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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2012  |  Volume : 23  |  Issue : 2  |  Page : 290-295
Assessment of nitrogen radicals and their scavenging activity in patients with end-stage renal failure

1 Department of Pharmacology, College of Medicine, Al-Mustansiriya University, Baghdad, Iraq
2 Department of Medicine, College of Medicine, Al-Mustansiriya University, Baghdad, Iraq

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Date of Web Publication28-Feb-2012


Reactive oxygen species are implicated in cardiovascular disease in patients with chronic renal failure (CRF). This study is aimed to assess the nitrogen radicals in patients with end-stage renal failure (ESRF) referred for peritoneal dialysis. A total of 36 patients with estimated creatinine clearance ≤ 15 mL/min were recruited from the Dialysis Unit at Al-Yarmouk Teaching Hospital in Baghdad, Iraq during 2009, and enrolled into the study. The main causes of CRF were hypertension and diabetes mellitus. The median levels of serum nitric oxide and peroxynitrite were 300 and 152 μmol, respectively. Nitric oxide was not detected in the sera of 10 patients. The sera of 12 patients showed ability to scavenge in vitro, the nitric oxide released by sodium nitroprusside. Significant inverse correlation was found between serum peroxynitrite and either systolic (r = -0.402) or diastolic (r = -0.387) blood pressure. Our study shows that production of nitrogen radicals is enhanced in ESRF and it is accompanied by limited activity of scavenging nitric oxide.

How to cite this article:
Al-Nimer MS, Jaleel NA. Assessment of nitrogen radicals and their scavenging activity in patients with end-stage renal failure. Saudi J Kidney Dis Transpl 2012;23:290-5

How to cite this URL:
Al-Nimer MS, Jaleel NA. Assessment of nitrogen radicals and their scavenging activity in patients with end-stage renal failure. Saudi J Kidney Dis Transpl [serial online] 2012 [cited 2021 Oct 21];23:290-5. Available from: https://www.sjkdt.org/text.asp?2012/23/2/290/93156

   Introduction Top

End-stage renal failure (ESRF) is a permanent reduction in glomerular filtration rate sufficient to produce detectable alterations in well-being and organ function. [1] In the United States, approximately 100-150 per million persons de­velop ESRF annually, i.e., 0.010-0.015 per year. The most common causes of ESRF are diabetic nephropathy, hypertensive nephrosclerosis, glo-merulonephritis, interstitial nephritis, and poly-cystic kidney disease. [2],[3],[4] Nitric oxide (NO) plays a pivotal role in kidney homeostasis at the level of renal vasculature, glomerulus, and renal tu­bules. All three subtypes of NO synthase (endo-thelial, neuronal, and inducible) are expressed in various kidney structures and participate in the control of glomerular and medullary hemo-dynamics, tubuloglomerular feedback responses, the renin-angiotensin system, and electrolyte/fluid balance through endogenous generation of NO. It had previously been found, both in experi-mental [5],[6] and clinical studies, [7],[8] that urinary ex­cretion of NO metabolites, an index of the generation of NO, is low in renal failure. Recently Caimi et al [9] reported that the production of NO was elevated in 42 patients with ESRF and it decreased after a hemodialysis session. It has been hypothesized that the cytotoxic sequel of NO production depends on the redox state of the cell and its ability to generate peroxynitrite (ONOO - ) anion. [10] Experimentally, intra-arterial infusion of ONOO- in anesthetized rats resulted in increase renal blood flow and glomerular filtration rate at low doses while high doses (≥20 μg/kg/min) produced the opposite effect. [11] Schmidt et al [12] reported that the production of ONOO- is increased in uremic patients as a result of inducible NO synthase enzyme induc­tion by the uremic toxin, phenylacetic acid. This study was aimed to investigate the status of nitrogen radicals in patients with established ESRF referred for peritoneal dialysis.

   Materials and Methods Top

This study was conducted in the Department of Pharmacology, College of Medicine, Al-Mustansiriya University in cooperation with the Dialysis Unit at the Al-Yarmouk Teaching Hospital in Baghdad, Iraq during the year 2009. The study protocol was approved by the Scien­tific Committee of the College of Medicine, Al-Mustansiriya University. Informed consent to participate in the study was obtained from patients or their proxy. The criteria of inclusion were that patients had established ESRF of whatever cause referred for peritoneal dialysis because of symptomatic renal failure, with the estimated creatinine clearance ( e C cr ) being ≤ 15 mL/min. A total of 36 patients (18 of each sex) with median age of 59 years were enrolled into the study. Each patient was examined thoroughly and venous blood was obtained for determi­nation of serum creatinine, estimated creatinine clearance, serum NO, serum peroxynitrite, and the endogenous substances that scavenge NO in serum. In this study, a commonly used surro­gate marker for actual creatinine clearance, namely the Cockcroft-Gault formula, which cal­culates the estimated creatinine clearance [ e C cr ] which in turn estimates the glomerular filtration rate, was used. [13],[14] The formula is as follows:

the constant for men 1.23 and for women, it is 1.04.

