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INTRODUCTION |
In the kidney, the activity of the Na+-H+
exchanger isoform NHE3, located on the apical microvillar membrane of
the proximal tubule, plays a major role in mediating transepithelial
bicarbonate and NaCl reabsorption (2-4). Numerous physiologic studies
of brush border Na+-H+ exchange have shown that
this activity is regulated by such hormones as angiotensin II (5) and
parathyroid hormone (6) as well as by systemic alterations in
acid-base balance (2, 3). Several laboratories have presented evidence
suggesting that NHE3 is regulated by posttranslational mechanisms that
may include membrane trafficking between an intracellular compartment
and the plasma membrane (7-9). Such models predict that NHE3 must be
localized in a nonmicrovillar membrane compartment, which functions as
a store of transporter, as well as on the microvillar membrane where
NHE3 is active.
We have recently reported an association between NHE3 and the putative
scavenger receptor megalin in renal brush border membranes (1).
Moreover, we found that renal brush border NHE3 exists in two states
with distinct sedimentation coefficients, a 9.6 S megalin-free form and
a 21 S megalin-bound form (1). The purpose of the present study was to
use membrane fractionation methods to determine whether these two
oligomeric forms of NHE3 are expressed in distinct microdomains of the
renal brush border and, if so, to compare the functional activity of
NHE3 in these microdomains. Our findings are consistent with a model in
which the 9.6 S form of NHE3 is present in the microvillar membrane and
is active, whereas the 21 S megalin-associated form is concentrated in
the intermicrovillar microdomain of the brush border and is inactive.
Thus, regulation of renal brush border Na+-H+
exchange activity may be mediated by shifting the distribution between
these forms of NHE3.
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MATERIALS AND METHODS |
Antibodies to NHE3--
In a previous paper, we described in
detail the development and characterization of isoform-specific
monoclonal antibodies (mAbs)1
to a restricted region of the carboxyl terminus of NHE3 (10). mAbs 2B9,
4F5, and 19F5 were raised to a fusion protein (fpNHE3-702-832) that
reproduced the C-terminal 131 amino acids of rabbit NHE3 (11). mAb 2B9
was used throughout the present study. This mAb was used as purified
IgG from hybridoma supernatants.
A polyclonal antibody, raised in a goat to fpNHE3-702-832, was also
used in this study. As shown in Fig. 1,
this antibody immunoprecipitates and immunoblots the same protein that
is precipitated and blotted by mAb 2B9, namely NHE3. Since this
antibody is also specific for NHE3, it was used to immunoblot NHE3 in
immune complexes precipitated with the mAbs (see Fig. 8).

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Fig. 1.
Characterization of goat anti-NHE3
antibody. Rabbit microvillar membrane vesicles (100 µg) were
solubilized in 1% Triton X-100 in TBS and prepared for
immunoprecipitation. Immunoprecipitation was performed with anti-NHE3
mAb 2B9 (lanes 1), goat polyclonal anti-NHE3
antibody (lanes 2), or a control antibody
(lanes 3). Immune complexes were prepared for
immunoblotting and probed with either mAb 2B9 (left
four lanes) or goat anti-NHE3 (right
four lanes). Lane 4 in each
group represents 25 µg of microvillar membrane vesicles applied to
the gel and used as a blotting control. Monomeric NHE3 is seen at 80 kDa (small arrow), while an aggregate of NHE3 is
seen near the top of the gel (arrowhead). The
lower bands (~55 kDa) represent IgG heavy chains. Molecular weights,
expressed as 10 3 × Mr, are presented on the left. Note
that both mAb 2B9 and goat anti-NHE3 immunoprecipitate and immunoblot
the same protein, NHE3.
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A polyclonal antibody raised to a synthetic peptide representing amino
acids 809-831 of rat NHE3 was a gift from Dr. Mark Knepper (National
Institutes of Health, Bethesda, MD) (12). This antibody was used as
affinity-purified IgG.
Antibodies to Megalin--
In a previous paper (1) we described
the development and characterization of mAb 10A3 to rabbit megalin.
This antibody was used for immunoblotting and immunoprecipitation in
rabbits. An additional mAb raised to megalin (mAb DC6) was a gift from Drs. Markus Exner and Dontscho Kerjaschki, Vienna, Austria. This mAb
has been described previously and labels megalin in numerous mammalian
species (14). These mAbs were used as purified IgG from hybridoma
supernatants. Finally, a polyclonal antibody (rabbit anti-megalin C
terminus) was a gift from Drs. Marilyn Farquhar and Robert Orlando
(University of California, San Diego). This antibody was raised to a
fusion protein representing the C-terminal cytoplasmic domain of rat
megalin (14). This antibody was used as diluted serum for
immunocytochemistry (see Fig. 4).
