ORIGINAL ARTICLE

Is oxidative stress related to childhood urolithiasis?

Nilufer Göknar & Faruk Oktem & Engin Arı &Aysegul Doğan Demir & Emel Torun

Received: 1 October 2013 /Revised: 10 January 2014 /Accepted: 22 January 2014# IPNA 2014

AbstractBackground Urolithiasis is a common condition in pediatricpopulations in Turkey. The role of oxidative stress in renalstone formation in pediatric patients has not been reported todate. The aim of this study was to assess oxidative stress inchildhood urolithiasis.Methods Seventy-four children diagnosed with urolithiasisand 72 healthy control subjects were enrolled in the study.Kidney stone formers were evaluated by analysis of metabolicconditions related to urolithiasis, such as hypercalciuria,hyperoxaluria, hypocitraturia and hyperuricosuria. Urine totalantioxidant status (TAS), and total oxidant status (TOS) weremeasured, and oxidative stress index (OSI) was calculated asan indicator of the degree of oxidative stress.Results Among the stone formers, metabolic analyses re-vealed that 30 % had hypercalciuria, 45 % had hypocitraturia,6 % had hyperoxaluria and 40 % had hyperuricosuria. Elevat-ed levels of the renal tubular damage marker urinary N-acetyl-beta-D-glucosaminidase (NAG) was elevated in 25 % of thepatient group, but microalbuminuria was not detected. Totaloxidant status and total antioxidant status were significantlyhigher in stone formers than in the controls (p=0.023 and0.004, respectively). In addition, urinary NAG was signifi-cantly correlated with TOS (r=0.427, p=0.019).Conclusions The results of this study show that oxidativestress may play an important role in the pathogenesis ofpediatric stone formers.

Keywords Urolithiasis . Oxidative stress . Children .

Metabolic conditions

Introduction

Urolithiasis is a common condition that affects 11 % of thepopulation in Turkey with considerable morbidity and recur-rence [1]. Genetic, metabolic, anatomic, dietary and environ-mental factors are common contributing factors to urolithiasis.Pediatric urolithiasis has different characteristics compared tothat in adults. Unlike adults, metabolic risk factors, such ashypercalciuria, hyperoxaluria and cystinuria, are well de-scribed in 33–95 % of all urolithiasis cases in childhood [2].

Humans excrete millions of urinary crystals every day, yetfew people develop kidney stones. Kidney stones can onlyform in the presence of urine supersaturation, crystal forma-tion and renal tubular damage. Supersaturation, although nec-essary for crystal formation, is by itself not sufficient for stoneformation [3]. Within the urinary tract, crystal formation,particularly of calcium phosphate and calcium oxalate(CaOx), is widespread. Persistent mild hyperoxaluria andCaOx crystals are not only injurious to the renal epitheliumbut can result in its death and degradation [4, 5]. Hydroxylradicals (OH−) and their subsequent products are the mostharmful reactive oxygen species (ROS) and are the mainfactors responsible for the oxidative injury of biomolecules;however, they are neutralized by antioxidants. Exposure toexcessive Ox and CaOx crystals produces more ROS that canbe compensated for by endogenous antioxidants; consequent-ly, the formation of these crystals also results in renal injury.Cellular damage promotes crystal retention through the pro-motion of nucleation, aggregation and attachment of crystalsto the renal epithelium. In addition to all of these factors, aninflammatory response is mandatory for stone formation [3].

N. Göknar : F. OktemDepartment of Pediatric Nephrology, Bezmialem Vakif UniversityMedical Faculty, Istanbul, Turkey

E. Arı :A. D. Demir : E. Torun (*)Department of Pediatrics, Bezmialem Vakif University Hospital,Adnan Menderes Avenue P.K, 34093 Fatih, Istanbul, Turkeye-mail: dr.emeltorun@gmail.com

Pediatr NephrolDOI 10.1007/s00467-014-2773-z

Oxidant status can be assessed by determining the totaloxidant status (TOS). The antioxidant status of individualsdepends on multiple factors, such as systemic antioxidants,antioxidant enzymes and dietary composition. The effects ofthese factors, which are additive, are termed the total antiox-idant status (TAS). Oxidative stress occurs when there is animbalance between pro-oxidants and antioxidants in favor ofoxidants, which can lead to inflammation and injury [6, 7].

