Animal preparations. Ten-week-old C57BL/6 male mice (Koatech, Gyounggido, Korea) were used. All experiments were approved and performed in accordance with the approved guidelines of the Institutional Animal Care and Use Committee of Kyungpook National University, Republic of Korea. The mice were provided free access to water, but water supply was stopped for some mice for 24 to 48 h. Tubastatin A (10 mg/kg BW; Selleckchem, Houston, TX, USA) or 2% DMSO/saline (vehicle) were intraperitoneal injected daily from 2 days before water restriction until sacrifice. Five mice were included in each group. The mice were euthanized by an over-dose of pentobarbital sodium (65 mg/kg body wt; Sigma-Aldrich, St. Louis, MO). To perform biochemical and histological experiments, the kidneys were either frozen in liquid nitrogen or perfusion-fixed with periodate-lysine-paraformaldehyde (PLP) (4% paraformaldehyde, 75 mM l-lysine, 10 mM sodium periodate; Sigma-Aldrich, St. Louis, MO, USA) immediately after retrieval.
Blood was withdrawn from the heart of mice using a heparinized syringe. Urine was collected with abdominal massage or by housing the mice individually in metabolic cage. Urine and plasma osmolalities were measured using cryoscopic osmometer (Osmomat 030-D, Gonotec, Berlin, Germany).
Cell culture. Madin-Darby canine kidney cells (MDCK, CCL-34™, ATCC Inc., Manassas, VA, USA) were cultured in MEM (Corning, NY, USA) with 5% FBS (Mediatech Inc., Herndon, VA, USA) with 100 units/mL of streptomycin/penicillin (S/P) (WelGENE Inc., Daegu, Korea). Four days after confluent growing, 10 µM of tubastatin A were treated to the cells 4 days after confluent-grown. Four h thereafter, the cells were treated with either 10 or 20 mM NaCl or 20 mM mannitol. At 24 h after these treatments, the cells were either fixed with 4% paraformaldehyde for immunofluorescence or lysed using lysis buffer for western blot analyses.
Immunofluorescence staining. Kidney paraffin-sections were stained with anti-acetylated-α-tubulin (ac-α-tubulin, 1:200; Sigma-Aldrich), anti-Na/K-ATPase (1:100 dilution; Santa Cruz, Santa Cruz, CA, USA), anti-aquaporin-1 (anti-AQP1; 1:200 dilution; Alomone Laboratories, Jerusalem, Israel), and anti-AQP2 (1:200 dilution, Alomone Laboratories) antibodies. The fixed cells were stained with anti- ac-α-tubulin for the measurement of primary cilia length. To detect the cell nuclei, DAPI was applied to the samples. Images were captured using Leica microscope (DM2500, Wetzlar, Germany).
Measurement of primary cilium length. Images were captured using a microscope. Primary cilia length was measured blindly. We randomly captured (x 400) 5–10 fields in the cortex, outer medulla, and inner medulla. More than 50 cells from each group were used to measure the length of the primary cilia using I-Solution software (IMT I-Solution, Rochester, NY, USA).
BrdU incorporation assay. 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg body weight; Sigma) incorporation assay was performed to determine cell proliferations. BrdU was administered to mice beginning on 1 day before cisplatin injection, every other day until sacrifice. Kidney sections were subjected to immunohistochemical staining using anti-BrdU (1:200 dilution; Serotec, Oxford, UK) antibody. Photomicrographs were obtained using a Leica microscope.
Scanning electron microscopy. Kidneys were perfusion-fixed with a fixing agent (0.5% glutaraldehyde and 0.5% paraformaldehyde) and then immersed in the fixing agent for 12 h. Kidneys were post-fixed with 1% osmium tetroxide for 1 h at 4 °C. After rinsing with 0.1-M phosphate buffer, the kidneys were immersed serially in 25% and 50% DMSO for 30 min each. The kidneys were rapidly frozen on a metal plate by chilling with liquid nitrogen and then cracked using a scalpel and hammer. The cracked kidneys were thawed with 50% DMSO, washed thrice with 0.1-M phosphate buffer, and placed in 1% osmium tetroxide for 1 h at 4 °C. Kidneys were transferred to 25% tannic acid for 2 h at room temperature and placed in 1% osmium tetroxide for 1 h at 4 °C. The kidneys were dehydrated using an ethanol series and isoamyl acetate. The dehydrated kidneys were subjected to critical point dry and mounted. Pictures were obtained using a scanning electron microscope (H-2500, Hitachi, Japan).
Western blot analyses. Western blot analyses were performed using the anti-ac-a-tubulin (Sigma-Aldrich), -α-tubulin (Sigma-Aldrich), -PCNA (DAKO, Carpinteria, CA, USA), -p21 (Santa Cruz), -GAPDH (NOVUS, Littleton, CO, USA), and -β-actin (Sigma-Aldrich).
Quantitative real time RT-PCR (qRT-PCR) analysis. RNA was extracted using PureHelix RNA extraction solution (Nanohelix, Seoul, Korea). For cDNA synthesis, 1 µg of RNA was used with the DiaStar RT Kit (SolGent, Daejeon, Korea). qRT-PCR was performed with AmpiGene qPCR Green Mix Hi-ROX (Enzo Life Science, Farmingdale, NY, USA) and AriaMx Real-Time PCR machine (Agilent Technology, Santa Clara, CA, USA). Mouse qRT-PCR primer sequences were 5’- AAC CCT GAG ACA AGA GTG CCA GTT-3’ and 5’- TCA GTT GCT CTC TGA TGG CAT GGA-3’ for HDAC6, sense and antisense, respectively.
Measurement of HDAC6 activity. Measurement of HDAC6 activity was based on deacetylase activity of HDAC6 towards a synthetic acetylated-peptide substrate resulting in the release of an AFC fluorophore. Activity level was determined using an HDAC6 activity assay kit (Biovision Incorporated, Milpitas, CA, USA). Briefly, kidney samples were homogenized in HDAC6 lysis buffer and incubated on ice for 5 min. After centrifugation of the homogenate at 16,000 g, 4℃ for 10 min, the supernatant was used for determination. In 96-well plate, samples were transferred and HDAC6 substrate mix was added and incubated at 37℃ for 30 min. To stop the reaction, developers were added to each well and incubated at 37℃ for 10 min. Then, the fluorescence intensity was determined using a fluorescence spectrometer (Molecular Devices, California, USA) for 30 min at 380/490 nm at 37℃.
Statistical analyses. Using GraphPad Prism 6 software (San Diego, CA, USA), all data were analyzed. Results are expressed as mean ± standard deviation. Statistical differences among the groups were assessed using Student's t-test and two-way ANOVA with repeated measures followed by post hoc Bonferroni’s multiple comparisons test. Differences were considered statistically significant at a p value < 0.05.