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Carbamylated erythropoietin protects the kidneys from ischemia-reperfusion injury without stimulating erythropoiesis
Biochemical and Biophysical Research Communications 353 (2007) 786–792 www.elsevier.com/locate/ybbrc
Carbamylated erythropoietin protects the kidneys from ischemia-reperfusion injury without stimulating erythropoiesis Ryoichi Imamura a, Yoshitaka Isaka b,*, Naotsugu Ichimaru a, Shiro Takahara b, Akihiko Okuyama a b
a Department of Urology, Osaka University Graduate School of Medicine, Suita, Japan Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Japan
Received 8 December 2006 Available online 22 December 2006
Abstract Several studies have shown that erythropoietin (EPO) can protect the kidneys from ischemia-reperfusion injury and can raise the hemoglobin (Hb) concentration. Recently, the EPO molecule modified by carbamylation (CEPO) has been identified and was demonstrated to be able to protect several organs without increasing the Hb concentration. We hypothesized that treatment with CEPO would protect the kidneys from tubular apoptosis and inhibit subsequent tubulointerstitial injury without erythropoiesis. The therapeutic effect of CEPO was evaluated using a rat ischemia-reperfusion injury model. Saline-treated kidneys exhibited increased tubular apoptosis with interstitial expression of a-smooth muscle actin (a-SMA), while EPO treatment inhibited tubular apoptosis and a-SMA expression to some extent. On the other hand, CEPO-treated kidneys showed minimal tubular apoptosis with limited expression of a-SMA. Moreover, CEPO significantly promoted tubular epithelial cell proliferation without erythropoiesis. In conclusion, we identified a new therapeutic approach using CEPO to protect kidneys from ischemia-reperfusion injury. 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Carbamylated erythropoietin; Ischemia-reperfusion injury; Kidney; Cell proliferation
Renal ischemia-reperfusion (I/R) injury, which is unavoidable in renal transplantation and is frequently associated with shock or surgery, is a major cause of acute renal failure [1]. Despite decades of laboratory and clinical investigations and the advent of renal replacement therapy, the overall mortality rate due to acute tubular necrosis has changed little. Recently, a broader concept of erythropoietin (EPO) as a tissue-protective molecule has emerged. EPO has been found to protect the brain and the spinal cord from ischemic injury [2,3], the peripheral nerve from diabetic damage [4,5], the kidney from ischemic [6,7] or toxic insults [8], and the heart from acute I/R injury [9,10]. The initial understanding of the biology of EPO-mediated tissue protection largely developed from the study of *
Corresponding author. Fax: +81 6 6879 3749. E-mail address: [email protected] (Y. Isaka).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.099
the nervous system, which is susceptible to ischemic injury due to its high basal metabolic rate. The findings derived from the nervous system studies are applicable to the kidney. The normal kidney, like the nervous system, is characterized by regions in which energy substrates are limited. Commonly, chronic renal hypoxia with subsequent tubulointerstitial injury leads to end-stage renal failure [11]. In contrast, early treatment with EPO slows the progression of renal failure [12]. In the kidney, a potential role for the non-hematopoietic activities of EPO was first suggested by the identification of the EPO receptor (EPO-R) protein that is expressed throughout the kidney, including both proximal and distal tubular cells [13]. However, the affinity of these receptors (1 nM) is well below the normal plasma EPO concentration (1–10 pM). Therefore, EPO’s cytoprotective effect may require higher doses than those used to treat anemia.
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However, recent clinical trials have suggested that higher doses of EPO are likely to be associated with adverse effects [14]. Furthermore, recent report demonstrated that highhemoglobin level in chronic kidney disease was associated with increased risk and no incremental improvement in the quality of life [15]. Recently, a second receptor for EPO that mediates EPO’s tissue protection has been identified as consisting of the EPO-R and the b-common (CD131) receptor (CbR). The EPO modified by carbamylation [carbamylated EPO (CEPO)] is reported to signal only through this receptor and not through the homodimeric EPO-R [16]. It has been shown that CEPO does not stimulate erythropoiesis, but that it prevents tissue injury in spinal cord neurons [16,17] and cardiomyocytes [18,19]. In this study, we examined whether treatment with CEPO could protect the kidney from tubular apoptosis that occurs after ischemiareperfusion injury and, thereby, inhibit subsequent tubulointerstitial fibrosis. Materials and methods Experimental design. Forty-two male Sprague–Dawley rats weighing 220–280 g were purchased from Japan SLC Inc. (Shizuoka, Japan) and were maintained under standard conditions until the experiments were done. All studies were performed in accordance with the principles of the Guideline on Animal Experimentation of Osaka University. The rats were randomly allocated into three groups: (1) the saline-treatment group (control group; n = 15); (2) the EPO-treatment group (EPO group; n = 15); and (3) the CEPO-treatment group (CEPO group; n = 12). Prior to I/R injury, control, EPO, and CEPO group rats received subcutaneous injections of 1 ml of saline, 100 IU/kg recombinant human EPO (Kirin Corp., Tokyo, Japan), and 100 IU/kg CEPO [16] every 2 days for 2 weeks (a total of