Carbamylated Erythropoietin Outperforms Erythropoietin in the Treatment of AKI-on-CKD and Other AKI Models

Abstract

Erythropoietin (EPO) may be a beneficial tissue-protective cytokine. However, high doses of EPO are associate with adverse effects, including thrombosis, tumor growth, and hypertension. Carbamylated erythropoietin (CEPO) lacks both erythropoietic and vasoconstrictive actions. In this study, we compared the renoprotective, hemodynamic, and hematologic activities and survival effects of identical EPO and CEPO doses in rat models of clinically relevant AKI presentations, including ischemia-reperfusion-induced AKI superimposed on CKD (5000 U/kg EPO or CEPO; three subcutaneous injections) and ischemia-reperfusion-induced AKI in old versus young animals and male versus female animals (1000 U/kg EPO or CEPO; three subcutaneous injections). Compared with EPO therapy, CEPO therapy induced greater improvements in renal function and body weight in AKI on CKD animals, with smaller increases in hematocrit levels and similarly improved survival. Compared with EPO therapy in the other AKI groups, CEPO therapy induced greater improvements in protection and recovery of renal function and survival, with smaller increases in systolic BP and hematocrit levels. Overall, old or male animals had more severe loss in kidney function and higher mortality rates than young or female animals, respectively. Notably, mRNA and protein expression analyses confirmed the renal expression of the heterodimeric EPO receptor/CD131 complex, which is required for the tissue-protective effects of CEPO signaling. In conclusion, CEPO improves renal function, body and kidney weight, and survival in AKI models without raising hematocrit levels and BP as substantially as EPO. Thus, CEPO therapy may be superior to EPO in improving outcomes in common forms of clinical AKI.

Keywords: acute renal failure; chronic kidney disease; erythropoietin.

Copyright © 2016 by the American Society of Nephrology.

Figures

Figure 1.

CEPO is more effective than EPO in improving renal function after AKI superimposed on CKD. CKD was induced in male Sprague–Dawley rats by right nephrectomy and ligation of two to three branches of the left renal artery. This led to an increase in (A) SCr and (B) BUN and a decrease in (C) animal weight and (D) hematocrit (Hct). (E) Systolic BP did not change over the first 10 days. Ten days after selective renal artery branch ligation, AKI was induced by clamping of the remaining renal pedicle (35 minutes), and animals were treated 1 day later (vehicle, 5000 U/kg EPO sc, or 5000 U/kg CEPO sc) on days 11–13 after CKD. Both EPO and CEPO treatments led to lower (A) SCr and (B) BUN levels compared with vehicle-treated animals, with CEPO-treated animals showing (A and B) small but significantly greater improvements in kidney function than the EPO-treated groups and (C) better preserved body weights. (D) EPO treatment resulted in significantly higher Hct levels than in vehicle or CEPO-treated groups, whereas CEPO therapy resulted only in a moderate rise in Hct. (E) Systolic BP levels and (F) 7-day survival were higher in both CEPO and EPO groups compared with those in the vehicle-treated group. Compared with controls, both EPO- and CEPO-treated animals had (G) improved total renal blood flows on day 17 as well as (H) higher remnant kidney weights. Black bars indicate EPO treatment, gray bars indicate CEPO treatment, and white bars indicate vehicle treatment. Black arrowheads indicate CKD or AKI surgery as labeled. Black arrows indicate treatments on days 1–3. *P<0.05 compared with the vehicle control; #P<0.05 for CEPO compared with EPO.

Figure 2.

