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(Hypertension. 1997;30:120-127.)
© 1997 American Heart Association, Inc.
Articles |
Reversal of Microvascular Rarefaction and Reduced Renal Mass Hypertension
From the Department of Physiology, Medical College of Wisconsin, Milwaukee.
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Abstract |
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Abstract This study examined the microcirculatory and renin-angiotensin system changes following the reversal of hypertension in reduced renal mass rats. Nine-week-old Sprague-Dawley reduced renal mass rats were placed on a low or high sodium diet for 4 or 8 weeks or a combination of 4 weeks of high sodium followed by 4 weeks of low sodium. Blood pressure was directly measured during the development of hypertension and its reversal. Plasma renin activity, angiotensin-converting enzyme activity, and angiotensin II concentrations were measured throughout the experiment. The cremaster and hindlimb muscles were removed, and microvascular density was determined by quantitative stereology. Four weeks of high sodium increased blood pressure (152±7 mm Hg) and reduced microvessel density (13.7%). Reduced renal mass hypertension was rapidly reversed after the rats were returned to a low sodium diet (124±7 mm Hg after 3 days), and microvascular density returned to control levels. After 4 weeks of high sodium, circulating plasma renin activity and angiotensin II fell by 94% and 82%, respectively. Plasma angiotensin-converting enzyme activity was increased after 2 weeks of high sodium but returned to control levels after 4 weeks of high sodium. This study demonstrates that microvascular density is reduced in reduced renal mass hypertensive rats following exposure to high sodium diet and this is associated with a fall in circulating plasma renin activity and angiotensin II levels. Microvascular density can return to normal levels after a reactivation of the circulating renin-angiotensin system. This study provides further evidence for the hypothesis that modulation of the renin-angiotensin system is important in the regulation of microvascular structure.
Key Words: hypertension, renal • renin-angiotensin system • sodium • angiotensin-converting enzyme
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Introduction |
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The development of hypertension has been shown to be accompanied by a rarefaction, or a reduction in microvascular density, of small arterioles and capillaries in both animal models1 2 3 4 5 and human studies.6 7 Although many studies have examined the effects of antihypertensive therapy on cardiac hypertrophy, coronary reserve, and large-vessel structure,8 the question of whether the changes in microvascular structure induced by hypertension can be restored by normalization of arterial pressure has yet to be answered.
In several different animal models of hypertension, the RAS is suppressed, resulting in low circulating levels of PRA and Ang II. In these models, a consistent reduction of microvessel density is found, implicating RAS suppression as a component of microvascular rarefaction. Several studies from our laboratory have also implicated the RAS in the regulation of microvascular structure. Munzenmaier and Greene9 have shown that subpressor Ang II infusion can initiate angiogenesis. Furthermore, we have shown that normotensive animals fed an HS diet and with suppressed PRA exhibit a reduction in microvascular density, which can be blocked by Ang II infusion.10 11 Other researchers have also demonstrated that blockade of the RAS by captopril administration can inhibit both large- and small-vessel growth.12
Numerous experimental models of renal hypertension (eg, one-kidney, one clip; two-kidney, one clip; aortic coarctation) have been used to study the effects of hypertension on the cardiovascular system. In these experimental models, hypertension is induced by reducing renal perfusion pressure and develops in order to normalize sodium and water balance. In animals with one-kidney, one clip hypertension, arterial pressure can be rapidly returned to control levels by removal of the clip.13 14 15 16 Renal hypertension can also be produced surgically by partial nephrectomy, which reduces the ability of the kidney to excrete sodium and water.17 Previously, we have shown that microvascular rarefaction occurs in the RRM hypertensive rat model18 19 20 ; however, the reversibility of hypertension in the RRM salt-sensitive model has not been well characterized.
The purpose of the present study was to examine the reversibility of RRM hypertension and test the hypothesis that normalization of blood pressure by return to an LS diet would reverse microvascular rarefaction. The results from this study, when interpreted in light of our previous work, suggest that reversal of RRM hypertension, by return to a normal sodium intake, results in activation of the RAS and leads to a restoration of microvascular density.
