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Does a low-salt diet exert a protective effect on endothelial function in normal rats?
Does a low-salt diet exert a protective effect on endothelial function in normal rats? A. H. BOONSTRA, S. GSCHWEND, M. J. A. KOCKS, H. BUIKEMA, D. DE ZEEUW, and G. J. NAVIS GRONINGEN, THE NETHERLANDS
Sodium restriction is often used as an adjunct in the treatment of conditions characterized by endothelial dysfunction, such as hypertension and heart or kidney disease. However, the effect of sodium restriction on endothelial function is not known. Therefore, male Wistar rats were studied after a fixed salt diet had been maintained (low-salt group: 0.05% NaCl, n = 10; normal-salt group: 0.3% NaCl, n = 10) for 6 weeks. Blood pressure and sodium excretion values were measured once a week. Subsequently the rats were killed, the aorta was removed, and rings were cut. Endothelium-independent (sodium nitrite [SN]) and endothelium-dependent (acetylcholine [ACh]) vasodilator responses were assessed in the presence of indomethacin (a cyclo-oxygenase inhibitor) and in the presence or absence of NG-monomethyl-L-arginine (L-NMMA; a competitive inhibitor of nitric oxide [NO] synthase). Endothelium-independent vasodilatation was not different for the two salt groups. Endothelium-dependent vasodilatation, on the other hand, was different. The response to ACh was almost completely abolished by L-NMMA in the normal-salt group, whereas vasodilatation was partially preserved during L-NMMA in the low-salt group. Accordingly, the L-NMMA–sensitive contribution to ACh-dependent vasodilatation was smaller in the low-salt group. Thus, salt restriction induced a non-NO and non-prostaglandin–dependent vasodilating pathway. By exclusion this could be endothelium-derived hyperpolarizing factor, a pathway of vasculoprotective potential. Accordingly, the relative contributions of the different vasoactive endothelial pathways were affected by salt intake. Further research will be needed to clarify the nature and importance of this non-NO, nonprostaglandin–dependent pathway in the clinical setting as well. (J Lab Clin Med 2001;138:200-5) Abbreviations: ACh = acetylcholine; AUC = area under the curve; EDHF = endotheliumderived hyperpolarizing factor; L-NMMA = NG-monomethyl-L-arginine; NO = nitric oxide; NTG = nitroglycerine; PhE = phenylephrine; SN = sodium nitrite
A
n intact endothelium is considered an important contributor in the defense barrier against several cardiovascular risk factors. Accordingly, endothelial dysfunction is associated with an increased vulnerability to cardiovascular risk factors.1 From the Department of Internal Medicine, Division of Nephrology, University Hospital Groningen; and the Department of Clinical Pharmacology, University of Groningen. Submitted for publication December 15, 2000; revision submitted May 23, 2001; accepted May 30, 2001. Reprint requests: A. H. Boonstra, MD, University Hospital Groningen, Department of Internal Medicine, Division of Nephrology, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Copyright © 2001 by Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/117556 doi:10.1067/mlc.2001.117556 200
Hypertension and heart and and kidney disease are often characterized by endothelial dysfunction. Excess sodium intake has deleterious effects in each of these conditions. These are partly explained by the effect of excess sodium intake on extracellular volume and blood pressure, but blood pressure–independent effects on left ventricular hypertrophy have been reported as well.2 Excess sodium has been shown to blunt the vasodilator response to ACh in several studies in isolated blood vessels, indicating impairment of endothelial function.3,4 These effects might be caused by the impact of sodium status on endocrine and paracrine systems involved in the regulation of vascular tone and function, such as the renin-angiotensin system, the NO system, and vasoactive prostaglandins.
