Sirtuin 3–dependent mitochondrial dynamic improvements protect against acute kidney injury (original) (raw)

Experimental design. Two-month-old C57BL/6J, C57BL6 × 129 (Charles River Italia Srl), and Sirt3–/– female mice (provided by Frederick Alt, Harvard Medical School, Boston, Massachusetts, USA) were used. Sirt3–/– mice were generated in a mixed genetic background as previously described (11). Animals were housed in a constant-temperature room (22–24°C) with a 12-hour dark/12-hour light cycle and fed a standard diet. AKI was induced by subcutaneous injection of _cis_-diamminedichloroplatinum (cisplatin 17.4 mg/kg; Ebewe Italia Srl) or by an intramuscular injection with hypertonic glycerol (8 ml/kg body wt of a 50% glycerol solution; Sigma-Aldrich) into the inferior hind limbs. To investigate the effects of AICAR (Toronto Research Chemicals Inc.) and ALCAR (Sigma-Tau Pharmaceuticals Inc.) in the cisplatin-induced AKI model, C57BL/6J and Sirt3–/– mice receiving cisplatin were i.p. injected 24 hours later with AICAR (500 mg/kg) (33) or ALCAR (200 mg/kg) (34) for 3 days. The mice were then sacrificed 4 days after cisplatin treatment, and kidney samples were used for histology and immunohistochemistry evaluations. Normal mice served as the control. To assess survival, an additional group of C57BL6 × 129 cisplatin-treated mice were compared with cisplatin-treated Sirt3–/– mice. The effect of AICAR or ALCAR on survival of Sirt3–/– mice with cisplatin-induced AKI was also studied.

Renal function was assessed as BUN using the Reflotron test (Roche Diagnostics) and as serum creatinine using the Cobas Mira Plus autoanalyzer (Roche Diagnostics) according to the manufacturer’s instructions.

Estimation of mitochondrial numerical density and mean mitochondrial volume. Glutaraldehyde-fixed fragments of cortical kidney tissue were washed repeatedly in cacodylate buffer, postfixed in 1% OsO4, dehydrated through ascending grades of alcohol, and embedded in Epon resin. Ultrathin sections were stained with uranyl acetate for examination using a Philips Morgagni transmission electron microscope (TEM). Numerical density of mitochondria (NV, n/μm3) was estimated using morphometric analysis according to Weibel (35), using an orthogonal grid digitally superimposed onto digitized electron microscope pictures of proximal tubules at ×7,100. Briefly, the mitochondrial profile area density (NA) was estimated using the ratio between the number of mitochondria and the proximal tubular area in the image calculated on the basis of grid points. Mitochondrial volume density (VV) was determined using the ratio of grid points falling over mitochondria divided by the total number of points of the grid container in the proximal tubule section. NV was then estimated for each animal using the following formula (35): NV = (1/β) (_NA_3/2/_VV_1/2), where β is the shape coefficient for ellipsoidal mitochondria, calculated using the ratio of the harmonic mean of major and minor axes of mitochondrial sections measured on digital images. The mean mitochondrial volume was calculated for each animal as the ratio of VV to NV.

Oxidative damage. To determine protein nitration of tyrosine residues, paraffin kidney sections were fixed with methanol and incubated with 30% H2O2 for 30 minutes and then with 0.3% Triton X-100 in PBS for 15 minutes. Sections were incubated with a rabbit polyclonal anti-nitrotyrosine primary antibody (1:2,000, Upstate Biotechnology), followed by biotinylated goat anti-rabbit IgG secondary antibody (1:200, Vector Laboratories). Signal was developed using the Vecta­stain ABC kit and DAB reagents (Vector Laboratories). Negative controls were performed omitting the primary antibody. At least 30–40 non-overlapping sequential fields were analyzed. Each section was scored for intensity (0: absent, 1: faint, 2: moderate, 3: intense).

Renal histology. Kidney samples were fixed in Duboscq-Brazil, and paraffin sections were stained with periodic acid–Schiff (PAS). Luminal hyaline casts and tubular necrosis (denudation of the tubular basement membrane) were assessed in non-overlapping fields (up to 28 for each section) using a ×40 objective (HPF; Primo Start, Zeiss). The number of casts and tubular profiles showing necrosis was analyzed twice in a single-blind fashion.

Immunofluorescence analysis in renal tissue. For SIRT3 staining in renal tissue, 3-μm periodate-lysine-paraformaldehyde–fixed (periodate-lysine-PFA–fixed) cryosections were air dried and washed with PBS. After blocking nonspecific sites with 1% BSA, slides were incubated with goat anti–mouse SIRT3 (1:25, Santa Cruz Biotechnology Inc.) and rabbit anti–mouse VDAC (1:50, Sigma-Aldrich) antibodies, followed by rabbit anti-goat FITC-conjugated (1:80, Jackson Immuno­Research Laboratories) or donkey anti-mouse Cy3-conjugated secondary antibodies (1:80, Jackson ImmunoResearch Laboratories), respectively. Nuclei were stained with DAPI (Sigma-Aldrich), and the renal structure was labeled with rhodamine-labeled lens culinaris agglutinin (1:400; Vector Laboratories). SIRT3 quantification was examined in at least 25 fields for each animal by using the appropriate software (Axio Vision, Apotome Axio Imager Z2, Zeiss) and expressed as the SIRT3-positive area (μm2) in each field.

