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.

No abstract

No abstract

Genetic variation in α-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of α-adducin in controlling Na/K pump activity, wild-type or mutated rat and human α-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, α-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension.

Essential hypertension is a complex, multifactorial disease associated with a high cardiovascular risk and whose genetic-molecular basis is heterogeneous and largely unknown. Although multiple antihypertensive therapies are available, the large individual variability in drug response results in only a modest reduction of the cardiovascular risk and unsatisfactory control of blood pressure in the hypertensive population as a whole. Two mechanisms, among others, are associated with essential hypertension and related organ damage: mutant α-adducin variants and high concentrations of endogenous ouabain. An antihypertensive agent, rostafuroxin, selectively inhibits these mechanisms in rodents. We investigated the molecular and functional effects of mutant α-adducin, ouabain, and rostafuroxin in hypertensive rats, human cells, and cell-free systems and demonstrated that both mutant α-adducin variants and the ouabain-Na,K-ATPase (Na(+)- and K(+)-dependent adenosine triphosphatase) complex can interact with the Src-SH2 (Src homology 2) domain, increasing Src activity and the Src-dependent Na,K-ATPase phosphorylation and activity. Wild-type α-adducin or Na,K-ATPase in the absence of ouabain showed no interaction with the Src-SH2 domain. Rostafuroxin disrupted the interactions between the Src-SH2 domain and mutant α-adducin or the ouabain-Na,K-ATPase complex and blunted Src activation and Na,K-ATPase phosphorylation, resulting in blood pressure normalization in the hypertensive rats. We have also shown the translatability of these data to humans in a pharmacogenomic clinical trial, as described in the companion paper.

The significance of the erythrocyte abnormalities described in rats and humans with spontaneous hypertension is far from clear. This study, in two highly inbred strains of rats, was designed to evaluate whether these abnormalities are primary and thus genetically related to hyper-tension. The Milan hypertensive strain (MHS) and its normotensive control strain (MNS) were used to carry out two types of experiments. In two groups of lethally irradiated (MHS x MNS) F, hybrids, bone marrow from MHS or MNS was transplanted. The differences in red cell function between the recipients of bone marrow from MHS and recipients of bone marrow from MNS were similar to those existing between the parental donor MHS and MNS: Na +-K + cotransport was increased (p < 0.02) and intracellular Na + content (p < 0.05) and cell volume (p < 0.02) were decreased in MHS. The same pattern was observed when this experiment was repeated in different groups of F, hybrids. In individuals of the segregating F 2 population, obtained by crossing the (MHS x MNS) F, hybrids, there was a positive correlation (p < 0.001) between the red blood cell Na +-K + cotransport and the mean blood pressure. These results indicate that the erythrocyte abnormalities may well be genetically associated with the primary cause of spontaneous hypertension in rats. Because of the many similarities demonstrated when young prehypertensive MHS or humans prone to develop hypertension are compared with their respective controls, it is possible that the findings described here in rats are relevant to human essential hypertension. (Hypertension 7: 319-325, 1985) KEY WORDS • red blood cell • genetic hypertension • cell volume • ion transport T HE cause of essential hypertension is not known. It is generally accepted that hyperten-sion develops when there is increased genetic sensitivity to certain environmental stimuli 1 and that red blood cell (RBC) membrane functions are abnormal in humans with essential hypertension 2 " 6 and rats with spontaneous hypertension. 7 '" It is not clear whether these alterations in red cell cation transport are the result of chance genetic selection unrelated to the cause of hypertension, are secondary to the hyperten-sive disease, or are the direct phenotypic expression of the same genetic abnormalities responsible for the increased susceptibility to hypertension. We must determine which of these possibilities is responsible if we wish to use the RBC transport systems as probes to study the genetic mechanisms of arterial hypertension. We have approached these questions by using two highly inbred strains of rats, the Milan hypertensive strain (MHS, inbreeding coefficient 0.988) and its nor-motensive control (MNS, inbreeding coefficient 0.989). Previous studies 12 " 14 with MHS and MNS showed many pathophysiological similarities between their hypertension and essential hypertension in humans. Kidney cross-transplantation and renal function studies suggested that the hypertension of MHS might originate from a primary increase in the transport of Na...

Abstract-In a previous study, by using a candidate gene approach, we detected in both Milan hypertensive rats and humans a polymorphism in the ␣-adducin gene (ADD1) that was associated with blood pressure and renal sodium handling. In the present study, a genomewide search with 264 informative markers was undertaken in 251 (Milan hypertensive strain ϫ Milan normotensive strain) F2 rats to further investigate the contribution of the adducin gene family (Add1, Add2, and Add3) and to identify novel quantitative trait loci (QTLs) that affect blood pressure. The influence of 2 different methods of blood pressure measurement, the intracarotid catheter and the tail-cuff method, was also evaluated. We found evidence that QTLs affected systolic blood pressure (SBP) measured at the carotid (direct SBP) on rat chromosome 1 with a logarithm of the odds (LOD) score peak of 3.3 on D1Rat121 and on rat chromosome 14 on Add1 locus (LODϭ3.2). A QTL for SBP measured at the tail (indirect SBP) was found on rat chromosome 10 around D10Rat33 (LODϭ5.0). All of these QTLs identified chromosomal regions not detected in other rat studies and harbor genes (Na ϩ /H ϩ exchanger A3; ␣-adducin; ␣ 1B -adrenergic receptor) that may be involved in blood pressure regulation. Therefore, these findings may be relevant to human hypertension, also in consideration of the biochemical and pathophysiological similarities between MHS and a subgroup of patients of primary hypertension, which led to the identification of ␣-adducin as a candidate gene in both species. (Hypertension. 2000;36:734-739.) Key Words: genes Ⅲ rats, inbred strains Ⅲ hypertension, essential A variety of strategies can be applied to demonstrate the molecular genetic determinants of human essential hypertension. Our approach was to study, in parallel, hypertensive patients and an animal model of genetic hypertension, the Milan hypertensive strain (MHS) rat with its normotensive control (Milan normotensive strain [MNS]). 1 In this way, it may be possible to reduce the enormous complexity that arises from the polygenic nature of hypertension, the environmental and genetic context dependency of each gene effect, and all of the consequences that these 2 confounding characteristics may have at the different levels of biological organization. The main drawbacks of this strategy are that (1) only the genes at work in a particular strain of rats can be detected, and other genes, with their interactions, that may be involved in human hypertension are ignored, and (2) the small degree of polymorphism between 2 strains of rats derived from common ancestors may hamper the detection of a number of polymorphic markers suitable to carry out a total genome search and the congenic strain selection. This last limitation considerably delayed the whole genome screening in the MHSϫMNS cross. The continuous improvements and the recent availability of new rat markers 2-4 allowed us to provide a systematic genome scan for blood pressure (BP) quantitative trait loci (QTLs) in an (MHSϫMNS) F2 population.Bioche...