leica DMRE light microscope, a Zeiss LSM510, or a Bio-Rad 1024 laser confocal microscope.

Functional expression of AE4 in HEK-293 cells. The full-length AE4 cDNA was subcloned into a mammalian expression vector under the CMV promoter (pCMV-SPORT, GIBCO BRL). HEK-293 cells were cultured in DMEM-HG media supplemented with 10% fetal calf serum and plated on glass coverslips. Two plasmids, one carrying AE4 and one carrying GFP, were transfected using the Lipofectamine reagent (GIBCO BRL) according to instructions provided by the manufacturer and using 1.5 µg of each plasmid. GFP was used to identify the transfected cells. The cells were used for intracellular pH (pH_i) measurements 48-72 h posttransfection.

Measurement of pH_i. pH_i was measured with the aid of BCECF, as detailed (19). In brief, coverslips with cells attached to them were assembled to form the bottom of a perfusion chamber. The cells were perfused with solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 glucose, and 10 HEPES (pH 7.4 with NaOH) and loaded with 2.5 µM BCECF-AM by a 10-min incubation at room temperature. The level of AE4 transfection was estimated from GFP fluorescence. BCECF fluorescence was at least 10-fold higher than the original GFP fluorescence. After BCECF loading, the cells were perfused with a HCO-buffered solution, and pH_i was measured by photon counting using a PTI recording setup (Delta Ram, New Brunswick, NJ). The HCO-buffered solution contained (in mM) 120 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 glucose, 5 HEPES, and 25 NaHCO₃ (pH 7.4 with NaOH) and was continuously gassed with 95% O₂-5% CO₂. Na⁺-free solution was prepared by replacing Na⁺ with N-methyl-D-glucamine, and Cl-free solution was prepared by replacing Cl with gluconate. High-K⁺ solutions were prepared by replacing between 100 and 140 mM NaCl with KCl. BCECF fluorescence was recorded from two to four cells, all of which expressed GFP and at excitation wavelengths of 490 and 440 nm at a resolution of 2/s (19). As reported before for CFTR (19), the correlation between expression of GFP and AE4 activity was close to 100%.

Manipulation of acid-base status of rats. Experiments were with male Sprague-Dawley rats weighing 250-300 g. The animals were allowed free access to food and drinking solution up to the time of the experiments. In each series, a group of experimental animals was compared directly with controls that were obtained from the same shipment and studied during the same period. Chronic metabolic acidotic (CMA) rats were obtained by following the procedure described in Ref. 5. Control and CMA rats were allowed to drink water or water supplemented with 0.28 M NH₄Cl for 7 days, respectively. Both groups received standard rat chow ad libitum. On the day of the experiment, rats were anesthetized with pentobarbital sodium. Blood was collected by aortic puncture for analysis of plasma pH, and the kidneys were rapidly removed and embedded in the OCT compound for obtaining frozen sections or immersed in 10% formaldehyde solution for further analysis. The blood pH of control rats was 7.411 and 7.466 (n = 2). The blood pH of CMA rats was 7.306 and 7.343 (n = 2), respectively. To obtain alkalotic rats, animals were placed in metabolic cages and allowed to acclimate on a synthetic diet consisting of (in g) 180 casein, 200 cornstarch, 500 sucrose, 35 corn oil, 35 peanut oil, 10 CaHPO₄, 6 MgSO₄, 5.25 NaCl, 8.3 K₂HPO₄, and 10 vitamin fortification mixture (ICN, Cleveland, OH) for 5 days. NaCl was then replaced with 7.55 g NaHCO₃ for chronic alkali feeding. Subsequently, control rats receiving the synthetic diet were pair fed with rats receiving the synthetic alkali diet. Rats were estimated to receive 6 mmol · kg body wt¹ · day¹ NaCl or NaHCO₃. In this protocol Na ingestion by control and experimental animals is the same. Serum HCO concentration in control rats averaged 26.1 ± 0.76 mM, and in alkalotic rats it averaged 30.48 ± 0.29 mM. Animals were killed after 7 days, and the kidneys were removed and embedded in OCT and processed as described above.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Characterization of AE4 cDNA and predicted protein. The AE4 cDNA cloned from the rat kidney has a composite nucleotide sequence of 3,178 bp and encodes a protein of 953 amino acids with a predicted molecular mass of 105 kDa (GenBank/EBI/DDBJ Data Bank, accession no. AB024339) (Fig. 1A). Hydropathy analysis (Kyte-Doolittle algorithm) of the predicted sequence suggests that AE4 has 12 putative membrane-spanning segments. It has four potential NH₂-linked glycosylation sites (Asn-546, Asn-570, Asn-932, and Asn-949, but only Asn-546 and Asn-570 are predicted as extracellular). The NH₂ terminus has a leucine-zipper motif (LQKLRGLLAEGIVLLDCPARSL, aa Leu-101 to Leu-126), which may mediate protein-protein interaction. Three putative protein kinase A phosphorylation sites (Ser-173, Ser-273, and Ser-764, with Ser-173 and Ser-273 predicted to be intracellular), six protein kinase C phosphorylation sites (with Thr-71, Thr-252, Ser-268, and Thr-352 predicted intracellular and Thr-655 and Ser-759 predicted extracellular), and 10 casein kinase II phosphorylation sites (Ser-31, Ser-37, Ser-121, Ser-173, Ser-248, Ser-548, Thr-593, Ser-900, Thr-917, and Ser-930, with only Ser-548 and Thr-593 predicted to be extracellular) can be identified in the sequence.

