Franny Jongbloed

95 4 A PROTEIN-FREE DIET PROTECTS AGAINST RENAL IRI Therefore, more research is warranted to further elucidate the role of these pathways in the induction of acute stress resistance by short-termDR, which may ultimately lead to “dietary restriction mimetic” therapeutic strategies that exploit the benefits of DR in humans. MATERIALS AND METHODS Study design Sample size calculation of our previous study was based on a 25% decrease of serum urea levels at time point 24 hours after renal IRI, with a standard deviation (SD) of 20% and a power of 0.8 1,11 . These experiments demonstrated that our renal IRI model was feasible and stable, and we therefore reduced the number of mice to six mice per group in the dietary intervention groups. Primary endpoint of this study was kidney function 24 hours after surgery, measured via serum urea concentrations. Secondary endpoints of this study were mortality rate in the first seven days after surgery and changes in gene expression profiles measured directly after each dietary intervention. Experimental data of two weeks 30% DR and three days of fasting groups were previously obtained 1 . Animals were euthanized and excluded from the experiment if their body weight decreased ≥20% of their preoperative weight or if they developed a moribund phenotype, including ruffled fur, hunched body posture, hypothermia, and decreased activity 11,12 . Animals C57BL/6 male mice, 10-12 weeks old (20-25 grams), were obtained from Harlan, the Netherlands. Animals were housed in individually ventilated cages (3-4 animals/cage) under standard conditions. All mice had ad libitum (AL) food and water (acidified with HCl to a pH of 2.4-2.7) except where noted. All experiments were performed with the approval of the Animal Experiments Committee of the Erasmus University Medical Center, Rotterdam, the Netherlands under the Dutch National Experiments on Animals Act and according to the ARRIVE Guidelines, Animal Research: Reporting of In Vivo Experiments 50 . Diets and experimental design The control chow for the fasting and dietary restriction groups was obtained from Special Diet Services (SDS, Witham, UK). All other diets were purchased from Research Diets, Inc. (New Brunswick, NJ, USA). The macronutrient composition and energy content of all diets are shown in Table S4. The control diet differed from the standard chow given (Special Diet Services, SDS) in the protein source, which consisted of lactic casein protein in case of control diet, while the SDS control consisted of crude protein. These two control diets are designated as “Control” and “SDS” throughout the manuscript.