AP214 ameliorates sepsis-induced acute kidney injury and mortality

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).

Effects of AP214 treatment on liver damage in lethal CLP

We previously reported liver damage in mouse CLP model.10 The lethal CLP model showed

significant increases of aspartate aminotransferase (AST) and alanine aminotransferase (ALT)

at 18 hr after surgery compared with sham-operated animals [AST: vehicle 680.9 ± 92.5 U/l

(n = 14), sham 126.4 ± 22.3 U/l (n = 5), p<0.05; ALT: vehicle 314.3 ± 30.1 U/l (n = 14), sham

40.6 ± 3.8 U/l (n = 5), p<0.05]. AP214 treatment at the dose of 10 μg significantly attenuated

liver damage in lethal CLP (Figure 4A,B) and did not cause any change in sham-operated mice

(data not shown). Delayed 10 μg AP214 treatment that was started at 6 hr after surgery also

showed a protective effect against liver damage by lethal CLP (Figure 4C,D).

AP214 treatment improved hypotension in lethal CLP

Blood pressure and heart rate measurement was performed in conscious animals by

radiotelemetery. Injection of AP214 on normal CD-1 mice at the dose of 10 μg did not alter

blood pressure and heart rate compared with vehicle injection (Figure 5A,B). Sepsis induced

by lethal CLP surgery caused severe hypotension and decreases of heart rate. AP214 treatment

prevented the progression of severe hypotension and bradycardia [blood pressure at 18 hr:

sham 112.4 ± 2.8 mmHg (n = 3), CLP + vehicle 54.4 ± 4.7 mmHg (n = 5), CLP + AP214 75.7

± 6.4 mmHg (n = 5), p<0.05 (vs CLP + vehicle); heart rate: sham 459.4 ± 77.0 bpm (n = 3),

CLP + vehicle 197.6 ± 14.2 bpm (n = 5), CLP + AP214 459.4 ± 77.0 bpm (n = 5), p<0.05 (vs

CLP + vehicle)]. (Figure 5C,D).

Serum TNF-α and IL-10 levels in lethal CLP were suppressed by AP214

We evaluated serum TNF-α and IL-10 levels by ELISA 18 hr after surgery in lethal CLP

animals. Both serum TNF-α and IL-10 levels increased in CLP animals [TNF-α: Vehicle 572.5

± 65.5 pg/ml (n = 14), sham 55.3 ± 5.3 pg/ml (n = 5), p<0.05, IL-10: Vehicle 1365.2 ± 182.4

pg/ml (n = 14), sham 20.2 ± 9.3 pg/ml (n = 5), p<0.05]. Administration of AP214 reduced

serum TNF-α and IL-10 levels with bell-shaped dose-response curves (Figure 6A,B). AP214

had a maximum effect on sepsis-induced AKI at the dose of 10 μg and the same dose of 10

μg also had a maximum effect on reducing serum TNF-α and IL-10. Delayed AP214 treatment

(10 μg) also reduced serum TNF-α and IL-10 (Figure 6C,D).

Nuclear factor-κB activation in kidney and spleen was inhibited by AP214

We examined nuclear factor-κB (NF-κB) activation in kidney and spleen because α-MSH has

been reported to inhibit NF-κB activation induced by several inflammatory signals.27 NF-κB

activation in kidney and spleen at 18 hr after lethal CLP surgery was significantly increased

compared with sham-operated animals. AP214 treatment at the dose of 10 μg significantly

inhibited the increase of NF-κB activation both in kidney and spleen (Figure 7A,B). Delayed

AP214 treatment (10 μg) also decreased NF-κB activation (Figure 7C,D).

AP214 reduced splenocyte apoptosis in lethal CLP

Lymphocyte apoptosis is reported to contribute to sepsis severity.28 Therefore, we investigated

the effect of AP214 on splenocyte apoptosis. Splenocyte apoptosis was evaluated by

immunohistochemical analysis of activated caspase-3. In sham operated animals, activated

caspase-3 positive stained cells were rarely found in spleen (Figure 8A). CLP increased

activated caspase-3 positive stained cells profoundly in the white pulp of spleen at 18 hr after

surgery and AP214 treatment at the dose of 10 μg significantly reduced splenocyte apoptosis

(Figure 8B,C,D). Delayed AP214 treatment (10 μg) also reduced splenocyte apoptosis (Figure

8E). No apoptotic cell was detectable in the kidney as previously reported 10.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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

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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-

Doi et al.

