Human kidney biopsies IgA nephropathy (IgAN) is the most common type of chronic kidney disease (CKD) and one of the major causes of renal fibrosis. Hence, we chose the IgA nephropathy patients samples to investigate the relationship between Twist1 and renal fibrosis. Renal biopsy samples and Clinical data from patients diagnosed with IgAN at the Xijing Hospital (Xi’an, China) are shown in Supplementary Information Table 2. Histological examination was performed at the Kidney Pathology Department of Xijing Hospital. The relevant clinical information was collected from patients’ records.
Animal model Male C57BL/6 mice weighing 20 ± 2 g were acquired from the specific pathogen-free laboratory animal center of the Fourth Military Medical University utilizing a 14-hour light and 10-hour dark cycle and maintained according to the guidelines of the Institutional Animal Care and Use Committee at Fourth Military Medical University. UUO was performed as previously reported. The mice were euthanized, kidneys and bone marrow were harvested at days at 0, 3, 7, and 14 after UUO.
Homozygous Twist1 floxed mice [B6; 129S7-Twist1tm2Bhr/Mmnc] and mice expressing the Cre fusion protein under the control of macrophage-specific mouse Lyz2 promoter were acquired from Jackson Laboratories (West Grove, PA, USA). All animals were housed in the specific pathogen-free laboratory animal center of Fourth Military Medical University, as described above. Mating Twist1 floxed mice with Lyz2-Cre transgenic mice generated mice that were heterozygous for the Twist1 floxed allele (genotype: Lyz2-Cre+, Twist1fl/wt). These mice were crossbred with homozygous Twist1 floxed mice (genotype: Lyz2-Cre+; Twist1fl/fl) to generate offspring with different littermates (Lyz2-Cre+; Twist1fl/fl, Lyz2-Cre+; Twist1fl/wt, Lyz2-Cre-; Twist1fl/wt, and Lyz2-Cre-; Twist1fl/fl). Lyz2-Cre+; Twist1fl/fl mice and the same-sex Lyz2-Cre-; Twist1fl/fl littermates (controls) were subjected to UUO. The sham group underwent the same procedure but without UUO. Genotyping was performed by PCR assay using DNA extracted from the mouse tail and using the following primers: Cre transgene, sense: 5’- CCGGTCGATGCAACGAGTGATGAGG-3’; antisense:5’-GCCTCCAGCTTGCATGATCTCCGG-3’; Twist1 floxed, sense: 5’- AGCGGTCATAGAAAACAGCC-3’; antisense:5’- CCGGATCTATTTGCATTTT ACCATGGGTCATC-3’.
Cell culture Raw264.7 cells were cultured in RMPI-1640 containing 20% (vol/vol) FBS (GIBCO, Invitrogen Inc, Carlsbad, CA, USA) and 1% (vol/vol) antibiotics (100 U/ml penicillin) at 37°C in 5% CO2. Raw264.7 stimulated with IL-4 (25 ng/ml; catalog no. 214 − 14; PeproTech, Rocky Hill, NJ, USA), or with IFN-γ (25 ng/ml; catalog no. 315-05; PeproTech, Rocky Hill, NJ, USA) and LPS (100 ng/ml; catalog no. L2630; Sigma, St Louis, MO, USA) for 24 hours. Adherent cells were washed and harvested with trypsin/EDTA (Lonza).
Bone marrow-derived macrophages (BMMs) were isolated, as previously described.46 BMMs obtained from Lyz2-Cre+Twist1fl/fl and Lyz2-Cre-Twist1fl/fl mice were cultured in RMPI-1640 containing 10% (vol/vol) FBS, 25 ng/ml mouse M-CSF (catalog no. 315-02; PeproTech, Rocky Hill, NJ, USA), and 1% (vol/vol) penicillin/streptomycin antibiotics for 5 days, Briefly, on day 5, cells were replated in triplicate (3 × 105 cells/well). BMMs were cultured with serum-free medium and treated with IL-4 (25 ng/ml; catalog no. 214 − 14; PeproTech, Rocky Hill, NJ, USA), or with IFN-γ (25 ng/ml; catalog no. 315-05; PeproTech, Rocky Hill, NJ, USA) and LPS (100 ng/ml; catalog no. L2630; Sigma, St Louis, MO, USA) for 24 hours. Adherent cells were washed and harvested with trypsin/EDTA (Lonza).
