Reversible effects of acute hypertension on proximal tubule
sodium transporters
Yibin
Zhang1,
Clara E.
Magyar1,
John M.
Norian1,
Niels-H.
Holstein-Rathlou2,
Austin K.
Mircheff1, and
Alicia A.
McDonough1
1 Department of Physiology and
Biophysics, University of Southern California School of Medicine, Los
Angeles, California 90033; and
2 Department of Medical
Physiology, The Panum Institute, DK-2200 Copenhagen N, Denmark
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ABSTRACT |
Acute hypertension provokes a rapid decrease in proximal tubule
sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of
membranes containing apical membrane sodium/hydrogen exchangers (NHE3)
[Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol.
39): F1004-F1014, 1996]. To determine the reversibility and
specificity of these responses, rats were subjected to
1) elevation of blood pressure (BP)
of 50 mmHg for 5 min, 2) restoration of normotension after the first protocol, or
3) sham operation. Systolic
hypertension increased urine output and endogenous lithium clearance
three- to fivefold within 5 min, but these returned to basal levels
only 15 min after BP was restored. Renal cortex lysate was fractionated
on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase
activity (but not subunit immunoreactivity) decreased one-third to
one-half after BP was elevated and recovered after BP was normalized.
After BP was elevated, 55% of the apical NHE3 immunoreactivity,
smaller fractions of sodium-phosphate cotransporter immunoreactivity,
and apical alkaline phosphatase and dipeptidyl-peptidase redistributed
to membranes of higher density enriched in markers of the
intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5),
whereas density distributions of the apical cytoskeleton protein villin
were unaltered. After 20 min of normalized BP, all the NHE3 and smaller
fractions of the other apical membrane proteins returned to their
original distributions. These findings suggest that the dynamic
regulation of proximal tubule sodium transport by acute changes in BP
may be mediated by rapid reversible regulation of sodium pump activity
and relocation of apical sodium transporters.
sodium-potassium-adenosinetriphosphatase; NHE3; membrane
trafficking
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INTRODUCTION |
ACUTE INCREASES IN arterial pressure elicit rapid
natriuretic and diuretic responses that occur in the absence of changes in renal blood flow (RBF) or glomerular filtration rate (GFR) (18).
This autoregulation of RBF and GFR is mediated by an increase in volume
flow to the macula densa, which provokes an increase in afferent
arteriolar resistance. The increased volume flow at the macula densa
during acute hypertension, in the face of a constant GFR, is due, at
least in part, to a very rapid (within 1.5-2 min) inhibition of
salt and water reabsorption in the proximal tubule (11, 12, 23); the
mediating signals remain to be determined.
Active sodium reabsorption across the proximal tubule is mediated
primarily by apical entry via sodium/hydrogen exchangers (NHE3) and
extrusion via basolateral sodium pumps (Na-K-ATPase). The rapid
decrease in sodium transporter activity in response to acute
hypertension may be due to 1)
decreased activity of transporters in the apical and/or
basolateral plasma membranes, 2)
trafficking of transporters from plasma membranes to endosomal stores,
or 3) rapid degradation of
transporters. There is evidence for all three types of transport
regulation in the proximal tubule:
1) phosphorylation of sodium pumps
has been reported to change ATPase and transport activity (3, 4),
2) there is evidence for trafficking
of apical membrane proteins between the brush border and a large pool
of subapical endosomes (33), as well as reversible wholesale internal
retraction of microvilli with ATP depletion and repletion (16), and
3) apical membrane sodium-phosphate (Na-Pi) cotransporters are
internalized and degraded following acute high-phosphate diet (26).
We recently reported that during a 5-min arterial hypertension
Na-K-ATPase catalytic activity in the basolateral membranes decreased
and the density of membranes containing NHE3 increased (47). In this
study, we test the hypothesis that the responses are reversible when
normal blood pressure is restored, and we examine the specificity of
the NHE3 redistribution. The findings demonstrate that transport
returns to control levels by 10-15 min after normalization of
blood pressure and suggest that the dynamic regulation of proximal
tubule sodium transport by fluctuations in blood pressure may be
mediated by changes in sodium transporter characteristics at both the
apical and basolateral membranes via 1) reversible inhibition of
basolateral Na-K-ATPase activity and 2) relocation of a set of apical
proteins, including NHE3 and Na-Pi
but not villin, consistent with redistribution to intermicrovillar cleft region and/or internalization to endosomal pools.
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EXPERIMENTAL PROCEDURES |
Animal preparations.
Experiments were performed on male Sprague-Dawley rats (300-350 g
body wt) that had free access to food and water before the experiment.
Rats were anesthetized intramuscularly with ketamine (Fort Dodge
Laboratories) and xylazine (Miles; 1:1, vol/vol) and then placed on a
thermostatically controlled warming table to maintain body temperature
at 37°C. Polyethylene catheters were placed into the carotid artery
for monitoring blood pressure, into the right jugular vein for infusion
of 0.9% NaCl at 50 µl/min during the entire experimental period to
maintain euvolemia, and into the ureter for urine collection.
