Micron and Microscopica Acta, Vol. 19, No.4, pp. 227—234, 1988. 0739—6260/88 $3.00+0.00Prided in Great Britain. © 1989 Pergamon Press plc

NEPHROTOXICITYOF CYCLOSPORINA IN DIABETIC BlOBREEDINGRATS

VINCENZO ESPOSITOandGIANPAOLO PAPACCIO

Institute of Anatomy—I,Schoolof Medicine,via L. Armanni, 5—80138,Naples,Italy

(Received5 July 1988; in revisedform 26 October 1988)

Ahstract—Aconsiderablenumberof studieswerecarriedOut Ofi patientsreceivingCyclosporinA (CSA)afterbonemarrow,heartandkidneytransplants.More recentlythis drughasbeenusedasan immunosuppressiveagentin themanagementoftypeI diabetes.Moreovertheincreaseofcreatininelevelsin CSA-treatedpatientsandanimalshasled theresearchersto believethatthisdrugmay be responsiblefor irreversiblenephrotubularsideeffects.

Our aim was,therefore,to studythehistopathologicaleffectsofCSA on kidneysof bio breeding(BB) rats,which developdiabetesspontaneously.

Animalsweretreatedfor 30 and60dayswith daily injectionsof 8 mg/kgbody wt ofCSA, dissolvedin 2 ml ofIntralipid 10% (Pierrel),given intraperitoneally(control animalsreceivedonly Intralipid). At theend of theexperimentsanimalsweresacrificedunderetheranaesthesiaandthekidneysremovedandprocessedfor lightmicroscopy,using standardprocedures.After a 30-day administrationof CSA, the tubular and glomerularstructuresappearedunchangedor, in somecases,only a few cells, in the proximal tubules,showedslightvacuolation.After 60 daysofCSA administration,theelementsof theproximalprofilesshoweda considerabledegreeof cytoplasmicvacuolation.Thesevacuolesresultedpositive to PFABB, SudanBlack B, PAS andalkalinetetrazoliumreactions.Distal tubularprofiles, loopsofHenle andglomeruli wereunaffected.

Our morphologicalfindings demonstratethatCSA causesnephrotubularmodifications,whenadministeredin therapeuticdosesof only 10 mg/kgbody wt, asinmanyclinicalschedules.Moreoverdatacouldbeconsistentwith a possiblereversionto thenormalstructuralappearance.

Index key words:CyclosporinA, kidney,bio breedingrats,histochemistry,ultrastructure,diabetes.

INTRODUCTION et al., 1981; Schulmanet al., 1981; Blair et a!.,1982; Myers et a!., 1984).

CyciosporinA (CSA) is a fungal derivative CSA toxicity hasbeeninvestigatedin severalthat selectivelyinhibits antigen-reactiveT-lym- animalmodels.In particularthe rat has provedphocytes(Green,1981). Its immunosuppressive useful in characterizingboth functional andactivity hasarousedmuch interest,andseveral structural abnormalities associatedwith thecentresarecurrentlyassessingits relativeproper- nephrotoxicityof the drug. In this speciesCSAties in suppressingaliograftsand,morerecently, toxicity hasbeenfoundtobedose(Whitinget a!.,the pancreaticinsulitis in type 1 diabetes(Mar- 1982),strain,sexandagedependent(Ryffel eta!.,liss eta!., 1982;Like eta!., 1984). 1983),reversibleupondrugwithdrawal(Svenyet

A considerablenumberof studieson patients a!., 1981) and proportionalto circulatingCSAreceiving CSA for bone marrow and renal levels (Whiting et a!., 1985).transplantshavesuggestedthat this immunosup- On the otherhand,Klintmalm et a!. (1981)pressivedrug is nephrotoxic(Caineet a!., 1978; report easily reversible nephrotoxicity of CSAKlintmaim et a!., 1981;Hows et a!., 1981;Starzl after 13—22 days of treatment in transplant

patients,andrecentlyEunet a!. (1987)foundno________________________________________specifichistopathoiogicalchanges,eventhougha

Addressfor correspondence:GianpaoloPapaccioM.D., minimal degenerationof the proximal convo-via G. Bonito, 21 80129, Naples,Italy. luted tubularepitheliumwasnoticed.

