Erythropoietin as a novel brain and kidney protective agent.
In this review, we assess the evidence supporting EPO as a general tissue protective drug and discuss the potential mechanisms by which it may achieve this general effect. We then focus on EPO's neurological and renal protective effects and the potential mechanisms through which it may confer this specific protection. Finally, we review the experimental studies and clinical trials of EPO in neurological and acute kidney injury (AKI), discuss risks, lessons learned and the need for further multi-centre randomised studies in humans before any change in clinical practice is considered.
PHYSIOLOGICAL PROPERTIES OF ERYTHROPOIETIN
Structure of the erythropoietin molecule
EPO is a 30.4 kD glycoprotein and class I cytokine consisting of 165 amino acids (3). EPO has four acidic oligosaccharide side chains (3 N-linked and 1 O-linked) and contains up to 14 sialic acid residues (Table 1 (3-8), see recombinant human EPO); its carbohydrate portion contributes 40% of its molecular weight (3). The N-linked polysaccharide side chains appear to be important for the biosynthesis and secretion of EPO. They are thought to enhance stability in the blood and to limit hepatic clearance, thus facilitating the systemic transit of EPO from the kidney (main production site of EPO) to target cells in the bone marrow (9).
The polypeptide skeleton of the human EPO molecule has a constant amino acid sequence; however, the carbohydrate side-chains are heterogenic in sugar content and structure. The variable nature of the sialic acid content gives rise to EPO isoforms with differences in charge. As the number of sialic acid groups on the carbohydrate portion of EPO increase, so does its serum half-life (Table 1), while receptor-binding capacity decreases (10-13). It is important, however, to note that clearance appears to have a stronger influence on in vivo activity than receptor-binding affinity.
Each EPO molecule has two EPO receptor (EPOR) binding sites, located on opposite faces of the molecule. There are two affinities of the EPOR for EPO in solution; one of high (~1 nM) and one of low affinity (~1 microM), perhaps reflecting two non-equivalent receptor binding sites on each EPO molecule (14).
Physiological stimuli for erythropoietin production/ release
Approximately 90% of systemic EPO in adults is produced by peritubular interstitial fibroblasts in the renal cortex and outer medulla of the kidney. Most of the remaining production comes from hepatocytes in the liver, with some expression in the brain, spleen, lungs, testes and bone marrow (15). A feedback mechanism involving oxygen delivery to the tissues appears to regulate EPO production (16). Hypoxiainducible factor regulates transcription of the EPO gene in the kidney and liver which determines EPO synthesis. This process is dependent on local oxygen tension. Hypoxia-inducible factor is continually transcribed at the messenger riboneucleic acid level but, at the protein level, it is only stable in hypoxic cells. Hypoxia-inducible factor is quickly destroyed by well-oxygenated cells through ubiquitylation (tagging for degradation in the proteasome) by the von Hippel-Landau tumour suppressor protein. However when oxygen delivery decreases, the von Hippel-Landau tumour suppressor protein ceases its proteolysis of hypoxia-inducible factor, increasing the levels of hypoxia-inducible factor which subsequently increases EPO production (15,17).
Structure of erythropoietin receptors
The EPOR is a 66kD membrane glycoprotein typically consisting of 484-amino acids and two peptide chains. It belongs to a large cytokine and growth factor receptor family, which includes IL-3, -4, -6 receptors, granulocyte macrophage colony stimulating factor receptor and the growth hormone receptor, and has some common signalling mechanisms (3). The EPOR has a single transmembrane domain, an extracellular domain composed of two parts and an intracellular domain (3). Binding studies have demonstrated that the EPOR has different affinities for EPO with perhaps an accessory component of the EPOR that increases the binding affinity of EPO. Alternatively this may be due to differences in the two distinct EPO molecule receptor binding sites; however, this is yet to be ascertained (14). It is thought that EPOR isoforms with higher affinity for EPO binding may be responsible for the erythropoietic effects of EPO, while isoforms with a lower affinity for EPO binding may have nonerythropoietic effects such as tissue protection (18).
The cytoplasmic domains of the EPOR contain a number of phosphotyrosines that are phosphorylated by the activation of a member of the Janus-type protein tyrosine kinase family (JAK2), which is bound to the common beta subunit of the EPOR (19). In addition to activating the mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) pathway (Figure 114), phosphotyrosines also serve as docking sites for signal transducer and activators of transcription (STAT) such as STAT5 (14). These pathways are further described in "Postreceptor (intracellular) effects of erythropoietin" below. Dephosphorylation of JAK can be induced by phosphatase with the consequent internalisation and degradation of the EPO/EPOR complex which marks the end of EPO activity. This prevents overactivation that may lead to excessive erythrocytosis (20).
[FIGURE 1 OMITTED]
The erythropoiesis stimulating effects of erythropoietin
The principal physiological function of EPO is red blood cell production, which results from a tightly controlled proliferation and differentiation pathway (21). Early haematopoietic progenitor cells differentiate into burst-forming unit-erythroid cells on which EPORs first appear. However, EPO is not required at this stage. Burst-forming uniterythroid cells differentiate into colony-forming unit-erythroid cells, dependent on EPO for survival, with a corresponding increase in EPOR expression (21). Continuous stimulation with EPO triggers the differentiation of colony-forming unit-erythroid cells into erythroblasts, which lose their nuclei to form reticulocytes. After a few days reticulocytes lose reticulin and become erythrocytes (red blood cells). Reticulocytes and erythrocytes stop expressing EPOR and cease being responsive to EPO (21). The steady-state lag time between effect-site EPO stimulation and reticulocyte appearance in the human systemic circulation was estimated at 10.8 hours (22) and the steady-state reticulocyte residence time reported in humans is one day, which increases two- to three-fold during stress erythropoiesis (23).
EPO-binding to EPORs on erythroid progenitor cells leads to activation of the JAK2-STAT5 signalling pathway and phosphorylation of PI3K and Akt (24) (Figure 1). Akt-mediated phosphorylation of Bad (a pro-apoptotic member of the B-cell lymphoma 2 protein family) in the Bad-B-cell lymphoma-extra large ([Bcl-x.sub.L]) complex releases the antiapoptotic protein [Bcl-x.sub.L] (B-cell lymphoma-extra large), which suppresses erythroid progenitor cell apoptosis (25). Akt is also involved in several pathways that promote cell survival and anti-apoptotic effects through inhibition of forkhead transcription factor (FOXO3a), inactivation of glycogen synthase kinase 3[beta] (GSK3[beta]), induction of x-chromosome-linked inhibitor of apoptotic protein (XIAP), inactivation of caspases, and prevention of cytochrome C release (Figure 2 (19,26,27)). These effects not only enhance the erythropoietic properties of EPO but appear to be important in the protection of other cell types as well and may contribute to the reported neuronal and renal protective effects (24).