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The effects of dietary salt restriction on endothelial vasodilator function, however, have not been investigated. This would be important because dietary salt restriction is a frequent adjunct in the treatment of hypertension and heart and kidney disease.5-7 In the present study we therefore investigated the effects of long-term dietary salt restriction on in vitro endothelial vasodilatory function in the isolated aorta of Wistar rats. To exclude effects of endothelial dysfunction caused by an underlying disorder, the experiments were conducted in normal rats. METHODS Experimental protocol. Studies were performed in male Wistar rats (240 to 250 g; Harlan, Zeist, The Netherlands). Before being entered in the study protocol, rats were placed on a reference diet for 1 week that contained 0.3% NaCl (Hope Farms BV, Woerden, The Netherlands). They had free access daily to fresh tap water. Subsequently rats were randomized into a low-salt group and into a normal-salt group whose diets contained, respectively, 0.05% NaCl and 0.3% NaCl. Each group consisted of 10 animals that were maintained on this diet for 6 weeks. During the study period the animals were housed in group cages in a temperature-controlled room with a fixed light/dark cycle. Once a week body weight, food intake, and 24-hour urine samples were collected in metabolic cages. Blood pressure was measured twice a week with the animals in the conscious state. At the end of the study period, rats were anesthetized with a mixture of oxygen and isoflurane, 2.5%. The aorta was rapidly excised for functional measurements in vitro. Blood pressure measurements. Before the experiments, animals were trained to get accustomed to the measurement procedure. To rule out interindividual differences and environmental influences, all measurements were performed by the same observer in a single room with no other animals around. Systolic blood pressure was measured with an automated multichannel system (Apollo 179; IITC, Life Science, Woodland Hills, CA). This system uses tail cuffs and photoelectric sensors to detect tail pulse. To this purpose the rats are placed in the test chamber in restrainers appropriate for their size and body weight. During each blood pressure measurement session, five measurements were recorded for each animal. Blood pressure was taken as the mean of the last three recordings. Dilatation measurements in the isolated aorta. Immediately after removal, the thoracic descending aorta was placed in a Krebs bicarbonate solution equilibrated with 95% O2 and 5% CO2 as described previously.8 After removal of connective tissue, rings of 2-mm length were cut with a sharp razor blade, with care not to disrupt the integrity of the endothelium. The rings were mounted in 15-mL organ baths filled with Krebs solution at 37.5°C and connected to a displacement transducer to determine isotonic changes. Rings were subjected to 14 mN and allowed to stabilize for 60 minutes, during which regular washing was performed. All measurements were performed in the presence of 10 µmol/L indomethacin, a cyclo-oxygenase inhibitor, to avoid potential production and interference of vasoactive prostanoids.
A
B Fig 1. Nitroglycerine-induced (NTG) vasodilatation as a percentage of SN in isolated aortic rings of Wistar rats, in the presence of indomethacin, receiving a salt-restricted diet versus a normal-salt diet, without (A) and with (B) L-NMMA.
Rings were primed by evoking a contraction with 1 µmol/L phenylephrine followed by repeated washings and repeated stabilization before being used in the protocol. Endothelium-dependent and endothelium-independent vasodilatation was tested, in a parallel design, as the dose-response to ACh (10 nmol/L to 0.1 mmol/L) and NTG (1 nmol/L to 10 µmol/L), respectively, in 1 µmol/L phenylephrine precontracted rings after preincubation with either vehicle (saline solution) or 100 µmol/L L-NMMA.9-11 In all cases, 10 mmol/L SN was given at the end of the dose-response curves so as to ensure assessment of maximal dilatation.
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sis of variance in the case of dose-response profiles) was used for primary evaluation of group means, in combination with post-hoc (Bonferroni-corrected) t statistics. Significance was assumed at P values < .05 (two-tailed). RESULTS
A
B Fig 2. ACh-induced vasodilatation as a percentage of SN in isolated aortic rings of Wistar rats, in the presence of indomethacin, receiving a salt-restricted diet versus a normal-salt diet, without (A) and with (B) L-NMMA.
Data analysis. The responses to ACh and NTG were calculated as the percentage change in maximum relaxation to SN for each ring. To compare the total response curves between the two salt groups in response to ACh, in the absence and presence of L-NMMA, the area size (AUC) was measured. The contribution of the NO cascade on the vasodilatory response to ACh was assessed by calculating the difference between the two in the presence and absence of LNMMA. The difference between the two was considered an estimate for the contribution of the NO cascade. Data are presented as mean ± SEM unless stated otherwise. One-way analysis of variance (and repeated-measures analy-
At baseline and during the study, no differences existed between the low-salt and control groups regarding weight and systolic blood pressure; that is, factors that might have an influence on endothelial function. Systolic blood pressure increased normally with age in both groups and was similar in the two groups throughout the study. In agreement with the diet, urinary sodium excretion was significantly lower throughout the study in the low-salt group as compared with the normal-salt group. Data at termination are given in Table I. Endothelium-independent and endothelium-dependent vasodilatation. Maximal endothelium-independent
vasodilatation to NTG was virtually 100% of the vasodilatation to SN for the two salt groups (Fig 1, A). As expected, the endothelium-independent vasodilatation was not affected by L-NMMA in both salt groups (Fig 1, B). The response to ACh in the absence of L-NMMA tended to be lower in the low-salt group as compared with the normal-salt group, albeit not significantly so (Fig 2, A). As expected, vasodilatation to ACh was almost completely abolished by the presence of L-NMMA in the aorta rings of the normal-salt group. In the low-salt group, to the contrary, vasodilatation to ACh was partially preserved during L-NMMA (Fig 2, B; P < .05 between the two groups). Fig 3 (left side of divider) shows the total dose response to ACh, depicted as the AUC, for the two salt groups, without and with L-NMMA. In accord with the data on the separate dose steps shown in Fig 2, the difference between the low-salt and the normal-salt group did not reach statistical significance for the responses without LNMMA, whereas with L-NMMA the AUC was significantly different between the groups (P = .021). Fig 3 (right side of divider) shows the magnitude of the modulating effect of L-NMMA on the response to ACh, depicted as the difference between the AUCs without and with L-NMMA. The modulating effect of L-NMMA was significantly larger in the normal-salt group (P = .037). DISCUSSION
This study is the first to demonstrate an effect of a low-salt diet per se on endothelial function. Whereas in rats on control salt intake the response to ACh was completely annihilated by L-NMMA, the low-salt group showed a preserved vasodilator response to ACh despite NO inhibition by L-NMMA. Because prostaglandin synthesis was also inhibited, it cannot be due to vasodila-
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Table I. Clinical characteristics Normal-salt group (n = 10)
Body weight (g) Systolic BP (mm Hg) UNa (µmol/24 h)
349 ± 7 129 ± 5 751 ± 50
Low-salt group (n = 10)
361 ± 9 131 ± 6 235 ± 56*
Body weight, blood pressure, and urinary sodium excretion at the end of the study (mean ± SEM). *P < .001 versus the normal-salt group.
