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Institut National de la Santé et de la Recherche Médicale (INSERM) U367, Département de Santé Publique et d'Informatique Médicale, Faculté de Médecine Broussais Hôtel Dieu and INSERM U36, Collège de France, Paris, France; and Department of Chemical Pathology, Imperial College School of Medicine, Charing Cross Hospital Campus and FRCP, Blood Pressure Unit, Department of Medicine, St. George's Hospital Medical School, London, United Kingdom
ABSTRACT I. INTRODUCTION II. SALT INTAKE AND BLOOD PRESSURE A. Relation of Habitual Salt Intake to Blood Pressure B. Exceptions to the General Finding That Habitual Salt Intake Controls Blood Pressure C. Effect of Acute Changes in Salt Intake on Blood Pressure D. Prolonged Reductions in Salt Intake and Blood Pressure E. Salt Intake and Blood Pressure in Other Mammalian Species III. MECHANISMS BY WHICH HABITUAL SALT INTAKE CONTROLS BLOOD PRESSURE A. Central Role of the Kidneys B. Links Between an Inadequate Renal Capacity to Excrete a High Salt Intake and Hypertension C. Genetic Aspects of Renal Salt Handling 1. {alpha}-Adducin 2. Epithelial sodium channel 3. Aldosterone synthesis and signaling 4. Aldosterone-induced and ENaC interacting proteins 5. Sodium-chloride cotransport 6. With no lysine (WNK) serine-threonine kinases 7. Sodium-potassium-chloride cotransport (NKCC2), potassium (ROMK1) and chloride (ClC-Kb) channels 8. Sodium/proton exchanger 3 (NHE3) 9. Renin-angiotensin system 10. Other regulatory systems D. Salt Sensitivity IV. EVOLUTIONARY VIEWPOINT V. CONSEQUENCES FOR PUBLIC HEALTH ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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II. SALT INTAKE AND BLOOD PRESSURE |
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For several million years the evolutionary ancestors of humans ate a diet that contained <1 g salt/day (38, 88). This implies that present-day humans are genetically programmed to a salt intake of that amount. The deliberate addition of salt to food only began 
5,000–10,000 years ago at the beginning of agriculture and farming so that the present consumption of 10 g/day on average is, in evolutionary terms, relatively recent. The earliest comment that relates dietary salt to blood pressure is that of a Chinese physician Huang Ti Nei Ching Su Wein (1,700 BC) who stated from the translation by Wan Ping (AD 762) "therefore if large amounts of salt are taken, the pulse will stiffen and harden." Twentieth century epidemiological evidence on the relation of dietary salt to blood pressure varies between the clear-cut absence of hypertension in populations who absorb <3 g/day to the high incidence of hypertension in populations that consume >20 g/day (hypertension is defined throughout the review as a systolic and/or diastolic pressure over 140/90 mmHg). The relation of dietary salt to blood pressure in populations lying between these two extremes has been more difficult to define. The reasons are not only the range of salt intake between individuals is narrower but also, in contrast to the relative steadiness of body weight for example, the existence of large fluctuations in day-to-day salt intake within individuals. Nevertheless, it appears clearly that in populations on a salt intake >3 g/day, the proportion of individuals with hypertension rises with age, and the phenomenon is more pronounced when the salt intake is higher.
Approximately 40 nonacculturated tribes have been recorded which consumed <3 g salt/day (80). Their blood pressure did not rise with age (Fig. 1A). They lived, or still live, in South America, Africa, the Pacific, and the Arctic. The most striking example are the Yanomamo Indians on the border between Venezuela and Brazil (231, 265). They have a mean salt intake of <0.5 g/day, and at the age of 50 years, the blood pressure of men is only 100/64 mmHg. This lack of rise of blood pressure with age is not due to a peaceful Arcadian existence accompanying "the certainty of behaviour in a society ruled by ritual and taboo," in contrast to the "uncertainties of Western Societies in which life is a series of individual choices" (279). On the contrary, the Yanomamo have a culture that encourages aggression and a life of chronic warfare with violence and tension (50). They probably represent the ultimate human example of the overriding importance of dietary salt on blood pressure. There are two other nonacculturated tribes, which demonstrate that it is not the absence of acculturation per se that is responsible for the lack of rise of blood pressure with age in the populations consuming a low intake of salt. The Quash'Qai in Iran are nomadic herdsmen who inhabit an area that contains many natural surface deposits of salt (268). Mean salt excretion is 11 g/day in men and 9 g/day in women. Blood pressure in this nonacculturated society, which consumes the same levels of salt as those of economically developed societies, increases with age. The situation was similar in an area of Northern Kashmir that was unexposed to Western influences of industrialization, diet, and economy but in which the inhabitants also ate a relatively high salt intake (246). The high salt intake was due to their custom of drinking boiled tea to which they added various amounts of salt. Dietary surveys of the mean salt intake in three villages varied between 9.9 and 10.1 g/day with a wide range between individuals of 4.4–20.5 g/day. Both the systolic and diastolic pressure correlated significantly with the individual salt intake.
