A nemia of chronic renal failure is a well-established entity, and depending on the primary renal disease, may vary in its severity ( 1 ).

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1 Is Erythropoietin Survivl Fctor for Red Blood Cells?1 Momir Polenkovic2 nd Aleksndr Sikole M. Polenkovic, A. Sikole, Deprtment of Nephrology, Medicl Fculty, University Sts. Cyril nd Methodius, Skopje. Republic of Mcedoni (J. Am. Soc. Nephrol. 1996; 7: ) ABSTRACT Recombinnt humn erythropoietin (rhuepo) therpy hs been reported to mintin corrected hemtocrit vlues by incresing the length of red blood cell (RBC) survivl. This rticle presents controlled study tht ssessed the RBC survivl before, during, nd fter termintion of prolonged rhuepo tretment of chronic hemodilysis ptients. Two groups of 20 ptients were studied. The hemtocrit vlue of ech ptient ws below 28 vol%. One group (Group A) ws treted with rhuepo for 1 yr nd then tretment ws stopped becuse of the unvilbility of the drug. The second group (Group B) ws treted for 2 yr. Epoetin bet ws dministered subcutneously. The initil dose ws 20 U per kg body weight three times weekly. Upon reching the trget hemtocrit vlue of 30 to 35 vol%, the dose ws individulized for ech ptient, to mintin trget rnge. RBC survivl ws determined by the chromlum-51 technique. In Group A, RBC survivl ws determined: ( 1) before, (2) t 12 months, nd (3) 1 yr fter cesstion of rhuepo tretment. In Group B, RBC survivl ws determined: ( 1) t 24 months of therpy, nd (2) 1 yr fter cesstion of rhuepo tretment. RBC survivl incresed significntly in both ptient groups under rhuepo tretment. After cesstion of therpy, the RBC survivl decresed to pretretment vlues. During the correction period, reticulocyte counts incresed significntly in both groups. Over the mintennce period, they slightly decresed, nd fter termintion of rhuepo therpy, they decresed to the pretretment vlues. The results of this study could suggest the possibility tht RBC survivl ws prolonged by the ction of EPO on the erythroid progenitors, resulting in the production of RBC with improved vibility. Key Words: Kidney filure (chronic), hemodilysis. renl nemi, erythropoietin. red blood cell survivl Received December 7, Accepted Februry 23, Correspondence to Dr. M.H. Polenkovic, Deprtment of Nephrology, Medicl Fculty, Vodnjnsk 17, 900 Skopje. Republic of Mcedoni / $03.OO/O Journl of the Americn Society of Nephrology Copyright C 1996 by the Americn Society of Nephrology A nemi of chronic renl filure is well-estblished entity, nd depending on the primry renl disese, my vry in its severity ( 1 ). The vrious fctors tht my underlie the nemi of chronic renl filure include: reduced vilbility of erythropoietin (EPO), Inhibitors of erythropoiesis, bone mrrow fibro- 515, nd hemolysis s result of extr corpusculr fctors. Renl nemi is of hypoprolifertive, normocytic, nd normochromic type, but certin deficiency sttes, such s iron, folte, or vitmin B12 deficiency, my lter its nture (1,2). One of the fetures of renl nemi is shortened red blood cell (RBC) survivl. Most rdiolsotopic studies confirm the presence of mild hemolysis in renl nemi cses. Although the cuse for the hemolysis Is not yet fully understood, severl fctors in the uremic ptient hve been Incriminted: prthyroid hormone, spermine, nd ribonuclese (1-3). Recombinnt humn erythropoietin (rhuepo) tretment hs been very effective in the correction of renl nemi, both by intrvenous nd subcutneous dministrtion. It hs been reported tht rhuepo mintins corrected hemtocrit (Hct) t desired level by Incresing the length of RBC survivl, nd not by sustined elevted reticulocyte count, despite mrked reduction of the rhuepo dose required to mintin the Hct level (4). The purpose of the study ws to ssess the RBC survivl before nd fter prolonged