AP214 improved survival both in lethal and sublethal CLP

We evaluated the effects of 10 μg AP214 treatment on survival after lethal CLP, in which 15

mm of the cecum was ligated. Drug or vehicle was administrated just after surgery, at 6 hr post

surgery and every 12 hr thereafter. All vehicle-treated animals in the lethal CLP model died

within 48 hrs after surgery; treatment with AP214 improved mortality (Figure 9). We also

evaluated the effect of AP214 on sublethal CLP model, in which only 8 mm of the cecum was

ligated. After sublethal CLP, 30-40% of control animals survived at 96 hr and treatment with

AP214 increased the survival rate to 65% (Figure 4). AKI after sublethal CLP was evaluated

at 18 hr after surgery. Compared with our lethal CLP model, the control animals in sublethal

CLP showed lower serum creatinine levels and treatment with AP214 at the dose of 10 μg

showed a trend towards decreased serum Cr [vehicle 0.29 ± 0.06 mg/dl (n = 15) vs AP214 0.19

± 0.04 mg/dl (n = 15), P= 0.089].NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author ManuscriptDiscussionWe have evaluated the effects of the α-MSH analogue AP214 on sepsis-induced AKI andmortality by using a modified rodent CLP sepsis model in outbred mice. AP214 showed aprotective effect on sepsis-induced AKI and improved survival in lethal and sublethal CLPmodels. We also demonstrated potential mechanisms of AP214 action on several pro- and anti-inflammatory pathways and splenocyte apoptosis.We developed a new CLP model as follows; we changed the mouse strain from inbred C57BL/6 to outbred CD-1 mice that are more representative of a genetically heterogeneous humanpopulation. In our previous reports, we used aged C57BL/6 mice because CLP surgery in youngC57BL/6 mice produced a wide range of creatinine values without much histologic evidenceof renal damage.7 In contrast, 8 week old CD-1 mice showed more pathological renal damagecompared with young C57BL/6 mice. Since the length of the ligated cecum determines theseverity in other CLP models,29, 30 we developed two different models: A lethal CLP model

(ligation 15 mm from the end of cecum) and a sublethal CLP model (8 mm) models (Figure

9). Finally, we reduced the volume of fluid resuscitation compared to our previous studies to

avoid volume overload and maintain constant body weight.

Mice subjected to lethal CLP were severely hypotensive and bradycardic (Figure 5). The

radiotelemetry data in the present study indicate that systemic circulation failure induced by

CLP may contribute to organ hypoperfusion, including hypoperfusion of kidney that has been

previously shown in CLP animals.8, 31 Severe systemic circulation failure might contribute

to sepsis-induced AKI in this model. AP214 significantly improved blood pressure and heart

rate. Recent studies revealed that melanocyte stimulating hormones including α-MSH have

important roles in cardiovascular regulation.32 It has been reported that α-MSH increases blood

pressure and heart rate by activation of MCR4 that is primarily expressed in the central nervous

system.33 Hence, AP214 might improve sepsis via a direct hemodynamic effect. However,

exogenous α-MSH increases blood pressure only when it is administered by

intracerebroventricular injection; intravenous injection does not alter blood pressure in rodents.33, 34 Similarly, intravenous AP214 did not increase blood pressure and heart rate in normal

mice. However, the present data does not clarify the precise mechanism by which intravenously

injected AP214 exerts its hemodynamic effects in the septic CLP mice, as it is not possible to

differentiate between a direct hemodynamic effect from an initial anti-inflammatory effect with

subsequent improvement in hemodynamics.

α-MSH has broad anti-inflammatory properties and inhibits organ injury in various

inflammatory animal models.35 We have already demonstrated that the native α-MSH

ameliorated AKI caused by ischemia reperfusion injury.13-15, 17, 18 Although one report

showed that native α-MSH improved survival of sepsis induced by CLP, its effect on sepsis-