- Email: [email protected]
Nitric oxide and salt sensitivity
AJH
2000;13:973–979
Nitric Oxide and Salt Sensitivity Luigi X. Cubeddu, Anna B. Alfieri, Irene S. Hoffmann, Elizabeth Jimenez, Carmen M. Roa, Roberto Cubeddu, Coromoto Palermo, and Rosa M. Baldonedo
Studies in laboratory animals suggest that altered nitric oxide (NO) production may be associated with salt sensitivity. In this investigation we determined whether the endogenous NO production was altered in salt-sensitive human subjects when salt intake was changed. Salt sensitivity was assessed from the magnitude of the blood pressure (BP) lowering obtained when the salt intake was reduced from high to a low intake. The combined urinary excretion of nitrites and nitrates, the major metabolites of NO, was employed as an index of endogenous NO production. Salt-sensitive subjects (n ⴝ 23) were older, heavier, and had greater waist-to-hip ratios and higher baseline BP than salt-resistant individuals (n ⴝ 25). In salt-sensitive subjects, mean blood pressure (MBP) decreased 11.8 ⴞ 0.7 mm Hg, and NO metabolite excretion increased from 823 ⴞ 102 to 1530 ⴞ 148 mmol/24 h, when salt intake was reduced from 316 to 28 mol/day. NO metabolite excretion was 45% lower during high salt (0.66 ⴞ 0.1 mol/mg creatinine) than during low salt intake (1.12 ⴞ 0.1 mol/mg creatinine) (P < .001). In contrast, when salt intake was reduced, salt-resistant subjects exhibited no significant mean
changes in BP or NO metabolite excretion. During low salt intake, NO metabolite excretion (mol/ day) was significantly higher in salt-sensitive individuals. The magnitude of decrease of systolic blood pressure, diastolic blood pressure, or MBP induced by reducing salt intake was not related to the increase in urinary excretion of NO metabolite levels (r2 ⴝ 0.009; P ⴝ .66). In summary, to the extent that urinary NO metabolite levels reflect the activity of the endogenous NO system, our results support the view that salt sensitivity may in part be determined by an inability to increase or to sustain NO production in response to high salt. Insufficient NO production during high salt may in turn lead to altered pressure-natriuresis relationships and to an increase in BP. The possibility that the increase in BP induced by high salt intake in salt-sensitive individuals could be the key factor in reducing NO metabolite levels can not be ruled out. Am J Hypertens 2000; 13:973–979 © 2000 American Journal of Hypertension, Ltd. KEY WORDS:
Nitric oxide, salt sensitivity, essential hypertension.
uman subjects are classified as salt sensitive or salt resistant based on their blood pressure (BP) response to changes in salt intake.1 Approximately 40% of patients with essential hypertension seem to be of the salt-
H
sensitive type, when salt sensitivity is defined as a BP increase of at least 10 mm Hg during a 250 mmol/day salt diet, compared with a diet containing only 10 –30 mmol/day of salt.1–3 Experimental evidence indicates that endogenous
Received August 23, 1999. Accepted February 8, 2000. From the Center for the Detection and Treatment of Silent Cardiovascular Risk Factors (SIL-DETECT), Clinical Pharmacology Unit, Department of Pharmacology, School of Pharmacy, Central University of Venezuela, and Division of Endocrinology, University Hospital, Caracas, Venezuela. This study was supported by grants from the Consejo Nacional de
Investigaciones Cientificas y Tecnologicas, CONICIT S1-96001890; and from The Consejo de Desarrollo Cientifico y Humanistico de la Universidad Central de Venezuela, CDCH 06-104214-98. Address correspondence and reprint requests to Luigi X. Cubeddu, MD, PhD, Nova Southeastern University, HPD, School of Pharmacy, 3200 S. University Dr., Ft. Lauderdale, FL 33328; e-mail: [email protected]
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
0895-7061/00/$20.00 PII S0895-7061(00)00283-1
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NO plays an important role in renal hemodynamics and sodium homeostasis, inducing renal vasodilation and natriuresis. In salt sensitive Dahl/Rapp and obese Zucker rats, the state of salt sensitivity may be caused by an inability to increase NO production in response to high salt.4 –11 Inhibition of NO synthesis in saltresistant rats induces hypertension when the animals are exposed to a high intake of salt.6,8 In addition, in salt-sensitive rats, l-arginine feeding increases NO levels, lowers BP, normalizes pressure-natriuresis, and corrects salt sensitivity.4,7,10 In human subjects, Higashi and colleagues12 demonstrated that the renal vascular endothelium of salt-sensitive hypertensives produced lesser amounts of NO compared to that of salt-resistant hypertensives. In addition, in black hypertensives, plasma NO metabolite levels decreased under conditions of high salt intake.13 In summary, the evidence described, although derived mainly from experiments conducted in laboratory animals, indicates that altered NO production may be associated with salt sensitivity. Therefore, it appeared of interest to determine whether NO production is impaired in salt-sensitive human subjects. In this study, we evaluated the effects of high- and low-salt diets on the endogenous production of NO in salt-sensitive and salt-resistant individuals. The combined urinary excretion of nitrites and nitrates, the major metabolites of NO, was employed as an index of endogenous NO production. The results of the study should provide additional understanding of the role of the l-arginine–NO system in the pathogenesis of human salt sensitivity. MATERIALS AND METHODS Forty-eight patients attending our Center for the Detection and Treatment of Silent Cardiovascular