Biomarkers
ISSN: 1354-750X (Print) 1366-5804 (Online) Journal homepage: http://www.tandfonline.com/loi/ibmk20
Urinary 8-hydroxy-2′-deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection Jien-Wen Chien, Lien-Yen Wang, Yu-Shan Cheng, Yi-Giien Tsai & Chin-San Liu To cite this article: Jien-Wen Chien, Lien-Yen Wang, Yu-Shan Cheng, Yi-Giien Tsai & Chin-San Liu (2014) Urinary 8-hydroxy-2′-deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection, Biomarkers, 19:4, 326-331 To link to this article: http://dx.doi.org/10.3109/1354750X.2014.910552
Published online: 21 Apr 2014.
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http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(4): 326–331 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.910552
RESEARCH ARTICLE
Urinary 8-hydroxy-20 -deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection Jien-Wen Chien1,2, Lien-Yen Wang3, Yu-Shan Cheng2, Yi-Giien Tsai1, and Chin-San Liu2,4,5# Departments of Pediatrics, 2Department of Vascular and Genomics Center, 3Department of Nuclear Medicine, 4Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan, and 5Graduate Institute of Integrative Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan
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Abstract
Keywords
Background: There are no good biomarkers to predict renal parenchymal involvement in children with urinary tract infection (UTI). Methods: Children (N ¼ 73) younger than 5 years with UTI were enrolled. Urinary levels of 8-hydroxy-20 -deoxyguanosine (8-oxodG) and total antioxidant capacity (TAC) were checked as markers of oxidative stress and antioxidant capacity, respectively. Tc99m-dimercaptosuccinic acid (DMSA) renal scintigraphy was used to find evidence of renal involvement. Results: Patients with positive DMSA findings had higher levels of urinary 8-oxodG (p ¼ 0.003) and higher urinary TAC (p ¼ 0.001) than patients with normal DMSA findings. Conclusions: High level of urinary 8-oxodG may be a risk factor of severe renal damage.
8-hydroxy-20 -deoxyguanosine, Tc99m-dimercaptosuccinic acid, total antioxidant capacity, urinary tract infection
Introduction Upper urinary tract infection (UTI) can result in renal damage in young children because of the growing nature of the kidneys. Isotopic uptake studies such as Tc99 m-dimercaptosuccinic acid (DMSA) scintigraphy have shown that the renal parenchyma is affected in about 55–75% of children with febrile UTI, and that about 20–40% of these children will develop permanent renal damage, e.g. renal scarring (Benador et al., 1994; Lin et al., 2003; Rushton et al., 1992). Reported risk factors for renal damage after UTI include young age, delayed initiation of antibiotic treatment, repeat upper UTI and renal structural anomalies such as high-grade vesicoureteral reflux (Peters & Rushton, 2010). The mechanisms governing renal damage after upper UTI involve host–pathogen interactions, such as extracellular release of reactive oxygen species (ROS) and oxidative stress. In patients with febrile UTI, oxidative damage occurs to lipids of cellular membranes, proteins and DNA. Guanine is the DNA base most prone to oxidation, both in the nucleus and in mitochondria. Upon oxidation, a hydroxyl group is added to the C-8 position of the guanine molecule, resulting
#Chin-San Liu is responsible for statistical design/analysis. E-mail: [email protected] Address for correspondence: Chin-San Liu, MD, PhD, Department of Neurology, Vascular and Genomic Research Center, Changhua Christian Hospital, 135 Nanhsiao Street, Changhua 500, Taiwan. Tel: 886-4-7238595 ext. 4752. Fax: 886-4-7238595 ext. 4063. E-mail: [email protected]
History Received 5 February 2014 Revised 26 March 2014 Accepted 28 March 2014 Published online 21 April 2014
in 8-hydroxy-20 -deoxyguanosine (8-oxodG), one of the most common forms of free radical-induced lesions of DNA. Oxidized nuclear DNA in general undergoes repair. The repair products of oxidative DNA lesions are fairly watersoluble and are excreted into urine without being further metabolized. Oxidatively modified DNA in the form of 8-oxodG can, therefore, be quantified to indicate the extent of free radical-induced oxidative DNA damage (Valavanidis et al., 2009). In recent years, 8-oxodG has been used widely in many studies as a biomarker for the measurement of endogenous oxidative DNA damage. In addition, it has also been shown to be a risk factor for many diseases, such as cancer, atherosclerosis and diabetes (Wu et al., 2004). DMSA renal scintigraphy is a very sensitive method for identifying acute changes in the kidney associated with pyelonephritis. DMSA scans are considered normal if homogenous uptake of the radioisotope is evident throughout the kidneys and the renal contour is preserved. In acute pyelonephritis (APN), the DMSA scan will show focal or diffuse areas of decreased uptake of dimercaptosuccinic acid. The scan will also allow visualization of the collecting system, allowing for estimation of the percentage of renal function of each kidney. Studies in animals (Gupta et al., 2004; Kaur et al., 1988) and in humans (Sobouti et al., 2012) have shown that ROS play an important role in renal damage and that free oxygen scavengers can prevent said damage after UTI. To the best of our knowledge, there are no reports regarding the association between oxidative stress during UTI and renal image studies. In this study, we explore the association between urinary
8-OxodG level
DOI: 10.3109/1354750X.2014.910552
8-oxodG, a marker of oxidative stress, and DMSA renal scans in patients with upper UTI.
Materials and methods
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Patients Children younger than 5 years who were treated for their first episode of febrile UTI at our hospital were enrolled in the study. Blood samples were taken from each patient for complete blood count and measurement of C-reactive protein. Urine samples were also taken for urinalysis and culture. The definition of febrile UTI included a core temperature 38 C; urine culture with growth of microorganisms 105 colonyforming units per mL from clean voided mid-stream urine in older children or 103 colony-forming units per mL after urinary catheterization, and 5 leukocyte cells per highpower field. Clinical data such as amplitude and duration of fever, age and sex were collected. All patients received renal echo and DMSA scanning during hospitalization. Patients were stratified into four groups according to their DMSA results: group 1, normal; group 2, one focal lesion; group 3, two or more focal lesions; and group 4, diffuse lesions with a renal functional difference 410%. Results of the DMSA studies were evaluated by an experienced nuclear medicine physician, who was blinded to the patients’ clinical information. All of the human experimental procedures followed the ethical standards of Changhua Christian Hospital and were approved by the hospital’s institutional review committee (Approval number is 100102). Informed consent for study enrollment was obtained from the parent of the study subjects. Urinary 8-oxodG Urine samples were collected for 8-oxodG measurement. For determination of the 8-oxodG level in urine samples, urine specimens were centrifuged at 10 000 g at 4 C for 10 min, and the supernatant was stored at 70 C before use. The amount of 8-oxodG in urine was measured using an 8-oxodG ELISA kit (Japan Institute for the Control of Ageing, Fukuroi, Japan) according to the manufacturer’s instructions. The specificity of the assay has been established, and the detection range is 0.5–200 ng/mL. Urinary 8-oxodG was adjusted to the level of creatinine in urine. Total antioxidant capacity measurement The total antioxidant capacity (TAC) assay is a cupric ion reducing antioxidant capacity spectrophotometric method. The amount of TAC in urine was measured using a TAC assay kit (Cell Biolabs, Inc., San Diego, CA) according to the manufacturer’s instructions. Briefly, a 20-mL aliquot of urine or uric acid standard and 180 mL of 1 reaction buffer was added to a microtiter plate and mixed. Initial absorbance was obtained by reading the plate at 490 nm. Then, 50 mL of 1 copper ion reagent was added to each well to initiate the reaction. The reaction mixture was then incubated for 5 min on an orbital shaker. Finally, 50 mL of 1 stop solution was added to each well to terminate the reaction. The plate was read again at 490 nm. The stress score is defined as 8-oxodG/ TAC ratio.
