The study protocols were approved by the Institutional Animal Care and Use Committee at Nanjing Medical University. A unilateral I/R-induced AKI model was established as previously reported (Wan et al., 2015). Male C57BL/6 mice aged 8–10 weeks were anesthetized using an intraperitoneal injection of chloral hydrate (10%, 0.35 ml/10 g). After a flank incision, the left renal pedicle was bluntly dissected and clamped by a micro vascular clamp for 45 min. During the procedure, mice were kept well hydrated by warm sterile saline at a constant temperature (37°C). After the clamps were removed, the wounds were sutured, and the mice were allowed to recover with free access to food and water. Sham-operated mice underwent identical procedures, except the clamping of the renal pedicles was omitted. The cohorts of mice were sacrificed at 1, 2, and 3 days after surgery. For 1 day group, the ligation of right kidney was done on the same day after IRI, and the other two groups (2 and 3 days) had their ligation of right kidney 1 day before sampling. The post-ischemic kidneys and the sham-operated kidneys were harvested and stored at −80°C until they were needed for further analysis. Blood samples were obtained at the time of euthanasia. Serum creatinine was measured using enzymatic method with creatininase coupled sarcosine oxidase (Creatinine (Cr) Assay kit, C011-2-1, jiancheng Bioengineering). The level of Blood Urea Nitrogen (BUN) was determined by urease method (Urea Assay kit, C013-2-1, jiancheng Bioengineering).

An in vivo virus transduction to overexpress Six1 in mice kidney was performed as described elsewhere (Rocca et al., 2014). In anesthetized mice, 10 μl of an AAV cocktail (AAV-Six1 or AAV-Control, 1.0×10¹² vector genome [vg]/ml) was directly injected into the left renal pelvis at five different sites using a glass micropipette (tip OD: 30–50 μm) with a 29 G needle, and renal pedicle was not obstructed. During injection, the position of the needle should avoid the renal vein, artery and ureter, and the depth of the needle should be about 0.5 cm. The mice were subjected to renal I/R injury 2 weeks after virus injection, and the strategy was described in Supplementary Figure S2B. The HBAAV9-CMV-mSix1-3× flag and the HBAAV9-Control vectors were purchased from HanBio Technology (Shanghai, China), and the construct of AAV-Six1 vector was shown in Supplementary Figure S2A.

Kidney sections from paraffin-embedded tissues were prepared at 4 μm thickness and stained with hematoxylin and eosin using standard procedures for histologic evaluation. The sections were blind-reviewed by arenal pathologist and scored using a previously described semiquantitative scale designed to evaluate the degree of tubulointerstitial injury according to tubular necrosis, tubular dilatation and/or atrophy, inflammatory cell infiltration, and cellar edema (Ventura et al., 2002; Cao et al., 2004). Injury was graded on a scale from 0 to 4. Higher scores represented more severe damage (maximum score, 4): 0, normal kidney; 1, minimal damage (<5% involvement of the cortex); 2, mild damage (5–25% involvement ofthe cortex); 3, moderate damage (25–75% involvement of the cortex); and 4, severe damage (>75% involvement of the cortex).

For the immunofluorescence analysis, immunostaining was processed in 3 μm OCT-embedded sections. Nonspecific adsorption was minimized by incubating the section in 1% (g/ml) Bovine Serum Albumin (BSA; Sigma-Aldrich, St. Louis, MO)in PBS for 1 h. The samples were incubated overnight at 4°C with antibodies Six1 (1:100, 10709-1-AP, Proteintech) and F4/80 (1:100, ab6640, Abcam). sections were incubated with fluorochrome-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature and stained with a 1:10000dilution of 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO) before the cells were mounted. The samples were viewed using a confocal imaging system (710; Carl Zeiss, Oberkochen, Germany). Image acquisition, analysis, and processing were standardized in each experiment.

