Abstract
Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.
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Introduction
According to the Centers for Disease Control and Prevention, more than 10 percent of American adults have chronic kidney disease (CKD). With increases in comorbidities, diabetes, obesity, atherosclerosis and hypertension, the estimated number of cases with CKD will continue to grow1,2. Chronic renal allograft injury (CAI), a major cause of CKD in transplant patients, is characterized by interstitial fibrosis/tubular atrophy (IF/TA) and is the most common cause of long-term renal allograft failure in the first post-transplant decade. Kidney inflammation and fibrosis are the common pathways mediating the progression of CKD and CAI3. However, the underlying mechanisms remain obscure, and the treatment is lacking. It has been shown that excessive and prolonged inflammation is a major driver of fibrosis4,5. Macrophage is known to play a major role in regulation of renal inflammation and fibrosis by producing pro-inflammatory cytokines and pro-fibrosis factors6. Although multiple profibrotic factors have been identified, these experimental interventions have not been translated into clinical practice. Recent clinical trial using anti-TGFβ monoclonal antibody has failed in patients with diabetic kidney disease. TGF-β also has anti-inflammatory properties7,8,9 and a complete blockade of TGF-β may enhance inflammatory response10. Therefore, there is an urgent need to develop better drug targets for treatment of renal inflammation and fibrosis in CKD patients.
SRC family kinases (SFKs) belong to a family of non-receptor protein tyrosine kinases and have been implicated in the regulation of numerous cellular signaling and biological effects11,12. Previous studies have demonstrated the important role of SFKs in the development of various fibrosis-related chronic diseases such as idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, and systemic sclerosis13. Our recent unbiased screening studies revealed that hematopoietic cell kinase (HCK), a SFK member, promotes renal fibrosis pathway and dasatinib, a non-selective inhibitor of HCK attenuates renal fibrosis in mice with unilateral ureteral obstruction and lupus nephritis14. However, how HCK contributes to renal fibrosis remains unclear. Several studies suggest that HCK plays a major role in regulating macrophage functions of polarization, migration, and proliferation11, The GoCAR study is a prospective, multicenter study from USA and Australia to investigate the association of differential gene expression and the development of chronic allograft injury. The study was approved by the institutional review boards of the participating institutions. Written informed consent was obtained from all enrolled patients. Details of the observational GoCAR cohort, including ethics approval, eligibility and exclusion criteria are published elsewhere17. RAW264.7 (TIB-71™), HEK293 (CRL-1573™) and L929 fibroblasts (CCL-1™) cells commercially obtained from ATCC. HEK293, RAW264.7 and L929 cells were expanded using DMEM (Gibco) media with 10% FBS and 1% Pen/Strep. Bone marrow-derived macrophages (BMDM) were isolated from mice following the protocols67,68 and cultured using full DMEM (Gibco) with 20% of L929 cell supernatant for 7 days. For M1 activation, 100 ng/ml LPS with 50 ng/ml IFNγ was added in culture medium; for M2 activation, 10 ng/ml IL-4 was used to treat for 24 h. Dasatinib was purchased from Selleckchem (Cat#: S1021) and was made stock at 10 mM at DMSO, then it was diluted to 100 nM to treat R264.7 and BMDM. Structure activity relationship (SAR) studies27,28 was performed by Dr. Bhaskar Das. BT424 was synthesized by Dr. Das’s lab and purified with HPLC after synthesis. A step-by-step experimental procedure of BT424 synthesis and its chemical characterization were provided in the supplementary Fig. S4. BT424 is insoluble in water. It was stocked at 100 mM in DMSO and diluted to 25 uM to treat cells. Plasmids were purchased from Dharmacon (https://horizondiscovery.com/) for HCK, FYN and SRC overexpression and shRNA knockdown. HCK overexpression (Clone ID: ccsbBroad304_00733) and knockdown (Clone ID: V2LHS_237454, V2LHS_133010, V2LHS_133007). FYN overexpression (Clone ID: ccsbBroad304_00600), SRC overexpression (Clone ID: ccsbBroad304_06992). Plasmids for control (for OVE, Cat#: OHS6085, for KD, Cat#: RHS4349). Transfecting in Raw264.7 was used Lipofectamine LTX Reagent (Cat #: A12621, Thermo Fisher Scientific) and in HEK293 used PolyJet™ (SignaGen