Uric acid-induced endothelial dysfunction is associated with mitochondrial alterations and decreased intracellular ATP concentrations

Abstract

Background/aims: Endothelial dysfunction is associated with mitochondrial alterations. We hypothesized that uric acid (UA), which can induce endothelial dysfunction in vitro and in vivo, might also alter mitochondrial function.

Methods: Human aortic endothelial cells were exposed to soluble UA and measurements of oxidative stress, nitric oxide, mitochondrial density, ATP production, aconitase-2 and enoyl Co-A hydratase-1 expressions, and aconitase-2 activity in isolated mitochondria were determined. The effect of hyperuricemia induced by uricase inhibition in rats on renal mitochondrial integrity was also assessed.

Results: UA-induced endothelial dysfunction was associated with reduced mitochondrial mass and ATP production. UA also decreased aconitase-2 activity and lowered enoyl CoA hydratase-1 expression. Hyperuricemic rats showed increased mitDNA damage in association with higher levels of intrarenal UA and oxidative stress.

Conclusions: UA-induced endothelial dysfunction is associated with mitochondrial alterations and decreased intracellular ATP. These studies provide additional evidence for a deleterious effect of UA on vascular function that could be important in the pathogenesis of hypertension and vascular disease.

Fig. 1

Uric acid increases intracellular oxidative stress in cells incubated with UA 714 μM (12 mg/dL) for 48 h. This effect was prevented by cotreatment with apocynin suggesting the participation of NADPH oxidase in this effect (A) (n=3, triplicates). Uric acid reduces NO bioavailability in HAEC incubated with UA 714 μM (12 mg/dL) for 48 h as suggested by a significant reduction in nitrates/nitrites measured by DAF2-DA fluorescence in fixed cells (B) (n=3, triplicates) and as secreted nitrates/nitrites in the cell culture media (C) (n=2, triplicates).

Fig. 2

Uric acid reduces mitochondrial mass in HAEC incubated with UA 714 μM (12 mg/dL) as showed by a significant decrement of mitDNA at 24 and 48 h (A) (n=3, triplicates). This effect was in association with a lower fluorescence intensity using the specific mitochondrial dye Mitotracker Orange^™ (B) at 48 h of incubation with UA (n=2, triplicates). Uric acid also alters the expression and bioactivity of mitochondrial enzymes and reduces ATP levels in HAEC incubated with UA 714 μM (12 mg/dL) for 48 h. Mitochondrial ECoAH-1 expression (C) and ACO-2 activity (D) were significantly reduced in UA treated cells (n=3, triplicates). UA incubation for 48 h also reduced basal intracellular ATP concentration (E) (n=3, triplicates).

Fig 3

Representative micrographs of renal sections (X400) from a control and an oxonic acid (OA) treated hyperuricemic rat showing inflammatory cell infiltration (anti-CD45 antibody) (arrows) (A). The concentration of UA in the renal cortex was significantly increased in OA-treated rats and was associated with increased oxidative stress. In addition, the intra-renal UA level correlated with the degree of oxidative stress (B). OA-treated animals had less relative mitDNA copy number and a proportional increment of damaged mitDNA in the renal cortex compared to control rats, as showed by a significant increased percentage of common mitDNA deletion (C).

Fig. 4

Potential mechanisms involved in uric acid-induced endothelial dysfunction and renal damage. Hyperuricaemia promotes augmented oxidative stress which in turn decreases endothelial NO bioactivity, induces mitochondrial dysfunction characterized by diminished mitochondrial mass and decreased expression or activity of the Kreb’s cycle enzymes ECoH and ACO-2 and reduced intracellular ATP. Reduced endothelial NO bioactivity may also reduce mitochondrial mass by altering biogenesis, and altered mitochondria may also contribute to reduced intracellular ATP levels. Therefore, reduced NO, mitochondrial alterations and decreased ATP results in the induction of endothelial dysfunction, which may promote arteriosclerosis and insulin resistance. In the kidney, endothelial dysfunction result in tissue hypoxia, inflammatory cell infiltration, and progressive renal disease as manifested by glomerulosclerosis and tubulointerstitial fibrosis.