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* CHAPTER 1

Regulation of oxygen sensingand erythropoietin production

Patrick H. Maxwell, Christopher W. Pugh

IRON2009_CAP.1(16-43):EBMT2008 4-12-2009 16:02 Pagina 16

1. Historical backgroundEvidence for a link between tissue oxygenation and haematocrit arose fromobservations in the late nineteenth century. An initial step was the recognitionby Bert that animals living at high altitude had an elevated haemoglobin level(1). An adaptive response to the effects of hypoxaemia was proposed by Viaultas a result of measuring changes in his own red cell concentration, and thehaematocrits of various animals, during an expedition in the Andes (2). Credit forthe concept that erythropoiesis was controlled by a humoral factor is generallyattributed to Carnot and Deflandre, following their observation in 1906 that transferof serum from an anaemic animal to an experimental animal resulted in a rise inhaematocrit in the recipient (3, 4). The experiment on which this hypothesis wasbased proved difficult to repeat and so for most of the first half of the twentiethcentury it was generally considered that anaemia had a direct effect on the bonemarrow. Notable exceptions to this opinion came from the work of Sandor (5) andKrumdieck (6) who respectively reported reticulocytosis and increased red cellprecursors in the bone marrow following the injection of serum from hypoxic donors.Further support for a circulating factor came from parabiotic rat experiments (7)and compelling evidence from the carefully controlled serum transfer experimentsof Erslev (8).The advent of reliable bioassays for erythropoietin (Epo) led to a large body ofevidence supporting the concept that oxygen delivery to the tissues regulatescirculating levels of Epo. Epo levels were shown to be elevated in patients with acuteor chronic anaemia and acute or chronic hypoxaemia (8-15). Further support camefrom studies of individuals with haemoglobin variants with increased oxygenaffinity, resulting in reduced peripheral release of oxygen into the tissues andincreased Epo production (16, 17). Similarly, the potent effect of carbon monoxidein stimulating Epo production could be explained by the fact that carboxyhaemoglobinis unavailable for oxygen transport, so that tissue oxygen delivery is reduced (18,19). In contrast to the effect of reducing oxygen delivery, uncoupling mitochondrialrespiration did not stimulate Epo production, suggesting that the underlyingcontrol mechanism might sense the concentration of oxygen itself, rather than anycompromise of mitochondrial function (20, 21). An intriguing observation was thatcobalt exposure increases Epo production, which turned out to be a major clue tothe underlying oxygen sensing mechanism (discussed below) (22, 23).Organ ablation studies proved that the kidney was important for Epo production(24-28). However, for some time it was considered that the “oxygen sensor”might be in a different location from the site of Epo production, and that the roleof the kidney might be to activate a pro-hormone produced elsewhere (29, 30).

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CHAPTER 1 • Regulation of oxygen sensing and erythropoietin production


Important experiments in this context were the demonstration that an isolatedperfused kidney could respond to reduced oxygen delivery by increasing EPOmessenger RNA expression, and hence Epo production (31-34). Conclusive proofthat both oxygen-sensing and Epo production could reside in a single cell type camefrom the demonstration by Goldberg and Bunn that the clonal human hepatomacell lines Hep3B and HepG2 were each capable of producing more Epo in responseto hypoxia (35, 36).A landmark achievement was the purification of Epo from the urine of patients withaplastic anaemia (37), which led to the cloning of the EPO gene in 1985 (38, 39).This provided a route to making recombinant human Epo, which has revolutionisedthe care of patients with kidney disease (40, 41). It also provided the necessarytools to investigate the tissue distribution of Epo production using mRNA analysis.This was important because Epo is released into the circulation as it is synthesised,rather than being stored intracellularly, and Epo production is regulated mainly atthe level of gene transcription. The ability to measure changes in EPO expressionaccurately, and the ability to identify and analyse regulatory sequences adjacentto the EPO gene were important steps towards understanding the genetic mechanismscontrolling which organs produce Epo at different stages in development and alsothe mechanisms underlying regulation of Epo production in response to stimuli suchas anaemia and hypoxia. The latter processes have been of particular interestsince the underlying oxygen sensing system and transduction mechanism iswidespread and has been shown to regulate expression of many other genes inresponse to changes in ambient oxygen tension (see below).

2. Sites of Epo production

2.1 The kidneyIn adult life the kidney is the dominant source of Epo. The importance of the kidneyis illustrated by human renal disease, since almost all individuals with substantiallyimpaired kidney function have defective production of Epo (28). Classical experimentshad shown that removing the kidneys virtually abolished the erythropoietic responseto hypoxia (25). Expression studies made it clear that the adult kidney contains moreEPO mRNA than other tissues in the basal state, and that this is greatly increasedwhen blood oxygen content is reduced (42-44).Within the normal kidney, the cells that produce Epo are the fibroblasts of the cortexand the outer medulla (45-47). In the basal state, a small number of fibroblasts atthe cortico-medullary junction express EPO mRNA. When stimulated, recruitmentspreads outwards, with some evidence that individual cells are recruited in an all-

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or-none fashion. In response to maximal stimulation it is likely that all thefibroblasts in the cortex and outer medulla express Epo, so it is clear that Epoproduction is not the task of a specialist subpopulation. It is slightly surprising thatthe part of the kidney where the mean oxygen tension is lowest – the innermedulla – does not produce Epo. It is known that oxygen gradients in the cortexare very steep, but as yet the actual oxygen tension at which fibroblasts produceEpo in vivo has not been determined.It is important to recognise that while direct monitoring of arterial pO2 providesan appropriate signal for controlling respiration via the carotid body, arterial pO2is not influenced by haemoglobin concentration and so would not provide thenecessary information for regulating Epo production. In contrast, measuring localpO2 in a tissue in which oxygen is being consumed at a constant rate will not onlybe altered by arterial pO2, but also by other changes influencing the amount of oxygenthat is delivered – including changes in haemoglobin concentration. Fibroblasts areprobably in a good position to use local pO2 as a signal for blood oxygen content,since oxygen is being continuously consumed by the adjacent renal tubules, andalso because some oxygen is removed before the blood reaches the peritubularcapillary network. If tubular oxygen consumption is decreased by administrationof acetazolamide, the Epo response to hypoxia is blunted (48-50). The potentialphysiological subtlety of Epo regulation is illustrated by the fact that although theskin does not produce Epo itself, activating the hypoxic response pathway in theskin alters Epo production in the liver and kidney, probably by altering visceral bloodflow (51).

2.2 Other sites producing EpoAnimal studies have shown that besides the kidney, the liver produces a significantamount of Epo. The cells responsible are the hepatocytes and stellate cells (52, 53).Expression is very low in normal animals, but is greatly increased in anaemia, withrecruitment of cells spreading outwards from the perivenous region – the leastoxygenated part of the liver lobule. Despite this, in humans and animals it is clearthat the liver does not produce enough Epo to maintain a normal haematocrit inkidney disease. In contrast, the liver is the dominant site of Epo production duringfoetal life. Interestingly, the timing of the switch from liver to kidney is species-dependent amongst mammals, implying that changes in oxygenation at birth arenot the key factor (54, 55).EPO mRNA has been found in many organs besides the kidney and liver – includingthe brain, retina, placenta and testis. In these sites it is also induced in responseto hypoxia (56).Epo receptor expression is not restricted to red cell precursors (57, 58), and there

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has been increasing interest in the idea that Epo may have other roles – notablyin retinal angiogenesis, and as a protective factor for neurons, photoreceptors, renaltubular cells and cardiomyocytes (59-63).

3. Regulation of tissue specific EPO gene expressionDNA sequences involved in the tissue specific and oxygen regulated expression ofthe EPO gene have been identified by studying mice bearing transgenes from thehuman EPO locus (64-68). Mice bearing a 4 kilobase human transgene including 0.5kilobases of 5’ flanking sequence and 0.7 kilobases of 3’ flanking sequence showpromiscuous over-expression in a variety of tissues, including those not normallyexpressing EPO, leading to polycythaemia. When the mice are made anaemic furtherinduction of transcripts from the transgene can be produced in the liver, but notthe kidney (66). In contrast, a 33 kilobase human transgene including 20 kilobasesof 5’ flanking sequence and 8.5 kilobases of 3’ flanking sequence reasonablyrecapitulates expression of the endogenous gene. The structure of the flankingsequence to the mouse gene appears more compact, since similar results wereobtained with a transgene including 9 kb of 5’ and 3.5 kilobases of 3’ flankingsequence (46). Basal expression from these large human and mouse transgenes islow and the mice do not become polycythaemic. On making the mice anaemicincreased EPO mRNA production is seen in both kidney and liver. Mapping studiesinvolving a variety of intermediate transgenes are summarised in Figure 1. Overallthe results suggest that there is a repressive element capable of suppressingheterologous overexpression of the EPO gene in a segment between 2.2 and 8.5kilobases 3’ to the human gene. A cis-acting element necessary for inducible renalexpression exists in a 14 kilobase 5’ flanking region but not in an 8.5 kilobase 5’flanking region, nor a 9.5 kilobase 5’ flanking region when the 3’ repressivesequence is present. In keeping with this a DNase I hypersensitive site that is inducedin anaemic animals is found approximately 10 kilobases 5’ to the open reading frame.Fine mapping of this renal specific element has produced inconclusive results, possiblybecause sequences that are necessary do not prove to be sufficient when tested inisolation. Alternatively it may be that the precise distance of the cis-actingelements from the promoter is crucial to recapitulate physiological regulation.Many of these transgenes produce constitutive expression in the brain, at least insome lines but the sequences responsible for this have not been mapped with thesame care as those responsible for kidney and liver expression. Expression of EPOin the testis of these transgenic animals is not reported so no conclusions can bedrawn on which cis-acting elements are relevant in this organ.Recently a more extensive 180 kb fragment from the mouse epo locus which



includes 60 kb of 5’ sequence has been used to generate transgenic mice in whichgreen fluorescent protein is expressed in an anaemia-responsive manner in renalfibroblasts and hepatocytes, which should provide a useful method for further studyof the mechanisms governing cell-type specific expression (69). Mutation of a singlenucleotide in the GATA motif in the promoter region led to expression in renal tubularepithelium, suggesting that GATA-2 and GATA-3 may be important in repressingexpression of EPO in these cells.

