JOURNAL OF ENDOUROLOGYVolume 22, Number 12, December 2008© Mary Ann Liebert, Inc.Pp. 2723–2731DOI: 10.1089/end.2008.0357

Protective Effect of a Potent Antioxidant, Pomegranate Juice, in the Kidney of Rats

with Nephrolithiasis Induced by Ethylene Glycol

Volkan Tugcu, M.D.,1 Eray Kemahli, M.D.,1 Emin Ozbek, M.D.,2 Yasar Volkan Arıncı,3 Mehmet Uhrı, M.D.,4

Pelin Erturkuner, M.D.,5 Gokhan Metın, M.D.,6 Ismail Seckın, M.D.,5 Cetin Karaca, M.D.,7

Nursen Ipekoglu, M.D.,3 Tuncay Altug, M.D.,7 Mustafa Baki Cekmen, M.D.,8 and Ali Ihsan Tascı, M.D.1

Abstract

Purpose: We aimed to study the protective effects of pomegranate juice (PJ) on ethylene glycol (EG)-inducedcrystal deposition in renal tubules, renal toxicity, and inducible nitric oxide synthase (iNOS) and nuclear fac-tor-�B activities in rat kidneys.Materials and Methods: Fifty-six rats were divided into four equal groups: Control, EG, EG � 50 �L PJ/d (PJ50),and EG � 100 �L PJ/d (PJ100). Rats were sacrified on days 10 and 45. Tissue sections were evaluated underlight and polarized microscopy for the presence and degree of crystal deposition and toxicity in the kidneys.Crude extracts of the cortex were used to determine reduced gluthatione (GSH), nitric oxide (NO), and mal-ondialdehyde (MDA) levels.Results: In the EG group, crystal depositions were more evident and mild crystalization was observed in prox-imal tubules on day 10; severe crystalization and granulovacuolar epithelial cell degeneration were observedon day 45. There was limited or no crystal formation in the EG � PJ-given groups. There were completely nor-mal renal and tubular structures in the control group. There was no significant difference between the fourgroups in serum levels of sodium, potassium, blood urea nitrogen, and creatinine in any sampling time. Hy-peroxaluria, a marked increase in MDA and NO levels, and decrease of GSH were observed in the EG-givengroups compared with the others. There were marked iNOS and p65 expressions in only the EG-given ratscompared with control and PJ groups, immunohistochemically.Conclusion: This experiment shows the protective effect of PJ in the EG-induced crystal depositions in renaltubules.

2723

Introduction

HYPEROXALURIA IS ONE of the major risk factors in humanidiopathic calcium oxalate (CaOx) stone disease, and its

experimental induction is necessary for the production ofCaOx urolithiasis in rats.1–3

Acute and chronic production of CaOx crystals induceslipid peroxidation; therefore, it has been suggested that thisprocess plays an important role in CaOx stone formation.4

Glycolate or glycoxylate is responsible for the produc-tion of oxalate (Ox), and rats given 7.5% ethylene glycol(EG) showed a significant increase in the activities of Ox-synthesizing enzymes, such as glycolic acid oxidase, inliver.5,6 By measuring malondialdehyde (MDA) and thio-thiobarbituric acid, Thamiselvan and associates7 found thatlipid peroxidation occurred in kidney tissue and urine sam-ples of male rats that were treated with 0.75% EG duringall periods.

1Department of Urology, Bakirkoy Research and Training Hospital, Istanbul, Turkey.2Department of Urology, Vakif Gureba Research and Training Hospital, Istanbul, Turkey.3Department of Chemistry Engineering, Istanbul Technical University, Istanbul, Turkey.4Department of Pathology, Bakirkoy Research and Training Hospital, Istanbul, Turkey.5Department of Histology, 6Department of Physiology, and 7Department of Animal Research Laboratory, Cerrahpasa Medical Faculty,

Istanbul University, Istanbul, Turkey.8Department of Biochemistry, Medical Faculty, Kocaeli University, Koceali, Turkey.

