The influence of oxazaphosphorine agents on kidney function in rats

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Original Research Article

The influence of oxazaphosphorine agents on kidney function in rats

Łukasz Dobrek

^a,

* , Beata Skowron

^a

, Agnieszka Baranowska

^a

, Klaudia P łoszaj

^b

, Dorota B ądziul

^c

, Piotr Thor

^a,c

aDepartmentofPathophysiology,FacultyofMedicine,JagiellonianUniversityMedicalCollege,Krakow,Poland

bMedicalanalyticsgraduatestudent,JagiellonianUniversityMedicalCollege,Krakow,Poland

cDepartmentofHumanPhysiologyandPathophysiology,FacultyofMedicine,UniversityofRzeszów,Rzeszow,Poland

*Correspondingauthorat:DepartmentofPathophysiology,FacultyofMedicine,JagiellonianUniversityMedicalCollege,Czysta18,31-121 Krakow,Poland.

E-mailaddress:lukaszd@mp.pl(Ł.Dobrek).

article info Articlehistory:

Received20October2016 Receivedinrevisedform 10April2017

Accepted2May2017 Availableonline2July2017

Keywords:

Cyclophosphamide Ifosfamide Kidney Nephrotoxicity Rats

abstract

Backgroundandobjective: Theapplicationofcytostaticoxazaphosphorinessuchascyclo- phosphamide(CP)andifosfamide(IF)isassociatedwiththeriskofkidneydamagethat, dependingonthetypeofdrug,doseandrouteofadministration,adoptsadifferentclinical entityandseverity.TheaimofourstudywastoassesstheinﬂuenceofCPandIFonthekidney histologyandfunctioninratsintraperitoneallytreatedwithfourdosesofeitherCPorIF.

Materialsandmethods:Atotalof30ratsweredividedintothreegroups(10ineachgroup):

group1(control),shamtreatedwithsalinesolution,group2(treatedwith75mg/kgb.w.of CP),andgroup3(treatedwith60mg/kgb.w.ofIF).Afterthetreatmentratsweresacriﬁced, bloodwascollectedandnephrectomyandcystectomywereperformed.Qualitativeand quantitativeparameters(includingneutrophilgelatinase-associatedlipocalin-1,NGAL-1)of kidneyfunctionwereassayedinurineandplasma.

Results:CP-treatedratswerecharacterizedbyasigniﬁcantpolyuria,decreasedurinepHand bydecreaseddailyurinaryexcretionofsodium,potassium,ureaanduricacidaccompanied byincreasedNGAL-1excretion.Asigniﬁcantdecreaseoftheplasmauricacidconcentration wasalsoobserved.IF-treatedanimalswerealsocharacterizedbydecreasedurinepHbut withnormaldailyurinaryexcretionofassessedsubstances(exceptforreduceduricacid excretion).

BothCPandIFtreatedratsdidnotshowanyhistopathologicalabnormalitiesintheir kidneys.

Conclusions: CPcausedmoreadvancedkidneydysfunctionandsomeindicessuggestedthe developmentofprerenalacutekidneyinjury.IntheCP-treatedgroupsomeparticularly markedurinaryandplasmauricaciddisturbancessuggestedcompensationofincreased oxidativestressasuricacidisconsideredtoexertalsoantioxidantproperties.

Availableonlineatwww.sciencedirect.com

ScienceDirect

journal homepage:http://www.elsevier.com/locate/medici

http://dx.doi.org/10.1016/j.medici.2017.05.004

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1. Introduction

Nephrotoxicity is deﬁned asa structural and/or functional kidneydamageresultingfromexposuretoanynoxiousfactor of toxic or ischemic character. Drugs are one of kidney- affecting agents, therefore the incidence of drug-induced nephrotoxicity has been continuously increasing [1]. Some general issues determining drug-induced kidney disease include glomerular or tubular dysfunction, impairment of renal blood ﬂow or disturbances of kidney metabolic and endocrine function,accompanied by microscopicstructural lesionsorgrossmorphologicalchanges[2].Thebackgroundfor nephrotoxicityaredetailedpathomechanisms,affectingrenal vasculature(e.g.hemodynamicacutekidneyinjuryorthrom- botic microangiopathy), glomeruli (e.g. minimal change disease, focalsegmental glomerulosclerosis) ortubulointer- stitium(acutetubularnecrosis,[ATN],crystalnephropathyor tubulopathies such as Fanconi syndrome, salt wasting, nephrogenic diabetes insipidus, syndromeof inappropriate antidiuresis)[3,4].

Nephrotoxicityremainsanimportantadversedrugreac- tion (ADR) in case of administration of many classes of pharmacologicalagents,includingparticularly:contrastme- dia,immunosuppressants(mostlycyclosporineA),aminogly- cosides,sulphonamides,amphotericinB,non-steroidalanti- inﬂammatorydrugs,goldandD-penicillamine,antihyperten- sives,withspecialattentiontoangiotensinconvertingenzyme inhibitors or diuretics [1]. Also, many cytostatic drugs are characterized by a signiﬁcant nephrotoxicity. Therefore, despitetheimprovementineffectivenessof chemotherapy, drug-inducedkidneydamageremainsacomplicationentan- glingtheentiretreatment[3].

Amongthechemotherapeuticsendowedwithnephrotoxi- city, oxazaphosphorine alkylating agents should be mentioned. These compounds include cyclophosphamide (CP), ifosfamideandlessfrequentlyusedtrofosfamide.CPisusedin chemotherapyofbothsolidtumorsandacuteleukemiaandis usedasan immunosuppressantinnonneoplasticdisorders (systemic lupus erythematosus and rheumatoid arthritis).