Laboratories, Cat #: SL100688) following manufacturer’s instructions. RNA was extracted using the TRIzol Reagent (Thermo Fisher Scientific) from cell and whole kidneys. One thousand nanograms of RNA was used to prepare cDNA. Quantitative real-time PCR was performed using Qiagen QuantiTect One Step RTPCR SYBR green kit (Qiagen). Data were analyzed by the 2 − ΔΔCT method and presented as fold change relative to a control sample after normalization against the expression of housekee** genes. Cells were lysed with Cell Lysis Buffer II (ThermoFisher Scientific, # FNN0021) containing 1% NP-40, added protease inhibitor cocktail (Sigma, #P1860), L-isozymes of alkaline phosphatase inhibitor cocktail (Sigma, #P2850) and tyrosine and serine/threonine phosphatase inhibitors (Sigma, #P5726). Lysates were subjected to immunoblot analysis using the following antibodies: HCK (CST #14643), V5 tag (GenScript Inc #A01724), phosphor-Y410 HCK (Abcam #ab61055), LC3A/B (CST #4108), Fyn (CST #4023 S), p62/SQSTM1 (NOVUS BIOLOGICALS NBP1-48320SS), Atg2A (CST #15011), c-Cbl (CST #2747), phospho-c-Cbl (Tyr700) (D16D7) (CST #8869), MMR/CD206 (R&D Systems #AF2535), iNOS (BioLegend #690902), Arginase 1 (BioLegend #678802), Phospho-PI3K p85 /p55 (CST #17366), Phospho-Akt (CST #4060), Akt (CST #4691), Phospho-p44/42 MAPK (Erk1/2) (CST #9101), p44/42 MAPK (Erk1/2) (CST #9102), EGFR (CST #4267), Phospho-EGFR (CST #3777), SYK (CST #13198), pY20 (Invitrogen # 14-5001-82), a-SMA (Sigma-Aldrich #A5228) and GAPDH mAb (CST #2118). Anti-V5-tag mAb-Magnetic Beads (MBL #M167-11) were used for immunoprecipitation. Antibody anti-GAPDH was diluted 5000 times with 3% BSA in PBST. All other antibodies mentioned above for WB were used dilution of 1:1000. The Anti-V5-tag mAb-Magnetic Beads were used 50 ul for each reaction with 400 ul incubation volume. The secondary antibodies were from Promega: Anti-Rabbit IgG (H+L), HRP Conjugate (#W4011) and Anti-Mouse IgG (H + L), HRP Conjugate (#W4021). The secondary antibodies were diluted with 1:5000. Image Studio Lite Ver5.2 for western blot image acquisition. Densitometry was performed on images of Western blots using Image J software. Autophagy LC3 HiBiT reporter (Promega, #GA2550) was transfected to HEK293 cells with PloyJet (SignaGen Laboratories, #SL100688) following the manufacture’s protocol. Then LC3 reporter was detected with Nano-Glo HiBiT fluorescent reagent (Promega, N3030). The fluorescent signal was adjusted by load protein amount. For Raw264.7 and BMDM, autophagy inducer PP242 at 1 uM, autophagosome degradation inhibitor Bafilomycin A1 (BafA1) at 100 nM, and autophagy inhibiter 3-Methyladenine (3MA) at 5 mM treated for 24 h for LC3 and P62 measurement with WB. HCK inhibitors BT424 and dasatinib were added to pretreat cells 2 h before autophagy stimulation. BMDMs from WT and HCK KO mice were starving overnight with 1% FBS DMEM with L929 on day 5. Then EGF at 100 ng/ml was used to treat cells for 15 and 30 min before harvesting on ice with cold PBS wash. The cell lysate was then used for western blot assay to measure EGFR signaling-related proteins. For the integrin signaling, BMDMs were plated to collagen pre-coated 10 cm dishes for 1 h following the paper16. F-actin and pY20 with SYK were measured by IF stain and western blot. Immunofluorescence (IF) staining follow our previous paper14. In brief, Raw264.7 and BMDMs were cultured on cover glasses and washed with PBS 3 times. Then fixed with 4% PFA for 10 min and blocking for 1 h. Primary antibody with 1:100 dilution incubated overnight, and the next day added fluorescence secondary antibodies. For the UUO and uIRIx mouse tissue staining, paraffin embedding tissue was used, antigen retrieval was performed first with steamer. The following antibodies were used: anti-phospho Y410 HCK antibody (Abcam, ab61055), anti-HCK antibody (Abcam, ab75839 or Cell Signaling Technology, #14643), anti-LC3A/B antibody (Cell Signaling Technology, #4108), anti-Ki67 antibody (Abcam, ab16667), anti-F4/80 antibody (Invitrogen, 14-4801-82), anti-COL1A1 antibody (Southern Biotech, #1310-01), anti-F-actin antibody (Novus Biologicals, #NB100-64792). The secondary antibodies were from Invitrogen: Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568, Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568. The secondary antibodies were diluted with 1:200. AxioImager. Z2(M) and ZEN 2.3 were used for IHC IF microscopy image acquisition. HCK and SRC family kinases activity was measured by Reaction Biology Corp, PA. The chemical array was performed with cultured medium from