RAPID COMMUNICATION
Regulation of proximal tubule sodium/hydrogen antiporter with
chronic volume contraction
Kenneth A.
Fisher1,
Sung H.
Lee2,
Jeffrey
Walker2,
Christine
Dileto-Fang2,
Leonard
Ginsberg2, and
Susan R.
Stapleton2
1 Michigan State University/Kalamazoo Center for Medical
Studies and 2 Department of Biological Science, Western
Michigan University, Kalamazoo, Michigan 49008
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ABSTRACT |
We developed a model of volume contraction
in rabbits by using a furosemide/low-salt diet to follow changes, if
any, in proximal tubule Na+/H+ exchanger 3 (NHE3) mRNA and brush-border protein. The rabbits' plasma renin,
aldosterone, and urine sodium content confirmed the volume-contracted
state. RNase protection assays demonstrated increases in treated-animal
NHE3 mRNA as a percentage of control with 172 ± 23, 154 ± 15, 153 ± 14, and 141 ± 7 (SE) % (P < 0.05) at 1, 5, 10, and 31 days, respectively. Western analysis of
brush-border membrane with NHE3 antibody revealed increased
immunoreactivity in treated animals as a percentage of control with
120 ± 30, 190 ± 59, 307 ± 72, and 427 ± 41%
(P < 0.05) at 1, 5, 10, and 31 days, respectively.
There was no significant difference in serum potassium, bicarbonate,
and cortisol in control vs. experimental animals. These data suggest
that there is chronic upregulation of NHE3 in the volume-contracted state.
sodium-hydrogen exchanger 3 induction; renal cortical; salt
restriction
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INTRODUCTION |
IN A WHOLE-ANIMAL MODEL,
CHRONIC dietary sodium depletion in the rat demonstrated an
increase in sodium/hydrogen countertransport activity in brush-border
membranes (14). Subsequently, this carrier has been
identified in the proximal tubule as the sodium/hydrogen antiporter
(NHE3) (1, 3, 8). The possible mechanisms that underlie
this change in activity, such as increases in transcription, translation, or activation, were not investigated. Two other recent chronic models investigating the regulation of renal cortical NHE3
activity and expression have been published. In one model, the chronic
administration of the glucocorticoid analog dexamethasone resulted in a
similar increase in both mRNA and activity of NHE3, suggesting
transcriptional regulation of NHE3 (6, 10). In the second
model, induction of chronic metabolic acidosis revealed no measurable
increase in cortical NHE3 mRNA; however, increases in NHE3 protein
abundance were evident (2). In this model it was suggested
that NHE3 regulation occurred at the translational or possibly
posttranslational level.
Because of the critical role of NHE3 in proximal tubule sodium
reabsorption, this study focuses on the adaptive changes in NHE3 mRNA
and protein abundance during volume contraction in rabbits. By using
diuresis and dietary sodium depletion to create a volume-contracted state, the expression of NHE3 mRNA and protein was quantitated at
several time points over 31 days. Results from this study demonstrate that both NHE3 mRNA and protein increase to different degrees in
response to volume contraction.
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METHODS |
Animal model.
New Zealand White female rabbits (1.5-2.0 kg; HRP, Denver, PA)
were housed in cages and given food (Quality Select Basic Blend 1600)
and water ad libitum. After an equilibration period, animals were given
an intravenous injection of 5 mg/kg furosemide (American Regent
Laboratories, Shirley, NY) to create a volume-contracted state, which
was maintained by administering a low-salt diet of pearled barley
(0.003% sodium, LaCrosse Milling, Cochrane, WI). Animals at 1, 5, 10, and 31 days of volume contraction along with age-matched controls were
injected with 50 mg/kg pentobarbital (iv). After confirmation of deep
anesthesia, the renal arteries were clamped at the hilum, and the
kidneys were removed before cardiac puncture. Slices of kidney cortex
were flash-frozen in liquid nitrogen and stored at
70°C for
RNA isolation and brush-border membrane preparation. Samples from
cardiac puncture were used for determination of plasma renin activity
(Mayo Medical Laboratories, Rochester, MN), cortisol, aldosterone,
electrolytes, and hematocrit, as well as sodium concentration from urine.
