Na+ Transport in Primary Hypertension

Na+ Transport in Primary Hypertension RICARDO GARAY, CLELIA ROSATI, AND PHILIPPE MEYER

Inserm U7 H6pital Necker 161 rue de Skvres Paris 75015 France I

INTRODUCTION One major environmental factor contributing to the onset of high blood pressure is excess N a + intake.' In recent years, the considerable progress in the understanding of N a + metabolism at a molecular level has contributed to the elucidation of this causal relati~nship.*-'~

MOLECULAR MECHANISMS OF Na+ TRANSPORT ACROSS CELL MEMBRANES Knowledge of N a + metabolism at a molecular level is not so advanced as that of oxygen, sugar, or lipid metabolism; this may be related to the fact that all known functional proteins of N a + metabolism are membrane transport proteins. They are therefore difficult to extract, purify, and almost impossible to crystallize. The human red cell has been widely used for molecular studies of N a + transport. FIGURE 1 shows three different N a + transport systems that have been well characterized in these cells: a ouabain-sensitive Na+,K+-pump,which catalyzes the exchange of internal N a + for external K+ coupled to the hydrolysis of ATP, thus generating electrochemical gradients of Na' and K + across the cell membrane; a furosemide (or bumetanide)-sensitive Na+,K+ cotransport system, which catalyzes inward and outward fluxes of N a + and K+; and a Na+,Na+countertransport system, which catalyzes a one-to-one exchange of internal for external N a t (Li+ and perhaps H + may replace N a + in this system). Unfortunately, N a + transport in other cells is not so well known as in the human 1 shows that the above three erythrocyte N a + transport systems erythrocyte. FIGURE are also present in different segments of the nephron: a ouabain-sensitive Na+,K+pump is present in basolateral membranes all along the nephron, a furosemide (or bumetanide)-sensitive Na+,K+-cotransport system is located in the luminal side of Henle's loop cells, and Na+,Na+-countertransportappears to be the same as the Na+:H+ exchange catalyzing a fraction of luminal N a + reabsorption in the proximal tubule. Conversely, the amiloride-sensitive N a + channel of distal and collecting tubules does not appear to be represented in the human red cell membrane. It is important to note that some other N a + transport systems, such as Na+:Ca2+exchange, that are present in other cells appear to be lacking in human red cell membranes. Cell N a + content of non-epithelial cells (like vascular smooth muscle cells) is the result of the activity of all membrane N a + transport systems (and channels). FIGURE 2 shows two different situations: (1) the stationary (basal) cell N a + content, which 187

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ACETAZOLAMIO~

THIAZIDES

SPIRONOLACTONE

FUAOSEMIOE BUMETllNlOE

FIGURE 1. Na' transport systems in human red cells and kidney. Three erythrocyte Na+ transport systems [the ouabain-sensitive Na+,K+-pump, the furosemide (or bumetanide)sensitive Na+,K+ cotransport system, and the Na+,Na+countertransport system] appear to be representative of those present in different segments of the nephron.

generally speaking depends on the balance between Na+ entry by passive N a + permeability (membrane N a + leak) and N a + extrusion by the Na*,K+-pump and (2) cell Na+-load, which is normally extruded by the Na+,K+-pump and the Na+,K+cotransport system.

NATRIURETIC HORMONES Excess Na' intake tends to induce volume expansion, which may be rapidly compensated by the transient secretion of a t least two natriuretic hormones, the atrial natriuretic factor (ANF), which is a peptide present in specific granules of atrial c a r d i o c y t e ~and ' ~ the endogenous "ouabain-like" factor(s) (OLF), of unknown chemical structure and site of production. The site of action of A N F is the renal glomeruli where it increases glomerular filtration rate.15 The mechanism of this effect may involve an increase in renal blood flow and in the area of glomerular filtration (through an indirect action on mesangial cells). At the molecular level the interaction of A N F with specific receptors in vascular smooth muscle cells provokes a cascade of events,

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stimulation of cyclic GMP production,16 decrease in cytosolic free Caz+content,” and vasorelaxation. The second natriuretic hormone, OLF, seems to inhibit the N a + , K ’ - p ~ m p . ~At ’~ the tubular level, this results in a natriuretic effect by decrease of renal Na’reabsorption. However, a similar pump inhibition at the vascular wall may transiently increase cell N a + content. FIGURE 2 shows that a normal membrane N a + transport function may rapidly ensure the extrusion of such cell N a + load.

