The male adult mice (8 to 10 weeks old; 20–25 g) used in these experiments were housed in specific-pathogen-free conditions and allocated to age- and sex-matched groups. The mice were obtained from SLAC Laboratory Animal Co., Ltd., Shanghai, China.

Western blot was performed as previously described (11). The primary antibodies used included anti-CD206 (ab64693, Abcam), anti-Arg-1 (ab233548, Abcam), anti-Ym1 (ab192029, Abcam), anti-SIRPα (ab191419, Abcam), anti-p-STAT3 S727 (ab32143, Abcam), anti-p-STAT3 Y705 (#9664, CST), anti-STAT3 (9662, CST), anti-α-SMA (ab7817, Abcam), anti-cleaved Caspase 3 (CST; 9662), anti-Caspase 3 (CST; #3498), anti-Bcl2 (CST; #3498), anti-E-cadherin (GTX100443, GTX), and anti-GAPDH (ab181602, Abcam). The secondary antibodies included Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (111035003, 115035003, Jackson, West Grove, PA, USA). Protein levels were quantified using the Image Lab software, version 3.0 (Bio-Rad, Hercules, CA, USA).

Total RNA from Raw264.7, BMDMs, and kidney tissues were extracted using TRIzol and reverse-transcribed into cDNA using the PrimeScript™ RT Reagent Kit. The 18S rRNA gene was used to normalize gene expression, including Fizz1, Arg-1, Ym-1, IL-10 (interleukin-10), IL-6 (interleukin-6), TNF-α (tumor necrosis factor-α), iNOS, Col1, and TGF-β1. The expression of miR-382 was reverse-transcribed by MultiScribe™ Reverse Transcriptase Kit (4311235, Applied Biosystems, Foster City, CA, USA) and detected using TaqMan probes (000572, Life Technologies, Carlsbad, CA, USA). miR-382 expression was normalized to that of U6. PCR primer sequences are shown in Supplementary Table 1 .

All experiments were performed in biological replicates. All the experiments were replicated at least twice. All in vivo and in vitro experiment samples were randomized. Data were analyzed using GraphPad Prism Software and expressed as the mean ± standard error of the mean. Two-tailed, unpaired Student’s tests were performed to determine the significance of differences between two groups. ANOVA was used to analyze intergroup differences. Statistical significance was set at P < 0.05.

This work was supported by the Science and Technology Commission of Shanghai (14DZ2260200) and the National Natural Science Foundation of China grants 91849123 (to XD) and 81870466 (to PJ).

In CKD, persistent M2 macrophage infiltration may promote kidney fibrosis (25). To study the role of the macrophages of kidneys in AAN, we determined their number and polarization via flow cytometry. Leukocytes were distinguished as CD45⁺, and macrophages were further analyzed as CD11b⁺/F4/80⁺. CD206⁺ macrophages were regarded as M2 and CD86⁺ as M1 macrophages (26). The number of CD45⁺ leukocytes increased during AAN ( Figure 1A ). A robust increase in CD11b⁺/F4/80⁺ macrophages through the course of AAN ( Figure 1B ). Interestingly, both CD86⁺ M1 and CD206⁺ M2 macrophages increased notably on days 7, 14, and 28 after AA injection ( Figures 1C, D ). Consistently, immunohistochemistry for anti-F4/80, anti-CD206, and anti-CD86 in renal sections showed the accumulation of macrophages and M2-like macrophages and M1-like macrophages in AAN ( Figure 1E ). Thus, our data showed significant activation of macrophage in kidney in the progression of AAN.

To explore the function of renal macrophages in AAN, liposomal clodronate (LC), which was previously reported to inhibit macrophage influx (27), was administered in AAN mice at 14 days (PBS liposomes were used as control treatment). Via flow cytometry, we confirmed that LC injection efficiently depleted renal macrophages (F4/80⁺/CD11b⁺ macrophages) ( Figure S1A ). Immunofluorescence for F4/80⁺ macrophages in renal tissue further confirmed the efficiency of LC ( Figure S1B ). Further, α-SMA protein levels in renal tissue were significantly downregulated after LC treatment ( Figure S1C ). Macrophage depletion suppressed the mRNA expression of TGF-β1 and collagen I ( Figure S1D ). Taken together, the current results suggest a central role of renal macrophages in the progression of AA-induced kidney fibrosis.

