erythropoietin in health and disease

European Journal of Clinical Investigation (1989) 19, 1 17-127

REVIEW

Erythropoietin in health and disease

K.-U. ECKARDT & C. BAUER, Physiologisches Institut, Universitat Zurich, Winterthurerstrasse 190, Zurich, Switzerland

Received 13 December 1988

Introduction

The renal hormone erythropoietin (EPO) that was not long ago alluded to as an elusive hormone [ I ] , is now on its way to becoming a major advancement in hormonal replacement therapy. This exciting development was possible after the gene coding for EPO was cloned a few years ago [2,3] and by the subsequent demonstration that ‘artificial’ EPO, produced by recombinant DNA technology, is effective in correcting the anaemia of renal disease [4-lo]. In addition, recombinant DNA technology has provided basic scientists with sufficient amounts of the pure hormone as well as with molecular probes, the experimental application of which has contributed considerably to understanding the biogenesis and function of EPO.

A humoral regulator of erythropoiesk. The concept that erythropoiesis is under humoral control dates back more than 80 years [l 1). The original experiments which led to the hypothesis of a hormonal regulation of erythropoiesis could, however, not be reproduced quantitatively [cf. 121 and it was not until the middle of this century that this concept was solidified. At that time Reissmann observed hyperplasia of the erythroid part of the bone marrow in normoxic rats whose parabiotic partner was submitted to hypoxia [ I 31. At about the same time Erslev demonstrated a significant reticulocytosis in rabbits infused with large volumes of plasma from anaemic donor animals [14].

The importance of the kidneys for the biogenesis of this erythropoietic factor-meanwhile named ‘erythropoietin’ [ 15]-was convincingly demonstrated in 1957 by Jacobson et al. [16]. However, the precise role of the kidneys for the generation of EPO remained contro- versial for almost three decades. Three different hypotheses were put forward [cf. 171:

Correspondence: Dr K.-U. Eckardt, Physiologisches Institut, Univenitlt Ztrich, Winterthurerstrasse 190, CH 8057 Zurich, Switzerland.

1 A proteolytic enzyme of renal origin (termed ‘eryth- rogenin’) cleaves EPO from a precursor molecule that is produced extrarenally [ 181. 2 A plasma enzyme splits EPO from a precursor molecule produced by the kidney [ 191. 3 EPO is directly synthesized by the kidneys.

The third concept received more and more experimental support since 1980 [cf. 1 1 and was finally proven by demonstrating the occurrence of EPO mRNA in the kidney [20-221. Comparatively small amounts of EPO mRNA were also found in the liver, but not in any other organ [20,23].

The major breakthroughs in the elucidation of the structure of EPO were based on the purification of a few milligrams of human EPO by Miyake, Kung & Goldwasser in 1977 [24], using large amounts of urine from patients with aplastic anaemia as starting material. From this purified material a partial amino acid sequence of EPO was derived and corresponding oligonucleotides were synthesized. These molecular probes were then used to isolate the EPO gene from a gene library. The subsequent expression of the gene for EPO in mammalian cell lines [2,3,25] has provided the basis for todays, principally unlimited, availability of EPO for therapeutic use.

Physiology of erythropoietin

The molecule EPO is a glycoprotein with a molecular weight of approximately 34 000 daltons. The protein backbone of the mature hormone consists of 165 amino acids* [26-281. Four complex carbohydrate chains, contain- ing a high amount of sialic acids are linked to the protein at three N-linked and one 0-linked glycosilation sites, constituting approximately 40% of the total

The cDNA coding for EPO predicts I66 amino acids. However, the recombinant molecule as well as EPO purified from human urine lacks a final arginine molecule [28]. The significance of this processing is unknown.

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Figure I. Simplified scheme of the cellular anatomy of erythropoiesis. The primary target cell for EPO is the c.f.u-E. (colony forming unit-erythroid). Earlier erythroid progenitors (b.f.u.-E. burst forming unit-erythroid) are only responsive to high EPO concentrations at a later developmental stage (late b.f.u.-E.). The early divisions of the b.f.u-E. are dependent upon other growth factors, referred to as burst-promoting activity (BPA), including interleukin 3 (IL 3) and granulocyte/macrophage colony-stimulating factor.

molecular weight [29]. This glycosilation of the molecule is required for its survival in the circulation (see below) [30,31], while it is not essential for or may even inhibit the biological activity on erythroid precursor cells [32].

The function of the hormone At physiological concentration EPO stimulates erythropoiesis by enhancing the mitotic frequency of late erythroid precursors in the bone marrow, the c.f.u.-E. (colony forming unit-erythroid) cells and their immed- iate successor, the proerythroblasts [33,34] (Fig. I). At high hormone concentrations the earliest recognizable erythroid progenitor in culture, the b.f.u.-E (burst forming unit-erythroid cell), also becomes responsive to EPO. However, the divisions of these progenitors are mainly dependent on other growth factors, referred to as having burst-promoting activity [34].

The number and binding affinity of the receptors to which EPO binds have been determined in some EPO- dependent erythroid precursor cells [32,35,36] as well as in permanent cell lines [37-391. In general, one single class of EPO receptors with an apparent molecular mass of about 100 kDa was found that binds the hormone with a dissociation constant of about 300 pM. EPO-dependent cells display between 1000 and 3000 receptor sites per cell [39], only a small percentage of which must be populated with EPO to initiate cell division [37,38] by a Ca2+-dependent mechanism [40].

Production sites The kidneys are the major source of EPO in the adult organism. Employing in situ hybridization the location of the EPO-producing cells within the kidneys of anaemic mice was confined to peritubular cells in the cortex and to a lesser extent in the outer medulla [41,42]. The precise nature of these EPO-producing cells has, however, not yet been determined. From their location they may either be endothelial cells, macrophages or other interstitial cells.

EPO mRNA has also been demonstrated in the liver 120,231, which is the predominant production site for EPO during fetal life 1431, and constitutes the main source of the so-called ‘extrarenal’ EPO that is produced in anephric adults [44]. The contribution of liver-derived EPO, however, is generally very small and is insufficient to compensate for loss of the renal source.