Published studies have addressed both oxidative stress inrenal stone patients and the use of antioxidants to treat adults,but to our knowledge no data have been published on pediatricstone formers who exhibit properties different from those ofadult ones. Oxidant and antioxidant parameters have beenmeasured individually in recent studies [8–12].Measurementsof single oxidant or antioxidant parameters are complex andexpensive and are not a practical means by which to accurate-ly determine the free radical burden among those in thepediatric age group. Neither TOS nor TAS has yet beeninvestigated in pediatric urolithiasis, and with Erel’s method,both can be measured easily and inexpensively. This methodcan also be used to determine the overall oxidative stress index(OSI), which may include those antioxidants or oxidants notyet recognized or not easily measured [6, 7].

The aim of our study was to define the total oxidative andantioxidative status of children with renal stone disease bymeasuring TAS, TOS and OSI. We were also interested in theresults of the metabolic analysis related to urolithiasis inchildren.

Materials and methods

The prospective study group comprised 74 children (48 girls,26 boys; mean age 5.5±4 years) who had been diagnosed withurolithiasis in our pediatric nephrology outpatient clinic. Uro-lithiasis diagnoses were determined by ultrasound (US), directurinary system radiography and computerized tomography.The patients were matched for age and gender with healthychildren (41 girls, 31 boys) who had no evidence of stones byUS and were assigned as the control group.

Each subject’s medical history included age, gender,weight, height, history of stone disease, family history,breastfed period and previous diagnosis of urinary tract infec-tion, as well as symptoms such as dysuria, enuresis, vomiting,flank pain and hematuria. For the metabolic investigation,each patient’s urine was analyzed for calcium, uric acid,oxalate, citrate, albumin and N-acetyl β-D-glucosaminidase(NAG), and blood was analyzed for calcium, phosphorus,potassium, uric acid, creatinine and sodium. Measurementsfor citrate and oxalate were made using standard enzymaticand photometric methods (Cobbas 6000 autoanalyzer, Roche,Milan, Italy and Trinity Biotech Oxalate®, Trinity Biotech,Wicklow, Ireland, respectively) in the hospital’s clinical

laboratory. Urinary NAG was analyzed using a photometricmethod (Cobbas 8000 autoanalyzer, Diazyme Labsoratories,Poway, CA).

Hypercalciuria, hyperoxaluria and hyperuricosuria weredefined as calcium, oxalate and uric acid excretion of>4 mg/kg/day, >40 mg/1.73 m2/day and >10.7 mg/kg/day,respectively. Hypocitraturia was defined as urinary citrateexcretion of <320 mg/1.73 m2/day. In order to rule out theinfluence of urinary dilution or concentration, a ratio of spotvalues to creatinine was used for children who could notsupply 24-h urine samples. Spot urine calcium/creatinine ra-tios of <0.21 mg/mg creatinine for children and <0.6 mg/mgfor infants were considered to be normal. A spot urine oxalate/creatinine ratio of <0.15 mg/mg creatinine for children youn-ger than 4 years and of <0.53 mg/dl glomerular filtration ratewere accepted as normal values. For hypocitraturia, a spoturine citrate/creatinine ratio of 0.51 g/g creatinine was used[13–15]. Urine albumin and NAGwere used as biomarkers ofrenal injury. Microalbuminuria was defined as any level>30 mg/g creatinine [16]; for urine NAG, the cutoff levelwas 5 U/g creatinine.

Second-morning urine samples obtained from both thepatient and control groups were centrifuged at 3,000 rpm for10 min and stored at −80 °C. Samples were assessed at thesame time. TOS and TAS were measured, and the OSI foreach sample was calculated. Values were divided by urinecreatinine to reduce variations due to dilution.

The study was approved by the local ethics committee.Written informed consent was obtained from parents.

Measurement of TOS

Total oxidant status was measured with an assay based on theoxidation of the ferrous ion-o-dianisidine complex to ferricion. The reaction was enhance with oxidation of the glycerolmolecule. The ferric ion produces a colored complex withxylenol orange, and the color can be measured spectrophoto-metrically. The assay was calibrated with hydrogen peroxide(H2O2), and the results are expressed as micromolar H2O2

equivalents per liter [17].