Peroxynitrite-mediated nitration of phenol was measured (an index of ONOO- release) as des­cribed by Beckman et al [15] and cited by VanUffelen et al. [16] Briefly, 50 μL was added to 5 mM phenol in a 50 mM sodium phosphate buffer pH 7.4 in a final volume of 3 mL. After incubation for two hours at 37°C, 50 μL of 0.1 M sodium hydroxide was added, and the ab-sorbance at 412 nm of each sample was imme­diately recorded. The yield of nitrophenol was calculated from ï = 4400/M/cm. All expe­riments were performed in duplicate.

Nitric oxide donating activity was determined as described by Newaz et al [17] utilizing Greiss reagent. Briefly 0.5 mL serum was added to 50 μL HCl (6.5 M) and 50 μL sulfunalic acid (37.5 mM). After incubation for 10 minutes, 50 μL naphthylethylenediamine dihydrochloride (12.5 mM) was added and incubated for further 30 minutes and centrifuged for 10 minutes at 1000 g. The reference NO donating compound was lithium nitrite. The absorbance at 540 nm was immediately recorded. All experiments were performed in duplicate.

Nitric oxide radical scavenging was estimated by the use of Greiss reaction. [18] Greiss reagent was modified by using naphthylethylenedia-mine dihydrochloride (0.1% w/v). The reaction mixture (3 mL) contained sodium nitroprusside (10 mM, 2 mL), phosphate buffer (0.5 mL) and serum or distilled water as negative control (0.5 mL), which was incubated in a dark room at 25°C for 150 minutes. After incubation, 0.5 mL of the reaction mixture was mixed with 1 mL of sulfunalic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for five minutes for complete diazotization. Following this, 1 mL of naphthylethylenediamine dihy-drochloride was added, mixed and allowed to stand for 30 min in a dark place at 25°C. A pink colored chromophore is formed. The absorbance of these solutions was measured at 540 nm against corresponding blank. All experiments were performed in duplicate. All chemicals used in the study were of analr grade obtained from local sources and were prepared freshly prior to the experiments.

   Statistical Analysis Top

The results are expressed as number, percen­tage, range, median, and mean ± SD. The data are analyzed using two-tailed student's " t" and simple correlation tests taking P ≤ 0.05 as the lowest limit of significance.

   Results Top

[Table 1] shows the characteristics of the study. Clinical, laboratory, and radiological investiga­tions revealed that the causes of ESRF were hypertension, diabetes mellitus, polycystic kidney, and chronic glomerulonephritis [Table 1]. The duration of the disease was varied according to the etiology and the established diagnosis of ESRF. High blood pressure was uncontrolled by antihypertensive drugs in 10 out of 18 known hypertensive patients enrolled in this study. Fourteen and 11 patients, respectively, presen­ted with high systolic and diastolic blood pres­sures [Table 1]. The median serum creatinine and estimated creatinine clearance were 587 μmol/L and 10.93 mL/min, respectively. Ultra-sonography and plain X-ray of abdomen re­vealed no radiological abnormalities in 18 pa­tients, symmetrical bilateral small kidneys with loss of corticomedullary differentiation and in­creased echogenicity in 15 and bilateral enlarged kidneys with multiple cysts in three patients.
Table 1: Characteristics of the study patients.

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Hydronephrosis, mild benign prostatic enlarge­ment and renal stone were found concomitantly, in one case each. Serum nitrogen species in the form of ONOO- was demonstrated in all cases with median value of 152 μmol [Table 2] and it significantly correlated inversely with systolic and diastolic blood pressures [Figure 1]. Serum ONOO- did not significantly correlate with serum creatinine (r = -0.229) or estimated creatinine clearance (r = 230). Serum NO was not detected in 10 out of 36 patients (27.8%). There was wide variation in the serum level of detected NO that ranged between 17 and 1622 μmol. There was significant positive correlation (r = 0.4) bet­ween serum ONOO- and NO levels [Figure 2]. The property of scavenging NO in the serum was not detected in 24 patients. In 12 patients (33.3%) with ESRF, the serum was able to scavenge an equivalent median value of 1.021 μmol of NO. Although the serum NO level di­rectly correlated (r = 0.244) with the scavenging property of the serum, it did not reach statis­tically significant level.
Figure 1: Correlation between serum peroxynitrite level and blood pressure in patients with end-stage renal failure.