[FIGURE 2 OMITTED]
PLEIOTROPIC EFFECTS OF ERYTHROPOIETIN
The tissue protective effects of EPO may be elicited through the EPOR homodimer via JAK2-STAT5 activation and mediation of apoptosis. However, the interaction and contribution of the various downstream cellular signalling pathways may differ for each different cell type. Alternatively or additionally, tissue protection may be mediated by a second EPOR isoform; a heterodimer composed of an EPOR monomer and the cytokine receptor, common beta subunit. This signal-transducing subunit is also common to the granulocyte-macrophage colony stimulating factor and the IL-3 and IL-5 receptors (28,29). Carbamylated EPO (CEPO) does not bind to the classical EPOR isoform, and is devoid of classic haematopoietic activity (5,30) (Table 1), yet has been shown to provide tissue protection in the brain, heart (31) and kidney (30), supporting the existence of a heteroreceptor EPO isoform, which mediates tissue protection.
Carbamylated erythropoietin and tissue protection
The administration of CEPO, provides renal tissue-protective effects. In an ischaemia-reperfusion rat model, CEPO markedly reduced apoptosis and increased tubular epithelial cell proliferation. Moreover, CEPO was more protective against ischaemia-reperfusion injury to tubular epithelial cells than EPO in this study (4). In an in vitro model developed by the same team, CEPO promoted more capillary formation than EPO and also appeared to protect the kidneys from ischaemia-reperfusion injury by promotion of angiogenesis (32). This protective effect requires mitogenesis, and endothelial progenitor cell differentiation, proliferation and migration. The anti-apoptotic, cell proliferative, angiogenesis-promoting properties of CEPO may indicate involvement of the following signalling pathways: JAK2-STAT5, PI3K/Akt and downstream anti-apoptotic pathways, and mitogen-activated protein kinase. Whether different conformations of the EPOR activate different intracellular pathways or whether the single EPOR chain (in the case of CEPO) dimerises with other membrane proteins to provide the protective effect is currently not known (18). Different classes of EPOR with respect to affinity for EPO-binding have also been described: high affinity EPOR appear to mediate the haematopoietic effects and low affinity receptors may be principally involved in tissue protection by EPO (18).
It is clear that the relationship of EPO with its receptor is extremely complex. Therefore further investigation is required to fully understand the EPOR heterodimer isoform, and the mechanisms and pathways involved in its tissue protective activity. These findings could inform the future development of tissue-protective cytokines.
Post-receptor (intracellular) effects of erythropoietin
There are a number of common pathways through which EPO exerts its erythropoietic effects that also appear to confer tissue protection. As mentioned, it is uncertain whether the pathways for tissue-protection are activated by EPOR homodimers or heterodimers, both, or another receptor conformation (18). EPO 'classically' binds to two EPORs which become joined as a homodimer and change. This activates JAK2, which is bound to the common beta subunit of the EPOR (19), and leads to phosphorylation of tyrosine residues of the EPOR, which activates a number of signalling pathways discussed below (Figures 1 and 2).
Signal transducer and activators of transcription 5
EPO classically signals through the STAT5 pathway. The STAT proteins are direct substrates of Janus kinases (JAK); activation of JAK results in tyrosine phosphorylation of the STATs (Figures 1 and 2). Activation of JAK2 also leads to phosphorylation of the PI3K and subsequent phosphorylation of Akt (also known as protein kinase B) (Figure 1).
Akt (also called protein kinase B) is important because it is the principal component in a variety of pathways that promote cell survival and antiapoptotic effects (Figure 2). It is involved in inactivating caspases which mediate apoptosis, mitochondrial dysfunction and subsequent release of cytochrome C (27), leading to cell injury and genomic DNA degradation. Cytochrome C binds to apoptotic protease-activating factor-1 which activates proapoptotic caspase pathways and results in cell injury and death (27). Akt reduces caspase activation by increasing the activity of B-cell lymphoma 2 and [Bcl-x.sub.L] which, in turn, prevent B-cell lymphoma 2-associated X protein translocation to the mitochondria, maintain mitochondrial membrane potential and prevent cytochrome C release from the mitochondria (19). EPO's ability to maintain cellular integrity and prevent inflammatory apoptosis is closely linked to maintenance of mitochondrial membrane potential; modulation of apoptotic proteaseactivating factor-1; inhibition of cytochrome C release and inhibition of caspase 1, 3, 8 and 9 activation.
Recent data also indicate that PI3K-regulated serum and glucocorticoid-regulated kinase-1 may contribute to the mediation of EPO's anti-apoptotic effects and possibly renal protective effects (33).
Mitogen-activated protein kinase
The phosphorylation of mitogen-activated protein kinases appears to also contribute to the cell protection which EPO confers (Figure 2).
Protein kinase C
Protein kinase C is also involved in the signal transduction pathways of EPO and inhibition of apoptosis and cell survival. It regulates the EPO induced erythroid proliferation and differentiation (34). Indeed, inhibition of protein kinase C activity interferes with phosphorylation of the EPOR which suggests that protein kinase C may be an upstream modulator of the EPOR (27).
EPO may be involved in modulation of cellular calcium homeostasis given that classical protein kinase C isoenzymes depend on calcium for activity (35). EPO can elicit a prompt rise in intracellular free calcium in bone marrow, stimulate calcium channel activity and enhance intracellular calcium concentration of neuronal cells (35-37). This suggests that EPO influences calcium homeostasis by increasing calcium influx (38). A quick, transient increase in calcium by EPO may be required for cytoprotection during excitotoxicity, since a reduction of intracellular calcium can cancel the protective effect of EPO during glutamate administration (39). EPO may enhance the function and viability of neurons through a calcium mechanism (37,40).
Nuclear factor-kappaB (NF-kB), a mediator of inflammatory and cytokine response is also implicated in EPO signalling. The expression and cytoprotection of EPO partly depend on Akt and subsequent NF-kB activation (Figures 1 and 2). NF-kB plays a role in the release of EPO during hypoxia-inducible factor-1 induction; Akt can increase NF-kB and hypoxia-inducible factor-1 activation with resultant increase in EPO expression (41).
Heat shock protein 70
Induction of heat shock protein 70 by EPO is related to renal protection in ischaemic kidneys (42). Heat shock protein 70 prevents apoptosis a) by inhibiting movement of apoptosis inducing factor to the nucleus (43) and b) by preventing apoptotic protease-activating factor-1/cytochrome C binding in the cytosol (44) (Figure 2).
Potential new tissue protective mechanisms
There are other mechanisms by which EPO may confer cytoprotection. It has recently been demonstrated that local nitric oxide bioavailability increases when EPO activates endothelial nitric oxide synthase and this effect on the endothelium may be critical for the renal tissue protective effects of EPO. EPO is an extremely potent stimulator of endothelial progenitor cells, whose function is partly dependent on nitric oxide bioavailability. Endothelial progenitor cells appear to be involved in endothelial recovery after injury and the formation of new blood vessels in ischaemic areas (18). In the kidney, AKI triggers apoptosis, inducing an inflammatory response. EPO limits these negative effects in part by stimulating vascular repair and by mobilising endothelial progenitor cells and increasing tubular cell proliferation (28). These findings suggest that EPO may exert protective effect via an interaction with the microvasculature.