Ifosfamide(IF)isalsoaneffectiveagentusedintreatmentof solidtumors,includingtesticularcancer,rhabdomyosarcoma, Wilms'tumor,Ewing'ssarcoma,bonesarcomas,osteosarco- maandneuroblastomaaswellassomeformsoflymphoma [3,5].ThemainADRofCPishemorrhagiccystitis,inducedby acrolein–atoxicproductreleasedduringCPbiotransforma- tion.Ifosfamideshareswithcyclophosphamideatoxicproﬁle characterizedbyurotoxicity.Moreover,IFisconsideredtobe morenephrotoxiccomparedtoCP[2,3,5].Thatisduetothefact that biotransformationof IF leadsto aprincipal release of chloroacetaldehydethatisattributedtoexertamorepowerful nephrotoxic effect compared to acrolein [6]. To sum up, nephrotoxicityofoxazaphosphorine agentsdependsonthe type of drug (CP vs. IF), the applied dose, the route of administration,thetotaltimeoftreatment,thepresenceof otherco-existingfactorspredisposingtonephrotoxicity[2,4].

That multitude of nephrotoxicity-determining factors also results in a varied clinical description of kidney damage evokedbyuseofthoseagents.Moreover,publishedreportsof kidneydamageevokedbyoxazaphosphorineadministration

are only partial, based on the different nature of the assessment (imagingstudies, selectedbiochemicalparame- ters),soanymorecomplexlaboratoryanalysisoftheissueis stillmissing.

Therefore,theaimofourstudywastoassesstheinﬂuence oftwooxazaphosphorines(CPandIF)ontherenalhistology and function,estimated by panelof laboratoryparameters assessed in urine and plasma, in rats treated with four successivedosesofeitherCPorIF.

2. Materials and methods

Themedicalexperimentdescribedinthispaperwasapproved bytheILocalEthicalCommitteeinKrakow.Theexperiment wascarriedoutinaccordancewithboththeDirective2010/63/

EUontheprotectionofanimalsusedforscientiﬁcpurposes andwiththePolishActof15January2015ontheprotectionof animals used for scientiﬁc or educational purposes (JL, February26,2015,Pos.266).

2.1. Examinedgroupsofanimalsandageneralplanofthe experiment

Theexperimentincluded3010-week-oldalbinoWistarrats,in equalquantitiesofmalesandfemales.Animalswereobtained fromtheCentralAnimalHouseoftheFacultyofPharmacyof theUJCMinKrakow.

UponarrivaltothelocalAnimalHouseoftheDepartment ofPathophysiologyoftheUJCM,theywerekeptduringtheﬁrst sevendaysinanisolatedroominordertoacclimatizetothe new living conditions. After that period, the rats were randomlyassignedtothestudygroupsofthesamesize(10 ineachgroup;5malesand5females):group1(controlrats), group2(ratswithCP-inducedchroniccystitis), andgroup3 (ratswithIF-inducedchroniccystitis).

All animals survived till the end of the experiment, although theoverall conditionof the animalsfrom groups 2 and 3 was deteriorating with subsequent CP/IF doses, respectively.

Duringtheexperiment(exceptwhenratswerehousedin individualmetaboliccages),animalswerekeptinsingle-sex cagesseparateforthestudygroups,withunlimitedaccessto waterandstandardfeed(Labofeed,Kcynia,Poland),intheair- conditionedroomwithaconstanttemperatureandhumidity, maintaininga12/12-hday/nightcycle.

Theplanoftheexperimentassumedinitialassessmentof vitalsigns,dailydiuresis,feedandwaterconsumptionofall studyanimalsperformedinindividualcages.Urinesamples werecollectedover24h forsubsequent laboratoryanalysis and for qualitativeand semi-quantitative analysis of urine withurinedipsticks.Then,animalsingroups2and3received CPorIFtreatment,respectively,toinducechroniccystitisin7 days.Controlindividualsweregivennormalsaline.Afterthe lastCP/IF/salinedoseallanimalswereoncemoremonitored during24hinmetaboliccages,withassessmentofthesame parametersasthosebeforethetreatmentandurinesamples werecollectedagain.Afterthesecondstayinmetaboliccages, blood samples for further biochemical assay were also collected.Finally,theanimalsweresacriﬁcedandcystectomy

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andnephrectomywereperformedforsubsequenthistopath- ologicalevaluationofurinarybladdersandkidneys.

2.2. Chroniccystitisinducedbycyclophosphamide(group 2)orifosfamide(group3)

Ratsinthegroup2weregivenCP(CPmonohydrate;Sigma Aldrich) intraperitoneally in the amount of 75mg/kg b.w., dissolvedin0.5mLofnormalsaline.Thedosewasadminis- tratedfourtimes,everytwodays,aftertheinitialmonitoring inmetaboliccagesontheﬁrstdayoftheexperiment.Afterthe last CP dose, animals were once again placed in separate metaboliccages. Accordingtoliterature data,the approved dosing schedule can induce chronic cystitis [7,8]. Besides administrationofCPanimalswerenotsubjectedtoanyother treatment.

In the group 3, the procedure was identical, but rats received60mg/kgb.w.ofifosfamide.ThechoiceofIFdosewas alsobasedonliteraturereports–therearestudiesreporting ifosfamide-inducednephrotoxicityinratsusing80mg/kgb.w.

for three days [9] or 50mg/kg b.w. for ﬁve days [10,11].

AccordingtoSpringateandVanLiew[12],therecommended urotoxicdoseofIFis40–80mg/kgb.w.Therefore,wedecidedto applythe doseof 60mg/kg b.w.of ifosfamideasasuitable dosage(thedosageregimenprovidedthesameglobalIFdose asintheabovecitedpapers).Controlanimals(group1)were subjectedtothesameprocedureasindividualsofgroups1and 2, but instead of using CP/IF, rats were sham-treated with normal saline, in accordance with the regimen, and the volumesusedingroups1and2.

