Ethnic Differences in Cellular Ions and Transport

Ethnic Differences in Cellular Ions and Transport Janice G. Douglas, MD, and Richard S. Cooper, MD INDEX WORDS: Sodium; calcium; race; black; white; Na pump.

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ECAUSE the cellular regulation of sodium and other ions has been implicated in the pathogenesis and maintenance of hypertension, the study of ion transport has captured the attention of many researchers interested in the pathophysiology of hypertension. Several investigators have proposed that sodium metabolism by the kidney, not sodium intake, is the critical issue underlying the observed differences in the prevalence and characteristics of hypertension between blacks and whites in the United States. I - 5 Evaluation of racial/ethnic phenotypic differences in ion transport has provided data that suggest mechanisms by which a genetic or environmental factors may contribute to the twofold excess of hypertension in US blacks compared with whites. A caveat inherent in the interpretation of studies of racial differences relates to the genetic admixture of populations in the United States. 6 Despite this limitation, however, certain trends have become apparent. Assessment of black/white differences in intracellular cations has pointed to sodium as the cation that is most consistently abnormal. Sodium interacts with other cations, notably calcium, and various cellular transport systems to increase vascular reactivity, often considered the basic pathway to essential hypertension. The primary mechanism proposed by a number of investigators whereby hypertension develops in this setting is that higher intracellular sodium increases activity of the sodium-calcium exchanger (Fig 1), thereby increasing intracellular calcium. The net effect is to enhance responsiveness to vasoactive From the Division ofEndocrinology and Hypertension, Case Western Reserve University School ofMedicine and University Hospitals of Cleveland, Cleveland, OH; and the Department ofPreventive Medicine and Epidemiology, Loyola University, Maywood, IL. Address reprint requests to Janice G. Douglas, MD, Division of Endocrinology and Hypertension, Case Western Reserve University, School of Medicine, Room W165, 10900 Euclid Ave, Cleveland, OH 44106-4982. © 1993 by the National Kidney Foundation, Inc. 0272-6386/93/2104-0109$3.00;0 46

agonists and increase tone of smooth muscle cells through calcium-calmodulin-dependent mechanisms. No unifying hypothesis has developed as to whether higher intracellular sodium is related to a defect in a specific transporter, a circulating transport inhibitor, or environmental factors that alter sodium handling. The purpose of this report is to review the scientific basis for racial/ethnic differences in intracellular ions and explore the relationship(s) to blood pressure regulation. INTRACELLULAR SODIUM CONCENTRATIONS

Excessive intracellular sodium assessed primarily in circulating blood cells is the most consistently reported hypertension-associated abnormality of cation metabolism. Measurements of sodium using circulating blood cells have been shown to be highly reproducible in the same subject and are not influenced by modest changes in dietary sodium intake in health-normotensive subjects. 7 A review by Hilton in 1986, based on data from case control studies that included 965 hypertensive subjects and 1857 normotensive control subjects, reported that hypertensive subjects had a 13% higher red blood cell sodium level. 8 However, population-based studies among non-black groups have demonstrated only a weak positive correlation between intracellular sodium and blood pressure. 9 ,10 A large number of studies have evaluated black/white differences in the United States and have consistently shown that black children and adults have 10% to 20% higher intracellular sodium level than white adults and children. 11-17 This observation has been reported not only in US blacks, but in other populations of African descendant sampled in the Caribbean, England, and West Africa. 18-21 In fact, racial admixture in the United States, usually estimated to be approximately 25% to 40%,6,22 should diminish the importance of race as a determinant of red blood cell sodium levels in African Americans compared with Africans. Cooper et al observed a graded increase in sodium among normotensive US whites (7.7 mEq/L cells, n = 27),

American Journal of Kidney Diseases, Vol 21, No 4, Suppl 1 (April), 1993: pp 46-52

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ETHNIC DIFFERENCES IN ION TRANSPORT

Ca++ channel

Fig 1. Hypothetical diagram relating calcium homeostasis to intracellular sodium.

