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NDT Advance Access originally published online on January 14, 2008
Nephrology Dialysis Transplantation 2008 23(5):1521-1528; doi:10.1093/ndt/gfm842
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Nonerythropoietic derivative of erythropoietin protects against tubulointerstitial injury in a unilateral ureteral obstruction model

Harumi Kitamura1, Yoshitaka Isaka1,2, Yoshitsugu Takabatake1, Ryoichi Imamura3, Chigure Suzuki1, Shiro Takahara2 and Enyu Imai1

1 Department of Nephrology 2 Department of Advanced Technology for Transplantation, 3 Department of Urology, Osaka University Graduate School of Medicine, Osaka, Japan

Correspondence and offprint requests to: Yoshitaka Isaka, Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871 Japan. Tel: +81-6-6879-3746; Fax: +81-6-6879-3749; E-mail: isaka{at}att.med.osaka-u.ac.jp



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Erythropoietin (EPO), a member of the cytokine type I superfamily, acts to increase circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors, is known to protect tissues and can raise haemoglobin (Hb) concentrations. Recently, a second receptor for EPO comprising the EPO receptor and β-common receptor has been reported to mediate EPO-induced tissue protection. EPO modified by carbamylation (CEPO) only signals through this second receptor. Accordingly, we hypothesized that treatment with CEPO, which would not increase Hb concentrations, would protect against tubular damage and thereby inhibit tubulointerstitial injuries.

Methods. We evaluated therapeutic effects of CEPO using a rat unilateral ureteral obstruction model.

Results. CEPO decreased tubular apoptosis and {alpha}-smooth muscle actin ({alpha}SMA) expression in the absence of polycythaemia, while the untreated obstructed kidneys exhibited increased tubular apoptosis with expanded ({alpha}SMA) expression. While EPO treatment similarly inhibited tubular apoptosis and {alpha}SMA expression, EPO treatment increased Hb concentrations and induced a wedge-shaped infarction.

Conclusion. We established a therapeutic approach using CEPO to protect against tubulointerstitial injury. The therapeutic value of this approach warrants further attention and preclinical studies.

Keywords: apoptosis; carbamylated erythropoietin; tubulointerstitial injury; ureteral obstruction



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Tubulointerstitial inflammation is a common pathological feature of progressive renal diseases [1–3] that leads to fibrosis via tubular atrophy, myofibroblast proliferation, macrophage infiltration and interstitial matrix accumulation [4–7]. Unilateral ureteral obstruction (UUO), a representative model of tubulointerstitial renal fibrosis, has numerous quantifiable cellular and molecular events, such as apoptosis and phenotypic alteration [5]. In progressive obstructive nephropathy, the tubules dilate and the renal tubular epithelial cells undergo apoptosis, leading to tubular atrophy.

Preliminary knowledge of the biology of erythropoietin (EPO)-mediated tissue protection was largely developed from studies of the nervous system [8,9], which is susceptible to ischaemic injury due to its high basal metabolic rate. As the normal kidney, like the nervous system, is characterized by regions in which energy substrates are limited, findings derived from nervous system studies are applicable to the kidney. Although chronic renal hypoxia with subsequent tubulointerstitial injury commonly leads to end-stage renal failure [10], early treatment with EPO slows the progression of renal failure [11]. On the other hand, EPO administration to rats with chronic renal failure was shown to accelerate the progression of chronic renal disease, especially in relation to the increased blood pressure [12].

A potential role for the non-haematopoietic activities of EPO in the kidney was suggested by the identification of the EPO receptor (EpoR) protein expression throughout the kidney, including both proximal and distal tubular cells [13]. As the affinity of these receptors (~1 nM) is well below the normal plasma EPO concentrations (~1–10 pM), the cytoprotective effects of EPO may require higher doses than those used to treat anaemia. However, recent clinical trials have suggested that higher doses of EPO are likely to be associated with adverse effects [14–17].