Immunohistochemical staining was performed using routine protocols with the antibodies of SIX1 (1:100, 10709-1-AP, Proteintech), Ki67 (1:200, ab15580, Abcam) and MMP9 (1:200, 10375-2-AP, Proteintech). After incubation with the primary antibody at 4 °C overnight, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. 3,3-Diaminobenzidine tetrahydrochloride was applied to the slides for developing brown color. Counterstaining was carried out with hematoxylin. All slides contained duplicate sections, from which one served as a control for secondary antibody binding specificity. The positive areas were measured in five randomly chosen fields, and quantified blindly using a Nikon 80i camera.

Human proximal tubular epithelial cells (HK2) were used in this study. HK2 cells were immortal cells (a gift from Dr. Xiubin Liang, Nanjing Medical University, China) that were cultured in Dulbecco’s Modified Eagle Medium/F-12 (Gibco, Grand Island, NY) supplemented by a 10% fetal bovine serum (FBS) (Gibco BRL) and a 1% penicillin/streptomycin solution (Gibco, Grand Island, NY) at 37°C in 5%CO₂. After synchronization, the cells were subjected to hypoxic conditions (1% O₂, 5% CO₂, 95% humidity) for 6 h, and then treated under the normal conditions (5% CO₂, 95% humidity) for 0, 4, 8, 12, and 16 h.

Mouse Six1 cDNA (NM-009189) was cloned into the BamH I site of pCAGDNA3. This construct is referred to as pCAGDNA3-mSix1. HK2 cells were stably transfected with pCAGDNA3 (Control) or pCAGDNA3-mSix1 using Lipofectamine 3000 (Life Technologies, Carlsbad, CA) following the manufacturer’s instructions. Stable transfectants, HK2-Six1 and Control, were selected with 200 μg/ml G418 (Gibco) in 10-cm dishes for about 10 days. Individual cell colonies were selected and cultured in 24-well plates and then transferred to 12-well plates.

Six1 knockout mouse cell lines were generated by CRISPR/Cas9. The CRISPRs were designed using a CRISPR design web tool (http://crispr.mit.edu). The sequences of sgRNAs targeting Six1 were: 5′-CAG​CCA​TGT​CGA​TGC​TGC​CGT​CGT​T-3′. The sgRNAs were cloned into the pX330-U6-Chimeric-BB-CBh-hSpCas9 plasmid (Addgene plasmid 42,230, Watertown, MA, USA). The resulting CRISPR/Cas9 plasmids for targeting Six1 were confirmed by sequencing. Mouse renal tubular epithelial cells (TCMK1) (iCell Bioscience Inc., Shanghai, China) were transfected with 8 μg Six1-Cas9/sgRNA plasmids with 2 μg pCMV-tdTomato vector (Clontech, Mountain View, CA) using Lipofectamine 3000 according to the manufacturer’s protocol. After a 24 h of transfection, the selected cells were placed with 150 μg/ml G418 in 10-cm dishes for 8 days. After the G418 selection, the resistant cell clones in each experimental group were pooled and collected.

To determine the growth rate of the Control and HK2-Six1 cells, 10,000 cells per well were plated in 24-well plates, in 500 μl of DMEM/F-12 containing 10% FBS. Cells in triplicate wells were counted daily for up to 4 days using a hemocytometer. Cell proliferation was also evaluated by the BrdU incorporation assay. Briefly, the Control and HK2-Six1 cells were cultured until 90% confluent and then treated with BrdU (30 μM) at 37°C for 1h. Single-cell suspensions (1×10⁶/tube) were fixed by 500 μl of fix buffer at 4°C overnight. The cells were then washed twice with cold wash buffer and permeabilized with Permeabilization Buffer in the dark for 2 min on ice. After treatment with DNase (300 μl) for 30 min at 37°C, the cell pellet was incubated in 200 μl BrdU antibody mix (PBS containing 5 μl of PE-BrdU antibody) (KeyGen Biotech, Nanjing, China) at 4°C for 30 min. The cells were then filtered through a 70 µm nylon cell strainer (BD Biosciences). A flow cytometry analysis (30,000 cells) was performed using a BD LSRII flow cytometer (BD Biosciences).