Risk Factors were evaluated for salt sensitivity. The exclusion criteria were age greater than 70 yr; a history of angina pectoris, myocardial infarction, congestive heart failure, valvular heart disease, cerebral infarction or hemorrhage, transient ischemic attacks, arteriosclerosis obliterans, pulmonary disease; any patient with active disease, evidences of renal or hepatic dysfunction, urinary tract infection, active inflammatory disease states, severe hypertension, diabetes mellitus; treatment with organic nitrates, women taking birth control pills; or serum creatinine concentration greater than 2 mg/dL. Any medication was discontinued at least 4 weeks before the sodium-sensitivity protocol was started. Determination of Salt Resistance and Salt Sensitivity Patients received a liberal sodium intake diet, in addition to a total of 12 tablets a day, each containing 1 g (17.1 mmol) sodium chloride. Subsequently, patients received a low-salt diet (20 – 40 mmol/day) for 7
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days. On days 6 or 7 of both sodium diets, patients returned to the Center for the following procedures: systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR), 24-h sodium excretion, 24-h nitrite and nitrate excretion, and serum and urinary creatinine levels. BP was measured with a standard mercury sphygmomanometer. Korotkoff sounds I and V were used to record SBP and DBP, respectively. The BP measurements were done with the patient in the supine position after the patient had rested for at least 30 min. The average of a least three determinations was used. The mean BP was calculated as one-third of the pulse pressure added to the diastolic pressure. Patients were classified as salt sensitive and salt resistant. If the difference in mean BP between highand low-sodium weeks was equal to or more than 10 mm Hg, the patient was deemed salt sensitive. Salt resistance was defined as increases of less than 3 mm Hg, no change, or decreases in mean BP. Determination of Nitrites and Nitrates in Urine Samples Nitrites plus nitrates were quantitated employing a modification of a commercially available NO assay kit (Oxford Biomedical Research Inc., Oxford, MI). Three days before the urine collection, patients were asked to refrain from canned foods, black tea, meat and meat derivates, and from processed food. Twenty-four– hour urine samples were collected at the end of the high- and low-sodium diets. Urines were frozen at ⫺60°C until required for assay. Urines were diluted from 1:6 to 1:12 (V:V) with distilled water before the assay. After precipitation of the protein content, the nitrates present in the urine supernatant were reduced 1:1 to nitrites by incubation with metallic cadmium beads for 24 h. The total nitrite concentration was then estimated by the Griess reaction, using a multiwell microplate for reading of sample absorbance at 540 nm. This value represents the total amount of urine NO end-products (nitrite plus nitrate). Urine samples were processed in duplicate. Statistics Categoric variables were compared by means of the 2 test. Continuous variables were compared by the Student t test for independent samples and paired t test for paired samples, or by a one-way ANOVA followed by a Duncan’s test. Results are shown as mean values ⫾ SEM; differences were considered significant at a value of P ⬍ .05. RESULTS Effects of Sodium Intake on BP The demographic characteristics of the subjects studied are shown in Table 1. Salt-sensitive subjects were older, heavier, and had greater waist-to-hip ratios than salt-resistant individuals. Baseline BP were also higher in the saltsensitive group (Table 1). Systolic blood pressure and
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TABLE 1. BASELINE CHARACTERISTICS OF SALTRESISTANT AND SALT-SENSITIVE PATIENTS
Age (years) Gender (F/M) Weight (Kg) BMI (Kg/m2) WHR MBP (mm Hg) SrCr (mg/dL)
SR (n ⴝ 25)
SS (n ⴝ 23)
37.1 ⫾ 2.5 18/7 72 ⫾ 2.9 26.9 ⫾ 0.9 0.88 ⫾ 0.01 86 ⫾ 2.0 0.81 ⫾ 0.03
48.3 ⫾ 2.4* 16/7 80 ⫾ 4† 31.2 ⫾ 1.2* 0.92 ⫾ 0.01† 100 ⫾ 3* 0.78 ⫾ 0.03
SR ⫽ salt resistant; SS ⫽ salt sensitive; BMI ⫽ body mass index; WHR ⫽ waist-to-hip ratio; MBP ⫽ mean blood pressure; SrCr ⫽ serum creatinine. Values shown are mean ⫾ SEM. * P ⬍ .01 and † P ⬍ .05 between SR and SS groups.
DBP averaged 132 ⫾ 4 and 84 ⫾ 3 mm Hg for saltsensitive and 112 ⫾ 3 and 74 ⫾ 2 mm Hg for saltresistant individuals (P ⬍ .01). The baseline daily urinary excretion of sodium for all patients averaged 146 ⫾ 8 mmol/day. Sodium excretion increased to 327 ⫾ 11 mmol/day during the high-salt diet and decreased to 23.5 ⫾ 2.3 mmol/day during the low-sodium diet. A higher intake of salt was also associated with higher urine volumes (2099 ⫾ 72 mL/day on high salt v 1640 ⫾ 53 mL/day on low salt; P ⬍ .001). No significant differences in urine sodium and urine volume were observed between salt-resistant and salt-sensitive individuals, either at baseline or with low- and high-salt diets (Table 2). The effects of dietary salt on BP are shown in Table 2. In salt-sensitive subjects, SBP, mean blood pressure (MBP), and DBP decreased 18.8 ⫾ 1.5, 11.8 ⫾ 0.7 and 8 ⫾ 0.08 mm Hg, respectively, when the salt intake was reduced from 316 to 28 mmol/day. In the salt-
FIG. 1. Urinary excretion of NO metabolites in salt-resistant and salt-sensitive individuals during high and low salt intake. The urinary excretion of nitrites ⫹ nitrates was quantitated during high and low salt intake and expressed as mol of nitrites ⫹ nitrates/mg creatinine in 24-h urine samples. Shown are mean values ⫾ SEM for n observations. SR ⫽ salt resistant; SS ⫽ salt sensitive. **Significantly different from SR at P ⬍ .001.