Angiogenesis and EPO's renal protective effects may also be influenced by vascular endothelial growth factor (VEGF). In a number of studies, the presence of VEGF appears to be associated with tissue protection (45-47). EPO was the first described hypoxia-inducible factor target gene but many more have been discovered including VEGF which has additional potential to provide renal protection (28). Nakano and colleagues found that the vascular EPO/EPOR system promoted post-ischaemic angiogenesis by upregulating the VEGF/VEGF receptor system, both directly by promoting neovascularisation and indirectly by mobilising endothelial progenitor cells and bone marrow-derived proangiogenic cells (48). It appears that angiogenesis is impaired and blood vessels are less responsive to VEGF in the absence of EPOR.
In animal studies, preconditioning of the hypoxiainducible factor system using short episodes of ischaemia has been found to protect the kidney from ischaemic and toxic injury. Hypoxia-inducible factor is capable of inducing a range of protective gene products including glucose transporters and heme oxygenase-1 with renal protective potential. Comparison of the protective capacities of EPO and hypoxia-inducible factor activation in the kidneys may be worthwhile (28). Moreover, it is possible that some effects of EPO such as the promotion of angiogenesis may be responsible for some of the benefit provided by ischaemic preconditioning. The tissue protective or 'pleiotropic' effects of EPO beyond erythropoiesis have been shown in the brain, heart and kidney in many animal and some clinical studies.
Traumatic brain injury and the potential protective effect of erythropoietin
Brain injury, particularly as a result of trauma, is a leading cause of mortality and long-term disability, particularly affecting young people. It is associated with significant human and financial costs. Complementary to measures to prevent injury, treatment can be directed at decreasing morbidity after primary injury (49). Extensive research shows that, after brain trauma (primary injury), several biochemical pathways are activated, leading to secondary brain injury (50). These pathways include inflammation, oxidative stress, increased vascular permeability and excitotoxic mechanisms (release of chemicals toxic to brain cells) (50). Secondary injury greatly increases brain cell death, stroke size and overall brain injury (51). Despite intense investigation, no specific treatment has conclusively been shown to attenuate secondary brain injury, and current management of traumatic brain injury (TBI) is supportive, seeking to prevent or rapidly treat complications which worsen secondary injury (52,53). It is therefore a priority to investigate promising therapies for traumatic or acute brain injury and EPO is one such intervention.
Human neurons, astrocytes and microglial cells produce EPO and express EPOR (54). The increase in production and secretion of EPO and enhanced expression of the EPOR in the human central nervous system in response to oxygen deficiency suggests a possible physiological role for EPO to attenuate secondary brain injury (27). The receptor complex mediating the neuroprotective effects of EPO may be associated with the common beta receptor subunit, CD131. Increasing evidence suggests that EPOR activation after EPO binding inhibits neuronal apoptosis (55,56). EPO-EPOR binding may induce JAK2 activation with phosphorylation of the inhibitor of NF-kB, leading to transcription of neuro-protective genes (57) (Figure 2). In addition, EPO seems to prevent neurological apoptosis through an Akt-dependent mechanism (58). Amongst a number of cell-survival promotion pathways, Akt blocks cell apoptosis by inhibiting glycogen synthase kinase 3p (59) (Figure 2). Furthermore, an in vitro study indicated that EPO attenuated glutamate excitotoxicity when given after its induction, and promoted nerve terminal sprouting in motor neurons by direct effect involving the JAK2 pathway (60).
EPO's neuroprotective mechanisms could also include activation of calcium channels to protect from glutamate toxicity, antioxidant enzyme production and neo-angiogenesis that improves blood flow and oxygenation in border zones of ischaemic areas (61). EPO also blocks free radicals, normalises cerebral blood flow, affects neuro-transmitter release (preventing excitotoxicity) and has anti-inflammatory effects, thus playing a crucial role in neuroprotection of the central and peripheral nervous system (62). It is therefore clear that EPO modulates a number of key pathways which could reduce neural injury and provide protective effects.
In animal models, EPO has been shown to decrease cerebral infarct volume, oedema and neuronal apoptosis, while neuronal survival and cerebral function increased (63-65). Following experimental subarachnoid haemorrhage, EPO was shown to reduce neuronal death and vasoconstriction and improve functional recovery and cerebral blood flow autoregulation (63-65). EPO's potential to prevent cortical injury has been investigated in spinal cord models, the ocular system and the peripheral nervous system in animals with promising results (66-70). Importantly, administration of EPO up to 24 hours after a traumatic brain injury has been shown to be protective and to improve functional neurological outcomes in rats (71). These results suggest that EPO not only attenuates gross anatomical injury but that it may also improve functional outcomes when administered many hours after the primary insult in animal models of brain injury.
In humans, EPO dramatically improved functional neurological outcomes in a randomised controlled trial when administered to patients with subarachnoid haemorrhage (72). Furthermore, in a double-blind proof-of-concept study, high dose EPO improved clinical outcome of patients at one month after acute ischaemic stroke and displayed a trend for reduction in infarct size. Moreover, EPO appeared to reduce neurocognitive dysfunction at two months after coronary artery bypass graft surgery and was considered safe to use in a double-blind Canadian randomised control trial (73). In chronic neurological disease EPO may also provide benefit. High-dose EPO appeared to improve cognitive performance in a small study of schizophrenic men with cognitive deficit, compared to placebo-treated patients (74). A small open label exploratory study of high-dose EPO for patients with chronic progressive multiple sclerosis also suggested potential benefit (75). EPO has the potential to provide benefit for neurological dysfunction in numerous settings.
A large multicentre intensive care unit (ICU) randomised controlled trial (53) found that EPO significantly decreased 29-day mortality compared to placebo (3.5% vs 6.6%) in a pre-planned subgroup analysis of trauma patients (many with traumatic brain injury). This occurred despite a lack of effect on transfusion requirement. However, an increase in thrombotic events was noted. In a recent German double-blind randomised controlled trial of EPO in acute ischaemic stroke, systemic thrombolysis (rtPA) was concurrently administered with EPO in most patients (63%)76. Contrary to existing clinical evidence (77) from a small single-centre study, this trial showed a negative effect, which may be related to the concurrent use of thrombolysis and/or factors related to the multi-centre nature of this trial. Notably, intracerebral haemorrhage was the main cause of death, and stroke severity was higher in those who died in the EPO group. Furthermore, pre-clinical combination therapy safety studies had not been performed (78). Of note, it is stated that one of the authors of this study holds a patent on the use of EPO for the treatment of cerebral ischaemia (76).
There is substantial experimental evidence, a plausible biological rationale and supportive clinical evidence from clinical trials to suggest a possible beneficial effect of EPO in acute and chronic brain injury. In addition, a recent small case controlled study suggests that EPO used in conjunction with hypothermia may increase survival and improve neurological outcomes (79). However, as these clinical studies suggest, EPO treatment may also result in clinically important side-effects.