Measurement of TAS

Themethod used for measuring TAS is based on the bleachingof the characteristic color of a more stable ABTS [2,2′-azino-bis (3-ethylbezothiazoline-6-sulfonic acid)] radical cation byantioxidants. The results are expressed in millimoles Trolox(Rel Assay) equivalent per liter [7].

Measurement of OSI

The OSI was determined by the ratio of TOS to TAS. The TASvalues were converted to micromoles per liter, the OSI value

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was calculated according to the following formula:

OSI arbitrary unitð Þ ¼ TOS μmol H2O2eqv=lð Þ=TAS μmol Trolox eqv=Lð Þ � 100

Statistical analysis

Statistical analysis was performed by the IBM SPSS Statisticsver. 17.0 software program. The results are expressed as themean ± standard deviation. Comparison analyses were per-formed with Student’s t test and the Mann–Whitney U test.Linear regression analysis and Pearson’s correlation coeffi-cient were used to compare the variables. Categorical datawere evaluated using the chi-square test. Only values ofp<0.05 were regarded as significant.

Results

The clinical and demographic data in Table 1 show that therewere no significant differences in gender or age between thepatient and control groups (p>0.05). The breastfeeding periodof the control group was about 2.5 months longer than that ofthe patient group, but this difference was not statisticallysignificant (p=0.123). There was no significant differencebetween time to start formula or complementary feeding(p>0.05), and there was no significant correlation betweenbreastfeeding period and urinary TAS (r=−0.162, p=0.103)and TOS (r=−0.186, p=0.06). As expected, urinary tract

infections were more prevalent in the patient group(p<0.001).

The results of the metabolic analyses of the patient grouprevealed that 30% of the patientshad hypercalciuria, 45% hadhypocitraturia, 6 % had hyperoxaluria in 6 % and 40 % hadhyperuricosuria; in addition, a significant number of the pa-tients had two or more lithogenic factors (Table 2). UrinaryNAG was elevated in 25 % of the patients, butmicroalbuminuria was not detected. The main clinical presen-tations were flank and abdominal pain (44 %), hematuria(40 %), restlessness (35 %), vomiting (27 %) and dysuria(33 %); 16 % were asymptomatic and were detected inciden-tally. Among the stone formers, a family history of urolithiasiswas identified in 44 % of the first degree relatives.

Thirty-four percent of the kidney stones were detected inthe right kidney, 42 % were in the left kidney, 20 % were fromboth kidneys, 1 % were in the bladder and 1 % were in theright ureter. Five patients had hydronephrosis, and one hadhydroureteronephrosis. Calculi ranged from 2 to 14.3 mm indiameter (mean 4.3±2.7).

As seen in Table 3, TOS and TAS were both significantlyhigher in the patient group than among the controls (p=0.023and 0.004, respectively). The OSI was also higher in thepatient group, but the difference was not statistically signifi-cant (p=0.368). There were no significant correlations be-tween hypercalciuria and TOS (p=0.961), TAS (p=0.400)and OSI (p=0.779); hyperoxaluria and TOS (p=0.333), TAS(p=0.511) and OSI (p=0.963); hyperuricosuria with TOS (p=0.828), TAS (p=0.959) and OSI (p=0.572); hypocitraturiaand TOS (p=0.162), TAS (p=0.654) and OSI (p=0.067) inPearson’s correlation test. Also, TOS, TAS and OSI were not

Table 1 Clinical anddemographic characteristics of thepatients and controls

Data are presented as the mean ±standard deviation unlessindicated otherwise

Clinical and demographic characteristics Patients (n = 74) Controls (n = 72) p

Age (months) 66.7±43 76.5±5 0.217

Male/female (n) 26/48 31/41 0.330

Urinary tract infection (%) 50.8 21.1 <0.001

Breastfed period (months) 12.5±10.1 15.0±8.9 0.132

Time to start complementary feeding (months) 6.2±2.4 6.5±1.6 0.123

Time to start formula feeding (months) 4.5±3.7 4.3±2.8 0.862

Table 2 Metabolic evaluation ofthe patient group

NAG, Urinary N-acetyl- beta-D-glucosaminidase

Values are presented as themean ± SD or as the percentage ofpatients, where appropriate

