Male BTBR (black and tan, brachyuric) Lepob/ob and BTBR wild-type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were kept on specific pathogen-free facility with constant temperature on a 12:12-hour light-dark cycle with free access to standard diet and water. At 8 weeks of age, when they had already developed albuminuria, BTBR ob/ob mice were randomly allocated to receive (n = 12 mice/group): vehicle (DMSO in saline solution) or honokiol (10 mg/kg in DMSO; BioVision, Milpitas, CA, USA) by once-daily intraperitoneal injection. The dose of honokiol was chosen according to available data in the literature49. Treatment lasted until mice were 14 weeks old. BTBR WT mice (n = 9) were followed for the same length of time as controls. Mice were euthanized through CO2 inhalation and their kidneys were collected and processed for analysis. Before sacrifice, mice were housed in metabolic cages for 24-hour urine collection for albuminuria assessment. Blood samples were collected for glucose, cholesterol and triglyceride measurements. The experimenters were not blind to the treatment, but they were blind for measurement of experimental outcomes. All animal experiments were conducted in accordance with institutional guidelines in compliance with national (D.L.n.26, March 4, 2014), and international laws and policies (directive 2010/63/EU on the protection of animals used for scientific purposes) and were approved by the Institutional Animal Care and Use Committees of Istituto di Ricerche Farmacologiche Mario Negri IRCCS.
Biochemical parameters were assessed as we previously described20,50. Blood glucose levels were assessed with a reflectance meter (OneTouch UltraEasy, LifeScan, Milpitas, CA, USA). Plasma cholesterol and triglycerides were measured using the Reflotron test (catalog 10745065202 and catalog 10745049202, Roche Diagnostic Corporation, Indianapolis, IN, USA). Urinary albumin excretion was measured with the ELISA test using the Bethyl test kit (catalog E101, catalog A90-134A and catalog A90-134P, Bethyl Laboratories Inc, Montgomery, TX, USA).
Quantitative (q) RT-PCR
Total RNA was isolated from whole kidney tissue as we previously described51. Briefly, TRIzol Reagent (catalog 15596026, Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer’s instructions. After treatment with DNase (catalog 6101, Promega Madison, WI, USA), cDNA was prepared using the SuperScript VILO cDNA synthesis kit (catalog 11754050, Thermo Fisher Scientific). qRT-PCR analyses were performed on ABI ViiA 7 Real-Time PCR system (Thermo Fisher Scientific). Sirt3, Sirt1, Sirt6 and Nampt were assessed using TaqMan Gene Expression Master Mix (catalog 4369016, Thermo Fisher Scientific) and the following TaqMan assays (Thermo Fisher Scientific): Mm01275638_m1, Mm01168521_m1, Mm01149042_m1 and Mm01293560_m1, respectively. Mouse ACTB Endogenous Control (VIC/MGB probe) was used to evaluate the housekeeping gene β-actin. Gene expression levels of Sirt3, Sirt1, Sirt6 and Nampt were normalized to β-actin levels. Nrf2 and Keap1 were assessed using SYBR Green PCR Master Mix (catalog 4367659, Thermo Fisher Scientific) and the following primers (300 nM): Nrf2 forward 5′-CCCAGCAGGACATGGATTTGA-3′ and reverse 5′-CATAGTCCTTCTGTCGCTGACT-3′; Keap1 forward 5′-ACGTCCTCGGAGGCTATGAT-3′ and reverse 5′- GGGTCACCTCACTCCAGGTA-3′ β-actin (forward 5′-CACTGTCGAGTCGCGTCC-3′ and reverse 5′-TCATCCATGGCGAACTGGTG -3′) was used as endogenous control. Relative quantities were calculated by the 2-ΔΔCt method. Data are presented as fold change relative to WT control group.
Kidney samples were fixed in Duboscq-Brazil (catalog P0094, Diapath, Bergamo, Italy), dehydrated, and embedded in paraffin, as we previously described50. Three-micrometer sections were stained with periodic acid-Schiff (PAS) reagent, and at least 50 glomeruli were examined per animal. The degree of glomerular mesangial matrix expansion was quantified using a score between 0 and 3 (0 = no mesangial matrix expansion; 1 = minimal; 2 = moderate; 3 = diffuse mesangial matrix expansion). The number of glomeruli exhibiting mesangiolysis in an entire kidney section was counted and expressed as a percentage. All biopsies were reviewed by a blinded pathologist. Samples were examined using ApoTome Axio Imager Z2 (Zeiss, Jena, Germany).