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Statistical methods Categorical variables were examined non-parametrically using the Mann–Whitney U test. The Kruskal–Wallis test was used to compare differences in scan results among the four DMSA groups. A generalized linear model test was applied to Figure 1(a–c), and the Spearman’s rank correlation coefficient was used. A receiver operating characteristic (ROC) curve was used to determine the optimal cut-off point of 8-oxodG that predicts renal damage. A p value50.05 was considered to indicate statistical significance. All statistical analyses were performed on a personal computer with the statistical package SPSS, version 15 (SPSS Inc., Chicago, IL).
Results A total of 73 children (43 boys (59%) and 30 girls (41%)) with a mean age of one year (0.95 ± 1.63) were included in the study. Children were classified into four groups based on the DMSA scan results: group 1, normal (N ¼ 26); group 2, one focal lesion (N ¼ 23); group 3, two or more focal lesions with renal functional difference 510% (N ¼ 11); and group 4, diffuse or multiple lesions and renal functional difference 410% (N ¼ 13). The mean duration of fever was 3.7 ± 1.8 d, the mean maximal body temperature was 39.4 ± 0.7 C, the mean white blood cell count was 16.0 ± 6.2 109/L, and the mean C-reactive protein level was 6.38 ± 5.71 mg/dL. The mean urinary 8-oxodG/creatinine level was 12.62 ± 11.28 ng/mg, and the mean urinary TAC/creatinine level was 2.05 ± 1.49 mg/ml. We examined the differences in clinical parameters (age, sex, duration of fever and highest body temperature) and laboratory findings (8-oxodG/urine creatinine, TAC/urine creatinine, white blood cell count, C reactive protein, pyuria, hematuria, bacteriuria and nitrite) between patients with normal DMSA renal scans and those with positive DMSA renal scans (Table 1), and then examined the differences between the four groups of patients (Table 2). We found that patients with positive DMSA findings had higher urinary 8-oxodG/urine creatinine level (p ¼ 0.003), higher urinary TAC/urine creatinine level (p ¼ 0.001), higher white blood cell count (p ¼ 0.006) and higher C-reactive protein (p ¼ 0.014) than patients with normal DMSA findings (Table 1). Parameters that differed significantly between the four groups included urinary 8-oxodG/urine creatinine level (p ¼ 0.02), urinary TAC/urine creatinine level (p ¼ 0.006), white blood cell count (p ¼ 0.044) and the presence of nitrite in urinalysis (p ¼ 0.013). The relationship between 8-oxodG and results of DMSA testing is shown in Figure 1(a). We found that the level of urinary 8-oxodG tended to be higher in patients with more severe DMSA findings. The difference was significant between group 1 (normal DMSA finding) and group 4 (diffuse DMSA finding with renal function difference 410%) (p ¼ 0.003). Figure 1(b) shows the relationship between urinary TAC/ urine creatinine level and the results of DMSA testing. Urinary TAC was significantly higher in group 2 than in group 1 (p ¼ 0.035). In addition, urinary TAC tended to be higher in groups 3 and 4 than in group 1. Figure 1(c) shows that patients in groups 2 and 3 had higher white blood cell
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Biomarkers, 2014; 19(4): 326–331
Figure 1. (a) This figure showed the relationship between 8-oxodG and results of DMSA. Urinary 8-oxodG was significantly higher in group 4 than that in group 1 (p ¼ 0.003). (b) The relationship between urinary TAC/urine creatinine level and the results of DMSA. Urinary TAC was significantly higher in group 2 than that in group 1 (p ¼ 0.035). Besides, urinary TAC tended to be higher in groups 3 and 4 than in group 1. (c) White cell count was significantly higher in group 3 and 4 than that in group 1 (p ¼ 0.014 and 0.011, respectively). Value in the bar indicated the mean ± standard deviation. *p 0.05, compared to negative group.
counts than patients in group 1 (p ¼ 0.014 and 0.011, respectively). Figure 2 shows the ROC curve that was generated to determine the cutoff values of urinary 8-oxodG, white blood cell count and C-reactive protein that can predict acute renal damage. We found that the best predictor of a positive DMSA scan was urinary 8-oxodG. The area under the curve for predicting acute renal damage was 0.732 (p ¼ 0.002). When the cutoff point of urinary 8-oxodG/urine creatinine was set at 5.60 ng/mg, the sensitivity and specificity were 0.822 and 0.64, respectively.
Discussion Prompt diagnosis and treatment of UTI in young children is very important because these infections can result in
permanent renal damage when adequate treatment is not given quickly. However, in children with febrile UTI, it is difficult to differentiate between APN and acute cystitis without parenchymal involvement based on routine clinical and laboratory parameters alone. A meta-analysis found a higher incidence of renal scarring after APN in Asia (Faust et al., 2009). Previous studies have revealed that DMSA is a highly sensitive and specific noninvasive imaging modality for detection of renal inflammation (Jakobsson et al., 1992a,b). In addition, DMSA scans performed during the acute phase of infection have been shown to provide prognostic information that helps determine whether APN lesions are at risk of developing into scars (Chiou et al., 2001). In our study, we found that urinary 8-oxodG level was higher in patients with DMSA scans showing severe renal
8-OxodG level
DOI: 10.3109/1354750X.2014.910552
damage, indicating that this biomarker can be used to predict childhood UTI with renal parenchymal involvement. The pathogenesis of UTI involves both bacterial virulence factors and host factors. In pyelonephritic areas, blood flow is reduced due to intravascular granulocyte aggregation, which leads to arteriolar or capillary occlusion. In addition, transport function of proximal tubular cell membranes is disturbed in pyelonephritic areas due to the release of toxic enzymes and superoxide by intratubular neutrophils (Majd & Rushton, 1992; Roberts et al., 1982; Stokland et al., 1999). Inflammatory cells including macrophages and neutrophils are endowed with a battery of enzymes that generate ROS to
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Table 1. Differences between patients with normal DMSA and positive DMSA.
