Abstract
Integrin αvβ6 holds promise as a therapeutic target for organ fibrosis, yet targeted therapies are hampered by concerns over inflammatory-related side effects. The role of αvβ6 in renal inflammation remains unknown, and clarifying this issue is crucial for αvβ6-targeted treatment of chronic kidney disease (CKD). Here, we revealed a remarkable positive correlation between overexpressed αvβ6 in proximal tubule cells (PTCs) and renal inflammation in CKD patients and mouse models. Notably, knockout of αvβ6 not only significantly alleviated renal fibrosis but also reduced inflammatory responses in mice, especially the infiltration of pro-inflammatory macrophages. Furthermore, conditional knockout of αvβ6 in PTCs in vivo and co-culture of PTCs with macrophages in vitro showed that depleting αvβ6 in PTCs suppressed the migration and pro-inflammatory differentiation of macrophages. Screening of macrophage activators showed that αvβ6 in PTCs activates macrophages via secreting IL-34. IL-34 produced by PTCs was significantly diminished by αvβ6 silencing, and reintroduction of IL-34 restored macrophage activities, while anti-IL-34 antibody restrained macrophage activities enhanced by αvβ6 overexpression. Moreover, RNA-sequencing of PTCs and verification experiments demonstrated that silencing αvβ6 in PTCs blocked hypoxia-stimulated IL-34 upregulation and secretion by inhibiting YAP expression, dephosphorylation, and nuclear translocation, which resulted in the activation of Hippo signaling. While application of a YAP agonist effectively recurred IL-34 production by PTCs, enhancing the subsequent macrophage migration and activation. Besides, reduced IL-34 expression and YAP activation were also observed in global or PTCs-specific αvβ6-deficient injured kidneys. Collectively, our research elucidates the pro-inflammatory function and YAP/IL-34/macrophage axis-mediated mechanism of αvβ6 in renal inflammation, providing a solid rationale for the use of αvβ6 inhibition to treat kidney inflammation and fibrosis.
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Introduction
Chronic kidney disease (CKD) is a global public health threat with high morbidity and mortality, affecting about 9.1-13.4% of the general population and causing over a million deaths annually worldwide [1]. Renal fibrosis is a common and dynamic pathological process that drives nearly all types of kidney dysfunction to progress to CKD, eventually resulting in renal failure [2]. However, current therapies for CKD primarily address symptoms rather than directly ameliorating kidney fibrosis [3]. Therefore, it is urgent to develop safe and effective treatments to halt this life-threatening process.
Integrin αvβ6 is a member of the integrin family, a group of transmembrane receptors that play crucial roles in cell adhesion and communication between cells and their surrounding extracellular matrix. Previous research showed that integrin αvβ6 is up-regulated during multiple organ fibrosis, e.g., lung, liver, and kidney, and promotes fibrosis via activating the key profibrotic mediator, transforming growth factor-β1 (TGF-β1). This has positioned it as a promising therapeutic target for organ fibrosis [4,5,6,7,Aristolochic acids (AA) injection-induced nephropathy (AAN) model A model of renal fibrosis induced by AA injection as previously described [77]. Briefly, male WT and Itgb6-/- mice were intraperitoneally injected with 5 mg/kg AA (Sigma-Aldrich, A9451) or PBS every other day, and kidneys and serum were collected 10 days later for detection. Blood urea nitrogen (BUN) and serum creatinine levels were measured by commercial reagents and biochemical analyzers (Roche). For in vivo IL-34 administration, each Itgb6-/--uIRI mice were intraperitoneally injected with 1 μg of rmIL-34 (RD, 5195) on days 0 and 3 after uIRI. Control animals received PBS. For in vitro IL-34 treament, after hypoxia for 24 h, serial concentration gradients (0–250 ng/mL) of rmIL-34 (RD, 5195-ML) were added to the co-culture system of TKPTS cells and RAW264.7 cells, and the cells were treated for 12 h. Renal biopsy sections from CKD patients were subjected to antigen retrieval and non-specific binding sites were blocked with 5% BSA. According to the experimental requirements, kidney sections were incubated with sheep anti-human integrin β6 antibody (Ab) (PA5-47588, Thermo Fisher Scientific), mouse anti-human CD20 Ab (ab9475, Abcam), rabbit anti-human CD3 Ab (ab5690, Abcam), or mouse anti-human CD68 Ab (ab955, Abcam) at 4 °C overnight. Primary antibodies were labeled by incubating biotin-linked secondary antibodies, respectively. Mouse kidneys used for immunohistochemistry experiments were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections. After being blocked with 5% BSA, kidney sections were stained with rat anti-mouse F4/80 Ab (BIO-RAD, MCA497G), goat anti-mouse integrin β6 Ab (RD, AF2389), sheep anti-mouse IL-34 Ab (RD, AF5195), or rabbit anti-mouse YAP Ab (CST, 14074S) at 4 °C overnight. 