4. Regulation of EPO transcriptionIn 1991 a hypoxia responsive enhancer was identified lying 3’ to the mouse andhuman EPO genes (70-72). Although anaemia-inducible DNase I hypersensitivity sites



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Figure 1: Transgenes used to define DNA elements responsible for inducibleerythropoietin expression in liver and kidney

A variety of human erythropoietin transgenes of varying lengths used in the mapping of kidney andliver specific control elements are illustrated. For each transgene the rectangle represents the DNA encodingthe erythropoietin exons, the line to the left of the rectangle represents the 5’ flanking sequence andthe line to the right represents the 3’ flanking sequence. The length of these flanking sequences in kilobasesis indicated numerically. The ability of each construct to confer inducible expression in kidney or liverand their ability to cause dysregulated expression leading to polycythaemia is indicated. The genomicregions responsible for kidney expression, liver expression, repression of polycythaemia and hypoxicregulation (HRE) are indicated at the bottom of the Figure.

Inducibleexpression Polycythaemia

Epo 33 Kidney & Liver No

Epo 22 Liver No

Epo 4 Liver Yes

Epo 10 Liver Yes

Epo 18 Kidney & Liver Yes

Epo 22 Kidney & Liver Yes

Epo 13 Liver Yes

Epo 15 Liver Yes

Epo 1.6 Liver Yes

Mouse Epo Kidney & Liver No

9.5

20

16.5

14

8.5

8.5

0.3 0.7

0.70.7

0.7

0.70.7

3.5

2.2

6.0

5.01.6

6.00.3

9

8.5

Kidneyelement

Liverelement

HRE Repression ofpolycythaemia


were identified in a variety of locations at the EPO locus it was transient transfectionstudies of EPO gene regulation in the HepG2 and Hep3B human hepatoma cell linesthat led to the precise identification of this enhancer element which up-regulatestranscription in hypoxia. The element is located approximately 120 bases pairs 3’to the polyadenylation signal and is the binding site for the subsequently identifiedhypoxia inducible factor (HIF) transcriptional complex (71, 73-77). In addition tohypoxia, enhancer activity can be induced by treatment of cells with the iron chelatordesferrioxamine or with transition metals including cobalt, nickel and manganeseions. Insensitivity of enhancer activity to cyanide, azide and rotenone has been takenas further evidence that the sensing mechanism responds directly to oxygen levelsrather than the metabolic compromise that is a consequence of hypoxia. Indeed ithas recently been suggested that by blocking oxygen consumption, these agentsmay increase local oxygen availability and thus reduce signalling via this pathway.Hypoxia inducible factor HIF-1 was cloned by Wang and Semenza in 1995 (76). Itconsists of a heterodimer of the basic helix loop helix PAS (for Per-Arnt-Sim)domain containing proteins, HIF-1 a and HIF-1 b, the latter having previously beenidentified as the aryl hydrocarbon receptor nuclear translocator (ARNT) (78, 79).Subsequent work has identified two further a chain genes, HIF-2 a (also known asendothelial PAS protein 1 (EPAS1) (80), HIF-like factor (HLF) (81), or HIF relatedfactor (HRF) (82)) and HIF-3 a (83-85) (one splice variant of which has beendesignated as inhibitory PAS protein (IPAS) (86), and a total of three ARNT loci arenow recognised (79, 87-90). Interestingly, mouse hypoxia-inducible factor-1 a isencoded by two different mRNA isoforms, one expressed from a tissue-specificpromoter in the testis and the other from a promoter that is active in most othertissues (91).The beta chains of HIF are constitutively expressed. Hypoxia affects the function ofHIF-a chains, and thus the complex as a whole, by two distinct, but related post-translational mechanisms (Figure 2). One system affects HIF a chain abundance whilstthe other affects transcriptional activity. When oxygen is present the a chainproteins are extremely labile, being subject to ubiquitylation via the von Hippel-Lindau(VHL) tumour suppressor protein E-3 ligase and consequent proteasomal degradation(92-95). The signal for VHL dependent targeting is hydroxylation of two critical prolylresidues within the mid section of the HIF a proteins (P402 and P564 in human HIF-1 a) (96-98). The involvement of the VHL tumour suppressor protein in the HIFdegradation process underlies the high HIF levels seen in VHL defective renalcancers and the inappropriately high Epo levels in patients with Chuvash polycythaemia(99), thereby explaining many, but not necessarily all, features of these diseases.Hydroxylation of the prolyl residues in HIF a is enzymatic, being accomplished bya family of three HIF prolyl hydroxylases (PHD 1-3) (100). In low oxygen conditions



enzyme activity is reduced, hydroxylation, and hence ubiquitylation, is reduced andthe HIF a protein stabilised, allowing dimerisation with ARNT and DNA binding. HIFtranscriptional activity is also dependent on recruitment of the p300/CBP co-activator (101). This process is also oxygen regulated, being blocked in normoxicconditions by hydroxylation of an asparagine residue in the carboxy terminalactivation domain of the HIF a chains (N803 in human HIF-1 a) (102). Thishydroxylation is another enzyme dependent process, the relevant enzyme being calledfactor inhibiting HIF (FIH) (103-105). Co-activator recruitment can also beinfluenced by expression of CITED2 (CBP/p300-interacting transactivator with glu-asp rich C-terminal domain 2) itself a hypoxia inducible protein (106). All four HIFhydroxylase enzymes are members of a family of oxoglutarate dependent dioxygenases



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HIF a

VHLOHOHOH

Poly- ubiquitin Proteasomal destruction

Co-activatorrecruitment

blocked

Active

HIFhydroxylase

Inactive

EPO gene

EPO mRNA

Epo protein

a b

ActiveHIF

complex

Basictranscriptional

machinery

P

P

N

HIF aHIF b

P

P

NCBP/P300

Co-activator

PHD 1,2,3

FIH

PHD 1,2No active

HIF

p300

Figure 2: Regulation of HIF transcriptional activity by HIF hydroxylases

Hydroxylation of critical amino acids of the HIF a chain leads to its inactivation by two mechanisms,von Hippel-Lindau mediated proteasomal destruction and blockade of co-activator recruitment. Whenthe HIF hydroxylases are inactivated hydroxylation is blocked allowing HIF a chains to heterodimerisewith HIF b, bind DNA and recruit the CBP/p300 co-activator to form an active complex leading totranscription of downstream genes including erythropoietin.

Inactive

ActiveNo active

HIF

ActiveHIF

complex


(Figure 3). They contain iron at their active site, coordinated between two histidineresidues and a carboxylate group located within a motif known as a b barrel jellyroll. The enzymes use oxoglutarate, oxygen and HIF as co-substrates, generatingcarbon dioxide, succinate and hydroxylated HIF as the products. The requirementfor molecular oxygen in this process explains the oxygen sensitivity of HIFtranscriptional activity and the presence of iron at the active site is thought to explainthe actions of iron chelators and transition metals on the erythropoietin response(reviewed in (107)). Nitric oxide is thought to bind at the active site of theenzymes perhaps explaining the effects of this gas on HIF activity (108). pHsensitivity of the enzymes or altered iron availability may contribute to thediminished Epo response in metabolic acidosis (109). Expression of differentamounts of the three enzymes is likely be important in fine-tuning the HIF response– for example in determining whether HIF-1 a or HIF-2 a is more active, and adjustingthe relationship between oxygen tension and HIF activation (110). PHD1 isexpressed in the nucleus and levels can be induced by estrogen exposure (111, 112).The mRNA and protein are most abundant in the testis, with marked expression inround spermatids (113). A recent report has suggested that PHD1 and PHD3 levelsare also determined by their rate of degradation, mediated at least in part by ubiquitinligases of the SIAH family (114, 115). PHD2 is the most abundant HIF hydroxylasein most cell types in normoxia, is largely localised in the cell cytoplasm and levelsincrease following hypoxic exposure (100, 110, 111, 116). PHD3 is found in boththe nucleus and cytoplasm, is particularly abundant in the heart, is also induced


Figure 3: Requirements for HIF hydroxylase activity

The HIF hydroxylases use oxygen, 2-oxoglutarate and HIF as co-substrates producing carbon dioxide,succinate and hydroxylated HIF as products. Iron is necessary for activity, being co-ordinated in thebeta barrel jelly roll motif at the active site of the enzyme.