six injections), respectively, because pre-treatment with EPO is clinically established in kidney transplantation. One day after the last injections, the rats were subjected to renal I/R injury. All rats were anesthetized with an intraperitoneal injection of sodium thiopentone (30 mg/kg). The animals were allowed to stabilize for 30 min (min) before they were subjected to 45 min of bilateral renal occlusion using artery clips to clamp the renal pedicles. Occlusion was confirmed visually by a change in the color of the kidneys to a paler shade. Reperfusion was initiated with the removal of the artery clips and was confirmed visually by noting a blush. The rats were sacrificed 24 hour (h), 72 h, and 1 week (wk) after reperfusion. Before the ischemic period and 24 h after reperfusion, 1 ml blood samples were collected from the anesthetized rats via their tail veins to measure hemoglobin and serum creatinine levels. Antibodies. To identify myofibroblasts, we used anti-human a-smooth muscle actin (a-SMA) antibody (EPOS System: Dako, Hamburg, Germany). To detect the pathways that protect the kidneys, we used the following antibodies for immunochemical testing: polyclonal Ki-67 antibody (1:200, Abcam, Cambridge, UK), monoclonal phosphatidylinositol-3 kinase (PI3K) p85a antibody (1:1000, Santa Cruz), polyclonal phospho-Akt (Ser473) antibody (1:1000, Cell Signaling Technology, Beverly, MA), polyclonal Akt antibody (1:1000, Cell Signaling), and polyclonal Ets-1 antibody (1:1000, Santa Cruz). We normalized protein levels with polyclonal b-actin antibody (1:1000, Cell Signaling). Morphology and immunohistochemical staining. Tissue samples were fixed in 4% (wt/vol) of buffered paraformaldehyde (PFA) for 16 h and then embedded in paraffin. Four micrometers of tissue sections were mounted on silane (2% 3-aminopropyltriethoxysilane)-coated slides (Muto Pure Chemicals, Tokyo, Japan) and deparaffinized with xylene. Immunohistochemical staining was done using the Envision system (Dako), according to the manufacturer’s instructions. Endogenous peroxidase activities were blocked with 3% H2O2 for 10 min. The first anti-
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bodies were diluted in 1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) with 0.1% Tween 20 (PBS-T) at specific concentrations as described above, and then incubated for 24 h at 4 C. This was followed by incubation with suitable secondary antibodies. Antigen retrieval was performed for 10 min in preheated 10 mmol/L sodium citrate (pH 7), using an autoclave. All incubations were performed in a humidified chamber. Chromogenic color was developed with 3,3 0 diaminobenzidine tetrahydrochloride (DAB; Dako). Negative controls, omitting the first antibodies, were carefully examined for each reaction. The nuclei were counterstained with hematoxylin. All histological slides were examined by light microscopy using a Nikon Eclipse 80 i (Nikon, Tokyo, Japan); pictures were taken with the Nikon ACT-1 ver. 2.63. The a-SMA-positive area relative to the total area of the field was calculated as a percentage by a computer-aided manipulator. Glomeruli and large vessels were not included in the microscope fields for image analysis. The scores of 10 fields per kidney were averaged, and the mean scores for each group were then averaged. For Ki-67 staining, the number of positive cell nuclei and the total numbers of cell nuclei stained with hematoxylin were counted in 10 random areas, and the percentages of the numbers of positive nuclei to the numbers of total cell nuclei were then compared. Terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining. TUNEL staining was performed using the in situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan), according to the manufacturer’s instructions. Briefly, the sections were deparaffinized and treated with antigen retrieval in preheated 10 mmol/L sodium citrate (pH 7), using a steamer for 40 min. They were then incubated with 3% H2O2 for 10 min, which was followed by incubation with TdT enzyme solution for 90 min at 37 C. The reaction was terminated by incubation in a stop/ wash buffer for 30 min at 37 C. The number of TUNEL-positive cell nuclei and the total numbers of cell nuclei stained with hematoxylin were counted in 10 random areas, and the percentages of the numbers of TUNEL-positive nuclei to the numbers of total cell nuclei were then calculated. Western blot Analysis. Kidney tissue was homogenized in a radioimmunoprecipitation (RIPA) Lysis Buffer with phenylmethylsulfonylfluoride (PMSF) solution, sodium orthovanadate solution, and protease inhibitor (Santa Cruz). Homogenates were centrifuged (12,000g for 10 min at 4 C), and the supernatant total protein was measured by the Lowry protein assay (Bio-Rad, Hercules, CA). Total protein lysate (15 lg) containing 1:1 denaturing sample buffer was boiled for 3 min and resolved on 5.0–10.0% SDS–polyacrylamide gels and electrophoretically transferred onto an immobilon PVDF membrane (Millipore, Bedford, MA). The filter was blocked with 5% (wt/vol) nonfat milk or 1% BSA in 10 mM Tris-buffered saline with 0.1% Tween 20 (TBS-T), followed by overnight incubation at 4 C with diluted primary antibodies in TBS-T or blocking buffer. After washing five times in TBS-T, the filter was incubated with secondary antibody (1:1000) (Cell Signaling) in TBS-T for 45 min at room temperature and developed to detect specific protein bands using ECL reagents (Amersham Bioscience Corp., Piscataway, NJ). The band density was analyzed by NIH image software. Statistical analysis. Data are expressed as means ± SD. Statistical significance, defined as p < 0.01 or <0.05, was evaluated using ANOVA.