Lipid peroxidation usually refers to the functional im-pairment of cellular components by reactive oxygen species(ROS), such as superoxide radicals, hydroxyl free radicals,and hydrogen peroxide.7 ROS act as mediators of nuclearfactor kappa-B (NF-�B) activation by inhibitor kappa-B (I-�B) degradation.8

In our previous study, we showed the MDA and nitric ox-ide (NO) concentrations were significantly higher and re-duced glutathione (GSH) concentration was lower in the kid-ney of EG-given groups compared with EG � pyrolidiumdithiocarbamate (PDTC) (NF-�B inhibitor).4

Inducible nitric oxide synthase (iNOS) is one of the threeNOS isoforms that is affected and increases by NF-� throughincreased tissue damage. The process of iNOS expression in-volves different signal transduction pathways, including nu-clear translocation of the transcription factor NF-�B.9 TheiNOS-mediated NO production is significantly elevated inincreased oxidative stress, and excessive NO production be-cause of elevated expression of iNOS may impose cytotoxiceffects on various organs, including the renal medulla.9,10

Huang and colleagues11 found that endothelial NOS ex-pression decreased and iNOS expression significantly in-creased in the renal medulla of the EG-treated rats.

In our previous study, we showed that iNOS and p65 (NF-�B) expressions were significantly increased in EG-treatedrats, and we found that NF-�B inhibitor (PDTC) preventedCaOx precipitation in rats with kidney stones.4

Pomegranate juice (PJ) is a rich source of potent polyphe-nolic, flavonoid antioxidants (anthocyanins). Edible parts ofpomegranate fruit (about 50% of total fruit weight) comprise80% juice and 20% seeds. Fresh juice contains 85% water, 10%total sugars, and 1.5% pectin, ascorbic acid, and polypheno-lic flavoids. Pomegranate seeds are a rich source of crudefibers, pectin, and sugars. The soluble polyphenol content inPJ varies within the limits of 0.2% to 1.0% depending on thevariety, and includes mainly anthocyanins that have beenshown to possess antiatherogenic properties.12 It has alsobeen shown that PJ can suppress NF-�B activation througha novel mechanism in vascular endothelial cells.13

Anthocyanins were shown to be effective inhibitors oflipid peroxidation, production of NO, and iNOS activity indifferent model systems.14,15

Therefore, we administered the NF-�B inhibitor PJ to ratsforming stones to investigate its possible inhibitory effectson urolithiasis and its mechanism of action.

Materials and Methods

Animals

Fifty-six adult male Sprague-Dawley rats (230–250 g) wereacquired from the Experimental Animal Laboratory of Is-tanbul University, Cerrahpasa Medical Faculty vivariumsources and maintained in a 14-hour light/10-hour dark cy-cle with free access to food and water.

Experimental conditions

Rats were first divided into four equal major groups: Ingroup 1 (control group), the rats received distilled drinkingwater. In group 2 (EG group), they received a hyperoxaluria-inducing diet of 0.75% EG in distilled water. In group 3 (PJ50group), rats were fed via gavage 50 �L PJ/d and received a

diet of 0.75% EG in distilled water. In group 4 (PJ100), ratswere fed via gavage 100 �L PJ/d and received a diet of 0.75%EG in distilled water.

All the major groups were divided into two groups ac-cording to the experimental sampling periods: 10 and 45 days.The animals were placed in cages 7 days before beginning theexperiment, to acclimate them to the cages. During the ex-perimental period, all groups had free access to regular ratchow and water, so water consumption was not monitored.

At the end of the experimental period, rats were placed inthe metabolic cages to determine 24-hour urine output, uri-nary pH, total urinary protein, and Ox levels. At 24 h afterthe gavage, rats were killed under anesthesia (intraperitonealsodium pentobarbital, 50 mg/kg body weight). The kidneyswere quickly removed and decapsulated, and the renal cor-tex was carefully separated from the medullaand homoge-nized as described previously.16 Small samples were fixed informaldehyde solution for histopathologic and immunohis-tochemical examination.