Regulation of erythropoietin formation Erythropoiesis is adapted to the oxygen requirements of the body through variations in the amount of circulating EPO. The physiological basis of this adap- tation is a negative feedback loop with the kidneys as the central regulatory organ (Fig. 2). This feedback control is geared to maintain the oxygen content of the blood at a constant level. Such a feedback loop requires an ‘oxygen sensor’ that monitors the available amount of oxygen, thereby providing the basis for the regulation of EPO levels. Since the kidneys contain no stores for EPO [45,4q, the information gathered by the ‘oxygen sensor’ must be directly translated into an altered production rate of the hormone.

Renal oxygen sensing. The current conceptual framework of the function of the ‘oxygen sensor’ that governs EPO formation is characterized by three major features: 1 The ‘oxygen sensor’ is thought to be mainly located in the kidney itself [cf. 473. However the extent to which extrarenal humoral or nerval factors modulate its function, has yet to be quantitated. 2 The parameter perceived by the sensor is the venous Po2 [48]. In this regard the oxygen-sensitive cells in the kidney differ principally from those in the carotid body, that are sensitive to changes in the arterial Po*. This sensitivity to venous Po2 enables the system that

ERYTHROPOIETIN IN HEALTH AND DISEASE 119

] f i l t e red sodium

Figure 2. Schematic presentation of the main determinants of renal EPO formation.

controls hypoxia-dependent EPO production to detect changes in the arterial oxygen saturation (e.g. hypo- ventilation or heart disease with right-left shunt) as well as changes in the oxygen-carrying capacity of blood at normal arterial P% (e.g. anaemia) where the diminished oxygen content can become apparent only after removal of oxygen along the capillary bed. 3 The function of the ‘oxygen sensor’ is probably related to proximal tubular function, as experimental

inhibition of Na reabsorption at the proximal tubular site results in a decreased sensitivity of the ‘oxygen sensor’ in terms of EPO production [49]. The apparent dependency of EPO formation on tubular sodium reabsorption allows a regulation of EPO that is, within a certain range, independent from renal blood flow, because a reduction in renal blood flow not only reduces oxygen supply, but also the sodium load and therefore renal oxygen consumption (Fig. 2).

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Figure 3. Two possibilities are considered how the proximal tubule might influence the EPO-producing cells. Upper: the oxygen consumption ofthe tubularcells might lower the pS in the renal interstitium. Lower: biochemical signals generated in the tubular cells upon hypoxia are transferred to the EPO-producing cells. (Reproduced with permission from the Annual Review of Physiology [ref. So], 0 1989 by Annual Reviews Inc.)

120 K.-U. ECKARDT & C. BAUER

It is not known by which mechanisms the proximal tubule exerts its influence on the adjacent EPO- producing cells. The main question is whether the EPO-producing cells possess an oxygen sensor of their own or if the adjacent tubuli generate biochemical signals upon hypoxia that are transferred to the EPO- producing cells (Fig. 3). The trans- or intracellular biochemical events that lead to an oxygen-dependent production of EPO in the kidney are not fully under- stood and many questions remain to be resolved [cf. 501.

Two different ways of regulation can be envisioned: First, hypoxia might activate metabolic pathways, from which certain messenger molecules arise that activate EPO formation. Three such signal molecules, prostaglandins, cyclic AMP and adenosine [cf. 50-521 have been implicated in this process, but the know- ledge about the chain of metabolic events leading to a hypoxia-dependent EPO production is quite fragmen- tary. Second, some sort of molecular ‘oxygen receptor’ like a haemoprotein could undergo conformational changes dependent on the oxygen availability [ 1211. Under physiological conditions, the oxygen affinity of such a haemoprotein would have to be much lower than that of most intracellular haemoproteins, in order to detect changes in Po2 values well above the critical Poz in renal tissue [53].

Metabolism of erythropoietin No evidence exists that the mature hormone is physiologically subjected to any processing after it has been released by the kidneys. Circulating EPO, as measured by radioimmunoassays usually exhibits full biological activity, as determined by in uiuo bioassays [54-571. Only in few individuals does some of the circulating immunoreactive EPO seem to lack biological activity, indicating either a modification or cleavage of the hormone [58].

Where does EPO disappear? The level of circulating EPO is, apart from the production rate, also determined by the rate of metabolic clearance. There is no indication that EPO metabolism is directly regulated in an oxygen-dependent manner. However, the half- life times that were determined in humans after bolus injection of recombinant EPO vary considerably from approximately 5 to 13 h [10,59,60]. The reason for this variation is not known and it is also not clear where exactly EPO is metabolized or eliminated. Three organs have to be considered: 1 The kidneys. Some of the circulating EPO is excreted by the kidneys and urine is, in fact, the only biological source from which human EPO has been purified so far 1241. However, the overall contribution of renal excre- tion or metabolism to the whole body clearance of EPO is small and the half-life time of circulating EPO is not apparently changed in end-stage renal failure [60] and only slightly prolonged after nephrectomy of experimental animals [6 1,621.

2 The bone marrow. The appealing concept had early been proposed that a considerable amount of the circulating EPO might be eliminated by uptake into the target cells 1631. If this holds true, the size of the erythron might indirectly contribute to the regulation of EPO levels in a sort of positive feedback: marrow hypoplasia would lead to an increase of EPO levels, whereas the expanded pool of EPO-responsive target cells would decrease circulating EPO levels. Support for this concept comes from in uitro studies in which receptor-mediated uptake of EPO into erythroid precursor cells was demonstrated [32,37]. The in uivo evidence, however, is inconclusive so far [64,65] and the quantitative contribution of this phenomenon has yet to be assessed. 3 The liver. The role of hepatic elimination of EPO has not yet been quantitated. It is only known that removal of the terminal sugars from the carbohydrate part of glycoproteins results in the rapid uptake by the liver via receptor-mediated endocytosis. This is particularly true when galactose residues are exposed after the loss of terminal sialic acid residues [30,31], but it is not known if such enzyme-dependent processes contribute to the physiological regulation of the disappearance rate of EPO.