To explore the role of miR-382 in the promotion of macrophage M2 polarization and AA-induced CKD, we subjected miR-382 knockout (KO) mice and macrophage-specific depletion miR-382 mice (MKO) to AA-induced nephropathy. We first performed a time-course study using mouse models of AAN (1, 3, 7, 14, and 28 days; NS as control). In AA-treated mice, renal miR-382 levels gradually increased throughout the experiment and peaked at 14 days of AAN ( Figure 2A ). Furthermore, we isolated CD45+CD11b+ F4/80+ cells via flow cytometry and found a large induction of miR-382 expression as well as Fizz1 and Ym-1 mRNA levels on macrophages from the fibrotic kidneys, compared with normal kidneys ( Figure 2B ), supporting the hypothesis of involvement of miR-382 in the regulation of M2 polarization.

We analyzed the injured kidney from WT and KO mice for 14 and 28 days of AAN via flow cytometry. As the results show, miR-382 deficiency did not affect the number of CD45⁺ leukocytes and CD86+ macrophages but decreased CD11b+F4/80 macrophages at 14 days of AAN ( Figures 2C, D, F ). Of note, ablation of miR-382 significantly suppressed CD206⁺ macrophages in the kidney at both 14 and 28 days of AAN ( Figure 2E ). In addition, we analyzed normal kidneys from WT and KO mice and we found no differences between them on CD45+, CD11b+F4/80+, CD86+, and CD206+ cells. Representative flow cytometry images are shown in Figure S2A , and quantification of flow cytometry data is shown in Figures 2C–F . AA injection could largely increase Fizz1, Arg-1, and Ym-1 expression in renal as early as 3 days and up until 28 days, but it was inhibited in KO mice at 14 and 28 days of AAN ( Figure 2G ), suggesting that miR-382 would promote renal M2-like macrophage polarization in AAN. Co-staining for F480 and CD206 in the kidney further demonstrated that the percentage of CD206-positive macrophages was inhibited notably in miR-382 KO mice after 14 days of AAN ( Figure 3A ). Immunohistochemistry for F4/80, CD206, Arg-1, and Ym-1 in renal sections indicated that alternatively activated macrophages were significantly suppressed in miR-382 KO kidneys ( Figure 3B ).

Moreover, miR-382 could also regulate AA-induced CKD. Serum creatinine of mice increased significantly at 7 and 14 days after AA injection, while depletion of miR-382 alleviated renal dysfunction ( Figure 3D ). Sirius Red staining revealed significantly enhanced collagen deposition in WT kidneys relative to KO kidneys following AA treatment ( Figures 3C, I ). Immunohistochemical staining showed a decline in α-SMA, fibronectin, and vimentin and collagen I expression in KO kidneys, compared with WT kidneys ( Figures 3C, E–H ).

Additionally, we generated mice with macrophage-specific miR-382 depletion (miR-382^flox/flox Lyz2-Cre+, or “MKO”) and wild-type littermate control subjects (miR-382^flox/flox Lyz2-Cre-, or “CKO”) and subjected these animals to the AAN model for 14 days. There were no differences between CKO and MKO mice in CD45+ leukocytes, CD11b+F4/80+ macrophages, and CD86+ M1-like macrophages ( Figure S3B-D ). Of note, consistent with our findings on KO mice, MFI of CD206+ M2-like macrophages in renal was inhibited notably in MKO mice, compared with CKO mice ( Figure S3E ). Compared to the CKO mice, the protein expression of α-SMA after AA induction was significantly inhibited in renal, indicating that depletion of miR-382 on macrophages would be reno-protective ( Figure S3F ). AA-induced tubule atrophy, deposition of collagen, or expression of α-SMA, collagen I, and fibronectin was relieved remarkably in MKO mice, compared with CKO mice ( Figure S3G ). Collectively, our data showed that renal macrophage plays a critical role in AA-induced kidney fibrosis. Ablation of miR-382 could inhibit renal macrophage M2 polarization and AA-induced CKD. Thus, it is our hypothesis that miR-382 would regulate AA-induced CKD by promoting M2-lile macrophage polarization.