RNAse protection assay.
Total RNA was isolated from the kidney cortex by using TRIzol reagent
(GIBCO, Grand Island, NY). For in vitro transcription of a
-32P-labeled cRNA probe, a template was constructed by
using a 317-bp region of NHE3 cDNA [1181-1498 nucleotides (nt)]
(15) cloned into the pGEM-4Z (Promega, Madison, WI) vector
(NHE3 cDNA was a gift from Drs. Chung-Ming Tse and Mark Donowitz, The
Johns Hopkins University School of Medicine). Transcription of the
antisense cRNA probe was performed by using the MaxiScript kit (Ambion, Austin, TX), Sp6 RNA polymerase, and [
-32P]UTP. A
250-bp
-actin probe was also synthesized similarly by using a
template supplied by the manufacturer. After treatment with RNase-free
DNase to destroy the templates, unincorporated nucleotides were removed
by precipitating the probe with ammonium acetate and ethanol. A RNase
protection assay (RPA) was performed by using an RPA II (Ambion) kit.
Labeled cRNA antisense probes (20,000-50,000 cpm/sample, where cpm
is counts/min) and 20 µg total RNA in 20 µl hybridization solution
were heated to 90°C for 5 min and hybridized overnight at 42°C.
After hybridization, the RNA-RNA hybridization mix was treated with 10 U RNase T1 at 37°C for 30 min to degrade the single-strand
unhybridized probe. The "protected" RNA-RNA hybrids were
precipitated with ethanol and resuspended in loading buffer
containing 95% formamide. After heating to 90°C for 5 min, the
samples were separated on an 8 M urea-8% polyacrylamide
Tris-borate-EDTA denaturing gel. A radiolabeled RNA ladder (Century
Marker Template, Ambion) was loaded onto each gel to confirm that the
RNA hybrids were of the expected size. After drying, the gel was
visualized by autoradiography, and the signals were quantitated by densitometry.
Western blot analysis.
Brush-border membrane (BBM) was isolated from kidney cortex samples by
using a previously established Mg2+ aggregation and
differential centrifugation technique (7). This technique
for BBM isolation was confirmed by assaying for cytosolic (acid
phosphatase), basolateral membrane (Na-K-ATPase), and apical membrane
(alanyl aminopeptidase) protein markers. The specific activities in
each batch of the protein markers obtained from the BBM preparations
were similar to those previously published for BBM isolations verifying
the technique (7). Protein concentration was determined by
the method of Lowry (13), and 150 µg of each sample were
then size fractionated by SDS-PAGE on 8% gels. After the samples were
electroblotted to a polyvinylidene fluoride Immobilon-P membrane
(Millipore, Bedford, MA), the membrane was blocked for 2 h in
blocking buffer (TBS with 0.1% Tween 20 and 5% milk protein). The
membrane was probed in the same buffer with mouse anti-rabbit NHE3
antibody (a gift from Dr. Daniel Biesmesderfer, Yale Univ. School of
Medicine) for 18 h at 4°C with shaking. The membrane was then
washed four times for 15 min in blocking buffer and incubated with
1:2,000 dilution of sheep horseradish peroxidase-conjugated anti-mouse
IgG (Amersham Life Science, Arlington Heights, IL) for 30 min at room
temperature. After being washed four times in blocking buffer, the
bound probe was detected with enhanced chemiluminescence (ECL Western
Blotting Reagents, Amersham Life Science). NHE3 protein abundance was
quantitated by densitometry.
Statistics.
Results are expressed as means ± SE. Differences among the groups
were assessed by either an F-test or paired Student's
t-test with unequal variance, as appropriate. Significance
was established as P < 0.05.
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RESULTS |
Animal model characteristics.
