The semaphorin 4A–neuropilin 1 axis alleviates kidney ischemia reperfusion injury by promoting the stability and function of regulatory T cells
Authors: Junnan Xu, Xiubin Li, Qing Yuan, Chenfeng Wang, Liang Xu, Xing Wei, Haitao Liu, Bo Yu, Zhekun An, Yuanyu Zhao, Xiang Li, Xu Zhang, Xin Ma, Ming Cai
Journal: Kidney International (2021)

Previous studies have suggested the role of CD4+Foxp3+ regulatory T cells (Tregs) in protection against kidney ischemia reperfusion injury via their immunosuppressive properties. Unfortunately, the associated mechanisms of Tregs in kidney ischemia reperfusion injury have not been fully elucidated. Semaphorin 4A (Sema4A) is essential for maintaining the immunosuppressive capacity of Tregs in tumors. However, whether Sema4A can alleviate kidney ischemia reperfusion injury through Tregs has not yet been demonstrated. Here, we investigated the effect and mechanism of Sema4A on the development of kidney ischemia reperfusion injury. Administration of recombinant human Sema4A-Fc chimera protein prior to ischemia reperfusion injury promoted the expansion and function of Tregs and decreased the accumulation of neutrophils and proinflammatory macrophages thereby attenuating functional and histological injury of the injured kidneys. Depletion of Tregs abrogated the protective effect of Sema4A on kidney ischemia reperfusion injury, suggesting Tregs as the main target cell type for Sema4A in the development of this injury. Mechanistically, Sema4A bound to neuropilin 1 (Nrp1), a cell surface receptor for Sema4A and other ligands and a key regulator of Tregs, which then promoted recruitment of phosphatase and tensin homologue and suppressed the Akt–mTOR pathway in Foxp3Cre mice but not in Nrp1f/f Foxp3Cre mice. Consistently, Treg-specific deletion of Nrp1 blocked the effect of Sema4A on the expansion and function of Treg cells. Thus, our results demonstrate that the Sema4A–Nrp1 axis alleviates the development of ischemia reperfusion injury by promoting the stability and function of Tregs in mouse kidneys. Previous studies have suggested the role of CD4+Foxp3+ regulatory T cells (Tregs) in protection against kidney ischemia reperfusion injury via their immunosuppressive properties. Unfortunately, the associated mechanisms of Tregs in kidney ischemia reperfusion injury have not been fully elucidated. Semaphorin 4A (Sema4A) is essential for maintaining the immunosuppressive capacity of Tregs in tumors. However, whether Sema4A can alleviate kidney ischemia reperfusion injury through Tregs has not yet been demonstrated. Here, we investigated the effect and mechanism of Sema4A on the development of kidney ischemia reperfusion injury. Administration of recombinant human Sema4A-Fc chimera protein prior to ischemia reperfusion injury promoted the expansion and function of Tregs and decreased the accumulation of neutrophils and proinflammatory macrophages thereby attenuating functional and histological injury of the injured kidneys. Depletion of Tregs abrogated the protective effect of Sema4A on kidney ischemia reperfusion injury, suggesting Tregs as the main target cell type for Sema4A in the development of this injury. Mechanistically, Sema4A bound to neuropilin 1 (Nrp1), a cell surface receptor for Sema4A and other ligands and a key regulator of Tregs, which then promoted recruitment of phosphatase and tensin homologue and suppressed the Akt–mTOR pathway in Foxp3Cre mice but not in Nrp1f/f Foxp3Cre mice. Consistently, Treg-specific deletion of Nrp1 blocked the effect of Sema4A on the expansion and function of Treg cells. Thus, our results demonstrate that the Sema4A–Nrp1 axis alleviates the development of ischemia reperfusion injury by promoting the stability and function of Tregs in mouse kidneys. Translational StatementIschemia-reperfusion injury (IRI) is associated with adverse outcomes for both native and transplanted