Plasma Reason

Increased Plasma Viscosity as a Reason for Inappropriate Erythropoietin Formation

A. Singh,*K. U.Eckardt,1A.Zimmermann,*K. H.

Gdtz,l

M. Hamann,'P.J.Ratcliffe,11 A.Kurtz,§and W. H. Reinhart*

*DepartmentofInternal Medicineand*InstituteofPathology, UniversityofBern, CH 3010Bern,Switzerland;4 InstituteofPhysiology University ofRegensburg, D-8400 Regensburg, Germany;andIIlnstituteofMolecular Medicine, JohnRadcliffHospital,

Oxford GB-OXJJAA, United Kingdom

Abstract

Theaimofthis studywas to examine whether alteredplasma viscositycouldcontributetotheinappropriatelylowproduction rate of erythropoietin (EPO) observed in patients suffering fromhypergammaglobulinemias associated withmultiplemy- eloma orWaldenstrom's disease. We found that the EPO for- mation in response toanemiain thesepatients was inversely relatedtoplasmaviscosity.Asimilar inverserelationship be- tweenplasma viscosityand EPOproductionwas seenin rats in which EPOformationhad beenstimulatedby exchange^trans- fusionand theplasma viscosityof whichwastherebyalteredby using exchange solutions of different composition to alter plasmaviscosityand thus whole bloodviscosityindependently fromhematocrit. Raising the gammaglobulin concentration to

- 40mg/ml plasmaintheratsalmosttotallyblunted the rise in serumEPOlevelsdespiteafall of the hematocrit to20%.

Determination

of renal EPO mRNA levels byRNaseprotec- tionrevealedthatthereductions inserumEPO levelsathigher plasmaviscositieswereparalleled by reductionsinrenal EPO mRNAlevels.

Takentogether,ourfindingssuggest thatplasma

viscosity

may be a significant inhibitory modulator of anemia-induced EPO formation. The increased plasma

viscosity

in

patients

with

hypergammaglobulinemias

may therefore contribute to the

inappropriate

EPOproduction,which isamajorreasonfor theanemia

developing

in these

patients. (J.

Clin. Invest. 1993.

91:251-256.)

Key words:

viscosity

*

erythropoietin

*parapro- teinemias*

gamma-globulins

^-dextrans

Introduction

Multiple myelomaand macroglobulinemiaWaldenstromare frequently associated with anemia ( 1). The pathogenesis of this type of anemia hasnotyet beenunequivocallyclarified but may involve bone marrow

displacement

and/or insufficient stimulation oferythropoiesis.Recent studies haveprovidedevi- dence thaterythropoietin (EPO)' levels in thosepatientsare

inappropriately

low and thatthistypeof anemiacanbe mark- edlyimproved by the applicationof recombinanthuman EPO Addresscorrespondence to W. Reinhart, M. D., Internal Medicine, Kantonsspital, CH-7000 Chur, Switzerland.

Receivedforpublication 24 March 1992 and in revisedform 20 July 1992.

1.Abbreviations used in this paper: EPO, erythropoietin; hct, hemato- crit.

(2, 3). In the adult EPO ispredominantly produced by the kidneys, and its production rate is mainly determined by the hemoglobin concentrationandthearterial oxygentension(4).

Thereasonswhy patients sufferingfromhypergammaglobulin- emias elaborate inappropriately low amountsoferythropoietin areunknown. Reduced renal functionwillcontribute butdoes notprovideacompleteexplanation.Anotherpossibility is that thealteredbloodrheology, namely,the increasedplasmavis- cosityin thosepatients couldbe involved. Since atpresentno information exists aboutapossible influence of blood viscosity on EPO formation itappeared reasonable to us to examine whetheralterations ofplasmaviscositycan have aninfluence ontheproductionoferythropoietin.

Tothisend we haveexaminedthecorrelationbetween EPO production and plasmaviscosityor renalfunctionin anemic patients suffering from hypergammaglobulinemias.Moreover, weinvestigatedtheinfluence ofplasmaviscosity on EPO for- mation innormal rats,in which EPOformationhadbeen stim- ulatedbyexchangetransfusion.