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NIH-PA Author ManuscriptPage 5induced AKI and multiple organ failure were not determined.24 In the current study, we foundthat a α-MSH analogue AP214 improved sepsis-induced multiple organ failure including AKIand mortality. Moreover, we found that AP214 had a bell-shaped dose-response curve forsepsis-induced AKI. There are several reports that demonstrated biphasic dose-response curvesin the anti-inflammatory influence of α-MSH. Both the large and small doses were ineffectivefor in vivo animal models of ear swelling induced by picryl chloride36, 3738 and crystal-inducedperitonitis, and in vitro cell culture models such as TNF-α production in microglia3940, cyclicAMP production in mast cells and glutathione peroxidase activity in melanocytes andkeratinocytes41. The cause of this phenomenon is unknown, but might be explained in part byreceptor downregulation to avoid excess effects. Equimolar native α-MSH also showed aprotective effect on sepsis-induced AKI, although binding affinity of AP214 for melanocortinreceptors in vitro were higher than native α-MSH. These data suggest that effects of AP214 invivo may not be straightforward and confounded by pharmacokinetic parameters and/orindirect regulatory influences. Further evaluations would be required to clarify the mechanismsof these biphasic responses of AP214 both in physiological and pathological in vivo conditions.Failure to regulate hyperactivation of the inflammatory system plays an important role inpathophysiology of sepsis and blocking inflammatory mediators has been often proposed as atreatment strategy for sepsis. However, many clinical trials with drugs that inhibit specificinflammatory mediators have not been successful. α-MSH has a breadth of anti-inflammatoryactivities and can improve inflammatory disorders through suppressing multiple pathways.Moreover, α-MSH modulates inflammatory responses thorough inhibition of the nucleartranscription factor NF-κB activation, which plays the central role in immune and inflammationsystems.27 In the present study, we demonstrated the inhibition of systemic TNF-α production(Figure 6) and renal and splenic NF-κB activation (Figure 7). AP214 reduced serum TNF-αlevels with a bell-shaped dose-response curve similar to its effects on serum creatinine levels.This indicates that modulation of the exacerbated inflammatory response is beneficial for AKIinduced by lethal CLP. AP214 can be an agonist of several melanocortin receptors (MCR1,

MCR3, MCR4, and MCR5). MCR1 and MCR3 are known to mediate anti-inflammatory

effects and MCR4 regulates hemodynamics in the central nerve system as described above,

whereas MCR5 has been implicated in multiple exocrine functions.42 Further investigation on

the contribution of each melanocortin receptor pathway will be necessary to further understand

the mechanisms of action of α-MSH in sepsis and to minimize unwanted effects from other

melanocortin receptors.

The action of α-MSH is reminiscent of corticosteroids because both of them have multiple anti-

inflammatory effects through several mechanisms including inhibiting NF-κB pathway.43 It

has been recently suggested that ‘physiologic’ but not high doses of corticosteroids might be

beneficial to septic patients.44 Our data indicates that AP214 similarly has a relatively narrow

therapeutic window since 10 μg treatment but not 50 μg showed protective effects in CLP.

Therefore, translation of AP214 to studies in human sepsis will likely require precise evaluation

of the pharmacokinetics and dose responses of AP214 in humans before large-scale clinical

trials can be initiated.

Serum IL-10 levels were increased in our mouse sepsis model and suppressed after AP214

treatment. Although the role of IL-10 as an anti-inflammatory cytokine is apparent in renal

ischemia/reperfusion and cisplatin-induced renal injury,16 the role of IL-10 and its relationship

to α-MSH in sepsis is less clear. IL-10 knockout mice showed higher mortality in CLP than

wild type mice and anti-IL-10 monoclonal antibody treatment worsened the survival after CLP

when injected at 0 or 2 hr before surgery.45, 46 On the other hand, anti-IL-10 monoclonal

antibody treatment was protective when it was injected 10 hr after CLP.46 Therefore, IL-10

appears to have dual effects on sepsis that depends on the timing. The finding that AP214

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.

