The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin ␣ ( F316Y ) and ␤ ( Q529R ) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: ( a ) actin assembly both in a cell-free system and within transfected cells; ( b ) topography of ␣ V integrin together with focal contact proteins; and ( c ) Na-K pump activity at V max (faster with the mutated isoforms, 1281 Ϯ 90 vs 841 Ϯ 30 nmol K/ h · mg pt., P Ͻ 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage. ( J. Clin. Invest. 1996. 97:2815-2822.)

The Milan hypertensive strain of rats (MHS) develops a genetic form of renal hypertension that, when compared to its normotensive control (MNS), shows renal dysfunction similar to that of a subset of human patients with primary hypertension. MHS and MNS were shown to be homozygous by multilocus miisatellite analysis and monolocus microsatellite markers. We show here that one point mutation in each of two genes coding for the membrane skeleton protein adducin is associated with blood pressure in the Milan strain of rats. Adducin is a heterodimer formed by a and 13 subunits that promotes the assembly of actin with spectrin. MHS and MNS differ, respectively, by the amino acids Y and F at position 316 of the a subunit. In the ,&adducin locus, MRS is always homozygous for R at position 529 while in MNS either R or Q occurs in that position. The R/Q heterozygotes showed lower blood pressure than any of the homozygotes. In vitro phosphorylation studies suggest that both of these amino acid substitutions occur within protein kinase recognition sites. Analysis of an F2 generation demonstrated that Y alleles segregated with a significant increment in blood pressure. This effect is modulated by the presence of the R allele of the 1 subunit. Taken together, these dings strongly support a role for adducin polymorphisms in causing variation of blood pressure in the Milan strain of rats.

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Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

Abstract-␣-Adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na ϩ ,K ϩ -ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na ϩ ,K ϩ -ATPase activity, and their Na ϩ ,K ϩ -ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na ϩ ,K ϩ -ATPase because of adaptin-2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na ϩ ,K ϩ -ATPase activity, 2-subunit hyperphosphorylation, and impaired Na ϩ ,K ϩ -ATPase endocytosis. Thus, increased renal Na ϩ ,K ϩ -ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na ϩ ,K ϩ -ATPase endocytosis in response to natriuretic signals. Key Words: high blood pressure Ⅲ sodium retention Ⅲ protein trafficking Ⅲ cytoskeleton Ⅲ endocytosis S pontaneous mutations in ␣-adducin gene favor renal sodium retention and the development of high blood pressure in humans and rodents. 1 The estimated frequency of the mutated allele fluctuates among 8% in African Americans, 15% to 20% in whites, and 50% in Japanese individuals. The resulting phenotype is similar in rats and humans despite species differences in the mutation sites (human G460W/ S586C; rats F316Y). These polymorphisms affect the pressure natriuresis curve, the lithium clearance (an indicator of proximal tubule [PCT] sodium transport), 2 and the chronic blood pressure response to diuretics. 3,4 Recent studies further substantiate the role of adducin in human hypertension and the associated renal or cardiac alterations. 5,6 Many of these findings have been obtained in low-renin hypertensive patients, 7 in whom renal Na ϩ retention is a particularly important pathogenetic factor, or in patients carrying the gene variants known to affect renal Na ϩ handling. 8 At the cellular level, adducin plays an active role in cytoskeleton organization by regulating actin dynamics. 9 The mutated hypertensive variant of the adducin ␣-subunit increases the rate of actin polymerization and bundling in a cell-free system compared with the wild-type variant. 10 In cultured renal cells stably transfected with the mutated ␣-adducin, the actin cytoskeleton network appears thicker and Na ϩ ,K ϩ -ATPase activity is higher than in cells transfected with the wild-type variant. 10 In renal epithelial cells, the Na ϩ ,K ϩ -ATPase is located at the basolateral membrane domain and is critical for vectorial sodium transport. Its regulation by catecholamines cont...

Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb+ occlusion, turnover number, alpha 1- and beta 1-subunit protein abundance, and alpha 1 and beta 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb+ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of alpha 1 and beta 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.

Twenty years of genetic studies have not contributed to improvement in the clinical management of primary arterial hypertension. Genetic heterogeneity, epistatic-environmental-biological interactions, and the pathophysiological complexity of hypertension have hampered the clinical application of genetic findings. In the companion article, we furnished data from rodents and human cells demonstrating two hypertension-triggering mechanisms-variants of adducin and elevated concentrations of endogenous ouabain (within a particular range)-and their selective inhibition by the drug rostafuroxin. Here, we have investigated the relationship between variants of genes encoding enzymes for ouabain synthesis [LSS (lanosterol synthase) and HSD3B1 (hydroxy-d-5-steroid dehydrogenase, 3b-and steroid d-isomerase 1)], ouabain transport {MDR1/ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1]}, and adducin activity [ADD1 (adducin 1) and ADD3], and the responses to antihypertensive medications. We determined the presence of these variants in newly recruited, never-treated patients. The genetic profile defined by these variants predicted the antihypertensive effect of rostafuroxin (a mean placebo-corrected systolic blood pressure fall of 14 millimeters of mercury) but not that of losartan or hydrochlorothiazide. The magnitude of the rostafuroxin antihypertensive effect was twice that of antihypertensive drugs recently tested in phase 2 clinical trials. One-quarter of patients with primary hypertension display these variants of adducin or concentrations of endogenous ouabain and would be expected to respond to therapy with rostafuroxin. Because the mechanisms that are inhibited by rostafuroxin also underlie hypertensionrelated organ damage, this drug may also reduce the cardiovascular risk in these patients beyond that expected by the reduction in systolic blood pressure alone.

BACKGROUND Endogenous ouabain (EO) has been linked with long-term changes in sodium balance and cardiovascular structure and function. The biosynthesis of EO involves, cholesterol side-chain cleavage (CYP11A1), 3-β-hydroxysteroid dehydrogenase (HSD3B) with sequential metabolism of pregnenolone and progesterone. Furthermore, the renal excretion of cardiac glycosides is mediated by the organic anion transporter (SLCO4C1) at the basolateral membrane and the P-glycoprotein (PGP) (encoded by MDR1) at the apical membrane of the nephron. METHODS Average 24-h ambulatory blood pressures were recorded in 729 untreated essential hypertensives. Aldosterone (Aldo), EO, urinary Na+, and K+ excretions were determined. Single-nucleotide polymorphism (SNP) and haplotype-based association study was performed with a total of 26 informative SNPs. RESULTS Plasma EO was significantly directly related to both day (r = 0.131, P < 0.01) and nighttime diastolic blood pressure (DBP) (r = 0.143, P < 0.01), and remained significantly related after correction for confounders (sex, body mass index, age). Genotype analysis for EO levels and daytime DBP gave significant results for CYP11A1 rs11638442 and MDR1 rs1045642 (T/C Ile1145) in which the minor allele tracked with higher EO levels (T/T 210.3 (147–272) vs. C/C 270.7 (193–366) pmol/l, P < 0.001). Association was found between HSD3B1 polymorphisms and/or haplotypes with blood pressure (systolic blood pressure (SBP) 140.3 (11.7) vs. 143.8 (11.2) mm Hg, P < 0.01) and plasma Aldo (P < 0.05). Haplotype-based analyses support the data of SNP analysis. CONCLUSIONS Among patients with essential hypertension, cholesterol side-chain cleavage and MDR1 loci are related to circulating EO and DBP, most likely by influencing EO synthesis and transmembrane transport, respectively. In contrast, variants in HSD3B1 are related with SBP probably via Aldo.