Effects of Chronic Kidney Disease on levels of Oxidative Stress and Trace Elements

Introduction: The relationship between trace elements and OS in CKD patients is still not completely elucidated. The aim of this work is to determine the serum levels of OS and the trace elements in CKD patients. Methods: A total of 91 patients were enrolled and then divided into 3 groups at the basis of CKD stages. Healthy volunteers were included as a control group. Serum levels of SOD, MDA, Zn, Cu, Ca, Fe, and Mg were determined , and the correlation among these datas were analyzed. Results: The MDA level in the patients’ sera was signicantly higher than the controls’ (P<0.05); however, their sera level of SOD was obviously lower (P<0.05). Meanwhile, the creatinine level in CKD patients’ sera was positively correlated with the MDA level (r = 0.534, P<0.01), while negatively with the expression of SOD (r = -0.427, P < 0.01). A lower expression of Zn and Ca values could be seen in the cases’ sera (P<0.05). Besides that, it showed a signicant negative correlation between serum levels of MDA and Ca (r=-0.282, P<0.01), MDA and Zn (r=-0.358, P<0.01), while a positive correlation between MDA and Cu (r = 0.236, P=0.022). Adversely, the expression of SOD in the sera of cases was positively related to the levels of Ca (r=0.273, P<0.01) and Zn (r=0.277, P<0.01). Conclusion: Abnormalities of OS and Zn (cid:0) Ca existed in CKD patients; and elevated OS may play a role in the trace elements imbalance. Dialysis seems to have no obvious effect on plasma trace elements.


Introduction
Chronic Kidney Disease (CKD) is characterized by progressive loss of kidney function, and has turned into a major global health problem. The incidence and mortality of CKD have increased over the past few decades [1]. A Chinese survey in 2012 found that the overall prevalence of CKD in adults was approximately 10.8%, placing a huge burden on health care resources [2].
Oxidative stress (OS) refers to an imbalance between formation of reactive oxygen species (ROS) and antioxidative defense mechanisms. In CKD patients, oxidative stress is an important risk factor for the development and progression of several complications. There is good evidence indicating that uremia in general is associated with enhanced oxidative stress, and treatment of uremic patients with HD has been suggested to particularly contribute to oxidative stress and reduced antioxidant levels [3,4].
Trace elements, which are measured in microgram per gram or less, occur in minute amounts and are indispensable for our body.
In some instances, they are the key components of many enzymatic pathways [5], thereby play important physiological and biochemical functions. Many studies con rmed that patients with CKD are often at risks of developing severe trace elements imbalances [6,7].
Dialysis, including hemodialysis and peritoneal dialysis, is the most common and signi cant method of treatment for end-stage renal disease (ESRD). Researches in regard to the changes of trace elements in the blood levels, have been reported in CRF patients, especially in those treated by hemodialysis [8].The effect of trace elements on metabolism may be exacerbated in ESRD patients on dialysis because these patients are incapable of excreting excess elements properly [9]. However, the incidence of abnormal trace element status in different stages of CKD patients, including CKD3, CKD4, CKD5, especially in dialysis patients, has not been comprehensively studied.
In this study, we investigate the plasma trace elements levels and OS parameters in CKD patients, determining plasma levels of Zn, Cu, Ca, Fe and Mg. We also evaluated plasma MDA and SOD ,which are the key markers of OS. The relation between OS and those trace elements are investigated as well.

Patients
All cases were recruited from the Department of Nephrology and Blood Puri cation Center in the Second A liated Hospital of Xi'an Jiaotong University. The primary disease for all patients was chronic glomerulonephritis, and the inclusion criteria were as follows: stable blood pressure, absence of complicated infection and heart failure within the last 3 months, not pregnant or breast feeding, absence of severe cardiovascular, and mental disorders. The patients were not taking any immunosuppressive agents.
A total of 91 patients were enrolled and divided into 3 groups based on the CKD stages calculated from glomerular ltration rate (GFR). Using the MDRD equation to calculate the estimated GFR. Patients are grouped into CKD3, CKD 4 and CKD5 which were further divided into 3 subgroups: Non-dialysis uremic group (ND), hemodialysis group (HD) and peritoneal dialysis group (PD).
The patients in the HD group had undergone regular hemodialysis treatment (4-hour treatment given 3 times a week) for more than 3 months using a dialysis solution sugar-free, Ca 2+ 1.5mmol/L, K + 2.0mmol/L). The dialysis membrane was polysulfone with an area of 1.4-1.8 m 2 , and the blood ow was set to 220-250 mL/min, dialysate ow 500 mL/min. The PD patients had undergone regular treatment for more than 3 months using low-calcium peritoneal dialysis solution ,which were the standard Tenckhoff catheter, and the daily volume of replacement uid was 8000 mL.
Twenty healthy volunteers were recruited to the control group.
This study was approved by the Ethics Committee in the Second A liated Hospital of Xi'an Jiaotong University. Written informed consent was obtained from all patients and healthy volunteers participating in the study.