kidneys; however, to date, no effective treatment exists for IRI. Previous reports have shown that regulatory T cells (Tregs) play a critical role in protecting against IRI, and semaphorin 4A (Sema4A)–neuropilin-1 (Nrp1) axis promoted the number and suppressive function of Tregs, which may represent a potential therapeutic strategy. In this study, we show that activation of the Sema4A-Nrp1 axis in mice promotes Treg survival and inhibits the protein kinase B–mechanistic target of rapamycin signaling pathway, further alleviating IRI. These findings strongly suggest that targeting the Sema4A-Nrp1 axis may be a strategy for kidney IRI prevention or treatment. Ischemia-reperfusion injury (IRI) is associated with adverse outcomes for both native and transplanted kidneys; however, to date, no effective treatment exists for IRI. Previous reports have shown that regulatory T cells (Tregs) play a critical role in protecting against IRI, and semaphorin 4A (Sema4A)–neuropilin-1 (Nrp1) axis promoted the number and suppressive function of Tregs, which may represent a potential therapeutic strategy. In this study, we show that activation of the Sema4A-Nrp1 axis in mice promotes Treg survival and inhibits the protein kinase B–mechanistic target of rapamycin signaling pathway, further alleviating IRI. These findings strongly suggest that targeting the Sema4A-Nrp1 axis may be a strategy for kidney IRI prevention or treatment. Renal ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury in native and transplanted kidneys. IRI contributes to adverse kidney events, including chronic kidney disease and renal fibrosis.1Akbari G. Role of zinc supplementation on ischemia/reperfusion injury in various organs.Biol Trace Elem Res. 2020; 196: 1-9Crossref PubMed Scopus (9) Google Scholar, 2Wang S. Liu A. 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Immune cells in experimental acute kidney injury.Nat Rev Nephrol. 2015; 11: 88-101Crossref PubMed Scopus (242) Google Scholar Cluster of differentiation (CD) 4+ Forkhead box protein (Foxp) 3+ regulatory T cells (Tregs), which are commonly identified by surface makers CD4 and CD25 and transcription factor Foxp3, are one of the most important immunosuppressive lymphocytes.12Whiteside T.L. FOXP3+ Treg as a therapeutic target for promoting anti-tumor immunity.Expert Opin Ther Targets. 2018; 22: 353-363Crossref PubMed Scopus (50) Google Scholar, 13Ono M. Control of regulatory T-cell differentiation and function by T-cell receptor signalling and Foxp3 transcription factor complexes.Immunology. 2020; 160: 24-37Crossref PubMed Scopus (42) Google Scholar, 14Shevyrev D. Tereshchenko V. Treg heterogeneity, function, and homeostasis.Front Immunol. 2019; 10: 3100Crossref PubMed Scopus (74) Google Scholar Many studies have demonstrated that Tregs are capable of protecting kidneys from IRI given their immune-suppressive properties. In fact, Treg expansion or activity can attenuate kidney IRI, whereas Treg depletion aggravates tissue damage.15Burne M.J. Daniels F. El Ghandour A. et al.Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure.J Clin Invest. 2001; 108: 1283-1290Crossref PubMed Scopus (372) Google Scholar, 16Burne-Taney M.J. Ascon D.B. Daniels F. et al.B cell deficiency confers protection from renal ischemia reperfusion injury.J Immunol. 2003; 171: 3210-3215Crossref PubMed Scopus (150) Google Scholar, 17Li L. Huang L. Sung S.S. et al.NKT cell activation mediates neutrophil IFN-gamma production and renal ischemia-reperfusion injury.J Immunol. 2007; 178: 5899-5911Crossref PubMed Scopus (256) Google Scholar, 18Kinsey G.R. Li L. Okusa M.D. Inflammation in acute kidney injury.Nephron Exp Nephrol. 2008; 109: e102-e107Crossref PubMed Scopus (289) Google Scholar, 19Huang Y. Rabb H. Womer K.L. Ischemia-reperfusion and immediate T cell responses.Cell Immunol. 