Role of rat alpha adducin in angiogenesis: null effect of the F316Y polymorphism.

OBJECTIVE: Rat alpha adducin point mutation (F316Y) has been associated with primary systemic arterial hypertension. As microcirculatory abnormalities are present in most forms of hypertension, the aim of the present study was to investigate whether rat alpha adducin may regulate endothelial cell (EC) functions in vitro and in vivo. METHODS AND RESULTS: The overexpression of rat wild type alpha adducin (WT-Add1) in ECs induced capillary-like structure development in Matrigel in vitro and enhanced capillary formation in Matrigel implants in vivo in CD1 mice. In contrast, the overexpression of the mutated form (MUT-Add1) of rat alpha adducin had a Null effect in vitro and lacked any significant activity in vivo. Further, adenovirus-mediated rat WT-Add1 but not MUT-Add1 gene transfer to murine ischemic hindlimb enhanced capillary formation in skeletal muscles. Gene profiling of human umbilical vein endothelial cells overexpressing alpha adducin was performed in order to identify putative effector molecules of alpha adducin-mediated activities on ECs. Interestingly, among a number of genes involved in angiogenesis regulation, retinoic acid-induced protein (RAI17) was found to be upregulated in WT-Add1 vs MUT-Add1 overexpressing cells, possibly representing a key molecule/axis for the functional Add1-induced effect. CONCLUSIONS: Rat WT alpha adducin enhanced EC functions both in vitro and in vivo. The expression of the F316Y variant, associated with the hypertensive phenotype, had a Null effect and might contribute to endothelial rarefaction/dysfunction in hypertension. RAI17 was found to be a putative effector molecule differentially regulated by the overexpression of the two forms of Add1 in endothelial cells.

Hypertension-linked mutation in the adducin alpha-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+,K+-ATPase trafficking in response to GPCR signals and intracellular sodium.

Alpha-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+,K+-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+,K+-ATPase activity, and their Na+,K+-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+,K+-ATPase because of adaptin-mu2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na+,K+-ATPase activity, mu2-subunit hyperphosphorylation, and impaired Na+,K+-ATPase endocytosis. Thus, increased renal Na+,K+-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+,K+-ATPase endocytosis in response to natriuretic signals.

RFX-1, a putative alpha Adducin interacting protein in a human kidney library.

Adducin regulates tubular absorption of sodium by modulating the expression levels of the sodium-potassium-ATPase in renal tubular cells. Adducin is a candidate gene in the pathogenesis of hypertension. Yeast two hybrid screen showed a specific interaction between human alpha Adducin and the regulatory factor for X box (RFX-1), a nuclear protein that down regulates the expression of several proteins in non neuronal cells. The interaction was confirmed in cells through co-immunoprecipitation and colocalization experiments. The binding of alpha Adducin to RFX-I and their nuclear co-localization suggests that Adducin can have a role in modulating the transcriptional regulating activity of RFX-I.

Haplotype analysis of carnitine transporters and left ventricular mass in human essential hypertension.

OBJECTIVE: The carnitine-associated alteration of myocardial fatty acid metabolism may be one of the molecular mechanisms underlying left ventricular hypertrophy (LVH) in essential hypertension. We tested the hypothesis that polymorphisms of the genes involved in carnitine transport, OCTN2, CPT1A, CPT1B, and CPT2, might be associated with LVH. DESIGN: Haplotype-based association analysis in an observational study. SETTING: Outpatients from the Nephrology Division of the University Hospital. PATIENTS: A total of 215 never-treated, middle-aged patients with mild essential hypertension. METHODS: Relationships between left ventricular mass index (LVMI) (measured with m-mode echocardiography) and haplotype combinations for 13 common genetic variants selected from single nucleotide polymorphism database (dbSNPs). RESULTS: The SNPs were selected to cover the genomic region of the four loci, and a total of 23 haplotypes were identified: 8 for OCTN2 (H1 to H8), 8 for CPT1A (H9 to H16), 3 for CPT1B (H17 to H19), and 4 for CPT2 (H20 to H23). In a multilocus haplotype analysis, after adjusting for sex, age, systolic blood pressure, diastolic blood pressure, body mass index, and duration of hypertension, a significant effect on LVMI was seen for H13 (+8.9, P = .05), H14 (-5.63, P = .05), H15 (-18.79, P = .0006), H18 (-1.66, P = .03), and H22 (-3.42, P = .004). These significant haplotypes were respectively 3.7%, 1.6%, 1.6%, 39.3%, and 29.7% of the total population. CONCLUSIONS: These results identify the carnitine-transporter gene family as candidate modifiers of LVMI in human hypertension. The use of common SNPs to define informative haplotypes associated with the phenotype of interest is the starting point for progress toward identification of the trapped contributing SNP(s).

Genetics of hypertension: the adducin paradigm.

The following were investigated: (1) how we became interested in studying adducin genes and what we know about adducin; (2) studies in animals and humans supporting the role of adducin polymorphisms in hypertension, including some methodological problems related to the dissection of the role of a given genetic molecular mechanism in a complex multifactorial polygenic disease like hypertension; (3) biochemical mechanisms underlying the effect of adducin and its interaction with the Na-K pump; and (4) future directions.

Mutation in the beta adducin subunit causes tissue-specific damage to myogenic tone.