Three groups of rats (n = 5 each) were
compared: 1) control (sham
operated), 2) hypertension (5 min of
acute systolic hypertension), and
3) restored or restoration
(normalizing blood pressure to control after 5 min of acute
hypertension). To induce acute hypertension, the total peripheral
resistance was increased as suggested by Roman and Cowley (38), without
hormone infusion. Mean arterial pressure was increased 40-50 mmHg
over basal levels for 5 min by constricting the superior mesenteric
artery, celiac artery, and abdominal aorta below the renal artery with
Schwantz vascular clamps (no. 18052-01, Fine Science Tool). Blood
pressure was restored to basal levels by releasing clamps around
arteries. Control sham-operated rats were processed in parallel, with
arteries dissected but not constricted.
Urine collection and endogenous lithium clearance.
Urine volume, collected from the ureter catheter, was determined
gravimetrically. A blood sample was collected after the kidneys were
removed. The concentrations of endogenous lithium in blood and urine
samples were measured by flameless atomic absorption spectrophotometry
(Perkin-Elmer 5100PC) as described previously (24).
Homogenization, differential sedimentation, and density gradient
centrifugation.
Kidneys were cooled in situ before excision by flushing the abdominal
cavity with ice-cold PBS solution to block further membrane trafficking. After excision, the renal cortices were rapidly dissected in isolation buffer (5% sorbitol, 0.5 mM Na2EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 mM
histidine-imidazole buffer, pH 7.5). The procedure for subcellular
fractionation of the renal cortex membranes has been described in
detail previously (20, 21, 47). Briefly, cortex was homogenized in two
rounds with a Tissuemiser (Tekmar Instrument) for 10 min at a thyristor
setting of 45 and centrifuged at 2,000 g for 10 min. The two low-speed supernatants (So) were pooled,
loaded at the interface between two hyperbolic sorbitol gradients
(ranging between 35 and 70% sorbitol), and centrifuged in a swinging
bucket rotor (100,000 g for 5 h).
Twelve fractions were collected with a Buchler AutoDensi Flow apparatus
from the top, and each fraction was diluted with isolation buffer,
pelleted by centrifugation (250,000 g
for 1.5 h), resuspended in 1 ml isolation buffer, and stored at
80°C, pending assays.
Phase partitioning.
Methods for phase partitioning are described in detail elsewhere (30).
Phase systems containing 5% dextran T-500 (Pharmacia, Piscataway, NJ),
3.5% polyethylene glycol (Carbowax 8000, Union Carbide, Danbury, CT),
5 mM sorbitol, 10 µM Na2EDTA,
and 8.33 mM imidazole, pH adjusted to 7.3 with HCl, were prepared the
day before use. Analyses were performed in an Albertsson thin-layer counter-current distribution apparatus. Samples were suspended in the
upper phase and added to chambers 1 and 2. After 18 transfers, contents of
adjacent chambers were pooled, producing 10 fractions. Thus membranes
that partitioned into the stationary, dextran-rich phase remained near
the origin, i.e., fraction 1, whereas
membranes that partitioned into the mobile, polyethylene glycol-rich
phase migrated toward fraction 10.
Membranes were sedimented by centrifugation at 250,000 g for 75 min, resuspended, and
analyzed in the same way as the density gradient fractions.
Na-K-ATPase and enzymatic marker measurements.
Na-K-ATPase activity was measured by the potassium-dependent
p-nitrophenylphosphatase
(K-pNPPase) reaction (32), since our previous analysis
demonstrated indistinguishable distribution patterns for K-pNPPase
activity and ouabain-sensitive ATPase activity in kidney cortex.
Standard assays were used for alkaline phosphatase (31) and protein
(27).
Immunoblot analysis and antibodies.
A constant volume of sample from each gradient fraction was prepared in
SDS-PAGE sample buffer (final concentration: 2% SDS, 1%
-mercaptoethanol, 0.25 mM
Na2EDTA, and 2.5 mM
H2PO4-HPO4
buffer, pH 7.0), which was denatured for 30 min at 37°C, resolved
on 7.5% SDS polyacrylamide gels, and transferred to polyvinylidene
difluoride membranes according to standard methods. The antibody
incubation protocol has been detailed previously (1). The monoclonal
antibody specifically against the rat Na-K-ATPase
1-subunit (464.6), generously provided by M. Kashgarian (Yale), was used at 1:200 dilution, and a
polyclonal anti-rat
1 fusion
protein, generated in our lab, was used at 1:500 dilution. An NHE3
monoclonal cell (2B9) culture supernatant provided by D. Biemesderfer
and P. Aronson (Yale) (7, 45) was used without dilution on blots and
detected with an enhanced chemiluminescence kit (from Amersham).