tory prostaglandins. The endothelium-independent vasodilatation was similar on the two salt intakes, indicating that the signaling mechanisms for relaxation of the vascular smooth muscle cells to NO were not affected by sodium intake. To interpret these findings, it is important to consider the level of salt intake in relation to endothelial function. Different animal models, species, and salt regimens—in some cases even up to an excessive amount of 8% NaCl— have been used to explore the influence of salt intake on endothelial function. Control groups in these studies usually receive approximately 0.30% of NaCl in their diets.4,12-14 In these studies, high sodium intake was associated with harmful effects on the endothelium. However, whether those results were caused by the sodium intake as such or by the confounding effect of a high blood pressure in some of the studies cannot be concluded. In our study we used healthy normotensive rats, and the normal-salt group was fed a 0.30% NaCl–containing diet versus a 0.05% NaCl–containing diet in the group of interest. By this approach we could avoid the confounding effect of an altered blood pressure on the endothelium. Of note, blood pressure was measured by the tailcuff method. With this method, small differences in blood pressure between the groups may have been missed. However, in our hands the reproducibility of group means is high. Substantial differences in blood pressure between the groups, therefore, are unlikely. Thus the observed effect of a low-salt diet—that is, induction of a non-NO and non-prostaglandin pathway—appears to be caused by dietary salt restriction as such. Several dilatory pathways, such as NO and the prostaglandin system, are involved in endothelial function. Nowadays it is known that neither NO nor prostacyclin is the sole mediator of endothelium-dependent vasodilatation. Another, as-yet-unidentified, factor (or factors) that hyperpolarizes the underlying vascular smooth muscle cells may contribute to the endothelium-dependent vasodilatation and has therefore been termed EDHF.15 We tested endothelium-dependent vasodilatation as the response to ACh. Under normal conditions, NO is a main mediator of the ACh-induced
Fig 3. Response size (displayed as AUC) of ACh-induced vasodilatation in isolated aortic rings of Wistar rats fed a normal- or lowsalt diet, without (open bars) and with (hatched bars) L-NMMA (left side of vertical dashed dividing line). The right side of the vertical dashed dividing line shows the magnitude of the modulating effect of L-NMMA for the normal- and the low-salt group. Data are expressed as mean ± SEM.
vasodilatation, as shown by the annihilation of the response by L-NMMA in the normal-salt animals. The preserved vasodilatation in response to ACh during LNMMA in the low-salt group strongly suggests an enhanced impact of another vasodilatory pathway induced by a low-salt diet. Because the experiments were conducted during prostaglandin synthesis inhibition, EDHF—by exclusion—might be involved. Kilpatrick and Cocks16 demonstrated that both NO and hyperpolarization contribute differentially to endothelium-dependent relaxation in vitro, and they postulated hyperpolarization to act as a reserve or “backup” system that becomes important when the main vasodilator system (ie, NO) fails. More recently, several authors observed a shifted contribution of components maintaining vascular tone in response to ACh in spontaneously hypertensive rats receiving excessive sodium intake—that is, an increase in NO and a decrease in EDHF.12,14 We showed that salt intake in healthy WKY rats is able to shift the contribution of the different vasoactive endothelial pathways, with salt restriction eliciting an increased non-NO, non-prostaglandin contribution. Other studies have shown that sodium restriction can enhance vascular NO formation,17,18 a finding that is in accord with an effect of sodium on vasoactive endothelial pathways as well. In the present study we have no formal proof that NO blockade was complete during low salt, which raises the possibility that incomplete blockade of NO was involved in the findings during low salt. However, the dose of L-NMMA used in this study has been shown to block NO formation (almost) completely in studies of different design.9,10 Previous studies in WKY rats showed that a high sodium diet can promote end organ damage indepen-