To further explore the role of miR-382 in macrophages in the AA-induced CKD, we depleted and reconstituted macrophages in mice. Bone marrow-derived macrophages (BMDMs) were transfected miR-382 mimic and then transferred into mice with AA induction ( Figure 4A ). In AA induction, the serum creatinine of miR-382 OE BMDM transfer mice was higher than that of NC BMDM transfer mice ( Figure 4B ). Interestingly, adoptive transfer miR-382 OE BMDMs could raise more CD11b+F4/80+ macrophages in kidney in both control and AA mice but preserved CD45+ leukocytes ( Figures 4C, D ). In addition, adoptive transfer miR-382 OE BMDMs also triggered macrophage M1-to-M2 transition ( Figures 4E, F ). Representative flow cytometry images are displayed in Figure 4G . Moreover, adoptive transfer miR-382 OE BMDMs could augment AA-induced renal injury and fibrosis according to HE staining, Masson staining, IHC for α-SMA, collagen I, and fibronectin ( Figure 4H ). These results supported the role of miR-382 in macrophages for promoting the recruitment of macrophages in kidney, macrophage M1-to-M2 transition, and aggravating of AA-induced injuries and fibrosis.

To further explore the effect of AA on macrophages, we treated BMDMs and Raw264.7 with AA (10 μg/ml). BMDMs were identified as CD11b⁺/F4/80⁺ via flow cytometry ( Figure S4A ). After 48 h of AA induction, the expression of Arg-1, Ym-1, and CD206 increased in Raw264.7 cells, accompanied by loss of SIRP-α and upregulation of p-STAT3, as immunofluorescence staining shown in Figure 5A . The mRNA levels of Ym-1, Fizz1, and IL-10 were notably elevated at 24 and 48 h ( Figures 5B–D ). Via flow cytometry, the mean fluorescence intensity (MFI) of CD206 was significantly enhanced at 12, 24, and 48 h after AA treatment ( Figure 5E ). Abundance of miR-382 increased significantly as AA induction ( Figure 5F ). In addition, the ratio of p-STAT3 S727/STAT3 and p-STAT3 Y705/STAT3 increased gradually in AA induction but SIRP-α decreased significantly at 12, 24, and 48 h after AA treatments ( Figures 5G–J ).

Of note, we found that both M1 and M2 macrophages coexisted in the kidney of AAN. Interestingly, in an in vitro experiment, we also found that the expression of CD86 increased gradually after AA treatment via flow cytometry and the mRNA levels of IL-6, TNFα, and iNOS were significantly enhanced as early as 6 h after AA treatment ( Figures S4B, C ), indicating that macrophage M1 polarization was induced in the early stage of AA induction. However, whether miR-382 could affect the phenotype of macrophage remained unknown.

To further explore the role of miR-382 on polarization of macrophage, locked nucleic acid (LNA)-modified anti-miR-382 oligo was used to knock down miR-382 and bone marrow-derived macrophages (BMDMs) were acquired from WT and miR-382 KO mice, respectively. According to the immunofluorescence staining and Western blot analysis, expressions of Arg-1, Ym-1, and CD206 were suppressed after miR-382 downregulation in AA induction, compared with the anti-scramble group ( Figures 6A, B ). Abundance of miR-382 declined significantly after anti-miR-382 transfection in Raw264.7 cells ( Figure 6C ). Knockdown of miR-382 within AA treatment could partly inhibit CD206 expression in Raw264.7 cells ( Figure 6D ). IL-4 treatment is a canonical cytokine that induces macrophage M2 polarization. We also found that miR-382 knockdown with IL-4 treatment largely decreased CD206 expression in Raw264.7 cells ( Figure 6E ). In BMDMs, AA induced the increase in CD206 and Fizz1 expression in WT BMDMs, but it was inhibited in miR-382 KO BMDMs ( Figures 6F, G ). Of note, knockdown of miR-382 with AA treatment upregulated the MFI of CD86, compared with the anti-scramble +AA group ( Figure S5A ). However, we found no difference in the CD86 expression between WT and KO BMDMs after AA treatment ( Figure S5B ). Thus, in both in vivo and in vitro experiments, out data showed that miR-382 may exert little effect on macrophage M1 polarization but is important in macrophage M2 polarization in AAN.

