Delayed administration of darbepoetin or erythropoietin protects against ischemic acute renal injury and failure
2006; Elsevier BV; Volume: 69; Issue: 10 Linguagem: Inglês
10.1038/sj.ki.5000356
ISSN1523-1755
AutoresDavid W. Johnson, Betty Pat, David A. Vesey, Zhengrong Guan, Zoltán Endre, Glenda C. Gobé,
Tópico(s)Dialysis and Renal Disease Management
ResumoAdministration of human recombinant erythropoietin (EPO) at time of acute ischemic renal injury (IRI) inhibits apoptosis, enhances tubular epithelial regeneration, and promotes renal functional recovery. The present study aimed to determine whether darbepoetin-alfa (DPO) exhibits comparable renoprotection to that afforded by EPO, whether pro or antiapoptotic Bcl-2 proteins are involved, and whether delayed administration of EPO or DPO 6 h following IRI ameliorates renal dysfunction. The model of IRI involved bilateral renal artery occlusion for 45 min in rats (N=4 per group), followed by reperfusion for 1–7 days. Controls were sham-operated. Rats were treated at time of ischemia or sham operation (T0), or post-treated (6 h after the onset of reperfusion, T6) with EPO (5000 IU/kg), DPO (25 μg/kg), or appropriate vehicle by intraperitoneal injection. Renal function, structure, and immunohistochemistry for Bcl-2, Bcl-XL, and Bax were analyzed. DPO or EPO at T0 significantly abrogated renal dysfunction in IRI animals (serum creatinine for IRI 0.17±0.05 mmol/l vs DPO-IRI 0.08±0.03 mmol/l vs EPO-IRI 0.04±0.01 mmol/l, P=0.01). Delayed administration of DPO or EPO (T6) also significantly abrogated subsequent renal dysfunction (serum creatinine for IRI 0.17±0.05 mmol/l vs DPO-IRI 0.06±0.01 mmol/l vs EPO-IRI 0.03±0.03 mmol/l, P=0.01). There was also significantly decreased tissue injury (apoptosis, P<0.05), decreased proapoptotic Bax, and increased regenerative capacity, especially in the outer stripe of the outer medulla, with DPO or EPO at T0 or T6. These results reaffirm the potential clinical application of DPO and EPO as novel renoprotective agents for patients at risk of ischemic acute renal failure or after having sustained an ischemic renal insult. Administration of human recombinant erythropoietin (EPO) at time of acute ischemic renal injury (IRI) inhibits apoptosis, enhances tubular epithelial regeneration, and promotes renal functional recovery. The present study aimed to determine whether darbepoetin-alfa (DPO) exhibits comparable renoprotection to that afforded by EPO, whether pro or antiapoptotic Bcl-2 proteins are involved, and whether delayed administration of EPO or DPO 6 h following IRI ameliorates renal dysfunction. The model of IRI involved bilateral renal artery occlusion for 45 min in rats (N=4 per group), followed by reperfusion for 1–7 days. Controls were sham-operated. Rats were treated at time of ischemia or sham operation (T0), or post-treated (6 h after the onset of reperfusion, T6) with EPO (5000 IU/kg), DPO (25 μg/kg), or appropriate vehicle by intraperitoneal injection. Renal function, structure, and immunohistochemistry for Bcl-2, Bcl-XL, and Bax were analyzed. DPO or EPO at T0 significantly abrogated renal dysfunction in IRI animals (serum creatinine for IRI 0.17±0.05 mmol/l vs DPO-IRI 0.08±0.03 mmol/l vs EPO-IRI 0.04±0.01 mmol/l, P=0.01). Delayed administration of DPO or EPO (T6) also significantly abrogated subsequent renal dysfunction (serum creatinine for IRI 0.17±0.05 mmol/l vs DPO-IRI 0.06±0.01 mmol/l vs EPO-IRI 0.03±0.03 mmol/l, P=0.01). There was also significantly decreased tissue injury (apoptosis, P<0.05), decreased proapoptotic Bax, and increased regenerative capacity, especially in the outer stripe of the outer medulla, with DPO or EPO at T0 or T6. These results reaffirm the potential clinical application of DPO and EPO as novel renoprotective agents for patients at risk of ischemic acute renal failure or after having sustained an ischemic renal insult. Ischemic acute renal failure (ARF) is a common, serious condition that culminates in patient death in over 