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The relationship of dietary salt to blood pressure in populations that consume >3 g/day and <20 g/day is intermediary between the situations described above. It has been difficult to find out whether there is a relationship between dietary salt and blood pressure if all populations, whatever the salt intake, are included. The overriding difficulty is the normal day-to-day within-individual variation (up to 10-fold fluctuations) in the amount of salt excreted in the urine, which reflects changes in dietary salt (225, 331). This difficulty is most relevant in the large number of populations that consume 10 g salt/day. Regression coefficients, which calculate relationships, are seriously underestimated in the presence of such large fluctuations within-individual variability in the independent variable (e.g., salt excretion), an effect known as regression dilution (202, 300). In other words, large within-subject variations mask the detection of differences between subjects. To minimize this effect, it is necessary to use large numbers of 24-h urine collections, and to obtain a true estimate of urinary salt excretion, it is necessary to collect at least half a dozen 24-h urine collections, which is impractical. The first attempt to relate the dietary intake of salt to the incidence of hypertension, which included populations on a moderate salt intake, was put forward by Meneely and Dahl (68). The data came from a total of five populations in only two of which was the intake of salt between 3 and 15 g/day. In addition to the smallness of the number of observations, information on the age of subjects and the definition of hypertension were unclear. Nevertheless, it is interesting that the relation of the salt intake to the blood pressure of these five populations was distributed along a straight line. Ten years later, Gliebermann (117) described the relation of the blood pressure of men aged 50–55 years to their salt intake in 27 populations (117). In 20 populations, dietary salt was >3 and <15 g/day, blood pressure appeared to rise with the salt intake, and the results lay between those obtained in subjects whose salt intake was <3 or >15 g/day. But the assessment of salt intake was haphazard, and no precautions had been taken to ensure that the measurement of blood pressure, the selection of the subjects, and the collection of urine were uniform. More importantly, the effect of several known confounding variables had not been taken into account such as potassium intake, body weight, and alcohol intake. The third set of populations studied was INTERSALT, an international epidemiological comparative study that began in 1981 (165). To remedy the drawbacks of the previous studies, standard methods were applied across a variety of populations, major confounding variables were studied simultaneously, and sufficient numbers were included to evaluate relationships in individuals. Altogether 10,079 subjects were involved in 52 centers worldwide. Each center recruited 200 men and women aged 20–59 years separated by age and sex into 8 equal 10-year groups. Urine samples were sent to a central laboratory. The study design allowed the relation between 24-h salt excretion and the blood pressure to be examined both in the 10,079 individuals within centers and across the 52 centers of study. Initial calculations found that salt excretion was significantly related to blood pressure in individuals but not across centers. On the other hand, salt excretion across centers was related to the slope of blood pressure with age (Fig. 1B). Various calculations included the finding that, after adjustment for age, sex, body mass index, and alcohol, a 5.7 g/day lower salt intake between the ages of 25 and 59 years was associated with a 9 mmHg lower rise of systolic pressure. As, however, an inquiry into the relationship of salt intake to the slope of the rise in blood pressure with age had not been anticipated in the original objectives of the INTERSALT study, some authorities considered this finding to be of doubtful importance (359). Subsequently, further calculations of the results to correct for the previous incomplete correction for the regression dilution problems posed by the day-to-day variations in individual salt excretion estimated the effect of a median salt excretion higher by 5.7 g/day. Over a 30-yr period (comparing age 55 to age 25) in cross population analyses there was a difference of 10 mmHg in systolic pressure and 6 mmHg in diastolic pressure (90). It was also found that higher salt excretions were associated with substantially greater differences in blood pressure in middle age compared with young adulthood. Another very large international epidemiological comparative study that has tested the relationship between 24-h urinary sodium excretion and blood pressure is the still ongoing CARDIAC (Cardiovascular Diseases and Alimentary Comparison) study (410). Overseen by the World Health Organization, very similar to the INTERSALT study in its settings, this study has so far examined the relation between 24-h sodium excretion and blood pressure in at least 3,681 men and 3,653 women aged 50–54 years from 60 centers in 25 countries worldwide. Cross-center correlation analyses showed that systolic blood pressure and diastolic blood pressure were positively associated with 24-h sodium excretion in both men and women, but the association was significant only in the men (410). The analysis of 2,212 women aged 48–56 years showed that after adjustment for age, body mass index, and 24-h urinary potassium excretion, 24-h sodium excretion was positively and significantly associated with systolic and with diastolic blood pressure in postmenopausal women (409). The associations were not significant in premenopausal women. Cross-center correlation analyses of the 21 centers, which had data on menopausal status indicated that 24-h sodium excretion was positively associated with systolic and with diastolic blood pressure in both pre- and postmenopausal women, but again this positive association was only significant in postmenopausal women. This suggests a tendency for salt sensitivity to increase at menopause (409).
Regional differences in habitual salt intake and blood pressure within a population have been documented. In Newfoundland, a survey of salt intake revealed that a county in the center of the island had a typical salt intake varying between 6.7 and 7.3 g/day. In contrast, the salt intake varied between 8.4 and 8.8 g/day in a relatively isolated coastal community where the diet traditionally contained a large quantity of salt (102). This difference in salt intake was accompanied by parallel changes in the incidence of hypertension defined as a diastolic pressure >100 mmHg. In individuals aged between 55 and 75 years, the incidence of hypertension in the inland community was 15% while it was 27% in the coastal community. Forty-five years before, a study was undertaken in Brazil on the dietary habits of two adjoining nonacculturated tribes, the Mundurucu and the Caraja. The Mundurucu had moved from a savannah-like forest to a Franciscan mission on the Cururu river where they had access to salt (80). Otherwise there was no substantial change in their diet. The Caraja lived on a nearby river and continued to eat their traditional low-salt diet. There was a rise in blood pressure with age in the men of the Mundurucu and a similar but not significant trend in the women. The blood pressure of the Caraja did not rise with age. Similar evidence has been obtained among the Solomon Islanders (267). In those tribes, which lived away from the coast and had a salt intake below 2 g/day, only 1% of the population had a raised blood pressure (systolic and/or diastolic over 140/90 mmHg). In two tribes with salt intakes between 3 and 8 g/day, 3% of the population had a raised blood pressure. In one tribe, which lived on the coast and cooked in "copious amounts of seawater" and had a salt intake between 9 and 15 g/day, 8% of the population had a raised blood pressure.
Migratory studies provide also evidences for a relation between habitual salt intake and blood pressure. There are several cases of groups of individuals from low salt-eating countries whose blood pressure rises when a change in their circumstances causes them to eat more salt. One good example was a carefully controlled study from Kenya where subsistence farmers ate a low-salt/high-potassium diet (282). Some of the farmers migrated to an urban community where they underwent a marked increase in salt intake with a fall of potassium intake to levels similar to the diet in Westernized countries (283). Blood pressure in these migrants rose after a few months (+6.9/6.2 mmHg for systolic and diastolic), whereas it did not increase in a control group who did not migrate. Another example of the effect of life-style changes including dietary sodium intake on blood pressure is that of the Yi people, an ethnic minority living in southwestern China. Blood pressure rose very little with increasing age (0.13 and 0.23 mmHg/yer for systolic and diastolic, respectively) in the Yi farmers who lived in their natural remote mountainous environment and consumed a sodium-poor diet. In contrast, Yi migrants and Han people who lived in urban areas consumed a sodium-rich diet and experienced a much greater increase in blood pressure with progressive aging (0.33 and 0.33 mmHg/yr for systolic and diastolic, respectively) (147). In a sample of 417 recent migrants (Yi) or native (Han) men living in the urban areas, a positive and statistically significant relationship was found between sodium intake and blood pressure (150). These findings suggest that changes in life-style, including higher intake of dietary sodium, contribute to the higher blood pressure among Yi migrants.