Until more is known about variations in the clearance rate and the factors influencing it, it cannot be ruled out that changes in circulating EPO levels might be induced at least partially by alterations in the clearance, rather than exclusively by changes in the hormonal production rate.

Erythropoietin formation in health and disease

a Appropriate eythropoietin-response to varying oxygen supply

Normal oxygen supply Normal serum values for EPO as determined by radioimmunoassays are in the range of 10-25 mU ml-’ [55,66]*. Assuming a specific activity of the hormone of about 100 000 iu mg-I, this corresponds to about 100-250 pg ml-’ or 3-8 pM. Diurnal variations in the EPO levels are not a consistent finding [67], though they have once been reported [68]. The median apparent volume of distribution of recombinant EPO was determined to be 0-073 1 kg-’ corresponding to 1-5 to two-fold the plasma volume [60]. Applying these data including a median half-life time of 9 h to the endogeneous production of EPO, results in an estimate of the production rate of about 2 iu kg-’ per 24 h, corresponding to less than 20 ng kg-’ per 24 h.

That this basal production of EPO is essential for the maintenance of normal erythropoiesis (see Fig. 4, left

* EPO is traditionally quantified as ‘International units’ (iu). one iu originally defined as the amount of erythropoietic stimulating material that produced a response equivalent to 5 pM cobaltous chloride in assay rats.


-High alt i tude -Respiratory

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Normal or increased oxygen s u ~ p l y Decreased oxygen supply

Figure 4. Schematic representation of the feedback system that regulates erythropoiesis, under conditions of normal or increased oxygen supply (left panel) and decreased oxygen supply (right panel).

panel) becomes apparent when animals autoimmu- nized against EPO develop a severe and finally lethal anaemia.

Increased oxygen supply Under conditions of an expanded packed red cell volume at unchanged oxygen saturation, the peripheral oxygen supply is increased. This occurs either after hypertransfusion, after a previous hypoxia has been corrected (e.g. after decline from high altitude) or when erythropoiesis proliferates autonomously and independent from stimulation by EPO as in polycythaemia Vera. Under these situations EPO levels decline (see Fig. 4, left panel) [54,55,69,70], indicating that the ‘basal’ production under normoxia does not occur at a fixed rate but is already the result of oxygen-dependent regulation.

Decreased oxygen supply The hypoxia triggering an increase in the formation of EPO mainly arises from either diminished arterial oxygen saturation or a reduced red cell mass. In the former the stimulation of the erythron by EPO results in an enhanced blood volume (secondary erythrocytosis) in an attempt to compensate for the reduced

saturation. In the latter, when blood volume is primarily reduced, the increased formation of EPO leads either to its restoration or a new equilibrium is approached as in chronic anaemias (see Fig. 4, right panel).

Diminished oxygen saturation. Approximately 1 .5 h after the acute onset of hypoxia, circulating EPO levels in humans begin to raise significantly (Fig. 5) . The hormonal production rate underlying this rise increases exponentially with the reduction in oxygen availability (Fig. 6) [71]. From this correlation it becomes apparent that EPO production is only margi- nally augmented when oxygen supply is moderately reduced, but strikingly enhanced at low oxygen tensions that are potentially harmful for the organism’s vital functions.

A striking feature is that circulating EPO reaches maximal levels 1-3 days after the onset of hypoxia and then declines despite continued hypoxic exposure to a level, that is still above baseline values, but several-fold lower than early peak concentrations [72-741. The mechanism underlying this temporal pattern of EPO levels as well as its significance for the stimulation of the erythron are not known.

Once a secondary erythrocytosis is established in response to ongoing hypoxia through the EPO stimu-


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Figure 5. Time-course of serum EPO levels as measured by radioimmunoassay in healthy male volunteers exposed to simulated altitudes of 3000 m and 4OOO m in a decompression chamber (mean+SE; n=6). EPO levels began to rise significantly after approximately 1.5 h of hypoxic exposure, as indicated by an asterix (P<O.OS). (Reproduced from [71] with permission. 0 1989 by Am Physiol Soc.)

lation (see Fig. 4, right panel), peripheral levels of the hormone appear to decline again down to the normal range in up to 75% of cases [69], while in the remaining cases EPO stays continuously elevated [55,69,70,75,76]. With regard to the sensitivity of the bone marrow it is noteworthy that the maintenance of erythrocytosis is not necessarily associated with a continuous rise in EPO levels.

The value of EPO determinations in the differential diagnosis of polycythaemias is, however, limited by the overlap between values in secondary erythrocytosis and the normal range [69], all the more as considerable overlap also exists between values in patients with polycythaemia Vera and normals [66,69,75].

Reduced blood cell mass. In chronic anaemias of non-renal origin an inverse correlation exists between EPO levels and the haematocrit or haemoglobin concentrations [55,66,77], with up to several hundred- fold increases of EPO in severe forms. The inter- individual variation of EPO values appears very high with up to ten-fold differences in EPO levels in patients with the same haematocrit [77] and an exact quantifi-

cation of the 'adequate' increase in EPO levels with decreasing red blood cell mass has so far not been achieved. Comparing several values of patients with anaemias of different aetiology has revealed, however, that in one form of anaemia EPO levels are generally much lower than in other patients with the same reduction in haematocrit and this is the anaemia associated with chronic renal failure [54,77,78]. Whether in addition a blunted EPO production occurs in other non-renal anaemias, e.g. in anaemias associated with rheumatoid arthritis, has not been settled conclusively [79,80].

b Disorders of erythropoietin formation

The role of EPO deficiency in the pathogenesis of renal anaemia In chronic renal failure a variety of factors challenge the physiological equilibrium between formation and destruction of red blood cells. These include chronic blood loss on dialysis [8l], a shortened red cell life [82], iron and folate deficiency, myelofibrosis associated with hyperparathyroidism [cf. 831 and an impaired iron transfer to erythroid progenitor cells due to aluminium toxicity [84]. Irrespective of the individual contribution of each of these factors, the main patho- genic cause for the moderate to severe anaemia associated with end-stage renal disease is, however, the failure of EPO to adequately increase in response to this challenge. EPO levels in chronic renal failure generally do not or only slightly exceed the normal range and are much lower than in non-renal anaemias of comparable severity [54,57,66,77,78,85]. Particularly low levels are found in anephric patients [77,86] and this is generally associated with even more severe anaemia.

