Methyl-2-acetamidoacrylate, an ethyl pyruvate analog, decreases sepsis-induced acute kidney injury in mice

Abstract

We tested the anti-inflammatory agent methyl-2-acetamidoacrylate (M2AA), an ethyl pyruvate analog, in a cecal ligation-and-puncture (CLP) model of sepsis in CD-1 mice. M2AA administration at the time of CLP improved survival, renal function, kidney histology, liver injury, and splenocyte apoptosis, and lowered cytokine levels (TNF-alpha, IL-6, IFN-gamma, and IL-10). When M2AA treatment was delayed 6 h (but not 12 h), M2AA still significantly reduced kidney dysfunction, liver injury, splenocyte apoptosis, and cytokine levels. NF-kappaB, a M2AA target, was transiently activated in spleen, peaking at 6 h; kidney and liver NF-kappaB increased steadily with a plateau at 12-24 h. M2AA reduced NF-kappaB activation in spleen at 6 h and in kidney and liver at 24 h. Splenectomy diminished the ability of M2AA to reduce cytokines, especially IL-6, but M2AA still decreased kidney and liver dysfunction, suggesting that splenic NF-kappaB is not central to M2AA action. In contrast, beneficial effects of chloroquine on cytokines and organ damage were neutralized by splenectomy, demonstrating a spleen-specific chloroquine target. Because M2AA and chloroquine act differently, we tested this combination. Survival at 96 h was highest with combination therapy (57%) vs. chloroquine (38%), M2AA (47.6%), or vehicle (5%). The benefit of combination therapy over chloroquine or M2AA alone did not reach statistical significance, indicating potential mechanistic overlap. We conclude that the transient target(s) for M2AA responsible for the narrow 6-h therapeutic window is not splenic NF-kappaB. Identifying this new target and downstream signaling pathways could lengthen the therapeutic window and improve combination therapy with chloroquine.

Figures

Fig. 1.

Methyl-2-acetamidoacrylate (M2AA) improves survival, kidney dysfunction, and liver injury after polymicrobial sepsis. A: survival analysis of control cecal ligation and puncture (CLP; solid line, n = 18) or M2AA at 0 h after CLP (shaded line, n = 18). *P < 0.05 vs. control. B–G: kidney function evaluated as serum creatinine (Cr) and blood urea nitrogen (BUN) levels (B and C), kidney tubular histology damage score in the cortex (D) and outer stripe of the outer medulla (OSOM; E), and liver function evaluated as increases in alanine transaminase (ALT; F) and aspartate transaminase (AST; G) at 24 h after surgery in sham group (n = 8) or groups treated with vehicle plus CLP (n = 8–11), M2AA 0 h after CLP (n = 9–11), M2AA 6 h after CLP (n = 9–11), or M2AA 12 h after CLP (n = 7–10). *P < 0.05 vs. vehicle plus CLP.

Fig. 2.

M2AA attenuated splenocyte apoptosis in CLP sepsis: effect of M2AA in splenocyte apoptosis at 24 h after CLP. Splenocyte apoptosis was detected by immunohistochemistry of active caspase-3 in the spleen at 24 h after surgery in sham group (n = 4) or groups treated with vehicle plus CLP (n = 4), M2AA 0 h after CLP (n = 5), M2AA 6 h after CLP (n = 5), or M2AA 12 h after CLP (n = 4). *P < 0.05 vs. vehicle plus CLP.

Fig. 3.

M2AA improves cytokine response. Serum proinflammatory cytokines (TNF-α, A; IL-6, B; and IFN-γ, C) and an anti-inflammatory cytokine (IL-10; D) at 24 h after surgery in sham group (n = 5) or groups treated with vehicle plus CLP (n = 8–9), M2AA 0 h after CLP (n = 9–10), M2AA 6 h after CLP (n = 11), or M2AA 12 h after CLP (n = 8–10). *P < 0.05 vs. vehicle plus CLP.

Fig. 4.

Time course of NF-κB activation, splenocyte apoptosis, kidney dysfunction, liver injury, and cytokine responses after CLP. Animals were killed at indicated times for measurement of each parameter. A: activated NF-κB p65 in spleen, kidney, and liver were measured at indicated time points and normalized by Raji nuclear extract. B: time course of splenocyte apoptosis detected by immunohistochemistry of active caspase-3 in the spleen. C–F: kidney function as measured by serum Cr and BUN (C), liver function as measured by ALT and AST (D), and cytokine levels (TNF-α and IL-10, E; Il-6 and IFN-γ, F) (n = 4–6 per group). *P < 0.05 vs. 0 h.

Fig. 5.

M2AA reduced NF-κB activation in spleen, kidney, and liver. We evaluated the effect of M2AA on NF-κB p65 activation in spleen at 6 h or in kidney and liver at 24 h. NF-κB activation (normalized by Raji cell nuclear extract) in sham group (n = 4–6) or groups treated with vehicle plus CLP (n = 4) or M2AA (n = 4). *P < 0.05 vs. control CLP.

Fig. 6.

Comparison of M2AA and chloroquine (CQ) to improve sepsis-induced organ injury and cytokine levels in splenectomized mice. Splenectomy was performed at the same time as CLP surgery. Kidney dysfunction (A and B), liver injury (C and D), and cytokine levels (E–H) were determined at 24 h after surgery in mice subjected to sham surgery (n = 4–6) or treatment with vehicle plus CLP (n = 7–8), CQ (50 mg/kg) 3 h before CLP (n = 7–8), or M2AA 0 h after CLP (n = 6–8). *P < 0.05 vs. vehicle plus CLP in nonsplenectomy group. #P < 0.05 vs. vehicle plus CLP in splenectomy group.

Fig. 7.

Effect of M2AA and CQ combination therapy on survival, kidney dysfunction, liver injury, and cytokine levels in CLP sepsis model. A: survival curves from mice given vehicle (CLP; n = 21), M2AA 0 h after CLP (n = 21), CQ 3 h before CLP (n = 18), or M2AA + CQ treatment (n = 21). Kidney (B and C) and liver function (D and E) and cytokine responses (F–I) at 24 h after surgery are shown in sham group (n = 6) and in groups treated with vehicle plus CLP (n = 8–11), M2AA (n = 8–11), CQ (n = 7–11), or combination therapy (n = 7–11). *P < 0.05 vs. vehicle plus CLP.