Mice were anesthetized with a three compound mixture containing 0.5 mg/kg medetomidine, 5 mg/kg midazolam and 0.05 mg/kg fentanyl to achieve analgesia, amnesia and hypnosis prior to sham surgery or unilateral renal pedicle clamping with a micro aneurysm clamp (Medicon, Germany) via a flank incision. Ischemia times applied in this study were 15, 25, 35 and 45 minutes, following reperfusion. Online rectal temperature recording (ORTR) was installed for every mouse after the onset of complete anesthesia. For incision/ clamping and suturing mice were placed on a platform according to the protocol as shown in Fig 1A . After renal pedicle clamping and after clamp removal the successful reperfusion was assessed by color change from pale (ischemia) to the original color [19 (link)]. After clamping the kidney was placed back inside the abdomen, with only the clamps handle remaining outside the body. Sham-operated mice were handled in the same manner, except for clamping. After clamp removal wounds were closed using absorbable sutures for peritoneal and cutaneous layer (Ethicon, Belgium). For adjustment of fluid losses, 200 µl of saline were administered into the peritoneal cavity before wound closure. Anesthesia was antagonized using 2.5 mg/kg atipamezole, 0.5 mg/kg flumazenil and 1.2 mg/kg naloxone. 0.05 mg/kg Buprenorphin was injected subcutaneously for pain control. The experiments ended after 24 hours and 5 weeks of reperfusion time, respectively, when the animals were sacrificed by cervical dislocation and kidneys were collected. Post-ischemic and contralateral kidneys were removed, decapsulated, and divided, with one part being stored in 4% buffered formalin, and one part being frozen in RNAlater (Ambion, Germany).
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