Rapid Microvessel Rarefaction With Elevated Salt Intake and Reduced Renal Mass Hypertension in Rats
To identify the sequence of events associated with the development of reduced vessel density (rarefaction) in hypertension, microvessel density and ultrastructure were assessed in the cremaster muscle of rats subjected to a 75% surgical reduction of renal mass and normotensive sham-operated control rats. Rats with reduced renal mass (RRM rats) and sham-operated rats were then maintained on either a high salt (4.0% NaCl) or a low salt (0.4% NaCl) diet for 3 days. Acute exposure to the high salt diet significantly increased mean arterial pressure in RRM rats but did not affect sham-operated control rats. Quantitative fluorescence microscopy of cremaster muscle whole mounts using rhodamine-labeled Griffonia simplicifolia I lectin revealed substantial rarefaction of microvessels in both RRM hypertensive rats and normotensive sham-operated rats on a high salt diet relative to corresponding control rats on a low salt diet. Confocal microscopy revealed a heterogeneous distribution of microvessels in RRM rats on a high salt diet, with some areas largely devoid of vessels. RRM and sham-operated rats on a high salt diet both exhibited changes in arteriolar ultrastructure, which included a loss of basement membranes and a dissociation of the endothelial and smooth muscle components of the vascular wall, resulting in a loss of vessel integrity. These observations demonstrate that a rapid loss of microvessels can occur not only in rats with RRM hypertension but also in normotensive rats on a high salt diet. This loss of microvessels results from structural alterations, which differ from the degenerative processes associated with microvascular rarefaction in rats with chronic RRM hypertension.
A number of studies have reported a reduction in the density of microvessels (rarefaction) in the established stage of many animal models of hypertension1 2 3 4 5 6 and in human hypertension.7 8 9 We have previously demonstrated structural degeneration of microvessels, ie, true anatomic rarefaction (in contrast to active closure or “functional rarefaction” of arterioles) in the cremaster muscles of rats with chronic reduced renal mass (RRM) hypertension.10
Anatomic rarefaction generally has been considered to be a form of “structural autoregulation,” reflecting the long-term adaptation of the microcirculation to the elevated blood pressure or the initial increase of blood flow in hypertension. In this respect, anatomic rarefaction would allow tissue blood flow to be regulated without consumption of the energy necessary for active vasoconstriction.1 11 12 Rarefaction of microvessels may have important consequences in hypertension, since it could contribute to the maintained elevation of total peripheral resistance11 and adversely affect oxygen delivery to the tissues.13
The time required for the onset of microvascular rarefaction in hypertension may differ substantially between vascular beds,1 2 and a number of structural and functional vascular changes have recently been shown to occur sufficiently rapidly to cause us to reassess our concept of rarefaction as a gradual long-term structural alteration in the vasculature occurring in response to a prolonged elevation in blood pressure. Moreover, previous studies by our group5 14 have demonstrated that a reduction in microvessel density also occurs in normotensive animals maintained on a high salt diet for prolonged periods of time.
The purpose of the present study was to determine whether acute (3-day) RRM hypertension and acute exposure to an elevated salt intake in normotensive rats lead to early alterations in microvessel density and ultrastructure. The results of the study demonstrate that rats with acute RRM hypertension and normotensive sham-operated control rats on a high salt diet for 3 days both exhibit a reduced microvessel density and structural degeneration of microvessels relative to sham-operated control rats or RRM rats on a low salt diet.
Materials and Methods
A total of 39 male Sprague-Dawley rats were used in the present study. One group of rats was subjected to a 75% reduction in renal mass via a two-stage surgical procedure, as previously described.6 The animals were 6 weeks old and weighed 180 to 190 g at the time of the initial surgery. Another group consisting of age-matched normotensive control rats underwent a sham operation in which the kidneys were exposed, cleared of perirenal fat, and returned to the abdominal cavity. Three to 5 days after the final reduction in renal mass, the RRM and sham-operated rats were placed on either a high salt rat chow (Dyets No. 113756, AIN-76A with 4% NaCl, Dyets, Inc) or a low salt chow (Dyets No. 113755, AIN-76A with 0.4% NaCl, Dyets, Inc) for 3 days. An additional normotensive control group for the ultrastructural studies consisted of unoperated age-matched Sprague-Dawley control rats receiving standard Purina chow (0.8% NaCl). All animals were allowed to drink water ad libitum. Sodium intake on standard Purina rat chow is ≈2 to 3 mEq per day. The high salt diet (4% NaCl) results in a sodium intake of ≈15 mEq per day, whereas the low salt diet (0.4% NaCl) results in a sodium intake of ≈1 mEq per day. On the standard rat chow, RRM rats will become hypertensive within 4 weeks. The high salt diet accelerates the development of hypertension, whereas the low salt diet prevents it.