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Figure 2: Correlation between serum peroxynitrite and nitric oxide levels in patients with end-stage renal failure.

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Table 2: The status of nitrogen species in 36 patients with end-stage renal failure.

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   Discussion Top

The results reported in this study show that there is imbalance between overproduction of nitrogen radicals and their scavenging property in patients with ESRF. Hypertension and dia­betes mellitus, as with other studies, are the main causes of ESRF and they shared the pro­perty of overproduction of nitrogen radicals. [19] In this work, a new observation is reported that the sera of 12 patients had the ability to scavenge the NO. This property of scavenging NO is limited compared to NO production. It is well known that in the sera of patients with ESRF, there are many toxins and inflammatory me­diators involved in the production of nitrogen radicals but the opposite has not been repor-ted. [20],[21],[22] The clinical significance of NO scaven­ging property is not clear because of the small sample size. Previous studies have shown that oxidative stress in ESRF may be implicated in the pathogenesis of atherosclerosis, [23] and the patients are at risk of cardiovascular disease. [24] On the other hand, a 1 mg/dL higher uric acid level, a natural scavenger of ONOO-, was asso­ciated with 17% increased risk of all-cause mortality and no association with renal failure. [25] As a result of nonenzymatic reaction of NO and superoxide anion, ONOO- is formed, i.e., higher the NO, higher is the ONOO - , and this ex­plained the significant correlation between NO and ONOO. Moreover, overproduction of nitro­gen radicals in uremic patients may be in­volved in reducing the blood pressure as de­monstrated in this study; there was a significant inverse correlation between blood pressure and ONOO. The limitations of the study included small sample size and the effect of peritoneal dialysis on the activity of NO scavenging of the serum. We conclude that production of nitrogen ra­dicals is enhanced in ESRF and it is accom­panied by limited activity of scavenging NO.

   References Top

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3.Hsu CY, Iribarren C, McCulloch CE, Darbinian J, Go AS. Risk factors for end-stage renal disease: 25-year follow-up. Arch Intern Med 2009;169:342-50.  Back to cited text no. 3
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8.Kari JA, Donald AE, Vallance DT, et al. Physiology and biochemistry of endothelial function in children with chronic renal failure. Kidney Int 1997;52:468-72.  Back to cited text no. 8
9.Caimi G, Carollo C, Montana M, Iatrino R, Bondì B, Lo Presti R. Nitric oxide metabolites, leukocyte activation markers and oxidative status in dialyzed subjects. Blood Purif 2009; 27:194-8.  Back to cited text no. 9
10.Lipton SA, Choi YB, Pan ZH, et al. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 1993;364: 626-32.  Back to cited text no. 10
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12.Schmidt S, Westhoff TH, Krauser P, Zidek W, van der Giet M. The uraemic toxin phenylacetic acid increases the formation of reactive oxygen species in vascular smooth muscle cells. Nephrol Dial Transplant 2008;23:65-71.  Back to cited text no. 12
13.Cockcroft DW, Gault MH. Prediction of crea-tinine clearance from serum creatinine. Nephron 1976;16:31-41.  Back to cited text no. 13
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18.Sundararajan R, Haja NA, Venkatesan K, et al. Cytisus scoparius link-a natural antioxidant. BMC Complement Altern Med 2006;6:8.  Back to cited text no. 18
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20.Roselaar SE, Nazhat NB, Winyard PG, Jones P, Cunningham J, Blake DR. Detection of oxidants in uremic plasma by electron spin resonance spectroscopy. Kidney Int 1995;48:199-206.  Back to cited text no. 20
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23.Galle J, Seibold S. Has the time come to use antioxidant therapy in uraemic patients? Nephrol Dial Transplant 2003;18:1452-5.  Back to cited text no. 23
24.Hage FG, Venkataraman R, Zoghbi GJ, Perry GJ, DeMattos AM, Iskandrian AE. The scope of coronary heart disease in patients with chronic kidney disease. J Am Coll Cardiol 2009;53: 2129-40.  Back to cited text no. 24
25.Madero M, Sarnak MJ, Wang X, et al. Uric acid and long-term outcomes in CKD. Am J Kidney Dis 2009;53:796-803.  Back to cited text no. 25

Correspondence Address:
Marwan S.M. Al-Nimer
Department of Pharmacology, College of Medicine, Al-Mustansiriya University, Baghdad
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PMID: 22382221

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