2007; 248: 4-11Crossref PubMed Scopus (115) Google Scholar Although current reports suggest a protective role of Tregs, the underlying mechanism regulating Treg activity in renal IRI requires further investigation. Semaphorin 4A (Sema4A), a class IV semaphorin, participates in a wide range of biofunctions, including neural development, angiogenesis, carcinogenesis, and immune responses.20Ito D. Kumanogoh A. The role of Sema4A in angiogenesis, immune responses, carcinogenesis, and retinal systems.Cell Adh Migr. 2016; 10: 692-699Crossref PubMed Scopus (20) Google Scholar Sema4A has been suggested as important for Treg maintenance in the immune system.21Delgoffe G.M. Woo S.R. Turnis M.E. et al.Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis.Nature. 2013; 501: 252-256Crossref PubMed Scopus (350) Google Scholar Moreover, Sema4A reportedly interacts with neuropilin-1 (Nrp1), a novel surface marker on Tregs that can enhance the function and survival of Tregs, particularly at inflammatory sites.22Romeo P.H. Lemarchandel V. Tordjman R. Neuropilin-1 in the immune system.Adv Exp Med Biol. 2002; 515: 49-54Crossref PubMed Scopus (44) Google Scholar,23Tordjman R. Lepelletier Y. Lemarchandel V. et al.A neuronal receptor, neuropilin-1, is essential for the initiation of the primary immune response.Nat Immunol. 2002; 3: 477-482Crossref PubMed Scopus (265) Google Scholar On binding to Nrp1 on Tregs, Sema4A inhibits protein kinase B (Akt)–mechanistic target of rapamycin (mTOR) signaling by recruiting phosphatase and tensin homologue (PTEN) to Nrp1; this phenomenon leads to Foxo3a nuclear localization, thereby promoting Treg function and stability.21Delgoffe G.M. Woo S.R. Turnis M.E. et al.Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis.Nature. 2013; 501: 252-256Crossref PubMed Scopus (350) Google Scholar However, the effect of Sema4A-Nrp1 interaction on Tregs within the context of renal IRI has not yet been elucidated. Therefore, the primary aim of this study was to investigate the in vivo effect of recombinant Sema4A–fragment crystallizable (Fc) on renal IRI and kidney Tregs in a mouse model. We then examined the role of Nrp1 in Treg regulation using Treg-specific Nrp1 conditional knockout mice (Nrp1f/fFoxp3Cre). We further explored the underlying mechanism downstream of the Sema4A-Nrp1 axis. Male C57BL/6J mice, aged 6 to 8 weeks and weighing 20 to 25 g, were used in our study. Nrp1 conditional knockout mice were generated using the Cre-loxP system. Specifically, to obtain Foxp3+-cell–specific Nrp1-gene–knockout (Nrp1f/fFoxp3Cre) mice, we bred mice with the loxP-flanked exon 2 of Nrp1 gene (C57BL/6-Nrp1em1[flox]Smoc, Nrp1f/f) with mice expressing the gene for Cre recombinase driven by the Foxp3 promoter (C57BL/6-Foxp3em3[IRES-tdTomato-2A-iCre]Smoc, Foxp3Cre). The genetic background of both Nrp1f/f and Foxp3Cre mice was C57BL6 (detailed information provided in Supplementary Methods). Knockout efficiency was confirmed by flow cytometry, real-time polymerase chain reaction (PCR), and Western blotting. All mice were maintained under specific pathogen-free conditions. All animal protocols were approved by the Ethics Committee of People's Liberation Army General Hospital. Bilateral renal IRI was established, as previously described.24Koo T.Y. Lee J.G. Yan J.J. et al.The P2X7 receptor antagonist, oxidized adenosine triphosphate, ameliorates renal ischemia-reperfusion injury by expansion of regulatory T cells.Kidney Int. 2017; 92: 415-431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Briefly, mice were kept on a heating pad to maintain their body temperatures at 36.5 °C to 37 °C. An abdominal median incision was made under anesthesia, and both renal pedicles were exposed and clamped for 30 minutes. Sham operation was performed without clamping the renal pedicles. Blood samples were collected on day 0 (immediately before the IRI protocol) and days 1, 2, and 3 after IRI. Kidneys and spleens