AP214 ameliorates sepsis-induced acute kidney injury and mor(6)

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