4.1. Ischemia-Reperfusion Injury Model

Female Sprague-Dawley rats (200–250 g; 8–10 weeks old) were housed at the KU Leuven animal facility. After at least 1 week of acclimatization, they were anesthetized by an intraperitoneal injection of 7.5 mg/kg of ketamin (Anesketin^®, Eurovet Animal Health BV, Bladel, The Netherlands) and 2.5 mg/kg of xylazin (Xyl-M 2%^®, Van Miert & Dams Chemie (VMD), Arendonk, Belgium). Both renal pedicles were dissected free through a midline abdominal incision and clamped “en bloc” with microaneurysm clamps to induce ischemia. Reperfusion was initiated by the removal of the clamps. Sham-operated rats underwent the same surgery without the clamping of the pedicles. Analgesics were administered daily (Vetergesic 0.1 mg/kg, CEVA, Libourne, France) following surgery. At the end of the experiment, pentobarbital (Nembutal 60 mg/kg, CEVA) was injected intraperitoneally and the rats were sacrificed by exsanguination, which allowed plasma sampling directly from the aorta. Plasma was spun down (1000× g; 10 min) and snap-frozen. Kidneys were collected and processed immediately, as described below. For the trehalose experiments, sterile PBS (Vehicle) or 2 g/kg bodyweight of trehalose in PBS (in a total volume of ca. 500 µL) was injected intraperitoneally 48 h and 24 h prior to surgery. The rat survival, behavior, and humane endpoints were monitored thrice daily. The animal care and experimental protocols were in accordance with the European guidelines and approved by the Ethical Committee for Animal Experimentation of KU Leuven (P053/2016).