Table 1.
Effect of high NaCl on expression of genes related to endothelial cell function in cultured HUVEC cells.
Fig 1.
Elevation of extracellular NaCl increases expression of pro-inflammatory mediators in endothelial cells.
HUVECs were exposed to media in which NaCl was elevated for 4 days from 270 mosmol/kg to the total osmolality indicated in the figure panels. mRNA for 84 genes was measured by real-time PCR as described in Methods. (A) mRNA of adhesion molecules VCAM-1, E-selectin and of chemokine MCP-1 showed biggest increase upon exposure to high NaCl. The graph plots fold changes relative to 270 mosmol/kg of mRNA for 84 genes related to endothelial cell biology. Each dot shows mean value from 3 independent experiments. (B) Statistical analysis for VCAM-1, E-selectin and MCP-1 (mean ±SEM, *P<0.05, N = 3)
Fig 2.
Elevation of NaCl in cell culture medium promotes adhesion of peripheral blood mononuclear cells (PBMC) to HUVECs and their transmigration through HUVEC monolayer.
HUVECs were exposed to media in which NaCl was elevated for 4 days from 270 mosmol/kg to the total osmolality indicated in the figure panels. (A) The table shows sodium concentrations in culture medium of different osmolarities used in experiments shown on Figs 1 and 2. Note, that blood sodium range 135–145 mmol/l is considered normal; up to 160 mmol/l can be seen in people without symptoms (12); and higher levels are seen only in cases of severe hypernatremia in critically ill patients. (B) High NaCl increases E-selectin, a mediator for PBMC adhesion, in plasma membrane of HUVECs (representative IF image: green (Alexa488)—E-selectin, blue—cell nuclei stained with DAPI). (C) High NaCl increases VCAM-1 protein. Upper Panel: Representative Western Blot. Lower Panel: Quantification, relative to 270 mosmol/kg, normalized to tubulin (mean ±SEM, *P<0.05, t test, N = 3). (D) Elevation of NaCl within physiological range causes graded increase of adhesive properties of HUVECs. Left panel: Quantification of adhesion. Data are plotted as number of PBMC adhered to 1000 HUVECs (mean ±SEM, *P<0.05, N = 4, linearly dependent on NaCl concentration, P = 0.03). Note: Treatment with TNFα, known inducer of the adhesion was used as a positive control. Right panel: Representative image of stained cells used for the quantification of PBMC (green, calcein AM) attached to HUVECs (blue, DAPI). See methods for more details. (E) Exposure to elevated NaCl promotes transmigration of PBMC through HUVEC monolayer. Number of PBMC transmigrated in 6h was quantified relative to 270 mosmol/kg (mean ±SEM, *P<0.05, N = 3). See methods for more details.
Fig 3.
Water restriction activates pro-inflammatory signaling in mouse tissues.
To elevate NaCl in vivo, mice were subjected to water restriction for 9 days. To limit the amount of water, mice were fed with gel food containing 30% water and were not given any additional water. Control group mice were fed the same gel food, but had free access to water. (A) Water restriction increases VCAM-1, MCP-1 and E-selectin mRNA in several tissues. Levels of the mRNA were measured by real-time PCR. Results are presented as mean ±SEM, *P<0.05, N = 5. (B, C) Water restriction increases VCAM-1 protein in endothelial cells in the liver. (B) Representative images from immunohistochemical staining for VCAM-1 protein and for endothelial cells marker CD31 (brown) in the liver tissue sections. Similar patterns of VCAM-1 and CD31 staining are consistent with endothelial expression of VCAM-1. (C) Quantification of VCAM-1 staining shown on B. See methods section for more information about the quantification. Left panels. Images of extracted CMYK yellow channel from images shown on B. The pattern and intensity of the signal in the CMYK yellow channel corresponds to pattern and intensity to the brown diaminobenzidine (DAB) staining of VCAM-1. Right panel. Result of the quantification (mean ±SEM, *P<0.05, N = 4). (D) Representative images from immunohistochemical staining for VCAM-1 protein of heart sections. Positive staining of inner surface of coronary arteries of water restricted mice (shown by arrowheads) is consistent with endothelial expression of VCAM1. See S1 Fig for more images.
Fig 4.
Water restriction accelerates atherosclerosis in ApoE-/- mice.
Mice were water restricted starting at 6 weeks of age for 7–9 weeks and atherosclerosis was analyzed in aortic root as described in methods. Data are presented as mean ±SEM, *P<0.05, N = 9. (A) Experiment design. Water restricted mice were fed with gel food made from “western” diet containing 40% of calories from fat and 30% water and were not given any additional water. Control group were fed the same gel food, but had free access to water. (B) Water restricted mice grow at the same rate as controls after transient growth retardation. (C-F) Atherosclerotic lesions are larger in water restricted mice. (C) Representative images of frozen sections through aortic root in which lipids in the atherosclerotic lesions are stained in red with Oil Red O dye (shown by arrows). (D) The graph pots mean areas of the lesions on serial sections spanning approx. 900 μm of the aortic root. The data are presented as a box-and-whisker plot (n = 9, t test, P = 0.01). (E) The graph plots mean areas of the lesions at different distances from the base of the aortic root. (F) Atherosclerotic lesions grow faster in water restricted mice as shown by steeper regression line on the scatterplot of mean lesion area vs time (see methods for more details). (G) Serum cholesterol was measured after 6 weeks on high fat diet and water restriction.
Fig 5.
Water restriction causes thickening of the walls of coronary arteries in ApoE-/- mice.
(A) Representative images from hematoxylin and eosin staining of heart sections showing coronary arteries. (B) Quantification of the arterial wall thickness, lumen diameter and wall thickness to lumen diameter ratio. Data are presented as mean ±SEM, *P<0.05, N = 7.
Table 2.
Basic descriptive statistics for the variables used in analysis of the association between sodium and 10 Years Risk of CHD in the ARIC Study (N = 12779).
Fig 6.
Plasma sodium is positively associated with 10 Years Risk of CHD in Atherosclerosis Risk in Community (ARIC) Study.
(A, B) Multivariable linear regression analysis was used to assess effect of serum sodium on 10 Years Risk of CHD (N = 12779). (A) Distribution histograms for variables used in the analysis. (B) Overview of the results. The results demonstrate that serum Na+, as well as glucose, significantly contributes to predicting the Risk of CHD. See text and Table 2 for details of the analysis. (C) 3D Mesh Plots, visualizing Ln(10 Years Risk of CHD), as functions of serum sodium concentration and age. At all ages, 10 Years Risk of CHD is increased in participants with higher levels of serum sodium.
Table 3.
Multivariable linear regression analysis of 10 Years Risk of CHD (Ln transformed) with serum Na+ and Glucose as predictor variables (ARIC Study).