B. Exceptions to the General Finding That Habitual Salt Intake Controls Blood Pressure
There is a 30-year study in 144 Italian nuns and 138 controls who were living in the vicinity of the convent (368, 369). All the activities of the nuns are performed in strict isolation from urban life and in near absolute silence. Over the years the urinary salt excretion of the two groups was similar (7.5–8.0 g/day). The blood pressure of the control group rose, whereas the blood pressure of the nuns did not change. At the end of 30 years, the difference in blood pressure between the two groups was 30/15 mmHg. These results suggest that the hypertensive effect of dietary salt can be avoided by living in a stress-free monastic environment characterized by silence, meditation, and isolation from society. It is noticeable, however, that though the first account of these nuns appeared 10 years ago, these observations do not appear to have been confirmed. A nonacculturated tribe, the Kuna Indians, who live in the isolated San Bas Island chain off the Caribbean coast of Panama, also appears to be an exception. They have no rise in blood pressure yet a dietary assessment indicates that the consumption of salt was probably >8 g/day (156). It has to be stressed that this assessment was mainly based on a rough measurement of salt excretion and each subject's recollection of how many teaspoons of salt they had added to their food. The relative isolation of the Kuna could have facilitated the occurrence of a founder effect or a genetic shift that might explain their protection from hypertension; thus they may provide an attractive population for examining the genetic mechanisms involved in salt sensitivity.
C. Effect of Acute Changes in Salt Intake on Blood Pressure
Studies in humans on the effect of an acute change in salt intake on blood pressure have been carried out for the past 100 years. For the first 50 years they were undertaken on patients with hypertension and subsequently on both hypertensive patients and normal subjects.
The hypotensive effect of lowering the intake of salt in hypertensive patients was first demonstrated by Ambard and Beaujard in 1904 (9). At that time, presumably because of Bright's observation that in patients with severe overt renal disease the blood pressure is raised, the generally accepted view was that hypertension, even when there was no rise in blood urea or proteinuria, was due to protein intoxication. Ambard and Beaujard (9) varied the salt and protein content of the diet fed to six hypertensive patients. They performed 24-h metabolic balances by measuring the salt content of the food and the urine. They found that when the salt intake was suddenly reduced the patients went into negative salt balance and the blood pressure fell, even if the protein intake was raised. Inversely, an increase of the salt intake triggered a positive salt balance and an elevation of blood pressure even in the presence of a decreased protein intake, showing that it was salt and not the protein content of the diet that primarily affected blood pressure. Subsequently a few French physicians advocated the use of a reduced intake of salt for hypertension while most Germans claimed that such a maneuver was ineffective. In 1931, however, Volhard (381) in a textbook on medicine confirmed that a low intake of salt could lower the blood pressure of hypertensive patients with renal involvement. In the 1920s Houghton (158) and Allen and Sherill (7) in the United States, against a general background of disbelief, published the results of reducing the intake of salt below 2 g and 0.5 g on 10 and 180 hypertensive patients, respectively. Among Allen and Sherrill's patients, the blood pressure was restored to normal in 19%, and in 42% there was some lowering of the pressure and relief of some associated symptoms; complete failure to change the blood pressure occurred in 14%. In spite of these results, the connection between salt intake, as opposed to protein intake in causing the blood pressure to rise, continued to be denied. In 1945, one of the foremost authorities on hypertension stated that any hypotensive results that had been obtained with salt restriction were due not to the restriction but to "rest in bed and the psychotherapy of constant attention." The position was finally clarified by Kempner in 1948 (188) who used a 2,000-calorie rice and fruit diet which contained 5 g fat, 20 g protein, and <0.5 g salt on 500 hypertensive patients. Among these patients, 229 had some evidence of "renal involvement." The diet had a "beneficial" effect on 62% of the 500 patients, i.e., there was a decrease in mean arterial pressure of at least 20 mmHg. A reduction in heart size with a change in the transverse diameter of 18% or more, a change in the electrocardiogram T wave from completely inverted to upright, and a disappearance of severe retinopathy were also observed. The diet was slightly less effective (56%) when there was some renal involvement. The probable reason that Kempner's paper had such an impact was that it was visually so compelling and that it confirmed several previous similar studies. There were blood pressure charts showing relentless falls in blood pressure, chest x-rays of reductions in heart size, echocardiograms showing T-wave inversions reverting to normal and photographs of retina showing loss of edema, hemorrhages, and exudes. Kempner himself believed that the diet's effectiveness was the rigid restriction of protein intake. It is ironic, therefore, that he is now remembered as the person who most convincingly established that a high blood pressure can often be lowered by a low-salt diet. Kempner's results were confirmed by the Medical Research Council in the United Kingdom (387). The outstanding revelation that at that time, in spite of its considerable drawbacks, this unpleasant diet was the only known therapeutic measure that could lower blood pressure in more than 50% of patients with hypertension. There do not appear to have been any attempts made to make the diet more appealing. Instead, it was stressed that even if the blood pressure was controlled, a sudden rise in dietary salt still caused an immediate rise in blood pressure. It is not surprising therefore that, when oral diuretics were developed in mid 1950s, they were considered to be a satisfactory alternative to Kempner's diet. The idea that lowering salt intake might reduce blood pressure was not revived until the 1970s. In view of the difficulties of lowering salt intake below 1 g/day and the implacable dreariness of such a diet, reducing salt intake to 5 g/day was now studied. The first double-blind controlled study of moderate salt restriction was performed in the 1980s in a group of unselected patients with mild to moderate hypertension (229). It clearly demonstrated that a reduction in salt intake from 10 to 5 g/day for 4 wk induced a substantial fall in blood pressure equivalent to that seen with a diuretic. This was followed by many further trials. In some, however, the duration of low salt intakes was of short duration, e.g., 5 days, the reduction in salt intake was relatively small, e.g., <1 g/day, urinary salt excretion was inadequately monitored or the conditions were not random or double blind. In only 16 trials there was a random allocation to the experimental condition, no concomitant intervention in either group, and a dietary salt reduction which induced a reduction in 24-h salt excretion >2.9 g/day (3.0–6.7 g) for at least 4 wk. In these cases, systolic and diastolic blood pressure in 658 patients fell by 4.2/2.4 ± 0.4/0.3 mmHg (143). Weighted linear regression analysis showed a dose-response relationship between the change in urinary salt and blood pressure and that a reduction of 5.8 g/day in salt intake predicts a fall in blood pressure of 6.1/3.5 mmHg.