Ca++ Na+ - Ca++ exchanger

African Americans (9.96 mEq/L, n = 21), and West African blacks (10.7 mEq/L, n = 27) in support of this hypothesis. 23 Direct estimates of gene admixture correlated with the lower sodium levels in US blacks compared with African blacks. A positive correlation between sodium and systolic blood pressure (r = 0.38, P < 0.01) was observed in all groups in this triethnic study, but was most strongly positive among the two black groups. Additional studies also support a positive correlation between sodium and blood pressure in blacks. For example, Cooper et al investigated the correlation of intracellular sodium and potassium concentrations to blood pressure in a large sample of 120 normotensive and 97 hypertensive blacks in the US (Chicago).24 The intracellular sodium concentration was 11 % higher in hypertensive than in normotensive subjects and there was a significant positive correlation between diastolic blood pressure and intracellular sodium concentration when measurements from both groups were combined (r = 0.14, P < 0.05; n = 217). Even though a significant correlation was found between sodium and potassium (r = 0.272, P < 0.001), no correlation existed between intracellular potassium and blood pressure. Despite the evidence supporting a strong positive correlation between sodium and blood pressure in persons of African descent, the correlation is much weaker for non-blacks. 8- 10 In summary, these epidemiologic studies support the hypothesis that a high concentration of intracellular sodium may be a risk factor for development of hypertension and precedes its onset. An increase in the intracellular sodium level may be a phe-

notypic marker for excess prevalence of hypertension in blacks. SPECIFIC MEMBRANE TRANSPORTERS THAT MAY BE ALTERED

Any of several membrane transport mechanisms may playa role in the phenotypic expression of high intracellular sodium in US blacks and other populations of African descent: sodium-hydrogen exchange, as measured directly or by sodium-lithium countertransport, sodiumpotassium-adenosine triphosphatase (ATPase) ("sodium pump"), and sodium-potassium-chloride cotransporter.

Sodium-Lithium Countertransport and Sodium-Hydrogen Antiporter Erythrocyte sodium-lithium countertransport is considered by some investigators to be an alternative operational mode of the sodium-hydrogen antiporter. 25 However, more recent evidence suggests that these transporters do not act in parallel and are probably not identical. 26 The physiologic importance of this sodium-lithium countertransport system for regulation of sodium is not completely understood. On the other hand, the sodium-hydrogen exchange is an important mechanism of pH regulation in many cell types as it exchanges external sodium for internal hydrogen ions. Many vasoactive agonists and growth factors have regulation of sodium-hydrogen antiporter as a primary component of their signalling mechanisms. Despite some limitations, erythrocytic sodiumlithium countertransport has been used as a con-

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venient way to assess sodium-hydrogen antiport abnormalities. In vitro erythrocytic sodium-lithium countertransport activity appears to have properties similar to sodium-hydrogen activity in the renal proximal tubular brush border. Accelerated erythrocyte countertransport thus would facilitate increased proximal tubular sodium-hydrogen exchange and therefore increase sodium resportion in the proximal tubule, a proposed mechanism for sodium-sensitive hypertension. If so, and if transport systems can be found that operate in parallel in renal tubular or vascular smooth muscle cells similar to those established for erythrocytes, increased countertransport activity would be a useful marker for sodium-sensitive hypertension. 27-29 Thus, it would be possible to relate ethnic and racial patterns of whole-body cation metabolism to cellular transport dysfunction. Greater exchange rates of sodium-lithium countertransport consistently have been demonstrated in hypertensive subjects and in the normotensive offspring of hypertensive parents, while normotensive subjects without a family history of hypertension had lower sodium-lithium countertransport. 11,30-35 The positive correlation between higher rates of this transporter and blood pressure has been a very consistent finding over the last decade and is characteristic of non-black populations. Interestingly, blacks consistently have on the average 30% lower rates of sodiumlithium countertransport than whites in a large number of studies. 14,15,17,36-40 Several recent studies have not simply relied on the assessment of the sodium-lithium countertransporter for determining relationships to hypertension, but also have assessed sodium-hydrogen anti porter activity directly using platelets instead of erythrocytes. Livne et al reported higher rates of sodium-hydrogen exchange in platelets of essential hypertensives. 41 Several additional studies confirmed this observation. 15 ,42 A genetic basis has been suggested since the mechanism of the increased activity of the sodium-hydrogen exchanger does not appear to be secondary to the lower cytosolic pH in hypertensives compared with normotensives. 42,43 Contrary to the relatively consistent findings of lower sodium-lithium countertransport activity in African Americans, assessments of sodium-hydrogen antiporter activity have varied significantly: a de-

