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JOURNAL OF ENDOUROLOGYVolume 21, Number 9, September 2007© Mary Ann Liebert, Inc.DOI: 10.1089/end.2007.0074

Rapid Communication

Protective Effect of a Nuclear Factor �B Inhibitor, Pyrolidium Dithiocarbamate, in the Kidney of Rats with Nephrolithiasis Induced by Ethylene Glycol

VOLKAN TUGCU, M.D.,1 EMIN OZBEK, M.D.,2, ERAY KEMAHLI, M.D.,1, MUSTAFA BAKI CEKMEN, M.D.,3 NAZLI CANER, M.D.,4 ADNAN SOMAY, M.D.,5

PELIN ERTURKUNER, M.D.,6 ISMAIL SECKIN, M.D.,6 CENNET GURAL DEMIR, M.D.,3

TUNCAY ALTUG, M.D.,4 and ALI IHSAN TASCI, M.D.1

ABSTRACT

Purpose: To study the protective effects of a selective nuclear factor kappa B (NF-�B) inhibitor, pyrolidiumdithiocarbamate (PDTC), on ethylene glycol-induced crystal deposition in the renal tubules, renal toxicity, aswell as inducible nitric oxide synthase (iNOS) and NF-�B activities in rat kidneys.

Materials and Methods: Rats were divided into three equal groups: control, ethylene glycol-treated (EG),and ethylene glycol � PDTC treated (EG�PDTC). Rats were sacrificed on day 7, 15, or 45, and tissue sec-tions were evaluated under light and transmission electron microscopy for the presence and degree of crys-tal deposition and toxicity in the kidneys. The iNOS and NF-�B activity were evaluated immunohistochemi-cally, with p65 being stained to define NF-�B activity. Crude extracts of the cortex were used to determinereduced glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA) concentrations.

Results: Crystal depositions were more evident in the proximal tubules on day 7 in the EG than in the othergroups. Mild crystallization was observed on day 15, and severe crystallization and granulovacuolar epithe-lial-cell degeneration were observed on day 45. There was limited or no crystal formation in the EG�PDTCgroup and completely normal renal and tubular structures in the control group. Whereas ethylene glycol ad-ministration stimulated iNOS and NF-�B/p65 activity in renal tubules, PDTC inhibited it. Rats given only ve-hicle demonstrated no significant alterations. Hyperoxaluria, a marked increase in MDA and NO concentra-tions, and a decrease in GSH were observed in the EG group.

Conclusion: This experiment has shown the role of transcription factors, NF-�B, and iNOS in ethylene gly-col-induced crystal depositions in renal tubules.

INTRODUCTION

CALCIUM-CONTAINING URINARY STONES are acommon clinical problem. The recurrence rate is high,

about 50% at 10 years and 75% at 15 years, if no prophylacticmeasures are used. Hyperoxaluria is one of the major risk fac-

tors in human idiopathic calcium oxalate (CaOx) stone disease,and its experimental induction with ethylene glycol can be usedto produce CaOx urolithiasis in rats. 1 Glycolate or glycoxylateis responsible for the production of oxalate, and rats given 7.5%ethylene glycol showed significant increases in the activities ofoxalate-synthesizing enzymes such as glycolic acid oxidase in

1Department of Urology, Bakirkoy Research and Training Hospital, Istanbul, Turkey.Departments of 2Urology and 5Pathology, Vakif GurebaResearch and Training Hospital, Istanbul, Turkey.3Department of Biochemistry, Kocaeli University, Medical Faculty, Kocaeli, Turkey.4Department of Animal Research Laboratory, Istanbul University Cerrahpasa Medical Faculty, Istanbul, Turkey.6Department of Histology, Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey.