Fe

HIFhydroxylases

O2

2-oxoglutarate

Prolyl/asparaginylresidue

CO2

Succinate

Hydroxyprolyl/asparaginylresidue


by hypoxia and may be particularly important in HIF degradation on re-oxygenation(110, 111). FIH is constitutively expressed in a wide variety of cell types,predominately localizing in the cytoplasm (117). Important unanswered questionsrelate to the extent to which these enzymes hydroxylate substrates other than HIF,and whether the hydroxylation of amino acids in HIF, or other substrates, isreversible. Recent work has expanded the number of components contributing toregulation of the HIF pathway with reports of OS-9 (for amplified in osteosarcoma-9), RSUME, involved in sumoylation, and NEMO (for NF-KB essential modulator)interacting with HIF and/or HIF hydroxylases and influencing activity of thesystem, although the precise scope and mechanisms of these actions requireclarification (118-120).Early work demonstrated that the EPO enhancer is oxygen-responsive in cell typesnot involved in EPO expression (121). This showed that the upstream oxygen-sensingmechanisms were present in most cells and suggested that they might regulate othergenes. Although we do not yet know the full extent of genes regulated by HIF, itis now clear that there are a very large number. HIF binding sites, containing aconsensus BRCGTGV nucleic acid sequence, have now been found in the regulatoryelements of many hypoxia-regulated genes, including those for vascular growthfactors, glucose transporters and glycolytic enzymes (122-126). HIF isoforms arewidely expressed and therefore understanding the underlying mechanisms of HIFregulation is proving to be of great importance in a wide variety of biological systemsand there is increasing interest in therapeutic manipulation of this pathway. Forexample, inactivating HIF in tumours might be useful in reducing angiogenesis andtumour growth (127), while increasing HIF activation may be useful in ischaemia(128).As yet, we know relatively little about what determines the different downstreameffects of activating HIF in different cell types – and particularly what governsthe ability to produce Epo. Although HIF-1 a and HIF-2 a are regulated verysimilarly (129), it is increasingly clear that they do not have equivalent effects(130). In fact, mice with homozygous HIF-1 a or HIF-2 a defects generally bothdie in utero, implying that any functional redundancy is limited (131). It seemedlikely that HIF-1 a would underlie EPO regulation, because this protein wasisolated from nuclear extracts using the EPO enhancer (76). However, HIF-2 a isthe isoform expressed in the renal interstitial fibroblasts which produce Epo(132), and is also highly expressed in hepatocytes (133) suggesting that HIF-2a may be the key regulator of EPO in vivo. Functional analysis supports a dominantrole for HIF-2 a in EPO production in both Hep3B and Kelly cells (134); nevertheless,HIF-1 a contributes to EPO expression in HepG2 cells (135). Recently the role of



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HIF-2 a has been directly tested in mice using conditional knockouts. Theerythrocytosis consequent on inactivating VHL in hepatocytes is corrected bydeletion of HIF-2 a in these cells, but not HIF-1 a (136). Using a global tamoxifen-dependent strategy Gruber et al. showed that deletion of HIF-2 a in adult miceresulted in anaemia (137). Taken together these studies suggest that HIF-2 aregulates EPO expression in vivo.Viable homozygous HIF-2 a defective mice have been generated by selectivebreeding, and show an impaired erythropoietin response to hypoxia. Interestingly,they are pancytopaenic, and bone marrow reconstitution experiments show that thisdefect is related to the host environment rather than an intrinsic marrow defect (138,139). This suggests that HIF-2 a regulates other key parameters important forhaemopoiesis besides Epo production.The role of other transactivating factors and processes such as methylation incontrolling tissue specific expression of the EPO gene has been studied (140).Blanchard (141, 142) systematically investigated elements within the promoter and3’ enhancer necessary for highly inducible Epo expression in Hep3B cells. Theyimplicated not only the HIF binding site but an adjacent tandem repeat of consensussteroid hormone receptor half sites in both the minimal inducible promoter andminimal enhancer element. Subsequent work suggested that at least in thehepatoma cell lines these sites were occupied by hepatic nuclear factor 4 and thatits transcriptional activity was antagonised by EAR 3 (for ERBA-related 3) (143).More recent work has suggested that the factors binding this site may differ duringdevelopment, suggesting that the retinoic acid receptor a may be important foractivity of this site in foetal liver between gestational days 9 and 12, but notthereafter (144).Whilst the regulation of the HIF complex, and thus Epo, by hypoxia is wellunderstood, it is clear that both HIF and EPO gene expression can respond to non-hypoxic influences (145-147). Some of these may explain aberrant Epo regulationin various disease processes – for example it is clear that cytokines have a potentinfluence on Epo production. The extent to which these regulatory effects aremediated via direct effects on the HIF hydroxylases or actions at other levels in thepathway is currently a subject of great interest. That biologically important effectsmay be mediated by actions on HIF hydroxylase activity is demonstrated by the recentfinding that tumours and cell lines from individuals with germline mutations infumarase or succinate dehydrogenase have reduced enzyme activity and elevatedHIF activity (148, 149). This is in keeping with the polycythaemia that is a featurein some patients with germline mutations in fumarase. Erythrocytosis has also beenreported in patients with paraganglioma, and it is notable that these tumours areassociated with succinate dehydrogenase defects. Conversely, it has recently been



suggested that anaemia associated with inappropriately low Epo levels arising duringprolonged amphotericin B treatment(150) arises because amphotericin stimulatesthe interaction between FIH and the C terminus of HIF, blocking co-activatorrecruitment (151).

5. Failure to produce Epo in diseaseIn almost all kidney diseases Epo production is reduced, but not abolished. Theseverity of the deficiency in Epo increases as the renal disease progresses. Thedeficiency of Epo is relative, rather than absolute. Commonly, circulating values ofEpo fall in the normal range. However, in normal individuals with the samehaematocrit much higher values would be expected (152). The consequence of thisrelative deficiency is that patients with renal disease have a stable reduction inhaematocrit. This is an important feature of renal disease, and patients commonlyrequire Epo (or an equivalent) to maintain a haemoglobin concentration of 11 g/dLwhen their GFR falls below about 20 mL/min. Dialysis patients, except for those withcystic kidneys, almost all need Epo. Interestingly, when individuals with renaldisease and Epo deficiency do become severely hypoxic they can mount anerythropoietic response (153). In humans it is not known to what extent this Epois produced by the remnant kidneys or by the normal liver. What did become clearin the early days of dialysis is that removal of the native kidneys in dialysispatients generally led to a marked increase in transfusion requirements. Thisimplied that the remnant kidneys do produce a significant amount of basal Epo under“normal” circumstances in dialysis patients, which was subsequently confirmed bydirect measurements (28).Why the Epo response is impaired in renal disease is not entirely clear. Since thereare more fibroblasts in renal disease, it seems unlikely that there is a loss of theEpo producing cells. Probable explanations are a change in their ability to produceEpo due to altered phenotype or microenvironmental signals. The possibility thatthe hypoxic signal is reduced in renal disease is unlikely; in fact parenchymal hypoxiais probably increased (summarised in 154). Potential clues to the decreased Epoproduction include the observation that in diabetic renal disease the Epo deficiencytends to be more marked for a given level of renal function, especially in those withautonomic neuropathy (155, 156). Conversely, patients with autosomal dominantpolycystic kidney disease often do not need exogenous Epo even when they are ondialysis (157). Interestingly, dialysis patients with other causes of renal disease whodevelop cystic changes in their remnant kidneys when on dialysis often regainsufficient Epo production, or may develop erythrocytosis due to Epo excess (158,159). It seems likely that the cysts themselves, or more likely an effect of the cysts



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on the fibroblasts, permits Epo production (160).In the past, there was considerable interest in the possibility that in renal patientsthere might be accumulation of a substance that decreased the effect of Epo onthe bone marrow (161). Some support for this is given by the observation thatincreasing the amount or efficiency of dialysis does seem to improve anaemia. Asit turns out, however, Epo resistance is not a major issue since moderate doses ofrecombinant Epo can be used to treat the vast majority of renal patients. On theother hand, in certain settings patients are resistant to Epo. Common causesinclude inflammation (162), iron deficiency (163), aluminium accumulation (164)and uncontrolled hyperparathyroidism (165). A much rarer cause is the developmentof antibodies to Epo (166).

