1. Volume 82, Issue 7, Pages 771-789 (October 2012)
Activation of peroxisome proliferator-activated receptor-γ coactivator 1α ameliorates mitochondrial dysfunction and protects podocytes from aldosterone-induced injury 
Yanggang Yuan, Songming Huang, Wenyan Wang, Yingying Wang, Ping Zhang, Chunhua Zhu, Guixia Ding, Bicheng Liu, Tianxin Yang, Aihua Zhang 
Kidney International 
Volume 82, Issue 7, Pages (October 2012)
DOI: /ki
Copyright © 2012 International Society of Nephrology Terms and Conditions

2. Figure 1
Aldosterone (Aldo)-induced mitochondrial dysfunction (MtD) in podocytes. (a–d) Aldo-induced reactive oxygen species (ROS) production of mitochondrial origin. (a) Confluent podocytes in chamber slides were exposed to vehicle or Aldo (100nmol/l) for 2h in the presence of dichlorodihydrofluorescein diacetate (DCFDA). (b, c) Quantitation of 2′,7′-dichlorofluorescein (DCF) fluorescence by flow cytometry. In the presence of DCFDA, confluent podocytes were treated with 100nmol/l Aldo for the indicated periods of time (b) or incubated with Aldo at the indicated concentrations for 2h (c). (d) Confluent podocytes were pre-treated with apocynin (Apo, 100μmol/l) or rotenone (ROT, 10μmol/l) for 30min, and then stimulated by 100nmol/l Aldo for further 2h in the presence of DCF. DCF fluorescence was detected by flow cytometry. (e) Mitochondrial ultrastructure morphology. Podocytes were treated with vehicle or Aldo for 24h (× 50,000). (f) Representative images of podocytes stained with JC-1. Podocytes were exposed to vehicle or Aldo for 24h. (g) Aldo induced MtD in podocytes. Podocytes were stimulated with the indicated concentrations of Aldo for 24h. Mitochondrial membrane potential (MMP), adenosine-5′-triphosphate (ATP) content, and mitochondrial DNA (mtDNA) copy number were detected as described in Materials and Methods section. Values represent the means±s.e.m. (n=6). *P<0.01 vs. control. #P<0.01 vs. the Aldo-treated group. Cntl, control; rRNA, ribosomal RNA.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

3. Figure 2
Aldosterone (Aldo)-induced podocyte injury. (a, b) Aldo-induced podocyte apoptosis. (a) Hoechst staining. Confluent podocytes were exposed to vehicle or Aldo for 48h. Arrow indicated apoptosis-induced chromatin condensation and fragmentation (× 200). (b) Quantification of apoptotic cells by flow cytometry. Podocytes were incubated with Aldo at the indicated concentrations (0–100nmol/l) for 48h. (c) Caspase-3, -8, and -9 activities. Podocytes were treated as in b. (d) Real-time reverse transcriptase (RT)-PCR analysis for nephrin and podocin expression. (e) Western blotting analysis for nephrin and podocin. The cells were treated with Aldo (0–100nmol/l) for 24h. Left: representative immunoblots. Right: densitometric analysis. The data are expressed as the means±s.e.m. (n=6). *P<0.01 vs. control. Cntl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

4. Figure 3
Aldosterone (Aldo)-induced mitochondrial dysfunction (MtD) via mineralocorticoid receptor (MR). (a) Reactive oxygen species (ROS) production. Confluent podocytes were pre-treated with eplerenone (EPL, 10μmol/l) or RU-486 (20μmol/l) for 30min, and then stimulated by 100nmol/l Aldo for further 2h in the presence of dichlorofluorescein (DCF). DCF fluorescence was quantified by flow cytometry. (b) Representative images of podocytes stained with JC-1 (× 200). (c) Effects of EPL and RU-486 on MMP, adenosine-5′-triphosphate (ATP) content, and mitochondrial DNA (mtDNA) copy number. (d) Western blotting analysis for nephrin expression. The podocytes were pre-treated with EPL (10μmol/l) or RU-486 (20μmol/l) for 30min, and then stimulated by 100nmol/l Aldo for further 24h. Results are presented as means±s.e.m. (n=6). *P<0.01 vs. vehicle group. #P<0.01 vs. the Aldo-treated group. Cntl, control.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

