Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 (product no. L3012) were purchased from Sigma-Aldrich; Merck KGaA. Recombinant mouse interferon-γ (IFN-γ; cat. no. 485-MI-100; R&D Systems, Inc.) was used for macrophage intervention. The levels of plasma blood urea nitrogen (BUN; cat. no. C013-2-1) and serum creatinine (SCr; cat. no. C011-2-1) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

The experimental animal procedures were approved (approval no. N2017-078) by the Ethics Committee of Soochow University (Suzhou, China). All the mice were housed under pathogen-free conditions in a standard laboratory with a controlled room temperature (22±1°C) and humidity (65-70%), and a 12:12-h light-dark cycle, with free access to food and water. For the ischemic AKI model (n=6), bilateral I/R injury was induced in mice by clamping the renal pedicle for 30 min as previously described (17). Mice were sacrificed by cervical dislocation at 24 h after I/R injury, and the renal cortex was harvested. Mice in the GW4869 group received intraperitoneal injection of GW4869 (2.5 mg/kg; Sigma-Aldrich; Merck KGaA; n=6) 2 h before I/R surgery. The intraparenchymal injection (n=6) was performed as previously described (18). Briefly, the inferior pole of the left kidney was exposed. Exosomes derived from M1 or M0 macrophages (20 µg in 60 µl of PBS) were injected into 2 sites (the left and right sides of the inferior pole of the kidney) via a 50-G needle. The in vivo miRNA-155 inhibitor (Suzhou GenePharma Co., Ltd.) was transfected into the mouse kidneys through tail vein injections using the in vivo-jetPEI (Polyplus-transfection SA). Briefly, the miR-155 inhibitor (50 µg) or negative control (NC; 50 µg) dissolved in 5% glucose solution was injected into each mouse at 24 h before surgery as instructed by the manufacturer's protocols. Mice were sacrificed at 24 h after I/R, and their serum and renal cortex were harvested. To ameliorate the suffering of mice throughout the experimental period, the mice were euthanized with isoflurane inhalation followed by the dislocation of the cervical vertebra, where isoflurane was used at 4% for induction and 2% for maintenance of anesthesia. Animal sacrifice was confirmed by respiratory and cardiac arrest, and no righting reflex.

Periodic Acid-Schiff (PAS) staining was performed. Kidneys were fixed in 10% buffered formalin at room temperature for 24 h, embedded in paraffin, cut into 3-µm sections, stained with Periodic Acid for 20 min and Schiff for 20 min at room temperature, and visualized at a magnification of ×400 under an optical microscope (Olympus Optical Co., Ltd.). To evaluate the tubular injury score, 10 random tissue section images per animal were assessed on PAS-stained sections in a blinded manner by nephropathologists, and semi-quantitatively scored as previously described (19).

For immunohistochemical staining, 10% buffered formalin-fixed at room temperature for 24 h, and paraffin-embedded tissue sections (4 µm) were blocked with 10% goat serum (Wuhan Servicebio Technology Co., Ltd.) for 2 h at room temperature and were incubated with primary antibodies against F4/80 (1:200; product code ab6640; Abcam) and kidney injury molecule-1 (KIM-1) (1:200; cat. no. MA5-28211; Invitrogen; Thermo Fisher Scientific, Inc.) for 12 h at 4°C and then analyzed using a streptavidin peroxidase detection system (50 µl; cat. no. KIT-9720; Fuzhou Maixin Biotech Co., Ltd.) at room temperature according to the manufacturer's protocol. DAB (Fuzhou Maixin Biotech Co., Ltd.) was used as an HRP-specific substrate. The images were visualized under an optical microscope (Olympus Corporation).

TECs were isolated for primary culture using an established method (20) and then were cultured in DMEM-Ham's-F12 medium (Hyclone; Cytiva) supplemented with 10% fetal bovine serum (FBS; ScienceCell Research Laboratories, Inc.), and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Ischemia/reperfusion injury (I/RI) conditions of TECs were modeled in vitro (1% oxygen, 94% N₂ and 5% CO₂ with glucose-free and FBS-free for 12 h and then regular culture medium with 21% oxygen for 2 h of reoxygenation). The mouse RAW264.7 macrophage cell line (American Type Culture Collection; ATCC no. TIB-71) was used for this study. RAW264.7 cells were cultured in RPMI-1640 (Hyclone; Cytiva) supplemented with 10% FBS and 1% penicillin-streptomycin. Both cell lines (TECs and RAW264.7 macrophages) were cultured in an atmosphere of 5% CO₂ and 95% air at 37°C.

