Abnormal Platelet Function and Calcium Handling in Dahl Salt-Hypertensive Rats
Abstract—The effect of dietary salt on platelet function and Ca2+ homeostasis was studied in Dahl (DS) rats, a genetic model of salt-sensitive hypertension. DS rats were fed a high-salt (DSHS) or a low-salt diet (DSLS) for up to 4 weeks, and the effects of salt loading on systolic blood pressure, platelet P-selectin expression, and platelet Ca2+ homeostasis were measured. The high-salt diet increased blood pressure and markedly increased the amount of ionomycin (IM)-releasable Ca2+ in platelet intracellular stores (Ca2+/IM). The alteration in Ca2+ stores was not prevented when the hypertension was prevented by treatment with hydralazine and reserpine. The Ca2+ store filling during platelet exposure to 1 mmol/L Ca2+ for 5 minutes and the rate of sarcoplasmic/endoplasmic Ca2+ ATPase–dependent Ca45 uptake were higher in DSHS compared with that in DSLS. There was a decrease in thrombin-induced Ca2+ influx in platelets from DSHS; consistent with this, agonist-induced P-selectin expression was decreased. In DSLS, nitric oxide accelerated reloading of platelet Ca2+ stores after their emptying by thrombin but failed to do so in DSHS. These results indicate that in DS rats, a high-salt diet increases sarcoplasmic/endoplasmic Ca2+ ATPase activity and the Ca2+/IM but decreases the reuptake of Ca2+ caused by nitric oxide. Decreases in Ca2+ influx and platelet P-selectin expression might be explained by changes in intracellular Ca2+ stores in DSHS rats, which apparently is a heritable response to a high-salt diet.
Epidemiological studies show that there is an association between a dietary high-salt diet and arterial hypertension in salt-sensitive individuals.1 2 The Dahl-Rapp salt-sensitive (DS) rat has been used as a genetic model of human salt-sensitive hypertension.3 When challenged with a high-salt diet, DS rats have an attenuated pressure natriuresis, renal medullary vasoconstriction, renal injury, and hypertension.4 However, the mechanism by which salt sensitivity alters vascular function and mediates hypertension is not clear.
Platelets play a key role in the development of vascular disease and related thrombotic events. Cytosolic calcium concentration ([Ca2+]i) is the main determinant of platelet activation and aggregation. Results from studies in hypertensive rats5 and individuals with essential hypertension6 7 indicate that hypertension is associated with altered platelet Ca2+ homeostasis. Platelets also have been used as a model reflecting smooth muscle Ca2+ homeostasis.5 7 In DS rats, the effect of hypertension on platelet Ca2+ homeostasis is controversial, with both increases and decreases in [Ca2+]i having been reported.8 9
Similar to other types of cells, the levels of [Ca2+ ]i in platelets are controlled by its influx and efflux as well as the content and release from intracellular Ca2+ storage sites (dense tubular system). In platelets, there are no voltage-regulated Ca2+ channels,10 and the mechanisms controlling receptor-regulated Ca2+ influx are not clear. Intracellular Ca2+ stores play an important role in Ca2+ homeostasis and calcium influx. In the resting state, most of the Ca2+ is in the intracellular Ca2+ stores, whose content is controlled by sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and inositol trisphosphate receptors. A number of studies11 including those on platelets12 indicate that intracellular stores regulate Ca2+ entry. We recently showed that nitric oxide (NO), a known inhibitor of platelet activation, inhibits thrombin-induced Ca2+ influx indirectly by accelerating SERCA-dependent refilling of the Ca2+ stores.13
This study determined the effect of long-term dietary salt loading on the amount of Ca2+ in intracellular stores, agonist-stimulated Ca2+ entry, SERCA activity, and P-selectin expression in platelets from DS and salt-resistant (DR) rats. The effect of a high-salt diet on the changes in platelet intracellular Ca2+ stores caused by NO was also investigated.