6. Increased Epo productionA common reason for erythrocytosis is reduced tissue oxygenation leading toincreased Epo production. In general, the increased haematocrit improves tissueoxygenation so that the Epo level usually falls back to within the normal range. Inthe light of the elevated haematocrit these “normal” values are actually supra-normal.The commonest cause of this kind of secondary erythrocytosis is hypoxaemiaresulting from respiratory diseases such as emphysema or cardiovascular conditionsincluding cyanotic congenital heart disease and primary pulmonary hypertension.Intermittent hypoxia, as occurs in obstructive sleep apnea, is sometimes, but notcommonly, associated with erythrocytosis due to Epo excess. Reduced tissue oxygendelivery, and consequent increased Epo production, may occur in patients withinherited disorders of 2,3-biphosphoglycerate metabolism or with altered haemoglobinoxygen affinity (17). Renal oxygenation may be limited in renal artery stenosis butthis is rarely associated with polycythaemia, probably because of a parallel reductionin renal oxygen consumption.Similarly, living at altitude provokes an elevation of haematocrit, which is usuallylimited. However, in chronic mountain sickness a pathological response to the effectsof altitude occurs (Monge’s disease) with an inappropriately high haematocritwhich has been regarded as producing such viscous blood that tissue oxygenationbecomes further impaired. Alternatively, it has been suggested that hypoventilation,resulting in exacerbations of the environmental hypoxaemia, underlies thepathogenesis of this disease (167). Interestingly, it has recently been reported thatcobalt poisoning, by inhibiting HIF hydroxylases and thereby stimulatingerythrocytosis, may contribute to polycythaemia in a significant proportion ofhigh altitude dwellers in the Andes who obtain their water from lakes contaminatedby waste from the mines in which they work (168).



Less commonly, erythrocytosis occurs because there is Epo production from the kidneyor another organ when tissue oxygen delivery is normal. For example, erythrocytosisquite commonly occurs following renal transplantation (169, 170). The evidencesuggests that this results from dysregulated Epo production from the native kidneys,which fail to suppress production in response to erythrocytosis (in addition to failingto produce enough Epo in response to anaemia when patients are supported bydialysis). Post-transplant erythrocytosis is ameliorated with an angiotensin convertingenzyme inhibitor or angiotensin II receptor antagonist (171-173). The mechanismof this effect is incompletely understood; some authors reporting a decrease in serumEpo in some, but not all, patients treated this way (174), whilst others havesuggested a direct effect via type I angiotensin receptors on erythroid precursorsin these patients (175). However, elegant genetic experiments have recently shownthat activation of the renin-angiotensin system enhances erythropoiesis throughactions on the AT1a receptor of kidney cells and not marrow precursors (176). Thereare several case reports of increased Epo production in patients with liver diseases.Examples include viral hepatitis (177, 178) and patients with primary hepatocellularcarcinomata (179-182). These phenomena have been observed in anephric patients,suggesting EPO production from the liver itself. In keeping with the role of the liverin Epo production in foetal life (183, 184), the liver often produces other markersof the foetal phenotype, including a-foetoprotein, in these circumstances, indicatinga degree of general dedifferentiation and/or recruitment of a stem cell population.Certain tumours are associated with erythrocytosis due to excess Epo production.In the case of haemangioblastoma (185, 186), phaeochromocytoma (187) and clearcell renal cell cancer (188), this is likely to relate to loss of function of the vonHippel-Lindau tumour suppressor protein. VHL loss-of-function results in constitutiveactivation of HIF (92), but almost certainly another genetic or epigenetic changeis required to enable EPO gene transcription within the tumour cells. This is onefactor explaining why erythrocytosis is only associated with a small proportion ofVHL defective tumours; also in many cases although EPO mRNA is expressed thisis not associated with erythrocytosis, probably because Epo production is insufficientto overwhelm normal homeostasis (189).Recently cases of polycythaemia associated with other defects in the oxygensensing system have been reported. Chuvash polycythaemia is a recessive conditionassociated with a partially disabling mutation in the von Hippel-Lindau proteinresulting in inappropriate elevation of HIF activity at a given partial pressure ofoxygen (99, 190). Mice homozygous for the R200W mutation that underlies Chuvashpolycythemia show activation of HIF target genes including epo in both the kidneyand liver, with some evidence for a selective effect on HIF-2 a (191).The dioxygenases regulating HIF activity are also potential candidates for mutations



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provoking erythrocytosis, and mutations in PHD-2 have been shown to causeautosomal dominant erythrocytosis (192) (OMIM no. 609820). Again, studies of micewith inactivation of PHD enzymes is possible, and acute global inactivation of PHD2in adult mice results in erythrocytosis confirming that inactivation of PHD2 issufficient to upregulate epo. Whereas lack of PHD2 is incompatible with developmentto birth, mice which lack PHD1 or PHD3 are viable; these mice do not havesignificant erythrocytosis, but deficiency of both PHD1 and PHD3 results inerythrocytosis and HIF-2 a accumulation in the liver (193).Most recently, activating mutations in HIF-2 a have also been shown to causeautosomal dominant erythrocytosis (194-197).Overall, these studies in mice and humans suggest that PHD2 and HIF-2 a are thekey isoforms regulating Epo production in mice and humans. Very recently PHDinhibitors have been shown to be effective in increasing erythropoiesis in rodents,primates and humans (198-200).

References1. Bert P. Sur la richesse en hemoglobine du sang des animaux vivant sur les hauts lieux.

C.R. Acad Sci Paris 1882; 94: 805-807.2. Viault F. Sur l’augmentation considerable du nombre des globules rouges dans le sang

chez les habitants des hauts plateaux de l’Amerique du Sud. C.R. Acad Sci Paris 1890;111: 918-919.

3. Carnot P. Sur le mécanisme de l’hyperglobulie provoquée par le serum d’animaux enrénovation sanguine. C.R. Soc Biol Paris 1906; 111: 344-346.

4. Carnot P. Deflandre C. Sur l’activité hémopoiétique du sérum au cours de la régénérationdu sang. C.R. Acad Sci Paris 1906; 143: 384-386.

5. Sandor G. Über die blutbildende Wirkung des Serums von Tieren, die in verdunnter Luftgehalten wurden. Z Gesamte Exp Med 1932; 82: 633-646.

6. Krumdieck N. Erythropoietic substance in the serum of anaemic animals. Proc Soc ExpBiol Med 1943; 54: 14-17.

7. Reissmann KR. Studies on the mechanism of erythropoietic stimulation in parabiotic ratsduring hypoxia. Blood 1950; 5: 372-379.

8. Erslev AJ. Humoral regulation of red cell production. Blood 1953; 8: 349-357.9. Caro J, Erslev AJ, Silver R et al. Erythropoietin production in response to anemia or hypoxia

in the newborn rat. Blood 1982; 60: 984-988.10. Milledge JS, Cotes PM. Serum erythropoietin in humans at high altitude and its relation

to plasma renin. J Applied Physiol 1985; 59: 360-364.11. Eckardt K-U, Boutellier U, Kurtz A et al. Rate of erythropoietin formation in humans

in response to acute hypobaric hypoxia. J Applied Physiol 1989; 66: 1785-1788.12. Necas E, Neuwirt J. Feedback regulation by red cell mass of the sensitivity of the

erythropoietin-producing organ to hypoxia. Blood 1970; 36: 754-763.13. Lenfant C, Torrance J, English E et al. Effect of altitude on oxygen binding by hemoglobin



and on organic phosphate levels. J Clin Invest 1968; 47: 2652-2656.14. Schooley JC, Mahlmann LJ. Evidence for the de novo synthesis of erythropoietin in hypoxic

rats. Blood 1972; 40: 662-670.15. Lorentz A, Jendrissek A, Eckardt K-U et al. Serial immunoreactive erythropoietin levels

in autologous blood donors. Transfusion 1991; 31: 650-654.16. Adamson JW, Parer JT, Stamatoyannopoulos G. Erythrocytosis associated with hemoglobin

Rainier: oxygen equilibria and marrow regulation. J Clin Invest 1969; 48: 1376-1386.17. Adamson JW, Finch CA. Hemoglobin function, oxygen affinity and erythropoietin. Annu

Rev Physiol 1975; 37: 351-369.18. Scaro JL, Carrera MA, Prado de Tombolesi AR, Miranda C. Carbon monoxide and

erythropoietin production in mice. Acta Physiol Lat Am 1975; 25: 204-210.19. Jelkmann W, Seidl J. Dependence of erythropoietin production on blood oxygen

affinity and hemoglobin concentration in rats. Biomed Biochim Acta 1987; 46: S304-S308.

20. Necas E, Thorling EB. Unresponsiveness of erythropoietin-producing cells to cyanide. AmJ Physiol 1972; 222: 1187-1190.

21. Necas E, Neuwirt J. The effect of inhibitors of energy metabolism on erythropoietinproduction. J Lab Clin Med 1972; 79: 388-396.

22. Necas E, Neuwirt J. Cobalt and erythropoietin production. In: The regulation oferythropoiesis and haemoglobin synthesis. Trávnícek T and Neuwirt J editors. UniversitaKarlova, Prague 1971: 91-97.

23. Miller ME, Howard D, Stohlman F, Flanagan P. Mechanism of erythropoietin productionby cobaltous chloride. Blood 1974; 44: 339-346.

24. Jacobson LO, Goldwasser E, Fried W, Plzak L. Role of the kidney in erythropoiesis. Nature1957; 179: 633-634.

25. Schooley JC, Mahlmann LJ. Erythropoietin production in the anephric rat. I. Relationshipbetween nephrectomy, time of hypoxic exposure and erythropoietin production. Blood1972; 39: 31-38.

26. Erslev AJ. Erythropoietin function in uremic rabbits II: Effect of nephrectomy on red cellproduction and iron metabolism. Acta Haematologica (Scandinavica) 1960; 3: 226-236.

27. Kominami N, Lowrie EG, Ianhez LE et al. The effect of total nephrectomy on hematopoiesisin patients undergoing chronic haemodialysis. J Lab Clin Med 1971; 78: 524-532.