Metabolic parameter Value Metabolic parameter Value

Blood sodium (mmol/l) 138.6±2.2 Urine NAG/creatinine (U/g) 4.7±4.4

Blood calcium (mg/dl) 9.8±0.4 24-h urine microalbumin (mg/day) 9.4±7.5

Blood phosphorus (mg/dl) 4.9±0.6 Hypercalciuria 30 %

Blood potassium (mmol/l) 4.4±0.4 Hypocitraturia 45 %

Blood uric acid (mg/dl) 3.6±2.5 Hyperuricosuria 40 %

Blood creatinine (mg/dl) 0.3±0.1 Hyperoxaluria 6 %

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significantly different in patients with or without metabolicrisk factors in the study group (p>0.05). As shown in Fig. 1,regression analysis disclosed that urinary NAGwas correlatedsignificantly with TOS (r=0.427, p=0.019).

Discussion

In children, oxidative stress is involved in the pathogenesis ofseveral disorders, such as bronchiolitis, asthma, congenitalheart disease and hypertension [18–21]. In order to evaluatethe relation of urolithiasis with oxidative stress in our patientgroup we checked their TAS, TOS and OSI levels. Our resultsshow that TAS and TOS were elevated in stone formerscompared to those who were stone-free. To the best of ourknowledge this is the first published report on oxidative stressand urolithiasis in a pediatric population.

ROS are chemically reactive molecules and free radicalsgenerated from molecular oxygen which have one or moreunpaired electrons. Major molecular ROS include superoxideanions (02

−), nitric oxide radicals (NO), hydroxyl radicals(OH−) and H202. Under normal conditions, ROS are generatedby tightly controlled enzymes, such as NADPH oxidase,

xanthine oxidase, lipooxygenase, cyclooxygenase and hemeoxygenase, and are found in the electron transport chain ofmitochondria during cellular respiration. ROS are short-livedand are cleared by antioxidants and scavengers. Uncontrolledgeneration of ROS and/or a decrease in antioxidant capacitycreates oxidative stress, which may lead to inflammation andinjury [22, 23].

ROS-induced renal cellular injury and inflammation arelikely to be involved in the pathogenesis of idiopathic stonedisease. Both tissue culture and animal studies have demon-strated that interaction between CaOx/calcium phosphate(CaP) crystals and renal epithelial cells provokes ROS gener-ation [8, 24]. Oxidative stress or an abundance of ROS causespermanent damage to macromolecules. In one study, renalenzymes indicative of renal tubular damage (e.g. γ-glutamyltranspeptidase, angiotensin 1 converting enzyme, β-galactosidase and NAG) were found to be excreted in theurine at high levels in idiopathic CaOx stone formers [25].In another study, stone formers had a significantly higher levelof malondialdehyde (a product of lipid peroxidation) in theurine, in addition to renal enzymes, indicating that renal injurywas most likely caused by ROS production. Urinary 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA

Table 3 Total antioxidant status, total oxidant status and oxidative stress index of patients and controls

Parameters Patients (n=54) Controls (n=53) p

TOS/creatinine (μmol H2O2 equivalent/mg) 46.91±103.12 13.77±12.66 0.023

TAS/creatinine (μmol Trolox equivalent/mg) 0.084±0.098 0.0413±0.044 0.004

OSI (arbitrary unit) 451.35±351.98 403.18±167.76 0.368

TAS, Total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index

Fig. 1 Urinary N-acetyl- beta-D-glucosaminidase (NAG)concentrations in stone formersaccording to the total oxidantstatus (TOS)/creatinine ratio(p=0.016)

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oxidative damage, has also been found to be higher in stone-forming patients; in the same study, urinary NAG excretioncorrelated positively with 8-OHdG concentration [26]. Vari-ous oxidative stress markers have been studied in patients withurolithiasis, and a general finding is that patients showedincreased oxidative stress. Although no prior study has mea-sured the TOS levels in urolithiasis patients, we found signif-icantly higher TOS levels among our patients. High TOS maybe accepted as a general indicator of the oxidant molecules inpatients with urolithiasis.