50% of cases.1.The Australian kidney National Epidemiological Survey of Diseases of the Kidney and Urinary Tract. The Australian Kidney Foundation, Adelaide1999Google Scholar Numerous studies have attempted to modify the outcome of ARF by either ameliorating renal tubular injury (e.g. arginine–glycine–aspartate peptides, anaritide, dopamine, mannitol) or promoting tubular regeneration (e.g. hepatocyte growth factor, epidermal growth factor, insulin-like growth factor-I).2.Hirschberg R. Kopple J. Lipsett P. et al.Multicenter clinical trial of recombinant human insulin-like growth factor I in patients with acute renal failure.Kidney Int. 1999; 55: 2423-2432Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 3.Schena F.P. Role of growth factors in acute renal failure.Kidney Int Suppl. 1998; 66: S11-S15PubMed Google Scholar, 4.Power D.A. Duggan J. Brady H.R. Renal-dose (low-dose) dopamine for the treatment of sepsis-related and other forms of acute renal failure: ineffective and probably dangerous.Clin Exp Pharmacol Physiol Suppl. 1999; 26: S23-S28PubMed Google Scholar, 5.Takala J. Ruokonen E. Webster N.R. et al.Increased mortality associated with growth hormone treatment in critically ill adults.N Engl J Med. 1999; 341: 785-792Crossref PubMed Scopus (988) Google Scholar However, to date, none of these agents has made any appreciable clinical impact on the high mortality and morbidity rates associated with this condition. Recently, human recombinant erythropoietin (EPO), a widely available treatment for uremic anemia, was found to reduce injury caused by ischemia–reperfusion (IR) of the brain,6.Bernaudin M. Marti H.H. Roussel S. et al.A potential role for erythropoietin in focal permanent cerebral ischemia in mice.J Cereb Blood Flow Metab. 1999; 19: 643-651Crossref PubMed Scopus (675) Google Scholar, 7.Brines M.L. Ghezzi P. 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McDonald M.C. et al.Erythropoietin attenuates the tissue injury associated with hemorrhagic shock and myocardial ischemia.Shock. 2004; 22: 63-69Crossref PubMed Scopus (142) Google Scholar and heart.15.Bogoyevitch M.A. An update on the cardiac effects of erythropoietin cardioprotection by erythropoietin and the lessons learnt from studies in neuroprotection.Cardiovasc Res. 2004; 63: 208-216Crossref PubMed Scopus (127) Google Scholar, 16.Lipsic E. van der M.P. Henning R.H. et al.Timing of erythropoietin treatment for cardioprotection in ischemia/reperfusion.J Cardiovasc Pharmacol. 2004; 44: 473-479Crossref PubMed Scopus (152) Google Scholar, 17.Cai Z. Manalo D.J. Wei G. et al.Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia–reperfusion injury.Circulation. 2003; 108: 79-85Crossref PubMed Scopus (490) Google Scholar Endogenous EPO is known to be primarily produced by renal cortical fibroblasts18.Bachmann S. Le Hir M. Eckardt K.U. Co-localization of erythropoietin mRNA and ecto-5′-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates that fibroblasts produce erythropoietin.J Histochem Cytochem. 1993; 41: 335-341Crossref PubMed Scopus (305) Google Scholar and it is considered that important paracrine cytoprotective functions could occur within the kidney, as functional EPO receptors (EPORs) are expressed on renal tubular epithelial, mesangial, and endothelial cells.19.Westenfelder C. Biddle D.L. Baranowski R.L. Human, rat, and mouse kidney cells express functional erythropoietin receptors.Kidney Int. 1999; 55: 808-820Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar During the early phase of ischemic ARF, EPO expression is virtually absent whereas EPOR expression is well maintained.20.Westenfelder C. Unexpected renal actions of erythropoietin.Exp Nephrol. 