Animals

All animal experiments complied with a protocol approved by the Institutional Animal Care and Use Committee of Nanfang Hospital Southern Medical University. Atg5flox/flox and LysM-Cre mice were obtained from a C57BL/6 background. Atg5flox/flox and LysM-Cre mice were purchased from RIKEN BioResource Research Center (RBRC02975) and Jackson Laboratory (004781), respectively. Atg5flox/flox mice were crossed with LysM-Cre+ mice to obtain LysM-Cre+Atg5flox/flox macrophage-conditional Atg5 knockout (MΦ atg5−/−) and LysM-Cre–Atg5flox/flox (WT) littermate mice. All mice were genotyped using PCR before and after all experiments. Genotyping of genomic DNA from the tail of a 4-week-old mouse was performed using PCR amplification with primers Atg5flox/flox (5’-GAATATGAAGGCACACCCCTGAAATG-3’, 5’-ACAACGTCGAGCACAGCTGCGCAAGG-3’, and 5’-GTACTGCATAATGGTTTAACTCT TGC-3’) and LysM-Cre (5’-CCCAGAAATGCCAGATTACG-3’, 5’-CTTGGGCTGCCAGA ATTTCTC-3’, and 5’-TTACAGTCGGCCAGGCTGAC-3’). Atg5flox/flox amplified a 700-bp fragment, while LysM-Cre+/− amplification of a 350-bp fragment and a 700-bp fragment. The mice were bred and maintained under pathogen-free conditions at an experimental animal center (Nanfang Hospital) under a natural light cycle with free access to water and food. Specifically, 8–10-week-old male mice were used in this study and subjected to surgical procedures.

Establishment of I/R and UUO mouse models

First, the mice were intraperitoneally anesthetized with barbital sodium (60 mg/kg) for the kidney I/R models. During surgery, a homeothermic blanket control unit with a rectal probe (Shanghai Yuyan Scientific Instrument Co., Shanghai, China, Y585650809) was used to maintain body temperature at 36.5 °C. The kidneys were subjected to bilateral flank incisions, and both pedicles were clamped for 26 min. After the clamp was released, color changes in the kidneys were noted, which suggested adequate ischemia and reperfusion. Animals were sacrificed at different times (1 day, 4 days, and 7 days). Therefore, to induce unilateral renal I/R, the left renal pedicle was clamped for 31 min. Subsequently, the clamp was released for reperfusion at different durations (14 and 28 days). In contrast, sham control mice underwent the same procedure without a clamped renal pedicle. In a part of the unilateral ischemia AKI experiments, left renal reperfusion was performed for 13 days, after which the right kidney was removed, and blood samples were collected 1 day later to determine blood urea nitrogen (BUN) and serum creatinine levels to evaluate left kidney function. For the UUO models, the left abdominal cavity of the mouse was opened, the left ureter was exposed and separated, and the middle ureter was ligated with a silk thread. Finally, the mice were sacrificed 14 days after UUO, and the contralateral and obstructed kidneys were harvested for further analysis.

Flow cytometry

Kidney cell suspensions were prepared from the mice subjected to I/R, UUO, or sham surgery. The kidneys were perfused with phosphate-buffered saline (PBS), minced into fragments, filtered through a 40-µm filter, and digested in Gey’s balanced salt solution containing 0.7 mg/mL Liberase (Roche, 5,401,127,001) for 30 min at 37 °C to obtain a single-cell suspension. Next, red blood cells were removed using erythrocyte lysis buffer (QIAGEN, 79,217). The cells were blocked in 2.5 mg/mL Fc blocking solution and then stained with the fluorescent antibodies PerCP/Cyanine5.5 anti-mouse CD45.2 (109,827), PE anti-mouse F4/80 (123,110), FITC anti-mouse CD11c (117,306), PE/Cyanine7 anti-mouse CD206 (141,720), and APC/Cy7 anti-mouse CD11b (557,657). These antibodies were purchased from BioLegend, except for APC/Cy7 anti-mouse CD11b, which was purchased from BD Biosciences. Macrophages were identified as CD45+ CD11c− CD11b+ F4/80+ cells. PE/Cyanine7 anti-mouse CD206 antibody was used to identify M2 macrophage polarization. Flow cytometry data were acquired on the BD Canto II system (BD Bioscience, Franklin Lakes, NJ) and analyzed using FCS Express 10.0 software.