227

228 V. EspositoandG. Papaccio

However,only few data(Bertani et a!., 1987) insulin and serum creatinine were assayedare available to correlate morphologic with weekly. At the end of the experiments,thefunctional abnormalities in animals receiving animalswereanaesthetizedandperfusedwith along-termCSA administration. 0.1 M solution of 3.25% paraformalde-

Discrepanciesfound in experimentaldataare hyde—0.25%glutaraldehydeat pH 7.4. Kidneysmostlyascribedto the employeddoses,that are were removedand processedfor light micro-oftenhigherthanthoseusedin clinical practiceto scopy. Sections of 7 j.tm were stained withsuppresstransplants in humans or in the haematoxylin—eosin,PA-Shiff, performic aci-managementof type 1 diabetes. d—Alcian Blue (PFAAB), Sudan Black B and

This study was planned to point out the alkalinetetrazoliumreactions.histopathologicaleffects on kidneys of CSA Two kidneys of eachgroupwerepostfixed ingiven,in therapeuticdoses,to bio breeding(BB) 1% 0s04dissolvedin a 0.1 M phosphatebufferrats (whose characteristic is related to the pH 7.4, 4°C,thendehydratedandembeddedinspontaneousdevelopmentof a type-I diabetes), epon resins.Semithinsection(0.5 j.~m)werecutin order to suppressthe isletitis. undera LKB ultratomeandstainedwith Tolu-

idine Blue. Ultrathin sectionsof 70—80nm wereexaminedin a Zeiss EM 109 electron micro-

MATERIALS AND METHODS scope.Thirty BB rats,15 malesand15 females,kindly The resultsaregiven asmeanvalue±SEM of

supplied by Dr Thibert (Animal Resources n-samplesinvestigatedat each time. StatisticalDivision, Ottawa,Canada),fed ad !ibitum (aged analysiswasperformedusingtheStudent’st-test.90—130 days,weighing l50-200g), were used.Ratsbelongingto the categoryA animals (pre-diabetics, with plasma glucose concentrations RESULTSexceeding150 mg/dl) were subdividedin threegroups. Laboratoryfindings

First group animals (n= 12) receiveda daily Plasma glucose, serum insulin and serumi.p. injection of CSA (Sandoz,Basel) 10 mg/kg creatinineconcentrationsbeforeandafter treat-bodywt/30days,dissolvedin0.5 ml of Intralipid mentsare summarizedin Table 1.10%, an emulsionof lipids and phospholipids Control animalsat the endof theexperiments(PierrelS.p.A.). showeda very significant (P<0.0000l)increase

Secondgroupanimals(n= 12) receiveda daily in plasmaglucoselevels,due to the fact that thei.p. injection of CSA 10 mg/kgbody wt/60 days, ratsusedwereof the BB rat strain.dissolvedin 0.5 ml of Intralipid. On the otherhand,both CSA-treatedgroups

Third group animals (n=6) receiveda daily showed only a slight (P<0.05) increase ini.p. injection of 0.5ml Intralipid alone for 30 plasmaglucoseconcentrations.(n=3) and 60 (n=3) days and were used as Serum insulin levels were significantly de-controls.Animalswerenot subjectedto insulin creased(P<0.00001)in controlanimalsafterthetreatment. treatment period with Intralipid, but little

Fasting serum glucose (Reflocheck-glucose- reducedin CSA-treatedgroups (P<0.05).system—Boehringer-Biochemia-Robin),serum Serum creatinine levels were actually

Table 1. Laboratoryfindings

Blood glucose(mg/dl) Seruminsulin (U/I) Creatinine(pl/l)Start End Start End Start End

Group treatment treatment treatment treatment treatment treatment

1 (CSA lOmg/30days) 160±8 172±9 59.7±3.8 47.2±4.2 50.2±3.5 62.6±2.12 (CSA 10 mg/60days) 162±7 178±9 58.5±3.5 46.8±2.5 51.1±2.3 64.4±1.23 (Controls)* 157±9 265±15 60.1±2.1 32.1±5.1 50.7±2.9 51.2±2.4

* Intralipid.

Valuesaremeans±SEM.

CSA Nephrotoxicityin BB Rats 229

unchangedin control andgroup 1 (30-dayCSA seemto be affected.No signs of damagewereadministration) animals, and only slightly detectablein arteriesandarterioles.enhanced(P.<0.05)aftera60-dayCSAadminis-tration. DISCUSSION

Biochemicalresultsconfirm that CSA is ableHistologicfindings to protectBB rats from diabetes.But control

Control animals (group 3) showeda normal animals,receiving only the vehicle Intralipid,kidney appearance(Fig. 1). Group 1 animals becamediabetics with plasma glucose levelsshowedin somecasesonly slight, butno specific, exceeding250 mg/dl.alterations: tubule structure often appeared On the contrary, CSA-treatedanimals hadunchangedor showed only minimal changes, only minimal changesin glycaemicand serumnamely eosinophilic cytoplasmic inclusions, insulin values,asreportedinTable1. Moreover,found in a few cells of the proximal profiles after a 60-dayCSA treatmentserumcreatinine(Fig. 2). showed increasedvalues, even though small,