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Methods |
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Surgical Preparation and Blood Pressure Measurement
Male Sprague-Dawley rats were purchased from SASCO (Madison, Wis) and housed in an animal care facility approved by the American Association for Accreditation of Laboratory Animal Care. Animals were fed standard rat chow containing either 4% NaCl (HS) or 0.4% NaCl (LS) (Dyets, AIN 76-A). Before preparation for renal mass reduction, all animals were placed on an LS diet. The rats were anesthetized with a ketamine (50 mg/kg IM) and acepromazine (5 mg/kg IM) mixture and subjected to a 75% reduction in renal mass by a two-step surgical procedure as previously described.20 The rats were 6 weeks old and weighed approximately 180 to 190 g at the time of initial surgery. After a 3- to 5-day recovery period, the animals were reanesthetized, and a polyvinyl catheter was implanted in the femoral artery under aseptic conditions. Catheters were tunneled subcutaneously and exteriorized at the back of the neck. A flexible spring was implanted to protect the catheter and was connected to a swivel placed above the rat's cage to allow unrestricted movement. The rats were given 5 days to recover from surgery before arterial pressure was measured. Arterial pressure was measured in conscious rats by connecting the arterial catheter to a pressure transducer. The output of the transducer was low pass–filtered (30 Hz), digitized at 100 Hz, and analyzed by software of our own design for computation of systolic, diastolic, and mean pressures and heart rate. Pressure measurements were taken at the same time of day in rats resting in their home cages and represent the average of a 1-hour recording period. All animals were allowed to drink water ad libitum. All surgical and experimental methods were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.
Experimental Groups
Eight groups of animals were studied in these experiments. Blood
pressure was measured in two groups: group 1 (4-week HS) was placed on
an HS diet for 4 weeks, and group 2 (8-week HS/LS) was placed on an HS
diet for 4 weeks and then returned to an LS diet for 4 weeks. The
development of the hypertension was studied in the 4-week HS group, and
the reversibility of the hypertension was studied in the 8-week HS/LS
group. In all groups, blood pressure was measured during 2 control
days, for 3 days after the diet was changed, and weekly thereafter for
4 weeks.
A second set of animals with the same dietary regimens as those for blood pressure measurements (4-week HS and 8-week HS/LS) were used to characterize changes in PRA, ACE activity, and Ang II concentration during the development and reversal of hypertension. These animals were instrumented with arterial catheters for blood sampling. All blood samples were drawn through the exteriorized arterial lines with the rats resting in their home cages. Samples were taken in the 4-week HS group during the control period, 2 and 4 weeks after the rats were switched to the HS diet. In the 8-week HS/LS group, samples were collected after 4 weeks of the HS diet and 2 and 4 weeks after the rats were returned to an LS diet.
Animals used for the determination of salt intake on microvascular density were not instrumented for blood pressure measurement to eliminate any potential effects of catheterization. These RRM animals were placed on an LS diet and were given 7 days to recover fully from surgery before beginning their specific dietary regimen. Rats were divided into the following experimental groups: control LS, 4-week LS, 4-week HS, 8-week LS, 8-week HS, and 4-week HS followed by 4-week LS (8-week HS/LS). At the end of each dietary regimen, the rats were euthanized with a sodium pentobarbital overdose, and samples from cremaster, lateral and medial gastrocnemius, plantaris, and soleus muscles were taken for study of microvascular density.
Determination of Microvascular Density
Hindlimb muscle samples were rinsed in
physiological salt solution, sectioned (100
µm thickness), and immersed in 30 µg/mL (263 nmol/L)
rhodamine-labeled Griffonia simplicifolia I lectin
(Sigma Chemical Co) for 40 minutes to define the microvascular bed.
Cremaster muscles were processed identically as whole mounts
without sectioning. After exposure of the tissue to lectin, the muscle
was rinsed thoroughly in physiological salt
solution three times for 15 minutes, 30 minutes, and 12 hours. Samples
were then mounted on a microscope slide with a water-soluble mounting
medium (SP ACCU-MOUNT 280, Baxter Scientific). Each section was studied
with a video (Cohu, 5000 Series Television Camera) fluorescent
microscope system (Olympus ULWD CD Plan) with epi-illumination and
examined at x300 so representative fields of view
could be found. Images were digitized and quantified with an automated
computer vessel-counting method previously described.21
Approximately 20 images were randomly identified from each section, and
the number of intersections of vessels with a fixed grid overlay was
averaged to give an estimate of microvascular density in each
muscle.
