Animal experiments. Both wild-type and Fn14-deficient BALB/c mice were used in this study. Fn14 deficiency was generated in mice by using the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 method (14). Mice were routinely housed at the specific-pathogen-free facility of the Medical Animal Center of Xi’an Jiaotong University. Eight-week-old male mice were used in the experiments. Mice were randomly divided into the sham operation group of Fn14+/+ mice, the sham operation group of Fn14−/− mice, the UUO operation group of Fn14+/+ mice, and the UUO operation group of Fn14−/− mice. Mice were intraperitoneally anesthetized with pentobarbital sodium (75 mg/kg). For the UUO operation, the left ureter was isolated through a median abdominal incision and ligated with a 4 − 0 silk suture (15). Sham-operated animals underwent the same surgical procedures, except that the left ureter was operated without ligation. Mice were euthanized on days 3, 5 or 7 after surgery. The medical ethics committee of the Second Affiliated Hospital of Xi’an Jiaotong University approved all animal study procedures.
Histological evaluation and immunohistochemistry. Renal tissues were routinely processed for paraffin sectioning, then further stained with hematoxylin and eosin (H&E), periodic-acid Schiff (PAS), Masson trichrome or Sirius red. All sections were scanned using a Nano Zoomer 2.0 HT slide scanner (Hamamatsu Photonics, Hamamtsu, Japan), then analyzed using the NDP.view2 software (Hamamatsu Photonics). Six to 8 cortex fields were randomly selected per section. Two pathologists blinded to the groupings evaluated the histological changes. Tubular injury (including atrophy, casts, dilatation, and inflammatory infiltration) was scored on the H&E- and PAS-stained sections using a six-point scale in which 0, 1, 2, 3, 4, and 5 indicated normal, very little, very mild, mild, moderate, and severe histological lesions, respectively (16). Renal interstitial fibrosis was assessed on Sirius red and Masson trichrome-stained sections, and positive signals were quantified using Image Pro-Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Paraffin sections were routinely processed for immunohistochemistry. After blocking with endogenous peroxidase blocking buffer (Beyotime Co., Shanghai, China), rabbit anti-collagen I, α-SMA, fibronectin, CD45, F4/80 or Ki67IgG (Abcam, Cambridge, MA, USA) were used as the primary antibodies. Horseradish peroxidase-conjugated goat anti-rabbit IgG was the secondary antibody (Abcam). 3,3′-Diaminobenzine-chromogen substrates was used for signal development (CWbio, Beijing, China). To evaluate collagen I, fibronectin, and α-SMA expression, ten fields per section were selected randomly, then quantified using Image Pro-Plus 6.0 software. Two pathologists counted the CD45+, F4/80+ and Ki67+ cells in a single-blinded manner.
Cell culture. Human PTECs (HK2) were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), and cultured in Hyclone Dulbecco’s modified Eagle’s medium (GE Healthcare, Port Washington, NY, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). Before the stimulation assays, HK2 cells were starved in 1% fetal bovine serum-supplemented medium for 12 h. Recombinant human TWEAK (0–250 ng/ml; Peprotech, Cranbury, NJ, USA) was then added for 24 h.
In some experiments, HK2 cells were transfected with Fn14 or control siRNA before the stimulation assays (17). Cells grown in six-well plates were added with the siRNA oligomer and Lipofectamine 3000 reagent complexes (Life Technologies, Carlsbad, CA, USA). The Fn14 siRNA sequences were siRNA1, GCAGGAGAGAGAAGUUCACTT (sense) and GUGAACUUCUCUCUCCUGCTT (antisense) and siRNA2, CACUGACUAAGGAACUGCATT (sense) and UGCAGUUCCUUAGUCAGUGTT (antisense). The transfection efficiency was verified via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (Fig.s1). The inhibitor of Notch1/Jagged1, DAPT (C23H26F2N2O4; Abcam), was added 1 h before TWEAK stimulation.
Semi-quantitative RT-PCR. Total RNA was extracted from fresh tissue samples or cell cultures using TRIzol reagent (Invitrogen, Grand Island, NY, USA). cDNA was synthesized using PrimeScript™ RT Master Mix (Takara Bio, Shiga, Japan). qRT-PCR was performed on the ABI StepOne System (Applied Biosystems, Carlsbad, CA, USA) using SYBR green master mix as the fluorescent dye (Takara). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the endogenous control. The PCR primer sequences were synthesized by Sangon Biotech (Shanghai, China; Table 1). The mRNA expression levels were calculated using the 2-ΔΔCt method, then normalized to the GAPDH values.