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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

with a 21-gauge needle and gently squeezed to confirm leakage of cecal contents. In sham-

operated animals, the cecum was located, but neither ligated nor punctured. The abdominal

incision was closed in two layers with 4-0 silk sutures, and 1 ml of prewarmed normal saline

(NS) was injected intraperitoneally. Treatment with fluid and antibiotic was started at 6 hr after

surgery with subcutaneous injection of imipenem/cilastatin (14 mg/kg) in 1 mL of NS,

treatment in the survival study was continued every 12 hr with imipenem/cilastatin (7 mg/kg)

in 1 mL of NS. AP214 (0.1, 1, 10, and 50 μg) or native α-MSH (6.8 μg) dissolved in NS 0.15

ml was administered intravenously as a bolus just after surgery, at 6 hr post-surgery and then

every 12 hr thereafter in the survival study. In the vehicle-treated CLP group, animals received

0.15 ml of NS intravenously at the indicated times. In another experiment, 10 μg of AP214

dissolved in 0.15 ml of NS or only vehicle was given intravenously 6 hr after surgery (delayed-

treatment group). Blood, kidneys and spleen were collected under isoflurane anesthesia at 18

hr after surgery except for survival study.

Morphologic evaluation of kidneys

Kidney specimens fixed in 10% formalin and embedded in paraffin were stained with periodic

acid-Schiff reagent (PAS). Histologic changes in the cortex and in the outer stripe of the outer

medulla (OSOM) were scored by a blinded observer.7

Measurement of blood chemistry, blood cell counts and cytokines

Serum creatinine was measured by HPLC.57 Aspartate aminotransferase (AST) and alanine

aminotransferase (ALT) were measured by autoanalyzer (Hitachi 917, Boehringer Mannheim,

Indianapolis, IN). Peripheral blood cell counts were analyzed by an automated hematology

analyzer (Abott Cell-Dyn 3500, GMI, Ramsey, MN). Tumor necrosis factor-α (TNF α) and

interleukin 10 (IL-10) were measured by ELISA (R&D Systems, Minneapolis, MN).

Measurement of blood pressure and heart rate

The mean blood pressure and heart rate were measured by radiotelemetry. Under anesthesia

with ketamine (50 mg/kg), xylazine (5 mg/kg) and acepromazine (1 mg/kg), a telemeter

catheter was implanted in the left carotid artery and advanced to reach the aortic arch. The

attached telemetry transmitter (model TA11PA-C10, Data Sciences International, St. Paul.

MN) was placed in a subcutaneous pocket on the left flank one week before CLP surgery or

AP214 administration. The telemeter signals were processed as previously described.58 Blood

pressure and heart rate data was analyzed from 6 hr before CLP surgery for 24 hrs, or 20 min

before injection of AP214 or vehicle until 90 min after.

NF-κB activation assayNIH-PA Author ManuscriptNIH-PA Author Manuscript

NIH-PA Author ManuscriptNF-κB p65 activity was quantified by using the TransAM Assay (Active Motif, Carlsbad, CA).

Briefly, a NF-κB consensus oligonucleotide bound to a 96-well plate was incubated with 100

μg of whole cell extracts from kidney and spleen, followed by p65 primary antibody and

secondary antibody conjugated with horseradish peroxidase. The optical density (OD) values

of peroxidase reaction product from each sample were normalized with the OD values of Raji

nuclear extract (Active Motif).

Immunohistochemical analysis of activated caspase-3 in spleen

Immunohistochemical staining of 4 μm paraffin sections was performed with anti-activated

caspase-3 antibody (Cell Signaling Technology, Beverly, MA). The sections were

deparaffinized, incubated in 10 mmol/L citrate-buffer, pH 6.0 for 10 min at 95°C and

preincubated with 3% hydrogen peroxide for 10 min. After blocking with goat serum, sections

were incubated for 1 hr at room temperature with primary antibodies. Two-step

immunohistochemical staining with horseradish peroxidase-conjugated secondary antibody

was conducted (EnVison+ System-HRP (DAB), DakoCytomation, Glostrup, Denmark) with

diaminobenzidine substrate, then counterstaining with hematoxylin. Positive stained cells were

counted from five randomly selected non-overlapping 200X fields.

