- Email: [email protected]
Recombinant erythropoietin rapidly treats anemia in ischemic acute renal failure
Kidney International, Vol. 59 (2001), pp. 246–251
Recombinant erythropoietin rapidly treats anemia in ischemic acute renal failure TAKASHI NEMOTO, NAOKO YOKOTA, WILLIAM F. KEANE, and HAMID RABB Department of Medicine, Hennepin County Medical Center, University of Minnesota Medical School, Minneapolis, Minnesota, USA
Recombinant erythropoietin rapidly treats anemia in ischemic acute renal failure. Background. The anemia associated with acute renal failure (ARF) is currently treated with blood transfusions, while the anemia of chronic renal failure is treated with recombinant erythropoietin (EPO). We hypothesized that EPO treatment during ARF could rapidly improve hemoglobin levels and be a useful therapeutic approach. In addition, as tubular epithelial cells have EPO receptors that can mediate proliferation, enhanced recovery of renal function may occur with EPO use. Methods. An established rat model of ischemic ARF was studied, using either moderate or severe ischemia. EPO was administered in a dose of 500 or 3000 U/kg starting at time of ischemia. Hematocrit (Hct), serum creatinine, reticulocyte count, and mortality rate were measured. Results. EPO treatment led to a rapid and significant increase in Hct at 48 and 72 hours after moderate ischemic renal reperfusion injury (IRI) in EPO (500 U/kg)-treated rats compared with control (saline treated) rats (mean ⫾ SE; 45.6 ⫾ 0.3% vs. 42.0 ⫾ 1.0%, P ⬍ 0.01) and (46.6 ⫾ 0.3 vs. 41.0 ⫾ 1.0, P ⬍ 0.01, N ⫽ 3 per group). In severe renal IRI, EPO treatment also led to significantly increased Hct at 48 (40.0 ⫾ 4.4% vs. 36.8 ⫾ 0.3%, P ⬍ 0.01, N ⫽ 3 per group) and 72 hours (43.5 ⫾ 1.5% vs. 34.7 ⫾ 2.3%, P ⬍ 0.01, N ⫽ 3 per group). Higher dose (3000 U/kg) EPO led to a more pronounced Hct increase after severe IRI at 48 hours compared with the 500 U/kg dose (43.5 ⫾ 0.3 vs. 40.3 ⫾ 0.3, P ⬍ 0.01, N ⫽ 3 per group). EPO treatment during moderate or severe renal IRI did not change the course of the renal dysfunction. EPO treatment (N ⫽ 19) had a significant protective effect on mortality during severe IRI. In addition, loss of body weight during ARF was not affected by EPO therapy. Conclusions. Recombinant EPO can rapidly increase Hct and improve mortality during ARF. Human studies are warranted to evaluate the clinical applicability of this important finding.
of most cases of ARF, it is commonly believed that adequate erythropoietin (EPO) response could take longer than the course of ARF itself. Thus, anemia of ARF, when treatment is warranted, is usually treated with blood transfusions. This contrasts with the anemia of chronic renal failure where recombinant human EPO is the current therapy of choice. In chronic renal failure, it may take weeks and even months to achieve the target rise in hematocrit (Hct). However, differences may exist in the pathogenesis of the anemia and response to EPO in ARF compared with chronic renal failure. We therefore hypothesized that EPO treatment during ARF could rapidly improve Hct levels and could be a useful therapeutic approach. In addition, because of the presence of EPO receptors on renal tubular epithelial cells [2], we hypothesized that EPO may also change the course of tubular repair during ARF and thus accelerate the course of renal recovery. These hypotheses were tested in an established rat model of ischemic ARF [3]. METHODS Ischemic acute renal failure Ischemic ARF was induced in male Sprague-Dawley rats weighing 150 to 200 g as previously described in depth [3]. Briefly, rats were anesthetized with pentobarbital sodium injection (50 mg/kg). A midline abdominal incision under sterile conditions was followed by clamping of the right kidney with a vascular clip (Roboz Surgical Instruments, Rockville, MD, USA) with simultaneous nephrectomy of the left kidney. After each occlusion, the clip was released at either 30 (moderate) or 45 (severe) minutes, and reperfusion was observed. Temperature was maintained constant, and warm saline (5% of body weight) was administered. After awakening, animals had free access to food and water. All experimental techniques were in accordance to National Institutes of Health guidelines for animal experimentation.
Acute renal failure (ARF) is associated with the development of anemia [1]. Because of the short-term course Key words: hematocrit, tubular epithelial cells, reperfusion injury, intensive care therapy. Received for publication March 28, 2000 and in revised form July 7, 2000 Accepted for publication July 24, 2000
Erythropoietin administration Recombinant human EPO (Epoetin Alpha; Amgen, Thousand Oaks, CA, USA) was administered. EPO was
2001 by the International Society of Nephrology
246
Nemoto et al: EPO therapy in ARF
Fig. 1. A gradual decline occurs in the hematocrit (Hct) after severe (45-minute clamp) renal ischemia in rats given saline injections (N ⫽ 6).
