Zoledronate and fenofibrate were purchased from Tokyo Chemical Industry (Tokyo, Japan); Human TGFβ1 was purchased from PeproTech (Rocky Hill, USA) and SB431542 was obtained from Harveybio (Beijing, China); 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Biotechnology (St. Louis, MO, USA); Annexin V-FITC Apoptosis Detection Kit was obtained from KeyGEN Biotech. Co., Ltd (Nanjing, China); Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal bovine serum (FBS), penicillin–streptomycin, 0.25% trypsin and phosphate buffer saline (PBS) were purchased from Gibco (Grand Island, NY, USA); Saline, hematoxylin and eosin (H&E), periodic acid schiff (PAS) and Masson’s trichrome were purchased from Solarbio (Beijing, China), the Oil Red O (ORO) solution (0.5% in isopropanol) was purchased from Sigma-Aldrich Biotechnology; Human TGFβ1 ELISA kit was purchased from BOSTER (Wuhan, China). The antibodies as follows: TGFβ (CST-3709), p-SMAD3 (CST-9520), SMAD3 (BOSTER BM3559), Fibronectin (FN1) (BOSTER BA1771), Collagen I (BOSTER BA0325), α-smooth muscle actin (α-SMA) (CST-19245), ROCK (Abcam ab45171), Ras (Abcam ab52939), Cofilin (Abcam ab134963), NOX4 (BBI D121050), SLC27A2 (Proteintech 14048-1-AP), CD36 (CST-14347), Rho-GTPase antibody sampler kit (CST-9968), Combo Rho A/Rac1/Cdc42 Activation Assay Kit (Cytoskeleton BK030), SLC2A1 (CST-12939), PDHB (CST-3205), CPT1A (Proteintech 15184-1-AP), PPARA (BOSTER BA1691), β-Tubulin (EASYBIO BE0025), Na+-K+-ATPase (Abcam ab76020).
HK-2 (CRL-2190) and HEK 293T (CRL-3216) were purchased from ATCC (Manassas, VA, USA); NRK-52E was given as a present by Prof. Baoxue Yang (Peking University). All the cells were cultured in DMEM supplemented with 10% FBS and 100 IU/ml penicillin–streptomycin at 37 °C in a humid atmosphere with 5% CO2.
Cell viability assay
Cell viability was measured by using MTT assay. Briefly, HK-2 cells were seeded into a 96-well plate and exposed to zoledronate (0, 0.1, 1, 5, 10, 50 µM). After treatment for varying periods of time (24, 36, 48, 60, or 72 h), 20 μl MTT at 5 mg/ml was added to each well. The cells were incubated at 37 °C for another 4 h and then dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, USA) was added to each well. The absorbance was detected at 570 nm with a microplate reader (Molecular Devices SpectraMax M5, PerkinElmer, USA). Cell viability was expressed as a percentage of the control culture.
Flow cytometry with Annexin V/PI staining
The HK-2 cells were treated with zoledronate (0, 1, 10, 50 µM) for 48 h. Apoptosis rates were determined by flow cytometry (BD Biosciences, San Jose, CA, USA). Briefly, cells were washed with PBS once and collected by centrifuging at 1,200×g for 5 min, and then incubated with Annexin V-FITC (dilution 1:50) and PI (dilution 1:50) (KeyGEN Biotech) in binding buffer for 15 min in the dark at room temperature. Double-stained cells were analyzed immediately using flow cytometry and identified as apoptotic cells.
Proteomics of HK-2 cells with and without zoledronate treatment
After treatment with zoledronate (0 and 50 µM) for 48 h, cells were lysed using urea lysis buffer (Solarbio), and then were scraped and transferred to a 1.5 ml tube, incubated at 4 °C for 30 min, centrifuged for 10 min and protein concentrations were measured using the Bradford method. 200 mg of proteins from sample were reduced with 1 mM dithiothreitol (DTT) and alkylated with 5.5 mM iodoacetamide. Proteins were digested with trypsin for overnight, and stopped by 10% trifluoroacetic acid. The peptides were desalted using C18 Sep-Pak cartridges and eluted with 1 ml methanol. After centrifugation, peptides were redissolved in tetraethylammonium bromide and labeled using TMT sixplex labeling reagent. The TMT-labeled peptides were combined and desalted by C18 Sep-Pak cartridges. The fractions were centrifuged and analyzed by LC–MS/MS. The instruments and the data analysis methods were the same as described previously (Zhao et al. 2017).