Our findings indicate thatinanemic patients with hyper- gammaglobulinemias insufficient EPO production is corre- lated with the increase ofplasma viscosity ratherthan with restriction of renal function.Moreover, we observed an inhibi- toryeffect

of

increasing plasma viscosityon EPOformationin anemicrats.

Methods

Patients. Patients with documented monoclonal paraproteinemia (Waldenstrom's macroglobulinemiaormultiple myeloma), without major impairment of renal function (serum creatinine < 150,mol/

liter, creatinine clearance >30 ml/min) and without evidence of chronic inflammatory disease (no fever and leukocyte count

<

10,000/,u

withessentially normal distribution)werestudied. Blood anticoagulated with K2-EDTA wasused. Hematocritwasmeasured withamicrocentrifuge, the plateletcount wasdetermined withan elec- tronicparticlecounter.Plasmawasobtained byafirst centrifugationat 1,500gfor 10 min, followed by gentle aspiration of plasma anda second10-mincentrifugation oftheplasmaat 1,500g. Plasmaviscos- itywasdetermined inaCouette-type viscometer (model LS30; Con- traves,Zurich,Switzerland)at37°C and shearratesof10.2 and 87s-' and the arithmeticmean wascalculated. Plasma samplesweredeep frozenat-20'Cfor EPOanalysis (see below).

Bloodexchangeexperiments in rats. Male Wistar and Sprague- Dawleyrats(bodyweight 180-350 g)wereanesthetized with 10mg Nembutal intraperitoneally. When required smalldosesofthe anes- theticweregivenrepeatedlyduring the experiment.Theanesthetized animalswereplacedon aheating table.A rectaltemperature probe connectedto athermostat held the coretemperatureof the animalat 37.0±0.5'Cbyregulating the heating ofthe table. Anarterialcatheter (either femoralorcarotid) andavenous catheter(eitherfemoralor jugular)wereinsertedavoidingasfaraspossibleany bloodloss. Blood pressure wasmonitored byaStathamtransducer connected to the arte-

rialcatheter.Blood exchange wascarried out with one of four different J.Clin.Invest.

©TheAmericanSocietyfor ClinicalInvestigation, Inc.

0021-9738/93/01/0251/06 $2.00 Volume 91, January 1993, 251-256

solutions: (a)ratplasma prepared from donor rats;(b)Ringer's solu- tion (130 mmol/literNa, 4 mmol/liter K, 1.5 mmol/liter Ca, 109 mmol/liter Cl, 28mmol/literlactate,l0g/literglucose);(c)Ringer's solution supplemented with graded doses of rat or bovine gammaglobu- lin (G 2885 and G 7516;Sigma Chemical Co., St. Louis, MO); or (d) Ringer'ssolution containing 260 g/liter dextran 40 (mol wt - 40,000;

Pharmacia AB, Uppsala, Sweden).

The plasmaexchangewasstartedby gentlyaspirating2 mlof blood into asyringeflushed with sodium heparin ( 10 U/ml), whichwasused for the determinations ofblood gases, plasmaviscosity, hematocrit, andEPO.Afterimmediate replacement of this volume withexchange solution, blood exchangewascontinuedoverthenext 10-20 minby simultaneous blood withdrawalonthearterialside andinfusion of the exchangesolutiononthevenousside. After thecompletionof theex- change(time: zero)aninfusion of thesamesolutionasusedfor the exchangewasstartedwithaflowrateof 0.6ml/htoreplaceusualfluid lossbyperspiration anddiuresis.4h after thecompletionof the ex- change(time:4h)2 mlofheparinizedbloodwasagain aspiratedfor the measurement ofhematocrit, plasmaviscosity, EPO, and arterial blood gasanalysis.

Atthe end of theexperimentthe animals werekilledbycervical dislocation or an overdose of nembutal. Thekidneyswere removed andweighed.Onekidneywassnapfrozen inliquid nitrogenandkeptat -70°C for EPOmRNAanalysis.The otherkidneywas cutinto thin slices,fixed in Bouin'ssolution,and later embedded in methacrylate.

Semi-thinsectionswerepreparedandexamined byapathologistwho wasunawareof theexperimental procedures.

The hematocrit level was determined with a microcentrifuge.

Plasmawasobtained bycentrifuging the blood for 10 minat 1,500g.

Plasma viscosity was measured asdescribed above. Aliquotsof the plasma were deepfrozenfor thedetermination of EPO.

Determination ofserum EPO. Serum EPO concentrations were measuredbyradioimmunoassayasdescribed(5).Forhumansamples humanrecombinant EPOwasusedasstandard, forratsamplesa rat EPO standardwasusedfor the standardcurve.

DeterminationofEPOmRNA.Kidneys fromratssubjectedtodif- ferenttreatments werehomogenizedinguanidinethiocyanate(4mol/

liter)containing sarcosyl (0.5%),EDTA(10 mmol/liter),sodium ci- trate(25mmol/liter),andmercaptoethanol(700mmol/liter).Total RNA waspurifiedbycentrifugationfor 20h at33,000rpmon acesium chloridegradient(5.7mol/literCsCl and100mmol/literEDTA)and EPO mRNA was measuredby RNAse protection asdescribed(6), usinga32P-labeledEPOprobe,whichwas aPstI/SacIfragmentof the ratEPO gene,containing 132 bp ofexonVandapproximately300bp of theadjoiningintron.Inbrief, 100 ,ug of totalRNAwashybridizedto 0.7x 106 cpmof radiolabeledprobein80%formamide/40mmol/liter piperazine-N,N'-bis(2-ethanesulfonicacid), pH 6.4/400 mmol/liter NaCl/liter mmol/liter EDTAat60°Covernight,and RNasedigestion wascarriedout at20°Cfor 30 min. Protectedfragmentswereseparated byelectrophoresison adenaturing 10%polyacrylamidegel.Autoradi- ographyof the driedgelwasperformedat-70°C.

Statistics. Student's unpaired t test wasused for comparison of groupsand analysisof varianceto testthe significanceof linear and exponentialregressions.P<0.05wasconsideredsignificant.

Results

16 patients with monoclonal paraproteinemia were studied, some ofthem repeatedly during ambulatory care. They are

summarizedinTable I. All the

patients

wereanemic(hemato- crit17-38%)butmostofthem had normalplateletcounts.The EPO levelsrangedfrom 15to246mU/ml.The threepatients with platelet counts< 100x 109/liter (patients7, 8, and 15) werethosewiththehighestEPO levels.Thissuggestsageneral depressionof the bonemarrowfunctionbyother factorswith an ensuing strong stimulation of EPO secretion.

Indeed,

in

patient7 averysevere bonemarrowinfiltration of70-100%

wasfound, inthe two others amarked infiltration of60-80%

wascombined with vitamin B12-deficiency (Vitamin B12: 19 and 131 pg/ml,respectively, normal values 150-630pg/ml).

Atfirst sight plasmaEPO levels appeared to berelatively lowwhen comparedwiththe degreeofanemia and it was there- fore of interest to see whether the inappropriately low EPO production correlated withotherclinicalparameters,in particu- larplasmaviscosityand serumcreatinine concentrations.EPO production and plasma EPO levels, however, are normally strongly dependentonthehematocrit and thehematocrit var- iedin awiderangeamongthepatients under study. To allow a correlation between the reduction of EPO production and otherclinicalparametersitwas necessarythereforetostandard- izeEPOproductioninrelationtothe degreeofanemia in those patients. Sinceanumberof studieshave shown that anegative semilogarithmic correlationexists between plasma EPO con- centrations and the hematocrit value in nonrenal anemias (8-10)we consideredthe value(log

EPOpatient

^- log

EPOnor.

ma)/(patient

-

hctnomal)

as astandardizedparameter which we termed "EPO response." Normal EPO concentration of healthy adults (mean hematocrit 45%) average 17 mU/ml when measuredwith theradioimmunoassayusedfor this study (10). For nonrenal anemias average valuesfor the EPO re- sponse arein the rangeof6to10(8-10).The EPO responsefor theindividual patients in thisstudywas setintorelation with theplasmaviscosityorwithcreatinine clearanceas anindirect indicator for renal function. In patients withmorethan one measurement(patients 1-4and 9) mean values were used. It appeared that the EPO response was correlated with both plasma

viscosity

andcreatinine clearancevalues(Fig. 1). The best fitbetween plasmaviscosityand EPO-response wasob- tainedwithan

exponential

inversecorrelation: EPOresponse

= 4.4 Xplasma

viscosity(cp)-'1'3(r

= -0.54; P< 0.01). The best fit between EPOresponseandcreatinine clearancewas a linearcorrelation: EPO response= -0.37 +0.05 Xcreatinine clearance(r= 0.62,P<0.02).

The questionarose as to whether there wasa causallink between EPOresponseandplasma

viscosity.

Toanalyze this we haveperformeda seriesof experiments with anesthetized rats. In theseanimalsEPOproduction wasstimulated by an acutereduction ofthe

hemoglobin

concentrationtohalfnor- mal values induced by volume exchange transfusion (Table II).Volumeexchangewasperformed with solutions of differ- entviscositiessuchas

Ringer's solution,

ratdonorplasma,or Ringer's solution supplemented with graded doses of bovine

gammaglobulin.

4 hafter conclusion of the volume

exchange

plasma EPO levelsweremeasured. As shown in

Fig.

2the in- crease ofplasma EPO levels reflecting EPO release into the circulation was dependent on the solution used for volume exchange. Highest EPO levels were measured with

Ringer's

solution followed by plasma and Ringer's solution supple- mented with gammaglobulin. A sham exchange

experiment

(replacement withwholeblood)didnotelevate the EPO level markedly(44mU/ml).

As with the patients we have calculated the EPO re- sponse (log EPO4hafterexch - log

EPOb,fore

exch) / (

hct4

hafter exch

-

hctbefore

exch) fortheindividual ratsand havecorrelated this parameter with the plasma

viscosity.

As shown in

Fig.

3 the EPO response ofthe rats was

inversely

correlated with the plasmaviscosityinaverysimilar fashionaswith the

patients

TableI. Patients with Waldenstrom'sDiseaseand Multiple Myeloma

Pat Serum Creatinine Platelet Plasma

No. Sex Age Diagnosis creatinine clearance count viscosity* Hematocrit EPO

,umol/liter ml/min X109/liter cp % mU/liter

1 f 74 Waldenstrom(IgMKappa) 75 45t 246 2.4 34.5 18

1.6 32.5 17

2 f 79 Myeloma(IgGLambda) 109 35t 181 4.0 20.5 21

2.3 22.5 84

3 m 76 Waldenstrom(IgM Kappa) 133 50t 128 10.4 22.5 17

7.4 23.5 17

7.2 24.0 16

6.0 16.5 40

7.8 19.5 17

4 m 54 Waldenstrom (IgMKappa) 106 66t 147 2.8 33.5 21

9.8 36.5 15

8.2 35.5 18

9.2 37.0 21

6.8 37.0 20

S f 58 Myeloma (IgA Lambda) 87 60$ 1.9 31.5 23

6 m 65 Waldenstrom (IgM Kappa) 89 72t 470 3.9 38.0 30

7 m 41 Waldenstrom(IgMKappa) 91 110 16 1.5 24.5 208

8 f 63 Myeloma(IgG Kappa) 66 103 89 1.4 26.5 246

9 f 77 Waldenstrom(IgM Kappa) 107 41t 2.4 36.0 23

1.4 30.5 18

10 m 83 Waldenstrom(IgM Kappa) 98 69 263 2.8 25.5 36

11 m 71 Myeloma(IgG Kappa) 117 52t 110 1.5 24.0 145

12 f 68 Waldenstrom(IgM Kappa) 80 67t 174 1.9 29.0 46

13 f 80 Myeloma (IgG Kappa) 87 36t 128 1.4 31.0 41

14 f 78 Waldenstrom (IgM Kappa) 125 32 421 1.4 31.0 51

15 m 64 Waldenstrom(IgMLambda) 90 71 35 2.2 17.0 180

16 f 74 Myeloma(Light chains) 67 97 430 1.7 25.0 99

*Normal range: 1.1- 1.4 cp. * Calculated accordingtoCockcroftand Gault (7).

lIog EPO

Ahct 7

6

5 4 3 2

plasma viscosity(cp)

10 30 50 7b 90 110

creatinineclearance(ml/min)

Figure 1. (Left panel) Relationship between plasmaviscosity and EPO responseinpatients suffering from multiple myelomas and Waldenstrom's disease. EPOresponseis definedas(logEPOpatient

-log

EPO,,.,,,a,)/(hctjati

^-

hctn.0,,m).

The dashed line indicatesthe regression line. Thesymbol withacrossrepresentsavaluefor nonrenalanemiasasestimated from reference 10. (Right panel)Re- lationship betweencreatinine clearance and EPOresponsein patients suffering from multiple myelomas and Waldenstrom's disease. The dashed lineindicates theregression line.

sufferingfromhypergammaglobulinemias.Thereasonforus-

ing bovine gammaglobulin for the exchange experimentsre-

sultedfromtherelatively^pooravailabilityof ratgammaglobu- lin. To test for the possibility that bovine gammaglobulin mighthave hadnonspecificeffectsonEPOproductioninthe rats,three animalswereexchangedwithRinger'ssolutionsup-

plemented with 60 mg/ml rat gammaglobulin. In these ani- mals hematocrit fell from 42.5±1.8% beforeto22.0±1.8% 4 h afterexchange. Serum EPOlevelswere21±4and 55±3 mU/

ml (mean±SD)before and 4 h after exchange, respectively.

These values are very similarto those obtained with bovine gammaglobulin, suggestingthatapossible incompatibilitywas

notthereasonfor thedose-dependentattenuation ofEPO for- mationbybovinegammaglobulinintherats.Alsootherparam- eters that might interfere with renal EPO formation such as

changes of the arterial oxygen (Table II) or carbon dioxide tensions or changes of hemodynamics were not different amongthedifferentgroups.

Since the plasma viscosity is causallylinked with plasma concentration of gammaglobulins, it could not be distin- guished whether the attenuation of the EPOresponsewasde- pendent on the increaseofviscosityor duetoanother effect inducedbyanincreased level ofgammaglobulins. We there-

omyeloma

*Waldenstrom's disease

00 ~~~~~~~~~~~~~0

jo

0

0.

*⁰⁶. ^0~~~~~~~~~

TableII. ExchangeTransfusionExperiments in Rats

Hct (%) Systolicblood pressure Arterial P02

Before Before Before

Exchangewith exchange 4 hafter exchange 4hafter exchange 4hafter Body weight

mmHg mmHg g

Ringer's 39.4±4.2 23.2±3.1 170.1±7.3 100.7±20.3 90.7±10.1 109.7±6.3 202.0±4.8

Plasma 35.3±14.8 21.5±4.1 174.0±18.2 116.3±17.0 86.7±4.7 112.2±9.4 217.6±23.3

Ringer's +30g/liter

gammaglobulin 41.6±4.9 20.7±2.0 160.8±22.0 109.2±11.6 80.8±7.3 96.2±9.8 223.8±15.1 Ringer's

+60g/liter

gammaglobulin 41.2±1.9 19.8±2.1 144.2±13.5 96.0±7.4 77.9±7.9 111.5±7.1 189.4±26.5

foreattemptedtoincreaseplasmaviscositywithout increasing the plasma concentration ofgammaglobulin. To this end a volume exchange transfusion with Ringer's solution supple- mented with260g/literdextran 40 wasperformed. Exchange transfusion with this solution led to a similardropinhemato- crit as withRinger's solutionalone;theplasmaviscosity,how- ever,increased from 1.13±0.07cpbefore to1.75±0.13cp after exchange.The rise ofplasmaEPO levels inducedbythereduc- tionofthehemoglobin concentrationwasmarkedlyattenuated in thepresenceof dextran (Fig. 4).

Histological examinations ofthekidneysbylight micros- copydidnotrevealobvious differencesamong thefourgroups.

(Twoexperiments withRinger's solution,twowith plasmaex- change,three withgammaglobulin [60 mg/ml]andfiveexper- iments with dextran). Glomeruliandinterstitiumwere normal in allspecimens. Slighttubularnecrosisorepithelial swelling was seeninoneof five and slightvacuolization oftubularepi- thelia in fourof five dextran-treated rats.

Kidneys

ofrats ex-

changed with gammaglobulin occasionally showed tubular castsin themedullary region.

The synthesis of EPO by thekidney was assessed bymea- suring EPO mRNA concentrations in the kidneys 4 h after conclusion of the blood volumeexchange. As shown inFig. 5 renal EPO mRNA levelswerediminished if volumeexchange wasperformed with solutions which increased plasma viscos- ity. Moreover, it was apparent that the diminuation of renal EPO mRNA levels went in parallel with the attenuation of plasma EPO concentrations.

Discussion

Although it is well established that altered blood oxygen avail- ability is theprincipal stimulus controlling EPO production, in

,&IlogEPO Ahct

6.0

5.01 Figure 2. Plasma EPO

concentrations inrats before and4hafterex- changetransfusion, whichwasperformed with Ringer'ssolution, donorplasma, and Ringer's solutionsup- plemented withbovine gammaglobulin.EPO values in animalsex- changed withplasmaor supplementedRinger's solutionsweresignifi- cantly lower than those inanimals exchanged withpureRinger'ssolu- tion.

4.01

3.0I

2.0

1.0 1.1

1.2 1.3 1.4

plasma

viscosity (cp)

Figure 3.RelationshipbetweenEPOresponse andplasmaviscosityin rats4 hafterexchangetransfusion.

EPO

mU/ml

Wbefore 1 1

*~~~xchange

Elz *hafter

jiTj ~~exchange iii~~~:::iiiger

i~~~~~.-lbi0mg/i

iiiiiiiii 1=Rng3

n=15

^-1 ii

\ =Ringer 2- Plasma

\n-12

3=Y-Glob. 30mg/mI

- ~:~-4=Y-Glob. 60mg/mI

"'- n=6 'S 3

*% n=8

\>4

3501 300[

2501 200[

1501

100l 50

n=15 n=12 n=6 n=8

mU/ml EP0

400F i

300F

before exchange 4 hafter exchange 1=Ringer 2=Ringer +

Dextran 40

1260g/1)

200F 2- 1001

B..... .

... ^...

...

ht 42.8 25.9 43L 25.8

hct(%)+14 ±059 ±21 ±2.1

n=11 n=9

Figure4. Plasma EPOconcentration inratsbefore and4 hafterex- changetransfusion withRinger'ssolutionorRinger'ssolution sup- plementedwith dextran 40(260g/liter).Dextransupplementation causedasignificantreduction in EPO values.

a number of situations EPO production is lessthan thatex-

pected for the reduction in hematocrit. This studyaimed to

examine if changesof plasma

viscosity

couldhaveaninfluence onEPO

production

andcould thus contributetothe

inappro-

priate EPO

production

observed in

patients

with increased plasmaviscosity(2,3)duetomonoclonal

gammopathies

such as multiple myelomaorWaldenstrom's diseaseorpolyclonal

gammopathies

ofseveredegree

occasionally

seen, e.g.,in rheu- matoidarthritis (11, 12). In agreementwith

previous

studies we found that serum EPOlevels are in factrelatively low in patients with multiple myeloma or Waldenstrom's disease when setintoproportiontothedegreeof anemia. This circum- stancebecomes moreobviousifthe EPO response,definedas therise ofserumEPO relatedtothe

degree

of

anemia,

iscom-

pared withthatofpatients

suffering

from

hyporegenerative

ane- mias. Ourfindingsshowaninversecorrelation betweenEPO responseandplasma

viscosity.

Astriking feature of thiscorre- lation is that comparably small changes of plasma viscosity correlatewith markedchangesof the EPO response(Fig. 1).

AnimpairedEPO response ischaracteristic fortheanemia associated with chronic renal insufficiency. Since hypergam- maglobulinemiasmayinducearestriction ofrenalfunction, it appeared reasonableto us toconsideranimpairment ofrenal function as apossible

pathophysiological

mechanism forthe inappropriateEPOformation inhypergammaglobulinemicpa- tients. The EPO response correlated with creatinine clearance, which is surprising because it has been shown that erythropoie-

Io

^I)extran_{1 1tIfI} ^Ringer

_{~ ~}

_II'^$-AIob._{- I} ^Plasma_----

1 2 3 4 5 6 7 8 9 1011 12 13 14

Figure5. RNaseprotection assays of renal EPO mRNA inrats ex- changed withRinger'ssolution (lanes 6-8),ratplasma(lanes12-14), Ringer's supplemented with 60 mg/mlgammaglobulin (lanes9-11), orRinger's supplemented with dextran 40 (260 g/liter) (lanes 3-5).

Lane1:size marker generated by HpaIIdigestofpBR322 DNA; lane 2:external ratEPOmRNAstandardconsistingof 5

jig

total RNA from kidneys of severely anemic rats(hematocrit < 10%).

sis is unaffected at creatinine clearance values above 30-40 ml/min and renal anemia is notfound abovethis threshold value ( 13, 14). Forthis reason it is unlikely that a restriction of renal function istheprimaryand only causefortheimpaired EPOformation.It wasof interest thereforeto seewhetherarise of plasma

gammaglobulins

andarelatedriseof plasma viscos- ityper seis capabletoattenuatetheEPOresponseinotherwise normalorganisms. Tothis end^wehaveinvestigatedwhether anassociation exists between thegammaglobulin concentra- tionortheplasma

viscosity

and theEPOresponsetoanemiain normalrats.Itturnedoutthat theriseinplasma EPO levels in response tothesamedegreeof anemiawasinversely relatedto thegammaglobulin concentration. Since gammaglobulinsare majordeterminants ofplasmaviscosity,theEPOresponse was, notsurprisingly, also inversely correlated withplasmaviscosity (Fig.3).Interestingly,thecorrelationbetweenplasmaviscosity andEPOresponsewasrathersimilarfor

hypergammaglobulin-

emicpatientsand thenormalrats,suggestingthat theimpaired EPOresponseinpatientsmayinfact be duetoeffectsrelatedto theincreaseof

gammaglobulins.

To distinguish whether the attenuation of the EPO re- sponseinthehypergammaglobulinemic rats waslinkedtothe alteration of plasmaviscosityor toothereffects relatedtohy- pergammaglobulinemia,inadditional experimentsplasmavis- cositywasincreased bydextran.Again thismaneuverledto a markedattenuation oftheEPO response(Fig. 4) and we there- foresuggest thattheplasmaviscositycouldbeafactor in deter- miningEPOproduction, and couldtherefore accountforthe impairedEPO response inhypergammaglobulinemic patients.

Nonetheless it was obvious that raising plasma viscosity by dextran waslesseffectiveon EPO levels thanariseinplasma viscosity induced by infusion of gammaglobulins and we can- notexplainthisobservationyet.

Sinceanincrease ofplasma viscositymayimpair capillary circulation andmaythusimpede thereleaseof EPO from the renocortical interstitium into the blood flow, we examined whetherviscosityrelatedeffectsonplasma EPO levels were due to aninhibition of EPO

synthesis

or dueto aninhibition of EPO release. EPO

production

rateis

primarily

determinedby the EPO mRNA levels within the EPO producing cells. We found that the impaired EPOresponsein therats wentinparal- lelwith decreased levelsof EPOmRNAin the

kidneys,

suggest- ingthatitwasthe

synthesis

of EPO rather than the release of EPO thatwasattenuated by the increase of plasma viscosity

(Fig. 5).

The present study cannotdetermine the mechanisms

by

which plasmaviscosityexertsinfluenceon thestimulation of EPOproduction

by

anemia. Since renal

capillary endothelial

cellsare considered as likely candidates for producing EPO (15, 16), onecould speculate that plasmaviscosityexerts an influenceon endothelial cells by changing the shearstress on theendothelial surface.Ithasbeenfound for instance that the release

of

histamin( 17),renin (

18),

endothelin

( 19),

prosta- cyclin (20,21), andendothelium-derived relaxing factor (22) are shear stress dependent,

indicating

that this parameter is capabletomarkedly modulate endothelial function. It islikely that mechanoreception, the most

widely

distributed sensory modality, isthemechanism involved (23).

Thepresent data have clinical implications, since it has become commonpracticetoadminister recombinantEPO to patients with blunted EPO response. Clinicians should be aware that low EPO levelsmay represent aphysiological re- sponseof theorganismtoimpending hyperviscosityand thatit maybe dangeroustoovercomethis negative feedbackcontrol byexogenousEPO. Inthiscontextit isnoteworthythat EPO levels are lowin sickle cellanemia (24), a classical condition with increasedwhole

blood

viscosity.

Acknowledgments

The expertsecretarial help provided by Hannelore Trommer and Aloi- syaBockis gratefully acknowledged.

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