Additional Primary Antibodies--
A mAb (mouse IgG) to villin
was purchased from AMAC, Inc. (Westbrook, ME). This mAb was raised to
the C-terminal headpiece region of purified pig villin (15). A
polyclonal antibody raised to the renal brush border Na-Pi
cotransporter, NaPi-2, was provided by Drs. Heini Murer and Jurg Biber
(Institute of Physiology, University of Zurich-Irchel, Zurich,
Switzerland) (16). A polyclonal antibody raised to
-glutamyltranspeptidase was a gift from Dr. David Castle (University of Virginia). A mAb raised to clathrin heavy chain was a
gift from Dr. Pietro DiCamilli (Yale University).
Antibody Conjugates--
For indirect immunofluorescence
microscopy, Alexa Fluor 594-conjugated goat anti-mouse or Alexa Fluor
488-conjugated goat anti-rabbit were purchased from Molecular Probes,
Inc. (Eugene, OR). For immunoblotting, horseradish peroxidase
(HRP)-conjugated rabbit anti-goat IgG (heavy and light chain specific),
goat anti-mouse (
-chain-specific), or goat anti-rabbit (heavy and
light chain-specific) were purchased from Zymed Laboratories
Inc. Laboratories (San Francisco CA).
Preparation of Renal Membrane Fractions--
Adult male New
Zealand White rabbits (Gabrielle Farms, Woodstock, CT) or Harlan
Sprague-Dawley rats (Charles River) were sacrificed by injection of
sodium pentobarbital (Butler Co., Columbus, OH). Microvillus membrane
vesicles (BBMV) were prepared from renal cortex using the
Mg2+ precipitation method described previously (17).
Postmitochondrial microsomes were prepared based on a modification of a
protocol described by Sabolic and Burckhardt (18). Briefly, renal
cortex was dissected cold from recently excised kidneys. Tissue from
one animal was homogenized in 35 ml of homogenization buffer (20 mM Tricine (pH 7.8), 8% sucrose, and protease inhibitors (as below). Homogenization was performed using a Potter-Elvehjem with a
loose-fitting Teflon pestle. The homogenate was centrifuged for 15 min
at 1900 × g using an SS-34 rotor. The supernatant was saved and again centrifuged for 20 min at 21,000 × g
using the same rotor. The supernatant and the upper, light portion of
the pellet were saved. These membranes, referred to as
postmitochondrial microsomes, were collected by centrifugation for 30 min at 48,000 × g in the same rotor. The membrane
pellets were resuspended in 5% OptiPrepTM (see below) at a
concentration of 10-20 mg/ml.
The protease inhibitors (Sigma) pepstatin A (0.7 µg/ml), leupeptin
(0.5 µg/ml), phenylmethylsulfonyl fluoride (40 µg/ml), and EDTA (1 mM) each were included in all membrane preparations. Protein concentrations were determined by the method of Lowry (19).
Density Separation of Postmitochondrial Microsomes--
The
density separation of cellular membranes was accomplished by isopycnic
centrifugation using OptiPrepTM (Nycomed Pharma, Oslo,
Norway) density gradients. OptiPrepTM was diluted to
appropriate concentrations from stocks using 20 mM Tricine
(pH 7.8) and 8% sucrose according to the manufacturer's protocols.
Preformed OptiPrepTM gradients were made using a Gradient
MasterTM (Biocomp Inc., New Brunswick, Canada). 1-5 mg of
postmitochondrial microsomes (see above) in 5% OptiPrepTM
were layered on the top of 15-25% OptiPrepTM gradients.
Gradients were centrifuged to equilibrium (at least 2 h) at
100,000 × g using an SW 41 rotor in a Beckman
ultracentrifuge. 1-ml fractions were manually collected from the top
and stored at
70 °C. For analysis by immunoblotting and
immunoprecipitation, equal volumes of each fraction were used.
Labeling Endosomes in the Proximal Tubule with HRP--
Renal
endosomes were labeled with HRP as follows. Harlan Sprague-Dawley rats
were anesthetized with sodium pentobarbital intraperitoneal. Following
an abdominal incision, 25 mg of HRP (Sigma) in 1 ml of
phosphate-buffered saline was injected into the mesenteric vein. This
vein was used because of its accessibility. 5-10 min after injecting
the tracer, the kidneys were perfused in a retrograde manner via the
abdominal aorta. When kidneys were used for membrane fractionation
studies, they were perfused with homogenization buffer (see above).
When the kidneys were used for morphological studies they were perfused
with paraformaldehyde-lysine-periodate (PLP) fixative as
described previously (20).
