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ORIGINAL ARTICLE
Year : 2013  |  Volume : 26  |  Issue : 2  |  Page : 78-85

Evaluation of nitric oxide and superoxide dismutase in different stages of chronic kidney disease


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, El-Shohadaa Hospital, Ministry of Health, Menoufia, Egypt

Date of Submission02-Feb-2013
Date of Acceptance14-May-2013
Date of Web Publication31-Jan-2014

Correspondence Address:
Asmaa M El-Gamacy
MBBCh, Denishway, beside Mahfouze Mousgue, El-Shohadaa, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.126099

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  Abstract 

Objective
The aim of this work was to evaluate nitric oxide (NO) and superoxide dismutase (SOD) as parameters of oxidative stress in different stages of chronic kidney disease (CKD) and their correlation with the lipid profile.
Background
CKD is a debilitating disease that leads to many complications, the most common being cardiovascular. Hence, this study was carried out to evaluate the oxidative stress that predisposes to cardiovascular diseases in these patients.
Methods
This study included 60 patients with CKD. In addition, 12 apparently healthy age-matched and sex-matched individuals were included as a control group. Patients were classified into five groups according to estimated glomerular filtration rate (eGFR) calculated using the CKD-EPI formula. We estimated serum creatinine, urea, lipid profile [total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c)], serum NO, and SOD levels.
Results
There was a progressive increase in the mean value of TC, TG, and LDL-c from group I to group V, whereas HDL-c decreased progressively from group I to group V. There was a highly significant progressive decrease in the mean value of NO and SOD from group I to group V when compared with the controls and on comparison of groups with each other. There was no significant correlation between NO and SOD with other parameters in group I, group II, and group III. However, there was a significant positive correlation between NO and SOD with eGFR, HDL-c and a significant negative correlation between NO and SOD with urea, creatinine, TC, TG, and LDL-c in group IV and group V.
Conclusion
This study suggests that the progressive decline in kidney function could lead to decreased antioxidants levels along with disturbance in the lipid profile.

Keywords: Chronic kidney disease, chronic kidney disease epidemiology collaboration, estimated glomerular filtration rate, nitric oxide, superoxide dismutase, total cholesterol, triglycerides


How to cite this article:
Abd-Elhalim EF, El-Saeed GK, Khodeer SA, Koura MA, El-Gamacy AM. Evaluation of nitric oxide and superoxide dismutase in different stages of chronic kidney disease. Menoufia Med J 2013;26:78-85

How to cite this URL:
Abd-Elhalim EF, El-Saeed GK, Khodeer SA, Koura MA, El-Gamacy AM. Evaluation of nitric oxide and superoxide dismutase in different stages of chronic kidney disease. Menoufia Med J [serial online] 2013 [cited 2020 Apr 3];26:78-85. Available from: http://www.mmj.eg.net/text.asp?2013/26/2/78/126099


  Introduction Top


Development of chronic kidney disease (CKD) has become a serious problem worldwide. CKD is characterized by a slow and progressive decline in the kidney function. Some of the manifestations of CKD include atherosclerosis, increased hemolysis, platelet dysfunction, and neuropathy. These sequelae of CKD have been attributed to the overproduction of free radicals and reactive oxygen species (ROS) in these patients [1] .

ROS are intermediary metabolites that are normally produced during the course of oxygen metabolism. Oxidative stress defines an imbalance between the formation of ROS and an antioxidative defense mechanism. In view of the profound biological effects of ROS, numerous clinical and experimental studies have focused on the detection of signs of oxidative stress in CKD patients [2] .

Cells defend themselves against free radical attacks by developing various antioxidant systems. Several enzymatic systems can detoxify free radicals: copper/zinc superoxide dismutase (Cu/Zn-SOD), which catalyzes the conversion of superoxide anion into hydrogen peroxide and works concomitantly with hydroperoxide, removing enzymes such as catalase and glutathione peroxidase [3] .

Nitric oxide (NO) is one of the main factors involved in the antiatherosclerotic effects of endothelium. Several investigators have focused their attention on a possible role played by NO in the development of uremic symptoms, but the results were controversial the topic is still debated [4] .

Lipid disturbances are a constant feature of CKD. They are a significant risk factor for vascular complications, leading to increased morbidity and mortality in this patient group [5],[6] .

Lipid abnormalities and enhanced oxidative stress in CKD patients accelerate the process of atherosclerosis, resulting in cardiovascular complications. Hence, this study was carried out to assess alterations in the serum lipid profile and oxidative stress to determine the risk of development of cardiovascular complications in CKD patients.