The Control and HK2-Six1 cells were plated into 6-well plates and cultured until they were 90% confluent. Cell monolayers were wounded with a sterile 1 ml pipettip and then incubated with DMEM/F-12 containing 1% FBS for 24 and 48 h in an atmosphere of 5% CO₂ at 37°C. Representative areas of the wounded monolayers containing wounds of the same width were marked and photographed. After incubation, the same areas were photographed. The extent of wound repair was evaluated by measuring the wound area using ImageJ (NIH). Each experiment was performed in triplicate wells and repeated three times.

The migration ability of the Control and HK2-Six1 cells was measured using transwell chambers (24-well plate) (Corning, Cambridge, MA) with polycarbonate membranes (8.0 μm pore size) coated with 100 μl of the extracellular matrix (ECM; Sigma-Aldrich, St. Louis, MO) on the top side of the membrane. The upper surface of the matrix was challenged with 20,000 cells, which were kept in basal medium containing 0.1% BSA (Sigma-Aldrich, St. Louis, MO). The lower chamber contained the complete medium. After 24 h and 48 h, the cells were fixed and stained. The cells and ECM on the upper surface of the membrane were carefully removed using a cotton swab. Cells that migrated through the membrane were fixed, stained with 1% crystal violet, and counted in five randomly chosen areas. The migration of cells was measured by directly counting the number of cells that migrated to the lower chamber. Each experiment was performed in triplicate wells and repeated three times.

Total RNA was isolated from the cells and frozen tissues using TRIzol (total RNA extraction reagent; Life Technologies Corporation, Carlsbad, CA). Reverse transcription was performed with 1 μg of total RNA using HiScript II One Step RT-PCR Kit (Vazyme Biotech, Nanjing, China), and quantitative real-time PCR was performed using ChamQ™ SYBR qPCR Master Mix (Vazyme Biotech). The sequence of the primers used in the quantitative real-time polymerase chain reaction (qRT-PCR) is shown in Supplementary Table S1. β-Actin was used as an internal control. All samples were assayed in triplicate.

Western blotting was performed as previously described (Wang et al., 2019). Whole cells and tissues lysates were prepared in RIPA buffer (Beyotime Biotechnology, Shanghai, China). Antibodies against the following proteins were used: SIX1 (1:1000, sc-514441, Santa Cruz Biotechnology, Dallas, TX); SIX1 (1:1000, D4A8K, Cell Signaling Technology); C-MYC (1:1000, D3N8F, Cell Signaling Technology); Cyclin A1 (1:1000, ab185619, Abcam); Cyclin D1 (1:1000, ab134175, Abcam); AES (1:1000, ab137060, Abcam); FUS (1:1000, ab124923, Abcam); GLUT1 (1:1000, 21829-1-AP, Proteintech); PGK1 (1:1000, 17811-1-AP, Proteintech); LDHA (1:1000, 19987-1-AP, Proteintech); MMP9 (1:1000, 10375-2-AP, Proteintech); GAPDH (1:3000, HRP-60004, Proteintech) was used as a loading control.

1×10⁵ HK2-Six1 and HK2-control (Control) cells, 1×10⁵ TCMK1-Six1^−/− and TCMK1 wild-type (Control) cells were seeded into each well of 6-well plates. The indicated plasmids were transfected into cells with the firefly luciferase reporter of NF-κB and the renilla luciferase reporter (pNFκB-TA-luc, pRL-SV40-N, Beyotime Biotechnology) using Lipofectamine 3000 (Life Technologies). Forthe cytokine treatment, after 24 h of transfection, the cells were treated with 25 ng/ml TNFα (Novoprotein Scientific, Shanghai, China) for 24 h. Luciferase activity was measured according to the manufacturer’s protocol (Dual Luciferase Reported Gene Assay Kit, Beyotime Biotechnology). The luminescence units were measured using multimode microplate readers (Varioskan LUX, Thermo Scientific). The relative luciferase activity was quantified by adjusting it to the renilla luciferase activity and normalizing it to the experimental control.