Tissue Preparation for Electron Microscopy and
Immunocytochemistry--
Rats were anesthetized with sodium
pentobarbital injected intravenously, and the kidneys were
perfusion-fixed with PLP fixative (21) as described previously
(20).
For the HRP tracer studies, 30-µm cryosections were prepared and
incubated in diaminobenzidine. Sections were then prepared for electron
microscopy as described previously (20). For light microscopic studies,
Epon sections (0.5 µm) were cut with a glass knife, stained with
toluidine blue, and examined with a Zeiss Axiophot microscope. For
electron microscopy, ultrathin sections of Epon embedded tissue were
cut and examined using a Zeiss 910 electron microscope.
Indirect immunofluorescence microscopy was performed using either
semithin cryosections of fixed tissue or paraffin sections of the same
tissue that was further subjected to antigen retrieval. Cryosections
were prepared and stained exactly as described previously (10). For
antigen retrieval, fixed tissue was embedded in paraffin. 0.5-2.0-µm-thick sections were cut using a glass knife mounted on
the sectioning stage of a Reichert ultramicrotome. Sections were
mounted on glass coverslips, deparaffinized using xylene, and
rehydrated in graded ethanols and phosphate-buffered saline. Sections
were next microwaved in buffer containing 10 mM citrate in
Tris-buffered saline at 40% power for 20 min. After washing in TBS,
the sections were further denatured using 1% SDS in TBS for 5 min.
After washing in TBS the sections were immunolabeled as described
previously (10).
Immunoprecipitation--
Immunoprecipitation of soluble renal
proteins was performed essentially as described previously (22). Renal
membranes were solubilized at 4 °C in TBS (pH 7.4) containing 1%
Triton X-100 and protease inhibitors phenylmethylsulfonyl fluoride,
leupeptin, pepstatin, and EDTA as described above. The samples were
cleared of insoluble material by centrifugation (15,000 × g for 10 min) using a table top centrifuge
(HermleTM model Z230M; National Labnet Co., Woodbridge,
NJ). To the above supernatants were added primary antibody, ~50 µg
for mAbs or 10 µl of serum. When immunoprecipitating across sucrose
gradients, the primary antibodies were added directly to the gradient
fractions. Primary antibodies were allowed to incubate at 4 °C for
1 h. Immune complexes were collected using 5 mg/sample of Protein
G-Sepharose 4B (Amersham Pharmacia Biotech). The beads were washed five
times in solubilization buffer and then incubated in 50 µl of
SDS-PAGE sample buffer for 1-3 min at 100 °C, and the samples were
prepared for SDS-PAGE and immunoblotting. Although we have found that
heating NHE3 increases its tendency to form aggregates (see Fig. 7),
heating is necessary in these experiments to completely reduce IgG to monomeric heavy and light chains.
Sucrose Velocity Gradient Centrifugation--
Velocity gradient
sedimentation was carried out according to Copeland et al.
(23). Membranes were solubilized in lysis buffer (pH 7.4) containing 20 mM MES, 30 mM Tris, 100 mM NaCl,
and 1% Triton X-100. The samples were applied to the top of 5-25%
continuous sucrose gradients. Sucrose solutions were prepared with
lysis buffer containing 0.1% Triton X-100. After centrifugation for 12 h at 4 °C at 200,000 × g in an SW 41 rotor,
the gradients were fractioned by hand from the top. Sucrose
concentrations of each fraction were calculated from the refractive
index. Sedimentation coefficients were determined by comparison with
standard proteins with known S values (aldolase,
s20,w = 7.3; catalase, s20,w = 11.3; horse spleen apoferritin,
s20,w = 16.5; and bovine thyroglobulin,
s20,w = 19.3) or by using the formula
s20,w =
I/
2t, where I is the
time integral,
is rotor speed (radians/s), and t is
time, as described by Griffith (24). Buffers (Tris and MES), Triton
X-100, and apoferritin were purchased from Sigma. Aldolase, catalase,
and thyroglobulin were from Amersham Pharmacia Biotech.
SDS-PAGE and Immunoblotting--
Protein samples were
solubilized in SDS-PAGE sample buffer and separated by SDS-PAGE using
7.5% polyacrylamide gels according to Laemmli (25). For
immunoblotting, proteins were transferred to polyvinylidene difluoride
(Millipore Immobilon-P) at 500 mA for 6-10 h at 4 °C with a
TransphorTM transfer electrophoresis unit (Hoefer
Scientific Instruments, San Francisco, CA) and stained with Ponceau S
in 0.5% trichloroacetic acid. Immunoblotting was performed as follows.