Recently, a second receptor that mediates EPO-induced tissue protection for EPO comprising the EpoR and the β-common (CD131) receptor (βCR) has been identified [18]. EPO modified by carbamylation, carbamylated EPO (CEPO), is reported to signal only through this receptor, not through the homodimeric EpoR [17]. It has been shown that CEPO does not stimulate erythropoiesis, but that it prevents tissue injury in spinal cord neurons [17,19] and cardiomyocytes [18,20]. The results that membrane proteins prepared from the rat kidney were greatly enriched in the EpoR covalently bound in a complex with βcR suggest that the EpoR physically interacts with βcR in the kidney [18]. In this study, we examined whether treatment with CEPO is able to protect the kidney from the tubular apoptosis that typically occurs after unilateral obstruction injury, thereby inhibiting subsequent tubulointerstitial fibrosis.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Experimental design
CEPO was synthesized from human EPO (Epoetin alfa; Kirin Corp., Tokyo, Japan) as described earlier [19]. Briefly, one volume of EPO (~1 mg/ml) was mixed with one volume of 1 M Na-borate (pH –8.8) and recrystallized KOCN was added to a final concentration of 1 M. After incubation, samples were immediately dialysed against milli-Q water, and subsequently against 20 mM sodium citrate in 0.1 M NaCl, pH 6.0. After dialysis, the samples were concentrated to ~2 mg/ml.

The therapeutic effects of CEPO on interstitial fibrosis were examined using a UUO model [5]. All procedures were handled in a humane fashion in accordance with the guidelines of the Animal Committee of Osaka University. Six-week-old male Sprague Dawley (SD, SLC Inc., Hamamatsu, Japan) rats were anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg), the left kidney and ureter were surgically exposed using a mid-line incision and the left proximal ureter was ligated with silk thread. Rats were randomly divided into three groups: (1) the saline-treatment group (control group; n = 9); (2) the EPO-treatment group (EPO group) and (3) the CEPO-treatment group (CEPO group). Rats with sham operation served as normal control (sham ope group). Control, EPO and CEPO group rats received subcutaneous injections of 0.5 ml of saline, 500 IU/kg (low dose; n = 6) or 1000 IU/kg (high dose; n = 11) recombinant human Epoetin alfa (Kirin Corp., Tokyo, Japan) in 0.5 ml of saline, or 500 IU/kg (low dose; n = 6) or 1000 IU/kg (high dose; n = 11) CEPO in 0.5 ml of saline [17], respectively, every 2 days after disease induction. In order to examine the effect of EPO or CEPO (high dose) treatment on blood pressure, three rats from each group were selected and blood pressures were monitored 1 day before the disease induction and 1 day before the harvest using a tail cuff and a pneumatic pulse transducer (BP-98 A; Softron, Tokyo, Japan). On day 7, kidneys were perfused with cold autoclaved PBS and samples of the cortex were taken for RNA preparation and histology. Tissues for RNA extraction were frozen using liquid nitrogen and homogenized with acid-guanidium thiocyanate-phenol-chloroform. Tissues for light microscopy were fixed with 4% paraformaldehyde overnight, dehydrated through a graded ethanol series and embedded in paraffin. Histological sections (2 µm) of the kidneys were stained using Masson's tricrome method. The fibrotic area in the interstitium, stained blue by Masson's Trichrome, was highlighted on digitized images using a computer-aided manipulator. The fibrotic area relative to the total area of the field was calculated as a percentage. The scores of 10 fields per kidney were averaged, after which the mean scores from animals in each group were averaged.

Antibodies
Anti-human {alpha}-smooth muscle actin ({alpha}SMA) antibody (EPOS System: Dako, Hamburg, Germany) was used to identify myofibroblasts. Pathways that protect the kidneys were detected using the following antibodies in immunohistochemical testing or western blotting: polyclonal EpoR antibody (1:1000, Santa Cruz), polyclonal IL-3/IL-5/GM-CSFRβ antibody (1:1000, Santa Cruz), polyclonal Ki-67 antibody (1:50, Dako), polyclonal phospho-Akt (Ser473) antibody (1:1000, Cell Signaling Technology, Beverly, MA, USA) and polyclonal Akt antibody (1:1000, Cell Signaling). We normalized protein levels using polyclonal β-actin antibody (1:1000, Cell Signaling).