Metabolomics of HK-2 cells with and without zoledronate treatment
After treatment with zoledronate (0 and 50 µM) for 48 h, HK-2 cells were washed three times by PBS, and then 1 ml of 80% methanol was added and incubated at −80 °C for 3 h. Cells were harvested and isolated by centrifugation at 14,000×g for 20 min at 4 °C. The protein concentration of the pellet was measured by the BCA assay kit (Solarbio) for normalization. The metabolite-containing supernatant of cells was transferred to a new tube and dried under nitrogen flow. The dried samples were stored in −80 °C freezer for subsequent analysis. The instruments and the data analysis methods were the same as described previously (Zhao et al. 2017).
Treatment of zoledronate or TGFβ1 and TGFβ receptor I inhibitor SB431542
HK-2 cells were seeded into 60 mm dishes and exposed to zoledronate (0, 50 µM) or human TGFβ1 (1 ng/ml) at 37 °C for 48 h, and then SB431542 (10 µM) treatment or DMSO control was added to the cell culture supernatant and harvested 1 h later for analysis.
Examination of ultrastructural changes by transmission electron microscopy (TEM)
HK-2 cells were treated 48 h with zoledronate and then chemically fixed with 2.5% glutaraldehyde buffered in 0.1 M PBS, pH 7.2. Cells were harvested with a cell scraper, washed with 0.1 M PBS, pH 7.2, and embedded in 2% agarose. Staining was performed with 1% osmium tetroxide for 50 min and with 1% uranyl acetate/1% phosphotungstic acid for 1 h. Dehydration of samples was done using graded acetone series. Specimens were embedded in Spurr epoxy resin and incubated for polymerization at 65 °C for 24 h. Sections were inspected with a TEM (H-7650, Hitachi, Japan).
Lipid droplets were stained with BODIPY 558/568 C12 (Thermo Fisher). After HK-2 cells were treated for 48 h by zoledronate, they were incubated with the dye at a final concentration of 20 μg/ml in PBS for 45 min at 37 °C, and rinsed to remove excess stain followed by a further incubation of 1 h at 37 °C in cell culture medium. Cells were fixed with 4% paraformaldehyde at 4 °C for 30 min. Fixed cells were washed with PBS before mounting on slides using Citifluor antifadent solutions (Citifluor Ltd, United Kingdom). Samples were analyzed using a BX63 fluorescence microscope.
Triglyceride (TG) measurement
All samples were measured using quantification kits (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s instructions, and then absorbance was determined by EnVision Microplate reader (Molecular Devices SpectraMax M5, PerkinElmer, USA).
Palmitic oxidation and palmitate uptake assay
Palmitic oxidation was performed according to Chen’s protocol (Chen et al. 2014). Briefly, HK-2 cells, followed by zoledronate (0, 50 μM), were incubated in the reaction medium (the same as Chen’s protocol). Reactions performed in a sealed flask were allowed to proceed for 30 min in a shaking water bath at 37 °C; 1 ml 3 M perchloric acid was added in the reaction medium to precipitate protein and nonmetabolized palmitate, and then incubated at room temperature for 2 h for collection of 14CO2 into a suspended well containing 500 μl of ethanolamine. Radioactivity was counted by a liquid scintillation counter (MicroBeta2, Perkin Elmer).
Palmitate uptake assay was similar to the aforementioned palmitate oxidation assay with the following changes (Pillon et al. 2015). HK-2 cells were cultured with [14C]-palmitate-containing medium for 1 h, washed three times with PBS, and then were trypsinized and transferred into glass tubes containing 3 ml methanol-chloroform (2:1), and the lipids were extracted by adding 1 ml of chloroform and 1 ml of NaCl (1 M). The lower layer was transferred into a glass tube, dried with nitrogen and redissolved with chloroform. Radioactivity was quantified by a liquid scintillation counter.
Intracellular ROS accumulation detection
The ROS production in different samples was determined by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (YEASEN, Shanghai, China). Briefly, HK-2 cells were seeded in the 6-well plates at 2 × 105 cells/well and pretreated with 0, 1, 10 and 50 µM zoledronate at 37 °C for 6 h, and then 500 µl DCFH-DA (10 µM final concentration) was added to each well incubated at 37 °C for 30 min. After washing three times with PBS, cells were harvested by cell scrapers and resuspended in PBS, were analyzed immediately using flow cytometry (Ex = 488 nm, Em = 525 nm).
Membrane and cytoplasmic fractions, Rho and Rac activation assays
For extracting membrane and cytosol proteins from the cell lysate Mem-PER plus membrane protein Extraction reagent kit was purchased from Thermo Fisher Scientific (89842). RhoA and Rac1 activation were assessed using commercially available kits from Cytoskeleton (Denver, CO USA), according to the manufacturer’s instructions, with modifications. HK-2 cells were harvested using provided lysis buffer containing protease inhibitors. To detect active RhoA, equal volumes of clarified lysates were incubated with Rho-GTP binding domain of the Rhotekin protein bound to GST beads. To detect active Rac1, lysates were incubated with Rac/Cdc42 binding domain of p21-activated kinase protein bound to GST beads for 1 h at 4 °C. Active GTP-Rho, GTP-Rac, and whole cell lysates were subjected to electrophoresis on 12% acrylamide gels and analyzed via western blot techniques.
To determine the effect of zoledronate on cell cytoskeletal organization, we utilized the phalloidin immunofluorescence staining method. HK-2 cells were cultured on glass coverslips. After washing with PBS, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Then the samples were incubated with FITC-phalloidin (40735ES75) in PBS for 30 min. Subsequently, the samples were counterstained with DAPI for 10 min. The images of samples coverslips were examined with BX63 fluorescence microscopy (Olympus, Japan).
Generation of Slc27a2
−/− mice model was generated using the CRISPR/Cas9 technology and was maintained on the C57BL/6J background. The candidate chimeric sgRNA targeting exon1 of Slc27a2 (Gene ID: 26458) was designed based on http://crispr.mit.edu/. The sequence of the 20 nucleotide guide RNA used was GCGGGCGCTGCACGATCAACTGG (Underlined part is the PAM motif), and a pair of oligonucleotides for the target sequence was: forward primer 5′-TAGGCGGGCGCTGCACGATCAAC-3′ and reverse primer 5′-AAACGTTGATCGTGCAGCGCCCG-3′. In order to obtain gRNA and hCas9 mRNA, in vitro transcription reactions were respectively performed using the MEGAshortscript kit (Ambion, Am1354, USA) and mMESSAGE mMACHINE® T7 Ultra Kit (Ambion, Am1345) according to the manufacturer’s instructions (Fu et al. 2016). And then, both the sgRNA and the Cas9 mRNA were purified using the MEGAclear kit (Ambion, AM1908) according to the manufacturer’s instructions. The sgRNA (30 ng/μl) was mixed with Cas9 mRNA (60 ng/μl), and the mixture was microinjected into the cytoplasm of the one-cell stage embryos. The embryos injected with RNAs were cultured in the M16 medium until the blastocyst stage, and then, approximately 15–25 blastocysts were implanted in pseudo-pregnant female mice. Genomic DNA was extracted from the tail tips of the newborn pups digested by proteinase K. The genomic sequences around the gRNA target sites were PCR amplified using the following primers: forward primer 5′-CTCCAAGATGTGCGGTACTT-3′ and reverse primer 5′-GCAGAGACTTGGCACGAATG-3′. The PCR products were purified using TIANquick Midi Purification Kit (Tiangen, Beijing, China) and sequenced directly. Over the ten generations of breeding, Slc27a2
−/− mice can be used for experiments.
The wild type C57BL/6 J mice (8 weeks old) were supplied by laboratory animal research center of Tsinghua University (Beijing, China). The male wild type and Slc27a2
−/− mice were housed in an animal care facility at Tsinghua University, under controlled conditions of temperature (22 ± 2 °C) and relative humidity (50 ± 20%) with a 12 h light/dark cycle with water and food freely available. The mice were randomly allocated to the vehicle (0.9% saline)–treated (control, n = 5), zoledronate (3 mg/kg/week)–treated (Zole, n = 5) and zoledronate (3 mg/kg/week)/fenofibrate (20 mg/kg) co-treated (Zole + fenofibrate, n = 5) groups. And the Slc27a2
−/− mice were randomly allocated to the vehicle (0.9% saline)–treated (control, n = 5) and zoledronate (3 mg/kg/week)–treated (Zole, n = 5) groups. 1 day before the zoledronate injection, the Pparα agonist fenofibrate (20 mg/kg for 3 days) was administered by oral gavage, and all animals were injected zoledronate via the tail vein each week and treated for 4 weeks.
Following final treatment and collection of urine, kidney tissues were harvested and cut in cross section. Part was fixed in 4% paraformaldehyde for morphological examination and the others kidney was snap frozen in liquid nitrogen and then stored at −80 °C. All procedures performed in studies involving animals were approved by the Institutional Animal Care and Use Committee of Tsinghua University. All animal experiments were repeated three times independently.
Biochemical markers of urine
After 2 or 4 weeks treatment, mice were maintained under fasting condition for 12 h and urine was collected using a metabolic cage that was placed over an ice bath to avoid degradation of metabolites. Urine samples were immediately centrifuged at 10,000×g for 10 min at 4 °C. And then the supernatant were immediately stored at −80 °C for subsequent analysis. For clinical chemistry measurements, the supernatant were analyzed for creatinine using a fully automatic biochemical analyzer (Toshiba, Tokyo, Japan).
Determination of TGFβ1 concentration in culture supernatant and serum
The TGFβ1 concentration of HK-2 cells culture supernatant or mice serum was measured by specific enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. This assay has <1% cross-reactivity for TGFβ2, TGFβ3 and TGFβ5.
RNA extraction and qPCR analysis
Total RNAs were prepared from cells or kidney tissues by using ultra-pure TRIzol reagent (Tiangen) according to the manufacturer’s instructions. Reverse transcription was performed on equal amounts of total RNA (2.5 μg) by using random hexanucleotide primers to produce a cDNA library for each sample. qPCR were run in the ABI ViiA™7 Real-Time System (Life Technologies) by using SYBR Green Master Mix (Transgen, Beijing, China) and gene-specific primers (Table S1 online). Each sample was run in triplicate, and the comparative threshold cycle (Ct) method was used to quantify fold increase (2−ΔΔCt) compared with lean controls.
Cells or kidney tissue were washed with PBS (only cells) sonicated in RIPA lysis buffer (Beyotime, Shanghai, China) with PMSF (Beyotime), and centrifuged at 14,000×g for 10 min. The 2X sample loading buffer was added to the protein sample and heated at 100 °C for 10 min. The proteins were separated by electrophoresis in 10% gels, and then transferred onto PVDF membrane (Merck Millipore, Germany) with 350 mA, blocked with 5% skimmed milk for 1 h, and incubated with antibody overnight at 4 °C. Protein expression was detected using a chemiluminescent staining reagent kit to visualize the signals.
H&E staining, ORO staining, PAS staining and Masson’s staining
Paraffin sections (5 μm) (Harada et al. 2016) stained with H&E, PAS and Masson’s trichrome, and frozen sections (6 μm) were used for ORO staining to determine the lipid accumulation. All stained sections were examined under a light microscope (Olympus,), and finally evaluated for injury by an experienced pathologist who was blinded to the treatment each animal had received.
Immunohistochemical (IHC) staining
Kidney tissue sections (5 μm) (Harada et al. 2016) were deparaffinized in xylene, using graded ethanol, and rinsed with distilled water. For antigen retrieval, the sections were incubated with citrate buffer solution (pH: 6.0, 100 °C). After blocking by using 0.3% hydrogen peroxide in methanol for 30 min, the sections were incubated overnight at 4 °C with rabbit primary antibody Fn1, collagen I, α-SMA and Slc27a2. The secondary antibody was added and the sections were incubated at 37 °C for 1 h. An Olympus microscope was used for image analysis.
Smad2/3 ChIP-Seq data were downloaded from Gene Expression Omnibus (GEO: GSE53233) and aligned to mouse genome (mm10) by using Bowtie1.1.2. Both peak calling and generation of ChIP-Seq visual files were implemented by using MACS1.4.2. Gene tracks of ChIP-Seq data are the snapshots of bedgraph files visualized in IGV.
Statistical analysis was performed using GraphPad Prism software, version 6.0. Data were expressed as mean ± standard error of mean. Unpaired t tests were performed for comparison between two groups. Data were analyzed using a one-way analysis of variance (ANOVA) followed by a Newman–Keuls multiple comparison test. Statistical significances were calculated and indicated.