Mouse cecal ligation and puncture (CLP) model

A total of 20 8-week-old male C57BL/6J mice (weight, 20-25 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd., and housed in a room at 22±1˚C, 40-60% humidity, with a 12-h light/dark cycle (8:00 a.m.-8:00 p.m.), with free access to food and drinking water. All operations on mice were performed by one individual who was skilled in animal modeling. As described previously (9), a 1-cm midline laparotomy was performed following anesthesia with 3% inhaled isoflurane. The base of the cecum was ligated and punctured at its distal tip using a single 21-gauge needle. Subsequently, a small amount of stool content (about 1 mm length) was extruded and put back into the abdominal cavity. The abdomen was sealed and 1 ml warm sterile 0.9% saline was injected subcutaneously to resuscitate mice. The sham group (n=8) underwent laparotomy and cecum exposure but no cecal ligation or puncture.

At 24 h or 48 h post-surgery, all mice were anesthetized by inhalation of 3% isoflurane, and then 50 µl retro-orbital venous blood was collected to measure Scr and BUN levels. Following collection of retro-orbital venous blood to assess Scr and BUN levels, 4 mice were sacrificed (24 h post-CLP, SA-AKI group) and 8 mice were given an additional 10 ml/kg warmed sterile 0.9% saline, and then sacrificed at 48 h post-CLP by cervical dislocation. AKI was defined as a serum creatinine (Scr) level >20 µmol/l and blood urea nitrogen (BUN) >28.3 mg/dl. Mice were grouped into persistent injury (PI; n=3) and recovery (RE; n=5) groups according to renal function at 48 h post-CLP. The RE group consisted of mice whose Scr and BUN levels returned to normal (<20 µmol/l and <28.3 mg/dl, respectively) at 48 h. The PI group contained mice with high levels of Scr (>20 µmol/l) and BUN (>28.3 mg/dl) at 48 h. Mice in the SA-AKI group were sacrificed to collect blood and kidneys at 24 h (n=4). Mice in the PI (n=3) and RE (n=5) group were sacrificed to collect blood and kidneys at 48 h. Mice in the sham group were sacrificed via cervical dislocation to collect blood and kidneys at 24 h (n=4) and 48 h (n=4), respectively.

All protocols of this study were approved by the Animal Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang (approval ID. 2016160; 25 February 2016), which follows the institutional guidelines.