2.3. Monitoringinmetaboliccages

Inthebeginning(beforereceivingtheﬁrstdose)andintheend oftheexperiment(afterthelast,fourthdoseofCP/IFingroups 2/3orsalineingroup1,respectively)animalswereplacedin isolated metabolic cages over 24h to measure daily urine outputandwaterandfeedconsumption.Duringthestaythey had a free, yet monitored access to water and food. Vital parameterswereassessed:bodyweight(g),bodytemperature (8C),dailywaterintake(mL/24h),dailyfoodconsumption(g/24h) anddailydiuresis(mL/24h).Bodytemperaturewasmeasured usingadigitalrectalthermometerforrodents(Vivari,UK).

2.4. Urinedipstickcontrol

Afterrecordingthevolumeofurine,samplesweresubjectedto generaltests.Dipstickswereusedfor thatpurpose(Insight, ACONLaboratories,REFU031-105),allowingassessmentofpH andspeciﬁcgravity(SG)ofurine,presenceofblood,leukocytes (whitebloodcells;WBC),protein,glucose,bilirubin,urobilinogen, ketones, nitrates. The analysis was performed strictly accordingtomanufacturer'sguidelines.

2.5. Laboratoryparametersestimatedinurineandplasma

After dailyurineoutput wasmeasured, urinesampleswas subsequentlycentrifuged(2000rpm/5min),anddividedinto portions. Resulting samples were stored at 208C until laboratory determinations completed with the ADVIA 1200

SIEMENSanalyzer.Initially,sodium,potassium,urea,creatinine (Cr), uricacid and protein concentrations weredeter- mined. Moreover, blood urea nitrogen (BUN) and BUN/

creatinineratiowerecalculated:

BUN¼urea ðmg=dLÞ 2:1428

Urinaryconcentration (ng/mL)ofnovelbiomarkerof kidney damage–neutrophilgelatinase-associatedlipocalin-1(NGAL- 1),using acommercially availableELISA kit(BioPortoDiag- nostics, Denmark), strictlyaccording tothe manufacturer's instructions,wasalsoassessed.

Then,havingresultsofdailydiuresis,24-heliminationof sodium, potassium, urea (mmol/24h), uric acid, creatinine (mmol/24h),protein(mg/24h)andNGAL-1(ng/24h)withurine was calculated. Creatinine clearance was also estimated, accordingtotheformula

CLcr¼Crurine ðmmol=LÞdiuresis ðmL=minÞ Crplasmaðmmol=LÞ

Fractional sodiumeliminationwascalculatedusingthefor- mula:

FENa¼½ðNaurine ðmmol=LÞCrplasmaðmmol=LÞÞ100

½ðNaplasma ðmmol=LÞCrurine ðmmol=LÞÞ

and the renal failure index (RFI) was calculated with the formula:

RFI¼½ðNaurineðmmol=LÞCrplasma ðmmol=LÞÞ

Crurine ðmmol=LÞ

Similarlytourine,collectedbloodwascentrifugedandresult- ingplasmasampleswerekeptfrozenuntilthetimeoflabora- torydeterminations.Plasmasodium,potassium(mmol/L)and creatinineanduricacid(mmol/L)levelsweredeterminedusing thesameanalyzerastheoneusedforurinesamples.Plasma proteinwasnotassayed.

2.6. Sacriﬁcinganimalsandcollectingkidneysand bladdersforhistopathologicalanalysis

Afterthesecondstayinmetaboliccages,animalswerekilled by intraperitoneal injection of a lethal dose of sodium pentobarbital(Morbital;Biowet,Pulawy;200mg/kgb.w.).After conﬁrmedcessationofvitalsigns,cystectomyandnephrec- tomywereperformedinordertoobtaintissueformicroscopic specimensnecessaryforhistopathologicalevaluation.

2.7. Histologicalevaluationofcollectedbladdersand kidneys

Bladdersandkidneysobtainedduringautopsywerestoredina formalinsolutionuntilpreparationofmicroscopicslides.The histopathologicalassessment wasperformedat theDepart- ment of Anatomopathology of the non-public healthcare institution ‘‘Prosmed’’in Krakow.Thesections werecut at preselected thickness of 4 microns using LEICA RM2135 microtome. In order to assess the severity of histological

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inﬂammation,thepreparationswerestainedwithhematoxy- lin–eosin(HE).Adetaileddescriptionofthehistopathological technique is described in one of our previously published paper[13].

2.8. Statisticalanalysis

Resultsofthequantitativeanalysisofurineperformedwith urinedipstickswereanalyzedusingthenonparametrictestof signs, examining changes in the direction of the assessed parameter, separately for each study group in the paired comparison(pre-andposttreatment).

Resultsofthevitalparametersanddailywaterand feed intakewereanalyzedbothforintragroup(atthebeginningand the end of the experiment) and intergroup differences, comparinggroups1and2aswellasgroups1and3.

Differences concerning results of plasma and urine laboratoryparameterswereestimated forintergroupdiffer- ences,comparinggroups1and2aswellasgroups1and3.

Inthestatisticalanalysisofvitalparametersandlaboratory parametersestimatedinplasmaorurine,intheﬁrststep,a distribution of obtained results was analyzed using the Shapiro–Wilk test.If resultswere notnormally distributed, thefurtheranalysiswasperformedusingtheMann–Whitney test,andiftestedresultsdemonstratedanormaldistribution, theStudentttestwasused.Thelevelofstatisticalsigniﬁcance wassetatP≤0.05.