Age, year Gender Male (%, N) 8-oxodG/urine creatinine, ng/mg TAC/urine creatinine, mg/ml Stress score Duration of fever, d Maximal body temperature, C White blood cell count, 109/L C-reactive protein, mg/dL Pyuria Hematuria Bacteriuria (%, N) Nitrite (%, N)
DMSA Normal (N ¼ 26) Mean ± SD
DMSA Positive (N ¼ 47) Mean ± SD
0.86 ± 0.82
1 ± 1.18
65%, 17 7.96 ± 6.51 1.45 ± 1.04 7.02 ± 5.86 3.4 ± 1.9 39.3 ± 0.7 13.2 ± 5.3 4 ± 3.63 23.9 ± 21.3 13.7 ± 17.3 42%, 11 15%, 4
55%, 26 15.21 ± 12.55 2.38 ± 1.6 7.57 ± 7.08 3.9 ± 1.8 39.4 ± 0.7 17.5 ± 6.2 7.64 ± 6.23 43 ± 31.5 14.7 ± 19.3 51%, 24 34%, 16
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combat invading pathogens (Rama et al., 2005). However, excessive production of ROS leads to damage of phagocytes themselves and if these oxygen metabolites leak out they will damage the surrounding tissue. In other words, microorganisms may initiate disease, but progression of the disease may be the result of inflammatory reactions, characterized by excessive production of ROS (Gupta et al., 1996). ROS are formed by the incomplete reduction of molecular oxygen and include super oxide anion (O 2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH) and singlet oxygen (1O2). Experimental and clinical studies have shown that ROS plays a role in the pathogenesis of renal damage during UTI. Non piliated or mannose-resistant piliated strains of Escherichia coli, which lack receptors for polymorphonuclear
p 0.917 0.336a 0.003b 0.001b 0.69 0.212 0.402 0.006b 0.014b 0.09 0.936 0.421a 0.06a
DMSA, Tc99m-dimercaptosuccinic acid; stress score, 8-oxodG/TAC ratio; pyuria, leukocyte per high power field; and hematuria, blood cells per high-power field. Values are mean (standard deviation) with p value from Mann–Whitney U test of variance. a Chi-square test. b As a significant increase between two groups (p50.05).
Figure 2. ROC curve analysis for the diagnostic accuracy of urine 8-oxodG, WBC and CRP levels predicts result of DMSA image study. The best predictor is 8-oxodG with the area under the curve (AUC) for predicting acute renal damage was 0.732 (p ¼ 0.002).
Table 2. Differences between four groups of patients based on the DMSA scan results. DMSA-positive
DMSA
Age, year Gender Male (%, N) 8-oxodG/urine creatinine, ng/mg TAC/urine creatinine, mg/ml Stress score Duration of fever, d Maximal body temperature, C White blood cell count, 109/L C-reactive protein, mg/dL Pyuria Hematuria Bacteriuria (%, N) Nitrite (%, N)
Normal (N ¼ 26)
One focal lesion (N ¼ 23)
Two or more focal lesions (N ¼ 11)
Renal function difference 410% (N ¼ 13)
0.86 ± 0.82
1.04 ± 1.19
0.68 ± 0.26
1.21 ± 1.61
65%, 17 7.96 ± 6.51 1.45 ± 1.04 7.02 ± 5.86 3.4 ± 1.9 39.3 ± 0.7 13.2 ± 5.3 4 ± 3.63 23.9 ± 21.3 13.7 ± 17.3 42%, 11 15%, 4
57%, 13 13.1 ± 11.91 2.35 ± 2.07 6.96 ± 6.4 4 ± 1.8 39.5 ± 0.7 17.9 ± 7.2 6.75 ± 6.11 50.6 ± 32.2 16 ± 20.3 39%, 9 48%, 11
36%, 4 14.73 ± 9.61 2.41 ± 0.89 6.49 ± 4.03 3.9 ± 1.9 39.8 ± 0.6 17.9 ± 4.6 8.08 ± 5.95 45 ± 38.1 18.5 ± 23.7 64%, 7 36%, 4
69%, 9 19.51 ± 15.68 2.4 ± 1.14 9.68 ± 10.03 3.9 ± 2 39 ± 0.6 16.4 ± 5.9 8.84 ± 6.89 23 ± 20.6 8.5 ± 11.3 62%, 8 8%, 1
p 0.998 0.336a 0.02b 0.006b 0.915 0.588 0.053 0.044b 0.07 0.102 0.658 0.424a 0.013a,b
DMSA, Tc99 m-dimercaptosuccinic acid; stress score, 8-oxodG/TAC ratio; pyuria, leukocyte per high power field; hematuria, blood cells per high power field. p, Kruskal–Wallis test. a Chi-square test. b p 0.05.
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leukocytes, making them resistant to phagocytosis, are more liable to generate ROS extracellular and hence are more pathogenic (Gupta et al., 1997). An in vitro study collected uropathogenic E. coli strains from two groups of patients: one with renal scars and one without renal scarring. Pathogens from both groups induced a pronounced neutrophil respiratory burst activity. However, when the intracellular and extracellular oxidative responses were measured separately, the response induced by the scarring strains was extracellular (Mundi et al., 1991). An in vivo mouse study also revealed that ROS mediate renal injury in pyelonephritis by causing lipid peroxidation and subsequent DNA damage and that antioxidant treatment resulted in marked improvement in histology. Among the antioxidants they studied, superoxide dismutase (SOD) showed a maximum protective effect. The authors, therefore, proposed that super oxide anion (O 2 ) is the main species involved in renal injury (Gupta et al., 1996). Kurutas et al. (2005) measured malondialdehyde (MDA) as indicator of lipid peroxidation levels in humans and found that patients with positive urine cultures had higher urinary MDA levels. Studies have also shown that ROS play an important role in reflux nephropathy (Okur et al., 2003), UTI during pregnancy (Ciragil et al., 2005) and UTI in patients with diabetes mellitus (Gul et al., 2005). In our study, we used a commercial ELISA kit to detect 8-oxodG in urine. Although there are other methods with which to detect 8-oxodG, such as HPLC-MS/MS, those methods are time consuming and not practical to perform in most clinical settings. Possible sources of urinary 8-oxodG include diet, cell death and DNA repair in kidney during APN (Cooke et al., 2008). However, there is evidence that 8-oxodG is not affected by diet, and that cell death contributes very little to urinary 8-oxodG levels (Cooke et al., 2008). The main source of urinary 8-oxodG appears to be DNA repair. We also checked the antioxidative status in urine using a TAC assay and found that urine TAC level was higher in patients with UTI with positive DMSA findings than in patients with normal DMSA findings. Antioxidants are diverse substances that exist in different human extracellular fluids, including urine (Halliwell & Gutteridge, 1990). The methods used to measure antioxidants are also diverse. In a previous study of human UTI (Kurutas et al., 2005), urine catalase and SOD levels were lower in patients with UTI. However, due to the highly variable composition of urine, it is more accurate to evaluate overall antioxidant activity. It has been shown that fluorescence assays positively correlate with the total antioxidant potential in urine samples, suggesting that antioxidant potential increases in response to oxidative stress (Kirschbaum, 2001). A study using urinary TAC level as an antioxidant marker also showed that patients with UTI have higher urinary TAC levels (Ciftci et al., 2008). In our study, we found that although the TAC level was higher in APN patients, there was no significant difference between the three groups. Interestingly, however, there were marked differences in 8-oxodG level between patients with various degrees of APN. These findings imply that TAC has only a moderate protective effect against renal damage (Figure 1). Many studies have shown that ROS scavengers can prevent renal parenchymal injury in patients with APN. Ganguly and coworkers showed that SOD and catalase can decrease the
Biomarkers, 2014; 19(4): 326–331
extent of lipid peroxidation in mice with experimental pyelonephritis, even in the presence of infection (Kaur et al., 1988). In an ascending non-obstructive mouse model with chronic pyelonephritis, Gupta et al. found that both catalase and dimethyl sulfoxide can decrease lipid peroxidation and prevent pathological changes. They concluded that renal tissue damage is the direct consequence of free radical generation during the infective process and that inhibition of these free radicals can neutralize the tissue damage to a great extent (Gupta et al., 2004). In a study by Sobouti et al. (2012), vitamin E or A was administered to 35 of 61 pediatric patients with DMSA evidence of APN. Follow-up DMSA performed six months later revealed a marked reduction in renal lesions in patients who received vitamin treatment. Besides ROS scavengers, early treatment with prednisolone has also been shown to prevent renal scar formation in rats (Haraoka et al., 1994) and in pediatric patients with APN. There are several limitations of this study. The first limitation is that the case number in each group was small. The second limitation is that we used ELISA to measure urinary 8-oxodG levels, which has been demonstrated to have a low specificity (Barregard et al., 2013). The third limitation is that the results of DMSA during or shortly after UTI indicate APN, not permanent renal damage. Repeat DMSA after six months is necessary.