subcutneous rhuepo tretment in chronic hemodllysis ptients. Furthermore, becuse of unfortunte circumstnces, we hd the unusul possibility to observe the chnge of the RBC survivl fter cesstion of rhuepo therpy. MATERIAL AND METHODS Ptients In prospective clinicl study, two groups of ptients were treted with rhuepo by the sme protocol, but for different time intervls (5). Forty ptients were selected for the study on the bsis of inclusion nd exclusion criteri, nd they signed informed consents to prticipte in the study. All ptients were older thn 18 yr of ge nd were nemic, with Hct vlue below 28 vol%. They were treted by chronic hemodilysis t lest 6 months before entering the study. The hemodilysis regime included 4 to 4.5 h ofdilysis three times per week, with n cette bth nd cellulosic membrne of 1. 1 to 1.3 m2. The blood flow rnged between 250 nd 280 ml/min. The vsculr ccess for hemodilysis ws from Cimino-Bresci rteriovenous fistul. The dilysis dose used before the study for ech ptient remined constnt throughout the entire study period. There hve been no significnt chnges in weight or In the predilysis serum ure nd cretlnine level In the ptients. The exclusion criteri were: iron, folic cid, nd vitmin B12 deficiency; severe infection; mlignnt or systemic disese; cute liver infection; uncontrolled hypertension; epilepsy; pregnncy; hemolytic-uremic syndrome; nd thrombocytosis (>500,000 / mm3) Volume 7 - Number

2 Polenkovic nd Sikole The ptients were rndomized into two groups of 20 ptients ech, their bsic clinicl nd demogrphic dt were recorded (Tble 1 ), nd they were then enrolled In the prospective Tretment study. Protocol Ech group of ptients ws treted by the following protocol. The initil dose of epoetin bet (Recormon; Boehringer- Mnnheim, Mnnhelm, Germny) ws 20 U per kilogrm body weight (U/kg body wt), dministered subcutneously three times weekly, mm before ech dilysis session. The dose could be incresed by 20 U/kg body wt per single dose every 4 wk, if Hct rise ws too slow or bsent. This period ws clled the correction period. After reching the trget Hct of 30 to 35 vol%, the lst dministered dose ws reduced by hlf nd djusted therefter individully, to keep the Hct vlue in the rnge of 30 to 35 vol%. It ws the mintennce period of the study. During the rhuepo therpy, body iron stores were regulrly monitored by determintion of serum ferritin, trnsferrin, nd totl iron binding cpcity. The ptients were treted with orl Iron therpy in the form of ferrous sulfte if serum ferritin level fell below 200 ng/ml or if trnsferrin sturtion index fell below 20%. One group of ptients (Group A) ws treted for 1 yr nd tretment ws then discontinued becuse of unvilbility of the drug. The second group (Group B) ws treted for 2 yr nd the tretment ws lso then discontinued for the sme reson. Determintion of RBC Survivl The following procedure ws used: t the onset of dilysis session, 1 0 ml of blood were withdrwn from the ptient into sterile 50-mL glss tube contining 2 to 3 ml cid-citrtedextrose (Grde I NIH). One hundred mci rdioctive sodium chromte (Cr5 i ) with high specific ctivity ws then dded to the mixture. After 30 to 40 mm of incubtion t room temperture with intermittent gentle gittion, the tube ws centrifuged (00 x g for 12 mm t 25#{176}C).The superntnt TABLE 1. Demogrphic nd bsic clinicl dt of ptients Ptients Group A Group B Age (yr) 48.3 (rnge, 47.6 (rnge, 23 to 69) 25 to 70) Sex Mle Femle 8 9 Hemodilysis Durtion 47.6 (rnge, 60.5 (rnge, (months) 21 to 111) 2Oto 133) Primry Renl Disese Chronic Glomerulonephritis 6 5 (N) Chronic Interstitil 2 4 Pyelonephritis (N) Clculous Pyelonephritis (N) 3 Blkn Nephropthy (N) 2 1 Hereditry Nephropthy (N) 2 2 Nephrosclerosls (N) 3 1 Uric Nephropthy (N) 2 Undifferentited 3 4 Nephropthy (N) ws discrded using sterile needle