Monoclonal antibody to villin was obtained from Immunotech, used at
1:1,000, and detected with
125I-labeled protein A. Polyclonal
antiserum to the Na-Pi
cotransporter from F. Ghishan (University of Arizona) (14, 42) and
polyclonal antisera to dipeptidyl-peptidase IV (DPPIV) and megalin
[provided by M. Farquhar (University of California at San
Diego)] and against Rab 5a and Rab 4 [obtained
from Santa Cruz Biotechnology (Santa Cruz, CA)] were all used at
1:1,000 dilutions. These antibody-antigen complexes were detected with
125I-protein A (ICN). The
resulting autoradiographic signals were quantified with a Bio-Rad
imaging densitometer with molecular analyst software. Multiple
exposures of autoradiograms were analyzed to ensure that signals were
within the linear range of the film.
Quantitation and statistical analysis.
Data are expressed as means ± SE. ANOVA was applied to determine
whether there was a significant effect of treatment on the overall
fractionation pattern of a given parameter. Treatment was one repeated
factor, and fraction was another repeated factor. If the interaction
between treatment and fraction was found to be significant
(P < 0.05), it was concluded that
the treatment had a significant effect. If so, the location of the
difference in the pattern was assessed by two-tailed Student's
t-test for paired samples, and
differences were regarded to be significant at
P < 0.05.
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RESULTS |
Physiological responses.
Figure 1 summarizes the mean
arterial blood pressure, urine output, and endogenous lithium clearance
following constriction of the celiac artery, superior mesenteric
artery, and the abdominal aorta and after the subsequent release of
arterial constriction. Arterial blood pressure increased immediately by
50 mmHg above control level when arteries were constricted. This
pressure is within the range in which GFR and RBF are autoregulated
(11, 12) during similar protocols. During the 5 min of acute
hypertension, urine output increased 4.8 ± 0.6-fold, and endogenous
lithium clearance, an inverse measure of proximal tubule sodium
reabsorption (43), increased 2.9 ± 0.3-fold. After release of
constriction around the arteries, blood pressure returned immediately
to the basal level of 100 mmHg. Recovery of sodium reabsorption,
indicated by endogenous lithium clearance and urine output, lagged
behind, returning to basal levels by 15 min after the return to normal blood pressure. The persistent elevation of urine output and endogenous lithium clearance after blood pressure was normalized indicates that
either the physical stimulus of elevated blood pressure is not itself
the signal that alone depresses sodium transport and that chemical
mediators that persist after pressure restoration are likely involved
or, alternatively, that reversing the modifications or redistribution
in the transporters requires 15 min. On the basis of this time course,
we chose a time point of 20 min after blood pressure restoration to
analyze the restoration response.

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Fig. 1.
Physiological responses to constricting and then releasing clamps
around celiac and superior mesenteric arteries and abdominal aorta as a
function of time, where 0-5 min is acute hypertension duration.
A: arterial pressure, recorded from
carotid artery. B: urine output
collected over 5-min intervals, expressed as urine weight in µg/5-min
collection period. C: endogenous
lithium clearance calculated for each 5-min collection period as
urinary Li+
concentration · urine
output · plasma
Li+
concentration 1 (µl/min).
Data are expressed as means ± SE,
n = 5. * P < 0.05 vs. control period
by paired Student's
t-test.
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Response of renal cortex Na-K-ATPase to acute hypertension and blood
pressure restoration.
The sodium pump drives active transepithelial sodium reabsorption and
transports sodium ions from the cell into the extracellular fluid. We
previously established that Na-K-ATPase activity decreases in response
to acute hypertension (47). We aimed to determine if activity returned
to control levels when normotension was restored. Figure
2 summarizes the subcellular distribution
of Na-K-ATPase activity in renal cortex membrane fractions from
control, acute hypertension, and restored protocols, measured under
maximal reaction velocity conditions. The peak of
Na-K-ATPase activity, the traditional marker for location of
basolateral membranes, was between fractions 3 and 5. Acute
hypertension did not change the density distribution pattern of
Na-K-ATPase activity (Fig. 2A) but
did decrease Na-K-ATPase activity by one-third in the basolateral peak
region of the gradient, not in other regions with Na-K-ATPase activity
(fractions 6-12) consistent
with our previous findings (47). After blood pressure was restored for
20 min, Na-K-ATPase activity increased significantly in
fractions 3-5 above the activity
of samples taken during acute hypertension, although activity was not
completely restored. When assayed in the cortex sample before
fractionation, the So Na-K-ATPase activity decreased ~30% during 5 min of hypertension and returned to
control levels after 20-min blood pressure restoration (Table 1).

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Fig. 2.
Distribution of Na-K-ATPase activity in membrane
fractions of renal cortex from control, acute
hypertension, and blood pressure-restored protocols following density
gradient fractionation. Na-K-ATPase activity was assessed by measuring
p-nitrophenylphosphatase (pNPPase)
activity as described in EXPERIMENTAL
PROCEDURES. Na-K-ATPase activity distribution,
expressed as percentage of the total in the gradient, was unaltered by
acute systolic hypertension. B:
Na-K-ATPase-specific activity, corrected for total protein in the 12 fractions, was decreased by acute hypertension in the peak basolateral
membrane fractions. Results are expressed as means ± SE,
n = 5 in each group.