The ChIP assay was performed according to the manufacturer’s instructions (Simple ChIP Plus Enzymatic Chromatin IP Kit, 9003, Cell Signaling Technology). In brief, 1.2×10⁷ cells were cross-linked with 1% formaldehyde for 10 min at room temperature and glycine was used to quench crosslinking. The cells were then washed twice with chilled PBS, and lysed in lysis buffer. The nuclei were then pelleted down by spinning at 3,000 rpm. for 5 min. Nuclei lysates were sonicated for three cycles at 20 s. on and 30 s off to yield fragments from 150 to 900 bp using Bioruptor (SONICS and MATERIALS). Nuclei lysates were then immunoprecipitated by ChIP grade antibodies against SIX1 (D4A8K, Cell Signaling Technology) or a normal IgG using protein G agarose beads. Then the DNA and transcriptional factor complexes were uncross-linked to obtain a pure DNA fragment. A quantitative PCR was then performed using the following primers: AES promoter (forward 5′-CTT CAG GTC GCT TTA GGG GTG GAG G-3’; reverse 5′-GGC AGG TCA GAT GGT GGG GAC AGT-3′) and FUS promoter (forward 5′-CAC AGG AAT CTC GGT TCC ACC C-3’; reverse 5′-CAG GAC TAG CCC ACG ATC TAT CTC C-3′) in human cells. Fold enrichment was normalized to the experimental IgG control.

The data were expressed as mean ± standard deviations (SD). A statistical analysis was performed using the student t-test when only two value sets were compared. A one-way ANOVA followed by Dunnett’s test or Bonferroni correction was conducted when the data involved three or more groups. The differences were evaluated using SPSS10.0 software (SPSS, Chicago, IL, USA). p < 0.05 was considered statistically significant.

An I/R-induced AKI model was established as previously reported (Wan et al., 2015). After I/R injury, mice were weak but still alive, and were sacrificed at three time points: 1 day (1d), 2 days (2d), and 3 days (3d). Serum creatinine and BUN were significantly increased in the I/R injury (IRI) mice at 1, 2, and 3 days (Figure 1A). Histologically, TEC injury and protein casts were found in kidneys at 1, 2, and 3 days (Figure 1B). The mRNA expression of Six1 was significantly increased in the injured kidney at 1 day (3.6-fold), and peaked at 2 days (7.3-fold), which preceded a significant decrease at 3 days (3.3-fold), but all increased significantly compared with the sham-operated kidney (Figure 1C). Six1 protein expression was also detected by Western blotting (Figure 1D). To determine which cells expressed Six1 in the injured kidney, we performed an immunofluorescence and immunohistochemistry analysis on kidney sections. In the IRI 2 days kidney, Six1⁺ cells were present in TECs, whereas the expression of Six1 was undetectable in the sham-operated kidney (Figures 1E,F).

To investigate cell proliferation and migration after I/R injury, we compared Ccna 1 and Mmp9 mRNA expression in the injured kidney with the sham-operated kidney. The expression of Ccna 1 and Mmp9 was significantly elevated (6.1-fold and 15.9-fold, respectively) in the IRI 2 days kidney (Figures 2A,B). Immunohistochemistry analysis further revealed that Ki67 and Mmp9 were expressed in TECs at 1, 2ays, and 3 days after I/R injury (Figures 2C,E). Concomitantly, the percent of Ki67⁺ TECs and Mmp9 positive areas was significantly increased in the injured kidney at 2 days (27.5 and 65%, respectively) and positively correlated with the expression of Six1 in the injured kidney at 1, 2, and 3 days (r = 0.7794, p = 0.0133 and r = 0.8914, p = 0.0012, respectively) (Figures 2D,F,J).

