CHARACTERIZATION OF RESPIRATORY FUNCTION OF ISOLATED RAT CORTICAL RENAL MITOCHONDRIA

Mitochondrial dysfunction is a major event in the pathophysiology of renal diseases, the kidney having the second highest mitochondria concentration after the heart. The aim of the present work was to standardize the isolation of rat cortical renal mitochondria in order to perform in vitro studies of high-resolution respirometry. Mitochondrial O-2 consumption was measured at 37C using the Oxygraph-2k (Oroboros). The multiple substrate-uncoupler-inhibitor titration (SUIT) protocol was used to assess mitochondrial respiration in the presence of Complex I (CI) substrates (glutamate and malate) and Complex II (CII) substrate (succinate plus the CI inhibitor, rotenone), respectively. The following parameters were measured: State 2 (basal respiration), State 3 (ADP-stimulated respiration or OXPHOS), State 4 (after the addition of oligomycin), and the uncoupled respiration (after the addition of FCCP); the ratio OXPHOS/State 4 or the respiratory control ratio (RCR) was further calculated. Mitochondrial integrity was verified after the addition of cytochrome c. A mean value of 4,67 +/- 0.1 for RCR (for CI substrates) and of 4.01 +/- 0.2 (for CII substrate) was obtained. The standardized rat cortical renal mitochondria isolation technique is a reliable method that will allow the characterization of mitochondrial respiratory dysfunction in renal pathology.