resistant group, comparable reductions in salt intake failed to elicit significant BP lowering (⌬SBP: 0.5 ⫾ 0.8 mm Hg and ⌬MBP: 0.16 ⫾ 0.4 mm Hg). The sodium sensitivity index, which is the reciprocal of the slope of the pressure/natriuresis curve, was 100-fold greater in salt-sensitive than in salt-resistant subjects (P ⬍ .001) (Table 2). Urinary Excretion of NO Metabolites During High and Low Salt Intake For all subjects (both groups combined), the urinary excretion of nitrites plus nitrates was significantly lower (P ⬍ .05) during high than during the low salt intake. Salt-resistant individuals excreted similar amounts of NO metabolites during low and high salt intake (Figures 1 and 2). How-
TABLE 2. BLOOD PRESSURE CHANGES AND URINARY SODIUM EXCRETION DURING HIGH AND LOW SALT INTAKE BY SALT-SENSITIVITY STATUS SR (n ⴝ 25)
SBP DBP MBP Urinary Na⫹ Urine volume SSI ⫻ 100
SS (n ⴝ 23)
High Salt
Low Salt
High Salt
Low Salt
111 ⫾ 3.2 74 ⫾ 1.8 86 ⫾ 2.2 345 ⫾ 18 2144 ⫾ 156
110 ⫾ 3.1 74 ⫾ 1.7 86 ⫾ 2.1 21 ⫾ 4* 1574 ⫾ 107*
137 ⫾ 3 88 ⫾ 2 105 ⫾ 2.2 316 ⫾ 18 2053 ⫾ 128
118 ⫾ 2.8* 80 ⫾ 2* 93 ⫾ 2* 28 ⫾ 4* 1734 ⫾ 127*
0.03 ⫾ 0.01
4.6 ⫾ 0.4*
SBP ⫽ systolic blood pressure; DBP ⫽ diastolic blood pressure; BP units ⫽ mm Hg; urinary sodium ⫽ sodium excretion in 24 h (mmol/24 h); urine volume: mL/24 h; SSI ⫽ salt sensitivity index (MBP on high salt minus MBP on low salt divided by 24-h sodium excretion on high salt minus 24-h sodium excretion on low salt); SSI units: mm Hg/mmol in 24-h; SSI values were multiplied by 100, to reduce decimal values; other abbreviations as in Table 1. Shown are mean values ⫾ SEM of n observations. * Significantly different from high salt at P ⬍ .01.
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FIG. 2. Changes in the urinary excretion of nitric oxide (NO) metabolites induced by changes in salt intake, in salt-resistant (SR) and salt-sensitive (SS) individuals. Shown are mean values ⫾ SEM for the differences in the urinary excretion of NO metabolites during high and low salt intake in SR and SS individuals. Negative values indicate greater urinary excretion of NO metabolites during low than during high salt intake. Mean changes in NO metabolite excretion were expressed as moles/mg creatinine (A), and as mol/24 h (B). ***Significantly different from baseline levels and from SR at P ⬍ .001.
ever, in salt-sensitive subjects, NO metabolite excretion was 45% lower during high than during low salt intake, ie, NO metabolite excretion nearly doubled when salt intake was reduced from an average of 316 mmol to an average of 28 mol/day (P ⬍ .001) (Figures 1 and 2). This effect was observed both for the absolute amount of NO metabolites excreted in 24 h (mol/day), as well as for the NO metabolite levels divided by urinary creatinine concentrations (mol/mg creatinine) (Figure 2; Table 3). In addition, during low salt intake, NO metabolite excretion
TABLE 3. EFFECTS OF SALT INTAKE ON THE URINARY EXCRETION OF NO METABOLITES BY SALT-SENSITIVITY STATUS Urinary NO Metabolites mol/mg Creatinine
SR SS
moles/24 h
High Salt
Low Salt
High Salt
Low Salt
0.93 ⫾ 0.17 0.66 ⫾ 0.1†
0.97 ⫾ 0.17 1.12 ⫾ 0.1*
1092 ⫾ 142 823 ⫾ 102
1057 ⫾ 116 1530 ⫾ 148*†
Abbreviations as in Table 1. Shown are mean values ⫾ SEM. The urinary excretion of nitrites ⫹ nitrates was determined during high and low salt intake and expressed either as mol of nitrites ⫹ nitrates/mg creatinine or as mol/24 h. * Significantly different from high salt at P ⬍ .001; † significantly different from SR at P ⬍ .05.
(mol/day) was significantly higher in salt-sensitive than in salt-resistant subjects, whereas when the results were expressed as mol of NO metabolites/mg creatinine the values did not reach statistical significance. No relationship was observed between the magnitude of the BP lowering (SBP, DBP, or MBP) induced by reducing salt intake and the changes in NO metabolite levels, in salt-sensitive or in salt-resistant individuals (r2 ⫽ 0.009; P ⫽ .66). Salt-induced changes in BP were not related to the absolute values for NO metabolite excretion in either group of subjects (data not shown). No significant relationships were encountered between absolute BP levels during low or high salt intake and the urinary excretion of NO metabolites for salt-resistant, salt-sensitive, or for both groups combined (P ⬍ .04). DISCUSSION The present study was designed to investigate the effects of salt intake on the endogenous production of NO, and to determine whether NO production was impaired in salt-sensitive individuals. Salt sensitivity was assessed from the magnitude of the BP lowering obtained when the salt intake was reduced from about 300 mmol (high salt) to nearly 30 mmol/day (low salt). Subjects in whom MBP was reduced by 10 mm Hg or more were considered as salt sensitive. Our results
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revealed that in salt-sensitive individuals, the urinary excretion of NO metabolites doubled when the intake of salt was reduced from high to low. In other words, during high salt intake, the urinary levels of NO metabolites were nearly half of those excreted during low salt intake. In salt-resistant subjects, on the other hand, no significant changes in BP or NO excretion were detected despite drastic changes in salt intake. It is important to emphasize that NO production was not decreased in salt-sensitive individuals. Therefore, it could be postulated that salt-sensitive subjects fail to sustain NO production during a salt load, which may in turn lead to altered pressure-natriuresis relationships. Recent evidence indicates that alterations in renal interstitial hydrostatic pressure and papillary hemodynamics seem responsible for the altered pressurenatriuresis relationships in salt-sensitive subjects.14,15 Because of the effects of NO on intrarenal hemodynamics and sodium homeostasis, alterations in NO production may be involved in salt sensitivity.4 –11 The following evidence supports the role of NO in saltsensitivity models. Compared with salt-resistant rats, high salt intake failed to increase NO production in salt-sensitive Dahl/Rapp rats.4 A mutation in the coding region for the NO synthase (NOS2) was demonstrated in salt-sensitive Dahl/Rapp rats.10 Inhibition of NO synthesis induces salt sensitivity in previously salt-resistant rats.6,8 In salt-sensitive Dahl/Rapp rats, l-arginine feeding increases NO levels, lowers BP, normalizes pressure-natriuresis, and corrects salt sensitivity.4,7,10 Finally, a reduced production of NO within the renal medulla of obese-Zucker rats seems to determine the state of salt sensitivity.11 All of this evidence, although derived from animal studies, suggests that salt sensitivity may be caused by an inability to increase NO production in response to high salt. To the extent that urinary NO metabolite levels reflect the activity of the endogenous NO system (see later), our results in human subjects support this view. The validity of measurements of urinary nitrite plus nitrate levels as indicators of endothelial function and/or of the l-arginine–nitric oxide pathway deserves further comment. Although most evidence favors the view that plasma and urinary NO metabolite levels derive mainly from the l-arginine–nitric oxide pathway,16,17 additional factors may affect the validity of the measurements. Consumption of cured meats, canned foods, black tea, and organic nitrates (exogenous sources) may increase the urinary excretion of these substances.18 In our study, dietary contamination was minimized by prescribing nitrite–nitrate free diets during the 2 weeks of salt sensitivity testing and by excluding patients taking organic nitrates. Presence of nitrate-producing bacteria may be another complicating factor. In our study, previous normal urine test,