All the procedures used in the present study conformed to institutional guidelines. All protocols were approved by the animal care committee of the Medical College of Wisconsin.
Blood Pressure, Heart Rate, and Plasma Ang II Measurements
Rats were anesthetized intraperitoneally with 40 mg/kg ketamine-HCl followed by 20 to 30 mg/kg sodium pentobarbital and placed on a heated table. A catheter was placed in the left carotid artery and connected to a pressure transducer (Statham P23AC) for the measurement of blood pressure and heart rate.
In a separate series of experiments, plasma Ang II levels were measured in conscious, chronically catheterized normotensive Sprague-Dawley rats that had been on high and low salt diets for 3 days. In those experiments, plasma Ang II levels were measured by radioimmunoassay after extraction from the plasma with a C18 column (Waters Associates) and separation from other angiotensin metabolites by high-performance liquid chromatography, as previously described.15
Immediately after measurement of blood pressure and heart rate, samples of the cremaster muscle were removed with a trephine and immersed in 30 μg/mL rhodamine–labeled Griffonia simplicifolia I (GS-I) lectin for 30 minutes to label the small (third- and fourth-order) arterioles and capillaries.5 16 17 18 After exposure of the tissue to the lectin, the muscle was rinsed thoroughly in physiological salt solution and mounted on a microscope slide, using a water-soluble mountant for the coverslip. Samples were studied as whole mounts using a video fluorescent microscope system with epi-illumination. Microvessel density was measured by counting the intersections of fluorescently labeled microvessels with a computer-generated square grid (20-μm mesh) overlying the microscope field observed at ×300. Two slides of each muscle were studied, and five fields from each slide were randomly selected and counted. The results from the 10 fields were averaged to give a single density for each muscle. The coefficient of variation of the vessel densities was computed for each rat as the ratio of the standard deviation to the mean.
For the electron microscope studies, the scrotum was opened, the spermatic cord was ligated, and the testes and surrounding cremaster muscle were removed and immersed in 2% cacodylate-buffered glutaraldehyde, in order to preserve the cremaster muscle in its most natural position. After an initial 5-minute fixation, the cremaster muscle was opened, and the testes were removed. The muscle was fixed overnight in the 2% cacodylate-buffered glutaraldehyde solution before transfer to a cacodylate buffer rinse. For subsequent sampling, segments of several second- and third-order arteriolar and venular pairs from each muscle were identified under a dissecting microscope and dissected out in a manner that improved the probability of orienting them for cross-sectional analysis. Samples were also taken from four or five sites between the visible arteriolar and venular pairs in order to obtain samples of the terminal vessels, which could not be identified under the dissecting microscope.
The samples were poststained in osmium tetroxide, embedded, and processed for light and electron microscopy as described previously.10 19 One-micron sections stained with toluidine blue were examined in 10 blocks from each cremaster. Four to 6 of these blocks were selected for subsequent ultrastructural examination.
The whole mounts of the cremaster muscle were also examined via fluorescence microscopy using both a standard microscope (Nikon Optiphot) and a Bio-Rad laser scanning confocal microscope. The objective of these studies was to identify structural changes in the microvessel network that would correlate with the quantitative measurements of microvessel density and with the ultrastructural analysis.
All results were expressed as mean±SEM. Differences between groups were analyzed by a two-factor ANOVA with no repeated measures. Significant differences between individual means were determined using Duncan's new multiple-range test or a Newman-Keuls test. Heterogeneity of vessel density was assessed by calculating the coefficient of variation (SD/mean) for each animal and obtaining a mean of the coefficient of variation values for each group. Values of P<.05 were considered to be statistically significant.