2002; 10: 294-298Crossref PubMed Scopus (57) Google Scholar, 21.Nielsen O.J. Thaysen J.H. Erythropoietin deficiency in acute tubular necrosis.J Intern Med. 1990; 227: 373-380Crossref PubMed Scopus (40) Google Scholar, 22.Tan C.C. Tan L.H. Eckardt K.U. Erythropoietin production in rats with post-ischemic acute renal failure.Kidney Int. 1996; 50: 1958-1964Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 23.Gong H. Wang W. Kwon T.H. et al.EPO and alpha-MSH prevent ischemia/reperfusion-induced down-regulation of AQPs and sodium transporters in rat kidney.Kidney Int. 2004; 66: 683-695Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar We24.Vesey D.A. Cheung C. Pat B. et al.Erythropoietin protects against ischaemic acute renal injury.Nephrol Dial Transplant. 2004; 19: 348-355Crossref PubMed Scopus (260) Google Scholar and others14.Abdelrahman M. Sharples E.J. McDonald M.C. et al.Erythropoietin attenuates the tissue injury associated with hemorrhagic shock and myocardial ischemia.Shock. 2004; 22: 63-69Crossref PubMed Scopus (142) Google Scholar, 23.Gong H. Wang W. Kwon T.H. et al.EPO and alpha-MSH prevent ischemia/reperfusion-induced down-regulation of AQPs and sodium transporters in rat kidney.Kidney Int. 2004; 66: 683-695Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 25.Sharples E.J. Patel N. Brown P. et al.Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia–reperfusion.J Am Soc Nephrol. 2004; 15: 2115-2124Crossref PubMed Scopus (382) Google Scholar, 26.Yang C.W. Li C. Jung J.Y. et al.Preconditioning with erythropoietin protects against subsequent ischemia–reperfusion injury in rat kidney.FASEB J. 2003; 17: 1754-1755Crossref PubMed Scopus (175) Google Scholar, 27.Patel N.S. Sharples E.J. Cuzzocrea S. et al.Pretreatment with EPO reduces the injury and dysfunction caused by ischemia/reperfusion in the mouse kidney in vivo.Kidney Int. 2004; 66: 983-989Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar have demonstrated that exogenous administration of EPO before25.Sharples E.J. Patel N. Brown P. et al.Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia–reperfusion.J Am Soc Nephrol. 2004; 15: 2115-2124Crossref PubMed Scopus (382) Google Scholar, 26.Yang C.W. Li C. Jung J.Y. et al.Preconditioning with erythropoietin protects against subsequent ischemia–reperfusion injury in rat kidney.FASEB J. 2003; 17: 1754-1755Crossref PubMed Scopus (175) Google Scholar, 27.Patel N.S. Sharples E.J. Cuzzocrea S. et al.Pretreatment with EPO reduces the injury and dysfunction caused by ischemia/reperfusion in the mouse kidney in vivo.Kidney Int. 2004; 66: 983-989Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar or at23.Gong H. Wang W. Kwon T.H. et al.EPO and alpha-MSH prevent ischemia/reperfusion-induced down-regulation of AQPs and sodium transporters in rat kidney.Kidney Int. 2004; 66: 683-695Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 25.Sharples E.J. Patel N. Brown P. et al.Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia–reperfusion.J Am Soc Nephrol. 2004; 15: 2115-2124Crossref PubMed Scopus (382) Google Scholar the time of bilateral renal ischemia–reperfusion injury inhibits apoptotic cell death, enhances tubular epithelial regeneration, and promotes renal functional recovery. Unfortunately, in the clinical setting, most cases of ischemic ARF are not identified until some time after the insult has already occurred. Thus, the clinical utility of any therapeutic agent for ARF would be greatly enhanced if delayed administration of the drug still proved to be renoprotective. In the setting of experimental brain injury, EPO administration remains beneficial up to 6 h after the onset of the inciting event.7.Brines M.L. Ghezzi P. Keenan S. et al.Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury.Proc Natl Acad Sci USA. 2000; 97: 10526-10531Crossref PubMed Scopus (1281) Google Scholar Whether delayed treatment with EPO or its analogues exerts similar benefits in ischemic renal injury (IRI) is presently unknown. Darbepoetin alfa (DPO), an erythropoietic agent with a serum half-life approximately three times as long as EPO,28.Macdougall I.C. Novel erythropoiesis stimulating protein.Semin Nephrol. 