Franny Jongbloed

93 4 A PROTEIN-FREE DIET PROTECTS AGAINST RENAL IRI similarity with the protective 3-day protein-free diet, but with gene expression levels that were lower. These results indicate that the directionality, the number of probe sets and the expression levels are of importance in order to induce protection against renal IRI. To provide structure in the infinite amount of data generated by microarray analysis, we used meta-analytic approximations to specify for overlapping pathways and factors in all diets. A specific meta-analysis in which the DEPS were oppositely regulated in the protective diets versus the non-protective CHO-free diet, resulted in only 160 DEPS and yielded no additional pathways of interest compared to a meta-analysis including the CHO-free diet. In this meta-analysis several TFs, including FOXO3 and HNF4A, remained significantly upregulated in the protective diets whilst not or oppositely regulated in the CHO-free diet (data not shown). Activation of nuclear receptor signaling pathways dominated the top 10 overrepresented pathways after three days of fasting, two weeks of 30% DR and three days of a protein- free diet (Table 2). Specific retinoid receptors, including the retinoid X receptor (RXR), the pregnane X receptor (PXR) and the peroxisome proliferator-activated receptor (PPAR) 32 , were also prominently upregulated in our analysis, pointing towards a pivotal role for nuclear receptor signaling. Nuclear receptors are transcription factors that can be activated by steroid hormones and lipid-soluble agents, such as the retinoid acids (RAs) 33 . Administration of RAs induces many of the beneficial effects observed after DR. DR is able to ameliorate age-related insulin resistance and degenerative brain diseases, and similar results have been described after treatment with RAs 34-36 . Both DR and administration of RAs are able to protect from ischemic stroke in the brain 37-39 . Signaling pathways activated by the interaction between RAs and nuclear receptors have also been considered to have tumor-, and immune suppressive effects, just as DR 9,33,40 . Involvement of nuclear receptor signaling is further supported by the upstream TF analysis (Table 3), since the majority of the activated or inhibited TF could be directly or indirectly linked to the activation of nuclear receptors. The activated TFs in all protective diets that were oppositely or not regulated in the non-protective CHO-free diet are FOXO3, HNF4A, HMGA1 and HSF1. A role for each of these four TFs in increased stress resistance has been previously observed. For example, FOXO3 phosphorylation via the c-Jun N-terminal kinase -pathway results in its nuclear inclusion and activation of various processes involved in cellular stress resistance, biosynthesis, cell cycle regulation as well as apoptosis and autophagy 41,42 . A fasting-induced interaction, mediated by insulin signaling, with members of the FOXO family and RXR has been described as well 43 . HMGA1 is a downstream