3. Results

3.1. Resultsofvitalparametersobtainedfrommonitoring inmetaboliccages

Consideringchangesofthestudyparametersinthebeginning andintheendofthestudy,controlanimalswerecharacter- izedbyasignificantincreaseoftheirbodyweight.Thebody temperatureanddailywaterandfeedintakewerecomparable throughout the experiment. Contrary to control ones, CP- treated animals demonstrated a statistically significant decrease of their body weight with a parallel reduction of theamountofdailyfeedintakeintheendofthestudy.Other parameters demonstrated no statistically significant differ- encesbetweenthebeginningandtheendoftheexperiment.

IF-treated animals were characterized by a signiﬁcant decrease of their body temperature after the last IF dose and,similarlytoCP-treatedones,demonstratedadecreased ﬁnal24-hfeedintake.Theirbodyweightanddailywaterintake didnotchangeoverthetime.

Consideringintergroup differences,no significant,initial differences were revealed between control and animals randomized to both groups 2and 3,thus showinglack of differences between individuals in the beginning of the experiment.Intheendofthestudy,CP-treatedratsdiffered withrespecttoallparameterscomparedtothecontrolones, whereas IF-treated animals demonstrated some significant differencesconcerningtheirfinalbodytemperatureanddaily feedintakecomparedtothecontrol.

Detailed results of parameters mentioned above are

presentedinTables1and2. Table1–Parametersoftheanimalstreatedwithfourdosesofcyclophosphamide(CP):intra-andintergroupanalysis. MetaboliccagemeasurementP BeginningoftheexperimentEndoftheexperiment BodyBody24-hwater weight,gtemperature,8Cintake, mL/24h 24-hfeed intake, g/24h Body weight,gBody temperature,8C24-hwater intake, mL/24h

24-hfeed intake, g/24h ABCDEFGH Control(group1)226.6644.8937.430.9326.384.0723.756.16257.4563.1937.930.2630.003.2720.382.39Evs.A,0.0001 Fvs.B,NS Gvs.C,NS Hvs.D,NS CP-treated animals(group2)240.0365.2737.220.2729.256.5921.132.36209.7374.8437.450.6022.0011.719.637.67Evs.A,0.0002 Fvs.B,NS Gvs.C,NS Hvs.D,0.002 PNSNSNSNS0.050.030.050.002 ValuesaremeanSD.NS,notsigniﬁcant.

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3.2. Resultsofthequalitativeandsemi-quantitative analysisofurineusingdipsticks

ResultsofurinepHandSGindicatedbythedipstickstestare presentedinTable4.

Inthe controlgroup,presence ofblood andglucosewas demonstratedinnoneofurinesamplesinbothtests,inthe beginningandintheendofthestudy.Intheﬁrsttestinthat group,leukocyteswereassessedat(15(WBC/mL))in88%of casesandat+(70(WBC/mL))in12%,nitratesassessedat+in 50% and they were not detected in the remaining 50% of samples, urobilinogen was measured at 0.2 (mg/dl) in all samples,proteinwasdeterminedat+(30(mg/dL))in50%of cases and ++ (100(mg/dL)) inother 50%, ketoneswerenot foundin88%samplesandwereassayedat(5(mg/dL))and bilirubinwasassessedat+(1(mg/dL))in88%andat++(2(mg/dL)) in12%.

Inthesecondtestperformedaftertheshamtreatmentwith normalsaline, the findingsregarding urobilinogen,protein, blood, bilirubin and glucose were identical to the initial assessment. Thepresenceof leukocytesinurinedecreased, and the analysis demonstrated no leukocytes in 63% of samplesandtheparameterwasassessedat(15(WBC/mL)) in25%andat+(70(WBC/mL))in13%.Inallsamplesnitrates presence estimated at + was confirmed. Ketones werenot detectedin50%andintheremaininghalfofthesamplesthey were measured at (5 (mg/dL)). Only changes in urine leukocyte,nitrateandketoneswerefoundtobestatistically significant.

Ingroup2,nobloodandglucoseweredemonstratedinthe beginning of the study. In all samples urobilinogen was assessedat0.2(mg/dL).Leukocyteswerenotshownin50%of samplesandwereestimatedat(15(WBC/mL))in25%andat+ (70 (WBC/mL)) in 25%. The presence of nitrates was not conﬁrmedin75%,andintheremaining25%itwasestimated at+.Theurineproteinwasmeasuredat(15(mg/dL))in38%, at + (30(mg/dL)) in12%and at ++ (100(mg/dL))in50%. In assessmentofketones,in12%ofcasestheywereestimatedat

(5(mg/dL))andotherurinesamplescontainednoketones.

Bilirubinwasassessedat+(1(mg/dL))in88%andat++(2(mg/

dL))in12%.Nosamplescontainedglucose.

InthesecondanalysisperformedinCP-treatedanimals,a presenceofurobilinogenassessedat+(1(mg/dL))andbilirubin assessedon+(1(mg/dL))wasfoundinallcases,andglucose andketoneswereabsent.In25%ofsamples,leukocyteswere estimatedat(15(WBC/mL)andin50%at+(70(WBC/mL))and theywerenodemonstratedin25%ofsamples.Nitratesmarked as+werepresentin88%,whereas12%ofsampleswerefree fromnitrates.Presenceoftheurinaryproteinwassimilartothe ﬁrstassessment–itwasestimatedat(15(mg/dL))in38%,at+ (30(mg/dL))in25%andat++(100(mg/dL))in38%ofcases.There wasnobloodfoundinhalfofurinesamplesandin12%ofthem itwasestimatedat+andat++in38%.Consideringdifferences intheurinedipsticksanalysisobservedbeforeandafterthe treatmentwith CP,only changesrelatedtothenitrates and bloodwereconsideredtobestatisticallysigniﬁcant.