It remains unclear whether, in addition to the low production of EPO, the sensitivity of the bone marrow towards EPO is diminished due to yet undefined uraemic inhibitors of erythropoiesis. This idea is supported by the observation that dialysis treatment, by which putative inhibitors may be removed, tends to improve the severity of anaemia [87,88]. However, despite much effort, the exact nature of these potential inhibitors has so far eluded positive identification. Definite answers to the question of impaired EPO responsiveness will arise from studies comparing the dose-response relationship of EPO in renal and non- renal anaemias. The success in correcting renal anaemia with recombinant EPO demonstrates so far that inhibitors, if they are present at all, are at least not of a potency that cannot be overcome by high doses of EPO.

The inappropriately low EPO production in renal anaemia is frequently attributed to the destruction of the renal production sites of the hormone. In addition, since the stimulation of EPO formation appears to arise from the intricate interaction between renal oxygen supply and the excretory renal function, not only the capability to produce EPO, but rather the


renal ‘oxygen sensor’ regulating EPO production, may be disturbed. Namely, when anaemic uraemic patients experience an additional acute blood loss, they are able to respond with some increase in EPO production [89], revealing that the production was not at its maximum. This might indicate that the sensitivity of the feedback circuit between oxygen supply and EPO production is diminished in renal disease. That the regulatory princi- ple is nevertheless intact is suggested by the observation that, conversely to acute blood loss, transfusion further suppresses EPO formation [57,89].

A few patients in chronic kidney failure do remain normocythaemic or, at least, are less severely anaemic and others occasionally experience a marked improvement of their anaemia during the course of renal failure. Some of those have cystic kidneys, either because of inherited polycystic kidney disease [90,9 I] or due to secondary cyst formation in the shrunken end-stage kidneys [92,93]. In both instances elevated EPO levels have been reported [90,93), but the mechanisms underlying this phenomenon remain unknown. In some patients an increase in erythropoiesis followed the onset of viral hepatitis [94,95] and this may result from an enhanced hepatic EPO production that has also been found experimentally in association with hepatic proliferation [96,97J.

Autonomous or inappropriate increase in erythropoietin

Erythrocytosis that is associated with neither hypoxia nor autonomous erythroid proliferation, may result from EPO production that has escaped the physiological feedback control of oxygen supply.

Neoplasms. Renal cell carcinomas occur with erythrocytosis in 2% of patients [98] and some tumors were demonstrated to retain EPO secretion when cultured in vitro [99,100]. Within the tumor the transcription of mRNA for EPO is confined to epithelial structures and the EPO-producing tumor cells appear therefore not to be derived from cells physiologically producing EPO in the kidney (P. Bruneval, C. Lacombe, J.L. Da Silva, B. Varet, personal communication). In other tumors sometimes associated with erythrocytosis, including hepatic carcinomas [ 101-1031, infratentorial haeman- gioblastomas and uterine myomas [cf. 1041, the role of tumor derived EPO in the pathogenesis of the erythrocytosis has been suggested, but not yet been proven.

Renal lesions. Apart from renal tumors, certain benign renal lesions may also rarely be associated with erythrocytosis due to ‘inappropriate’ EPO secretion. These are chiefly single [lo51 or multiple renal cysts [ 1061 and vascular disorders, including renal artery stenosis [ 107, I083 and microvascular abnormalities [109]. Enhanced EPO secretion resulting from renal artery stenosis is, though ‘inappropriate’ in terms of whole body oxygen supply, nevertheless due to intact renal oxygen sensing. That increased EPO secretion

40 60 80 100 120

alveolar Poz (mmHg)

Figure 6. Relationship between alveolar oxygen tensions and relative increases in EPO production rate (mean f SE; n = 6) under simulated altitude, as calculated from the raise in peripheral EPO levels, shown in Fig. 5. (Reproduced from [71] with permission. 0 1989 by Am Physiol Soc.). .

occurs only in a few patients with renal artery stenosis is most likely due to the simultaneous decline in renal sodium load that usually goes along with the limited blood flow, thus maintaining the balance between renal oxygen supply and oxygen consumption.

About 10% of patients develop erythrocytosis after renal transplantation and in some cases a selective catheterization has revealed an enhanced EPO secretion from the native diseased kidneys [110,111]. The extent to which this secretion is still responsive to changes in oxygen supply is unknown.

Erythropoietin as a therapeutic The correction of renal anaemia. The therapeutic potential of exogenously administered EPO in renal anaemia was first experimentally demonstrated by Eschbach and coworkers in 1984. They observed an improvement in the renal anaemia of uraemic sheep, when infusing them with plasma harvested from non- uraemic anaemic donor animals that contained high concentrations of EPO [112].

After recombinant EPO became available for clinical trials in humans, numerous studies since 1986 have reported that the application of the recombinant


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Figure 7. Median haematocrit levels during the dose-finding period of a multicenter trial with recombinant human EFQ (rhEPO) in anaemic dialysis patients. Patients were randomly divided into three groups receiving either 40 U (G 40), 80 U (G 80) or 120 U (G 120) rhEP0 per kg body weight intravenously three times weekly. In patients of groups (G 40) and (G 80) not reaching target haematocrit levels (30-35%) after 12 weeks of treatment, rhEP0 dose.was increased to the higher dose levels. The increment in haematwrit in groups (G 80) and (G 120) was significantly higher than in group (G 40). (Reproduced from [I] with permission 0 1988 by S. Karger AG.)

hormone effectively corrects the anaemia of end-stage renal disease in almost all patients [4-101 (Fig. 7). This correction is associated with a marked improvement in patients’ general well-being and an increase in the physical work capacity [l 13,1141. There is no evidence for the formation of anti-EPO antibodies or a reduction in the responsiveness under maintenance therapy.