Arterial Pressure, Heart Rate, and Body Weight
Mean arterial pressure, heart rate, and body weight in the various groups are summarized in Table 1⇓. Arterial pressure was significantly elevated in RRM rats after 3 days on a high salt diet. All other groups had normal blood pressures. Heart rates were elevated (P<.05) in both groups of rats on a high salt diet relative to corresponding control rats on a low salt diet. Although body weights in the RRM rats on a given diet were significantly less than those of the corresponding sham-operated control rats, body weights in RRM and sham-operated control rats on a high salt diet were not significantly different from those of corresponding groups on a low salt diet (Table 1⇓), indicating that the high salt diet did not lead to edema in either the RRM rats or the sham-operated control rats.
Microvessel Density Changes
Microvessel density was significantly reduced in the cremaster muscle of both the hypertensive and normotensive rats fed a high salt diet relative to RRM rats and sham-operated control rats fed a low salt diet (Fig 1⇓). The reductions in microvessel density averaged ≈22% in the RRM group and 24% in the sham-operated group on a high salt diet. There were no significant differences in microvessel density between sham-operated rats on a low salt diet and RRM rats on a low salt diet. Statistical analysis of the distribution of rarefaction (Table 2⇓) revealed a large increase in the heterogeneity of vessel density (indicated by a significantly higher mean coefficient of variation) in the hypertensive RRM rats fed a high salt diet but not in sham-operated control rats fed a high salt diet. Further analysis of many fields using confocal microscopy at low magnification revealed focal regions completely lacking microvessels in RRM hypertensive rats or having a noticeable loss of vessels within the network (Fig 2⇓), whereas other regions were similar to control regions or only moderately reduced in vessel density.
The ultrastructural features of microvessels of sham-operated rats and RRM rats on a low salt diet were similar to those of unoperated normotensive age-matched control rats and were essentially the same as those reported earlier.10 No evidence of rarefaction or degeneration was observed. Endothelial cells were moderately electron-dense, with few organelles and smooth cell boundaries. Endothelial cell basement membranes were distinct and intact. The cells typically had abundant pinocytotic vesicles associated with both the luminal and abluminal aspects. Vascular smooth muscle cells had a normal contractile phenotype. The smooth muscle cells were closely associated with the endothelial cells but were separated from them by an elastic lamina in second- and third-order arterioles, as described previously.19
Structural alterations occurring in microvessels of RRM hypertensive rats and sham-operated rats on a high salt diet for 3 days were very similar and will be described together. Most of the microvessels observed in the tissue were structurally intact, but their ultrastructural appearance was generally different from that seen in either the unoperated age-matched control rats or the low salt control rats. Endothelial cells of RRM rats and sham-operated rats on a high salt diet generally differed from those of low salt control rats or age-matched unoperated rats by being more electron-lucent and filled with organelles, particularly ribosomes. Various types of vesicles and occasional centrioles were also observed. Numerous clear, dense, and coated varieties of vesicles were found within the cytoplasm. However, the density and distribution of pinocytotic vesicles at the luminal and abluminal boundaries of endothelial cells of rats on a high salt diet appeared to be greatly reduced relative to RRM or sham-operated control rats on a low salt diet or to unoperated rats on a normal salt diet.
Most endothelial cells of RRM hypertensive rats and high salt sham-operated rats appeared thickened relative to those of the low salt sham-operated rats or unoperated control rats, and endothelial cells often bulged into the lumen of the vessel (Fig 3⇓). The luminal and the abluminal boundaries of endothelial cells in RRM and sham-operated animals on a high salt diet were often irregular. Endothelial cells bulged into the lumen in some terminal arterioles of RRM hypertensive rats and high salt sham-operated rats, completely closing the vessel (Fig 3⇓). In those vessels, closure of the lumen may have been due to a combination of endothelial cell swelling and smooth muscle contraction.
Smooth muscle cells of some arterioles in RRM hypertensive animals and high salt sham-operated animals appeared to be normal (Fig 4a⇓). However, smooth muscle cells in other arterioles (Fig 4b⇓) had conspicuous ribosomes and an abundant rough endoplasmic reticulum. Basement membranes surrounding the smooth muscle cells were amorphous in many microvessels of RRM hypertensive rats and high salt sham-operated rats, and there was often an increased amount of fibrillar collagen and other amorphous material separating the endothelial cells from the vascular smooth muscle cells (Fig 4b⇓).