For kidney exosome extraction, 100 mg of the kidney cortex derived from the ischemic kidney was collected. Renal exosomes were isolated using ultracentrifugation as previously reported (21). For in vitro experiments, RAW264.7 macrophages were cultured with the presence or absence of LPS (100 ng/ml) plus IFN-γ (20 ng/ml) in serum-free RPMI-1640 medium. The medium was then used for exosome purification using differential ultracentrifugation (Type 70 Ti Rotor; Beckman Coulter Optima L-80 XP) as previously described (21). Briefly, the medium was centrifuged at 2,000 × g for 20 min at 4°C to eliminate the cells and debris and at 13,500 × g for 20 min at 4°C to eliminate the microvesicles, followed by ultracentrifugation at 200,000 × g for 120 min at 4°C. The exosome pellet was washed in 20 ml of PBS and collected by subsequent ultracentrifugation at 200,000 × g for 120 min at 4°C.

The size and morphology were detected by electron microscopy and the specific surface markers (Alix, CD63, CD9) of the isolated exosomes were also detected by western blotting for characterization of the exosomes.

The exosome sample was diluted 10 times with PBS and then stained with 2% phosphotungstic acid for 10 min at room temperature. The samples were detected using a TEM (Hitachi HT 7700; Hitachi, Ltd.) at 80 kV.

To study the process of macrophage-derived exosomes communicating with TECs, a Transwell Permeable Support system (Corning, Inc.) with a 0.4-µm pore-size filter was used according to the manufacturer's protocol. Macrophages (2×10⁶) were seeded into the upper chamber containing RPMI-1640 medium supplemented with 10% FBS. Subsequently, recipient TECs were then seeded in 12-well plates (lower chamber) in DMEM-Ham's-F12 medium supplemented with 10% FBS. Both cell lines were cultured for 12 h at an atmosphere of 5% CO₂ and 95% air at 37°C. Macrophages with LPS plus IFN-γ treatment were then assessed in the upper chamber followed by DiO-labeling for 6 h at 37°C. All co-cultured experiments were then conducted in hypoxia or normoxia for 12 h. Uptake of DiO-labeled exosomes by TECs was visualized by the confocal microscope (FV1000; Olympus Corporation).

m iR NA ta rgets were predicted using 5 online databases: TargetScan (Human 8.0/Mouse 8.0; https://www.targetscan.org/mmu_80/), miRDB (http://www.mirdb.org/), miRanda (http://www.microrna.org/microrna/home.do), DIANA-TarBase (v7.0; http://www.microrna.gr/tarbase), and PicTar (http://www.pictar.org/).

RAW264.7 cells were transfected with NC inhibitor (forward, 5′-CCC CCC CCC CCC CCC CCC CC-3′ and reverse, 5′-CCC CCC CCC CCC CCC CCC CC-3′) or miR-155 inhibitor (forward, 5′-ACC CCU AUC ACG AUU AGC AUU AA-3′ and reverse, 5′-ACG UGA CAC GUU CGG AGA ATT-3′) at a concentration of 100 nM using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer′s protocol. Following transfection for 6 h at 37°C, the medium was removed and fresh medium was added. The subsequent experiments were performed after 18 h of transfection.

Total RNA was extracted using TRIzol following the manufacturer's instructions (Takara Bio, Inc.). mRNA was reverse-transcribed using PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions and PCR was performed using SYBR Premix Ex Taq and 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The amplification level was programmed with a denaturation step at 95°C for 30 sec, followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec. miRNA was reverse-transcribed and detected with All-in-One miRNA First-Strand cDNA Synthesis kit and All-in-One miRNA qPCR kit (GeneCopoeia, Inc.) according to the manufacturer's instructions. The housekeeping genes β-actin and U6 were used as controls. The 2^−ΔΔCq method was used as previously reported (22). All the primers for RT-qPCR are listed in Table I.