The only relevant adverse effect of EPO treatment so far reported is the development or aggravation of hypertension in approximately 30% of patients [cf. 1 IS]. This increase in blood pressure appears not to be a direct effect of EPO, but rather arises from the haemodynamic changes associated with the correction of the anaemia. It is mainly due to an increase in peripheral vascular resistance [116], that results from a rise in blood viscosity and a reversal of the pre-existing hypoxic vasodilation. As the incidence of hypertension appears to be correlated with the dose-dependent rate of rise in haematocrits, a slow correction of the anaemia by EPO treatment has been suggested [115].

Outlook

Clinical research

Future studies on EPO treatment in renal anaemia will have to concentrate on the long-term cardiovascular consequences of correcting the anaemia and it must be assessed whether the benefit of improved myocardial oxygenation might in the long term be reversed by myocardial hypertrophy in response to the increased vascular resistance [ 1 171.

Future indications. With regard to EPO therapy in anaemias other than those associated with renal disease, encouraging preliminary results have been

reported in anaemic patients with rheumatoid arthritis [118]. It is also possible that other anaemias, though not primarily a result of EPO deficiency, might respond to pharmacological doses of the hormone.

In addition, a ‘supraphysiological’ stimulation with EPO may be of value in increasing the yield of pre- operative autologous blood donation programmes prior to elective surgery [ 1 19,1201.

Basic sciences Areas which will prove fruitful for future research are the mechanisms of action of EPO as well as the regulation of its biogenesis. The former includes the structure/function relationship of the molecule, isolation and structural analysis of the EPO receptor and mechanisms of EPO-induced mitosis of erythroid precursors. The latter encompasses the determination of the cellular production sites of EPO within the kidney and of the mechanisms and signals by which hypoxia finally influences the EPO gene expression. The biological significance of these questions extends beyond the physiology of EPO and will contribute to the general understanding of complex biological regulation.

Acknowledgments We are very grateful to Armin Kurtz for many helpful discussions and to Steve Reshkin for editorial advice. We also want to express our thanks to Olga Stoupa for devoted secretarial work and to Werner Gehret, who did the artwork. The research done in the authors laboratory is supported by the Swiss National Science Foundation (grant no. 3.165-88), the Hartmann


Mueller Stiftung fur medizinische Forschung and the Roche Research Foundation. K.-U. E. is a recipient of a fellowship from the German Research Foundation.

References 1 Alfrey CP, Riggs SA. Erythropoietin-an elusive hormone.

J Lab Clin Med 1981;97:141-3. 2 Lin F-K, Suggs S, Lin C-H et al. Cloning and expression of the

human erythropoietin gene. Proc Natl Acad Sci USA 1985;82:7580-4.

3 Jacobs K, Shoemaker C, Rudersdorf R et al. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 1985;3 13:806-10.

4 Winearls CG, Oliver DO, Pippard MJ, Reid C, Downing MR, Cotes PM. Effect of human erythropoietin derived from recombinant DNA on the anaemia of patients maintained by chronic haemodialysis. Lancet 1986;ii: 1175-7.

5 Eschbach JW, Egrie JC, Downing MR, Browne JK, Adamson JW. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. New Engl J Med

6 Casati S, Passerini P, Campise MR et al. Benefits and risks of protracted treatment with human recombinant erythropoietin in patients having haemodialysis. Brit Med J 1987;295:1017-20.

7 Stutz B, Rhyner K, Vdgtli J, Binswanger U. Erfolgreiche Behandlung der Animie bei Himodialyse-Patienten mit rek- ombiniertem humanem Erythropoietin. Schweiz med Wochenschr 1987;117: 1397402.

8 Bommer J, Kugel M, Schoeppe W et al. Dose-related effects of recombinant human erythropoietin on erythropoiesis. Contr Nephrol 1988;6685-93.

9 Schaefer RM, Kdrner B, Zech M, Krahn R, Heidland A. Therapie der renalen Anmie mit rekombinantem humanem Erythropoietin. Dt med Wochenschr 1988;113: 125-9.

10 Urabe A, Takaku F, Mizoguchi H et al. Effect of recombinant human erythropoietin on the anemia of chronic renal failure. Int J Cell Cloning 1988;6179-91.

11 Carnot P, Deflandre C. Sur I'activite hemopoietique des differents organes au cours de la regeneration de sang. C R Seances Acad Sci, Paris 1906;143:432-5.

12 Jelkmann W. Erythropoietin research, 80 years after the initial studies by Carnot and Deflandre. Resp Physiol1986;63:257-66.

13 Reissmann KR. Studies on the mechanism of erythropoietic stimulation in parabiotic rats during hypoxia. Blood

14 Erslev AJ. Humoral regulation of red cell production. Blood

15 Bonsdorff E, Jalavisto E. A humoral mechanism in anoxic erythrocytosis. Acta physiol Scand 1948;16:150-70.

16 Jacobson LO, Goldwasser E, Fried W, Plzak LF. The role of the kidney in erythropoiesis. Nature 1957; 179633-4.

17 Jelkmann W. Renal erythropoietin: properties and production. Rev Physiol Biochem Pharmacol 1986;104:140-215.

18 Gordon AS, Cooper GW, Zanjani ED. The kidney and erythropoiesis. Semin Hematol 1967;4337-58.

19 Peschle C, Condorelli M. Biogenesis oferythropoietin: evidence for pro-erythropoietin in a subcellular fraction of the kidney. Science 1975; 190:91&12.

20 Bondurant MC, Koury MJ. Anemia induces accumulation of erythropoietin mRNA in the kidney and liver. Mol Cell Biol 1986;6273 1-3.

21 Beru N, McDonald J, Lacombe G, Goldwasser E. Expression of the erythropoietin gene. Mot Cell Biol 1986;6:2571-5.

22 Schuster SJ, Wilson JH, Erslev AJ, Car0 J. Physiologic regulation and tissue localization of renal erythropoietin messenger RNA. Blood 1987;70:316-18.