Conclusion Young children with UTI involving the renal parenchyma who had positive DMSA findings had higher urinary 8-oxodG and TAC levels, higher white blood cell counts and higher C-reactive protein levels than patients with normal DMSA. In patients with positive DMSA findings, patients with more severe renal involvement had higher urinary 8-oxodG levels. Thus, urinary 8-oxodG may serve as a biomarker of renal parenchymal involvement in pediatric UTI patients, and high level of urinary 8-oxodG may be a risk factor of severe renal damage.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by grant 99-CCH-IRP-40 from the Changhua Christian Hospital, Changhua, Taiwan.
References Barregard L, Møller P, Henriksen T, et al. (2013). Human and methodological sources of variability in the measurement of urinary 8-oxo-7,8-dihydro-20 -deoxyguanosine. Antioxid Redox Signal 18: 2377–91. Benador D, Benador N, Slosman DO, et al. (1994). Cortical scintigraphy in the evaluation of renal parenchymal changes in children with pyelonephritis. J Pediatr 124:17–20. Chiou YY, Wang ST, Tang MJ, et al. (2001). Renal fibrosis: prediction from acute pyelonephritis focus volume measured at 99mTc dimercaptosuccinic acid SPECT. Radiology 221:366–70. Ciftci H, Verit A, Yeni E, Savas M. (2008). Decreased oxidative stress index of urine in patients with urinary tract infection. Urol Int 81: 312–15. Ciragil P, Kurutas EB, Gul M, et al. (2005). The effects of oxidative stress in urinary tract infection during pregnancy. Mediators Inflamm 2005:309–11.
Downloaded by [University of Pennsylvania] at 01:34 06 November 2015
DOI: 10.3109/1354750X.2014.910552
Cooke MS, Olinski R, Loft S. (2008). Measurement and meaning of oxidatively modified DNA lesions in urine. Cancer Epidemiol Biomarkers Prev 17:3–14. Faust WC, Diaz M, Pohl HG. (2009). Incidence of post-pyelonephritic renal scarring: a meta-analysis of the dimercapto-succinic acid literature. J Urol 181:290–7; discussion 97–8. Gul M, Kurutas E, Ciragil P, et al. (2005). Urinary tract infection aggravates oxidative stress in diabetic patients. Tohoku J Exp Med 206:1–6. Gupta A, Sharma S, Nain CK, et al. (1996). Reactive oxygen speciesmediated tissue injury in experimental ascending pyelonephritis. Kidney Int 49:26–33. Gupta R, Gupta S, Ganguly NK. (1997). Role of type-1 fimbriae in the pathogenesis of chronic pyelonephritis in relation to reactive oxygen species. J Med Microbiol 46:403–6. Gupta R, Verma I, Sharma S, Ganguly NK. (2004). Prevention of tissue injury in an ascending mouse model of chronic pyelonephritis – role of free radical scavengers. Comp Immunol Microbiol Infect Dis 27: 225–34. Halliwell B, Gutteridge JM. (1990). The antioxidants of human extracellular fluids. Arch Biochem Biophys 280:1–8. Haraoka M, Matsumoto T, Takahashi K, et al. (1994). Suppression of renal scarring by prednisolone combined with ciprofloxacin in ascending pyelonephritis in rats. J Urol 151:1078–80. Jakobsson B, Nolstedt L, Svensson L, et al. (1992a). 99mTechnetiumdimercaptosuccinic acid scan in the diagnosis of acute pyelonephritis in children: relation to clinical and radiological findings. Pediatr Nephrol 6:328–34. Jakobsson B, Soderlundh S, Berg U. (1992b). Diagnostic significance of 99mTc-dimercaptosuccinic acid (DMSA) scintigraphy in urinary tract infection. Arch Dis Child 67:1338–42. Kaur A, Garg UC, Sethi AK, et al. (1988). Effect of various oxygen free radical scavengers in preventing tissue injury caused by Escherichia coli in pyelonephritic mice. Biochem In 16:1083–93. Kirschbaum B. (2001). Total urine antioxidant capacity. Clin Chim Acta 305:167–73. Kurutas EB, Ciragil P, Gul M, Kilinc M. (2005). The effects of oxidative stress in urinary tract infection. Mediators Inflamm 2005:242–4.
8-OxodG level
331
Lin KY, Chiu NT, Chen MJ, et al. (2003). Acute pyelonephritis and sequelae of renal scar in pediatric first febrile urinary tract infection. Pediatr Nephrol 18:362–5. Majd M, Rushton HG. (1992). Renal cortical scintigraphy in the diagnosis of acute pyelonephritis. Semin Nucl Med 22: 98–111. Mundi H, Bjorksten B, Svanborg C, et al. (1991). Extracellular release of reactive oxygen species from human neutrophils upon interaction with Escherichia coli strains causing renal scarring. Infect Immun 59: 4168–72. Okur H, Kose O, Kula M, et al. (2003). The role of infection and free oxygen radical damage in reflux nephropathy: an experimental study. J Urol 169:1874–7. Peters C, Rushton HG. (2010). Vesicoureteral reflux associated renal damage: congenital reflux nephropathy and acquired renal scarring. J Urol 184:265–73. Rama G, Chhina DK, Chhina RS, Sharma S. (2005). Urinary tract infections-microbial virulence determinants and reactive oxygen species. Comp Immunol Microbiol Infect Dis 28:339–49. Roberts JA, Roth Jr JK, Domingue G, et al. (1982). Immunology of pyelonephritis in the primate model. V. Effect of superoxide dismutase. J Urol 128:1394–400. Rushton HG, Majd M, Jantausch B, et al. (1992). Renal scarring following reflux and nonreflux pyelonephritis in children: evaluation with 99mtechnetium-dimercaptosuccinic acid scintigraphy. J Urol 147:1327–32. Sobouti B, Hooman N, Movahed M. (2012). The effect of vitamin E or vitamin A on the prevention of renal scarring in children with acute pyelonephritis. Pediatr Nephrol 28:277–83. Stokland E, Hellstrom M, Jakobsson B, Sixt R. (1999). Imaging of renal scarring. Acta Paediatr Suppl 88:13–21. Valavanidis A, Vlachogianni T, Fiotakis C. (2009). 8-hydroxy-20 deoxyguanosine (8-OHdG): a critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 27:120–39. Wu LL, Chiou CC, Chang PY, Wu JT. (2004). Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta 339: 1–9.