were harvested on day 1 or 3 after IRI. For Sema4A-Fc administration, 20 μg of recombinant Sema4A-Fc (R&D Systems) or control IgG was administered to mice via tail vein injection on day 1 and immediately before the IRI protocol. Anti-CD25 monoclonal antibodies (PC61, 300 μg; BioXCell) or IgG was i.v. injected into mice on day 5 and day 1 before renal IRI to perform the in vivo Treg depletion experiment. The depletion efficiency was evaluated via flow cytometry. Blood samples were collected on days 1, 2, and 3 after reperfusion, and were centrifuged at 3000 rpm and 4 °C for 20 minutes. Serum in the upper layer was used for analysis. The serum creatinine and blood urea nitrogen levels were assessed using a QuantiChrom creatinine assay kit (BioAssay Systems) and 7070 Hitachi analyzer in accordance with the manufacturer's instructions. After perfusion with phosphate-buffered saline (Thermo Fisher Scientific), mice kidneys were processed using Stromacher 80 Biomaster (Stewart). Renal homogenates were passed through a 70-μm BD Falcon cell strainer (BD Biosciences) to obtain single-cell suspensions. Subsequently, the cells were resuspended in 35% Percoll (GE Healthcare Bio-Science), and were subjected to gradient centrifugation at 400 × g for 30 minutes. Flow cytometry assays were performed to evaluate the absolute number and proportion of leukocytes in mouse kidneys, blood, or spleens. The fluorophore-labeled antibodies used in flow cytometry assays are described in the Supplementary Methods. For intracellular staining of Foxp3, BD Cytofix/Cytoperm Fixation/Permeabilization kits (BD Biosciences) were used in accordance with the manufacturer's protocol. Caspase-3 was detected using a CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen). Bromodeoxyuridine staining was detected using an FITC BrdU Flow Kit (BD Biosciences) in accordance with manufacturer's instructions. Flow cytometry analyses were performed on BD FACS Canto II (BD Biosciences) using the FlowJo software (Tree Star Inc.). Total RNA was extracted from kidney tissues or mouse primary lymphocytes using the TRIzol RNA isolation system (Life Technologies) and was reverse-transcribed into cDNA using PrimeScript RT master mix (Takara). The 2-ΔΔCT method was applied to analyze PCR results with β-actin as the internal reference. Real-time PCR was performed with an Applied Biosystems 7500 Realtime PCR system using SYBR Premix Ex Taq (Takara). Primer pair sequences used in quantitative real-time PCR are described in the Supplementary Methods. For assessment of protein levels in the kidneys, kidneys were harvested and rinsed with phosphate-buffered saline and homogenized in radioimmunoprecipitation a buffer (Pierce) (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecylsulfate) containing protease and phosphatase inhibitor cocktails. Isolated Tregs were lysed in 1× Laemmli buffer (BioRad). The extracted protein concentrations were quantified via bicinchoninic acid assay (Pierce) and were mixed with the loading buffer. Proteins were separated on 4% to 15% sodium dodecylsulfate–polyacrylamide gel electrophoresis gels and were electrotransferred onto polyvinylidene difluoride membranes. Primary antibodies were incubated at 4 °C overnight, after which horseradish peroxidase–conjugated secondary antibodies were incubated at room temperature for 1 hour (antibodies used in Western blotting are described in the Supplementary Methods). Antibody binding was visualized via chemiluminescence, and images were visualized using the Odyssey imaging system (LI-COR Bioscience). Kidneys or blood samples were harvested and lysed in lysis buffer at 4 °C for 30 minutes. The levels of proinflammatory cytokines, including Sema4A, Nrp1, tumor necrosis factor-α, interleukin (IL)-6, interferon-γ, and CC chemokine ligand 2, as well as those of the anti-inflammatory cytokine IL-10 were analyzed using commercially