Animal and in vitro studies
EPORs have been found in both vascular and non-vascular renal tissue (80). Many animal studies have shown that EPO administration protects kidney tissue from damage and improves renal function in ischaemia-reperfusion and contrast-induced injury models of AKI (Tables 2 and 3 (4,8,24,32,42,46,47,81-102)). These investigations invariably found that EPO reduced kidney dysfunction by decreasing apoptosis. It has also been shown that different intracellular 'survival' pathways in the kidney such as the PI3K/Akt pathway are activated by EPO (103,104). Furthermore, in a recent in vitro study (24), EPO was shown to reduce cisplatin-induced apoptosis in human proximal tubule epithelial cells, while phosphorylation of STAT5 and Akt increased. Involvement of the JAK2-STAT5 pathway has also been implicated in cell protection (8,24). STAT5 activation can modulate proliferation and protect against cell apoptosis. In addition, EPO has been shown to reduce the expression of pro-inflammatory mediators, TNF-alpha and IL2, in ischaemia-reperfusion renal injury and reverse the effect of endotoxin on the anti-oxidant, renal superoxide dismutase (94). These anti-inflammatory properties of EPO also suggest involvement of the NF-kB pathway in its kidney protection.
Erythropoietin in acute kidney injury
AKI as classified by the RIFLE criteria (an extensively used and validated classification system for renal function) is common in the ICU and occurs in approximately 36% of critically ill patients (105,106). AKI is independently associated with increased mortality and with prolonged length of stay. It escalates both the human and financial costs of care. Therefore, it seems desirable to investigate treatments with potential to ameliorate or prevent AKI.
Some injury pathways for AKI in the critically ill include exposure to endogenous and exogenous toxins, metabolic factors, ischaemia and reperfusion insults, neurohormonal activation, inflammation and oxidative stress. Of these, ischaemia-reperfusion may be the most common. EPO can prevent or reduce injury and assist renal repair and recovery through limitation of apoptosis, promotion of neovascularisation, anti-inflammatory action and tissue regeneration.
Investigation of potential treatments for AKI has had limited success to date. However, from the results of animal and some limited preliminary human studies, therapeutic use of EPO seems promising for those 'at risk' for AKI (8,86,96,107).
Clinical trials of erythropoietin in acute kidney injury
One randomised clinical pilot trial of preoperative EPO/placebo in 71 patients undergoing elective coronary artery bypass graft surgery showed renal protective results (107) (Table 4 (107,108)). EPO 300 //kg intravenous given immediately preoperatively seemed to reduce the incidence of AKI, (8% EPO vs 29% placebo, P=0.035); and improved postoperative renal function as indicated by a smaller increase in SCr (%SCrt at 24 hours: EPO 1[+ or -]3, placebo 15[+ or -]7, P=0.04) and a smaller decline in estimated glomerular filtration rate (% estimated glomerular filtration rate [down arrow] at 24 hours: EPO 3[+ or -]3, placebo -5[+ or -]4, P=0.04) postoperatively (Table 4). A more recent and slightly larger (n=162) study assessed EPO's effect in ICU patients at risk for AKI (defined by a cut-off value of two proximal tubular enzymes in urine: [gamma]-glutamyl transpeptidase and alkaline phosphatase) (Table 4). EPO 500 [micro]/kg intravenous was given after a high [gamma]-glutamyl transpeptidase Xalkaline phosphatase product was detected and again 24 hours later. The primary outcome was the average percent SCr increase from baseline over four to seven days. This trial found no renal protective effect of EPO as determined by the average percent SCr increase from baseline over four to seven days. Table 4 compares the 'coronary artery bypass graft' and 'EARLY ARF' EPO trials. The reasons for these contradictory findings may be related to the differences in design and methods used.
Potential risks of erythropoietin
Pure red cell aplasia
Despite the numerous benefits of EPO there are also some risks. Pure red cell aplasia is a rare adverse event characterised by anaemia, low reticulocyte count, absence of erythroblasts, resistance to EPO and neutralising antibodies against EPO (109,110). Only three cases were reported in EPO patients from 1988 to 1998, after which incidence increased to a peak in 2001. This was attributed to a number of factors that were subsequently addressed. Since the introduction of Teflon-coated plungers (2003), and changes to EPO formulation and the regulation of EPO administration (2002 to 2004), there have been only six reported cases of pure red cell aplasia, converting it once again into an extremely rare occurrence.
EPO administration in patients with cancer, given to reduce chemotherapy or radiotherapy induced anemia, has been associated with increased mortality and enhanced tumour growth (17,111). The underlying mechanisms remain uncertain, but EPO may serve as a growth factor to cancer cells and may promote tumour angiogenesis (17). Furthermore, patients with certain malignancies may be in a hypercoagulable state, making EPO administration unadvisable. Consequently patients with a known malignancy should be excluded from future EPO trials.
Recent studies and clinical trials have found an increased rate of thrombosis with EPO (53,112,113) which has mainly been observed in patient groups with higher than conventional levels of haemoglobin (>120 g/l) (113). Putative mechanisms are increased blood viscosity, increased platelet count and reactivity, decreased protein C and S plasma levels, enhanced thrombin generation and factor VIII antigen plasma levels. Exclusion of patients with haemoglobin >120 g/l from clinical trials of EPO minimises the risk for thrombosis113. Nonetheless, systematic assessment for thrombosis should be performed in any EPO trials of critically ill patients as they have an increased risk for thrombosis.
Hypertension occurs in about 30% of patients receiving long-term EPO treatment (3). This was partly attributed in the past to the rise in haematocrit, but now appears to involve increased endothelin release, upregulation of tissue renin and angiotensin production, changes in the balance of vasoactive substances (prostaglandin/prostacyclin/ thromboxane), and an elevation of calcium by EPO (at least in chronic kidney disease) that impairs the vasodilating action of nitric oxide (27). It is advisable that patients with uncontrolled hypertension do not participate in trials of EPO in AKI.
Carbamylated EPO, a cytoprotective, nonerythropoietic derivative of EPO (Table 1), may not exhibit the same risks as EPO and holds great interest as a future tissue-protective therapy. However, it requires further experimental testing before it can be safely evaluated in clinical trials.
The EPO-TBI trial and EPO-AKI substudy
EPO-TBI, a randomised, double-blind, controlled trial of EPO in ICU patients with TBI in Australia, New Zealand and Saudi Arabia, has recently commenced recruitment (ACTRN12609000827235) at the Alfred and Royal Melbourne hospitals in Victoria. The trial will soon roll out to a total of 19 sites. EPO-TBI is endorsed by the Australian and New Zealand Clinical Trials Group and has National Health and Medical Research Council and Victorian Neurotrauma Initiative funding. With a cohort of 606 patients, it will be the largest randomised controlled trial of EPO in patients with TBI ever performed. Furthermore, it is one of the largest TBI trials currently being conducted. Participants are moderate and severe TBI patients admitted to ICU with a GCS [less than or equal to] 12, aged between 15 and 65 years, with a haemoglobin <120 g/l, within 24 hours of injury and expected to stay [greater than or equal to] 48 hours. Patients will be randomised to receive either EPO 40,000 IU subcutaneously or placebo in a 1:1 ratio, weekly for up to three weeks while in ICU. Pharmaceutical and mechanical venous thrombo-embolism prophylaxis will be prescribed if not contraindicated, and compression Doppler ultrasound examinations to monitor patients for the development of proximal deep vein thrombosis will be performed prior to or within 48 hours of the first EPO/placebo dose, then twice in each week following each dose. The primary outcome is the proportion of unfavourable neurological outcomes at six months: defined as severe disability (Glasgow outcome scale, extended=2 to 4) or death (Glasgow outcome scale, extended=1).
