decreased serum IL-10 levels at 18 hr after surgery in the present study indicates that excessive

IL-10 at the late phase might worsen sepsis-induced AKI.

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author ManuscriptReagentsThe relationship between α-MSH and IL-10 is also unclear. In vitro, α-MSH modestlystimulates IL-10 production in human peripheral blood mononuclear cells.47, 48 There is onereport in vivo showing no effect of [Nle4, D-Phe7] α-MSH on lipopolysaccaride (LPS)-stimulated plasma IL-10 levels in monkeys.49 Our results clearly showed that the increase ofserum IL-10 by CLP was attenuated by α-MSH. One possible explanation for this discrepancyis that LPS, especially at the subclinical dose used in the primate study,49 causes an increasein IL-10 that is more resistant to α-MSH. The observation that LPS signaling via Toll-likereceptor 4 does not contribute to AKI in polymicrobial sepsis by CLP supports this view.10The mechanisms of α-MSH to decrease IL-10 production in vivo during sepsis need furtherinvestigation. Because the effect of IL-10 disruption on sepsis outcomes depends on timing,the success of AP214 as a clinical therapeutic may require optimization of the timing ofadministration.Apoptosis was detected predominantly in lymphocytes in human sepsis,50 and severalstrategies to decrease immune cell apoptosis have been reported to improve the survival rateof CLP animals.51-53 CLP animals in the present study showed splenocyte apoptosis, whichwas reduced after AP214 treatment. α-MSH has been reported to activate anti-apoptoticpathways in cultured melanocytes54, 55 and reduce renal tubular cell apoptosis in a bilateralureteral obstruction model.56 It might be possible that AP214 acts on splenocytes andsuppresses apoptotic pathways.ConclusionThe α-MSH analogue AP214 ameliorated severe septic shock and sepsis-induced AKI andmortality in outbred CD-1 mouse models of polymicrobial sepsis. We found the biphasic dose-response curves of AP214 against AKI and cytokine productions, indicating that strictregulation of α-MSH dosing might be important in sepsis. We also demonstrated that AP214improved systemic hemodynamics, pro- and anti-inflammatory actions, renal and splenic NF-κB activation, and splenocyte apoptosis. These results suggested that AP214 might be usefulfor the prevention or treatment of sepsis in humans.Materials and Methods

AP214 is an analogue of α-MSH modified with six lysine residues at N-terminus of native α-

MSH (Ac-KKKKKKSYSMEHFRWGKPV-NH2). AP214 was provided by Action Pharma A/

S (Aarhus and Copenhagen, Denmark). AP214 has a higher binding affinity for melanocortin

receptors compared with the native α-MSH [Inhibition constants (AP214 vs MSH): MCR1:

2.9 vs 5.7 nM; MCR3: 1.9 vs 22 nM; MCR4: 3.7 vs 24 nM and MCR5: 110 vs 1300 nM

(Jonassen et al. J Am Soc Nephrol S17: S326A, 2006)]. Native α-MSH was purchased from

Phoenix Pharmaceuticals (Mountain View, CA).

Cecal ligation and puncture (CLP) model

All animal experiments were conducted in accordance with an animal study protocol approved

by NIDDK animal care and use committee. 8 week old male CD-1 mice (Charles River

Laboratories, Wilmington, MA) were allowed food and water ad libitum. CLP was performed

as previously described with some modifications.7 Under isoflurane anesthesia, a 1.5 cm

midline incision was made and the cecum was exposed. A 4-0 silk ligature was placed 15 mm

from the cecal tip in lethal CLP and 8 mm in sublethal CLP. The cecum was punctured twice