STABLE Na+ TRANSPORT ABNORMALITIES IN PRIMARY HYPERTENSION In the past ten years, the extensive investigation of N a + transport mechanisms in circulating cells of humans and rats with primary hypertension strongly suggested that different genetic abnormalities can be associated with high blood pressure. Studies in Human Hypertensives FIGURE3 shows four different and stable N a + transport abnormalities found in erythrocytes from essential hypertensive patients. These may induce transitory (or compensatory) changes in the same or in other Na’ transport systems. The four stable Na’ transport abnormalities can be summarized as follows.

Condition :

Basal Na

Exces Na+ intake

NLTRIURETIC FllGTOR P

NORMAL

__v__

P

Coo HYPERTENSIVES

L e a k @ HYPERTENSIVES

SHR-RATS

HYPERTENSIVE-PRONE SABRA RATS

FIGURE 2. Abnormal cell Na+ homeostasis under excess Na’ intake in some forms of primary hypertension. A key step in the development of primary hypertension is the disturbance in cell Na homeostasis induced by the interaction of “ouabain-like” factors with vascular cells having genetic abnormalities in membrane Na’ transport. +

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Increased Passive Entry of Na+ (Leak(+)] This first N a + transport abnormality was suspected by Wessels et al. in 1967, measuring unidirectional **Na influx and recently confirmed by more specific studies 10.1I.I3 in about 15 to 30%of human hypertensives (Leak(+) hypertensives). A leak( +) abnormality may be compensated by an increase in the maximal rate of the Na+,K+ pump and the Na+,K+ cotransport system.'' Indeed, internal N a + contents (and blood pressure levels) in Leak( +) hypertensives are inversely correlated to these compensatory phenomena.'' Decreased Apparent Afinity of the Na+,K+Cotransport System for Internal Na+ /Co(-)] About 30 to 40% of human hypertensives (Co(-) hypertensives) exhibited a decreased apparent affinity of the Na+,K+cotransport system for internal Na+.@This

COUNTER 0 Increased maximal rate

Increased p a s s i v e

Na+

Na+

K +* K+ PUMP 0 Decreased apparent a f f i n i t y for i n t e r n a l N a +

Decreased apparen!

Na+

K+

FIGURE 3. Stable Na' transport abnormalities in erythrocytes from essential hypertensive

patients. results in a decreased ability of the Na+,K+cotransport system to extrude a cell N a + load. The maximal rate of the Na+,K+cotransport system may be decreased, normal, or increased as a function of the severity of high blood pressure and several other factors besides hypertension.6 In addition, most Co( -) hypertensives exhibit a normal interaction with external K+.18 Increased Maximal Rate of Na+:Li+ Countertransport (Counter( +)] Firstly described by Canessa et al. in 1980, this abnormality is present in about 20 to 40% of human hypertensives (Counter( +) hypertensives) and is frequently associated with an increased maximal rate of outward Na+,K+c ~ t r a n s p o r t . ~ ~ ' ~

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Decreased Apparent Afinity of the Na+,K+Pump for Internal Na+ [Pump(-)] We recently observed a fourth stable N a + transport abnormality, decreased apparent affinity of the Na+,K+ pump for internal Na+ in about 5-10% of human hypertensives.” This abnormality appears to be compensated by an increase in the maximal rates of the Na+,K+ pump and Na+, K + cotransport system.

Studies in Genetically Hypertensive Rats Studies of N a + transport systems in erythrocytes from genetically hypertensive rats showed varied results. Spontaneously Hypertensive Rats of the Okamoto strain (SHR) had a stable Co( -) abnormality (apparent affinity of the Na+,K+ cotransport system for internal N a + reduced by 50% in S H R rats aged from 2 to 26 weeks). Sabra Rats of the Hypertensive-prone substrain (HPS) had a stable Leak( +) abnormality that is compensated by increased maximal rates of the Na+,K+pump and the Na+,K+ cotransport ~ y s t e mMilano .~ Hypertensive Rats exhibited increased maximal rates of the Na+,K+ cotransport system.’

PHYSIOPATHOLOGICAL IMPLICATIONS The physiological relevance of each N a + transport system differs from cell to cell. It appears therefore that the physiopathological disturbances induced by each N a + transport abnormality may differ from organ to organ. For instance, Weder has shown that increased erythrocyte Na+:Li+ countertransport in human hypertensives is correlated with lower renal fractional lithium clearance.” This suggests that Counter( + ) hypertensives have increased proximal tubular sodium reabsorption and thus their target organ is the kidney. Increased sodium reabsorption may induce volume expansion, permanent secretion of OLF, inhibition of the pump in vascular cells, and hypertension.’ In Co(-) hypertensives (and S H R rats) the target cells may be noradrenergic neurons. Indeed, a Co(-) abnormality in noradrenergic endings may lead to an abnormal recovery of cell N a + content after an action potential, thus explaining the increased noradrenergic activity of S H R and of Co( -) hypertensives.” In addition, the transitory secretion of endogenous O L F following excess N a + intake may unmask the Co(-) defect in vascular smooth muscle cells leading to a more pronounced increase in cell N a + content (FIGURE2).3 Leak( +) hypertensives and HPS rats may present a similar disturbance in N a + handling by vascular smooth muscle cells than Co( -) hypertensives and S H R rats (FIGURE2) . 3

