Circulating purine compounds, uric acid, and xanthine oxidase/dehydrogenase relationship in essential hypertension and end stage renal disease

Abstract

Purine nucleotide liberation and their metabolic rate of interconversion may be important in the development of hypertension and its renal consequences. In the present study, blood triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) breakdown pathway was evaluated in relation to uric acid concentration and xanthine dehydrogenase/xanthine oxidase (XDH/XO) in patients with essential hypertension, patients with chronic renal diseases on dialysis, and control individuals. The pattern of nucleotide catabolism was significantly shifted toward catabolic compounds, including ADP, AMP, and uric acid in patients on dialysis program. A significant fall of ATP was more expressed in a group of patients on dialysis program, compared with the control value (p < 0.001), while ADP and AMP were significantly increased in both groups of patients compared with control healthy individuals (p < 0.001), together with their final degradation product, uric acid (p < 0.001). The index of ATP/ADP and ATP/uric acid showed gradual significant fall in both the groups, compared with the control value (p < 0.001), near five times in a group on dialysis. Total XOD was up-regulated significantly in a group with essential hypertension, more than in a group on dialysis. The activity of XO, which dominantly contributes reactive oxygen species (ROS) production, significantly increased in dialysis group, more than in a group with essential hypertension. In conclusion, the examination of the role of circulating purine nucleotides and uric acid in pathogenesis of hypertension and possible development of renal disease, together with XO role in ROS production, may help in modulating their liberation and ROS production in slowing progression from hypertension to renal failure.

• ADP
• AMP
• ATP
• circulating purine nucleotides
• dialysis
• essential hypertension
• renal failure
• xanthine dehydrogenase
• xanthine oxidase

Introduction

It was documented that renal failure, followed by the end-stage renal disease (ESRD) would be primarily caused by hypertension in about of 30% cases.1–5 Despite the fact that essential hypertension may occurs in the absence of any known cause, a number of metabolic disturbances, including hyperuricemia, obesity as well as hyperactive sympathetic nervous system may be responsible for pathophysiological and hemodynamic features.6,7 Hyperuricemia may induce renal failure through the crystal-dependent and crystal-independent mechanisms, by inducing endothelial dysfunction, renal vasoconstriction, and activation of the renin–angiotensin system.8–11 Purine nucleotide degradation pathway may be important in the development of hypertension and its renal consequences.

Adenine nucleotides can be found in circulation, after their release from peripheral tissues, endothelial cells, stimulated blood platelets, immune cells, and during stimulation of sympathetic adrenergic and purinergic nerves.12–16 Tissue damage, necrosis, metabolic stress, hypoxia, endothelial ischemia during shear stress, myocardial ischemic attack, muscle exercise, or smooth muscle cells contraction, may contribute to their liberation into extracellular space. Their release may occur via exocytosis or diffusion across damaged membrane. From the other side, hyperactive sympathetic nervous system, or adrenal medulla stimulation may produce an increase in adenine nucleotide levels and platelet activation (aggregation).17–20 Once present in the circulation, they may exert vasoactive, immunomodulatory, and prothrombotic responses. Very strong vasoactive properties of different purine nucleotides mainly depend on their phosphate form, whether they are in triphosphate, diphosphate, and monophosphate forms.18,19 Their vasoactive effects occur mainly via purinergic system.20–23 Different pharmacologic drugs (like acetyl-choline or bradykinin) may also lead to purine nucleotide liberation.24 Primary disorders of purine synthesis or salvage pathway may increase uric acid metabolism, leading to gout and renal calculi. Secondary hyperuricemia may be frequently present in anemia, central nervous system dysfunction, obesity or diabetes, commonly contributing to the development of serious renal failure.9,25

Human enzyme xanthine oxidoreductase (XOD) is a rate-limiting enzyme involved in terminal catabolic pathway of purine bases, adenine and guanine, through hypoxanthine and xanthine to uric acid. In tissues and circulation, it can exist in two interconvertible forms: xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The interconversion from XDH to XOD can be stimulated by the oxidation of the present sulfhydryl residues or by stimulated proteolysis.25,26 Since XO reaction, simultaneously with uric acid, produces reactive oxygen species (ROS), this enzyme represents a main source of ROS liberation in circulation. XO activity in circulation is mainly elevated in ischemic–reperfusion conditions, coronary disease, endothelial dysfunction, inflammatory conditions followed by cytokine liberation (IL-1, IL-6, and TNF-α), and bacterial lipopolysaccharide presence, as well as following steroid hormone treatment.25–30

Since the excess accumulation of different purine nucleotides, together with increased XOD expression and XO activity, can be regarded as an independent pathogenic factor for endothelial damage, alteration of metabolic processes in the kidneys and pro-coagulant phase, in the present study the adenine nucleotide breakdown pathway in blood was evaluated in relation to uric acid concentration and XDH/XO ratio in patients with essential hypertension and patients with chronic renal diseases on dialysis.

