Salt, Salt Sensitivity, and the Endothelium
A Pathway to Discovery of Molecular Mechanisms
See related article, pp 951–956
This issue of Hypertension contains a report of molecular mechanisms transducing dietary salt intake1 that may have broad clinical relevance. These molecular mechanisms are complex, but to begin to understand this study we have to understand the importance of dietary salt intake and salt sensitivity. This background will be discussed in some detail before discussing the molecular mechanisms.
Although cohort studies have called into question the role of dietary salt intake in causing hypertension,2 cause and effect relationships are most convincing when demonstrated using randomized clinical trials. It would be unethical to perform a randomized clinical trial in which the dietary intake of salt (sodium chloride) is increased for years in humans to observe an increase in blood pressure. However, such an experiment in primates has been performed by Denton et al,3 who studied in Gabon, Africa, 26 chimpanzees. Chimpanzees have a native diet that is vegetarian and is very high in potassium content. The baseline sodium intake in these primates was only 6 mEq/d; in contrast, that of K was 235 mEq/d. Blood pressure was measured in the 26 animals for 1 year. Subsequently, among half the animals, increasing amounts of salt (in increments of 5 g/d sodium) were added to a diet to a steady-state level of 15 g/d. This dietary intake was maintained for 16 months. After this period of dietary supplementation, the diet was switched to baseline and observations made for another 6 months. A dose–response relationship between salt supplementation and blood pressure increment was seen. With 15 g/d sodium supplementation for 67 weeks, the change from baseline in blood pressure was 33/10 mm Hg. In comparison, 5 g/d sodium supplementation for 19 weeks provoked blood pressure change of 12/0 mm Hg. Notably, plasma renin activity was reduced with salt supplementation, suggesting that volume expansion may have been a mechanism to induce hypertension. These experiments support the notion that dietary salt supplementation increases blood pressure in primates.
It has also been recognized that response to increasing dietary salt intake is not always accompanied by hypertension. Some people, despite increasing salt intake to very high levels, remain normotensive. This was recognized a long time ago by Dahl,4 who hypothesized that the blood pressure response to an increase in salt intake is an inherited trait. He reasoned that if such a trait existed, it would be possible to segregate these genes by inbreeding. However, if such a trait was environmental, inbreeding experiments would not be able to separate out animals who are predisposed and those who are protected for hypertension in response to a high-salt intake. In this classic article published in 1962, Dr Dahl4 produced evidence to demonstrate that genetic factors play an important role in susceptibility to experimental hypertension. The blood pressure response to high-salt diet in animals is normally distributed. Thus, there would be individuals who have extreme increase in blood pressure or little increase in blood pressure. Dr Dahl reasoned that these extreme responses may be genetically determined. If response to blood pressure was inherited, then it should be possible to segregate the genes by inbreeding experiments. As a first step, he fed Sprague Dawley rats a high-salt diet and measured serial blood pressures. The blood pressure response was compared with animals given a low-salt control diet to demonstrate the response of salt on blood pressure. He then discovered that thyroid injections and a high-salt diet would uncover the blood pressure response within 4 days. Blood pressure in thyroid-salt–treated animals was much higher compared with a control group. Also, those animals on the high-salt diet stayed hypertensive at the end of 1 month and 1 year compared with controls. In the high-salt diet–fed animals, those that remained normotensive or hypertensive were selected and inbred for 3 generation. In the normotensive group, increasing resistance in response to a high-salt intake could be demonstrated in blood pressure response for 3 generations. In the hypertensive group, increasing blood pressure susceptibility to high-Na intake could be demonstrated with successive generations.
These experiments strongly support the role of genetic susceptibility to the development of experimental hypertension from excess salt ingestion. Dahl, in his original experiments, also reported that pneumonia swept the rat colony, preferentially killing the salt-sensitive rats. Could there be more than volume to the salt-sensitivity story? What is the underlying pathophysiology of hypertension in these animals?
These questions were asked by the group of Dr Sanders5 at the University of Alabama at Birmingham in the early 1990s. Young Dahl rats were randomized in a 2×2 design to receive either a low-salt or high-salt diet. Blood pressure was measured at baseline and also after infusing a drug, NG-monomethyl-l-arginine (l-NMMA), to block endothelial nitric oxide synthase. Elevation in blood pressure would then be a reflection of nitric oxide synthase activity. Thus, l-NMMA response to blood pressure was used as a biomarker of NO activity.
In the Dahl salt-resistant rats, baseline levels of mean arterial pressure (MAP) were similar, despite high salt feeding for 2 weeks; an expected response, given that these animals are resistant to salt feeding. A 21% increase in MAP was seen in animals treated with low-Na diet but a 31% increase with high-Na diet. These data suggest that NO production was stimulated with high salt feeding in salt-resistant animals. This fact was uncovered by greater increase in MAP with l-NMMA in high-Na–treated animals.
Further experiments revealed that salt-sensitive animals seem to be different from salt-resistant animals in that no NO stimulation occurs with Na feeding. This raises the possibility that these animals have a defect in endothelial function that underlies the salt sensitivity.