DOUGLAS AND COOPER

creased activity was observed in two studies, no change was observed in one study, and increased activity was observed in a fourth study. Decreased activity in blacks compared with whites was observed by Cooper and Borke44 using the same groups in which they had observed higher red blood cell sodium levels (West Africans and African Americans v US whites) and by Gretler et al. 45 No change was observed by Schmouder et al in blacks compared with whites, although they confirmed higher activity in hypertensive subjects compared with normotensive subjects. 46 One potential problem with this latter study was the small sample size; thus, it may not have been an adequate representation of the population at large (eight black hypertensive subjects and 12 white hypertensive subjects).46 The one study that reported an increase in sodium-hydrogen exchanger in normotensive US blacks compared with whites used serially passaged fibroblasts in tissue culture rather than acutely harvested platelets. 47 Important questions are raised as to whether the fibroblasts underwent phenotypic changes in tissue culture and/or whether there are inherent differences in the expression of the sodium-hydrogen exchanger in platelets compared with fibroblasts. Thus, the observed differences in countertransport rates between hypertensive and normotensive patients support the hypothesis that the sodium-hydrogen exchanger as measured directly and through the sodium-lithium countertransport system may somehow be involved in the hypertensive process; however, it cannot account for racial differences. In fact, these studies implicate some other transporter to be responsible for the higher levels of intracellular sodium observed consistently in blacks. Further studies are needed to better understand regulatory mechanisms, the relationship to intracellular calcium, and effects on the body's metabolism of sodium. Sodium-Potassium-Adenosine Triphosphatase

The sodium-potassium-A TPase (sodium pump) was one of the earliest transporters to be studied to examine possible racial differences. Because this pump is primarily responsible for extruding sodium from cells, diminished activity of this transporter could be responsible for the increased levels of intracellular sodium that

ETHNIC DIFFERENCES IN ION TRANSPORT

characterize blacks. A number of reports confirm this hypothesis. A recent study of 247 subjects by Rygielski et al 13 from the University of Medicine and Dentistry of New Jersey found a highly significant inverse correlation between systolic blood pressure and red blood cell sodium pump activity (r = -0.24, P < 0.001). Because a similar study from France48 did not confirm the observations of Rygielski et al, it is not possible to develop a unifying hypothesis regarding the relationship between the sodium pump and blood pressure, as was possible with the sodium-lithium countertransporter. However, the large number of subjects in the report by Rygielski et al makes the data compelling. Furthermore, racial heterogeneity (inclusion of 50% blacks) in the report by Rygielski et al may account for the differing conclusions. Lower levels of red blood cell sodium-potassium-ATPase activity have been reported in blacks in the majority of reports in the literature since the earliest report by Balfe et al in 1968. 49 The lower levels appear to be secondary to a diminished maximal initial reaction velocity (Vmax). 50 These observations agree with the racial! ethnic differences observed by Beutler et al. 51 The magnitude of decreased sodium pump activity is estimated to be 11.3% using an unweighted mean. 13. 15 ,2I,52,53 Of interest is the fact that the pattern is not unique to US blacks, but encompasses blacks from Zaire living in Belgium compared with Europeans21 and immigrants from India compared with whites in England. 51 Only one group failed to demonstrate racial differences in the sodium pump using both normotensive and hypertensive subjects that were analyzed independently. 16 However, gender differences were not taken into account in this latter report, as differences in black and white females have been shown to be much greater than in men. 50 The mechanism responsible for the decreased Vmax of the sodium pumps has not been determined. One possibility is that parathyroid hormone may be responsible; Bell et al 54 reported increased parathyroid hormone in blacks in the United States. Accetto and Weder reported that parathyroid hormone inhibits erythrocyte sodium-potassium-ATPase in a dose-dependent manner;55 this may be responsible for the decreased number of sodium pumps. Again, the sample size in this study was small and thus may