  Methods Top


This study was carried out at the Clinical Pathology Department, Faculty of Medicine, Menoufia University, in the period between December 2010 and December 2012. Patients were selected from the Nephrology Outpatient Clinics, In-patients of the Internal Medicine and Hemodialysis Unit, from Menoufia University Hospitals.

This study included 60 patients with CKD (30 men and 30 women). Their ages ranged from 22 to 62 years. In addition, 12 apparently healthy age-matched and sex-matched individuals were included in this study as a control group (six men and six women), their age ranged from 22 to 62 years. Patients were classified into five groups according to estimated glomerular filtration rate (eGFR) by CKD-EPI in which the values of the constants of a, b, and c vary on the basis of race, sex, and serum creatinine [7] .



The variable a takes on the following values on the basis of race and sex:

  1. Black women = 166.
  2. Black men = 163.
  3. White women and women of other races = 144.
  4. White men and men of other races = 141.


The variable b takes on the following values on the basis of sex:

  1. Women = 0.7.
  2. Men = 0.9.


The variable c takes on the following values on the basis of sex and serum creatinine:

  1. Women
    1. Serum creatinine <0.7 mg/dl = −0.329.
    2. Serum creatinine >0.7 mg/dl = −1.209.


  2. Men

    1. Serum creatinine <0.9 mg/dl = −0.411.
    2. Serum creatinine >0.9 mg/dl = −1.209.


Group I included 12 patients with eGFR (90-119 ml/min/1.73 m 2 ), six men and six women ranging in age from 28 to 62 years. Group II included 12 patients with a mild reduction in eGFR (60-89 ml/min/1.73 m 2 ), six men and six women ranging in age from 30 to 62 years. Group III included 12 patients with a moderate reduction in eGFR (30-59 ml/min/1.73 m 2 ), six men and six women ranging in age from 22 to 62 years. Group IV included 12 patients with a severe reduction in eGFR (15-29 ml/min/1.73 m 2 ), six men and six women ranging in age from 22 to 62 years. Group V included 12 patients with established kidney failure eGFR (<15 ml/min/1.73 m 2 ), six men and six women ranging in age from 28 to 62 years. The control group included 12 healthy individuals with normal eGFR (>120 ml/min/1.73 m 2 ), six men and six women ranging in age from 22 to 62 years.

All participants in the study were subjected to the following investigations: kidney function tests (serum creatinine, urea), lipid profile [total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c)], serum NO, and SOD levels.

Samples were collected by sterile venipuncture after a 12 h overnight fast. Each sample was 4 ml and was divided into three parts:

  1. 3 ml for assay of kidney function tests (serum creatinine was assessed using the Jaffe method and urea using the urease method) and lipid profile (TC and TG were assessed using the enzymatic method, HDL-c using the precipitation method, and LDL-c using the Friedewald equation).
  2. 0.5 ml was left to clot in a plain vacutainer tube at room temperature and sera were separated by centrifugation and stored at −20°C to −80°C for estimation of NO level. The NO assay kit was supplied by Bio-Diagnostics (Giza, Egypt). This assay is based on the colorimetric detection of nitrite as an azo dye product of the Griess Reaction. The Griess Reaction is based on a two-step diazotization reaction in which acidified nitrite produces a nitrosating agent, which reacts with sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine to form the chromophoric azo derivative, which absorbs light at 540 nm and is expressed as μmol/l.
  3. 0.5 ml in a heparinized EDTA tube for estimation of SOD level. This part was washed immediately four times by NaCl 0.9% for preparation of the erythrocyte lysate. The SOD assay kit was supplied by Bio-Diagnostics. This assay relies on the ability of the enzyme SOD to inhibit the phenazine methosulfate-mediated reduction of nitroblue tetrazolium dye. Spectrophotometric readings were taken at 560 nm and expressed as U/g Hb.


The statistical analysis included the Mann-Whitney U-test (nonparametric test) and correlation coefficient (r).


  Results Top


[Table 1] shows a highly statistically significant difference between the groups studied in the mean values of eGFR, urea, and creatinine when compared with the controls (P < 0.001) and when compared with each other. However, there was no statistically significant difference in the mean value of urea in group II compared with group I (P > 0.05).
Table 1: Statistical comparison between the studied groups in the mean eGFR, urea, and creatinine

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[Table 2] shows a highly significant difference in the mean value of TC in all groups when compared with the controls (P < 0.001). There was a progressive increase in the mean value of TC from group I to group V, but no statistical difference in the mean value of TC in group V when compared with group IV (P > 0.05).
Table 2: Statistical comparison between the studied groups in the mean of total cholesterol, triglycerides, LDL-c, and HDL-c

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There was a highly significant difference in the mean value of TG in all groups when compared with the controls (P < 0.001). There was a progressive increase in the mean of TG from group I to group V. However, there was no statistical difference in the mean value of TG in group II when compared with group I and also in group III when compared with group II (P > 0.05).