ISSN: 1354-750X (Print) 1366-5804 (Online) Journal homepage: http://www.tandfonline.com/loi/ibmk20
Urinary 8-hydroxy-2′-deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection Jien-Wen Chien, Lien-Yen Wang, Yu-Shan Cheng, Yi-Giien Tsai & Chin-San Liu To cite this article: Jien-Wen Chien, Lien-Yen Wang, Yu-Shan Cheng, Yi-Giien Tsai & Chin-San Liu (2014) Urinary 8-hydroxy-2′-deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection, Biomarkers, 19:4, 326-331 To link to this article: http://dx.doi.org/10.3109/1354750X.2014.910552
Published online: 21 Apr 2014.
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http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(4): 326–331 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.910552
RESEARCH ARTICLE
Urinary 8-hydroxy-20 -deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection Jien-Wen Chien1,2, Lien-Yen Wang3, Yu-Shan Cheng2, Yi-Giien Tsai1, and Chin-San Liu2,4,5# Departments of Pediatrics, 2Department of Vascular and Genomics Center, 3Department of Nuclear Medicine, 4Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan, and 5Graduate Institute of Integrative Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan
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Abstract
Keywords
Background: There are no good biomarkers to predict renal parenchymal involvement in children with urinary tract infection (UTI). Methods: Children (N ¼ 73) younger than 5 years with UTI were enrolled. Urinary levels of 8-hydroxy-20 -deoxyguanosine (8-oxodG) and total antioxidant capacity (TAC) were checked as markers of oxidative stress and antioxidant capacity, respectively. Tc99m-dimercaptosuccinic acid (DMSA) renal scintigraphy was used to find evidence of renal involvement. Results: Patients with positive DMSA findings had higher levels of urinary 8-oxodG (p ¼ 0.003) and higher urinary TAC (p ¼ 0.001) than patients with normal DMSA findings. Conclusions: High level of urinary 8-oxodG may be a risk factor of severe renal damage.
8-hydroxy-20 -deoxyguanosine, Tc99m-dimercaptosuccinic acid, total antioxidant capacity, urinary tract infection
Introduction Upper urinary tract infection (UTI) can result in renal damage in young children because of the growing nature of the kidneys. Isotopic uptake studies such as Tc99 m-dimercaptosuccinic acid (DMSA) scintigraphy have shown that the renal parenchyma is affected in about 55–75% of children with febrile UTI, and that about 20–40% of these children will develop permanent renal damage, e.g. renal scarring (Benador et al., 1994; Lin et al., 2003; Rushton et al., 1992). Reported risk factors for renal damage after UTI include young age, delayed initiation of antibiotic treatment, repeat upper UTI and renal structural anomalies such as high-grade vesicoureteral reflux (Peters & Rushton, 2010). The mechanisms governing renal damage after upper UTI involve host–pathogen interactions, such as extracellular release of reactive oxygen species (ROS) and oxidative stress. In patients with febrile UTI, oxidative damage occurs to lipids of cellular membranes, proteins and DNA. Guanine is the DNA base most prone to oxidation, both in the nucleus and in mitochondria. Upon oxidation, a hydroxyl group is added to the C-8 position of the guanine molecule, resulting
#Chin-San Liu is responsible for statistical design/analysis. E-mail: [email protected] Address for correspondence: Chin-San Liu, MD, PhD, Department of Neurology, Vascular and Genomic Research Center, Changhua Christian Hospital, 135 Nanhsiao Street, Changhua 500, Taiwan. Tel: 886-4-7238595 ext. 4752. Fax: 886-4-7238595 ext. 4063. E-mail: [email protected]
History Received 5 February 2014 Revised 26 March 2014 Accepted 28 March 2014 Published online 21 April 2014
in 8-hydroxy-20 -deoxyguanosine (8-oxodG), one of the most common forms of free radical-induced lesions of DNA. Oxidized nuclear DNA in general undergoes repair. The repair products of oxidative DNA lesions are fairly watersoluble and are excreted into urine without being further metabolized. Oxidatively modified DNA in the form of 8-oxodG can, therefore, be quantified to indicate the extent of free radical-induced oxidative DNA damage (Valavanidis et al., 2009). In recent years, 8-oxodG has been used widely in many studies as a biomarker for the measurement of endogenous oxidative DNA damage. In addition, it has also been shown to be a risk factor for many diseases, such as cancer, atherosclerosis and diabetes (Wu et al., 2004). DMSA renal scintigraphy is a very sensitive method for identifying acute changes in the kidney associated with pyelonephritis. DMSA scans are considered normal if homogenous uptake of the radioisotope is evident throughout the kidneys and the renal contour is preserved. In acute pyelonephritis (APN), the DMSA scan will show focal or diffuse areas of decreased uptake of dimercaptosuccinic acid. The scan will also allow visualization of the collecting system, allowing for estimation of the percentage of renal function of each kidney. Studies in animals (Gupta et al., 2004; Kaur et al., 1988) and in humans (Sobouti et al., 2012) have shown that ROS play an important role in renal damage and that free oxygen scavengers can prevent said damage after UTI. To the best of our knowledge, there are no reports regarding the association between oxidative stress during UTI and renal image studies. In this study, we explore the association between urinary
8-OxodG level
DOI: 10.3109/1354750X.2014.910552
8-oxodG, a marker of oxidative stress, and DMSA renal scans in patients with upper UTI.
Materials and methods
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Patients Children younger than 5 years who were treated for their first episode of febrile UTI at our hospital were enrolled in the study. Blood samples were taken from each patient for complete blood count and measurement of C-reactive protein. Urine samples were also taken for urinalysis and culture. The definition of febrile UTI included a core temperature 38 C; urine culture with growth of microorganisms 105 colonyforming units per mL from clean voided mid-stream urine in older children or 103 colony-forming units per mL after urinary catheterization, and 5 leukocyte cells per highpower field. Clinical data such as amplitude and duration of fever, age and sex were collected. All patients received renal echo and DMSA scanning during hospitalization. Patients were stratified into four groups according to their DMSA results: group 1, normal; group 2, one focal lesion; group 3, two or more focal lesions; and group 4, diffuse lesions with a renal functional difference 410%. Results of the DMSA studies were evaluated by an experienced nuclear medicine physician, who was blinded to the patients’ clinical information. All of the human experimental procedures followed the ethical standards of Changhua Christian Hospital and were approved by the hospital’s institutional review committee (Approval number is 100102). Informed consent for study enrollment was obtained from the parent of the study subjects. Urinary 8-oxodG Urine samples were collected for 8-oxodG measurement. For determination of the 8-oxodG level in urine samples, urine specimens were centrifuged at 10 000 g at 4 C for 10 min, and the supernatant was stored at 70 C before use. The amount of 8-oxodG in urine was measured using an 8-oxodG ELISA kit (Japan Institute for the Control of Ageing, Fukuroi, Japan) according to the manufacturer’s instructions. The specificity of the assay has been established, and the detection range is 0.5–200 ng/mL. Urinary 8-oxodG was adjusted to the level of creatinine in urine. Total antioxidant capacity measurement The total antioxidant capacity (TAC) assay is a cupric ion reducing antioxidant capacity spectrophotometric method. The amount of TAC in urine was measured using a TAC assay kit (Cell Biolabs, Inc., San Diego, CA) according to the manufacturer’s instructions. Briefly, a 20-mL aliquot of urine or uric acid standard and 180 mL of 1 reaction buffer was added to a microtiter plate and mixed. Initial absorbance was obtained by reading the plate at 490 nm. Then, 50 mL of 1 copper ion reagent was added to each well to initiate the reaction. The reaction mixture was then incubated for 5 min on an orbital shaker. Finally, 50 mL of 1 stop solution was added to each well to terminate the reaction. The plate was read again at 490 nm. The stress score is defined as 8-oxodG/ TAC ratio.
