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

Discussion 

General Observations

Oxidoreductases, due to their dual activities, are an intriguing and highly important enzyme that allows us to uncover the mechanisms of oxidative stress in the human body. However, the dual activity of XOR also means the interpretation of its functions is more challenging than for other antioxidant enzymes.

In the present study, the highest XOR activity was observed in the control group in PPP and in PRP (second result), meaning that this group is best at oxidative stress compensation. The highest antioxidant isoform (XD) activity observed in PPP and the high activity of the XO isoform confrmed that. This means that the pro-oxidative and antioxidant balance is preserved. Hemodialysis patients before and shortly after kidney transplantation are highly exposed to oxidative stress as a result of the underlying disease as well as the types of renal replacement therapy. This highlights that peritoneal dialysis causes lower exposure of patients to the harmful effects of reactive oxygen species than other renal replacement therapies.

Dołęgowska et al. (2010) investigated XOR isoform activity in plasma in individuals after kidney transplantation, divided into three groups: early, slow, and delayed function of the transplanted organ. They showed an increase in XO and XOR activity in all groups after 1 and 5 minutes after transplantation. XD activity increased in the slow and delayed function groups also after 1 and 5min. The highest activity was found in the case of the XD isoform (antioxidant) and the lowest in the case of the XO isoform (prooxidative). This was probably due to increased oxidative stress as a result of organ transplantation, which the body tried to compensate for by the increased activity of the XD isoform [3, 23].

Increased conversion of XD to XO after kidney transplantation was presented by Kwiatkowska et al (2010). During the 6-month observation of patients after transplantation, it was shown that the average level of XOR in serum is constantly increasing from the frst day after surgery. This suggests that the XOR level reflects the degree of damage to the transplanted organ during ischemia/reperfusion (I / R). Further increases in XOR levels can be explained by immunosuppressive therapy, which includes steroids [23–25]. Herken et al. (2007) showed that after kidney transplantation, the transformation of the XD isoform into the XO isoform is initiated in the ischemic period and is then continued after reperfusion [25].

HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK ON KIDNEY DISEASE PATIENTS

In this study, the activity of XOR in PPP after transplantation significantly decreases, similar to the XD and XDO isoforms. The activity of the oxidase isoform also decreases both in the poor and the platelet-rich plasma, but there are no statistically signifcant relationships. This indicates lower oxidative stress after kidney transplantation because the conversion of the XD isoform to XO is not increased (lower XDO activity after kidney transplantation). These results may indicate small damage to the transplanted organ during the I/R period. Attention should also be paid to the significantly lower activity of XOR and its isoform in patients before and after kidney transplantation in comparison to other forms of renal replacement therapy as well as in comparison to the control group. It suggests a lower efficiency of the antioxidant system in this group of patients.

The optimal type of renal replacement therapy must be selected for each patient to achieve the best treatment outcomes. Many different factors impact the success of the therapy used, one of which is oxidative stress. Therefore, it is crucial to study the effect of dialysis on XOR activity.

In our study, XOR activity decreased after hemodialysissis in PPP and PRP. However, it is higher in comparison to the other groups studied (except for control groups). This indicates a higher exposure to oxidative stress in this group of patients. The activity of the XD isoform in the platelet-poor plasma increases after hemodialysis, and the XDO isoform decreases, similarly to the XO isoforms. However, these were not statistically signifcant changes. In turn, in platelets, XD activity is significantly lower after hemodialysis, while XO activity is lower in the HD A group, and the activity of the intermediate isoform is higher after hemodialysis (albeit with no statistical significance). This indicates a low conversion of the anti-oxidant to prooxidative isoform, which may mean less oxidative stress after hemodialysis.

The influence of hemodialysis on XOR activity and its isoforms in platelet-poor plasma and platelets was studied by Cecerska-Heryć et al (2017). They are demonstrated the effect of hemodialysis on the activity of XOR and its isoforms in PPP and platelets. Besides, they showed a decrease in oxidative stress after hemodialysis, as evidenced by a decrease in XO activity and an increase in XD activity in PPP plasma [26]. This study also confirms the results obtained by other scientists [3, 23–25]. Hemodialysis causes intense oxidative stress, hence the increased conversion of the pro-oxidative to antioxidant isoform after hemodialysis.