Morphology and immunohistochemical staining
Tissue samples were fixed in 4% (wt/vol) buffered paraformaldehyde (PFA) for 16 h and then embedded in paraffin. Tissue sections (4 µm) were mounted on silane (2% 3-aminopropyltriethoxysilane)-coated slides (Muto Pure Chemicals, Tokyo, Japan), deparaffinized with xylene and stained with periodic acid-Schiff (PAS). Immunohistochemical staining was carried out using the Envision system (Dako), according to the manufacturer's instructions. To examine the expression of {alpha}SMA or Ki-67, endogenous peroxidase activities were blocked with 3% H2O2 for 10 min and then incubated with anti-{alpha}SMA and anti-Ki-67 antibodies for 60 min at room temperature. After labelling, the endogenous peroxidase activity in tissue sections was blocked by incubating in methanol with 0.3% hydroxyoxidase for 30 min. The sections were then processed using an avidin-biotinylated peroxidase complex method (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA, USA) with diaminobenzidine as the chromogen. All histological slides were examined by light microscopy using a Nikon Eclipse 80 i (Nikon, Tokyo, Japan), and pictures were taken with Nikon ACT-1 version 2.63.

The {alpha}SMA-positive area relative to the total field area was calculated as a percentage using a computer-aided manipulator. The mean score for 15 fields per kidney was determined, and the mean scores for kidneys in each group were then averaged. For Ki-67 staining, the number of positive cell nuclei counted in 15 random areas was averaged.

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, sections were deparaffinized and subjected to antigen retrieval in preheated 10 mmol/l sodium citrate (pH 7) using a microwave. They were then incubated with 3% H2O2 for 10 min, followed by incubation with a TdT enzyme solution for 90 min at 37°C. The reaction was terminated by incubation in stop/wash buffer for 30 min at 37°C. The number of TUNEL-positive cell nuclei was counted in 15 random areas and averaged.

Western blot analysis
Kidney tissues were homogenized in Cell Lysis Buffer (Cell Signaling) with protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Homogenates were centrifuged (12 000 x g for 10 min at 4°C), and supernatant total protein was measured by the Lowry protein assay (Bio-Rad, Hercules, CA). Total protein lysate (15 µg) containing 1:1 denaturing sample buffer was boiled for 5 min and resolved on 6.0–8.0% SDS-polyacrylamide gels, and electrophoretically transferred onto an immobilon PVDF membrane (Millipore, Bedford, MA, USA). The filter was blocked with 5% (wt/vol) nonfat milk in 10 mM tris-buffered saline with 0.1% Tween-20 (TBS-T), followed by overnight incubation at 4°C with diluted primary antibodies (EpoR antibody, polyclonal IL-3/IL-5/GM-CSFRβ antibody, polyclonal phospho-Akt antibody and polyclonal Akt antibody as the above concentration) in TBS-T containing 5% BSA. After washing three 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, USA).

Statistical analysis
Data are expressed as means ± SD. Statistical significance, defined as P < 0.01 or <0.05, was evaluated using one-way ANOVA.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effects on erythropoiesis
Compared to saline-treated (control group) rats, both high dose and low dose of EPO treatment (EPO group) significantly increased Hb (saline, 14.3 ± 0.4 g/dl, median 14.2 g/dl, range 13.7–14.8 g/dl, first to third quartile 14.0–14.7 g/dl; high dose of EPO, 16.1 ± 0.9 g/dl, median 16.3 g/dl, range 14.3–16.9 g/dl, first to third quartile 15.7–16.8 g/dl, P < 0.01 versus saline group; low dose of EPO, 16.0 ± 0.29 g/dl, median 16.0 g/dl, range 15.8–16.1 g/dl, first to third quartile 15.8–16.1 g/dl, P < 0.05 versus saline group), Ht (saline, 43.2 ± 5.5%, median 45.6%, range 33.4–46.4%, first to third quartile 39.0–46.3% versus EPO, 58.0 ± 2.5%, median 59.2%, range 53.4–60.1%, first to third quartile 55.8–59.9%, P < 0.01) and reticulocyte concentrations (saline, 40.6 ± 5.2{per thousand}, median 40{per thousand}, range 35–49{per thousand}, first to third quartile 36.5–45.0{per thousand}; high dose of EPO, 116.8 ± 2.6{per thousand}, median 117.5{per thousand}, range 113–119{per thousand}, first to third quartile 114–118.8{per thousand}, P < 0.05 versus saline group; low dose of EPO, 95.5 ± 9.2{per thousand}, median 95.5{per thousand}, range 89–102{per thousand}, first to third quartile 89–102{per thousand}, P < 0.05 versus saline group) as shown in Figure 1. On the other hand, the high dose of CEPO treatment (CEPO group) neither enhanced nor reduced Hb, Ht and reticulocyte counts (13.9 ± 0.4 g/dl, median 13.8 g/dl, range 13.4–14.3 g/dl, first to third quartile 13.5–14.2 g/dl, P < 0.01 versus EPO group, 43.5 ± 1.5%, median 43.5%, range 41.4–46.3%, first to third quartile 42.3–43.9%, P < 0.01 versus EPO group and 37.5 ± 3.7{per thousand}, median 37{per thousand}, range 34–42{per thousand}, first to third quartile 34.3–41.3{per thousand}, respectively, P < 0.05 versus EPO group), suggesting that CEPO, unlike EPO, does not stimulate erythropoiesis.