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Statistical methods Categorical variables were examined non-parametrically using the Mann–Whitney U test. The Kruskal–Wallis test was used to compare differences in scan results among the four DMSA groups. A generalized linear model test was applied to Figure 1(a–c), and the Spearman’s rank correlation coefficient was used. A receiver operating characteristic (ROC) curve was used to determine the optimal cut-off point of 8-oxodG that predicts renal damage. A p value50.05 was considered to indicate statistical significance. All statistical analyses were performed on a personal computer with the statistical package SPSS, version 15 (SPSS Inc., Chicago, IL).
Results A total of 73 children (43 boys (59%) and 30 girls (41%)) with a mean age of one year (0.95 ± 1.63) were included in the study. Children were classified into four groups based on the DMSA scan results: group 1, normal (N ¼ 26); group 2, one focal lesion (N ¼ 23); group 3, two or more focal lesions with renal functional difference 510% (N ¼ 11); and group 4, diffuse or multiple lesions and renal functional difference 410% (N ¼ 13). The mean duration of fever was 3.7 ± 1.8 d, the mean maximal body temperature was 39.4 ± 0.7 C, the mean white blood cell count was 16.0 ± 6.2 109/L, and the mean C-reactive protein level was 6.38 ± 5.71 mg/dL. The mean urinary 8-oxodG/creatinine level was 12.62 ± 11.28 ng/mg, and the mean urinary TAC/creatinine level was 2.05 ± 1.49 mg/ml. We examined the differences in clinical parameters (age, sex, duration of fever and highest body temperature) and laboratory findings (8-oxodG/urine creatinine, TAC/urine creatinine, white blood cell count, C reactive protein, pyuria, hematuria, bacteriuria and nitrite) between patients with normal DMSA renal scans and those with positive DMSA renal scans (Table 1), and then examined the differences between the four groups of patients (Table 2). We found that patients with positive DMSA findings had higher urinary 8-oxodG/urine creatinine level (p ¼ 0.003), higher urinary TAC/urine creatinine level (p ¼ 0.001), higher white blood cell count (p ¼ 0.006) and higher C-reactive protein (p ¼ 0.014) than patients with normal DMSA findings (Table 1). Parameters that differed significantly between the four groups included urinary 8-oxodG/urine creatinine level (p ¼ 0.02), urinary TAC/urine creatinine level (p ¼ 0.006), white blood cell count (p ¼ 0.044) and the presence of nitrite in urinalysis (p ¼ 0.013). The relationship between 8-oxodG and results of DMSA testing is shown in Figure 1(a). We found that the level of urinary 8-oxodG tended to be higher in patients with more severe DMSA findings. The difference was significant between group 1 (normal DMSA finding) and group 4 (diffuse DMSA finding with renal function difference 410%) (p ¼ 0.003). Figure 1(b) shows the relationship between urinary TAC/ urine creatinine level and the results of DMSA testing. Urinary TAC was significantly higher in group 2 than in group 1 (p ¼ 0.035). In addition, urinary TAC tended to be higher in groups 3 and 4 than in group 1. Figure 1(c) shows that patients in groups 2 and 3 had higher white blood cell
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Biomarkers, 2014; 19(4): 326–331
Figure 1. (a) This figure showed the relationship between 8-oxodG and results of DMSA. Urinary 8-oxodG was significantly higher in group 4 than that in group 1 (p ¼ 0.003). (b) The relationship between urinary TAC/urine creatinine level and the results of DMSA. Urinary TAC was significantly higher in group 2 than that in group 1 (p ¼ 0.035). Besides, urinary TAC tended to be higher in groups 3 and 4 than in group 1. (c) White cell count was significantly higher in group 3 and 4 than that in group 1 (p ¼ 0.014 and 0.011, respectively). Value in the bar indicated the mean ± standard deviation. *p 0.05, compared to negative group.
counts than patients in group 1 (p ¼ 0.014 and 0.011, respectively). Figure 2 shows the ROC curve that was generated to determine the cutoff values of urinary 8-oxodG, white blood cell count and C-reactive protein that can predict acute renal damage. We found that the best predictor of a positive DMSA scan was urinary 8-oxodG. The area under the curve for predicting acute renal damage was 0.732 (p ¼ 0.002). When the cutoff point of urinary 8-oxodG/urine creatinine was set at 5.60 ng/mg, the sensitivity and specificity were 0.822 and 0.64, respectively.
Discussion Prompt diagnosis and treatment of UTI in young children is very important because these infections can result in
permanent renal damage when adequate treatment is not given quickly. However, in children with febrile UTI, it is difficult to differentiate between APN and acute cystitis without parenchymal involvement based on routine clinical and laboratory parameters alone. A meta-analysis found a higher incidence of renal scarring after APN in Asia (Faust et al., 2009). Previous studies have revealed that DMSA is a highly sensitive and specific noninvasive imaging modality for detection of renal inflammation (Jakobsson et al., 1992a,b). In addition, DMSA scans performed during the acute phase of infection have been shown to provide prognostic information that helps determine whether APN lesions are at risk of developing into scars (Chiou et al., 2001). In our study, we found that urinary 8-oxodG level was higher in patients with DMSA scans showing severe renal
8-OxodG level
DOI: 10.3109/1354750X.2014.910552
damage, indicating that this biomarker can be used to predict childhood UTI with renal parenchymal involvement. The pathogenesis of UTI involves both bacterial virulence factors and host factors. In pyelonephritic areas, blood flow is reduced due to intravascular granulocyte aggregation, which leads to arteriolar or capillary occlusion. In addition, transport function of proximal tubular cell membranes is disturbed in pyelonephritic areas due to the release of toxic enzymes and superoxide by intratubular neutrophils (Majd & Rushton, 1992; Roberts et al., 1982; Stokland et al., 1999). Inflammatory cells including macrophages and neutrophils are endowed with a battery of enzymes that generate ROS to
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Table 1. Differences between patients with normal DMSA and positive DMSA.
