Monica Florio

Renal Na,K-ATPase in Genetic Hypertension

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.

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.

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.

Characteristics of a ouabain-like factor from Milan hypertensive rats

Ouabain-like factor (OLF) has been extracted from hypothalamus and adrenal glands of the ox and rats of the Milan hypertensive strain (MHS) and their normotensive controls (MNS). OLF was identified by its ability (a) to inhibit ouabain-sensitive 86Rb uptake into human erythrocytes, (b) to displace [3H]ouabain binding, and (c) to inhibit purified dog kidney Na-K-ATPase. Rat and bovine OLF have similar characteristics. Those that are close to ouabain are (a) ligand conditions for maximal inhibitory activity, (b) high-performance liquid chromatography retention time, (c) reversibility of inhibitory activity on Na-K-ATPase, (d) reduced Na-K-pump inhibitory activity by K+, (e) high affinity for Na-K-ATPase, and (f) no activity on Ca(2+)-ATPase. OLF does not resemble ouabain because the capacity of OLF to inhibit ouabain low-affinity Na-K-ATPase isoform is greater than that of ouabain. The yield of OLF is greater from MHS than MNS hypothalamic and adrenal extracts. These findings represent the first direct evidence that a higher amount of OLF is present in tissues from genetically hypertensive rats than from their inbred normotensive controls, maintained under the same dietary and environmental conditions. This further supports previous observations on the role of OLF in the pathogenesis of MHS hypertension.

Age-dependency and dietary influence on the hypothalamic ouabain-like factor in Milan hypertensive rats

Aim Previous studies have demonstrated that the hypothalamus of the adult Milan hypertensive rat strain (MHS) contains a higher proportion of ouabain-like factor than Milan normotensive (MNS) controls. The present study was designed to demonstrate that the rat standard diet contains a ouabain-like factor similar to that extracted from rat tissue and to investigate the influence of low or high dietary ouabain-like factor content on tissue ouabain-like factor levels at different ages in MHS and MNS rats. Materials and methods MHS and MNS rats were reared on two controlled batches of standard rat diet containing low (batch A 0.09 μg/kg) and high (batch B 0.7 μg/kg) concentrations of ouabain-like factor. The mothers of these rats had also been fed with the diet throughout pregnancy and lactation. The hypothalamic content of ouabain-like factor was measured in both strains at 21, 30 and 90 days of age by high performance liquid chromatography fractionation. Results (1) The dietary ouabain-like factor content did not influence either the hypothalamic ouabain-like factor yield or systolic blood pressure, either in MHS or MNS rats. (2) As a function of age, the hypothalamic ouabain-like factor content was constant between 21 and 30 days of age in MHS rats, and then decreased by 60% at 90 days. In MNS rats, ouabain-like factor was decreased by 80 and 90%, respectively, at 30 and 90 days, compared to the age of 21 days. (3) At the age of 21 days, MHS rats had 30% lower levels of ouabain-like factor than MNS rats, but 60% higher levels at 30 and 90 days. Conclusions Hypothalamic ouabain-like factor and systolic blood pressure are not influenced by dietary ouabain-like factor, thus excluding a process of passive tissue accumulation. Different mechanisms regulate the age-dependent endogenous ouabain-like factor production and accumulation in MHS and MNS rats, suggesting that the maintenance of higher ouabain-like factor levels in MHS than in MNS at the age of 30 and 90 days contributes to the development and maintenance of hypertension in this strain.