The effects of I/R on Mcp-1 and inflammation genes (Tnfα and Il-1β) mRNA expression were examined using quantitative real-time PCR. As shown in Figure 2G, there was a significant elevation of Mcp-1, Tnfα, and Il-1β mRNA levels (160-fold, 7.1-fold, and 194-fold, respectively) in the IRI 3 days kidney compared with the sham-operated kidney. The immunofluorescence analysis further revealed that more F4/80⁺ Mophs were present in the IRI 3 days kidney (Figures 2H,I). Although there was no significant negatively correlated between the number of F4/80⁺ Mophs and the expression of Six1 in the injured kidney (r = –0.5045, p = 0.166), Six1 expression could obviously reduce the number of infiltrated F4/80⁺ Mophs (Figure 2J). Moreover, stronger expression of Six1 was observed in the IRI 2 days kidney, whereas there was little Mophs infiltration. These results implied that the TECs proliferation/migration and Mophs infiltration was associated with the time-course expression of tubular Six1 in the I/R injury kidney.

To determine whether hypoxia/reoxygenation (H/R) affected the expression of SIX1, we measured the mRNA and protein levels of SIX1 in human proximal epithelial tubule cells (HK2) subjected to H/R. This protocol reproduced in vitro the stimuli and effects of renal I/R in vivo (Sáenz-Morales et al., 2006). The expression of SIX1 mRNA was significantly increased in the HK2 cells at 4–12 h of reoxygenation before decreasing to a low level at 16 h as compared with the HK2 cells under normal conditions (NC) (Figure 3A). A similar pattern of protein expression was observed by Western blotting (Figure 3B).

Initially, we examined mouse Six1 overexpression to determine how it affected the proliferation of HK2 cells by counting them. As shown in growth curves (Figure 3C), there were higher cell numbers in the Six1 overexpression cell line (HK2-Six1) (Supplementary Figure S1A–C) groups at different time points compared to the control cell line (Control). We next assessed the effects of Six1 on cell cycle progression by 5-bromodeoxyuridine (BrdU) uptake. After the cells were treated with BrdU, the number of BrdU-labeled cells was significantly higher in HK2-Six1 than in the Control (28.8 vs. 21.7%) (Figure 3D). The Western blot analysis further revealed that the protein levels of human Cyclin A1, Cyclin D1, and c-Myc were dramatically increased in the HK2-Six1 cells (Figures 3E,F). Moreover, the transfection of mouse Six1 stimulated the protein expression of human glycolytic genes (GLUT1, PGK, and LDHA) (Figures 3G,H). These results demonstrated that the overexpression of mouse Six1 promoted cell proliferation by elevating cell cycle progression and energy metabolism.

Because Mmp9 expression was strongly associated with the time-course expression of Six1 in vivo, we sought to identify changes in cell migration involved with the overexpression of mouse Six1. In the wound healing assay, HK2-Six1 exhibited enhanced cell migration compared with the Control (Figures 4A,B). We next conducted a cell migration assay for 24 h and 48 h using transwells. Based on our observations, the number of migrated cells was significantly increased in HK2-Six1 at 24 h and 48 h compared with the control cells (Figures 4C,D). To fully explore the underlying mechanism by which Six1 overexpression in HK2 cells affects its migration, we examined the human MMP-9 protein expression level in HK2-Six1 cells. A higher level of expression of MMP-9 was found in HK2-Six1 cells compared to the control cells (Figures 4E,F). These findings suggest that Six1 might promote cell migration by increasing the expression of MMP9 directly or indirectly.