28. Caro J, Brown S, Miller O et al. Erythropoietin levels in uremic nephric and anephricpatients. J Lab Clin Med 1979; 93: 449-458.

29. Gordon AS, Cooper GW, Zanjani ED. The kidney and erythropoiesis. Sem Hematol 1967;4: 337-358.

30. Gordon AS, Kaplan SM. Erythrogenin (REF). In: Kidney Hormones. Fisher JW editor.Academic Press, London 1977: 187-230.

31. Kuratowska Z, Lewartowski B, Michalak E. Studies on the production of erythropoietinby isolated perfused organs. Blood 1961; 18: 527-534.

32. Pagel H, Jelkmann W, Weiss C. Erythropoietin production in the isolated perfusedkidney. Biomed Biochim Acta 1990; 49: S271-274.

33. Scholz H, Schurek H-J, Eckardt K-U et al. Oxygen-dependent erythropoietin production



31


by the isolated perfused rat kidney. Pflugers Archives 1991; 417: 1-6.34. Ratcliffe PJ, Jones RW, Phillips RE et al. Oxygen-dependent modulation of erythropoietin

mRNA levels in isolated rat kidneys studied by RNAase protection. J Exp Med 1990; 172:657-660.

35. Goldberg MA, Glass GA, Cunningham JM, Bunn HF. The regulated expression oferythropoietin by two human hepatoma cell lines. Proc Natl Acad Sci USA 1987; 84: 7972-7976.

36. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: Evidence thatthe oxygen sensor is a heme protein. Science 1988; 242: 1412-1415.

37. Miyake T, Kung KH, Goldwasser E. Purification of human erythropoietin. J Biol Chem 1977;252: 5558-5564.

38. Jacobs K, Shoemaker C, Rudersdorf R et al. Isolation and characterization of genomicand cDNA clones of human erythropoietin. Nature 1985; 313: 806-810.

39. 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: 7580-7584.

40. Winearls CG, Oliver DO, Pippard MJ et al. Effect of human erythropoietin derived fromrecombinant DNA on the anaemia of patients maintained by chronic haemodialysis. Lancet1986; ii: 1175-1178.

41. Eschbach JW, Egrie JC, Downing MR et al. Correction of the anemia of end-stage renaldisease with recombinant human erythropoietin: Results of the Phase I and II clinicaltrial. New Engl J Med 1987; 316: 73-78.

42. Bondurant MC, Koury MJ. Anemia induces accumulation of erythropoietin mRNA in thekidney and liver. Mol Cell Biol 1986; 6: 2731-2733.

43. Tan CC, Eckardt K-U, Firth JD, Ratcliffe PJ. Feedback modulation of renal and hepaticerythropoietin mRNA in response to graded anemia and hypoxia. Am J Physiol 1992; 263:F474-F481.

44. Fandrey J, Bunn HF. In vivo and in vitro regulation of the erythropoietin mRNA:measurement by competitive polymerase chain reaction. Blood 1993; 81: 617-623.

45. Koury ST, Koury MJ, Bondurant MC et al. Quantitation of erythropoietin-producing cellsin kidneys of mice by in situ hybridization: Correlation with hematocrit, renalerythropoietin mRNA and serum erythropoietin concentration. Blood 1989; 74: 645-651.

46. Maxwell PH, Osmond MK, Pugh CW et al. Identification of the renal erythropoietin-producing cells using transgenic mice. Kidney Int 1993; 44: 1149-1162.

47. Bachmann S, Le Hir M, Eckardt K-U. Co-localization of erythropoietin messenger RNA andecto-5’-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicatesthat fibroblasts produce erythropoietin. J Histochem Cytochem 1993; 41: 335-341.

48. Fisher JW, Knight DB, Couch C. The influence of several diuretic drugs on erythropoietinformation. J Pharmacol Exp Ther 1963; 141: 113-121.

49. Eckardt K-U, Kurtz A, Bauer C. Regulation of erythropoietin production is related toproximal tubular function. Am J Physiol 1989; 256: F942-947.

50. Eckardt K-U, Kurtz A, Scholz H, Bauer C. Regulation of erythropoietin formation in vivo.Does excretory renal function play a role in the endocrine production of erythropoietin?Contrib Nephrol 1989; 76: 33-38.



51. Boutin AT, Weidemann A, Fu Z et al. Epidermal sensing of oxygen is essential forsystemic hypoxic response. Cell 2008; 133: 223-234.

52. Koury ST, Bondurant MC, Koury MJ, Semenza GL. Localization of cells producingerythropoietin in murine liver by in situ hybridization. Blood 1991; 77: 2497-2503.

53. Maxwell PH, Ferguson DJP, Osmond MK et al. Expression of a homologously recombinederythropoietin-SV40 T antigen fusion gene in mouse liver: Evidence for erythropoietinproduction by Ito cells. Blood 1994; 84: 1823-1830.

54. Peschle C, Marone G, Genovese A et al. Erythropoietin production by the liver in fetal-neonatal life. Life Sci 1975; 17: 1325-1330.

55. Eckardt K-U, Ratcliffe PJ, Tan CC et al. Age-dependent expression of the erythropoietingene in rat liver and kidneys. J Clin Invest 1992; 89: 753-760.

56. Tan CC, Eckardt K-U, Ratcliffe PJ. Organ distribution of erythropoietin messenger RNAin normal and uremic rats. Kidney Int 1991; 40: 69-76.

57. Masuda S, Nagao M, Takahata K et al. Functional erythropoietin receptor of the cells withneural characteristics. Comparison with receptor properties of erythroid cells. J Biol Chem1993; 268: 11208-11216.

58. Anagnostou A, Liu Z, Steiner M et al. Erythropoietin receptor mRNA expression inhuman endothelial cells. Proc Natl Acad Sci USA 1994; 91: 3974-3978.

59. Calvillo L, Latini R, Kajstura J et al. Recombinant human erythropoietin protects themyocardium from ischemia-reperfusion injury and promotes beneficial remodeling. ProcNatl Acad Sci USA 2003; 100: 4802-4806.

60. Grimm C, Wenzel A, Groszer M et al. HIF-1-induced erythropoietin in the hypoxic retinaprotects against light-induced retinal degeneration. Nature Med 2002; 8: 718-724.

61. Sakanaka M, Wen T-C, Matsuda S et al. In vivo evidence that erythropoietin protectsneurons from ischemic damage. Proc Natl Acad Sci USA 1998; 95: 4635-4640.

62. Shingo T, Sorokan ST, Shimazaki T et al. Erythropoietin regulates the in vitro and in vivoproduction of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci2001; 21: 9733-9743.

63. Zaman K, Ryu H, Hall D et al. Protection from oxidative stress-induced apoptosis in corticalneuronal cultures by iron chelators is associated with enhanced DNA binding ofhypoxiainducible factor-1 and ATF-1/CREB and increased expression of glycolyticenzymes, p21 (waf1/cip1), and erythropoietin. J Neurosci 1999; 19: 9821-9830.

64. Semenza GL, Dureza RC, Traystman MD et al. Human erythropoietin gene expression intransgenic mice: multiple transcription initiation sites and cis-acting regulatory elements.Mol Cell Biol 1990; 10: 930-938.

65. Semenza GL, Koury ST, Nejfelt MK et al. Cell type-specific and hypoxia-inducibleexpression of the human erythropoietin gene in transgenic mice. Proc Natl Acad Sci USA1991; 88: 8725-8729.

66. Semenza GL, Traystman MD, Gearhart JD et al. Polycythaemia in transgenic miceexpressing the human erythropoietin gene. Proc Natl Acad Sci USA 1989; 86: 2301-2305.

67. Madan A, Lin C, Hatch SL et al. Regulated basal, inducible, and tissue-specific humanerythropoietin gene expression in transgenic mice requires multiple cis DNA sequences.Blood 1995; 85: 2735-2741.



33


68. Kochling J, Curtin PT, Madan A. Regulation of human erythropoietin gene induction byupstream flanking sequences in transgenic mice. Br J Haematol 1998; 103: 960-968.

69. Obara N, Suzuki N, Kim K et al. Repression via the GATA box is essential for tissue-specificerythropoietin gene expression. Blood 2008; 111: 5223-5232.

70. Pugh CW, Tan CC, Jones RW et al. Functional analysis of an oxygen-regulated transcriptionalenhancer lying 3’ to the mouse erythropoietin gene. Proc Natl Acad Sci USA 1991; 88:10553-10557.

71. Semenza GL, Nejfelt MK, Chi SM et al. Hypoxia-inducible nuclear factors bind to anenhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad Sci USA1991; 88: 5680-5684.

72. Beck I, Ramirez S, Weinmann R et al. Enhancer element at the 3’-flanking regioncontrols transcriptional response to hypoxia in the human erythropoietin gene. J BiolChem 1991; 266: 15563-15566.

73. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesisbinds to the human erythropoietin gene enhancer at a site required for transcriptionalactivation. Mol Cell Biol 1992; 12: 5447-5454.

74. Wang GL, and Semenza GL. Desferrioxamine induces erythropoietin gene expression andhypoxia-inducible factor 1 DNA-binding activity: Implications for models of hypoxia signaltransduction. Blood 1993; 82: 3610-3615.

75. Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation ofDNA binding activity by hypoxia. J Biol Chem 1993; 268: 21513-21518.

76. Wang GL, Jiang B-H, Rue EA et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995; 92: 5510-5514.

77. Wang GL, Semenza GL. Purification and characterisation of hypoxia-inducible factor 1.J Biol Chem 1995; 270: 1230-1237.

78. Reyes H, Reisz-Porszasz S, Hankinson O. Identification of the Ah receptor nucleartranslocator protein (Arnt) as a component of the DNA binding form of the Ah receptor.Science 1992; 256: 193-1195.

79. Hoffman EC, Reyes H, Chu F-F et al. Cloning of a factor required for activity of the Ah(Dioxin) receptor. Science 1991; 252: 954-958.

80. Tian H, McKnight SL, Russell DW. Endothelial PAS domain protein 1 (EPAS1), a transcriptionfactor selectively expressed in endothelial cells. Genes Dev 1997; 11: 72-82.

81. Ema M, Taya S, Yokotani N et al. A novel bHLH-PAS factor with close sequence similarityto hypoxia-inducible factor 1a regulates the VEGF expression and is potentially involvedin lung and vascular development. Proc Natl Acad Sci USA1997; 94: 4273-4278.

82. Flamme I, Fröhlich T, von Reutern M et al. HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1a anddevelopmentally expressed in blood vessels. Mech Dev 1997; 63: 51-60.

83. Hara S, Hamada J, Kobayashi C et al. Expression and characterization of hypoxiainduciblefactor (HIF)-3a in human kidney: Suppression of HIF-mediated gene expression by HIF-3a. Biochem Biophys Res Commun 2001; 287: 808-813.



84. Heidbreder M, Frohlich F, Johren O et al. Hypoxia rapidly activates HIF-3a mRNAexpression. FASEB J 2003; 17: 1541-1543.

85. Maynard MA, Qi H, Chung J et al. Multiple splice variants of the human HIF-3a locusare targets of the von Hippel-Lindau E3 ubiquitin ligase complex. J Biol Chem 2003; 278:11032-11040.

86. Makino Y, Kanopka A, Wilson WJ et al. Inhibitory PAS domain protein (IPAS) is ahypoxia-inducible splicing variant of the hypoxia-inducible factor-3a locus. J BiolChem 2002; 277: 32405-32408.

87. Hirose K, Morita M, Ema M et al. cDNA cloning and tissue-specific expression of a novelbasic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the arylhydrocarbon receptor nuclear translocator (Arnt). Mol Cell Biol 1996; 16: 1706-1713.

88. Ikeda M, Nomura M. cDNA cloning and tissue-specific expression of a novel basichelixloop-helix/PAS protein (BMAL1) and identification of alternatively spliced variantswith alternative translation initiation site usage. Biochem Biophys Res Commun 1997;233: 258-264.

89. Hogenesch JB, Guy Y-Z, Jain S et al. The basic-helix-loop-helix-PAS orphan MOP3 formstranscriptionally active complexes with circadian and hypoxia factors. Proc Natl AcadSci USA 1998; 95: 5474-5479.

90. Hogenesch JB, Gu Y-Z, Moran SM et al. The basic helix-loop-helix-PAS protein MOP9 isa brain-specific heterodimeric partner of circadian and hypoxia factors. J Neurosci2000; 20: 1-5.

91. Wenger RH, Rolfs A, Kvietikova I et al. The mouse gene for hypoxia-inducible factor-1a.Genomic organization, expression and characterization of an alternative first exon and5’ flanking sequence. Eur J Biochem 1997; 246: 155-165.

92. Maxwell PH, Wiesener MS, Chang G-W et al. The tumour suppressor protein VHL targetshypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999; 399: 271-275.

93. Cockman ME, Masson N, Mole DR et al. Hypoxia inducible factor-a binding andubiquitylation by the von Hippel-Lindau tumor suppressor protein. J Biol Chem 2000;275: 25733-25741.

94. Ohh M, Park CW, Ivan M et al. Ubiquitination of hypoxia-inducible factor requires directbinding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol 2000; 2: 423-427.

95. Tanimoto K, Makino Y, Pereira T et al. Mechanism of regulation of the hypoxia induciblefactor-1a by the von Hippel-Lindau tumor suppressor protein. EMBO J 2000; 19: 4298-4309.

96. Ivan M, Kondo K, Yang H et al. HIFa targeted for VHL-mediated destruction by prolinehydroxylation: Implications for O2 sensing. Science 2001; 292: 464-468.

97. Jaakkola P, Mole DR, Tian Y-M et al. Targeting of HIF-a to the von Hippel-Lindauubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001; 292: 468-472.

98. Masson N, Willam C, Maxwell PH et al. Independent function of two destruction domainsin hypoxia-inducible factor-a chains activated by prolyl hydroxylation. EMBO J 2001; 20:5197-5206.



35


99. Ang SO, Chen H, Hirota K et al. Disruption of oxygen homeostasis underlies congenitalChuvash polycythemia. Nat Genet 2002; 32: 614-621.

100.Epstein ACR, Gleadle JM, McNeill LA et al. C. elegans EGL-9 and mammalian homologuesdefine a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001; 107:43-54.

101.Arany Z, Huang LE, Eckner R et al. An essential role for p300/CBP in the cellularresponse to hypoxia. Proc Natl Acad Sci USA 1996; 93: 12969-12973.

102.Lando D, Peet DJ, Whelan DA et al. Asparagine hydroxylation of the HIF transactivationdomain: A hypoxic switch. Science 2002; 295: 858-861.

103.Lando D, Peet DJ, Gorman JJ et al. FIH-1 is an asparaginyl hydroxylase enzyme thatregulates the transcriptional activity of hypoxia-inducible factor. Genes Dev 2002; 16:1466-1471.

104.Hewitson KS, McNeill LA, Tian Y-M et al. Hypoxia inducible factor (HIF) asparaginehydroxylase is identical to Factor Inhibiting HIF (FIH) and is related to the cupin structuralfamily. J Biol Chem 2002; 277: 26351-26355.

105.Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-1a andVHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 2001; 15: 2675-2686.

106.Bhattacharya S, Michels CL, Leung M-K et al. Functional role of p35srj, a novel p300/CBPbinding protein, during transactivation by HIF-1. Genes Dev 1999; 13: 64-75.

107.Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 2004;5: 343-354.

108.Metzen E, Zhou J, Jelkmann W et al. Nitric Oxide impairs normoxic degradation of HIF1a by Inhibition of Prolyl Hydroxylases. Mol Biol Cell 2003; 14: 3470-3481.

109.Eckardt K-U, Kurtz A, Bauer C. Triggering of erythropoietin production by hypoxia isinhibited by respiratory and metabolic acidosis. Am J Physiol 1990; 258: R678-R683.

110.Appelhoff RJ, Tian YM, Raval RR et al. Differential function of the prolyl hydroxylasesPHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J Biol Chem 2004;279: 38458-38465.

111.Metzen E, Berchner-Pfannschmidt U, Stengel P et al. Intracellular localisation of humanHIF-1a hydroylases: Implications for oxygen sensing. J Cell Science 2002; 116: 1319-1326.

112.Seth P, Porter D, Polyak K. Novel estrogen and tamoxifen induced genes identified bySAGE (Serial Analysis of Gene Expression). Oncogene 2002; 21: 836-843.

113.Willam C, Nicholls LG, Ratcliffe PJ et al. The prolyl hydroxylase enzymes that act as oxygensensors regulating destruction of hypoxia-inducible factor a. Adv Enzyme Regul 2004;44: 75-92.

114.Habelhah H, Laine A, Erdjument-Bromage H et al. Regulation of 2-oxoglutarate(alphaketoglutarate) dehydrogenase stability by the RING finger ubiquitin ligase Siah.J Biol Chem 2004; 279: 53782-53788.

115.Nakayama K, Frew IJ, Hagensen M et al. Siah2 regulates stability of prolyl-hydroxylases,controls HIF1a abundance, and modulates physiological responses to hypoxia. Cell2004; 117: 941-952.



116.Berra E, Benizri E, Ginouves A et al. HIF prolyl-hydroxylase 2 is the key oxygen sensor settinglow steady-state levels of HIF-1a in normoxia. EMBO J 2003; 22: 4082-4090.

117.Stolze IP, Tian YM, Appelhoff RJ et al. Genetic analysis of the role of the asparaginylhydroxylase FIH in regulating HIF transcriptional target genes. J Biol Chem 2004; 279:42719-42725.

118.Baek JH, Mahon PC, Oh J et al. OS-9 interacts with hypoxia-inducible factor 1alpha andprolyl hydroxylases to promote oxygen-dependent degradation of HIF-1alpha. Mol Cell2005; 17: 503-512.

119.Carbia-Nagashima A, Gerez J, Perez-Castro C et al. RSUME, a small RWD-containing protein,enhances SUMO conjugation and stabilizes HIF-1alpha during hypoxia. Cell 2007; 131:309-323.

120.Bracken CP, Whitelaw ML, Peet DJ. Activity of hypoxia-inducible factor 2alpha isregulated by association with the NF-kappaB essential modulator. J Biol Chem 2005; 280:14240-14251.