In the NHANES III study, antioxidant levels of kidneystone formers were examined and mean levels of antioxidants,α-carotene, β-carotene and β-cryptoxanthin were found to besignificantly low [27]. The widespread use of antioxidants andinhibitors of ROS for stone prophylaxis and treatment hasbeen reported in the literature, and antioxidants such as N-acetylcysteine, vitamin E, methionine and gluthathione havebeen evaluated for this purpose [9, 28, 29]. Antioxidants havebeen shown to reduce oxidative stress; however, chemicalsthat inhibit the generation of ROS provide considerable ben-efits over general antioxidants because antioxidants appear tobe less efficient. Interest in ROS inhibitors has increased,especially NADPH oxidase inhibitors such as apocynin,dihphenyleneiodonium chloride and Pefabloc [22]. Studiesin adults have revealed that antioxidant therapy would re-duced the oxidative stress in urolithiasis. However, moreevidence supported by longitudinal, prospective studies onthe recommended use of prophylactic antioxidants in child-hood urolithiasis is needed.

In this study we showed that TOS was increased in pediat-ric stone formers; however, TAS was not decreased relative tohealthy controls. In general, when oxidative stress increases,the body’s natural response is to increase the production ofantioxidants. If the condition is long-lasting, antioxidant levelswill increase; therefore, high values may indicate persistentoxidative stress. Patients with asthma and chronic tonsillitishave been found to have elevated levels of both TAS and TOS[19, 30, 31], as was the case in our study. The authors of thesestudies suggested that elevated TAS was the body’s responseto the oxidant system [19, 30, 31]. We suggest that since stoneformation is a chronic and long-lasting condition, the endresult would be increased TAS. As both TAS and TOS wereelevated in our patients, OSI (TAS/TOS) was similar in bothstone formers and the control group.

In this study we were also interested in the metabolic andanatomic evaluation of pediatric stone formers. The majorityof stones in children are composed of calcium oxalate (45–65 %) and calcium phosphate (14–30 %), while uric acid,struvite and cystine stones account for 5–10 % of pediatricnephrolithiasis. In recent decades more metabolic causes ofstone formation have been identified compared with infec-tious causes [13]. Hypercalciuria is the most commonly en-countered metabolic disorder in children with urolithiasis,

accounting for 34–97 % of all cases. Hyperoxaluria andhyperuricosuria have been reported in 2–36 % and 2.7–53.4 % of cases, respectively. Citrate is known to act as aninhibitor of CaOx and calcium phosphate crystallization [2].We found that 30 % of the patients had hypercalciuria, 45 %had hypocitraturia, 6 % had hyperoxaluria and 40 % hadhyperuricosuria. In our study, the oxidative status in patientswith or without metabolic risk factors did not differ among ourpatients with urolithiasis, possibly suggesting that the oxida-tive stress caused by urolithiasis was not enhanced by meta-bolic disorders among the stone formers in our study. Func-tional and anatomical obstructions of the urinary tract increasethe potential of stone formation by promoting urinary stasisand infection. In our study four patients had hydronephrosisand one had hydroureteronephrosis.

The eating behavior of children might affect urinary min-eral excretion [32]. Breast milk is low in phosphorus andcitrate contents, and infant formulas are high in both calciumand phosphorus. Both have a high oxalate content [33]. In-fants with microlithiasis have higher ratios of feeding withformula [34]. In our study, although the breastfeeding periodof the control group was longer by about 2.5 months than thatof the patients with urolithiasis, there was no significantcorrelation between length of breastfeeding period or time tothe start of formula feeding/complementary feeding and uri-nary TAS and TOS.

One of the limitations of the present study is its cross-sectional design, which permitted only an examination ofassociation but not causation. The lack of supersaturation dataas a result of the restricted laboratory facilities and the lack ofmetabolic data in the controls are also limitations of the study.

Conclusions

Our results show that TOS and TAS were elevated in thepediatric stone formers enrolled in our study. Since oxidativestress appears to be a critical event in children with urolithia-sis, this condition should also be taken into considerationwhen choosing the appropriate treatment and prophylaxis.

Acknowledgement S. Delacroix (English Editor)

Conflict of interest None.

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