Results Effect on erythropoiesis and renal function When compared to saline-treated rats, EPO-treatment (100 IU/kg · 3/wk · 2wk) significantly increased Hb concentration (saline, 13.5 ± 0.9 g/dl vs. EPO, 14.9 ± 1.0 g/ dl; p < 0.01). On the other hand, CEPO-treatment neither enhanced nor reduced Hb concentration (13.1 ± 0.2 g/dl; p = 0.73), suggesting that CEPO, unlike EPO, does not stimulate erythropoiesis. Saline-treated rats demonstrated
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the increment of serum creatinine (1.54 ± 0.68 mg/dl) 24 h after I/R injury, and EPO-treatment showed the tendency to suppress the increase of creatinine (1.25 ± 0.80 mg/dl; p = 0.096 vs. saline group), while CEPO-treatment significantly suppressed (0.53 ± 0.20 mg/dl; p < 0.05 vs. saline group). Effects on tubular apoptosis and proliferation in the I/R injury kidney To elucidate the protective mechanisms by which EPO or CEPO administration ameliorated tubular injury, we did TUNEL immunostaining to quantify the number of apoptotic cells. In the saline-treated I/R injury model rats, TUNEL-positive, apoptotic cells increased among the tubular epithelial cells at 24 h (TUNEL-positive cell number per field, 64.3 ± 10.8) (Fig. 1a), while TUNEL-positive, apoptotic cells were significantly decreased by EPO-treatment (5.28 ± 1.16) (Fig. 1b). Moreover, CEPO-treatment decreased the number of apoptotic cells (2.14 ± 0.23) significantly more than EPO-treatment, suggesting that CEPO has a greater anti-apoptotic role than EPO (Fig. 1c and d). Ki-67 antigen is a large nuclear protein that is preferentially expressed during the active phase of the cell cycle (G1, S, G2, and M phases), but is absent in resting cells (G0). To assess the regeneration of tubular epithelial cells, cortical Ki-67-positive tubular cells were counted at 400· magnification in a minimum of 10 fields. There were few Ki-67positive cells in saline-treated kidneys (2.2 ± 0.64% of
tubular cells at 24 h and 2.6 ± 0.48% at 1 wk) (Fig. 2a and d). On the other hand, there were significantly more Ki-67 cells in EPO-treated rats at 24 h (16 ± 2.7% of tubular cells at 24 h and 4.0 ± 1.2% at 1 wk; p < 0.01 vs. salinetreatment at 24 h) (Fig. 2b and e). CEPO-treatment further increased the Ki-67-positive cells (63 ± 13% of tubular cells at 24 h and 23 ± 6.5% at 1 wk; p < 0.01 vs. saline or EPOtreatment) (Fig. 2c and f). This suggests that CEPO is more likely to have a greater regenerative effect on tubular epithelial cells than EPO. Effect on cell signaling and Ets-1 expression To elucidate the intracellular erythropoietin signaling implicated in tubular protection, we examined the expression of PI3K and the activation of Akt (Fig. 3a–c). Western blot analysis demonstrated that treatment with EPO-significantly increased PI3K at 1 wk (p < 0.05 vs. saline group), but not at 24 h. However, treatment with CEPO induced marked expression of PI3K at 24 h (p < 0.01 vs. saline and EPO group), and this expression remained until 1 wk after I/R injury (Fig. 3a and b). Phosphorylation of Akt is dependent on PI3K expression and was markedly up-regulated in CEPO-treated kidneys 24 h after I/R injury (p < 0.01 vs. saline and EPO group); it remained activated until 1 wk after I/R injury. However, activation of Akt was weak in EPOtreated kidneys, confirming that CEPO up-regulated the cytoprotective signaling more strongly than EPO (Fig. 3a and c).
Fig. 1. Effects of EPO and CEPO on apoptosis at 24 h (400·). The dark brown dots correspond to representative TUNEL-positive nuclei. (a) control group, (b) EPO group, and (c) CEPO group. (d) Quantification (%) of TUNEL-positive cells in the kidney sections. Data shown are means ± SD for 10 independently performed experiments. **p < 0.01.
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Fig. 2. Immunohistochemical staining of Ki67 (400·). Control group (a,d), EPO group (b,e), CEPO group (c,f) at 24 h (a–c) and 1 wk (d–f). (g) Quantification (%) of Ki-67-positive cells in the kidney sections. White bars indicate 24 h after I/R injury and dark bars indicate 1 wk after I/R injury. Data shown are means ± SD for 10 independently performed experiments. **p < 0.01.
Fig. 3. Western blot analysis of the pathway of cell proliferation. (a) To elucidate the intracellular erythropoietin signaling implicated in tubular protection, we examined the expression of PI3K, Akt, and Ets-1 24 h and 1 wk after I/R injury. Relative protein levels of PI3K (b), p-Akt (c), and Ets-1 (d) were measured using NIH image. White bars indicate 24 h after I/R injury and dark bars indicate 1 wk after I/R injury. Data represent means ± SD. **p < 0.01, *p < 0.05.
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Fig. 4. Effect on phenotypic changes at 1 wk. Interstitial phenotypic changes are assessed by immunohistochemical staining of a-SMA in the control (a), EPO (b), and CEPO (c) groups. (d) Quantification (%) of the a-SMA-positive fields in the kidney sections. Data shown are means ± SD for 10 independently performed experiments. **p < 0.01.