prepared at two different dosages: 500 or 3000 U/kg. All injections were diluted with sterile saline and adjusted to 0.5 cc, with similar volumes of sterile saline given to control rats. EPO was administrated intravenously at the time of ischemia and then subcutaneously at 24 and 48 hours postischemia. Experimental groups The following are the experimental groups used: group 1, sham surgery, saline-treated; group 2, sham surgery, EPO-treated; group 3, moderate ischemia, saline-treated; group 4, moderate ischemia, EPO-treated; group 5, severe ischemia, saline-treated; group 6, severe ischemia, EPO-treated; and group 7, severe ischemia, high-dose EPO-treated. Outcomes measured Blood samples were obtained from the tail vein. Serum creatinine (SCr) was measured as a marker of renal function using a Synchron LX20 Clinical system (Beckman Culture Inc., Fullerton, CA, USA). Hct was used as an indicator of anemia using a Hct tube and centrifugation at 10,000 r.p.m. for five minutes. Body weights were obtained daily, and mortality was also monitored. Statistical analysis All values were presented as means ⫾ SE. Statistical analysis comparing saline-treated control and two different EPO doses was performed by analysis of variance and Fisher’s least-significant difference test. Mortality was also compared using the Kaplan–Meier method. Statistical significance was set at P ⬍ 0.05. RESULTS Renal ischemia reperfusion injury results in anemia Rats (N ⫽ 6) underwent 45 minutes of ischemia to a single kidney, nephrectomy of the contralateral kidney,
247
Fig. 2. Hematocrits (Hcts) of erythropoietin (EPO)-treated (500 U/kg) and saline-treated groups that underwent sham surgery. Significant Hct increasing was observed from 96 hours after surgery in EPO-treated group. Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 5); (䊉) salinetreated (N ⫽ 5). **P ⬍ 0.01.
and then reperfusion. A steady decline in Hct was observed over the next three days (Fig. 1). Thus, this current model can be used to simulate the anemia of ARF. Effect of EPO on sham surgery Hematocrit remained stable in saline-treated, shamoperated animals. However, in EPO-treated (500 U/kg) rats, a significant rise in Hct (%) was observed that became statistically significant 96 hours after surgery (47.3 ⫾ 1.3 vs. 41.5 ⫾ 0.3, P ⬍ 0.01, N ⫽ 5 per group; Fig. 2). Effect of EPO in moderate renal ischemic renal reperfusion injury Moderate ischemic renal reperfusion injury (IRI; 30 minutes of ischemia) simulates reversible acute tubular necrosis. This was not accompanied by development of significant anemia (Fig. 3). Administration of EPO at 500 U/kg led to a significant rise in Hct at 48 and 72 hours compared with saline-treated rats with IRI (45.6 ⫾ 0.3 vs. 42.0 ⫾ 0.6, P ⬍ 0.01, N ⫽ 3 per group, and 46.6 ⫾ 0.3 vs. 41.0 ⫾ 0.6, P ⬍ 0.01, N ⫽ 3 per group). Effect of EPO in severe renal IRI Anemia developed rapidly in the saline-treated rats that underwent severe IRI (Fig. 4). In contrast, Hct was significantly higher at 48 and 72 hours after IRI in EPO (500 U/kg)-treated rats compared with saline-treated rats (41.0 ⫾ 0.6 vs. 36.5 ⫾ 0.4, P ⬍ 0.01, N ⫽ 3 per group, and 42.3 ⫾ 1.2 vs. 36.5 ⫾ 0.4, P ⬍ 0.01, N ⫽ 3 per group). When higher doses of EPO (3000 U/kg) were administered to rats with 45 minutes of ischemia, no further increments in Hct were observed. However, at 48 hours postischemia, a more marked response in Hct
248
Nemoto et al: EPO therapy in ARF
Fig. 3. Hematocrits of EPO-treated (500 U/kg) and saline-treated groups that underwent moderate ischemic reperfusion injury (IRI; 30-minute clamp). Significant increases in Hct were observed at 48 and 72 hours after IRI in the EPO-treated group. Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3). **P ⬍ 0.01.
Fig. 5. Effect of erythropoietin dose on hematocrit (Hct) during severe renal ischemia. Hct was higher at 48 hours after IRI in the high-dose (3000 U/kg) EPO-treated rats compared with the lower-dose (500 U/kg) rats. Symbols are: (䊊) high-dose EPO-treated (3000 U/kg, N ⫽ 3); (䊐) high-dose EPO-treated (500 U/kg, N ⫽ 3); **P ⬍ 0.01.
Fig. 4. Hematocrits of erythropoietin-treated (500 U/kg) and salinetreated group. Both groups underwent severe IRI (45-minute clamp). Hct was significantly higher at 48 and 72 hours after IRI in EPO-treated rats compared with saline controls. Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 3); (䊉) Control (N ⫽ 3); **P ⬍ 0.01.
Fig. 6. Serum creatinine (SCr) value (mg/dL) in the EPO-treated (500 U/kg) and saline-treated groups. Both groups underwent moderate IRI (30 minutes). Baseline creatinine in both groups was ⬍0.3 mg/dL. No different was observed at any time point. Symbols are: (䊊) EPOtreated (500 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3).
was observed compared with the 500 U/kg dose (43.5 ⫾ 0.3 vs. 40.3 ⫾ 0.3, P ⬍ 0.01, N ⫽ 3 per group; Fig. 5).
Effect of EPO on body weight postischemia
Effect of EPO on serum creatinine postischemia Growth factor administration can enhance the resolution of experimental ARF [4]. Thus, EPO, whose receptors are found on tubular epithelial cells, may modulate renal tubular cell recovery after ischemia [2]. However, no effect of EPO was observed on either the rise or decline in SCr during moderate renal IRI (Fig. 6). Similarly, no EPO effect was observed on the course of SCr after severe IRI (Fig. 7). Even at higher doses (3000 U/kg), EPO lacked effect on the course of SCr during severe IRI (data not shown).
Erythropoietin can affect the hypothalamic-hormonal axis and may have many effects independent of merely correction of anemia, including reducing catabolism [5]. However, EPO treatment did not influence the loss of body weight during severe renal IRI (Fig. 8). EPO treatment during moderate IRI was also ineffective in mitigating the loss of body weight (data not shown). Effect of EPO on mortality after renal ischemia The high mortality associated with ARF continues to be a serious clinical problem [6]. Mortality rates were compared in EPO and untreated animals. All rats survived after moderate IRI. However, after severe IRI,
Nemoto et al: EPO therapy in ARF
Fig. 7. Serum creatinine (SCr) values in (mg/dL) EPO-treated (500 U/kg) and saline-treated groups after severe renal ischemia (45-min clamp). No difference was observed at any time point. No effect on SCr was seen even when higher dose EPO (3000 U/kg) was used (data not shown). Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3).
