Human kidney tissues were obtained from Children’s Hospital of Nanjing Medical University and proper informed consent was obtained from all human subjects. The study was approved by the Research Ethics Board of Children’s Hospital of Nanjing Medical University.
Male mice on C57BL/6 background aged 8–12 weeks (20–25 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were housed in the animal facility of Nanjing Medical University under controlled conditions (23–25 °C) and a 12/12 h light–dark cycle. Food and water were available ad libitum. All animal study protocols were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. Mice were randomly divided into four groups, and the researchers were blinded to these groups as much as possible, but due to the requirements of the Plk1 global knockout mice system, it was not possible to blind the mouse groups.
On day 0, mice were anesthetized with pentobarbital sodium (50 mg/kg) intraperitoneally and then subjected to UUO surgery, whereby the left ureter was exposed through a mid-abdominal incision and ligated twice using a 4-0 silk suture. For the sham group, the left ureter was exposed without ureteral ligation. On day 2, mice subjected to Plk1 silencing were injected with 2 mL of Plk1-shRNA plasmid (pPLK/GFP+Puro-mPlk1 shRNA, Public Protein/Plasmid Library, China) through a lateral tail vein in 10 sec according to the hydrodynamic-based gene delivery approach . For the drug treatment group, Plk1 inhibitor, BI6727 (Selleckchem, S2235, USA) (15 mg/kg, dissolved in corn oil) was administered by gavage on day 3 and 5 after UUO surgery. Dosing was based on published studies . Plk1 wild-type and knockout mice were subjected to UUO surgery, and contralateral kidneys were used as the control. All mice were euthanized on day 7 after surgery for tissue analysis.
Plk1 global knockout mice were generated using CRISPR/Cas9 technology. According to the structure of Plk1 gene, exons 2–3 of Plk1 were selected for deletion (Supplementary Fig. S6A). Cas9 and sgRNA were microinjected into the fertilized eggs of mice with C57BL/6 background. Fertilized eggs were transplanted to obtain positive F0 mice that were confirmed by polymerase chain reaction (PCR) and sequencing. A stable F1 generation mouse model was obtained by mating positive F0 generation mice with C57BL/6 mice. Reportedly, homozygous ablation of Plk1 leads to embryonic lethality. Hence, heterozygous mice were used for the experiments. All procedures of generation for Plk1 knockout mice were conducted in Model Animal Research Center of Nanjing University (GemPharmatechCo., Ltd). The two pairs of primers were used for tail genotyping: 5’-ATGGAAAGGCTTCTAGCGAGGG-3’, 5’-CCCTCAGAAGGATGCAAGTGAC-3’with PCR product 303 bp for mutant and 5’-TAACCTAGCACTGAGCCAGACCC-3’, 5’-CCCTGAACCCTTCCACTGTACTG-3’ with PCR product 446 bp for wild-type (WT). Hence, WT mice show one band (446 bp) and knockout (KO) mice show two bands (303 and 446 bp).
Rat kidney fibroblast cells NRK49F or mouse primary tubular epithelial cells mPTC were purchased from ATCC and were cultured at 37 °C under 5% CO2 in DMEM or DMEM/F12 medium containing 10% FBS supplemented with penicillin-streptomycin (100 IU/mL and 100 mg/mL). At 70–80% confluency, the cells were transferred to 2% FBS media for 24 h. Then, the cells were exposed to different treatments as indicated. Fibroblast activation or tubular cell pEMT were induced by human recombinant TGF-β1 (Peprotech, 100-21, NJ, USA) at 10 ng/mL and 15 ng/mL in vitro, respectively. BI6727 (Selleck, S2235, TX, USA), Chloroquine diphosphate (CQ, Apexbio, A8628, USA), Concanamycin A (MedChemExpress, HY-N1724, USA) and Bafilomycin A1 (Baf1-A1, Selleckchem, S1413, USA) was added at different concentration 24 h before TGF-β1 stimulation. Plk1-shRNA (pLKO.1-mPlk1 shRNA, species of mouse, Public Protein/Plasmid Library, China), Plk1 siRNA (species of rat), ATP6V1A-shRNA (pLKO.1-rAtp6v1a shRNA for NRK49F, pLKO.1-mAtp6v1a shRNA for mPTC, Public Protein/Plasmid Library, China), or CyclinB1-shRNA plasmid (pLKO.1-rCcnb1-shRNA, Public Protein/Plasmid Library, China) was transfected using Lipofectamine 2000 reagent (Invitrogen, 11668019, USA) 24 h before TGF-β1 stimulation.