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the liver.2,3 Acute and chronic production of CaOx crystals in-duces lipid peroxidation, and therefore, it has been suggestedthat this process plays an important role in CaOx stone forma-tion.3

“Lipid peroxidation” usually refers to the functional impair-ment of cellular components by reactive oxygen species (ROS)such as superoxide radicals, hydroxyl free radicals, and hydro-gen peroxide.4 The ROS act as mediators of nuclear factorkappa-B (NF-�B) activation by inhibitor kappa-b degradation(I�-B).5

Inducible nitric oxide synthase (iNOS) is one of the threeNOS isoforms that is affected by NF-� as a result of tissue dam-age. The process of iNOS expression involves different signal-transduction pathways, including nuclear translocation of thetranscription factor NF-�B.6 The contribution of NO to tissueinjury can be secondary to a direct effect mediated by the NOmolecule itself and an indirect effect mediated by reactive ni-trogen species produced by the interaction of NO with super-oxide anions or oxygen.7 The iNOS-mediated NO productionis significantly elevated when there is increased oxidativestress,7,8 and excessive NO production secondary to elevatedexpression of iNOS may impose cytotoxic effects on variousorgans, including the kidney.9 In an attempt to limit the oxida-tive damage, a number of antioxidants have been tested, as ithas been proposed that antioxidants, which maintain the con-centration of reduced glutathione (GSH), may restore the cel-lular defense mechanism and block lipid peroxidation.10

Malondialdehyde (MDA) is one of the important markers oflipid peroxidation.11 Excessive MDA produced as a result oftissue injury and DNA damage could combine with free aminogroups of proteins, resulting in the formation of MDA-modi-fied protein adducts.

Pyrrolidine dithiocarbamate (PDTC) is a metal chelator andantioxidant with potent anti-inflammatory properties, in part be-cause of its ability to suppress NF-�B.12

Superoxide dismutase (SOD) accelerates the conversion ofO2

� to H2O2 as the first line of enzymatic antioxidant defense.Selenium-containing glutathione peroxidase reduces all organiclipid peroxides and requires GSH as a hydrogen donor. Themost active non-enzymatic antioxidant is GSH itself, as it is ascavenger of H2O2, OH�, and chlorinated antioxidants.13

Therefore, we administered the NF-�B inhibitor pyrolidiumdithiocarbamate (PDTC) to rats forming stones to investigateits possible inhibitory effects on urolithiasis and its mechanismof action.

MATERIALS AND METHODS

Animals

A group of 72 adult male Sprague-Dawley rats (230–250 g)were obtained by the Experimental Animal Laboratory of Is-tanbul University, Cerrahpasa Medical Faculty. The rats weremaintained in a 14-hour light/10-hour dark cycle with free ac-cess to food and water.

Experimental conditions

Rats were divided into three equal groups. In group 1 (con-trol group), the rats received distilled drinking water. In group

2 (EG group), they received hyperoxaluria-inducing 0.75% eth-ylene glycol in distilled water. In group 3 (EG�PDTC group),rats were injected intraperitoneally with PDTC (Sigma-AldrichChemical Corp. MO) (100 mg/kg/day) and received 0.75% eth-ylene glycol in distilled water.

Each of the major groups was then divided into three groupsaccording to the experimental sampling periods; i.e., 7, 15, and45 days. The animals were placed in cages 7 days before theinitiation of the experiment with a view to acclimating them.During the experimental period, all the groups had free accessto regular rat chow.

After the last injection, rats were placed in metabolic cages todetermine 24-hour urine output, urinary pH, and total urinary pro-tein and oxalate concentrations. At 24 hours after the last injec-tion, rats were killed under anesthesia (intraperitoneal sodiumpentobarbital, 50 mg/kg of body weight). The kidneys werequickly removed and decapsulated. The renal cortex was care-fully separated from the medulla and homogenized as describedpreviously.14 Small samples were fixed in formaldehyde solutionfor histopathologic and immunohistochemical examination.

Histopathologic examination

The tissues were prepared for routine examination by lightmicroscopy (Nikon, Tokyo, Japan). The kidney sections wereanalyzed semiquantitatively using the technique of Houghtonet al.15

Immunohistochemical evaluation

For the immunohistochemical evaluation, specimens wereprocessed for light microscopy. Sections were incubated at60oC overnight and then dewaxed in xylene for 30 minutes. Af-ter soaking in a decreasing series of ethanol, sections werewashed with distilled water and phosphate-buffered saline(PBS) for 10 minutes. Sections were then treated with 2%trypsin in 50 mM Tris buffer (pH 7.5) at 37oC for 15 minutesand washed with PBS. Sections were delineated with a Dakopen (Dako, Glostrup, Denmark) and incubated in a solution of3% H2O2 for 15 minutes to inhibit endogenous peroxidase ac-tivity. Then sections were incubated with NF-�B/P65 (Rel A)antibody (Ab-1; Neomarkers R-B-1638-R7) and iNOS antibody(Ab-1; Neomarkers R-B-1605-R7). The Ultra-vision HRP-AECstaining protocol was used.