connected to vcuum. Lbeling efficiency ws over 80% /dose mesured In dose clibrtor. Approximtely four-fifths of the Cr5 1 -lbeled RBC ws injected intrvenously into the ptient t the end of the sme dilysis session, nd the needle ws flushed by sterile sline. A hepninized blood smple of 5 ml ws withdrwn fter 24 h nd ws used s the Dy 0 smple. Subsequently, blood smples were drwn three times weekly t pproprite intervls for the next 3 wk or until the rdiolsotpe concentrtion in the blood hd decresed to one-hlf its originl (Dy 0) level. The dt were plotted on semilog grphs nd RBC survivl ws determined by regression nlysis (6,7). RBC survivl ws determined In both ptient groups (8,9). In Group A, it ws determined before the onset of rhuepo therpy in 18 ptients. Two ptients were unvilble for determintion. All 18 ptients were tested gin t 12 months of rhuepo therpy. The third determintion of RBC survivl ws performed 1 yr fter cesstion of rhuepo tretment In 1 2 ptients of this group. In Group B, RBC survivl ws determined In 18 ptients fter 24 months of rhuepo therpy. The second determintion ws mde 1 yr fter termintion of therpy in 1 1 ptients from this group. Hemtocrit (vol%) level ws determined weekly, nd uncorrected reticulocyte count (% peripherl RBC) ws determined monthly over the entire study period, using stndrd lbortory methods. Throughout the study, ll lbortory prmeters were determined from blood tken before hemodilysis sessions. Results were expressed s men (M) ± SD. The t test ws pplied to observe the difference between different time intervls of the study. RESULTS Group A During the correction period of 16 wk, Hct vlue hd risen to the trget rnge of 30 to 35 vol% In ll ptients, nd ws therefter mintined within the trget limits by low subcutneous doses of rhuepo. The highest men rhuepo dose during the correction period for the whole group reched 35 U/kg body wt per single dose. The men mintennce dose vried between 20 nd 30 U/kg body wt pplied three times weekly. The men reticulocyte vlue before the study ws ± 0.55%. DurIng the correction period, It rose to the mximum level of2.22 ± 0.73% (P < 0.001). In the mintennce phse of the study, the men reticulocyte count rnged between 1.32 ± 0.64% nd 1.9 ± 0.86%, verging 1.79 ± 0.16% (P < 0.001). After termintion of rhuepo therpy, the men reticulocyte vlue ws 1.26 ± 0.84% (P = not significnt ENSI). The difference between the mximum level of 2.22 ± 0.73% nd the verge level ofthe mintennce period ( 1.79 ± 0. 16%) ws lso significnt (P < 0.05). The RBC survivl t different time intervls of the study for this group ofptients re presented in Figure 1. Before the onset of epoetin tretment, RBC survivl, Le., men Cr5 1 hlf-life (t#{189})ws 23.3 ± 2.6 dys, t men Hct vlue of 23.8 ± 2. 1 vol%. At 1 2 months of tretment nd men Hct level of 33.2 ± 1.8 vol%, the Journl of the Americn Society of Nephrology 1 179

3 EPO nd RBC Survivl 40- norml rnge ences between the three Intervls were ll nonsignificnt. The predilysis serum ure level ws 33 ± 5.17 mmol!l before rhuepo therpy, ± 4.52 mmol!l t 12 months oftretment, nd ± 6.29 mmol!l 1 yr fter cesstion of rhuepo tretment. These differences were lso nonsignificnt. These prmeters support the fct tht the dilysis dose did not chnge during the study. Cl) 11 I U 0 E +4 0) (5. C 4 C., n ±2.1 n12 I I I before EPO t l2mo. EPO l2mo. fter EPO p<o01 norml rnge 27.2±4.1 - n ±2.6 n= ±3.6 n=12 before EPO t l2mo. EPO l2mo. fter EPO Figure 1. The hemtocrit dt of Group A ptients t the time of determintion of RBC survivl re presented in the upper grph. The dt of the Cr51 hlf-life of Group A ptients re shown on the lower grph, determined t three intervls of the study: ( 1) before the strt of erythropoietin tretment, (2) t 12 months of rhuepo therpy, nd (3) 12 months fter cesstion of rhuepo therpy. The vlues re expressed s men :t SD. Pired t test ws used for sttisticl nlysis. men Cr5 1 tv2 rose to 27.2 ± 4. 