For each animal the mean, SD, and CV (CV=SD/Mean) of the microvascular density estimate were calculated. In each group, the CV values were averaged. The CV from each group was compared for determination of whether the spatial distribution of microvessels changed between groups. An increase in the CV would reflect an increase in the heterogeneity of the microvascular structure, as previously described for flow.18
Measurement of Circulating RAS Components
Plasma Renin Activity
The method used for measurement of PRA is a modification of the
assay developed by Sealey and Laragh,22 who kindly
provided us with the Ang I antibody. Whole blood was collected from the
femoral artery catheter in chilled, sterile tubes containing
K3EDTA and immediately centrifuged (Sorvall 3B) at
1500g and 4°C. The plasma was separated and stored frozen
at -35°C until assayed. The samples were slowly thawed on ice, and
neomycin sulfate (0.1%), phenylmethylsulfonyl fluoride
(0.25%), and maleic anhydride (0.2 mol/L) were added to 50 µL
of sample to inhibit converting enzyme and protease activity.
This mixture was incubated at 37°C in a water bath for 3 hours
and then rapidly frozen to stop Ang I generation. After generation, the
Ang I concentration was determined in duplicate by radioimmunoassay.
Ang I standard (Peninsula Laboratories) was run in triplicate at eight
concentrations ranging from 10 to 800 pg per tube. Aliquots (20 µL)
of the incubation mixture or Ang I were added to 0.1 mol/L Tris buffer
(pH 7.5; with lysozyme, neomycin, and bovine serum albumin)
containing both the Ang I antibody and 5000 cpm 125I–Ang I
(Amersham Corp). The tubes were incubated overnight (18 hours) at
4°C. Dextran-coated charcoal was then added to separate bound from
free Ang I, and the supernatant was counted (model 1185 gamma counter,
Tracor). Typical binding with no standard added (B0)
averaged 49.8±2.2% (n=15 assays, CV=4.5), and Ang I concentration at
50% of the B0 (B50) averaged 96.4±6.5 pg
(n=15, CV=6.7%). The value of a rat plasma control pool averaged
3.22±0.34 ng Ang I/mL per hour, with an interassay variation of 10%
(n=15). All results are reported as nanograms Ang I per milliliter per
hour.
Plasma ACE Activity
At each of the time points, 200 µL blood was drawn from the
femoral artery catheter and placed on ice. Samples were immediately
centrifuged at 3800g and 4°C for 15
minutes;plasma was removed and stored at -35°C until
assayed. Plasma ACE activity was measured with a modified fluorometric
technique by incubating 10 µL of a 1:10 dilution of plasma at 37°C
for 60 minutes according to the method described by Santos et
al.23 The amount of His-Leu product formed was
measured by adding 50 µL (20 mg/mL; 74.5 mmol/L) of
o-phthaldialdehyde reagent (Sigma). The fluorescence
of the samples was read with 365 nm excitation and 485 nm emission
using a scanning fluorometer (F-2000 fluorescence
spectrophotometer, Hitachi). ACE activity was expressed as milliunits
per milliliter, where 1 U=1x10-6 mol His-Leu
product produced per minute at 37°C.
Plasma Ang II Concentration
Arterial blood samples (1 mL) were drawn into
chilled tubes containing 50 µL/mL of 0.125 mol/L Na2EDTA,
0.025 mol/L phenanthroline, and 0.5 mmol/L neomycin sulfate.