View larger version (60K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of anion exchanger isoform 4 (AE4) and phylogenic tree of mammalian HCO transporters. A: sequence alignments of AE4 from rat (rAE4; GenBank accession no. AB024339), rabbit (rabAE4; AB038263), and human (hAE4; AB032762). The putative transmembrane (TM) domains predicted by Kyte-Doolittle algorithm are underlined with a solid line and marked TM1-TM12. The putative DIDS binding motifs are in black boxes. Double underline represents peptide sequence used to raise the antibodies. B: phylogenic tree was constructed according to Higgins' method (13a). The length of the horizontal lines indicates the degree of amino acid divergence. For most transporters rat sequences were used. For NDCBE1, NCBE, NBC4, and BTR1 the human sequences were used because the rat sequences for these transporters are not available at present. GenBank accession nos.: rAE1, P23562; rAE2, A34911; rAE3, A42497; rAE4, AB024339; rNBC1, AF004017; rNBC2, AF070475; NDCBE1, AF069512; NCBE, AB040457; and BTR1, AF336127.

Expression profile of AE4 in rat tissues. Northern blot analysis revealed the presence of a prominent 3.4-kb AE4 mRNA in the kidney (Fig. 2A) and gastrointestinal (GI) tract (Fig. 2B), which was absent or below detection levels in all other tissues examined. A higher molecular weight mRNA of lower intensity was present in the kidney (Fig. 2A). Expression of AE4 mRNA in the kidney and GI tract was further analyzed by subdividing the kidney into segments and using various tissues of the GI tracts (Fig. 2B). High levels of AE4 mRNA were found in the cortex with diminished amount in the outer medulla and absence from the inner medulla. Interestingly, high level of AE4 mRNA was also expressed in the cecum, but it was absent in other segments of GI tracts. To localize mRNA expression more precisely, we performed RT-PCR analysis with mRNA prepared from microdissected nephron segments. Figure 2C shows that AE4 mRNA is expressed at high levels in the CCD.

View larger version (69K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis, RT-PCR, and Western blot analysis of AE4. A: Clontech membrane with poly(A) RNA from multiple rat tissues was hybridized with randomly primed full-length AE4 cDNA. Ht, heart; Br, brain; Sp, spleen; Lg, lung; Lv, liver; Ms, muscle; Kd, kidney; Te, testes. B: Northern blot analysis of total RNA prepared from rat tissues and hybridized as in A. Nos. indicate preparation of mRNA from stomach (1), jejunum (2), ileum (3), cecum (4), colon (5), pancreas (6), renal cortex (7), renal outer medulla (8), and renal inner medulla (9). C: primers listed in EXPERIMENTAL PROCEDURES were used to amplify AE4 mRNA by RT-PCR from microdissected nephron segments, and the result of a Southern blot with the AE4 probe is shown. Arrow-marked probe indicates migration of probe amplified from the cDNA. The segments used are glomeruli (GL), proximal convoluted tubule (PCT), proximal striated tubule (PST), thick ascending limb of the loop of Henle (TAL), and cortical collecting duct (CCD). Identity of product amplified from CCD was verified by sequencing. For Western blots HEK-293 cells were transfected with green fluorescent protein [GFP; control (Con) lane in each blot in D and E] or with AE4. Microsomes were prepared from HEK-293 cells (D and E) or the kidney outer medulla (OM) and cortex (Cx) (F) and were used for SDS-PAGE. For HEK-293 cells, each lane contained ~20 µg protein, and for kidney each lane contained ~10 µg protein. The blots in E and F were probed with anti-AE4 antibodies A, and the blot in D was probed with anti-AE4 antibodies B. The primary antibodies were used at a dilution of 1:1,000 and the secondary antibody at a dilution of 1:1,000. The right blot in F was probed with anti-AE4 antibody A that was preabsorbed with the peptide (10 µg/ml) used to raise the antibodies. The specific 125- to 135-kDa band (arrow) is expressed at higher level in the kidney Cx and at low level in the OM. Note the band at 125- to 135-kDa disappeared as a result of preabsorption of the antibodies with a competing peptide.