BMDM from WT and HCK KO mice using the Proteome Profiler Mouse Chemokine Array Kit (R&D system, ARY006) following the manufacturer’s instructions. BMDMs at 7-day were polarized to M1 with 100 ng/ml LPS with 50 ng/ml IFNγ for 24 h, WT BMDM were pre-treated with HCK inhibitor dasatinib for 2 h before M1 polarization. HCK-V5, FYN-V5 and SRC-V5 plasmids were overexpressed in HEK293 cells. Protein lysates after 48 h transfection was immunoprecipitated with anti-V5-tag magnet beads and run with PAGE gels. Three resultant lanes were sent for mass spectrometry (Center for Advanced Proteomics Research, RUTGERS New Jersey Medical School). Proteins identified in control and HCK, FYN and SRC overexpression lanes were selected for further filtering analysis. Proteins in overexpression samples with Spectra # <2 were omitted and ratio of overexpression and control lanes were ranked for identified interacting proteins. Macrophages proliferation was measured with different assays with BMDMs, Raw264.7 and in mice. Click-iT™ EdU Cell Proliferation Kit from ThermoFisher Scientific (#C10340) was used to detect proliferating cells following the manufacture protocol. EdU (5-ethynyl-2’-deoxyuridine) was intraperitoneally injected in mouse (40 mg/kg) or treated BMDM and RAW264.7 (10uM) for 2 h for incorporated into newly synthesized DNA. Kidney tissue and cells were fixed with 4% formaldehyde and coupling of EdU with Alexa Fluor™ 647 dye in Click-iT™ kit following the manufacturer’s instructions. For the UUO and uIRIx mouse tissue staining, frozen tissue was used following the manufacture’s protocol like cell staining. BMDMs cultured for 4 days were used for MTT and PI flow assay. eBioscience™ Propidium Iodide staining solution (#00-6990-50) and MTT (#M2003, Sigma) were used and following the manufactory’s protocols. Attune Flow Cytometer and FlowJo v10.8.0 were used for flow data analysis. BT424 treated Raw 264.7 for 24 h before the cell proliferation assay. Scratch Assays. RAW 264.7 cells and BMDMs were plated on 6 well plates one day prior to the assay day and starved overnight. Starving medium with 0.5% FBS was used to reduce cell proliferation. Scratches were made by using 200 ul pipet tips. Starving medium with 0.5 FBS was replaced for full culture medium to reduce the cell proliferation. Images were taken at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h after the scratch. The distance of unhealed area was measured with ImageJ package “Wound_healing_size_tool”. Transwell assay. 2D transwell migration assays were performed in 24-transwells chambers (5um pores, Corning, Cat #: 3421) according to the manufacturer’s instructions. Briefly, BMDMs were starved for 3 h and then seeded 1E5 cells in the upper chambers. The lower chamber was filled with 750 ul of full DMEM medium with 20% of L929 medium. After 24 h, cells in the upper chambers were removed with cotton swab. Cells on the lower side of the insert membrane were fixed with 4% glutaraldehyde for 10 min at room temperature. Then 0.1% crystal violet was used to stain the cells for 20 min at room temperature following two times of PBS washing. Images were taken after microscope after the insert completely dry. Image J was used to analysis the cell number. 3D transwell assay. The transwell assay was performed following the paper16. Briefly, 100 μl of Matrigel diluted to 0.4 mg/ml (Corning® Matrigel® Matrix, Cat #: 354277) was poured into 24 transwell (8 μm pores) and polymerized in cell incubator for 30 min. 700 μl of Full DMEM medium with 20% of L929 medium was put to lower chamber to hydrate the Matrigel and the membrane for 3 h. BMDMs (2E5) in starving medium (0.5% FBS) were seeded in the upper chamber and after 24 h of migration. Cells passed through the transwells were counted as in regular transwell assay above. 3D-random migration analysis was performed following the paper16. Briefly, BMDMs (1E5) suspended in Matrigel (Corning, Cat #: 354277) at 4 °C were plated in ibidi 8 well high glass bottom chamber (ibidi, Cat.No:80807) and polymerized in cell incubator for 30 min. 200 ul of full DMEM medium with 15% of L929 supernatant was added to each chamber and kept in 37 °C in 5% CO2 atmosphere to record cell migration. 