Urine sodium, plasma renin, and aldosterone levels were measured to
assess the rabbits' volume-contracted state. The urine sodium
concentration and plasma renin activity for control and treated animals
are shown in Figs. 1 and
2, respectively. The urine sodium
concentration was significantly less, and plasma rennin activity was
greater, in the sodium-restricted animals. Table
1 compares serum potassium, bicarbonate,
cortisol, and aldosterone for control and sodium-restricted animals.
The data reported in Table 1 are means ± SD of all values
comparing experimental and control groups at days 1,
5, 10, and 31. There was no
significant difference among time points. There was also no significant
difference when sodium-restricted animals are compared with control
animals, except that serum aldosterone was significantly higher
(P < 0.003) in the volume-contracted group.

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Fig. 1.
Effect of diuresis and chronic dietary sodium restriction
on urine sodium concentration. The average urine sodium concentration
(mmol/l) in control (n = 4) and volume-contracted
rabbits at 1, 5, 10, and 31 days (n = 3 at each time
point) is shown. Error bars, SE. *Significant decrease in the average
urine sodium concentration of volume-contracted animals compared with
controls, P < 0.05.
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Fig. 2.
Effect of diuresis and chronic dietary sodium restriction
on plasma renin activity. The average plasma renin activity
(ng · ml 1 · h 1) for control
and volume-contracted rabbits at 1, 2, 7, and 31 days and the no. of
samples are shown in each bar. Error bars, SE. *P < 0.05.
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Table 1.
Comparison of serum potassium, bicarbonate, cortisol, and
aldosterone for control and sodium-restricted rabbits
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Effect of volume contraction on NHE3
mRNA abundance.
An RPA was chosen to quantitate the mRNA in kidney cortex not only
because of the sensitivity of the assay but also to alleviate concern
that accurate measurements might not be made if mRNA from the kidney is
highly degraded. In Northern analysis, single cleavage of 20% of an
mRNA species will result in a 20% signal loss, whereas in an RPA the
loss of as little as 1% of the signal would result from such
degradation (12). This increase in sensitivity compared with Northern analysis makes an RPA a more appropriate assay for quantitating mRNA in this instance. In Fig.
3, an autoradiograph from a typical RPA
indicates the location of
-actin and NHE3 bands on an 8 M urea-8%
polyacrylamide gel. The detection of
-actin in each lane was used to
normalize the mRNA loading because it has been determined that the
expression of
-actin does not change in a volume-contracted state
(data not shown). Results of the NHE3 mRNA content in the kidney cortex
of volume-contracted animals are shown in Fig.
4. A maximal increase in NHE3 of 172 ± 23% of control was detected at 1 day after volume contraction. The
NHE3 mRNA at days 5, 10, and 31 is
also significantly increased compared with controls at 153 ± 15, 152 ± 14, and 141 ± 7%, respectively. All of the NHE3 mRNA
increases in the volume-contracted animals are significantly higher
than in the controls; however, the magnitude of this increase in NHE3
mRNA did not change with time after day 1.

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Fig. 3.
Na+-H+ cotransporter 3 (NHE3)
mRNA expression during chronic volume contraction. Results of a typical
RNase protection assay indicating NHE3 and -actin bands from 20 µg
of total RNA from renal cortex of age-matched and volume contracted
rabbits at 1, 5, 10, and 31 days are shown. Isolated RNA was hybridized
to a 317-bp NHE3 probe and a 250-bp -actin probe and size
fractionated on an 8% polyacrylamide-Tris-borate-EDTA denaturing
gel.
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Fig. 4.
Using -actin to normalize for all measurements, the average NHE3
mRNA is shown as a percentage of the age-matched control
(n = 6 at each time point). Error bars, SE.
*Significant increase in NHE3 mRNA in the renal cortex of
volume-contracted animals compared with controls, P < 0.05.
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Effect of volume contraction on NHE3 protein
abundance.
BBM were prepared from kidney cortex, and NHE3 protein abundance was
assessed by Western blot analysis. As shown in Figs. 5 and 6,
the NHE3 protein abundance progressively increased over time in the
volume-contracted rabbits. A typical blot (Fig. 5) demonstrates that,
as the volume-contracted time is increased, the rabbits respond by
increasing the amount of NHE3 in the BBM. No significant increase in
NHE3 abundance was observed at day 1 of volume contraction;
however, after 5 days of volume contraction protein abundance did
significantly increase (P < 0.05) compared with
controls (190 ± 59%). This increase climbed to 307 ± 72% at 10 days and 427 ± 41% at 31 days after volume contraction. The stepwise increase in NHE3 protein at 5, 10, and 31 days reflects not only a significant increase from controls but also significant increases at each successive measurement compared with the previous time period.

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Fig. 5.
NHE3 protein expression during chronic volume
contraction. Typical Western blot of 150 µg of cortical brush border
from age-matched control and volume-contracted rabbits at 1, 5, 10, and
31 days is shown. By using anti-NHE3 antibody, bands were detected
between the 80- and 110-kDa markers.
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Fig. 6.
The average NHE3 protein abundance is shown as a percentage of the
age-matched control (n = 6 at each time point). Error
bars, SE. Exp, experimental.*Significant increase in NHE3 protein
abundance from the brush border of volume-contracted animals compared
with controls, P < 0.05.
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DISCUSSION |
This study tested the hypothesis that chronic extracellular volume
depression would have a significant effect on NHE3 mRNA and protein
content in the renal cortex. NHE3 is the isoform that is heavily
expressed on proximal tubule brush border and is responsible for a
majority of proximal tubule sodium reabsorption (1). Significant increases in expression of this protein would probably reflect adaptive changes in proximal tubule sodium-hydrogen exchange that have previously been described in salt-depleted rats
(14).
Using the volume-contracted rabbit as our model, we demonstrated a
significant increase in NHE3 mRNA by day 1. This increase was maintained throughout the 31-day experimental period. Additionally, levels of proximal tubule BBM NHE3 protein steadily increased, starting
at day 5 and continuing through day 31.
Mechanisms causing this increase are as yet unknown.
Glucocorticoids have been shown to increase NHE3 mRNA and NHE3
activity, at least in part, via glucocorticoid-response elements within
the NHE3 promoter region (5, 9, 11). However, in our
experiments there was no detectable increase in serum cortisol.
Acidosis also has an effect on proximal tubular NHE3. In rats, chronic
acidosis increased NHE3 protein levels in proximal tubule BBM, but
increased NHE3 mRNA was not detected (2). In an opossum
kidney cell line, however, acid incubation increased NHE3 mRNA and
protein levels (4). However, we could not detect a change
in acid-base status in our animals, as determined by there being no
significant difference in serum bicarbonate between control and
experimental animals.
We have demonstrated that chronic volume contraction in the rabbit
significantly increases NHE3 mRNA (~150% for at least 31 days) and
NHE3 protein abundance in the apical surface of the proximal tubule
(>400% at 31 days). Further experiments are necessary to clarify the
cellular mechanisms involved in this process and the signals informing
the proximal tubule cell of the presence of chronic volume contraction.
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ACKNOWLEDGEMENTS |
The authors thank Drs. Mark Donowitz and Chung-Ming Tse for
supplying NHE3 cDNA. We also thank Dr. Daniel Biesmesderfer for supplying NHE3 antibody and Dr. Carolyn Lamonica for help in obtaining serum samples.
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FOOTNOTES |
This work was supported through a grant from Michigan State
University/Kalamazoo Center for Medical Studies and initiated by a
grant from the National Kidney Foundation of Illinois.
Address for reprint requests and other correspondence: K. A. Fisher, Sect. of Nephrology and Hypertension, CFP-5, Henry Ford Hospital and Medical Centers, 2799 W. Grand Blvd., Detroit, MI 48202-2689.
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.
Received 14 August 2000; accepted in final form 13 December 2000.
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