Animals and Isolation of Rat Platelets
Male DS and DR rats (8 to 9 weeks of age, from Harlan Sprague-Dawley) were divided into 2 groups. The low-sodium diet group was fed a 0.12% NaCl diet. The high-sodium diet group was fed a diet supplemented with 8% NaCl. Some DS rats receiving 8% NaCl also received antihypertensive drugs in the drinking water (hydralazine, 80 mg/L; reserpine, 1.4 mg/L).14 Systolic arterial blood pressure and body weight were measured before and 3 to 4 days, 2 weeks, or 4 weeks after initiation of salt diets. Blood pressure was measured by tail-cuff method. Blood (8 to 10 mL) was drawn from the abdominal aorta of pairs of rats that were fed low-salt or high-salt diets on the same day and was put into tubes containing 1/10 volume of 3.8% trisodium citrate. Platelet-rich plasma was obtained by centrifuging the blood for 10 minutes at 350g at room temperature. Platelet pellets were obtained by centrifugation at 750g for 10 minutes and then resuspended and washed in HEPES buffer consisting of (mmol/L): 137 NaCl; 2.7 KCl; 1 MgCl2 · 6 H2O; 3.3 NaH2PO4; 5.5 glucose; and 3.8 HEPES (pH 7.4).
Measurement of Platelet Cytosolic Ca2+ Concentrations
Before each individual experimental run, isolated platelets (108/mL) were loaded with the Ca2+ indicator Fura-2 AM (2.5 μmol/L) at 37°C for 10 minutes. The extracellular dye was removed by centrifugation, and platelets were resuspended in 2 mL HEPES buffer immediately before Ca2+ measurements. Fluorescence was measured at 37°C under constant stirring in a Hitachi F-4500 spectrofluorometer, with excitation wavelength alternating between 340 and 380 nm every 0.5 second and emission wavelength at 510 nm. Recordings were corrected for autofluorescence that was determined in unloaded platelets. Platelet cytosolic Ca2+ concentration was estimated by the ratio of fluorescence detected at the two excitation wave lengths (R340/R380).
As shown in Figures 1⇓ and 2⇓, the amount of Ca2+ in platelet intracellular stores was estimated by 2 protocols. In the first protocol, Fura-2–loaded platelets were suspended in Ca2+-free medium and a maximum dose of ionomycin (IM, 5 μmol/L) was applied to release all releasable Ca2+ from intracellular stores (Ca2+/IM). In the second protocol, platelets were suspended in 1 mmol/L Ca2+ buffer for 5 minutes to augment platelet intracellular stores that may have depleted during isolation in Ca2+ chelating buffers. After removing the extracellular Ca2+ with EGTA, ionomycin was added to measure Ca2+/IM. In both protocols, Ca2+/IM was estimated from the amount of Ca2+ released by ionomycin in two different ways: (1) the peak [Ca2+]i increase after IM (expressed as the change in R340/380 between the baseline and peak) and (2) the integral of changes in the fluorescence ratio during the first 5 minutes of the IM release transient.
Platelet P-Selectin Expression
Platelet P-selectin expression was used as a marker of platelet activation and was measured in whole blood by flow cytometry as described.15 P-selectin expression was measured with no added stimulus (spontaneous) and after activation by agonists. Briefly, 5 μL of whole blood was placed immediately into 4 tubes containing 50 μL PBS as negative control, 50 μL PBS plus 10 μL rabbit anti–P-selectin to determine spontaneous activation, and either 5 μmol/L ADP plus 500 μmol/L epinephrine or thrombin (Thr, 0.5 U/mL) and anti–P-selectin antibody for agonist-induced activation. Each sample was mixed and washed with PBS and centrifuged at 1700g for 5 minutes. Then, 20 μL of a solution containing secondary antibody was added and incubated in the dark. Finally, the samples were resuspended in 500 μL of PBS and analyzed by flow cytometry as described.15 Results are expressed as percentage of platelets staining for P-selectin compared with unstained samples.