The effect of an acute reduction in salt intake on blood pressure of normotensive individuals has been studied, as it was in hypertensive patients, in many trials in which the conditions were not suitable. Furthermore, to add to the difficulties, the effect on normotensive individuals appears to be less than in hypertensive patients. Nevertheless, in 10 randomly controlled trials representing 2,104 normotensive individuals in whom the reduction in 24-h salt excretion was greater than 2.3 g/day (2.3–6.8 g),systolic and diastolic blood pressure fell by 1.6/0.6 ± 0.3/0.2 mmHg (143). There too, weighted linear regression analysis showed a dose-response relationship between the changes in urinary salt and blood pressure. A reduction of 5.8 g/day in salt intake predicts a fall in blood pressure of 2.7/0.9 mmHg. The most recent randomized trial included both normotensive and hypertensive individuals and was the most meticulous in the manner with which the dietary salt intake was monitored. It was a multicentered trial that studied the effect of three levels of dietary salt intake on 412 individuals whose blood pressure exceeded 120/80 mmHg (305). One diet contained 8 g/day salt, another diet 6 g, and the third diet 4 g. Each intake of salt was maintained for 30 days. The outstanding feature of the trial was the provision to the participants of all their food, including snacks and cooked meals, and taste tests were performed to ensure that the diets were palatable. The participants' adherence to the diet was monitored, not only by measuring the salt content of 24-h urine at the end of each period on a fixed salt intake, but also their daily food diaries were inspected and they ate their weekday lunches or dinners "on site." In addition to studying the effect of the three dietary salt intakes on blood pressure when the participants were otherwise on their habitual diet, the effect of the various salt intakes was also studied on a diet rich in vegetables, fruits, and low-fat dairy products. This diet by itself, known as a "Dietary Approaches to Stop Hypertension (DASH)" diet, reduces blood pressure (13). There was a very significant difference in systolic pressure (–6.7 mmHg) and of diastolic pressure (–3.5 mmHg) between participants on the 8 g/day diet and those on the 4 g/day (Fig. 2A). The pressures were all significantly lower on the DASH diet. There was a greater reduction in systolic pressure when blood pressure was initially high and in women, but most importantly the blood pressure-lowering effect of reducing the salt intake was observed in all categories of the population, in particular in normotensive as well as in hypertensive people (Fig. 2B). This observation, which confirms the previous studies, is very important for the public health issue. Indeed, it is known that most of the deaths related to high blood pressure occur in normotensive individuals with moderately elevated pressure (systolic and/or diastolic between 120/80 and 140/90 mmHg) and not in hypertensive people because the number of these latter in the population is much smaller even though their individual risk is higher (380).
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There are some reports on the effect on blood pressure of normotensive individuals of acutely raising the salt intake. One was a nonacculturated tribe in Papua, New Guinea in which the habitual salt intake was 0.6 g/day (294). Two groups, each of five individuals, were given diets with a raised dietary intake of salt for 10 days. In one group the urinary salt excretion rose to 7.5 g/day and in the other it rose to 15 g/day. On the lower rise in salt intake, there was an increase in blood pressure that was not significant, but on the higher salt intake, there was a significant rise in both systolic and diastolic pressure from 92/56 to 102/60 mmHg. There do not appear to be any studies of the effect of a prolonged increase in salt intake on blood pressure in normotensive humans previously accustomed to a low intake of salt. Dietary salt has been increased for relatively short periods in young adults on their normal high-salt diet (8.7 g/day). This has rarely induced a change in blood pressure. In four of five studies, increases in salt intake for up to 4 wk in young and middle aged (<47 yr) normotensive subjects did not cause a rise in blood pressure (77, 226).
D. Prolonged Reductions in Salt Intake and Blood Pressure
One of the most clear-cut examples of the effect of reducing the salt intake on the blood pressure of a community occurred in Portugal, which is notorious for its high consumption of salt (103). The trial was carried out in two communities within the same district, each with 800 inhabitants who had salt intakes of 21 g/day and a 30% incidence of hypertension. In the intervention community there was a vigorous, widespread health education effort to reduce the intake of salt especially from those foods that had previously been identified as the major sources of salt. The reduction in salt intake in one of the communities to 12 g/day was associated with a highly significant difference in blood pressure. By the end of the second year, there was a small rise in systolic pressure in the control community and a significant fall in both systolic and diastolic pressure in the community on the low salt intake, the difference between the two villages reached 13/6 mmHg. The fall in blood pressure involved the whole community, normotensives and hypertensive individuals alike, and the response did not differ between the young and the old or between men and women. Those with the greatest fall in salt excretion tended significantly to be also those who showed the greatest fall in blood pressure. Another long-term trial was carried out in Tianjin in China as part of a community-based intervention program to reduce noncommunicable diseases (367). This intervention was based on examinations of independent cross-sectional population samples in 1989 (1,719 persons) and 1992 (2,304 persons) in the intervention and matched reference areas. Food weighing and consecutive 3-day food records were used to measure dietary intake. The mean reduction in salt intake was 1.3 g/day in men and 0.7 g/day in women in the intervention area from 1989 to 1992. During the same period, the sodium intake increased significantly in men of the reference area. The reduction was significant in men (P = 0.001) and near significance in women (P = 0.05). This reduction in salt intake was similar in different educational and occupational groups, suggesting that the intervention had reached the whole community. In the intervention area, the mean systolic blood pressure decreased by 3 mmHg for the total population and by 2 mmHg for normotensive people. The decrease in systolic blood pressure was significant for both hypertensive and normotensive subjects. A third example of a long-term trial is the intervention that took place in two Belgian towns of 12,000 and 8,000 inhabitants, situated within 50 km of each other (345). The low salt intervention in one of the towns was mainly directed at women and implemented through mass media techniques. Cross-sectional random sampling at baseline and at 5 yr examined a total of 2,211 subjects. No significant difference was observed in the evolution of mean systolic and diastolic pressures that declined to the same extent in the two towns during the trial. The reduction in salt intake was considerably smaller than in the Portuguese trial but of the same order of magnitude than in the Chinese trial. In women of the intervention town, 24-h urinary salt excretion decreased by 1.5 g, whereas in the control town it rose by 0.5 g. This negative result may be explained by the small reduction in salt consumption that would be insufficient to observe a net effect on blood pressure in the Belgian environment, whereas such reduction was high enough to demonstrate an effect in the Chinese environment. In Japan between 1955 and 1989, as a result of a wide-ranging endeavour by public health authorities, the average salt consumption of the whole country fell from 13.5 to 12.1 g/day. In those localities where the salt intake was highest, as in the province of Akita in the north of the country, the intake of salt fell from 18 to 14 g/day. A gradual fall in blood pressure and a marked decline in mortality accompanied the reduction in salt consumption from strokes. In particular, there was a considerable gradual fall in blood pressure that occurred between 1957 and 1973 in each of the yearly intakes into three grades of school children aged between 12 and 15 yr (315).