In addition, KO BMDMs exhibited significantly decreased phosphorylation of STAT3 in Y705 and S727 after AA treatment, compared with WT BMDMs, while there was no difference in total STAT3 expression between WT and KO BMDMs in AA induction, indicating that miR-382 would regulate the phosphorylation of STAT3 instead of transcription or translation of STAT3 ( Figures 6A, H ). Accordingly, SIRP-α in macrophages was decreased in AA induction but was upregulated significantly after miR-382 knockdown, compared with the anti-scramble group ( Figures 6A, I ). However, the relationship among miR-382, SIRP-α, and STAT3 remained vague.

To explore whether miR-382 alone could promote macrophage M2 polarization, miR-382 mimic was transfected into Raw264.7 cells and NC served as control. The expression of miR-382 elevated significantly after transfecting miR-382 mimic ( Figure 6K ). Overexpression of miR-382 alone upregulated phosphorylated STAT3 in Y705 and S727 but preserved total STAT3 expression, which was consistent with results above ( Figures 6J, L, M ). Additionally, overexpression of miR-382 suppressed the protein of SIRP-α in Raw264.7 cells ( Figures 6J, N ), indicating the reciprocal suppression relationship between miR-382 and SIRP-α. Moreover, overexpression of miR-382 promoted macrophage M2 polarization but exerted no effect on M1 polarization without AA induction ( Figure 6O , Figure S5C ). Overexpression of miR-382 could also upregulate the mRNA level of Arg-1, Fizz1, and Ym-1 in Raw264.7 cells ( Figure 6P ).

Signal-regulatory protein alpha (SIRP-α) is a receptor expressed on macrophage, which could combine with CD47 and regulate the phagocytosis of macrophage (28). The protective role of SIRP-α was reported in both acute and chronic kidney diseases (29–31). Enhanced STAT3 phosphorylation was observed in tumor-exposed SIRP-α knockdown macrophage (32). It was predicted that SIRP-α is a target of miR-382 using TargetScan (http://www.targetscan.org/). Therefore, it was hypothesized that miR-382 activates STAT3 possibly by targeting SIRP-α. To prove the relationship between miR-382 and SIRP-α, a dual-luciferase reporter assay was performed in Raw264.7 cells. In comparison with the NC+ pMIR-SIRP-α plasmid group, luciferase activity was suppressed significantly in the miR-382-5p mimic+ pMIR-SIRP-α plasmid group, indicating that miR-382-5p could downregulate SIRP-α. As opposed to the miR-382-5p mimic+ pMIR-SIRP-α plasmid group, the downregulation of luciferase activity was hindered in the miR-382-5p mimic+ pMIR-SIRP-α-mut plasmid group, suggesting that miR-382-5p targets SIRP-α via binding to CAACUUA in the 3′UTR ( Figure 7A ).

Knockdown of miR-382 hindered AA-induced macrophage M2 polarization but was recovered after SIRP-α siRNA transfection ( Figure 7B ), demonstrating that miR-382 regulated the polarization of macrophage possibly via suppression of SIRP-α. In addition, knockdown SIRP-α alone significantly upregulated miR-382 expression and increased Fizz1 ( Figure 7C ), suggesting that downregulation of SIRP-α promotes macrophage M2 polarization and SIRP-α may have a positive feedback on miR-382.

To address whether SIRP-α plays a role on the activation of STAT3 signaling, SIRP-α expression in Raw264.7 cells was suppressed via siRNA transfection. Both mRNA and protein levels of SIRP-α were inhibited significantly via SIRP-α siRNA transfection ( Figures 7D–F ). Furthermore, SIRP-α knockdown also increased the ratio of p-STAT3 S727/STAT3 and p-STAT3 Y705/STAT3, while total STAT3 was preserved ( Figures 7D, G–I ). Collectively, miR-382 suppressed SIRP-α by combining its 3′UTR and downregulation of SIRP-α further promoted phosphorylation of STAT3 signaling.