On the other hand, Miric et al. (2013) showed higher XOR activity before hemodialysis compared to control, and increased XOR activity during renal replacement therapy in patients with GNRI (Geriatric Nutritional Risk Index)≤90 (high risk of complications and mortality due to malnutrition), and a reduction in XOR activity in patients with GNRI>90 (low risk of death due to malnutrition) in which hemodialysis is performed. This may mean that oxidoreductase is involved in hemodialysis-induced oxidative damage, which may contribute to accelerated protein destruction in patients with GNRI ≤90 [27].

Boban et al. (2014) showed that total XOR activity was higher in the group of patients suffering from essential hypertension, compared to patients on dialysis. The highgest activity in this group was also demonstrated by the XD isoform about the control or dialysis patients. On the other hand, the activity of XO, which mainly contributes to the production of ROS, was the highest in dialysis patients [28].

In the case of peritoneal dialysis, the results achieved by us are ambiguous. In platelet-poor plasma, XD activity is significantly lower than in hemodialyzed patients, as is XO activity. This could indicate a much lower exposure to oxidative stress of patients on peritoneal dialysis than those subjected to hemodialysis. However, the pattern is different in platelets. XD activity is significantly lower in patients from the PD group than in hemodialysis, but XO's acuteness is significantly higher in peritoneal dialysis patients.

DXO intermediate activity was higher in patients undergoing peritoneal dialysis, which may indicate an increased conversion to the antioxidant isoform. However, it should be remembered that under the influence of RFT, there is a leakage of oxidoreductase from cells to plasma. Therefore, the activity of XOR isoforms in platelets related to hemodialysis and peritoneal dialysis may disrupt this picture. During hemodialysis, there is increased activation of platelets accompanied by strong oxidative stress, resulting in ROS that can cause XOR to be released to the plasma, which may not happen in the case of peritoneal dialysis: hence, much higher XO and XDO activity in patients from the PD group. Patients treated conservatively are also exposed to oxidative stress caused by the underlying disease, which can be demonstrated by the highest activity of the XDO isoform and the high activity of the XO isoform in PPP.

The activity of xanthine oxidoreductase isoforms according to gender, duration of dialysis, patient age, cause, and stage of CKD 

Our study revealed no relationship between XOR activity and sex, except for a signifcant association between XDO activity in platelets. In men, XDO activity was significantly lower than in women. This supports the theory that the activity of antioxidant enzymes may be higher in women. However, the primary purpose of measuring XDO activity is to analyze XD to XO transformations. This confirms the results reported in previous studies, where such a relationship was not observed [19, 25]. Previously, only Decker et al. (1982) reported higher XOR activity in male rats compared to females [29].

An age effect was observed in the activity of all isoforms of XOR PPP and platelets in all groups. Patients in groups PD and TE were, on average, younger, and XD and XO activity in platelet-poor plasma was lower in these patients than in the other groups. Only XO isoform activity in platelets was higher in these (younger) groups. These results are different from those obtained for other antioxidant enzymes. This might mean that this group of patients has not yet lost XOR activity under the influence of RFT. However, it is more likely that the type of implemented renal replacement therapy affects XOR activity more than age. Tis seems to be confrmed by the results of our multivariate regression, as well as studies carried out by Cecerska-Heryć et al.(2017).

Table 8 The influence of particular parameters on the activity of xanthine oxidoreductase isoforms 

The table presents p values defining statistical significance. The relationship between gender, duration of dialysis, age, stage of chronic kidney disease and the causes of chronic kidney disease and the activity of XOR isoforms was assessed using one-way ANOVA HD and duration of dialysis - the relationship between the type of therapy (hemodialysis, peritoneal dialysis, patients before kidney transplantation), duration of dialysis and the activity of XOR isoforms HD and age - dependence between the studied groups (hemodialysis, peritoneal dialysis, conservative treatment, patients before kidney transplantation and control group), patients' age and XOR isoform activity The stage of chronic kidney disease - the relationship between the severity of chronic kidney disease based on eGFR and the activity of XOR isoforms The causes of chronic kidney disease - the relationship between selected causes of chronic disease and the activity of XOR isoforms NS no statistically signifcant relationship was found