Functional characterization of AE4. To characterize the activity of AE4 it was necessary to show that the recombinant protein is targeted to the plasma membrane. Therefore, we expressed AE4 in HEK-293 and LLC-PK₁ cells and immunolocalized it using the antibody that was used in Fig. 2, E and F. Figure 3, A and B, establishes the specificity of the antibody for immunolocalization. Thus the anti-AE4 antibody A detected the transfected cells (Fig. 3A), and the staining was completely eliminated by preincubation of the antibodies with the blocking peptide (Fig. 3B). The high-magnification images in Fig. 3, C and D, show that in HEK-293 cells AE4 localized largely at the plasma membrane with no noticeable expression in the endoplasmic reticulum (ER) or the Golgi. Figure 3, E and F, shows punctate expression of AE4 in the plasma membrane of LLC-PK₁ cells, including at cell-cell contacts. Control experiments with antibodies adsorbed with the antigenic peptide revealed complete absence of labeling (not shown). Targeting of AE4 to the plasma membrane made it possible to use expression of AE4 in HEK-293 cells for functional characterization.

View larger version (87K): [in this window] [in a new window]   Fig. 3.   Immunolocalization of AE4 expressed in HEK-293 and LLC-PK1 cells. AE4 was transiently expressed in HEK-293 (A-D) or LLC-PK1 cells (E and F). A and B: cells were immunostained with anti-AE4 antibody A (green) and counterstained with 4,6-diamidino-2-phenylindole to label the nuclei. In B the antibodies were preadsorbed with the competing peptide before use for immunostaining. Scale bar, 25 µm. C and D: 2 examples of localization of AE4 to the plasma membrane of HEK-293 cells. Scale bar, 5 µm. E and F: similar targeting to the plasma membrane of LLC-PK1 cells. Scale bar, 5 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Properties of Cl/HCO exchange activity in HEK-293 cells expressing AE4. HEK-293 cells were transfected with GFP (A; control). In all other panels (B-E) the cells were cotransfected with GFP and AE4. The cells in A (control) and B (AE4) were incubated in HEPES-buffered media, and, where indicated by bars, the cells were exposed to Cl-free medium. The same protocol was repeated after incubating the cells in HCO-buffered media. In C-E the cells were maintained in HCO-buffered media throughout. In the first portion of each experiment, the cells were transiently incubated in Cl-free medium before exposure to media containing 5 mM Na+ (C), 10 µM ethylisopropyl amiloride (EIPA; D), or 100 mM K+ and 2 µM valinomycin (Val) (E). As indicated by the bars, the cells were exposed to Cl-free media under each of the conditions. Each trace represents 1 of 3 experiments with similar results.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   DIDS inhibits AE4-mediated Cl/HCO exchange activity. A: HEK-293 cells transfected with AE4 were incubated in HCO-buffered solutions and in the presence or absence of the indicated concentration of 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS). At the times indicated by the bars, the cells were exposed to Cl-free solutions. B: summary of the results of 4-7 experiments under each H2DIDS concentration between 0 and 200 µM.

Localization of AE4 in the kidney. The RT-PCR analysis in Fig. 2 indicated expression of AE4 mainly in the CCD. We extended this analysis to localize the AE4 in specific regions of the CCD by immunocytochemistry. Figure 6 shows that the anti-AE4 antibody A stained exclusively the basolateral membrane of cells in the CCD. Staining was absent from all other segments of the nephron, including intercalated cells in collecting ducts in the inner stripe of the outer and inner medulla (Fig. 6, D and E). In control experiments preincubation with the peptide used to raise the antibodies eliminated the staining (Fig. 6F). Localization of AE4 in the basolateral membrane of the rat CCD cells (Fig. 6) was unexpected in view of the reported expression of AE4 in the luminal membrane of rabbit CCD -intercalated cells (38). As a first protocol to verify basolateral localization of AE4 in the rat CCD we used the anti-AE4 antibody B (see EXPERIMENTAL PROCEDURES and Fig. 2). Figure 6, G and H, shows that anti-AE4 antibody B also stained exclusively the basolateral membrane of the rat CCD.

View larger version (138K): [in this window] [in a new window]   Fig. 6.   Localization of AE4 in the rat CCD. AE4 was localized in rat kidney cortex (A-C), outer medulla (inner stripe) (D), and inner medulla (E) using anti-AE4 antibody A (serum, 1:6,000). A: survey section showing anti-AE4 labeling in CCD. No labeling of other parts of the nephron was observed. CD, collecting duct; Glo, glomerulus. B and C: AE4 labeling is associated with the basal (large arrows) and lateral (small arrows) plasma membrane, whereas the luminal membrane was unlabeled. P, proximal tubule. D and E: in the inner stripe of the outer medulla and in the inner medulla no labeling was observed. F: control using anti-AE4 antibody preabsorbed with immunizing peptide (in all cases 0.1 mg/ml) shows no labeling. G and H: localization of AE4 in rat kidney cortex using anti-AE4 antibody B (IgG, 1:15,000). G: as with antibody A, the labeling with anti-AE4 antibody B was associated with the basal (large arrows) and lateral (small arrows) plasma membrane. H: control using anti-AE4 antibody B preabsorbed with immunizing peptide and showing no labeling. Magnification: A, ×130; B-H, ×360.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Laser-confocal localization of AE4 in collecting ducts in rabbit and rat kidneys. Labeling was with biotinylated anti-AE4 antibody A (affinity purified, 1:200). A: CCD in rabbit kidney showing AE4 labeling in the apical (large arrows) and lateral (small arrows) plasma membrane in the intercalated cells. L, lumen. B: control using anti-AE4 antibody preabsorbed with immunizing peptide (in all cases 0.1 mg/ml) shows no labeling. CD, collecting duct. C: AE4 (green) was present in the apical and lateral plasma membrane of CCD -intercalated cells. -Intercalated cells were identified as the cells expressing the vacuolar H+- ATPase in the apical plasma membrane (red labeling, large arrows). L, lumen. D: in contrast to the rabbit CCD, the rat CCD showed basal (large arrows) and lateral (small arrows) labeling using the same antibody as in A-C. Inset: control using biotinylated anti-AE4 antibody preabsorbed with immunizing peptide and showing no labeling. Magnification: A-D, ×600; inset, ×150.

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Localization of AE4 and the vacuolar H+ pump in rat kidney cortex, outer medulla, and inner stripe of the outer medulla. A and B: AE4 labeling (green) is present in the basolateral (arrows) and absent from the luminal membrane of -intercalated cells in the CCD. -Intercalated cells were identified by expression of the H+ pump in the luminal membrane (red labeling, arrowheads). CD, collecting duct. C and D: AE4 (green) was also observed in the basolateral membrane of -intercalated cells (arrows) in the collecting duct. -Intercalated cells were identified as cells expressing H+ pump (red) in the basolateral membrane. E and F: little or no AE4 was observed in collecting duct intercalated cells in the inner stripe of the outer medulla. By contrast, these cells exhibit abundant H+ pump (arrowheads). Magnification: A, C, E, and F, ×1,300; B and D, ×650.

View larger version (93K): [in this window] [in a new window]   Fig. 9.   Localization of AE4 in mouse kidneys and submandibular gland (SMG) and lack of effect of the metabolic state of the rats on localization of AE4. Mouse kidneys (A and B) and SMG (C) were stained with anti-AE4 antibody A (A and C) or anti-AE4 antibody B (B). Arrows mark AE4 in the basolateral membrane. In D-F the effect of metabolic acidosis and alkalosis on localization of AE4 in the rat kidney was examined. Kidneys from 2 control (D), 2 acidotic (E), and 4 alkalotic (F) rats were processed for immunolocalization and stained with anti-AE4 antibody A. Note that in all kidneys AE4 was found only in the basolateral membrane of the CCD.