'EPO-AKI' and 'EPO-Biomarkers' comprise the Intensive Care Foundation-funded renal sub-study of the EPO-TBI trial. This sub-study assesses the effect of EPO on the development of acute kidney injury and the response to treatment using multiple renal biomarkers with different time profiles. All of these biomarkers have been extensively investigated and patterns in response to injury are known. Having recently sustained a timed physical injury and with a moderate incidence of AKI (114,115), this homogeneous cohort lends itself to such a study. AKI will be classified using methods based on the RIFLE criteria; a classification system used extensively and validated to classify renal function in several populations with studies cumulatively involving over 250,000 subjects. Baseline renal function will be taken from a consistent source for all patients. With a cohort of 606 patients, this will also be the largest study of EPO to protect against AKI ever performed, increasing the probability of detecting a treatment effect. Furthermore, it will be the first EPO trial to incorporate active risk assessment for thrombotic episodes. Weekly intervals separate the doses of EPO (up to three) in this trial to allow time for clearance and avoid excessively high levels of EPO. In addition, patients with a known malignancy and/ or uncontrolled hypertension will be excluded, thus minimising risk to patients. The EPO-TBI trial provides a unique opportunity to clarify the potential benefit of EPO as a brain protective and kidney protective agent. This trial may also provide valuable insight into the mechanisms of EPO in AKI and pave the way for further dedicated large scale trials of EPO in AKI.
DECLARATION OF INTEREST
RB and AN are investigators of a National Health and Medical Research Council/Victorian Neurotrauma Initiative funded clinical trial of Erythropoietin in Traumatic Brain Injury (NCT00987454).
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E. M. MOORE *, R. BELLOMO ([dagger]), A. D. NICHOL ([double dagger])
Department of Epidemiology and Preventive Medicine, Australian and New Zealand Intensive Care Research Centre, Monash University, Melbourne, Victoria, Australia
* R.N., Pgrad. Dip. (Crit. Care Nursing), M.P.H., Ph.D. Student.
([dagger]) M.B., B.S., M.D., F.R.A.C.P., F.A.C.C.P., F.J.F.I.C.M., Professor.
([double dagger]) B.A. (Health Sc.), M.B., B.Ch., Dip. Man., F.C.A.R.C.S.I., Ph.D., F.J.F.I.C.M.I., F.C.I.C.M., Associate Professor.
Address for correspondence: Dr R. Bellomo, ANZIC Research Centre, Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, The Alfred Centre, 99 Commercial Road, Melbourne, Vic. 3004.
Accepted for publication on December 20, 2010.
TABLE 1 Erythropoeitin analogues (3-8) rHuEPO Darbepoietin Molecular weight, kDa 30.4 38 N-linked 3 5 oligosaccharide chains Sialic acid residues 10-14 [less than or equal to] 22 IV half life, h 5 25.3 SC half life, h 25 48.8 Clearance, ml/h/kg 4-15 ([up arrow] 1.6 [+ or -] 0.3 with dose) Erythropoietic Yes Yes activity Use Anaemia Rx-CKD Anaemia Rx-CKD AsialoEPO Carbamylated erythropoietin Molecular weight, kDa 30-34 40 N-linked 0 Carbamylation of oligosaccharide lysines in EPO-- chains homocitrulline residues Sialic acid residues <0.02 sialic Carbamylation of acids/molecule lysines in EPO-- homocitrulline residues IV half life, h 0.023 3.3 SC half life, h 2.5 3-6 (rats--same as EPO) Clearance, ml/h/kg rapid -- Erythropoietic No No activity Use Potential: neuro Potential: neuro-, injury cardio-, renoprotective AsialoEPO (7,8): Desialylation [right arrow]high clearance. Half life is too short for erythropoiesis as continuous EPOR stimulation is required. Has same or increased affinity for EPOR. Neuroprotection appears to be retained. Carbamylated erythropoietin (4-6): No interaction with classical EPOR. Less affinity for EPOR but retains cytoprotection. May interact with alternative receptor e.g. heterodimer with only 1 EPOR. Colony-stimulating factor levels comparable to EPO. rHuEPO=recombinant human erythropoietin, asialoEPO=asialo erythropoietin, EPO=erythropoietin, [right arrow]=leads to, IV=intravenous, SC=subcutaneous, [down arrow] reduced, Rx=treatment, CKD=chronic kidney disease, EPOR=receptor. TABLE 2 Animal studies of erythropoietin in acute kidney injury relevant to non-haematopoietic functions: ischaemia-reperfusion models Year, Type AKI model/follow-up EPO protocol reference 2009 (98) Dogs IR: nephrectomy, 2 EPO 500 U-kg IV pre- weeks recovery, then ischaemia [+ or -] 90 renal artery occlusion, min abdominal ischaemia 1 h; insufflation reperfusion 28 days 2009 (86) Rats IR: transplanted with EPO 5000 U 30 min male bone marrow pre-ischaemia cells; reperfusion 2 or 4 weeks 2008 (93) Rats IR: occlusion of EPO 1000 U/kg s.c. infrarenal abdominal 5 min pre-ischaemia aorta, ischaemia 30 min, reperfusion 60 min vs sham 2007 (4) Rats IR: bilateral renal EPO 100U/kg or 100U/kg pedicle occlusion 1 day CEPO s.c. every 2 after last EPO days for 2 weeks injection, ischaemia 45 (6 injections) vs min; reperfusion 24, saline 72 h and 1 week 2007 (46) Rats IR: bilateral renal EPO 100 U/kg s.c. every pedicle occlusion 1 day 2 days for 2 weeks after last EPO (6 injections) vs injection, ischaemia 60 saline min; reperfusion 24, 72 h 2008 (32) Rats In vitro: endothelial EPO 100 U/kg or 100 tube formation assay; U/kg CEPO s.c. every 2 IR: bilateral renal days for 2 weeks pedicle occlusion 1 day (6 injections) vs after last EPO dose, saline ischaemia 45 min; reperfusion 24, 72 h and 1 week 2007 (88) Pigs IR: unilateral EPO 5000 U/kg IV at nephrectomy; occlusion ischaemia, then 1000 renal artery for 1 h 1 U/kg s.c. daily, week later, reperfusion reperfusion 5 days vs 5 days no treatment 2006 (100) Rats IR: bilateral renal EPO 500 U/kg i.p. 20 pedicle occlusion, min pre-ischaemia ischaemia 45 min; reperfusion 48 h vs sham/saline 2006 (92) Rats