available enzyme-linked immunosorbent assay kits (R&D Systems) in accordance with manufacturer's instructions. Signals were determined by measuring the absorbance at 450 nm. Statistical analysis was performed using SPSS 25.0 (IBM Corp.) and GraphPad Prism 7.0 (GraphPad Prism). Results were described as mean ± SD. The Student t test was performed for comparisons between 2 groups. Analysis of variance was performed for multiple comparisons (>2 groups). P < 0.05 was considered statistically significant. We established a mouse bilateral renal IRI model and administered recombinant Sema4A-Fc (or IgG for control) via the tail vein to evaluate the in vivo effect of Sema4A on renal IRI. Blood samples were collected at 3 different time points (days 1, 2, and 3 after IRI) and were used for renal function analysis. Kidneys were harvested on day 3 after IRI and were used for subsequent histologic analysis (Figure 1a). Renal function analysis showed that serum creatinine and blood urea nitrogen levels were significantly lower in Sema4A-treated mice than in IgG-treated mice (on days 1, 2, and day 3 after IRI; Figure 1b and c). As revealed by histologic analysis with hematoxylin and eosin and periodic acid–Schiff staining, Sema4A-Fc administration significantly attenuated tubular injury after day 3 of IRI development (Figure 1d). Terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling staining was performed to assess tubular apoptosis, and showed that Sema4A-Fc administration led to a remarkable reduction in terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling–positive cells in IRI kidneys (Figure 1e). Moreover, the mRNA expression levels of proapoptotic factors, including caspase-3, caspase-9, and Bax, significantly decreased in the Sema4A/IRI group (Supplementary Figure S1). Sema4A-Fc administration also reduced Kidney injury molecule-1 (Kim-1) expression level (Supplementary Figure S2). These results suggested that Sema4A-Fc administration before IRI exerted a protective effect against functional and histologic renal injury following IRI. Interestingly, we also tried to administer Sema4A-Fc at 0.5 and 24 hours after IRI development in our study; the results showed that neither renal function nor tissue damage showed any significant difference (Supplementary Figure S3). Therefore, it is likely that only Sema4A-Fc administration before IRI had therapeutic effects on kidney IRI. We analyzed cytokine expression and inflammatory immune cell accumulation in the kidneys to assess the intensity of renal inflammation. Given the increased severity of inflammatory responses at the early stage of IRI, kidneys were harvested after 24 hours of IRI or sham surgery and analyzed via enzyme-linked immunosorbent assay or flow cytometry assay. Enzyme-linked immunosorbent assay indicated that the expression levels of renal proinflammatory cytokines, including tumor necrosis factor-α, IL-6, and CC chemokine ligand 2, significantly decreased following Sema4A-Fc administration (Figure 2a–c). However, the expression of INF-γ and anti-inflammatory cytokines, such as IL-10 and transforming growth factor (TGF)-β1, was not significantly different (Figure 2d–f). We further analyzed proinflammatory cell accumulation via flow cytometry. Results show that when gated on CD45+, the absolute number and proportion of innate immune cells, which were represented by neutrophils (CD11b+Gr-1+) and proinflammatory macrophages (CD11bhighF4/80low) in Sema4A-treated mice (vs. IgG-treated mice), significantly decreased on day 1 after IRI development (Figure 2g–k). However, no significant difference was observed in the absolute number or proportion of dendritic cells or activated T cells (CD44+/CD69+CD4+ and CD44+/CD69+CD8+ T cells; Supplementary Figure S4). These data indicated the suppressive role of Sema4A in innate immune responses during the early stage of kidney