It is well known that NF-κB is activated in kidney during I/R, which is regulated by several factors, including SIX proteins (Marko et al., 2016; Liu et al., 2019). To determine whether mouse Six1 affects the activation of NF-κB in TECs, we analyzed luciferase reporter expression driven by four NF-κB binding sites (4 × κB) (Figure 5A [top]). The stable transfection of HK2 cells with mouse Six1 inhibited the basal activities of NF-κB and the TNFα-stimulated activation of NF-κB, respectively (Figure 5A [bottom]). In addition, mouse renal TEC Six1 knockout cell lines (TCMK1-Six1^−/−) (Supplementary Figure S1D,E) enhanced the stimulation of 4 × κB-LUC under both quiescent and stimulated conditions (Figure 5B). These results suggest that Six1 represses the basal and TNFα-stimulated activities of NF-κB.

To reveal the mechanism of the inhibition of NF-κB by Six1, we examined the expression and activation of human RELA (p65), a member of NF-κB family responsible for transcriptional activation (Schmitz et al., 1995). There was no significant difference in the RNA and protein level of RELA as well as phosphorylated-RELA (p-RELA) level between the HK2-Six1 cells and the control cells (Figures 5C–E). Previous studies indicated that specific protein-protein interactions with RELA determine its transcriptional competence (Tetsuka et al., 2000; Uranishi et al., 2001). We next examined the RNA expression of corepressors and coactivators of RELA. The results showed that human amino-terminal enhancer of split (AES, a corepressor gene of RELA) RNA expression was markedly elevation, but fused in sarcoma (FUS, a coactivator gene of RELA) RNA expression was suppressed dramatically in the HK2-Six1 cells (Figure 5C). A Western blot analysis further confirmed that the pattern of protein expression of AES and FUS was in keeping with RNA expression in HK2-Six1 cells (Figures 5D,E). As SIX1 DNA binding motifs has been defined for human, we searched and obtained SIX1 DNA binding motifs for human on the website of JASPAR, and found that SIX1 DNA binding compatible motif (ACCTGA) is in AES and FUS gene promoter regions by sequence alignments (counting position from the gene transcription start sites, the location of compatible motif was -1008 in AES and -2948 in FUS respectively) (Figure 5F). The chromatin immunoprecipitation (ChIP) analysis showed that SIX1 protein bound the promoter regions of the AES and FUS genes, indicating that it might be primed for transcriptional activation and inhibition, respectively (Figure 5F). These results showed that mouse Six1 inhibited the transactivation function of the RELA subunit of NF-κB by regulating the expression of corepressor gene AES and coactivator gene FUS.

Our previous laboratory studies clearly demonstrated that enhanced MCP-1 expression was responsible for the activation of NF-κB (Cao et al., 2004). To clarify whether MCP-1 expression was regulated by the SIX1-mediated NF-κB pathway, we examined human MCP-1 and mouse Mcp-1 RNA expression in mouse Six1 overexpression and Six1 knockout cell lines under both quiescent and TNFα-stimulated conditions, respectively. As shown in Figure 5G, the human MCP-1 mRNA level was more attenuated in the HK2-Six1 cells compared with the control cells. However, mouse Mcp-1 was significantly increased in TCMK1-Six1^−/− (Figure 5H). Thus, mouse Six1 repressed the basal and TNFα-stimulated expression of the chemokine MCP-1 in TECs.