Pomegranate processing

Fresh pomegranates were washed, crushed, thensqueezed, and treated enzymatically with pectinase to yieldPJ and by-products, which included the inner and outer peelsand seeds. Pectinase hydrolyses �-1,4-galacturonide bondsin pectin and thus improves extraction and filtration and pre-vents the formation of pectin gels. The juice was filtered, pas-teurized, concentrated, and stored at �18°C as described pre-viously.17,18 Concentrated PJ was diluted in water (10 mLand 20 mL of concentrated juice in 500 mL of distilled wa-ter). The average of 2.5 mL diluted PJ contains 50 �L and100 �L PJ, which are equivalent to 1.4 �mol and 2.8 �moltotal polyphenols per day.

Histopathologic examination

In histopathologic examination, the tissues are preparedfor routine examination by light microscopy (Nikon, Tokyo,Japan). The kidney sections were analyzed semiquantita-tively using the technique of Houghton and coworkers.19

Photomicroscopic assessment

Tissues were prefixed in 1% glutaraldehyde and 4%formaldehyde in phosphate-buffered saline for 2 hours. Thenthe tissues were postfixed in phosphate buffered 1% osmiumtetraoxide for 1 hour (pH � 7.2), dehydrated in a graded se-ries of ethanol solutions, and embedded in Araldite®. Ultra-thin sections were cut using a Reinheirt Om U3 ultramicro-tome with glass knives for 0.5 �m, then stained withtoloudine blue and examined using a photomicroscope(Nikon, Tokyo, Japan).

Tissue sections were scored on a four-point scale by twohistologists using a double-blind study, applying the scaleto both medulla and cortex. The scoring was: 0, no Ox crys-tals in any field; 1, no more than two crystals in any field; 2,more than two crystals in any field; and 3, all fields withmultiple collections of crystals.

Immunohistochemical evaluation

For immunohistochemical evaluation, specimens wereprocessed for light microscopy, and sections were incubated

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FIG. 1. (a) Intense granulovacuolar cell degeneration inbasal and apical portions of proximal tubules (EG day 10,PM � 800). EG � ethylene glycol; PM � photomicroscopy.(b) Varying sizes of crystals within proximal tubule cellsand lumen (EG day 45, PM � 1000). EG � ethylene glycol;PM � photomicroscopy. (c) Normal glomerulary and prox-imal tubules (Control day 45, PM � 600). PM � photomi-croscopy. (d) (e) Cell structure is observed and only poorcrystallizations are seen on proximal tubules (PJ50 day 45,PM � 750, PJ100 day 45, PM � 800). PJ � pomegranatejuice.

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PROTECTIVE EFFECTS OF POMEGRANATE JUICE IN NEPHROLITHIASIS 2727

at 60°C overnight and then dewaxed in xylene for 30 min-utes. After soaking in ethanol, sections were washed withdistilled water and phosphate-buffered saline (PBS) for 10minutes. Sections were then treated with 2% trypsin in 50mM Tris buffer (pH 7.5) at 37°C for 15 minutes washed withPBS. Sections were delineated with a Dako pen (Dako,Glostrup, Denmark) and incubated in a solution of 3% H2O2

for 15 minutes to inhibit endogenous peroxidase activity.Then, sections were incubated with NF-�B/P65 (Rel A) Ab-1 (Neomarkers R-B-1638-R7) and iNOS Ab-1 (NeomarkersR-B-1605-R7) antibodies histochemically. Ultravision HRP-AEC staining protocol was used in this stage.

Sections prepared for each case were examined by lightmicroscopy by two pathologists using a double-blind study.Sections of rat lung were used for control of immunohisto-chemical staining specificity according to data provided bythe antibody-producing company.