The Link between Abnormal Cell Na+ Handling and Hypertension The final step in the sequence of events leading to primary hypertension involves cytosolic free Ca2+content2and catecholamines. In the vascular wall, pump inhibition may partially depolarize cell membranes thus opening potential-dependent Ca2+ channels,’* increase catecholamine output in noradrenergic endings23 thus opening receptor-dependent Ca’+ channels, and stimulate CaZ+influx through Na+:Ca’+ exchange.2 The final consequence is an increase in both cytosolic free Ca2+content and peripheral arterial resistance.

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CLINICAL ASPECTS It is still too early to draw the clinical and therapeutical characteristics of the different subgroups of hypertensives. Nevertheless preliminary studies suggest that with elevated Co( -) hypertensives present labile or moderate hyperten~ion~.”.’~ plasma norepinephrine.” Conversely, Counter( +) hypertensives exhibit more severe hypertension and increased renin activity.24 PHARMACOLOGICAL ASPECTS The above studies suggested that a key step in the development of some forms of primary hypertension is the disturbance in cell N a + homeostasis induced by the interaction of “ouabain-like” factors with vascular cells having genetic abnormalities in membraneNa’ transport. We therefore proposed that drugs that may “protect” the digitalis receptor site of vascular cells against pump inhibition are of potential interest as a new approach to the treatment of high blood p r e s s ~ r e . ~ ’ Several arguments have suggested to us that canrenone, an antihypertensive drug, may interfere with the above mechanism. Canrenone is the active metabolite of

TABLE 1. Na+,K+-Pump in Human Erythrocytes Preincubated with Canrenone

Preincubation 14 hrl 1 pM Canrenone 10 pM Canrenone

Na+,K+-PumpActivity“ Control 2310 15 2370 * 10 2365 2 15

42.5 nM Ouabain 2075 r 30 2190 L 10 2265 i 30

% Inhibition

10.2% 7.6% 4.2%

Values in this table are given as mean r range of duplicates. A similar result was obtained in two further experiments. “Ouabain-sensitive Na efflux, in pmoles (1 . cells x hr)-l.

spironolactone. Its action mechanism was supposed to involve a competition with aldosterone for a common cytosolic receptor in distal and collecting tubules of the nephron. However, several authors have found that, in addition to this mechanism, ~ ~ .interaction ~~ is the canrenone is able to interact with the Na+,K+pump in ~ i t r o .This one expected for a partial agonist a t the digitalis receptor site. Under basal conditions, canrenone slightly inhibits Na+,K+-pump activity. This effect could explain the previous observation that canrenone administration (as K-canrenoate) potentiates the inotropic effect of digitalis in dogs.*’ If the pump is blocked by high doses of ouabain, canrenone is able to restimulate it. This effect is likely correlated with the ability of canrenone to protect against digitalis-induced cardiac toxicity” and to reverse the inhibitory effect of digoxin on basal and furosemide-stimulated renin secretion.*’ It is important to note that the above experiments in vitro required canrenone concentrations one order of magnitude higher than those observed in plasma of treated patients (which are of about 8-1 0 pM3’). We therefore investigated if preincubation with canrenone may elicit effects in vitro a t pharmacological doses. TABLE1 shows the effect of a 4 hr preincubation of human red cells with pharmacological doses of canrenone. It can be seen that the ability of low doses of ouabain to inhibit the pump is reduced by about one fourth and one half at canrenone concentrations of 1 and 10 pM,

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respectively. Under those conditions canrenone behaves like an antagonist at the digitalis receptor site. Interestingly canrenone is able to partially antagonize in vivo the secondary effects of endogenous “ouabain-like” factors on cell N a + handling and blood pressure in rats with reduced renal mass under excess Na’ intake.3’ These results suggest that chronic administration of canrenone to subjects with primary hypertension may induce a lowering of blood pressure by antagonism with endogenous “ouabain-like” factors at the vascular wall.