Sheets of polyvinylidene difluoride containing transferred protein from
entire gels were incubated first in Blotto (5% nonfat dry milk in
phosphate-buffered saline, pH 7.4) for 1-3 h to block nonspecific
binding of antibody, followed by overnight incubation in primary
antibody. Primary antibodies, diluted in Blotto, were used at dilutions
ranging from 1:1000 to 1:5000. The sheets were then washed in Blotto
and incubated for 1 h with an appropriate HRP-conjugated secondary antibody diluted 1:2000 in Blotto. After washing three times in Blotto,
once in phosphate-buffered saline (pH 7.4), and once in distilled
water, bound antibody was detected with the ECLTM
chemiluminescence system (Amersham Pharmacia Biotech) according to the
manufacturer's protocols. In some experiments, polyvinylidene difluoride blots were reprobed with additional primary antibodies (see
Fig. 2) after stripping away the first
antibody. This was accomplished by incubating the polyvinylidene
difluoride sheets in 2% SDS, 100 mM
-mercaptoethanol,
50 mM Tris (pH 6.9) for 60 min at 70 °C.

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Fig. 2.
Density separation of NHE3-rich
membranes. Rat cortical microsomes were separated by isopycnic
centrifugation using 15-25% OptiPrepTM density gradients.
1-ml fractions were collected across the gradient, and 50 µl of each
was prepared for SDS-PAGE and immunoblotting. Fraction 1 represents the
top (light fractions) and fraction 12 represents the bottom (dense
fractions) of the gradient. The same blot was probed successively with
antibodies for NHE3, villin, NaPi-2, megalin, and clathrin.
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Measuring Na+-H+ Exchange in Membrane
Vesicles--
Na+-H+ exchange was measured by
monitoring the fluorescence change of the weak base dye acridine orange
(26). These experiments were designed so that the H+
modifier site (27) would be equally activated regardless of the
topological orientation of the membrane vesicles. The vesicles were
loaded with either 100 mM Na2SO4 or
100 mM (NH4)2SO4,
buffered with 10 mM Hepes, pH 6.5, by preincubation in
these solutions for 2 h at 37 °C. In all experiments, the
protein concentrations of the samples were matched. The final protein
concentration was ~10 mg/ml. After loading, samples (10 µl) were
added to cuvettes containing 9.5 µM acridine orange, 100 mM K2SO4, and 10 mM
Hepes, pH 6.5. Thus, an outward NH
or
Na+ gradient was imposed in the absence of an initial pH
gradient. Generation of an inside acid pH gradient was monitored by
measuring fluorescence quenching at room temperature by use of a
PerkinElmer Life Sciences spectrofluorometer (excitation, 493 nm;
emission, 535 nm).
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RESULTS |
Separation of NHE3-containing Dense Membranes--
To examine the
subcellular location of NHE3 in the proximal tubule, we evaluated the
distribution of NHE3 in rat renal cortical microsomes following their
separation by density gradient centrifugation. In the experiment
illustrated in Fig. 2, fractions from 15-25% OptiPrepTM
density gradients were analyzed by immunoblotting using antibodies to
proteins known to be markers for specific subcellular domains within
cells of the proximal tubule. As shown in Fig. 2, NHE3 was detected in
membranes with two distinct densities. A relatively light fraction was
found at the top of the gradient and had the same density as that of
microvillar markers such as villin and the Na-Pi
cotransporter, NaPi-2. A second peak of NHE3 was found in very dense
membranes, which lacked a distinct peak of other microvillar markers
but which overlapped peaks of megalin and clathrin. This experiment
implied that in the proximal tubule a major fraction of total NHE3 is
located in a nonmicrovillar membrane compartment represented by the
dense membranes.
NHE3-rich Dense Membranes Are Not Derived from
Endosomes--
Studies from other laboratories have suggested that
regulation of NHE3 might involve trafficking to the plasma membrane
(microvilli) from an endosomal compartment (7, 28, 29). To determine if
the NHE3-rich dense membranes are derived from endosomes, we labeled
the endosomal compartment of the proximal tubule with HRP (Fig.
3, A and B) and
examined the density of labeled endosomes in the OptiPrepTM
gradients. As seen in Fig. 3 (A and B), when the
tracer was localized ultrastructurally it was found only in endosomes.
It is important to note that the tracer was never found in tubular
lumens, where it might be artificially trapped during homogenization
(see Fig. 3A). Importantly, as seen in Fig. 3C,
the HRP-labeled endosomes were predominantly found at the top of the
gradient and did not have the same density as the NHE3-rich dense
membranes. These data indicate that the NHE3-rich, dense membranes are
not derived from endosomes.

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Fig. 3.