23 Koury MJ, Bondurant MC, Graber SE, Sawyer ST. Erythro- poietin messenger RNA levels in developing mice and transfer of '*'I-erythropoietin by the placenta. J Clin Invest 1988;82: 154-9.

24 Miyake T, Kung CKH, Goldwasser E. Purification of human erythropoietin. J Biol Chem 1977;2525558-64.

1987;316:73-8.

1950;5:372-80.

1953;8:349-57.

25 Powell JS, Berkner KL, Lebo RV, Adamson JW. Human erythropoietin gene: High level expression in stably transfected mammalian cells and chromosome localization. Proc Natl

26 Lai P-H, Everett R, Wang F-F, Arakawa T, Goldwasser E. Structural characterization of human erythropoietin. J Biol Chem 1986;261:3116-21.

27 McDonald JD, Lin F-K, Goldwasser E. Cloning, sequencing, and evolutionary analysis of the mouse erythropoietin gene. Mol Cell Biol 1986;6:842-8.

28 Recny MA, Scoble HA, Kim Y. Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. J Biol Chem 1987;262:17156-63.

29 Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells bv a human ervthroooietin cDNA. J Biol Chem

Acad Sci USA 1986;83:6465-9.

30

31

32

33

34

- . 1987;2b2: 12059-76. Lukowsky WA, Painter RH. Studies on the role of sialic acid in the physical and biological properties of erythropoietin. Can J Biochem 1972;50:909-17. Smith Dordal M, Wang FF, Goldwasser E. The role of carbohydrate in erythropoietin action. Endocrinology

Mufson RA, Gesner TG. Binding and internalization of recombinant human erythropoietin in murine erythroid precursor cells. Blood 1987;69: 1485-90. Nijhof W, Wierenga PK. Isolation and characterization of the erythroid progenitor cell CFU-E. J Cell Biol 1983;96:386-92. Yen YP, Zabala P, Doney K ef al. Hematopoietic growth factors in human serum. Erythroid burst-promoting activity in normal subiects and in patients with severe aplastic anemia. J

1985;116:2293-9.

Lab Clin hied 1985;106384-92. 35 Fukamachi H, Saito T, Tojo A, Kitamura T, Urabe A, Takaku

F. Binding of erythropoietin to CFU-E derived from fetal mouse liver cells. Exp Hematol 1987;15:833-7.

36 Mayeux P, Billat C, Jacquot R. The erythropoietin receptor of rat erythroid progenitor cells. Characterization and affinity cross-linkage. J Biol Chem 1987;262 13985-90.

37

38

39

40

41

42

43

44

45

Sawyer S, Krantz S, Luna J. Identification of the receptor for erythropoietin by cross-linking to Friend virus-infected erythroid cells. Proc Natl Acad Sci USA 1987;8436904. Sawada K, Krantz S, Kans J et al. Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin. J Clin Invest 1987;80:357-66. Broudy VC, Lin N, Egrie J et al. Identification of the receptor for erythropoietin on human and murine erythroleukemia cells and modulation by phorbol ester and dimethyl sulfoxide. Proc Natl Acad Sci USA 1988;85:6513-17. Miller BA, Scaduto RC, Tillotson DL, Botti JJ, Cheung JY. Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors. J Clin Invest 1988;82:309-15. Lacombe C, Da Silva J-L, Bruneval Pet al. Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney. J Clin Invest 1988;81:620-3. Koury ST, Bondurant MC, Koury MJ. Localization oferythropoietin synthesizing cells in murine kidney by in situ hybridization. Blood 1988;71:524-7. Zanjani ED, Peterson EN, Gordon AS, Wasserman LR. Erythropoietin production in the fetus: role of the kidney and maternal anemia. J Lab Clin Med 1974;83:281-7. Fried W. The liver as source ofextrarenal erythropoietin. Blood

Sherwood JB, Goldwasser E. Extraction of erythropoietin from normal kidnevs. Endocrinoloev 1978: 103:866-70.

1972;4O:67 1-7.

46 Jelkmann W: Bauer C. hkonstrat ion of high levels of erythropoietin in rat kidneys following hypoxic hypoxia. F'fliigers Arch 1981;393:34-9.

47 Bauer C. Chemoreception of oxygen in the kidney and erythropoietin production. In: Rich IN, ed. Molecular and Cellular Aspects of Erythropoietin and Erythropoiesis. NATO AS1 Series H 1986;8:311-27.

48 Kurtz A, Ekkardt K-U, Tannahill L, Bauer C. Regulation of erythropoietin production. Contr Nephrol 1988;66:1-16.


49 Eckardt K-U, Kurtz A, Bauer C. Erythropoietin formation is related to proximal tubular function. Am J Physiol 1989 (in press).

50 Bauer C, Kurtz A. Oxygen sensing in the kidney and its relation to erythropoietin formation. Ann Rev Physiol 1989;51: 845- 56.

51 Paul P, Rothmann SA, Meagher RC. Modulation of erythropoietin production by adenosine. J Lab Clin Med

52 Ueno M, Brookins J, Beckman B, Fisher JW. A, and A2 adenosine receptor regulation of erythropoietin production. Life Sci 1988;43:229-37.

53 Leichtweiss HP, Lubbers DW, Weiss C, Baumgiirtl H, Reschke W. The oxygen supply of the rat kidney. Measurements of intrarenal Pe. Pflgers Arch 1969;309:32849.

54 Rege AB, Brookins J, Fisher JW. A radioimmunoassay for erythropoietin: serum levels in normal human subjects and patients with hemopoietic disorders. J Lab Clin Med 1982; 100:82943.

55 Garcia JF, Ebbe SN, Hollander L, Cutting HO, Miller ME, Cronkite EP. Radioimmunoassay of erythropoietin: circulating levels in normal and polycythemic human beings. J Lab Clin Med 1982;99:624-35.