200X objective of an automated Leica DMIRBE microscope equipped with a CoolSnapEz Camera (Roper Scientific) acquired images every 3 min for 10 h with software LAS X 3.7.4 to record cells migration. The Z stacks imaging was used between 1500 and 2000 μm (30 μ m intervals) above the glass surface. Migrating cells were tracked from projected stacks to analyze velocity and persistence over time with ImageJ software TrackMate plugins. The first 15 images were removed to avoid light variation and cell recovery at beginning, and 8-bit image and lower and upper threshold levels were set as 20 and 120. Laplacian of Gaussian (LoG) detector was used. Detailed parameters were set as: Estimated object diameter: 40-micron, Quality threshold: 0.02, Initial thresholding: 0.03. Simple LAP tracker was used to track the cell movement. Parameters were set as: Linking max distance: 30-micron, Gap-closing max distance: 30-micron, Gap-closing max frame gap: 2, number of spots in track: 12.28. TrackMate output files were opened in MATLAB for velocity analysis and AVI format of video files were saved for cell movement tracking. The cells that moved less than 1 um/min were considered as dead cells and omitted. HCK exon3 loxp flanked KO mouse were developed at EuMMCR in Germany (HCK ES Cell Clone: HEPD0510). These mice were crossed with tissue specific Cre mice (CMV-cre B6.C-Tg (CMV-cre)1Cgn/J006054; LysM-cre B6.129P2-Lyz2tm1(cre)Ifo/J, 004781, the Jackson Laboratory) to generate HCK KO specifically in tubular cells and macrophages. C57BL/6 J WT mice also from Jackson Laboratory. All mice were maintained in our animal facility (Center for Comparative Medicine and Surgery, CCMS) at Mount Sinai under controlled environmental conditions: 12/12 light/dark cycle, ambient temperature 20–25 °C. More husbandry operation information can be found through CCMS website: https://icahn.mssm.edu/research/ccms/services-rates/husbandry. All mouse experiments were performed per the guidelines and were approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai (New York, NY). UUO model in WT, HCK KO, and HCK inhibitor mice was performed following our previous paper14. BT424 was made in 250 mg/ml in DMSO as stock, then was diluted to 2.5 mg/ml with PBS before gavage mice for final concentration of 25 mg/kg body weight. Unilateral IRI with contralateral nephrectomy (uIRIx) model was performed following the papers69,70, briefly, artery and vein of right kidney was tied, and the right kidney was removed. Then the left kidney’s artery and vein were clamped for 25 min and released. At the end of mice models, the mice were IP injected with 100 mg/kg ketamine and 10 mg/kg xylazine and then perfused with cold PBS for tissue collection. Data were expressed as mean ± SEM (X ± SEM). 2-tailed unpaired t test was used to analyze data between two groups after determination of data distribution. The ANOVA test was used when more than two groups were present. Statistical significance was considered when P < 0.05. All statistical analyses and figures were performed using GraphPad Prism software (version 6). Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.Methods
GoCAR Study
Cells
Compounds
Plasmids and transduction
Quantitative PCR
Western blotting
Autophagy measurement
EGFR and integrin signaling pathway studies
Immunofluorescence staining
Chemokine array
Mass spectrometry
Click-iT™ EdU cell proliferation assay
MTT and propidium iodide flow for cell proliferation
2D and 3D migration assay
Animal studies
Statistics
Reporting summary
Data availability
All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files. Source data are provided with this paper.
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Acknowledgements
We would like to publish this study in the loving memory of Dr. Barbara Murphy who contributed to the first part of this study. She passed away on June 30, 2021. J.C.H. is supported by NIH/NIDDK R01DK109683, R01DK122980, R01DK129467, P01DK56492, and VA Merit Award I01BX000345; K.L. is supported by NIH/NIDDK R01DK117913-01 and R01DK129467; C. Wei is supported by ASN Career Development Grant.
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C.W., J.C.H. and B.D. designed the research project. C.W., M.C., M.M., W.W., J.F., Z.Y., J.L., Z.L., L.M., K.B., S.L., Y.D., N.A., E.A. performed the experiments. B.D. designed and synthesized the compounds. C.W., J.C.H., W.Z., K.L., Z.Y., Z.S. analyzed the data. C.W., J.C.H., M.M., W.Z. and B.D. drafted and revised the manuscript. All authors approved the final version of the manuscript.
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Chen, M., Menon, M.C., Wang, W. et al. HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy. Nat Commun 14, 4297 (2023). https://doi.org/10.1038/s41467-023-40086-3
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DOI: https://doi.org/10.1038/s41467-023-40086-3
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