ACKNOWLEDGMENTS We are greatly indebted to G. Dagher, M. De Mendonqa, C. Nazaret, P. Hannaert, M.L. Grichois, M. Price, and J. Diez who worked in our laboratory in the past eight years. REFERENCES 1 . DAHL,L. K. 1977. Salt intake and hypertension. I n Hypertension. J. Genest, E. Koiw & 0. Kuchel, Eds.: 548-559. McGraw-Hill. New York. M. 1977. Sodium ions, calcium ions, blood pressure regulation and hyperten2. BLAUSTEIN, sion: A reassessment and a hypothesis. Am. J. Physiol. 2 3 2 C165-C173. M., A. KNORR,M. L. GRICHOIS, D. BEN-ISHAY,R. GARAY& P. MEYER. 3. DEMENDONCA, 1982. Erythrocytic ion transport systems in primary and secondary hypertension of the rat. Kidney Int. 21 (Suppl. 1 I): S69-S75. 4. BUCKALEW, V. M. & K. A. GRUBER.1983. Natriuretic hormone. I n The Kidney in Liver Disease. M. Epstein, Ed.: 479-499. Elsevier Biomedical. New York. 1983. The natriuretic hormone and its possible H. E. & G. A. MACGREGOR. 5. DEWARDENER, relationship to hypertension. In Hypertension. J. Genest, 0. Kuchel, P. Hamet & M. Cantin, Eds.: 84-95. McGraw Hill. New York. 6. GARAY,R. P., C. NAZARET,P. HANNAERT& M. PRICE.1983. Abnormal Na+,K+ cotransport function in a group of patients with essential hypertension. Eur. J. Clin Invest 13:311-320. 7. CANESSA,M., SPALVINS, & B. FALKNER. 1984. Red cell sodiumcountertransN. ADRAGNA port and cotransport in normotensive and hypertensive blacks. Hypertension 6 344-35 I. & G. BIANCHI. 1984. Cation 8. CUSI, D., C. BARLASSINA,P. FERRARI,M. FERRANDI transport abnormalities in human and rat essential hypertension. In Topics in Pathophysiology of Hypertension. H. Villarreal & M. P. Sambhi, Eds.: 136-146. Martinus-Nijhoff Publishers. Boston. Y.V. & S. N. ORLOV.1985. Ion transport across plasma membrane in primary 9. POSTNOV, hypertension. Physiol. Rev. 6 5 904-945. 1985. New aspects concerning the ”Na influx into red cells in 10. WESSELS,F. & H. ZUMKLEY. essential hypertension. Klin. Wochenschr. 63(SupplIII): 38-41. 1 1 . GARAY,R. P. & C. NAZARET.1985. Na’ leak in erythrocytes from essential hypertensive patients. Clin. Sci. 6 9 613-624. R. LORENZ,P. C. WEBER& J. DUHM.1985. Red cell Na+-K+ 12. BEHR,J., H. WITZGALL, transport in various forms of human hypertension. Role of cardiovascular risk factors and plasma potassium. Klin. Wochenschr. 63(Suppl 111): 63-65. J. G. 1985. Erythrocyte cation fluxes in essential hypertension of children and 13. MONGEAU, adolescents. Jnt. J. Ped. Nephrol. 6 41-46. 14. DE BOLD,A. 1985. Atrial natriuretic factor: A hormone produced by the heart. Science 230: 767-770. M. J. CAMARGO, A. JANUSZEWICZ, J. E. SEALEY, J. H. 15. ATLAS,S. A,, H. D. KLEINERT, J. A. LEWICKI,L. K. JOHNSON & T. MAACK.1984. LARAGH,J. W. SCHILLING,