IRI. We next aimed to determine the effect of Sema4A-Fc on Tregs in the kidneys. Mice kidneys were harvested on day 1, 2, or 3 after IRI, and intrarenal leukocytes were isolated for flow cytometry analysis. Results showed that Sema4A-Fc treatment substantially increased the absolute number and proportion of CD4+Foxp3+ cells (gated on CD45+) in the kidneys on days 1, 2, and 3 after IRI, with the most significant difference observed on day 1 after IRI (Figure 3a–d and Supplementary Figure S5). Furthermore, we investigated Treg functions in the kidney by evaluating the production of suppressive cytokines, including IL-10 and TGF-β1. As expected, Sema4A-Fc treatment significantly promoted IL-10 and TGF-β1 (Figure 3e and f) production by Tregs. These results indicated that Sema4A-Fc administration promoted the expansion of CD4+Foxp3+ Tregs with a boosted immunosuppressive property. Although the above results suggested that Sema4A-Fc had a positive regulatory effect on kidney Tregs, whether Tregs play a pivotal role in the attenuation of kidney IRI by Sema4A remains unknown. Therefore, we conducted an in vivo Treg depletion experiment with anti-CD25 monoclonal antibody (PC61), which induced selective depletion of Tregs in vivo (Figure 4a). Flow cytometry assays showed that, compared with IgG administration, PC61 administration resulted in a 60% reduction in the number of CD4+Foxp3+ Tregs in the blood, spleen, and kidneys (Supplementary Figure S6). Renal function analysis further revealed that blood urea nitrogen and serum creatinine were significantly elevated in PC61-treated and PC61 + Sema4A-treated mice, with no significant difference observed between the 2 groups (Figure 4b and c). Similarly, the beneficial effects of Sema4A on tissue damage, cell apoptosis, and inflammatory response in the kidneys were all eliminated following PC61 treatment (Figure 4d–g). These results indicated that the protective effects of Sema4A against renal IRI were primarily mediated by Tregs. Nrp1 is a homogeneously expressed cell marker of Tregs that acts as a key receptor of Sema4A.25Bruder D. Probst-Kepper M. Westendorf A.M. et al.Neuropilin-1: a surface marker of regulatory T cells.Eur J Immunol. 2004; 34: 623-630Crossref PubMed Scopus (369) Google Scholar, 26Corbel C. Lemarchandel V. Thomas-Vaslin V. et al.Neuropilin 1 and CD25 co-regulation during early murine thymic differentiation.Dev Comp Immunol. 2007; 31: 1082-1094Crossref PubMed Scopus (40) Google Scholar, 27Yadav M. Louvet C. Davini D. et al.Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo.J Exp Med. 2012; 209 (S1711–1719): 1713-1722Crossref PubMed Scopus (463) Google Scholar Nrp1 conditional knockout mice (Nrp1fl/flFoxp3Cre) were generated by crossing Nrp1f/f with Foxp3Cre mice, and the knockout efficiency was confirmed via real-time PCR, Western blotting, and flow cytometry. Results showed that the knockout efficiency of Nrp1 was >90% (Supplementary Figure S7). Foxp3Cre mice were used as the control to determine whether Nrp1 was indispensable for renal IRI alleviation by Sema4A. Comparing the renal functions, histologic injury, and inflammatory responses of Foxp3Cre mice with those of Nrp1fl/flFoxp3Cre mice revealed that all the beneficial effects of Sema4A on renal IRI were completely abrogated in Nrp1f/fFoxp3Cre mice (Figure 5a–i). Furthermore, Kim-1 immunofluorescence confirmed the protective role of Sema4A-Nrp1 on kidney IRI (Supplementary Figure S2). These data suggested that Nrp1 was indispensable for the alleviation of renal IRI by Sema4A. We next sought to determine whether Sema4A promotes Treg accumulation and function via Nrp1 binding. As shown by flow cytometry results, among kidney CD4+Foxp3+ cells, the number and proportion of Sema4A+ cells were significantly lower in Nrp1f/fFoxp3Cre + Sema4A mice than in Foxp3Cre + Sema4A mice (Supplementary Figure S8). This result strongly