Effects of continuous erythropoietin receptor activator in sepsis-induced acute kidney injury and multi-organ dysfunction

Abstract

Background: Despite advances in supportive care, sepsis-related mortality remains high, especially in patients with acute kidney injury (AKI). Erythropoietin can protect organs against ischemia and sepsis. This effect has been linked to activation of intracellular survival pathways, although the mechanism remains unclear. Continuous erythropoietin receptor activator (CERA) is an erythropoietin with a unique pharmacologic profile and long half-life. We hypothesized that pretreatment with CERA would be renoprotective in the cecal ligation and puncture (CLP) model of sepsis-induced AKI.

Methods: RATS WERE RANDOMIZED INTO THREE GROUPS: control; CLP; and CLP+CERA (5 µg/kg body weight, i.p. administered 24 h before CLP). At 24 hours after CLP, we measured creatinine clearance, biochemical variables, and hemodynamic parameters. In kidney tissue, we performed immunoblotting--to quantify expression of the Na-K-2Cl cotransporter (NKCC2), aquaporin 2 (AQP2), Toll-like receptor 4 (TLR4), erythropoietin receptor (EpoR), and nuclear factor kappa B (NF-κB)--and immunohistochemical staining for CD68 (macrophage infiltration). Plasma interleukin (IL)-2, IL-1β, IL-6, IL-10, interferon gamma, and tumor necrosis factor alpha were measured by multiplex detection.

Results: Pretreatment with CERA preserved creatinine clearance and tubular function, as well as the expression of NKCC2 and AQP2. In addition, CERA maintained plasma lactate at normal levels, as well as preserving plasma levels of transaminases and lactate dehydrogenase. Renal expression of TLR4 and NF-κB was lower in CLP+CERA rats than in CLP rats (p<0.05 and p<0.01, respectively), as were CD68-positive cell counts (p<0.01), whereas renal EpoR expression was higher (p<0.05). Plasma levels of all measured cytokines were lower in CLP+CERA rats than in CLP rats.

Conclusion: CERA protects against sepsis-induced AKI. This protective effect is, in part, attributable to suppression of the inflammatory response.

Figure 1. Immunoblots for renal Na-K-2Cl cotransporter, revealing a 146- to 176-kDa band (centered at 150 kDa).

Semiquantitative immunoblots prepared from kidney samples. Densitometric analysis of all samples from control rats, rats submitted to cecal ligation and puncture only, and rats treated with continuous erythropoietin receptor activator prior to undergoing cecal ligation and puncture. Differences among the means were analyzed by analysis of variance followed by the Student-Newman-Keuls test. P>0.05 for control vs. CLP+CERA. CLP, cecal ligation and puncture; CERA, continuous erythropoietin receptor activator; NKCC2, renal Na-K-2Cl cotransporter.

Figure 2. Aquaporin 2 expression was preserved in the rats pretreated with continuous erythropoietin receptor activator.

Semiquantitative immunoblots prepared from kidney samples. Densitometric analysis of all samples from control rats, rats submitted to cecal ligation and puncture only, and rats treated with continuous erythropoietin receptor activator prior to undergoing cecal ligation and puncture. Differences among the means were analyzed by analysis of variance followed by the Student-Newman-Keuls test. P>0.05 for control vs. CLP+CERA. CLP, cecal ligation and puncture; CERA, continuous erythropoietin receptor activator; AQP2, aquaporin 2.

Figure 3. Erythropoietin receptor expression was partially protected in the rats pretreated with continuous erythropoietin receptor activator.

Semiquantitative immunoblots prepared from kidney samples. Densitometric analysis of all samples from control rats, rats submitted to cecal ligation and puncture only, and rats treated with continuous erythropoietin receptor activator prior to undergoing cecal ligation and puncture. Differences among the means were analyzed by analysis of variance followed by the Student-Newman-Keuls test. p>0.05 for control vs. CLP+CERA. CLP, cecal ligation and puncture; CERA, continuous erythropoietin receptor activator; EpoR, erythropoietin receptor.

Figure 4. Immunoblots reacted with anti-Toll-like receptor 4 antibody (1∶100), revealing an 89-kDa band.

Semiquantitative immunoblots prepared from kidney samples. Densitometric analysis of samples from control rats, rats submitted to cecal ligation and puncture only, and rats treated with continuous erythropoietin receptor activator prior to undergoing cecal ligation and puncture. Differences among the means were compared by analysis of variance followed by the Student-Newman-Keuls test. p>0.05 for control vs. CLP+CERA. CLP, cecal ligation and puncture; CERA, continuous erythropoietin receptor activator; TLR4, Toll-like receptor 4.

Figure 5. Immunoblots reacted with anti-nuclear factor kappa B antibody (1∶500), revealing a 65-kDa band.

Semiquantitative immunoblots prepared from kidney samples. Densitometric analysis of samples from control rats, rats submitted to cecal ligation and puncture only, and rats treated with continuous erythropoietin receptor activator prior to undergoing cecal ligation and puncture. Differences among the means were compared by analysis of variance followed by the Student-Newman-Keuls test. p>0.05 for control vs. CLP+CERA. CLP, cecal ligation and puncture; CERA, continuous erythropoietin receptor activator; NF-κB, nuclear factor kappa B.

Figure 6. Number of CD68-positive cells/0.087 mm² field in the tubulointerstitium at 24 h after CLP.

A, photomicrographs of immunohistochemical staining in control rats, rats submitted to cecal ligation and puncture only, and rats treated with continuous erythropoietin receptor activator prior to undergoing cecal ligation and puncture (magnification, ×40). B, Graphic representation of CD68-positive cell counts. Data are mean±SEM. p>0.05 for control vs. CLP+CERA. CLP, cecal ligation and puncture; CERA, continuous erythropoietin receptor activator.