Measurements of 45Ca2+ Uptake
SERCA activity was measured by 45Ca2+ uptake in platelet lysates. Washed platelets were disrupted by sonication, and the total lysates were used for 45Ca2+ uptake assay. Paired samples from DS rats receiving a high-salt diet (DSHS) and DS rats receiving a low-salt diet (DSLS) were studied on the same day. Uptake was assayed in samples from both groups of rats without or with added thapsigargin (TG, 10 μmol/L), a specific inhibitor of SERCA, which was added, mixed, and incubated at 37°C for 15 minutes before assay. Ca2+ uptake buffer (30 mmol/L Tris-HCl, pH 7.0, 100 mmol/L KCl, 5 mmol/L sodium azide, 6 mmol/L MgCl2, 0.15 mmol/L EGTA, 0.12 mmol/L CaCl2, 10 mmol/L oxalate) was mixed with 1 μCi/mL 45CaCl2 and 2 mmol/L ATP at 37°C. The reaction was started by adding 300-μg aliquots of protein into the uptake mixture (500 μL) and was stopped at 10, 30, and 60 minutes by filtration of 0.1 mL of the mixture through Whatman GF/C glass filters. The filters were washed twice with 2.5 mL of wash buffer consisting of 30 mmol/L imidazole, 250 mmol/L sucrose, and 0.5 mmol/L EGTA and counted by liquid scintillation. 45Ca2+ uptake was calculated by counting the radioactivity standardized by protein concentration, which was determined by the Bradford method. The rate of TG-sensitive 45Ca2+ uptake was considered as SERCA activity and was compared in DSHS and DHLS.
NO gas was obtained from Matheson. Saturated NO solution was prepared as described16 and stored at 4°C. A flexible, plastic 1-L intravenous bag was filled with 750 mL distilled water and bubbled with nitrogen gas to remove oxygen. Then, 30 mEq Bio-Rad analytical grade anion exchange resin was mixed in the water before the bag was purged with nitrogen gas for 30 minutes. (Any nitrite or nitrate that is formed by the reaction of NO with residual oxygen is retained by the resin.) The bag was then purged with NO gas to give a dissolved gas concentration at 4°C of 3.1±0.6 mmol/L (n=5) and was stable for 1 week as measured by a chemiluminescent method (Siever NOA model 270 NO analyzer). Subsequent NO dilutions were made from the saturated NO solution by drawing off the solution from the bag with a gas-tight syringe and dissolving in sealed tubes filled with deoxygenated solution (bubbled with nitrogen for 1 hour).
45CaCl2 was obtained from New England Nuclear. Fura-2/AM was from Molecular Probes. Thr, ADP, epinephrine, and IM were from Sigma Chemical.
Calculations and Statistical Analysis
The Hitachi software was used for data collection, calculation of Fura-2 emission ratio, and data analysis. An unpaired t test was used to determine the significance of differences in the means of data between the rats with low-salt and high-salt diets. A repeated-measures ANOVA was performed to compare 45Ca2+ uptake in DSHS and DSLS platelets over time. Data were expressed as mean±SEM; n indicates the number of animals. A value of P<0.05 was considered significant.
Effect of High Salt on Systemic Blood Pressure
Before initiating salt diets, there was no significant difference in systemic blood pressure or body weight between DS and DR rats. After 4 weeks of the high-salt (8% NaCl) diet, the blood pressure in DS rats increased from 120±2 to 200±6 mm Hg (n=8, P<0.05). There was no significant change in systemic blood pressure in DR rats after the same period of the high-salt diet (from 122±2 to 123±2 mm Hg, n=5). Also, in DS rats after 4 weeks of the low-salt (0.12% NaCl) diet, the blood pressure did not increase significantly (from 122±2 to 125±2 mm Hg, n=8). In DS rats, 2 weeks of the high-salt diet also caused a marked increase in blood pressure (200±8 mm Hg, n=3). Antihypertensive treatment normalized the blood pressure in DS rats fed the 8% NaCl diet for 2 weeks (110±6 mm Hg, n=3, P<0.05 compared with those not treated with antihypertensive agents).
Amount of Ca2+ in IM-Releasable Stores
The amount of Ca2+ in Ca2+/IM is shown for platelets from DS rats given the low-salt and high-salt diets in Ca2+-free conditions (Figure 1⇑). In addition, the Ca2+/IM was also assessed after Ca2+ store refilling, which also changed the kinetics of Ca2+ release from the stores (Figure 2⇑). With both protocols and regardless of whether the stores were quantified by the integral of the Fura-2 [Ca2+ ]i transient or its peak, platelets from the DS rats fed the high-salt diet showed significantly greater IM-evoked [Ca2+]i release than those of DS rats fed with low-salt diet. These data indicate that in DS rats, a high-salt diet increased the amount of releasable Ca2+ in intracellular Ca2+ stores.
Figure 1⇑ also shows the time course of the change in IM-releasable stores in DS rats fed a high-salt diet as well as the effect of antihypertensive treatment. Compared with the IM-releasable stores in DS rats given the low-salt diet, the Ca2+/IM was increased in DS rats after the high-salt diet for 3 to 4 days, 2 weeks, or 4 weeks. Despite the fact that antihypertensive treatment prevented the rise in blood pressure, the antihypertensive drugs did not affect the change in platelet calcium stores caused by the high-salt diet.
