Background
The prevalence of increased plasma uric acid (UA), hyperuricemia, is high in patients with chronic renal insufficiency (CRI). The detrimental effects of hyperuricemia have been linked to cardiovascular complications, as high plasma UA levels commonly predict the development of hypertension [
1] and the loss of renal function [
2]. To date, however, the contribution of UA to cardiovascular disease has still remained controversial [
3].
Previous experimental studies, carried out in rats made hyperuricemic by the inhibition of the UA degrading enzyme uricase using dietary 2.0% oxonic acid, have suggested a causal relationship between high UA and cardiovascular disease [
4-
7]. UA has been associated with stimulation of the renal renin-angiotensin system (RAS) and reduced nitric oxide (NO) synthesis. These mechanisms may have participated in the subsequent hypertrophic remodeling of the preglomerular arteries, tubulointerstitial damage, and thus predisposed to enhanced sodium retention [
4-
6,
8]. Previously, incubation of the rat aortic rings with UA was found to reduce vasodilatation in response to acetylcholine (Ach) [
9], while the endothelial NO production
in vitro was reduced in hyperuricemic rats [
10]. This suggests that the detrimental effects of hyperuricemia may partly result from endothelial dysfunction.
There is an ongoing debate about the role of UA in vascular disease. This arises from the ability of UA to reduce oxidative stress by preventing the superoxide radical from reacting with NO to generate peroxynitrite [
11]. Peroxynitrite can impair NO-mediated relaxation by inhibiting a critical cofactor of the endothelial NO synthase [
12], while it also can modulate vascular tone via smooth muscle [
13]. In the aortas of apolipoprotein E-deficient mice, the ability of UA to reduce peroxynitrite levels was associated with improved Ach-elicited relaxation [
12]. This result is in agreement with our earlier report that oxonic acid-induced hyperuricemia improved NO-mediated relaxation of the carotid artery in experimental CRI by alleviating oxidative stress [
14].
In experimental CRI, reduced vasodilatation via Ca
2+-activated K
+-channels (BK
Ca), observed in isolated mesenteric arterial branches, may precede the elevation of blood pressure (BP) [
15,
16]. However, until now the vascular effects of UA have only been studied in arteries in which the endothelium-mediated dilatation is mainly mediated via NO [
12,
14]. Here we examined the tone of mesenteric arteries
in vitro from the 5/6 nephrectomized (NX) and Sham-operated rats allocated to 2.0% oxonic acid diet for 9 weeks. Our findings suggest that oxonic acid diet impaired relaxation via BK
Ca in arterial smooth muscle, but did not significantly affect the endothelium-dependent responses, resistance artery structure, or cardiac load in experimental CRI.
Discussion
Oxonic acid-induced hyperuricemia has been widely studied in recent years, but consensus on the putative harmfulness of increased circulating UA concentration still remains elusive. In this study we employed the 5/6 nephrectomy rat model of CRI to investigate the influence of experimental hyperuricemia on the tone and morphology of the mesenteric artery. The NX rats showed several characteristic findings of moderate renal insufficiency [
16,
29], whereas experimental hyperuricemia did not influence vasoconstrictor responses, renal function, cardiac load, or small artery morphology. To our knowledge, the present study is the first to suggest that hyperuricemia may impair vasorelaxation via BK
Ca, indicating alteration in smooth muscle hyperpolarization.
Oxonic acid feeding inhibits the oxidation of UA to its metabolite, allantoin, resulting in hyperuricemia. In the present study, oxonic acid diet elevated plasma UA 2.4 to 3.6-fold, which is in line with the 1.3 to 2.8-fold elevations observed in previous experimental studies [
4-
6]. The development of stage 2–3 renal insufficiency was confirmed by the elevated levels of plasma creatinine and urea, impaired endothelium-mediated vasodilatation, hypertrophic remodeling of the resistance vessels, and modest elevation of BP [
15,
29]. Increased right and left ventricular weights, higher ANP and BNP mRNA content, as well as increased left ventricular SkαA and β-MHC mRNA content, indicated permanent volume and pressure overload after subtotal renal ablation [
15]. However, hyperuricemia did not significantly influence any of the indicators of cardiac load either in the Sham or NX rats.
Arterial contractions were examined here in order to reveal differences in vasoconstrictor sensitivity that would potentially interfere with the interpretation of the relaxation experiments. No differences were found in responses elicited by membrane depolarisation with KCl (Table
2). In the main branch, NX rats exhibited slightly higher sensitivity but no significant change in maximal response to NE. However, in the small artery the NX groups exhibited slightly higher maximal response without changes in sensitivity to NE. Importantly, oxonic acid feeding did not influence the vasoconstrictor responses either in the second order or main branches of the mesenteric artery. Thus, changes in the vasodilator responses induced by hyperuricemia were not explained by alterations in vasoconstrictor responses.
Oxonic acid feeding did not induce changes in the morphology of small mesenteric arteries, while hypertrophic arterial remodeling was clearly observed in the NX rats [
15]. Previously, oxonic acid diet was found to activate renal RAS, as indicated by increased expression of juxtaglomerular renin, and result in hypertrophic remodeling of the glomerular afferent arterioles [
5-
7,
30]. Although local vascular RAS components were not examined here, the oxonic acid model of hyperuricemia is characterized by elevated plasma levels of aldosterone with subsequent sodium retention [
8]. The present results suggest that despite possible activation of the circulating RAS, high UA level does not influence the morphology of small arteries and heart.