For immunoperoxidase experiments, as we previously performed50, formalin-fixed, 3-μm paraffin-embedded kidney sections were incubated with Peroxidazed 1 (catalog PX968H, Biocare Medical, Pacheco, CA, USA) to quench endogenous peroxidase, after antigen retrieval in a decloaking chamber with DIVA, Rodent or Borg decloaker buffer (catalog DV2004MX, catalog RD913M and catalog BD1000MM, Biocare Medical) to increase the reactivity of antibodies to antigens. After blocking for 30 minutes with Rodent Block M (catalog RBM961G, Biocare Medical), sections were incubated with rabbit anti-SOD2/MnSOD (acetyl K68) (catalog ab137037, Abcam, Cambridge, UK, 1:50 and 1:200), rabbit anti-nitrotyrosine (catalog 06-284, Merck Millipore, Burlington, MA, USA, 1:100), rat anti-Mac 2 (clone M3/38, Cedarlane, Burlington, ON, Canada, 1:600), rabbit anti-PGC-1α (catalog ab54481, Abcam, 1:100) and rabbit anti-NRF2 (catalog ab31163, Abcam, 1:100) antibodies, followed by Rat on Mouse HRP-Polymer or Rabbit on Rodent HRP-Polymer (catalog RT517 and catalog RMR622G, Biocare Medical) for 30 minutes at room temperature. Stainings were visualized using diaminobenzidine (catalog BDB2004H, Biocare Medical) substrate solutions. Slides were counterstained with Mayer’s hematoxylin (catalog MHS80-2.5 L, Bio Optica, Milan, Italy), mounted with Eukitt mounting medium (catalog 09-00250, Bio Optica) and finally observed using light microscopy (ApoTome, Axio Imager Z2, Zeiss). Negative controls were obtained by omitting the primary antibody on adjacent sections. Glomerular acetylated SOD2 and nitrotyrosine stainings were quantified with a semiquantitative score between 0 and 3 (0: absent glomerular staining, 1: weak staining in a few glomerular cells, 2: moderate glomerular staining, 3: intense glomerular staining). At least 15–20 glomeruli/section for each animal were randomly analyzed. Tubular acetylated SOD2 was quantified with a semiquantitative score between 0 and 3 in proximal tubules (0: absent staining, 1: weak staining, 2: moderate staining, 3: intense staining). At least 10-15 fields/section for each animal were randomly analyzed. Mac-2-positive monocyte/macrophages within glomeruli were counted in a minimum of 50 glomerular cross-sections and expressed as average number of cells per glomerulus. Glomerular PGC-1α was evaluated by expressing the positive-PGC-1α nuclei as a percentage of the total nuclei per tuft (ImageJ software). At least 15–20 glomeruli/section per animal were randomly analyzed.
OCT-frozen kidney sections were fixed with cold acetone, blocked in 1% bovine serum albumin (BSA) and then incubated with the following primary antibodies: rat anti-mouse CD31 (catalog 550274, BD Pharmingen, San Jose, CA, 1:100), Cy3-conjugated mouse anti- α-smooth muscle actin (α-SMA) (catalog c6198, clone 1A4, Sigma Aldrich, St. Louis, MO, USA, 1:200), goat anti-nephrin (catalog sc-19000, clone N-20, Santa Cruz Biotechnology Inc, Dallas, TX, USA, 1:100) or rat anti-nestin (catalog ab81462, clone 7A3, Abcam, 1:300) followed by appropriate Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Cambridge, UK). For nephrin staining, the antigen retrieval in citrate buffer was performed. Negative controls were obtained by omitting the primary antibody on adjacent sections. Samples were examined using an inverted confocal laser microscope (LSM 510 Meta, Zeiss). Glomerular CD31-positive staining was quantified in 15 glomeruli in each section and expressing the positive glomerular areas as a percentage of the total area (ImageJ software)20. As we previously analyzed20, α-SMA, nephrin and nestin stainings were quantified by score from 0 to 3 (α-SMA 0–0.5: absent or weak signal, 1: mild, 2: moderate, 3: intense signal; nephrin, 0–0.5: absent or weak and fragmented signal, 1: linear and thin signal, 2: linear signal, 3: linear and intense signal; nestin, 0–0.5: absent or weak staining in a few podocytes, 1: moderate staining in podocytes with altered distribution, 2: moderate podocyte staining, 3: intense podocyte staining). At least 20 glomeruli/section for each animal were randomly analyzed. Immunohistochemical analysis and scoring assays were done by individuals unaware of sample identity.