E. Salt Intake and Blood Pressure in Other Mammalian Species
The available data in other terrestrial mammalian species confirm largely the existing relationship between habitual salt intake and blood pressure levels. Thus, in natural populations of genetically nonselected animals absorbing chronically varying amounts of salt for long periods of time, blood pressure appears to increase with the magnitude of the salt intake. For example, in groups of young adult rats fed different amounts of salt in their diet (0.15, 2.8, 5.6, 7.8, or 9.8%) with free access to distilled water, the average blood pressure after 9 mo increased proportionally to the salt content of the diet (19). The individual blood pressure values of the rats were quite scattered and overlapped from one group to another illustrating the large variation that exists in the susceptibility of each animal to the salt intake. This shows that the relationship between the habitual salt intake and blood pressure is essentially valid on a population and not individual basis. A similar study has been performed in pigs fed a diet containing either 0.5 or 3% salt for 8 mo after weaning with free access to pure water. The average diastolic and systolic blood pressures became progressively higher from the second to the eighth month in the group of pigs with the high salt intake (62). In baboons, adding 4% of salt to the diet resulted in increased blood pressure levels after 1 yr of exposure performed either from birth, during the sexual maturation, or in adults (55). The effect on systolic and diastolic blood pressures was observed in males and females and was substantially accentuated in the adults when the exposure time to the high salt intake was increased from 1 to 2.5 yr. In addition, at the termination of the experiment, the interruption of the high salt intake after 1 yr of exposure was accompanied by a return of blood pressure levels to normal in a few months. In African green monkeys, a gradual increase of dietary salt from 0 to 6% over a 1-yr period showed that, as a group, this primate species responds to salt intake with elevated systolic and diastolic blood pressures (344). Like in rats, significant individual variations in salt sensitivity were observed that tended to be consistent on the different salt diets, suggesting the involvement of the genetic makeup. In a colony of adult chimpanzees, our closest relatives on a genetic viewpoint, in a study where the salt intake was progressively increased for 20 mo from 0 to 15 g/day, both diastolic and systolic pressures became elevated compared with the group control (79). Moreover, when the salt was removed from the diet, blood pressure levels fell back to the values of the control group after 6 mo. It was also obvious that some chimpanzees reacted more than others to these changes of the salt intake; 60% of the cohort became hypertensive, whereas 40% remained resistant to high salt intake.
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III. MECHANISMS BY WHICH HABITUAL SALT INTAKE CONTROLS BLOOD PRESSURE |
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The primary functional disturbances that link salt intake to the arterial pressure lie in the kidneys. In several hereditary strains of hypertensive rats, renal cross-transplantation experiments with normotensive strains have shown that the rise in arterial pressure is due to abnormal kidneys (33, 69, 70, 125, 153, 250, 290). When a kidney from a normotensive rat is inserted into a young bilaterally nephrectomized hypertensive rat, the blood pressure of the hypertensive rat does not rise, and conversely, when a kidney from a young hypertensive rat (before it has developed hypertension) is inserted into a bilaterally nephrectomized normotensive rat, the blood pressure of the normotensive rat will rise. Similarly the high blood pressure of patients with essential hypertension and terminal nephrosclerosis became normal (over a mean follow-up of 4.5 yr) when, following bilateral nephrectomy, they were transplanted with a kidney from a young normotensive donor (67). The finding that the blood pressure of a bilaterally nephrectomized hypertensive rat does not rise when cross-transplanted with a kidney from a normotensive rat, and the comparable results which have been observed in humans with essential hypertension, indicate that whatever functional abnormalities may occur at other sites, the primary disturbance that initiates the rise in blood pressure in these hereditary forms of hypertension resides in the kidneys.
The primacy of the kidneys in the regulation of blood pressure has been confirmed by the experimental and conceptual work developed by Guyton (133) on the pressure natriuresis and diuresis response. The body has several systems for controlling blood pressure, which largely differ in their time of activation after the pressure suddenly becomes abnormal. Some systems based on neural receptors react within seconds while others like the hormonal systems respond within minutes. But the system whose contribution is by far the greatest is the kidney-fluid volume system, which reacts within hours or days. This system, which is able to restore the pressure to its exact original level, operates as follows. When the arterial pressure rises above normal, the excess pressure causes the kidneys to excrete more water and salt in the urine than are entering the body. Therefore, the extracellular and blood volumes decrease. This causes the heart to pump less blood, and the arterial pressure falls. Conversely, when the pressure falls below normal, the incoming salt and water overbalance the excreted fluid, and the pressure rises.
B. Links Between an Inadequate Renal Capacity to Excrete a High Salt Intake and Hypertension
In normotensive first degree relatives of patients with essential hypertension, compared with control subjects, volume expansion with saline leads to a lower rate of sodium excretion and a rise in blood pressure (126–128, 397). The effect on the blood pressure of a person's habitual salt intake, as measured by 24-h urinary sodium excretion, has also been studied in normotensive offspring of two hypertensive parents, one hypertensive parent, and two normotensive parents (379). Twenty-four-hour urinary sodium excretion was similar in the three groups, but while there was a positive association between urinary sodium excretion and systolic pressure in the offspring of hypertensive parents, no such association was apparent in the offspring of two normotensive parents. In the spontaneously hypertensive rat (SHR), sodium is retained between 4–6 wk of age when the sodium excretion of the SHR is significantly less than that of the control, the Wistar-Kyoto (WKY) rat (253, 371). Urinary sodium excretion has also been monitored during the development of hypertension in the Milan hypertensive rat. At 24 days, there is a statistically significant retention of sodium associated with a transient fall in urinary excretion of sodium but accompanied by an increased fecal content of sodium. The overall result, however, is an average retention of 2.5 mmol sodium (31, 139).
It should be mentioned that in normal humans the kidney's capacity to excrete sodium declines with age, and smaller increases in salt intake induce a rise in arterial pressure (30, 227). There is an accelerating fall in glomerular filtration rate (GFR) with age which begins around the age of 30 yr (215, 302). At the age of 80 yr, the fall in GFR is 40%. Individual variations are wide, and in one longitudinal study of 254 subjects who were followed serially for 8 or more years, one-third had no fall in GFR. The overall deterioration in GFR is more marked in blacks (245). The redistribution in GFR with age is accompanied by a decline in the number of functioning nephrons and is associated with the progressive development of glomerulosclerosis which eventually leads to glomerular obsolescence (185, 224). As there is generally no decline in salt consumption with age, sodium balance is maintained by raising fractional excretion of sodium. This is achieved, in part, by increasing the circulating concentrations of atrial natriuretic peptide, reducing plasma renin and aldosterone, and raising the blood pressure (263, 374). It is probable that the gradual rise in blood pressure that occurs with age in all populations on diets that contain more than 60 mmol sodium/day is due, in part, to these senescent involutional changes in renal structure superimposed on one or more primary renal structural and functional abnormalities (48). In the normal rat, GFR begins to decrease at 3 mo with a mean fall of 30% at 24 mo (63).