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not be representative of the population at large. A second possibility is that there may be some other circulating inhibitor of transport (the natriuretic digitalis-like substance) that is more abundant in blacks, as proposed by de Wadener et al 56 and recently reviewed. s7 A third possibility is that the diminished number of transporters is an inherited defect. Kuriyama et al S8 examined cultured skin fibroblasts from 15 black and white subjects with established essential hypertension and from 15 matched normotensive controls to determine the behavior of sodium-potassium transport systems. In this study, fibroblasts in tissue culture from blacks had higher intracellular sodium turnover rates than those from whites. Fibroblasts from blacks also expressed accelerated sodium-potassium-ATPase activity, a higher rate of intracellular accumulation of sodium in the presence of ouabain, and a higher intracellular sodium level than those from whites. Interestingly, these results differed from the finding found using acutely harvested cells published by this same group 1 year earlier.13 Whether the increased sodium pump activity observed by these investigators represented phenotypic alterations secondary to placing the cells in tissue culture or different expression in cell types has not been determined. Alternatively, if blacks had a circulating inhibitor of the sodium pump in vivo, such as parathyroid hormone 54,55 or the "digitals-like" substance, 59 the suppressed pump activity could be expressed in acutely harvested cells but not in cells maintained long-term in tissue culture. A circulating inhibitor of the sodium pump in subjects with essential hypertension and documented higher levels of this digitalis-like substance were observed in low renin hypertension, a well-accepted characteristic of black hypertensives. 57 ,59 The as yet elusive circulating substance has properties identical to digoxin as it binds to the sodium pump and inhibits sodium extrusion from cells. Unfortunately, the inhibitor has not been purified and its sequence is unknown, thus limiting assessment oflevels in large population studies and correlations with sodium and blood pressure. Such an inhibitor, if higher in black prehypertensive and hypertensive subjects, could be responsible for higher intracellular sodium and suppression of measurable sodium pumps. However, one detailed kinetic analysis by Lasker et

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al 50 suggests the possibility that a circulating inhibitor of sodium transport is not responsible for the decreased number of sodium pumps. Large population-based studies are needed to ascertain the significance of these observations that currently derive from different sources. Sodium-Potassium-Chloride

The sodium-potassium cotransporter is an ATP-independent, furosemide-sensitive bidirectional transporter. Despite initial enthusiasm following early reports by Garay et a160,61 suggesting an inverse relationship between transporter activity and blood pressure, other reports demonstrated the opposite relationship. 16,31 Thus, in non-blacks there appears to be no cinsistent relationship to blood pressure; however, interesting racial differences have emerged. Studies examining the role of this transporter in blacks have concluded that Vmax values are substantially lower than in whites. Mean values (unweighed) are 45% lower in blacksY-17,31,44 In addition, there is a modest correlation with the presence of hypertension in black as hypertensive subjects have a 30% lower mean Vmax than normotensive subjects. 16,17,31 Two additional studies demonstrated that hypertensive black women had 9% lower mean Vmax values than normotensive black womenY While the coupling rate between sodium and potassium is generally accepted as 1 for this transporter, additional data suggest that the ratio exceeds 1 in blacks compared with whites. 44 The significance of this latter observation remains unclear at the present time. Thus, racial/ethnic differences in this transporter are more pronounced than the correlation between the presence and absence of hypertension in black subjects. Cooper and Burke44 concluded that these observations oflow group men Vmax values of the sodium-potassium cotransporter indicate that this transporter could play a role in group susceptibility to hypertension among blacks. An inherited defect expressed phyenotypically as low Vmax could impair intracellular sodium removal in that this transporter functions as a backup to the sodium pump. However, before one can make firm conclusions about between-group differences, better evidence of an association between sodium-potassium cotransport and blood pressure is needed as has emerged for intracellular sodium and blood pressure in blacks. 44

DOUGLAS AND COOPER

INTRACELLULAR CALCIUM

A number of studies have demonstrated elevated intracellular calcium concentrations in platelets and erythrocytes of patients and animal models with essential hypertension 62-64 and have documented a strong positive correlation between intracellular calcium and blood pressure. An increase in the intracellular calcium concentration has been though to contribute to increased contraction of vascular smooth muscle cells, thereby increasing vascular resistance and raising blood pressure. The intracellular calcium level can increase because of accelerated influx of calcium from extracellular stores, mobilization from intracellular stores, or increase exchange of calcium for sodium (Fig 1). Thus, one would predict that if this hypothesis is correct, blacks would have higher cytosolic calcium that would be secondary to higher intracellular sodium that has been observed. While higher resting cytosolic intracellular calcium has been documented in untreated black hypertensives63 as compared with black normotensives, resting intracellular calcium has been reported to be 16% lower in US blacks compared with US whites. 23 In this triethnic study that used African and US blacks, intracellular sodium was significantly higher in blacks compared with whites and positively correlated with blood pressure. Thus, there appears to be consistent observations that support higher intracellular calcium in hypertensive subject versus both black and white normotensive subjects. However, based on the limited data available, blacks have lower levels of intracellular calcium despite higher intracellular sodium. Thus, these two cations do not appear to be as tightly coupled as has been hypothesized. In summary, the most consistent transport defects observed in blacks compared with whites include (1) higher levels of intracellular sodium, (2) lower resting cytosolic Ca2+, (3) lower red blood cell sodium-potassium-ATPase, (4) lower sodium-potassium cotransport, and (5) lower red blood cell sodium-lithium countertransport (Table 1). Thus, higher intracellular sodium may be an important phenotypic marker for hypertension in blacks as there is a strong positive correlation with blood pressure in multiple racial/ethnic groups. Current estimates suggest that impaired