2000; 20: 375-381PubMed Google Scholar might have a pharmacokinetic profile that is clinically advantageous in ARF if it were shown to demonstrate similar effects to that observed with EPO. DPO ameliorated apoptosis of porcine tubular and murine mesangial cells induced by toxic and hypoxic stimuli.29.Fishbane S. Ragolia L. Palaia T. et al.Cytoprotection by darbepoetin/epoetin alfa in pig tubular and mouse mesangial cells.Kidney Int. 2004; 65: 452-458Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar However, DPO has not yet been studied in in vivo models of ischemic ARF. The aims of the present study therefore were to determine in an in vivo model of IRI whether (a) DPO administration exhibited comparable renoprotection to that afforded by EPO, and (b) delayed administration of EPO or DPO up to 6 h following IRI still ameliorated subsequent renal injury and dysfunction. The role of pro and antiapoptotic Bcl-2 proteins in any such amelioration of renal dysfunction by DPO or EPO was also investigated. Examples of histopathology of IRI animals with or without EPO or DPO are given in Figure 1a–f. Figure 1a and b is an example of nontreated IR kidneys at 1 and 2 days, respectively, and shows tubular epithelial cell desquamation, primary and secondary (after loss of adhesion to basement membrane) apoptosis, luminal casts, and necrosis. Figure 1c–f is at comparable magnification and represents IR plus EPO (c, d) or DPO (e, f) at 1 and 2 days. EPO and DPO treatments show lessening of the extent of renal pathology in the outer stripe of the outer medulla (OSOM), in comparison with IR alone. Terminal dideoxy-uridine transferase-mediated nick end labeling (TUNEL) staining is demonstrated in Figure 2a and b (negative control for the procedure is shown in Figure 2c). TUNEL-positive nuclei are demonstrated in the tubular epithelium and lumen. Luminal cells, similar to those arrowed in Figure 1a and b, were TUNEL-positive and apoptotic. Figure 2d–f demonstrates proliferating cell nuclear antigen (PCNA). Figure 2d represents PCNA in control kidney sections. Figure 2e is an example of increased PCNA with IR with and without EPO and DPO treatments (negative control for PCNA is shown in Figure 2f). Necrosis, mainly of proximal straight segments, was seen at around 15% of tubular epithelial cells at 24 and 48 h, decreasing at later times to almost negligible levels at 7 days post-IR. Details of assessment of necrosis are detailed elsewhere.24.Vesey D.A. Cheung C. Pat B. et al.Erythropoietin protects against ischaemic acute renal injury.Nephrol Dial Transplant. 2004; 19: 348-355Crossref PubMed Scopus (260) Google Scholar No amelioration of necrosis was seen with either EPO or DPO treatments. Both the proximal straight segment and thick ascending limb had lower levels of apoptosis with EPO and DPO. Figure 3 summarizes histologic quantification, and demonstrates mean±standard error of the mean (s.e.) for counts of apoptosis and mitosis in the OSOM and cortex, in control, IR, IR plus EPO delivered at time of surgery (T0) and 6 h post-IR treatment (T6), and IR plus DPO at T0 and T6. Because there were often zero counts for control animals, the control bar is absent from some sets of bar graphs.Figure 2Verification of apoptosis and mitosis. In (a and b), TUNEL-positive nuclei are demonstrated in the tubular epithelium and tubular lumen. Note that cells similar to those arrowed as apoptotic in (a and b) are TUNEL positive. In (d and e), examples of PCNA positivity in a control renal section (d, arrows) can be compared with the increase in the PCNA positivity in the outer medulla after IR (e, examples arrowed). This was seen with and without EPO and DPO, but more so with the EPO and DPO treatments. Negative controls for TUNEL (c) and PCNA (f) are demonstrated, and morphological examples of apoptosis (c) and mitosis (f) are arrowed in these photomicrographs.View Large Image Figure ViewerDownload (PPT)Figure 3Means±s.e. of quantified histological characteristics in the medulla and cortex. In each case, sets of bar graphs represent control animals (open bar), IR alone (IR, solid bar), IR plus EPO at T0 and T6 (full and open dot bars, respectively), and IR plus DPO at T0 and T6 (hatched and striped bars, respectively). In the outer stripe of the outer medulla (outer medulla), IR induced a significant increase in cell death (apoptosis) and mitosis (P<0.005) at all times. There was also a small but significant increase in these parameters for IR alone in some instances in the cortex (P<0.05). Both EPO and DPO, whether administered at T0 or T6, significantly ameliorated the extent of injury in the outer medulla at all times (P<0.05). In many cases, as demonstrated for cell proliferation by #, there was a significant increase in cell proliferation in the EPO- or DPO-treated animals compared with the level of proliferation in the IR-alone animals (P<0.05). Cortical proximal tubule mitotic figures were significantly increased in EPO- or DPO-treated animals with IRI at 1 and 2 days post-IR, compared with IR alone (P<0.05).View Large Image Figure ViewerDownload (PPT) Compared with control animals, rats with bilateral IRI demonstrated significant derangements in renal histology within 24 h, including increased tubular epithelial apoptosis and necrosis in the OSOM (P<0.005 for apoptosis). Only the counts for apoptosis are demonstrated. Increased apoptosis was maintained over the period of study. Both EPO and DPO, whether administered at T0 or T6, significantly ameliorated the extent of injury. In many cases, as demonstrated in Figure 3, there was a significant increase in cell proliferation in the EPO- or DPO-treated animals compared with the level of proliferation in the IR-alone animals (P<0.05). Cortical proximal tubule mitotic figures were significantly increased in EPO- or DPO-treated animals with IRI at 1 and 2 days post-IR, compared with IR alone. There was no pathology induced by EPO, or DPO alone and also no significant increase in proliferation by these treatments without IR (data not demonstrated). Both DPO and EPO administration at time of bilateral IRI in rats significantly abrogated subsequent renal functional impairment (Figure 4). Plasma creatinine elevations peaked at day 2 following bilateral IRI and were significantly lower in EPO- and DPO-treated rats compared with vehicle-treated rats (DPO 0.08±0.03 vs EPO 0.04±0.01 vs vehicle 0.17±0.05 mmol/l, P=0.01). DPO- and EPO-treated rats subjected to bilateral IRI had peak plasma creatinine concentrations (0.08±0.03 vs 0.04±0.01 mmol/l, respectively) that were not significantly different from those of sham-operated controls (whether treated with vehicle, EPO, or DPO) or each other (P=0.22). Similar results were seen for plasma urea concentrations, which also peaked on day 2 (DPO-treated IRI 28.6±10.2 vs EPO-treated IRI 18.2±4.8 vs vehicle-treated IRI 46.7±11.8 vs DPO-treated sham 5.9±0.2 vs EPO-treated sham 4.6±0.6 vs vehicle-treated sham 5.0±0.2 mmol/l, P<0.01). Administration of DPO or EPO 6 h after onset of ischemia in rats subjected to bilateral IRI still significantly abrogated subsequent renal functional impairment (Figure 5). Plasma creatinine elevations peaked at day 2 and were significantly lower in delayed DPO- and EPO-treated rats compared with vehicle-treated rats (DPO 0.06±0.01, EPO 0.03±0.03 vs vehicle 0.17±0.05 mmol/l, P=0.01). Delayed DPO- and EPO-treated rats subjected to bilateral IRI had peak plasma creatinine concentrations (0.05±0.01 vs 0.03±0.03 mmol/l, respectively) that were not significantly different from those treated with vehicle, EPO or DPO or both (P=0.21). Similar results were seen for plasma urea (delayed DPO-treated IRI 18.2±4.8 vs delayed EPO-treated IRI 10.5±3.0 vs vehicle-treated IRI 46.7±11.8 vs DPO-treated sham 5.9±0.2 vs EPO-treated sham 4.6±0.6 vs vehicle-treated sham 5.0±0.2 mmol/l, P<0.01). Hematocrits gradually and significantly increased over the 7-day study period in IRI animals receiving either DPO (net increase 9.0±1.5%) or EPO (8.0±0.8%). Similar results were observed in sham-operated animals receiving either DPO (11.4±2.1%) or EPO (8.1±0.4%). Using