AP214 ameliorates sepsis-induced acute kidney injury and mor

NIH Public AccessAuthor ManuscriptKidney Int. Author manuscript; available in PMC 2008 June 1.Published in final edited form as: Kidney Int. 2008 June; 73(11): 1266–1274.

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AP214, an analogue ofα-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortalityKent Doi, M.D., Ph.D.1, Xuzhen Hu1, Peter S.T. Yuen, Ph.D.1, Asada Leelahavanichkul, M.D. 1, Hideo Yasuda, M.D., Ph.D.1, Soo Mi Kim, Ph.D.1, Jürgen Schnermann, Ph.D.1, Thomas E.N. Jonassen, M.D.2,4, Jørgen Frøkiær, M.D.3,4, Søren Nielsen, M.D., Ph.D.3,4, and Robert A. Star, M.D.1 1National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 2Department of Pharmacology, University of Copenhagen, Copenhagen, Denmark 3The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark 4Action Pharma, Aarhus, Denmark

AbstractSepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury.α-Melanocyte-stimulating hormone is a potent antiinflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a newα-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 hr after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-α and IL-10 levels with a bell-shaped doseresponse curve. Additionally; NF-κB 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. Sepsis is one of the major causes of mortality, and the incidence of sepsis in the United States is still increasing.1 Acute kidney injury (AKI) is also associated with a high mortality rate2 and AKI and sepsis increase mortality synergistically; septic AKI patients show worse mortality compared with non-septic AKI patients,3 whereas renal dysfunction in septic patients increases mortality.4 However therapies to treat or prevent sepsis-induced AKI are largely ineffective, and novel drugs available at clinical settings are urgently required. Several pre-clinical animal models have been developed to evaluate the possible efficacy of drugs on sepsis-induced AKI.5 Among them, the ceca

l ligation and puncture (CLP) model is widely used because the resulting polymicrobial sepsis mimics many features of human sepsis. 6 We have already demonstrated the efficacy of several drugs by a clinically relevant sepsisinduced AKI model based on CLP.7-9 In this model, animals were treated with fluid resuscitation and antibiotics similar to septic patients in an intensive care unit. AKI can be

Address for correspondence: Robert A. Star, M.D., Renal Diagnostics and Therapeutics Unit, NIDDK, NIH, 10 Center Drive, Room 3N108, Bethesda, MD 20892-1268, Phone: 301-402-6749, Fax: 301-402-0014, E-mail: Robert_Star@nih.gov.

detected within 6 hr after surgery by a sensitive MRI technique9 and within 12 hr after CLP

by measurement of serum creatinine.7 Moreover, our model shows not only kidney injury but

multiple organ failure such as liver damage and splenocyte apoptosis.7, 10 In the present study,

we further developed our CLP model with some modifications in terms of mouse strain, length

of ligated cecum, and volume of fluid resuscitation.

α-Melanocyte stimulating hormone (α-MSH) darkens skin pigmentation in amphibians,

rodents, and humans; however it is also a potent anti-inflammatory cytokine that decreases

inflammatory cytokines, nitric oxide production, and expression of a neutrophil adhesion

molecule.11, 12 α-MSH inhibits multiple forms of inflammation and shows protective effects

on AKI induced by ischemia/reperfusion and cisplatin injection.13-18 α-MSH also showed

protective effects in other experimental animal models such as brain ischemic injury,19

mesenteric ischemia-reperfusion injury,20 endotoxin-induced hepatitis,21 cutaneous

vasculitis,22 and inflammatory bowel disease.23 Lipton et al. showed a protective effect of

native α-MSH on survival of CLP animals although they did not measure sepsis-induced AKI

or other organ failure.24 The purpose of the present study was to examine the effect of the α-

MSH analogue AP214 that has an additional six lysine residues at the amino terminus on sepsis-

induced AKI and mortality in a newly modified mouse sepsis model of CLP.NIH-PA Author Manuscript

Results

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NIH-PA Author ManuscriptEffects of AP214 treatment on AKI induced by lethal CLP in young CD-1 miceLethal polymicrobial sepsis was induced in 8 wk old outbred CD-1 mice by placing a ligatureat 15 mm from the tip of cecum and puncturing it twice with a 21-G needle. Serum creatinineincreased 18 hr after surgery demonstrating the occurrence of acute kidney injury. Renalmorphologic evaluation performed 18 hr after surgery showed mild tubular damage withvacuolization but no thrombosis, tubular necrosis, infiltrating inflammatory cells or castformation both in cortex and the outer stripe of outer medulla (OSOM) (Figure 1). These

histological damages were similar to our previous studies in aged C57BL/6 mice.7-10 Body

weight did not increase in CD-1 mice, in contrast to our previous report that showed 7%

increase,7 because the volume of fluid therapy that started at 6 hr after surgery and every 12

hr thereafter was reduced from 1.5 ml to 1.0 ml (data not shown).

Treatment with AP214 attenuated AKI with a bell-shaped dose-response curve; 10 μg treatment

had a maximum effect, 1 μg showed a partial improvement, whereas, neither 0.1 μg nor 50

μg treatment showed any effect against AKI (Figure 2A). Native α-MSH treatment with 6.8

μg (an amount equimolar to 10 μg AP214) also showed a protective effect on AKI (Figure 2B).

AP214 treatment at the dose of 10 μg significantly ameliorated tubular damage in both the

cortex and the OSOM (Figure 1, 3A,B). Treatment with the maximally efficacious dose of 10

μg reduced AKI and histological damage even when started 6 hrs after surgery (Figure 2C,

3C,D and Supplementary Figure 10). Treatment of sham-operated mice with 10 μg of AP214

did not cause any change in serum creatinine (data not shown).

Severe neutropenia was improved by AP214

The present lethal CLP model showed severe neutropenia at 18 hr after surgery in accordance

with previous reports.25, 26. AP214 treatment at the dose of 10 μg significantly improved

neutropenia in peripheral blood in lethal CLP, although there was no significant difference in

the total white blood cell count between the CLP and AP214 treatment groups. AP214 did not

cause any change in sham-operated mice (Table 1).