491
Renal Apical Membrane Sodium-Hydrogen
Exchange in Genetic Salt-Sensitive Hypertension
James L. Lewis, David G. Warnock
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Abstract Inbred Dahl/Rapp salt-sensitive and salt-resistant
rats differ in their blood pressure response to dietary salt. We
studied sodium-hydrogen (Na-H) exchanger kinetics in renal
brush border membrane vesicles prepared from both strains
on either a 1% or 8% NaCl diet. Kinetics measurements were
made with the acridine orange fluorescence quenching technique in vesicles prepared at pH 6.0. The initial Na-H exchange rate was measured using preparations with similar
initial quench values. The maximal transport rate (Km,, fluorescence units per second per milligram protein [±SEM]) in
salt-sensitive rats on a 1% NaCl diet was significantly lower
than that in salt-resistant rats (36.9±4.4 versus 51.8±5.5,
respectively, P<.0005). With the 8% NaCl diet for 1 week, the
V^ of salt-resistant rats decreased and became similar to that
of salt-sensitive rats. The affinity for sodium (Km, millimoles
per liter [±SEM]) was also lower in salt-sensitive rats than in
salt-resistant rats while on a 1% NaCl diet (11.8±1.0 versus
19.6±2.3, respectively, P<.002). These values converged when
both strains were fed an 8% NaCl diet for 1 week. Inhibition
by 25 ^imol/L amiloride was less in salt-sensitive rats than in
salt-resistant rats on the 1% NaCl diet. These results show that
salt-sensitive rats have lower renal apical membrane Na-H
exchange activity than salt-resistant rats on a 1% NaCl diet.
Salt-sensitive rats do not modulate renal apical membrane
Na-H exchange in response to an 8% NaCl diet, whereas
salt-resistant rats show a more physiologically appropriate
response to increased dietary salt. The maladaptive response
by salt-sensitive rats on a high salt diet may well contribute to
the development of severe hypertension under these conditions. The differences between these two strains in sodium
affinity and amiloride inhibition suggest structural differences
in the renal apical membrane Na-H exchangers at the substrate and amiloride binding sites or altered cellular regulation
of these transport proteins. {Hypertension. 1994^24:491-498.)
Key Words • ion exchange • rats, inbred strains •
hypertension, genetic • amiloride
T
In addition to this so-called housekeeping role in
nonpolarized cells, Na-H exchangers play an additional role in epithelial cells by promoting vectoral
transport across the epithelium. Na-H exchange occurring in the apical membrane of the renal proximal
tubule results in the majority of proximal tubular
sodium reabsorption as well as the reclamation of
most of the filtered load of bicarbonate.1 An enhanced
Na-H exchange rate in the proximal tubular apical
membrane has been suggested as an additional mechanism by which increased Na-H exchange could be
causally related to essential hypertension.9
The cloning, sequencing, and expression of the cDNA
encoding an amiloride-sensitive form of the Na-H exchanger by Sardet et al in 198910 and the later mapping
of the gene now denoted APNH to chromosome I 11
have made possible further studies exploring the relation of Na-H exchange and essential hypertension on a
genetic level. Despite what seems to be an intuitive link
to hypertension, Lifton et al12 did not find a linkage
between increased sodium-lithium exchange and alleles
at the APNH locus in defined pedigrees from Utah.
Furthermore, by using affected sib-pair analysis, they
excluded APNH as a candidate gene for essential
hypertension in these pedigrees.12
For several years, several lines of evidence, including
differential amiloride sensitivities and regulation mechanisms, have suggested that there are at least two
functional types of the Na-H exchanger: an amiloridesensitive form, which is generally distributed in nonpolarized cells and restricted to the basolateral membrane
in epithelial cells, and an amiloride-insensitive form,
which is present primarily on the apical membrane of
he sodium-hydrogen (Na-H) exchangers make
up a family of integral membrane proteins
present in nearly all mammalian cells. Using
the energy stored in the inwardly directed sodium
gradient, these transport proteins mediate the electroneutral exchange of extracellular sodium for intracellular protons. Na-H exchangers are involved in a number
of cellular homeostatic processes and are thought to
play a central role in the control of intracellular pH and
cell volume.1-2 It has been postulated that an abnormality in cellular sodium uptake is an important factor in
the development of essential hypertension.3 Since Na-H
exchange mediates a large fraction of cellular sodium
uptake, the role of Na-H exchangers in the development
of human hypertension, particularly of the salt-sensitive
variety, has been an area of intense research over the
past several years. Several studies have examined platelet and leukocyte Na-H exchange as well as red blood
cell sodium-lithium exchange in humans and have found
enhanced exchange rates in patients with essential
hypertension. 48
Received October 18, 1993; accepted in revised form Jury 11,
1994.