Patients and methods

Three groups of patients were included in the study: I, patients with essential hypertension without renal pathology (30; 21/9 females/males); II, patients with chronic renal diseases on dialysis (28; 16/12 females/males); and III, controls healthy persons (20; 12/8 females/males). Control group was allocated from randomly selected, healthy individuals, blood donor volunteers, and age-matched. All participants gave written informed consent for analysis. Patients with essential hypertension and the end stages of chronic kidney diseases on dialysis were recruited from the Clinic for Nephrology and Hemodialysis Medical Faculty, University of Nis. The diagnosis of disease was based on a survey, which included standard clinical and imaging methods and laboratory examination of blood and urine. Routine biochemical methods were used to determine serum concentrations of creatinine, urea, and other clinically relevant parameters for renal function in establishing a diagnosis of stages of chronic kidney disease. They were selected according to the National Kidney Foundation (NKF) criteria.31 Patients were checked for history of previous or present infections as well. None of them had the above-mentioned exclusion criteria.

Normal blood pressure was defined as a systolic blood pressure of less than 130 mmHg and a diastolic blood pressure of less than 80 mmHg, while systolic pressures of 130–139 mmHg and diastolic pressures of 80–89 mmHg were considered as borderline hypertension. Hypertension was defined as a systolic blood pressure of at least 140 mmHg, a diastolic blood pressure of at least 90 mmHg, or both, measured in a sitting position, repeated at least three times, according to the World Health Organization (WHO) classification.6,32

Blood collection was done from cubital vein and patients and control group were advised not to perform any intensive physical training or to eat meat at least 2 d before the analysis. For each participant, a 10 mL blood sample was collected after a 30-min sitting period. Routine biochemical analyses were determined on separated plasma on Automatic analyzer A24 for In Vitro Diagnostics (manufactured by Biosystems SA, Barcelona, Spain). For the evaluation of purine nucleotides concentration, fresh blood was immediately processed for their isolation and the content of intermediates of purine metabolism was determined in the whole by HPLC analysis.33

XOD and XO were measured in plasma according to the liberation of uric acid by using xanthine as substrate,34 in the presence of NADH (for XOD) or absence of NADH (for XO) when only molecular oxygen was electron acceptor.28 The XDH activity was calculated by subtracting from XOD the XO activity.

Results

The results of clinical and biochemical parameters are shown in Table 1. Increased creatinine and urea, together with glomerular filtration rate (GFR) fall, were expected to be present in a group on dialysis. Hypertension was confirmed in both clinical groups.

Table 1. Plasma urea, creatinine, and blood pressure in the investigated groups.

Investigated parameters	Control20	Essential hypertension30	Dialysis28
Plasma U urea  (mmol/L)	4.09 ± 1.35	6.06 ± 2.08	22.48 ± 7.43*** ^ooo
Plasma creatinine  (µmol/L)	72.06 ± 5.18	84.37 ± 13.97	597.03 ± 101.88*** ^ooo
Systolic TA  (mmHg)	134.45 ± 12.44	163.18 ± 24.56	150.34 ± 23.28
Dyastolic TA  (mmHg)	79.98 ± 9.99	94.65 ± 17.36	89.99 ± 20.98

***p < 0.001, statistical significance compared with the control.

^ooop < 0.001, statistical significance compared with the patients with essential hypertension.

Figure 1 represents the concentration of circulating blood nucleotides, triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in investigated groups. In the fresh whole blood samples, the ATP, ADP, AMP, and uric were detected as circulating purine compounds. A significant fall in the level of ATP was documented in both clinical groups of patients, more in a group of patients on dialysis program, compared with control value (p < 0.001). Both degradation nucleoside phosphate products, ADP and AMP, were significantly increased in both groups of patients compared with control healthy individuals (p < 0.001), together with their final degradation product, uric acid (p < 0.001). Since simultaneous purine nucleotide degradation may consequently lead to the production of uric acid, the index of ATP/ADP and ATP/uric acid was evaluated and it is shown in Figure 2. Gradual significant fall in both indexes was observed in both groups, compared with the control value (p < 0.001), which was almost five times in a group on dialysis. Significant difference was observed between two clinical groups, where group with essential hypertension had almost two times higher index of ATP/ADP than group on dialysis and also higher index ATP/uric acid. Figure 3 represents the activity of total XOD activity and both XO and XDH activities in plasma of the investigated groups. Total XOD activity was the most significantly expressed in a group with essential hypertension, more than in a group on dialysis. But this group still remained higher XDH activity, significantly more than control or dialysis group. From the other side, the activity of XO, which dominantly contributes to uric acid and ROS production, was significantly increased in dialysis group, more than in a group with essential hypertension.