IR: bilateral renal EPO 5000 U/kg or DPO 25 and in pedicle occlusion, [micro]g/kg i.p. at vitro ischaemia 45 min; time of ischaemia or 6 reperfusion 1-7 days vs h post reperfusion sham/ vehicle 2005 (83) Rats IR: right nephrectomy, EPO 1000U/kg and clamp left pedicle 45 genistein (tyrosine min and reperfusion 45 kinase inhibitor) 10 min and 24 h mg/kg 2 h before ischaemia 2004 (102) Rats IR: uni/bilateral renal EPO 5000 U/kg i.p. and in artery occlusion, 30 30 min pre-ischaemia. vitro min ischaemia, 24 or 48 In vitro: 6.25-400 U/ml h reperfusion vs sham/ rHuEPO vs vehicle, vehicle. In vitro: incubated for 5 or 24 h exposure of human PTCs to 1 or 21% O2 for up to 24 h; or 1% O2 16 h then 21% O2 24 h 2004 (97) Mice IR: bilateral renal EPO 1000 U/kg/day artery occlusion, 30 s.c. 3 days pre IR, or min ischaemia, 24 h EPO 1000 U/kg sc on reperfusion vs reperfusion sham/vehicle 2004 (99) Rats IR: bilateral renal EPO 300 U/kg IV 30 pedicle occlusion, 45 min pre-ischaemia, 5 min ischaemia, 6 h min pre-reperfusion, or reperfusion vs 30 min post reperfusion sham/saline 2004 (90) Rats IR: bilateral renal 1. EPO 200 U/kg i.p. at artery occlusion, 40 start ischaemia and 6, min ischaemia, 48 (1) 24 h post reperfusion; or 96 (2) h reperfusion or 2. 200 U/kg IV and vs sham/vehicle 4, 10, 24, 48 h post reperfusion 2003 (42) Rats IR: bilateral renal EPO 3000 U/kg 24 h and in artery occlusion, 45 pre IR injury vitro min ischaemia, [less than or equal to] 72 h reperfusion vs sham/saline 2001 (95) Rats IR: right kidney EPO 500 or 3000 U/kg occlusion, 30 or 45 min IV at ischaemia then ischaemia, simultaneous s.c. 24, 48 h post left nephrectomy, [less than or equal to] 96 h reperfusion vs sham/vehicle Year, Outcome reference 2009 (98) [down arrown] microalbuminuria, [up arrown] trenal function recovery at 4 weeks; IV EPO better than mannitol for renal IR injury protection 2009 (86) [up arrow] GFR (4 weeks), [right arrow] proteinuria/Hb (2 and 4 weeks), [down arrow] tubulointerstitial changes. EPO did not enhance bone marrow cell recruitment 2008 (93) [down arrow] MDA levels, [down arrow] SOD activity, [down arrow] catalase ([down arrow] oxidative stress so not required), [down arrow] histopathological changes: [down arrow] focal glomerular necrosis, [down arrow] dilation of Bowman's capsule, [down arrow] degeneration/necrosis in tubular epithelium, [down arrow] interstitial inflammatory infiltration and [down arrow] blood vessel congestion 2007 (4) CEPO: (no erythropoiesis) [down arrow] apoptosis, [down arrow] [alpha]-SMA expression, [up arrow] tubular epithelial cell proliferation, [down arrow] SCr. EPO: [up arrow] [down arrow] b, Jin apoptosis and [alpha]-SMA (not as marked as CEPO). CEPO more therapeutic than EPO 2007 (46) [up arrow] HIF-1alpha-positive cells, [up arrow] VEGF mRNA expression, [down arrow] tubular hypoxia, [down arrow] apoptotic and [alpha]-SMA-positive interstitial cells 2008 (32) EPO: tendency of increased tube formation; CEPO: more capillary-like formation than EPO; [up arrow] peritubular capillary endothelial cells. CEPO may protect kidneys from IR injury by promoting angiogenesis 2007 (88) [down arrow] renal dysfunction, [down arrow] cell death (histology at 5 days) 2006 (100) [down arrow] SCr, [down arrow] urea, [down arrow] histological injury, [down arrow] tubular apoptosis 2006 (92) EPO and DPO at T0 and T6--[down arrow] tubular apoptosis, [down arrow] plasma Cr/urea, [up arrow] tubular regeneration (cell proliferation and mitosis) 2005 (83) [down arrow] SCr, [down arrow] urea, [down arrow] TNF-[alpha] and IL-2 expression (pro-inflammatory mediators of IR injury), [down arrow]-LDH (indicates lipid peroxidation), [down arrow] histological injury; genistein reversed benefits of EPO 2004 (102) [down arrow] apoptosis, [down arrow] regeneration, [down arrow] casts, [down arrow] plasma Cr. In vitro: [down arrow] apoptosis, [down arrow] mitosis/DNA synthesis 2004 (97) [down arrow] plasma Cr, [down arrow] plasma AST, [down arrow] histological injury, [down arrow] kidney MPO and MDA levels 2004 (99) [down arrow] tubular apoptosis, [down arrow] tubular (NAG) and reperfusion (AST) injury, [down arrow] histological injury, [down arrow] SCr, better urine flow, [down arrow] creatinine clearance 2004 (90) [down arrow] plasma Cr, [down arrow] polyuria, [down arrow] FENa, [down arrow] AQP/NHE/TSC expression (prevented down-regulation of AQPs and Na+ transporters--may imrove ischaemia-induced urine concentrating defects and impairment of tubular Na+ reabsorption) 2003 (42) [down arrow] SCr, [down arrow] tubular necrosis, [down arrow] tubular apoptosis, Jtubular cell proliferation (renoprotection may be independent of growth promotion), [up arrow] bcl-2 protein, [down arrow] caspase 3 activity, [down arrow] JNK expression, dose-dependent [up arrow] HSP70 expression, inhibition of HSP70 expression lead to [down arrow] renoprotection 2001 (95) [up arrow] HCt, [up arrow] mortality (severe ischaemia group), [right arrow] SCr/ weight AKI = acute kidney injury, EPO = erythropoeitin, IV = intravenous, IR = ischaemia-reperfusion, [down arrow] = decreased, [up arrow] = increased, IV = intravenous, GFR = glomerular filtration rate, [right arrow] = the same level, MDA = malondialdehyde (indicates free radical generation), SOD = superoxide dis- mutase (an antioxidant), CEPO = carbamylated EPO, [alpha]-SMA = alpha-smooth muscle actin (associated with renal injury), SCr = serum creatinine, HIF-1 alpha = hypoxia inducible factor-alpha, VEGF = vascular endothelial growth factor, mRNA = messenger RNA, i.p. = intraperitoneal, DPO = darbepoietin, Cr = creatinine, TNF-[alpha] = tumour necrosis factor-alpha, IL-2 = interleukin-2, LDH = lactate dehy- drogenase, rHuEPO = recombinant human erythropoietin, PTC = proximal tubule cell, MPO = myeloperoxidase, NAG = N-acetylglutamate, AST = aspartate aminotransferase (indicates