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use of sterile collection containers, maintaining samples refrigerated, and keeping urine aliquots at ⫺60°C before assay were employed to minimize such a possibility. Physical activity is another factor to consider when assessing NO metabolite levels. Plasma and urine NO metabolite levels have been shown to increase in direct relationship with the levels of exercise.19 –21 Marked increases in plasma and urinary NO metabolite levels have been reported in highly trained runners.19 In our work, with the exception of one subject, all study individuals were sedentary or undertook very low levels of physical activity. To minimize the contribution of NO metabolites derived from inducible NO synthase, subjects with active inflammatory disease were excluded from this study. In summary, appropriate dietary restrictions and exclusion criteria are required when employing measurements of urinary NO metabolites as possible indicators of NO derived from vascular endothelium and, perhaps, from renal tissues.17–19 It should be emphasized, however, that the contribution of neuronal NO synthase to the plasma and urine levels of NO metabolites has not been established. However, recent findings suggest that NO derived from neuronal NO synthase activity may play a role in salt-sensitive Dahl/Rapp rats.22 Other laboratories have also evaluated the role of NO in salt sensitivity in human subjects, employing plasma and/or urine levels to assess the activity of the l-arginine–NO pathway. Campese and coworkers13 reported that hypertensive blacks and higher plasma NO metabolite levels that normotensives, and that plasma NO metabolite levels were reduced during high salt intake, both in salt-sensitive and salt-resistant black hypertensives. In agreement with these investigators, we observed that a high intake of salt was associated with a lower urinary excretion of NO metabolites in all patients combined. However, our findings diverged from those of Campese and coworkers13 in that the high salt intake reduced NO levels in salt-sensitive, but not in salt-resistant individuals. Several factors may account for the discrepancy in the results. These include racial differences (blacks versus Hispanics) and baseline level of salt intake (not reported in Campese et al’s study and around 150 mmol/day in our study subjects). It is feasible that our study patients developed adaptive changes in NO excretion as a result of the long-term intake of high amounts of salt.16 Another factor is the biological fluid in which NO metabolites were measured (plasma versus urine). Theoretically, plasma levels may better reflect systemic production, although we can not rule out that increases in NO production in the renal parenchyma would not lead to increases in plasma NO metabolites. Urinary excretion may reflect both systemic and renal production; therefore, differences in the origin of NO metabolites may explain the discrep-
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ancies between studies. Finally, we must consider baseline BP levels. Our salt-sensitive subjects had higher baseline BP levels than the salt-resistant individuals, and experienced a further increase in BP when placed on a high-salt diet. Hypertensives are known to have higher basal NO levels, which decrease when the intake of salt is increased.13 Therefore, the differential effects induced by changes in salt intake on NO metabolite excretion in salt-sensitive and saltresistant subjects may be partly due to the higher BP and the further BP increases induced by the high-salt diet in the salt-sensitive group (present study). If NO is the main determinant of the pressurenatriuresis relationship, a relationship between saltinduced changes in NO excretion and in BP should be observed. In this case, one should expect that reductions in BP induced by salt restriction would be related to the increases in NO metabolite levels. However, in salt-sensitive subjects, the changes in urinary NO metabolites induced by reducing salt intake were not related to the changes in BP produced by the salt manipulation. Because salt-resistant individuals by definition experienced no changes in BP with changes in salt intake, no correlation was observed between changes in NO and in BP. These findings suggest that although salt sensitivity is associated with lower NO metabolite excretion during high than during low salt intake, the magnitude of the BP changes (degree of salt sensitivity) may be determined by more than one factor. In addition, there may be no cause-effect relationship between these two variables. However, Facchini and coworkers23 reported in healthy normotensive subjects that the changes in BP induced by the highsalt diet (174 mmol/day) were inversely related to the changes in urinary nitrate excretion. Unfortunately, only five of their 19 subjects could be classified as salt sensitive. Further studies are required to clarify this aspect. Salt sensitivity has been reported to cluster with other cardiovascular risk factors. Obesity, black race, older age, insulin resistance, and hypertension have been associated with salt sensitivity.3,24 –29 Body weight reduction in obese adolescents has been shown to lower BP and to correct salt sensitivity.30 Because, in this study, patients were classified solely on their BP response to salt intake, it was not unexpected to find that our salt-sensitive individuals were older, heavier, and had higher BP than the salt-resistant subjects. It is thus also possible that older age, higher weight, and BP could play a role, in addition to salt sensitivity, in determining the marked suppression of NO metabolite excretion observed during high salt. In conclusion, an increase in salt intake reduced the urinary excretion of NO metabolites in all patients combined, an effect due to a major effect of salt intake in salt-sensitive individuals. We demonstrated that
lowering salt intake produced large reductions in BP associated with increases in the urinary excretion of NO metabolites in salt-sensitive individuals. In these subjects, the urinary excretion of NO metabolites was 45% lower during high than during low salt intake. In salt-resistant individuals, reductions in salt intake were not associated with mean changes in NO metabolite excretion. To the extent that urinary NO metabolite levels reflect the activity of the endogenous NO system, our results support the view that salt sensitivity may in part be determined by an inability to increase or to sustain NO production in response to high salt. Insufficient NO production during high salt may, in turn, lead to altered pressure-natriuresis relationships leading to moderate to marked increases in BP. The possibility that the increase in BP induced by high salt intake in salt-sensitive individuals could be the key factor in reducing NO metabolite levels can not be ruled out. REFERENCES 1.