Age, year Gender Male (%, N) 8-oxodG/urine creatinine, ng/mg TAC/urine creatinine, mg/ml Stress score Duration of fever, d Maximal body temperature, C White blood cell count, 109/L C-reactive protein, mg/dL Pyuria Hematuria Bacteriuria (%, N) Nitrite (%, N)
DMSA Normal (N ¼ 26) Mean ± SD
DMSA Positive (N ¼ 47) Mean ± SD
0.86 ± 0.82
1 ± 1.18
65%, 17 7.96 ± 6.51 1.45 ± 1.04 7.02 ± 5.86 3.4 ± 1.9 39.3 ± 0.7 13.2 ± 5.3 4 ± 3.63 23.9 ± 21.3 13.7 ± 17.3 42%, 11 15%, 4
55%, 26 15.21 ± 12.55 2.38 ± 1.6 7.57 ± 7.08 3.9 ± 1.8 39.4 ± 0.7 17.5 ± 6.2 7.64 ± 6.23 43 ± 31.5 14.7 ± 19.3 51%, 24 34%, 16
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combat invading pathogens (Rama et al., 2005). However, excessive production of ROS leads to damage of phagocytes themselves and if these oxygen metabolites leak out they will damage the surrounding tissue. In other words, microorganisms may initiate disease, but progression of the disease may be the result of inflammatory reactions, characterized by excessive production of ROS (Gupta et al., 1996). ROS are formed by the incomplete reduction of molecular oxygen and include super oxide anion (O 2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH) and singlet oxygen (1O2). Experimental and clinical studies have shown that ROS plays a role in the pathogenesis of renal damage during UTI. Non piliated or mannose-resistant piliated strains of Escherichia coli, which lack receptors for polymorphonuclear
p 0.917 0.336a 0.003b 0.001b 0.69 0.212 0.402 0.006b 0.014b 0.09 0.936 0.421a 0.06a
DMSA, Tc99m-dimercaptosuccinic acid; stress score, 8-oxodG/TAC ratio; pyuria, leukocyte per high power field; and hematuria, blood cells per high-power field. Values are mean (standard deviation) with p value from Mann–Whitney U test of variance. a Chi-square test. b As a significant increase between two groups (p50.05).
Figure 2. ROC curve analysis for the diagnostic accuracy of urine 8-oxodG, WBC and CRP levels predicts result of DMSA image study. The best predictor is 8-oxodG with the area under the curve (AUC) for predicting acute renal damage was 0.732 (p ¼ 0.002).
Table 2. Differences between four groups of patients based on the DMSA scan results. DMSA-positive
DMSA
Age, year Gender Male (%, N) 8-oxodG/urine creatinine, ng/mg TAC/urine creatinine, mg/ml Stress score Duration of fever, d Maximal body temperature, C White blood cell count, 109/L C-reactive protein, mg/dL Pyuria Hematuria Bacteriuria (%, N) Nitrite (%, N)
Normal (N ¼ 26)
One focal lesion (N ¼ 23)
Two or more focal lesions (N ¼ 11)
Renal function difference 410% (N ¼ 13)
0.86 ± 0.82
1.04 ± 1.19
0.68 ± 0.26
1.21 ± 1.61
65%, 17 7.96 ± 6.51 1.45 ± 1.04 7.02 ± 5.86 3.4 ± 1.9 39.3 ± 0.7 13.2 ± 5.3 4 ± 3.63 23.9 ± 21.3 13.7 ± 17.3 42%, 11 15%, 4
57%, 13 13.1 ± 11.91 2.35 ± 2.07 6.96 ± 6.4 4 ± 1.8 39.5 ± 0.7 17.9 ± 7.2 6.75 ± 6.11 50.6 ± 32.2 16 ± 20.3 39%, 9 48%, 11
36%, 4 14.73 ± 9.61 2.41 ± 0.89 6.49 ± 4.03 3.9 ± 1.9 39.8 ± 0.6 17.9 ± 4.6 8.08 ± 5.95 45 ± 38.1 18.5 ± 23.7 64%, 7 36%, 4
69%, 9 19.51 ± 15.68 2.4 ± 1.14 9.68 ± 10.03 3.9 ± 2 39 ± 0.6 16.4 ± 5.9 8.84 ± 6.89 23 ± 20.6 8.5 ± 11.3 62%, 8 8%, 1
p 0.998 0.336a 0.02b 0.006b 0.915 0.588 0.053 0.044b 0.07 0.102 0.658 0.424a 0.013a,b
DMSA, Tc99 m-dimercaptosuccinic acid; stress score, 8-oxodG/TAC ratio; pyuria, leukocyte per high power field; hematuria, blood cells per high power field. p, Kruskal–Wallis test. a Chi-square test. b p 0.05.
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leukocytes, making them resistant to phagocytosis, are more liable to generate ROS extracellular and hence are more pathogenic (Gupta et al., 1997). An in vitro study collected uropathogenic E. coli strains from two groups of patients: one with renal scars and one without renal scarring. Pathogens from both groups induced a pronounced neutrophil respiratory burst activity. However, when the intracellular and extracellular oxidative responses were measured separately, the response induced by the scarring strains was extracellular (Mundi et al., 1991). An in vivo mouse study also revealed that ROS mediate renal injury in pyelonephritis by causing lipid peroxidation and subsequent DNA damage and that antioxidant treatment resulted in marked improvement in histology. Among the antioxidants they studied, superoxide dismutase (SOD) showed a maximum protective effect. The authors, therefore, proposed that super oxide anion (O 2 ) is the main species involved in renal injury (Gupta et al., 1996). Kurutas et al. (2005) measured malondialdehyde (MDA) as indicator of lipid peroxidation levels in humans and found that patients with positive urine cultures had higher urinary MDA levels. Studies have also shown that ROS play an important role in reflux nephropathy (Okur et al., 2003), UTI during pregnancy (Ciragil et al., 2005) and UTI in patients with diabetes mellitus (Gul et al., 2005). In our study, we used a commercial ELISA kit to detect 8-oxodG in urine. Although there are other methods with which to detect 8-oxodG, such as HPLC-MS/MS, those methods are time consuming and not practical to perform in most clinical settings. Possible sources of urinary 8-oxodG include diet, cell death and DNA repair in kidney during APN (Cooke et al., 2008). However, there is evidence that 8-oxodG is not affected by diet, and that cell death contributes very little to urinary 8-oxodG levels (Cooke et al., 2008). The main source of urinary 8-oxodG appears to be DNA repair. We also checked the antioxidative status in urine using a TAC assay and found that urine TAC level was higher in patients with UTI with positive DMSA findings than in patients with normal DMSA findings. Antioxidants are diverse substances that exist in different human extracellular fluids, including urine (Halliwell & Gutteridge, 1990). The methods used to measure antioxidants are also diverse. In a previous study of human UTI (Kurutas et al., 2005), urine catalase and SOD levels were lower in patients with UTI. However, due to the highly variable composition of urine, it is more accurate to evaluate overall antioxidant activity. It has been shown that fluorescence assays positively correlate with the total antioxidant potential in urine samples, suggesting that antioxidant potential increases in response to oxidative stress (Kirschbaum, 2001). A study using urinary TAC level as an antioxidant marker also showed that patients with UTI have higher urinary TAC levels (Ciftci et al., 2008). In our study, we found that although the TAC level was higher in APN patients, there was no significant difference between the three groups. Interestingly, however, there were marked differences in 8-oxodG level between patients with various degrees of APN. These findings imply that TAC has only a moderate protective effect against renal damage (Figure 1). Many studies have shown that ROS scavengers can prevent renal parenchymal injury in patients with APN. Ganguly and coworkers showed that SOD and catalase can decrease the
Biomarkers, 2014; 19(4): 326–331
extent of lipid peroxidation in mice with experimental pyelonephritis, even in the presence of infection (Kaur et al., 1988). In an ascending non-obstructive mouse model with chronic pyelonephritis, Gupta et al. found that both catalase and dimethyl sulfoxide can decrease lipid peroxidation and prevent pathological changes. They concluded that renal tissue damage is the direct consequence of free radical generation during the infective process and that inhibition of these free radicals can neutralize the tissue damage to a great extent (Gupta et al., 2004). In a study by Sobouti et al. (2012), vitamin E or A was administered to 35 of 61 pediatric patients with DMSA evidence of APN. Follow-up DMSA performed six months later revealed a marked reduction in renal lesions in patients who received vitamin treatment. Besides ROS scavengers, early treatment with prednisolone has also been shown to prevent renal scar formation in rats (Haraoka et al., 1994) and in pediatric patients with APN. There are several limitations of this study. The first limitation is that the case number in each group was small. The second limitation is that we used ELISA to measure urinary 8-oxodG levels, which has been demonstrated to have a low specificity (Barregard et al., 2013). The third limitation is that the results of DMSA during or shortly after UTI indicate APN, not permanent renal damage. Repeat DMSA after six months is necessary.