An increase in GFR increases the rate of delivery of tubular fluid to the macula densa, the cells of which then signal the adjoining afferent arteriole to constrict. This reduces the filtration rate and the delivery of tubular fluid to the macula densa and reduces urinary sodium excretion (136). In the normal animal, the sensitivity and reactivity of tubuloglomerular feedback increases when there is a need to conserve sodium, as in hemorrhage and dehydration (186, 276), and diminishes when there is a prolonged need to increase sodium excretion, as in chronic salt loading (135, 322) and DOCA administration (248, 321). In the 6-wk-old SHR when there is most evidence of sodium retention, tubuloglomerular feedback is increased (82, 280). This paradoxical increase that should enhance sodium reabsorption is independent of the associated rise in blood pressure. Conversely, if the SHR is chronically salt loaded, the resultant fall in tubuloglomerular feedback is less than in the salt-loaded WKY rat. By measuring tubuloglomerular feedback activity when perfusing the tubule with harvested tubular fluid from SHR and control rats, one group has demonstrated that the increase in tubuloglomerular feedback activity in the SHR is due to the defective action of a feedback inhibitory substance in the tubule fluid (378). The situation is similar in the Milan hypertensive strain rat. At 3.5–5 wk when the Milan hypertensive strain rat is in a state of slight volume expansion, tubuloglomerular feedback activity is appropriately absent. Two weeks later, however, when the blood pressure starts to rise, tubuloglomerular feedback increases inappropriately to high levels, so diminishing the kidney's ability to excrete sodium (237).
Disturbances of renal circulation in essential hypertension and hereditary strains of hypertensive rats may participate to the kidney's incapacity to excrete sodium. Investigation of renal hemodynamics in normotensive children of hypertensive parents has yielded inconsistent results, but the majority suggests that increased vascular resistance precedes the development of hypertension (116, 157, 377). In the SHR, renal blood flow and GFR are reduced before the rise in blood pressure (83). The kidney of immature prehypertensive SHR demonstrates a blunted pressure natriuresis that worsens with maturity so that by the age of 10–20 wk an increase in perfusion pressure of 54 mmHg gives rise to only a fourfold rise in sodium excretion compared with a ninefold increase in controls (297). Medullary hemodynamics in the SHR are abnormal (65). Measurements of papillary blood flow from the third to the sixteenth week show that whereas cortical and total blood flow in the SHR and WKY rat are similar, papillary blood flow in the SHR, at 6–9 wk onwards, is consistently less than in the WKY rat. Roman and Kaldunski (298) suggested that the increased medullary vascular tone prevents the normal increase in renal interstitial pressure upon which the mechanism of pressure natriuresis depends and that the decreased papillary blood flow in the 6- to 9-wk old rat would enhance sodium reabsorption (298). There is much evidence in the SHR that the circulatory disturbances described are related to local disturbances of arachidonic acid metabolism, particularly cytochrome P-450-dependent monooxygenase activity. The observation by Sacerdoti et al. (304) of higher levels of both cytochrome P-450 and its products in microsomal fractions from 5- to 13-wk-old SHR kidneys compared with WKY rat impelled them to study the effect of renal cytochrome P-450 depletion on the blood pressure of the SHR. Treatment with stannous chloride for 4 days caused a reduction of the blood pressure of 7-wk-old SHR that was maintained for at least 7 wk and was associated with a natriuresis and reduction in the renal content of cytochrome P-450 and its arachidonic acid metabolites (stannous chloride stimulates renal heme oxygenase production and so reduces the availability of heme for the formation of other hemoproteins including cytochrome P-450 monooxygenases). Stannous chloride did not affect the blood pressure of 20-wk-old hypertensive SHR or WKY rats. The same investigators administered stannous chloride to SHR from 5 to 13 wk of age and found that the development of hypertension was prevented during treatment and for 7 wk thereafter (93). There is also evidence of enhanced renal vascular tone and reactivity in the Dahl salt-sensitive rat, which both precede and accompany the rise in arterial pressure. The impaired pressure natriuresis is due principally to a defect in the sensitivity of the tubule to alter sodium reabsorption in response to changes in interstitial pressure (295, 296). In the Dahl salt-sensitive rat, in contrast to the SHR, one factor responsible for the development of salt-induced hypertension is an inability to increase vasodilatory nitric oxide production (54). In addition, there is also an absence of vasodilatation to atrial natriuretic peptide and nitroprusside, and an increased vasoconstrictive response to norepinephrine and angiotensin II (334).
Most hypotheses on how dietary salt increases the blood pressure incorporate the premise that the initial rise in arterial pressure is associated with an increase in extracellular fluid volume (Fig. 3). In view of the impaired ability to excrete sodium in normotensive children of hypertensive parents (397) and in young prehypertensive genetically hypertensive rats (31), this premise is theoretically reasonable, but measurements of the extracellular fluid volume in hypertension are inconsistent. Beretta-Piccoli and co-workers (27, 28) found a significant correlation between exchangeable sodium, related to body surface, in men but not in women, but in hypertensive men below the age of 36 years exchangeable sodium was significantly decreased. In the SHR, the extracellular volume or exchangeable sodium is significantly greater than in the WKY rat (139, 253, 371). In the Milan hypertensive rat however, exchangeable sodium is not significantly different from that of the Milan normotensive rat (31, 139). Perhaps the most striking evidence in favor of the proposition that in hypertension there is a state of continuous correction of a slightly expanded extracellular fluid volume is the exaggerated natriuretic response of normotensive children of hypertensive parents, to a rapid infusion of saline. In these groups, rapid volume expansion leads to the phenomenon of accelerated natriuresis, which does not occur in normotensive children of normotensive parents (398). Such a response is well documented in circumstances in which there is a tightly controlled state of volume expansion. It occurs in normal individuals given aldosterone, even when it may not be possible to detect an increase in extracellular volume, in primary hyperaldosteronism (36, 301); it also occurs in established hypertension and in the SHR (25, 399). In addition, the reduced levels of plasma renin (238), the raised levels of atrial natriuretic hormone (397), and the increase in the plasma's capacity to inhibit Na+-K+-ATPase (76) are consistent with an increase in extracellular fluid volume.
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Others have proposed that the pressor mechanism induced by dietary salt in essential hypertension and the SHR is, in part, due to an increase in the plasma's capacity to inhibit Na+-K+-ATPase, which raises the blood pressure by inhibiting the sodium-calcium exchange pump in vascular smooth muscle (39, 76). This hypothesis is based on the demonstration that in normal dogs acute volume expansion increases the plasma's capacity to inhibit Na+-K+-ATPase and that this increase is also detectable in essential hypertension, the SHR, and the Milan hypertensive rat. The nature of the substance responsible for the Na+-K+-ATPase inhibition in hypertension has been difficult to elucidate. One study in 27 untreated patients with essential hypertension has demonstrated that plasma marinobufagenin immunoreactivity, which rises with acute volume expansion, is raised in essential hypertension (17, 119). A variable increase in plasma ouabain immunoreactivity, and of ouabain extracted from plasma, has also been reported in essential hypertension though volume expansion does not raise plasma ouabain (119, 234).
