Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice.

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.

Expression and function of the human mineralocorticoid receptor: lessons from transgenic mouse models.

The human mineralocorticoid receptor (hMR), a ligand-dependent transcription factor (NR3C2) which belongs to the nuclear receptor superfamily, mediates most of the known effects of aldosterone. Beside its involvement in the regulation of sodium balance and the control of blood pressure, aldosterone-hMR tandem also exerts important regulatory functions on the cardiovascular and central nervous systems. To study the molecular mechanisms involved in the tissue-specific expression of hMR and explore its functional implication in pathophysiology, transgenic mouse models have been generated using both targeted oncogenesis and MR overexpression. We have previously demonstrated that the transcription of hMR is directed by two alternative promoters, P1 and P2, which correspond to the 5'-flanking regions of the untranslated exons 1alpha and 1beta of the hMR gene, respectively. Utilization of P1 and P2 to drive expression of the SV40 large T antigen (TAg) in transgenic mice led us (i) to determine distinct tissue-specific patterns of promoter usage; (ii) to identify novel sites of MR expression including brown adipose tissue, thus providing a new functional link between aldosterone and energy homeostasis; (iii) to generate original immortalized cell lines derived from numerous aldosterone-sensitive tissues most notably distal nephron, brown fat, skin, liver, lung, brain, heart, blood vessels and inner ear. These differentiated cell lines represent suitable models to further explore cell-specific mineralocorticoid responses and cross-talk with other signaling pathways. Generation of transgenic mice in which hMR expression was directed by P1 promoter demonstrated the importance of MR in the cardiac and renal function. Morphological and functional alterations of the renal tubule were observed with basal decreased sodium/potassium ratio exacerbated under sodium depletion. Hypokinetic dilated cardiomyopathies were associated with tachycardia, arrhythmia but normal arterial blood pressure emphasizing the direct role of MR on cardiomyocyte function. Taken together, transgenic animal models constitute valuable experimental systems to gain new insights into the widespread and pleiotropic in vivo functions of MR.

Panton-valentine leukocidin enhances the severity of community-associated methicillin-resistant Staphylococcus aureus rabbit osteomyelitis.

BACKGROUND: Extensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection. METHODOLOGY/PRINCIPAL FINDINGS: We compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACDeltapvl), using a low and a high inoculum (8x10(5) and 4x10(8) CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACDeltapvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACDeltapvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACDeltapvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC. CONCLUSIONS/SIGNIFICANCE: PVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.

Exploring the role of galectin 3 in kidney function: a genetic approach.

Galectin 3 belongs to a family of glycoconjugate-binding proteins that participate in cellular homeostasis by modulating cell growth, adhesion, and signaling. We studied adult galectin 3 null mutant (Gal 3-/-) and wild-type (WT) mice to gain insights into the role of galectin 3 in the kidney. By immunofluorescence, galectin 3 was found in collecting duct (CD) principal and intercalated cells in some regions of the kidney, as well as in the thick ascending limbs at lower levels. Compared to WT mice, Gal 3-/- mice had approximately 11% fewer glomeruli (p < 0.04), associated with kidney hypertrophy (p < 0.006). In clearance experiments, urinary chloride excretion was found to be higher in Gal 3-/- than in WT mice (p < 0.04), but there was no difference in urinary bicarbonate excretion, in glomerular filtration, or urinary flow rates. Under chronic low sodium diet, Gal 3-/- mice had lower extracellular fluid (ECF) volume than WT mice (p < 0.05). Plasma aldosterone concentration was higher in Gal 3-/- than in WT mice (p < 0.04), which probably caused the observed increase in alpha-epithelial sodium channel (alpha-ENaC) protein abundance in the mutant mice (p < 0.001). Chronic high sodium diet resulted paradoxically in lower blood pressure (p < 0.01) in Gal 3-/- than in WT. We conclude that Gal 3-/- mice have mild renal chloride loss, which causes chronic ECF volume contraction and reduced blood pressure levels.

Pharmacokinetics of antibiotics in the middle ear.

The middle ear mucosa is described as a non-specialized site where drug substances diffuse passively. However, precise pharmacokinetic studies on antibiotic penetration into middle ear effusion (MEE) are difficult to conduct in humans because of ethical and methodological problems: (1) studies must involve the population in which acute otitis media (AOM) is most frequent (i.e. in children from 3 months to 3 years of age); (2) studies are commonly performed during surgery for chronic otitis media with effusion, whereas drug penetration into MEE is higher during AOM; (3) kinetics are deduced from data per individual obtained in groups with marked interindividual variations. Furthermore, the results of different studies are difficult to compare because of differences in protocols, antibiotic assay methods and interpretation. Clinical studies have nonetheless shown the poorly predictive nature of serum concentrations, given the large differences in drug delivery to the MEE among antibiotic classes, among members of the same class, and among individuals receiving a given antibiotic. Experimental studies can help to narrow the gap between in vitro and clinical data, as the more precise pharmacokinetic data they yield can throw light on the link between drug/host/pathogen factors and bacteriologic efficacy.

Impaired 11-beta hydroxysteroid dehydrogenase type 2 activity in sweat gland ducts in human essential hypertension.

The enzyme 11-beta hydroxysteroid dehydrogenase type 2 plays a major role in blood pressure regulation. It metabolizes glucocorticoid hormones into derivatives with low affinity for the mineralocorticoid receptor, preventing its permanent occupancy by circulating cortisol, which is 100- to 1000-fold more abundant than aldosterone in the plasma. Inactivating mutations of the enzyme result in severe hypertension, as seen in children with apparent mineralocorticoid excess syndrome. In patients with essential hypertension, however, attempts to evidence enzyme deficiency have been inconclusive. In this pilot study, its catalytic activity was measured directly in aldosterone-sensitive sweat gland ducts collected from skin biopsy samples of 10 male normotensive subjects and 10 subjects with essential hypertension (more than 140 to 90 mm Hg) with no sign of hypermineralocorticism. Isolated ducts were assayed for nicotinamide-dinucleotide-dependent dehydrogenase activity (transformation of tritiated corticosterone into tritiated-11 dehydrocorticosterone, as measured by high-pressure liquid chromatography). Hypertensive patients exhibited significantly lower 11-beta hydroxysteroid dehydrogenase type 2 activity (9.7+/-4.7 femtomoles per 3 mm length of duct and per 10 minutes incubation, median+/-SD) than did normotensive subjects (15.9+/-2.6). Such defect was undetectable using the classical urinary corticosteroid metabolism indexes, probably because of compensatory mechanisms. Relations between these findings and blood pressure levels should benefit from direct enzyme measurements in the vasculature. In conclusion, this cross-sectional study points to partial 11-beta hydroxysteroid dehydrogenase type 2 deficiency as a novel feature of essential hypertension, which should stimulate search for new signaling pathways and therapeutical targets.