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).

In vivo and in vitro treatment of recombinant mouse IL-34 (rmIL-34)

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.

Immunohistochemical staining

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).

Quantitative real-time PCR (qRT-PCR)

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.

Preparation of kidney single-cell suspension

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.

Flow cytometry

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.

Western blot

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.

Collagen fiber detection

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.

Immunofluorescence

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.

Hypoxia/reoxygenation (H/R) injury cell model

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.