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dent of arterial blood pressure and intravascular volume.19 In addition, a more recent study by Dworkin et al20 showed that dietary sodium restriction per se could prevent organ damage in Wistar rats with established kidney disease. Our data provide a possible mechanism for the vascular protective effects of low salt, because EDHF is suggested to have protective effects on the vascular bed.12 Moreover, our finding may be useful for the methodologic aspects in further exploring the role of vasodilator pathways alternative to NO and prostaglandins—hence EDHF—in vascular function. As to the possible protective effect of low salt, it is important to mention that our results were obtained in healthy rats, whereas the hemodynamic and vascular effects of a low-salt diet may well depend on the presence of disease.21,22 Moreover, whether a particular hemodynamic or vascular effect of low sodium is beneficial may also depend on the disease condition. For instance, renal vasodilation is induced by low sodium in diabetes, which may turn out to be unfavorable, considering the ensuing hyperfiltration.23-25 Furthermore, low sodium has been shown to decrease the vascular sensitivity for insulin in rats and human subjects,26,27 indicating an increased insulin resistance. Other clinical evidence, particularly in salt-sensitive individuals or hypertensive patients, showed that a high-sodium diet can affect end organ circulation.28,29 The Intersalt study recommended furthermore a reduction in sodium intake to prevent and control adverse blood pressure levels.30 It would therefore be of interest to investigate whether the observations made in this study also apply in human beings. We conclude that a long-term low-salt diet can affect endothelial function independent of blood pressure by inducing a non-NO and non-prostaglandin-dependent vasodilatation that could, by exclusion, be EDHF. It would be important, next, to establish whether such an effect is also present in disease states. For methodologic purposes, our data show that a low-salt diet can be used as a tool to induce vasodilator pathways alternative to NO and prostaglandin for studies aimed at exploring the effects of EDHF in vascular function. REFERENCES
1. Vaskonen T, Mervaala E, Krogerus L, Teravainen TL, Laasko J, Karppanen H, et al. Cardiovascular effects of chronic inhibition of nitric oxide synthesis and dietary salt in spontaneously hypertensive rats. Hypertens Res 1997;20:183-92. 2. Schmieder RE, Langenfeld MR, Friedrich A, Schobel HP, Gatzka CD, Weihprecht H. Angiotensin II related to sodium excretion modulates left ventricular structure in human essential hypertension. Circulation 1996;94:1304-9. 3. Boegehold MA. Flow-dependent arteriolar dilation in normotensive rats fed low- or high-salt diets. Am J Physiol 1995;269:H1407-14. 4. Liu Y, Rusch NJ, Lombard JH. Loss of endothelium and
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receptor-mediated dilation in pial arterioles of rats fed a short-term high salt diet. Hypertension 1999;33:686-8. He J, Whelton PK. Role of sodium reduction in the treatment and prevention of hypertension. Curr Opin Cardiol 1997;12:202-7. Navis G, de Jong P, Donker AJ, van der Hem GK, de Zeeuw D. Diuretic effects of angiotensin-converting enzyme inhibition: comparison of low and liberal sodium diet in hypertensive patients. J Cardiovasc Pharmacol 1987;9:743-8. Heeg JE, de Jong PE, van der Hem GK, de Zeeuw D. Efficacy and variability of the antiproteinuric effect of ACE inhibition by lisinopril. Kidney Int 1989;36:272-9. Buikema H, Grandjean JG, van-den-Broek S, van-Gilst WH, Lie KI, Wesseling H. Differences in vasomotor control between human gastroepiploic and left internal mammary artery. Circulation 1992;86(suppl 5):II205-9. Rees DD, Palmer RM, Schulz R, Hodson HF, Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 1990;101:746-52. Moore PK, al Swayeh OA, Chong NW, Evans RA, Gibson A. L-NG-nitro arginine (L-NOARG), a