Fig. 8. Change in body weight after severe renal ischemia. More severe weight loss is seen in the severe renal IRI compared with moderate IRI. However, no significant difference was seen in the saline-treated group compared with the EPO-treated group (500 U/kg). The higherdose EPO (3000 U/kg) group did not differ from saline group either (data not shown). Symbols are: (䊊) EPO-treated (3000 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3).
substantial mortality ensues (Fig. 9). EPO treatment significantly improves survival when rats were followed 96 hours postischemia. Effect of EPO on reticulocyte count after renal ischemia Reticulocyte counts, reflective of erythrocyte production, were decreased 48 hours after 45 minutes of renal ischemia (7.5 ⫾ 0.7 vs. 5.6 ⫾ 0.4, P ⬍ 0.05). However, EPO treatment significantly increased the reticulocyte count during ARF (6.9 ⫾ 0.3, P ⬍ 0.05).
249
Fig. 9. Effect of EPO on mortality after IRI. Mortality was pronounced after severe (45 minutes) renal IRI. EPO (3000 U/kg) treatment significantly improved mortality (P ⬍ 0.05) after severe IRI. All rats survived after moderate IRI at 72 hours (data not shown). Symbols are: (䊊) EPO-treated (N ⫽ 19); (䊉) Control (N ⫽ 17).
DISCUSSION Anemia is often observed during ARF [7]. We hypothesized that recombinant EPO would be a useful therapy in ischemic ARF. We found that EPO can rapidly raise Hct during moderate and severe ischemic ARF in rats. The likely mechanism is by increasing erythrocyte production, as evidenced by an increased reticulocyte count after EPO treatment. Importantly, EPO treatment resulted in a significant improvement in mortality after severe ischemic injury. However, EPO administration did not significantly influence the course of the SCr or body weight. It is useful to compare the Hct responses in our studies to another model of ARF, cisplatin-induced ARF. In this model, EPO was administrated during cisplatin-induced ARF in rats [8]. There were no significantly differences in Hct between the two groups of rats on day 4. However, the EPO-treated group showed a significantly higher Hct level compared with untreated ARF group on day 9 after cisplatin. This cisplatin study also showed that renal function and body weight was improved in the EPOtreated group nine days after cisplatin [8]. In our current study using an IRI model of ARF, we found a much more rapid response after EPO, and no effect on body weight. However, cisplatin toxicity is likely to have additional effects, particularly on the bone marrow, than what is observed after IRI. The rapid response in Hct to EPO in experimental ARF that we observed was quite different from that seen in chronic renal failure. During chronic renal failure, EPO production is depressed with inappropriately low plasma levels throughout the uremic phase [9]. Many
250
Nemoto et al: EPO therapy in ARF
other factors contribute to anemia, including reduced red cell life span, iron deficiency, chronic inflammation, and contaminants of hemodialysis. The pathophysiology of anemia in ARF is likely to be complex. A significant reduction in serum haptoglobin and an increase in serum lactate dehydrogenase in ARF suggest that that hemolysis is a contributory factor in the initial fall in Hct in ARF [8]. Production of EPO has also been demonstrated to be reduced in rats and humans during ARF [10, 11]. The pathogenesis is not well understood. Other factors underlying the anemia include bleeding, hemolysis, blood sequestration, and resistance to EPO [7]. It is also possible that saline treatment during our surgical protocol may have contributed to a dilutional component to the anemia, but in this regard, all rats received similar quantities of saline. Despite the partial knowledge underlying the anemia of ARF, EPO therapy could still be a useful therapy in ARF as it is for CRF. We found that EPO at 500 U/kg for three consecutive doses increased Hct rapidly in rats with normal renal function. Previous studies have also looked at EPO’s effect in normal rats. EPO administered twice weekly (150 U/kg) had no significant effects on Hct after one week compared with controls [12]. In another study in which EPO was administrated to normal rats, a significant increase in Hct was observed at day 3 [13]. When the Hct response to EPO is rapid, changes can be detected early in reticulocytes. In a study in which EPO was administrated 250 U/kg and 1250 U/kg, an increase in reticulocytes began at 24 hours and reached the peak at 72 to 96 hours after injection of EPO [14]. In the current study, we did not specifically investigate the mechanisms for the anemia of ARF. However, the increase in reticulocyte count in the EPO-treated groups makes it likely that the effect we observed was based on increased red blood cell production. Alternatively, an early rise in reticulocyte count may also be due to early release of these cells from the bone marrow induced by EPO. We also tested the hypothesis that EPO treatment could potentially alter the course of ARF. This hypothesis was based on previous data in both the brain and kidney. EPO mRNA expression increases in rodent and primate brain after hypoxia [15]. EPO receptors are found in neural cell lines [15], neurons [16], and renal tubular epithelial cells [2]. Furthermore, EPO administration enhances proliferation of renal tubular epithelial cells [2], thought to be important in the recovery after renal IRI. In addition, EPO administration prevented ischemia-induced learning disability and rescued hippocampal neurons from ischemia [17], while infarct volume was improved in EPO-pretreated mice in cerebral ischemia [18]. Although renal dysfunction was not improved by EPO therapy in our current study, it did not worsen it either. We cannot exclude the possibility that given the short duration of our study, prolonged EPO treatment in
