Franny Jongbloed

37 2 FASTING PROTECTS AGED-OVERWEIGHT MICE light/12-hour dark) with either one animal (male) or two animals (females) per cage and were allowed free access to water and food (Special Diet Services, UK) unless noted otherwise. The conditions of and procedures done in the young male C57BL/6J mice are described elsewhere 9 . Preoperative fasting Mice were pair-matched based on body weight and sex. Subsequently, the animals were randomly assigned to one of two groups. The first group had unlimited access to food and water preoperatively. The average amount of food eaten in the ad libitum fed male and female group was approximately 5.7 and 4.4 grams/day respectively. The second group was fasted for three days preoperatively with no access to food and unlimited access to water. Fasted animals did not show any morbidity due to the fasting. Directly after surgery, all mice had unlimited access to food and water. For the microarray analysis, 10 other male mice were also assigned to either ad libitum food or fasting for three days, after which the mice were sacrificed. Surgical procedure Renal IRI was performed as described previously 9,14 . In brief, mice were anesthetized by isoflurane inhalation (5% isoflurane initially and then 2-2.5%with oxygen for maintenance). A midline abdominal incision was performed and the renal artery and vein of both kidneys were exposed. In the male mice, the pedicles of both kidneys were occluded for 37 minutes. As female mice are more resistant to ischemic renal damage 33,34 , the ischemic time in female mice was extended to 60 minutes. Purple discoloration of the kidneys confirmed ischemia macroscopically. After removal of the clamps, reperfusion was confirmed when the kidney color turned back to normal. The incision was closed in two layers with 5/0 sutures. After closure, the animals were subcutaneously injected with 0.5 ml PBS supplemented with 2.4 µg buprenorphine in order to compensate for fluid loss and provide analgesia. An identical analgesic dose of buprenorphine was administered in the morning after surgery to ameliorate suffering. The mice used for microarray analysis did not undergo renal IRI, instead were sacrificed right after the dietary intervention. Follow-up and serummeasurements The animals were followed up to 28 days postoperatively unless they died or were sacrificed as a result of morbidity (ruffled fur, decreased mobility, cold to the touch, excessive weight loss), or noted otherwise. During follow-up, they were inspected at least daily. Body weight was measured daily in the first postoperative week and on day 10, 14, 17, 21 and 28 if applicable. At day 28, all remaining animals were euthanized after which the abdominal cavity was opened for inspection of the kidneys. A blood sample of 50 μl was collected