Abstract
Hypertension, defined as a chronically high blood pressure, is a significant risk factor for heart attacks and strokes. Despite hypertension affecting 28% of individuals over the age of 20 years old, little is known about the changes which occur in the vascular endothelial cells that contribute to the progression of this condition. Increased dietary sodium and purine/fructose intake have both been implicated as independent risk factors for developing cardiovascular disease (CVD) and hypertension.
Under high serum sodium concentrations production of the potent vasodilator nitric oxide (NO) from vascular endothelial cells is decreased. A downregulation of NO production promotes vasoconstriction and a subsequent increase in blood pressure. Significant increases in serum sodium concentrations have been found to increase endothelial sodium channel (ENaC) expression within vascular endothelial cells, facilitating sodium uptake, promoting the downregulation of NO production. Purine metabolite, uric acid (UA), has also been shown to downregulate endothelial cell NO synthesis when serum concentrations are elevated. High UA concentrations also increase ENaC expression in mouse kidney cells. However, it is unknown whether elevated UA concentrations upregulate vascular endothelial cell ENaC expression. The aims of this study were to determine whether elevating either sodium or UA treatment concentrations induces a significant change in ENaC α, β, γ, or δ subunit expression in human umbilical vein endothelial cells (HUVEC) incubated in either static conditions or under shear stress. We hypothesised an increase in either sodium or UA would induce a significant increase in all four ENaC subunit expressions, regardless of whether the cells were grown in static conditions or under shear stress.
For the present study, cells were incubated in either a control (300 μM UA), high sodium (+20 mM Na+), high UA (500 μM UA) or high sodium/UA solution (+20 mM Na+, 500 μM UA). HUVECs were seeded onto either 6-wells plates for 48 hrs of static treatment exposure, or I0.6 Luer μ-slides for 24 hrs of exposure to the treatments and shear stress using an Ibidi fluidic system (Ibidi, Cat.No. 10906). Following treatments, cells were lysed and analysed using western blot analysis to determine changes in α, β, γ, and δENaC expression between the treatment groups. Lysed cells from the static control treatment group were used to determine whether HUVECs express UA transporters (human glucose transporter 9 (GLUT9) and multidrug resistance protein 4 (MRP4)), and UA synthesising enzyme xanthine oxidase (XO).
In comparison to the control treatment group there was no significant difference in α, β, γ, and δENaC subunit expression when HUVECs were treated with elevated sodium or uric acid concentrations within a static system. HUVECs were shown to express GLUT9, MRP4 and XO. Due to obtaining only one set of results from the HUVEC experiments conducted within the fluidic system, whether exposure to increased sodium or UA concentrations impacts ENaC subunit expression when HUVECs are under shear stress remains inconclusive.
The finding that increasing sodium or UA concentrations within HUEVC cells did not significantly influence ENaC subunit expression was unexpected. We hypothesise this finding may be due to either experimental procedure or an intrinsic sodium concentration threshold that regulates ENaC subunit transcription/translation that was not met by any of the treatment solutions. Future experiments employing a range of sodium and UA treatment concentrations with both cell and animal models would aid in confirming this theory.