Conclusion Young children with UTI involving the renal parenchyma who had positive DMSA findings had higher urinary 8-oxodG and TAC levels, higher white blood cell counts and higher C-reactive protein levels than patients with normal DMSA. In patients with positive DMSA findings, patients with more severe renal involvement had higher urinary 8-oxodG levels. Thus, urinary 8-oxodG may serve as a biomarker of renal parenchymal involvement in pediatric UTI patients, and high level of urinary 8-oxodG may be a risk factor of severe renal damage.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by grant 99-CCH-IRP-40 from the Changhua Christian Hospital, Changhua, Taiwan.
References Barregard L, Møller P, Henriksen T, et al. (2013). Human and methodological sources of variability in the measurement of urinary 8-oxo-7,8-dihydro-20 -deoxyguanosine. Antioxid Redox Signal 18: 2377–91. Benador D, Benador N, Slosman DO, et al. (1994). Cortical scintigraphy in the evaluation of renal parenchymal changes in children with pyelonephritis. J Pediatr 124:17–20. Chiou YY, Wang ST, Tang MJ, et al. (2001). Renal fibrosis: prediction from acute pyelonephritis focus volume measured at 99mTc dimercaptosuccinic acid SPECT. Radiology 221:366–70. Ciftci H, Verit A, Yeni E, Savas M. (2008). Decreased oxidative stress index of urine in patients with urinary tract infection. Urol Int 81: 312–15. Ciragil P, Kurutas EB, Gul M, et al. (2005). The effects of oxidative stress in urinary tract infection during pregnancy. Mediators Inflamm 2005:309–11.
Downloaded by [University of Pennsylvania] at 01:34 06 November 2015
DOI: 10.3109/1354750X.2014.910552
Cooke MS, Olinski R, Loft S. (2008). Measurement and meaning of oxidatively modified DNA lesions in urine. Cancer Epidemiol Biomarkers Prev 17:3–14. Faust WC, Diaz M, Pohl HG. (2009). Incidence of post-pyelonephritic renal scarring: a meta-analysis of the dimercapto-succinic acid literature. J Urol 181:290–7; discussion 97–8. Gul M, Kurutas E, Ciragil P, et al. (2005). Urinary tract infection aggravates oxidative stress in diabetic patients. Tohoku J Exp Med 206:1–6. Gupta A, Sharma S, Nain CK, et al. (1996). Reactive oxygen speciesmediated tissue injury in experimental ascending pyelonephritis. Kidney Int 49:26–33. Gupta R, Gupta S, Ganguly NK. (1997). Role of type-1 fimbriae in the pathogenesis of chronic pyelonephritis in relation to reactive oxygen species. J Med Microbiol 46:403–6. Gupta R, Verma I, Sharma S, Ganguly NK. (2004). Prevention of tissue injury in an ascending mouse model of chronic pyelonephritis – role of free radical scavengers. Comp Immunol Microbiol Infect Dis 27: 225–34. Halliwell B, Gutteridge JM. (1990). The antioxidants of human extracellular fluids. Arch Biochem Biophys 280:1–8. Haraoka M, Matsumoto T, Takahashi K, et al. (1994). Suppression of renal scarring by prednisolone combined with ciprofloxacin in ascending pyelonephritis in rats. J Urol 151:1078–80. Jakobsson B, Nolstedt L, Svensson L, et al. (1992a). 99mTechnetiumdimercaptosuccinic acid scan in the diagnosis of acute pyelonephritis in children: relation to clinical and radiological findings. Pediatr Nephrol 6:328–34. Jakobsson B, Soderlundh S, Berg U. (1992b). Diagnostic significance of 99mTc-dimercaptosuccinic acid (DMSA) scintigraphy in urinary tract infection. Arch Dis Child 67:1338–42. Kaur A, Garg UC, Sethi AK, et al. (1988). Effect of various oxygen free radical scavengers in preventing tissue injury caused by Escherichia coli in pyelonephritic mice. Biochem In 16:1083–93. Kirschbaum B. (2001). Total urine antioxidant capacity. Clin Chim Acta 305:167–73. Kurutas EB, Ciragil P, Gul M, Kilinc M. (2005). The effects of oxidative stress in urinary tract infection. Mediators Inflamm 2005:242–4.
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Lin KY, Chiu NT, Chen MJ, et al. (2003). Acute pyelonephritis and sequelae of renal scar in pediatric first febrile urinary tract infection. Pediatr Nephrol 18:362–5. Majd M, Rushton HG. (1992). Renal cortical scintigraphy in the diagnosis of acute pyelonephritis. Semin Nucl Med 22: 98–111. Mundi H, Bjorksten B, Svanborg C, et al. (1991). Extracellular release of reactive oxygen species from human neutrophils upon interaction with Escherichia coli strains causing renal scarring. Infect Immun 59: 4168–72. Okur H, Kose O, Kula M, et al. (2003). The role of infection and free oxygen radical damage in reflux nephropathy: an experimental study. J Urol 169:1874–7. Peters C, Rushton HG. (2010). Vesicoureteral reflux associated renal damage: congenital reflux nephropathy and acquired renal scarring. J Urol 184:265–73. Rama G, Chhina DK, Chhina RS, Sharma S. (2005). Urinary tract infections-microbial virulence determinants and reactive oxygen species. Comp Immunol Microbiol Infect Dis 28:339–49. Roberts JA, Roth Jr JK, Domingue G, et al. (1982). Immunology of pyelonephritis in the primate model. V. Effect of superoxide dismutase. J Urol 128:1394–400. Rushton HG, Majd M, Jantausch B, et al. (1992). Renal scarring following reflux and nonreflux pyelonephritis in children: evaluation with 99mtechnetium-dimercaptosuccinic acid scintigraphy. J Urol 147:1327–32. Sobouti B, Hooman N, Movahed M. (2012). The effect of vitamin E or vitamin A on the prevention of renal scarring in children with acute pyelonephritis. Pediatr Nephrol 28:277–83. Stokland E, Hellstrom M, Jakobsson B, Sixt R. (1999). Imaging of renal scarring. Acta Paediatr Suppl 88:13–21. Valavanidis A, Vlachogianni T, Fiotakis C. (2009). 8-hydroxy-20 deoxyguanosine (8-OHdG): a critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 27:120–39. Wu LL, Chiou CC, Chang PY, Wu JT. (2004). Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta 339: 1–9.
