Franny Jongbloed

96 CHAPTER 4 Long-term dietary intervention Upon arrival, mice were allowed ad libitum (AL) access to the SDS chow for seven days. At the start of the dietary intervention period, all mice were transferred to clean cages at 4:00 pm. Mice were randomly divided into a group with 30% DR (n=5) or AL access to a carbohydrate-free (CHO-free) diet (n=6) or a fat-free diet (n=6) for 14 days, or a protein- free diet (n=6) for 10 days (Figure S3). Mice in the control group for DR had AL access to the control SDS chow (n=10), the control group for CHO-free and fat-free had AL access to Control diet (n=12). The effect of food intake was measured using pair-fed (PFed) control groups. Pair-feeding of each group was accomplished by giving the PFed groups the identical isocaloric amount of the control diet as the mice on the experimental diet had consumed the day before. The CHO-free, fat-free and protein-free diets were PFed in this manner (n=6/group). Mice with 30% DR were given 70% of the normal daily intake of mice on the control diet, which was administered once daily at 4:00 pm. Since the phenotypical effects of two weeks 30% DR were reported previously, microarray analysis was used as the only outcome for this group 1 . Short-term dietary intervention Upon arrival, the same procedure was followed as for the long-term experiment. Mice were randomly divided into groups with AL access to control diet (n=4), a protein-free diet (n=6 per group) for three days or 30% DR for three days (n=6), or into groups with AL access to SDS chow or fasting for three days (n=5 per group). Since the phenotypical effects of three days fasting were reported previously, transcriptome analysis was the only parameter for this group 1 . For a graphical overview of the experimental setup see Figure S3. Dietary intake and body weight Food intake and body weight were measured daily. To determine calorie intake, the daily food intake was corrected for the variation in the energy content (per gram of food) in the diets as follows: food intake per mouse times the number of calories per gram of food. Change in body weight was addressed in percentages calculated by dividing the body weight measurements during the dietary intervention through body weight at onset of the intervention period times 100. Surgical procedure Following each dietary intervention, bilateral renal IRI was induced as previously described 1 . In brief, mice were anesthetized via inhalation of isoflurane (5% isoflurane initially followed by 2-2.5% with oxygen for maintenance). Via midline abdominal incisions, the renal arteries and veins were exposed followed by occlusion of both renal pedicles for 37 minutes using non-traumatic vascular clamps. Purple discoloration of the