The cases were evaluated for diffuseness and staining. Ac-cording to staining diffuseness, sections were graded as fol-lows: 0 � no staining; 1 � staining less than 25%; 2 � stain-ing between 25% and 50%; 3 � staining between 50% and75%; 4 � staining more than 75%. According to staining in-tensity, sections were graded as follows : 0 � no staining;1 � weak but detectable above control; 2 � distinct; 3 � in-tense staining.

Immunohistochemical values were obtained by addingdiffuseness and intensity scores.

Biochemical determinations

All tissues were washed two times with cold saline solu-tion and immediately stored at �80°C for the measurementof MDA, GSH, and NO levels. Tissues were homogenized infour volumes of ice-cold buffer containing 20 mM Tris, 10mM ethylenediaminetetraacetic acid (pH 7.4)

Total nitrite (NOx) was quantified by the Griess reaction,after incubation of supernatant with Escherichia coli nitratereductase to convert NO3 to NO2.

Thiobarbituric acid (TBA) reacts with lipoperoxidationaldehydes, such as MDA, as the most common method toassess lipid peroxidation in biologic samples. The procedurewas modified from Buege and Aust.20

The GSH level was determined by the spectrophotomet-ric method of Ellman16 based on the development a yellowcolor when DTNB (5,5� dithiobis-2-nitrobenzoic acid) isadded to compounds containing sulfhydryl groups.

Blood samples were also taken to assess the serum levelsof urea, creatinine, blood urea nitrogen (BUN), sodium,potassium, and gamma-glutamyl transpeptidase. All bio-

chemical variables were determined using an Olympus Au-toanalyser (Olympus Instruments, Tokyo, Japan).

All animals were placed in metabolic cages 1 day beforesacrifice. Twenty-four–hour urine samples were collectedfrom all groups. Urine samples were centrifuged at 2000 xgravity for 10 minutes to remove debris. Supernatants wereused to determine the urinary pH and total urinary protein.Urinary Ox level was assayed using the Ox oxidase enzy-matic method with a commercial Ox assay kit (Sigma-Aldrich Chemical Corp, St Louis, MO) and expressed in mg.The urinary assays were performed in Guven Laboratories(Istanbul, Turkey).

Statistical Analysis

Statistical analyses of histopathologic and immunohisto-chemical evaluation of groups were compared by chi-squaretest, and biochemical values were compared by the Mann-Whitney U test. Probability values of less than 0.05 were con-sidered significant.

Results

Biochemical variables in urine, serum, and tissue

There was no significant difference between the controland experimental groups for body weight, daily urine out-put, and total urinary protein. Urinary pH levels were sig-nificantly higher in EG-given groups compared with controland EG � PJ-given groups for days 10 and 45 (P � 0.05)(Table 1).

The 24-hour urinary Ox excretion in EG-given groups wassignificantly higher than those of control and EG � PJ-givengroups (P � 0.05 in PJ50 and PJ100 at day 10, and P � 0.01 forday 45, Table1). In the EG � PJ-given groups, 24-hour urinaryOx excretion was increased gradually after administration andwas higher than those of controls, but differences were notsignificant (P 0.05 on days 10 and 45, Table 1).

There were no marked changes in serum sodium, potas-sium, creatinine, and BUN levels at any sampling time in allgroups (P 0.05) (Table 1).

There were significantly lower MDA and NO levels (P �0.01) and higher GSH levels (P � 0.05) in the kidney cortexin the EG � PJ-given groups compared with only EG-givengroups (EG days 10 and 45) (Table 1). Also, there was a sig-nificant difference in these three parameters in the EG groupscompared with control groups (P � 0.01). There was no sig-nificant difference in MDA, GSH, and NO levels betweencontrols and EG � PJ-given groups for any sampling times(P 0.05) (Table 1). There was no significant difference be-