a protracted ARF model could have led to improvement in SCr. In a study of cis-platinum–induced ARF in which EPO was administered for nine consecutive days, renal functional changes were apparent after nine days but not at four days [19]. Given the high rate of mortality and catabolism associated with native kidney ARF [20], we were interested in the possible effect of EPO on mortality and body weight. It has been shown that that the administration of EPO provided a beneficial effect on the body weight, Hct, and healing rate in rats seven days following a large bowel operation [21]. In our current study, both in moderate and severe IRI, the decrease in body weights during ARF was not affected by EPO. However, a marked improvement in mortality was observed in EPO-treated rats after severe IRI. It is unclear as to the mechanisms of the decreased mortality. One possibility is that although there were no significant differences in SCr, more sensitive tests such as inulin clearances would have detected small increases in glomerular filtration rate (GFR) after EPO treatment. These small increases in GFR may have been adequate to improve mortality. Another possibility is that EPO may have reduced the lung dysfunction and systemic inflammatory state that has been associated with renal IRI [22]. Our current data demonstrate that EPO treatment during ARF rapidly improves Hct levels and could be a useful therapeutic approach, especially in the intensive care unit. In humans, at least one case report exists describing EPO therapy for anemia with ARF [23]. A properly designed clinical trial with an adequate number of patients will be required prior to justifying the routine use of EPO in patients with ARF. ACKNOWLEDGMENTS T.N. and N.Y. were supported by the Showa University, and H.R. was supported by a NKF Clinical Scientist Award, Hermundslie Trust and Minneapolis Medical Research Foundation. The authors thank Ms. Jan Lovick for assistance in manuscript preparation. Novel EPO indication submitted for patent. Reprint requests to Hamid Rabb, M.D., Nephrology, D-5, Hennepin County Medical Center, 701 Park Avenue, Minneapolis, Minnesota 55446, USA. E-mail: [email protected]
REFERENCES 1. Rainford DJ, Stevens PE: The investigative approach to the patient with acute renal failure, 8.1, in Oxford Textbook of Clinical Nephrology (vol 2), edited by Cameron S, Davison AM, Grunfeld J, et al: Oxford University Press, 1992, pp 969–982 2. Westenfelder C, Biddle DL, Baranowski RL: Human, rat, and mouse kidney cells express functional erythropoietin receptors. Kidney Int 55:808–820, 1999 3. Rabb H, Mendiola CC, Dietz J, et al: Role of CD11a and CD11b in ischemic acute renal failure in rats. Am J Physiol 267:F1052–F1058, 1994 4. Humes HD, MacKay SM, Funke AJ, et al: Acute renal failure:
Nemoto et al: EPO therapy in ARF
5.
6. 7.
8.
9.
10.
11.
12. 13.
14.
Growth factors, cell therapy, and gene therapy. Proc Assoc Am Physicians 109:547–557, 1997 Ramirez G, Bittle PA, Rabb H, et al: Effect of haemoglobin and endogenous erythropoietin on hypothalamic-pituitary thyroidal and gonadal secretion: An analysis of anemic (high EPO) polycythanemic (low EPO) patients. Clin Endocrinol (Oxf) 43:167–174, 1995 Star RA: Treatment of renal failure. Kidney Int 54:1817–1831, 1998 Steinman TI, Lazarus JM: Organ-system involvement in acute renal failure, in Acute Renal Failure (2nd ed), edited by Brenner BM, Lazarus JM, New York, Churchill Livingstone, 1988, pp 705–739 Baldwin MD, Zhou XJ, Ing TS, et al: Erythropoietin ameliorates anemia of cisplatin induced acute renal failure. ASAIO J 44:44–47, 1998 Zhang F, Laneuville P, Gagnon RF, et al: Effect of chronic renal failure on the expression of erythropoietin message in a murine model. Exp Hematol 24:1469–1474, 1996 Tan CC, Tan LH, Eckardt KU: Erythropoietin production in rats with post-ischemic acute renal failure. Kidney Int 50:1958–1964, 1996 Morgera S, Heering P, Szentandrasi T, et al: Erythropoietin in patients with acute renal failure and continuous veno-venous haemofiltration. Int Urol Nephrol 29:245–250, 1997 Vaziri ND, Zhou XJ, Smith J, et al: In vivo and in vitro pressor effects of erythropoietin in rats. Am J Physiol 269:F838–F845, 1995 Eggena P, Willsey P, Jamgotchian N, et al: Influence of recombinant human erythropoietin on blood pressure and tissue reninangiotensin systems. Am J Physiol 261:E642–E646, 1991 Masunaga H, Takahira R, Hirabayashi M, et al: Pharmacokinetics and the time course of pharmacodynamics of recombinant hu-
15. 16.
17. 18. 19. 20. 21.
22. 23.