1 dys. One yer fter termintion of rhuepo therpy, Cr5 1 T #{189} decresed to ± 3.6 dys. The Hct level t tht time hd lso dropped to 25.9 ± 5. 1 vol%. These chnges were significnt. The men body weight of this group before the strt of therpy ws 66.5 ±.42 kg. At 12 months of therpy, It ws 66.8 ± kg (P = NS). After 1 yr of termintion ofrhuepo tretment, It ws ± 8.98 kg in the remining 12 ptients (P = NS). The differ- Group B This group ws treted with rhuepo for 2 yr nd the Hct vlue ws mintined t 30 to 35 vol%. The mximum rhuepo dose for the group during the correction period reched 49 U!kg body wt, wheres it rnged between 1 7 nd 27 U! kg body wt during the mintennce period. Figure 2 presents dt on the Hct vlue nd the RBC survivl t the time of determintion. The men Cr51 t#{189} t 24 months of rhuepo therpy ws 27.3 ± 3.7 dys, t men Hct level of 33.5 ± 2.6 vol%. One yer fter termintion of therpy nd reduced Hct level of 24 ± 2.9 vol%, Cr5 1 tv2 ws significntly lower t 22.6 ± 2.3 dys. During the study, reticulocytes vried similrly to those of Group A. Men reticulocyte percentge before the onset of therpy ws 1.2 ± 0.43%. It rose to mximum of 2.58 ± % (P < ) during the correctionperiod nd rnged between 1.39 ± 0.72% nd 1.84 ± 0.84% during the mintennce period, with men verge of 1.71 ± 0.26% (P < 0.001). After termintion of rhuepo therpy, It decresed to 1.3 ± 0.53% (P = NS). The difference between the mximum level (2.58 ± 1. 16%) nd the verge mintennce level ( 1.71 ± 0.26%) ws lso significnt (P < 0.01). Men body weight of this group t 24 months of rhuepo therpy ws ± 9.51 kg. In the 11 ptients of this group fter yer of termintion of rhuepo, it ws 57.3 ± 9.08 kg. This chnge ws not significnt. Serum predilysis ure ws ± 3.99 mmol!l t 24 months of therpy, nd ± 3.25 mmol!l 1 yr fter termintion of rhuepo. The chnges of these prmeters were nonsignificnt, indicting tht the dilysis dose ws not chnged during the study period. DISCUSSION Our results suggest tht subcutneous rhuepo tretment effectively corrects nemi in uremic dily- 515 ptients, nd tht corrected Hct vlue is mintmed with reltively low doses of rhuepo, provided tht the iron stores in the body re well preserved with dequte supplementtion. In both ptient groups, RBC survivl, s expressed by the Cr5 1 technique, ws significntly incresed during tretment, with rhuepo pproching norml rnge. The RBC survivl of Group A t 1 2 months of tretment ws comprble to tht of Group B t 24 months of tretment, but both vlues were significntly incresed when compred with the pretretment vlue of Group A Volume 7 Number

4 Polenkovic nd Sikole.44 U 0 (5 E I +4 >, (5 (.4 0) C.) flo011 5±2 6 norml rnge 24±2.9 n=11 t 24mo. EPO l2mo. fter EPO norml rnge 22.6±2.3 n=11 t 24mo. EPO l2mo. fter EPO Figure 2. The hemtocrit vlues of Group B ptients t the times of determintion of Cr51 hlf-life of the red blood cells re presented in the upper grph. The RBC survivl of Group B ptients, determined t two intervls, is presented in the lower grph: ( 1) t 24 months of rhuepo therpy nd (2) 12 months fter cesstion of rhuepo therpy. The vlues re expressed s men ± SD. Pired ttesf ws used for sttisticl nlysis. One yer fter cesstion of therpy, RBC survivl ws significntly reduced nd comprble in both Group A nd Group B. Furthermore, men Cr51 hlf-life vlues were comprble to the pretretment vlue of Group A. Thus, EPO tretment significntly incresed RBC survivl of uremic dilysis ptients nd this effect ws continuously mintined under tretment but diminished fter cesstion of therpy, indicting tht EPO directly influenced the longevity of RBC. Our results re In greement with those of Schwrtz et l. (4, ), Hughes et t. ( 1 1 ), nd Eschbch (1). Some