Xanthine Oxidoreductase Activity in Plateletpoor And Rich Plasma As A Oxidative Stress Indicator in Patients Required Renal Replacement Therapy

Abstract Background: Xanthine oxidoreductase (XOR) is a hydroxylase enzyme involved in the metabolism of purines. XOR activity can vary: the homodimer protein can be converted into two different isoforms XD (antioxidant) and XO (prooxidant). Oxidative stress and infammation that accompany chronic kidney disease (CKD), dialysis, and kidney transplantation, resulted in platelet activation. The present study aimed to determine the influence of applied renal replacement therapy on xanthine oxidoreductase and its isoform activity.

Materials and Methods: The study group consisted of 117 patients, divided into 4 groups: hemodialysis - 30 patients, peritoneal dialysis - 30 patients, kidney transplant patients - 27, and conservative treatment - 30 patients. The control group consisted of 30 healthy volunteers. Results: Signifcant diferences were found in XOR activity in platelet-poor plasma (PPP) within the groups studied (p=0.001). There was a relationship between the type of renal replacement therapy of all oxidoreductase isoforms in PPP (p<0.001 all isoforms) and XD (p=0.008), XO (p<0.001) in platelet-rich plasma (PRP). A relationship was observed between the activity of all oxidoreductase isoforms in PPP and PRP, the type of renal replacement therapy the duration of dialysis, and the age of patients. The cause of chronic kidney disease was also reflected in diferences in XD and XO activity in PPP. 

Conclusions: The type of renal replacement therapy used in CKD patients, age of patients, duration of dialysis, CKD causes, and stage of progression significantly affect the activity of XOR and its isoforms. 

Keywords: Xanthine oxidoreductase, Platelets, Renal replacement therapy, Chronic kidney disease, Antioxidant enzymes

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Background 

Xanthine oxidoreductase (XOR) is a hydroxylase enzyme involved in the metabolism of purines. It catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid (UA). The XOR activity can vary: the homodimer protein can be converted into twodifferentt isoforms. Xanthine dehydrogenase (XD) is expressed predominantly in healthy tissue, and xanthine oxidase (XO) is generated by post-translational XD modification, oxidation cysteine residues as well as limited proteolysis, playing a dominant role in cells and tissues during injuries [1–3]. The activities of the mentioned isoforms oppose each other [4]. XOR acts in the presence of NAD+ as a dehydrogenase, and with molecular oxygen as an oxidase. The ability of XOR to rapidly convert from antioxidant to oxidant, in various types of tissue damage, is an essential element for a rapid innate immune response, beneficial in, for example, bacterial or fungal infection [5].

A reaction intermediates of xanthine dehydrogenase (XDO) and oxygen can react with both NAD+ and O2 but present a higher affinity for NAD+ [6]. This intermediate isoform has not been isolated, but the determination of its activity facilitates the tracking of the transformation of XD to XO [7].

The serum XOR activity in various diseases has been widely investigated. The reason for the interest in this enzyme is the dualism of its action: the ability to produce antioxidants, and on the other hand, reactive oxygen species creation. An increase in XOR activity occurs in pathological conditions such as viral hepatitis, infectious mononucleosis, autoimmune diseases, pneumonia, schizophrenia, and type II diabetes. The increase in XOR activity is also observed in the serum of patients after renal or liver transplantation [8]. It has been shown that the gene coding for XOR may be responsible for renal maturation, and adipogenesis in the kidneys, and may prevent the transformation of epithelial cells into mesenchymal tissue [9]. However, due to our best knowledge, there are no published reports investigating XOR activity in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in patients undergoing renal replacement therapy. The importance of the XOR activity in this group of patients is related to increased platelet activation, which is caused by oxidative stress and infammation that accompany chronic kidney disease (CKD), dialysis, and kidney transplantation. Besides, during dialysis and organ transplantation, tissues and blood vessels are damsaged, and platelets are the frst cells to reach the site of tissue damage, actively participating in the initial stages of the inflammatory process and healing [10].

