ABSTRACT: Obstructive nephropathy (ON) is a common clinical entity caused by different etiologies such as urolithiasis, prostatic hyperplasia in males, tumors, congenital stenosis, and others. Unilateral ureteral obstruction (UUO) in rodents is an experimental model widely used to explore the pathophysiology of ON, replicating vascular alterations, tubular atrophy, inflammation, and fibrosis development. On the other hand, mitochondria have been the subject of great attention, as morphological and functional alterations have been demonstrated in kidney diseases. Therefore, in this study, we explored the differences of the renal mitochondrial proteome during a time-course of 7, 14, and 21 days after the UUO in a rat model; and subsequently performed an overrepresentation analysis of biological processes to gain insight into the most relevant mitochondrial processes during UUO. We first isolated mitochondria from each UUO group and the control (sham) group, then the mitochondria were lysed in lysis buffer (8 M Urea, 20 mM HEPES, 1mM EDTA, pH 8.0) and digested using iST Sample Preparation Kit®. Peptides were separated using a UHPLC ACQUITY M-Class. Spectral data were acquired in an MS with electrospray ionization and ion mobility separation Synapt G2-Si operated with data-independent acquisition and ion mobility spectrometry in HDMSE mode. A total of 954 proteins were identified and quantified by label-free with Progenesis QI software and the database UP000002494 . Subsequently, after cross-checking with the MitoMiner database, we were left with 379 mitochondrial or mitochondrial transit proteins. Then, only proteins that had abundance values in at least 50% of the samples per study group and did not show atypical abundance values were selected, thus conserving 308 mitochondrial proteins for principal component analysis (PCA). PCA showed that 11 components can explain the total variation in protein abundance among samples and that the first two components can differentiate between the control group (sham) and the UUO groups. Considering the results obtained from the PCA analysis, a heat map was constructed with 243 proteins that strongly correlated (r ≤ -0.5 or r ≥ 0.5) with the first two principal components. The results show the abundance patterns for each UUO day and for the sham group and that these proteins are mainly involved in three metabolic pathways, oxidative phosphorylation (OXPHOS), the tricarboxylic acid cycle (TCA), and fatty acid (FA) metabolism.
Emmanuel Rios-Castro 