From the Department of Medicine, Division of Nephrology and
Nephrology Research and Training Center, The University of
Alabama at Birmingham, and the Department of Veterans Affairs
Medical Center, Birmingham, Ala.
Portions of these results have been reported in abstract form
(Clin Res. 1990;38:275A; 11th International Congress of Nephrology, Tokyo, Japan, July 16-20, 1990:477A; and J Am Soc Nephrvl.
1990;l:654A).
Reprint requests to James L. Lewis, MD, The University of
Alabama at Birmingham, 618 Zeigler Research Bldg, 703 S 19th
St, Birmingham, AL 35294-0007.
O 1994 American Heart Association, Inc.
492
Hypertension
Vol 24, No 4 October 1994
specialized epithelia.13'14 Indeed, the recent cloning and
expression of several Na-H exchanger protein isoforms
have advanced this concept. 1417 Although direct tissue
localization of the protein product of each cloned Na-H
exchanger awaits the development of isoform-specific
antibodies, Northern analysis has been done. mRNA for
the amiloride-sensitive isoform cloned by Sardet et al10
(NHE-1) has been found in nearly all tissues.18 Other
isoforms (NHE-2 through NHE-4) have more restricted
mRNA distribution but include kidney cortex.15-16 Previous linkage studies by Lifton et al11 made use of
probes derived from NHE-1. Therefore, these results do
not directly test the relationship between hypertension
or salt sensitivity and increased activity of amilorideinsensitive isoforms of the Na-H exchanger located in
the apical membrane of the proximal tubule.
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Direct testing of Na-H exchange rates in renal epithelia from large numbers of human subjects is not
feasible, so work in this area must rely on animal models
of hypertension. We hypothesized that increased Na-H
exchange activity would be found in the proximal tubule
apical membrane in salt-sensitive hypertension, thus
leading to increased proximal sodium reabsorption. To
examine this thesis, we measured Na-H exchanger activity in salt-sensitive SS/Jr (S) and salt-resistant SR/Jr
(R) Dahl/Rapp rat kidney cortex brush border membrane vesicles (BBMVs). The Dahl/Rapp rats were
chosen over other hypertensive rat strains because of
the clear-cut salt sensitivity of the hypertension in the S
strain. In addition, pure inbred stocks of each strain
were available from a single source. The homogeneity of
these strains was also attractive because the small size of
the rat kidney relative to the amount of tissue needed
for fluorescence-based vesicle studies necessitated the
pooling of tissue from several rats.
Methods
Experiments were designed to compare apical membrane
Na-H exchanger kinetics between S and R rats on either a low
or high NaCl diet. Since vesicle preparation and fluorescent
measurements can display a fairly wide day-to-day variation, a
paired design was incorporated from the inception of these
studies. In addition, since performing complete Na-H exchange kinetics requires a greater amount of renal BBMV
than can be collected from an individual rat, renal BBMVs
were pooled from groups of four to eight rats. In every
instance, pairs of S and R rat groups were of equal number and
age and arrived in the same shipment from the vendor.
Preparation of BBMVs and fluorescence studies were carried
out concurrently on each paired set, permitting paired statistical analysis as previously described.19
Experimental Animals
All studies were done in accordance with The University of
Alabama at Birmingham Institutional Animal Care and Use
Committee guidelines. Inbred Dahl/Rapp S and R rats were
used (Harlan Sprague Dawley, Inc).20 A total of 288 S and R
rats were obtained during the years 1989 to 1991, at a time
when these strains had been inbred for more than 20 generations.21 The resulting extremely high level of genetic homogeneity justified the pooling of samples from individual S and R
rats as described above. Rats were housed at the University of
Alabama at Birmingham Animal Resources Facility in a
climate-controlled room and received either a standard ( 1 %
NaCl) or high salt (8% NaCl) diet (Agway Inc) for 1 week
before death and vesicle preparation. The two diets differed
significantly only in their NaCl content. Throughout the di-
etary manipulation period, all rats had free access to food and
water.