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2000;13:973–979
Nitric Oxide and Salt Sensitivity Luigi X. Cubeddu, Anna B. Alfieri, Irene S. Hoffmann, Elizabeth Jimenez, Carmen M. Roa, Roberto Cubeddu, Coromoto Palermo, and Rosa M. Baldonedo
Studies in laboratory animals suggest that altered nitric oxide (NO) production may be associated with salt sensitivity. In this investigation we determined whether the endogenous NO production was altered in salt-sensitive human subjects when salt intake was changed. Salt sensitivity was assessed from the magnitude of the blood pressure (BP) lowering obtained when the salt intake was reduced from high to a low intake. The combined urinary excretion of nitrites and nitrates, the major metabolites of NO, was employed as an index of endogenous NO production. Salt-sensitive subjects (n ⴝ 23) were older, heavier, and had greater waist-to-hip ratios and higher baseline BP than salt-resistant individuals (n ⴝ 25). In salt-sensitive subjects, mean blood pressure (MBP) decreased 11.8 ⴞ 0.7 mm Hg, and NO metabolite excretion increased from 823 ⴞ 102 to 1530 ⴞ 148 mmol/24 h, when salt intake was reduced from 316 to 28 mol/day. NO metabolite excretion was 45% lower during high salt (0.66 ⴞ 0.1 mol/mg creatinine) than during low salt intake (1.12 ⴞ 0.1 mol/mg creatinine) (P < .001). In contrast, when salt intake was reduced, salt-resistant subjects exhibited no significant mean
changes in BP or NO metabolite excretion. During low salt intake, NO metabolite excretion (mol/ day) was significantly higher in salt-sensitive individuals. The magnitude of decrease of systolic blood pressure, diastolic blood pressure, or MBP induced by reducing salt intake was not related to the increase in urinary excretion of NO metabolite levels (r2 ⴝ 0.009; P ⴝ .66). In summary, to the extent that urinary NO metabolite levels reflect the activity of the endogenous NO system, our results support the view that salt sensitivity may in part be determined by an inability to increase or to sustain NO production in response to high salt. Insufficient NO production during high salt may in turn lead to altered pressure-natriuresis relationships and to an increase in BP. The possibility that the increase in BP induced by high salt intake in salt-sensitive individuals could be the key factor in reducing NO metabolite levels can not be ruled out. Am J Hypertens 2000; 13:973–979 © 2000 American Journal of Hypertension, Ltd. KEY WORDS:
Nitric oxide, salt sensitivity, essential hypertension.
uman subjects are classified as salt sensitive or salt resistant based on their blood pressure (BP) response to changes in salt intake.1 Approximately 40% of patients with essential hypertension seem to be of the salt-
H
sensitive type, when salt sensitivity is defined as a BP increase of at least 10 mm Hg during a 250 mmol/day salt diet, compared with a diet containing only 10 –30 mmol/day of salt.1–3 Experimental evidence indicates that endogenous
Received August 23, 1999. Accepted February 8, 2000. From the Center for the Detection and Treatment of Silent Cardiovascular Risk Factors (SIL-DETECT), Clinical Pharmacology Unit, Department of Pharmacology, School of Pharmacy, Central University of Venezuela, and Division of Endocrinology, University Hospital, Caracas, Venezuela. This study was supported by grants from the Consejo Nacional de
Investigaciones Cientificas y Tecnologicas, CONICIT S1-96001890; and from The Consejo de Desarrollo Cientifico y Humanistico de la Universidad Central de Venezuela, CDCH 06-104214-98. Address correspondence and reprint requests to Luigi X. Cubeddu, MD, PhD, Nova Southeastern University, HPD, School of Pharmacy, 3200 S. University Dr., Ft. Lauderdale, FL 33328; e-mail: [email protected]
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
0895-7061/00/$20.00 PII S0895-7061(00)00283-1
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NO plays an important role in renal hemodynamics and sodium homeostasis, inducing renal vasodilation and natriuresis. In salt sensitive Dahl/Rapp and obese Zucker rats, the state of salt sensitivity may be caused by an inability to increase NO production in response to high salt.4 –11 Inhibition of NO synthesis in saltresistant rats induces hypertension when the animals are exposed to a high intake of salt.6,8 In addition, in salt-sensitive rats, l-arginine feeding increases NO levels, lowers BP, normalizes pressure-natriuresis, and corrects salt sensitivity.4,7,10 In human subjects, Higashi and colleagues12 demonstrated that the renal vascular endothelium of salt-sensitive hypertensives produced lesser amounts of NO compared to that of salt-resistant hypertensives. In addition, in black hypertensives, plasma NO metabolite levels decreased under conditions of high salt intake.13 In summary, the evidence described, although derived mainly from experiments conducted in laboratory animals, indicates that altered NO production may be associated with salt sensitivity. Therefore, it appeared of interest to determine whether NO production is impaired in salt-sensitive human subjects. In this study, we evaluated the effects of high- and low-salt diets on the endogenous production of NO in salt-sensitive and salt-resistant individuals. The combined urinary excretion of nitrites and nitrates, the major metabolites of NO, was employed as an index of endogenous NO production. The results of the study should provide additional understanding of the role of the l-arginine–NO system in the pathogenesis of human salt sensitivity. MATERIALS AND METHODS Forty-eight patients attending our Center for the Detection and Treatment of Silent Cardiovascular Risk