Samples were immediately centrifuged, and plasma was separated
and frozen at -70°C until extracted. Angiotensins were
first extracted from plasma by loading the sample on a C18 Sep-Pak
column (Waters). The column was rinsed with 3 mL water, and the peptide
fragments were eluted with 10 mL methanol. The sample was then dried,
reconstituted in 150 µL of 0.1 mol/L CH3COOH, and passed
through a Millex LCR13 filter unit. Ang II was separated from Ang I,
Ang III, and Ang II metabolites by reversed-phase
high-performance liquid chromatography (HPLC)
using a µBondapack C18 10-µm column (300x2 mm) (Waters) and a
column heater (45°C) and was eluted at a flow rate of 0.5 mL/min with
a mobile phase consisting of 66.8% of 0.85% phosphoric acid and
33.5% methanol. Retention times of Ang I, Ang II, and the primary
angiotensin metabolites Ang II-(1-7), Ang II-(2-8), Ang
II-(3-8), and Ang II-(2-10) were determined by injecting mixtures
containing 1 to 5 ng of each peptide and monitoring with a UV detector
at 210 nm. All peptides were obtained from Peninsula Laboratories. A
typical chromatogram using this system is presented in Fig 1
. The retention time of Ang II was 6.0 minutes, and it
was well separated from Ang I and Ang II-(1-7). Ang II was also well
separated by at least 1 minute from the other Ang II peptides, with the
closest retention times including Ang II-(2-8) (retention time, 4.5
minutes), Ang II-(4-8) (retention time, 4.3 minutes), and Ang I-(2-10)
(retention time, 7.7 minutes). Therefore, we chose to assay samples
with retention times of 1 minute on either side of the Ang II peak (5
to 7 minutes) as the Ang II peak. At the beginning of each day, the
retention time of the Ang II peak was established by injecting 3000 cpm
of [3H]Ang II standard (35 Ci/[mmol/L]) and monitoring
the elution profile with a radioactive flow detector (Flowone A120,
Radiomatic Instruments). The retention time was verified at the end of
the day after analysis of the samples, and any group of samples
in which the Ang II retention time shifted by more than 0.5 minute from
the beginning to the end of the run was chromatographed again.
The entire eluate volume from this 2-minute window was collected. Two
preinjection blanks were collected before injection of each unknown.
All HPLC fractions were lyophilized and stored at -35°C until
assayed (within 5 days). Recovery of [3H]Ang II from
plasma processed through the entire extraction and HPLC separation
procedure averaged 83±1.7% (n=6, CV=4.9%). After HPLC separation,
Ang II concentration in the 5- to 7-minute Ang II window was determined
by radioimmunoassay using a polyclonal antibody (kindly provided by Dr
Charles Wood) raised in rabbits that recognizes the carboxy-terminal
end of Ang II. The antibody exhibits no cross-reactivity with Ang
II-(1-7), Ang I, or Ang I-(2-10); between 0.1 and 100 pg, however, it
does fully cross react with Ang II-(2-8), Ang II-(3-8), and Ang
II-(4-8).
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For radioimmunoassay, samples were reconstituted in 100 µL of assay
buffer (0.1 mol/L Tris buffer containing 0.3% bovine serum
albumin, 0.3% lysozyme, 0.2% neomycin sulfate, and 0.003%
phenylmercuric acetate) and Ang II antibody and incubated overnight at
4°C. The standard curve range was 0.34 to 86.5 pg. Labeled Ang II
(7000 cpm, 125I–Ang II; Amersham) was then added, and the
incubation continued overnight at 4°C. After incubation, bound
125I–Ang II was separated from free 125I–Ang
II with dextran-coated charcoal, and the supernatant was counted. A
typical standard curve is presented in the Fig 1
inset.
B0 averaged 51.5±1.0% (n=13 assays), and B50
averaged 12.8±0.22 pg (n=13 assays, CV=6.2%). Intra-assay variability
of plasma pools taken through the entire extraction separation and
assay procedure averaged less than 8%.
Statistical Analysis
Measurements of blood pressure, microvascular density, PRA, ACE
activity, Ang II levels, and CV values of microvascular density are
expressed as mean±SEM and were analyzed by a one-way ANOVA.
Significant differences between groups were determined with Dunnett's
post hoc test. Values of P<.05 were considered
significant.