novel, L-argininereversible inhibitor of endothelium-dependent vasodilatation in vitro. Br J Pharmacol 1990;99:408-12. Buikema H, Monnink SH, Tio RA, Crijns HJ, de Zeeuw D, van Gilst WH. Comparison of zofenopril and lisinopril to study the role of the sulfhydryl-group in improvement of endothelial dysfunction with ACE inhibitors in experimental heart failure. Br J Pharmacol 2000;130:1999-2007. Kagota S, Tamashiro A, Yamaguchi Y, Nakamura K, Kunitomo M. Excessive salt or cholesterol intake alters the balance among endothelium-derived factors released from renal arteries in spontaneously hypertensive rats. J Cardiovasc Pharmacol 1999;34:533-9. Fujii K, Ohmori S, Onaka U, Abe I, Fujishima M. Effects of salt loading on membrane potentials in mesenteric arteries of spontaneously hypertensive rats. Hypertens Res 1999; 22:181-6. Ghisdal P, Godfraind T, Morel N. Effect of nitro-L-arginine on electrical and mechanical responses to acetylcholine in the superior mesenteric artery from stroke-prone hypertensive rat [erratum appears in Br J Pharmacol 2000;129:1056]. Br J Pharmacol 1999;128:1513-23. Feletou M, Vanhoutte PM. The alternative: EDHF. J Mol Cell Cardiol 1999;31:15-22. Kilpatrick EV, Cocks TM. Evidence for differential roles of nitric oxide (NO) and hyperpolarization in endotheliumdependent relaxation of pig isolated coronary artery. Br J Pharmacol 1994;112:557-65. Tojo A, Kimoto M, Wilcox CS. Renal expression of constitutive NOS and DDAH: separate effects of salt intake and angiotensin. Kidney Int 2000;58:2075-83. Fujiwara N, Osanai T, Kamada T, Katoh T, Takahashi K, Okumura K. Study on the relationship between plasma nitrite and nitrate level and salt sensitivity in human hypertension: modulation of nitric oxide synthesis by salt intake. Circulation 2000;101:856-61. Paradise NF, Sadri F, Ely DL. Left ventricular mass and fluid compartmentalization in Wistar-Kyoto and spontaneously hypertensive rats fed high- or low-sodium diet. J Hypertens 1986;4(suppl):S189-91. Dworkin LD, Benstein JA, Tolbert E, Feiner HD. Salt restriction inhibits renal growth and stabilizes injury in rats with established renal disease. J Am Soc Nephrol 1996;7:437-42. Williams GH, Dluhy RG, Lifton RP, Moore TJ, Gleason R,
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Williams R, et al. Non-modulation as an intermediate phenotype in essential hypertension. Hypertension 1992;20:78896. Miyoshi A, Suzuki H, Fujiwara M, Masai M, Iwasaki T. Impairment of endothelial function in salt-sensitive hypertension in humans. Am J Hypertens 1997;10:1083-90. Miller JA. Renal responses to sodium restriction in patients with early diabetes mellitus. J Am Soc Nephrol 1997;8:74955. Vallon V, Wead LM, Blantz RC. Renal hemodynamics and plasma and kidney angiotensin II in established diabetes mellitus in rats: effect of sodium and salt restriction. J Am Soc Nephrol 1995;5:1761-7. Luik PT, Van der Kleij FGH, Beusekamp BJ, Dullaart RP, de Jong PE, de Zeeuw D, et al. Sodium restriction elicits an unfavourable renal hemodynamic profile in well-controlled, normotensive, normoalbuminuric type I diabetes mellitus (DM) [abstract]. J Am Soc Nephrol 2000;9:118A. Vecchione C, Morisco C, Fratta L, Argenziano L, Trimarco
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B, Lembo G. Dietary sodium restriction impairs endothelial effect of insulin. Hypertension 1998;31:1261-5. Feldman RD, Schmidt ND. Moderate dietary salt restriction increases vascular and systemic insulin resistance. Am J Hypertens 1999;12:643-7. Barba G, Cappuccio FP, Russo L, Stinga F, Iacone R, Strazzullo P. Renal function and blood pressure response to dietary salt restriction in normotensive men [comment]. Hypertension 1996;27:1160-4. van Paassen P, de Zeeuw D, Navis G, de Jong PE. Does the renin-angiotensin system determine the renal and systemic hemodynamic response to sodium in patients with essential hypertension [comment]? Hypertension 1996;27:202-8. Elliott P, Stamler J, Nichols R, Dyer AR, Stamler R, Kesteloot H, et al. Intersalt revisited: further analyses of 24 hour sodium excretion and blood pressure within and across populations. Intersalt Cooperative Research Group [comment] [erratum appears in BMJ 1997;23:315]. BMJ 1996; 312:1249-53.