Samples of cells and cortical tissues were lysed in cold RIPA lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with protease inhibitor cocktail and the protein concentration was determined using a BCA protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). Proteins (20-40 µg) were subjected to 10% SDS-PAGE (Thermo Fisher Scientific, Inc.) and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk for 2 h at room temperature. The primary antibodies used were anti-Alix (1:500; cat. no. sc-53540; Santa Cruz Biotechnology, Inc.), anti-CD63 (1:1,000; product code ab213090; Abcam), anti-suppressor of cytokine signaling-1 (SOCS-1; 1:1,000; product code ab62584; Abcam), and anti-β-actin (1:3,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) and the membranes were incubated with the primary antibodies for 12 h at 4°C. Goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:3,000; product nos. 7076 and 7074, respectively; Cell Signaling Technology, Inc.) were used for detection for 2 h at room temperature. The signals were then detected using an enhanced chemiluminescent kit (GE Healthcare; Cytiva). Finally, ImageJ software (v1.8.0; National Institutes of Health) was used for densitometry.

The plasmids were all obtained from Shanghai Genechem Co., Ltd. TECs were transfected with 3′UTR luciferase reporter constructs (3′UTR-NC and 3′UTR-SOCS-1), miRNA (miR-155 mimic, 5′-TTA AUG CTA ATC GTG ATA GGG GT-3′; and miR-NC, 5′-CCC CCC CCC CCC CCC CCC CC-3′) and Renilla luciferase using Lipofectamine 3000, according to the manufacturer′s instructions (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 6 h at 37°C, the medium was removed and fresh medium was added. To assess the binding specificity, the sequences in the 3′UTR-SOCS-1 (3′UTR-SOCS-1-mut) that interact with the miR-155 seed sequence were mutated. After 48 h of transfection, the luciferase activity of cells was detected using a Dual Luciferase Assay kit (cat. no. E1910; Promega Corporation). Renilla luciferase activity was normalized to Firefly luciferase activity.

Data were obtained from at least three independent experiments and expressed as the means ± SEM. Statistical analysis was performed using unpaired Student's t-tests or one-way analysis of variance (ANOVA) followed by Bonferroni correction. Statistical analyses were performed using SPSS version 20.0 (IBM Corp.). A 2-sided P-value of <0.05 was considered to indicate a statistically significant difference.

In the present study, markedly elevated levels of SCr and BUN were observed in the I/R-treated mice (Fig. 1A). Histologically, TEC injury (necrosis, detachment, cellular debris, and cast formation) and increased inflammatory cell infiltration were observed in the I/R-treated kidneys (Fig. 1B). In addition, KIM-1, a marker of tubular injury, was detected to reveal tubular injury (Fig. 1C and D). Concomitantly, there were significant increases in the mRNA expression of renal inflammatory cytokines [monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β)] (Fig. 1E). Notably, the number of F4/80⁺ macrophages was also revealed to be significantly increased (Fig. 1F). Thus, these results suggested that macrophage infiltration was associated with tubular injury in I/R-induced AKI.

In this experiment, GW4869 was used to block exosome production (23). Notably, it was determined that the levels of SCr and BUN were markedly decreased in GW4869-treated mice (Fig. 2A). Histologically, ameliorated TEC injury was observed in GW4869-treated kidneys (Fig. 2B). In addition, the results of PCR and immunohistochemical analysis of KIM-1 also confirmed similar results (Fig. 2C and D). Concomitantly, the renal mRNA expression of inflammatory factors (MCP-1, TNF-α, and IL-1β) was significantly decreased in the GW4869-treated group compared with the vehicle (Fig. 2E). Thus, the findings suggested that exosome secretion is an important mechanism of tubular injury in I/R-induced AKI.

To explore the potential mechanism for the effect of exosomes on tubular injury, exosomes from I/R-injured kidneys were first isolated and characterized via western blotting (using Alix and CD63, as exosome markers) and TEM (Fig. 3A and B). To mimic the microenvironment in which exosomes are released from the activated macrophages to promote tubular injury, a schematic diagram depicting the process of the experiment is provided in Fig. 3C. As anticipated, it was determined that exosomes released from macrophages with LPS and IFN-γ treatment (activated macrophages, namely M1 macrophages) transferred to TECs (Fig. 3D). To study the effect of exosomes derived from M1 macrophages on tubular cells, TECs with exosomes derived from M1 macrophages were cultured under hypoxia (exo-M1). It was determined that exo-M1 promoted tubular injury compared with the exosomes derived from non-activated macrophages (exo-M0) (Fig. 3E and F). Concomitantly, the mRNA expression of the inflammatory factors was significantly increased in the exo-M1-treated group (Fig. 3G). These findings indicated that exosomes secreted from activated macrophages promote tubular injury.