* P < 0.05 vs. control and
+ P < 0.05 vs. acute hypertension, assessed by ANOVA and followed by
paired t-test.
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Regarding the specificity of the inhibition of enzyme activity, acute
hypertension also significantly decreased activity of the apical
membrane marker alkaline phosphatase by 40% but did not decrease the
activity of the apical enzyme DPPIV (Table 1). Blood pressure
restoration returned alkaline phosphatase enzyme activity to control
levels. These results demonstrate that both the basolateral Na-K-ATPase
and apical alkaline phosphatase activities are affected by alterations
in blood pressure in a manner that persists through membrane isolation,
supporting the hypothesis that during hypertension these membrane
proteins (or their regulators) undergo reversible structural
modification or translocation to lipid domains where their activity is
decreased and that the decreases are not due to degradation.
We previously reported that there was a significant, albeit minor,
redistribution of Na-K-ATPase subunits to heavier densities during 5 min of acute hypertension that would contribute to the decrease in
ATPase activity in fractions 3-5.
In this study, we again measured Na-K-ATPase
- and
-subunit
immunoreactivity after control, hypertension, and blood
pressure-restored protocols (Fig. 3).
Compared with the peak in Na-K-ATPase activity profile in Fig. 2,
immunoreactivity is broadly distributed near 100 kDa between
fractions 3 and
10, whereas the
immunoreactivity
pattern near 50 kDa has a distinct peak between
fractions 3 and
6, similar to ATPase activity. In this
series of experiments, no significant decrease in immunoreactive
-
or
-subunits in fractions 3-5
occurred that would account for the corresponding decrease in enzymatic activity. The difference between these and the previous findings may
reflect a subtle difference in methodology: in our previous study,
animals were killed directly after 5 min of hypertension without
chronic infusion, whereas in this study all of the animals were
infused, as described in EXPERIMENTAL
PROCEDURES, to maintain euvolemia during the more
lengthy protocols.

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Fig. 3.
Relative immunoreactive pools of Na-K-ATPase
1- and
1-subunits in membrane
fractions of renal cortex from control, acute hypertension, and blood
pressure-restored protocols fractionated on sorbitol density gradients.
A constant volume of each fraction was resolved by SDS-PAGE, blotted,
probed with 1- and
1-specific antibodies, and
quantitated by scanning densitometry. Subunit relative immunoreactivity
is expressed as the percentage of total signal in all 12 fractions.
Left: summary of 5 independent
experiments. Results are expressed as means ± SE,
n = 5 in each group.
Right: autoradiograms of a typical
experimental set, with detected at 97.4 kDa and at ~50 kDa.
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The density gradient results do not rule out the possibility that
during hypertension the inactivated sodium pumps were transported out
of the basolateral membranes to internal membranes that share the same
density distribution as basolateral membranes. Indeed, there are
previous reports that sodium pump activity can be decreased by
internalization (reviewed in Refs. 2, 4, and 10). To test this
possibility, we made use of a fractionation strategy that separates
membranes on the basis of their partitioning in an aqueous,
dextran-polyethylene glycol two-phase system.
Figure 4 summarizes the distributions of
Na-K-ATPase catalytic activity and
- and
-subunit
immunoreactivities after phase-partitioning analysis of density
gradient fraction 4 (in which
Na-K-ATPase was decreased 35% without detectable changes in
or
immunoreactivity). In the baseline blood pressure control sample, all
three markers exhibited peaks, with maxima in partitioning
fraction 8, evidently marking the
basolateral membranes. The
- and
-subunits both exhibited minor
peaks with maxima in fraction 2, but
these were without a peak in catalytic activity. Hypertension was
associated with leftward shifts in the major peaks of all three
markers, from maxima in fraction 8 to
maxima in fraction 7, and a >50% decrease in Na-K-ATPase activity localized to fraction
8. A similar leftward shift and decrease in catalytic
activity was observed in a second experiment, performed with a
different phase system pH. The most economical interpretation of this
result is that a change in the basolateral membrane physical properties
that determine phase-partitioning behavior accompanies the modification that decreases Na-K-ATPase catalytic activity.

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Fig. 4.
Phase-partitioning analysis of Na-K-ATPase in fraction
4 from paired control and acute hypertension samples in
a polyethylene glycol-dextran two-phase system as described in
EXPERIMENTAL PROCEDURES.
Top: Na-K-ATPase activity is corrected
for total protein applied to the two-phase system. Na-K-ATPase subunit
immunoreactivity is expressed as the percentage of the total
1- or
1-subunit immunoreactivity
detected in all 10 samples. Bottom:
autoradiograms scanned for quantitation. Experiment shown is
representative of the 2 separate independent assays conducted.
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Response of renal cortex apical membrane proteins to acute
hypertension and blood pressure restoration.
