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/HCO
1 Department of Physiology and 3 Department of Medicine, Division of Nephrology, University of Texas Southwestern Medical Center, Dallas, Texas 75390; 2 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK 8000 Denmark; and 4 Department of Pharmacology, Jichi Medical School, Minamikawachi, 329-0498 Tochigi, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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The renal cortical collecting duct (CCD)
plays an important role in systemic acid-base homeostasis. The
-intercalated cells secrete most of the HCO
/HCO/HCO
-intercalated cells. Importantly, localization of AE4 was not affected by the systemic acid-base status
of the rats. Therefore, we conclude that expression and possibly
function of AE4 is species specific. In the rat and mouse AE4 functions
as a Cl/HCO-intercalated cells and may participate in
HCO
anion exchanger isoform 4; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; submandibular
gland; -intercalated cells; kidney; cortical collecting duct
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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SYSTEMIC AND
TISSUE-SPECIFIC HCO-, -, 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/HCO-intercalated cells to
mediate HCO-intercalated cells display the opposite polarity in terms of
expression of the H+/HCO-intercalated cells express a
Cl/HCO-intercalated cells appear to express Cl/HCO
An unresolved and intriguing question is the identity of the protein(s)
responsible for Cl/HCO-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-intercalated cells. These studies were interpreted to suggest that
AE4 is the protein responsible for HCO-intercalated cells.
A different view emerged from examination of HCO/ 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/ mice absorbed,
rather than secreted, HCO
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/HCO-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-intercalated cells.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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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 (pHi) measurements 48-72 h posttransfection.
Measurement of pHi.
pHi 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-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
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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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.
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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.
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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.
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-dependent HCO-free medium
had minimal effect on pHi. On the other hand, exposing the
cells to Cl-free medium in the presence of
HCO. At higher expression
levels AE4 also showed Cl/OH exchange
activity (not shown). The cytosolic alkalinization on removal of
Cl and the acidification on readdition of
Cl to the incubation medium were similar in media
containing 150 or 5 mM Na+ (Fig. 4C). Incubating
the cells with 10 µM ethylisopropyl amiloride (EIPA), a concentration
sufficient to inhibit Na+/H+ exchange activity
and the activity of the newly discovered novel, luminal
Na+-dependent OH/HCO/HCO-dependent changes in
pHi (Fig. 4E). Averaging the rates of
pHi/min under each condition showed no statistically
different effect of any of the conditions on AE4 activity. Hence, the
results in Fig. 4 show that AE4 mediates a Na+-independent,
Cl-dependent HCO
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uptake (38).
This is a critical point with respect to the possible function of AE4
because the Cl/HCO-intercalated cells is DIDS insensitive
(8, 9, 40). Therefore, we carefully examined the effect of
4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid
(H2DIDS) on AE4 activity. Individual examples and the
summary of the results are shown in Fig.
5. H2DIDS potently inhibited AE4 activity with 50% inhibition at ~5 µM H2DIDS. We
also tested the effect of 200 µM DIDS, which also completely
inhibited AE4 activity.
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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.
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-intercalated cells because they also expressed
the vacuolar H+ pump in the luminal membrane. Only
-intercalated cells express the H+ pump in the luminal
membrane (3, 11, 22, 30, 31, 37, 39). Finally, the
biotinylated anti-AE4 antibodies stained the basolateral
membrane of the rat CCD as was found with the nonbiotinylated antibodies (Fig. 7D). In Fig.
8 the rat kidney was double-stained with
anti-AE4 and anti-H+ pump antibodies to identify the cells
expressing AE4 in the basolateral membrane. Figure 8, A and
B, and Fig. 8, C and D, respectively, show that both -intercalated cells and -intercalated cells in the
rat CCD express AE4 only in the basolateral membrane.
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/HCO/HCO/HCO/HCO/Cl exchange in the absence of
HCO/HCO. Second, we find that AE4 is expressed in the
basolateral membrane of rat kidney -intercalated cells and the mouse
CCD and SMG duct, whereas previous work suggested expression of AE4 in
the luminal membrane of rabbit kidney -intercalated cells
(38). This turned out to be at least in part due to
species-specific expression of the protein. Hence, the antibodies used
in the present work to localize AE4 in the basolateral membrane of the
CCD localized AE4 to the luminal and lateral membranes of the rabbit
CCD. The physiological significance of such species-specific
localization is not obvious. A naturally more alkaline rabbit diet can
explain expression of AE4 in the luminal membrane. However, why such
expression exists in -intercalated cells together with the
H+ is puzzling. One possibility is that
HCO-intercalated cells aid the
-intercalated cells in HCO/HCO-intercalated cells suggests that the major role of this transporter
in these species is mediating HCO-intercalated cells of the
CCD also express a splice variant of AE1 in the basolateral membrane
(2, 3, 12). Identification of mutations in AE1 that leads
to distal renal tubular acidosis (6, 32, 36) clearly
indicates that AE1 plays an important role in clearance of cytosolic
HCO-intercalated cells.
It is, however, possible that AE4 is complementary with AE1 in
mediating basolateral HCO-intercalated
cells, and expression of AE4 allows some acid-base regulation in
patients with mutations in AE1. Alternatively, expression of AE4 may
overlap with that of AE1, and both proteins share the same
physiological role. In any case, it is interesting that the function of
AE4 and AE1 does not appear to be redundant, although both
Cl/HCO| |
ACKNOWLEDGEMENTS |
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We are indebted to Dr. P. Preisig for access to the metabolic cages and for overseeing the preparation of the acidotic and alkalotic rats. We thank E. A. Salam and A. Y. Umpierre for expert assistance in setting the metabolic state of the rats and in determining blood gases and electrolytes, respectively.
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FOOTNOTES |
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This work was supported by a grant from the Salt Science Foundation to K. Ishibashi and by National Institutes of Health Grant DE-12309 and a grant from the Cystic Fibrosis Foundation to S. Muallem. S. B. H. Ko was supported by a fellowship from the Uehara Memorial Foundation.
Address for reprint requests and other correspondence: S. Muallem, Dept. of Physiology, Univ. of Texas Southwestern Medical Center, Dallas, TX 75390 (E-mail: shmuel.muallem{at}utsouthwestern.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.
June 5, 2002;10.1152/ajpcell.00512.2001
Received 25 October 2001; accepted in final form 8 May 2002.
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TOP
ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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