PRP and PPP are fractions of blood plasma with different platelet concentrations. The platelet content of PRP and PPP is platelets/ml and platelets/ml, respectively. PPP and PRP are obtained by repeatedly centrifuging and washing the whole blood of humans at different centrifugegal speeds [11, 12].

PPP, as a centrifugation byproduct of anticoagulated blood, has a lower platelet concentration than normal blood. The main components of PPP are fibrinogen, fibronectin, and thrombin. The biological effects of PPP are participating in hemostasis and coagulation, acting as a cell attachment vector, and promoting mitosis of fibroblasts and epithelial cells [13]. Although PPP is not as concentrated in platelets as PRP, it has been demonstrated that PPP can also sustain cell growth and survival. PPP promotes wound healing-associated cell functions and accelerates cell migration and proliferation of fibroblasts [14, 15]. Platelet-rich plasma has more concentrated platelets than normal plasma (approximately 150–400× 103 cells/ dL). It is one of the most common definitions of PRP in the literature [16]. To date, limited studies characterized XOR and its isoform activity in platelet-rich or poor plasma in patients suffering from chronic kidney disease. Tan et al. showed cells damaged by reactive oxygen species (ROS) (in the case of chronic renal replacement therapy) "leak" XOR isoforms, leading to the increase of the enzyme level in plasma [17]. This mechanism explains the lower activity of XOR and its isoforms in platelets compared to PPP. Therefore, the oxidoreductases activincity in PPP and PRP are of great interest, as they could help to uncover the cellular processes that occur daily, sis. Such investigations could also indirectly highlight the severity of oxidative stress in this group of patients.

Berry et al. describe also that xanthine oxidoreductase is distributed in the liver, small intestine, mammary gland, and endothelial cells. Subcellular localization methods have demonstrated the presence of xanthine oxidoreductase both in the cytoplasm and on cell membranes. They also indicate that plasma xanthine oxidasereductase may be due to xanthine oxidoreductase shading from cell membranes or leaking from the cytoplasm. This is one of the reasons we test the activity of XOR in PPP and PRP to distinguish the enzyme activity in platelets and other blood cells contained in the plasma [18]. Based on the activity of antioxidant enzymes, we can also determine which type of renal replacement therapy is less likely to expose the patient to oxidative stress. Due to the dual nature of XOR, the understanding of the relationship between the type of renal replacement therapies and the activity of XOR isoforms can be very interesting and helpful in the selection of the type of renal replacement therapy.

Materials and methods 

Ethical approval and consent 

The Bioethical Commission at the Pomeranian Medical University in Szczecin approved the research carried out (no KB−0012/36/11). All participants, including the healthy volunteers in the control group, were informed about the purpose and scope of the study and gave their consent to donate samples and publication of the resulting data.

Study group

There were 147 participants: a control group of 30 healthy volunteers (NK), and 117 patients with chronic kidney disease (CKD) attending the Nephrology, Transplanetology, and Internal Diseases Clinic of the Pomeranian Medical University in Szczecin. Te patients were divided into 4 groups based on the treatment they received: 30 patients before and after hemodialysis (HD A and HD B): 30 patients received peritoneal dialysis (PD); 27 patients before and after kidney transplantations (5–7 days after surgery) (TE, TE A); 30 patients received conservative treatment (CT) (CKD stage 2–5). The gender, age, duration of dialysis, cause and stage of chronic kidney disease, and creatinine concentration in the test and control groups are given in Tables 1 and 2.