The NHE is a major transporter for sodium entry across the proximal
tubule apical membrane, and NHE3 is responsible for virtually all the
NHE activity in this region (1, 6, 45). We previously reported that an
acute hypertension provoked a rapid redistribution of apical membrane
NHE3 immunoreactivity to higher-density membranes. In this study, we
aimed both to determine whether this response was reversible and to
characterize the specificity of the response. NHE3, detected at 80 kDa
by immunoblot (7), distributes to a major peak at
fractions 4-5 after the control
protocol, containing 75% of the total NHE3, defined as the apical
membrane population (Fig. 5). After 5 min
of acute hypertension, the immunoreactive pool of NHE3 in
fractions 4-5 is reduced to 20%
of total due to redistribution to a peak centered around
fraction 6, with a small shoulder
appearing at fractions 8-10.
After 20 min of blood pressure restoration, the apical membrane NHE3
immunoreactivity returns to its starting level and distribution in
fractions 4-5. This result
demonstrates reversible redistribution of NHE3 associated with the
changes in sodium transport provoked by blood pressure fluctuations.

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Fig. 5.
Top: Distribution of Na/H exchanger
(NHE3) in membrane fractions of renal cortex from control, acute
hypertension, and blood pressure-restored protocols. A constant volume
of each fraction was resolved by SDS-PAGE, blotted, and probed with
NHE3-specific monoclonal antibody. Antibody antigen complexes were
detected by enhanced chemiluminescence. NHE3 immunoreactivity in each
fraction is expressed as the percentage of total signal in all 12 fractions. Results are expressed as means ± SE,
n = 5 in each group.
* P < 0.05 vs. control, and
+ P < 0.05 vs. acute hypertension, assessed by ANOVA and followed by
paired Student's t-test.
Bottom: representative immunoblots
from a typical experiment with NHE3 detected at 80 kDa.
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To determine the specificity of the redistribution phenomenon with
acute hypertension, the distribution of the renal
Na-Pi cotransporter, a type II
Pi transporter, was investigated.
Like NHE3, the Na-Pi cotransporter
is expressed in the proximal tubule apical brush border. The
Na-Pi cotransporter has been shown
to move from apical membranes to internal membranes in response to acute high-phosphate diet (26). Figure
6A is an
autoradiogram illustrating the immunoblot detection of the
Na-Pi cotransporter as a series of
bands between 75 and 90 kDa and smaller bands at 37 kDa, as previously
reported (14). The autoradiogram in Fig. 6A also establishes that the density
distribution of the Na-Pi cotransporter coincides with that of NHE3, with a single major peak in
fractions 4-6 and baseline
expression restricted to fractions 4-7. Figure 6B
shows the results of two independent experimental sets of control,
hypertension, and restoration samples, specifically fractions 4-7 between 75 and 90 kDa, that were analyzed by immunoblot. In both sets, there was a
pronounced decrease in the immunoreactivity in
fraction 4, increases in signals in
fractions 6-7, and a shift of the
peak from fraction 5 to
6, analogous to the redistribution of
NHE3 with acute hypertension. With blood pressure restored, the
distribution shifted back toward the lower densities (although not
completely restored as observed for NHE3 in the same experimental sets).

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Fig. 6.
Distribution of Na-Pi
cotransporter immunoreactivity in membrane fractions of renal cortex
from control, acute hypertension, and blood pressure-restored protocols
resolved by density gradient fractionation. A constant volume of each
fraction was resolved by SDS-PAGE and blotted and probed with
Na-Pi cotransporter-specific
polyclonal antibody. Antibody-antigen complexes were detected with
125I-labeled protein A. A: entire blot of a fractionation of
control, sham-operated renal cortex, which illustrates immunodetection
of a series of bands between 75 and 90 kDa and a smaller molecular mass
bands at 37 kDa, as previously reported for this antiserum (14).
Essentially, all of the Na-Pi is
restricted to fractions 4-7 in
the 12 fraction set. Brush-border membranes (bbm) are included as a
positive control in the last lane. B:
series of immunoblots of fractions
4-7 from control, acute hypertension, and blood
pressure-restored cortices from 2 independent experiments; upper series
of bands between 75 and 90 kDa, not the bands at 37 kDa, are shown.
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The distribution response of two classical apical membrane markers that
are not sodium transporters was investigated as well. Alkaline
phosphatase activity and DPPIV immunoreactivity (detected by immunoblot
at 105 kDa) both have distribution patterns with a main peak centered
at fractions 5 coincident with the
peaks of NHE3 and the Na-Pi
cotransporter, indicative of apical plasma membranes (6). However, both
are broader than the sodium transporter peaks, suggesting expression in
multiple membrane populations. A dotted line is provided in Fig. 7 as a
reference to the location of the apical sodium transporter peaks.
During hypertension, the alkaline phosphatase activity and DPPIV
immunoreactivity in fractions 1-5
decreased 50% compared with control. During hypertension, the position
of the peak shifted to fraction 6 and
the fraction of DPPIV in the higher-density shoulder increased
significantly, as also seen for the NHE3 pattern. Twenty minutes after
blood pressure restoration, both alkaline phosphatase activity and
DPPIV immunoreactivity in fractions
1-5 increased back to 70% of control levels (Fig.