56 Eckardt K-U, Kurtz A, Hirth P, Scigalla P, Wieczorek L, Bauer C. Evaluation of the stability of human erythropoietin in samples for radioimmunoassay. Klin Wochenschr

57 Naets JP, Garcia JF, Tousaaint C, Buset M, Waks D. Radioimmunoassay of erythropoietin in chronic uraemia or anephric patients. Scand J Haematol 1986373904.

58 Sherwood JB, Carmichael LD, Goldwasser E. The heteroge- neity of circulating human serum erythropoietin. Endocrino-

59 Egrie JC, Eschbach JW, McGuire T, Adamson JW. Pharma- cokinetics of recombinant human erythropoietin (rHuEPO) administered to hemodialysis @ID) patients. Kidney Int 1988;33:262a.

60 Kindler J, Eckardt K-U, Ehmer B et ul. Single dose pharmaco- kinetics of recombinant human erythropoietin (rHuEFQ) in patients with various degrees of renal failure. Nephrol Dial Transp 1989 (in press).

61 Fu J-S, Lertora JJL, Brookins J, Rice JC, Fisher JW. Pharma- cokinetics of erythropoietin in intact and anephric dogs. J Lab Clin Med 1988;111:669-76.

62 Emmanouel DS, Goldwasser E, Katz AJ, Metabolism of pure human erythropoietin in the rat. Am J Physiol 1984;247: 168- 76.

63 Stohlman F Jr, Brecher G. Humoral regulation of erythropoiesis. V. Relationship of plasma erythropoietin level to bone marrow activity. Proc Soc Exp Biol Med 1959;100:4043.

64 Naets JP, Wittek M. Effect of erythroid hypoplasia on utiliza- tion of erythropoietin. Nature 1965;206:726-7.

65 Bozzini CE. Influence of erythroid activity of the bone marrow on the plasma disappearance of injected erythropoietin in dogs. Nature 1966;209: 1140-1.

66 Cotes PM. Immunoreactive erythropoietin in serum. Brit J Haem 1982;50427-38.

67 Miller ME, Garcia JF, Cohen RA, Cronkite EP, Moccia G, Acevedo J. Diurnal levels of immunoreactive erythropoietin in normal subjects and subjects with chronic lung disease. Brit J Haem 1981;49189-200.

68 Cotes PM, Brozovic B. Diurnal variation of serum immunoreactive erythropoietin in a normal subject. Clin Endocr

69 Cotes PM, Dore CJ, Yin JAL er uf. Determination of serum immunoreactive erythropoietin in the investigation of erythrocytosis. New Engl J Med 1986;315:283-7.

70 Erslev AJ, Caro J. Pure erythrocytosis classified according to erythropoietin titers. Am J Med 1984;76:57-61.

71 Eckardt K-U, Boutellier U, Kurtz A, Schopen M, Koller EA, Bauer C. Rate of erythropoietin formation in humans in response to acute hypobaric hypoxia. J Appl Physiol 1989 (in press).

1988;112: 168-73.

1988;6624 1-5.

logy 1988;122: 1472-7.

1982; 17~419-22.

72 Faura J, Ramos J. Reynafarje C, English E, Finne P, Finch CA. Effect of altitude on erythropoiesis. Blood 1969;33:668-76.

73 Abbrecht PH, Littell JK. Plasma erythropoietin in men and mice during acclimatization to different altitudes. J Appl Physiol 1972;3254-8.

74 MiUedge JS, Cotes PM. Serum erythropoietin in humans at high altitude and its relation to plasma renin. J Appl Physiol 1985;59360-4.

75 Koemer HP, Goldwasser E. Erythropoietin radioimmunoassay in evaluating patients with polycythemia. Ann Int Med 198 1;94&7.

76 Wedzicha JA, Cotes PM, Empey DW, Newland AC, Royston JP, Tam RC. Serum immunoreactive erythropoietin in hypoxic lung disease with and without polycythaemia. Clin Sci

77 Erslev AJ, Wilson J, Caro J. Erythropoietin titers in anemic, nonuremic patients. J Lab Clin Med 1987;109:429-33.

78 Urabe A, Saito T, Fukamachi H, Kubota M, Takaku F. Serum erythropoietin titers in the anemia of chronic renal failure and other hematological states. fnt J Cell Cloning 1987;5:202-8.

79 Baer AN, Dessypris EN, Goldwasser E, Krantz SB. Blunted erythropoietin response to anaemia in rheumatoid arthritis. Brit J Haem 1987;66559-64.

80 Birgegard G, Hiillgren R, Car0 J. Serum erythropoietin in rheumatoid arthritis and other inflammatory arthritides: relationship to anaemia and the effect of anti-inflammatory treatment. Brit J Haem 1987;65:479-83.

81 Lindsay RM, Burton JA, King P, Davidson JF, Boddy K, Kennedy A. The measurement of dialyser blood loss. Clin Nephrol 1973; 1:24-8.

82 Shaw AB. Haemolysis in chronic renal failure. Brit Med J

83 Eschbach JW, Adamson JW. Anemia of end-stage renal disease (ESRD). Kidney Int 1985;28:1-5.

84 Mladenovic J. Aluminium inhibits erythropoiesis in virro. J Clin Invest 1988;8 I: 1661 -5.

85 Mdjonigle RJS, Husserl F, Wallin JD, Fisher JW. Hemodialy- sis and continuous ambulatory peritonal dialysis effects on erythropoiesis in renal failure. Kidney Int 1984;25:430-6.

86 Caro J, Brown S, Miller 0, Murray T, Erslev AJ. Erythropoie- tin levels in uremic nephric and anephric patients. J Lab Clin

87 Radtke HW, Frei U, Erbes PM, Schoeppe W, Koch KM. Improving anemia by hemodialysis: Effect on serum erythropoietin. Kidney Int 1980;17382-7.

88 Eschbach JW, Funk D, Adamson J, Kuhn J, Scribner BH, Finch CA. Erythropoiesis in patients with renal failure under- going chronic dialysis. New Engl J Med 1967;276653-6.

89 Walle AJ, Wong GY, Clemons GK, Garcia JF, Niedermayer W. Erythropoietin-hematocrit feedback circuit in the anemia of end-stage renal disease. Kidney Int 1987;31:1205-9.