Table 1
Primer
|
Sequence
|
Human α-SMA
|
Forward: 5′-GCTGACAGAGGCACCACTGAAC-3′
|
Reverse: 5′-AGTCACACCATCTCCAGAGTCCAG-3′
|
Human collagen
|
Forward: 5′-GCGAGAGCATGACCGATGGATTC-3′
|
Reverse: 5′-GCCTTCTTGAGGTTGCCAGTCTG-3′
|
Human fibronectin
|
Forward: 5′-ATGCAACGATCAGGACACAAGGAC-3′
|
Reverse: 5′-TGCCTCTCACACTTCCACTCTCC-3′
|
Human Fn14
|
Forward: 5′-CTCTGAGCCTGACCTTCGTG-3′
|
Reverse: 5′-GGGGGCACATTGTCACTGGA-3′
|
Human GAPDH
|
Forward: 5′-CAGGAGGCATTGCTGATGAT-3′
|
Reverse: 5′-GAAGGCTGGGGCTCATTT-3′
|
Human Jagged1
|
Forward: 5′-TCAGGCAGAAGCAGAGGTAGGC-3′
|
Reverse: 5′-TGTGGCTTGGATCTGTTGCTTGG-3′
|
Human Notch1
|
Forward: 5′-TGCGAGACCAACATCAACGAGTG-3′
|
Reverse: 5′-ACGTTGTTGGTGGTGTTGTCCTC-3′
|
Mouse α-SMA
|
Forward: 5′-GCTGGTGATGATGCTCCCA-3′
|
Reverse: 5′-GCCCATTCCAACCATTACTCC-3′
|
Mouse collagen I
|
Forward: 5′-TGACTGGAAGAGCGGAGAGT-3′
|
Reverse: 5′-GTTCGGGCTGATGTACCAGT-3′
|
Mouse CTGF
|
Forward: 5′-CTTCTGCGATTTCGGCTCC-3′
|
Reverse: 5′-TACACCGACCCACCGAAGA-3′
|
Mouse fibronectin
|
Forward: 5′-ACAGTCCAGCAAGCAGCAAGC-3′
|
Reverse: 5′-TGGTGGTCACTCTGTAGCCTGTC-3′
|
Mouse GAPDH
|
Forward: 5′-TGTGTCCGTCGTGGATCTGA-3′
|
Reverse: 5′-TTGCTGTTGAAGTCGCAGGAG-3′
|
Mouse IL-6
|
Forward: 5’-TGATGGATGCTACCAAACTGGA-3’
|
Reverse: 5’-CTGAAGGACTCTGGCTTTGTCT-3’
|
Mouse IP-10
|
Forward: 5′-GGCCATAGGGAAGCTTGAAA-3′
|
Reverse: 5′-CAGACATCTCTGCTCATCATTCT-3′
|
Mouse MCP-1
|
Forward: 5′-GGCTCAGCCAGATGCAGTTA-3′
|
Reverse: 5′-ATTTGGTTCCGATCCAGGTT-3′
|
Mouse TNF-α
|
Forward: 5’-GATCGGTCCCCAAAGGGATG-3’
|
Reverse: 5’-TTTGCTACGACGTGGGCTAC-3′
|
Abbreviations: α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor; Fn14, fibroblast growth factor inducible 14; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP-10, interferon gamma-induced protein 10; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor |
Western blot analysis. Fresh tissue or cell cultures were ground with a homogenizer in RIPA lysis buffer with protease inhibitors (HEART, Xi’an, China). The protein samples were 20 µg per well. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (HEART), then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies targeting GAPDH (Cell Signaling, Danvers, MA, USA), collagen I (Cell Signaling), α-SMA (Abcam), fibronectin (Abcam), Notch1 (Abcam) or Jagged1 (Abcam). The horseradish peroxidase-conjugated secondary antibodies were purchased from Proteintech (Wuhan, China). The signals were detected with an electrochemiluminescence kit (Millipore). The band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Enzyme-linked immunosorbent assay (ELISA). ELISAs were performed with tissue lysates. Fresh tissue was ground in phosphate-buffered saline for the protein lysates. All lysate samples were normalized according to the original tissue weight. The commercial immunoassay kits (targeting mouse IL-6 and TNF-α) were purchased from BD Bioscience (San Jose, CA, USA). The commercial kits (targeting mouse MCP-1) were purchased from R&D Systems (Minneapolis, MN, USA). The commercial kits (targeting mouse IP-10) were purchased from Elabscience Biotechnology (Wuhan, China). The protocols were performed per the manufacturer’s instructions.
Statistical analysis. Data are expressed as the means ± SEM and were statistically analyzed using SPSS software (SPSS Inc., Chicago, IL, USA). Bonferroni multiple comparison tests and analysis of variance were used for multiple comparisons. Statistical differences were considered significant at P < 0.05.