Conversely, after a 60-day treatment with indicating a possiblerenal injury. Studies ofCSA,cellsof proximal tubulesshowedclearsigns Whiting et a!. (1985)indicatedthat,in the adultof cytoplasmicvacuolation:the affectedtubular Sprague—Dowleyrats,impairmentof renalfunc-cells displayed varying degreesof vacuolation tion by CSA is observedusing oral dosesof(Fig. 3). The extent of this damagecan differ ~ 25 mg/kg/day.Functionalabnormalitieswere,markedlybetweenadjacentcells.The vacuoles, however, accompaniedby structural changespositive to the PAS reaction, are sometimes observed within 4 days of the start of thelarger than the nuclei (Fig. 4). The cytoplasmic treatment(Blair et a!., 1982;Whiting et a!., 1982;inclusionsare,moreover,eosinophilicandposi- Ryffel et a!., 1983).tive to the PFAAB, SudanBlack and alkaline ‘Isometric’ vacuolationis the mostconsistenttetrazoliumreactions.Distaltubularprofilesand finding in experimentalCSA toxicity andis veryglomeruliwerealwaysunaffected(Fig. 5). common in human material. It particularly

Semithinsectionsshowthat thevacuolesoften involvesthe straightpart of theproximalconvo-occupya greatpart of the affected cells andare luted tubules(Wallace,1985;Solezet a!., 1985).mainly distributed in their upper portion Tubular damage has been shown beyond(Fig. 6). In some instanceseven the basalcell reasonabledoubt to occur with CSA therapyportionshowsareasofslight vacuolation.Never- (Wallace, 1985). This conclusionwas reached,theless,the structuralintegrityof the cellsseems however, following experimentsin which theto be, on the whole,preserved, dosesof CSA administeredto the experimental

Ultrastructurally,after a 60-day CSA treat- animalsweregreaterthan the therapeuticdosesment almost all of the cells lining the proximal usedin man.Theearlylesionsdescribedincludetubulesshowlargevacuoles(Fig. 7),whilst,after degenerationandnecrosis,tubularmicrocalcifi-a 30-day CSA treatment,only fewer cells are cationanda mild lymphocyticinterstitialnephri-involved (focal tubular cell vacuolation).These tis withoutedema.Latelesionsarecharacterizedvacuolesare often locatedcloseto the nuclei in by tubular atrophy with basementmembranethe uppercell portion. Sometimesthey havean thickening,interstitialfibrosis,arterialandarter-elongatedshapeandare very extended.In the iolar thickening.subnucleararea a fine cytoplasmic stippling Otherauthors(Sibleyet a!., 1983;Myerset a!.,(basallipid droplets) is observable. 1984) havedescribedsix lesionsas being highly

In addition, focal areas of several densely characteristicof CSA nephrotoxicity,in ultra-stainedblebsof differentsizesfilling theproximal structural reports: ‘isometric’ vacuolation oftubular lumen were seen. Both smooth and proximal tubules, the presenceof eosinophilicroughendoplasmicreticulumare dilated. cytoplasmicinclusions(‘giant mitochondria’) in

Mitochondria,with regularcristae,are leng- proximal tubules, arteriolar thickening (CSAthenedand seemto be undamagedevenif they arteriolopathy),the accumulationof mononuc-are surroundedby the vacuoles. Signs of cell lear cells in peritubular capillaries, vasculardestructionwere not seen. In a few cases the thrombosisand‘striped’ interstitial fibrosis.Thenumberof lysosomesare increased, two lesionswhicharea consistentfinding in pure

Distal tubular profiles and glomeruli do not CSA nephrotoxicitymodels—isometrictubular

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I tg. I. Light nucrographofacontrolanimal showingthenormalkidneyappearance.Haernatoxvlin—easmstains’ ~S61.

I ig 2. Light inicrographbelonging to a 30—da~(‘SA—treatcdanimal~ho~ing a fe~~cellsof theproximal tubulecontainingcosunophmlmc vacimoles(arro~s).Haematox’shncosinstain ( x 22~I

CSA Nephrotoxicityin BB Rats 231

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~ ,~1~ww ~r _ _

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Fig. 3. Light micrographbelongingto a 60-dayCSA-treatedanimal showingnumerouscellsof theproximalprofiles containingPASpositive inclusions(arrows). PAS+ haematoxylineosinstain (x 223).