Awake unrestrained salt-sensitive rats did not develop hypertension when given l-arginine orally, despite consuming a high-salt diet. In contrast, d-arginine–treated animals were significantly hypertensive by day 2. Prevention of hypertension occurred in a dose–response fashion. l-Arginine did not lower blood pressure in the salt-resistant animals. In separate experiments, l-arginine supplementation did not prevent the development of hypertension in the spontaneously hypertensive rats indicating this mechanism to be specific to the Dahl rats. These observations were confirmed further by demonstrating that intraperitoneal injections of l-arginine in high-salt diet–fed salt-sensitive animals did not result in the development of hypertension. In contrast, injections of d-arginine did not protect from increased MAP. In l-arginine–treated animals, l-NMMA infusion increased MAP 45% from baseline. In contrast, among d-arginine–treated animals MAP increased only 18%. These data suggested that stimulation of NO production occurred with l-arginine feeding that restored the endothelial response to high salt feeding strongly pointing to endothelial dysfunction being causal in the pathogenesis of salt-sensitive hypertension.
Translation of the above findings to humans was provided by a study from Spain, in which 19 patients with salt-resistant hypertension confirmed by ambulatory blood pressure monitoring were compared with 26 patients with salt-sensitive hypertension.6 Although maximal vasodilatory responses to sodium nitroprusside were similar in the 2 groups, endothelium-dependent vasodilatation was less in the salt-sensitive group. l-NMMA produced a greater change in forearm blood flow in the salt-resistant group. The endothelium may also mediate the renal and systemic hemodynamic responses among healthy humans given a high-salt diet.7 Specifically, compared with a low-salt diet period, among 12 healthy volunteers participating in a crossover trial, l-NMMA resulted in a greater change from baseline in renal and systemic hemodynamics in subjects given a high-salt diet. Taken together, these data suggest that (1) the endothelium modulates the renal and systemic response to salt and (2) that the endothelial function is impaired in people with salt-sensitive hypertension.
From the discussion to date it is clear that the response to salt intake in populations is variable. Some members of the population remain normotensive, despite a high-salt intake. The missing link seems to be the vascular response. Those who are unable to vasodilate have a hypertensive response. Thus, although the endothelium can see the salt, how does it do so?
Although the endothelium has many eyes, my discussion is focused to a few pathways. The Sanders laboratory has demonstrated that the production of the profibrogenic cytokine, transforming growth factor-β1 (TGF-β1) is increased in rodents fed a high-salt diet within 2 to 4 days at a time point when volume expansion may not have occurred.8 This leads to an important and testable hypothesis whether high-salt intake initiate damage to the vasculature independently of blood pressure. To dissociate the effects of pressure from direct damage, molecular techniques in cultured cells have provided important insights.
In the current issue of Hypertension, the group led by Paul Sanders1 examines such molecular mechanisms using multiple lines of investigations from using whole animal models to cells in culture. They first demonstrate that compared with rats fed a low-salt diet (0.3%), within 4 days of feeding young Sprague Dawley rats with a high-salt diet (8%), several endothelial effects were seen. These included an increase in several endothelial proteins, such as TGF-β and phosphorylated Smad2 (mothers against decapentaplegic homolog 2) (Figure). Downstream, phosphorylation of Smad2 provoked phosphorylation of both the Akt (protein kinase B) and the endothelial isoform of nitric oxide synthase (NOS3) but decreased the concentration of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). After feeding high-salt diets for 2 days to these animals followed by another 2 days of a specific inhibitor of the TGF-β receptor 1/activin receptor–like kinase 5, phosphorylation of Smad2 was blocked to levels seen in animals fed a low-Na diet (0.3%). Furthermore, downstream events were also abrogated.
Next, to further understand the role of TGF-β1 on blood vessels, experiments in macrovascular endothelial cells were performed. Treatment of these cells with TGF-β1 increased phosphorylated NOS3 and as expected the concentration of nitric oxide metabolites in the medium. However, such effects were abolished by blocking PTEN via siRNA. Akt activation and NOS3 phosphorylation increased when PTEN was blocked; supplementing TGF-β1 in this setting provided no additional effects on Akt, NOS3, or nitric oxide production.
These experiments reveal the complex interaction of salt with endothelial cells on TGF-β1 production, stimulation of PTEN, and neutralization of the TGF-β1 effect on the vasculature by increased production of nitric oxide. Observations made in this study may be of great clinical relevance. For example, several molecular lesions can be postulated that may provoke vascular injury in response to a high-salt diet. These include a robust production in the vasculature of TGF-β1, lack of PTEN response, impaired Akt activation, and NOS3 phosphorylation culminating in reduced nitric oxide production. These pathological responses that likely will provoke vascular injury may occur because of genetic polymorphisms or acquired defects in these pathways. The latter may be seen with chronic kidney disease, increasing age, or other conditions associated with salt sensitivity. However, these pathways may be amenable to pharmacological intervention and increase our ability to protect the vasculature among the elderly or those with chronic kidney disease. Furthermore, it illustrates the perseverance of a group of investigators who with continued federal support have increased our understanding of the molecular basis of salt sensitivity.
Sources of Funding
R. Agarwal is supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases grant 2R01-DK062030-10 and a grant from the VA Merit review board.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- © 2013 American Heart Association, Inc.
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