There was highly significant difference in the mean values of HDL-c in all groups when compared with the controls (P < 0.001). There was a progressive decrease in the mean value of HDL-c from group I to group V. However, there was no statistical difference in the mean value of HDL-c in group III when compared with group II (P > 0.05).

There was a highly significant difference in the mean values of LDL-c in all groups when compared with the controls (P < 0.001). There was a progressive increase in the mean values of LDL-c from group I to group V. However, there was no statistical difference in the mean value of LDL-c in group III when compared with group II and in group V when compared with group IV (P > 0.05).

[Table 3] and [Figure 1] show that there was a highly significant progressive decrease in the mean value of NO from group I to group V when compared with the controls (P < 0.001) and on comparison of the groups with each other.
Figure 1:

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Table 3: Statistical comparison between the studied groups in the mean level of nitric oxide

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[Table 4] and [Figure 2] show that there was a highly significant progressive decrease in the mean value of SOD in the chronic renal failure (CRF) patients in group I and group V compared with the control group (P < 0.001) and when compared with each other. However, there was no statistically significant difference in the mean value of SOD in group II when compared with group I (P > 0.05).
Figure 2:

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Table 4: Statistical comparison between the studied groups in the mean level of superoxide dismutase

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[Table 5] shows that there was a highly significant positive correlation between NO and SOD on the one hand with eGFR and HDL-c on the other (P < 0.001), whereas there was a highly significant negative correlation between NO and SOD with urea, creatinine, TC, TG, and LDL-c (P < 0.001) in all the individuals studied.
Table 5: Correlation between NO and SOD and the other parameters in the total groups

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  Discussion Top


In this work, we study the oxidative stress in CKD patients through two of its biomarkers, NO and SOD, and its relation to lipid abnormalities.

In our study, there was a highly significant difference in the mean values of TC, TG, HDL-c, and LDL-c in all groups compared with the controls (P < 0.001). There was a progressive increase in the mean value of TC, TG, and LDL-c from group I to group V, whereas there was a progressive decrease in the mean value of HDL-c from group I to group V.

Many studies have focused on lipid abnormalities in CKD; some results were in agreement with our study such as Sumathi et al. [6] , who reported that the mean values of TC, TG, LDL-c, TC/HDL-c, and LDL-c/HDL-c ratio were increased in cases of CKD when compared with controls, except HDL-c [6] . Also, Nagane and Ganu [8] reported a significant increase in TC, LDL-c, and TG of prehemodialytic samples of CRF patients as compared with controls. The mean values of HDL-c were found to be significantly reduced in prehemodialytic samples as compared with controls [8] .

Hypercholesterolemia in CRF is suggested to be because of the associated proteinuria and renal insufficiency. Proteinuria leads to alterations in gene expression for HMG-CoA reductase, resulting in increased activity of HMG-CoA reductase, leading to hypercholesterolemia [5],[8] . Hypertriglyceridemia is caused by the presence of insulin resistance in renal failure, which activates hormone-sensitive lipase, causing increased FFA, which in turn stimulates the production of apoB-100-containing lipoproteins such as VLDL, leading to hypertriglyceridemia. However, several authors suggested that hypertriglyceridemia in CKD may be because of the defective metabolism of TG-rich lipoproteins by lipoproprotein lipase and hepatic lipase [5],[8] , whereas the reason for decreased concentrations of HDL-c in CKD is not fully understood. It may be because of decreased activities of lipoproprotein lipase, hepatic triglyceride lipase, and lecithin cholesterol acyl transferase, increased concentration of cholesterol ester transfer protein, and decreased apolipoprotein concentrations [8] .

In contrast to these results, the study of Padalkar et al. [9] reported that levels of TC, HDL-c, and LDL-c were decreased, whereas TG was elevated in CKD patients as compared with the control group. However, these values were not statistically significant [9] .

To evaluate the oxidative state in CKD patients in this study, we have focused on two of the oxidative stress markers: NO and SOD. In our study, there was a highly significant progressive decrease in the mean value of NO from group I to group V when compared with the controls (P < 0.001).

Many studies have published the relation between NO and CKD and reported that there are considerable clinical, in-vitro, and in-vivo data to suggest that systemic, endothelial, and renal NO deficiency is a common feature of CKD irrespective of the primary genesis of the disease. Some evidence suggests that this NO deficiency contributes significantly to the progressive nature of the CKD and the endothelial NO deficiency certainly contributes to the cardiovascular damage in patients with renal failure. Strategies that reverse eNOS inhibition and/or can boost the ability of the damaged kidney to produce NO might help preserve residual renal function and/or reduce the rate of progression to end stage [10],[11] .

