Feedforward activation of endothelial ENaC by high sodium.

Kidney epithelial sodium channels (ENaCs) are known to be inactivated by high sodium concentrations (feedback inhibition). Recently, the endothelial sodium channel (EnNaC) was identified to control the nanomechanical properties of the endothelium. EnNaC-dependent endothelial stiffening reduces the release of nitric oxide, the hallmark of endothelial dysfunction. To study the regulatory impact of sodium on EnNaC, endothelial cells (EA.hy926 and ex vivo mouse endothelium) were incubated in aldosterone-free solutions containing either low (130 mM) or high (150 mM) sodium concentrations. By applying atomic force microscopy-based nanoindentation, an unexpected positive correlation between increasing sodium concentrations and cortical endothelial stiffness was observed, which can be attributed to functional EnNaC. In particular, an acute rise in sodium concentration (+20 mM) was sufficient to increase EnNaC membrane abundance by 90% and stiffening of the endothelial cortex by 18%. Despite the absence of exogenous aldosterone, these effects were prevented by the aldosterone synthase inhibitor FAD286 (100 nM) or the mineralocorticoid receptor (MR)-antagonist spironolactone (100 nM), indicating endogenous aldosterone synthesis and MR-dependent signaling. Interestingly, in the presence of high-sodium concentrations, FAD286 increased the transcription of the MR by 69%. Taken together, a novel feedforward activation of EnNaC by sodium is proposed that contrasts ENaC feedback inhibition in kidney.

Long-term application of the aldosterone antagonist spironolactone prevents stiff endothelial cell syndrome.

Aldosterone triggers the stiff endothelial cell syndrome (SECS), characterized by an up-regulation of epithelial sodium channels (ENaCs) and mechanical stiffening of the endothelial cell cortex accompanied by endothelial dysfunction. In vivo, aldosterone antagonism exerts sustained protection on the cardiovascular system. To illuminate the molecular mechanisms of this time-dependent effect, a study on endothelial cells in vitro and ex vivo was designed to investigate SECS over time. Endothelia (from human umbilical veins, bovine aortae, and explants of human arteries) were cultured in aldosterone-supplemented medium with or without the mineralocorticoid receptor (MR) antagonist spironolactone. MR expression, ENaC expression, cortical stiffness, and shear-mediated nitric oxide (NO) release were determined after 3 d (short term) and up to 24 d (long term). Over time, MR expression increased by 129%. ENaC expression and surface abundance increased by 32% and 42% (13.8 to 19.6 molecules per cell surface), paralleled by a 49% rise in stiffness. Spironolactone prevented this development and, after 3 wk of treatment, increased NO release by 50%. Thus, spironolactone improves endothelial function long-lastingly by preventing a time-dependent manifestation of SECS. This emphasizes the key role of vascular endothelium as a therapeutical target in cardiovascular disorders and might explain blood pressure independent actions of MR antagonism.

Endothelial sodium channels trigger endothelial salt sensitivity with aging.

The epithelial sodium channel is also expressed in vascular endothelium (endothelial sodium channel [EnNaC]). Depending on ambient sodium concentration, EnNaC is associated with mechanical stiffening of the endothelial cell cortex, leading to endothelial dysfunction. Because the incidence of both salt sensitivity and endothelial dysfunction increases with age, we investigated the abundance of EnNaC in aging mice. To assess EnNaC functionality and endothelial salt sensitivity, stiffness was measured while ambient sodium was varied. Aortae of young (3 months) and old (15 months) C57BL/6J wild-type mice were kept ex vivo on a physiological concentration of aldosterone (0.45 nmol/L). Spironolactone (10 nmol/L) and amiloride (1 µmol/L) were applied for aldosterone antagonism and EnNaC blockage, respectively. EnNaC at the endothelial cell surface was quantified by immunofluorescence staining. Cortical stiffness was monitored by atomic force microscopy when ambient sodium was raised from 135 to 150 mmol/L. In ex vivo aortae of older mice, endothelial cells had significantly higher EnNaC numbers than those of younger mice (+23%). In parallel, cortical stiffness was found increased (+8.5%). Acute application of high sodium led to an immediate rise in stiffness in both groups but was pronounced in endothelium of older mice (+18% versus +26%). Spironolactone and amiloride lowered EnNaC abundance and prevented endothelial stiffening under all conditions. We conclude that EnNaC mediates endothelial salt sensitivity in the aging process. This mechanism might contribute to the development of age-related cardiovascular disease and suggests the usage of spironolactone and amiloride specifically in the elderly.

Nanomechanics and sodium permeability of endothelial surface layer modulated by hawthorn extract WS 1442.

The endothelial glycocalyx (eGC) plays a pivotal role in the physiology of the vasculature. By binding plasma proteins, the eGC forms the endothelial surface layer (ESL) which acts as an interface between bloodstream and endothelial cell surface. The functions of the eGC include mechanosensing of blood flow induced shear stress and thus flow dependent vasodilation. There are indications that levels of plasma sodium concentrations in the upper range of normal and beyond impair flow dependent regulation of blood pressure and may therefore increase the risk for hypertension. Substances, therefore, that prevent sodium induced endothelial dysfunction may be attractive for the treatment of cardiovascular disease. By means of combined atomic force-epifluorescence microscopy we studied the impact of the hawthorn (Crataegus spp.) extract WS 1442, a herbal therapeutic with unknown mechanism of action, on the mechanics of the ESL of ex vivo murine aortae. Furthermore, we measured the impact of WS 1442 on the sodium permeability of endothelial EA.hy 926 cell monolayer. The data show that (i) the ESL contributes by about 11% to the total endothelial barrier resistance for sodium and (ii) WS 1442 strengthens the ESL resistance for sodium up to about 45%. This mechanism may explain some of the vasoprotective actions of this herbal therapeutic.