Factors were evaluated for salt sensitivity. The exclusion criteria were age greater than 70 yr; a history of angina pectoris, myocardial infarction, congestive heart failure, valvular heart disease, cerebral infarction or hemorrhage, transient ischemic attacks, arteriosclerosis obliterans, pulmonary disease; any patient with active disease, evidences of renal or hepatic dysfunction, urinary tract infection, active inflammatory disease states, severe hypertension, diabetes mellitus; treatment with organic nitrates, women taking birth control pills; or serum creatinine concentration greater than 2 mg/dL. Any medication was discontinued at least 4 weeks before the sodium-sensitivity protocol was started. Determination of Salt Resistance and Salt Sensitivity Patients received a liberal sodium intake diet, in addition to a total of 12 tablets a day, each containing 1 g (17.1 mmol) sodium chloride. Subsequently, patients received a low-salt diet (20 – 40 mmol/day) for 7
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days. On days 6 or 7 of both sodium diets, patients returned to the Center for the following procedures: systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR), 24-h sodium excretion, 24-h nitrite and nitrate excretion, and serum and urinary creatinine levels. BP was measured with a standard mercury sphygmomanometer. Korotkoff sounds I and V were used to record SBP and DBP, respectively. The BP measurements were done with the patient in the supine position after the patient had rested for at least 30 min. The average of a least three determinations was used. The mean BP was calculated as one-third of the pulse pressure added to the diastolic pressure. Patients were classified as salt sensitive and salt resistant. If the difference in mean BP between highand low-sodium weeks was equal to or more than 10 mm Hg, the patient was deemed salt sensitive. Salt resistance was defined as increases of less than 3 mm Hg, no change, or decreases in mean BP. Determination of Nitrites and Nitrates in Urine Samples Nitrites plus nitrates were quantitated employing a modification of a commercially available NO assay kit (Oxford Biomedical Research Inc., Oxford, MI). Three days before the urine collection, patients were asked to refrain from canned foods, black tea, meat and meat derivates, and from processed food. Twenty-four– hour urine samples were collected at the end of the high- and low-sodium diets. Urines were frozen at ⫺60°C until required for assay. Urines were diluted from 1:6 to 1:12 (V:V) with distilled water before the assay. After precipitation of the protein content, the nitrates present in the urine supernatant were reduced 1:1 to nitrites by incubation with metallic cadmium beads for 24 h. The total nitrite concentration was then estimated by the Griess reaction, using a multiwell microplate for reading of sample absorbance at 540 nm. This value represents the total amount of urine NO end-products (nitrite plus nitrate). Urine samples were processed in duplicate. Statistics Categoric variables were compared by means of the 2 test. Continuous variables were compared by the Student t test for independent samples and paired t test for paired samples, or by a one-way ANOVA followed by a Duncan’s test. Results are shown as mean values ⫾ SEM; differences were considered significant at a value of P ⬍ .05. RESULTS Effects of Sodium Intake on BP The demographic characteristics of the subjects studied are shown in Table 1. Salt-sensitive subjects were older, heavier, and had greater waist-to-hip ratios than salt-resistant individuals. Baseline BP were also higher in the saltsensitive group (Table 1). Systolic blood pressure and
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TABLE 1. BASELINE CHARACTERISTICS OF SALTRESISTANT AND SALT-SENSITIVE PATIENTS
Age (years) Gender (F/M) Weight (Kg) BMI (Kg/m2) WHR MBP (mm Hg) SrCr (mg/dL)
SR (n ⴝ 25)
SS (n ⴝ 23)
37.1 ⫾ 2.5 18/7 72 ⫾ 2.9 26.9 ⫾ 0.9 0.88 ⫾ 0.01 86 ⫾ 2.0 0.81 ⫾ 0.03
48.3 ⫾ 2.4* 16/7 80 ⫾ 4† 31.2 ⫾ 1.2* 0.92 ⫾ 0.01† 100 ⫾ 3* 0.78 ⫾ 0.03
SR ⫽ salt resistant; SS ⫽ salt sensitive; BMI ⫽ body mass index; WHR ⫽ waist-to-hip ratio; MBP ⫽ mean blood pressure; SrCr ⫽ serum creatinine. Values shown are mean ⫾ SEM. * P ⬍ .01 and † P ⬍ .05 between SR and SS groups.
DBP averaged 132 ⫾ 4 and 84 ⫾ 3 mm Hg for saltsensitive and 112 ⫾ 3 and 74 ⫾ 2 mm Hg for saltresistant individuals (P ⬍ .01). The baseline daily urinary excretion of sodium for all patients averaged 146 ⫾ 8 mmol/day. Sodium excretion increased to 327 ⫾ 11 mmol/day during the high-salt diet and decreased to 23.5 ⫾ 2.3 mmol/day during the low-sodium diet. A higher intake of salt was also associated with higher urine volumes (2099 ⫾ 72 mL/day on high salt v 1640 ⫾ 53 mL/day on low salt; P ⬍ .001). No significant differences in urine sodium and urine volume were observed between salt-resistant and salt-sensitive individuals, either at baseline or with low- and high-salt diets (Table 2). The effects of dietary salt on BP are shown in Table 2. In salt-sensitive subjects, SBP, mean blood pressure (MBP), and DBP decreased 18.8 ⫾ 1.5, 11.8 ⫾ 0.7 and 8 ⫾ 0.08 mm Hg, respectively, when the salt intake was reduced from 316 to 28 mmol/day. In the salt-
FIG. 1. Urinary excretion of NO metabolites in salt-resistant and salt-sensitive individuals during high and low salt intake. The urinary excretion of nitrites ⫹ nitrates was quantitated during high and low salt intake and expressed as mol of nitrites ⫹ nitrates/mg creatinine in 24-h urine samples. Shown are mean values ⫾ SEM for n observations. SR ⫽ salt resistant; SS ⫽ salt sensitive. **Significantly different from SR at P ⬍ .001.