The endothelium-derived NO is critically involved in the regulation of a wide variety of vascular functions. It had been hypothesized that a deficiency of vascular NO might be involved in the accelerated atherosclerosis and high cardiovascular mortality observed in patients with CKD [12] . There are multiple ways in which NO deficiency develops in CKD. At end stage, there are uremic factors in plasma that inhibit l-arginine transport into cells and this may cause a 'net' substrate deficiency. Also, increases occur in endogenous NO synthetase inhibitors, in particular, asymmetric dimethylarginine (ADMA) [13] .

In contrast to these results, studies of Brunini et al. [14],[15] reported that basal NOS activity was increased in uremic compared with control platelets. After stimulation by ADP, NOS activity increased significantly in control platelets but failed to increase further the elevated NOS activity in uremic platelets. However, Nagane et al. [16] reported that there was no significant change in the mean values of serum NO in stage 1 and 2 in CKD patients as compared with controls, but a significant and progressive decrease was observed in the mean values of serum NO from stage 3, 4, and 5 in CKD patients as compared with controls.

Free radicals are highly reactive molecules generated by biochemical redox reactions that occur as a part of normal cell metabolism and during the course of free radical-mediated diseases such as renal diseases. Free radicals are eliminated from the body by their interaction with antioxidant enzymes such as glutathione peroxidase, SOD, catalase, etc. Patients with CKD, including those on regular hemodialysis, have a high incidence of premature cardiovascular disease. Oxidative stress that occurs when there are excessive free radical production or low antioxidant levels has recently been implicated as a causative factor in atherogenesis [17] .

In our study, there was a highly significant progressive decrease in the mean value of SOD from group I to group V CKD patients as compared with the controls (P < 0.001). However, there was no statistically significant difference in the mean values of SOD in group II when compared with group I (P > 0.05).

This is in agreement with Vaziri et al. [18] , who reported that erythrocyte SOD levels were lower in CRF patients than in controls. However, Nagane et al. reported that there was no significant change in the mean values of serum SOD in stage 1 and 2 in CRF patients as compared with controls, but a significant and progressive decrease was observed in the mean values of serum (SOD) from stage 3, 4, and 5 in CKD patients as compared with controls [17] .

Mechanisms involved in decreased SOD activity in CKD patients may be because of increased production of ROS such as H2O2 which is known to suppress SOD activity. Decreased SOD activity among hemodialyzed patients could be because of decreased levels of Cu 2+ and Zn 2+ as they are cofactors of cytoplasmic SOD. Increased lipid peroxidation causes consumption of antioxidant enzymes, particularly in hemodialysis patients, and may also be one of the reasons for decreased SOD levels. The reduction in cellular SOD isoforms in the CKD animals was accompanied by a significant increase of the gp91 phox subunit of NADPH oxidase, which is the major source of superoxide production in the cardiovascular tissues. Almost all cardiovascular cell types produce ROS that play an important role in numerous physiological processes as well as in the pathogenesis of cardiovascular disease [1] .

In contrast to these results, Chugh et al. [19] and Menevfie et al. [20] reported that erythrocyte SOD levels were higher in CRF patients than in controls. However, Schettler et al. [21] and Akiyama et al. [22] reported a significant increase in SOD activity in leukocytes in CKD patients, but this increase affects only Cu/Zn-SOD. Moreover, Cu/Zn-SOD mRNA expression in leukocytes was also significantly higher as compared with controls. The increased activity of antioxidant enzyme SOD may be a compensatory mechanism in response to increased oxidative stress in these patients or because of limited number of cases in these studies.

When comparing these different studies, as different sources of cells were used, considering that erythrocytes in CKD patients could have a shorter life span because of anemia, it is possible that this shorter life span could explain the difference in SOD levels between these studies. This raises the question of a suitable cell for the assessment of SOD.

We also found that there was no significant correlation between NO and SOD with other parameters in groups I, II, and III. However, there was a highly significant positive correlation between NO and SOD with eGFR, HDL-c, whereas there was a highly significant negative correlation between NO and SOD with urea, creatinine, TC, TG, and LDL-c in group IV and group V.

From this work, it can be concluded that progression of CKD from stage I to stage V through progressive reduction of eGFR and progressive increase in urea and creatinine is associated with reduced activity of NO and SOD along with increased levels of TG, TC, and LDL-c and reduction of HDL-c. These changes may lead to cardiovascular diseases.