Given that Ets-1, which is transiently up-regulated after I/R injury, has been reported to induce tubular regeneration [20], we also examined the expression of Ets-1 by Western blot analysis. Western blot analysis demonstrated that the administration of EPO slightly increased Ets-1 protein expression at 24 h, and dramatically up-regulated Ets-1 protein expression 1 wk after I/R injury (p < 0.01 vs. saline group). Consistent with the activation of Akt, CEPO induced Ets-1 expression at 24 h (p < 0.05 vs. saline group), and the up-regulation was sustained 1 wk after I/R injury (p < 0.01 vs. saline and EPO group) (Fig. 3a and d). Effects on interstitial phenotypic changes in the I/R injury kidney To detect interstitial myofibroblasts, which are associated with interstitial damage and fibrosis, the expression of a-SMA was examined immunohistochemically. The interstitial expression of a-SMA increased 1 wk after I/R injury in the saline-treated rats (Fig. 4a), while EPO-treatment significantly suppressed interstitial expression of a-SMA (Fig. 4b). CEPO-treatment suppressed interstitial expression of a-SMA even further so that the expression of a-SMA was limited to the blood vessels (Fig. 4c). Discussion In this paper, we examined whether EPO or CEPO may have therapeutic effects on tubulointerstitial injury in a rat model of I/R injury. Untreated kidneys exhibited increased tubular apoptosis and interstitial a-SMA expression, while
EPO-treatment inhibited tubular apoptosis and a-SMA expression to some extent. On the other hand, CEPO-treated kidneys showed minimal tubular apoptosis and limited a-SMA expression. In addition, CEPO administration did not increase the Hb concentration, while EPO-treatment increased the Hb concentration. Based on our results, both EPO and CEPO appear to have a protective function against I/R injury; however, CEPO has more protective effects against I/R injury to tubular epithelial cells than EPO. Several key findings support this conclusion. (1) There were fewer TUNEL-positive, apoptotic cells among the tubular epithelial cells in CEPO-treated kidneys than in EPO-treated kidneys. (2) Compared to EPO, CEPO significantly promoted tubular epithelial cell proliferation assessed by Ki-67 staining. (3) While EPO-treatment significantly suppressed the interstitial phenotypic alteration assessed by a-SMA expression, CEPO-treatment suppressed this even further, so that interstitial a-SMA expression was not observed. (4) CEPO-treatment significantly suppressed the increase of serum creatinine 24 h after I/R injury. These observations suggest that CEPO has a greater therapeutic value in I/R injury than EPO. We demonstrated that CEPO-treatment markedly suppressed I/R injury-induced tubular epithelial apoptosis compared to EPO-treatment. Furthermore, though EPO-treatment significantly suppressed the interstitial phenotypic alteration assessed by a-SMA expression, CEPOtreatment further suppressed a-SMA expression, so that there was no interstitial expression of a-SMA. One possible signal transduction pathway by which tubular
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apoptosis, as well as later interstitial phenotypic changes, could be suppressed may involve the activation of PI3K. In fact, on Western blot analysis, we found that EPOtreatment increased PI3K at 1 wk, but not at 24 h. However, CEPO-treatment induced marked expression of PI3K at 24 h, which continued until 1 wk after I/R injury. Thus, phosphorylation of Akt was markedly up-regulated in CEPO-treated kidneys 24 h after I/R injury and remained activated until 1 wk. However, activation of Akt was weak in EPO-treated kidneys. Kashii et al. [21] reported that PI3K is activated by EPO in the EPO-dependent UT-7 leukemia cell line, where it recruits Akt. The PI3K-Akt pathway also leads to the up-regulation of Bcl-xL and the inhibition of apoptosis in Baf-3 cells [22]. Furthermore, using the EPO-dependent human erythroid progenitor cell line, Silva et al. [23] showed that EPO-treatment maintained the cells’ viability by repressing apoptosis through up-regulation of Bcl-xL, an antiapoptotic gene of the Bcl-2 family. These results suggest that the earlier upregulation of PI3K-Akt pathway in CEPO-treated kidneys suppressed tubular epithelial apoptosis, likely due to the induction of the anti-apoptotic genes of the Bcl-2 family. As well, CEPO was found to significantly increase the number of proliferating tubular epithelial cells as assessed by Ki-67 expression. Tanaka et al. [20] reported that Ets1 expression is up-regulated in the early phase of ischemic acute renal failure; Ets-1 expression was localized exclusively in the PCNA-positive regenerative proximal tubules, suggesting that Ets-1 protein may induce the transformation of regenerative renal tubular cells. In our study, compared to EPO-treatment, CEPO-treatment significantly increased the number of Ets-1-positive tubular epithelial cells. In fact, CEPO-treatment was found to further increase the number of Ki-67-positive regenerative tubular epithelial cells more than EPO-treatment. In a recent paper, Oikawa et al. [24] reported that hypoxia induced Ets-1 via the activity of HIF-1a. In their study, the Ets-1 promoter contained a hypoxia-responsive element-like sequence, and HIF-1a bound to it under hypoxic conditions. Given that we have documented the up-regulation of HIF-1a by EPO or CEPO treatment (unpublished data), it is likely that EPO or CEPO induced Ets-1 in tubular epithelial cells and, thus, promoted tubular proliferation via HIF-1a expression. In conclusion, CEPO can protect the kidneys from ischemia-reperfusion injury; thus, the therapeutic use of CEPO warrants further attention and preclinical studies.