3-3-diaminobenzidine (DAB) was used for color development in immunohistochemistry. The slides were then examined on a pathological section scanner (Kfbio, KF-PRO-020). Immunohistochemistry was quantified by counting the positive areas in 10 high-power fields (HPF). QRT-PCR was performed on mouse kidneys and cells. Trizol was used to lyse and extract total RNA from kidney homogenates, TKPTS cells, and RAW264.7 cells. The RNA extracted from trizol was extracted by chloroform, further precipitated in isopropanol, and washed with absolute ethanol. Finally, the RNA was dissolved in DEPC water. The concentration and quality of RNA were measured by NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, USA). The RNA was reverse transcribed into cDNA according to a commercial reverse transcription kit (Vazyme, China). A PCR system was constructed using SYBR green dye, specific primers, and cDNA, and detection was performed in Applied Biosystems 7500 (Thermo Fisher Scientific, USA). Primer sequences are shown in Table S2. Mice were anesthetized with 1% pentobarbital and perfused with PBS until their kidneys became pale. The kidneys were mechanically cut into chunks and minced in RPMI 1640 containing 2% FBS at low temperatures before digestion. Digestion buffers were prepared with 1 mg/mL collagenase type II (Thermo Fisher Scientific, 17101015) and 0.5 mg/mL dispase type II (Thermo Fisher Scientific, 17105041) in RPMI 1640 containing 2% FBS. The kidneys were digested in a 200 rpm oscillator at 37 °C for 30 min. Post-digestion, the digestive fluid was filtered with a 70 μm filter and centrifuged. Red blood cells were lysed by 1 ml ACK Lysis Buffer (A1049201, Thermo Fisher Scientific). Centrifugation after the termination of fission was performed and the cell pellets were resuspended with PBS to obtain single-cell suspensions of mouse kidneys. Single-cell suspensions from mouse kidneys were prepared, and extracellular antigens were stained with flow cytometry antibodies. The antibodies used for Flow cytometry analysis are listed in Table S3. An AttuneNxT acoustic focusing cytometer (Thermo Fisher Scientific) was used for flow cytometry analysis, and FlowJo v.10 was used to process flow cytometry results. RIPA lysis buffer was used for protein extraction from mouse kidney homogenate, TKPTS cells, and RAW264.7 cells after supplementing protease inhibitors and phosphatase inhibitors. After centrifugation to remove structural proteins, the protein concentration was detected by the BCA method. Equal amounts of protein were separated by SDS-PAGE and electro-transferred to PVDF membranes. After blocking with 5% skim milk or 5% BSA, the PVDF membranes were incubated with primary antibodies overnight at 4 °C. The antibodies used in western blot were as follows: goat anti-mouse integrin β6 antibody (RD, AF2389), mouse anti-mouse α-SMA Ab (Sigma-Aldrich, A5228), rabbit anti-mouse Fibronectin Ab (BOSTER, BA1772), sheep anti-mouse IL-34 Ab (RD, AF5195), rabbit anti-mouse YAP Ab (CST, 14074 S), rabbit anti-mouse p-YAP (S127) Ab (CST, 4911 S), mouse anti-mouse GAPDH Ab (Abcam, ab8245), mouse anti-mouse α-Tubulin Ab (CST, 12351 S), and mouse anti-mouse β-actin Ab (Abcam, ab8226). After incubation was complete, unbound antibodies were washed with TBST (TBS: Tween, 1000:1). The horseradish peroxidase-conjugated secondary antibody derived from the primary antibody was incubated with the PVDF membrane for 1 h at room temperature, and the enhanced chemiluminescence (ECL) kit was used to develop specific protein bands. The image development of specific protein bands was quantitatively analyzed by ImageJ software. Kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick kidney sections. Kidney sections were stained with Sirius red dye. The severity of tubulointerstitial fibrosis was assessed by a renal pathologist who was blinded to the experimental group, and the criterion was the area of Sirius red-positive area. Scoring was performed in 10 successive HPF fields in a blinded manner. Mouse kidney paraffin sections were permeabilized with 0.2% Triton X-100 after completion