Samples 

Blood samples (K2EDTA (8 ml), 3.8% trisodium citrate (9: 1; v / v), and serum (8 ml)) were drawn from all study participants. Hemodialyzed patient blood was drawn from their arteriovenous fistula; peripheral venipuncture was used for all other participants. Samples were taken from hemodialysis patients before (HD A) and about 10 min after the pump was stopped (HD B). Transplant patient blood was collected before transplantation (TE) and 5–7 days after surgery (TE A). Patients recruited for the TE group did not belong to the group of hemodialysis or peritoneal dialysis patients in this study. They were patients qualified for transplantation from all over Poland. The vast majority of patients with kidney transplantation had prior modeanalysis. K2EDTA and clotted blood samples were centrifuged at 2600 rpm for 10 min at 20 °C to obtain plasma and serum, respectively. To obtain platelet-rich plasma (PRP) and platelet-poor plasma (PPP), blood collected with citrate was centrifuged under the conditions at 1100 rpm for 10 min at 20 °C. The resulting PRP was transferred to a new tube and centrifuged at 6000 rpm for 10 min at 20 °C: platelet-poor plasma (PPP) was transferred to a separate tube; the platelet pellet was rinsed twice and suspended in Tyroda buffer (pH7.4). Plasma, serum, PPP, and PRP were frozen at −80 °C until the assays were performed. In hemodialysis patients, blood was collected before heparin administration and after dialysis lasting 4–5 h on average (heparin half-life - 4 h) to eliminate any possible influence of heparin on XOR activity.

Xanthine oxidoreductase activity in platelet‑poor plasma and platelets 

Determinations were carried out with a Perkin Elmer UV/VIS Lambda 40P spectrophotometer. Extinction changes were recorded at 340nm (XD) and 302nm (XDO, XO) for 5min at 30°C. The enzymatic activity was measured as the formation of uric acid and NADH (increases in A340 and A302) and expressed in mU× mL−1 (milliunits per milliliter). The enzymatic activity was calculated, taking into account the initial rates of reaction. The uric acid formation was measured at 302nm (isoforms XDO and XO) because its absorbance is still high there, whereas changes in NAD+ concentration do not contribute. Thee extinction coefficient for NADH+ H+ ε340=6.22× 103 [L∙mol−1 cm−1 ] was used tocalculatee the activity of isoforms of xanthine oxidoreductase NADH+ H+: ε302=2.30× 103 [L∙mol−1 cm−1 ] [7, 19–22].

Table 1 General characteristics of hemodialysis patients (HD), peritoneal dialysis (PD) treated conservatively (CKD), kidney transplantation (TE), and control group (C) participating in the study (mean±SD)

P * - statistical significance for diferences between HD, PD and CKD groups, TE and C exact Fisher test for qualitative variables; for quantitative variables - one-way ANOVA and; P ** - statistical significance for diferences between HD, PD and CKD groups and TE exact Fisher test for qualitative variables for quantitative variables one-way ANOVA or; DM - diabetic nephropathy; HA - hypertension; GIK - glomerular infammation kidney; ADPKD - polycystic kidney disease inherited autosomal dominant; NS - no statistically signifcant diferences.

Table 2 General characteristics of hemodialysis patients (B - before HD, A - after), peritoneal dialysis (PD) treated conservatively (CKD) before and after kidney transplantation (TE B and TE A) and control group (NK) taking part in the study (mean±SD)

P * - statistical significance for diferences between HD A, HD B, PD and CKD groups, TE and C for quantitative variables - Kruskal Wallis ANOVA, one-way ANOVA or Student's t-test P ** - statistical significance for diferences between HD A, HD B, PD and CKD and TE groups for Kruskal Wallis's ANOVA quantitative variables or ANOVA one-way analysis Kt / V - dialysis index (volume fraction V purified by clearance K at time t) NS no statistically signifcant relationships were found

Statistical analysis To assess distributions, the K-S test (Kolmogorov-Smirnov) was used, which in the case of some variables (the activity of XD and XDO isoforms in PRP) showed a non-normal distribution of parameters. Exact Fisher and Chi-square tests were used to analyze quantitative data. Using Student's t-test and ANOVA analysis for univariate systems, the diferences between associated (paired) and unrelated (unpaired) variables were evaluated in the case of variables with a normal distribution. In the case of variables with non-normal distributions, Kruskal-Wallis ANOVA analysis was performed to evaluate diferences between the parameters, as well as the Mann-Whitney U nonparametric test for unpaired data or Wilcoxon for paired data. A linear multiple regression model was used to determine the multifactor evaluation of relationships between the parameters studied. Statistical analysis of results was performed using Statistica 12 (StatSoft).