Semiquantitative analysis of the fibrotic area in kidney tissue Mouse kidney sections of 3 µm in thickness were stained with the Masson Trichrome kit (catalog no. HT15-1KT; Sigma, St Louis, MI, USA), according to the manufacturer’s protocol. Accumulated collagen in the interstitial area was stained with aniline blue. Ten ×400 fields were randomly selected in the cortical area for each kidney section. The percentage of interstitial fibrotic area to the selected field was analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and an average percentage of fibrotic kidney area for each section was calculated.
Histology and immunohistochemistry Paraffin-embedded mouse kidney sections (3-µm thickness) were stained with PAS (catalog no. G1280; Solarbio, Beijing, China), Masson (HT15-1KT; Sigma-Aldrich, St Louis, MI, USA), and Sirius red (catalog no. G1472-2; Solarbio, Beijing, China). The antibodies for immunohistochemistry were: anti-α-SMA (catalog no ab32575, Abcam, Cambridge, United Kingdom), anti-fibronectin (catalog no. 610154; Transduction Laboratories, Lexington, KY, USA), anti-type I collagen (catalog no. AB765P; Millipore Corp., Billerica, MA, USA) and anti-CCR2 (catalog no. ab176390; Abcam, Cambridge, United Kingdom) and anti-galectin-3 (catalog no.3027070; Millipore Corp., Billerica, MA, USA). After incubation with the primary antibodies at 4°C overnight, the slides were stained with the secondary antibody for 1 hr at room temperature. The sections were incubated with the ABC reagents for 1 hr at room temperature before DAB staining (Vector Laboratories, Burlingame, CA, USA). Images were captured using a light microscope (Olympus, Tokyo, Japan).
Immunofluorescence Kidney cryosections at 3 µm thickness were fixed for 15 min with 4% paraformaldehyde followed by permeabilization with 0.3% Triton X-100 in 1× PBS for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were stained with the following antibodies: anti-Twist1 (catalog no. 50581; Abcam, Cambridge, United Kingdom), anti-CD68 (catalog no. 31630; Abcam, Cambridge, United Kingdom),anti-F4/80 (catalog no. 6640; Abcam, Cambridge, United Kingdom), anti-cleaved caspase3 (catalog no. 9664; Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-galectin-3 (catalog no. 3027070; Millipore Corp., Billerica, MA, USA), followed by staining with Alexa 488- or Cy3-conjugated secondary antibodies. For the quantitative analysis of Twist1 expression in macrophages in kidney tissues, ten ×400 fields were randomly selected in the cortical area from each kidney section.
Kidney monocyte/macrophage enrichment Mice were sacrificed by i.p. injection of Beuthanasia-D (Merck). After perfusion with cold 1× PBS, the mouse kidneys were removed, minced into fragments, and digested in HBSS containing 1 mg/ml collagenase (catalog no. c5138; Sigma, St Louis, MI, USA) for 1 hr 37°C with intermittent agitation. The fragments were filtered through a 70 µm mesh (DKW33-N25, Dakewei, Shanghai, China) to achieve a single-cell suspension. RBC Lysis Buffer (eBioscience) was used to lyse RBCs at room temperature and cell counts were performed on the cell suspensions from the kidney digests. In some experiments, the kidney tissues were inflated with 10% formalin, removed, and fixed in 10% formalin prior to paraffin embedding. Sections were stained with H&E, Masson, PAS, Siris red. Macrophages were enriched from the single-cell suspension with F4/80 Microbeads and BD magnetic frame (BD, Bergisch-Gladbach, Germany), according to the manufacturer’s instruction.