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Carbamylated erythropoietin protects the kidneys from ischemia-reperfusion injury without stimulating erythropoiesis Ryoichi Imamura a, Yoshitaka Isaka b,*, Naotsugu Ichimaru a, Shiro Takahara b, Akihiko Okuyama a b
a Department of Urology, Osaka University Graduate School of Medicine, Suita, Japan Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Japan
Received 8 December 2006 Available online 22 December 2006
Abstract Several studies have shown that erythropoietin (EPO) can protect the kidneys from ischemia-reperfusion injury and can raise the hemoglobin (Hb) concentration. Recently, the EPO molecule modified by carbamylation (CEPO) has been identified and was demonstrated to be able to protect several organs without increasing the Hb concentration. We hypothesized that treatment with CEPO would protect the kidneys from tubular apoptosis and inhibit subsequent tubulointerstitial injury without erythropoiesis. The therapeutic effect of CEPO was evaluated using a rat ischemia-reperfusion injury model. Saline-treated kidneys exhibited increased tubular apoptosis with interstitial expression of a-smooth muscle actin (a-SMA), while EPO treatment inhibited tubular apoptosis and a-SMA expression to some extent. On the other hand, CEPO-treated kidneys showed minimal tubular apoptosis with limited expression of a-SMA. Moreover, CEPO significantly promoted tubular epithelial cell proliferation without erythropoiesis. In conclusion, we identified a new therapeutic approach using CEPO to protect kidneys from ischemia-reperfusion injury. 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Carbamylated erythropoietin; Ischemia-reperfusion injury; Kidney; Cell proliferation
Renal ischemia-reperfusion (I/R) injury, which is unavoidable in renal transplantation and is frequently associated with shock or surgery, is a major cause of acute renal failure [1]. Despite decades of laboratory and clinical investigations and the advent of renal replacement therapy, the overall mortality rate due to acute tubular necrosis has changed little. Recently, a broader concept of erythropoietin (EPO) as a tissue-protective molecule has emerged. EPO has been found to protect the brain and the spinal cord from ischemic injury [2,3], the peripheral nerve from diabetic damage [4,5], the kidney from ischemic [6,7] or toxic insults [8], and the heart from acute I/R injury [9,10]. The initial understanding of the biology of EPO-mediated tissue protection largely developed from the study of *
Corresponding author. Fax: +81 6 6879 3749. E-mail address: [email protected] (Y. Isaka).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.099
the nervous system, which is susceptible to ischemic injury due to its high basal metabolic rate. The findings derived from the nervous system studies are applicable to the kidney. The normal kidney, like the nervous system, is characterized by regions in which energy substrates are limited. Commonly, chronic renal hypoxia with subsequent tubulointerstitial injury leads to end-stage renal failure [11]. In contrast, early treatment with EPO slows the progression of renal failure [12]. In the kidney, a potential role for the non-hematopoietic activities of EPO was first suggested by the identification of the EPO receptor (EPO-R) protein that is expressed throughout the kidney, including both proximal and distal tubular cells [13]. However, the affinity of these receptors (1 nM) is well below the normal plasma EPO concentration (1–10 pM). Therefore, EPO’s cytoprotective effect may require higher doses than those used to treat anemia.
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However, recent clinical trials have suggested that higher doses of EPO are likely to be associated with adverse effects [14]. Furthermore, recent report demonstrated that highhemoglobin level in chronic kidney disease was associated with increased risk and no incremental improvement in the quality of life [15]. Recently, a second receptor for EPO that mediates EPO’s tissue protection has been identified as consisting of the EPO-R and the b-common (CD131) receptor (CbR). The EPO modified by carbamylation [carbamylated EPO (CEPO)] is reported to signal only through this receptor and not through the homodimeric EPO-R [16]. It has been shown that CEPO does not stimulate erythropoiesis, but that it prevents tissue injury in spinal cord neurons [16,17] and cardiomyocytes [18,19]. In this study, we examined whether treatment with CEPO could protect the kidney from tubular apoptosis that occurs after ischemiareperfusion injury and, thereby, inhibit subsequent tubulointerstitial fibrosis. Materials and methods Experimental design. Forty-two male Sprague–Dawley rats weighing 220–280 g were purchased from Japan SLC Inc. (Shizuoka, Japan) and were maintained under standard conditions until the experiments were done. All studies were performed in accordance with the principles of the Guideline on Animal Experimentation of Osaka University. The rats were randomly allocated into three groups: (1) the saline-treatment group (control group; n = 15); (2) the EPO-treatment group (EPO group; n = 15); and (3) the CEPO-treatment group (CEPO group; n = 12). Prior to I/R injury, control, EPO, and CEPO group rats received subcutaneous injections of 1 ml of saline, 100 IU/kg recombinant human EPO (Kirin Corp., Tokyo, Japan), and 100 IU/kg CEPO [16] every 2 days for 2 weeks (a total of six injections), respectively, because pre-treatment with EPO is clinically established in kidney