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Results |
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The time course of changes in arterial pressure after exposure of RRM rats to an HS diet is presented in Fig 2A
. Baseline arterial pressure averaged
112±2.0 mm Hg during the control period; pressure rose
significantly to 131±7.0 mm Hg after 2 weeks of HS diet. After 4
weeks of HS diet, mean arterial pressure averaged
152±7.0 mm Hg. In rats placed on an HS diet for 4 weeks and
then switched to an LS diet (Fig 2B), control arterial
pressure (151±7.0 mm Hg) was similar to that seen in the first
group of rats. After replacement of the HS diet with LS diet, blood
pressure fell rapidly and was significantly reduced after only 1 day of
the LS diet (135±5.0 mm Hg). After 4 weeks of LS, it reached an
average steady-state pressure of 126±5.6 mm Hg.
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Changes in microvascular density in the cremaster and hindlimb muscles
over the 4 weeks of the development of hypertension and its reversal
are summarized in Fig 3. Rats fed an HS diet for 4 or 8
weeks experienced a significant reduction in microvascular density
(13.7% and 14.9%) in the cremaster compared with control LS animals
(Fig 3A). Microvessel density in RRM rats fed an HS diet was
significantly reduced from that in rats fed an LS diet for 4 weeks.
Animals switched to an LS diet after 4 weeks of HS diet showed a
complete reversal of the rarefaction in the cremaster muscle back to
control levels. Animals maintained on an LS diet throughout showed a
progressive reduction in microvascular density, exhibiting
significant rarefaction in the cremaster (17.3%) after 8 weeks
compared with LS control animals (Fig 3A).
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P<.05, significantly different from 4-week LS;
P<.05, significantly different from 8-week HS. Data are
mean ±SEM.
In the hindlimb, microvascular density in each experimental group was
compared among muscle types (medial and lateral gastrocnemius, soleus,
and plantaris) with a one-way ANOVA. No significant differences were
found between each of the muscle densities; therefore, they were
averaged for an estimate of whole hindlimb density. Similar to the
cremaster muscle, 4 weeks of HS diet accompanied by hypertension
produced a significant reduction (10.4%) in hindlimb density compared
with LS controls (Fig 3B). Eight weeks of HS diet resulted in a 12.8%
reduction in microvascular density (Fig 3B). In addition, animals
maintained on an LS diet for 8 weeks also exhibited a significant
time-dependent reduction (11.0%) in hindlimb density compared with LS
controls (Fig 3B). As seen in the cremaster muscle, a complete reversal
of the rarefaction to control levels was accomplished by a dietary
change to LS at 4 weeks (Fig 3B).
For each animal in every group, the average SD, average CV, and mean of
the microvascular density estimate were calculated (Table 1). Animals that exhibited a significant reduction in
microvascular density compared with control LS animals showed a
consistently higher CV than animals with nonrarefied
microcirculation. The average CV for a rarefied group of animals
(4-week HS, 8-week LS, and 8-week HS) was 23.7% higher than that of
the control LS group, whereas the nonrarefied groups (4-week LS, 8-week
HS/LS) were only 7.8% greater than control. Regression
analysis showed a significant inverse correlation
(r2=.78) between microvascular density and
CV.
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Circulating levels of RAS components were measured while the animals
were on both HS and LS diets and are summarized in Fig 4. Control levels of PRA and Ang II in rats on an LS
diet were 9.1±1.5 ng Ang I/mL per hour and 18.8±4.1 pg/mL,
respectively. Increasing dietary sodium intake for 2 weeks suppressed
PRA by 85% to 1.3±0.6 ng Ang I/mL per hour, and plasma Ang II
concentration fell by 79% to 3.9±0.9 pg/mL. An additional 2 weeks of
HS diet caused no further suppression of either PRA or Ang II. Animals
previously placed on an HS diet for 4 weeks and switched back to an LS
diet exhibited a significant increase in PRA and Ang II back toward
control LS levels. Plasma ACE activity was significantly
increased (from 182±14 mU/mL during control to 309±46 after 2 weeks)
during an HS diet, but it returned to control levels after 4 weeks of
HS. Switching rats back from an HS to LS diet had no significant effect
on plasma ACE activity.