Renal function, histology, immunohistochemistry, and quantitative image analysis

Blood samples were collected at the indicated time points to examine renal function. Whole blood was allowed to settle at room temperature (∽ 25 °C) for 30 min. After coagulation, the blood samples were centrifuged at 1,000 × g for 10 min at 4 °C, and the supernatant was collected as the serum sample. BUN and serum creatinine levels were measured on an automatic biochemical analyzer (Mindray, Shenzhen, China, BS-240VET) using BUN (Mindray, 0041-30-53008) and creatinine (Mindray, 105-000456-00) assay kits.

For histology, kidney tissues were washed in cold PBS, fixed in 4% formaldehyde for 24 h, and sliced into 4-µm sections. Parts of the paraffinized sections were used for Masson’s trichrome staining (Leagene, DC0032) or SR/Fast green staining (Leagene, DC0040) following the manufacturer’s procedures. Finally, the fibrosis area (%) was measured in 10 randomly selected fields from each section using ImageJ software.

Immunohistochemistry was performed on 4-µm paraformaldehyde-fixed paraffin-embedded kidney tissue sections. Briefly, the sections were incubated in 3% hydrogen peroxide, and Tris-EDTA antigen repair solution (pH 9.0) (absin, abs9342) was used after dewaxing and rehydration. The samples were blocked with 3% bovine serum albumin blocking solution and incubated overnight at 4 °C with the following primary antibody solutions: CD11b (Abcam, ab10079; 1:250), vimentin (Proteintech, 10366-1-AP; 1:250), α-SMA (actin alpha 2, Abcam, ab5694; 1:250), or fibronectin 1 (FN1, Abcam, ab2413; 1:250). Finally, tissue staining was visualized under a microscope (Nikon, Tokyo, Japan, ECLIPSE Ci-S), and the positive area (%) was analyzed in 10 randomly selected fields from each section using ImageJ software.

Quantitative real-time PCR

Total RNA was extracted from the kidney or isolated renal myeloid cells with TRIzol Reagent (Ambion, 15,596,026) following the manufacturer’s protocol. The RNA was reverse-transcribed into single-stranded cDNA using a commercial reverse transcription kit (Roche, 4,897,030,001). Quantitative real-time PCR was performed on the LightCycler 480 System (Roche, Basel, Switzerland) using the SYBR Green I Master kit (Roche, 4,887,352,001), following the manufacturer’s instructions. Data were calibrated to glyceraldehyde phosphate dehydrogenase values. The sequences of primers used in the experiments included Atg5, α-SMA (Acta2), Collagen 1a1 (Col1a1), CCR6 (Ccr6), CXCR2 (Cxcr2), CCL20 (Ccl20),TNFα (Tnfα), Il6 (Il-6), iNOS (iNOS), TGFβ1 (Tgfb1), and GADPH (Gapdh) (Table S1).

Isolation and treatment of BMDMs

MΦ atg5−/− and WT mice were anesthetized with isoflurane, sacrificed, and soaked in 75% alcohol for 3 min for disinfection. Subsequently, the tibia and femur were harvested. Next, the tibia and femur cells were flushed using a 1-mL syringe with RPMI 1640 medium (Solarbio, 31,800) supplemented with 10% fetal bovine serum (Gibco, 10099-141). The cell suspension was passed through a 40-µm cell strainer, centrifuged, resuspended in 5 mL of erythrocyte lysis buffer (QIAGEN, 79,217), and incubated for 5 min. The cells were recentrifuged and resuspended in RPMI 1640 medium with 5 mM HEPES followed by monocyte isolation using a monocyte isolation kit for mice (Miltenyi Biotec, 130-100-629) to obtain BMDMs. Finally, the BMDMs were plated in a 6-well plate at a density of 4 × 106 cells per well and stimulated with 1 µg/mL LPS or PBS for 4 h at 37 °C.