5. Figure 4
Aldosterone (Aldo)-reduced peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and silent mating type information regulation 2 homolog 1 (SIRT1) expression in podocyte. (a) Expressions of PGC-1α in kidney and podocyte. Left: representative reverse transcriptase (RT)-PCR, right: immunoblots. (b) Immunofluorescence detection of PGC-1α in podocytes (× 200). (c, d) Aldo decreased PGC-1α expression. Podocytes were treated with Aldo (0–100nmol/l) for 24h. (c) Real-time RT-PCR analysis. (d) Western blotting analysis. Left: representative immunoblots. Right: densitometric analysis. (e) Effect of actinomycin D (ActD) on Aldo-induced decreases of PGC-1α mRNA expression. Podocytes were pre-treated with ActD (1μmol/l) and then stimulated with Aldo for 24h. PGC-1α mRNA expression were determined by real-time RT-PCR. (f) The time course of PGC-1α acetylation treated by Aldo. Podocytes were treated with 100nmol/l Aldo for the indicated periods of time. PGC-1α was immunoprecipitated (IP) and then immunoblotted with an anti-acetylated lysine antibody. (g) Aldo decreased SIRT1 expression. Podocytes were treated with Aldo (0–100nmol/l) for 24h, and SIRT1 mRNA and protein expression was determined by real-time RT-PCR (left) and immunoblotting analyses (right), respectively. (h) Aldo decreased PGC-1α and SIRT1 expression via mineralocorticoid receptor (MR). The podocytes were pre-treated with eplerenone (EPL, 10μmol/l) or RU-486 (20μmol/l) for 30min, and then stimulated by 100nmol/l Aldo for further 24h. The data are expressed as the means±s.e.m. (n=5). *P<0.01 vs. control. #P<0.01 vs. the Aldo-treated group. Cntl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IgG, immunoglobulin G.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

6. Figure 5
Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) silencing-induced mitochondrial dysfunction (MtD). Podocytes were transfected with PGC-1α short hairpin RNA (shRNA) for 24h, and PGC-1α mRNA and protein expression, mitochondrial membrane potential (MMP), adenosine-5′-triphosphate (ATP), and mitochondrial DNA (mtDNA) copy number were detected as described in Materials and Methods section. (a) Real-time reverse transcriptase (RT)-PCR analysis. (b) Western blotting analysis. Left: representative immunoblots. Right: densitometric analysis. (c) Representative images of podocytes stained with JC-1 (× 200). (d) MMP, ATP content, and mtDNA copy number. Values represent the means±s.e.m. (n=6). *P<0.01 vs. control and vehicle groups. Cntl, control; rRNA, ribosomal RNA; Vehi, vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

7. Figure 6
Short hairpin RNA (shRNA) silencing of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)-induced podocytes injury. Podocytes were transfected with PGC-1α shRNA for 24h, and podocyte apoptosis, caspase-3, -8, -9 activities, and nephrin and podocin expression were determined as described in Materials and Methods section. (a) Hoechst staining (× 200). Arrow indicated apoptosis-induced chromatin condensation and fragmentation. (b) Quantification of apoptosis by flow cytometry. (c) Caspase-3, -8, and -9 activities. (d) Western blotting analysis for nephrin and podocin expressions. Left: representative immunoblots. Right: densitometric analysis. Results are presented as means±s.e.m. (n=6). *P<0.01, #P<0.05 vs. control and vehicle groups. Cntl, control; Vehi, vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

8. Figure 7
Overexpression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) prevents aldosterone (Aldo)-induced mitochondrial dysfunction (MtD). (a) PGC-1α expression in infected podocytes. Podocytes were infected with Ad-PGC-1α at multiplicities of infection (MOI) 50 or empty vector (vehicle), with untreated cells used as the control (Cntl). Expression levels of PGC-1α in the indicated transfectants were analyzed by western blotting. Left: representative immunoblots. Right: densitometric analysis. (b) Mitochondrial ultrastructure morphology (× 50,000). After the infection for 24h, podocytes were treated with Aldo (100nmol/l) for another 24h. (c) Podocytes in chamber slides were infected with Ad-PGC-1α, then exposed to Aldo (100nmol/l) for 120min in the presence of dichlorodihydrofluorescein diacetate (DCFDA). (d) Quantitative analysis of reactive oxygen species (ROS). (e) Representative images of podocytes stained with JC-1. After infection for 24h, podocytes were treated with or without Aldo for another 24h before being stained with JC-1. (f) Mitochondrial membrane potential (MMP), adenosine-5′-triphosphate (ATP) content, and mitochondrial DNA (mtDNA) copy number were measured. Results are presented as means±s.e.m. (n=6). *P<0.01 vs. vehicle group. #P<0.01 vs. Aldo-treated group. Ad-, adenovirus; rRNA, ribosomal RNA; Vehi, vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