7). These data provide evidence that
several apical brush-border proteins are found in higher-density
membranes after an acute increase in blood pressure.

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Fig. 7.
Distribution of apical membrane proteins in membrane fractions of renal
cortex from control, acute hypertension, and blood pressure-restored
protocols. Top left: alkaline
phosphatase activity, corrected for total protein recovered from all 12 fractions. Top right:
dipeptidyl-peptidase IV (DPPIV) immunoreactivity detected at 105 kDa by
immunoblot (bottom) of a constant
volume of each fraction with specific antibodies, expressed as
percentage of total signal density from all 12 fractions. A dotted line
at fraction 5 is provided as a
reference, indicating the separation between the apical plasma
membranes and higher-density intracellular membranes. Data are
expressed as means ± SE; n = 5. * P < 0.05 vs. control and
+ P < 0.05 vs. acute hypertension, assessed by ANOVA and followed by
paired t-test.
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Redistribution of a set of apical membrane proteins in response to
acute hypertension could result from
1) modification in the array of
proteins associated with microvilli that changes its density
equilibrium without leaving the apical plasma membrane, 2) wholesale internal retraction of
microvilli, as recently reported to occur during ATP depletion (16),
3) concerted trafficking of several
apical proteins to a new membrane population with a different density,
or 4) redistribution of NHE3 within
the apical membrane from the microvillar domain to the intermicrovillar
cleft membrane. To address this question, we examined the distribution patterns of apical proteins relative to that of
1) villin, a 95-kDa microvillar
cytoskeleton bundling (and severing) protein associated with F-actin
and restricted to the microvillar cytoskeleton,
2) megalin (gp330, 330 kDa), a
scavenger receptor for filtered proteins found mainly in the
intermicrovillar domain, coated pits, endocytotic vacuoles, and
lysosomes of the early proximal tubule, with patchy distribution in the
brush-border domain in later proximal tubules (5, 13), and
3) Rab 5a and Rab 4 (both detected
at ~25 kDa), monomeric GTPases associated with endocytotic and
exocytotic vesicles, respectively (8, 25, 40, 44).
Villin was broadly distributed between fractions
4 and 12 (Fig.
8), in contrast to the sharper peak of NHE3
and Na-Pi cotransporter, but
overlapping with that of alkaline phosphatase and DPPIV. The overall
distribution of villin and the percentage of total villin in the sodium
transporter peak (fractions
4-6) are unchanged by acute hypertension. That
is, sodium transporters redistribute out of fractions
4 and 5 to higher
densities without an accompanying shift in villin from these fractions,
indicating that the shift is not likely to result from a change in the
density of microvillar membranes associated with villin and containing
NHE3 and the Na-Pi cotransporter.

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Fig. 8.
Top: distribution of villin
immunoreactivity in membrane fractions of renal cortex from control and
acute hypertension protocols. A constant volume of each fraction was
resolved by SDS-PAGE and blotted and probed with villin-specific
monoclonal antibody. Antibody-antigen complexes were detected with
125I-protein A. Villin relative
abundance in each fraction is expressed as the percentage of total
signal in all 12 fractions. Results are expressed as means ± SE;
n = 5 in each group. Distributions
were not significantly different, assessed by ANOVA.
Bottom: representative immunoblots
from a typical experiment with villin at 95 kDa.
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Distribution of megalin (gp330) is also distinct from that of NHE3 and
other apical proteins and overlaps with the distribution of villin
(Fig. 9). As expected from the complex
pattern of subcellular expression previously described and discussed
above (5, 13), there are multiple peaks of megalin in the gradient.
Statistical analysis (by two-way repeated ANOVA) did not detect a
significant difference in the overall distribution pattern of megalin
immunoreactivity among the three treatment groups, but there was a
tendency for fractional expression to be lower in
fractions 4 and
5 and greater in
fractions 8-10 with acute
hypertension, reminiscent of the NHE3 redistribution with hypertension.
This could reflect redistribution of the megalin expressed in the
microvillar domain in the later proximal tubule. The major peak of
megalin, which likely marks the peak in intermicrovillar membranes, is
at fraction 6 in all three protocol
groups. This location is coincident with the peaks of redistributed
apical proteins, suggesting that these proteins move from the
microvilli to the intermicrovillar cleft region when blood pressure is
elevated. This is a plausible path, since it has been demonstrated for
internalization of another microvillar protein, the insulin receptor
(33) using electron microscopy. Rab 4 is detected in
fractions 8-10 and Rab 5a is
detected in fractions 10 and
11 (Fig. 9), a pattern unaffected by
blood pressure perturbations (not shown). Thus classical endosomal
membranes are localized to fractions
8-11, coincident with the high-density shoulder of
apical proteins increased with hypertension and decreased with pressure
restoration.

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|
Fig. 9.
Distribution of intermicrovillar cleft and endosomal protein markers in
membrane fractions of renal cortex from control, acute hypertension,
and blood pressure-restored protocols.
