Citrate synthase activity assay

Kidney samples were washed twice in 0.9% (w/v) sodium chloride solution and homogenized in a CelLytic MT buffer (C3228; Sigma-Aldrich, St. Louis, MO, USA) supplemented with protease inhibitor cocktail (P8340; Sigma-Aldrich), and tissue disruption was completed by using a blunt-ended needle and a syringe. The lysates were centrifuged 16,000× g for 10 min at 4 °C, and supernatants were collected. The total protein concentration was determined using a Pierce™ BCA Protein Assay Kit (23227; ThermoFisher, Waltham, MA, USA). Ten micrograms of proteins was analysed by a citrate synthase activity assay (CS072; Sigma-Aldrich) according to the manufacturer’s protocol. The activity of citrate synthase was assessed by using the multimode microplate reader TECAN Infinite M200^® PRO (Tecan Group Ltd., Mannedorf, Switzerland) at 412 nm under a controlled temperature with a kinetic program.

To assess citrate synthase activity, tissue was homogenized in CelLytic Mt Cell Lysis Reagent (Sigma-Aldrich, Saint Louis, Missouri) with protease inhibitors (Sigma-Aldrich, Saint Louis, Missouri) and the activity level was measured using a colorimetric Citrate Synthase Activity Assay (Sigma-Aldrich, Saint Louis, Missouri). Specific activity was calculated as relative to protein mass, as measured by Bradford Assay.
Complex I and complex II activity levels were measured using immunocapture colorimetric assays (Abcam, Cambridge, Massachusetts); samples were prepared according to directions after having been flash frozen upon dissection. Specific activity was calculated as relative to protein mass, as measured by Bradford Assay.