Fig 1.
PGC-1α expression was decreased in the kidney of diabetic rats and correlated with renal lesions.
A: Real-time RT-PCR assay for PGC-1α mRNA relative to the control values. B: Western blotting analysis of PGC-1α expression in the control rats (control) and diabetic rats (DM). Equal protein loading was confirmed by staining with the tubulin antibody. C: Result of the correlation analysis between PGC-1α protein expression and 24-h microalbuminuria level. D: Glomerular H&E staining in the control and diabetes groups. Glomerular hypertrophy was obvious in the diabetes group. Original magnification: ×400. E: Immunohistochemical detection of fibronectin (FN) in glomeruli from control and diabetic rats. Data are expressed as the mean ± SD values from 5–10 rats per group, and the experiments were repeated independently at least 3 times with similar results (**P < 0.01 vs. the control; ***P < 0.001 vs. the control). Scale bar: 25 μm.
Table 1.
Physiological and biochemical data for the experimental groups (mean ± SD).
Fig 2.
Decreased PGC-1α expression is associated with a hyperglycemia-induced increase in mitochondrial fragmentation and ROS generation.
A: Kidney mitochondrial ROS production in the control and diabetic rats (DM). ROS production was identified by the fluorescent probe Mitochondrial ROS. B: Immunofluorescent micrographs of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the control and diabetic rats (DM). (Original magnification: ×400). Scale bar: 25 μm. C: Intracellular ROS production was determined by the mitochondrial fluorescent probe in RMCs exposed to normal glucose (NG), mannitol (Man), and high glucose (HG). Scale bar: 100 μm. D, E and F: Mitochondrial morphology was determined using the MitoTracker probe in RMCs exposed to NG, HG, and Man. D: Mitochondrial morphology at x200. Quantitative analysis of mitochondrial morphology was conducted using a computer-assisted morphometric analysis application for the calculation of form factor (FF) and aspect ratio (AR) values. Scale bar: 10 μm. G: Real-time RT-PCR assay for PGC-1α mRNA relative to the NG values. H: Western blotting analysis of PGC-1α expression in NG, HG, and Man. I: A correlation analysis between ROS generation and the mitochondrial morphology parameter, FF. J: A correlation analysis between ROS generation and PGC-1α expression. Data are expressed as the mean ± SD values for 5–10 rats per group or three cells per group, and the experiments were repeated independently at least three times with similar results (**P < 0.01 vs. control or vs. NG).
Fig 3.
PGC-1α inhibited hyperglycemia-induced elevation of ROS production as well as mitochondrial fragmentation.
A: Mesangial cells were transfected with PGC-1α short hairpin RNA (shRNA) for 48 h, and PGC-1α protein expression was detected by western blotting. PGC-1α expression was inhibited (~66% reduction) by PGC-1α shRNA. B: PGC-1α expression in transfected mesangial cells. Mesangial cells were transfected with the pcDNA3-PGC-1α (PGC-1α expression) plasmid or empty vector (pcDNA3), with untreated cells used as the control. Expression levels of PGC-1α in the indicated transfectants were analyzed by Western blotting. (C-F) ROS production, mitochondrial morphology changes, and computer-assisted morphometric analyses of mitochondrial morphology in RMCs exposed to normal glucose (NG) and high glucose (HG) conditions, RMCs transfected with PGC-1α shRNA plasmid (NG + PGC-1α shRNA) and shRNA control plasmid (NG+shRNA-con) and exposed to NG conditions, RMCs transfected with pcDNA-PGC-1α plasmid (HG + pcDNA3-PGC-1α) and empty plasmid pcDNA3 (HG + pcDNA3) and exposed to HG conditions, RMCs transfected with PGC-1α shRNA plasmid and DRP1-shRNA plasmid (NG + PGC-1α shRNA+DRP1-shRNA). ***P < 0.001, ** P < 0.01 versus NG, ## P < 0.01, # P < 0.05 versus HG. Scale bar: 10 μm.
Fig 4.
Inhibitory action of PGC-1α on mitochondrial fragmentation occurs via the downregulation of DRP1.
A: Western blotting analysis of DRP-1 and PGC-1α expression in the NG, NG+shRNA-con, NG+PGC-1α shRNA, HG, HG+pcDNA3, and HG+pcDNA3-PGC-1α groups. Equal protein loading was confirmed with tubulin antibody staining. Data are presented as the mean ± SD values for three cells per group, and experiments were repeated independently at least three times (*P < 0.05 vs. NG, # P < 0.05 vs. HG). B: Mesangial cells were transfected with DRP1 short hairpin RNA (shRNA) for 48 h, and DRP1 protein expression was detected by western blotting. DRP1 expression was inhibited (~50% reduction) by DRP1 shRNA. C-F: ROS production, mitochondrial morphology changes, and computer-assisted morphometric analyses of mitochondrial morphology in RMCs exposed to normal glucose (NG), NG incubated with DRP1 protein and high glucose (HG) conditions, RMCs transfected with DRP1 shRNA to silence the expression of DRP1 under HG conditions (HG+DRP1-shRNA), and RMCs transfected with pcDNA-PGC-1α to overexpress PGC-1α and exogenous DPR1 protein under HG conditions (HG+pcDNA3-PGC-1α+DRP1). ***P < 0.001,** P < 0.01 versus NG, ## P < 0.01, # P < 0.05 versus HG.
Fig 5.
PGC-1α suppresses mesangial cell hypertrophy induced by hyperglycemia.
The ratio of total amount of protein to cell number (A) and cell morphology (B) in RMCs incubated in normal glucose (NG) and high glucose (HG) conditions, RMCs transfected with PGC-1α shRNA or shRNA-con under NG conditions (NG+PGC-1α shRNA, NG+shRNA-con), and RMCs transfected with pcDNA3-PGC-1α or pcDNA3 under to HG conditions (HG+pcDNA3-PGC-1α, HG+pcDNA3). Data are represented as the mean ± SD values from three cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG, ## P < 0.01 vs. HG). Scale bar: 10 μm.