AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality

Abstract

Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. alpha-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new alpha-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 h after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-alpha and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-kappaB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.

Figure 1. Renal pathology

Animals were sacrificed 18 hr after lethal CLP surgery. Representative renal histology of the cortex and the outer stripe of the outer medulla (OSOM) in each group were shown. Original magnification: X400.

Figure 2. Effect of AP214 on AKI induced by CLP

Mice were sacrificed 18 hr after lethal CLP surgery for measurement of serum creatinine by HPLC method. AP214 was injected at 0, 6 hr (A, B) or only at 6 hr (delayed-treatment) (C). Equimolar native α-MSH (6.8 μg) was injected at 0 and 6 hr (B). Values are mean ± SEM (n = 4∼5 in sham-operated group, n = 10∼14 in CLP group, n = 7∼10 in each AP214 treatment group). #, P < 0.05 vs. CLP.

Figure 3. Pathological score

The tubular damage score (see Methods section) was measured in the cortex and the OSOM. Values are mean ± SEM (n = 4 in sham-operated group, n = 8 in CLP group, n = 6 in each AP214 group). #, P < 0.05 vs. CLP.

Figure 4. Effect of AP214 on liver damage induced by CLP

Serum aspartate transaminase (AST) and alanine transaminase (ALT) were measured 18 hr after lethal CLP surgery. AP214 was injected at 0 and 6 hr (A, B) or only at 6 hr (delayed-treatment) (C). Values are mean ± SEM (n = 5 in sham-operated group, n = 14 in CLP group, n = 7 in AP214 group). #, P < 0.05 vs. CLP.

Figure 5. AP214 prevented hypotension and bradycardia by CLP

Telemetric recordings of mean arterial pressure (MAP) and heart rate (HR) after injection of AP214 (closed circle) or vehicle (open circle) to normal CD-1 mice are shown in (A) and (B). Values are mean ± SEM (n = 5 in vehicle group, n = 5 in AP214 group). MAP and HR in CLP mice with AP214 treatment (closed circle) or vehicle injection (open circle), and sham-operated group (open square) are shown in (C) and (D). Values are mean ± SEM (n = 3 in sham-operated group, n = 5 in CLP group, n = 5 in AP214 group).

Figure 6. AP214 reduced serum TNF-α and IL-10

Serum TNF-α and IL-10 levels were measured by ELISA. AP214 was injected at 0 and 6 hr (A, B) or only at 6 hr (delayed-treatment) (C, D). Animals were sacrificed 18 hr after lethal CLP surgery. Values are mean ± SEM (n = 5 in sham-operated group, n = 14 in CLP group, n = 7 in each AP214 group). #, P < 0.05 vs. CLP.

Figure 7. NF-κB activation in kidney and spleen

NF-κB activation in kidney and spleen was evaluated by ELISA. AP214 was injected at 0 and 6 hr (A, B) or only at 6 hr (delayed-treatment) (C, D). OD value of each sample was normalized with that of Raji cell nuclear extract. Values are mean ± SEM (n = 4 in sham-operated group, n = 6-7 in CLP group, n = 6-7 in AP214 group). #, P < 0.05 vs. CLP.

Figure 8. Immunohistochemical analysis of activated caspase-3 in spleen

Immunohistochemistry with anti-activated caspase-3 antibody in sham-operated group (A), CLP group (B), AP214-treated group (C); Original magnification: X400. Counts of positive stained cells were shown. AP214 was injected at 0 and 6 hr (D) or only at 6 hr (delayed-treatment) (E). Values are mean ± SEM (n = 3 in sham-operated group, n = 5 in CLP group, n = 5 in AP214 group). #, P < 0.05 vs. CLP.

Figure 8. Immunohistochemical analysis of activated caspase-3 in spleen

Immunohistochemistry with anti-activated caspase-3 antibody in sham-operated group (A), CLP group (B), AP214-treated group (C); Original magnification: X400. Counts of positive stained cells were shown. AP214 was injected at 0 and 6 hr (D) or only at 6 hr (delayed-treatment) (E). Values are mean ± SEM (n = 3 in sham-operated group, n = 5 in CLP group, n = 5 in AP214 group). #, P < 0.05 vs. CLP.

Figure 9. AP214 improved survival of CLP

AP214 treatment at the dose of 10 μg improved the survival rate both in lethal and sublethal CLP model. Open squares indicate lethal CLP group (n = 16), closed squares AP214 treatment group in lethal CLP (n = 16), open circles sublethal CLP group (n = 20), and closed circles AP214 treatment group in sublethal CLP (n =20). #, P < 0.05 vs. lethal CLP. ##, P < 0.05 vs. sublethal CLP.