Top: distribution of megalin (a marker
for the intermicrovillar cleft, detected by immunoblot of a constant
volume of each fraction probed with specific antibodies), expressed as
percentage of total signal density from all 12 fractions, was unaltered
by increasing or restoring blood pressure. Data are expressed as means ± SE, n = 5. Distributions were
not significantly different, assessed by ANOVA.
Middle: representative immunoblots of
megalin at 330 kDa from a typical experiment.
Bottom: distribution patterns of the
endosomal markers Rab 4 and Rab 5a, detected by immunoblot at 25 kDa of
a constant volume of each fraction probed with specific antibodies.
|
|
 |
DISCUSSION |
We previously demonstrated cellular mechanisms that could, at least in
part, account for the decrease in proximal tubule sodium transport
during acute hypertension: sodium pump activity decreases and apical
NHE3 shifts to membranes with higher density. This current study
demonstrates that these responses are reversed after blood pressure is
returned to basal levels. Specifically, 20 min after blood pressure
restoration, Na-K-ATPase activity is restored to control levels and
NHE3 immunoreactivity shifts back from a higher- to a lower-density
position in the gradient, which is typical of apical microvilli
markers. The study also demonstrates that the response to acute
hypertension is not restricted to Na-K-ATPase and NHE3: alkaline
phosphatase activity is reversibly inhibited and the distributions of
apical alkaline phosphatase and DPPIV are reversibly shifted.
Two assays demonstrate that the inactivation of Na-K-ATPase enzymatic
activity in hypertension is reversible: in density gradient fractions
containing basolateral membranes (fractions
3-5), mean activity decreases to 67% of control
with hypertension and increases back to 83% of control with
restoration, whereas, in the starting So before
fractionation, activity decreases to 72% with
hypertension and fully increases back to control levels with
restoration. It should be noted that the ATPase activities, although
reported in the same units (µmol
Pi · mg
protein
1 · h
1),
are calculated slightly differently in the two assays. In the assays of
So Na-K-ATPase, specific activity
is expressed as the Pi liberation
divided by the amount of protein in that
So sample, whereas, in the density
gradient fractions, Pi liberated
in each fraction is divided by the total protein recovered in all 12 fractions to normalize for variation in protein content between sample
sets. Within the peak basolateral membranes (fraction
3) pNPPase activity is enriched about fivefold (to 7 µmol Pi · mg
protein
1 · h
1)
compared with activity in So.
There is a burgeoning literature on mechanisms responsible for
short-term regulation of Na-K-ATPase activity (reviewed in Refs. 2 and
4). Pathways linked to both generation of protein kinase C (PKC)
and/or cAMP-dependent protein kinase A (3, 4) are postulated to
regulate Na-K-ATPase activity by changing the
catalytic subunit
phosphorylation status. However, phosphorylation has been associated
with both decreased activity (3, 29, 39) and increased activity (9, 28,
36) and no change in activity (15). There is also evidence that PKC
causes a withdrawal of sodium pumps from the basolateral membranes
independent of their PKC phosphorylation site [demonstrated by
mutating at Thr-15 and Ser-16 to Ala (S16A/T15A)] (2). Proximal
tubule Na-K-ATPase activity is also inhibited (whether directly or
indirectly is not known) by activation of phospholipase
A2, which stimulates production of
arachidonate metabolites of cytochrome
P-450 such as
20-hydroxyeicosatetraenoic acid (34, 35, 37), and sodium pump transport
activity is inhibited by apical ATP mediated by purinergic receptors
(22). Although the precise signaling mechanisms for the response to
altered blood pressure remains to be elucidated, our results indicate
that the inhibition of the sodium pump activity is due to structural
modification of the pump itself or an associated regulator, rather than
solely mediated by trafficking of active pumps to a new location; the
data demonstrate significant changes in total ATPase activity that
persist through membrane fractionation and phase-partitioning analysis.
However, the minor change in the partitioning properties of the
Na-K-ATPase
- and
-subunits may reflect either a modification of
the basolateral membranes containing the inhibited pumps or a transfer
of inhibited pumps to membranes with different partitioning properties.
The findings of this study add to a growing body of evidence for rapid
regulation of renal proximal tubule solute transport by trafficking of
the transporters between surface and internal membrane domains (2, 10,
20, 26) In isolated proximal tubules, Hensley et al. (20) provided
evidence, using similar subcellular fractionation strategy, for
redistribution of NHE activity from apical to internal membranes
mediated by parathyroid hormone (PTH) stimulation, although the
trafficking route and isoform were not identified. Proximal tubule
Na-Pi cotransporter, present in
both subapical and apical pools, is rapidly recruited to the apical
brush border by acute dietary Pi
restriction mediated by microtubule-dependent translocation of
presynthesized Na-Pi cotransporters, and surface expression is rapidly downregulated with
acute high-Pi diet independent of
microtubules (26). By high-resolution immunocytochemistry, Biemesderfer
and colleagues (7) found NHE3 in subapical vesicles in the proximal
tubule consistent with possible regulation by membrane recycling. In the present study, we demonstrate that the decrease in proximal tubule
sodium transport provoked by acute hypertension is associated with a
redistribution of both NHEs and
Na-Pi cotransporters to membranes
of higher density. Immunofluorescence studies have demonstrated that
both the NHE3 and the renal Na-Pi
cotransporter are highly enriched in the apical brush border under
control conditions (7, 26). The observation that there is no detectable
shift of the brush-border cytoskeletal protein villin to higher
densities with hypertension argues against the interpretation that the
density of the apical membranes has increased. We postulate that the
apical sodium transporters have redistributed out of the brush border to the subapical vesicles containing NHE3 demonstrated by Biemesderfer and colleagues (7).