9. Figure 8
Overexpression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) inhibited aldosterone (Aldo)-induced podocytes injury. (a) Measurement of apoptosis. After infection of Ad-PGC-1α for 24h, podocytes were treated with or without Aldo for another 48h. Top: Hoechst staining. Bottom: representative photographs of double-fluorescence labelling of Hoechst nuclear staining (blue) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) (green). (b) Cytochrome c (Cyt c) release. The cells were treated as in a and apoptosis was determined by flow cytometry. After treatment with Ad-PGC-1α for 24h, podocytes were treated with or without Aldo for another 48h. Mitochondria and cytosolic proteins were isolated and Cyt c release was detected by immunoblotting. (c) Quantitation of apoptosis. The cells were treated as in a and apoptosis was determined by flow cytometry. (d) Caspase-3 and -9 activities. After the infection of Ad-PGC-1α for 24h, podocytes were treated with or without Aldo for another 24h. (e, f) Nephrin and podocin expression. After the infection of Ad-PGC-1α for 24h, podocytes were treated with or without Aldo for another 24h. (e) Real-time reverse transcriptase (RT)-PCR analysis. (f) Western blotting analysis. Left: representative immunoblots. Right: densitometric analysis. Results are presented as means±s.e.m. (n=5). *P<0.01 vs. vehicle. #P<0.01 vs. Aldo treatment group. Cntl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Vehi, vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

10. Figure 9
Overexpression of silent mating type information regulation 2 homolog 1 (SIRT1) prevented aldosterone (Aldo)-induced mitochondrial dysfunction (MtD) via peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). (a) SIRT1 expression in Ad-SIRT1-infected cells. Podocytes were infected with increasing multiplicities of infection (MOI: 5, 10, 20, and 50) of Ad-SIRT1, and the expression of SIRT1 was examined by immunoblotting after 24-h infection. (b) PGC-1α mRNA expression. Podocytes were infected with Ad-SIRT1 for 24h at increasing MOI of 10, 20, and 50, and PGC-1α expression was detected by real-time reverse transcriptase (RT)-PCR analysis. (c) PGC-1α luciferase reporter activity. Podocytes were co-transfected with SIRT1 and PGC-1α reporter vector for 24h. (d) PGC-1α and mitochondrial transcription factor A (TFAM) expression. After the infection for 24h, podocytes were treated with or without Aldo for another 24h at increasing MOI of 10, 20, and 50, and PGC-1α and TFAM expression was detected by immunoblotting. (e) Podocytes were co-infected with Ad-SIRT1 and PGC-1α short hairpin RNA (shRNA) for 24h and stimulated with Aldo (100nmol/l) for another 24h. TFAM expression was detected by immunoblotting. (f) PGC-1α acetylation. After the infection for 24h, podocytes were treated with or without Aldo for another 24h. PGC-1α was immunoprecipitated (IP) and then immunoblotted with an anti-acetylated lysine antibody. (g) Mitochondrial membrane potential (MMP), adenosine-5′-triphosphate (ATP) production, and mitochondrial DNA (mtDNA) copy number. Podocytes were co-infected with Ad-SIRT1 and PGC-1α shRNA for 24h and stimulated with Aldo (100nmol/l) for another 24h. Results are presented as means±s.e.m. (n=5). *P<0.01 vs. vehicle. #P<0.01 vs. Aldo treatment group. ΔP<0.01 vs. Aldo plus SIRT1-overexpressed group. A+S, Aldo plus SIRT1 overexpressed group; Cntl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Vehi, vehicle; WB, western blotting.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