For this experiment, the role of exosomal miR-155 in ischemia-induced kidney injury was explored. Interestingly, the kidneys with I/R injury secreted exosomes that were highly enriched in miR-155 (Fig. 4A). M1 macrophage exosomes were then isolated for miR-155 detection. As expected, exosomes derived from M1 macrophages were highly enriched in miR-155 (Fig. 4B). To examine the effects of exosomal miRNA-155, miR-155 was silenced in M1 macrophages, and exosomes derived from M1 macrophages were isolated. The miR-155-silenced exosomes (Exo-miR-155 inhibitor) were then used to treat TECs. Notably, compared with the Exo-miR-NC group, Exo-miR-155 inhibitor administration significantly ameliorated tubular injury (Fig. 4C and D). In addition, the mRNA expression of inflammatory factors (MCP-1, TNF-α, and IL-1β) was decreased in the Exo-miR-155 inhibitor-treated group (Fig. 4E). Therefore, the level of miR-155 in exosomes parallels tubular injury, suggesting that the secretion of miR-155-laden exosomes by M1 macrophages may be associated with the pathological mechanism of tubular injury.

To determine the effect of miR-155 on I/R-induced kidney injury, miR-155 inhibitor or scrambled NC was administered before I/R injury to the kidney. Interestingly, compared with the scrambled NC-treated mice, the miR-155 inhibitor efficiently reversed the upregulation of SCr and BUN levels (Fig. 5A). Notably, miR-155 inhibitor could ameliorate TEC injury and significantly reduce protein casts (Fig. 5B). The results of PCR and immunohistochemical analysis of KIM-1 also confirmed similar results (Fig. 5C and D). In addition, the mRNA expression of inflammatory factors was similarly attenuated in the I/RI + miR-155 inhibitor group (Fig. 5E). Thus, these data indicated that miR-155 inhibition could attenuate I/R-induced tubular injury.

To further investigate the exact molecular mechanism of exosomal miR-155 on tubular cells, possible miR-155 targets that contribute to tubular injury were predicted using 5 online databases: TargetScan (Human 8.0/Mouse 8.0), miRDB, miRanda, DIANA-TarBase (v7.0), and PicTar. Overlap analysis revealed that the possible target of miR-155 was SOCS-1 (Fig. 6A), a negative regulatory factor of NF-κB. It was determined that exosomes released from M1 macrophages decreased the levels of SOCS-1 protein in tubule cells (Fig. 6B and C). In addition, the in vitro study revealed that the miR-155-silenced exosomes (Exo-miR-155 inhibitor) were used to treat TECs. As expected, SOCS-1 expression was increased in the Exo-miR-155 inhibitor group (Fig. 6D and E), indicating that SOCS-1 was modulated by exosome-containing miR-155. To examine whether miR-155 could regulate the expression of SOCS-1 in I/R-induced kidney injury, as revealed in Fig. 6F, miR-155 inhibitor significantly increased SOCS-1 protein expression. Notably, luciferase reporter assay (Fig. 6G) showed that the activity of luciferase reporters was markedly reduced by miR-155 overexpression as compared with the control. Furthermore, the activity of SOCS-1-3′-UTR-mut luciferase reporter was not affected by the miR-155 overexpressed vector compared with the control, demonstrating that miR-155 could directly interact with the 3′-UTR of SOCS-1. These results indicated that increased miR-155 in macrophage-derived exosomes contributed to tubular injury via targeting SOCS-1.

To determine the role of exosomes derived from M1 macrophages in vivo, exosomes derived from activated macrophages (exo-M1) were transferred into the kidneys by direct renal parenchyma injection. As anticipated, it was determined that exo-M1 treatment promoted the damage of kidney function (Fig. 7A). Histologically, increased TEC injury was observed in the exo-M1-treated kidneys (Fig. 7B). In addition, the results of PCR and immunohistochemical analysis for KIM-1 also confirmed the effect of tubular injury promotion (Fig. 7C and D). Furthermore, the renal mRNA expression of inflammatory factors (MCP-1, TNF-α, and IL-1β) was similarly elevated in the exo-M1-treated group (Fig. 7E). Additionally, immunohistochemical analysis revealed a reduction of SOCS-1 expression in the exo-M1 recipients (Fig. 7D). Thus, the findings demonstrated that exosomes derived from M1 macrophages contribute to tubular injury.