  Acknowledgements Top


The authors thank all patients and personnel who participated in this study.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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2.Galle J. Oxidative Stress in CRF. Nephrol Dial Transplant 2001; 16 :2135-2137.  Back to cited text no. 2
    
3.Aymelek G, Yesim A, Mehmat NO, Bolkan S. Lipid peroxidation and antioxidant systems in hemodialyzed patients. Dial Transplant 2002; 31 :88-96.  Back to cited text no. 3
    
4.Sarkar SR, Kaitwatcharachai C, Levin NW. Nitric oxide and hemodialysis. Semin Dial 2004; 17 :224-228.  Back to cited text no. 4
    
5.Rutkowski B, Chmielewski M. Lipid disturbances in chronic renal failure - patomechanisms and treatment. Annu Acad Med 2004; 49 :99-105.  Back to cited text no. 5
    
6.Sumathi ME, Tembad MM, Jayaprakash DS. Preethi BP study of lipid profile and oxidative stress in CRF. Biomed Res 2010; 21 :451-456.  Back to cited text no. 6
    
7.Levey AS, Stevens LA. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150 :604-612.  Back to cited text no. 7
    
8.Nagane NS, Ganu JV. Lipid profile and serum paraoxonase1 activity in CRF patients pre- and post-hemodialysis. Al Ameen J Med Sci 2011; 4 :61-68.  Back to cited text no. 8
    
9.Padalkar RK, Shinde AV, Patil SM. Lipid profile, serum malondialdehyde, SOD in CRF and type 2 diabetes mellitus. Biomed Res 2012; 23 :207-210.  Back to cited text no. 9
    
10.Saran R, Novak JE, Desai A, Abdhulhayoglu E, Warren JS, Bustami R, et al. Impact of vitamin E on plasma asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD): a pilot study. Nephrol Dial Transplant 2003; 18 :2415-2420.  Back to cited text no. 10
    
11.Baylis C. Nitric oxide deficiency in CRF. Eur J Clin Pharmacol 2006; 13 :123-130.  Back to cited text no. 11
    
12.Passauer J, Pistrosch F, Büssemaker E. Nitric oxide in chronic renal failure. Kidney Int 2005; 67 :1665-1667.  Back to cited text no. 12
    
13.Baylis C. Nitric oxide deficiency in CRF. Am J Physiol Renal Physiol 2008; 294 :1-98.  Back to cited text no. 13
    
14.Brunini TMC, Yaqoob MM, Malagris NLE, Ellory JC, Mann GE, Mendes Ribeiro AC. Increased nitric oxide synthesis in uremic platelets is dependent on L-arginine transport. Pflugers Arch 2003; 445 :547-550.  Back to cited text no. 14
    
15.Brunini TMC, Mendes-Ribeiro AC, Ellory JC, Mann GE. Platelet nitric oxide synthesis in uremia and malnutrition: a role for l-arginine supplementation in vascular protection. Cardiovasc Res 2007; 73 :359-367.  Back to cited text no. 15
    
16.Nagane NS, Ganu JV, Gandhi R. Oxidative stress, serum homocysteine and serum nitric oxide in different stages of chronic renal failure. Biomed Res 2009; 20 :71-74.  Back to cited text no. 16
    
17.Moujerloo M. Variations of lipid peroxidation and SOD activity due to haemodialysis in Gorgan. J Clin Diagn Res 2010; 4 :2763-2767.  Back to cited text no. 17
    
18.Vaziri ND. Dyslipidemia of chronic renal failure: the nature, mechanisms and potential consequences. Am J Physiol Renal Physiol 2006; 290 :262-272.  Back to cited text no. 18
    
19.Chugh SN, Jain S, Agrawal N, Sharma A. Evaluation of oxidative stress before and after haemodialysis CRF. J Assoc Physicians India 2000; 48 :981-984.  Back to cited text no. 19
    
20.Menevfie E, Svrkaya A, Karagozolu E, Tiftik AM, Turk S. Study of elements, antioxidants and lipid peroxidation in hemodialysis patients. Turk J Med Sci 2006; 36 :279-284.  Back to cited text no. 20
    
21.Schettler V, Kühn W, Kleinoeder T, Armstrong VW, Oellerich M, Müller GA, et al. No acute impact of haemodialysis treatment on free radical scavenging enzyme gene expression in white blood cells. J Intern Med 2003; 253 :201-207.  Back to cited text no. 21
    
22.Akiyama S. Dysregulation of superoxide dismutase in chronic kidney disease. Nephron Clin Pract 2005; 99 :c107-c114.Evaluation of nitric oxide and superoxide dismutase in different stages of chronic kidney disease  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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