Immunoelectron microscopy. Fragments of periodate-lysine-PFA–fixed renal cortex were infiltrated with 2.3 M sucrose for at least 1 hour, sectioned, and transferred to nickel grids coated with Formvar (Electron Microscopy Sciences). After blocking with 1% BSA for 15 minutes, sections were incubated with goat anti–mouse SIRT3 antibody (1:400, Santa Cruz Biotechnology Inc.), followed by 12 nm gold-conjugated donkey anti-goat IgG secondary antibody (1:50, Jackson Immuno­Research Laboratories), then stained with 2% aqueous uranyl acetate and embedded in methylcellulose before being examined with TEM.

Cell culture and incubation. Human RPTECs were purchased from Lonza and grown in renal epithelial cell basal medium (REBM) supplemented with REGM SingleQuots (Lonza). To assess SIRT3 mRNA expression, RPTECs were exposed to 5 μM cisplatin for 24 hours in the presence or absence of AICAR (2 mM) or ALCAR (1 mM) added 1 hour before and during cisplatin incubation.

SIRT3 silencing. RPTECs were transfected with Silencer Select predesigned siRNA human SIRT3 (s23768, Life Technologies) (100 pmol) or with control nontarget siRNA (Ambion, Silencer Select Negative Control #2siRNA) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Forty-eight hours after transfection, cells were incubated with 5 mM cisplatin for 24 hours in the presence or absence of AICAR (2 mM) or ALCAR (1 mM) added 1 hour before and during cisplatin incubation for MitoTracker Red (Molecular Probes, Invitrogen) assessment. SIRT3 silencing resulted in 70 % inhibition of SIRT3 mRNA expression (si_NULL_: 0.93 ± 0.17, vs. si_SIRT3_: 0.29 ± 0.05 arbitrary units, P < 0.01).

SIRT3 overexpression. Cells seeded on 35-mm2 tissue culture plates at 70% confluence were transfected with plasmid DNA (GFP-tagged pCMV-hSIRT3, Origene Technologies) using Lipofectamine 2000 (Invitrogen, Life Technologies) as described in the manufacturer’s instructions. Briefly, cells were incubated with 5 μl Lipofectamine 2000 reagent and 2.5 μg plasmid DNA mixed in 2 ml medium for 6 hours followed by fresh medium for 24 hours. Then, the RPTECs were exposed to medium with or without cisplatin (5 μM; Ebewe Italia Srl) for 24 hours.

Immunofluorescence analysis in cultured cells. Cells were fixed in 2% PFA (Electron Microscopy Science) and 4% sucrose (Sigma-Aldrich), followed by permeabilization with 0.3% Triton X-100 (Sigma-Aldrich). Nonspecific binding sites were blocked with 2% FBS, 2% BSA, and 0.2% bovine gelatin. Cells were incubated with a mouse monoclonal anti-OPA1 antibody (1:50, BD Biosciences) or with a rabbit polyclonal anti-PINK1 antibody (1:50, Santa Cruz Biotechnology Inc.), followed by incubation with a donkey anti-mouse or goat anti-rabbit Cy3-conjugated secondary antibody (1:80, Jackson ImmunoResearch Laboratories), respectively. For colocalization studies, cells were then incubated with a mouse monoclonal anti–human mitochondria antibody (hMT, 1:50, Millipore) and a goat anti-mouse FITC-conjugated secondary antibody (1:80, Jackson Immuno­Research Laboratories). Nuclei were counterstained with DAPI (Sigma-Aldrich). Negative controls were obtained by omitting primary antibodies. Samples were examined using a confocal inverted laser microscope (LSM 510 Meta, Zeiss). The quantification of the area corresponding to OPA1 staining was performed in 15 random fields per sample, expressed as pixel2 (ImageJ 1.40g software; http://imagej.nih.gov/ij/), and normalized for the number of DAPI-positive cell nuclei. The quantification of the merged fluorescence (yellow area) of PINK1 expression (red) and hMT-positive mitochondria (green) was performed in 15 random fields per sample.