transplantation. One day after the last injections, the rats were subjected to renal I/R injury. All rats were anesthetized with an intraperitoneal injection of sodium thiopentone (30 mg/kg). The animals were allowed to stabilize for 30 min (min) before they were subjected to 45 min of bilateral renal occlusion using artery clips to clamp the renal pedicles. Occlusion was confirmed visually by a change in the color of the kidneys to a paler shade. Reperfusion was initiated with the removal of the artery clips and was confirmed visually by noting a blush. The rats were sacrificed 24 hour (h), 72 h, and 1 week (wk) after reperfusion. Before the ischemic period and 24 h after reperfusion, 1 ml blood samples were collected from the anesthetized rats via their tail veins to measure hemoglobin and serum creatinine levels. Antibodies. To identify myofibroblasts, we used anti-human a-smooth muscle actin (a-SMA) antibody (EPOS System: Dako, Hamburg, Germany). To detect the pathways that protect the kidneys, we used the following antibodies for immunochemical testing: polyclonal Ki-67 antibody (1:200, Abcam, Cambridge, UK), monoclonal phosphatidylinositol-3 kinase (PI3K) p85a antibody (1:1000, Santa Cruz), polyclonal phospho-Akt (Ser473) antibody (1:1000, Cell Signaling Technology, Beverly, MA), polyclonal Akt antibody (1:1000, Cell Signaling), and polyclonal Ets-1 antibody (1:1000, Santa Cruz). We normalized protein levels with polyclonal b-actin antibody (1:1000, Cell Signaling). Morphology and immunohistochemical staining. Tissue samples were fixed in 4% (wt/vol) of buffered paraformaldehyde (PFA) for 16 h and then embedded in paraffin. Four micrometers of tissue sections were mounted on silane (2% 3-aminopropyltriethoxysilane)-coated slides (Muto Pure Chemicals, Tokyo, Japan) and deparaffinized with xylene. Immunohistochemical staining was done using the Envision system (Dako), according to the manufacturer’s instructions. Endogenous peroxidase activities were blocked with 3% H2O2 for 10 min. The first anti-
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bodies were diluted in 1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) with 0.1% Tween 20 (PBS-T) at specific concentrations as described above, and then incubated for 24 h at 4 C. This was followed by incubation with suitable secondary antibodies. Antigen retrieval was performed for 10 min in preheated 10 mmol/L sodium citrate (pH 7), using an autoclave. All incubations were performed in a humidified chamber. Chromogenic color was developed with 3,3 0 diaminobenzidine tetrahydrochloride (DAB; Dako). Negative controls, omitting the first antibodies, were carefully examined for each reaction. The nuclei were counterstained with hematoxylin. All histological slides were examined by light microscopy using a Nikon Eclipse 80 i (Nikon, Tokyo, Japan); pictures were taken with the Nikon ACT-1 ver. 2.63. The a-SMA-positive area relative to the total area of the field was calculated as a percentage by a computer-aided manipulator. Glomeruli and large vessels were not included in the microscope fields for image analysis. The scores of 10 fields per kidney were averaged, and the mean scores for each group were then averaged. For Ki-67 staining, the number of positive cell nuclei and the total numbers of cell nuclei stained with hematoxylin were counted in 10 random areas, and the percentages of the numbers of positive nuclei to the numbers of total cell nuclei were then compared. Terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining. TUNEL staining was performed using the in situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan), according to the manufacturer’s instructions. Briefly, the sections were deparaffinized and treated with antigen retrieval in preheated 10 mmol/L sodium citrate (pH 7), using a steamer for 40 min. They were then incubated with 3% H2O2 for 10 min, which was followed by incubation with TdT enzyme solution for 90 min at 37 C. The reaction was terminated by incubation in a stop/ wash buffer for 30 min at 37 C. The number of TUNEL-positive cell nuclei and the total numbers of cell nuclei stained with hematoxylin were counted in 10 random areas, and the percentages of the numbers of TUNEL-positive nuclei to the numbers of total cell nuclei were then calculated. Western blot Analysis. Kidney tissue was homogenized in a radioimmunoprecipitation (RIPA) Lysis Buffer with phenylmethylsulfonylfluoride (PMSF) solution, sodium orthovanadate solution, and protease inhibitor (Santa Cruz). Homogenates were centrifuged (12,000g for 10 min at 4 C), and the supernatant total protein was measured by the Lowry protein assay (Bio-Rad, Hercules, CA). Total protein lysate (15 lg) containing 1:1 denaturing sample buffer was boiled for 3 min and resolved on 5.0–10.0% SDS–polyacrylamide gels and electrophoretically transferred onto an immobilon PVDF membrane (Millipore, Bedford, MA). The filter was blocked with 5% (wt/vol) nonfat milk or 1% BSA in 10 mM Tris-buffered saline with 0.1% Tween 20 (TBS-T), followed by overnight incubation at 4 C with diluted primary antibodies in TBS-T or blocking buffer. After washing five times in TBS-T, the filter was incubated with secondary antibody (1:1000) (Cell Signaling) in TBS-T for 45 min at room temperature and developed to detect specific protein bands using ECL reagents (Amersham Bioscience Corp., Piscataway, NJ). The band density was analyzed by NIH image software. Statistical analysis. Data are expressed as means ± SD. Statistical significance, defined as p < 0.01 or <0.05, was evaluated using ANOVA.
