and Gene

Age-dependent Expression of the Erythropoietin Gene in Rat Liver and Kidneys

Kai-UweEckardt,* Peter J.Ratcliffe,*Chorh C. Tan,* ChristianBauer, and Armin Kurtz

PhysiologischesInstitutder UniversitdtZurich, CH 8057Zurich,Switzerland;and *InstituteofMolecularMedicine, JohnRadcliffeHospital, OX39DUOxford, UnitedKingdom

Abstract

UsingRNAseprotection, wehavemadequantitativemeasure- mentsoferythropoietin (EPO) mRNA in liver andkidneysof developingrats(days1-54),todetermine the relative contribu- tion of both organs to the total EPO mRNA, to monitor changes which occur with development, and to compare the hypoxia-induced accumulation of EPO mRNA with the changesin serum EPO concentrations. To determine whether developmental and organ-specificresponsiveness isrelated to the type ofhypoxic stimulus, normobaric hypoxiawas com- paredwithexposure tocarbonmonoxide(functional anemia).

Underboth stimuli EPOmRNAconcentrationinliverwas maximal on day 7 and declined during development. In con- trast, EPO mRNA concentration in kidneyincreased during developmentfromday 1whenitwas30-65% thehepaticcon- centrationtoday 54whenitwas 12-foldhigherthan in liver.

Whenorganweightwasconsidered the liverwasfoundto con- tain themajority of EPOmRNAin thefirst threetofour weeks oflife, and although, in stimulated animals, the hepaticpropor- tion declinedfrom 85-91%onday1,itremained ^- 33%at day 54 andwassimilar for thetwotypesofstimuli. When normal- ized for body weightthe sumofrenal and hepaticEPOmRNA in animals of a particular age was related linearly to serum hormone concentrations. However, the slopeofthisregression increased progressively with development, suggestingage-de- pendentalterationsintranslationalefficiencyorEPOmetabo- lism. (J. Clin. Invest. 1992. 89:753-760.)Keywords: RNase protection radioimmunoassay*normobaric hypoxia* carbon monoxide*bilateral nephrectomy

Introduction

Erythropoietin(EPO)' isaglycoprotein hormone that ispro- duced ininverserelationshiptobloodoxygen contentand de- termines therateofred cellformation.ThesitesofEPO produc- tionwereoriginallydeducedfrom the resultsoforganablation studies and more recently from the distribution of EPO mRNA. Although these studies have firmly established the liver and the kidneysasproduction sites for EPO,discrepancies Address correspondence toK.-U. Eckardt, M.D., Physiologisches In- stitut der Universitat Regensburg, Universitatsstrasse 31, DW 8400 Regensburg, Federal Republic of Germany, which is his present ad- dress.

Receivedfor publication 3 April 1991 and in revisedform 3 October 1991.

1.Abbreviationused in this paper:EPO, erythropoietin.

exist and therelativeimportanceof eachorgan hasnot been determinedunequivocally.

Organablation studies have revealed that in adult animals theincrease in serum EPO levels inresponsetohypobarichyp- oxiais reducedby- 80-90%afterbilateralnephrectomy (1- 3), the remaining EPO formation being abolished when nephrectomy is combined with subtotalhepatectomy (4). In fetal and neonatal animals, however, bilateral nephrectomy wasfoundtohave littleor noeffectonEPOformation, whereas hepatectomy almost completely prevented an increase in serum EPOconcentrationsunderhypoxia(5-8).From agrad- ual change in the effect ofnephrectomy and/or hepatectomy onEPO formationduring developmentitwas inferredthatin sheepashiftofEPO production from livertokidney isinitiated during late gestation, withahepatic contributiontoEPO pro- ductionof 70% aroundbirth(9).Inrats,moreover,thekid- neysappearedtoplayno orlittle role in EPO formation forup totwoweeks afterbirth, thereaftergradually gaining increasing importance (2, 3, 10).

Detection of EPOmRNA by Northernblotting ofextracts fromdifferentorgansconfirmed liver andkidneyasthe sitesof EPOgeneexpression (I1, 12).Inadult animals the abundance of EPO mRNA inkidneyswasmuchhigher than that in liver (11,12).Furthermore, ina recentstudy Kouryetal. measured EPO mRNA levels in pooled extracts ofkidneys and livers fromdevelopingnormal and anemic miceusingbothNorthern blotting and RNAse protectionassay(13).They found thepro- portion of EPO mRNA in the liverdecreasing withage,but,in contrast towhatmaybeanticipated from theorgan ablation studies, they observed that the kidneys contain most EPO mRNA soon after they become macroscopically identifiable, i.e., during late gestation and all stagesof postnatal develop- ment.

Several possibilities could explain this apparent discrep-

ancybetween theablationstudies and the quantitative analysis ofEPO mRNA with regard to the role of theliver in EPO formation. Thus it is possible that EPO formation after nephrectomy and/or hepatectomymay not accurately reflect the situation inintact animals. Furthermore, the role of the liver for EPOformationmaybespecies specific and be of less importanceinmicethan inrats,goats, orsheep,or maydepend onthekindofstimulus used and be lesssignificantinanemia than inhypoxichypoxia. Alternatively,thefindingsmayindi- catethattherelationshipbetween EPOproduction rates and EPOmRNAcontentvariesbetweenliver andkidney,i.e.,that hepaticEPO mRNA is moreefficientlytranslated than renal EPOmRNA.

Therespective ability of liverandkidneystoproduce EPO andtheresponsiveness ofboth organs to different stimuliap- pearsofmajorimportancenotonly fortheregulationoferyth- ropoiesisaroundbirth,but may also haveimplicationsfor EPO formationunderconditions ofcompromised kidney function as inchronic renaldisease. We have thereforeaddressed the J.Clin. Invest.

© TheAmericanSociety for Clinical Investigation, Inc.

0021-9738/92/03/0753/08 $2.00 Volume 89, March 1992, 753-760

abovepossibilities by studyingtheagedependenceofEPOgene expression inratkidneys and liver underbasalconditions and following theexposure tonormobaric(hypoxic) hypoxia and carbon monoxide(functional anemia). Both stimuliwereap- plied forashortperiod of four hourstoallowmeasurementsat preciselytimed and closelyspacedpoints duringthe early post- natal phase and to avoid possible confounding effects from adaptiveprocesseswhichhavebeen observed withmorepro- longedexposure tohypoxichypoxia (14, 15). To investigate if the duration ofstimulation is of centralimportance in deter- miningorgan-specific responsiveness, additional experiments wereperformedonadult animals studied after three daysof repetitive bleeding. Therat waschosenasexperimental animal toenableadirectcomparison with the evidence available from organ ablation studies. To further judge the significance of theseorgan ablation studies for EPOformation in the intact organism,wehave alsoinvestigated the effect of nephrectomy onhepaticEPO geneexpression.To allowadirectcomparison betweencirculating EPO levels and EPO mRNA,serumEPO levels were determined by radioimmunoassay and EPO mRNA concentration was measured in kidneys and liver fromindividual animals usinga quantitative RNAseprotec- tionassay.

Methods

Animals.ZUR:SIVstrainratsof differentages werebredinthelocal animalhouse.Experimentswereperformedatages 1d,7d,14d, 28 d, and 54dpostnatally.Atages1,7,and 14 d rats werekepttogether with their mother untilimmediately beforetheexperiment. Afterthe sexof theanimalsbecameidentifiable,i.e., after14d,maleanimals were used only.

Stimulation ofEPOproduction. Forstudy oftheage-dependent changes of EPOgeneexpressionnormobarichypoxic hypoxiaorfunc- tional anemiacausedby carbon monoxideinhalation were usedto stimulate EPO formation. Animalswereexposedtothesestimuli for4 hinchambers that weregassed withamixture of normal airandnitro- gen ornormalair with addedcarbonmonoxide(0.1%). Duringhypoxic hypoxiaoxygen wasgraduallywashedoutofthechambers,reproduci- blyreachingaconcentration of11%after1 handaconcentrationof 8.5%after4h,asdetermined bymeansofafyriteoxygenindicator (Bacharach Inc., Pittsburgh,PA). Littermatesoftheanimalsexposed tohypoxiawerekeptatnormoxia andusedforstudyofEPOformation underbasalconditions.

Experimentswithanemicanimals.Forstudy oftheeffect ofmore prolonged stimulationonEPO geneexpressionadult rats(6-7wkold) werephlebotomizedonthreeconsecutive days.Underbrief anesthesia withfenatylcitrateandfluanisone animalswerebled ^- 6.5, 5.5,and 5 mlfrom femoralveinsondays 1-3,respectively, usingsalinefor vol- umereplacement.

Experiments withnephrectomized animals. Bilateral nephrecto- mies werecarriedoutinadultrats(6-7wkold)underlight methofane anesthesiathrough bilateral flank incisions. 7 h afterthe operation nephrectomizedrats wereexposedfor 4 hto0.1% carbonmonoxide togetherwithsham-operatedcontrol animals.

24 hafterthe lastphlebotomy(anemic animals),within 15 minafter theendofcarbon monoxide exposure, andwithin30minaftertheend ofhypoxic hypoxia, animalswerekilledbydecapitation(age 1-14d) followedbydirect collectionofblood orby cervicaldislocation(age 28-54d),after which blood was collectedfromtheabdominalaorta.

Kidneys and livers were removed and after determination ofwet weight kidneyswerecompletely and livers were in part or completely homogenized in guanidine thiocyanate (4 M) containing sarcosyl (0.5%),EDTA(10 mM), sodiumcitrate(25 mM),andmercaptoeth- anol(700mM).Differentamountsofguanidinethiocyanatewereused

tomaintainaconstant concentrationof - 0.04 g tissue per ml;except forthe kidneys ofl-doldanimals, when the concentration was 0.02 g tissue per ml.After severaldeterminationsin earlierexperimentsdem- onstrated that theconcentration of EPO mRNA is the same in all lobes of theliver(16), weighedportions of the right lateral lobe were used in thoseexperiments where only a portion of the liver was homogenized.

Tissuehomogenateswerefrozen at -80'C until preparation of RNA.

RNAseprotection assay. RNAseprotectionassay was performed as described (17).RNA waspurifiedbycentrifugationfor 20 h at 33,000 rpm on acesiumchloride gradient (5.7 M CsCl and 100 mM EDTA).

Aftercentrifugation RNA pellets were resuspended in 300UITE(10 mM Tris, 1 mMEDTA)containing0.1%SDS,precipitated with 3 M sodium acetate (0.1 vol) and ethanol (3 vol), and stored at -800C beforeanalysis.

Therat EPO probe was aPstI/SacI fragmentcontaining 132 bp of exon Vand - 300 bpofthe adjoining intron, inserted into pSP 64 for generation of RNAtranscripts.Transcriptswerecontinuously labelled with alpha32P-GTP(410Ci/mmol; Amersham International,Amer- sham, Bucks., UK). Forhybridizationaliquots of total RNA were dis- solved inbuffer (80% formamide, 40 mMpiperazine-N,N-bis(2-ethane sulfonicacid),400 mMsodium chloride, 1 mM EDTA pH 8) and the RNAconcentrationsdetermined by measurement of optical density at 260 nm. Theconcentration wasadjusted to yield

50-,ul

samples con- taining100-150,gtotal RNA.

Hybridization was performed overnight at60°Cwith0.5-1.0 X106 cpmradiolabelled EPO probe. RNAse digestion with RNAse A and TI was carried out at20°Cfor 30minandterminatedby theaddition of proteinase K and SDS beforepurificationofthe protectedfragmentsby phenol/chloroform extraction and ethanol precipitation and electro- phoresis on adenaturing 10% polyacrylamidegel.

Autoradiography of the dried gel was performed at -70°C. Pro- tected EPO mRNA bands were excised from the dried gel and counting wasperformed using a flat-bed liquidscintillation counter(1205;Beta- plater', Pharmacia-Wallac OY, Turku, Finland). The number of counts perminute obtained from each EPO mRNA sample was di- vided bythe quantity of total RNA analyzed to yield the EPOmRNA abundance(EPOmRNA/,ugtotal RNA). An external standard, con- sistingof pooled RNA extracted from the kidneys of severely anemic rats, was used tocorrect for any difference inquantitation arising be- tween assays.20,ugofthestandardwas run witheach assay and sample EPOmRNA levels wereexpressed relative to the EPO mRNA count in thecorrespondingstandard, which was assigned an arbitrary value of 1.0. Inearlier experiments the results of duplicate RNAse protection assaysof RNA prepared from the same organ showed a high degree of reproducibilityandassays ofdifferentconcentrationsofRNAprepared from thesame organ exhibited linearity ofthe assay overtheexperimen- tal range(18).

Thetotal organ quantity of RNA in kidney and liver was calculated from theamount of RNA extracted from aliquots ofhomogenate de- rived fromknown weights of tissue. There was areproduciblelinear relationship between the RNA yield and the weight of tissue homoge- nized.

EPOradioimmunoassay. SerumEPOconcentrations were deter- mined as described (19)with the use of a rabbitantiserum raised against purerecombinanthuman EPO andiodinatedrecombinanthu- manEPO(Amersham) as tracer. A rat serum pool enriched in EPO was prepared byexposing donor animals to hypoxia and was used as stan- dardaftercalibrationagainsttheII. International ReferencePrepara- tion by in vivo bioassay (19).Student's unpaired t test was used for comparisonof groups and analysis of variance todeterminesignifi- cance levelsoflinearregressions. P < 0.05 wasconsidered significant.

Results

Bodyweight, kidney, and liverweight oftheanimals studiedat different ages,the amount oftotal RNAextracted pergram tissue,andtheestimates oftheamountoftotalRNAin both

organs are given in Table I. In both liver andkidneysthe con- centrationsof total RNA declinedslightlyduringdevelopment andat day 54 were 42% lower inliverand36%lowerin kidney thaninneonatal animals. At each age level the amount of total RNAper gramtissue was about2-fold higherin liver than in kidney, and considering the differences in organ weight, the liver contained approximately 5- to 8-fold moretotal RNA than both kidneysduring the first three weeks and approxi- mately10-foldmore ondays 28 and 54.

Measurementsofserumerythropoietin concentrations.The serumEPO levels at thedifferentages underbasal conditions and afterexposure to normobarichypoxiaorcarbon monoxide aregiven in Fig. 1. Under basal conditions EPO levels were maximal one dayafterbirth (62.6±19.2 mU/ml, mean±SE, n

= 7) andthereafter declinedabout3-foldtoreach a level two weeksafterbirth that was similartothatof adults (17.7±5.5 mU/ml, mean±SE, n = 3). At all agesanimalsresponded to both stimuli with anincrease in EPOlevels. Theamplitudeof thisincrease, however,wasclearlyagedependent, with1.6-fold and 2.1-fold increases in newborn animals and 12-fold and 87-fold elevations in adults. Consequentlythe absolutelevels of serumEPO were 2- and 12-foldhigherin adult than in 4-d oldanimals(208±55versus98.1±12.7fornormobaric hypoxia and1,543±141 versus133.8±28.3 forcarbon monoxide stimu- lation, respectively;mean±SE, n= 8 [newbornanimals] or3 [adultanimals]).

MeasurementsoferythropoietinmRNA.To assess theaccu- mulation ofEPO mRNAunderlyingthese age-andstimulus- dependent variations in serum EPO concentrations, EPO mRNA in kidneyand livertissue ofeachanimalwasdeter- mined by RNAseprotection assay. Forquantitative analysis theabundance of EPO mRNA in eachsample(EPOmRNA per ,ug total RNA) was calculated from the radioactivity of protected fragments, and the value expressed relative to the EPO mRNA count ofan external standard that was run on eachgel. Figs. 2and 3 showexamplesofautoradiographsob- tained withRNA from liversand kidneysof rats atdifferent stagesofdevelopment. The mean valuesof therelativeabun- dance of EPO mRNA inliverandkidneys ofallanimalsare giveninFig.4A.Sincetheconcentration oftotal RNAinliver and kidney is differentandchangeswithage, theresponsive- nessofEPO geneexpression in liver andkidney tissueis more clearly represented when EPO mRNA is related to tissue weight.Accordingly,measurements of the concentrationofto- talRNAin eachorgan were used tocalculate theconcentration

2000-

1000

E E 0IL

E a

10

*

*

*

* *

01 7 14 28

age (days)

54

Figure1.Serumimmunoreactive EPO (irEPO) levels indeveloping

ratsunderunstimulated conditions(.),after 4 hof normobarichyp- oxia(A),andafter4 hofcarbonmonoxide exposure(m)(mean±SE, n =7-8 for 4-doldanimals,6for74 oldanimalsexposedtocarbon monoxide,and3for allothergroups).*Significant difference from unstimulatedcontrols.

ofEPO mRNA per gram wet weight oftissue. In addition, organweightswereusedtocalculate the total EPO mRNA in each organ.

Table II shows themeanvalues of EPO mRNAconcentra- tions per gramtissueweightatdifferent ages in stimulated and unstimulated rats. Under basal conditions the EPO mRNA concentration onday 1 was eightfoldhigher in liver than in kidney. Whereas in liver the basal EPO mRNA concentration

Table I.Body Weight and Weight and Total RNA Content ofLiver and Kidneys duringDevelopment

Liver Kidneys

TotalRNA/ Total organ Total RNA/ Total organ

Age Bodyweight Organweight gramtissue RNA Organ weight gram tissue RNA

days g mg g mg

1(n=23) 7.8±0.6 0.30±0.03 6.53±1.18 1.92±0.31 0.10±0.01 2.63±0.39 0.25±0.05

7(n= 12) 15.7±2.3 0.46±0.08 5.76±1.40 2.70±0.98 0.21±0.03 2.84±0.58 0.59±0.17

14(n=9) 35.1±2.2 1.01±0.09 4.79±0.35 4.84±0.27 0.36±0.04 2.17±0.20 0.79±0.10

28 (n=9) 93.4±12.4 4.51±0.77 4.54±0.77 20.4±3.9 1.03±0.13 1.95±0.17 2.00±0.18

54(n=9) 227.7±11.2 10.2±0.9 3.77±0.79 38.0±7.2 2.03±0.18 1.68±0.25 3.40±0.50

Mean±SD.

normobaric hypoxia carbonmonoxide

1 7 14 28 54

'P.

1 7 14 28 54

_11

1 7 14 28 54

Figure2. Autoradiograph,after6-dexpo- sure,ofanRNaseprotectionassayofEPO mRNA in thelivers ofratsunder unstimu- latedconditions,after 4 h of normobaric

hypoxiaorexposuretocarbon monoxide.

150 ,gof total RNA extracted from livers ofindividual animalswasanalyzedby

RNAseprotectionafterhybridizationto a

32P-labelledtranscript complementaryto 132bpofexonV and300bpof thead-

joiningintron of theratEPOgene.Note that EPO mRNAsignalsinliversaremax- imalonday1 (unstimulatedconditions)

orday7(normobaric hypoxiaand carbon monoxide)andthereafter declineprogres- sivelywithincreasingage.

declinedrapidly andwasbelowthequantifiablerangeby day 28, in kidney the basal EPOmRNA concentration declined fivefold within the first week andthereafterremained within thequantifiablerange andvaried onlyslightly. Afterstimula- tionEPO mRNAconcentration increasedinbothorgans at all agesbuttheamplitude of the increase and the levelattainedat differentstagesofdevelopmentwasquite differentbetween the twoorgans. Undernormobarichypoxiaandafterexposure to carbon monoxide the renal EPO mRNA concentration achievedincreasedprogressively withage,with the exception only ofaslightdecline in 7-d-old animals exposedto normo- barichypoxia, sothatby day 54it wasthree- andsevenfold higher thaninneonates. Incontrast theconcentration of EPO mRNA achieved in liver tissue in response to both stimuli showed littlechange between days 1 and14butthereafter de- creasedprogressivelysothatbyday54thehepaticEPOmRNA concentration was 10-fold less than that in kidney foreach stimulus. Whencomparing themean increases of renal and hepatic EPO mRNA concentrations afterstimulation, it be-

comesapparentthat the response of EPO mRNAconcentra- tionto stimulation byexposuretocarbon monoxide was al- ways greaterthan byexposure tonormobarichypoxia,butthe differencegenerally becamemoremarked withincreasingage.

However, withtheexception of7-dold animals, ineach age group the relative efficacy ofexposure to carbon monoxide versus exposureto normobaric hypoxia wassimilarbetween liverandkidneys.

The total amount ofEPOmRNA in liver andkidneys and therelativecontributionof thelivertoEPOformationin the whole animalareshown inFig.4B and Table III. It can be seen that the liveraccountsforaremarkablylargeproportion of the totalEPO mRNA and contains themajorityofEPOmRNA for thefirst threetofour weeks in bothstimulatedand unstim- ulatedrats.Duringthisperiod, however,thepredominanceof theliverwas mostpronounced under basal conditions.Thus theliver accounts for more than 94% of EPO mRNA inun- stimulatedanimalsduringthe first threeweeks,whereas under normobarichypoxiaand carbon monoxide, thehepaticcontri-

unstimulated

1 7 14 28 54

normobaric hypoxia

1 7 14 28 54

carbon monoxide

1 7 14 28 54

*._

__.

... .

.::-:<.:._Do < X_o

a. e

Figure3. Autoradiograph,after 14-hexpo- sure,of anRNAseprotection assayof EPO mRNA in thekidneysof ratsunder unstim- ulatedconditions,after 4 h of normobaric hypoxiaorexposuretocarbon monoxide.

150l goftotalRNA from unstimulated animals and animalsexposedtonormo- barichypoxiawasassayedand 100 ug of total RNAfromanimals exposedtocarbon monoxide. Insetshowssignals of protected fragmentsinunstimulated animals after 7-dexposureof theautoradiograph.While theabundance of EPOmRNA decreased withageinkidneysofunstimulatedani- mals,understimulation EPO mRNAsig- nalsgenerallyincreased withage.

Age (days)

Age (days)

a

756 K-U.Eckardt,P.J. Ratcliffe, C. C. Tan,C.Bauer,andA.Kurtz

unstimulated

unstimulated normobaric hypoxia carbon monoxide

** * *

*r

~~~~~*

*

200

B

100

*

10

1

0.1 0.03n.q.

* L

II

,;

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_{%% 6~~~~}

1 714 28

541714

28

541714

28

age

(days)

bution was91% and 85%in neonates. Althoughthispropor-

tiondecreasedwithageunder bothstimuli,atday54theliver stillcontainedonaverage32and 33%ofthe total EPOmRNA.

This hepatic contributionofaboutone-third in adult ani- mals isconsiderably higherthan thatwhich has been previ- ouslymeasured in anemic mice(13)orhas beeninferred from studies in bilaterally nephrectomized animals (1-3). Addi- tionalexperimentsweretherefore performed toinvestigate if EPO geneexpression inrat liver under the above short-term stimulation(a) differsfromthat during prolongedreductions

*

*

Figure4.Changeswithdevelopmentin the abundanceofEPOmRNA(A)andtheto- talamountofEPO mRNA(B)inratkid- neys(closedsymbols)andlivers(opensym-

bols).EPO mRNAwasquantifiedbyscin- tillationcountingofprotected

EPO

mRNA bands and isexpressedasEPO mRNA countsminusbackgroundcountspermi- crogramofRNAloaded,dividedbystan- dardcountsin eachgel

(Xl

0-4)(seeMeth- ods). Valuesaremean±SD,numbers of animalsasgivenin thelegendofFig. 1;

n.q.,notquantifiable. *In(A)indicatessig-

nificantdifferencefrom theabundanceof EPOmRNA inunstimulated animals and 54 in(B)indicatessignificantdifferencebe-

tweenthe totalamountofEPO^{mRNA in} kidneyandliver.

atocrit and

(b)

differs from that in

acutely nephrecto-

animals. For thispurposeseparategroupsofadultrats

;udied afterinduction ofanemia

by repetitive phlebot-

idafterexposuretocarbon monoxide

(0.1%)

afterbilat-

phrectomy.

7eriments with anemic animals.

Repetitive phleboto-

threeconsecutive

days (days 1-3)

resulted inadecrease atocritvalues from

40.4±3.3%

to

23.4±0.6%, 16.4±2%

±

1%

^on

days 2-4,

and a

corresponding

increase in EPOvaluesfrom

16±1.6 mU/ml

to

385±112 mU/ml,

TableII.Renal andHepatic EPO mRNA Concentrations(EPOmRNA/gTissue)duringDevelopment

Normobarichypoxia _{Carbonmonoxide}

Relativeincrease

(1.1-fold) 14.00±8.67 (2.7-fold) 20.30±13.23 (6.1-fold) 19.80±1.52

11.95±0.13 6.04±0.28

Unstimulated Normobarichypoxia

Relative increase Relative increase

(1.8-fold) 1.00±0.92 2.78±1.79 (2.8-fold) (6.1-fold) 0.19±0.08 1.49±0.30 (7.8-fold) (15.7-fold) 0.25±0.14 7.05±1.83 (28.2-fold)

0.36±0.15 7.97±4.39 (22.1-fold) 0.18±0.06 8.92±1.46 (49.6-fold)

EPO mRNA wasquantifiedbyscintillation countingofprotectedfagmentsinRNAseprotectionassays andrelatedtoanexternal standard(see Fig.4andMethods).EPO mRNAconcentrationsper gramwetweightoftissuewerecalculatedfrom theabundanceofEPOmRNApergram of total RNA(Fig.4A)andtheconcentrationsoftotalRNA per gram wetweightoftissue(TableI).mean±SD,n=7-8 for

4-d

^oldanimals,6 for74 oldanimals exposedtocarbonmonoxide,and 3 for allothergroups.nq, notquantifiable.

DevelopmentalChanges ofErythropoietin 500

1A

100-

10-

1-

n.q.J

z

w loo0

z

E

0 w

zcc

E

0

w

c

a 0of 0

*0

Age Unstimulalted days

1 7.96±7.27 7 3.33±0.91 14 1.26±0.39

28 nq

54 nq

9.10±4.16 9.04±2.33 7.71±2.73 2.00±0.84 0.91±0.43

,_Caro

9.07±5.76 33.96±26.41 26.06±11.82 90.30±7.59 59.89±12.29

Relativeincrease (9.0-fold)

(182.6-fold) (106.0-fold) (254.4-fold) (327.3-fold)

II v I I

Liver Kidneys

I I

Carbon monoxide

TableIII. TotalAmountofEPO mRNA (SumofRenal and Hepatic EPO mRNA) and Proportion of HepaticEPO mRNA duringDevelopment

Unstimulated Normobaric hypoxia Carbon monoxide

Hepatic Hepatic Hepatic

Total EPO mRNA/ Total EPO mRNA/ Total EPOmRNA/

Total EPO mRNA/ total Total EPO mRNA/ total Total EPO mRNA total

Age EPO mRNA bodyweight EPO mRNA EPOmRNA body weight EPOmRNA EPOmRNA body weight EPOmRNA

days rf % ^r % 8 %

1 2.41±2.19 0.322±0.30 95±3.2 3.01±1.24 0.371±0.15 91±5.1 4.77±2.74 0.616±0.39 85±10

7 1.72±0.66 0.133±0.05 98±0.5 3.76±0.83 0.289±0.10 93±1.4 18.57±12.89 1.021±0.69 64±18 14 1.44±0.52 0.041±0.02 94±1.2 9.75±2.17 0.284±0.08 73±9.1 30.02±6.88 0.351±0.19 69±11

28 0.34±0.14 0.004±0.00 17.99±5.80 0.189±0.07 55±16 141.4±19.71 1.548±0.03 35±2.4

54 0.34±0.11 0.001±0.00 29.28±6.77 0.140±0.07 32±12 182.1±32.62 0.818±0.14 33±3.9

Mean±SD; n=7-8forl-d old animals, 6 for 7-d oldanimalsexposed to carbonmonoxide, and 3 for all other groups.

1,278+511 mU/ml and 2,631±362 mU/ml, respectively (mean±SD, n=5).ThetotalorganEPO mRNA on day 4 was 157.7±42.8inkidneysand80.9±32.8inlivers(mean±SD);the resultingproportion ofhepaticEPO mRNA inrelationtothe total EPO mRNAwas34.4±11.5%(mean±SD)andnotsignifi- cantly different fromthatfollowingshort-termexposuretohyp- oxichypoxiaorcarbonmonoxide.

Experimentswithnephrectomized animals.Theamountof EPO mRNA and serum EPO levels in adult ratsexposedto carbonmonoxide7hafter bilateralnephrectomyareshownin Fig. 5. It can be seen that the serum EPO levels reachedwere only17%ofthosereachedin thesham-operatedcontrols. How- ever, hepatic EPO mRNA accumulation in the liver of nephrectomized animals was also significantly reduced to

- 55%ofthatobserved in thesham-operatedcontrols.

Relationship oferythropoietinmRNAto serumerythropoie- tin concentrationsduringdevelopment. Tofacilitatecompari- sonofEPO mRNA contentwith serumEPOconcentration, thetotal EPO mRNAcontent(thesum of renal andhepatic EPO mRNAcontent) for stimulatedandunstimulatedanimals atdifferentageswasnormalizedtothebody weight (Table III).

Whenexpressedinthisway, thetotal EPO mRNA inanimals ofaparticularagewasrelatedlinearlytotheserumEPOcon-

centration. This relationship was seen over a wide range of A

250^- 200 z

E 0 100 w

B

2000- E

1-E 0 tL1000-

E

00

control NTX

stimulated values irrespective of whether stimulation was by normobaric hypoxia or byexposure to carbon monoxide, as shown inFig.6. However, the slopeof the regression was not thesameforeach age group andincreased progressivelyduring development,being approximately ninefoldgreater in 54-d-old animalsthan in 1-doldanimals.Thus, when normalized for bodyweight,the totalEPOmRNAcontentachievedunder the mostpowerful stimulus was approximately constant with devel- opment(Table III),whereasthecorrespondingserumEPO lev- elsincreasedmarkedly(Fig. 1).

2000 1800 1600 1400 6 g 100- E o 800- 00

600-

400-

200-

O-

*L

control NTX

Figure 5. EPOmRNA(A)andserumimmunoreactiveEPO(irEPO) (B)inratsexposedtocarbonmonoxide7hafter bilateralnephrec- tomy(NTX) (n=7)andinsham-operatedcontrols(n=7).(A)dark shadowingillustrates EPO mRNA in livers andlightshadowingEPO mRNAinkidneysof controlanimals.QuantitationofEPO mRNA asinFig.4.*Significantdifference in theamountofhepaticEPO mRNA(A)andserumhormonelevels(B).

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 EPOmRNA/bodyweight(1/g)

Figure 6. Relationshipbetweenthe totalamountof EPO mRNA in theanimals, i.e.,thesumof renal andhepaticEPOmRNA, normal- izedforbodyweightand theserumimmunoreactive EPO(ir EPO) concentrations.Ateach age levelserumhormone levels correlated linearlywith EPO mRNA(day 1: P <0.0001,r:0.86; day7:P<

0.0001,r2:0.89;day 14: P<0.002,r2: 0.77; day28: P<0.0001,

r9:

1.00;day54: P<0.0001,r2: 0.93).Individual valuesareincluded onlyfor the youngest(o)and oldestanimals(o)investigated.

I

Discussion

A major control mechanism for EPO formation has been found to operate bymodulation of EPOmRNA(1 1, 12, 20) and this investigation demonstrates significant alterations in theamount of EPO mRNA in ratkidneys and liver during development, which is basically in accordance with the age dependenceofthe effects ofbilateralnephrectomy and hepatec- tomy on EPOformation (2, 5, 6, 10). In contrast toanemic mice, as recently shown by Koury et al. (13), in thepresent study thehepatic proportion of EPO mRNAwasconsiderable, accounting forthemajority ofEPOmRNAduringearlypost- natallife andfor about one-thirdofEPOmRNAduringadult- hood (Fig. 4B, Table III). The predominanceoftheliver in neonatal and juvenile animals was found irrespective of whether theanimalswerestimulated or notandregardlessof whether thestimuluswasnormobarichypoxiaor exposure to carbonmonoxide. Irrespectiveof changeswithagethehepatic proportion of the total EPO mRNA was infactvery similar under thesetwostimuliat 1, 14, and 54days, and somewhat lower only at 7 and 28 days in the carbon monoxidegroup (Table III).Furthermore, although thesestimuliwereapplied forashorttime only, the liverappearsnot torespond primarily in the early phaseof hypoxia, sincethehepatic contribution was also aboutone-third in adultanimalswithmoreprolonged reductions in hematocrit. Thus,despitethedifferences in the modeof limitationofoxygendelivery during normobarichyp- oxia,exposure tocarbon monoxide,oranemia, therewas no striking differencebetween the relativesensitivityof liver and kidney to each stimulus and under all conditions tested the liveraccountedforasurprisinglyhigh proportion ofthe total.

Clearly, however, allstimuliwererelativelysevere, sothatour resultsmaydemonstrate thepotentialforhepaticgene expres- sion rather than what is achieved under lessseverestimulation.

Foragiven stimulus,therewas acontinuousdeclineofthe proportionof hepatic EPOmRNAwithincreasingage,result- ing in a "shift" of the predominance from liver to kidneys betweenthree andfour weeks. Thisshiftwas notdueto a re- duction in the totalamount ofEPOmRNAin the liver,but rather resultedfrom a greaterincreasein the totalamountof EPOmRNAin the kidneys(Fig.4B). Whenorganweight is taken intoconsideration,it isevidentthat theconcentrationof EPOmRNA per gramlivertissuedecreasedwith increasingage oftheanimals, whereas the converse was true forrenalEPO mRNAconcentrations (TableII).Recently,EPOgene expres- sionwasdemonstrated by insituhybridizationinparenchymal aswellas nonparenchymalmurine liver cells (21). However, thetypeandsize of the hepatic cell populations producing EPO atdifferentstagesof developmentare yetunknown.Ifthenum- berofhepaticEPO-producing cells increases in proportionto liver growth, than the resultssuggestthatforagiven stimulus these cells produce progressively less EPO after about two weeksof life(TableII).Alternatively, the findings may indicate thatsubpopulations of hepatic cellsthat produce EPOprolifer- ateless rapidly than the majority of livercells. In thekidneys EPO mRNA increased out of proportion to the increase in organweight during development (TableII). Interestingly, in this regard, in adult mice withvaryingdegreesof anemia, the modulation ofEPO mRNA has been shown to reflect varia- tions inthe numberof cells in the renalcortical interstitium that express the EPO gene,withindividualcellsexpressingthe gene inan essentially all-or-none manner(22). Whether the

age-dependent increase in EPO mRNA also results from an increasing recruitment of cells remainstobeinvestigated.

When considering the significance of the accumulationof EPO mRNA, animportant question arises with regardtothe efficiency with which EPO mRNA in liver and kidneys istrans- latedtoachievearise inserumhormone concentrations under different stimuli andatdifferentstagesofdevelopment. Ifone assumesthatthedistribution volume for EPO, that has been shown in adultratsto correspondto plasma volume (23), is proportionaltobodyweight, itappearsjustifiabletocompare serumEPO levels with the totalamountof EPOmRNA, i.e., the sumof renal and hepatic EPO mRNA, after it has been normalized by body weight.Ateachagelevel,this comparison revealed significant linear relationships (Fig. 6), a result in keepingwiththeviewthat mRNAaccumulation is themajor controlmechanism of EPO formation.Interestingly,however, theslope of thisrelationship increases about ninefold during development. Thusserum EPO levelsat agivenstimulus in- creased with age (Fig. 1), an observation in accordance with previous studies (2,5, 10, 24, 25), although the totalamountof EPOmRNAwhennormalized forbodyweightwaseithercon- stant or evendeclined withage(Table III).

This age-dependent change in the relationship between EPO mRNA and the circulating hormone concentrations could arise by several mechanisms which the present study cannotclearly differentiate. Age-dependent alterations in both EPOproductionandmetabolism,e.g., adecrease indistribu- tion volume or clearance rate of the hormone are possible.

With regardto EPO clearancerate someevidence has in fact recentlybeenprovided in humans that the halflife timeofEPO inneonates(26)isshorter than thatin adults (27, 28). On the other hand, the fact that the increase in the ratio between serumEPO levels and EPO mRNAcoincides withtheshifting ofEPOgeneexpression fromlivertokidneys, raisestheques- tion whether thetranslational efficiencyin theliver isless than that in thekidneys,orwhetheraproportionofthe newlysyn- thesizedhormonein thelivercannot enterthesystemic circula- tion,since the liver is probablyamajor site of EPO clearance.

Interestinglyinthis regard,noEPOwasdetected byFriedetal.

(29)orCaroetal.(25) bybioassayfor EPO in liverextractsof hypoxic newborn and developingrats; andClemonset al.,us- ingaradioimmunoassay, found less EPO intheliverthanin thekidneys of hypoxicrats asearlyas twodaysafter birth (24).

Furthermore, although theagedependenceofthe effect ofbilat- eralnephrectomyonEPOformationshown by others (2,5,6, 10) is completely in accordance withtheshift ofEPO mRNA accumulationasobserved inthisstudy, aputativediscrepancy appears toarise with regardtothe quantitative contributionof theliver;whilewefoundmorethan 30%oftotal EPO mRNA in the livers ofadult rats (Fig. 4, Table III), several studies showedthatserumEPO levelsafter bilateralnephrectomyare reduced to ^- 10-15% (1-3, 30). To further investigate the causeofthisdifferencewemeasured EPO mRNAaccumula- tion in the livers in aseparate groupof bilaterallynephrecto- mizedrats.

Interestingly, as shown in Fig. 5, the amount ofhepatic EPOmRNAinthenephrectomized animals wassignificantly reducedto ^- 55%. Thissuggests that, due to an inhibitionof EPOgeneexpression inthe liver, studies in nephrectomized animalsmay haveunderestimatedthe contributionoftheliver toEPOformation.Furthermore,sinceserumEPO levelsafter nephrectomywereonly slightly morereduced thanthetotal

DevelopmentalChanges ofErythropoietin

amount of EPOmRNA, it appearsunlikely that the relation- ship between EPO mRNA and effective EPO productionrate intheliver isgenerally much lower than in thekidneys. Thus thedisproportionality betweenEPOmRNA and serum EPO levels during developmentis probably notprimarilydue to a switch in the site of production.

Inconclusion, this study shows that: (a) despite amarkedly increasingresponsiveness of developing rats in terms of serum EPO levels underhypoxia, the amountofEPO mRNA, when related to body weight, is rather constant or even decreasing during development;(b)theliver contributesmorethan 50% to thetotal amount of EPO mRNA for up to four weeks of life;

and (c) in adult animals the contribution of the liver under strong stimulation is moresignificant than previously recog- nized.

Acknowledgments

The technical assistance of U. Bolliger and C. Gasser is gratefullyac- knowledged.

This workwassupported by theSwiss National Science Foundation (grant 31-9433.88), the HartmannMuller Foundation for Medical Re- search, and the National Kidney Research Fund, Medical Research Council andWellcome Trust, United Kingdom.

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