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Discussion |
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The primary goal of this study was to determine the potential reversibility of structural changes in the microcirculation after reversal of RRM hypertension. We hypothesized that in the RRM rat, microvascular rarefaction would be reversed after a reduction in arterial blood pressure and reactivation of the circulating RAS. Previously, we reported that microvascular density is reduced by 22.8% and 20% after 4 weeks of hypertension in RRM rats11 and by 15% after 5 to 6 weeks of RRM hypertension.20 In the present study, microvascular density was significantly lowered 4 weeks after the induction of renal hypertension in rats on an HS diet and remained reduced after 8 weeks of this diet. After a 4-week HS diet, hypertensive animals were switched to an LS diet for 4 weeks. As we hypothesized, this change in dietary sodium reduced blood pressure, reactivated the RAS, and reversed the microvascular rarefaction that occurred during hypertension. This study is the first to demonstrate the reversibility of microvascular rarefaction after the reversal of hypertension.
The RRM rat provides an excellent model for study of the microvascular consequences of hypertension reversal for several reasons. First, as demonstrated in this and other studies that RRM hypertension in the rat can be reversed without pharmacological intervention simply by normalizing sodium intake. Second, reversibility is very rapid, with blood pressure falling by 16 mm Hg on the first day of return to an LS diet. Third, RRM hypertension is associated with substantial microvascular rarefaction.
Our current study shows a correlation between blood pressure and microvascular density, whereas others have shown that rarefaction may also occur by pressure-independent mechanisms.24 In the RRM rat returned to an LS diet (8-week HS/LS), microvascular density was normalized but arterial pressure remained slightly elevated (15 mm Hg) compared with control. This suggests that in our studies, other mechanisms besides changes in arterial pressure may also be responsible for the modulation of microvascular structure. Despite this, some evidence suggests that hemodynamic factors such as pressure may effect changes in microvascular density. For example, Hogan and Hirschmann25 showed that a 30% reduction in local perfusion pressure led to proliferation of second- and third-order arterioles without a change in flow.
A role for the RAS has also been hypothesized in the regulation of microvascular structure. Animal models of hypertension with low renin levels (eg, RRM; one-kidney, one clip; spontaneously hypertensive rat) all have a consistent reduction in microvessel density4 11 12 20 ; however, in the two-kidney, one clip hypertensive animal with high plasma renin levels, microvascular rarefaction is not present after 4 weeks of hypertension.26 In addition, infusion of pressor doses of Ang II into otherwise normal animals leads to hypertension but does not cause rarefaction.9 Therefore, in a variety of models, the effects of hypertension on microvascular remodeling can be overcome by maintenance of plasma Ang II levels. In normotensive animals placed on an HS diet, we have observed rarefaction at levels equal to that seen in low-renin forms of hypertension. In these animals, Ang II infusion at subpressor doses can block microvascular rarefaction.10 Subpressor Ang II infusion also has been shown to increase microvascular density and can potentiate angiogenesis by selective blockade of the angiotensin type 2 receptor.9 Finally, blockade of the RAS by ACE results in microvascular rarefaction in normotensive rats.27 Taken together, these studies suggest that circulating levels of plasma RAS components can determine microvascular density levels, which in turn may be modulated by changes in mean arterial pressure. The current study, showing a correlation of microvascular density and PRA and Ang II during suppression and reactivation of the RAS, gives further evidence and support to this hypothesis.
Even though PRA and Ang II levels become suppressed after the initiation of RRM hypertension, the RAS does seem to have a role in the development and maintenance of elevated blood pressure. Various RAS inhibitors have been used to block the initiation of hypertension or reduce established hypertension in the RRM rat. Administration of the Ang II type 1 receptor blocker losartan completely blocks the development of hypertension.28 ACE inhibitors also are effective at reducing blood pressure during RRM hypertension.29 30 Even though suppressed Ang II levels seem to contribute to microvascular degeneration, further pharmacological suppression of the RAS implicates even low circulating levels in the pathology of hypertension in this model.
The circulating RAS components are well known to be affected by both changes in dietary sodium intake and hypertension. The time course of changes in circulating PRA and Ang II levels during the development of hypertension supported our hypothesis that sodium would suppress their levels despite large reductions in renal mass. After 2 weeks of hypertension, both PRA and plasma Ang II levels were dramatically suppressed and maintained over the 4 weeks of hypertension. On reversal of hypertension, both PRA and Ang II levels were significantly increased, and PRA was not different from that in animals maintained on a continuous LS diet (8-week LS=3.9±0.8 ng Ang I/mL per hour). These results show the tight correlation between changes in PRA and Ang II levels during both hypertension and its reversal in RRM rats.