Cell proliferation and cell cycle analysis
Cell proliferation was analyzed by CCK8 (Beyotime Biotechnology, C0037, China) and Edu-488 kit (Beyotime Biotechnology, C0071S) according to the manufacturer’s instructions. For cell cycle analysis, cells were treated as needed, trypsinized, and collected by centrifugation. After phosphate-buffered saline (PBS) washes, cells were fixed in 4% paraformaldehyde for 10 min at 4 °C and permeabilized with ice-cold 0.1% Triton X-100 for 5 min. Then, the cells were stained with DAPI (Beyotime Biotechnology, C1005, China) for 30 min at room temperature. Cell cycle distribution was analyzed by flow cytometry (FACS, BD Biosciences, USA).
Quantitative PCR (qPCR) analysis
Total RNA was isolated from kidney tissue or cells using TRIzol reagent (Takara Bio Inc., 9109, Japan). Reverse transcription was performed using HiScript II Q RT SuperMix (Vazyme Biotech Co., R222-01, China). qRT-PCR was carried out on the ABI7500 instrument (Applied Biosystem, USA) with SYBR green master mix (Vazyme Biotech Co., Q131-02). The primer sequences are listed in Table 1. Melting curves were utilized to ensure the specificity of the PCR product. The data were calculated using the 2(-Delta Delta C(T)) method, and the relative expression of the target gene was normalized to that of GAPDH.
Kidney or cell samples were homogenized in RIPA lysis buffer (Beyotime Biotechnology, P0013K, China) containing a protease inhibitor cocktail (Roche, 04693132001, Switzerland). The supernatants were obtained by centrifugation of the homogenates at 12,000 rpm, 4 °C for 15 min), and the protein concentration was measured using the BCA protein assay kit (Thermo Scientific, 23227, USA). Total protein was separated by SDS-PAGE and blotted onto PVDF membranes. Then, the blots were probed at 4 °C overnight in the primary antibody before exposing to horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate (Tanon, 180-501, China). Densitometry analysis was carried out using Image Lab software (Bio-Rad, USA) using GAPDH (Abclonal, AC033, China) as a reference. The primary antibodies were as follows: anti-Collagen III (Bioss antibodies, bs-0549R, China), anti-α-SMA (Abcam, ab7817, UK), anti-pH3 (Cell Signaling Technology, 53348T, USA), anti-fibronectin (Abcam, ab2413, UK), anti-cyclin B1 (Cell Signaling Technology, 4138T, USA), anti-Plk1 (Abcam, ab17057, UK), anti-LC3 (Novus Biological, NB100-2220, USA), anti-P62 (Cell Signaling Technology, 5114S, USA for cell Western blotting; Abcam, ab91526, UK for mice Western blotting), anti-Smad2 (Cell Signaling Technology, 5339, USA), anti-p-Smad2 (Cell Signaling Technology, 3108T, USA), anti-ATP6V1A (Abcam, ab199326, UK), anti-p-p65-NF-κB (Cell Signaling Technology, 3033S, USA). The original data is available in Supplementary Material.