resistant group, comparable reductions in salt intake failed to elicit significant BP lowering (⌬SBP: 0.5 ⫾ 0.8 mm Hg and ⌬MBP: 0.16 ⫾ 0.4 mm Hg). The sodium sensitivity index, which is the reciprocal of the slope of the pressure/natriuresis curve, was 100-fold greater in salt-sensitive than in salt-resistant subjects (P ⬍ .001) (Table 2). Urinary Excretion of NO Metabolites During High and Low Salt Intake For all subjects (both groups combined), the urinary excretion of nitrites plus nitrates was significantly lower (P ⬍ .05) during high than during the low salt intake. Salt-resistant individuals excreted similar amounts of NO metabolites during low and high salt intake (Figures 1 and 2). How-
TABLE 2. BLOOD PRESSURE CHANGES AND URINARY SODIUM EXCRETION DURING HIGH AND LOW SALT INTAKE BY SALT-SENSITIVITY STATUS SR (n ⴝ 25)
SBP DBP MBP Urinary Na⫹ Urine volume SSI ⫻ 100
SS (n ⴝ 23)
High Salt
Low Salt
High Salt
Low Salt
111 ⫾ 3.2 74 ⫾ 1.8 86 ⫾ 2.2 345 ⫾ 18 2144 ⫾ 156
110 ⫾ 3.1 74 ⫾ 1.7 86 ⫾ 2.1 21 ⫾ 4* 1574 ⫾ 107*
137 ⫾ 3 88 ⫾ 2 105 ⫾ 2.2 316 ⫾ 18 2053 ⫾ 128
118 ⫾ 2.8* 80 ⫾ 2* 93 ⫾ 2* 28 ⫾ 4* 1734 ⫾ 127*
0.03 ⫾ 0.01
4.6 ⫾ 0.4*
SBP ⫽ systolic blood pressure; DBP ⫽ diastolic blood pressure; BP units ⫽ mm Hg; urinary sodium ⫽ sodium excretion in 24 h (mmol/24 h); urine volume: mL/24 h; SSI ⫽ salt sensitivity index (MBP on high salt minus MBP on low salt divided by 24-h sodium excretion on high salt minus 24-h sodium excretion on low salt); SSI units: mm Hg/mmol in 24-h; SSI values were multiplied by 100, to reduce decimal values; other abbreviations as in Table 1. Shown are mean values ⫾ SEM of n observations. * Significantly different from high salt at P ⬍ .01.
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FIG. 2. Changes in the urinary excretion of nitric oxide (NO) metabolites induced by changes in salt intake, in salt-resistant (SR) and salt-sensitive (SS) individuals. Shown are mean values ⫾ SEM for the differences in the urinary excretion of NO metabolites during high and low salt intake in SR and SS individuals. Negative values indicate greater urinary excretion of NO metabolites during low than during high salt intake. Mean changes in NO metabolite excretion were expressed as moles/mg creatinine (A), and as mol/24 h (B). ***Significantly different from baseline levels and from SR at P ⬍ .001.
ever, in salt-sensitive subjects, NO metabolite excretion was 45% lower during high than during low salt intake, ie, NO metabolite excretion nearly doubled when salt intake was reduced from an average of 316 mmol to an average of 28 mol/day (P ⬍ .001) (Figures 1 and 2). This effect was observed both for the absolute amount of NO metabolites excreted in 24 h (mol/day), as well as for the NO metabolite levels divided by urinary creatinine concentrations (mol/mg creatinine) (Figure 2; Table 3). In addition, during low salt intake, NO metabolite excretion
TABLE 3. EFFECTS OF SALT INTAKE ON THE URINARY EXCRETION OF NO METABOLITES BY SALT-SENSITIVITY STATUS Urinary NO Metabolites mol/mg Creatinine
SR SS
moles/24 h
High Salt
Low Salt
High Salt
Low Salt
0.93 ⫾ 0.17 0.66 ⫾ 0.1†
0.97 ⫾ 0.17 1.12 ⫾ 0.1*
1092 ⫾ 142 823 ⫾ 102
1057 ⫾ 116 1530 ⫾ 148*†
Abbreviations as in Table 1. Shown are mean values ⫾ SEM. The urinary excretion of nitrites ⫹ nitrates was determined during high and low salt intake and expressed either as mol of nitrites ⫹ nitrates/mg creatinine or as mol/24 h. * Significantly different from high salt at P ⬍ .001; † significantly different from SR at P ⬍ .05.
(mol/day) was significantly higher in salt-sensitive than in salt-resistant subjects, whereas when the results were expressed as mol of NO metabolites/mg creatinine the values did not reach statistical significance. No relationship was observed between the magnitude of the BP lowering (SBP, DBP, or MBP) induced by reducing salt intake and the changes in NO metabolite levels, in salt-sensitive or in salt-resistant individuals (r2 ⫽ 0.009; P ⫽ .66). Salt-induced changes in BP were not related to the absolute values for NO metabolite excretion in either group of subjects (data not shown). No significant relationships were encountered between absolute BP levels during low or high salt intake and the urinary excretion of NO metabolites for salt-resistant, salt-sensitive, or for both groups combined (P ⬍ .04). DISCUSSION The present study was designed to investigate the effects of salt intake on the endogenous production of NO, and to determine whether NO production was impaired in salt-sensitive individuals. Salt sensitivity was assessed from the magnitude of the BP lowering obtained when the salt intake was reduced from about 300 mmol (high salt) to nearly 30 mmol/day (low salt). Subjects in whom MBP was reduced by 10 mm Hg or more were considered as salt sensitive. Our results
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revealed that in salt-sensitive individuals, the urinary excretion of NO metabolites doubled when the intake of salt was reduced from high to low. In other words, during high salt intake, the urinary levels of NO metabolites were nearly half of those excreted during low salt intake. In salt-resistant subjects, on the other hand, no significant changes in BP or NO excretion were detected despite drastic changes in salt intake. It is important to emphasize that NO production was not decreased in salt-sensitive individuals. Therefore, it could be postulated that salt-sensitive subjects fail to sustain NO production during a salt load, which may in turn lead to altered pressure-natriuresis relationships. Recent evidence indicates that alterations in renal interstitial hydrostatic pressure and papillary hemodynamics seem responsible for the altered pressurenatriuresis relationships in salt-sensitive subjects.14,15 Because of the effects of NO on intrarenal hemodynamics and sodium homeostasis, alterations in NO production may be involved in salt sensitivity.4 –11 The following evidence supports the role of NO in saltsensitivity models. Compared with salt-resistant rats, high salt intake failed to increase NO production in salt-sensitive Dahl/Rapp rats.4 A mutation in the coding region for the NO synthase (NOS2) was demonstrated in salt-sensitive Dahl/Rapp rats.10 Inhibition of NO synthesis induces salt sensitivity in previously salt-resistant rats.6,8 In salt-sensitive Dahl/Rapp rats, l-arginine feeding increases NO levels, lowers BP, normalizes pressure-natriuresis, and corrects salt sensitivity.4,7,10 Finally, a reduced production of NO within the renal medulla of obese-Zucker rats seems to determine the state of salt sensitivity.11 All of this evidence, although derived from animal studies, suggests that salt sensitivity may be caused by an inability to increase NO production in response to high salt. To the extent that urinary NO metabolite levels reflect the activity of the endogenous NO system (see later), our results in human subjects support this view. The validity of measurements of urinary nitrite plus nitrate levels as indicators of endothelial function and/or of the l-arginine–nitric oxide pathway deserves further comment. Although most evidence favors the view that plasma and urinary NO metabolite levels derive mainly from the l-arginine–nitric oxide pathway,16,17 additional factors may affect the validity of the measurements. Consumption of cured meats, canned foods, black tea, and organic nitrates (exogenous sources) may increase the urinary excretion of these substances.18 In our study, dietary contamination was minimized by prescribing nitrite–nitrate free diets during the 2 weeks of salt sensitivity testing and by excluding patients taking organic nitrates. Presence of nitrate-producing bacteria may be another complicating factor. In our study, previous normal urine test,
