Uric acid promotes vascular stiffness, maladaptive inflammatory responses and proteinuria in western diet fed mice

Abstract

Objective: Aortic vascular stiffness has been implicated in the development of cardiovascular disease (CVD) and chronic kidney disease (CKD) in obese individuals. However, the mechanism promoting these adverse effects are unclear. In this context, promotion of obesity through consumption of a western diet (WD) high in fat and fructose leads to excess circulating uric acid. There is accumulating data implicating elevated uric acid in the promotion of CVD and CKD. Accordingly, we hypothesized that xanthine oxidase(XO) inhibition with allopurinol would prevent a rise in vascular stiffness and proteinuria in a translationally relevant model of WD-induced obesity.

Materials/methods: Four-week-old C57BL6/J male mice were fed a WD with excess fat (46%) and fructose (17.5%) with or without allopurinol (125mg/L in drinking water) for 16weeks. Aortic endothelial and extracellular matrix/vascular smooth muscle stiffness was evaluated by atomic force microscopy. Aortic XO activity, 3-nitrotyrosine (3-NT) and aortic endothelial sodium channel (EnNaC) expression were evaluated along with aortic expression of inflammatory markers. In the kidney, expression of toll like receptor 4 (TLR4) and fibronectin were assessed along with evaluation of proteinuria.

Results: XO inhibition significantly attenuated WD-induced increases in plasma uric acid, vascular XO activity and oxidative stress, in concert with reductions in proteinuria. Further, XO inhibition prevented WD-induced increases in aortic EnNaC expression and associated endothelial and subendothelial stiffness. XO inhibition also reduced vascular pro-inflammatory and maladaptive immune responses induced by consumption of a WD. XO inhibition also decreased WD-induced increases in renal TLR4 and fibronectin that associated proteinuria.

Conclusions: Consumption of a WD leads to elevations in plasma uric acid, increased vascular XO activity, oxidative stress, vascular stiffness, and proteinuria all of which are attenuated with allopurinol administration.

Keywords: Arterial stiffness; Macrophage polarization; Obesity; Proteinuria; Uric acid.

Figure 1. XO inhibition prevents WD-induced increases in aortic EC and vascular smooth muscle/extracellular matrix stiffness

A. Representative force curve (red trace) from CD, WD, WDA group showing increased EC stiffness. The steeper the slope in the deflection trace, the stiffer the tissue. Blue curve represents retraction force that varies depending on the underlying region but not accounted for force curve generation. The bar graph summarizes the increased EC stiffness in WD-fed mice and its suppression by XO inhibitor in ex vivo aortic explants. B. Representative force curve from CD, WD, WDA group for VSMC/ECM stiffness and bar graph representing VSMC/ECM or sub-endothelial stiffness was obtained after denudation of endothelium of aortic explants. C. Representative immune-staining for the expression of EnNaC from CD, WD and WDA group. N = 3 to 5 per group. CD - control diet, WD - Western Diet, WDA - Western diet plus allopurinol. Data are expressed as means ± SE. * P<0.05 vs CD; # p<0.05 vs WD.

Figure 2. XO inhibition reduces the levels of ROS and XO activity and decreases plasma uric acid levels induced by WD

A. Representative images for quantification of 3-nitrotyrosine by immunostaining. B–D. The bar graph showing increased oxidative stress in EC (B), VSMC (C) and adventitial layer (D) of aorta and its prevention by XO inhibition. E. XO activity in aortic tissue was significantly increased in WD group but not in WD plus XO inhibitor group. N = 3 to 6 per group. CD - control diet, WD - Western Diet, WDA - Western diet plus allopurinol. Data are expressed as means ± SE. * P<0.05 vs. CD; # p<0.05 vs WD.

Figure 3. XO inhibition suppresses WD-induced inflammatory gene expression in aorta

A. mRNA expression of MCP-1, a marker of EC activation, is increased in WD-fed mice which was prevented by XO inhibition B. CD11b as indicator of macrophage recruitment is increased in WD-fed group but not in WD plus XO inhibitor group. C. CD 86 expression is increased in WD-fed mice indicating M1 polarization and this response was blunted by XO inhibition. D. IL-6 expression is a marker of M1 polarization and enhanced pro-inflammatory response to WD feeding. This response is prevented by XO inhibitor treatment in WD-fed mice. N = 3 per group. CD - control diet, WD - Western Diet, WDA - Western diet plus allopurinol. Data are expressed as means ± SE. * P<0.05 vs CD; # p<0.05 vs WD.

Figure 4. Effect of allopurinol treatment on WD-induced mesenteric artery stiffness

A. Comparison of strain-stress relationship curves between arteries isolated from WD and WDA-treated mice. B. Comparison of the incremental moduli of elasticity between the same arteries as in A. Data are expressed as means ± SE. # Data points were significantly different between WD and WDA groups at p<0.05. N=3 to 4 number of animals (vessels) per treatment group. WD -Western Diet, WDA - Western diet plus allopurinol.

Figure 5. XO inhibition suppresses WD-induced kidney fibrosis and proteinuria

A. The bar graph shows increased excretion of protein in WD-fed mice and its prevention of treatment with XO inhibitor. B. WD tended increased fibronectin expression that is decreased by XO inhibition. C. TLR4 receptor expression is increased in WD-fed mice and tended to decrease in XO inhibitor treated group. N =4 to 8 per group. CD - control diet, WD - Western Diet, WDA - Western diet plus allopurinol. Data are expressed as means ± SE. * P<0.05 vs CD; # p<0.05 vs WD.