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ANNALS NEW YORK ACADEMY OF SCIENCES Purification, sequencing and synthesis of natriuretic and vasoactive rat atrial peptide. Nature 309: 717-719. S. PANG,R. GARCIA, G . THIBAULT, J. GUTKOWSKA, M. CANTIN HAMET,P., J. TREMBLAY, & J. GENEST. 1984. Effect of native and synthetic Atrial Natriuretic Factor on cyclic GMP. Biochem. Biophys. Res. Commun. 123 515-527. F. RODRIGUE,B. DUNHAM,P. MARCHE,J. GENEST,P. GARAY,R., P. HANNAERT, C. BIANCHI,M. CANTIN& P. MEYER.1985. Atrial natriuretic factor inhibits BRAQUET, Ca-dependent, K-fluxes in cultured vascular smooth muscle cells. J. Hypertension J(suppl3): S297-S298. PRICE.M.. P. HANNAERT. G. DAGHER& R. P. GARAY.1984. The interaction of internal Na' and external K+ with the erythrocyte N a + , K+ cotransport system in essential hypertension. Hypertension 6 352-359. H. SOLOMON, E. SLATER & D. C. TOSTESON.1982. Red cell ADRAGNA, N., M. CANESSA, lithium-sodium countertransport and sodium-potassium cotransport in patients with essential hypertension. Hypertension 4 795-804. GARAY,R., J. DIEZ & P. BRAQUET.1985. Na,K-pump in essential hypertension. In The Sodium Pump. I. Glynn & C. Ellory, Eds.: 657-662. The Company of Biologists Limited. Cambridge, UK. WEDER,A. B. 1986. Red cell lithium countertransport and renal lithium clearance in hypertension. N. Eng. J. Med. 314 198-201. M. J. 1985. Changes in sodium pump activity and vascular contraction. J. MULVANY, Hypertension 3 429-436. Y., A. OHGA& Y. ONODA.1978. The effect of ouabain on noradrenaline NAKAZATO, output from peripheral adrenergic neurons of isolated guinea pig vas deferens. J. Physiol. (Lond.) 278 45-54. C.,R. CORROCHER, L. FORONI,M. STEINMAYR, F. BONFANTI& G. DE BRUGNARA, SANDRE. 1983. Lithium-sodium countertransport in erythrocytes of normal and hypertensive subjects: relationship with age and plasma renin activity. Hypertension 5: 529534. G. DAGHER & J. P. ABITBOL. 1985. The interaction of GARAY,R. P., J. DIEZ,C. NAZARET, canrenone with the Na,K-pump in human red blood cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 329 3 11-3 15. FINOTTI,P. & P. PALATINI.1981. Canrenone as a partial agonist at the digitalis receptor site of sodium-potassium activated adenosine triphosphatase. J. Pharmacol. Exp. Ther. 217:784-790 B. CAVALLARO & C. SPONZILLI.1983. MARCHETTI, G., E. VITOLO,G. F. DIFRANCESCO, Positive inotropic effect of K-Canrenoate: an investigation in anaesthetized dogs untreated or treated with digitalis. Arch. Int. Pharmacodyn. Ther. 266: 250-263. YEH,B. K., B. N. CHIANG& P. K. SUNG.1976. Antiarrhythmic activity of K-Canrenoate in man. Am. Heart J. 9 2 308-314. 1982. Canrenoate reversal of inhibitory effects of digoxin on FINOTTI,P. & A. ANTONELLO. basal and furosemide-stimulated renin secretion. Clin. Pharmacol. Ther. 3 2 1-6. & H. SCHOLZZ. 1984. Negative inotropic effects of aldosterone MUGGE,A,, W. SCHMITZ antagonists in isolated human and guinea-pig ventricular heart muscle. Klin. Wochenschr. 6 2 717-723. B. THORMANN, P. MEYER,M. A. DEMENDONCA, M., M. L. GRICHOIS, M. G. PERNOLLET, DEVYNCK& R. P. GARAY.1985. Hypotensive action of canrenone in a model of hypertension where a ouabain-like factor is present. J. Hypertension. 3 (suppl. 3): 573575.

DISCUSSION OF THE PAPER Q. AL-AWQATI(Columbia University, New York, N Y ) : Does canrenone displace labeled ouabain from membranes and if so what is t h e EC,,?

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GARAY:Erdmann has shown that canrenone displaces [3H]ouabain from heart cell membranes. Finotti and Palatini showed the same in purified Na+,K+-ATPase from guinea pig brain. The EC,, was between lo-' and I 0-4 M. G. BIANCHI(University of Milan, Milan): Did you try other diuretics to see whether for the same increase in N a + excretion the fall in blood pressure was greater with canrenone? GARAY:We compared the diuretic effect of canrenone in rats with reduced renal mass with that of furosemide and thiazides. In contrast with those diuretics, the diuretic effect of canrenone is slight and only lasts 24 hr. Blood pressure fall is not correlated with such diuretic effects. L. H. OPIE(University of Cape Town, Observatory, South Africa): (1) Did the leak abnormalities in hypertensions change with therapy? (2) Was there an overlap between leak and cotransport abnormalities in hypertensive patients? GARAY:(1) We studied the effect of a new diuretic (cicletanine) on ion transport in 55 hypertensives treated during two years. None of the four abnormalities (Pump( -), Leak( +), Co( -), or Counter( +)) was changed by this treatment. I do not know what happens with other antihypertensive drugs. (2) In our first studies we only observed patients with one (or none) stable abnormality. However, in recent studies we have found some patients presenting two abnormalities. We also found a patient (and only one) with three abnormalities (Co( -), Pump( -), and Leak( +)).