Macrophage polarization is regulated by various transcriptional factors, such as STATs, PPARs, KLFs, and C/EBP β (8). Herein, STAT3 promotes M2-type macrophages in the progression of carcinoma (33–35). The phosphorylation of STAT3 at Y705 and S727 is necessary for the activation of STAT3 (36). We first examined phosphorylated STAT3 and total STAT3 in kidneys from the time course of AAN in mice. Both p-STAT3 Y705 and total STAT3 protein increased significantly after 14 and 28 days of AAN, and p-STAT3 S727 significantly increased at 28 days of AAN ( Figure 8A ). AA was administrated in WT and KO mice for 28 days separately, injected with saline as controls. We found that AA induced the activation of STAT3 signaling but was remarkably impaired in KO mice after 28 days of AAN ( Figure 8B ). Immunohistochemistry (IHC) staining for STAT3 signaling showed that total STAT3 is distributed in the cytoplasm and nucleus of renal cells and that p-STAT3 is mainly located in the nucleus. The expression of STAT3 signaling significantly decreased in KO mice after 28 days of AAN, compared with WT 28-day AAN ( Figure 8C ). More details of IHC for STAT3 signaling among the control group and 14 days of AAN and 28 days of AAN from WT and KO mice are displayed in Figure S6A . Moreover, co-staining for F4/80 and p-STAT3 in renal sections indicated that p-STAT3 expression in renal macrophages was inhibited in KO mice, compared with WT mice ( Figure 8D ). Thus, our data indicated that miR-382 would regulate the activation of STAT3 signaling in the AAN model.

To further explore the relationship of STAT3, macrophage M2 polarization, and kidney fibrosis, STA-21, an inhibitor of STAT3 (37),was administrated in the AAN model. The strategy for STA-21 injection is shown in Figure S7A . Sirius Red staining showed a significant decrease in collagen deposition in renal interstitial fibrosis after inhibition of STAT3 signaling ( Figure 8E ). At the protein level, both p-STAT3 and total STAT3 were downregulated in renal after administration of STA-21 in 14 days of AAN ( Figures 8F, G ). The protein expression of α-SMA decreased remarkably with STA-21 treatment in 14 days of AAN, as detected by Western blot assay ( Figures 8F, H ). Also, immunohistochemistry staining for α-SMA, collagen I, fibronectin, and vimentin showed relieved kidney fibrosis after STA-21 treatment ( Figure S7C, D ). In addition, we also evaluated the polarization of macrophage in the kidney after STA-21 treatment. Compared with DMSO +AA 14 days, macrophage M2 polarization was reduced remarkably while macrophage M1 polarization was preserved in STA-21 +AA 14 days ( Figure 8I , Figure S7B ). Therefore, activation of STAT3 signaling was involved in AA-induced macrophage M2 polarization as well as progression of kidney fibrosis.

Given that activation of STAT3 regulated AA-induced kidney fibrosis and renal macrophage M2 polarization, we further explored the roles of STAT3 in the activation of macrophage in vitro. The genetic knockdown or pharmacological inhibition of STAT3 was performed by STAT3 siRNA (NC as control) and STA-21 (DMSO as control) in vitro. The efficiency of STA-21 (STAT3 inhibitor) was identified via immunofluorescence for p-STAT3 in Raw264.7 between DMSO+AA and STA-21+AA groups ( Figure 9A ). In BMDMs, AA induced a significant expression of CD206 and Fizz1 but was inhibited by STA-21 ( Figures 9B, C ). In Raw264.7, AA increased the mRNA levels of stat3, Fizz1, and MR but they were suppressed by STAT3 siRNA ( Figures 9D–F ). The pharmacological inhibition of STAT3 in BMDMs also hindered macrophage M1 polarization by downregulating CD86 and iNOS ( Figure S8A, B ). Additionally, STAT3 was overexpressed by transfecting STAT3 OE plasmid in Raw264.7 cells. Both protein expression of p-STAT3 S727, p-STAT3 Y705, and STAT3 and mRNA level of stat3 were upregulated significantly after STAT3 OE transfection ( Figures 9G, H and Figures S9A–C ). Overexpression of STAT3 alone could upregulate Arg-1 and Ym-1 as well as abundance of miR-382 but exerted no effect on the expression of SIRP-α ( Figure 9H ).