Figure 1
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  		Fig. 1 Effects on erythropoiesis, renal function and blood pressure. Box plots show the changes in Hb (a), reticulocyte concentrations (b) and serum creatinine (c). Shown are the mean (black line), range (vertical bars) and first to third quartile (box). Both high dose and low dose of EPO treatment (n = 11) significantly increased Hb and reticulocyte concentrations (*P < 0.01 versus saline group and CEPO group, **P < 0.05 versus saline group and CEPO group). In contrast, CEPO treatment (n = 11) did not enhance Hb or reticulocyte count. EPO or CEPO treatment did not significantly affect the serum creatinine levels compared to control groups (c, *P < 0.05 versus sham ope group). Changes in the systolic blood pressure are shown (d). Treatment with EPO or CEPO had no significant effect on the systolic blood pressure, but one of three rats (#) treated with EPO showed markedly elevated blood pressure (173/133 mmHg).

 
We also examined the effect of EPO or CEPO treatment on serum creatinine, which reflects the renal function of the contralateral right kidney, because obstructed kidneys are non-functioning kidneys. The serum creatinine level was increased in saline-treated disease control rats (0.35 ± 0.07 mg/dl), compared with the normal rats with sham operation (sham ope group) (0.22 ± 0.01 g/dl). Treatment with a high dose of EPO or CEPO did not significantly affect the creatinine levels (0.30 ± 0.03 g/dl and 0.31 ± 0.03 g/dl, respectively, Figure 1c), suggesting that EPO or CEPO has no significant effect on the contralateral glomerular filtration rate.

We then checked the effect of EPO or CEPO treatment on the blood pressure, because treatment with EPO was reported to worsen systemic blood pressure [12]. Treatment with EPO (139.4 ± 25.5 mmHg; n = 3) or CEPO (119.2 ± 6.9 mmHg; n = 3) did not significantly increase the systolic blood pressure compared with untreated rats (126.4 ± 9.1 mmHg; n = 3, Figure 1d). One of three rats treated with EPO, however, showed markedly elevated blood pressure (173/133 mmHg). Of interest is that this rat exhibited a wedge-shaped infarction in the kidney.

Effects on tubular apoptosis and proliferation in UUO kidney
Quantification of the number of apoptotic cells by TUNEL immunostaining on EPO- or CEPO-treated obstructed kidneys showed an increase in TUNEL-positive, apoptotic cells among tubular epithelial cells at 1 week (average of TUNEL-positive cell number per 15 fields, 55 ± 16) in the control group (Figure 2a, d). Apoptotic cells were significantly decreased in the high dose of EPO (35 ± 14, P < 0.05, Figure 2b, d) and CEPO (35 ± 10, P < 0.05, Figure 2c, d) groups (no significance between the EPO and CEPO groups). In addition, the low dose of EPO or CEPO also had a significant effect on the number of TUNEL-positive apoptotic cells (31 ± 5 and 24 ± 8, respectively, Figure 2d), suggesting that both EPO and CEPO have similar anti-apoptotic actions.


Figure 2
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  		Fig. 2 Effects on tubular apoptosis. Representative TUNEL immunostaining in control group (a), high dose of EPO group (b) and high dose of CEPO group (c) are shown. The dark brown dots correspond to representative TUNEL-positive nuclei. Box plots demonstrate that EPO or CEPO [both high dose (n = 11) and low dose (n = 6)] treatment significantly decreased TUNEL-positive, apoptotic cells (d, *P < 0.05 versus control group). Shown are the mean (black line), range (vertical bars) and first to third quartile (box) (magnification, x400).