Density separation of endosomes.
Endosomes in the proximal tubules of rats were labeled in
vivo by injecting HRP intravenously 5-10 min before sacrifice. In
A and B, the kidneys were perfusion-fixed and
prepared for electron microscopy. A represents a toluidine
blue-stained section of the Epon-embedded tissue and is presented to
demonstrate the lack of free HRP in the lumen of the tubules.
B is an electron micrograph of the same tissue showing the
apical (brush border) region of a proximal tubule. In C, the
kidneys were perfused with homogenization buffer (see "Materials and
Methods") and renal microsomes prepared and separated based on
density as in Fig. 2. In C, fractions from the density
gradients were prepared for immunoblotting and labeled with an anti-HRP
antibody. Star, lumen of proximal tubules. The
arrows in B identify HRP-labeled endosomes.
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Identification of an Intermicrovillar Pool of NHE3 by
Immunocytochemistry--
The most direct approach to identify
NHE3-containing membranes is to localize the transporter using
immunocytochemistry. Indeed, we previously demonstrated by
immunoelectron microscopy with anti-NHE3 monoclonal antibodies that
NHE3 is almost exclusively expressed on the microvilli of the renal
brush border surface (10). However, we subsequently found that the
epitopes to which these antibodies bind on NHE3 are not available when
NHE3 was complexed with megalin (1). Hence, under the usual conditions
for immunocytochemistry, these antibodies may have failed to detect
sites of NHE3 expression where NHE3 is complexed with megalin.
Accordingly, we sought to utilize antigen retrieval as a method to
expose the megalin-associated NHE3 in the proximal tubule. Figs.
4 and 5
compare staining of rat kidney with the same anti-NHE3 mAb (2B9) using
cryosections (Fig. 4), as described previously (10), or on paraffin
sections following denaturing by antigen retrieval (Fig. 5). In this
experiment, double labeling was performed with anti-NHE3 mAb (2B9) and
polyclonal antibodies to megalin or the microvillar enzyme
-glutamyltranspeptidase. As shown previously, in the proximal
tubule, staining for NHE3 on cryosections is restricted to the
microvilli and colocalizes with staining for
-glutamyltranspeptidase (Fig. 4A). In contrast, double labeling with an anti-megalin
antibody shows little if any overlap (Fig. 4B). However,
following antigen retrieval, intense staining for NHE3 was seen at the
base of the microvilli (Fig. 5, A and B). In
double labeling experiments on these sections, the staining for NHE3
more closely overlapped that of megalin (Fig. 5B) than that
of
-glutamyltranspeptidase (Fig. 5A). These data strongly
suggest that the dense, NHE3-rich membranes identified by density
centrifugation are derived from the intermicrovillar region of the
brush border.

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Fig. 4.
Indirect immunofluorescence microscopy
localizing NHE3 in the proximal tubule using cryosections.
PLP-fixed rat kidney was prepared for cryosectioning. Sections were
double-labeled for NHE3 using mAb 2B9 and either
-glutamyltranspeptidase (A) or megalin
(B) using polyclonal antibodies. Staining for NHE3 is
seen here in red (B, arrowheads)
except where it colocalizes with -glutamyltranspeptidase
(A), where the overlap of the red and
green stains appears yellow (arrows).
Staining for megalin seen in B appears green
(small arrows).
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Fig. 5.
Indirect immunofluorescence microscopy
localizing NHE3 in the proximal tubule following antigen
retrieval. A portion of the same PLP-fixed rat kidney that was
used in Fig. 4 was subjected to antigen retrieval (see "Materials and
Methods"). As in Fig. 4, sections were double-labeled for NHE3
using mAb 2B9 and either -glutamyltranspeptidase (A) or
megalin (B). Staining for NHE3 is seen here in
red (A, small arrows)
except where it colocalizes with megalin (B,
arrowheads), where the overlap of the red and
green stains appears yellow
(arrows). Staining for -glutamyltranspeptidase seen in
A appears green (arrowheads).
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To verify the existence of a pool of NHE3 in the intermicrovillar
region of the brush border in the absence of antigen retrieval, we also
performed immunocytochemical studies utilizing a rabbit anti-NHE3
polyclonal antibody developed by Knepper and co-workers (12). As shown
in the double labeling experiment in Fig.
6, this anti-NHE3 antibody (seen in
green) is distinct from the anti-NHE3 mAb 2B9 (seen in
red) and stains a pool of NHE3 that was at the base of the
microvilli. This staining pattern is consistent with that previously
described by Kwon et al. using the same antibody (31). This
observation indicates that the findings in Fig. 4 were not artifactual
due to the antigen retrieval procedure and confirms the identification
of an intermicrovillar plasma membrane pool of NHE3.

