Lucia Torielli

HYPERTENSION-ASSOCIATED POINT MUTATIONS IN THE ADDUCIN ALPHA AND BETA SUBUNITS AFFECT ACTIN CYTOSKELETON AND ION TRANSPORT

The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (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 alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 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.

Red blood cell abnormalities and spontaneous hypertension in the rat. A genetically determined link.

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 hypertension. 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) F1 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 less than 0.02) and intracellular Na+ content (p less than 0.05) and cell volume (p less than 0.02) were decreased in MHS. The same pattern was observed when this experiment was repeated in different groups of F1 hybrids. In individuals of the segregating F2 population, obtained by crossing the (MHS X MNS) F1 hybrids, there was a positive correlation (p less than 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.

Genetic Mapping of Blood Pressure Quantitative Trait Loci in Milan Hypertensive Rats

In a previous study, by using a candidate gene approach, we detected in both Milan hypertensive rats and humans a polymorphism in the &agr;-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 × 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 (LOD=3.2). A QTL for SBP measured at the tail (indirect SBP) was found on rat chromosome 10 around D10Rat33 (LOD=5.0). All of these QTLs identified chromosomal regions not detected in other rat studies and harbor genes (Na+/H+ exchanger A3; &agr;-adducin; &agr;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 &agr;-adducin as a candidate gene in both species.

α-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption

Genetic variation in alpha-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 alpha-adducin in controlling Na/K pump activity, wild-type or mutated rat and human alpha-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, alpha-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.

Relationship between erythrocyte volume and sodium transport in the Milan hypertensive rat and age-dependent changes

The relationship between differences in red blood cell (RBC) volume and ion transport across the erythrocyte cell membrane were investigated in the Milan Hypertensive (MHS) and Milan Normotensive (MNS) rat strains, under different experimental conditions and during ageing. The results obtained indicate that: the difference in Na+/K+ cotransport between MHS and MNS disappear when the RBC volume of the two strains becomes equal under hypotonic swelling; MHS RBCs are osmotically more fragile than those of MNS, probably because of a different membrane structure rather than a different amount of membrane surface, and the smaller volume and the lower Na+ content of MHS RBCs are maintained throughout the life span, while Na+/K+ pump activity and Na+/K+ cotransport undergo age-dependent changes, related to the development of hypertension. All these findings suggest that a primary abnormality of the cell membrane structure of MHS, probably located in the cytoskeleton, is responsible for the cell functional alterations that we previously demonstrated to be genetically associated with MHS hypertension.

NKCC2 is activated in Milan hypertensive rats contributing to the maintenance of salt-sensitive hypertension

The Milan hypertensive strain of rats (MHS) develops hypertension as a consequence of the increased tubular Na+ reabsorption sustained by enhanced expression and activity of the renal tubular Na–K–ATPase. To verify whether the Na–K–2Cl cotransporter (NKCC2) is involved in the maintenance of hypertension in MHS rats, we have analysed the phosphorylation state and the activation of NKCC2 in Milan rats. Western blotting and immunofluorescence experiments were performed using specific antibodies against the regulatory phospho-threonines in the NKCC2 N terminus (R5 antibody). The phosphorylation levels of NKCC2 were significantly increased in the kidney of MHS rats. Moreover, the administration of furosemide in vivo decreased the blood pressure and increased the urine output and natriuresis in MHS rats demonstrating the actual involvement of NKCC2 activity in the pathogenesis of hypertension in this strain of rats. The up-regulation of NKCC2 activity is most probably mediated by a STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylation at serine-325 since it was significantly increased in MHS rats. Interestingly, aldosterone treatment caused an increase in NKCC2 phosphorylation in NKCC2-expressing MDCK cells. In conclusion, we demonstrated an increase in the activity of NKCC2 along the TAL that significantly contributes to the increase in systemic blood pressure in MHS rats. The elevated plasma levels of aldosterone, found in MHS rats, may influence Na+ balance through a SPAK-dependent regulation of NKCC2 accounting for the maintenance of the hypertensive state in MHS rats.

SODIUM TRANSPORT KINETICS IN ERYTHROCYTES AND INSIDE-OUT VESICLES FROM MILAN RATS

This paper describes the kinetics of the Na(+)-K+ pump and the Na(+)-K(+)-Cl- cotransport in sodium-loaded erythrocytes and of the Na(+)-K(+)-Cl- cotransport in erythrocyte inside-out vesicles (IOV) from Milan hypertensive (MHS) and normotensive (MNS) rats in order to evaluate the possible role of intracellular factor(s). In intact erythrocytes, no difference was detectable in the Na(+)-K+ pump kinetics between the two strains while the apparent affinity (Km) of Na(+)-K(+)-Cl- cotransport for internal sodium was significantly greater and the maximal rate of sodium transport lower in MHS when compared with MNS rats. IOV, which are depleted of cytoskeleton, showed a bumetanide-sensitive potassium- and chloride-dependent sodium uptake. However, the erythrocyte differences in Na(+)-K(+)-Cl- cotransport activity and the Km between strains disappeared in IOV, suggesting tha the altered sodium transport of MHS erythrocyte might be due to some intracellular factor or a membrane skeleton protein abnormality.