Statistical analysis

Results of statistical analyses are expressed as means ± SEM. Differences among experimental

groups were confirmed by one-way ANOVA followed by Dunnett's test for individual

comparison of group means vs the control group. Survival analyses were compared by a log-

rank test. These calculations were performed with SigmaStat v3.10 (Systat Software Inc,

Richmond, CA). The null hypothesis was rejected when P < 0.05.NIH-PA Author Manuscript

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NIH-PA Author ManuscriptSupplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgementsWe thank Dr. Yan Qin for technical assistance.Disclosure This research was supported by the Intramural Research Program of the NIH, NIDDK. Thomas Jonassen,Jørgen Frøkiær, and Søren Nielsen have a financial interest in Action Pharma, which supplied no research funding,other than providing AP214.References1. Martin GS, Mannino DM, Eaton S, et al. The epidemiology of sepsis in the United States from 1979through 2000. N Engl J Med 2003;348:1546–1554. [PubMed: 12700374]2. Levy EM, Viscoli CM, Horwitz RI. The effect of acute renal failure on mortality. A cohort analysis.Jama 1996;275:1489–1494. [PubMed: 8622223]3. Neveu H, Kleinknecht D, Brivet F, et al. Prognostic factors in acute renal failure due to sepsis. Results

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NIH-PA Author ManuscriptFigure 1. Renal pathology

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

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NIH-PA Author ManuscriptFigure 2. Effect of AP214 on AKI induced by CLP

Mice were sacrificed 18 hr after lethal CLP surgery for measurement of serum creatinine byHPLC 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 treatmentgroup). #, P < 0.05 vs. CLP.

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NIH-PA Author ManuscriptFigure 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 eachAP214 group). #, P < 0.05 vs. CLP.

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NIH-PA Author ManuscriptFigure 4. Effect of AP214 on liver damage induced by CLPSerum aspartate transaminase (AST) and alanine transaminase (ALT) were measured 18 hrafter 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.

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NIH-PA Author ManuscriptFigure 5. AP214 prevented hypotension and bradycardia by CLP

Telemetric recordings of mean arterial pressure (MAP) and heart rate (HR) after injection ofAP214 (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 CLPmice with AP214 treatment (closed circle) or vehicle injection (open circle), and sham-operatedgroup (open square) are shown in (C) and (D). Values are mean ± SEM (n = 3 in sham-operatedgroup, n = 5 in CLP group, n = 5 in AP214 group).

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NIH-PA Author ManuscriptFigure 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 lethalCLP 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.

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NIH-PA Author ManuscriptFigure 7. NF-κB activation in kidney and spleen

NF-κB activation in kidney and spleen was evaluated by ELISA. AP214 was injected at 0 and6 hr (A, B) or only at 6 hr (delayed-treatment) (C, D). OD value of each sample was normalizedwith 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.

Doi et al.

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NIH-PA Author ManuscriptPage 19Figure 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 positivestained 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.

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NIH-PA Author ManuscriptFigure 9. AP214 improved survival of CLPAP214 treatment at the dose of 10 μg improved the survival rate both in lethal and sublethalCLP model. Open squares indicate lethal CLP group (n = 16), closed squares AP214 treatmentgroup in lethal CLP (n = 16), open circles sublethal CLP group (n = 20), and closed circlesAP214 treatment group in sublethal CLP (n =20). #, P < 0.05 vs. lethal CLP. ##, P < 0.05 vs.sublethal CLP.

NIH-PA Author ManuscriptSham + NS (n = 4)Sham + AP214 (n = 4)CLP + NS (n = 7)CLP + AP214 (n = 7)

#P < 0.05 vs. CLP + NS

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NIH-PA Author ManuscriptTable 1WBC (/μl)Neutrophil (/μl)Lymphocyte + Monocyte (/μl)5673 ± 1301247 ± 924318 ± 1635125 ± 5501307 ± 1303758 ± 4291520 ± 33588 ± 691413 ± 3531049 ± 247679 ± 131#347 ± 116#

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