Sirtuin 3 sirt3

AKF-PD (lot No. 20190810) was purchased from Haikou Pharma (Haikou, China). ATP assays were obtained from Beyotime Biotechnology (Shanghai, China). Recombinant human transforming growth factor-β (TGF-β) was provided by PeproTech (Rocky Hill, USA). MitoSOX Deep Red was purchased from Invitrogen (New York, USA). The following antibodies for immunohistochemistry and western blotting were used: sirtuin 1 (SIRT1), NOX4, E-cadherin, superoxide dismutase 2 (SOD2), and sirtuin 3 (SIRT3) antibodies, which were purchased from Proteintech (San Diego, USA). Other antibodies were from Abcam (Cambridge, UK): collagen I, collagen III, fibronectin (FN), total OXPHOS complexes, and 4-hydroxynonenal (4HNE). Anti-alpha smooth muscle actin (α-SMA) antibody and anti-GAPDH antibody were obtained from Sigma (St. Louis, MO, USA) and Cell Signaling Technology (Boston, MA, USA), respectively. All other chemicals were of analytical grade.

The kidney cortexes were homogenized in RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) containing a protease inhibitor cocktail. Equal amounts of kidney cortex lysates were loaded on and electrophoresed through 7% or 10% SDS-PAGE gels and were then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against fibronectin (FN, 1 : 250), type IV collagen (Col-IV, 1 : 250) (Abcam, Cambridge, MA, USA), α-smooth muscle actin (α-SMA, 1 : 1000), QPRT (1 : 500), β-actin (1 : 5000) (Sigma-Aldrich, St Louis, MO, USA), sirtuin 3 (SIRT3, 1 : 1000) (Proteintech, Wuhan, China), dynamin-related protein 1 (Drp-1, 1 : 1000) (Cell Signaling Technology, Beverly, MA, USA), and optic atrophy 1 (OPA-1, 1 : 2000) (BD Biosciences, San Jose, CA, USA) at 4°C overnight. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1 : 2000) (Life Technologies, Carlsbad, CA, USA) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA), the blots were visualized using the ChemiDoc MP Imaging System (BioRad Laboratories, Hercules, CA, USA). Bands were quantified by densitometry using Image Lab software version 5.1 (BioRad Laboratories, Hercules, CA, USA).