251
man erythropoietin (SNB-5001). Nippon Yakurigaku Zasshi 99:213– 229, 1992 Masuda S, Nagao M, Takahata K, et al: Functional erythropoietin receptor of the cells with neural characteristics. J Biol Chem 268: 11208–11216, 1993 Morisita E, Masuda S, Nagao M, et al: Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 76:105–116, 1997 Sakanaka M, Wen TC, Matsuda S, et al: In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci USA 95:4635–4640, 1998 Bernaudin M, Marti HH, Roussel S, et al: A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab 19:643–651, 1999 Vaziri ND, Zhou XJ, Liao SY: Erythropoietin enhances recovery from cisplatin-induced acute renal failure. Am J Physiol 266:F360– F366, 1994 Price SR, Reaich D, Marinovic AC, et al: Mechanisms contributing to muscle-wasting in acute uremia: Activation of amino acid catabolism. J Am Soc Nephrol 9:439–443, 1998 Fatouros M, Dalekos GN, Mylonakis E, et al: Alterations in body weight, breaking strength, and wound healing in Wistar rats treated pre- and postoperatively with erythropoietin or granulocyte macrophage-colony stimulating factor: Evidence of a previously unknown anabolic effect of erythropoietin? J Lab Clin Med 133:253– 259, 1999 Kramer A, Postler G, Salhab K, et al: Renal ischemia/reperfusion leads to macrophage-mediated increase in pulmonary vascular permeability. Kidney Int 55:2362–2367, 1999 Walter J, Kun K, Doman J: Low-dose erythropoietin treatment of anemia associated with operative transfusion hemolysis and acute renal failure. Nephrol Dial Transplant 7:559–560, 1992
Recombinant erythropoietin rapidly treats anemia in ischemic acute renal failure TAKASHI NEMOTO, NAOKO YOKOTA, WILLIAM F. KEANE, and HAMID RABB Department of Medicine, Hennepin County Medical Center, University of Minnesota Medical School, Minneapolis, Minnesota, USA
Recombinant erythropoietin rapidly treats anemia in ischemic acute renal failure. Background. The anemia associated with acute renal failure (ARF) is currently treated with blood transfusions, while the anemia of chronic renal failure is treated with recombinant erythropoietin (EPO). We hypothesized that EPO treatment during ARF could rapidly improve hemoglobin levels and be a useful therapeutic approach. In addition, as tubular epithelial cells have EPO receptors that can mediate proliferation, enhanced recovery of renal function may occur with EPO use. Methods. An established rat model of ischemic ARF was studied, using either moderate or severe ischemia. EPO was administered in a dose of 500 or 3000 U/kg starting at time of ischemia. Hematocrit (Hct), serum creatinine, reticulocyte count, and mortality rate were measured. Results. EPO treatment led to a rapid and significant increase in Hct at 48 and 72 hours after moderate ischemic renal reperfusion injury (IRI) in EPO (500 U/kg)-treated rats compared with control (saline treated) rats (mean ⫾ SE; 45.6 ⫾ 0.3% vs. 42.0 ⫾ 1.0%, P ⬍ 0.01) and (46.6 ⫾ 0.3 vs. 41.0 ⫾ 1.0, P ⬍ 0.01, N ⫽ 3 per group). In severe renal IRI, EPO treatment also led to significantly increased Hct at 48 (40.0 ⫾ 4.4% vs. 36.8 ⫾ 0.3%, P ⬍ 0.01, N ⫽ 3 per group) and 72 hours (43.5 ⫾ 1.5% vs. 34.7 ⫾ 2.3%, P ⬍ 0.01, N ⫽ 3 per group). Higher dose (3000 U/kg) EPO led to a more pronounced Hct increase after severe IRI at 48 hours compared with the 500 U/kg dose (43.5 ⫾ 0.3 vs. 40.3 ⫾ 0.3, P ⬍ 0.01, N ⫽ 3 per group). EPO treatment during moderate or severe renal IRI did not change the course of the renal dysfunction. EPO treatment (N ⫽ 19) had a significant protective effect on mortality during severe IRI. In addition, loss of body weight during ARF was not affected by EPO therapy. Conclusions. Recombinant EPO can rapidly increase Hct and improve mortality during ARF. Human studies are warranted to evaluate the clinical applicability of this important finding.
of most cases of ARF, it is commonly believed that adequate erythropoietin (EPO) response could take longer than the course of ARF itself. Thus, anemia of ARF, when treatment is warranted, is usually treated with blood transfusions. This contrasts with the anemia of chronic renal failure where recombinant human EPO is the current therapy of choice. In chronic renal failure, it may take weeks and even months to achieve the target rise in hematocrit (Hct). However, differences may exist in the pathogenesis of the anemia and response to EPO in ARF compared with chronic renal failure. We therefore hypothesized that EPO treatment during ARF could rapidly improve Hct levels and could be a useful therapeutic approach. In addition, because of the presence of EPO receptors on renal tubular epithelial cells [2], we hypothesized that EPO may also change the course of tubular repair during ARF and thus accelerate the course of renal recovery. These hypotheses were tested in an established rat model of ischemic ARF [3]. METHODS Ischemic acute renal failure Ischemic ARF was induced in male Sprague-Dawley rats weighing 150 to 200 g as previously described in depth [3]. Briefly, rats were anesthetized with pentobarbital sodium injection (50 mg/kg). A midline abdominal incision under sterile conditions was followed by clamping of the right kidney with a vascular clip (Roboz Surgical Instruments, Rockville, MD, USA) with simultaneous nephrectomy of the left kidney. After each occlusion, the clip was released at either 30 (moderate) or 45 (severe) minutes, and reperfusion was observed. Temperature was maintained constant, and warm saline (5% of body weight) was administered. After awakening, animals had free access to food and water. All experimental techniques were in accordance to National Institutes of Health guidelines for animal experimentation.
Acute renal failure (ARF) is associated with the development of anemia [1]. Because of the short-term course Key words: hematocrit, tubular epithelial cells, reperfusion injury, intensive care therapy. Received for publication March 28, 2000 and in revised form July 7, 2000 Accepted for publication July 24, 2000
Erythropoietin administration Recombinant human EPO (Epoetin Alpha; Amgen, Thousand Oaks, CA, USA) was administered. EPO was
2001 by the International Society of Nephrology
246
Nemoto et al: EPO therapy in ARF
Fig. 1. A gradual decline occurs in the hematocrit (Hct) after severe (45-minute clamp) renal ischemia in rats given saline injections (N ⫽ 6).
prepared at two different dosages: 500 or 3000 U/kg. All injections were diluted with sterile saline and adjusted to 0.5 cc, with similar volumes of sterile saline given to control rats. EPO was administrated intravenously at the time of ischemia and then subcutaneously at 24 and 48 hours postischemia. Experimental groups The following are the experimental groups used: group 1, sham surgery, saline-treated; group 2, sham surgery, EPO-treated; group 3, moderate ischemia, saline-treated; group 4, moderate ischemia, EPO-treated; group 5, severe ischemia, saline-treated; group 6, severe ischemia, EPO-treated; and group 7, severe ischemia, high-dose EPO-treated. Outcomes measured Blood samples were obtained from the tail vein. Serum creatinine (SCr) was measured as a marker of renal function using a Synchron LX20 Clinical system (Beckman Culture Inc., Fullerton, CA, USA). Hct was used as an indicator of anemia using a Hct tube and centrifugation at 10,000 r.p.m. for five minutes. Body weights were obtained daily, and mortality was also monitored. Statistical analysis All values were presented as means ⫾ SE. Statistical analysis comparing saline-treated control and two different EPO doses was performed by analysis of variance and Fisher’s least-significant difference test. Mortality was also compared using the Kaplan–Meier method. Statistical significance was set at P ⬍ 0.05. RESULTS Renal ischemia reperfusion injury results in anemia Rats (N ⫽ 6) underwent 45 minutes of ischemia to a single kidney, nephrectomy of the contralateral kidney,
247
Fig. 2. Hematocrits (Hcts) of erythropoietin (EPO)-treated (500 U/kg) and saline-treated groups that underwent sham surgery. Significant Hct increasing was observed from 96 hours after surgery in EPO-treated group. Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 5); (䊉) salinetreated (N ⫽ 5). **P ⬍ 0.01.
and then reperfusion. A steady decline in Hct was observed over the next three days (Fig. 1). Thus, this current model can be used to simulate the anemia of ARF. Effect of EPO on sham surgery Hematocrit remained stable in saline-treated, shamoperated animals. However, in EPO-treated (500 U/kg) rats, a significant rise in Hct (%) was observed that became statistically significant 96 hours after surgery (47.3 ⫾ 1.3 vs. 41.5 ⫾ 0.3, P ⬍ 0.01, N ⫽ 5 per group; Fig. 2). Effect of EPO in moderate renal ischemic renal reperfusion injury Moderate ischemic renal reperfusion injury (IRI; 30 minutes of ischemia) simulates reversible acute tubular necrosis. This was not accompanied by development of significant anemia (Fig. 3). Administration of EPO at 500 U/kg led to a significant rise in Hct at 48 and 72 hours compared with saline-treated rats with IRI (45.6 ⫾ 0.3 vs. 42.0 ⫾ 0.6, P ⬍ 0.01, N ⫽ 3 per group, and 46.6 ⫾ 0.3 vs. 41.0 ⫾ 0.6, P ⬍ 0.01, N ⫽ 3 per group). Effect of EPO in severe renal IRI Anemia developed rapidly in the saline-treated rats that underwent severe IRI (Fig. 4). In contrast, Hct was significantly higher at 48 and 72 hours after IRI in EPO (500 U/kg)-treated rats compared with saline-treated rats (41.0 ⫾ 0.6 vs. 36.5 ⫾ 0.4, P ⬍ 0.01, N ⫽ 3 per group, and 42.3 ⫾ 1.2 vs. 36.5 ⫾ 0.4, P ⬍ 0.01, N ⫽ 3 per group). When higher doses of EPO (3000 U/kg) were administered to rats with 45 minutes of ischemia, no further increments in Hct were observed. However, at 48 hours postischemia, a more marked response in Hct
248
Nemoto et al: EPO therapy in ARF
Fig. 3. Hematocrits of EPO-treated (500 U/kg) and saline-treated groups that underwent moderate ischemic reperfusion injury (IRI; 30-minute clamp). Significant increases in Hct were observed at 48 and 72 hours after IRI in the EPO-treated group. Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3). **P ⬍ 0.01.
Fig. 5. Effect of erythropoietin dose on hematocrit (Hct) during severe renal ischemia. Hct was higher at 48 hours after IRI in the high-dose (3000 U/kg) EPO-treated rats compared with the lower-dose (500 U/kg) rats. Symbols are: (䊊) high-dose EPO-treated (3000 U/kg, N ⫽ 3); (䊐) high-dose EPO-treated (500 U/kg, N ⫽ 3); **P ⬍ 0.01.
Fig. 4. Hematocrits of erythropoietin-treated (500 U/kg) and salinetreated group. Both groups underwent severe IRI (45-minute clamp). Hct was significantly higher at 48 and 72 hours after IRI in EPO-treated rats compared with saline controls. Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 3); (䊉) Control (N ⫽ 3); **P ⬍ 0.01.
Fig. 6. Serum creatinine (SCr) value (mg/dL) in the EPO-treated (500 U/kg) and saline-treated groups. Both groups underwent moderate IRI (30 minutes). Baseline creatinine in both groups was ⬍0.3 mg/dL. No different was observed at any time point. Symbols are: (䊊) EPOtreated (500 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3).
was observed compared with the 500 U/kg dose (43.5 ⫾ 0.3 vs. 40.3 ⫾ 0.3, P ⬍ 0.01, N ⫽ 3 per group; Fig. 5).
Effect of EPO on body weight postischemia
Effect of EPO on serum creatinine postischemia Growth factor administration can enhance the resolution of experimental ARF [4]. Thus, EPO, whose receptors are found on tubular epithelial cells, may modulate renal tubular cell recovery after ischemia [2]. However, no effect of EPO was observed on either the rise or decline in SCr during moderate renal IRI (Fig. 6). Similarly, no EPO effect was observed on the course of SCr after severe IRI (Fig. 7). Even at higher doses (3000 U/kg), EPO lacked effect on the course of SCr during severe IRI (data not shown).
Erythropoietin can affect the hypothalamic-hormonal axis and may have many effects independent of merely correction of anemia, including reducing catabolism [5]. However, EPO treatment did not influence the loss of body weight during severe renal IRI (Fig. 8). EPO treatment during moderate IRI was also ineffective in mitigating the loss of body weight (data not shown). Effect of EPO on mortality after renal ischemia The high mortality associated with ARF continues to be a serious clinical problem [6]. Mortality rates were compared in EPO and untreated animals. All rats survived after moderate IRI. However, after severe IRI,
Nemoto et al: EPO therapy in ARF
Fig. 7. Serum creatinine (SCr) values in (mg/dL) EPO-treated (500 U/kg) and saline-treated groups after severe renal ischemia (45-min clamp). No difference was observed at any time point. No effect on SCr was seen even when higher dose EPO (3000 U/kg) was used (data not shown). Symbols are: (䊊) EPO-treated (500 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3).
Fig. 8. Change in body weight after severe renal ischemia. More severe weight loss is seen in the severe renal IRI compared with moderate IRI. However, no significant difference was seen in the saline-treated group compared with the EPO-treated group (500 U/kg). The higherdose EPO (3000 U/kg) group did not differ from saline group either (data not shown). Symbols are: (䊊) EPO-treated (3000 U/kg, N ⫽ 3); (䊉) control (N ⫽ 3).
substantial mortality ensues (Fig. 9). EPO treatment significantly improves survival when rats were followed 96 hours postischemia. Effect of EPO on reticulocyte count after renal ischemia Reticulocyte counts, reflective of erythrocyte production, were decreased 48 hours after 45 minutes of renal ischemia (7.5 ⫾ 0.7 vs. 5.6 ⫾ 0.4, P ⬍ 0.05). However, EPO treatment significantly increased the reticulocyte count during ARF (6.9 ⫾ 0.3, P ⬍ 0.05).
249
Fig. 9. Effect of EPO on mortality after IRI. Mortality was pronounced after severe (45 minutes) renal IRI. EPO (3000 U/kg) treatment significantly improved mortality (P ⬍ 0.05) after severe IRI. All rats survived after moderate IRI at 72 hours (data not shown). Symbols are: (䊊) EPO-treated (N ⫽ 19); (䊉) Control (N ⫽ 17).
DISCUSSION Anemia is often observed during ARF [7]. We hypothesized that recombinant EPO would be a useful therapy in ischemic ARF. We found that EPO can rapidly raise Hct during moderate and severe ischemic ARF in rats. The likely mechanism is by increasing erythrocyte production, as evidenced by an increased reticulocyte count after EPO treatment. Importantly, EPO treatment resulted in a significant improvement in mortality after severe ischemic injury. However, EPO administration did not significantly influence the course of the SCr or body weight. It is useful to compare the Hct responses in our studies to another model of ARF, cisplatin-induced ARF. In this model, EPO was administrated during cisplatin-induced ARF in rats [8]. There were no significantly differences in Hct between the two groups of rats on day 4. However, the EPO-treated group showed a significantly higher Hct level compared with untreated ARF group on day 9 after cisplatin. This cisplatin study also showed that renal function and body weight was improved in the EPOtreated group nine days after cisplatin [8]. In our current study using an IRI model of ARF, we found a much more rapid response after EPO, and no effect on body weight. However, cisplatin toxicity is likely to have additional effects, particularly on the bone marrow, than what is observed after IRI. The rapid response in Hct to EPO in experimental ARF that we observed was quite different from that seen in chronic renal failure. During chronic renal failure, EPO production is depressed with inappropriately low plasma levels throughout the uremic phase [9]. Many
250
Nemoto et al: EPO therapy in ARF
other factors contribute to anemia, including reduced red cell life span, iron deficiency, chronic inflammation, and contaminants of hemodialysis. The pathophysiology of anemia in ARF is likely to be complex. A significant reduction in serum haptoglobin and an increase in serum lactate dehydrogenase in ARF suggest that that hemolysis is a contributory factor in the initial fall in Hct in ARF [8]. Production of EPO has also been demonstrated to be reduced in rats and humans during ARF [10, 11]. The pathogenesis is not well understood. Other factors underlying the anemia include bleeding, hemolysis, blood sequestration, and resistance to EPO [7]. It is also possible that saline treatment during our surgical protocol may have contributed to a dilutional component to the anemia, but in this regard, all rats received similar quantities of saline. Despite the partial knowledge underlying the anemia of ARF, EPO therapy could still be a useful therapy in ARF as it is for CRF. We found that EPO at 500 U/kg for three consecutive doses increased Hct rapidly in rats with normal renal function. Previous studies have also looked at EPO’s effect in normal rats. EPO administered twice weekly (150 U/kg) had no significant effects on Hct after one week compared with controls [12]. In another study in which EPO was administrated to normal rats, a significant increase in Hct was observed at day 3 [13]. When the Hct response to EPO is rapid, changes can be detected early in reticulocytes. In a study in which EPO was administrated 250 U/kg and 1250 U/kg, an increase in reticulocytes began at 24 hours and reached the peak at 72 to 96 hours after injection of EPO [14]. In the current study, we did not specifically investigate the mechanisms for the anemia of ARF. However, the increase in reticulocyte count in the EPO-treated groups makes it likely that the effect we observed was based on increased red blood cell production. Alternatively, an early rise in reticulocyte count may also be due to early release of these cells from the bone marrow induced by EPO. We also tested the hypothesis that EPO treatment could potentially alter the course of ARF. This hypothesis was based on previous data in both the brain and kidney. EPO mRNA expression increases in rodent and primate brain after hypoxia [15]. EPO receptors are found in neural cell lines [15], neurons [16], and renal tubular epithelial cells [2]. Furthermore, EPO administration enhances proliferation of renal tubular epithelial cells [2], thought to be important in the recovery after renal IRI. In addition, EPO administration prevented ischemia-induced learning disability and rescued hippocampal neurons from ischemia [17], while infarct volume was improved in EPO-pretreated mice in cerebral ischemia [18]. Although renal dysfunction was not improved by EPO therapy in our current study, it did not worsen it either. We cannot exclude the possibility that given the short duration of our study, prolonged EPO treatment in
a protracted ARF model could have led to improvement in SCr. In a study of cis-platinum–induced ARF in which EPO was administered for nine consecutive days, renal functional changes were apparent after nine days but not at four days [19]. Given the high rate of mortality and catabolism associated with native kidney ARF [20], we were interested in the possible effect of EPO on mortality and body weight. It has been shown that that the administration of EPO provided a beneficial effect on the body weight, Hct, and healing rate in rats seven days following a large bowel operation [21]. In our current study, both in moderate and severe IRI, the decrease in body weights during ARF was not affected by EPO. However, a marked improvement in mortality was observed in EPO-treated rats after severe IRI. It is unclear as to the mechanisms of the decreased mortality. One possibility is that although there were no significant differences in SCr, more sensitive tests such as inulin clearances would have detected small increases in glomerular filtration rate (GFR) after EPO treatment. These small increases in GFR may have been adequate to improve mortality. Another possibility is that EPO may have reduced the lung dysfunction and systemic inflammatory state that has been associated with renal IRI [22]. Our current data demonstrate that EPO treatment during ARF rapidly improves Hct levels and could be a useful therapeutic approach, especially in the intensive care unit. In humans, at least one case report exists describing EPO therapy for anemia with ARF [23]. A properly designed clinical trial with an adequate number of patients will be required prior to justifying the routine use of EPO in patients with ARF. ACKNOWLEDGMENTS T.N. and N.Y. were supported by the Showa University, and H.R. was supported by a NKF Clinical Scientist Award, Hermundslie Trust and Minneapolis Medical Research Foundation. The authors thank Ms. Jan Lovick for assistance in manuscript preparation. Novel EPO indication submitted for patent. Reprint requests to Hamid Rabb, M.D., Nephrology, D-5, Hennepin County Medical Center, 701 Park Avenue, Minneapolis, Minnesota 55446, USA. E-mail: [email protected]
REFERENCES 1. Rainford DJ, Stevens PE: The investigative approach to the patient with acute renal failure, 8.1, in Oxford Textbook of Clinical Nephrology (vol 2), edited by Cameron S, Davison AM, Grunfeld J, et al: Oxford University Press, 1992, pp 969–982 2. Westenfelder C, Biddle DL, Baranowski RL: Human, rat, and mouse kidney cells express functional erythropoietin receptors. Kidney Int 55:808–820, 1999 3. Rabb H, Mendiola CC, Dietz J, et al: Role of CD11a and CD11b in ischemic acute renal failure in rats. Am J Physiol 267:F1052–F1058, 1994 4. Humes HD, MacKay SM, Funke AJ, et al: Acute renal failure:
Nemoto et al: EPO therapy in ARF
5.
6. 7.
8.
9.
10.
11.
12. 13.
14.
Growth factors, cell therapy, and gene therapy. Proc Assoc Am Physicians 109:547–557, 1997 Ramirez G, Bittle PA, Rabb H, et al: Effect of haemoglobin and endogenous erythropoietin on hypothalamic-pituitary thyroidal and gonadal secretion: An analysis of anemic (high EPO) polycythanemic (low EPO) patients. Clin Endocrinol (Oxf) 43:167–174, 1995 Star RA: Treatment of renal failure. Kidney Int 54:1817–1831, 1998 Steinman TI, Lazarus JM: Organ-system involvement in acute renal failure, in Acute Renal Failure (2nd ed), edited by Brenner BM, Lazarus JM, New York, Churchill Livingstone, 1988, pp 705–739 Baldwin MD, Zhou XJ, Ing TS, et al: Erythropoietin ameliorates anemia of cisplatin induced acute renal failure. ASAIO J 44:44–47, 1998 Zhang F, Laneuville P, Gagnon RF, et al: Effect of chronic renal failure on the expression of erythropoietin message in a murine model. Exp Hematol 24:1469–1474, 1996 Tan CC, Tan LH, Eckardt KU: Erythropoietin production in rats with post-ischemic acute renal failure. Kidney Int 50:1958–1964, 1996 Morgera S, Heering P, Szentandrasi T, et al: Erythropoietin in patients with acute renal failure and continuous veno-venous haemofiltration. Int Urol Nephrol 29:245–250, 1997 Vaziri ND, Zhou XJ, Smith J, et al: In vivo and in vitro pressor effects of erythropoietin in rats. Am J Physiol 269:F838–F845, 1995 Eggena P, Willsey P, Jamgotchian N, et al: Influence of recombinant human erythropoietin on blood pressure and tissue reninangiotensin systems. Am J Physiol 261:E642–E646, 1991 Masunaga H, Takahira R, Hirabayashi M, et al: Pharmacokinetics and the time course of pharmacodynamics of recombinant hu-
15. 16.
17. 18. 19. 20. 21.
22. 23.
251
man erythropoietin (SNB-5001). Nippon Yakurigaku Zasshi 99:213– 229, 1992 Masuda S, Nagao M, Takahata K, et al: Functional erythropoietin receptor of the cells with neural characteristics. J Biol Chem 268: 11208–11216, 1993 Morisita E, Masuda S, Nagao M, et al: Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 76:105–116, 1997 Sakanaka M, Wen TC, Matsuda S, et al: In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci USA 95:4635–4640, 1998 Bernaudin M, Marti HH, Roussel S, et al: A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab 19:643–651, 1999 Vaziri ND, Zhou XJ, Liao SY: Erythropoietin enhances recovery from cisplatin-induced acute renal failure. Am J Physiol 266:F360– F366, 1994 Price SR, Reaich D, Marinovic AC, et al: Mechanisms contributing to muscle-wasting in acute uremia: Activation of amino acid catabolism. J Am Soc Nephrol 9:439–443, 1998 Fatouros M, Dalekos GN, Mylonakis E, et al: Alterations in body weight, breaking strength, and wound healing in Wistar rats treated pre- and postoperatively with erythropoietin or granulocyte macrophage-colony stimulating factor: Evidence of a previously unknown anabolic effect of erythropoietin? J Lab Clin Med 133:253– 259, 1999 Kramer A, Postler G, Salhab K, et al: Renal ischemia/reperfusion leads to macrophage-mediated increase in pulmonary vascular permeability. Kidney Int 55:2362–2367, 1999 Walter J, Kun K, Doman J: Low-dose erythropoietin treatment of anemia associated with operative transfusion hemolysis and acute renal failure. Nephrol Dial Transplant 7:559–560, 1992