BeforetheIFtreatment,allurinesamplesfromratsinthe group3werecharacterizedbyabsenceoftheglucose,ketones, bloodandleukocytesin70%ofcases.In30%,leukocyteswere estimatedat+(70(WBC/mL)).Nitrateswerefoundin12%and Table2–Parametersoftheanimalstreatedwithfourdosesofifosfamide(IF):intra-andintergroupanalysis. MetaboliccagemeasurementP BeginningoftheexperimentEndoftheexperiment Body weight,gBody temperature,8C24-hwater intake, mL/24h

24-hfeed intake, g/24h Body weight,gBody temperature,8C24-hwater intake, mL/24h

24-hfeed intake, g/24h ABCDEFGH Control(group1)226.6644.8937.430.9326.384.0723.756.16257.4563.1937.930.2630.003.2720.382.39Evs.A,0.001 Fvs.B,NS Gvs.C,NS Hvs.D,NS IF-treated animals(group3)254.3551.4937.440.3726.008.5422.003.13251.5362.6236.980.3821.7015.2911.703.59Evs.A,NS Fvs.B,0.03 Gvs.C,NS Hvs.D,0.001 PNSNSNSNSNS0.001NS0.001 ValuesaremeanSD.NS,notsigniﬁcant.

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alltheotherurinesamplesweredeprivedofthesubstance.

Urobilinogenwasassessedat0.2(mg/dL)andbilirubinat+ (1(mg/dL))inallcases.Urineproteininratsinthegroup3was notdetectedin80%ofcasesandestimatedat(15(mg/dL)) in10%andat++(100(mg/dL))in10%,beforethetreatment withIF.

TheIFtreatmentresultedinincreasedurineproteinlevelof

(15(mg/dL))in90%ofcasesandof++(100(mg/dL))inthe remaining10%ofthecases.Moreover,in25%ofsamples,blood was alsodetected. All of theother parameters(leukocytes, urobilinogen, bilirubin, ketones, nitrates, glucose) were assessed inthe samemanner asin theinitial assessment.

However,theperformedstatisticalanalysisindicatedthatthe abovediscusseddifferenceswerenotstatisticallysigniﬁcant.

3.3. Resultsoftheassayedplasmaparameters

AdministrationoffourdosesofCPresultedbothinsigniﬁcant decrease of the plasma uric acid and increase in plasma creatininelevels.However,BUNandBUN/creatininerationdid not differ signiﬁcantly in control and CP-treated animals.

Plasmasodium, urea,levels werecomparableinCP-treated andcontrolrats.

Similarly toadministration of CP, the treatmentwithIF causedasigniﬁcantincreaseinplasmacreatininelevel,which was accompaniedby ablood urea nitrogenvalue increase.

OtherparametersassessedinplasmaofIF-treatedanimalsdid notdiffersigniﬁcantlyfromthoseincontrolsubjects.

DetailedresultsarepresentedinTable3.

3.4. Resultsofassessedurineparameters

CP-treatedratsdifferedintermsofthemajorityofestimated urine parametersfrom control ones.First of all,they were characterizedbyahighervalueofdailydiuresis(bothtotaland minute)withasubstantialacidiﬁcationofurine.CPadminis- tration resulted also in a signiﬁcant decrease in urinary excretionofsodium,potassium,ureaanduricacid.Incontrast, the daily urinary excretion of protein and creatinine was similarinCP-treatedanimalsandcontrolones.

TheIFtreatmentresultedonlyinasigniﬁcantdecreaseof urinepH,withsimultaneousincreaseof itsspeciﬁcgravity.

The daily urinary excretion of assayed substances didnot differsigniﬁcantlyfromvaluesofthoseparametersassessed in the control group, except for the uric acid, that was – similarlytoCP-treatedrats–foundintheurineindiminished amountcomparedtocontrolgroup.

Urinary NGAL-1 concentrations were not signiﬁcantly differentinallstudiedgroups.

DetailedresultsarepresentedinTable4.

Thevalueofcalculatedcreatinineclearancewassimilarin both CP- and IF-treated rats (0.850.26 and 0.920.44, respectively)anddidnotdifferfrom thevaluefoundinthe control group (1.10.37). Moreover, animals receiving CP demonstratedasigniﬁcantdecreaseofcalculatedvaluesofthe fractionalsodiumexcretionandRFIcomparedtocontrolrats.

Table3–Plasmaparametersinratstreatedwithcyclophosphamide(CP)andifosfamide(IF).

Control (group1)

CP-treatedrats (group2)

IF-treatedrats (group3)

P

Group1vs.2 Group1vs.3

Na,mmol/L 142.061.79 143.801.9 142.571.21 NS NS

Urea,mmol/L 6.340.75 6.761.07 7.281.06 NS NS

BUN,mg/dL 17.762.10 18.953.00 19.972.87 NS 0.05

Uricacid,mmol/L 157.9635.14 106.157.56 189.8194.21 0.02 NS

Creatinine,mmol/L 27.951.77 31.992.81 34.193.5 0.05 0.001

BUN/creatinine,mg/dL 54.344.40 52.678.94 52.5210.37 NS NS

ValuesaremeanSD.BUN,bloodureanitrogen;NS,notsigniﬁcant.

Table4–Urineparametersinratstreatedwithcyclophosphamide(CP)andifosfamide(IF).