Histology and immunostaining
Kidney samples were fixed in 10% formalin and embedded in paraffin. In all, 4-μm-thick sections were used for Masson and hematoxylin-eosin (HE) staining. For immunohistochemical staining, paraffin-embedded kidney sections were deparaffinized and hydrated, followed by antigen retrieval. Endogenous peroxidase activity was quenched by 3% H2O2. Then, the sections were blocked with 10% normal donkey serum, followed by incubation with anti-Collagen III (Bioss antibodies, bs-0549R, China), anti-fibronectin (FN, Abcam, ab2413, UK), anti-F4/80 (Cell Signaling Technology, 70076T, USA), anti-Plk1 (Abclonal, A2548, China). Images were analyzed and quantified using Image-ProPlus Software. Immunofluorescence staining was performed using cryosections and primary antibody anti-α-SMA (Cell Signaling Technology, 19245S, USA), anti-FSP1 (Proteintech, 66489-1-Ig, USA), and anti-Plk1 (Abcam, ab17057, UK). The staining was detected by Alexa Fluor 488-conjugated (green, Molecular Probes, Invitrogen, A21202, USA) or Alexa Fluor 555-conjugated (red, Molecular Probes, Invitrogen, A31572, USA) anti-IgG as secondary antibody. Nuclei were stained with DAPI (Beyotime Biotechnology, C1005, China). Fluorescein-labeled Lotus tetragonolobus lectin (LTL) (Vector Laboratories, FL-1321, USA) was stained for proximal tubule location. Fluorescence images were captured under a fluorescence microscope (Olympus, USA).
For F-actin staining, 48 h after Plk1-siRNA transfection, NRK49F cells were fixed with 4% paraformaldehyde at room temperature for 20 min, followed by treatment with 0.1% triton X-100 for 10 min at room temperature. Subsequently, phalloidin (Yeasen, 40737es75, China) and anti-α-SMA (Abcam, ab7817, UK) antibodies were applied.
Autophagic flux assay
RFP-GFP-LC3 plasmid (Invitrogen, P36239, USA) was transfected with Lipofectamine 2000 (Invitrogen, 11668-019, USA) into cells according to the manufacturer’s instructions. After 4 h, cells were pretreated with BI6727 or transfected with Plk1-siRNA in fresh medium for another 4–6 h, followed by TGF-β1 stimulation for 24–48 h.
LysoSensor DND-189 (Invitrogen, L7535, USA) was used to measure the lysosomal pH. After treatment with BI6727 for 24 h, NRK49F or mPTC cells were incubated in prewarmed (37 °C) probe-containing medium for 40 min. Then, the probe-containing medium was replaced with loading buffer, and the cells were observed under a fluorescence microscope.
NRK49F cells were treated with BI6727 for 24 h and homogenized. Cell extracts were immunoprecipitated with rat anti-ATP6V1A antibody (Abcam, ab199326, UK) and protein A/G agarose beads (Bimake, TX, B23201, USA). Then, the precipitates were analyzed by Western blotting with rabbit p-Serine (p-Ser) (Santa Cruz Biotechnology, sc-81514, CA, USA) and p-Threonine (p-Thr) antibodies (Santa Cruz Biotechnology, sc-5267, CA, USA).
NRK49F cells were treated with BI6727 or Plk1 siRNA for 24–48 h. The number of cells was more than 106 per well. Cell culture medium was discarded, and cells were digested with trypsin (appropriate amount of FBS was added to terminate digestion), Then equal volume electron microscope fixative was added to cells for 5 min. Cell suspension was transferred into 1.5 ml sharp bottom EP tube and centrifuged at 800–1200 rpm for 3 min to precipitate cells into clusters. Fresh electron microscope fixative were added and cells were kept at room temperature for more than 30 min. Cell sections were finally analyzed using transmission electron microscope.
Data depicted in the graphs were represented as means±standard error of mean (SEM) for each group. Multiple group comparison was made using one-way analysis of variance (ANOVA). The differences between the two groups were determined by Student’s t test. Statistically significant differences were detected between mean values in each graph. P < 0.05 showed significant difference. The statistical analyses were conducted by using GraphPad (Prism 7).