Quantitative real-time PCR Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and RNA concentrations were determined using a NanoDrop 1000 (Thermo Fisher Scientific). Then, cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa, Dalian, China). The SYBR Premix Ex Taq II (TaKaRa) was used to amplify the double-stranded cDNA of interest. RT-PCR primers for Twist1, galectin-3, PDGFA, PDGFB, PDGFC, PDGFD, TGFβ1, TGFβ2, TGFβ3, VEGFA, CCN2, YM1, Fizz, and ACTB (β-actin) were purchased from Ruibo Bio (Guangzhou, China). RT-PCR primers for iNOS, IL-6, TGF-β, CCL2, TNF-α IL-1β, IL-10, Arg1, and MR were synthesized by TaKaRa (Dalian, China). The levels of ACTB were used as internal controls for mRNA. The 2-ΔΔCt method was used to determine the relative expression level of RNA between groups. The primer sequences are listed in Supplementary Information Table 3.
Protein isolation and Western blots Protein lysates were collected in RIPA lysis buffer (Beyotime, Shanghai, China) containing a complete protease inhibitor cocktail (Roche, Manheim, Germany). Lysates were centrifuged for 5 minutes at 14,000 g to clear lysates and the supernatant was collected. Total protein was quantified by BCA assay following manufacturers protocols and 5–10 µg of total protein was used for each sample. 10× Reducing Agent and 4× LDS Sample Buffer and heated at 70°C for 10 minutes. Bolt Bis-Tris gradient gels (4–12%) were used for electrophoresis and proteins were transferred onto 0.2-µm PVDF at 20 V for 75 minutes using Bolt transfer buffer containing 10% methanol. Wash buffer was TBS containing 0.05% Tween 20, and 5% BSA was added for blocking and incubation steps in primary and secondary antibodies. The proteins were visualized using a Dura Super Signal Substrate (Pierce Chemical, Dallas, TX, USA). Bands were detected by chemiluminescence using Supersignal West Femto (Pierce) on an Omega Ultra Lum imaging system. The blots were scanned using a Molecular Imager ChemiDox XRS + Imaging System with Image Lab software (Bio-Rad, Hercules, CA, USA).
The following antibodies were used: anti-Twist1 [#49254(1:2000), Abcam, Cambridge, United Kingdom], anti-YM1 [#60130(1:1000), Stemcell Technologies Inc, Vancouver, Canada], anti-α-SMA [ab32575(1:500), Abcam, Cambridge, United Kingdom], anti-type I collagen [AB765P (1:500); Millipore Corp, Billerica, MA, USA)]and anti-ACTN [β-actin (1:200) ZSGB-BIO, Shanghai, China).
Luciferase reporter assay The mRNA 3′-UTR luciferase reporter vectors were constructed as previously described. For the 3′-UTR luciferase reporter assays, the indicated cells were co-transfected with Galectin-3 promoter plasmids and pGL3-basic (RiboBio Co., Guangzhou, China) and the indicated wild-type using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). For luciferase reporter assays of promoter activity, the indicated cells were co-transfected with the pGL3-galectin-3 promoter fragments, pRL-SV40 Renilla luciferase reporter, and siRNA-Twist1 or control. The DualLuciferase Assay (Promega, Madison, WI, USA) was used to detect Renilla and firefly luciferase activities. Renilla luciferase activity was normalized to the firefly activity and presented as the relative luciferase activity. All assays were performed in triplicate three times.