Blood Pressure and Body Weight Measurements
Blood pressure and body weight were measured on two
separate occasions during the week the rats were receiving
their prescribed diets. After several days were allowed for rats
to acclimatize after arrival from the vendor, blood pressure
was measured in conscious, restrained rats using the tail-cuff
method with a temperature-controlled restraining cage and
tail electrosphygmomanometer (Narco Biosystems) to familiarize the animals with the restraining cage device. At the end
of the week of dietary manipulation, the rats were weighed
and blood pressure was once again measured. The median of
five successive measurements was recorded as systolic blood
pressure as described by Chen et al.22
BBMV Preparation
Renal BBMVs were prepared by modifications of the Mg2+
aggregation technique of Booth and Kenny. 2328 After 1 week
on the respective diets, rats were given an injection of pentobarbital (5 mg/100 g body wt IP) and decapitated. The kidneys
were perfused by rapid injection into the distal aorta after the
aorta was occluded above the renal arteries with 10 mL iced
homogenizing solution containing (mmol/L) sucrose 50,
Hepes/KOH 10 (pH 6.0), and EGTA/KOH 5 (pH 6.0).
Throughout the next steps the tissue was kept at 5°C either by
chilling on ice or in a refrigerated centrifuge. The renal
cortices from four to six rats were then harvested and homogenized in 150 mL of the same buffer with a Sorvall Omnimixer
(Omni Corp International) at full speed for 4 minutes. The
homogenate was centrifuged for 15 minutes at 8000 rpm with
a JA-20 rotor and J2-21 refrigerated centrifuge (Beckman
Instruments, Inc). The supernatant was then centrifuged at
17 000 rpm for 30 minutes. The resulting pellet was resuspended in a second sucrose solution that contained 250
mmol/L sucrose and 10 mmol/L Hepes/KOH, pH 6.0, by
passage through a 22-gauge needle and centrifuged at 8000
rpm for 15 minutes. The supernatant was then centrifuged at
20 000 rpm for 25 minutes, and the resulting pellet was
resuspended by passage through a 27-gauge needle. The
resulting suspension was further concentrated by a fifth centrifugation at 20 000 rpm for 20 minutes, after which the
supernatant was discarded and the final pellet stored at -70°C
to preserve Na-H exchange activity until assays were
performed.25
Na-H Exchanger Assays
Na-H exchange rates were determined with a ratio spectrofluorometer (excitation, 493 nm; emission, 530 nm) using the
acridine orange fluorescence quenching technique as previously described.24 The BBMVs (which had been formed and
stored at pH 6.0) were placed in an acridine orange buffer that
contained 6 fimo\/L acridine orange, 150 mmol/L sucrose, 150
mmol/L N-methyl-glucamine gluconate (pH 7-5), and 10
mmol/L Hepes/KOH (pH 7.5). The actual amount of BBMVs
used was adjusted to always give an initial quenching of
acridine orange fluorescence between 50% and 60%. This
essential precaution ensured that the actual transmembrane
pH gradient was always the same.24 Na-H exchange rates,
expressed as fluorescence units (FU) per second, were determined by calculating the initial rate of the reappearance of
fluorescence after the addition of sodium gluconate. Sodium
gluconate was added by rapid injection using a 100-/xL syringe
(Hamilton) to give final concentrations of 90, 50, 25, 10, and 5
mmol/L. The initial recovery rates of fluorescence from five
trials at each sodium concentration were averaged to determine the Na-H exchange rate for that sodium concentration.
Amiloride inhibition was determined in BBMVs using 0, 25,
50, and 100 /xmol/L amiloride at the four higher sodium
concentrations listed above.
Lewis and Warnock
Renal Na-H Exchange in Hypertension
493
TABLE 1. Average Systolic Blood Pressures and Body Weights of Salt-Resistant and Salt-Sensitive Rats
on 1 % or 8% NaCI Diets
R
S
8%
1%
(n=35)
8%
(n=24)
( 1 % V8 8%)
P
1%
(n=34)
(n=23)
( 1 % vs 8%)
P
Systolic BP, mm Hg
112±3
113±3
NS
133±3*
142±3*
.05
Body weight, g
144+7
155±8
NS
152±7
166+8
NS
R indicates salt-resistant rats; S, salt-sensitive rats; and BP, blood pressure. Rats had been on an 8% NaCI diet for 5 to
6 days. Values are mean+SEM.
*P<.0001 compared with R rats on similar diet.