Fig. 4. Light micrographof a kidneybelongingto a 60-dayCSA-treatcdanimal. The proximal tubularcellscontainlargePASpositive inclusions,someofwhich arelargerthanthenuclei (arrows). PASreactionI x 890).

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I ig 5. Light micrographoFa 60-dab(‘SA-treatedrat kmdnc~showingseveralinclusionsin theproximaltubulesand unaffecteddistal tubularprofiles. Haematox~lineostnstain I ~ 223).

I ig 6. Light mierographof a kmdne~semithmnsectionbelonging to 60—day (‘SA—treated rat sho~~ing largesacuoles.mawR distributedin theupperportion of thecells.A slight acuolatmonof the basalcell portion can

I so he seen.Tt’l wilt ne BI tie stain I / 591))

CSA Nephrotoxicityin BB Rats 233

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Fig. 7. Electronmicrographof a proximaltubuleobtainedafter60 daysofCSA administration,showinglargevacuoles,locatedin theuppercell portion, anda fine cytoplasmicstipplingin thebasalarea(x 1647).

vacuolationandeosinophiliccytoplasmicinclu- specific lesionsof the proximal tubularprofiles.sions—donot correlatewell with the degreeof No abnormalitieswere found in distal tubularrenal functional impairment (Racusen et a!., cells nor in glomerularand vascularstructures1985). even when the CSA administrationwas pro-

Distal tubular cell damage following CSA longedto 60 days.treatment(40 mg/kg body wt) has rarely been Moreover as the creatininevalueswereonlyobservedin rats (Nemlanderet a!., 1982) and slightly modified,CSA seemsto affect tubularonly Thomson et a!. (1984) have described cells before they havedisplayedany consistentglomerularabnormalitiesin normalCSA-treated functionalabnormality.animals. Our resultsconfirmthat thestructuralmodifi-

In this studywehavefoundthataftera60-day cations increase with the duration of CSAadministrationof 10 mg/kg bodywt of CSA, administrationandthat the tubular injury is angivenas a therapeuticdose in orderto suppress importantfactor in CSA-inducedrenal failure.the inflammatory infiltrate of the islands of In clinical practice it has proved difficult toLangerhansin the early type 1 diabetes, a distinguishCSA-inducedrenalstructuralabnor-consistentnumberof the proximal tubule cells malities from those associatedwith low gradecontain vacuolesor other cytoplasmic inclu- rejection.sions. Theseinclusions wereeosinophilic, of a On theotherhand,wehavedemonstratedthatremarkablesize (larger than the nuclei) and the use of CSA (administeredin dosesandfor apositive to the PAS, alkaline-tetrazoliumor period analogousto clinical schedules)in sup-PFAAB reactions.This may indicate that they pressingthe BB rat isletitis is responsibleforcontainlipids, accordingto Solezet a!. (1985). tubular injuries involving the cells of the proxi-

Electronmicroscopyhasfurtherconfirmedthe mal profiles.

234 V. EspositoandU. Papaccio

Moreover due to the fact that the lesions relevanceto type I (insulin-dependent)diabetesin man.observeddo not seriouslyaffectthe cell’s struc- Diahetologia,22: 225--232.ture we may concludethat the damagemay be Myers, B. D., Ross, J., Newton, L., Leutscher. J. and

Periroth,M., 1984. Cyclosporin-associatedchronicneph-easilyreverstble.To this regardCunninghamet ropathy.NewEngi. J. Med., 311: 699—705.a!. (1984)andBertanieta!. (1987)havealready Nemlander,A., Socots,A., von Willebrand,E., Tallquist,U.

describeda spontaneousremissionwith appar- andHayry,P., 1982. Effect ofCyclosporin A on the in situent reversionto the normal structuralappear- inflammatory responseof rat renal allograft rejection.

Scand.J. Immun.. 16: 91-102.ance. Racusen,L. C., McGraw, D. J. and Solez,K., 1985. Renal

Heterogeneityand TargetCell Toxicity.Bach, P. H. (ed).Sussex,England,pp. 419—427.

Acknowledgements—MrGiuseppePaparo’stechnicalassist- Ryffel, B., Donatseh, P., Madörin, M., Matter, B Eance is acknowledged.Dr Giuseppe Corbetta (Sandoz, Rüttimann,G., Schdn,H., Stoll, R. and Wilson, J.. 1983.Milan) is acknowledgedfor supplying CSA. Mr Mike Toxicologicalevaluationof CyclosporinA.Arch. Tox., 53:Latronico for linguistic assistanceis alsoacknowledged. 107--141.

Schulman,H., Striker,G., Deeg,H. J., Kennedy.M., Storb.R. and Thomas,E. D., 1981. Nephrotoxicity of Cyclo-

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