90 Chandra M, Miller ME, Garcia JF, Mossey RT, McVicar M. Serum immunoreactive erythropoietin levels in patients with polycystic kidney disease as compared with other hemodialysis patients. Nephron 1985;3926-9.

91 Anderson ET, Walker BR. Polycystic kidney disease, polycythemia and azotemia. J Am Med Ass 1969;32:557-63.

92 Goldsmith HJ, Ahmad R, Raichura N er a[. Association between rising haemoglobin concentration and renal cyst formation in patients on long term regular haemodialysis treatment. Proc Europ Dial Transpl Assoc 1982;19:313-19.

93 Shalhoub RJ, Rajan U, Kim W, Goldwasser E, Kark JA, Antoniou LD. Erythrocytosis in patients on long-term hemodialysis. Ann Int Med 1982;97:686-90.

94 Kolk Veghter AJ, Bosch E, van L.eeuven AM. Influence of serum hepatitis on hemoglobin levels of patients on regular hemodialysis. Lancet 1971; i56.

95 Chan N, Barton CH, Mirahmadi MS, Gordon S, Vaziri ND. Erythropoiesis associated with viral hepatitis in end stage renal disease. Am J Med Sci 1984;287:56-8.

96 Fried W, Barone J, Schade S, Anagnostou A. Effect of carbon tetrachloride on extrarenal erythropoietin production. J Lab Clin Med 1979;93:702-7.

1985;69413-22.

1967;2213-16.

Med 1979;93:449-58.


97 Neughton BA, Kaplan SM, Roy M, Pilivo SJ, Gordon AS. Hepatic regeneration and erythropoietin production in the rat. Science 1977;196:301.

98 Donati RM, McCarthy JM, Lange RD, Gallagher NF. Eryth- rocythemia and neoplastic tumors. Ann Intern Med

99 Sherwood JB, Shouval D. Continuous production of erythropoietin by an established human renal carcinoma cell line: Development of the cell line. Proc Natl Acad Sci USA

100 Sytkowski AJ, Richie JP, Bicknell KA. New human renal carcinoma cell line established from a patient with erythrocytosis. Cancer Res 1983;43:1415-19.

101 Gordon AS, Zanjani ED, Zalusky R. A possible mechanism of the erythrocytosis associated with hepatocellular carcinoma in man. Blood 1970;35:151-7.

102 Nakao K, Kimura K, Miura Y, Takaku F. Erythrocytosis associated with carcinoma of the liver (with erythropoietin assay of tumor extract). Am J Med Sci 1966;251:161-5.

103 Sauter MA, Waldmann TA, Fallon AJ. Erythrocytosis and hyperlipemia as manifestations of hepatic carcinoma. Archs Intern Med 1967;120:735-9.

104 Hammond D, Winnick S. Paraneoplastic erythrocytosis and ectopic erythropoietins. Ann NY Acad Sci 1974;230:219-27.

105 Vertel RM, Morse BS, Prince JE. Remission of erythrocytosis after drainage of a solitary renal cyst. Archs Intern Med 1967; 120:M-8.

106 Rosse WF, Waldmann TA, Cohen P. Renal cysts, erythropoietin and polycythemia. Am J Med 1963;34:76-81.

107 Fisher JW, Schofield K, Porteous DD. Effects of renal hypoxia on erythropoietin production. Brit J Haem 196x1 1:382-8.

108 Luke RG, Kennedy AC, Stirling WB, MacDonald GA. Renal artery stenosis, hypertension and polycythemia. Brit Med J 1965;l: 164-6.

109 Kisker CT, Kleinman LI. A primary benign erythrocytosis and abnormal renal perfusion. Lancet 1971;i11624.

I10 Dagher FJ, Ramos E, Erslev AJ, Alongi SV, Karmi SA, Caro J.

1963;58:47-55.

1986;83: 165-9.

Are the native kidneys responsible for erythrocytosis in renal allorecipients? Transplantation 1979;28:496-8.

111 Thevenod F, Radtke HW, Griitzmacher P et ul. Deficient feedback regulation of erythropoiesis in kidney transplant patients with polycythemia. Kidney Int 1983;24227-32.

112 Eschbach JW, Mladenovic J, Garcia JF, Wahl PW, Adamson JW. The anemia of chronic renal failure in sheep. Response to erythropoietin-rich plasma in viuo. J Clin Invest 1984;74:434- 41.

113 Mayer G, Thum J, Cada EM, Stummvoll HK, Graf H. Working capacity is increased following recombinant human erythropoietin treatment. Kidney Int 1988;34:525-8.

114 Biicker A, Reimers E, Nonnast-Daniel B e? uZ. Effect of erythropoietin treatment on 0 2 affinity and performance in patients with renal anemia. Contr Nephrol 1988;66165-75.

115 Frei U, Nonnast DB, Koch KM. Erythropoietin und Hyperto- nic. Win Wochenschr 1988;66:914-19.

116 NonnastDB,CreutzigA,KiihnKetuZ.Effectoftreatment with recombinant human erythropoietin on peripheral hemodyna- mics and oxygenation. Contr Nephrol 1988;66185-94.

11 7 Raine AEG. Hypertension, blood viscosity, and cardiovascular morbidity in renal failure: Implications of erythropoietin therapy. Lancet 1988;i:97-9.

118 Means RT, Olsen NJ, Krantz SB et al. Treatment of the anemia of rheumatoid arthritis with recombinant human erythropoietin: Clinical and in uitro results. Blood 1987; 70 (suppl 1):139a abstr.

119 Levine EA, Rosen AL, Could SA e? ul. Recombinant human erythropoietin and autologous blood donation. Surgery

120 Kickler TS, Spivak JL. Effect of repeated whole blood dona- tions on serum immunoreactive erythropoietin levels in autologous donors. J Am Med Ass 1988;260:65-7.

121 Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: Evidence that the oxygen sensor is a heme protein. Science 1988;242 14 12- 1 5 .

1988;104:365-9.

erythropoietin in health and disease

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