Na+/K+/Cl− cotransport in resealed ghosts from erythrocytes of the Milan hypertensive rats

The erythrocytes (RBC) of the Milan hypertensive rats (MHS) have a smaller volume and faster Na+/K+/Cl- cotransport than RBC from normotensive controls (MNS). The difference in Na+/K+/Cl- cotransport is no longer present in inside-out Vesicles (IOV) of RBC membrane. To differentiate between cytoplasmic or membrane skeleton abnormalities as possible causes of these differences. Resealed ghosts (RG) were used to measure ion transport systems. The following results have been obtained: (1) RG from MHS have a smaller volume than MNS (mean +/- S.E. 20.7 +/- 0.45 vs. 22.09 +/- 0.42 fl, P < 0.05). (2) RG showed a bumetanide-sensitive Na efflux that retains the characteristics of the Na+/K+/Cl- cotransport of the original RBC: it is K(+)- and Cl(-)-sensitive and dependent on the intracellular Na+ concentration. (3) The Na+/K+/Cl- cotransport was faster in RG from MHS than in those from MNS (mean +/- S.E. 0.095 +/- 0.01 vs. 0.066 +/- 0.01 rate constant h-1, P < 0.01). These results, together with those of IOV, support the hypothesis that an abnormality in the membrane skeletal proteins may play a role in the different Na+/K+/Cl- cotransport modulation between MHS and MNS erythrocytes.

Antihypertensive Compounds That Modulate the Na-K Pump

Abstract: A primary impairment of the kidney sodium excretion has been documented both in hypertensive patients (EH) and genetic animal models (Milan hypertensive rat [MHS]) carrying mutations of the cytoskeletal protein adducin and/or increased plasma levels of endogenous ouabain (EO). Ouabain (OU) itself induces hypertension in rats and both OU and mutated adducin activate the renal Na/K‐ATPase function both in vivo and in cultured renal cells (NRK). A new antihypertensive agent, PST 2238, able to selectively interact with these alterations has been developed. PST lowers blood pressure (BP) by normalizing the expression and activity of the renal Na‐K pump selectively in those rat models carrying the adducin mutation (MHS) and/or increased EO levels (OS) at oral doses of 0.1‐10 μg/kg. In NRK cells either transfected with mutated adducin or incubated with 10−9 M OU, PST normalizes the Na‐K pump activity. Recently, an association between EO and cardiac complications has been observed in both EH and rat models consistent with a prohypertrophic activity of OU. OS rats showed a 10% increase of left ventricle and kidney weights as compared with controls, and PST 2238 (1 μg/kg OS) prevented both ventricle and renal hypertrophy. This effect was associated with the ability of PST to antagonize the OU‐dependent activation of growth‐related genes, in the membrane subdomains of caveolae. In conclusion, PST is a new antihypertensive agent that may prevent cardiovascular complications associated with hypertension through the selective modulation of the Na‐K pump function.

Altered expression of renal aquaporins and α-adducin polymorphisms may contribute to the establishment of salt-sensitive hypertension.

BACKGROUND Sodium-sensitive hypertension is caused by renal tubular dysfunction, leading to increased retention of sodium and water. Previous findings have suggested that single-nucleotide polymorphisms of the cytoskeletal protein, α-adducin, are associated with increased membrane expression of the Na/K pump and abnormal renal sodium transport in Milan hypertensive strain (MHS) rats and in humans. However, the possible contribution of renal aquaporins (AQPs) to water retention remains undefined in MHS rats. METHODS Kidneys from MHS rats were analyzed and compared with those from age-matched Milan normotensive strain (MNS) animals by quantitative-PCR, immunoblotting, and immunoperoxidase. Endocytosis assay was performed on renal cells stably expressing AQP4 and co-transfected either with wild-type normotensive (NT) or with mutated hypertensive (HT) α-adducin. RESULTS Semiquantitative immunoblotting revealed that AQP1 abundance was significantly decreased only in HT MHS whereas AQP2 was reduced in both young pre-HT and adult-HT animals. On the other hand, AQP4 was dramatically upregulated in MHS regardless of the age. These results were confirmed by immunoperoxidase microscopy. Endocytosis assays clearly showed that the expression of mutated adducin strongly reduced the rate of constitutive AQP4 endocytosis, thereby increasing its abundance at the plasma membrane. CONCLUSIONS We provide here the first evidence that AQP1, AQP2, and AQP4 are dysregulated in the kidneys of MHS animals. In particular, we provide evidence that α-adducin mutations may be responsible for AQP4 upregulation. The downregulation of AQP1 and AQP2 and the upregulation of AQP4 may be relevant for the onset and maintenance of salt-sensitive hypertension.