immunohistochemistry (IHC) and Western immunoblot, expression of antiapoptotic Bcl-2 and Bcl-XL was not significantly different among control and IRI kidneys with and without EPO and DPO. Using IHC, proapoptotic Bax expression was significantly reduced in both EPO and DPO treatments compared with IR alone, at 2 days. Bax expression in control kidneys was minimal (Figure 6a), except in collecting ducts, which had moderate expression in all kidneys. In IR kidneys, Bax was expressed mainly in proximal and distal tubular epithelium, strongest in the proximal straight (S3) segment and thick ascending limb in the OSOM. The OSOM had the greatest reduced Bax expression with EPO and DPO treatment. Kidneys from IR-treated animals had consistently higher levels of Bax (Figure 6b) compared with control, and EPO- and DPO-treated IR kidneys (Figure 6c and d, respectively). Means±s.e. for IHC Bax expression are demonstrated in Figure 6e. Bax was significantly increased in IR-alone animals compared with controls. EPO and DPO treatments were associated with decreased Bax in each instance, with significance reached for the T6 EPO and the T0 DPO treatments (P<0.05). A similar Bax expression pattern was also found with Western immunoblots (Figure 7).Figure 7Western immunoblot of Bax. (a) Representative Western immunoblot of renal Bax is given for nontreated, EPO and DPO controls, IR at 24 and 48 h for the animals treated at T0 and T6. Bax expression (top band of the doublet is 22 kDa) increased with IR, was greatest at 24 h, and both EPO and DPO treatments were associated with lower Bax expression than that seen in the IR animals.View Large Image Figure ViewerDownload (PPT) The present study clearly demonstrated that the administration of either DPO or EPO at the time of bilateral IRI significantly inhibited subsequent apoptotic cell death, enhanced tubular epithelial regeneration, minimized the severity of renal dysfunction, and promoted more rapid renal functional recovery. A key novel observation was that delaying the administration of either DPO or EPO for up to 6 h after the onset of ischemia maintained renoprotection by these agents. This is of potential therapeutic importance, as most cases of ischemic ARF in the clinical setting are only diagnosed after the inciting injury has already occurred. Indeed, our investigation did not exclude the possibility of a significant renoprotective benefit when EPO or DPO administration is delayed by more than 6 h after the onset of IRI, such that further studies of more prolonged deferment of EPO/DPO therapy following renal injury are warranted. The finding of a protective effect of erythropoietic compounds, even after delayed administration, is supported by experimental brain injury studies whereby systemic administration of recombinant human EPO before or up to 6 h after focal brain ischemia reduced cerebral injury by approximately 50–75%.7.Brines M.L. Ghezzi P. Keenan S. et al.Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury.Proc Natl Acad Sci USA. 2000; 97: 10526-10531Crossref PubMed Scopus (1281) Google Scholar Similarly, treatment of rats with EPO (5000 IU/kg) 45 min after the onset of coronary occlusion was at least as effective at reducing the ratio of infarct area/area at risk and the degree of myocyte apoptosis as giving EPO 2 h before the start of ischemia.16.Lipsic E. van der M.P. Henning R.H. et al.Timing of erythropoietin treatment for cardioprotection in ischemia/reperfusion.J Cardiovasc Pharmacol. 2004; 44: 473-479Crossref PubMed Scopus (152) Google Scholar In a rat model of renal IRI, a single intravenous injection of EPO (300 U/kg) reduced glomerular dysfunction, tubular injury, caspase activation, and apoptotic cell death, regardless of whether the EPO was given 30 min before the commencement of ischemia, 5 min before the onset of reperfusion, or 30 min following reperfusion commencement.25.Sharples E.J. Patel N. Brown P. et al.Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia–reperfusion.J Am Soc Nephrol. 