Sample preparation
Fasting hemospasia was performed in all participants. Blood samples in the HD group were collected prior to hemodialysis on the day of treatment. Routine blood test that included a complete blood count and routine clinical chemistry tests were performed.
10mL of peripheral blood was centrifuged at 3000 rpm for 5 min, and the supernatant was collected and stored at -80ºC.
Enzyme-linked immunosorbent assay (ELISA) All serum samples were stored at -80ºC and processed all at once. Serum levels of Cu, Ca, Fe, Zn, Mg Xi'an microelement inspection institute , SOD and MDA (Westang, Shanghai, China) were determined by ELISA using speci c detection reagents following the manufacturer's instruction. Each assay was performed in triplicates and the experiments were repeated once.

Statistical analysis
Data were analyzed using SPSS 16.0 Statistical Analysis software (SPSS Inc., Chicago, IL, USA). Measurement data are expressed as mean ± standard deviation (SD). Comparison of means among groups was made by one-way analysis of variance (ANOVA), and comparison between two groups was made by Fisher's Least Signi cant Difference (LSD) test. Spearman's rankorder correlation was used for bivariate correlation analysis. A P value less than 0.05 (P<0.05) was considered statistically signi cant, and P<0.01 indicated a more signi cant difference.
Routine blood and clinical chemistry tests were performed. Complete blood count was measured. Results were analyzed for each group and comparison was made between groups (Table 1). Signi cant differences between the controls and different stage of nitrogen, cystatin-C, parathyroid hormone among CKD groups (Table 1).
There were no signi cant differences in age and gender distributions between the controls and patients in different CKD stages (Table 1).

Expression pro les of serum cytokines
We evaluated the correlation of serum levels of the OS markers with creatinine levels. Serum levels of SOD ( Figure 1A) and MDA ( Figure 1B) were respectively signi cantly lower and higher in CKD patients than in controls (P<0.05), and positively correlated with advancing CKD stage(P<0.05). Serum levels of SOD were altered by dialysis treatment compared with no-dialysis patients. In contrast, serum levels of MDA were not altered by dialysis treatment. Moreover, serum creatinine levels in serum CKD patients correlated positively with serum MDA levels (r =0.534, P<0.01), and negatively with serum SOD levels (r =-0.427, P < 0.01; Figure  1C and D), re ecting an association between the extent of OS and the severity of renal damage.

Discussion
With recent advances studies of pathophysiology, in ammation and dysregulated immune responses have emerged as important factors contributing to the gradual loss of renal function in CKD patients [10]. Moreover, the risk of heart diseases and cancers increases markedly in CKD patients [11,12]. Also, the CKD patients are at the risk of developing severe trace elements imbalances [13,14]. Trace elements are indispensable and often combined with proteins, coenzyme, hormones and vitamins. In ESRD patients, many different factors affect the serum concentrations of trace elements, such as increased oral intake, failure of renal excretion, loss of appetite and metabolic alterations and so on. During dialysis processes some trace elements can accumulate because of dialysis uid impurities, and others may move from blood to the dialysate, leading to de ciency [15].
In our study, plasma Fe, Cu and Mg trace element levels of CKD patients, were compared with control group and compared with each other. We found that plasma Fe have no statistically differences between patients and the control group. This may be attributed to the fact that we often supplement iron in CKD patients because most of them have anemia, especially in CKD5.
Again there were no difference in plasma Cu and Mg, we analyze that the control and patients had the similar regional changes and dietary habits which soil and water contamination. Adversely, Serum Zn and Ca values were signi cantly lower in our CKD patients than the controls, and the tendency became more obvious as the disease progressed, especially in CKD5 patients. Zn is an essential component of various proteins and biomembrane [16]. Recent studies suggest that, both of the increased urinary zinc excretion and decreased intestinal zinc absorption may be the primary cause of hypozincemia in patients. Also dietary restrictions and anorexia lead to an insu cient micronutrient intake. Shrimpton [17] argue zinc de ciency is a leading cause of diseases in developing countries and is associated with immune de ciencies by impaired cell proliferation, abnormal T-cell function, and cytokine expression, all of which might contribute to the high risk of infection observed in uraemia patients. Meanwhile we found Zn amounts in HD and PD lower than other CKD patients. We think it is because of these patients are more seriously and renal replacement therapies consume more zinc. Ca is one of the most abundant mineral elements in human body, and the normal Page 5/12 function of tissues and organs regulating various systems of human body depends on its existence. The person can become sensitive, emotional instability and attention de cit if they lack of Ca. In CKD patients the kidney damage leading to the body of phosphorus excretion disorder, on the other hand the diet excessive intake of phosphorus, so the combined with Ca induced deposition, it caused the loss of calcium and insu cient production of 1.25-(OH) 2 VitD3 which reduce calcium absorption. We found that the decrease in blood calcium is more signi cant in PD patients which were more likely to use low calcium dialysis uid. These consequences consistent with several other investigators [18]. Miura et al. [19] compared serum Mg and Cu levels between HD patients and control groups and found no statistically signi cant differences. The results of Miura study also found Zn is lower in HD patients. But Krachler et al. [20]found elemental composition changes in plasma and observed a decrease in Mg levels over a 6-month period in uremia. Regulating trace element levels is essential to prevent some complications in HD patients.
Differing results have been reported with respect to plasma levels of trace elements in HD patients. Tonelli et al [21] found low plasma Zn and high plasma Cu levels in HD patients compared with controls. The distribution of trace elements can be affected by insu cient dietary intake, anorexia, degrees of gastrointestinal absorption, and drug treatment, it lead to some clinical abnormalities in uremic patients (D'Haese [22] ).The uremic state, medication, the dialysis process, dietary intake, and the quality of water used for dialysis may contribute to such clinical abnormalities of trace elements [23]. Although recent improvements of technology in the dialysis can prevent some serious complications by changes in trace elements, problems still remained [24]. In our study, although dialysis treatment improved the survival of CKD patients, it had a limited effect on trace elements. Dialysis augments reduced renal function by removing metabolic waste products and maintaining the electrolyte balance and acid-base equilibrium. However, due to the possible risks present in HD, such as blood loss during treatment, dietary restrictions, anorexia, aluminum intoxication [25] and dialyzer biocompatibility [26], there is no effect in trace elements balance compared with Nodialysis patients. Although PD has advantages over HD in these respects [27], problems such as increased protein loss, excessive glucose intake and triglyceride abnormalities make it ineffective in restoring immune balance. Further investigation is required to identify new therapeutic agents or strategies to improve the patient's microenvironment.
OS refers to an imbalance in the body oxidation and antioxidation, results in neutrophils in ammatory in ltrates, proteases secretion to increase, and produces a large number of oxide intermediate. Several studies have con rmed that the occurrence of complications in CKD such as accompany hypertension [26], disease of heart and nervous system lesions, OS is one of the important risk factors affecting prognosis of CKD [28]. SOD and MDA can be used to evaluate the body's resistance to oxidation and peroxidation level. In our study the Serum levels of MDA were signi cantly higher and SOD signi cantly lower in CKD patients than in controls. Meanwhile we found that Serum levels of MDA and SOD were be closely related with serum creatinine levels. The results reiterate that the severity of CKD correlates with the degree of OS. However, as the renal function deteriorates, OS response might impair renal function and the extent of renal damage could conceivably increase during progression of CKD. So we think that the OS reaction which caused by metabolic disorder and apoptosis may be involved in the development process of renal brosis. But serum levels of SOD were altered by dialysis treatment compared with no-dialysis, so we think replacement can improve the imbalance in the body oxidation and antioxidation system of CKD patients.
We also compared trace element levels and oxidant-antioxidant parameters and found that in patient groups, there was a signi cant positive correlation between serum levels of SOD with Ca (Fig. 3B, r = 0.273, P < 0.01), Zn (Fig. 3A, r = 0.277, P < 0.01). Adversely, there was a signi cant negative correlation between serum levels of MDA with Ca (Fig. 3C, r=-0.282,P < 0.01, Zn Fig. 3D, r=-0.358 P < 0.01)as well as a positive correlation between MDA and serum Cu, but the relationship did not quite reach statistical signi cance. According to the above description, OS is one of the important risk factors affecting prognosis of CKD, so we have reasons to regard that Zn and Cu are closely in uenced by OS. Cu is a transition element and a cause of radical ROS formation through Fenton reaction. These ROS radicals cause lipid peroxidation, an end product of MDA. Consistent with our study, Rijkgauer et al [29], analyzed trace elements and MDA concentrations in healthy persons and compared Cu levels and MDA concentrations, and found no correlations between them. Other trace elements (Mg. Fe) have no obvious correlation with SOD and MDA.
According to our results, elevate OS may play a role in the trace elements imbalance in CKD. Dialysis itself seems to have no obvious effect on plasma trace elements concentrations compared with No-dialysis CKD5 patients. Therefore, decreasing sources of oxidative stress and at the same time increasing oxidative capacity can be bene cial to these patients. These ndings show that whereas prolonged replacement treatment leads to improved oxidative stress damage, trace element concentrations are not affected. Since both de ciency and excess of trace elements are potentially harmful yet amenable to therapy, the hypothesis that trace element status in uences the risk of adverse clinical outcomes is worthy of investigation. And it is critical if micronutrient interventions are to be not only effective, but also targeted to those with the greatest need.

Declarations Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Human Research Committee for Research Involving Human subjects in the Second A liated Hospital of Xi'an Jiaotong University and the ethical standards of the institutional and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent
The study was approved by the local ethical committee and all patients gave informed consent.

Con ict of Interests
The authors declare no nancial or commercial con ict of interests.