of antigen retrieval, and nonspecific sites were blocked with 10% donkey serum. LTL-fluorescein (Vector, FL-1321-2) was used to label proximal renal tubules, PNA-fluorescein (Vector, FL-1071-5) was used to label distal renal tubules, DBA-fluorescein (Vector, FL-1031-2) was used to label collecting ducts. Rabbit anti-mouse YAP Ab (CST, 14074 S), sheep anti-mouse IL-34 Ab (RD, AF5195), or rabbit anti-mouse KIM-1 Ab (Novus, NBP1-76701SS) was used to label the localization of YAP, IL-34, or KIM-1. The above primary antibodies were incubated overnight at 4 °C. After the unbound primary antibody was eluted with PBS, the corresponding FITC- or PE-labeled fluorescent secondary antibody was incubated for 1 h at room temperature. 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was used to label the cell nucleus. Confocal fluorescence microscopy (ZEISS, LSM880 with Airyscan) was used to capture fluorescent signals, and ImageJ software was used to perform quantitative statistics on the co-localization of fluorescent signals. In order to simulate the ischemia-reperfusion injury model in vivo, we used an in vitro H/R injury cell model. The resume of the H/R injury cell model was performed as described previously [Quantification and statistical analysis GraphPad Prism v9.0 software was used for data statistics and visual presentation. Experimental data were presented as mean ± SEM. An unpaired Student’s t-test was used to compare the two groups. In more than two groups of comparison using general one-way ANOVA for statistics. All experiments were performed in at least three biologically independent replicates. The p-value < 0.05 indicated a statistically significant difference. The statistical significance was respectively expressed as: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001; ns, not significant.In vivo and in vitro treatment of recombinant mouse IL-34 (rmIL-34)
Immunohistochemical staining
Quantitative real-time PCR (qRT-PCR)
Preparation of kidney single-cell suspension
Flow cytometry
Western blot
Collagen fiber detection
Immunofluorescence
Hypoxia/reoxygenation (H/R) injury cell model
Data availability
The Raw and processed transcription sequencing data of TKPTS have been deposited at the GEO with the project number: GSE253494. All other study data are included in the article and/or the supplement. Any additional data in this work are available from the corresponding authors upon request.
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Acknowledgements
We are grateful to Guangzhou Genedenovo Biotechnology Co., Ltd for assisting in sequencing and/or bioinformatics analysis. We thank the authors of the GSE180394 and GSE139506 datasets for their data sharing.
Funding
This work was supported by First affiliated hospital of Sun Yat-sen University (SYSU-FAH) in Guangzhou, the National Natural Science Foundation of China 82270764, 82022009 to Y.Z., the Guangdong Natural Science Fund 2017A030306013 to Y.Z., Guangdong Special Support Program 2017TQ04R549 to Y.Z., the Medical Scientific Research Foundation of Guangdong Province of China A2024182 to Z.L., NHC Key Laboratory of Clinical Nephrology (Sun Yat-Sen University) and Guangdong Provincial Key Laboratory of Nephrology 2020B1212060028.
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CZ, RZ, and ZL designed, performed and interpreted all experiments. CZ and RZ carried out data analysis. CZ, RZ, ZL, XHan, ZT, FL, XHu, RL, JS, QP, and RW performed all animal work. ZP and GW provided Itgb6 knock-out mice. CZ, ZL, FL, and RL collected human kidney biopsies. CZ and RZ wrote the original manuscript, and ZL, WC, and YZ revised and finalized the manuscript. All authors contributed to the article and approved the submitted version.
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All human participants in this study signed informed consent and were approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University (approval numbers 2022(602), 2016(215)). All animal experiments in this study were approved by the Animal Ethics Committee of Sun Yat-sen University of Medical Sciences.
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Zhu, C., Zheng, R., Han, X. et al. Knockout of integrin αvβ6 protects against renal inflammation in chronic kidney disease by reduction of pro-inflammatory macrophages. Cell Death Dis 15, 397 (2024). https://doi.org/10.1038/s41419-024-06785-5
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DOI: https://doi.org/10.1038/s41419-024-06785-5
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