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use of sterile collection containers, maintaining samples refrigerated, and keeping urine aliquots at ⫺60°C before assay were employed to minimize such a possibility. Physical activity is another factor to consider when assessing NO metabolite levels. Plasma and urine NO metabolite levels have been shown to increase in direct relationship with the levels of exercise.19 –21 Marked increases in plasma and urinary NO metabolite levels have been reported in highly trained runners.19 In our work, with the exception of one subject, all study individuals were sedentary or undertook very low levels of physical activity. To minimize the contribution of NO metabolites derived from inducible NO synthase, subjects with active inflammatory disease were excluded from this study. In summary, appropriate dietary restrictions and exclusion criteria are required when employing measurements of urinary NO metabolites as possible indicators of NO derived from vascular endothelium and, perhaps, from renal tissues.17–19 It should be emphasized, however, that the contribution of neuronal NO synthase to the plasma and urine levels of NO metabolites has not been established. However, recent findings suggest that NO derived from neuronal NO synthase activity may play a role in salt-sensitive Dahl/Rapp rats.22 Other laboratories have also evaluated the role of NO in salt sensitivity in human subjects, employing plasma and/or urine levels to assess the activity of the l-arginine–NO pathway. Campese and coworkers13 reported that hypertensive blacks and higher plasma NO metabolite levels that normotensives, and that plasma NO metabolite levels were reduced during high salt intake, both in salt-sensitive and salt-resistant black hypertensives. In agreement with these investigators, we observed that a high intake of salt was associated with a lower urinary excretion of NO metabolites in all patients combined. However, our findings diverged from those of Campese and coworkers13 in that the high salt intake reduced NO levels in salt-sensitive, but not in salt-resistant individuals. Several factors may account for the discrepancy in the results. These include racial differences (blacks versus Hispanics) and baseline level of salt intake (not reported in Campese et al’s study and around 150 mmol/day in our study subjects). It is feasible that our study patients developed adaptive changes in NO excretion as a result of the long-term intake of high amounts of salt.16 Another factor is the biological fluid in which NO metabolites were measured (plasma versus urine). Theoretically, plasma levels may better reflect systemic production, although we can not rule out that increases in NO production in the renal parenchyma would not lead to increases in plasma NO metabolites. Urinary excretion may reflect both systemic and renal production; therefore, differences in the origin of NO metabolites may explain the discrep-
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ancies between studies. Finally, we must consider baseline BP levels. Our salt-sensitive subjects had higher baseline BP levels than the salt-resistant individuals, and experienced a further increase in BP when placed on a high-salt diet. Hypertensives are known to have higher basal NO levels, which decrease when the intake of salt is increased.13 Therefore, the differential effects induced by changes in salt intake on NO metabolite excretion in salt-sensitive and saltresistant subjects may be partly due to the higher BP and the further BP increases induced by the high-salt diet in the salt-sensitive group (present study). If NO is the main determinant of the pressurenatriuresis relationship, a relationship between saltinduced changes in NO excretion and in BP should be observed. In this case, one should expect that reductions in BP induced by salt restriction would be related to the increases in NO metabolite levels. However, in salt-sensitive subjects, the changes in urinary NO metabolites induced by reducing salt intake were not related to the changes in BP produced by the salt manipulation. Because salt-resistant individuals by definition experienced no changes in BP with changes in salt intake, no correlation was observed between changes in NO and in BP. These findings suggest that although salt sensitivity is associated with lower NO metabolite excretion during high than during low salt intake, the magnitude of the BP changes (degree of salt sensitivity) may be determined by more than one factor. In addition, there may be no cause-effect relationship between these two variables. However, Facchini and coworkers23 reported in healthy normotensive subjects that the changes in BP induced by the highsalt diet (174 mmol/day) were inversely related to the changes in urinary nitrate excretion. Unfortunately, only five of their 19 subjects could be classified as salt sensitive. Further studies are required to clarify this aspect. Salt sensitivity has been reported to cluster with other cardiovascular risk factors. Obesity, black race, older age, insulin resistance, and hypertension have been associated with salt sensitivity.3,24 –29 Body weight reduction in obese adolescents has been shown to lower BP and to correct salt sensitivity.30 Because, in this study, patients were classified solely on their BP response to salt intake, it was not unexpected to find that our salt-sensitive individuals were older, heavier, and had higher BP than the salt-resistant subjects. It is thus also possible that older age, higher weight, and BP could play a role, in addition to salt sensitivity, in determining the marked suppression of NO metabolite excretion observed during high salt. In conclusion, an increase in salt intake reduced the urinary excretion of NO metabolites in all patients combined, an effect due to a major effect of salt intake in salt-sensitive individuals. We demonstrated that
lowering salt intake produced large reductions in BP associated with increases in the urinary excretion of NO metabolites in salt-sensitive individuals. In these subjects, the urinary excretion of NO metabolites was 45% lower during high than during low salt intake. In salt-resistant individuals, reductions in salt intake were not associated with mean changes in NO metabolite excretion. To the extent that urinary NO metabolite levels reflect the activity of the endogenous NO system, our results support the view that salt sensitivity may in part be determined by an inability to increase or to sustain NO production in response to high salt. Insufficient NO production during high salt may, in turn, lead to altered pressure-natriuresis relationships leading to moderate to marked increases in BP. The possibility that the increase in BP induced by high salt intake in salt-sensitive individuals could be the key factor in reducing NO metabolite levels can not be ruled out. REFERENCES 1.
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