Endothelial cells as vascular salt sensors

Dietary sodium and potassium contribute to the control of the blood pressure. Endothelial cells are targets for aldosterone, which activates the apically located epithelial sodium channels. The activity of these channels is negatively correlated with the release of nitric oxide (NO) and determines endothelial function. A mediating factor between channel activity and NO release is the mechanical stiffness of the cell's plasma membrane, including the submembranous actin network (the cell's ‘shell’). Changes in plasma sodium and potassium, within the physiological range, regulate the viscosity of this shell and thus control the shear-stress-dependent activity of the endothelial NO synthase located in the shell's ‘pockets’ (caveolae). High plasma sodium gelates the shell of the endothelial cell, whereas the shell is fluidized by high potassium. Accordingly, this concept envisages that communications between extracellular ions and intracellular enzymes occur at the plasma membrane barrier, whereas 90% of the total cell mass remains uninvolved in these changes. Endothelial cells are highly sensitive to extracellular sodium and potassium. This sensitivity may serve as a physiological feedback mechanism to regulate local blood flow. It may also have pathophysiological relevance when sodium/potassium homeostasis is disturbed

Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line

The Madin-Darby canine kidney (MDCK) cell line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent line forms an epithelium which expresses many of the characteristics of principal cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal cells and is an ideal model for studying hormonal modulation of Na+ transport

Nanomechanics of the endothelial glycocalyx in experimental sepsis

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis

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

Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions

Salt Sensitivity Determined From Capillary Blood

Background/Aims: A significant rise of blood pressure in response to a given salt load is a weak indication of high salt sensitivity, supposed to foster the development of arterial hypertension and related diseases in later life. In search of an alternative method we recently developed the salt blood test (SBT), a new concept for quantifying salt sensitivity (SS). Based on this concept, namely that red blood cells (RBC) report on salt sensitivity, the SBT-mini was developed. Methods: The SBT-mini utilizes a droplet of capillary blood mixed with a ‘smart' Na+ cocktail. Red blood cells (RBC) of this mixture are allowed to sediment by gravity in a glass tube. SS is quantified by measuring RBC sedimentation rate. 90 healthy volunteers (39 males, 51 females; mean age: 23±0.5 years) were evaluated and ‘standard values' for males and females were derived. Results: Sodium buffer capacity of female blood is about 20 % smaller as compared to male blood due to the lower hematocrit of females. SS of an individual is related to the mean standard value (set to 100 %) of the respective male/female cohort. High SS (> 120 %) has been found in 31 % of males and 28 % of females. Conclusions: SS can be estimated derived from the individual RBC sodium buffer capacity as measured by the SBT-mini. About one third of a healthy test cohort exhibits a high sensitivity to salt. Reduction of sodium consumption to at least two grams per day (equals five grams of NaCl per day as suggested by the WHO) is recommended, particularly for individuals with high salt sensitivity