Enzyme Assays
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Brush border membrane enrichment and recovery (relative
to the crude homogenate) were determined by measuring
leucine aminopeptidase (LAP) activity as a marker of apical
membrane and protein concentration in both brush border
membrane preparations and cortical homogenates. LAP activity was determined by monitoring the appearance of 4-nitroaniline at 410 nm in a Lambda 6 UV/Vis spectrophotometer (Perkin-Elmer Corp) at 0 and 6 minutes in 1 tnL incubation
medium containing 100 mmol/L mannitol, 20 mmol/L Hepes/
Tris (pH 7.4), and 0.3 mg L-leucine 4-nitroanilide per milliliter.29 Brush border membrane preparation was done and
cortical homogenate protein concentrations were determined
with the bicinchoninic acid method using bovine serum albumin as a standard.30 Because of the viscosity of the BBMV
concentrates, all samples were aliquoted with a positivedisplacement capillary micropipette (American Hospital Supply Corp) as previously described.24 The BBMV preparations
were assessed by comparing the specific activity and enrichment of LAP activity in the vesicle preparation to that of the
crude homogenate. Percent recovery of LAP activity in the
vesicle preparation was also determined.26
Statistical Analysis
Blood pressure and body weight measurements were taken
in individual animals, and therefore statistical significance was
determined by unpaired Student's t test. Percent recovery and
enrichment factor were determined for BBMV samples prepared from paired groups of S and R rats; therefore, statistical
analysis was performed with Student's t test for paired samples. The maximal transport rate (KM,) and sodium affinity
(Km) were obtained by linear regression analysis using an
Eadie-Hofstee transformation (velocity versus velocity divided
by sodium concentration). In the analysis of Na-H exchange
kinetics, n refers to the number of S and R group pairs. A total
of 15 age-matched groups of S and R rats on a 1% NaCI diet
were compared. For studies involving 8% NaCI, two groups
each of S and R rats were fed either a 1% or 8% NaCI diet.
Vesicle preparation and transport assays in all four groups
were done simultaneously to eliminate day-to-day variability
as a source of error. Statistical significance for Na-H exchanger
kinetics was determined using a nested two-by-two ANOVA
with dependent variables of strain and diet for studies involving the 8% NaCI diet and dependent variables of strain and
amiloride concentration for studies with amiloride.
Materials
All chemicals were obtained from Sigma Chemical Co
unless otherwise noted and were of the highest purity available. Af-Methyl-glucamine gluconate was made by titrating the
free base with 1 mol/L rf-gluconic acid lactone to pH 7.5 as
previously described.24 Acridine orange was obtained from the
Eastman Kodak Co.
Results
Blood Pressure and Body Weight Measurements
Systolic blood pressure was measured in groups of
awake S and R rats on two separate occasions. As shown
in Table 1, none of the groups had severely elevated
systolic blood pressure at the time of the study. Blood
pressure was significantly higher in S than in R rats
within both dietary groups. After less than 1 week on
the high salt diet, S rats developed significantly higher
blood pressures than S rats that remained on the 1%
NaCI diet (P<.05). Body weight was determined in each
rat and is also reported in Table 1. There was no
significant difference in body weight between the S and
R rats on either diet.
Enzyme Assays
The total protein concentrations of the brush border
membrane preparations were similar, as were the protein concentrations of the crude homogenates. As
shown in Table 2, the percent recovery, specific activity,
and enrichment of LAP activity were similar in groups
of S and R rats on both a 1% and 8% NaCI diet. Even
though LAP specific activity appeared to be slightly
greater in BBMVs prepared from S rats compared with
those from R rats, LAP enrichment was identical for
both strains and unaffected by dietary NaCI content.
BBMV Na-H Exchange Studies
BBMV preparations from groups of S and R rats on
either a 1% or 8% NaCI diet were used to measure Na-H
exchange activity with the acridine orange fluorescent
quenching technique. Initial rates of the increase in acridine orange fluorescence with addition of five incremental
sodium concentrations were measured and are expressed
as FU per second per milligram protein (±SEM). Fig 1
shows the kinetic parameters of Na-H exchange in
BBMVs from 15 groups of S and R rats on a 1% NaCI
diet. The V^ in BBMVs from S rats was 36.9 ±4.4 and in
BBMVs from R rats was 51.8±5.5. This difference is
highly statistically significant (P<.0005). The Km for sodium in S rats (millimoles per liter sodium [±SEM]) was
11.8 ±1.0 and in R rats was 19.6 ±2.3. This difference is
also statistically significant (P<.002) and reflects a lower
affinity for substrate in BBMVs from R rats compared
with those from S rats. There were no obvious correlations
between either V^ or Km and age over the range of 4 to
9 weeks, but power to detect a difference was limited by
the low numbers of groups studied at each age.