To study the effects of Six1 overexpression on I/R injury, we injected the adeno-associated viral vector serotype 9 (AAV9)-Six1into an uninjured renal pelvis at 14 days before I/Rinjury. The RNA and protein expression of Six1 were both increased in the AAV-Six1 kidney compared with the phosphate buffer saline (PBS-Control) and the AAV empty vector control kidney (AAV-Control) (Figures 6A,B). Immunofluorescence and immunohistochemistry analysis of kidney sections revealed some Six1⁺ cells were present in cortex and medulla TECs in AAV-Six1 group, but the expression of Six1 was undetectable in AAV-Control group (Figures 6C,D). Animals that underwent 45 min of unilateral renal ischemia and reperfusion at 1, 2, and 3 days exhibited a significant degree of renal damage, which was evident in renal edema, compared with the sham-operated kidney. Compared with the AAV-Control group, the AAV-Six1 infected group exhibited remission of renal edema (Figure 6E). As expected, the histological analysis of kidney sections at 1, 2, and 3 days after unilateral I/R injury revealed significant tubulointerstitial damage, including tubular atrophy, flattening and sloughing of TECs, protein cast formation, and inflammatory cell infiltration, all of which were markedly ameliorated in the AAV-Six1 group (Figures 6F,G).

Notably, compared with AAV-Control, the administration of AAV-Six1 resulted in a significantly lower level of serum creatinine at 1 day (168.5 ± 8.918 vs. 204.4 ± 10.70, n = 6), 2 days (193.4 ± 4.297 vs. 244.6 ± 11.56, n = 6), and 3 days (205.2 ± 3.603 vs. 251.1 ± 10.99, n = 6) after I/Rinjury (Figure 6H). The serum creatinine levels were decreased 17.6% at IRI 1 day, 20.9% at IRI 2 days and 18.3% at IRI 3 days after Six1 overexpression in the animal model. In the AAV-Control group, the level of serum creatinine was significantly increased at IRI 3 days compared with IRI 1 day after analysis by Bonferroni correction. Meanwhile, the level of BUN was also significantly decreased in AAV-Six1 group compared to AAV-Control group at 1 day (35.02 ± 0.9528 vs 39.25 ± 1.037, n = 6), 2 days (35.49 ± 1.719 vs 44.15 ± 0.7915, n = 6), and 3 days (35.51 ± 1.045 vs 44.58 ± 1.518, n = 6) (Figure 6I). In the AAV-Control group, the levels of BUN were significantly increased at IRI 2d and 3 days compared with IRI 1 day after analysis by Bonferroni correction. These results suggested that Six1 overexpression in mouse kidney reduced the damage caused by I/R at 1, 2, and 3 days.

Based on our findings in the cell models, we analyzed cell proliferation and migration to determine whether they were increased by upregulating mouse cyclins, glycolytic genes, and Mmp9 in the AAV-Six1 kidney. As shown in Figure 7A, the RNA expression levels of cyclins (Ccna 1, Ccnd 1, and C-myc), glycolytic genes (Glut1, Pgk, and Ldha), and Mmp9 were increased in the AAV-Six1 kidney compared to the AAV-Control kidney after I/R injury. The infiltration of Mophs was assessed by immunostaining analysis. Compared with the AAV-Control group, the number of interstitial cells demonstrating F4/80 staining of Mophs was significantly decreased at 1, 2, and 3 days after I/R injury in the AAV-Six1 group (Figure 7B). We also found that the RNA expression of genes (Mcp-1, Tnfα, and Il-1β) correlated with Mophs infiltration was significantly attenuated in the AAV-Six1 kidney. Moreover, mouse Aes RNA expression was promoted and Fus RNA expression was suppressed at 1, 2, and 3 days after I/R injury in the AAV-Six1 kidney (Figure 7C). A Western blot analysis further confirmed that the protein expression levels of Cyclin D1 and Aes were positively correlated with Six1 overexpression in IRI 1 day, IRI 2 days, and IRI 3 days kidney (Figures 7D,E). In addition, the RNA expression of genes (Ccna 1, Ccnd 1, C-myc, Mmp9, Glut1 and Aes) were significantly increased in the AAV-Six1 kidney compared with the AAV-Control kidney before I/R injury (Supplementary Figure S2C). Thus, these results indicated that Six1 overexpression promoted cell proliferation and migration by upregulating cyclins, glycolytic genes, and Mmp9 and suppressed Mophs infiltration by repressing the expression of Mcp-1 in IRI kidney.