Vasorelaxation was investigated in the main branch of the mesenteric artery, which functionally resembles the second-order branches in the same arterial bed [
15,
16]. Unlike the rat aorta, where endothelium-dependent vasodilatation is largely mediated via NO, the endothelium-derived relaxation of the mesenteric artery is also mediated by hyperpolarization of smooth muscle [
15]. Relaxation to Ach was impaired in NX rats, and the response was practically abolished in the presence of L-NAME in both NX groups, indicating that it was mediated via NO. Inhibition of NOS reduced the relaxation to Ach also in Sham rats, but the vessels showed more pronounced relaxations in the presence of L-NAME than those from NX rats [
15,
16]. The L-NAME resistant vasorelaxation in the rat mesenteric artery has been attributed to endothelium-dependent hyperpolarization [
15-
17]. In Sham and NX rats hyperuricemia was without significant influence on the response to Ach, indicating that endothelium-dependent relaxation was not affected by oxonic acid feeding. Vasorelaxation to the exogenous NO donor NP was slightly impaired in the NX groups when compared with the Sham groups, but hyperuricemia did not influence the sensitivity of arterial smooth muscle to relaxation via cGMP.
The present study showed that the endothelium-independent vasodilatation induced by the BK
Ca channel opener NS-1619 was impaired in the NX+Oxo group. Notably, this impairment was not observed in Sham+Oxo rats. NS-1619 induces relaxation by triggering intracellular Ca
2+ sparks, which induce K
+-efflux via BK
Ca and lead to subsequent hyperpolarization [
31-
33]. The finding of decreased vasorelaxation sensitivity via BK
Ca solely in the hyperuricemic NX rats suggests that the effect was specifically associated with the combination of uremic milieu and increased plasma UA concentration.
Several mechanisms could result in alterations of BK
Ca mediated vascular tone. The BK
Ca channel, which consists of α- and β1-subunits, is the most prominent type of calcium-activated K
+ channel in arterial smooth muscle [
13]. The β1-subunit is responsible for tuning the Ca
2+-sensitivity [
34]. Interaction between the α-subunit and β1-subunit enhances Ca
2+-sensitivity of BK
Ca channels, whereas the loss of the β1-subunit decreases Ca
2+ sensitivity [
35]. In a recent study using diabetic mice [
36], BK
Ca expression in arterial myocytes was strongly influenced by the calcineurin pathway, which inhibits the expression of the regulatory β1-subunit.
Endogenous BK
Ca inhibitors may influence K
+-channel activity. Arachidonic acid metabolites, especially 20-hydroxyeicosatetraenoic acid (20-HETE), can inhibit BK
Ca [
37]. 20-HETE reduces the open-state probability of the channel [
38]. Another endogenous inhibitor of BK
Ca is hydrogen sulfide, which binds to the α-subunit and increases the voltage needed for channel activation [
39]. BK
Ca are also modulated by reactive oxidative species (ROS), which can activate or inactivate BK
Ca [
13]. Such mechanisms are relevant, as elevated UA level following 2.0% oxonic acid feeding has increased total peroxyl radical-trapping capacity and reduced oxidative stress markers in the rat [
14]. Hyperuricemia may increase superoxide dismutase (SOD) activity [
40], which catalyzes the dismutation of superoxide (O
2−) into oxygen and hydrogen peroxide (H
2O
2). UA itself is able to scavenge BK
Ca-inhibiting radicals, and increase the production of H
2O
2 by preventing the H
2O
2-induced inactivation of SOD [
40]. H
2O
2 can even induce vasodilatation directly via BK
Ca activation [
41], an effect known to be more pronounced under conditions of reduced NO availability [
13]. The latter is a characteristic feature of the uremic milieu [
42]. Taken together, a multitude of processes can influence vasorelaxation via BK
Ca, including changes in channel protein gene expression and structure, changes in cellular Ca
2+ sparks, levels of ROS, and endogenous BK
Ca inhibitors.
UA is produced from xanthine by the enzyme xantine oxidase, which has been found to play an important role in a variety of tissue and vascular injuries [
43]. Although therapeutic interventions with the aim to lower UA with xanthine oxidase inhibitors may be beneficial in treating the vascular disorders associated with renal disease, debate is still ongoing whether the effect is related to lowering UA levels
per se, or to reduced xanthine oxidase activity. The present protocol did not include the treatment of hyperuricemia, since the UA-lowering drugs allopurinol, febuxostat and uricosuric agents have been well documented to prevent the pathophysiological changes induced by the oxonic acid feeding [
4-
6,
10,
30,
44-
46].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
VK and TV contributed equally to this article. VK, TV and PP participated in the animal work. VK and TV carried out the in vitro vascular function studies, performed statistical analyses, and drafted the manuscript. AE participated in the animal and laboratory work, statistical analyses, and completed the manuscript with IP. JJ carried out the arterial morphology studies. HR and HT were responsible for the natriuretic peptide, as well as α-actin and ß-myosin determinations. ON carried out the blood and urine analyses. JM participated in the study design, financing, and coordination. IP conceived the study design, participated in the animal work and in vitro vascular function experiments, financed the study, and completed the manuscript. All authors read and approved the final manuscript.