121.Maxwell PH, Pugh CW, Ratcliffe PJ. Inducible operation of the erythropoietin 3’ enhancerin multiple cell lines: Evidence for a widespread oxygen sensing mechanism. Proc NatlAcad Sci USA 1993; 90: 2423-2427.

122.Firth JD, Ebert BL, Ratcliffe PJ. Hypoxic regulation of lactate dehydrogenase A:Interaction between hypoxia inducible factor 1 and cAMP response elements. J Biol Chem1995; 270: 21021-21027.

123.Firth JD, Ebert BL, Pugh CW et al. Oxygen-regulated control elements in thephosphoglycerate kinase 1 and lactate dehydrogenase A genes: Similarities with theerythropoeitin 3’ enhancer. Proc Natl Acad Sci USA 1994; 91: 6496-6500.

124.Ebert BL, Firth JD, Ratcliffe PJ. Hypoxia and mitochondrial inhibitors regulate expressionof glucose transporter-1 via distinct cis-acting sequences. J Biol Chem 1995; 270: 29083-29089.

125.Gleadle JM, Ebert BL, Firth JD et al. Regulation of angiogenic growth factor expressionby hypoxia, transition metals, and chelating agents. Am J Physiol 1195; 268: C1362-C1368.

126.Liu Y, Cox SR, Morita T et al. Hypoxia regulates vascular endothelial growth factor geneexpression in endothelial cells. Circ Res 1995; 77: 638-643.

127.Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3: 721-732.128.Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: Role of the HIF system.

Nat Med 2003; 9: 677-684.129.O’Rourke JF, Tian YM, Ratcliffe PJ et al. Oxygen-regulated and transactivating domains

in endothelial PAS protein 1: Comparison with hypoxia inducible factor-1a. J BiolChem 1999; 274: 2060-2071.

130.Wang V, Davis DA, Haque M et al. Differential gene up-regulation by hypoxia-induciblefactor-1alpha and hypoxia-inducible factor-2alpha in HEK293T cells. Cancer Res 2005;65: 3299-3306.

131.Giaccia AJ, Simon MC, Johnson R. The biology of hypoxia: the role of oxygen sensingin development, normal function, and disease. Genes Dev 2004; 18: 2183-2194.

132.Rosenberger C, Mandriota SJ, Jurgensen JS et al. Expression of hypoxia-inducible factor



37


1a and -2a in hypoxic and ischemic rat kidneys. J Am Soc Nephrol 2002; 13: 1721-1732.133.Wiesener MS, Jürgensen JS, Rosenberger C et al. Widespread hypoxia-inducible expression

of HIF-2alpha in distinct cell populations of different organs. FASEB J 2003; 17: 271-273.

134.Warnecke C, Zaborowska Z, Kurreck J et al. Differentiating the functional role ofhypoxiainducible factor (HIF)-1alpha and HIF-2alpha (EPAS-1) by the use of RNAinterference: erythropoietin is a HIF-2alpha target gene in HepB and Kelly cells. FASEBJ 2004; 18: 1462-1464.

135.Warnecke C, Weidemann A, Volke M et al. The specific contribution of hypoxia-induciblefactor-2alpha to hypoxic gene expression in vitro is limited and modulated by cell type-specific and exogenous factors. Exp Cell Res 2008; 314: 2016-2027.

136.Rankin EB, Biju MP, Liu Q et al. Hypoxia-inducible factor-2 (HIF-2) regulates hepaticerythropoietin in vivo. J Clin Invest 2007; 117: 1068-1077.

137.Gruber M, Hu CJ, Johnson RS et al. Acute postnatal ablation of Hif-2alpha results inanemia. Proc Natl Acad Sci USA 2007; 104: 2301-2306.

138.Scortegagna M, Ding K, Oktay Y et al. Multiple organ pathology, metabolic abnormalitiesand impaired homeostasis of reactive oxygen species in Epas1-/- mice. Nat Gen 2003;35: 331-340.

139.Scortegagna M, Ding K, Zhang Q et al. HIF-2alpha regulates murine hematopoieticdevelopment in an erythropoietin-dependent manner. Blood 2005; 105: 3133-3140.

140.Wenger RH, Kvietikova I, Rolfs A et al. Oxygen-regulated erythropoietin gene expressionis dependent on a CpG methylation-free hypoxia-inducible factor-1 DNA-binding site.Eur J Biochem 1998; 253: 771-777.

141.Blanchard KL, Acquaviva AM, Galson DL, Bunn HF. Hypoxic induction of the humanerythropoietin gene: cooperation between the promoter and enhancer, each of whichcontains steroid receptor response elements. Mol Cell Biol 1992; 12: 5373-5385.

142.Hu B, Wright E, Campbell L et al. In vivo analysis of DNA-protein interactions on thehuman erythropoietin enhancer. Mol Cell Biol 1997; 17: 851-856.

143.Galson DL, Tsuchiya T, Tendler DS et al. The orphan receptor hepatic nuclear factor 4functions as a transcriptional activator for tissue-specific and hypoxia-specificerythropoietin gene expression and is antagonized by EAR3/COUP-TF1. Mol Cell Biol 1995;15: 2135-2144.

144.Makita T, Duncan SA, Sucov HM. Retinoic acid, hypoxia, and GATA factors cooperativelycontrol the onset of fetal liver erythropoietin expression and erythropoietic differentiation.Dev Biol 2005; 280: 59-72.

145. Jelkmann J, Wolff M., Fandrey J. Modulation of the production of erythropoietin bycytokines: in vitro studies and their clinical implications. Contrib Nephrol 1990; 87: 68-77.

146.Jelkmann W, Huwiler A, Fandrey J et al. Inhibition of erythropoietin production by phorbolester is associated with down-regulation of protein kinase C-alpha isoenzyme inhepatoma cells. Biochem Biophys Res Commu 1991; 179: 1441-1448.

147.Jelkmann W, Pagel H, Wolff M et al. Monokines inhibiting erythropoietin production inhuman hepatoma cultures and in isolated perfused rat kidneys. Life Sci 1992; 50: 301-308.



148.Pollard PJ, Briere JJ, Alam NA et al. Accumulation of Krebs cycle intermediates andoverexpression of HIF1alpha in tumours which result from germline FH and SDHmutations. Hum Mol Genet 2005; 14: 2231-2239.

149.Isaacs JS, Jung YJ, Mole DR et al. HIF overexpression correlates with biallelic loss offumarate hydratase in renal cancer: Novel role of fumarate in regulation of HIF stability.Cancer Cell 2005; 8: 143-153.

150.MacGregor R, Bennett JE, Erslev AJ. Erythropoietin concentration in amphotericin Binduced anemia. Antimicrob Agents Chemother 1978; 14: 270-273.

151.Yeo EJ, Ryu JH, Cho YS et al. Amphotericin B blunts erythropoietin response to hypoxiaby reinforcing FIH-mediated repression of HIF-1. Blood 2006; 107: 916-923.

152.Cotes PM, Pippard MJ, Reid CDL et al. Characterization of the anaemia of chronic renalfailure and the mode of its correction by a preparation of human erythropoietin (r-Hu-EPO). An investigation of the pharmacokinetics of intravenous erythropoietin and itseffects on erythrokinetics. Q J Med 1989; 70: 113-137.

153.Kato A, Hishida A, Kumagai H et al. Erythropoietin production in patients with chronicrenal failure. Ren Fail 1994; 16: 645-651.

154.Heyman SN, Khamaisi M, Rosen S, Rosenberger C. Renal parenchymal hypoxia, hypoxiaresponse and the progression of chronic kidney disease. Am J Nephrol 2008; 28: 998-1006.

155.Bosman DR, Winkler AS, Marsden JT et al. Anemia with erythropoietin deficiency occursearly in diabetic nephropathy. Diabetes Care 2001; 24: 495-499.

156.Winkler AS, Marsden J, Chaudhuri KR et al. Erythropoietin depletion and anaemia indiabetes mellitus. Diabet Med 1999; 16: 813-819.

157.Chandra M, Miller ME, Garcia JF et al. Serum immunoreactive erythropoietin levels inpatients with polycystic kidney disease as compared with other hemodialysis patients.Nephron 1985; 39: 26-29.

158.Edmunds ME, Devoy M, Tomson CRV et al. Plasma erythropoietin levels and acquired cysticdisease of the kidney in patients receiving regular haemodialysis treatment. Br JHaematol 1991; 78: 275-277.

159.Ratcliffe PJ, Dunnill MS, Oliver DO. Clinical importance of acquired cystic disease of thekidney in patients undergoing dialysis. Br Med J 287: 1855-1858.

160.Eckardt K-U, Möllmann M, Neumann R et al. Erythropoietin in polycystic kidneys. J ClinInvest 1989; 84: 1160-1166.

161.McGonigle RJ, Husserl F, Wallin JD et al. Hemodialysis and continuous ambulatoryperitoneal dialysis effects on erythropoiesis in renal failure. Kidney Int 1984; 25: 430-436.

162.Macdougall IC, Cooper AC. Erythropoietin resistance: The role of inflammation andproinflammatory cytokines. Nephrol Dial Transplant 2002; 17: 39-43.