CEPO is superior to EPO for the treatment of AKI in either young or old F344 rats without raising the hematocrit (Hct) or BP. In young or old male F344 rats, AKI was induced with bilateral clamping of the renal pedicles (60 minutes for young rats and 38 minutes for old rats), and then, rats were treated (vehicle, 1000 U/kg EPO, or 1000 U/kg CEPO) at days 1–3 after AKI. In both (A and B) young and (D and E) old animals, I/R injury induced moderate AKI with elevated SCr and BUN levels. CEPO treatment reduced (A and D) SCr and (B and E) BUN levels significantly and more effectively than EPO. (C and F) On day 7, body weights of both treatment groups were significantly higher than those in controls. EPO treatment resulted in significantly higher (G and J) Hct and (H and K) systolic BP levels compared with vehicle control and CEPO groups. (I and L) Survival in CEPO-treated rats was significantly improved compared with that in vehicle or EPO-treated animals. Black bars indicate EPO treatment, gray bars indicate CEPO treatment, and white bars indicate vehicle treatment. Black arrows indicate treatments on days 1–3. *P<0.05 compared with the vehicle control; #P<0.05 for CEPO compared with EPO.

Figure 3.

EPO and CEPO reduce tissue injury and apoptosis and increase renal cell proliferation in young and old F344 rats after AKI. In young and old male F344 rats, both CEPO and EPO (A and D) significantly reduced kidney injury scores, (B and E) decreased terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick–end labeling+ (TUNEL+) cells, and (C and F) increased proliferating cell nuclear antigen+ (PCNA+) cells on day 3 after AKI. Black bars indicate EPO treatment, gray bars indicate CEPO treatment, and white bars indicate vehicle treatment. FOV, field of view. *P<0.05 compared with the vehicle control.

Figure 4.

CEPO is more effective than EPO for the treatment of AKI in either male or female rats. In adult male and female F344 rats AKI was induced with bilateral clamping of the renal pedicles (35 minutes for male rats and 50 minutes for female rats), and then, rats were treated (vehicle, 1000 U/kg EPO sc, or 1000 U/kg CEPO sc) on days 1–3 after AKI. In both male and female rats, EPO or CEPO treatment given on days 1–3 after AKI significantly reduced (A and E) SCr and (B and F) BUN levels compared with vehicle controls, with additional benefit obtained with CEPO compared with EPO. (C and G) CEPO treatment resulted in better maintained body weight after I/R AKI. (D) Both CEPO and EPO significantly improved survival in male rats compared with that in controls. In both male and female rats, EPO treatment significantly elevated (H and L) hematocrit (Hct) and (I and M) systolic BP levels on day 3 compared with those in vehicle or CEPO groups. Both EPO and CEPO increased (J and N) renal blood flows (RBFs) and (K and O) iothalamate clearances (Cls) compared with those in vehicle-treated controls. (J and N) However, total RBFs in CEPO-treated animals were always higher than those in EPO-treated rats. Black bars indicate EPO treatment, gray bars indicate CEPO treatment, and white bars indicate vehicle treatment. Black arrows indicate treatments on days 1–3. *P<0.05 compared with the vehicle control; #P<0.05 for CEPO compared with EPO.

Figure 5.

CD131 is expressed in the kidney. (A) In adult female Fisher rats, quantitative RT-PCR analysis of renal tissue at several time points after AKI showed that Epo mRNA levels were detectable in baseline (B.L.) controls (dashed line), dropped below detectable levels after AKI, and returned to baseline levels at 3 and 7 days post-AKI. (B) Epor mRNA levels fell below baseline levels at 2 and 24 hours and 3 days after AKI, and then, they returned toward baseline levels on day 7. (C) CD131 mRNA levels decreased and remained below baseline levels at 24 hours and 3 days, with a small but insignificant increase 7 days after AKI. *P<0.05 compared with the baseline control. (D) A Western blot for CD131 showed high expression in kidney and spleen tissues but not in the presence of a CD131–specific blocking peptide. CD131 has a molecular mass of approximately 130 kD (black arrow). Immunohistochemistry of kidney tissue showing (E) a negative control (Neg. Control) and (F) staining with CD131. (G) A magnified view of the area in the black box from E shows the negative control. (H) A magnified view of the area in the black box from F shows intensive CD131 staining of proximal tubular cells. n.d., not detectable. Scale bars, 100 μm in E and F; 200 μm in G and H.