Figure 1. The concentration of circulating blood nucleotides, ATP, ADP, AMP, and uric acid in investigated groups. For the evaluation of purine nucleotides concentration, fresh blood was immediately processed for their isolation after adding stabilizing solution of retard ATP degradation. ***p < 0.001 compared with the control; *p < 0.05 compared with the control; ^ooop < 0.001 compared with the essential hypertension group.

Figure 2. The index of ATP/ADP and ATP/uric acid in investigated groups. For the evaluation of purine nucleotides concentration, fresh blood was immediately processed for their isolation after adding stabilizing solution o retard ATP degradation. ***p < 0.001 compared with the control; ^ooop < 0.001 compared with the essential hypertension group.

Figure 3. The activities of total Xanthine oxidoreductase (XOD), xanthine oxidase (XO), and xanthine dehydrogenase (XDH) in plasma of investigated groups. Xanthine oxidoreductase (XOD) and xanthine oxidase (XOD) were measured in plasma according to the liberation of uric acid,³⁴ in the presence of NADH (XOD) or absence of NADH (XO) when only molecular oxygen was electron acceptor. ***p < 0.001 compared with the control; *p < 0.05 compared with the control; ^ooop < 0.001 compared with the essential hypertension group.

Discussion

Obtained results from our study showed disturbed circulating blood purine nucleotide level, which exerted strong shift toward the increased degradation products, such as ADP and AMP in relation to ATP form. The concentration of their terminal degradation product-uric acid reflected this condition as well (Figure 1) what was expressed on evaluated indexes, ATP/ADP, and ATP/ uric acid (Figure 2). The activity of plasma XDH/XO exerted strong shift toward XO activity, especially in a dialysis group (Figure 3).

Follow-up studies before effective antihypertensive therapy documented that between one-third and two-thirds of subjects with essential hypertension may develop proteinuria, with one-third developing renal insufficiency and ESRD, about 10% dying from uremia.1–3,35–37 Extracellular purine nucleotides, ATP, ADP and AMP represent potent vasoactive and prothrombotic compounds of the vascular wall, by triggering signaling via G-protein-coupled P2Y and via ligand-gated P2X receptors. Since vascular tone in blood vessels can be controlled by perivascular nerves and endothelial cells, released ATP may exert dual function, depending on the source of its liberation. If being released as a neurotransmitter by sympathetic nerves, it can act via P2X receptors of vascular smooth muscle cells, by producing vasoconstriction. From the other side, the ATP released from endothelial cells by shear stress, ischemia, or hypoxia may be recognized by P2Y receptors on endothelial cells, simultaneously releasing nitric oxide, which produces vasodilatation.20–23 In the healthy conditions, when vessel wall and endothelium are intact, the main effect of ATP is vasodilatory, presumably due to nitric oxide release. During atherosclerosis or pro-thrombotic states, the main source of ATP is aggregated thrombocytes. Since released ATP may be able to act through damaged endothelium on vascular smooth muscle cells, the effect is usually vasoconstrictory. Enhanced release of ATP, beside hypoxia, may be provoked by some pharmacologic agents, like calcium agonists, pro-thrombotic compounds (thrombin), and bacterial toxins (lipopolysaccharide).16–20 The ATP degradation products, such as AMP and uric acid, may mediate vasoconstrictory effect via stimulation of angiotensin system and may contribute to damage of the endothelial barrier.9–11