Results Effect on erythropoiesis and renal function When compared to saline-treated rats, EPO-treatment (100 IU/kg · 3/wk · 2wk) significantly increased Hb concentration (saline, 13.5 ± 0.9 g/dl vs. EPO, 14.9 ± 1.0 g/ dl; p < 0.01). On the other hand, CEPO-treatment neither enhanced nor reduced Hb concentration (13.1 ± 0.2 g/dl; p = 0.73), suggesting that CEPO, unlike EPO, does not stimulate erythropoiesis. Saline-treated rats demonstrated
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the increment of serum creatinine (1.54 ± 0.68 mg/dl) 24 h after I/R injury, and EPO-treatment showed the tendency to suppress the increase of creatinine (1.25 ± 0.80 mg/dl; p = 0.096 vs. saline group), while CEPO-treatment significantly suppressed (0.53 ± 0.20 mg/dl; p < 0.05 vs. saline group). Effects on tubular apoptosis and proliferation in the I/R injury kidney To elucidate the protective mechanisms by which EPO or CEPO administration ameliorated tubular injury, we did TUNEL immunostaining to quantify the number of apoptotic cells. In the saline-treated I/R injury model rats, TUNEL-positive, apoptotic cells increased among the tubular epithelial cells at 24 h (TUNEL-positive cell number per field, 64.3 ± 10.8) (Fig. 1a), while TUNEL-positive, apoptotic cells were significantly decreased by EPO-treatment (5.28 ± 1.16) (Fig. 1b). Moreover, CEPO-treatment decreased the number of apoptotic cells (2.14 ± 0.23) significantly more than EPO-treatment, suggesting that CEPO has a greater anti-apoptotic role than EPO (Fig. 1c and d). Ki-67 antigen is a large nuclear protein that is preferentially expressed during the active phase of the cell cycle (G1, S, G2, and M phases), but is absent in resting cells (G0). To assess the regeneration of tubular epithelial cells, cortical Ki-67-positive tubular cells were counted at 400· magnification in a minimum of 10 fields. There were few Ki-67positive cells in saline-treated kidneys (2.2 ± 0.64% of
tubular cells at 24 h and 2.6 ± 0.48% at 1 wk) (Fig. 2a and d). On the other hand, there were significantly more Ki-67 cells in EPO-treated rats at 24 h (16 ± 2.7% of tubular cells at 24 h and 4.0 ± 1.2% at 1 wk; p < 0.01 vs. salinetreatment at 24 h) (Fig. 2b and e). CEPO-treatment further increased the Ki-67-positive cells (63 ± 13% of tubular cells at 24 h and 23 ± 6.5% at 1 wk; p < 0.01 vs. saline or EPOtreatment) (Fig. 2c and f). This suggests that CEPO is more likely to have a greater regenerative effect on tubular epithelial cells than EPO. Effect on cell signaling and Ets-1 expression To elucidate the intracellular erythropoietin signaling implicated in tubular protection, we examined the expression of PI3K and the activation of Akt (Fig. 3a–c). Western blot analysis demonstrated that treatment with EPO-significantly increased PI3K at 1 wk (p < 0.05 vs. saline group), but not at 24 h. However, treatment with CEPO induced marked expression of PI3K at 24 h (p < 0.01 vs. saline and EPO group), and this expression remained until 1 wk after I/R injury (Fig. 3a and b). Phosphorylation of Akt is dependent on PI3K expression and was markedly up-regulated in CEPO-treated kidneys 24 h after I/R injury (p < 0.01 vs. saline and EPO group); it remained activated until 1 wk after I/R injury. However, activation of Akt was weak in EPOtreated kidneys, confirming that CEPO up-regulated the cytoprotective signaling more strongly than EPO (Fig. 3a and c).
Fig. 1. Effects of EPO and CEPO on apoptosis at 24 h (400·). The dark brown dots correspond to representative TUNEL-positive nuclei. (a) control group, (b) EPO group, and (c) CEPO group. (d) Quantification (%) of TUNEL-positive cells in the kidney sections. Data shown are means ± SD for 10 independently performed experiments. **p < 0.01.
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Fig. 2. Immunohistochemical staining of Ki67 (400·). Control group (a,d), EPO group (b,e), CEPO group (c,f) at 24 h (a–c) and 1 wk (d–f). (g) Quantification (%) of Ki-67-positive cells in the kidney sections. White bars indicate 24 h after I/R injury and dark bars indicate 1 wk after I/R injury. Data shown are means ± SD for 10 independently performed experiments. **p < 0.01.
Fig. 3. Western blot analysis of the pathway of cell proliferation. (a) To elucidate the intracellular erythropoietin signaling implicated in tubular protection, we examined the expression of PI3K, Akt, and Ets-1 24 h and 1 wk after I/R injury. Relative protein levels of PI3K (b), p-Akt (c), and Ets-1 (d) were measured using NIH image. White bars indicate 24 h after I/R injury and dark bars indicate 1 wk after I/R injury. Data represent means ± SD. **p < 0.01, *p < 0.05.
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Fig. 4. Effect on phenotypic changes at 1 wk. Interstitial phenotypic changes are assessed by immunohistochemical staining of a-SMA in the control (a), EPO (b), and CEPO (c) groups. (d) Quantification (%) of the a-SMA-positive fields in the kidney sections. Data shown are means ± SD for 10 independently performed experiments. **p < 0.01.