Although the present study clearly demonstrates the reversibility
of microvascular rarefaction after hypertension reversal, it does not
directly separate several potential mechanisms for this restoration of
microvessel architecture. Table 2 summarizes the current
data and that from three previous studies in which vessel density and
blood pressure were measured. If we consider blood pressure, sodium
intake, and plasma Ang II concentration as three potential factors that
determine vessel density, then we can use Table 2 to correlate these
potential determinants with vessel density. Shown in boldface type are
situations in which the correlation between the potential determinants
and vessel density does not hold. For example, under many conditions,
blood pressure and vessel density are inversely correlated. However, in
the case of normotensive rats on an HS diet, both blood pressure and
vessel density are low, causing us to reject the hypothesis that blood
pressure is the sole determinant of vessel density in these
experiments. Similarly, for sodium intake, in which an inverse
correlation also appears to hold, previous studies demonstrate that in
rats fed HS and infused with 5 ng/kg per minute of Ang
II,9 vessel density is high, opposite to what we would
predict on the basis of a sodium intake hypothesis. When plasma Ang II
concentration is hypothesized to be the determinant of vessel density,
there are no models in which the correlation fails, suggesting that
under each of the conditions measured, only Ang II remains a potential
determinant of vessel density.
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In addition to plasma Ang II level, we also measured plasma ACE activity in an attempt to gauge overall RAS activity. In our studies, circulating levels of ACE activity remained constant except for a significant increase after 2 weeks of HS diet. This transient increase in plasma ACE activity may be modulated by hemodynamic factors during the early development of hypertension. Previous studies of RRM hypertension have indicated that hypertension develops during two distinct phases.20 31 In the early phase, cardiac output increases and total peripheral resistance decreases, with a resultant increase in mean arterial pressure. In the later phase, cardiac output returns to normal and a chronic increase in total peripheral resistance causes the sustained increase in mean arterial pressure. It is possible that vascular ACE expression or cleavage from the endothelial membrane may be regulated by the early changes in blood flow and contribute to the transient rise in plasma ACE activity. Ang II also has been shown to act through intrinsic feedback mechanisms to maintain plasma ACE levels,32 which may cause plasma ACE activity to overshoot at this time. Feedback of elevated circulating Ang II levels has also been shown to suppress ACE gene expression in the lung,33 and a similar mechanism may be acting to increase plasma ACE activity in our study in the face of the early fall in Ang II levels (2 weeks). During the chronic phase (4 weeks) of RRM hypertension, ACE activity was normal and did not change through the remainder of the study. Normal plasma ACE levels have been observed with suppression of both PRA and Ang II levels in other species.34 Other studies suggest that ACE levels are unchanged during the induction phase of various forms of hypertension in the rat,35 whereas chronic hypertension has shown elevated levels of ACE activity.35
In the current study, animals fed an LS diet for 8 weeks also exhibited a sustained reduction in microvascular density. This finding was somewhat surprising for several reasons. In the 8-week LS group, PRA was not different from that in animals with a reversal of hypertension (8-week HS/LS). The gradual reduction in vessel density may be due to an age-dependent change in skeletal muscle structure. It has been suggested that muscle mass increases more quickly than capillary number during development, causing an apparent reduction in microvascular density.12 The age-dependent rarefaction with an LS diet (8-week LS) also suggests a possible role for sodium intake on microvascular structure. Chronic maintenance of dietary sodium levels may have a permissive effect and adversely contribute to the development of rarefaction. This rarefaction process may be due to a sensitization to high circulating Ang II or may affect the density of Ang II receptor subtypes by chronic sodium intake.36 Because these animals continuously maintained on an LS diet underwent rarefaction, we conclude that the change of the dietary sodium may be critical to the reversal of microvascular rarefaction seen in long-term hypertension (8-week HS) and may provide a dramatic stimulus for microvessel growth.