The same protocol was used to measure the effect of dietary salt on platelets from DR rats. The Table⇓ indicates that in DR rats, regardless of salt diet fed for 4 weeks, there was no significant difference in Ca2+/IM either before or after Ca2+ refilling.
Thr-Induced Ca2+ Influx and Platelet Activation
Thr-induced Ca2+ response was compared in DS rats given low-salt and high-salt diets. Figure 3⇓ shows the effect of dietary salt on the Thr-induced [Ca2+]i response in the presence of 1 mmol/L extracellular Ca2+. In platelets from DS rats given a high-salt diet, thrombin-evoked [Ca2+]i response was decreased and the time to reach the peak was increased compared with that in DS rats given the low-salt diet (Figure 3⇓, P<0.05). In the presence of extracellular Ca2+, the Thr-induced [Ca2+]i response is a reflection of net Ca2+ release, efflux, influx, and reuptake. To experimentally separate Ca2+ influx from the Ca2+ release from intracellular Ca2+ stores, Thr (0.5 U/mL) also was applied in Ca2+-free buffer to platelets whose stores had not been reloaded with Ca2+ (Figure 4⇓). Thr caused a small increase in [Ca2+]i representing only Ca2+ release from intracellular stores, which then declined to a steady-state level. Addition of 1 mmol/L extracellular Ca2+ 5 minutes after thrombin further increased [Ca2+]i as a result of Ca2+ influx. There was no significant difference in thrombin-induced Ca2+ release in Ca2+-free buffer in DS rats fed a low-salt or a high-salt diet. However, during Ca2+ influx, [Ca2+]i increased to significantly lower levels in DS rats fed the high-salt diet compared with those fed the low-salt diet.
Figure 5⇓ shows the effect of a high-salt diet on platelet P-selectin expression in DS rats. There was no significant difference in P-selectin expression under basal conditions, but P-selectin expression both by the combination of ADP (5 μmol/L) and epinephrine (500 μmol/L) as well as Thr (0.5 U/mL) decreased significantly in DS rats fed the high-salt diet compared with those fed the low-salt diet.
Ca2+ Store–Refilling Capacity
The above results indicate that in DS rats, a high-salt diet increased the amount of IM-releasable Ca2+ in platelet intracellular stores and diminished Thr-induced Ca2+ influx and agonist-stimulated P-selectin expression. One possible cause for such changes could be a change in reuptake into the intracellular stores. To test this hypothesis, two different approaches were used.
As shown in Figures 1⇑ and 2⇑, when compared in platelets from the same rats, the amount of releasable Ca2+ in intracellular stores was significantly larger when measured after incubating 5 minutes in 1 mmol/L extracellular Ca2+. This indicates that during the exposure of platelets to extracellular Ca2+, Ca2+ entered the cell and was taken up into intracellular stores. The increase in the integral of IM-induced Ca2+ transient between that determined in Ca2+-free buffer (Figure 1⇑) and after exposure to 1 mmol/L Ca2+ (Figure 2⇑) was analyzed as an indicator of Ca2+ store filling. In DS rats fed the high-salt diet, the increase in IM-releasable Ca2+ stores expressed as the integral before and after Ca2+ refilling was significantly higher than that in DS rats fed the low-salt diet (200±26 versus 60±42, P<0.05).
Because SERCA is mainly responsible for refilling intracellular Ca2+ stores, we hypothesized that increased activity of this enzyme is responsible for the larger amount of Ca2+ in intracellular stores and greater refilling of intracellular Ca2+ stores in platelets from DS rats fed the high-salt diet compared with those fed the low-salt diet. Platelet SERCA activity was estimated by measuring the TG-sensitive Ca2+ uptake in crude platelet lysates. As shown in Figure 6⇓, in DS rats fed the high-salt diet, TG-sensitive 45Ca2+ uptake was significantly greater compared with that in DS rats fed the low-salt diet (ANOVA, P<0.05, n=5). The rate of 45Ca2+ uptake over the first 10 minutes was also significantly greater in lysates from platelets of DS rats fed the high-salt diet when compared with that of DS rats fed the low-salt diet (Figure 6⇓).