BACKGROUND: Damage to renal artery myogenic tone is universally associated with progressive kidney damage. Recently, we have observed that mutations in the beta adducin subunit are associated with proteinuria in the Milan rat. Because of the role of adducin as a component of the cytoskeleton we hypothesized that this mutation may be associated with changes in myogenic tone. METHODS AND RESULTS: Congenic rats were generated with beta adducin subunit mutation (NB rats) and compared with a previously studied rat model with alpha adducin subunit mutation (NAs rats). Blood pressure and urinary protein excretion were studied at two time points: 6 weeks and 4 months of age, and at these time points, small renal, middle cerebral and skeletal (cremaster) arteries were isolated and studied using pressure myography. Agonist-induced vasoconstriction was not different between the two groups at any age. However, myogenic tone in renal arteries was significantly damaged in the NB rat compared to its NAs counterpart and this was associated with a decrease in vascular distensibility. There was a smaller reduction in myogenic tone in the middle cerebral arteries from the NB rat, whereas in the skeletal arteries there was no difference between the two strains. In the NB rat, this tissue-specific damage to myogenic tone was associated with progressive proteinuria despite lower blood pressure than the NAs rat. CONCLUSIONS: Mutations in the beta subunit of the adducin protein result in damage to renal artery myogenic tone and this is associated with renal damage as manifest by proteinuria.

Adducin- and ouabain-related gene variants predict the antihypertensive activity of rostafuroxin, part 2: clinical studies.

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-δ-5-steroid dehydrogenase, 3ß- and steroid δ-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 hypertension-related organ damage, this drug may also reduce the cardiovascular risk in these patients beyond that expected by the reduction in systolic blood pressure alone.

Steroid biosynthesis and renal excretion in human essential hypertension: association with blood pressure and endogenous ouabain.

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-beta-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.

Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris.

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.

Angiotensin-converting enzyme I/D and alpha-adducin Gly460Trp polymorphisms: from angiotensin-converting enzyme activity to cardiovascular outcome.

The angiotensin-converting enzyme (ACE) I/D and the alpha-adducin (ADD1) Gly460Trp polymorphisms are associated with cardiovascular risk factors. In a prospective population study and in cell models, we investigated the combined effects of these 2 polymorphisms. We randomly recruited 1287 white subjects (women: 50.0%; mean age: 55.9 years). We obtained outcomes from registries and repeat examinations (median 3). Over 9.0 years (median), 178 fatal or nonfatal cardiovascular events occurred. In ADD1 Trp allele carriers, the multivariate-adjusted hazard ratios associated with ACE DD versus I were 1.72 (P=0.007) for total mortality, 2.35 (P=0.02) for cardiovascular mortality, 2.02 (P=0.005) for all cardiovascular events, and 2.59 (P=0.03) for heart failure. In contrast, these hazard ratios did not reach significance in ADD1 GlyGly homozygotes (0.08

Tissue-specific modulation of beta-adducin transcripts in Milan hypertensive rats.

Genetic variants in Adducins, a family of cytoskeleton proteins (alpha, beta, and gamma) encoded by three genes, have been associated with primary hypertension in humans and in Milan hypertensive (MHS) rats. The present paper describes the identification of a rat beta 4 alternative splicing isoform differing from beta subunit for an in-frame insertion of 18 amino acids and showing a polymorphic site (R592W) between MHS and its normotensive control (MNS). Furthermore, we established a quantitative real-time PCR assay for analyzing the tissue expression of adducin gene family and determining whether any subunit transcript demonstrates altered expression during the development of MHS hypertension, especially in tissues relevant for the control of cardiovascular phenotypes (i.e., kidney, left ventricle, and large arteries). Among the three adducins only beta transcripts were modulated, in a tissue-specific manner, during the development of hypertension in MHS, compared to age-matched MNS controls. A 43% decrease in renal outer medulla was already present at the prehypertensive phase; a 70% decrease in femoral artery and 66% increase in left ventricle were observed after the development of hypertension. Surprisingly beta 4-Add, which is a minor component of total beta transcripts, is drastically reduced up to 88% in all MHS tissues. Alteration in beta-Add expression levels may account, at least in part, for the observed phenotypic changes in MHS hypertension.

Effect of Add1 gene transfer on blood pressure in reciprocal congenic strains of Milan rats.

Genetic variants of alpha adducin (ADD1) taken alone or in interaction with those of beta (ADD2) and gamma (ADD3) subunits have been associated with primary hypertension in humans and in Milan hypertensive (MHS) rats. In this study, we report the dissection of the individual contribution of each rat Add gene to blood pressure, by congenic substitution mapping. Congenic strains were developed by introgressing Add1, Add2, and Add3 genes (and chr14, chr4, and chr1 associated segments) of MHS in the Milan normotensive rat (MNS) genetic background (MNS.H-Add1, MNS.H-Add2, and MNS.H-Add3) and vice versa (MHS.N-Add1, MHS.N-Add2, and MHS.N-Add3). Systolic blood pressure (SBP) of MNS.H-Add1 rats was significantly higher (+10 mmHg) than that of MNS, whereas SBP of MHS.N-Add1 was significantly lower (-10 mmHg) than that of MHS. The differences account for 43% of the blood pressure differences between MHS and MNS. In contrast, SBPs of Add2 and Add3 congenic strains were not different from those of the correspondent recipient parental strain. The fine mapping of chr14 congenic segment supports the identity of blood pressure QTL with Add1 gene.

Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences.

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11beta-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [(14)C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.