2004; 15: 2115-2124Crossref PubMed Scopus (382) Google Scholar Moreover, delayed (4 h) treatment with EPO dramatically prevented the decrease in renal expression of sodium transporters (Na-K-ATPase and Na-K-2Cl co-transporter) associated with renal IRI.23.Gong H. Wang W. Kwon T.H. et al.EPO and alpha-MSH prevent ischemia/reperfusion-induced down-regulation of AQPs and sodium transporters in rat kidney.Kidney Int. 2004; 66: 683-695Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar In contrast, daily pretreatment with EPO (1000 IU/kg/day subcutaneously for 3 days) afforded greater renoprotection in a mouse model of renal IRI than a single bolus (1000 IU/kg subcutaneously) delivered at the time of reperfusion.27.Patel N.S. Sharples E.J. Cuzzocrea S. et al.Pretreatment with EPO reduces the injury and dysfunction caused by ischemia/reperfusion in the mouse kidney in vivo.Kidney Int. 2004; 66: 983-989Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar However, differences observed between the two groups may have been primarily explained by the greater cumulative dose administered to the pretreatment group (3000 vs 1000 IU/kg) rather than the timing of administration. Although there have been no in vivo studies to determine the optimal renoprotective dose of erythropoietic agents, a previous in vitro study by our group demonstrated that the doses of EPO needed to promote maximal antiapoptotic and mitogenic effects in human proximal tubule cells were quite high (200–400 IU/ml).24.Vesey D.A. Cheung C. Pat B. et al.Erythropoietin protects against ischaemic acute renal injury.Nephrol Dial Transplant. 2004; 19: 348-355Crossref PubMed Scopus (260) Google Scholar Despite the fact that no toxicity has been observed in animal models of ARF with the relatively high doses of EPO (5000 IU/kg) and DPO (25 μg/kg) employed in our studies, adverse sequelae in the clinical setting (e.g. thrombosis related to hematocrit increases) cannot entirely be excluded. In this respect, it is reassuring to note that a recent pilot safety study for a multi-center, randomized controlled trial of EPO in acute stroke demonstrated no adverse effects (including no significant change in hematocrit over 30 days) from a single EPO dose of 100 000 U (approximately 1500 U/kg), despite a transient 500-fold increase in serum EPO concentrations and a 100-fold increase in cerebrospinal fluid EPO concentrations.30.Ehrenreich H. Hasselblatt M. Dembowski C. et al.Erythropoietin therapy for acute stroke is both safe and beneficial.Mol Med. 2002; 8: 495-505Crossref PubMed Google Scholar This suggests that a similar study of renoprotection in humans is feasible. Alternative strategies for reducing systemic exposure might also be possible (e.g. by direct perfusion of donor kidneys in renal transplantation). Our results also extend previously reported findings of a renoprotective benefit of EPO14.Abdelrahman M. Sharples E.J. McDonald M.C. et al.Erythropoietin attenuates the tissue injury associated with hemorrhagic shock and myocardial ischemia.Shock. 2004; 22: 63-69Crossref PubMed Scopus (142) Google Scholar, 23.Gong H. Wang W. Kwon T.H. et al.EPO and alpha-MSH prevent ischemia/reperfusion-induced down-regulation of AQPs and sodium transporters in rat kidney.Kidney Int. 2004; 66: 683-695Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 24.Vesey D.A. Cheung C. Pat B. et al.Erythropoietin protects against ischaemic acute renal injury.Nephrol Dial Transplant. 2004; 19: 348-355Crossref PubMed Scopus (260) Google Scholar, 25.Sharples E.J. Patel N. Brown P. et al.Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia–reperfusion.J Am Soc Nephrol. 2004; 15: 2115-2124Crossref PubMed Scopus (382) Google Scholar, 26.Yang C.W. Li C. Jung J.Y. et al.Preconditioning with erythropoietin protects against subsequent ischemia–reperfusion injury in rat kidney.FASEB J. 2003; 17: 1754-1755Crossref PubMed Scopus (175) Google Scholar, 27.Patel N.S. Sh
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