 
The 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 x400 magnification in a minimum of 15 fields. The mean number of positive cell nuclei per field did not significantly differ among the three groups (control group, 13 ± 2, high dose of the EPO group, 15 ± 4, high dose of the CEPO group, 17 ± 4), but EPO and CEPO showed a tendency to promote tubular cell proliferation.

Effects on cell signaling
Working on the assumption that tissue protection is mediated via the EpoR and βcR heteroreceptors, we first examined the expression of EpoR and βcR (Figure 3a). Western blot analysis demonstrated that the expression of EpoR and βcR were low in control group obstructed kidneys, while the EpoR was upregulated in the EPO and CEPO groups. In addition, the expression of βcR was slightly increased in the EPO group, but strongly activated in the CEPO group.


Figure 3
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  		Fig. 3 Effects of EPO or CEPO treatment on Akt activation. Western blot analysis demonstrated that the expression of EpoR and βcR were low in control UUO kidneys, while the EpoR was upregulated in EPO and CEPO groups. In addition, the expression of βcR was strongly activated in CEPO group (a). Treatment with 1000 IU EPO or CEPO significantly increased phosphorylation of Akt at 1 week, but that 500 IU EPO or CEPO had a weak effect on activation of Akt (b). The band density of p-Akt expressed as the mean ± S.D. is illustrated (c, *P < 0.05 versus control group).

 
Intracellular EPO signaling, which is implicated in tubular protection, was examined through the activation of Akt (Figure 3b, c). Western blot analysis demonstrated that treatment with 1000 IU of EPO or CEPO significantly increased phosphorylation of Akt at 1 week (no significance between the EPO and CEPO groups). Compared with the anti-apoptotic effects (Figure 2d), treatment with 500 IU EPO or CEPO had a weak effect on activation of Akt, suggesting that a high dose of EPO or CEPO is necessary to protect the kidneys through activation of Akt.

Effects on interstitial phenotypic changes in the UUO kidney
One of the most important events associated with interstitial fibrosis, enhanced expression of {alpha}SMA, which is a marker of interstitial phenotypic changes, was assessed by immunohistochemistry and was shown to be strong in the interstitial area of control group obstructed kidneys (Figure 4a). However, immunostaining for {alpha}SMA was weak in the high dose of the EPO (Figure 4b, d) or CEPO (Figure 4c, d) group obstructed kidneys (no significance between the EPO and CEPO groups). In contrast, treatment with 500 IU EPO or CEPO had no significant effect on inhibiting {alpha}SMA expression (Figure 4d).


Figure 4
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  		Fig. 4 Effects of EPO or CEPO on phenotypic changes. Interstitial phenotypic changes were assessed by immunohistochemical staining for {alpha}SMA in the control group (a), high dose of EPO group (b) and high dose of CEPO group (c). Quantification (%) of the {alpha}SMA-positive areas is shown (d). Shown are the mean (black line), range (vertical bars) and first to third quartile (box). While the high dose of EPO (n = 11) and CEPO (n = 11) significantly suppressed {alpha}SMA expression, the low dose of EPO (n = 6) and CEPO (n = 6) did not (*P < 0.05 versus control group) (magnification, x400).

 
In order to determine the effects on interstitial fibrotic changes in obstructed kidneys, histological analysis was performed using Masson's trichrome staining. The area of the fibrotic lesion of the cortical interstitium was determined in the sections stained light blue. Saline-treated obstructed kidneys exhibited increased tubular dilation, with a marked expansion of the interstitium (Figure 5a). In contrast, the high dose of the EPO or CEPO group obstructed kidneys showed lower interstitial expansion, although they exhibited the same extent of tubular dilation (Figure 5b, c). However, a wedge-shaped infarction (Figure 5d, arrow) was observed in 4 of 11 EPO group kidneys, while no kidneys in the CEPO group showed infarctions. The Hb level in the EPO group with infarction was not higher than that in the EPO group without infarction (15.9 ± 1.4 g/dl versus 16.2 ± 0.5 g/dl, respectively).


Figure 5
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  		Fig. 5 Effects of EPO or CEPO on interstitial fibrotic changes. Interstitial fibrosis was assessed by Masson's trichrome staining in the control group (a), high dose of EPO group (b) and low dose of CEPO group (c) (magnification, x200). Representative wedge-shaped infarction (arrow) in EPO-treated kidneys is shown (magnification, x4) (d). Quantification (%) of the fibrotic area is shown by box plots (e, *P < 0.05 versus control group). Shown are the mean (black line), range (vertical bars), and first to third quartile (box).