In the present study, a signifcant relationship was found between the duration of dialysis the type of renal replacement therapy used, and the activity of all XOR isoforms in both the platelet-poor plasma and platelets. However, in PPP from longer-lasting dialysis, a decrease in the activity of the XD and XO isoforms was observed. Additionally, the activity of XD, XDO, and XO isoforms increases in the platelets. The XOR activity increased in platelets due to the continuous activation as a result of long-term dialysis and progressive renal disease that causes aggravation of oxidative stress, and ROS production, which in turn destroys the blood platelets. However, the lowering of XOR isoform activity in PPP confirms the results obtained by other researchers on the decrease in antioxidant enzyme activities along with long-term dialysis.

Based on the multivariate regression analysis, it was found that parameters such as the type of renal replacement therapy used, the patient's age, duration of dialysis, and CKD stage affected the activity of XO in PPP, XD in PRP, and XO in PRP, respectively, about 33% of XO in platelet-poor plasma, 39% of XD in platelets and 32% of XO in platelets. (Table 9).

Our study demonstrated the effect of chronic kidney disease on the activity of XD and XO in PPP. The highest activity of XOR isoforms occurred in patients with hypertension and diabetic nephropathy, and the lowest in patients with ADPKD (polycystic kidney disease inherited autosomal dominant). Multivariate regression analysis indicates an independent negative correlation between XO activity in PRP and the severity of chronic kidney diseases.

Because XOR plays an important role in the production of uric acid the relationship between the XOR activity and hypertension or cardiovascular risk is widely described. Elevated levels of uric acid in the serum are associated with oxidative damage in the vessel wall, inflammatory and proliferative changes of the vessels, hypertension, and impaired renal function. Raised uric acid levels might be a factor in the increased risk of cardiovascular events. Therefore, the attempts are focused on blocking the XOR activity to reduce the concentration of UA. The positive effect of inhibition of XOR production on the cardiovascular system was documented [33]. Our study showed a negative correlation between XD activity in PPP and uric acid concentration and a positive correlation between PPP and UA concentration XO activity. These results confirm the physiological importance of XOR also in the protection against cardiovascular diseases.

Nakatani et al. (2017) showed a positive correlation between glucose, uric acid concentration, and XOR activity in patients undergoing hemodialysis. In the same study, multivariate regression analysis showed positive, independent correlations between glucose concentration, and type 2 diabetes diagnosed with XOR activity. In contrast, the uric acid concentration correlated positively with XOR activity in hemodialyzed patients with undiagnosed type II diabetes. This study indicates that glycemic control might decrease oxidoreductase-mediated ROS production possibility in hemodialysis patients [34].

The high activity of XO and XD in PPP observed in this study in patients with diabetic nephropathy and hypertension confirm an increased activity of XOR in this type of disease, due to the distorted level of glucose and uric acid, which may exacerbate chronic kidney disease. For hypertensive patients, high XO and XD activity may be an indicator of an increased risk of cardiovascular events. There is no relationship between the activity of XOR and ADPKD in the literature.

Conclusion 

1. 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. 

2. Peritoneal dialysis patients are exposed to less oxidative stress than hemodialysis patients

Limitations Medicines used by patients Patients in the study group (mainly hemodialysis) took fromide, which may increase the level of uric acid, a strong antioxidant. It is also the last product in the hypoxanthine degradation pathway, catalyzed by XOR. However, our study showed no correlation between uric acid levels and XOR activity, except for the negative correlation of the XD and XO isoforms in PPP. This correlation only confirms the physiological properties of oxidoreductase; the higher uric acid concentration, the lower XO oxidative isoform activity. 

Kidney transplantation Initially, we planned to collect biological material from patients after kidney transplantation 5–7 days after transplantation, followed by one month, three months, and six months after transplantation. Unfortunately, due to logistical problems, it was not possible to provide good quality material to our unit at all time points. Therefore, we decided to collect material only approximately 7 days after transplantation.

References 

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