reperfusion injury), FENa-fractional excretion of Na+, AQP = aquaporin, NHE = Na+/H+ exchanger, TSC = thiazide-sensitive sodium chloride cotransporter, bcl-2 = oncogene activated by chromosome translocation in human B-cell lymphomas, JNK = c-Jun N-terminal kinase, HSP70 = heat shock protein 70. TABLE 3 Animal studies of erythropoietin in acute kidney injury relevant to non-haematopoietic functions: other models Year, Type AKI model/follow-up EPO protocol reference 2010 (96) Rats Brain death + 10 [micro]g/kg EPO or perfused kidney model CEPO IV, 4 h brain death, then kidney reperfusion in perfused kidney model 2010 (91) Mice Aristolochic acid DPO 0.1 [micro]g/kg nephropathy weekly from day of aristolochic acid administration or on day 28 2009 (82) In vitro Oxidative stress: EPO/alpha 100 and human proximal tubule 400 U/ml cells 2 h in [H.sub.2][O.sub.2] 2008 (8) Rats and CIN: Ioversol 2.9 g/ In vitro and EPO in vitro kg iodine + 10,000 U/kg or inhibition of asialoEPO 80 ng/g IV prostaglandin and NO 1 h before Ioversol synthesis. In vitro: Ioversol 100 mg/ml iodine to induce proximal tubular cell injury 2008 (24) Rats and In vitro/CIN: EPO 5000 U-kg IV OR in vitro cisplatin 5.5 mg/kg equivalent peptide IV mass of inactive EPO OR DPO 25 [micro]g- kg pre-cisplatin OR saline 2008 (85) Mice and CIN: i.p. cisplatin EPO 1000 U/kg i.p. in vitro injection (10 mg-kg- daily [less than or day) for 2 days vs equal to] 3 days placebo. Follow-up before cisplatin vs 6 days vehicle (or bone marrow isolation/ blood cultures) 2007 (94) Mice Endotoxaemia: 2.5 EPO 4000 U/kg 30 min mg/kg endotoxin i.p. before endotoxin vs (lipopolysaccharide); vehicle follow-up 16 h later 2006 (89) Rats CIN: (iothalamate), EPO 3000 U/kg and 600 following inhibition U/kg IV 24 and 2 h of NO and pre CIN induction vs prostaglandin saline synthesis with indomethacin and N[omega] nitro-L- arginine methyl ester 2005 (47) Rats and Chronic kidney DPO s.c. 0.4 in vitro disease [micro]g/kg/week into 5/6 remnant kidney rats after renal mass reduction, sacrificed at 1, 2 and 12 weeks 2004 (87) Pig/mouse: In vitro: exposure of DPO 6.25/100 ng/ml in vitro LLC-PK1 (pig kidney incubation for 16 h epithelial cell line) and mouse mesangial cells to protaglandin [D.sub.2] synthase, campothecin, hydrogen peroxide or hypoxia 2004 (81) Rats Haemorrhagic shock EPO 300 U/kg IV pre and endotoxic shock resuscitation 2001 (84) Rats CIN: cisplatin EPO 100 U/kg i.p. pre toxicity--i.p. cisplatin then daily cisplatin injection for 9 days vs placebo 6 mg/kg vs placebo 1994 (101) Rats CIN: cisplatin EPO 100 U/kg i.p. toxicity--i.p. post cisplatin then cisplatin injection daily for 9 days vs 7 mg/kg vs placebo placebo Year, Outcome reference 2010 (96) EPO and CEPO: [down arrow] expression of proinflammatory genes, [down arrow] infiltration of polymorphonuclear cells in kidney, preserved vascular integrity. CEPO more effective than EPO. Kidney function fully restored with EPO and CEPO 2010 (91) [up arrow] survival of tubular cells lead to [down arrow] acute tubular injury, interstitial inflammation and interstitial fibrosis 2009 (82) EPO 100 U-ml: little effect; 400 U-ml: [up arrow] damage due partly to [down arrow] activation of cell survival- prolifer'n signalling pathways; [down arrow] phosphorylation of Akt, GSK-3B, mTOR, ERK1-ERK2, and forkhead transcription factor of [H.sub.2][O.sub.2]- treated cells 2008 (8) EPO and asialoEPO: [down arrow] renal dysfunction and histological injury, [down arrow] apoptosis, [down arrow] caspase3/activated apoptosis in renal porcine epithelial cells in vitro with [up arrow] JAK2/STAT5 phosphorylation and HSP70 expression; [up arrow] JAK2/STAT5 phosphorylation and HSP70 expression in rat kidneys in vivo 2008 (24) In vitro: in human RPTE cells EPO [down arrow] apoptosis at [greater than or equal to] 100 U-ml, [up arrow] STAT5 and Akt-PKB phosphorylation. JAK2 inhibitor, tyrphostin AG-490 reduced EPO protection. Rats: EPO [up arrow] HCt, [down arrow] SCr; DPO also [up arrow] HCt, [down arrow] SCr. Clearance studies: GFR and renal blood flow confirmed DPO renal protection. [down arrow] tubular apoptosis and necrosis with DPO. DPO 48 h post cisplatin was renoprotective 2008 (85) [down arrow] urea, [up arrow] casts, tMSC numbers in vitro and in vivo (in bone marrow and spleen and mobilised into the peripheral circulation). Is protective effect due to [up arrow] in MSC numbers after EPO or their mobiliaation into circulation? 2007 (94) [up arrow] GFR (inulin clearance), [right arrow] MAP, [right arrow] renal bood flow, [up arrow] CRP, [right arrow] serum NO, EPO reversed the endotoxin effect on renal SOD activity (SOD [down arrow] in control group) 2006 (89) Cr clearance preserved, results inconclusive but may indicate protective trend 2005 (47) [up arrow] microvascular density, [up arrow] endothelial proliferation, preserved renal function ([down arrow] SCr), [down arrow] scarring, [up arrow] VEGF expression; In vitro: [up arrow] cell proliferation, [up arrow] VEGF, [down arrow] hypoxia-induced apoptosis 2004 (87) [down arrow] tubular apoptosis (for toxic and hypoxic stimuli) 2004 (81) [down arrow] renal dysfunction in haemorrhagic but not endotoxic shock 2001 (84) [up arrow] renal bld flow/GFR at 9 days, [up arrow] tubular regeneration, [up arrow] tubular cell proliferation, [up arrow] functional recovery 1994 (101) [up arrow] functional recovery, [up arrow] tubular regeneration, [up arrow] DNA synthesis AKI = acute kidney injury, EPO = erythropoietin, CEPO = carbamylated EPO, [down arrow] = decreased, IV = intravenous, DPO = darbepoietin, [up arrow] = increased, [H.sub.2][O.sub.2] = hydrogen peroxide, Akt/JAK2/STAT5/ = signalling pathways of EPO, GSK-3[beta] = glycogen synthase kinase 3 beta, mTOR = mammalian target of rapamycin, ERK = extracellular signal-regulated kinase, CIN = contrast induced nephropathy, i.p. = intraperitoneal, NO = nitric oxide, asialoEPO = asialoEPO, HSP70 = heat shock protein 70, RPTE = renal proximal tubular epithelial, PKB = protein kinase B, SCr = serum creatinine, MSC = marrow stem cell, GFR = glomerular filtration rate, [right arrow] = the same level, MAP = mean arterial pressure, CRP = C-reactive protein, SOD = superoxide dismutase (an anti-oxidant), Cr = creatinine, VEGF = vascular endothelial growth factor. TABLE 4 Comparison of articles: EARLYARF vs EPO in CABG surgery EPO in CABG (107) Sample size 71 (EPO=36, placebo=35) Patient population Elective CABG Country, centre(s) Seoul, Korea, single centre Study design Prospective randomised double-blind, placebo- controlled trial of EPO EPO type and dose 1 dose preop: 300 [micro]/kg EPO (Recormon, Roche) or normal saline IV Inclusion criteria >18, elective CABG Exclusion criteria Emergent CABG, pre-existing AKI, on RRT, uncontrolled HT, nephrotoxic drugs within 3 days of operation, previous use of EPO Measurements Baseline SCr preop and 24, 72, 120 h post Age (mean) 66.7 [+ or -] 9.6 Study groups: Baseline and intraop: no significant differences, EPO vs placebo most OPCABG (77%) (+3 valves in EPO group) Primary outcome Incidence of AKI after CABG Secondary outcomes Changes in SCr and eGFR (first 5 days postop), ICU and hospital LOS, in-hospital mortality AKI: definition [greater than or equal to] 50% [up arrow] in SCr from preop baseline (first 5 days postop); eGFR: Cockroft-Gault equation AKI: proportion EPO 8%, placebo 29%; P=0.035 (after CABG/ GGTxALP >46.3) Results %SCr [up arrow] at 24 h: EPO 1 [+ or -] 3, placebo 15 [+ or -] 7 (P=0.04). %SCr [up arrow] at 120 h: EPO 7 [+ or -] 4, placebo 27 [+ or -] 8 (P=0.01). %eGFR [down arrow] at 24 h: EPO 3 [+ or -] 3, placebo -5 [+ or -] 4 (P=0.04). %eGFR [down arrow] at 120 h: EPO -4 [+ or -] 3, placebo -13 [+ or -] 5 (P=0.01). ICU LOS h: EPO 65 [+ or -] 77, placebo 84 [+ or -] 113 (P=0.262). Hosp LOS days 10.1 [+ or -] 7, placebo 11.1 [+ or -] 5.5 (P=0.442) Onset of injury Initiation of CABG operation Possible mechanism CVS compromise, CPB exposure, of injury tcatecholemines, atheroembolism, IR injury, nephrotoxic agents, inflammation Safety No symptomatic thrombosis or other adverse events in EPO patients EARLYARF (108) Sample size 162 (EPO=84, placebo=78) Patient population Aim: ICU patients at high risk of AKI; Obtained: critically ill patients Country, centre(s) Christchurch and Dunedin, New Zealand, 2 centres Study design 2 parts: a) Prospective randomised double-blind placebo-controlled, parallel group trial of EPO; b) Observational study to assess GGTxALP to early identify patients at high risk of AKI EPO type and dose 2 doses: EPO/beta 500 U/kg to max 50,000 U or normal saline IV; 1st within 6 h of [up arrow] GGTxALP; 2nd 24 h later Inclusion criteria [up arrow] in GGT and ALP urine concentration product by >46.3 Exclusion criteria <16 y, no IDC, haematuria, rhabdomyolysis, myoglobinuria, polycythemia, cytotoxic chemotherapy, RRT or needs in 48 h, stay [less than or equal to] 24 h, survival [less than or equal to] 72 h, prior RIFLE 'failure' Measurements Baseline Cr: various versions of preop/pre-ICU Cr including lowest on ICU admit/last ICU Cr/minimum at 12 months. Triage: GGTxALP within 1 h of ICU admission, at 12 and 24 h, daily to 7 days; Intervention: Blood for Cr and Cyst C and start for 4/24 Cr clearance were at the same timepoints Age (mean) 61.6 [+ or -] 15.4 Study groups: EPO group older (P=0.011), [down arrow] likelihood EPO vs placebo for neuro surgery, injury, seizure or ICH (P <0.05) and [up arrow] likelihood for sepsis (P <0.05). More placebo pts had AKI (not significant) Primary outcome A priori: average % plasmaCr [up arrow] from baseline over 4-7 days Secondary outcomes Intervention trial: AKIN and RIFLE AKI definitions, plasma cystatin C, need for dialysis, death within 7/30/90 days; triage trial: mean time to randomisation after collecting urine sample with [up arrow] GGTxALP, identification of patients at [up arrow] risk of AKI/dialysis/death AKI: definition AKIN (Cr and UO) and RIFLE (Cr) definitions AKI: proportion AKIN Cr: EPO 45.2%, placebo 47.4%; RIFLE Cr: EPO (after CABG/ 23.8%, placebo 19.2%; AKIN UO: EPO 70.2%, placebo GGTxALP >46.3) 51.3% (P=0.016) Results No significant difference in 1[degrees] outcome or 2[degrees] outcomes except AKI (AKIN UO). Of randomised patients without AKI initially (n=104) EPO patients had higher %plasmaCr [up arrow]: EPO 8.5 [+ or /] 27 (n=61), placebo /4.6 [+ or /] 18 (n=47) (P=0.004). No difference in ICU or hosp stay. GGTxALP: poor predictive value for AKI, low for RRT/death Onset of injury Heterogenous. Time of injury estimated for subdivision analysis. Samples from 6-12 h after putative insult more predictive for AKI (AUC=0.69), dialysis and death Possible mechanism Heterogenous of injury Safety No evidence for [up arrow] intravascular thrombosis. EPO not associated with [up arrow] in adverse events EPO=erythropoietin, CABG=coronary artery bypass graft, ICU=intensive care unit, AKI=acute kidney injury, preop=preoperatively, GGT=[gamma]-glutamyl transpeptidase, ALP=alkaline phosphatase, IV=intravenous, RRT=renal replacement therapy, HT=hypertension, IDC=indwelling catheter, RIFLE and AKIN=AKI classification systems, post=postoperatively, (S)Cr=(serum) creatinine, Cyst C=cystatin C, intraop=intraoperatively, OPCABG=off pump CABG, ICH=intra-craneal haemorrhage, [up arrow]=increased, eGFR=estimated glomerular filtration rate, LOS=length of stay, UO=urine output criteria, [down arrow]=decreased, AUC=area under curve, CVS=cardiovascular system, CPB=cardiopulmonary bypass, IR=ischaemia reperfusion.
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|Author:||Moore, E.M.; Bellomo, R.; Nichol, A.D.|
|Publication:||Anaesthesia and Intensive Care|
|Date:||May 1, 2011|
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