Another hypothesis on the origin of the rise in arterial pressure that is initiated by an impaired ability to excrete sodium suggests that an associated increase in extracellular volume is responsible for the documented increase in the right and left (pulmonary wedge) pressures in the auricles. It was proposed that this increase in pressure induced an increase in afferent stimuli from the auricular walls to the hypothalamus and was thus responsible for the observed hypothalamic changes that lead to the documented pressor increase in sympathetic activity (75).
A body of evidence also suggests that the pressor and other harmful effects of dietary salt are due in part to a rise in plasma sodium. An acute experimental increase in plasma sodium in animals can raise the blood pressure, in spite of a fall in extracellular volume (106, 107). And a substantial acute increase in cerebrospinal fluid (CSF) sodium (+15 mM) induced in dogs by infusing hypertonic saline into the third ventricle raises the blood pressure within minutes (11), whereas a prolonged infusion which only raises the CSF sodium by 4–5 mM may take 6–10 days to raise the blood pressure (327). In cultured vascular smooth muscle, an increase in sodium concentration of 2–10 mM increases mRNA expression of many hypertrophy-related factors and the number of AT1 receptors; again, some of these changes take several days (130). In both normotensive and hypertensive humans, acute changes in salt intake are accompanied by parallel changes in plasma sodium (144, 146, 152, 177, 187, 226, 299, 308, 357). In hypertensive individuals, an acute increase in salt intake raises CSF sodium (121, 187).
There do not appear to be any direct observations on plasma sodium in large groups of humans whose habitual dietary intake of sodium is known. But it is possible that in normal circumstances such a rise may be difficult to detect for a rise in plasma osmolarity of only 1.6 ± 11% (which is equivalent to a change in plasma sodium of <1%) will stimulate the thirst center in the hypothalamus of the rat (101). The coefficient of variation for contemporary methods of detection of sodium is <1.5% (45). The close relation that exists between dietary sodium and urine volume in normal and hypertensive humans suggests that it is due to its effect on plasma sodium's control of thirst (145). The suggestion that in essential hypertension and the SHR there is a rise in plasma osmolarity (sufficient to affect the hypothalamus) is also consistent with the finding that in both these forms of hypertension, there is evidence which, though it suggests a state of continuous correction of a slightly expanded extracellular fluid volume (36, 301, 398, 399), which would tend to lower vasopressin secretion, yet both plasma and urinary arginine vasopressin are raised (75). This is consistent with a rise in plasma sodium. There are two studies in which the sodium concentration of the blood has been measured in a large number of hypertensives and controls. In one study the sodium concentration distribution curve in the patients with essential hypertension was shifted by 2 mM towards the higher value (193); in the other study there was a strong positive association between sodium and systolic pressure in the hypertensives and no relationship in the normotensive subjects (385). There is one study in the SHR and WKY rats in which plasma sodium was measured at 1- to 2-h intervals throughout the 24 h (94). Plasma sodium was 1–3 mmol/kg greater in the SHR than in the WKY rat throughout the 24 h. Overall therefore, acute experimental increases in plasma or CSF sodium concentration >5 mmol/kg can raise the blood pressure, independent of the extracellular fluid volume. The rate of rise in the blood pressure in such experiments is related to the extent of the rise in sodium concentration. It is proposed that with the 1- to 3-mmol/kg rise in sodium concentration that appear to occur in hypertension, the delay is likely to be considerably longer and that such an increase in plasma sodium not only tends to increase the extracellular fluid volume but may itself be a primary factor in the pressor effect of dietary salt (78).
C. Genetic Aspects of Renal Salt Handling
With the use of association or linkage studies and positional cloning during the last decade, over 20 genes associated with essential hypertension or responsible for rare Mendelian diseases with high or low blood pressure have been identified in humans to date (213). Remarkably, most of these genes encode proteins that either mediate or are involved in the control of renal sodium handling, i.e., ion channels and transporters or regulatory pathways that control their activity (Fig. 4). Moreover, it appears from these studies that mutations increasing renal sodium reabsorption raise blood pressure, whereas those diminishing sodium reabsorption lower blood pressure. More than 20 genome-wide searches for genes regulating blood pressure have also been reported (239). Quantitative trait loci have been suggested on almost all chromosomes with a poor replication from one study to another. As a result, although several genes encoding proteins that exert a direct or indirect effect on sodium homeostasis are located within the loci that seem the more convincing (1q, 2p, 2q, 4q, 6q, 12q, 17q), no particular common polymorphism or haplotype has been yet characterized by using this approach. Another approach that could help for the identification of the genes involved in the renal response to varied salt intake is the use of microarrays, although the technique is clearly limited by the availability of human samples (22).
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Adducin is a cytoskeletal protein interacting with the inner face of the plasma membrane that could modulate the activity of sodium transport systems like the Na+-K+-ATPase in the tubular renal cells (373). Several concordant studies have established a relation between the gene encoding the adducin 
-subunit and salt sensitivity and blood pressure, even though its effect is probably mild at the population level (32). In a study involving French and Italian hypertensive sib pairs, significant linkage was found between several markers surrounding the -adducin locus and essential hypertension (49). A positive association was found when the genotype frequencies of the G460W polymorphism of the -adducin gene were compared in 190 hypertensive patients and 126 controls. This effect may be due to epistatic interactions with other loci, especially the angiotensin I converting enzyme I/D polymorphism (346). The blood pressure response to the chronic administration of hydrochlorothiazide was also significantly more important in subjects bearing the 460W allele, suggesting that this variant could predispose to salt sensitivity and to hypertension (49). A better response to hydrochlorothiazide in subjects bearing the -adducin 460W allele, as well as a significant cardiovascular benefit, was suggested by the retrospective analysis of a large cohort (287). The association of the 460W allele with low-renin status was recently confirmed in a multicenter international study investigating intermediate phenotypes in hypertension (124). Moreover, in this study, the systolic blood pressure response to changes in dietary sodium was significantly greater in subjects homozygous for the 460W allele (25 ± 4 mmHg) compared with subjects heterozygous for 460W (12 ± 2 mmHg) or homozygous for the 460G allele (14 ± 1 mmHg). It is interesting to note that one (2p14) of the six chromosomal regions significantly linked to blood pressure in the HERITAGE Family Study contains the 
-adducin gene (291) that has been shown to be a modulator of a missense mutation in the -adducin gene (412). Homozygous -adducin-deficient mice have been generated. They display a sharp decrease of -adducin and a lesser reduction in 
-adducin levels and have higher systolic blood pressure, diastolic pressure, and pulse pressure compared with wild-type controls (236). In the Milan hypertensive rat strain, there is an increase in Na+-K+-ATPase activity which may be related to an abnormality of the adducin gene (35). In the same strain, Bianchi et al. (34) found a mutation in two of the genes that code for adducin. The interaction of these missense mutations could explain up to 50% of blood pressure differences between the Milan hypertensive and its normotensive control. Adducin is involved in the assembly of actin and inactin and actin binding proteins, which are coupled to a variety of transmembrane proteins including most ion transport molecules in epithelial cells. One such coupling is to the Na+-K+-ATPase 1-catalytic subunit. Interestingly, the activity of the enzyme in 5- to 8-wk-old SHR is significantly higher in dissected proximal tubules but significantly lower in the thick ascending limb of the loop of Henle than in the WKY rat, although these differences are no longer present at 20 wk (110). Gurich and Beach (131) have also demonstrated an abnormality in G protein control of Na+-K+-ATPase in suspensions of SHR renal proximal tubules and suggest that this could increase sodium reabsorption. In the Dahl salt-sensitive hypertensive male rat, there is a functional mutation of the 1 Na+-K+-ATPase subunit in the form of a leucine substitution for glutamine leading to a 3:1 sodium-potassium transport ratio instead of the normal 3:2 ratio in the normotensive salt-resistant rat (154). This change would lead to an excess of sodium ions reabsorbed by the tubule for each potassium ion transported.