other uthors hve reported tht RBC survivl is not ffected during rhuepo tretment, nd tht the correction of nemi depends solely on incresed RBC production (12-14). The bsic objection to the ltter studies would be tht they were too short in follow-up time becuse the effect of rhuepo on RBC survivl might be relted to the durtion of tretment. The reticulocyte counts vried similrly in both ptient groups. They rose during the correction period to mximum level. Then, s the rhuepo dose ws individulized nd tpered down to keep the Hct vlue within the limits of 30 to 35 vol% over the mintennce period, the reticulocyte counts decresed nd leveled off. The difference between pretretment counts nd mintennce counts could indicte the presence of erly reticulocyte relese nd minute prolongtion of RBC survivl. After cesstion of therpy, reticulocytes further decresed just slightly. It Is importnt to note tht the increse in reticulocyte counts in our study ws considerbly smller thn, for exmple, tht in the study of Schwrtz et t. (4). This difference could be explined by the different rhuepo doses nd routes of dministrtion used in the studles. One of the ims of our study ws the slow correction of the Hct vlue, to void occurrence of serious dverse events. Therefore, the doses used in the correction period were smller nd the trget Hct vlue ws lso lower (38 vol% in the study ofschwrtz et t. [41 versus 30 to 35 vol% in ours). For these resons, the correction period in our study lsted longer. However, In principle, the dynmics of reticulocytosis in our study entirely resembled the pttern of the study of Schwrtz et t. It Is not yet fully elucidted how rhuepo influences normliztion of erythropolesls. Koury nd Bondurnt ( 15) nd Spivk et t. ( 16) hve shown tht EPO cts s both mitogen nd survivl fctor. They proposed tht the possible mechnism of ction of EPO on the colony-forming units-erythroids ws stimultion of DNA synthesis, retrdtion of DNA brekdown, nd prevention of poptosis (progrmmed cell deth). Bsed on in vitro experiments, Bondurnt et t. ( 1 7) hve postulted tht three possibly interrelted processes occur simultneously during colony development: cell growth nd repliction, extensive differentition nd mintennce of ll functions necessry for survivl. The lst re hs only recently been identified s mechnism of hormonl regultion. Their model of erythropolesls proposes tht individul progenitors within the EPO-dependent popultion exhibit rnge of sensitivities to EPO such tht there is n extended dose rnge of EPO over which individul progenitors my survive nd continue prolifertion nd differentition. Thus, the level of EPO ultimtely controls RBC production by regulting the number of dependent progenitors tht survive or die. So. t given level of EPO, some cells die Journl of the Americn Society of Nephrology 1181

5 EPO nd RBC Survivl wheres others survive nd progress through differentltion. Our results, s well s those of Schwrtz et t. (4, ), suggest tht the vibility of the RBC increses with EPO therpy. EPO my exert number of moleculr effects on erythroid cells whose ultimte effect would be the incresed vibility of RBC ( 1 7). These processes my stbilize the erythrocyte membrne nd mke the RBC more resistnt to the ggressive uremic environment. Lerche et t. ( 18) hve provided evidence for this effect of EPO. They found significnt improvement of the whole RBC deformbility fter 30 wk of rhuepo tretment of 20 dults nd ten children with ESRD. They concluded tht the disturbed erythropolesis in uremic ptients lso cused impired mechnicl membrne properties tht could not be ttributed to the direct influence of uremic toxins on RBC lone, nd furthermore, tht the performed experiments did not exclude the direct ction of rhuepo on the mechnics of the membrne. Our results, s well s the work