Fig 2 shows VmM1 data for six groups of BBMVs from
S and R rats on either a 1% or 8% NaCI diet. The V^
was 37.0±4.9 in BBMVs from S rats on a 1% NaCI diet
and 53.5±9.1 in BBMVs from R rats. This difference is
significant (P<.04) and similar to that shown in Fig 1.
V^ was 34.7±6.2 in BBMVs from S rats on an 8% NaCI
diet and 38.7±3.4 in BBMVs from R rats on an 8%
NaCI diet. These values are not significantly different.
494
Hypertension
Vol 24, No 4
October 1994
TABLE 2. Protein Determinations, Percent Recovery, and Enrichment of Leucine Aminopeptidase Activity In BBMV
Preparations From Salt-Resistant and Salt-Sensitive Rats on 1 % or 8% NaCI Diets
1%
8%
R (n=8)
S (n=8)
P (R vs S)
R (n=4)
S (n=4)
P (R vs S)
4.54±0.62
4.39±0.65
m
5.48±0.51
5.69±0.67
NS
28.14±1.83
27.00+1.37
NS
27.74±1.41
30.42+3.40
NS
66.5±8.3
68.5±6.7
NS
79.1 ±9.7
66.6±7.0
NS
.03
0.068±0.012
0.101+0.007
NS
NS
24.2±3.4
22.4±2.4
NS
Protein, mg/mL
Homogenate
BBMV
BBMV LAP activity
Recovery, %
Specific activity,
(^imol/mg protein)/min
Enrichment factor
0.076±0.008
21.9±2.9
0.101 ±0.005
21.8+2.4
BBMV indicates brush border membrane vesicle; R, salt-resistant rats; S, salt-sensitive rats; and LAP, leucine aminopeptidase. Values
are mean±SEM.
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Two-by-two ANOVA shows a main effect for strain
(F=6.58, P<.02) and a trend toward a main effect for
diet (F=3.56, P<.079).
Fig 3 shows Km data obtained in the same six groups
presented in Fig 2. The Km (millimoles per liter sodium
[±SEM]) for sodium was 12.0 ±2.1 in BBMVs from S
rats and 19.7±3.1 in BBMVs from R rats on a 1% NaCI
diet. This difference is statistically significant (P<.02)
and is also similar to the difference seen in Fig 1. The
Km for sodium was 13.7±3.4 in S rats on an 8% NaCI
diet and 11.3±2.1 in R rats. As illustrated by the
differences in the slopes of the two lines in Fig 3, there
is an interaction between diet and strain on Km (F=5.48,
P<M). This implies that in BBMVs from S rats Na-H
exchange Km reacts in a significantly different fashion to
increases in salt intake than in BBMVs from R rats.
Amiloride Inhibition Studies
Amiloride inhibition of Na-H exchange was studied in
BBMVs from both S and R rats on a 1% NaCI diet.
Two-by-two nested ANOVA demonstrated main effects
for strain and amiloride for V^ (F= 14.48, P<.005 and
F=8.92, P<.02, respectively). It also should be noted
that there was a trend toward an interaction between
strain and amiloride (F=3.58, P<.091) that did not
reach statistical significance. Figs 4 and 5 illustrate these
data, which are summarized in Table 3. Despite the
significantly decreased V^ in BBMVs from R rats of
roughly 30% (P<.02), 25 yumol/L amiloride had no
significant effect on Km, as seen with the mixed-type
inhibition previously reported with the acridine orange
assay.25-28 As in BBMVs from R rats, there was no
significant effect on Km in BBMVs from S rats. At higher
amiloride concentrations of 50 and 100 /xmol/L, the V^
in BBMVs from S rats was found to decrease 57% and
60%, respectively, whereas the Vnai in BBMVs from R
rats decreased 63% and 76%, respectively (data not
shown). Therefore, Na-H exchange in BBMVs from S
and R rats demonstrates a noncompetitive effect to
amiloride. In addition, Na-H exchange in BBMVs from
656055-
•
RRat
o
SRat
n-6
50-
45
40
3530-
Dietary NaCI, percent
FIG 1. Bar graph shows V™, and Km in salt-sensitive (S) and
salt-resistant (R) rats fed 1 % NaCI diet. Error bars denote SEM.
*P<.0005, tP<-002 vs R rats. Data represent results obtained
from 15 groups of R and S rats with four to six rats In each group.