11. Figure 10
Overexpression of silent mating type information regulation 2 homolog 1 (SIRT1) protected against aldosterone (Aldo)-induced podocytes injury via peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). (a) Quantification of apoptotic cells. Podocytes were co-infected with Ad-SIRT1 and PGC-1α short hairpin RNA (shRNA) for 24h and stimulated with Aldo (100nmol/l) for another 48h. (b, c) Nephrin and podocin expression. Podocytes were co-infected with Ad-SIRT1 and PGC-1α shRNA for 24h and stimulated with Aldo (100nmol/l) for another 24h. (b) Real-time reverse transcriptase (RT)-PCR analysis. (c) Western blot analysis. Upper: representative immunoblots. Lower: densitometric analysis. Results are presented as means±s.e.m. (n=5). *P<0.01 vs. vehicle. #P<0.01 vs. Aldo treatment group. ΔP<0.01 vs. Aldo plus SIRT1-overexpressed group. A+S, Aldo plus SIRT1 overexpressed group; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Vehi, vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

12. Figure 11
Effect of resveratrol (RSV) on aldosterone (Aldo)-induced podocyte injury and mitochondrial dysfunction (MtD). Podocytes were pre-treated with RSV (10–50μmol/l) or RSV (50μmol/l) plus nicotinamide (NAM) (10mmol/l) for 30min followed by incubation with Aldo (100nmol/l) for further 12h (for PGC-1α acetylation, panel d), 24h (for real-time reverse transcriptase (RT)-PCR analysis, b; immunoblotting analysis, c; and mitochondrial function detection, e, or 48h (for apoptosis analysis, a)). (a) Apoptosis analysis. (b) Real-time RT-PCR analysis. (c) Immunoblotting analysis. Upper: representative immunoblots. Lower: densitometric analysis. (d) PGC-1α acetylation. (e) Mitochondrial membrane potential (MMP), adenosine-5′-triphosphate (ATP) production and mtDNA copy number. Results are presented as means±s.e.m. (n=6). *P<0.01 vs. control. #P<0.01 vs. Aldo treatment group. ΔP<0.01 vs. Aldo plus RSV (50μmol/l) group. Cntl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mtDNA, mitochondrial DNA; rRNA, ribosomal RNA; TFAM, mitochondrial transcription factor A.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

13. Figure 12
Podocyte apoptosis in aldosterone (Aldo)-infused mice. (a) Triple fluorescence labelling using 4,6-diamidino-2-phenylindole (DAPI), mouse anti-synaptopodin immunoglobulin G (IgG) and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay in renal cortex sections of sham and Aldo-infused mice. Arrow indicated synaptopodin and TUNEL-positive podocytes. (b) Bar graphs show the mean number of synaptopodin and TUNEL-positive podocytes per glomerular cross-section. Values represent the means±s.e.m. (n=8 for each group). *P<0.01 vs. sham group.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

14. Figure 13
Effect of resveratrol (RSV) on podocyte injury in aldosterone (Aldo)-infused mice. (a) Kidney histology (× 200). (b) Urinary albumin excretion. (c) Foot processes of podocytes by transmission electron microscopy (TEM) (× 50,000). (d) Quantitative analysis of the slit width. (e) Representative immunoblotting of nephrin, podocin, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), mitochondrial transcription factor A (TFAM) and silent mating type information regulation 2 homolog 1 (SIRT1) expression in control, Aldo-infused kidneys and RSV-treated Aldo-infused kidneys. (f) Densitometric analysis of the representative immunoblots in e (upper panel) and real-time reverse transcriptase (RT)-PCR analysis (lower panel). (g) PGC-1α acetylation. Values represent the means±s.e.m. (n=8 for each group). *P<0.01 vs. control. #P<0.01 vs. Aldo-infused mice.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

15. Figure 14
Effect of resveratrol (RSV) on mitochondrial function in aldosterone (Aldo)-infused mice. (a) Mitochondrial morphology in glomerular podocytes (× 5000). (b) Mitochondrial membrane potential (MMP), adenosine-5′-triphosphate (ATP) production, and mitochondrial DNA (mtDNA) copy number. (c) Mitochondrial respiratory chain enzyme complexes I, II, III, and IV activities. (d) Urinary F2-isoprostane levels, malondialdehyde (MDA) levels in kidneys, glomerular reactive oxygen species (ROS) production, and ROS induction in isolated glomerular mitochondria. Values represent the means±s.e.m. (n=8 for each group). *P<0.01 vs. control group. #P<0.01 vs. Aldo-infused mice. rRNA, ribosomal RNA.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions