Animals
The protocol of the animal research was reviewed and authorized by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University (2022527004). The C57BL/6 J mice (25–28 g) aged 6–8 weeks were purchased from GemPharmatech Co., Ltd. (Nanjing, Jiangsu, China). ACSS2 knockout mice and TEC-specific ACSS2 mice were acquired from Gempharmatech Co. Ltd. (Nanjing, Jiangsu, China). The mice were maintained in a temperature-controlled environment with a 12-hour light/dark cycle (lights-on at 7:00 a.m.) and had free access to food and water. All animals were randomly grouped (n = 3–7 mice per group). For unilateral ureteric obstruction (UUO) model, under anesthesia, the left ureter was isolated, and completely ligated with 3-0 silk suture at two points and cut between the ligatures to prevent retrograde urinary tract infection. The sham operation mice underwent an identical surgical intervention except for ureter ligation. Briefly, the abdominal cavity was exposed via a midline incision and the left ureter was isolated and ligated. The folic acid (FA) group was intraperitoneally injected with 250 mg/kg of folic acid which was diluted in 0.3 M sodium bicarbonate just once and the control mice received an injection of an equivalent volume of sodium bicarbonate alone.
Human kidney biopsy slides
Human renal tissue, fixed in formaldehyde, and embedded in paraffin, was selected from the files of the Service of Pathology of West China Hospital, Sichuan University: control normal renal tissue was obtained from a patient with nephrectomy performed for neoplasia, involving the possibility of tumor-related immune exhaustion. Each patient gave informed consent before enrollment. The institutional ethical committee board approved the clinical protocol. Th research was performed according to the Helsinki’s declaration principles.
Drug studies for in vivo experiments
For the inhibitor studies, littermate mice were randomly injected with either the ACSS2 inhibitor or antimurine IL-1β antibodies. The ACSS2 inhibitor VY-3-249 (S8588, Selleck, Shanghai, China) was diluted in 0.9% saline and orally administered at a dose of 50 mg/kg/d for 7 consecutive days after UUO surgery or FA injection. Seven days after surgery or FA injection, the mice were sacrificed. Antimurine IL-1β antibody or control IgG was administered at 10 mg/kg body weight intraperitoneally once a day for 7 consecutive days after UUO surgery. Recombinant IL-1β was injected intraperitoneally at 1 μg/mouse, and ACSS2 inhibitor was administered 6 hours earlier, for a consecutive 7 days after UUO surgery. Mice were sacrificed on the 7th day after UUO surgery, and kidneys were harvested. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals.
For the pharmacokinetic experiments, blood samples were collected from animals at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 h after ACSS2 inhibitor administration. Approximately 0.02 mL of blood samples were obtained from mice through the orbital venous plexus. Heparin sodium-containing sample tubes were used to immediately transfer the blood samples, which were then centrifuged at 3500 rpm for 10 min at 4 °C. The supernatant was collected as plasma. 100 μL of acetonitrile containing internal standard was added to 5 μL of plasma for protein precipitation, which were then centrifuged at 18900 g for 10 min at 4°C. The supernatant was transferred to sample tubes for UFLC-MS/MS analysis. The non-compartmental model of DAS2.0 software was applied to calculate the pharmacokinetic parameters after the plasma concentration was analyzed.
Serum biochemistry assays
Blood samples were taken and centrifuged at 1000 g for 20 min, and then serum was collected. The levels of serum creatinine (Scr), blood urea nitrogen (BUN), glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) were measured by an automatic biochemical analyzer (Mindray BS-240, Shenzhen, China).
Histologic examination
Remove half of the kidney, freeze in a liquid nitrogen and keep under a temperature of −80 °C. One-quarter of the kidney was excised and then fixed in 10% formaldehyde (50-00-0, Chron Chemicals, Chengdu, China), dehydrated, embedded into the paraffin and sectioned at a thickness of 4 μm for H&E, Masson and Sirius Red staining. An additional quarter of the kidney were embedded into OCT compound, which was frozen under a temperature of −80 °C. With Vectra® Polaris™ Automated Quantitative Pathology Imaging System and AxioCamHRc digital camera (Carl Zeiss, Jena, Germany), kidney sections were scanned and pictured at 100×, 200× and 400× magnification.
Immunohistochemistry (IHC) staining analysis
The paraffin-embedded kidneys were sectioned to a thickness of 4 μm, de-paraffinized, rehydrated, and next antigen-retrieved. Subsequently, these sections were blocked with 2.5% normal goat serum, and inoculated by the primary antibodies Pan-Kcr, H3K9cr, ACSS2 or IL1b diluted 200:1 in PBS under a temperature of 4 °C overnight, separately. Slides were cleaned in PBS 3 times and stained with the VECTASTAIN ABC kit (Vector, Burlingame, CA, USA), which were then visualized through utilizing AxioCamHRc digital camera (Carl Zeiss, Jena, Germany) with ZEN 2012 microscopy software (blue version).
Immunofluorescence (IF) staining analysis
Sections of OCT-embedded kidneys were 4 μm in thickness and subsequently inoculated at room temperature utilizing 5% horse serum for 60 min for blocking the non-specific binding sites. The slides were next inoculated overnight in a humid chamber under a temperature of 4 °C after dilution 1:200 in PBS with primary antibody Pan-Kcr, H3K9cr, α-SMA, COL6, γH2AX. The equivalent secondary antibody (1:500 dilution, 111-025-003, Jackson ImmunoResearch, West Grove, PA, USA) was utilized for 60 min. Slides were re-cleaned, stained with DAPI (dilution 1:500, D8200, Solarbio, Beijing, China), and sealed through coverslips. For cells, formaldehyde was used for fixation for 15 min, and the primary antibody γH2AX was added to the slides at 4 °C overnight. Secondary antibodies (1:500 dilution, 111-545-144, Jackson ImmunoResearch, West Grove, PA, USA) was applied for 60 min at room temperature. After washing, the cells were stained with DAPI (dilution 1:500, D8200, Solarbio, Beijing, China). The co-staining of IL-1β with H3K9cr, IL-1β with iNOS and F4/80 were performed by Opal reagent (Y6084S, Y6088S, Y6094S, Uelandy, Suzhou, China) according to the guidelines of the manufacturer. Images were gathered under an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany) via utilizing the ZEN 2012 microscopy software (blue version).
SA-β-gal staining
The activity of SA-β-gal was investigated in accordance with the guidelines of the manufacturer utilizing a kit (C0602, Beyotime Biotechnology, Shanghai, China). Images were gathered randomly with an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany).
Measurement of IL-1β by ELISA
Blood samples were centrifuged at 1000 g for 15 min and stored at −80oC until use. Frozen kidney tissues (10 mg) were added 100 µL PBS, homogenized with an electric homogenizer, centrifuged for 20 min at 13,000 rpm at 4 °C and stored at −80 °C until use. Pipette cell culture media into a centrifuge tube, centrifuge at 1,500 rpm for 10 min at 4 °C, and stored at −80 °C until use. Levels of IL-1β of mice serum, mice kidney tissue, and cell culture supernatant were detected by solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) kits (Ruixin Biotechnology, Quanzhou, China) specific for these factors, and absorbance was measured at 450 nm using a plate reader (BioTek ELx800, USA).
Western blotting
Nearly, 20-30 mg kidney tissue was homogenized in SDS-lysis buffer (#7722, CST) containing 42 mM DTT following the user manual. The same samples were loaded to the SDS-PAGE gels and run at 100 v for 1 h 40 min at RT in Tris-Glycin-SDS containing buffer. Blots were blocked with 5% non-fat dry milk powder in tris-buffer saline containing Tween-20 (TBST) for 30 min at RT. The blots were incubated in primary antibody overnight at 4 °C. After primary antibody (all the antibodies were shown in supplementary Data 4) incubation, blots were washed three times with TBST. Horseradish peroxidase-labeled goat anti-rabbit IgG (HA1001, 1:2000 dilution; HuaBio, Hangzhou, China) or goat anti-mouse IgG (HA1006, 1:2000 dilution; HuaBio, Hangzhou, China) were probed for 1 h at RT prepared in non-fat dry milk containing Tween-20. Finally, blots were washed with TBST for three minutes each 10 min at RT, and observed through Odyssey infrared imaging system. (Fluorescence Chemiluminescence Imaging System, Clinx Science, Shanghai, China) and quantified through utilizing ImageJ software (version 6.0; Wayne Rasband, National Institutes of Health, USA).
Histone extraction, western blotting, and LC-MS/MS analysis
To extract histones from kidneys, we first isolated the nuclei and proceeded with the acid-histone extraction method. Two micrograms of histone lysates were loaded onto 15% SDS-PAGE gels; Coomassie brilliant blue was used for gel staining to demonstrate histone purification. Rest histone lysates were used for LC-MS/MS to double-check histone purification.
Briefly, nearly 40 mg of kidney tissue was washed with ice-cold PBS and minced in the nuclei isolation buffer (NIB-250 is 15 mM Tris-HCl at pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 250 mM sucrose, to which 0.1% Nonidet P-40, 1x protease inhibitor cocktail, 1 mM DTT, and 10 mM sodium butyrate). Kidney pieces were collected into glass Dounce homogenizer on ice. After 5 min of incubation on ice, the homogenates were spin down, nuclei pellet was washed twice with NIB-250 without NP-40 and proceeded with histone extraction.
Histones were extracted with 0.4 N H2SO4 at 5:1 ratio for 2 h at 4 °C. Acidified nuclei were spin down at 11000rcf for 10 min at 4 °C, and the soluble fraction containing histones was collected into new tube and precipitated with 20% trichloro acetic acid at final concentration overnight at 4 °C overnight. Samples were spin down at 11000rcf for 10 min at 4 °C to sediment the histone pellet at the bottom. Histone pellets were washed with ice-cold 1 mL acetone containing 0.1% 12 N HCl then the pellet was washed twice with ice-cold 100% acetone, air-dried, and dissolved in RIPA buffer.
Histone liquid chromatography-mass spectrometry
Trypsin Digestion The sample was slowly added to the final concentration of 20% v/v TCA to precipitate protein, then vortexed to mix and incubated for 2 h at 4 °C. The precipitate was collected by centrifugation at 4500 g for 5 min at 4 °C. The precipitated protein was washed with pre-cooled acetone for 3 times and dried for 2 h. The protein sample was then redissolved in 200 mM TEAB and ultrasonically dispersed. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight. The sample was reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. Finally, the peptides were desalted by C18 SPE column. LC-MS/MS Analysis: The tryptic peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile/in water), directly loaded onto a home-made reversed-phase analytical column (25-cm length, 75/100 μm i.d.). Peptides were separated with a gradient from 6% to 22% solvent B (0.1% formic acid in acetonitrile) over 40 min, 22% to 30% in 12 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 450 nL/min on a nanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to a capillary source followed by the timsTOF Pro (Bruker Daltonics) mass spectrometry. The electrospray voltage applied was 1.60 kV. Precursors and fragments were analyzed at the TOF detector, with an MS/MS scan range from 100 to 1700 m/z. The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode. Precursors with charge states 0 to 5 were selected for fragmentation, and 10 PASEF-MS/MS scans were acquired per cycle. The dynamic exclusion was set to 24 s. The peptide segment was dissolved in the mobile phase A of liquid chromatography and separated by the NanoElute ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; Mobile phase B is a solution containing 0.1% formic acid and 100% acetonitrile. Liquid phase gradient setting: 0–40 min, 6–22% B; 40–52 min, 22% ~ 30% B; 52-56 min, 30% ~ 80% B; 56–60 min, 80% B, flow rate maintained at 450nL/min. The peptide segment is separated by the ultra-high performance liquid phase system and injected into the Capillary ion source for ionization and then analyzed by the timsTOF Pro mass spectrometry. The ion source voltage is set at 1.6 kV, and the peptide parent ion and its secondary fragments are detected and analyzed using high-resolution TOF. The scanning range of secondary mass spectrometry is set to 100–1700. The data acquisition mode uses parallel cumulative serial fragmentation (PASEF) mode. After a primary mass spectrum is collected, the secondary spectrum with the charge number of the parent ion in the range of 0-5 is collected by 10 times in PASEF mode. The dynamic elimination time of the tandem mass spectrum scanning is set to 24 s to avoid repeated scanning of the parent ion. Database Search The resulting MS/MS data were processed using MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the Mus_musculus_10090_SP_20220119_Histone_H. fasta database (50 entries) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in first search and 20 ppm in main search, and the mass tolerance for fragment ions was set as 0.02 Da. FDR was adjusted to <5%.
RNA isolation and quantitative real-time PCR
Total RNA was extracted from kidney and spleen tissues using a total RNA extraction kit (Foregene, Chengdu, China) according to the protocols and was reverse transcribed into cDNA using a PrimeScriptTM RT reagent kit (Takara, Kusatsu, Japan). The mRNA concentration was measured using a Scan Drop 100 (Analytik Jena, Thuringia, Germany) instrument. Quantitative real-time PCR was performed by using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) in a PCR system (CFX Connect; Bio-Rad, Hercules, CA, USA). The primers used for target mRNA detection are listed in Supplementary Data 5. Relative gene expression was normalized to that of GAPDH by comparison with the control groups using CFX ManagerTM Software (Bio-Rad, Hercules, CA, USA).
RNA sequencing
Frozen kidney samples from groups (n = 3 per group) were chosen randomly for sequencing. With TRIzol reagent (Invitrogen, Carlsbad, CA, USA), the total RNA of the samples could be extracted, and then the samples were examined for purity, quality, and integrity. Through LC-BIO Bio-Tech Ltd (Hangzhou, China), the construction and sequencing of libraries were conducted. With Illumina NovaSeq 6000 platform, such libraries were subsequently sequenced and paired-end reads with a 2×150 bp read length were produced.
Sample collection and preparation of ChIP sequencing
ChIP assays were performed by Shandong Xiuyue Biotechnology Co., Ltd, according to the standard crosslinking ChIP protocol with modifications. Briefly, cells were harvested and crosslinker with 1% formaldehyde for 10 min at room temperature. After sonication, immunoprecipitation was performed with anti-H3K9cr (PTM) and anti-H3K9ac (Active Motif). The immunoprecipitated complex was washed, and DNA was extracted and purified by Universal DNA Purification Kit (TIANGEN, #DP214). The ChIP-Seq library was prepared using original Ultra II DNA Library Kits (NEB, #E7645) according to the manufacturer’s instructions. For ChIP-seq, extracted DNA was ligated to specific adapters followed by deep sequencing in the Illumina Novaseq 6000 using 150 bp paired-end.
Data analysis of ChIP sequencing
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyzes were based on the clean data with high quality. Clean reads were mapped to the reference genome using Bowtie2 software. And reads from organelle, mapping quality smaller than 30, PCR duplicated reads all removed. Those high-quality mapping reads were subjected to further peak calling. Macs2 was used to call peaks with q value < 0.05. Peaks were annotated by ChIP seeker package.
We analysis differential accessible peak through 3 steps. First, merge the peak files of each sample using the bed tools software. Second, the counts of the reads over the bed were determined for each sample using bed tools multicov. Finally, differential accessible peak was assessed using DESeq2. The region were called differentially accessible if the absolute value of the log2 fold change was 1 at an p value < 0.05. Gene ontology (GO) analysis was performed to facilitate elucidating the biological implications of unique genes in the significant or representative profiles of the gene in the experiment [Ashburner M, et al. Gene ontology: a tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000 May;25(1):25- 9.]. We downloaded the GO annotations from NCBI, UniProt (http://www.uniprot.org/) and the Gene Ontology (http://www.geneontology.org/). Fisher’s exact test was applied to identify the significant GO categories and FDR was used to correct the p-values. Pathway analysis was used to find out the significant pathway of the genes according to KEGG database. We turn to Fisher’s exact test to select the significant pathway, and the threshold of significance was defined by P-value and FDR.
ChIP-qPCR assay
Proteins and DNA interaction was evaluated by ChIP-qPCR using the ChIP assay kit (Millipore, MA, USA). The experiment protocols were according to the manufacturer’s instructions. The antibodies used for the ChIP assay were as follows: anti-H3k9cr (PTM), anti-H3k9ac (Active Motif), anti-H3k4cr (PTM), anti-H3k14cr (PTM), anti-H3k18cr (PTM), anti-H3k23cr (PTM), anti-H3k27cr (PTM), anti-H3k36cr (PTM), anti-H2Bk34cr (PTM), and control IgG (Millipore). The primers used for ChIP were as follows: Il1b-F 5’- ACCCAGACAGGGCTTTTAGC-3’, Il1b-R 5’- TCCATTCCTAACACTGAGCCC-3’; Il1r1-F 5’- TCACTCAGGTCCTCTCAGTCC-3’, Il1r1 -R 5’- TCCAATTGTGGGCAGCAATGA-3’. The calculation formula for enrichment efficiency was elaborated on previously.
Isolation and culture of primary tubular epithelia cells
Renal cortex was isolated from mouse kidney and divided into tiny pieces, then added with 1 mg/ml type I collagenase (17100-017, Gibco, MA, USA), and incubated at 80 rpm in a 37 °C shaker for 30 min. Subsequently, PTCs were separated by strainer (100, 70, and 40 μm), and erythrocytes were removed with red blood cell lysis buffer (R1010, Solarbio, Beijing, China). Finally, the PTCs were cultured in RPMI1640 (HyClone, Logan, UT, USA) containing 10% fetal bovine serum, 1×insulin transferrin selenium additive, 1% penicillin/streptomycin and 20 ng/mL epidermal growth factor, and incubated at 37 °C in a humidified atmosphere of air/CO2 (95:5).
Cell viability assay
Cell viability was determined by the Cell Counting Kit-8 assay (CCK-8, APExBIO, Houston, TX, USA) according to the manufacturer’s instructions. Briefly, TCMK-1 cells in the logarithmic growth phase were seeded in 96-well culture plates at a density of 5000 cells/well. After treating with Il1b, a 10 μl CCK-8 solution was added to each well and incubated in the dark for 1 h at 37 °C. In the end, the absorbance at 450 nm was detected using a microplate reader (Synergy Mx, Biotek, Vermont, USA).
Cell culture and treatments
TCMK-1, HEK-293T, and RAW264.7 macrophage were acquired from the American Type Culture Collection (Manassas, VA). TCMK-1, HEK-293T and RAW264.7 cells were cultured in DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified atmosphere of air/CO2 (95:5). For IL-1β-induced aging research, TCMK-1 and HEK-293T were incubated in MEM medium supplemented with different doses of IL-1β for 24 h. For assessing the treatment effect of IL-1β antibody, cells were treated with IL-1β (5 ng/ml) or cell culture supernatant with IL-1β antibody (5 µg/ml) at the same time for 24 h. For TGFβ-induced fibrosis research, HEK-293T and primary tubular epithelia cells were exposed to TGFβ (20 ng/ml) for 48 h. For plasmid transfection research, gene-expressing plasmid and blank plasmid (pVector) were obtained from MiaoLing Plasmid (Wuhan, China). TCMK-1 and HEK-293T cells transfection with plasmids was conducted using Lipofectamine 2000 (12566014, Invitrogen, CA, USA) for 24 h, according to the manufacturer’s instructions.
Primary culture of mouse TECs
Kidneys were collected from 3- to 6-week-old mice, minced, and digested by collagenase I (2 mg/ml) for 30 min at 37 °C and then filtered successively through 100-, 70-, and 40-µm mesh to collect single TECs. Cells were cultured in RPMI 1640 supplement with 10% fetal bovine serum (FBS), epidermal growth factor (20 ng/ml), and 100× insulin-transferrin-selenium at 5% CO2, 37 °C. At 80% confluence, cells were treated in fed condition (RPMI 1640 + 10% FBS).
Statistical analysis
Statistical analysis was performed using the GraphPad Prism 9 software. One-way ANOVA and the unpaired t-test were performed on variables. Sample size estimation was not performed, and sample size was determined by the number of animals in the colony of a determined age and gender. The number of replicates (including the number of animals used in each experiment) are indicated in the figures and/or figure legends. All data are expressed as mean ± SEM. The statistical parameters can be found in the figures and the figure legends. p < 0.05 was considered significant. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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