Sodium restriction is often used as an adjunct in the treatment of conditions characterized by endothelial dysfunction, such as hypertension and heart or kidney disease. However, the effect of sodium restriction on endothelial function is not known. Therefore, male Wistar rats were studied after a fixed salt diet had been maintained (low-salt group: 0.05% NaCl, n = 10; normal-salt group: 0.3% NaCl, n = 10) for 6 weeks. Blood pressure and sodium excretion values were measured once a week. Subsequently the rats were killed, the aorta was removed, and rings were cut. Endothelium-independent (sodium nitrite [SN]) and endothelium-dependent (acetylcholine [ACh]) vasodilator responses were assessed in the presence of indomethacin (a cyclo-oxygenase inhibitor) and in the presence or absence of NG-monomethyl-L-arginine (L-NMMA; a competitive inhibitor of nitric oxide [NO] synthase). Endothelium-independent vasodilatation was not different for the two salt groups. Endothelium-dependent vasodilatation, on the other hand, was different. The response to ACh was almost completely abolished by L-NMMA in the normal-salt group, whereas vasodilatation was partially preserved during L-NMMA in the low-salt group. Accordingly, the L-NMMA–sensitive contribution to ACh-dependent vasodilatation was smaller in the low-salt group. Thus, salt restriction induced a non-NO and non-prostaglandin–dependent vasodilating pathway. By exclusion this could be endothelium-derived hyperpolarizing factor, a pathway of vasculoprotective potential. Accordingly, the relative contributions of the different vasoactive endothelial pathways were affected by salt intake. Further research will be needed to clarify the nature and importance of this non-NO, nonprostaglandin–dependent pathway in the clinical setting as well. (J Lab Clin Med 2001;138:200-5) Abbreviations: ACh = acetylcholine; AUC = area under the curve; EDHF = endotheliumderived hyperpolarizing factor; L-NMMA = NG-monomethyl-L-arginine; NO = nitric oxide; NTG = nitroglycerine; PhE = phenylephrine; SN = sodium nitrite
A
n intact endothelium is considered an important contributor in the defense barrier against several cardiovascular risk factors. Accordingly, endothelial dysfunction is associated with an increased vulnerability to cardiovascular risk factors.1 From the Department of Internal Medicine, Division of Nephrology, University Hospital Groningen; and the Department of Clinical Pharmacology, University of Groningen. Submitted for publication December 15, 2000; revision submitted May 23, 2001; accepted May 30, 2001. Reprint requests: A. H. Boonstra, MD, University Hospital Groningen, Department of Internal Medicine, Division of Nephrology, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Copyright © 2001 by Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/117556 doi:10.1067/mlc.2001.117556 200
Hypertension and heart and and kidney disease are often characterized by endothelial dysfunction. Excess sodium intake has deleterious effects in each of these conditions. These are partly explained by the effect of excess sodium intake on extracellular volume and blood pressure, but blood pressure–independent effects on left ventricular hypertrophy have been reported as well.2 Excess sodium has been shown to blunt the vasodilator response to ACh in several studies in isolated blood vessels, indicating impairment of endothelial function.3,4 These effects might be caused by the impact of sodium status on endocrine and paracrine systems involved in the regulation of vascular tone and function, such as the renin-angiotensin system, the NO system, and vasoactive prostaglandins.
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The effects of dietary salt restriction on endothelial vasodilator function, however, have not been investigated. This would be important because dietary salt restriction is a frequent adjunct in the treatment of hypertension and heart and kidney disease.5-7 In the present study we therefore investigated the effects of long-term dietary salt restriction on in vitro endothelial vasodilatory function in the isolated aorta of Wistar rats. To exclude effects of endothelial dysfunction caused by an underlying disorder, the experiments were conducted in normal rats. METHODS Experimental protocol. Studies were performed in male Wistar rats (240 to 250 g; Harlan, Zeist, The Netherlands). Before being entered in the study protocol, rats were placed on a reference diet for 1 week that contained 0.3% NaCl (Hope Farms BV, Woerden, The Netherlands). They had free access daily to fresh tap water. Subsequently rats were randomized into a low-salt group and into a normal-salt group whose diets contained, respectively, 0.05% NaCl and 0.3% NaCl. Each group consisted of 10 animals that were maintained on this diet for 6 weeks. During the study period the animals were housed in group cages in a temperature-controlled room with a fixed light/dark cycle. Once a week body weight, food intake, and 24-hour urine samples were collected in metabolic cages. Blood pressure was measured twice a week with the animals in the conscious state. At the end of the study period, rats were anesthetized with a mixture of oxygen and isoflurane, 2.5%. The aorta was rapidly excised for functional measurements in vitro. Blood pressure measurements. Before the experiments, animals were trained to get accustomed to the measurement procedure. To rule out interindividual differences and environmental influences, all measurements were performed by the same observer in a single room with no other animals around. Systolic blood pressure was measured with an automated multichannel system (Apollo 179; IITC, Life Science, Woodland Hills, CA). This system uses tail cuffs and photoelectric sensors to detect tail pulse. To this purpose the rats are placed in the test chamber in restrainers appropriate for their size and body weight. During each blood pressure measurement session, five measurements were recorded for each animal. Blood pressure was taken as the mean of the last three recordings. Dilatation measurements in the isolated aorta. Immediately after removal, the thoracic descending aorta was placed in a Krebs bicarbonate solution equilibrated with 95% O2 and 5% CO2 as described previously.8 After removal of connective tissue, rings of 2-mm length were cut with a sharp razor blade, with care not to disrupt the integrity of the endothelium. The rings were mounted in 15-mL organ baths filled with Krebs solution at 37.5°C and connected to a displacement transducer to determine isotonic changes. Rings were subjected to 14 mN and allowed to stabilize for 60 minutes, during which regular washing was performed. All measurements were performed in the presence of 10 µmol/L indomethacin, a cyclo-oxygenase inhibitor, to avoid potential production and interference of vasoactive prostanoids.
A
B Fig 1. Nitroglycerine-induced (NTG) vasodilatation as a percentage of SN in isolated aortic rings of Wistar rats, in the presence of indomethacin, receiving a salt-restricted diet versus a normal-salt diet, without (A) and with (B) L-NMMA.
Rings were primed by evoking a contraction with 1 µmol/L phenylephrine followed by repeated washings and repeated stabilization before being used in the protocol. Endothelium-dependent and endothelium-independent vasodilatation was tested, in a parallel design, as the dose-response to ACh (10 nmol/L to 0.1 mmol/L) and NTG (1 nmol/L to 10 µmol/L), respectively, in 1 µmol/L phenylephrine precontracted rings after preincubation with either vehicle (saline solution) or 100 µmol/L L-NMMA.9-11 In all cases, 10 mmol/L SN was given at the end of the dose-response curves so as to ensure assessment of maximal dilatation.
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sis of variance in the case of dose-response profiles) was used for primary evaluation of group means, in combination with post-hoc (Bonferroni-corrected) t statistics. Significance was assumed at P values < .05 (two-tailed). RESULTS
A
B Fig 2. ACh-induced vasodilatation as a percentage of SN in isolated aortic rings of Wistar rats, in the presence of indomethacin, receiving a salt-restricted diet versus a normal-salt diet, without (A) and with (B) L-NMMA.
Data analysis. The responses to ACh and NTG were calculated as the percentage change in maximum relaxation to SN for each ring. To compare the total response curves between the two salt groups in response to ACh, in the absence and presence of L-NMMA, the area size (AUC) was measured. The contribution of the NO cascade on the vasodilatory response to ACh was assessed by calculating the difference between the two in the presence and absence of LNMMA. The difference between the two was considered an estimate for the contribution of the NO cascade. Data are presented as mean ± SEM unless stated otherwise. One-way analysis of variance (and repeated-measures analy-
At baseline and during the study, no differences existed between the low-salt and control groups regarding weight and systolic blood pressure; that is, factors that might have an influence on endothelial function. Systolic blood pressure increased normally with age in both groups and was similar in the two groups throughout the study. In agreement with the diet, urinary sodium excretion was significantly lower throughout the study in the low-salt group as compared with the normal-salt group. Data at termination are given in Table I. Endothelium-independent and endothelium-dependent vasodilatation. Maximal endothelium-independent
vasodilatation to NTG was virtually 100% of the vasodilatation to SN for the two salt groups (Fig 1, A). As expected, the endothelium-independent vasodilatation was not affected by L-NMMA in both salt groups (Fig 1, B). The response to ACh in the absence of L-NMMA tended to be lower in the low-salt group as compared with the normal-salt group, albeit not significantly so (Fig 2, A). As expected, vasodilatation to ACh was almost completely abolished by the presence of L-NMMA in the aorta rings of the normal-salt group. In the low-salt group, to the contrary, vasodilatation to ACh was partially preserved during L-NMMA (Fig 2, B; P < .05 between the two groups). Fig 3 (left side of divider) shows the total dose response to ACh, depicted as the AUC, for the two salt groups, without and with L-NMMA. In accord with the data on the separate dose steps shown in Fig 2, the difference between the low-salt and the normal-salt group did not reach statistical significance for the responses without LNMMA, whereas with L-NMMA the AUC was significantly different between the groups (P = .021). Fig 3 (right side of divider) shows the magnitude of the modulating effect of L-NMMA on the response to ACh, depicted as the difference between the AUCs without and with L-NMMA. The modulating effect of L-NMMA was significantly larger in the normal-salt group (P = .037). DISCUSSION
This study is the first to demonstrate an effect of a low-salt diet per se on endothelial function. Whereas in rats on control salt intake the response to ACh was completely annihilated by L-NMMA, the low-salt group showed a preserved vasodilator response to ACh despite NO inhibition by L-NMMA. Because prostaglandin synthesis was also inhibited, it cannot be due to vasodila-
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Table I. Clinical characteristics Normal-salt group (n = 10)
Body weight (g) Systolic BP (mm Hg) UNa (µmol/24 h)
349 ± 7 129 ± 5 751 ± 50
Low-salt group (n = 10)
361 ± 9 131 ± 6 235 ± 56*
Body weight, blood pressure, and urinary sodium excretion at the end of the study (mean ± SEM). *P < .001 versus the normal-salt group.
tory prostaglandins. The endothelium-independent vasodilatation was similar on the two salt intakes, indicating that the signaling mechanisms for relaxation of the vascular smooth muscle cells to NO were not affected by sodium intake. To interpret these findings, it is important to consider the level of salt intake in relation to endothelial function. Different animal models, species, and salt regimens—in some cases even up to an excessive amount of 8% NaCl— have been used to explore the influence of salt intake on endothelial function. Control groups in these studies usually receive approximately 0.30% of NaCl in their diets.4,12-14 In these studies, high sodium intake was associated with harmful effects on the endothelium. However, whether those results were caused by the sodium intake as such or by the confounding effect of a high blood pressure in some of the studies cannot be concluded. In our study we used healthy normotensive rats, and the normal-salt group was fed a 0.30% NaCl–containing diet versus a 0.05% NaCl–containing diet in the group of interest. By this approach we could avoid the confounding effect of an altered blood pressure on the endothelium. Of note, blood pressure was measured by the tailcuff method. With this method, small differences in blood pressure between the groups may have been missed. However, in our hands the reproducibility of group means is high. Substantial differences in blood pressure between the groups, therefore, are unlikely. Thus the observed effect of a low-salt diet—that is, induction of a non-NO and non-prostaglandin pathway—appears to be caused by dietary salt restriction as such. Several dilatory pathways, such as NO and the prostaglandin system, are involved in endothelial function. Nowadays it is known that neither NO nor prostacyclin is the sole mediator of endothelium-dependent vasodilatation. Another, as-yet-unidentified, factor (or factors) that hyperpolarizes the underlying vascular smooth muscle cells may contribute to the endothelium-dependent vasodilatation and has therefore been termed EDHF.15 We tested endothelium-dependent vasodilatation as the response to ACh. Under normal conditions, NO is a main mediator of the ACh-induced
Fig 3. Response size (displayed as AUC) of ACh-induced vasodilatation in isolated aortic rings of Wistar rats fed a normal- or lowsalt diet, without (open bars) and with (hatched bars) L-NMMA (left side of vertical dashed dividing line). The right side of the vertical dashed dividing line shows the magnitude of the modulating effect of L-NMMA for the normal- and the low-salt group. Data are expressed as mean ± SEM.
vasodilatation, as shown by the annihilation of the response by L-NMMA in the normal-salt animals. The preserved vasodilatation in response to ACh during LNMMA in the low-salt group strongly suggests an enhanced impact of another vasodilatory pathway induced by a low-salt diet. Because the experiments were conducted during prostaglandin synthesis inhibition, EDHF—by exclusion—might be involved. Kilpatrick and Cocks16 demonstrated that both NO and hyperpolarization contribute differentially to endothelium-dependent relaxation in vitro, and they postulated hyperpolarization to act as a reserve or “backup” system that becomes important when the main vasodilator system (ie, NO) fails. More recently, several authors observed a shifted contribution of components maintaining vascular tone in response to ACh in spontaneously hypertensive rats receiving excessive sodium intake—that is, an increase in NO and a decrease in EDHF.12,14 We showed that salt intake in healthy WKY rats is able to shift the contribution of the different vasoactive endothelial pathways, with salt restriction eliciting an increased non-NO, non-prostaglandin contribution. Other studies have shown that sodium restriction can enhance vascular NO formation,17,18 a finding that is in accord with an effect of sodium on vasoactive endothelial pathways as well. In the present study we have no formal proof that NO blockade was complete during low salt, which raises the possibility that incomplete blockade of NO was involved in the findings during low salt. However, the dose of L-NMMA used in this study has been shown to block NO formation (almost) completely in studies of different design.9,10 Previous studies in WKY rats showed that a high sodium diet can promote end organ damage indepen-
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dent of arterial blood pressure and intravascular volume.19 In addition, a more recent study by Dworkin et al20 showed that dietary sodium restriction per se could prevent organ damage in Wistar rats with established kidney disease. Our data provide a possible mechanism for the vascular protective effects of low salt, because EDHF is suggested to have protective effects on the vascular bed.12 Moreover, our finding may be useful for the methodologic aspects in further exploring the role of vasodilator pathways alternative to NO and prostaglandins—hence EDHF—in vascular function. As to the possible protective effect of low salt, it is important to mention that our results were obtained in healthy rats, whereas the hemodynamic and vascular effects of a low-salt diet may well depend on the presence of disease.21,22 Moreover, whether a particular hemodynamic or vascular effect of low sodium is beneficial may also depend on the disease condition. For instance, renal vasodilation is induced by low sodium in diabetes, which may turn out to be unfavorable, considering the ensuing hyperfiltration.23-25 Furthermore, low sodium has been shown to decrease the vascular sensitivity for insulin in rats and human subjects,26,27 indicating an increased insulin resistance. Other clinical evidence, particularly in salt-sensitive individuals or hypertensive patients, showed that a high-sodium diet can affect end organ circulation.28,29 The Intersalt study recommended furthermore a reduction in sodium intake to prevent and control adverse blood pressure levels.30 It would therefore be of interest to investigate whether the observations made in this study also apply in human beings. We conclude that a long-term low-salt diet can affect endothelial function independent of blood pressure by inducing a non-NO and non-prostaglandin-dependent vasodilatation that could, by exclusion, be EDHF. It would be important, next, to establish whether such an effect is also present in disease states. For methodologic purposes, our data show that a low-salt diet can be used as a tool to induce vasodilator pathways alternative to NO and prostaglandin for studies aimed at exploring the effects of EDHF in vascular function. REFERENCES
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