The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step of salt reabsorption in the terminal part of the nephron. The three genes encoding the -, - and -ENaC subunits have been found to harbor mutations or polymorphisms related to gain or loss of function of the channel, increased or decreased sodium reabsorption in the terminal part of the nephron, and high or low blood pressure. Gain of function mutations have been found in - and -subunits and are associated with a rare clinical phenotype of low renin form of dominant hypertension with suppressed aldosterone secretion (known as Liddle's syndrome) in which the severity of hypertension is worsened by high salt intake and improved by salt restriction or by amiloride treatment alone (210). These mutations have been described originally as truncations or frame-shifts deleting a critical proline-rich region of the cytosolic tail that interacts with a regulatory protein called Nedd4 (2) and later as missense mutations of critical amino acids in this proline-rich region (138). Truncations as well as missense and splice-site mutations in any of the three genes encoding ENaC subunits that result in a loss of function of the channel provoke pseudohypoaldosteronism type I, an inherited and recessive hypotensive disorder characterized by salt wasting, elevated plasma renin activity, and aldosterone level and unresponsiveness to mineralocorticoids (51, 352). The presence of gain or loss of function mutations in the genes coding for the ENaC subunits suggests the possibility of the existence of more subtle polymorphisms in these genes that might modify ENaC activity, especially in salt-sensitive patients. Several groups have screened for mutations in the genes encoding - and -ENaC subunits among patients with essential hypertension in various ethnic populations. In a series of more than 400 hypertensive subjects, seven missense mutations were found in the gene coding for the -ENaC subunit, almost all of them in patients of African descent (277). Whereas these variants led to no significant increase in sodium current after expression in Xenopus oocytes, data obtained in human B lymphocytes (354) suggest that at least one of them (T594M) could have an effect on sodium reabsorption. In a case-control study involving black residents in London, a significant increase of the 594M frequency was found in the 206 hypertensive patients (8.3%) compared with the normotensive subjects (2.1%), the statistical significance persisting after adjustment for sex and body mass index (18). In the subset of patients in whom plasma renin activity was measured, the T594M polymorphism was also associated with a low renin profile, suggesting that it could raise blood pressure in affected people by increasing renal tubular sodium reabsorption. The association between this polymorphism and blood pressure was not replicated in a case-control study performed in 519 hypertensive patients and 514 normotensive individuals of African ancestry (262). Several polymorphisms have also been detected in the -ENaC subunit. Four neutral polymorphisms have been found in the third exon of the gene (T387C, T474C, C549T) and in the last exon (C1990G), but they had similar frequencies in 453 hypertensive and 245 normotensive Caucasian subjects as well as in patients with low-renin profile (278). All these polymorphisms in the genes encoding - and -ENaC subunits have not been shown to have a demonstrable effect in the in vitro expression systems used to examine ENaC activity (277, 278). Polymorphisms in the promoter region of the -ENaC gene have also been identified, one of them G(–173)A is associated with enhanced in vitro promoter activities and blood pressure in a large Japanese cohort (168). It remains therefore possible that some of the ENaC polymorphisms might be associated with higher ENaC activity in vivo and contribute to ethnic differences in sodium retention and the subsequent risk of developing low renin hypertension (10, 286). It is worth noting that a sib pair linkage study performed on 286 white families from the general population in Australia showed significant linkage between systolic blood pressure and micro-satellites at chromosome 16p12, located in the vicinity of the genes encoding the - and -subunits of ENaC (404). The analysis of the -ENaC subunit in Caucasian hypertensive subjects showed an interesting missense polymorphism (W493R) located in the extracellular loop of the subunit and in a rather well-conserved sequence not far from the amino acids responsible for the sensitivity to amiloride. However, in Caucasians, this polymorphism was found at similar allele frequency in hypertensive and normotensive individuals and did not change the amiloride-sensitive current when expressed in Xenopus oocytes (64). Another common coding polymorphism in the -ENaC gene (T663A) has been described, but conflicting results have been reported concerning its association with essential hypertension (10, 355). The expression of T663A in oocytes has been associated with higher currents and higher levels of cell surface expression of ENaC, suggesting that it might affect channel trafficking (310). Other polymorphisms in the -ENaC promoter region have been identified and studied, especially in a Japanese population (169). One of them (G2139A) has been suggested to be associated with higher in vitro promoter activities and with blood pressure levels. Other noncoding polymorphisms have been described on each ENaC subunit. Most of them have not been convincingly linked to hypertension or to salt sensitivity (281). The truncation of the -subunit found in the original Liddle pedigree was reproduced in the mouse using gene targeting and Cre/loxP techniques (285). Under normal salt diet, these mice have a blood pressure not different from wild-type mice despite evidence for chronic hypervolemia such as increased sodium reabsorption in distal colon and low plasma aldosterone. Under high-salt diet, the mice develop hypokalemic metabolic alkalosis, high blood pressure, and cardiac hypertrophy, thus reproducing to a large extent the human syndrome. A mouse model for pseudohypoaldosteronism type 1 has also been generated by disrupting the gene encoding the -subunit (284). On a normal salt intake, -subunit-deficient mice exhibit elevated plasma aldosterone level and compensated metabolic acidosis compared with wild-type mice, but no change in blood press
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