163.Macdougall IC, Hutton RD, Cavill I et al. Poor response to treatment of renal anaemiawith erythropoietin corrected by iron given intravenously. Br Med J 1989; 299: 157-158.

164.Altmann P, Plowman D, Marsh F et al. Aluminium chelation therapy in dialysis patients:Evidence for inhibition of haemoglobin synthesis by low levels of aluminium. Lancet 1988;i: 1012-1014.

165.Drueke TB, Eckardt KU. Role of secondary hyperparathyroidism in erythropoietin



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resistance of chronic renal failure patients. Nephrol Dial Transplant 2002; 17: 28-31.166.Casadevall N, Nataf J, Viron B et al. and N.E.J.M.F. 14. Pure red-cell aplasia and

antierythropoietin antibodies in patients treated with recombinant erythropoietin. NewEngl J Med 2002; 346: 469-475.

167.Sun SF, Huang SY, Zhang JG et al. Decreased ventilation and hypoxic ventilatoryresponsiveness are not reversed by naloxone in Lhasa residents with chronic mountainsickness. Ame Rev Respir Dis 1990; 142: 1294-1300.

168.Jefferson JA, Escudero E, Hurtado M-E et al. Excessive erythrocytosis, chronic mountainsickness, and serum cobalt levels. Lancet 2002; 359: 407-408.

169.Dagher FJ, Ramos E, Erslev AJ et al. Are the native kidneys responsible for erythrocytosisin renal allorecipients? Transplantation 1979; 28: 496-498.

170.Wickre CG, Norman DJ, Bennison A et al. Postrenal transplant erythrocytosis: A reviewof 53 patients. Kidney Int 1983; 23: 731-737.

171.Gaston RS, Julian BA, Diethelm AG et al. Effects of enalapril on erythrocytosis after renaltransplantation. Ann Intern Med 1991; 115: 954-955.

172.Conlon PJ, Farrell J, Donohoe J et al. The beneficial effect of enalapril on post renaltransplant erythrocytosis. Transplantation 1993; 56: 217-219.

173.Midtvedt K, Stokke ES, Hartmann A. Successful long-term treatment of post-transplanterythrocytosis with losartan. Nephrol Dial Transplant 1996; 11: 2495-2497.

174.Danovitch GM, Jamgotchian NJ, Eggena PH et al. Angiotensin-converting enzymeinhibition in the treatment of renal transplant erythrocytosis. Clinical experience andobservation of mechanism. Transplantation 1995; 60: 132-137.

175.Glicklich D, Kapoian T, Mian H et al. Effects of erythropoietin, angiotensin II, andangiotensin-converting enzyme inhibitor on erythroid precursors in patients withposttransplantation erythrocytosis. Transplantation 1999; 68: 62-66.

176.Kato H, Ishida J, Imagawa S et al. Enhanced erythropoiesis mediated by activation ofthe renin-angiotensin system via angiotensin II type 1a receptor. FASEB J 2005; 19: 2023-2025.

177.Simon P, Meyrier A, Tanquerel T et al. Improvement of anaemia in haemodialysed patientsafter viral or toxic hepatic cytolysis. Br Med J 1980; 280: 892-894.

178.Klassen DK, Spivak JL. Hepatitis-related hepatic erythropoietin production. Am J Med1990; 89: 684-686.

179.McFadzean AJS, Todd D, Tso SC. Erythrocytosis associated with hepatocellular carcinoma.Blood 1967; 29: 808-811.

180.Muta H, Funakoshi A, Baba T et al. Gene expression of erythropoietin in hepatocellularcarcinoma. Intern Med 1994; 33: 427-431.

181.Kew MC, Fisher JW. Serum erythropoietin concentrations in patients with hepatocellularcarcinoma. Cancer 1986; 58: 2485-2488.

182.Matsuyama M, Yamazaki O, Horii K et al. Erythrocytosis caused by an erythropoietin-producing hepatocellular carcinoma. J Surg Oncol 2000; 75: 197-202.

183.Zanjani ED, Poster J, Burlington H et al. Liver as the primary site of erythropoietinformation in the fetus. J Lab Clin Med 1977; 89: 640-644.



184.Zucali JR, Stevens V, Mirand EA. In vitro production of erythropoietin by mouse fetalliver. Blood 1975; 46: 85-90.

185.Waldmann TA, Levin EH, Baldwin M. The association of polycythemia with a cerebellarhemangioblastoma. The production of an erythropoiesis stimulating factor by the tumor.Am J Med 1961; 31: 318-324

186.Kamitani H, Masuzawa H, Sato J et al. Erythropioetin in haemangioblastoma:immunohistochemical and electron microscopy studies. Acta Neurochir 1987; 85: 56-62.

187.Waldmann TA, Bradley JE. Polycythemia secondary to a pheochromocytoma withproduction of an erythropoiesis stimulating factor by the tumor. Proc Soc Exp Biol Med1961; 108: 425-427.

188.Da Silva JL, Lacombe C, Bruneval P et al. Tumour cells are the site of erythropoietin synthesisin human renal cancers associated with polycythemia. Blood 1990; 75: 577-582.

189.Wiesener MS, Münchenhagen P, Gläser M et al. Erythropoietin gene expression in renalcarcinoma is considerably more frequent than paraneoplastic polycythemia. Int J Cancer2007; 121: 2434-2442.

190.Percy MJ, McMullin MF, Jowitt SN et al. Chuvash-type congenital polycythemia in 4 familiesof Asian and Western European ancestry. Blood 2003; 102: 1097-1099.

191.Hickey MM, Lam JC, Bezman NA et al. von Hippel-Lindau mutation in mice recapitulatesChuvash polycythemia via hypoxia-inducible factor-2alpha signaling and splenicerythropoiesis. J Clin Invest 2007; 117: 3879-3889.

192.Percy MJ, Zhao Q, Flores A et al. A family with erythrocytosis establishes a role for prolylhydroxylase domain protein 2 in oxygen homeostasis. Proc Natl Acad Sci USA 2006; 103:654-659.

193.Takeda K, Aguila HL, Parikh NS et al. Regulation of adult erythropoiesis by prolylhydroxylase domain proteins. Blood 2008; 111: 3229-3335.

194.Percy MJ, Furlow PW, Lucas GS et al. A gain-of-function mutation in the HIF2A gene infamilial erythrocytosis. New Engl J Med 2008; 358: 162-168.

195.Gale DP, Harten SK, Reid CD et al. Autosomal dominant erythrocytosis and pulmonaryarterial hypertension associated with an activating HIF2 alpha mutation. Blood 2008;112: 919-921.

196.Percy MJ, Beer PA, Campbell G et al. Novel exon 12 mutations in the HIF2A gene associatedwith erythrocytosis. Blood 2008; 111: 5400-5402.

197.Martini M, Teofili L, Cenci T et al. A novel heterozygous HIF2AM535I mutation reinforcesthe role of oxygen sensing pathway disturbances in the pathogenesis of familialerythrocytosis. Haematologica 2008; 93: 1068-1071.

198.Bunn HF. New agents that stimulate erythropoiesis. Blood 2007; 109: 868-873.199.Hsieh MM, Linde NS, Wynter A et al. HIF prolyl hydroxylase inhibition results in

endogenous erythropoietin induction, erythrocytosis, and modest fetal hemoglobinexpression in rhesus macaques. Blood 2007; 110: 2140-2147.

200.Abstract numbers 301, 302, 364 at Keystone Conference “Molecular, Cellular, Physiological,and Pathogenic Responses to Hypoxia” held in Vancouver, British Columbia, January 15-20, 2008.



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Multiple Choice Questionnaire

To find the correct answer, go to http://www.esh.org/iron-handbook2009answers.htm

1. Please select the main site of erythropoietin productionin adult mammals:a) The stromal cells of the bone marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b) The fibroblasts in the kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c) The medulla of the adrenal gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) The Kupffer cells in the liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Which of the following statements about erythropoietin is true?a) It is a glycoprotein with molecular weight 34 kDa . . . . . . . . . . . . . . . . . . . . . . . . .

b) Administration of recombinant human erythropoietin usuallyincreases the platelet count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c) It acts on erythroid precursors through a G-proteincoupled receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) Circulating levels are often increased in patientswith colon carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Please select the mechanism responsible for the increasein circulating erythropoietin in response to anaemia:a) Reduced capture of erythropoietin by receptors

on mature erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b) Exocytosis of pre-formed erythropoietin fromWiebel-Palade bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c) Increased transcription of the erythropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) Activating cleavage of proerythropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. Only one of the statements listed below about the hypoxiainducible factor complex is true: which one?a) It includes 5 subunits; a, b, g, d and e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b) The b subunit of the HIF complex is also known as RXR3 . . . . . . . . . . . . . . . . . .

c) Activation in hypoxia is mainly due to increased transcriptionof HIF a mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



d) It is regulated mainly by oxygen dependent destructionof the a subunit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Only one of the statements listed below about renal diseaseis true: which one?a) Proteinuria (> 1g/24 hours) is usually associated with

increased erythropoietin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b) The anaemia associated with renal disease usually respondsto recombinant human erythropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c) Anaemia in renal disease is commonly due to completedeficiency of erythropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) Anaemia in end stage renal failure is mainly due to a decreasein red cell half life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



43


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