Although the majority of microvessels examined in these animals appeared to be structurally intact, some arterioles of RRM rats and sham-operated rats on a high salt diet exhibited distinct ultrastructural changes, which indicate that the structural components of the arteriolar wall were dissociating (Fig 4b⇑). These changes in vessel structure in animals on a high salt diet for 3 days were distinct from the frank degenerative changes that we previously reported in rats with chronic RRM hypertension.10 Endothelial cells of microvessels from animals exposed to an acute elevation of salt intake appeared to be migratory and possibly proliferative (Figs 5⇓ and 6). Endothelial and smooth muscle cells were spatially separate from each other, resulting in isolated thin tortuous endothelial tubes and disconnected individual smooth muscle cells or clusters of smooth muscle cells. Some of these tubes contained erythrocytes, whereas others appeared empty (Figs 5 and 6⇓⇓).
Overt degeneration of vessels was extremely rare. An area of necrotic muscle and empty basement membranes was found in one sample (Fig 7⇓). There was no inflammation of the interstitium or evidence of breakdown by macrophages, but clusters of neutrophils and extravasated erythrocytes were found within the basement membranes of the smooth muscle layer, where cells were either absent or atrophied. The endothelium of these vessels was intact.
Once specific areas of apparent microvessel dissociation were identified, the GS-I–labeled whole mounts were reexamined to determine whether the changes observed by electron microscopy corresponded to alterations in the topology of the microcirculation. Various types of dissociative changes were observed in cremaster muscle whole mounts from RRM hypertensive rats and the high salt sham-operated rats that did not occur in control rats. In some cases, an abrupt cessation of microvessels was observed, leaving a relatively blunt end with discontinuous microvessel segments or cell clusters in the surrounding area (Fig 8⇓). Other microvessels exhibited saclike and funnel-like dilations, forming sinusoidal vessels that were often many times larger than the original vessels. These topographic alterations in the microvessels appear to correspond to the changes observed at the ultrastructural level, since dissociative changes in microvessels of RRM hypertensive animals and sham-operated animals on a high salt diet generally tended to occur in focal areas. However, two samples had fairly extensive areas in which altered microvessels could be identified.
Rarefaction of microvessels has been reported in a number of vascular beds in various experimental models of hypertension1 2 3 4 5 6 10 and in human hypertensive subjects.7 8 9 Ultrastructural studies of the cremaster muscle of rats with chronic RRM hypertension have revealed that microvascular rarefaction is mediated by atrophy and structural degeneration of microvessels.10 The resulting loss of third- and fourth-order arterioles and capillaries may have important effects on vascular resistance,11 blood flow heterogeneity,11 and tissue oxygen delivery.13
One unexpected finding in our initial studies of microvessel rarefaction in rats with chronic (4- to 6-week) RRM hypertension was that microvessel density was also reduced in normotensive sham-operated control rats on a high salt diet relative to control rats on a low salt diet.5 However, the exact nature of the structural alterations contributing to the reduced density of microvessels in normotensive animals during high salt intake and the exact time course of altered microvessel density occurring in RRM hypertension and during elevated salt intake were not determined in that study.
The results of the present study demonstrate that exposure to a high salt diet for only 3 days leads to a significant reduction of microvessel density in both RRM hypertensive rats and normotensive sham-operated control rats, relative to RRM or sham-operated rats on a low salt diet or unoperated age-matched rats on normal rat chow. These findings are important because they demonstrate that microvascular rarefaction, which has been previously studied only in chronic hypertension or with a long-term elevation in salt intake, can occur very rapidly after exposure of either normal or RRM rats to a high salt diet. The present study also demonstrates that the reduction of microvessel density occurring in response to a high salt diet is mediated by structural degeneration of microvessels rather than changes in tissue geometry resulting from alterations in muscle mass or tissue edema.
Even though the reduction of microvessel density occurring in RRM rats and sham-operated control rats after 3 days on a high salt diet is mediated by structural changes in the vessels, the mechanisms contributing to the loss of microvessels during acute exposure to a high salt diet appear to be fundamentally different from those operating in chronic hypertension. The primary structural alterations in microvessels during acute exposure to a high salt diet appear to involve a loss of vessel integrity due to dissociation of the endothelial and smooth muscle components of the arteriolar wall. These “dissociative” changes occurring during short-term exposure to a high salt diet are similar in RRM hypertensive rats and normotensive sham-operated control rats and differ from the degenerative changes seen in chronic hypertension.10 The latter are characterized by frank degeneration of endothelial and vascular smooth muscle cells. In contrast, instances of overt degeneration were rare in the present study of animals exposed to an acute elevation of salt intake.
