Franny Jongbloed

89 4 A PROTEIN-FREE DIET PROTECTS AGAINST RENAL IRI denominators might be TFs showing the same directionality in the protective diets, but are oppositely directed or not regulated in the non-protective CHO-free diet. The TFs complying with these criteria, in descending order of absolute z-score, were forkhead box O3 (FOXO3), heat shock factor protein 1 (HSF1), and high mobility group AT-hook 1 (HMGA1). Furthermore, hepatocyte nuclear factor 4-alpha (HNF4A) was highly activated in the protective diets, but only minimally in the non-protective CHO-free diet. Also, sterol regulatory element-binding transcription factor 1 (SREBF1) and 2 (SREBF2) were significantly downregulated after three days of fasting and in the protein-free diet, activated in the CHO-free diet but not regulated after two weeks 30% DR. The non-protective three days 30% of DR showed similar results as the protective diets, since all TFs after three days of 30% DR were similarly regulated as after three days of fasting, two weeks 30% DR and the three days of a protein-free diet. The validity of these findings was further examined by determiningmRNA expression levels. The expression levels of Foxo1 were significantly higher after all diets except the fat-free diet (Figure S4A), while Foxo3 was significantly upregulated in all diets except after two weeks of 30% DR (Figure S4B). Foxo4 was only significantly upregulated after three days of protein- free and three days of a carbohydrate-free diet (Figure S4C). Hnf4α was not significantly regulated after any of the dietary interventions (Figure S4D). The mRNA expression level of downregulated transcription factor Srebf1 was only significantly downregulated after three days of fasting (Figure S4E), while Srebf2 was significantly downregulated after three days of fasting and three days of a protein-free diet (Figure S4F). Target pathways possibly involved in the protective effect of renal IRI Various pathways have been proposed to be involved in the protective response against renal IRI induced by DR and protein restriction. One of these is the eukaryotic translation factor 2 (eIF2α) signaling pathway, in which eIF2α is phosphorylated by the general control nonderepressible 2 (Gcn2) kinase, thereby inhibiting initiation of translation 19 . The role of Gcn2 and the eIF2α pathway is subject of debate, and the relevance of this pathway still needs to be elucidated 19,21 . Our microarray analyses showed a significant upregulation of eIF2α transcription factor only after three days of fasting, while the Gcn2 gene and other target genes of the eIF2α pathway were not significantly differentially regulated after any of the dietary interventions. The mammalian Target of Rapamycin (mTOR) signaling pathway mediates between growth factors, hormones and nutrients to regulate essential cellular functions including survival and protein translation. Inhibition of the mTOR pathway has been demonstrated to increase lifespan in various animal species 22,23 mTOR is part of mTOR complex 1 (mTORC1), a nutrient sensor complex that is involved in induction of oxidative stress resistance 24 . We found a downregulation of mTOR after two weeks 30% DR (-0.6;