Chromatin immunoprecipitation (ChIP) ChIP assays were performed as previously described. Briefly, the recovered supernatants were incubated with a rabbit anti-Twist1 antibody (#50887, Abcam, Cambridge, United Kingdom) or an isotype control IgG (BD Biosciences, Franklin Lake, NJ, USA) for 2 hr in the presence of herring sperm DNA and protein A/G magnetic beads. The DNA was recovered and subjected to PCR to amplify the Twist1-binding sites. The primers are shown in Supplementary Information Table 4.
Plasmid construction The galectin-3 promoter construct was generated as previously described. Briefly, -2000 to -1 galectin-3 was generated from mouse genomic DNA. This construct, corresponding to the sequence from − 2000 to -1 (relative to the transcriptional start site) of the 5′- flanking region of the mouse gene, was generated with the forward and reverse primers incorporating MluI and XhoI sites at the 5′ and 3′ ends, respectively. The MluI and XhoI sites of the pGL3-Basic Vector (Promega, Madison, WI, USA) were inserted for the ultimate PCR product. Constructs including a deletion of the 5′-flanking region of the galectin-3 promoter (-2000/-1) galectin-3-1, (-1961/-1) galectin-3-2, (-1636/-1) galectin-3-3, (-1000/-1) galectin-3-4, (-931/-1) galectin-3-5, and (-272/-1) galectin-3-6 were generated in manner analogous to that for the (-2000/-1) galectin-3 construct. The QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to generate the constructs for site-directed mutation. All constructs were verified by sequencing. All primers are listed in Supplementary Information Table 5.
Oligonucleotide transfection The sense strand sequences of the Twist1 siRNAs designed to target mouse cells were: Twist1 siRNA no. 738 (siRNA-1), 5′- CGGACAAGCUGAGCAAGAUTT -3′; Twist1 siRNA no. 780 (siRNA-2), 5′- GGUACAUCGACUUCCUGUATT-3′, and Twist1 siRNA no.832 (siRNA-3), 5′-GAUGGCAAGCUGCAGCUAUTT-3′. Successful knockdown of Twist1 was confirmed by western blotting (Supplementary Information Fig. 3a). Transfection of the siRNA was performed using the RNA iMAX Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions.
Flow cytometry FACS analysis was performed according to previous reports. Briefly, Staining of cells for flow cytometry was performed in suspension cell from kidney tissue using between 1 × 105 and 1 × 106 cells per tube. For phospho-flow staining, kidney cells were resuspended in ice-cold PBS and immediately an equal volume of prewarmed BioLegend fixation buffer (catalog 420801 San Diego, CA, USA) was added and samples were incubated at 37°C for 15 minutes. The BioLegend intracellular staining with True-Phos Perm Buffer (catalog 425401 San Diego, CA, USA) protocol was followed and all washes were performed with BioLegend Cell Staining Buffer (catalog 425401 San Diego, CA, USA). BioLegend Trustain FcX for mouse (clone 93, catalog 101320 San Diego, CA, USA) were used to block samples before staining. Phospho-flow experiments were collected on a BD LSRII and kidney homogenate analyses were collected using a BD FACS Canto RUO. FlowJo software (BD) was used for analysis of flow cytometry data.
The following antibodies were used: anti-CD86-PE (105007; Biolegend, San Diego, CA, USA), anti-F4/80-FITC (101205; Biolegend, San Diego, CA, USA), anti-CD206-PE (141720; Biolegend, San Diego, CA, USA), and anti-galectin-3-PECy7 (125418; Biolegend, San Diego, CA, USA)
Statistical analyses GraphPad Prism 8 software was used to perform statistical analyses and specific statistical tests used are listed in individual figure legends. Multiple t tests were performed with corrections for multiple comparisons using the Holm-Sidak method, while 2-tailed unpaired t tests were used where indicated in figure legends. P < 0.05 was considered statistically significant and specific P-value identifiers are listed in each figure legend. Some data sets were checked for statistical outliers using the GraphPad Prism outlier calculator with an α of 0.05; if a data point was determined to be a significant outlier it was not included in the graphs or when calculating statistical significance.