Control (group1)

CP-treatedrats (group2)

IF-treatedrats (group3)

P

Group1vs.2 Group1vs.3

Diuresis,mL/24h 5.721.31 11.944.14 8.275.31 0.005 NS

Diuresis,mL/min 0.0040.001 0.0080.003 0.0060.004 0.001 NS

SG 1.0090.001 1.0080.006 1.0130.006 NS 0.05

pH 9.00.00 8.131.03 8.250.86 0.02 0.01

Protein,mg/24h 5.714.15 3.942.47 5.294.61 NS NS

Na,mmol/24h 0.760.25 0.480.19 0.630.37 0.04 NS

K,mmol/24h 2.080.55 1.020.66 1.680.85 0.005 NS

Urea,mmol/24h 5.931.28 4.451.14 5.052.36 0.02 NS

Uricacid,mmol/24h 9.032.85 6.071.25 5.262.99 0.02 0.01

Creatinine,mmol/24h 45.5614.14 39.3812.52 44.7019.92 NS NS

NGAL-1,ng/mL 1.530.80 3.342.42 1.020.90 NS NS

ValuesaremeanSD.SG,speciﬁcgravity;NGAL-1,neutrophilgelatinase-associatedlipocalin-1;NS,notsigniﬁcant.

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Dependencies concerning some signiﬁcant intergroup differencesforabovementionedparametersofFENaandRFI arepresentedinFigs.1and2.

Moreover, CP-treated rats were characterized by signiﬁ- cantly greater 24-h urinary NGAL-1 excretion (ng/24h)

compared to control (41.4435.32 vs. 8.644.36). In the IF-treatedgroup,dailyNGAL-1urinaryexcretionwassimilar tothevalue foundin thecontrol animals(6.245.45vs.

8.644.36).Theresultsdescribedabovearepresentedin Fig.3.

Fig.1–Fractionalexcretionofsodium(FENa)inCP-treated(left)andIF-treated(right)rats.P<0.05forCP-treatedrats;NS,not significantforIF-treatedrats.

Fig.2–Renalfailureindex(RFI)inCP-treated(left)andIF-treated(right)rats.P<0.05forCP-treatedrats;NS,notsignificantfor IF-treatedrats.

Fig.3–24-hurinaryexcretion(ng/24h)ofneutrophilgelatinase-associatedlipocalin-1(NGAL-1)inCP-treated(left)andIF- treated(right)rats.P<0.05forCP-treatedrats;NS,notsignificantforIF-treatedrats.

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3.5. Descriptionofthehistopathologicalassessmentof bladdersandkidneys

SpecimensofurinarybladderscollectedfromCP-treatedrats hadalargelyhomogeneouspresentation.Reactiveprolifera- tionofurothelialepitheliumwithmultiplicationoflayersupto 6andsignsofdyskaryosis(nuclearpolymorphism),mostlyin the basallayer of the urotheliumwerefoundinthe entire group.Moreover, aninﬂammatoryinﬁltrationwasfoundin stromainallspecimens.KidneysinallCP-treatedratswereat thelimitofanormalpresentation.

Specimens of urinary bladders collected from IF-treated animalspresentedaninflammatoryinfiltrationandfibrosisof thestroma.KidneysofIF-treatedanimalsappearednormal.

4. Discussion

Themainﬁndingsofthestudymaybesummarizedasfollows:

1. RatstreatedwithCPpresentedasignificantpolyuriaand acidificationofurine.TheadministrationofCPresultedalso indecreased24-hurinaryexcretionofsodium,potassium, ureaanduricacidwhichwasaccompaniedbyasignificant- lyhigherexcretionofNGAL-1.Disturbancesofparameters estimatedinurinewereaccompaniedbyasignificantand profoundreductionoftheplasmauricacidlevel.Moreover, thecalculatedFENaandRFIhadlowervaluesinCP-treated rats compared to control ones. An overall qualitative assessment of urine with dipsticks also demonstrated somemoreemphasizeddisturbancesinthegroupofrats treated with CP. The administration of CP was also associatedwithasignificantwastingofanimals,reflected byasignificantreductionoftheirfinalbodyweightandfood intake.

2. Ratstreatedwithifosfamide alsopresenteda significant urine acidification. However, in contrast to CP-treated animals,the24-hurinaryexcretionofmajorityofanalyzed substances (including NGAL-1) did not differ from that observedinthecontrolgroup–rats treatedwithIFwere characterized only by a reduced uric acid excretion. IF administrationwasalsoassociatedwithincreasedcreati- nineandBUNplasmalevel,butthecalculatedcreatinine clearanceandBUN/creatinineratioweresimilartothosein thecontrolgroup.Moreover,animalstreatedwithIFwere characterized by lesser abnormalities in the qualitative analysis of urine compared to CP-treated rats. Also, IF administration resulted in a much smaller cachexia compared to rats receiving CP (no final body weight decrease,lesslossofthedailyfeedintake).

AsalreadymentionedintheIntroduction,ifosfamidebeing themain source of chloroacetaldehyde,isregardedtobea compound associated with a signiﬁcant nephrotoxicity.

Kidneys metabolize nephrotoxic chloroacetaldehyde into a less toxic chloroacetate and the rate of that detoxication determinesthedegreeofkidneydamage[14].Bothoxazapho- sphorineagents(CPandIF)arehydrophilic.Forthatreason theydonoteasilydiffusethroughcellmembranesandrequire apresenceofspeciﬁccellcarriers–organicanion(OAT)and

cation(OCT)transporters[15].Inthekidney,aswellasinother tissues,OATisoforms1and3andOCTisoform2arelocatedin thebasolateralmembrane[16].BothCPandIFhaveafﬁnityfor OATs,butIFispreferablytransportedfurtherbyrenalOCT-2, whichexplains agreateraccumulationof thecompoundin kidneysandahighernephrotoxiceffect[15].

ThekidneydysfunctioncausedbytheuseofIF,describedin the literature, adopts a wide spectrum, ranging from the generalized proximal tubulopathy (Fanconi syndrome) to partial sodium, potassium, glucose,amino acids and other smallmoleculecompounds're-absorptiondefects[3,4,17]The CP-induced nephrotoxicityalso involves a variable tubular dysfunction[2–5].Moreover,theCP-inducedkidneydysfunc- tion results in reduction of tubular ﬁltration rate and developmentofaninappropriateantidiuresissyndrome[18].

Resultsofourstudyindicatedanabnormaldailyurinary excretionofassessedsubstancesbyCP-treatedrats(including increasedNGAL-1excretion)andasignificanturineacidifica- tion.IF-treatedanimalsdemonstratedonlyanabnormallylow urinepHaturinaryNGAL-1excretionremainingatasimilar level as compared to control rats. Therefore, our study demonstrated asignificant nephrotoxicity potential for CP.

Which proves that acrolein, which is a unique compound releasedduring metabolic processingof CP,exertsnotonly mainlyurotoxiceffectsbutalsosigniﬁcantnephrotoxicones.

Themostlikelymechanismofthe acrolein-relatednephro- toxicityiscomplexandbasedonthesameissuescontributing to urotoxic properties of that compound, and includes a reduction of the kidney cellular glutathione dependent antioxidantsystemandanincreaseinfreeradicalgeneration, mostly peroxynitrite.Thecompoundisresponsibleforlipid peroxidation–adeleteriousdamageoftherenaltubularcells [19]. Moreover, asignificant drasticdecrease (insteadof an increase)intheactivitiesoflysosomalenzymeswasobserved inkidneysofCP-treated rats.Adecreaseintheactivitiesof lysosomalproteindigestiveenzymesmaycontributetoCYP- induced renal damage – the accumulation of abnormal amounts of the protein in kidneysmaybeat least inpart due to a defect in lysosomal enzyme activity and may contributetorenaldamage[20].Tosumup,thedosageand routeofadministrationappliedinourstudyshowedamore emphasizedkidneydysfunctioninCP-treatedrats,withthe absenceof anyrenalhistopathologicalabnormalities.More- over, resultsobtained for CP-treated animalssuggest some possibledisturbancesofproximaltubules(impairmentofre- absorption), as well as of distal ones (abnormal urine acidification).ItseemsthatIFaffectsonlythedistalnephron function,becausetheagentcausedonlyadropofurinepHand the analysis of NGAL-1 did not reveal any significant abnormalities.Thestatementofdysfunctionofdistaltubules issupportedbythefindingthatH⁺secretionintourineand, therefore,determinationofthefinalurinepH,takesplacejust in the distal tubules [21,22]. Taking into account data presentedinTables1and2,itshouldbeconcludedthatthe 24-hwaterandfoodintakeinbothCP-andIF-treatedanimals duringmonitoringinindividualmetaboliccageswasalmost identical (both in the beginning and in the end of the experiment).Therefore,inouropinion,itisunlikelythatthe finalpHdropobservedinthetreatedanimalsresultedfrom intergroupdifferencesinwaterorfeedconsumption.

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Moreover, rats treated with CP, in addition to urine acidificationandabnormalNGAL-1excretion,developedalso a significant polyuria, which may be considered as an additional premise for the assumption of the distal tubule dysfunction.Polyuriademonstratedinourstudyiscontraryto literaturereportsindicatingdevelopmentofthe‘‘antidiuresis syndrome’’aftertheapplicationofCP.Thatdisorderwasdue totheincreased,CP-inducedexpressionofaquaporinsviathe vasopressin-independent mechanism [23,24]. On the other hand,however,developmentof IF-evokednephrogenicdia- betesinsipiduswasalsoreported[3].Thus,theinfluenceofthe oxazaphosphorine agents on diuresis is ambiguous and requiresfurtherresearch,butabovementionedstudies(also includingthecurrentlydiscussedone) suggestdisturbances withinthedistalnephronduetoCP/IFadministration.

Takingintoaccountalltheresultsobtainedinthegroupof animalstreatedwithCP,itisworthnotingthatsomeofthem (FENa,RFI)maysuggestdevelopmentofacutekidneyinjury (AKI).AKI,previouslyreferredtoasacuterenalfailure,canbe definedasanabruptdeteriorationof the all renalfunction (excretory,endocrine, metabolic). In other words, it means sudden lossof the ability of the kidneysto excrete waste, concentrateurine,conserveelectrolytes,andmaintainfluid balance[25].AKIisconditionedbyvariousfactorsofpre-,post- or toxic/inflammatory intra-renal origin that lead to acute kidneyfunctiondecompensation[25,26].Clinically,fractional excretion of sodium and renal failure index are regarded laboratory indices suggesting prerenal background of AKI [25,27].LowFENaisaneffectivemarkerofhypoperfusionof therenalartery[28].ThoseparametersobtainedinCP-treated rats achieved lowervalues compared tothe control group.

Therefore, an additional assumption may be set that CP administration also contributes to prerenal AKI features resultingfromkidneyhypoperfusion,asthatistheunderlying mechanismofthatformofAKI[26].Thepresenceofpolyuria demonstrated in CP-treated rats, although is not a typical ﬁnding,doesnotexcludethepossibilityofAKIdevelopment, asthedisorderparadoxicallymaybeassociatedwithpolyuria (althoughintheclassic formischaracterizedbyoliguriaor evenanuria)[25–27].

Anotherevidencesupportingkidneydamagedevelopment inCP-treatedanimalsistheanalysisofurinaryexcretionof novelbiomarkerof kidneydysfunction–NGAL-1.Whilethe urinaryconcentrationsofthebiomarkerwerecomparablein allgroups,the24-hexcretion ofthecompoundwassigniﬁ- cantly higher in CP-treated animals. NGAL-1 is a protein synthesizedandreleasedinto theplasmabymanytissues:

kidneys, prostate, trachea, lung, stomach, large intestine, uterusandbonemarrow.Inaddition,itisreleasedingreater amountsfromneutrophilsandmacrophagesduringinﬂam- matory response and endothelial injury [29–31]. NGAL-1 exhibits pleiotropic biological properties: extracellularly, it bindsiron-complexedsiderophoresandregulatesintracellular metabolismoftheiron,enhancestheproteolyticactivityof matrix metalloproteinases (MMP-9), therebypreventing the degradation of these enzymes by tissue MMP-9 inhibitor (TIMP-1).Thisphenomenonisofparticularimportanceinthe processes of progression of carcinogenesis [29,32]. The augmentationofMMP-9activitybyNGAL-1isalsoimportant inthe developmentof acute coronarysyndromes resulting

from atherosclerotic plaque destabilization [29,33]. Plasma NGAL-1levelisalsoelevatedinpatientswithtype2diabetes, thussuggestingthepotentialroleofNGAL-1inthepathogen- esisofinsulinresistance[34].NGAL-1isfreelyﬁlteredinthe kidneysandreabsorbedinproximaltubules.Inaddition,this protein issynthesizedbyHenle'slobeand distaltubulesin responsetovariouskidneydamagingfactors[31,35].There- fore, theincreased24-hurinaryexcretionof NGAL-1inCP- treatedratscouldhaveresultedfromproximaltubulesfailure and reduced reabsorption of this protein and/or increased tubularsynthesis.Moreover,thehypothesisofamorepotent nephrotoxicityofCPcomparedtoifosfamideisalsoconsistent withtheresultsofoneofourotherexperiments.Inthatstudy, weassessedtheurinaryconcentrationand24-hexcretionof anothernovelbiomarker–kidneyinjurymolecule-1(KIM-1).

Similarresultswererevealed–again,CP-treatedanimalswere characterized bysigniﬁcantly higherurinaryKIM-1concen- trationandincreased24-hexcretionoftheproteinwiththe urine[13].KIM-1isaglycoproteinsecretedbytheproximal tubule cells inresponsetoanynoxiousfactors –therefore, playsaspecialroleofoneofthe‘‘renaltroponins’’usedfor earlyandreliabledetectionofkidneyinjury.Underphysiolog- icalconditions,urinedoesnotcontainKIM-1–soitspresence in the urine is regarded tobe a quantitative biomarker of kidneydamage,whichappearsrapidlyintheurineinresponse totheproximaltubulardamage[13].

Tosumup,theincreased 24-hurinaryexcretionofboth NGAL-1andKIM-1biomarkersisanotherproofofCP-induced nephrotoxicity.

Thenextcharacteristicdisturbancedemonstratedinour experimentwasareducedurinaryexcretionofuricacidthatin thecaseofCP-treatedanimalswasassociatedwithdecreased plasmalevelofthecompound.Thedecreaseofserumuricacid maycontributetothedevelopmentofpolyuria,resultingfrom abnormalities in cortico-medullary osmotic gradient and disturbances of the countercurrent amplifier mechanism [36,37]. Thereduction of serumuricacid level (and, conse- quently,reducedurinaryexcretionofthecompound)mayalso reflectacompensationofanexcessiveoxidativestress,which is, as mentioned above,an essential, potentialelement of pathomechanismoftheCP-inducednephrotoxicity.Uricacid isoneofthemostimportant,lowmolecularmasscompounds thatisnowbelievedtoexertstrongantioxidanteffect[38].In vitroexperimentsdemonstratedthaturicacidisapowerful scavengeroftheperoxylandhydroxylradicalsandofsinglet oxygen[39].Moreover,uricacidisthoughttobeaninhibitorof radicals generated by decomposition of peroxynitrite – a compoundalsogeneratedbytheactionofacrolein[40,41].An importantpremisefortheantioxidantroleofuricacidisthe observation that the loss of urate oxidase, resulting in increased plasma uric acid level, improves antioxidant defense[38].Therefore,itcanbeconcludedthattheadvanced oxidativestressinkidneysinducedbyacroleinreleasedduring biotransformation of peroxynitrite, causes secondary ‘‘ex- haustion’’ of the compensatory antioxidant mechanisms, including reduction of plasma uricacid level.The indirect confirmationoftheroleofuricacidasanantioxidantinthe course of kidney and bladder damage is provided also by clinicalobservations:anegativecorrelationbetweenplasma levelof uricacidandtheseverityofhemorrhagiccystitisin

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childrentreatedwithCP[42].Thus,resultsofourstudyrelated to marked plasma uric acid decrease in CP-treated rats constituteone moreevidencefor arole of uricacid inthe pathogenesisofkidneyandbladderdamagecausedbyCP.

5. Conclusions

Tosumup,bothCPandifosfamideadministratedfourtimes intraperitoneally causedkidneydysfunctionthatwas more advancedinanimals treatedwithCPanddemonstrated by reduced24-hexcretionofsodium,potassium,ureaanduric acid,withamarkedacidiﬁcationofurineandincreasedNGAL- 1urinaryexcretion.Moreover,someindices(FENa,RFI)also suggestprerenaldevelopmentofacutekidneyinjury.

IF-treatedanimalswerecharacterizedonlybydecreased urinepHandreduceddailyuricacidexcretion.

DecreasedserumuricacidlevelsinratstreatedwithCPand decreaseddailyurinaryexcretionofthecompoundfoundin bothstudiedgroupsmayreﬂectparticipationofthatcompound intheantioxidantcompensationphenomenathataccompany renalfailureinducedbyoxazaphosphorinemetabolites.

Conﬂict of interest

All the authors herebydisclose thatthere isno conﬂict of interestamongtheauthors.

Funding

This work was supported by the Jagiellonian University MedicalCollege(UJCMstatutorygrantK/ZDS/006404).

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