TABLE 2. CRYSTALLIZATION SCORES OF CONTROL, EG, PJ 50, AND PJ100 GROUPS ON DAYS 10 AND 45

Day 10 Day 45

Crystallization Control EG EG � PJ50 EG � PJ100 Control EG EG � PJ50 EG � PJ100scores 10 10 10 10 45 45 45 45

0 7 3 5 4 7 — 5 51 — 3 2 3 — 1 2 22 — 1 — — — 3 —3 — — — — — 3 —

FIG. 2. (a) Poor iNOS staining in proximal tubules (Con-trol day 45, IHC � 400). iNOS � inducible nitric oxide syn-thase; IHC � immunohistochemical. (b) Mild iNOS stain-ing in proximal tubules (PJ50 day 45, IHC � 400). iNOS �inducible nitric oxide synthase; PJ � pomegranate juice;IHC � immunohistochemical. (c) Mild iNOS staining inproximal tubules (PJ100 day 45, IHC � 400). iNOS � in-ducible nitric oxide synthase; PJ � pomegranate juice;IHC � immunohistochemical. (d) Diffuse iNOS staining inproximal tubulary cells (EG day 45, IHC � 400). iNOS �inducible nitric oxide synthase; EG � ethylene glycol;IHC � immunohistochemical. (e) Diffuse p65 staining inproximal tubules (EG day 45, IHC � 400). EG � ethyleneglycol; IHC � immunohistochemical.

a

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e

b

PROTECTIVE EFFECTS OF POMEGRANATE JUICE IN NEPHROLITHIASIS 2729

tween PJ50 and PJ100 groups in any sampling times for anybiochemical variables.

Histologic examination

In light microscopy, there were tubular granulovacuolarepithelial cell degeneration and granular material in lumensof some tubules and interstitium. Also, there were mononu-clear inflammatory cells infiltrate in interstitium, which weremore apparent in only EG-given rats than the others, espe-cially on day 45 in the proximal tubules. This degenerativeappearance, increased interstitial cells, and interstitial mate-rial within the proximal tubules and interstitium were alsoseen more clearly with photomicroscopy (Fig. 1a and b).

Crystal depolarizations in the kidney were clearly visual-ized by photomicroscope in EG-given groups in proximaltubules of the cortex on days 10 and 45 (Fig. 1a and b) Ani-mals in only EG-given groups had calcium crystals in renalsections with different crystal amounts on day 10 (Fig. 1a).Crystals were found in all renal cortices of rats in the EG day45 group (Fig. 1b) No calcium crystal deposit was detected inthe control groups (control days 10, 45) (Fig. 1c). There waslimited or no crystal formation in the EG � PJ-given groups(PJ50 and PJ100 on days 10 and 45) (Fig. 1d and e) (Table 2).

Immunohistochemical studies

In immunohistochemical evaluation, there were more in-tensive positive expressions of iNOS and p65 staining withinthe renal cortex within proximal tubules in EG-given groups(EG days 10, 45) compared with others (Figs. 2a–e) (Table 3).Immunohistochemical scores of EG day 45 were higher thanthose of EG day 10.

There were poor or mild positive expressions of iNOS andp65 staining in PJ50 and PJ100 groups and control groupscompared with EG-given groups in any sampling time(Table 3). Also, there was no significant difference betweenPJ50 and PJ100 in any sampling period (Table 3).

Discussion

Urolithiasis resembles arteriosclerosis in the mechanism, cal-cification composition, epidemiology, and gene relationship.The vascular endothelial growth factor gene polymorphism isa suitable genetic marker of urolithiasis.21 Calcification in ar-

teriosclerosis has been inhibited by antioxidants. Magnesium,citrate, and antioxidant therapies with vitamin E and green teahave prevented CaOx precipitation in rat kidneys and de-creased Ox excretion in patients with kidney stones.22–24

PJ is rich in antioxidants of the polyphenolic class, whichincludes tannins and anthocyanins. These antioxidants aremore potent, on a molar basis, than many other antioxidants,including vitamins C and E, coenzyme Q-10, and �-lipoicacid. The antioxidant level in PJ was found to be higher thanin other natural juices, such as blueberry, cranberry, and or-ange, as well as in red wine.14