Metabolic deterioration of the macro energetic ATP compound in cells may reflect on current changes in blood purine nucleotides. The relative amounts of the three adenine nucleotides (adenylate pool) may summarize the energy status of a cell, known as the adenylate energy charge (ATP + ½ADP)/(ATP + ADP + AMP). Concerning the ATP degradation, the ATP/ADP ratio and the ATP/AMP ratio should be physiologically maintained as high as possible in order to perform a variety of energy-dependent cellular activities, such as muscle contraction, motility, and ion pumping. If the ratio physiologically tended to rise above 0.7, it may reflect potential for high energy phosphoryl transfer, while fall in this charge represents the cell energy crisis state.38 During shear stress and ischemia, the extracellular ATP concentration may increase up to 10-fold, but the adenine nucleotides are sequentially dephosphorylated by membrane-bound ecto-nucleotidases and circulating purine catabolic degrading enzymes. ADP represents one of the major contributors to the thrombotic effects, since ADP antagonists act as potential anti-thrombotic agents.14,24 The results obtained in our study documented significant fall in whole blood ATP level with consequent production of ADP, AMP, and uric acid (Figures 1 and 2). Concentration of ADP was documented to be significantly (about 50%) higher in patients with essential hypertension, but almost three times higher in patients on dialysis (Figure 1). Increased concentration of uric acid in this way may be result of consequent final degradation of purine compounds. The impairment of fractional excretion of uric acid was also observed in patients with essential hypertension.39,40 It may explain their role in augmenting not only hypertension but also platelet activity contributing to atherosclerosis acceleration and development of renal complications.10,11 Recent studies documented that extracellular purine nucleotides and uric acid may represent important extrarenal stimuli for the development of hypertension and ESRD, due to diverse metabolic functions and due to participation in the regulation of renal function, rennin–angiotensin system, vascular tone, and coagulation cascade.9,40 Since their concentration was especially disturbed in patients on dialysis, our results may suggest their contribution to the development and progression of chronic kidney disease. The harmful effect of hyperuricemia was confirmed, by reducing uric acid level, what may slow down renal progression in subjects with hypertension. Results obtained in experimental hyperuricemia have shown that the development of the preglomerular arteriolar lesions resulted in hemodynamic changes followed by reduced renal blood flow and glomerular hypertension. High level of uric acid may activate angiotensin II, may potentiate renal vasoconstriction, and may up-regulate angiotensin type 1 receptors on vascular smooth muscle cells, stimulating in this way, the renin–angiotensin system.9–11,40 The possible causal relationship of blood pressure with the uric acid level was documented by using xanthine oxidase inhibitors or uricosuric agents (allopurinol or benziodarone) to control essential hypertension.41 Hominoid evolution of primates led to uricase mutation and deletion, where uric acid appeared as the terminal purine degradation product. The hypothesis was that the uricase mutation helped to maintain uric acid high enough to maintain blood pressure acutely (via stimulation of the renin–angiotensin system) and chronically (by inducing salt-sensitivity) in order to develop and maintain the human upright posture and locomotion.42,43 During experimental hyperuricemia, the renal interstitial inflammation and tubular injury were documented to induce renal microvascular and interstitial disease. Uric acid is able to stimulate vascular smooth muscle cell proliferation through the activation of the mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk1/2), stimulation of platelet-derived growth factor (PDGF), cyclooxygenase-2 (COX-2) expression, and production of thromboxane.44–47

The results about plasma XOD activity and its components XDH and XO are shown in Figure 3. Total XOD activity showed up-regulated activity in a group with essential hypertension, more than in a group on dialysis. It may suggest that some endothelial factors may contribute to the increased expression of enzyme, since this group still remained higher XDH activity, significantly more than control or dialysis group. From the other side, the activity of XO, which dominantly contributes to uric acid and ROS production, significantly increased in a dialysis group, more than in a group with essential hypertension. It was documented that circulating XO may bind to glycosaminoglycans on the surface of endothelial cells, which contribute to its activation and longer stability.25,26,29 Taking into consideration both XO-catalytic reaction products, uric acid and ROS, our results are very close to the role of angiotensin and oxidative stress in essential hypertension what was commonly documented.9–11 The role of ROS in the development of renovascular hypertension was documented as well and our results pointed out the role of XO in their generation.48 Deposited XO via ROS generation may directly damage endothelium, contributing further to renal damage.

Conclusion

By simultaneously investigating circulating blood nucleotide level in patients with essential hypertension and patients on dialysis program, we showed that the pattern of nucleotide catabolism was significantly shifted toward catabolic compounds, including ADP, AMP, and uric acid in patients on dialysis program. Further, we observed significant increase in plasma XO activity, mainly due to the interconversion between dehydrogenase form (XDH) in patients on dialysis, but due to general up-regulation of total XOD activity, mainly in group with essential hypertension. The examination of the role of circulating purine nucleotides and uric acid in pathogenesis of hypertension and possible development of renal disease, together with XO role, may help in modulating their liberation and ROS production in slowing progression from hypertension to renal failure.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

The work is supported by the Ministry of Science Serbia TR31060.