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Fig. 6.
Indirect immunofluorescence microscopy
localizing NHE3 in the proximal tubule using antibodies to two epitopes
within the C terminus of NHE3. PLP-fixed rat kidney was prepared
for cryosectioning. Sections were double-labeled for NHE3 using
mAb 2B9 (the epitope lies between amino acids 702 and 756) and rabbit
anti-NHE3 synthetic peptide (the epitope lies between amino acids 809 and 831). Staining with mAb 2B9 is seen in red
(arrowheads), while the staining with the polyclonal
antibody is seen in green (small
arrows). Note that there is little overlap in the staining
patterns of the two anti-NHE3 antibodies.
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Biochemical Comparison of NHE3-rich Dense Membranes and
Microvilli--
The preceding membrane fractionation and
immunocytochemistry experiments indicated that there are two pools of
NHE3 in the brush border of the rat proximal tubule. One population is
located in the microvillar membrane, while the other is located in the intermicrovillar membrane, the latter corresponding to the dense membranes found with density fractionation. We next evaluated biochemical properties of these two membrane fractions.
It should be noted that we have observed proteolytic activity in the
rat but not the rabbit kidney that degrades NHE3 when solubilized in
nonionic detergents (data not shown). Therefore, these and subsequent
studies were performed using membrane preparations isolated from rabbit kidney.
For the experiment illustrated in Fig. 7,
renal cortical microvilli were isolated by divalent cation aggregation
(17), and dense membranes were isolated by centrifugation on an
OptiPrepTM gradient as above (fractions 5-7 in Fig. 2).
Then equal quantities of protein from each preparation were separated
by SDS-PAGE and subjected to immunoblotting using antibodies to the
microvillar protein villin, to the predominantly intermicrovillar
protein megalin, and to NHE3. As shown in Fig. 7, although villin was enriched in the microvillar fraction and megalin in the dense membrane
fraction, staining of NHE3 was approximately equal in both membrane
preparations. This experiment shows that although the microvillar and
intermicrovillar microdomains of the proximal tubule brush border are
biochemically distinct, as first demonstrated by Rodman et
al. (32), the level of expression of NHE3 appears to be nearly
equal between these two domains.

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Fig. 7.
Immunoblots comparing the protein composition
of dense membranes with microvilli. 50 µg of either microvillar
membrane vesicles (M) or dense membranes (DV)
(fractions 5-7 in Fig. 2) were separated by SDS-PAGE and prepared for
immunoblotting. Lanes were probed using mAbs to villin, megalin, and
NHE3.
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We previously demonstrated that renal brush border NHE3 exists in two
states with distinct sedimentation coefficients, a 9.6 S megalin-free
form, and a 21 S megalin-bound form (1). We next sought to determine
the relative abundance of these oligomeric forms of NHE3 in the
microvillar and intermicrovillar microdomains of the brush border.
To determine if the two oligomeric forms of NHE3 are differentially
expressed in microvilli and dense (intermicrovillar) membranes, we
performed immunoprecipitation experiments using equal quantities of
both membrane preparations. Megalin-associated NHE3 (21 S form) was
identified by coprecipitation with the anti-megalin mAb 10A3 (1). NHE3
not associated with megalin (9.6 S form) was identified by
precipitation with the anti-NHE3 mAb 2B9, which binds to an epitope
that is not available when NHE3 is complexed with megalin (1). As shown
in Fig. 8, the anti-NHE3 mAb precipitated
more NHE3 from the microvilli than from the dense membranes. In
contrast, the anti-megalin mAb coprecipitated more NHE3 from the dense
membranes than from microvilli. Indeed, Fig. 8 demonstrates that in the dense membranes more NHE3 could be precipitated by the anti-megalin mAb
than by the anti-NHE3 mAb, indicating that the majority of NHE3 in this
membrane domain is complexed with the receptor.

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Fig. 8.
Immunoprecipitation of NHE3 and the
NHE3-megalin complex from microvilli and dense membranes. 100 µg
of microvillar membrane vesicles (Microvilli) or NHE3-rich,
dense membranes (Dense Vesicles) made from
rabbits were solubilized in 1% Triton X-100 in TBS and prepared for
immunoblotting. Immunoprecipitations were performed with antibodies to
NHE3 (mAb 2B9) or to megalin (mAb 10A3). Immune complexes were prepared
for immunoblotting, and the blot was probed with goat anti-NHE3.
Molecular weights, expressed as 10 3 × Mr, are presented on the left. NHE3
is seen as either a monomer (arrowhead) or an aggregate
(small arrow). Note that in the dense membranes
more NHE3 coprecipitates with the anti-megalin antibody (10A3) than can
be precipitated with the anti-NHE3 antibody (2B9).