The blockade of NF-�B activation by antioxidants has beensuggested to be an effective strategy for the management ofurolithiasis and arteriosclerosis.4,14 In our previous study, wefound that PDTC has prevented CaOx precipitation in an ex-perimental rat model with kidney stones that were inducedby EG administitarion.4

It is clear from previous in vitro and in vivo studies thatOx-induced peroxidative injury is involved in the nucleationaggregation and development of CaOx stone disease.25,26

The cell is endowed with several antioxidant systems, in-cluding enzymatic (superoxide dismutase [SOD], catalase,and GSH peroxidase) and nonenzymatic—eg, reduced GSH,vitamins A, E, and C to limit the extend of lipid peroxida-tion.27 Thus, oxidant-antioxidant balance is a critical deter-minant of cell sensitivity to free-radical injury. Several stud-ies reported that Ox causes renal tubular injury by increasedgeneration of free radicals.25–27

In this study, we have shown that NF-�B inhibitor (PJ)treatment decreased urinary Ox excretion and CaOx depositformation compared with the stone-forming group. Thisstudy could be the evidence that hyperoxaluria-induced per-oxidation of renal tubular membrane binds individual CaOxcrystals and initiates kidney stone formation. It is clear thatPJ could protect the tubular membrane against the perox-idative damage.

Another possible protective mechanism of PJ might be thepossible suppressor effects on the activities of Ox-synthesiz-ing enzymes, such as glycolic acid oxidase in the liver. Bythis possible mechanism, Ox production could be sup-pressed. In our study, we did not measure the enzyme ac-tivities as mentioned above, and we did not measure theserum Ox concentration. If we could measure the enzymeactivities and serum Ox concentration, we could more clearly

TABLE 3. THE IMMUNOHISTOCHEMICAL STAINING SCORES OF GROUPS (IHC STAINING: INOS AND P65)

Day 10 Day 45

IHC staining Control EG EG � PJ50 EG � PJ100 Control EG EG � PJ50 EG � PJ100iNOS/p65 10 10 10 10 45 45 45 45

0 4/3 — -/- -/- 4/4 — 1/2 2/21 2/3 — 3/2 3/4 3/2 — 4/4 3/42 1/1 — 3/4 4/3 -/1 — 2/1 2/13 — 3/2 1/1 — — — — —4 — 3/2 — — — — — —5 — 1/2 — — — 2/3 —6 — -/1 — — — 3/3 —7 — — — — — 2/1 —

IHC � immunohistochemical; iNOS � inducible nitric oxide synthase; PJ � pomegranate juice.

say that the protective effects of PJ are on the liver or kid-ney or both. If we had performed these examinations dur-ing the study procedure, it would be more scientific to sayit is possible that PJ prevents PEG-induced hyperoxaluria,exclusive of its antioxidant activity.

Thamiselvan and Menon27 have shown that urinaryMDA concentrations were elevated in EG-given groups.Itoh and coworkers24 have shown that green tea (antioxi-dant) treatment increased SOD activity, which acceleratesthe dismutation rate of O2

� to H2O2 as the first line of en-zymatic antioxidant defense compared with the stone-forming group.

We found that MDA and NO concentrations were signif-icantly lower and the GSH concentration was higher in thekidney cortex of the EG � PDTC groups compared with theEG groups.4

The results of these studies are similar to our findings andshow the possible effects of antioxidants on the urolithiasismechanism. They therefore suggest new treatment choicesin urinary stone disease. We think that PJ can be used inurolithiasis treatment and in the prophylaxis of recurrent uri-nary stone disease.