Effect of NO on IM-Releasable Stores
In human platelets, NO inhibits store-operated Ca2+ influx indirectly by accelerating the SERCA-dependent refilling of intracellular stores because its effect is blocked by BHQ, 2,5-di-(tert-butyl)-1,4-benzohydroquinone, another specific inhibitor of SERCA.13 The same protocol was used in rat platelets, and the effect of NO on Ca2+ stores in platelets from DS rats fed high-salt and low-salt diets was evaluated. In the absence of extracellular Ca2+, Thr (2.5 U/mL) released intracellular Ca2+, and, 5 minutes later, a maximal dose of IM was added to release the remaining portion of Ca2+ from the stores. When NO (10−6 μmol/L) was applied 30 seconds before Thr, the amplitude of the Thr response was decreased and the amplitude of the IM-induced Ca2+ release was enhanced compared with control, as shown in Figure 7⇓. This indicates that after Ca2+ is released from the stores by Thr, NO can promote Ca2+ reuptake into the stores. To examine the effect of dietary salt on the ability of NO to promote Ca2+ reuptake into intracellular stores, the effect of NO on IM-releasable Ca2+ store in platelets from DS rats fed low-salt and high-salt diets are compared in Figure 7⇓. In DS rats fed the low-salt diet, NO significantly increased the amount of Ca2+ in IM-releasable stores by 38%, whereas the initially larger Ca2+ store in platelets of DS rats fed the high-salt diet was not significantly affected by NO.
This study demonstrates that a high-salt diet increases blood pressure and the amount of Ca2+ in intracellular stores in platelets from DS rats. We also found that the increase in Ca2+ in the stores could be explained by an increase of their refilling by higher SERCA activity. The increase in Ca2+ stores was associated with a decrease in agonist-induced Ca2+ entry as well as an apparent decrease in the ability of NO to further refill intracellular Ca2+ stores. Antihypertensive treatment prevented the rise in blood pressure that occurred with the high-salt diet, but the changes in platelet Ca2+ stores still occurred. This indicates that the change in platelet Ca2+ stores is unrelated to the increased blood pressure itself. Furthermore, because this alteration in Ca2+ homeostasis did not occur in salt-resistant rats given a high-salt diet, this result suggests that the change in platelet Ca2+ stores is linked to the genetic susceptibility to salt-induced hypertension in the DS rat.
Because of the similarities in Ca2+ homeostasis between platelets and smooth muscle and their easy accessibility from patients, platelets have been used as an attractive model to explore the relationship between Ca2+ homeostasis and vascular diseases. Earlier studies found that resting platelet [Ca2+]i is increased in both human essential hypertension6 and spontaneously hypertensive rats and that there is a positive relationship between resting platelet Ca2+ concentration and blood pressure.5 However, the changes in platelet resting [Ca2+]i in DS rats fed the high-salt diet are controversial. Vasdev et al8 17 found that in DS rats fed a high-salt diet, platelet resting [Ca2+]i increased. On the other hand, Ishida et al9 found that high dietary salt decreases resting [Ca2+]i in platelets from DS rats.
Recently, it was suggested that in human platelets, the resting [Ca2+]i is neither an indicator of blood pressure level nor of platelet Ca2+ homeostasis.18 These investigators showed that in a heterogeneous population of patients, the blood pressure positively correlated with the Ca2+ in the dense tubular system, Vmax of SERCA, and SERCA protein expression. They also showed that platelets from black Americans have a greater amount of Ca2+ in intracellular stores than those from white Americans.19 Our results provide evidence in rat platelets that the amount of Ca2+ in intracellular stores increases in salt-sensitive hypertension, which is also a characteristic type of hypertension in blacks. Thus, it may be that intracellular Ca2+ stores are a better indication of platelet Ca2+ homeostasis both in human and rat platelets in pathological conditions.
Apart from the increase in the amount of Ca2+ in platelet intracellular Ca2+ stores, we found a decrease in agonist-stimulated Ca2+ influx in platelets of salt-sensitive rats fed a high-salt diet. This is consistent with a store-operated mechanism governing Ca2+ influx in rat platelets. In a variety of cell types, Ca2+ entry is thought to be dependent on agonist-induced Ca2+ release and depletion of Ca2+ stores.11 Although the mechanism that relays the extent of the filling of Ca2+ stores in the dense tubules to the plasma membrane to regulate external Ca2+ influx is not fully understood, Kimura et al20 showed that in normal human platelets, overloading intracellular Ca2+ stores decreases agonist-evoked external Ca2+ entry. Our study indicates that pathological overloading of platelet intracellular Ca2+ stores, at least in those of hypertensive DS rats, may decrease agonist-induced Ca2+ entry.