Tracking monocyte cell migration into the injured kidney

BMDMs from MΦ atg5−/− and WT mice were labeled using CellTracker™ green 5-chloromethylfluorescein diacetate (CMFDA) dye (Invitrogen, c70251). Each C57BL/6 recipient mouse was injected with 2 × 106 CMFDA-labeled BMDMs from MΦ atg5−/− or WT mice via the tail and subjected to unilateral renal pedicle clamping for 31 min the next day. Three days later, the animals were sacrificed, and the injured kidneys were collected for cryosections. Next, kidney macrophage infiltration was evaluated by counting double-positive labeling with CMFDA (green) and F4/80 (red) using a confocal microscope (Carl Zeiss AG, Oberkochen, Germany, LSM980), which was identified as 4’,6-diamidino-2-phenylindole+nuclei (DAPI, Solarbio, C0065) as described in the literature [22]. Finally, flow cytometry was used to analyze the number of macrophage migration to the injured kidney.

Isolation of kidney macrophages

Single-cell suspensions were obtained from kidney tissues using enzymatic digestion with Liberase (Roche, 05401127001) diluted in Gey’s balanced salt solution. Fascia removal was performed using a 40-µm cell strainer. Next, F4/80+ cells were extracted from the cell suspension using the EasySep™ Mouse Biotin Positive Selection Kit II (STEMCELL, 17,665) according to the manufacturer’s instructions. Briefly, the kidney cells were transferred to a 5-mL (12 × 75 mm) polystyrene round-bottom tube and resuspended in RoboSep Buffer (STEMCELL, 20,104), blocked with FcR, and incubated with anti-mouse F4/80 antibody (STEMCELL, 60027BT) for 15 min. Subsequently, the cells were centrifuged, resuspended, incubated with the selection cocktail for 15 min, and reacted with RapidSpheres for 10 min. Finally, the tubes (without lids) were placed into a magnet (STEMCELL, 18,103) for 5 min, the supernatant was discarded, and the cells were collected from the sides of the tube.

Cell migration in vitro

The in vitro migration of BMDMs from MΦ atg5−/− and WT mice was evaluated using Transwells as previously described [22, 23]. Freshly isolated BMDMs (1 × 106) were seeded in the top chamber of a 24-well polyester membrane (8-mm pore size; BD Biosciences, 353,097) in serum-free medium. The cells translocated to the lower chamber in response to exposure to RPMI 1640 medium containing 10% fetal bovine serum and 50 ng/mL CXCL3 (R&D Systems, 453-KC-010) or CCL20 (ORIGENE, TP723274). After incubation for 4 h, the cells in the upper chamber were gently wiped with a cotton swab, and the filter membrane was fixed with 4% methanol and stained with 0.2% crystal violet (Solarbio, g1061). Next, the filters were photographed using a microscope (OLYMPUS, Shinjuku, Tokyo, Japan, BX63), and the cell numbers were counted in each field of view under ×200 magnification.

Western blotting

BMDMs were seeded in a 6-well plate at 3.5 × 106 cells per well. Cells in the experimental group were treated with 200 ng/mL CXCL3 or CCL20 collected after 30 min incubation at 37 °C. Subsequently, the stimulated BMDMs were homogenized in RIPA lysis buffer (CWBIO, CW23335) containing protease inhibitors for 30 min at 4 °C and centrifuged at 12,000 × g for 15 min. The supernatant was collected as the total protein for Western blotting. The primary and secondary antibodies were as follows: β-actin (Sigma, A5361; 1:1000), AKT (Cell Signaling Technology, 4691; 1:1000), p-AKT (Cell Signaling Technology, 4060; 1:1000), ERK1/2 (Cell Signaling Technology, 9102; 1:1000), p-ERK1/2 (Cell Signaling Technology, 4307; 1:1000), PI3K (Cell Signaling Technology, 4257; 1:1000), p-PI3K (Cell Signaling Technology, 4228; 1:1000), goat anti-rabbit IgG-HRP (Asbio, As006; 1:2000), and goat anti-mouse IgG-HRP (Asbio, As005; 1:2000). ECL detection reagents (Cytiva, RPN2232) were used for color development and Tanon gel imaging, and the gray value was obtained after analysis using ImageJ software.

Statistical analyses

All statistical analyses were performed using GraphPad Prism version 8.0.1 (GraphPad). Data are expressed as the mean ± SEM. To assess the significant differences, two-tailed unpaired Student’s t tests were used to compare 2 independent groups, and one-way ANOVA or two-way ANOVA was used to compare 3 or more groups. A P value of less than 0.05 was considered statistically significant.