FIG 2. Plot shows Na-H exchange Vma in paired groups of
salt-sensitive (S) and salt-resistant (R) rats fed either 1 % or 8%
NaCI diet for the week preceding brush border membrane
vesicle harvesting. Error bars denote SEM. *P<.04 vs R rats on
1% NaCI diet.
Lewis and Warnock
Renal Na-H Exchange in Hypertension
495
25
20-
15-
10-
Dietary NaCI, percent
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FIG 3. Plot shows Km for substrate sodium in groups of saltsensitive (S) and salt-resistant (R) rats presented in Fig 2. Error
bars denote SEM. *P<.02 vs R rats on 1 % NaCI diet.
S rats is less sensitive to amiloride than Na-H exchange
in BBMVs from R rats.
Discussion
In the present study we have demonstrated a difference in renal apical membrane Na-H exchange V^ and
Km between S and R rats on a 1% NaCI diet (Fig 1).
Contrary to predictions made by the hypothesis that
enhanced apical membrane Na-H exchanger activity in
the kidney plays a central role in the pathogenesis of
hypertension, our results show that maximal Na-H
exchange rates are lower in BBMVs from hypertensive
8
a.
I
Amiloride, \imoV\
FIG 4. Bar graph shows amiloride inhibition of Na-H exchange
Vmtx in groups of salt-sensitive (S) and salt-resistant (R) rats on
1% NaCI diet. Error bars denote SEM. *P<.02 vs R rat V,™
without amiloride.
Amllonde, nmol/1
FIG 5. Bar graph shows Na-H exchange apparent Km in groups
of salt-sensitive (S) and salt-resistant (R) rats presented in Fig 4.
Error bars denote SEM.
S rats than in those from R rats. The Na-H exchange Km
for sodium is also lower in BBMVs from S rats than in
those from R rats, reflecting a higher affinity for sodium
in the hypertensive strain. However, it seems unlikely
that this difference in affinity for substrate should have
any major physiological effect, as the proximal tubule
luminal concentration of sodium (approximately 140
mmol/L) is severalfold greater than these Km values.
Thus, the proximal tubule Na-H exchanger is saturated
with respect to sodium in both S and R rats, and V^
governs the rate of apical Na-H exchange.
The relationship of these findings to sodium balance
or hypertension is unclear for a number of reasons.
Although glomerular filtration rate (GFR) has been
shown not to differ between 5- to 7-week-old S and R
rats on a low salt diet,31-32 differences in GFR and thus
the filtered load of sodium presented to the proximal
tubule certainly exist between S and R rats by 16 to 20
weeks of age, with significantly decreased GFR in S
rats.33 In addition, differences between S and R rats
have been described in distal nephron sodium reabsorption. Prehypertensive and hypertensive S rats have been
shown to have increased reabsorption of chloride in the
loop of Henle as well as alterations in more distal
nephron segments in sodium and chloride handling.33-34
Thus, it is difficult to predict the effect that differences
in proximal Na-H exchange will have on either the net
renal handling of a salt load or the blood pressure
response to a high salt diet.
Even in the absence of distal nephron adjustments to
sodium reabsorption, the present association between
suppressed proximal tubule Na-H exchange rates in S
rats and hypertension cannot be taken to infer a causal
relationship between hypertension and suppressed
Na-H exchange. The developing hypertension in S rats
could just as likely be responsible for suppressing Na-H
exchange as the converse. Alternatively, hypertension
and suppressed BBMV Na-H exchange might not be
related at all but simply occur together in S rats because
496
Hypertension
TABLE 3.
Vol 24, No 4 October 1994
Inhibition wtth 25 /unol/L Amlloride in Salt-Resistant and Salt-Sensitive Rats on 1 % NaCI Diet
R
S
Amlloride, /unol/L
Amlloride,,nmol/L
0 (n=4)
25 (n=4)
P
(0 vs 25)
I'm,, (FU/s)/mg protein
47.0±9.6
32.5±6.9
.01
31.0±5.7
27.8±6.2
NS
Apparent Km, mmol/L Na
19.8±4.3
22.0±4.8
NS
12.3 + 1.3
12.8±3.6
NS
0 (n=4)
25 (n=4)
P
(0 vs 25)
R indicates salt-resistant rats; S, salt-sensitive rats; and FU, fluorescence units. Results were obtained from EadieHofstee transformations and are expressed as mean±SEM.