 
On morphometric analysis, fibrotic areas of the interstitium were significantly increased in control group obstructed kidneys (5.87 ± 2.20%). However, fibrotic areas in the EPO (4.01 ± 1.86 and 3.45 ± 0.22% in high dose and low dose, respectively) or CEPO (3.91 ± 1.16 and 3.69 ± 0.76% in high dose and low dose, respectively) group of obstructed kidneys were significantly smaller when compared with control group kidneys (Figure 5e) (no significance between the EPO and CEPO groups).



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this paper, we examined whether EPO or CEPO has therapeutic effects on tubulointerstitial injury in a rat UUO model. Based on our results, both high doses of EPO and CEPO appear to have similar protective functions against tubulointerstitial injury. Previous studies have also shown that EPO and CEPO have a similar efficacy for tissue protection. When delivered in a single dose (10 µg/kg = 2000 IU/kg), the potency of CEPO and EPO seems to be comparable in rat models of stroke and spinal cord compression [19]. However, when multiple doses are administered, CEPO is more potent than EPO in the spinal cord compression model [19]. Detailed dose-response studies remain to be carried out to compare the performance of CEPO with EPO; however, CEPO may be more useful than EPO.

The key findings that support this conclusion are as follows: (1) fewer TUNEL-positive, apoptotic cells were present among the tubular epithelial cells in both 1000 IU EPO- and CEPO-treated kidneys than in saline-treated kidneys, but this effect was also observed in 500 IU EPO- or CEPO-treated kidneys; (2) treatment with 1000 IU EPO or CEPO increased phosphorylation of Akt at 1 week, while 500 IU EPO or CEPO had a weak effect on activation of Akt; (3) both high dose and low dose of EPO treatment significantly increased Hb, Ht and reticulocyte concentrations, while CEPO treatment neither enhanced nor reduced Hb, Ht and reticulocyte counts; (4) a wedge-shaped infarction was observed in 4 of 11 EPO-treated kidneys, while no kidneys in CEPO-treated rats showed infarctions; (5) Hb levels in EPO-treated rats with infarction were not higher than those in EPO-treated rats without infarction, while one of three rats treated with EPO showed markedly elevated blood pressure.

We demonstrated that high dose of EPO and CEPO treatments markedly suppressed obstruction-induced tubular epithelial apoptosis and interstitial phenotypic alteration as assessed by {alpha}SMA expression. Renal tubular apoptosis in UUO has been suggested to be related to renal tissue loss and dysfunction [21]. Western blot analysis demonstrated the increase of phosphorylated Akt by EPO and CEPO treatment as previously reported [22]; activation of Akt may be one possible signal transduction pathway by which tubular apoptosis, as well as later interstitial phenotypic changes, is suppressed. Once activated, Akt activates multiple targets with anti-apoptotic effects, including phosphorylation of Bad, Bax, caspase-9 and GSK-3β, maintenance of mitochondrial membrane potential and preservation of glycolysis and ATP synthesis [23]. However, this activation of Akt was weak in kidneys with EPO or CEPO treatment at 500 IU. This observation could be related to the fact that the affinity of the EpoR in tubular epithelial cells is well below normal plasma EPO concentrations [13].

Kashii et al. [24] reported that phosphatidylinositol-3 kinase (PI3K) is activated by EPO in the EPO-dependent UT-7 leukaemia cell line, where it recruits Akt. The PI3K-Akt pathway also leads to the upregulation of Bcl-xL and the inhibition of apoptosis in Baf-3 cells [25]. Furthermore, using the EPO-dependent human erythroid progenitor cell line, Silva et al. [26] showed that EPO treatment maintains cell viability by repressing apoptosis through the upregulation of Bcl-xL, an anti-apoptotic gene of the Bcl-2 family. These results suggest that the upregulation of the PI3K-Akt pathway in EPO- or CEPO-treated kidneys suppresses tubular epithelial apoptosis, likely due to the induction of anti-apoptotic genes of the Bcl-2 family.