The proximal tubule has such a rich array of vesicles under the apical
membrane, also referred to as dense apical tubules, that it is
difficult to interpret trafficking events when studied by light
microscopy. However, Yip (46) has provided preliminary evidence that
NHE3 is actually internalized in response to acute hypertension. Using
electron microscopic techniques, Nielsen (33) studied early events in
trafficking of labeled insulin receptors of the apical membrane and
provided evidence that it involves two steps: first, lateral migration
of membrane receptors from microvilli to the intermicrovillar cleft
region and, subsequently, internalization into endocytotic vacuoles and
dense apical tubules. Our results with subcellular fractionation
suggest a similar route for redistribution of apical proteins in the
response to acute hypertension. Within 5 min of acute hypertension, the
apical proteins move to a region of the gradient in which there is the
greatest percentage of megalin (gp330), a marker for the
intermicrovillar cleft and coated pits (13), and to regions containing
endosomal Rab markers. However, it should be noted that Biemesderfer
and co-workers (7) did not observe NHE3 in coated pits in unstimulated rat kidney.
The fact that several apical proteins moved to higher densities during
acute hypertension suggested the possibility that the response involved
internal retraction of the brush-border microvilli, analogous to the
response seen in cultured cells during ATP depletion (16) with PTH
treatment (17) and that the restoration response involved the insertion
of preformed microvilli seen during recovery from ATP depletion in
cultured renal cells (16) or with epidermal growth factor treatment of
enterocytes (19). Although this has not been ruled out, the results do
not support this mechanism, since no accompanying shift in the
microvilli marker villin was detected with acute hypertension, and
there was, likewise, no detectable change in villin distribution in the
density gradient fractions in which there was >50% decrease in NHE3
and Na-Pi with hypertension.
During membrane recycling, endocytosed proteins can be either returned
to the plasma membrane or routed to lysosomes for degradation. For
example, there is evidence that
Na-Pi is colocalized with lysosomes during the transition from low- to
high-Pi diet (26). The
demonstration that the redistribution and inactivation of transporters
and apical markers are reversible and occur without a change in
starting pool size in the So
fraction argues that changes in the degradation rate do not correlate
to rapid decreases in sodium transport provoked by acute hypertension.
The onset of proximal tubule responses to acute hypertension (11, 12,
47) and the onset of pressure natriuresis (41) are almost instantaneous
with the increase in blood pressure. In this study, we observed that
the reversal of the natriuretic responses is gradual even though the
restoration of blood pressure to basal levels is nearly instantaneous.
The time courses of return in lithium clearance and urine output were
indistinguishable, suggesting that the two parameters are linked to
similar signaling mechanisms. The sustained elevation in lithium
clearance, an inverse indicator of sodium handling in the proximal
tubule (43) in the absence of the physical stimulus of elevated
pressure, suggests mediation by chemical regulators. For example, acute
hypertension may stimulate the release of a mediator (or may inhibit a
tonically released regulator) from cells that sense the elevated blood
pressure, and, when blood pressure is rapidly restored and the stimulus is removed, the return of the mediator to basal levels around the
responding cells will be a function of the rate of removal from (or
addition to) the regional pool. The rate of recovery of proximal tubule
sodium reabsorption and urine output will also be a function of the
rate of reversibility of sodium transporter trafficking and inhibition.
Another explanation for the lag in response is that the reversal of the
sodium pump modifications or rerouting of apical proteins requires 20 min.
In conclusion, this study demonstrates rapid, reversible redistribution
and inactivation of apical and basolateral sodium transporters in the
proximal tubule in response to acute hypertension and blood pressure
restoration. This complex coordinated set of cellular mechanisms can
potentially account for the altered proximal tubule sodium reabsorption
in response to blood pressure fluctuations.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
DK-34316 to A. A. McDonough and HL-45623 to N.-H. Holstein-Rathlou.
 |
FOOTNOTES |
Y. Zhang was supported by a fellowship award from the American Heart
Association, Greater Los Angeles Affiliate.
Portions of this work were presented at the 1995 and 1996 Annual
Meetings of the American Society of Nephrology.
Address for reprint requests: A. A. McDonough, Dept. of Physiology and
Biophysics, Univ. of Southern California School of Medicine, 1333 San
Pablo St., Los Angeles, CA 90033.
Received 8 September 1997; accepted in final form 19 December
1997.
 |
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