Sections prepared for each case were examined by light mi-croscopy. Sections of rat lung were used for the control of im-munohistochemical staining specificity according to the dataprovided by the antibody-producing company.

According to the diffuseness of the staining, sections weregraded as 0 � no staining; 1 � staining �25%; 2 � stainingbetween 25% and 50%; 3 � staining between 50% and 75%;or 4 � staining �75%. According to staining intensity, sectionswere graded as 0 � no staining; 1 � weak but detectable stain-ing; 2 � distinct staining; or 3� intense staining. Immunohis-tochemical values were obtained by adding the diffuseness andintensity scores.

Transmission electron microscopy

Tissues were prefixed in 1% glutaraldehyde and 4%formaldehyde in PBS for 2 hours. Then, the tissues were post-

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fixed in phosphate-buffered osmium tetraoxide for 1 hour (pH7.2), dehydrated in a graded series of ethanol, and embeddedin Epon. Ultrathin sections were cut using a Reinheirt Om U3ultramicrotome with glass knives, stained with acetate and leadcitrate, and examined using a Carl Zeiss EM 9 S-2. Tissue sec-tions were scored on a four-point scale by two histologists un-aware of the treatment given and applying the scale to both themedulla and the cortex. The scoring was as follows: 0 � no ox-alate crystals in any field; 1 � no more than two crystals in anyfield; 2 � more than two crystals in any field; and 3 � multi-ple collections of crystals in all fields.

Biochemical assessments

All tissues were washed two times with cold saline and im-mediately stored at �80°C for later measurement of MDA,GSH, and NO concentrations. Tissues were homogenized infour volumes of ice-cold buffer containing 20 mM Tris and 10mM EDTA (pH 7.4)

Total nitrite (NOx) was quantified by the Griess reaction af-ter the incubation of supernatant liquid with Escherichia colinitrate reductase to convert NO3 to NO2. Thiobarbituric acidreaction with lipoperoxidation aldehydes such as MDA is themethod most commonly used to assess lipid peroxidation in bi-ological samples. The procedure was modified from Buege andAust.16 The GSH concentration was determined by the spec-trophotometric method of Elman14 based on the developmentof a yellow color when 5,5’ dithiobis-2-nitrobenzoic acid(DTNB) is added to compounds containing sulfhydryl groups.

Blood samples were taken to assess the serum concentrationsof urea, creatinine, blood urea nitrogen (BUN), Na�, K�, andgamma-glutamyl transpeptidase. All biochemical variableswere determined using an Olympus Autoanalyzer (Olympus In-struments, Tokyo, Japan).

Statistical analysis

Statistical analyses of the histopathologic and immunohisto-chemical evaluation of the groups were carried out by the chi-square test and analyses of the biochemical data by the Mann-Whitney U test. Probability values of �0.05 were consideredsignificant.

RESULTS

Biochemical variables in urine, serum and tissue

There was no significant difference between the control andexperimental groups in terms of daily urine output or total uri-nary protein. The urinary pH was significantly higher (P �0.05)in the EG groups compared with the control and EG�PDTCgroups at all time points (Table 1).

The 24-hour urinary oxalate excretion was significantlyhigher in the EG groups than in the control and EG�PDTCgroups (P �0.05 for day 7 and P �0.01 for days 15 and 45)(Table 1). In the EG�PDTC groups, 24-hour urinary oxalateexcretion increased gradually after administration of the sub-stance began. The excretion in this group was higher than inthe controls, but the differences were not significant (P �0.05on days 7, 15, and 45) (Table 1). There were no marked changes

in serum Na, K, creatinine, or BUN at any sampling time inany of the groups (Table 1).

The MDA and NO concentrations were significantly lower(P �0.01) and the GSH concentration was higher (P �0.05) inthe kidney cortex of the EG�PDTC groups compared with theEG groups on days 7, 15, and 45) (Table 1). Besides, there wasa significant difference in the MDA concentration of the EGgroups (P �0.01). The mean tissue MDA concentration on day7 was 84.4 � 12.4 nanomol/g of wet tissue. On day 15, the con-centration had increased to 122.3 � 16.3 nmol/g, and on day45, it was 148.8 � 18.4 nmol/g. There was no significant dif-ference between the controls and EG � PDTC groups at anysampling time (Table 1).