In this study, we found that EpoR and βcR levels were significantly higher in CEPO-treated kidneys than in control group obstructed kidneys. Recently, it was reported that membrane proteins prepared from the rat brain, heart, liver or kidney were greatly enriched in the EpoR covalently bound in a complex with βcR and that knockout of the βcR fully abolished tissue protective properties of EPO or CEPO in the nervous system and heart [18]. Although the precise protein interactions of the EpoR and βcR have not been determined, CEPO-mediated upregulation of EpoR and βcR levels may contribute to the dimerization of the EpoR and βcR, and thereby lead to tissue protection.

A number of important factors must be considered prior to clinical application of EPO for cytoprotection. Recently, the Food and Drug Administration (FDA) issued a public health advisory outlining new safety information, including revised product labeling about erythropoiesis-stimulating agents (ESAs). This issue arises from the findings of several important studies: (1) an increased number of deaths and of non-fatal heart attacks, strokes, heart failure and blood clots when ESAs were adjusted to maintain higher RBC levels in patients with chronic kidney failure [16,17]; (2) faster tumour growth when ESAs were adjusted to maintain Hb levels above 12 g/dl in patients with head and neck cancer undergoing radiation therapy [14,27]; (3) earlier deaths and did not have fewer blood transfusions when ESAs were given according to the dosing recommendations for cancer patients receiving chemotherapy in cancer patients not receiving chemotherapy [28]; (4) more blood clots in patients scheduled for orthopaedic surgery who received ESAs to reduce blood transfusions during and after surgery than those not given an ESA.

We also demonstrated that high dose of EPO treatment increased Hb concentration, thereby inducing wedge-shaped infarction in 4 of 11 rats. However, Hb levels in EPO-treated rats with infarction were not higher than those in EPO-treated rats without infarction. In addition, low dose of EPO treatment developed similar increments of Hb as high dose of EPO, although we observed no infracted lesion in low dose of EPO-treated kidney. As EPO administration to rats with chronic renal failure was shown to accelerate the progression of chronic renal disease, especially in relation to the increased blood pressure [12], we also examined the effect of high dose of EPO or CEPO treatment on blood pressure. One of three rats treated with EPO showed markedly elevated blood pressure concomitantly with a wedge-shaped infarction in the kidney. Therefore, elevated blood pressure due to EPO administration may contribute to the infarction. In addition, it is known that EPO is pro-thrombotic in a dose-dependent manner, which is partly mediated by augmented expression of P- and E-selectins [29]. In addition, EPO induces the production of young, hyper-reactive platelets [30], which is particularly problematic for chronic administration of EPO and for the high doses that would be required for renoprotection [13,31]. Clinical studies indicate that patients with chronic kidney diseases [16,17] and patients with cancer [28] are at a higher risk of adverse events with EPO. Furthermore, the frequency and severity of adverse effects increased when larger doses of EPO were used to target higher haematocrit levels that were still within the normal range [32].