Unlike reports indicating that Thr-evoked Ca2+ entry is increased in rats with spontaneous hypertension5 or patients with diabetes,21 Dicha et al22 showed that in both salt-dependent hypertensive Sabra or DS rats, there was neither a marked elevation of basal [Ca2+]i nor an augmented response to Thr. Ishida et al23 described significantly lower [Ca2+]i values in resting platelets of DOCA salt–treated rats and unchanged responses to thrombin. It appears that alterations in platelet Ca2+ handling are different in salt-dependent and spontaneous forms of genetic hypertension. The mechanism for this difference is not clear, although our study suggests that increased Ca2+ in the intracellular stores may explain decreased agonist-induced Ca2+ influx.
An increased amount of Ca2+ in the intracellular stores may be due to decreased Ca2+ release or increased Ca2+ reuptake. This study provides two lines of evidence to suggest that augmented Ca2+ reuptake is responsible for the increase in the amount of releasable Ca2+ in the intracellular stores. The first is that in DS rats fed a high-salt diet, the capacity to directly refill Ca2+ stores is increased. When platelets from DS rats were suspended in Ca2+-free medium and then in the presence of extracellular Ca2+, the increase in IM-releasable Ca2+ from the store was greater than that in DS rats fed a low-salt diet. Because SERCA is the main mechanism responsible for Ca2+ reuptake, it is logical to propose that sequestering mechanisms of Ca2+ into intracellular Ca2+ stores are more active in DS rats fed a high-salt diet. Second, increased SERCA activity was directly demonstrated by measuring 45Ca2+ uptake in platelet lysates. Several studies have reported that platelet SERCA activity is related to blood pressure and is increased in spontaneously hypertensive rats and hypertensive patients. Resink et al24 have documented a substantial increase in platelet SERCA activity in patients with essential hypertension. Papp et al25 showed that the total Ca2+ ATPase activity in mixed platelet membranes isolated from spontaneously hypertensive rats was higher than that in Wistar-Kyoto rats. Our finding that SERCA activity is increased in platelets of DS rats fed a high-salt diet is consistent with an increased expression of SERCA protein. The fact that antihypertensive treatments did not prevent the changes in platelet Ca2+ stores suggests that the changes observed in SERCA activity may be due to the genetic response to a high-salt diet rather than to hypertension itself.
Higher SERCA activity might also explain the observation that the effect of NO on intracellular Ca2+ stores is decreased in platelets from DS rats fed a high-salt diet. Previous studies in our laboratory showed that NO inhibits store-operated Ca2+ influx in human platelets by promoting SERCA-dependent refilling of Ca2+ stores.13 This effect of NO could explain the entirety of its ability to reduce [Ca2+]i because its effects were blocked by inhibitors of SERCA. In rat platelets, we also found that after Ca2+ is released from the stores by Thr, NO increases the amount of Ca2+ in the IM-releasable stores. The change caused by NO was less in DS rats fed the high-salt diet. This might be interpreted to indicate that in DS rats fed a high-salt diet, SERCA activity is upregulated and the amount of Ca2+ in intracellular stores is already greater, so when NO was applied, the augmentation in store by NO was relatively smaller. This could mean that the amount of Ca2+ in intracellular stores is near maximal in hypertensive DS rats.
This study demonstrates that a high-salt diet, associated with the development of hypertension in DS rats, increased platelet SERCA activity and the amount of Ca2+ in intracellular stores and decreased agonist-induced Ca2+ entry and the response to NO. The modifications in Ca2+ stores may also occur in other cell types. The increased amount of Ca2+ in platelet intracellular stores and decreased response to NO in platelets may be involved in the pathogenesis of salt-induced hypertension and in the development of cardiovascular complications.
This study was supported by National Institutes of Health grants SCOR HL-55993, R01-HL-31607, and R01-HL-54150.
- Received August 18, 2000.
- Revision received September 19, 2000.
- Accepted September 26, 2000.
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