Glomerular podocyte count
Formalin-fixed, 3-μm paraffin-embedded kidney sections were incubated with Peroxidazed 1, after antigen retrieval in a decloaking chamber with Rodent decloaker buffer. After blocking for 30 minutes with Rodent Block M, sections were incubated with rabbit anti-WT1 (catalog ab89901, Abcam, 1:600) antibody followed by Rabbit on Rodent HRP-Polymer for 30 minutes at RT. Stainings were visualized using diaminobenzidine substrate solutions. Slides were counterstained with Mayer’s hematoxylin, mounted with Eukitt mounting medium and finally observed using light microscopy (ApoTome, Zeiss). Negative controls were obtained by omitting the primary antibody on adjacent sections. At least 20 glomeruli/section for each animal were randomly acquired. The estimate of the average number of podocytes per glomerulus and the glomerular volume were determined through morphometric analysis, as previously described52.
Mitochondrial morphology was observed by transmission electron microscopy, as we previously performed51. Fragments of kidney tissue were fixed overnight in 2.5% glutaraldehyde (catalog 340855, Sigma Aldrich, Darmstadt, Germany) in 0.1 M cacodylate buffer (pH 7.4) (catalog 11652, Electron Microscopy Sciences, Hatfield, PA, USA) and washed repeatedly in the same buffer. After postfixation in 1% OsO4, specimens were dehydrated through ascending grades of alcohol and embedded in Epon resin. Ultrathin sections were stained with uranyl acetate replacement (catalog 22405, UAR, Electron Microscopy Sciences, Hatfield, PA 19440, USA) and lead citrate (catalog 22410, Electron Microscopy Sciences) and examined using transmission electron microscopy (Fei Morgagni 268D, Philips, Hillsboro, OR, USA).
In situ hybridization
Sirt3 in situ hybridization (ISH) was performed on 3-µm formalin-fixed and paraffin-embedded kidney sections using the RNAscope 2.5 HD Brown Assay kit (catalog 321720, ACD Bio-techne, Minneapolis, MN, USA) according to the manufacturer’s instructions (https://acdbio.com/manual-assays-rnascope). Briefly, deparaffinized slides were treated with hydrogen peroxide, heat and protease before hybridization with Sirt3 target probe (40 °C for 2 hours; HybEZ Oven). Sirt3 target probe (catalog 300031, ACD Bio-techne, Minneapolis) is designed on mouse Sirt3, transcript variant 3, mRNA reference sequence (NM_001177804 at the National Center of Biotechnology Information) and detects all the murine Sirt3 transcript variants. After hybridization, slides were washed and processed for standard signal amplification steps. Chromogenic detection was performed using 3, 3’-diaminobenzidine (DAB) followed by counterstaining with 50% Mayer’s hematoxylin (Bio Optica, Milan, Italy). Probes against the housekeeping gene PPIB (peptidylprolyl isomerase B) and the bacterial gene DapB were used as positive and negative controls, respectively. Sirt3 mRNA molecules were visualized as brown, punctate dots. Ten fields of tubule cells and twenty glomeruli for each animal were randomly acquired using bright-field microscopy (ApoTome, Axio Imager Z2, Zeiss). Image-based quantitative software analysis was performed to evaluate tubular and glomerular expression of Sirt3. Fiji Image J software (https://imagej.net/Fiji) was used for the quantification of the number of Sirt3 mRNA dots (representing single mRNA molecules). Orbit Image analysis software (https://orbit.bio) was used to count the number of cells. Sirt3 mRNA levels were expressed as average number of dots per cell.
Results were expressed as mean ± SEM and in bar chart with individual data points. Data analysis was performed using Graph Pad Prism software (Graph Pad, San Diego, CA, USA). Comparisons were made using one-way ANOVA with Tukey’s multiple comparisons post hoc test, and the statistical significance was defined as a p value of <0.05.