Given that Ets-1, which is transiently up-regulated after I/R injury, has been reported to induce tubular regeneration [20], we also examined the expression of Ets-1 by Western blot analysis. Western blot analysis demonstrated that the administration of EPO slightly increased Ets-1 protein expression at 24 h, and dramatically up-regulated Ets-1 protein expression 1 wk after I/R injury (p < 0.01 vs. saline group). Consistent with the activation of Akt, CEPO induced Ets-1 expression at 24 h (p < 0.05 vs. saline group), and the up-regulation was sustained 1 wk after I/R injury (p < 0.01 vs. saline and EPO group) (Fig. 3a and d). Effects on interstitial phenotypic changes in the I/R injury kidney To detect interstitial myofibroblasts, which are associated with interstitial damage and fibrosis, the expression of a-SMA was examined immunohistochemically. The interstitial expression of a-SMA increased 1 wk after I/R injury in the saline-treated rats (Fig. 4a), while EPO-treatment significantly suppressed interstitial expression of a-SMA (Fig. 4b). CEPO-treatment suppressed interstitial expression of a-SMA even further so that the expression of a-SMA was limited to the blood vessels (Fig. 4c). Discussion In this paper, we examined whether EPO or CEPO may have therapeutic effects on tubulointerstitial injury in a rat model of I/R injury. Untreated kidneys exhibited increased tubular apoptosis and interstitial a-SMA expression, while
EPO-treatment inhibited tubular apoptosis and a-SMA expression to some extent. On the other hand, CEPO-treated kidneys showed minimal tubular apoptosis and limited a-SMA expression. In addition, CEPO administration did not increase the Hb concentration, while EPO-treatment increased the Hb concentration. Based on our results, both EPO and CEPO appear to have a protective function against I/R injury; however, CEPO has more protective effects against I/R injury to tubular epithelial cells than EPO. Several key findings support this conclusion. (1) There were fewer TUNEL-positive, apoptotic cells among the tubular epithelial cells in CEPO-treated kidneys than in EPO-treated kidneys. (2) Compared to EPO, CEPO significantly promoted tubular epithelial cell proliferation assessed by Ki-67 staining. (3) While EPO-treatment significantly suppressed the interstitial phenotypic alteration assessed by a-SMA expression, CEPO-treatment suppressed this even further, so that interstitial a-SMA expression was not observed. (4) CEPO-treatment significantly suppressed the increase of serum creatinine 24 h after I/R injury. These observations suggest that CEPO has a greater therapeutic value in I/R injury than EPO. We demonstrated that CEPO-treatment markedly suppressed I/R injury-induced tubular epithelial apoptosis compared to EPO-treatment. Furthermore, though EPO-treatment significantly suppressed the interstitial phenotypic alteration assessed by a-SMA expression, CEPOtreatment further suppressed a-SMA expression, so that there was no interstitial expression of a-SMA. One possible signal transduction pathway by which tubular
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apoptosis, as well as later interstitial phenotypic changes, could be suppressed may involve the activation of PI3K. In fact, on Western blot analysis, we found that EPOtreatment increased PI3K at 1 wk, but not at 24 h. However, CEPO-treatment induced marked expression of PI3K at 24 h, which continued until 1 wk after I/R injury. Thus, phosphorylation of Akt was markedly up-regulated in CEPO-treated kidneys 24 h after I/R injury and remained activated until 1 wk. However, activation of Akt was weak in EPO-treated kidneys. Kashii et al. [21] reported that PI3K is activated by EPO in the EPO-dependent UT-7 leukemia cell line, where it recruits Akt. The PI3K-Akt pathway also leads to the up-regulation of Bcl-xL and the inhibition of apoptosis in Baf-3 cells [22]. Furthermore, using the EPO-dependent human erythroid progenitor cell line, Silva et al. [23] showed that EPO-treatment maintained the cells’ viability by repressing apoptosis through up-regulation of Bcl-xL, an antiapoptotic gene of the Bcl-2 family. These results suggest that the earlier upregulation of PI3K-Akt pathway in CEPO-treated kidneys suppressed tubular epithelial apoptosis, likely due to the induction of the anti-apoptotic genes of the Bcl-2 family. As well, CEPO was found to significantly increase the number of proliferating tubular epithelial cells as assessed by Ki-67 expression. Tanaka et al. [20] reported that Ets1 expression is up-regulated in the early phase of ischemic acute renal failure; Ets-1 expression was localized exclusively in the PCNA-positive regenerative proximal tubules, suggesting that Ets-1 protein may induce the transformation of regenerative renal tubular cells. In our study, compared to EPO-treatment, CEPO-treatment significantly increased the number of Ets-1-positive tubular epithelial cells. In fact, CEPO-treatment was found to further increase the number of Ki-67-positive regenerative tubular epithelial cells more than EPO-treatment. In a recent paper, Oikawa et al. [24] reported that hypoxia induced Ets-1 via the activity of HIF-1a. In their study, the Ets-1 promoter contained a hypoxia-responsive element-like sequence, and HIF-1a bound to it under hypoxic conditions. Given that we have documented the up-regulation of HIF-1a by EPO or CEPO treatment (unpublished data), it is likely that EPO or CEPO induced Ets-1 in tubular epithelial cells and, thus, promoted tubular proliferation via HIF-1a expression. In conclusion, CEPO can protect the kidneys from ischemia-reperfusion injury; thus, the therapeutic use of CEPO warrants further attention and preclinical studies.
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