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Since we had previously shown that the sedimentation coefficient for
megalin-free NHE3 is 9.6 S, whereas that for megalin-associated NHE3 is
21 S (1), we performed density velocity centrifugation to confirm the
differential localization of these forms in microvilli and dense
membranes. In the experiment illustrated in Fig.
9, equal quantities of solubilized
microvilli and dense membranes were subjected to density velocity
centrifugation, and then each fraction was analyzed by immunoblotting
for the presence of NHE3. Shown in Fig. 9, the predominant form of NHE3
in microvilli had an S value of 9.6, while the majority of NHE3 in the
dense membranes had an S value of 21. Taken together with the preceding
immunoprecipitation data, these findings indicate that distinct
oligomeric forms of NHE3 are differentially expressed in the
microdomains of the renal brush border: the megalin-free 9.6 S form in
microvilli and the 21 S megalin-associated form in dense
(intermicrovillar) membranes.

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Fig. 9.
Sucrose velocity gradient centrifugation to
assess size of NHE3 oligomers in microvilli and dense membranes.
500 µg of microvillar membrane vesicles (Microvilli) or
NHE3-rich, dense membranes (Dense Vesicles) were
solubilized in 1% Triton X-100 in Tris/MES buffer (see "Materials
and Methods"). After centrifugation, the supernatant was applied to
the top of 5-25% sucrose gradients, and the gradients were
centrifuged for 12 h at 200,000 × g using
an SW 41 rotor. 0.75-ml fractions were collected across the gradients,
and 50 µl of each were prepared for SDS-PAGE and immunoblotting.
Blots were probed for NHE3 using mAb 2B9.
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Physiologic Comparison of NHE3-rich Dense Membranes and
Microvilli--
The fact that there are two distinct oligomeric forms
of NHE3 that are differentially located within microdomains of the
renal brush border led us to postulate that these forms may have
different functional characteristics. Therefore, we next sought to
compare the transport activity of the 9.6 S microvillar form of NHE3
with the 21 S intermicrovillar oligomer of the transporter. Again, rabbit microvilli were prepared using the divalent cation precipitation method, and rabbit dense vesicles were collected from appropriate fractions of OptiPrepTM density gradients. To avoid
variability between animals, microvilli were prepared from one kidney
and the dense vesicles from the other kidney of the same animals.
Na+-H+ exchange transport activity was assayed
as the rate of generation of an inside acid pH gradient in response to
an outward Na+ gradient. Generation of a pH gradient was
monitored by measurements of fluorescence of the weak base dye probe
acridine orange (26). Because the topology of the dense
(intermicrovillar) membranes is not known, membrane vesicles were
initially equilibrated at pH 6.5 to ensure that the cytoplasmic
H+ modifier site (27) was equally activated whether the
membranes were right-side-out, as is known to be the case for
microvilli (17), or inside-out.
As shown in the right panel of Fig.
10, when Na+-loaded dense
membranes were added to the cuvette, the outward Na+
gradient failed to generate a significant inside acid pH gradient. Importantly, rapid generation of an inside acid pH gradient was observed when NH
-loaded dense
membranes were added to the cuvette, resulting from efflux of
NH3 by nonionic diffusion. This pH gradient was collapsed
by the addition of NH
to the external
medium after 200 s. One trivial explanation for the inability of
the outward Na+ gradient to generate a pH gradient was that
the dense membranes were leaky or not in the form of sealed vesicles.
The fact that an outward NH
gradient
was capable of generating an inside acid pH gradient indicates that
sealed membrane vesicles were indeed present. Thus, the inability of an
outward Na+ gradient to generate an inside acid pH gradient
in the dense membranes indicates that Na+-H+
exchange activity is low or absent in this preparation. In contrast, as
shown in the left panel of Fig. 10, an outward
Na+ gradient generated a large pH gradient in microvillar
membranes, indicating brisk Na+-H+ exchange
activity as previously observed by use of similar assays (26). Thus,
although the level of expression of NHE3 is similar in microvilli and
dense membranes (as shown in Fig. 7), Na+-H+
exchange activity was only detected in the microvillar membranes. These
findings imply that the 9.6 S megalin-free form of NHE3 that
predominates in microvilli is active, whereas the 21 S megalin-bound form of NHE3 that predominates in the dense (intermicrovillar) membranes is inactive.

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Fig. 10.
