*2.3. Mitochondrial Function*

Mitochondrial function was studied using the Seahorse XFp analyser (Agilent Technologies, Santa Clara, CA USA). The left kidney was excised, minced and mitochondria were isolated as previously described [17] in mitochondrial isolation bu ffer (containing 70 mM sucrose, 210 mM mannitol, 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid), 1 mM EGTA (ethylene glycol-bis (betaaminoethylether)-N,N,N'N'-tetraacetic acid) and 0.5% (w/v) fatty acid-free BSA(bovine serum albumin), pH 7.2). The protein content was determined using the Bio-Rad protein assay. Mitochondrial coupling or electron flow experiments were carried out at 37 ◦C according to Roger et al. [24] and results were analysed on the Wave 2.3.0 software (Agilent Technologies, Santa Clara, CA USA).
