Franny Jongbloed

82 CHAPTER 4 RESULTS Absence of dietary protein induces protection against renal IRI To determine the effect of short-term macronutrient deficiency on renal IRI, we provided 10-day, 14-day or 3-day regimens of protein-free, carbohydrate (CHO)-free and fat-free diets before inducing renal IRI. As previously shown, mice that were fed a protein-free diet for six or 14 days tend to voluntarily decrease their food intake as compared to animals that were fed a normal diet 19 . We indeed found that mice fed a protein-free diet for 10 days decreased their dietary food intake by approximately 30% (Figure S1A), resulting in significant body weight loss up until 20% on day 10 (Figure S1B). The reduction in food intake and body weight was less substantial in mice fed a carbohydrate (CHO)-free diet for 14 days. A fat-free diet for 14 days led to a small increase in body weight. Only the protein-free diet improved survival (Figure S1C) and kidney function (Figure S1D) following renal IRI. However, due to the reduction in food intake and body weight, the effect of the absence of protein per se could not be disentangled from the effect of calorie restriction. Subsequently, to separate the effect of the absence of protein-from calorie restriction, mice were fed a protein-free diet for three consecutive days. We first showed that survival and kidney function of mice receiving 30% DR for three days did not differ from mice fed ad libitum (Figure 1A, B) 11 . Mice fed the protein-free diet for three days had significantly improved survival (Figure 1C) and kidney function compared to control mice (P<0.05) (Figure 1D). The energy intake during the 3-day protein-free dietary intervention was decreased, but did not significantly differ from the intake of animals fed the control diet for three days ( P= 0.13) (Figure 1E). Body weight of mice on the protein free diet decreased by 9%, while body weight of mice that were fed a control diet did not change. Mice receiving 30% DR for three days lost about 8% of their body weight (Figure 1F). Common gene expression profiles between macronutrient-free diets, fasting and DR To examine, in an unbiased manner, the transcriptomic response of the kidney, microarray analysis was performed on 45,141 probe sets in kidney samples after three days of fasting, three days and two weeks of 30% DR, and three days of a protein-, fat- or carbohydrate-free diet. Each diet was subsequently compared to its corresponding AL fed control group.