After rarefaction of the microcirculatory bed, the spatial distribution
of microvessels was changed. In the present study, vessel
distribution within the microcirculation was hypothesized to become
more heterogeneous in animals exhibiting microvascular
rarefaction. As a means of quantifying this change in the cremasteric
microvascular structure, we compared the CV values of each of the
experimental groups. As shown in Table 1, the average SD of the
microvascular density estimate was not different in any of the
experimental groups; therefore, any change in the CV would be due to a
reduction in the density estimate. However, increases in the CV in any
particular group would indicate a potentially more
heterogeneous microvasculature because of the relatively
larger distribution of the density estimate about its mean. In all
groups that underwent rarefaction, the percentage increase (23.7%) in
the CV was significantly greater than the percentage increase in the CV
of the nonrarefied groups (7.8%) compared with the nonrarefied control
LS group. A heterogeneous reduction in vessel density has
been hypothesized to contribute to abnormal oxygen delivery, leading to
areas of tissue hypoxia greater than would occur with a
homogeneous rarefaction of the
microcirculation.37
The present study also characterized the time course of changes in mean arterial pressure during the development of RRM hypertension in conscious, unrestrained rats fed an HS diet. Mean arterial pressure gradually increased and was significantly elevated after 2 weeks of an HS diet, reaching 152±7 mm Hg after 4 weeks. Previously, we measured the time course of RRM hypertension and found significant increases in mean arterial pressure approaching 150 mm Hg after only 5 days of an HS diet.11 However, the time course of hypertension development in the present study is similar to that found by Ylitalo et al38 under corresponding conditions. In the current study, the development of RRM hypertension was slower than that previously measured by our group, probably because pressure was measured in conscious animals while they were maintained in their home cages and sodium intake was elevated by raising the amount of sodium in the food. Other studies have examined the development of RRM hypertension for shorter time periods,28 with sodium intake elevated by NaCl infusion17 20 or with blood pressures measured at single time points with animals under anesthesia.20
This study is the first to demonstrate that RRM hypertension in the rat can be rapidly reversed by lowering dietary NaCl intake. One day after salt intake was reduced, pressure fell significantly by 16 mm Hg, presumably because of a negative sodium balance. Moreover, it remained approximately 25 mm Hg below the pressure seen in HS-fed rats over the 4-week course of the experiment. This rapid reversal of hypertension is very similar to that seen in two-kidney, one clip hypertensive rats after clip removal.13 14 15 16 Arterial pressure did not return all the way back to control levels in these animals; instead, it remained approximately 15 mm Hg above control over the duration of the experiment. This elevated pressure suggests that the ability of the remnant kidney to maintain sodium balance at a control level of arterial pressure was diminished, perhaps because of irreversible renal damage sustained during the 4 weeks of hypertension before the rats were returned to an LS diet. A similar induction and reversal of hypertension has been shown in RRM dogs given alternating infusions of normal tap water and 0.9% NaCl drinking water over 2-week periods; however, pressure returned to control levels after each reversal.39
In summary, we have confirmed that hypertension develops in RRM rats fed an HS diet and demonstrated that this hypertension can be rapidly reversed by changing dietary sodium back to a control LS diet. Microvascular rarefaction was observed in both the cremaster and hindlimb muscles after the development of hypertension in RRM rats, and it occurred heterogeneously throughout the muscle tissue. In addition, microvascular rarefaction could be completely reversed in hypertensive RRM rats after pressure was reduced by lowering sodium intake. These results indicate that hypertension produces transient structural changes in microcirculation that can be completely normalized by returning blood pressure to control levels. This may have important implications in the treatment of human essential hypertension with various pharmacological agents.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by National Heart, Lung, and Blood Institute grant HL-29587. The authors would like to gratefully acknowledge the technical assistance of Meredith Skelton, Lisa Henderson, Camille Torres, and Kelly Zanoni in the measurement of PRA, ACE, and Ang II levels. We would like to thank Dr Charles Wood, University of Florida, for providing us with the antibody used for the measurement of Ang II and Dr Jean Sealey for the antibody used for the measurement of Ang I.
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Footnotes |
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Reprint requests to Andrew S. Greene, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
Received February 2, 1996; first decision March 12, 1996; accepted December 17, 1996.
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References |
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