ETHNIC DIFFERENCES IN ION TRANSPORT

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Table 1. Summary Hypertensives v Normotensives Intracellular sodium Sodium-lithium countertransport Sodium-hydrogen exchanger Sodium-potassium-A TPase (sodium pump) Sodium-potassium cotransport Intracellular calcium

t12% t44% t45%

± ± t20%

Blacks v Whites

t30% ~30%

± ~11.3% ~46% ~16%

activity and/or numbers of the sodium pump and the sodium-potassium cotransporter may be the most important contributing factors. Whether these reflect genetic defects in ion transport or input from environmental factors remains to be determined.

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47. Hatori N, Gardner JP, Tomonari H, Fine BP, Aviv A: Na+-H+ antiport activity in skin fibroblasts from blacks and whites. Hypertension 15:140-145, 1990 48. Garay RP, Nazaret C: Na+ leak in erythrocytes from essential hypertensive patients. Clin Sci 69:613-624, 1985 49. Balfe JW, Cole C, Smith EKM, Graham JB, Welt LS: A hereditary sodium transport defect in the human red blood cell. J Clin Invest 47:4A, 1968 (abstr) 50. Lasker N, Hopp L, Grossman S, Bamforth R, Aviv A: Race and sex differences in erythrocyte Na+, K+ and Na+-K+adenosine triphosphatase. J Clin Invest 75:1813-1820, 1985 51. Beutler E, Kruhl W, Sachs P: Sodium and potassium ATPase activity is influenced by ethnic origin and not by obesity. N Eng! J Med 309:756-760, 1983 52. Woods KL, Beevers DG, West MJ: Racial differences in the red cell cation transport in white and black hypertensives. Postgrad Med J 57:769-771, 1981 53. Ahmad MN, Leeds AR: More on sodium-potassiumATPase and obesity. N Eng! J Med 310:1390-1391, 1984 54. Bell NH, Greene A, Epstein S, Oexmann MJ, Shaw S, Shary J: Evidence for alteration of the vitamin D-endocrine system in blacks. J Clin Invest 76:470-473, 1985 55. Accetto R, Weder AB: Parathyroid hormone and verapamil inhibit the Na+-K+ pump in human erythrocytes. Clin Res 35:437A, 1987 (abstr) 56. de Wadener HE, Mills IH, Clapham WF, Hayter CJ: Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dogs. Clin Sci 21:249-258, 1961 57. Blaustein MP, Hamlyn JM: Pathogenesis of essential hypertension-A link between dietary salt and blood pressure. Hypertension 18:III-184-III-195, 1991 58. Kuriyama S, Hopp L, Tamura H, Lasker N, Aviv A: A higher cellular sodium turnover rate in cultured skin fibroblasts from blacks. Hypertension 11:301-307, 1988 59. Hamlyn JM, Ringel R, Schaeffer J, Levinson PO, Hamilton BP, Kowarski AA, Blaustein MP: A circulating inhibitor of (Na+-K+)ATPase associated with essential hypertension. Nature 300:650-652, 1982 60. Garay RP, Dagher G, Pernollet M, Devynck M, Meyer P: Inherited defect in a Na-K cotransport system in erythrocytes from essential hypertensive patients. Nature 284:281283, 1980 61. Garay RP, Hyannaert P, Dagher G, Nazaret C, Maridonneau Y, Meyer P: Abnormal erythrocyte Na-K cotransport system: A proposed genetic marker of essential hypertension. Clin Exp Hypertens 3:861-870, 1981 62. Bruschi G, Bruschi ME, Caroppo M, Orlandini G, Spaggiari M, Cavatorta A: Cytosolic free (Ca2 +) is increased in platelets of spontaneously hypertensive rats and essential hypertensive patients. Clin Sci 68: 179-184, 1985 63. Cooper R, Shamsi N, Katz S: Increased intracellular calcium and sodium in hypertensive patients compared to normotensives. Hypertension 9:224-229, 1987 64. Erne P, Bolli P, Burgisser E, Buhler FR: Correlation of platelet calcium with blood pressure. N Eng! J Med 310: 1084-1088, 1984