A striking difference in microvessel structure during short-term exposure of RRM and sham-operated control rats to a high salt diet (versus chronic RRM hypertension) was the fate of the basement membranes and the extracellular matrix underlying the endothelium. In rats with chronic RRM hypertension, basement membranes of microvessels persisted during structural degeneration and atrophy.10 However, in the acute RRM hypertensive rats and in the normotensive sham-operated rats on a high salt diet, some regions of the microcirculation lacked an intact basement membrane. In contrast, intact and discrete basement membranes were always present in the age-matched control rats and in rats on a low salt diet.
During acute exposure to a high salt diet, endothelial cells of both RRM hypertensive rats and normotensive sham-operated rats appear to become migratory and to move away from the smooth muscle cells, presumably because of the loss of the basement membrane for attachment. The smooth muscle cells appeared to be nonmigratory and sometimes contained numerous ribosomes and a prominent rough endoplasmic reticulum. In some cases, this difference in the response of endothelial and smooth muscle cells to elevated salt intake led to a separation of the endothelium and vascular smooth muscle, resulting in the formation of sinusoidal vessels, which were perfused but could no longer be identified as arterioles. The absence of erythrocytes in some of these sinusoidal vessels and the finding of deformed erythrocytes in others suggests that perfusion may have been interrupted at an upstream site. In other cases, the endothelium appeared to dissociate into a nontubular structure, leading to loss of the microvessel.
We have previously reported the presence of thin-walled sinusoidal vessels containing red blood cells in rats with chronic RRM hypertension.10 These were originally interpreted as atrophic and degenerating vessels. However, the present study suggests that these vessels may result from the dissociation of vascular wall components, which leaves the vessel as a simple endothelial tube. These structural changes may have important effects on the hemodynamics of the microcirculation, since loss of arteriolar smooth muscle would adversely affect the ability of the vessels to actively control their tone and thereby regulate tissue blood flow and vascular resistance.
The rapid loss of microvessels during acute exposure to high salt intake in RRM and sham-operated rats appears to be mediated by suppression of Ang II in response to the high salt diet. Previous studies by our group14 and by other investigators20 have implicated the renin-angiotensin system in the maintenance of the microcirculation in both normal and hypertensive rats. For example, Wang and Prewitt20 reported that blockade of angiotensin-converting enzyme with captopril led to a significant reduction in microvessel density in the cremaster muscle of normotensive rats. Hernandez et al14 subsequently demonstrated that the decrease in microvessel density occurring in normotensive rats during chronic elevations in dietary salt intake could be prevented by the infusion of subpressor doses of Ang II. A role for Ang II suppression in mediating the reduction of microvessel density in these experiments is supported by the observation that plasma Ang II levels in animals on a high salt diet for 3 days (5.4±1.5 pg/mL, n=6) are significantly lower than those of animals on a low salt diet for 3 days (12.7±4.0 pg/mL, n=6). Taken together, these observations indicate that suppression of Ang II in response to elevated salt intake is responsible for the reduction of microvessel density that occurred in both the RRM rats and in the sham-operated control rats on a high salt diet.
In summary, the present experiments demonstrate that acute exposure of RRM rats and normotensive sham-operated control rats to a high salt diet leads to a rapid reduction of cremasteric microvessel density, which is mediated by structural alterations in microvessels that are distinct from the degenerative changes occurring in chronic RRM hypertension. Two particularly intriguing findings of the present study are that elevated salt intake alone can alter arteriolar structure and reduce microvessel density independent of an elevated blood pressure and that these alterations in the structure of the microcirculation can occur very rapidly, ie, within 3 days of exposure to the high salt diet.
This study was supported by National Institutes of Health grants HL-29587 and HL-37374 and American Heart Association Grant-in-Aid 890822. The authors thank our technical staff for their skilled and dedicated assistance.
Reprint requests to Julian H. Lombard, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail firstname.lastname@example.org.
- Received December 4, 1995.
- Accepted April 15, 1996.
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