Na+-H+ exchange
activity measured by acridine orange fluorescence in microvilli and
dense membranes. Microvillar membrane vesicles (A) and
NHE3-rich, dense membranes (B) were prepared from rabbits,
and the samples were matched for total protein. In both
panels fluorescence units (abscissa) are
expressed as a function of time (ordinate).
Squares, recordings from membranes that were loaded with
Na2SO4; circles, recordings from
membranes that were loaded with
(NH4)2SO4. Note that in
A an outwardly directed Na+ gradient acidifies
the intravesicular space of the microvilli and results in quenching of
the dye. There is no such activity in B when a similar
gradient is imposed using the NHE3-rich, dense membranes.
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DISCUSSION |
This study describes the localization of two oligomeric forms of
NHE3 to distinct microdomains of the renal brush border. Using density
centrifugation combined with specific brush border antibodies, we have
shown that a 9.6 S oligomer of NHE3 is predominately expressed on
microvilli, while a 21 S, megalin-associated form is concentrated in
the intermicrovillar region of the brush border. As shown by Rodman
et al. (32), this is a distinct microdomain of the apical
plasma membrane of the proximal tubule. In addition, transport studies
indicate that NHE3 is active in microvilli but inactive in
intermicrovillar membranes.
The mAb to NHE3 (2B9) that was used in this study is specific to a
region within the C terminus of the transporter that lies between amino
acids 702 and 756 (10). This region of NHE3 is blocked from antibody
binding when the transporter is complexed with megalin (1). The fact
that epitopes within the C-terminal region of NHE3 are blocked when
NHE3 is complexed with megalin raises questions regarding the
interpretation of previous immunocytochemical studies using antibodies
made to the C terminus of NHE3. Most (10, 33, 34) but not all (12) of
these studies localized NHE3 largely to the microvillar membrane. In
fact, when used for immunoelectron microscopy, our mAbs failed to label
the intermicrovillar microdomain of the renal brush border (10). We
hypothesized that the apparent discrepancy between previous
immunocytochemical studies and the present membrane fractionation
study, showing large amounts of NHE3 in intermicrovillar membranes, may
have resulted from the masking of C-terminal epitopes on NHE3 when the
transporter is complexed with megalin. Our present immunocytochemical studies using antigen retrieval show this to be the case. When kidney
sections were denatured during the antigen retrieval procedure, we
visualized a large pool of NHE3 that colocalized with megalin in the
intermicrovillar region of the brush border, consistent with our
membrane fractionation data showing a major pool of NHE3 in
megalin-rich, nonmicrovillar (and nonendosomal) dense membranes. Immunocytochemical localization of this pool of NHE3 was not an artifact of the antigen retrieval procedure, since similar staining was
found without antigen retrieval by use of a polyclonal anti-NHE3 antibody raised to a peptide encompassing the C-terminal 22 amino acids
of NHE3 (12).
Previous studies have localized Na+/H+ exchange
activity (35) and NHE3 protein (10) within endosomes in the proximal
tubule. In contrast, the data presented here indicate that the
membranes enriched in the NHE3-megalin complex are derived from
the intermicrovillar region of the brush border (apical plasma
membrane) and not from an endosomal compartment. Since the present
study did not directly examine the NHE3 protein present in endosomes,
the relationship between endosomal NHE3 and that found in the
intermicrovillar microdomain of the brush border remains unclear.
However, because the NHE3 antibodies that we used in a previous study
to detect NHE3 in endosomes (10) do not react with the NHE3-megalin
complex (1), it seems likely that endosomal staining with these mAbs was due to the presence of the 9.6 S form of NHE3 in the endosomes and
not the NHE3-megalin complex.
Our finding that the 21 S megalin-associated form of NHE3 concentrated
in the intermicrovillar membrane is inactive raises the possibility
that association with megalin may play a role in regulating NHE3
activity in response to physiological stimuli. For example, it is
possible that the association of NHE3 with megalin causes inactivation
of transport activity. However, it is known that phosphorylation of
NHE3 by protein kinase A causes inhibition of NHE3 activity (36, 37).
It is possible that phosphorylation of NHE3 causes both its
inactivation and its association with megalin. In addition, endocytosis
of NHE3 in response to protein kinase A-induced phosphorylation has
been described in cultured cell models (38, 39). It is therefore also
possible that phosphorylation of NHE3 leads to association with megalin and sequestration in the coated pits as a prelude to endocytosis in
proximal tubule cells.
In conclusion, we have demonstrated that renal brush border NHE3 exists
in two oligomeric states: a 9.6 S active form present in microvilli and
a 21 S, megalin-associated, inactive form in the intermicrovillar
microdomain. Future studies will be needed to examine whether
regulation of renal brush border Na+-H+
exchange activity may be mediated by shifting the distribution between
these forms of NHE3.