Mitochondrial morphology and membrane potential detection. Mitochondria were labeled by incubating living cells with the fluorescent probe MitoTracker Red (250 nM for 30 minutes, Molecular Probes, Invitrogen). Mitochondrial membrane potential was evaluated by exposing cells to JC-1 (5 μM for 30 minutes, Molecular Probes, Invitrogen), a dye exhibiting potential-dependent accumulation in mitochondria indicated by a fluorescence shift from green (cytoplasm) to red (mitochondria). At the end of incubations, cells were examined by confocal inverted laser microscopy; quantification of JC-1 red and green areas (pixel2) was performed in 15 random fields per sample; and mitochondrial membrane potential was expressed as the ratio of red to green fluorescence areas.

Real-time PCR. Mouse renal tissue and human RPTECs were harvested in TRIzol reagent (Invitrogen, Life Technologies), and total RNA was extracted according to the manufacturer’s instructions. Contaminating genomic DNA was removed by RNase-free DNase (Promega) for 1 hour at 37°C. The first-strand cDNA (2 μg) was produced using a SuperScript VILO cDNA Synthesis Kit (Life Technologies) according to the manufacturer’s procedure. No enzyme was added for reverse transcriptase–negative controls (RT–).

To amplify cDNA of mouse Nampt, Pgc1a, and Sirt3, we used TaqMan Universal PCR Master Mix (Applied Biosystems) and inventoried TaqMan assays of the Nampt gene (FAM/MBG probe Mm0129­3560m1), the Pgc1a gene (FAM/MBG probe Mm00447181m1), the Sirt3 gene (FAM/MBG probe Mm01275638m1), and a mouse α-actin endogenous control (VIC/MGB probe) according to the manufacturer’s instructions. PCR was performed on the Viia7 Real-Time PCR System (Applied Biosystems). The amplification profile consisted of 2 minutes at 50°C and 10 minutes at 95°C; the samples were cycled 40 times at 95°C for 15 seconds and 60°C for 60 seconds. Data were analyzed using the 2–ΔΔCT method and presented as fold changes relative to mouse wild type (control).

cDNA of human SIRT3 and GAPDH was amplified using inventoried TaqMan assays (FAM/MBG probe Hs_00202030­m1 and 4333764F, respectively) according to the manufacturer’s instructions.

Protein extraction from renal tissue and mitochondria. Renal tissues were homogenized in buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM NaVO4, 10 mM Na fluoride, 1 mM β-glycerophosphate, 20 mM H3NaO7P2, 10% glycerol, 1% Triton X-100, supplemented with protease inhibitor cocktail, Sigma-Aldrich) and centrifuged 15,000 g for 15 minutes at 4°C to remove detergent-insoluble material. Mitochondria were isolated from mouse renal tissue or human RPTECs using the Qproteome Mitochondria Isolation Kit (QIAGEN) according to the manufacturer’s protocol. Isolated mitochondria were solubilized in an appropriate buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 10 mM nicotinamide, 500 nM trichostatin A, 1% _n_-dodecyl-β-maltoside). Mitochondrial and total protein concentration was determined using a DC assay (Bio-Rad).

Western blot analysis. Equal amounts of proteins were separated on SDS-PAGE under reducing conditions and transferred to PVDF membranes (Bio-Rad). After blocking with 5% BSA in TBS supplemented with 0.1% Tween-20, membranes were incubated with the following antibodies: rabbit anti–phospho-AMPKα (1:1,000, Cell Signaling Technology), anti–total AMPKα antibody (1:1,000, Cell Signaling Technology), rabbit anti–acetylated lysine antibody (1:1,000, Cell Signaling Technology), mouse anti-DRP1 antibody (1:2,000, BD Biosciences), rabbit anti-MFF (1:1,000, Origene Technologies), mouse anti-OPA1 antibody (1:1,000, BD Biosciences), and rabbit anti-VDAC antibody (1:1000, Cell Signaling Technology). VDAC was used as a sample loading control in isolated mitochondria after stripping the same membranes. The signal was visualized using the corresponding horseradish peroxidase–conjugated secondary antibodies (1:30,000; Sigma-Aldrich) and enhanced chemiluminescence–Western Blotting Detection Reagent (Pierce, Thermo Fisher Scientific Inc.). Bands were quantified by densitometry using ImageJ 1.40g software.

Statistics. Data are expressed as mean ± SEM and in box-plot diagrams. Data analysis was performed using Prism software (GraphPad Software Inc.). Comparisons were made using ANOVA with the Bonferroni post hoc test or unpaired Student’s t test as appropriate. Statistical significance was defined as P < 0.05. Survival curves were calculated according to the log-rank test.

Study approval. All procedures involving mice were performed in accordance with institutional guidelines that are in compliance with national (D.L. n.116, G.U., suppl. 40, February 18, 1992, circolare no. 8, G.U., July 14, 1994) and international laws and policies (EEC Council Directive 86/609, OJL, no. 358, December 1987; Guide for the Care and Use of Laboratory Animals, National Academy Press, 1996) and were approved by the Institutional Animal Care and Use Committees of Mario Negri Institute, Milan, Italy.