of other uthors, suggest tht RBC survivl my be prolonged becuse of the ction of EPO on the erythroid progenitors. The consequence of the moleculr effects of EPO on the erythroid cells could result in incresed vibility of the peripherl red blood cells. ACKNOWLEDGMENTS Determintion ofthe red cell survivl ws performed t the Institute of Pthophyslology nd Nucler Medicine. Medicl Fculty, University Sts. Cyril nd Methodius of Skopje. Republic of Mcedoni. Erythropoietin bet ws provided by Boehringer Mnnheim GmbH. Mnnhelm. Germny. The uthors thnk Professor Gerld Schulmn of Vnderbilt University for his criticl review of the mnuscript. REFERENCES 1. Eschbch JW: The nemi of chronic renl filure: Pthophysiology nd the effects of recombinnt erythropoietin. Kidney mt 1 989;35: Mssry SG: Pthogenesis of the nemi of uremi: Role of secondry hyperprthyroidism. Kidney Int 1983; l6lsuppl 241: Bogin E, Mssry 5G. LevI J, Djldelll M, Bristol G, Smith J: Effect of prthyroid hormone on osmotic frgility of humn erythrocytes. J Clin Invest 1982;69: Schwrtz AB, Kelch B, Terzin L, et t.: One yer of rhuepo therpy prolongs RBC survivl nd my stbilize RBC membrnes despite nturl progression of chronic renl filure to uremi nd need for dilysis. ASAIO (Am Soc Artif Intern Orgns) Trns 1992;M Polenkovic M, Sikole A, Msin G, et t.: Clinicl use of humn recombinnt erythropoietin (rhuepo) for tretment of the nemi of end-stge renl disese-one yer experience with Recormon. Mked Med Pregled 1993; lsuppl 121: ICSH-Interntionl Committee of Stndrdiztion in Hemtology: Recommended methods for red blood cell survivl studies. Br J Hemtol 1980;45: Bogdnov V, Georgievsk 5, Tdzer IS: A rpid method of estimtion red blood cell survivl time. Mcedonin Acdemy of Sciences nd Arts. Section of Biologicl nd Medicl Sciences, Contributions XI, 1-2, Sikole A, Polenkovic M, Spsovski G, Msin G: Redcell survivl in chronic hemodllysis ptients treted with rhuepo (Abstrctl. Nephrol Dil Trnsplnt i993; 8:964A. 9. Polenkovic M, Sikole A: Is erythropoietin survivl fctor for the red blood cells (Abstrctl. Int J Artif Orgns 1995; l8lsuppl 8l:428A.. Schwrtz AB, Khn SB, Kelch B, Kim KE, Pequignot E: RBC improved survivl due to recombinnt humn erythropoietin explins effectiveness ofless frequent, low dose subcutneous therpy. Clin Nephrol 1992; 38tSuppl 51: Hughes RT, Cotes PM, Pipprd MJ, et t: Subcutneous dministrtion of recombinnt humn erythropoietln to subjects on continuous mbultory peritonel dilysis: An erythrokinetic ssessment. Br J Hemtol i990;75: Njen Y, Moynot A, Deschryver F, et t.: Kinetics of erythropolesis in dilysis ptients receiving recombinnt erythropoietin tretment. Nephrol Dil Trnsplnt i 989; 4: Zehnder C. Blumberg A: Humn recombinnt erythropoletin tretment in trnsfusion dependent nemic ptients on mintennce hemodilysis. Clin Nephrol 1989; 31: Cotes PM, Pipprd MJ, Reid CDL, Winerls CG, Oliver DO, Royston PJ: Chrcteriztion of the nemi of chronic renl filure nd the mode of its correction by preprtion of humn erythropoietin (r-huepo): An investigtion of the phrmcokinetics of intrvenous erythropoietin nd its effects on erythrokinetics. Q J Med 1989:7OlSuppl N2621: Koury MJ, Bondurnt MC: Erythropoietin retrds DNA brekdown nd prevents progrmmed deth in erythrold cells. Science (Wshington DC) 1990;248: Spivk JL, Phm T, Iscs M, Hnkins WD: Erythropoletin is both mitogen nd survivl fctor. Blood : Bondurnt M, Kelley L, Koury MJ. Is erythropoletin differentition fctor, mitotic fctor, or survivl fctor? In: Buer C, Koch KM, Scigll P. Wieczorek L, Eds. Erythropoietln: Moleculr Physiology nd Clinicl Applictions. New York: Mrcel Dekker Inc.; 1993: Lerche D, Schmidt R, Zoeliner K, et t.: Rheology in whole blood nd in red blood cells under recombinnt humn erythropoietin therpy. Contrib Nephrol 1989; 76: Volume 7 - Number