indicated that Sema4A was bound to Nrp1 on kidney Tregs. Moreover, following Sema4A-Fc administration, the accumulation (Figure 6a–c) and function (Figure 6d–g) of kidney Tregs in Nrp1f/fFoxp3Cre mice were not enhanced in contrast to those in Foxp3Cre mice. In addition, flow cytometric analysis showed a significant decrease in the number of caspase-3+ Tregs and an elevation in the number of Bcl-2+ and Ki67+ bromodeoxyuridine-positive Tregs in the kidneys of Foxp3Cre + Sema4A mice but not in those of Nrp1f/fFoxp3Cre + Sema4A mice, demonstrating that the Sema4A-Nrp1 axis inhibited apoptosis and induced the proliferation of kidney Tregs (Supplementary Figure S9). Taken together, these data provided evidence for the key role of Nrp1 in mediating Sema4A to potentiate Treg expansion and capacity. We further explored the signaling pathway downstream of the Sema4A-Nrp1 axis. Given the importance of Akt-mTOR signaling in Treg functions, combined with results of a previous study that reported the Sema4A-Nrp1 axis to inhibit the Akt-mTOR pathway,21Delgoffe G.M. Woo S.R. Turnis M.E. et al.Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis.Nature. 2013; 501: 252-256Crossref PubMed Scopus (350) Google Scholar we postulated that its promoting effects on kidney Tregs mediated by Sema4A-Nrp1 were also modulated via the Akt-mTOR pathway. As expected, Sema4A significantly decreased Akt and mTOR phosphorylation in CD4+Foxp3+ Tregs in the kidneys of Foxp3Cre mice. Conversely, in Nrp1f/fFoxp3Cre mice, no significant difference was observed in Akt-mTOR activation regardless of Sema4A-Fc treatment (Figure 7a). We next investigated the Akt-mTOR pathway using an ex vivo Treg model. CD4+Foxp3+ cells were isolated from Foxp3Cre or Nrp1f/fFoxp3Cre mouse spleens at 24 hours of reperfusion and were stimulated with anti-CD3/CD28 monoclonal antibody plus recombinant mouse IL-2 and recombinant human TGF-β1. Following this, Tregs were administrated with/without Sema4A-Fc (100 ng/ml). Western blotting was conducted to evaluate protein expression, and the results were consistent with those of flow cytometry analysis (Figure 7b). Previous studies have indicated that PTEN recruitment is essential for Akt-mTOR regulation by Nrp1. Specifically, the recruitment of PTEN by Nrp1 leads to the inhibition of Akt phosphorylation.21Delgoffe G.M. Woo S.R. Turnis M.E. et al.Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis.Nature. 2013; 501: 252-256Crossref PubMed Scopus (350) Google Scholar,28Stambolic V. Suzuki A. de la Pompa J.L. et al.Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar Accordingly, we proposed that the Sema4A-Nrp1 axis inhibited Akt-mTOR activation through PTEN. Coimmunoprecipitation based on an ex vivo Treg model was conducted to assess the interaction between Nrp1 and PTEN in Tregs. As expected, Sema4A significantly enhanced the combination between Nrp1 and PTEN in Foxp3Cre mice but not in Nrp1f/fFoxp3Cre mice (Figure 7c). Taken together, these results revealed that under IRI, the Sema4A-Nrp1 axis restrained the Akt-mTOR pathway via PTEN recruitment to Nrp1, and consequently, promoted Treg accumulation and function in the kidneys. The roles and mechanisms of Sema4A and its receptor Nrp1 in renal IRI have not been elucidated to date. In this study, we provided the first critical in vivo evidence demonstrating that in murine kidneys, the Sema4A-Nrp1 axis alleviates the development of renal IRI by promoting Treg expansion and function. Sema4A is a member of the semaphorin family, which is characterized by a common structure known as the Sema domain, and its receptor Nrp1 has been identified as a novel surface marker of Tregs.25Bruder D. Probst-Kepper M. Westendorf A.M. et al.Neuropilin-1: a surface marker of regulatory T cells.Eur J Immunol. 2004; 34: 623-630Crossref PubMed Scopus (369) Google Scholar,29Kolodkin A.L. Matthes D.J. Goodman C.S. The s