Franny Jongbloed

94 CHAPTER 4 target of the insulin receptor pathway and could function as an important nuclear factor mediating the binding of FOXO proteins to other nuclear receptors, including the retinoid nuclear receptor family and thereby regulating insulin target genes 44 . HNF4A is a nuclear TF that is involved in the development as well as in the metabolism of mainly the liver and kidney 45 . Upregulation of HNF4A occurs via co-stimulation of LXR and FXR and usually depends on the presence of low levels of stressors, such as interleukin-1 and tumor necrosis factor alpha. Transcriptional activity of HNF4A is also regulated indirectly by insulin through the action of FOXO1 45 . Activation of HNF4A results in inhibition of the activity of metabolic sensors, including SREBF and the mammalian target of Rapamycin (mTOR) 46,47 . This leads to downregulation of metabolism, in particular cholesterol metabolism, and may contribute to increased stress resistance via the downregulation of mTOR 47,48 . We showed that the activity of mTOR, as determined by the phosphorylation of the ribosomal protein S6, was significantly downregulated after three days of fasting. A trend towards lower phosphorylation levels was seen after the other two protective diets: two weeks 30% DR and a 3-day protein-free diet. These data indicate that mTOR may play an important role in the protection against IRI, which may vary according to the type of nutrient deprivation that is offered. Another pathway involved in nutrient sensing is GCN2, of which eIF2α is an important downstream target. GCN2 becomes transcriptionally activated by deprivation of amino acids and phosphorylates eIF2α, which leads to the activation of pathways involved in stress resistance. We did not find transcriptional regulation of the Gcn2 gene in any of the three protective diets. Although our data do not preclude posttranscriptional regulation of this pathway, they corroborate with those of Robertson et al 49 , who observed that GCN2 signaling was not required for protection against renal IRI by protein restriction. In summary, we demonstrated that three days of a protein-free diet in mice protects against renal IRI, similar to two weeks of 30% DR and three days of fasting. Comparative transcriptional analysis of kidney tissue following these dietary interventions demonstrated a significant overlap in differentially expressed genes and pathways, which are involved in resistance against oxidative damage induced by renal IRI. A meta-analysis of pathways and TFs indicates that DR upregulates at least four TFs that activate a transcriptional response, which, in turn, increases nuclear receptor signaling dependent and independent- cellular stress resistance. However, our attempt to understand the beneficial effects of different dietary restriction regimens on renal IRI by transcriptome analysis suggests that pivotal molecular changes also occur beyond the transcriptional level, and that additional ‘omics” analyses, including proteomics, are needed to come to a complete understanding.