The development of tissue injury probably depends on thebalance between the production of ROS and the tissue anti-oxidant defense mechanism. Reactive oxygen intermediateshave been shown to be involved in NF-�B activation in ourprevious studies.4,28,29

NF-�B is a sequence-specific transcription that is knownto be involved in the inflammatory and innate immune re-sponses.30,31 NF-�B is sequestered in the cytoplasm in an in-active form through interaction with I-�B. Phosphorylationof I-�B by I-�B kinase causes ubiquitination and degradationof I-�B, thus releasing NF-�B, which then translocates to thenucleus, where it binds to specific �B binding sites in thepromoter regions of several genes.32

Khan and colleagues33 found that treatment of pome-granate fruit extracts (PFE) in A549 human lung carcinomacells significantly inhibited I-�B kinase � and phosphoryla-tion and degradation of I-�B � protein. Because PFE blocksI-�B � phosphorylation and degradation, this study suggeststhat the effects of PFE on NF-�B/p65 are through inhibitionof phosphorylation and subsequent proteolysis of I-�B �.

The expression of iNOS is mainly controlled by the acti-vation of its transcriptional factors, including NF-�B. Zhangand associates34 observed that homocysteine, at pathophys-iologic concentrations, was able to activate NF-�B causingenhanced iNOS expression in macrophages. Leung andcoworkers35 have shown that urinary NO levels dramaticallyincreased in tubulary injury. Nakashima and colleagues36 re-ported that inhibition of active NF-�B by dexamethasone,acetylsalicylic acid alkaline or PDTC was caused by the sup-pression of only the p65 subunit of NF-�B.

We aimed to study the protective effects of NF-�B inhib-itor, PJ, on EG-induced crystal deposition in renal tubules,as well as iNOS and NF-�B activities elevated by increasedROS and lipid peroxidation in rat kidneys.

Treatment with PJ decreased the formation of CaOx de-posits in kidney tissue and expression of iNOS and p65. Wehave shown immunohistochemically that iNOS and p65 ex-pressions increased only in the EG groups in all the sam-pling times. On the other hand, iNOS expression decreasedin the rats treated with EG � PJ.

This study demonstrates that CaOx crystalluria and hy-

peroxaluria alone can activate NF-�B and induce iNOS ex-pression in the kidney. The blockade of NF-�B activation byantioxidants has been suggested to be an effective strategyfor the treatment and prophylaxis of urolithiasis.

Acidic urine is usually found in humans with idiopathicrenal CaOx stone formation, whereas it is not the same inrats. It has been shown with previous studies, as in our pres-ent study, that hyperoxaluric rats had alkaline urine.1,6 Themechanism of alkaline urine production after EG treatmentand its possible correlation with nephrolithiasis and may berelated to renal tubular acidosis in this rat model. This mech-anism is still unclear and needs further studies.6

To the best of our knowledge, our study is the first thathas evaluated the effect of PJ on renal crystallization. Our re-sults encourage us to say that PJ might be useful when usedclinically. There is need for further studies, however, on in-dications and dosages before clinical application becomespossible. Animal models may not reflect human conditionsat all times.

Disclosure Statement

No competing financial interests exist.

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Address reprint requests to:Dr. Volkan Tugcu

Gül D-5 Blok D:3534538 BahçesehirIstanbul, Turkey

E-mail: volkantugcu@yahoo.com

PROTECTIVE EFFECTS OF POMEGRANATE JUICE IN NEPHROLITHIASIS 2731

Abbreviations Used

BUN � blood urea nitrogenCaOx � calcium oxalate

EG � ethylene glycol-GT � gamma-glutamyl transpeptidaseGSH � glutathioneI-�B � inhibitor kappa-B

iNOS � inducible nitric oxide synthaseMDA � malondialdehyde

NF-�B � nuclear factor kappa-BNO � nitric oxide

NOx � nitriteOx � oxalate

PBS � phosphate-buffered salinePDTC � pyrolidium dithiocarbamate

PFE � pomegranate fruit extractsPJ � pomegranate juice

ROS � reactive oxygen speciesSOD � superoxide dismutase