Moreover, the protein expression of p-STAT3 and STAT3 was inhibited after knockdown of miR-382 and was recovered after STAT3 OE ( Figure 9I ). Furthermore, the abundance of miR-382, MFI of CD206, and mRNA levels of Fizz1 and Arg-1 were suppressed after downregulation of miR-382 and was recovered after STAT3 OE except for miR-382 ( Figures 9J–M ), which suggests that miR-382 would be upstream of STAT3 in macrophages. Interestingly, the MFI of CD86 and mRNA level of iNOS were preserved after knockdown of miR-382 but were upregulated after STAT3 OE ( Figures S10A, B ).

As described above, miR-382 ablation significantly ameliorated kidney fibrosis and inhibited M2 polarization in renal tissue. Depletion of miR-382 in BMDMs resulted in lower M2 polarization than in wild-type BMDMs following AA exposure. As previously reported, the apoptosis and epithelial to mesenchymal transition (EMT) of tubular epithelia (38)would drive kidney fibrosis. Therefore, we hypothesized that depletion of miR-382 in macrophages may also protect tubular cells from apoptosis and EMT. Therefore, we cocultured macrophages with tubular epithelial fibrosis ( Figure 10A ). In the coculture system, we detected apoptosis-related proteins, such as cleaved caspase 3, total caspase 3, and Bcl-2, as well as EMT-related marker proteins, such as epithelial cell marker E-cadherin and mesenchymal marker α-SMA. The conditional medium (CM) from AA-treated macrophages upregulated cleaved caspase 3 and total caspase 3 protein expression in MTECs ( Figures 10B, C ). MiR-382 knockdown in macrophages downregulated the protein expression of cleaved caspase 3 and Bcl-2 in MTECs ( Figures 10D, F ). Knockdown of miR-382 in macrophages resulted in the preservation of the epithelial marker E-cadherin and the suppression of the mesenchymal marker α-SMA in MTECs ( Figures 10E, F ). In turn, overexpression of miR-382 in macrophages upregulated cleaved caspase-3 ( Figures 10G, I ). Overexpression of miR-382 in macrophages induced a decrease in E-cadherin and promoted α-SMA expression ( Figure 10H, I ). Therefore, these results indicated that upregulation of miR-382 in macrophages would promote apoptosis and EMT of renal tubular epithelial cells.

To further explore the potential regulators by which AA-stimulated macrophages promoted injuries of tubular epithelial cells, Luminex liquid suspension chip detection was applied to compare the differential expression of 23 common cytokines or chemokines in conditional medium (CM) from Raw264.7 in ctrl, AA, anti-scramble+ AA, and anti-miR-382+AA groups. CCL-5, IL-1β, IL-17A, MIP-1α, IL-4, and IL-10 were the six most highly upregulated proteins in AA-CM, compared with Ctrl-CM ( Figure 10J ). IL-2, CCL-5, IL-5, and IL-4 were downregulated notably in anti-miR-382+AA-CM, compared with anti-scramble+ AA-CM. IL-1β and G-CSF were upregulated significantly in anti-miR-382+AA-CM, compared with anti-scramble+ AA-CM ( Figure 10K ). As reported in recent literature, CCL-5 and IL-4 were modulated by NGAL produced from macrophages and were proved to play a critical role in renal fibrosis (39). As our results show, CCL5 and IL-4 were elevated in AA-CM (Ctrl-CM as control) but were suppressed in anti-miR-382+AA-CM (anti-scramble+ AA-CM as control), suggesting that CCL-5 and IL-4 would be the critical mediators in the involvement of miR-382 on AA-induced CKD. More specific evidence will be learned in our following study.