Transmission electron microscopy

Crystal depositions in the kidney were clearly seen by TEMin the EG groups in proximal tubules of the cortex at all times(Fig. 1A, B). In the kidney sections of the EG animals on day7, CaOx particles were found in the renal cortex (Fig. 1A). Onlythe animals in the EG groups had calcium crystals in the renalsections, with different crystal amounts being found on day 15(Fig. 1B). Crystals were found in all the renal cortex tips in allthe rats in the EG group on day 45. No crystal deposits weredetected in the control groups at any time (Fig. 1C). There waslimited or no crystal formation in the EG�PDTC groups ondays 7, 15, and 45; and there were more crystal formations inthe cytoplasm of cells from the EG � PDTC animals on days7 and 15 compared with day 45 (Table 2).

Histologic examination

Light microscopy revealed tubular granulovacuolar epithelial-cell degeneration, as well as granular material in the lumens ofsome tubules and interstitium. Furthermore, there were mononu-clear inflammatory cells infiltrating the interstitium, which weremore apparent in the EG rats than the others and were especiallyprominent in the proximal tubules on day 45 (Fig. 2A, C). Thisdegenerative appearance, increased interstitial cells, and com-pacted material within the proximal tubules and interstitium wereobserved more clearly with TEM (Fig. 2D–F).

Immunohistochemical studies

There were more intense expressions of iNOS and p65 stain-ing within the renal cortex within the proximal tubules in theEG groups at all three times (Fig. 3A–D) (Table 3). Immuno-histochemical scores of the EG animals on day 15 were higherthan those on day 7, but there was no significant difference be-tween these two groups.

There were poor or slight expressions of iNOS and p65 inthe EG � PDTC and the control groups at all three times com-pared with the EG groups (Table 3).

DISCUSSION

Previous animal experiments have shown that hyperoxaluriaand crystalluria are accompanied by enzymuria and membra-nuria, a finding consistent with damage to renal tubularcells.4,17,18 Considerable evidence has implicated reactive free

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radicals in the pathophysiology of a wide spectrum of disor-ders, including atherosclerosis, ischemia–reperfusion injury, in-flammatory disorders, cancer, and aging.19

Urolithiasis resembles arteriosclerosis in its mechanism, cal-cification composition, epidemiology, and genetic relations. Ithas been reported that the vascular endothelial growth factorgene polymorphism is a suitable marker of urolithiasis.20 Cal-cification in arteriosclerosis has been inhibited by antioxidants.Magnesium citrate and antioxidant therapy with vitamin E hasprevented CaOx precipitation in rat kidneys and decreased uri-nary oxalate excretion in patients with kidney stones.21,22

The blockade of NF-�B activation by antioxidants has been

suggested to be an effective strategy for the treatment of urolithi-asis and arteriosclerosis.23 Itoh and collaborators18 have shownthat green tea (antioxidant) treatment decreased urinary oxalateexcretion and CaOx deposit formation and also increased SODactivity compared with the stone-forming group. The results ofthese studies are similar to our findings and show the possible ef-fects of antioxidants on the urolithiasis mechanism. They there-fore suggest new treatment choices in urinary-stone disease. Wethink that PDTC can be used in urolithiasis treatment and in theprophylaxis of recurrent urinary-stone disease.

Pragasam et al3 found that EG rats showed significant in-creases in the activities of oxalate-synthesizing enzymes such

PROTECTIVE EFFECT OF A NUCLEAR FACTOR �B INHIBITOR 1101

A B

C

FIG. 1. Deposits of CaOx crystals in rats fed ethylene glycol,as seen by TEM. (A) Day 7 EG animal. Formations showingconcentric rings and electron density (➮) in some regions withvesicles in proximal tubules. (Original magnification �10,000.)(B) Day 15. Vesicles of various sizes (✩) containing hypodenseand hyperdense material are seen within proximal tubule cells.Vesicles containing electron-dense materials are visible in lu-men (➮). (C) Day 7 control animal. Proximal tubules are seen.There is no apparent degeneration. (Original magnification�4300).

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TUGCU ET AL.1102

A B

FIG. 2. Histologic and ultrastructural findings. (A) Day 45 EG animal. Apparent granulovacuolar degeneration of tubule ep-ithelial cells, damaged brush borders in proximal tubules, and mononuclear inflammatory-cell infiltration are apparent. (H&E;original magnification �400.) (B) Day 45 EG�PDTC animal. Mild granulovacuolar degeneration of epithelial cells and mononu-clear inflammatory-cell infiltrate is noticeable in cortical interstitium. (H&E; original magnification �400.) (C) Day 45 controlanimal. Normal glomerular and tubular structures. (H&E; original magnification �400.) (D) Day 45 EG animal. Degenerativeappearance of proximal tubule. Vesicles (➮) concentrated in apical portion of cell containing huge electron-dense deposits withseparation of basal cellular infoldings (✩) are apparent. (TEM; original magnification �4000.) (E) Day 45 EG animal. Increasedinterstitial cells (➮) and compacted interstitial material (✩) between tubules in cortical region. (TEM; original magnification�6500.) (F) Day 15 EG animal. There are widening gaps between basal infoldings. Interstitial cells, abundant macrophages (➮),and leukocytes (✩) are apparent in interstitial space. (TEM; original magnification �6300.)

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PROTECTIVE EFFECT OF A NUCLEAR FACTOR �B INHIBITOR 1103

E F

FIG. 2. (Continued)

TABLE 2. CRYSTALLIZATION RATES OF GROUPS

Day 7 Day 15 Day 45

Crystallization Control EG EG�PDTC Control EG EG�PDTC Control EG EG�PDTCRates 7 7 7 15 15 15 45 45 45

0 8 — 6 8 — 5 8 — 61 — 3 2 — — 3 — — 22 — 5 — — 4 — — 2 —3 — — — — 4 — — 6 —

TABLE 3. HISTOPATOLOGICAL EFFECTS OF EG AND PDTC IN RAT KIDNEYS (IHC STAINING: iNOS&p65)

Day 7 Day 15 Day 45

iHC Staining Control EG EG�PDTC Control EG EG�PDTC Control EG EG�PDTCiNOS/p65 7 7 7 15 15 15 45 45 45

0 3/3 — 1/— 2/2 — 1/— 3/2 — 1/—1 3/2 — 3/3 5/4 — 3/2 4/3 — 3/22 2/3 — 4/5 1/2 — 3/4 1/3 — 2/33 — 4/3 — — 2/2 1/2 — — 2/34 — 4/5 — — 2/1 — — — —5 — — — — 4/5 — — 2/1 —6 — — — — — — — 3/4 —7 — — — — — — — 3/3 —

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as glycolic acid oxidase in the liver. Lactate dehydrogenase ac-tivity in the liver and kidneys also increased. The activity ofthe free radical-producing enzyme xanthine oxidase and thetissue oxalate and calcium concentrations increased signifi-cantly. Depletion of the antioxidant enzymes, membrane-boundATPases, and thiol status were observed in these rats. We didnot investigate the effects of PDTC on the activities of the ox-alate-synthesizing or free radical-producing enzymes in the

liver, but we think that PDTC must have a systemic effect andcan suppress these mechanisms by its antioxidant effect.

Thamilselvan and Menon24 have shown that urinary MDAconcentrations are elevated in EG groups. In this present study,we found that there were significant differences in the MDAconcentrations in the renal tissues of the EG groups, which canbe indicative of induced ROS activity and oxidative tissue dam-age. We did not assay urinary MDA, but previous studies25

TUGCU ET AL.1104

A B

C D

FIG. 3. Proximal tubules after immunohistochemical staining. (A) Day 45 EG animal. Diffuse iNOS staining. (B) Day 45 EGanimal. Diffuse p65 staining. (C) Day 45 E� PDTC animal. Mild p65 staining. (D) Day 45 control animal. Little iNOS stain-ing is visible. (Original magnification for all panels �400.)

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showed that urinary MDA concentrations are elevated throughincreased lipid peroxidation.

The development of tissue injury probably depends on thebalance between the production of ROS and tissue antioxidantdefense mechanisms. Reactive oxygen intermediates wereshown to be involved in NF-�B activation in our previousstudy.25 In this study, we sought to determine if iNOS is acti-vated by NF-�B in EG animals, and we found that iNOS andp65 expressions were significantly reduced in EG � PDTC ratscompared with EG rats.

The expression of iNOS is controlled mainly by the activa-tion of its transcriptional factors, including NF-�B. Zhang andcoworkers26 observed that homocysteine at pathophysiologicconcentrations was able to activate NF-�B, causing enhancediNOS expression in macrophages. Leung and associates27 haveshown that urinary nitric oxide concentrations increase dra-matically after tubular injury. Nakashima et al28 reported thatinhibition of active NF-�B by dexamethasone, acetylsalicylicacid, or PDTC was caused by the suppression of its p65 sub-unit. The p50 subunit was constitutively expressed and was notinhibited by the aforementioned NF-�B inhibitors. We studiedonly p65 immunohistochemically.

Reverse transcriptase-polymerase chain reaction or Westernblotting analyses are functional assays by which to measure theactual activity of iNOS. Western blotting provides a more quan-titative way of measuring iNOS and p65 activity. Therefore, itis a limitation of this study that Western blotting analyses werenot performed. However, there are a number of studies in theliterature that have made use of immunohistochemical gradingof iNOS to evaluate NO activity.29 We also used this methodin one of our recent studies.25

We aimed to study the protective effects of a selective NF-�B inhibitor, PDTC, on ethylene glycol-induced crystal de-position in renal tubules, as well as iNOS and NF-�B activitieselevated by increased ROS and lipid peroxidation in rat kidneys.Treatment with PDTC decreased the formation of CaOx depositsin kidney tissue and expression of iNOS and p65. We have shownimmunohistochemically that iNOS and p65 expressions increasedonly in the EG groups at all sampling times. On the other hand,iNOS expression decreased in the rats treated with EG�NF-�Binhibitor. The results suggest that iNOS expression can be inhib-ited by PDTC, as shown in our previous study. 25

Hyperoxaluria is a relatively uncommon cause of stone dis-ease, occurring in �10% of stone formers, and the hyperox-aluric rat model does not necessarily reflect the human condi-tion. This study demonstrates that CaOx crystalluria andhyperoxaluria alone can activate NF-�B and induce iNOS ex-pression in rat kidneys. In a study carried out by Holmes andAssimos in 2004,30 it was demonstrated that when 8 mmol ox-alate was administered, oxidative stress in normal adult kidneysdid not increase. In another study carried out by the same au-thors in 2007,31 two groups of six individuals were formed. Thefirst group consisted of normal healthy adults and the secondof stone formers. The members of both groups were given 0,2, 4, and 8 mmol oxalate. There was no significant differencebetween the groups when 0, 2, or 4 mmol oxalate was given.However, when 8 mmol oxalate was administered, oxalate ab-sorption increased significantly in two patients in the stone-for-mer group and one case in the control group. To our thinking,

the increases in oxidative stress and oxalate absorption mightbe affected by the dosage. Therefore, we think that intake ofhigher doses of oxalate might increase oxidative stress in thekidneys. On the other hand, Tungsanga and colleagues32 andHuang and associates33 have shown that in humans, there is in-creased oxidative stress and renal-cell injury in stone-formingpatients, which is similar to our findings.

CONCLUSION

According to our model, blockade of NF-�B activation by an-tioxidants could be an effective strategy for the treatment and pro-phylaxis of urolithiasis in conditions such as primary hyperox-aluria, in which urinary oxalate may be elevated to pathological/pharmacologic levels. To the best of our knowledge, our study isthe first that has evaluated the effect of PDTC on renal crystal-lization. Our results encourage us to say that PDTC might be use-ful clinically. However, there is a need for further studies on thisissue before clinical application becomes possible.

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Address reprint requests to:Volkan Tugcu, M.D.Gül D-5 Blok D:35

34538 BahçesehirIstanbul, Turkey

E-mail: volkantugcu@yahoo.comeraykemahli@mynet.com

ABBREVIATIONS USED

BUN � blood urea nitrogen; CaOx � calcium oxalate;DTNB � 5,5’ dithiobis-2-nitrobenzoic acid; EG � ethyleneglycol; GSH � reduced glutathione; H&E � hematoxylin andeosin stain; iNOS � inducible nitric oxide synthase; MDA �malondialdehyde; NF � nuclear factor; PBS � phosphate-buffered saline; PDTC � Pyrrolidine dithiocarbamate; ROS �reactive oxygen species; SOD � superoxide dismutase;TEM � transmission electron microscopy.

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