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of a random chance event occurring during the initial
selection and inbreeding of this line.35 The elimination
of this possibility requires the demonstration of a statistically significant association between Na-H exchange
rates and hypertension in a population with random
segregation of genetic material, such as the progeny of
an F 2 cross between S and R rats. Nonetheless, the
question of causality would exist unless an association
could be found between a polymorphism in the actual
gene encoding the renal apical membrane Na-H exchanger responsible for the observed kinetic differences
and hypertension.36 A cosegregation between a polymorphism in the renal apical Na-H exchanger gene and
a measurable kinetic parameter of Na-H exchange or
hypertension itself would suggest a causal relationship
between this genetic polymorphism and the phenotypic
trait.
Although differences in the absolute number of functioning Na-H exchange proteins present in the apical
membrane as well as the turnover rate of each exchanger unit can account for differences in V^, the
observed Km disparity implies either important differences in the regulation of apical Na-H exchange or
structural differences influencing the substrate binding
site or sites. This latter possibility implies the existence
of polymorphisms in the coding sequence of the renal
apical membrane Na-H exchanger gene between S and
R rats. As discussed below, the different responses to
high salt diet as well as the differences in amiloride
inhibition between the two strains are consistent with
this possibility. Determination of whether alterations in
amino acid sequence are responsible for the functional
differences observed between S and R rat renal Na-H
exchange will have to await gene sequence information
for the apical Na-H exchangers in S and R rats, the
discovery of polymorphisms between them, and the
measurement of Na-H exchange kinetics in individuals
of an F2 population so the relationship between these
polymorphisms and Na-H exchange can be determined.
As interesting as the baseline Na-H exchange rate
differences between S and R rats is the response to a
high salt diet. We observed a downregulation of BBMV
Na-H exchange rate with a high salt diet in R rats, which
was not seen in S rats (Fig 2). Decreases in apical
membrane Na-H exchange rate in response to a high
salt diet would decrease proximal sodium reabsorption,
resulting in augmented distal nephron sodium delivery
and thus allowing R rats to maintain sodium balance
without necessarily a change in GFR. S rats, on the
other hand, would need to increase the filtered load of
sodium to maintain the same distal sodium delivery,
because there appears to be no change in proximal
Na-H exchange rate in response to a high salt diet. An
inability of S rats to adequately modulate GFR when
faced with a high salt intake could lead to positive
sodium balance and an increase in blood pressure.
Indeed, data suggest that young S rats increase their
GFR less than R rats do when placed on an 8% NaCI
diet.37 Although these data were obtained in younger
rats than in the present studies, this may offer some
insight into the pathogenesis of the accelerated hypertension seen in S rats on a high salt diet. However,
rather than apical Na-H exchange activity, this observation implies that a variety of other physiological
factors controlling renal blood flow and filtration rate
are involved in the development of salt-sensitive hypertension in this model. Still, the failure of S rats to
decrease proximal tubule Na-H exchange activity on an
8% NaCI diet could be interpreted as a maladaptive
response that would contribute to renal salt retention
and potentiate the hypertensive response to a high salt
diet.
On the other hand, different responses to an 8% diet
in Na-H exchange between S and R rats could be caused
by differences in intracellular regulatory mechanisms.
Mechanisms controlling Na-H exchange activity directly
or indirectly, for instance, through the manipulation of
intracellular pH could account for the observed responses. Differences in rat lymphocyte Na-H exchange
have been attributed to changes in intracellular pH
(pHj).3*-39 LaPointe and Batlle40 have demonstrated
that S rats have a significantly lower pH, and higher net
acid excretion rate than R rats on a high salt diet.
Although this raises several intriguing questions regarding differences between S and R cellular metabolism, it
is unlikely that such a finding would explain the observed differences between S and R rat BBMV Na-H
exchange because we used a fixed pH gradient in the
present studies. In addition, since a variety of hormonal
signals are known to be involved in the control of Na-H
exchange activity, alterations in the elaboration of or
sensitivity to the effects of hormones such as angiotensin II could alter Na-H exchange kinetics in response to
a high salt diet. Angiotensin II has been shown to have
more than one effect on Na-H exchange in a single cell
type.41 Its effects on Na-H exchange in opossum kidney
cells are either stimulatory, as mediated via the angiotensin type 2 receptor(s), or inhibitory, as mediated
through the angiotensin type 1 receptor and protein
kinase C activation. A difference between the relative
vigor of these effects could explain the observed differences between S and R rats in response to a high salt
diet.