Compared to EPO, CEPO showed similar renoprotective effects on tubulointerstitial injury in the absence of polycythaemia. Recently, we also reported the therapeutic effects of EPO and CEPO on ischaemia-reperfusion injury, which is a transient insult on renal tubular epithelial cells. In the previous paper, single administration of EPO or CEPO (100 IU/kg) is sufficient to prevent ischaemia-reperfusion injury, while a high dose of EPO or CEPO is necessary to prevent the kidney from ureteral obstruction in this paper. In the previous study, we did not observe a wedge-shaped infarction in EPO-treated kidneys, probably due to the administration of a low dose of EPO. The likely situation to require a high dose of EPO may couple with the dose-dependent adverse effects of EPO, especially thrombosis or hypertension. In this case, the engineered non-erythropoietic tissue-protective EPO, such as CEPO, may be promising. Thus, CEPO may be able to protect the kidneys from tubulointerstitial injury. Further research is required concerning dose-ranging and pharmacodynamic properties after CEPO administration and related adverse effects using preclinical models appropriate for supporting potential clinical studies, but the therapeutic use of CEPO warrants further attention and preclinical studies.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Nath KA. Am J Kidney Dis (1992) 20:1–17.[Web of Science][Medline]
  2. Phillips AO, Steadman R. Histol Histopathol. (2002) 17:247–252.
  3. Uchiyama-Tanaka Y, Mori Y, Kimura T, et al. Am J Kidney Dis (2004) 43:e18–e25.[CrossRef][Medline]
  4. Eddy AA. J Am Soc Nephrol (1996) 7:2495–2508.[Abstract]
  5. Klahr S, Morrissey J. Am J Physiol Renal Physiol (2002) 283:F861–F875.[Abstract/Free Full Text]
  6. Klahr S, Schreiner G, Ichikawa I. N Engl J Med (1988) 318:1657–1666.[Abstract]
  7. Sharma AK, Mauer SM, Kim Y, et al. Kidney Int (1993) 44:774–788.[Web of Science][Medline]
  8. Sakanaka M, Wen TC, Matsuda S, et al. Proc Natl Acad Sci USA (1998) 95:4635–4640.[Abstract/Free Full Text]
  9. Siren AL, Fratelli M, Brines M, et al. Proc Natl Acad Sci USA (2001) 98:4044–4049.[Abstract/Free Full Text]
  10. Nangaku M. J Am Soc Nephrol (2006) 17:17–25.[Abstract/Free Full Text]
  11. Kuriyama S, Tomonari H, Yoshida H, et al. Nephron (1997) 77:176–185.[Web of Science][Medline]
  12. Garcia DL, Anderson S, Rennke HG, et al. Proc Natl Acad Sci USA (1988) 85:6142–6146.[Abstract/Free Full Text]
  13. Westenfelder C, Biddle DL, Baranowski RL. Kidney Int (1999) 55:808–820.[CrossRef][Web of Science][Medline]
  14. Henke M, Laszig R, Rube C, et al. Lancet (2003) 362:1255–1260.[CrossRef][Web of Science][Medline]
  15. Phrommintikul A, Haas SJ, Elsik M, et al. Lancet (2007) 369:381–388.[CrossRef][Web of Science][Medline]
  16. Drueke TB, Locatelli F, Clyne N, et al. N Engl J Med (2006) 355:2071–2084.[Abstract/Free Full Text]
  17. Singh AK, Szczech L, Tang KL, et al. N Engl J Med (2006) 355:2085–2098.[Abstract/Free Full Text]
  18. Brines M, Grasso G, Fiordaliso F, et al. Proc Natl Acad Sci USA (2004) 101:14907–14912.[Abstract/Free Full Text]
  19. Leist M, Ghezzi P, Grasso G, et al. Science (2004) 305:239–242.[Abstract/Free Full Text]
  20. Savino C, Pedotti R, Baggi F, et al. J Neuroimmunol (2006) 172:27–37.[CrossRef][Web of Science][Medline]
  21. Truong LD, Petrusevska G, Yang G, et al. Kidney Int (1996) 50:200–207.[Web of Science][Medline]
  22. Imamura R, Isaka Y, Ichimaru N, et al. Biochem Biophys Res Commun (2007) 353:786–792.[CrossRef][Web of Science][Medline]
  23. Cantley LC. Science (2002) 296:1655–1657.[Abstract/Free Full Text]
  24. Kashii Y, Uchida M, Kirito K, et al. Blood (2000) 96:941–949.[Abstract/Free Full Text]
  25. Chavakis T, Kanse SM, Lupu F, et al. Blood (2000) 96:514–522.[Abstract/Free Full Text]
  26. Silva M, Grillot D, Benito A, et al. Blood (1996) 88:1576–1582.[Abstract/Free Full Text]
  27. Henke M, Mattern D, Pepe M, et al. J Clin Oncol (2006) 24:4708–4713.[Abstract/Free Full Text]
  28. Wright JR, Ung YC, Julian JA, et al. J Clin Oncol (2007) 25:1027–1032.[Abstract/Free Full Text]
  29. Stohlawetz PJ, Dzirlo L, Hergovich N, et al. Blood (2000) 95:2983–2989.[Abstract/Free Full Text]
  30. Wolf RF, Peng J, Friese P, et al. Thromb Haemost (1997) 78:1505–1509.[Web of Science][Medline]
  31. Brines M, Cerami A. Kidney Int (2006) 70:246–250.[CrossRef][Web of Science][Medline]
  32. Peterson L. FDA oncologic drugs advisory committee (ODAC) meeting on the safety of erythropoietin in oncology. Trends Med (2004) 1–4.
Received for publication: 18. 5.07
Accepted in revised form: 29.10.07


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Nephrol. Dial. Transplant., September 1, 2008; 23(9): 3033 - 3034.
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