Lewis and Warnock Renal Na-H Exchange in Hypertension
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Another aspect of the difference between S and R rat
BBMV Na-H exchange is inhibition by amiloride. As
Table 3 shows, 25 /imol/L amiloride inhibits 30% of
Na-H exchange rate in BBMVs from R rats, whereas
inhibition could be demonstrated in BBMVs from S rats
only with higher concentrations. Km was not altered
significantly by 25 ^unol/L amiloride in either strain.
Renal apical Na-H exchange has shown a pattern of
mixed-type inhibition with amiloride in rabbit BBMVs
when measured with the acridine orange fluorescence
quenching method and anions other than chloride in the
external buffer.25-28 A possible explanation for the presence of a noncompetitive effect with 25 /xmol/L
amiloride in BBMVs from R rats but not in BBMVs
from S rats is that differences exist between the
amiloride binding sites of S and R rat renal apical
membrane Na-H exchangers. A second possibility involves the differential sensitivities to amiloride characteristically ascribed to the different Na-H exchanger
isoforms. Chambrey et al42 have recently expressed
individual isoforms of the Na-H exchanger in mouse
fibroblasts deficient in Na-H exchange. The IQo for
amiloride differed greatly between cells transfected with
NHE-1 (6 fimolfL) and NHE-3 (450 jtmol/L) cDNAs.
The inhibition pattern with amiloride seen in BBMVs
from R rats could be explained by the presence of an
isoform that is relatively sensitive to amiloride, such as
NHE-1 or -2.43 On the other hand, the inhibition
pattern seen in BBMVs from S rats could result from a
relative increase in expression of an isoform such as
NHE-3 on the apical membrane of proximal tubule
cells. The present study did not incorporate a measure
of basolateral contamination of the BBMV preparations, and therefore another possible explanation is that
the degree of basolateral contamination differed between BBMVs from S and R rats. Although possible,
this seems unlikely given the parallel manner in which
BBMVs from S and R rats were prepared. More work is
needed to further our understanding of the mechanisms
behind the observed differences in BBMV Na-H exchange but will require an experimental approach to
separate the individual contributions of each NHE
isoform.
Other researchers have studied Na-H exchange in
Dahl rats in a variety of nonrenal tissue. LaPointe and
Batlle40 have shown that Na-H exchange in isolated
vascular smooth muscle cells from hypertensive S rats is
actually reduced when compared with that of cells from
R rats. These findings show that suppressed Na-H
exchange in S rats is present in another cell type besides
renal cortical epithelia, providing evidence of a generalized epithelial cell transport defect involving the Na-H
exchanger. Na-H exchange has also been studied in
erythrocytes from Dahl/Rapp S and R rats by Pontremoli et al.44 Both blood pressure and red blood cell
Na-H exchange were found to be higher in S than R rats
on a high (8%) salt intake. No differences in Na-H
exchange were found between S and R rats on a low
(0.02%) salt diet.44 Despite the apparent discrepancy
between these observations and our data, one cannot
compare studies measuring Na-H exchange in nonpolarized and polarized tissues because of the existence of
different isoforms of the Na-H exchange protein and the
differential expression of these various isoforms in
different tissue types.1517-43'46 Nevertheless, it is inter-
497
esting to note that in these studies erythrocyte Na-H
exchange was modulated by dietary manipulation only
in S rats, whereas in our studies BBMV Na-H exchange
was modulated only in R rats. This supports the possibility of altered differential tissue expression of NHE
isoforms between S and R rats rather than a single
generalized defect in cellular regulation in the S strain.
In conclusion, we have shown that there are differences in apical membrane Na-H exchange in the renal
cortices of the two inbred Dahl/Rapp rat strains. These
differences probably do not explain or cause hypertension in S rats or prevent it in R rats; however, the
inability of S rats to decrease their Na-H exchange rate
in response to a high salt diet may exacerbate the
development of salt-sensitive hypertension. The differences observed in the response to sodium and the
inhibitory responses to amiloride and an 8% NaCl diet
raise the possibility that more than one isoform of the
Na-H exchanger are expressed in renal BBMVs from
Dahl/Rapp rats.
Acknowledgments
These studies were supported by the VA Research Service and
grants 7RO1 DK-19407-14 and 5P50 DK-39258-04 from the
National Institutes of Health (NIH), Bethesda, Md. James Lewis
was supported by NIH training grant 5T32 DK-07545-03.
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J L Lewis and D G Warnock
Hypertension. 1994;24:491-498
doi: 10.1161/01.HYP.24.4.491
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