VISTA attenuates ischemia reperfusion-induced renal injury and fibrosis by macrophage polarization reprogramming

VISTA attenuates ischemia reperfusion-induced renal injury and fibrosis by macrophage polarization reprogramming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article VISTA attenuates ischemia reperfusion-induced renal injury and fibrosis by macrophage polarization reprogramming Cheng Yang, Cuidi Xu, Juntao Chen, Siyue Chen, Lifei Liang, Hao Zeng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6575812/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Renal ischemia reperfusion (IR) injury is one of the major causes of acute kidney injury (AKI) and may contribute to the development of chronic kidney disease. However, the precise immunological mediators orchestrating these pathophysiological processes remain poorly defined. In the present study, we investigate the protective role of the V-domain Ig suppressor of T cell activation (VISTA), which is and immune checkpoint molecule and highly expressed in macrophages, in attenuating IR-induced renal injury. Using genetic deficiency models, we demonstrate that miace lacking VISTA ( Vsir −/− ) and macrophages-specific VISTA knockout ( Vsir fl/fl Lyz2 Cre ) exhibited significantly exacerbated renal dysfunction and histopathological damage post-IR injury. Mechanistically, hypoxia-inducible factor-1α, as a transcriptional regulator, induced VISTA expression in macrophages post IR injury. VISTA deficiency in macrophages reprogramed these cells toward a pro-inflammatory phenotype via NF-κB nuclear translocation, which amplified Th1 differentiation through IL-12 upregulation while simultaneously suppressing regulatory T cell expansion. Notably, neutralizing IL-12 activity rescued renal injury in VISTA-deficient mice, underscoring its role as a key effector in this pathway. Therapeutically, exogenous VISTA administration attenuated renal inflammation, fibrosis, and functional impairment, highlighting its direct renoprotective capacity. Therefore, VISTA emerges as a sentinel checkpoint protein that balances macrophage polarization and T cell immunity during AKI. Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages Biological sciences/Immunology/Translational immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Acute kidney injury (AKI) is a clinical complex disorder characterized by multifactorial pathogenesis and high morbidity, with significant global health burdens 1 , 2 . Due to the lack of effective therapeutic interventions, AKI often progresses to irreversible tubular dysfunction and fibrotic matrix deposition in the renal interstitium, linking to the transition toward chronic kidney diseases (CKD) 3 , 4 . Despite its clinical prevalence, the precise immunopathological mechanisms underlying AKI remain incompletely defined. Renal ischemia reperfusion (IR), which involves hypoxia, inflammation, apoptosis, and oxidative stress, serves as a widely utilized experimental model to investigate the mechanism bridging AKI and CKD progression 5 , 6 . The dynamic interplay of infiltrating immune cells, including macrophages and T cells, is increasingly recognized as a critical driver of this transition, as these cells orchestrate both the acute inflammatory response and the subsequent fibrotic remodeling observed in the kidney 7 . Notably, while immune cells are known to persist throughout the AKI-to-CKD trajectory, the specific mechanisms by which they mediate this pathological shift, particularly their cytokine-driven polarization, remain poorly understood. Rabb et al. demonstrated that the early phase of renal IR injury is marked by the activation of mononuclear phagocytes, which amplify T cell infiltration and modulate T cell immunometabolic profiles 8 , 9 . Macrophages play a central role in the inflammatory response to AKI and persist throughout the transition to CKD, significantly influencing the outcome of renal injury 10 , 11 . Notably, Mami et al. revealed that macrophages could be trained toward a pro-inflammatory phenotype and exacerbated AKI, and depletion of macrophages by clodronate liposome attenuated renal injury and fibrosis post IR injury 12 . V-domain Ig suppressor of T cell activation (VISTA),, also known as PD-1H, DD1α, Gi24, and Dies-1, is a type I transmembrane protein that acts as an immune-regulatory checkpoint on hematopoietic cells and shares homology with both the B7 family ligand programmed death-ligand 1 (PD-L1) and the CD28 family receptor PD-1 13 . As a protein encoded by a member of the B7/CD28 immune modulatory gene family, VISTA expressed on APCs exhibits an inhibitory effect on T cell function 14 . Blocking VISTA aggravated the progression of T-cell-mediated autoimmune diseases 14 . In a study that utilized mice bearing CT26 colon carcinoma tumors with complete resistance to PD-1/CTLA-4 blockade, the addition of VISTA blockade led to the rejection of more than half of the tumors 15 . Among all subsets of hematopoietic cells, myeloid cells, particularly monocytes, granulocytes, and dendritic cells (DCs), display the highest expression of VISTA. It is acknowledged that VISTA is highly constitutively expressed in resident macrophages. Park et al. reported that VISTA-expressing kidney-resident macrophages promote repair during ischemia injury 16 . In contrast to most other immune checkpoints, which are upregulated during different stages of stimulation or activation, VISTA is expressed by immune cells at steady state. This constitutive expression across a wide range of immune cells is unique and suggests that VISTA plays a role in maintaining immune system homeostasis. Although the expression of VISTA is modest, T cells are sensitive to respond to VISTA. VISTA–Fc fusion protein and cellular overexpression of VISTA are suppressive to T-cell activation, proliferation, and cytokine production 17 . Therefore, we speculate that VISTA may serve as a potential target to attenuate acute inflammation and chronic fibrosis in renal IR injury. Results VISTA expression dynamics in renal IR injury Macrophages, including resident macrophages (RM: CD45 + Ly6G − CD11b int F4/80 hi ) and infiltrating macrophages (IM: CD45 + Ly6G − CD11b hi F4/80 int ), are critical mediators of AKI and CKD pathogenesis. In the renal IR injury model 18 , we characterized immune cell populations at key timepoints. Flow cytometric analysis revealed that macrophages (both RM and IM) constituted a significant proportion of CD45 + immune cells at 6h, 1d, and 7d post-IR injury (Fig. 1 A, 1 B). Notably, macrophages exhibited the highest expression VISTA among all cell types in the kidney. To validate this observation, we performed single-cell RNA sequencing (scRNA-seq) using public datasets in the GEO database (GSE161201) from four groups: control kidneys (Con), 6h post-IR injury (IRI 6h), 1d post-IR injury (IRI 1d), and 7d post-IR injury (IRI 7d). Analysis confirmed robust Vsir (VISTA) transcription in both RM and IM, whereas T cells showed low expression and other cells showed no expression (Fig. 1 C-F). Gene expression profiling further revealed dynamic changes in renal injury markers: Havcr1 and Vcam1 (markers of acute injury) peaked at 1d post-IR injury, while collagen genes ( Col1a1 and Col2a1 , markers of fibrosis) increased by 7d post-IR injury. The expression of Vsir followed a biphasic pattern, rising sharply at 6h and 1d post-IRI before declining at 7d post-IR injury (Fig. 1 G). To investigate the VISTA expression in specific macrophage subsets, flow cytometry was performed. Baseline analysis showed that 35% of RM expressed VISTA, while only 10% of IM exhibited detectable VISTA (Fig. 1 H). Post-IR injury, RM maintained consistent VISTA expression at 48h, whereas IM macrophages, CD4 + T cells, and CD8 + T cells exhibited transient upregulation of VISTA. Notably, VISTA expression across all cell types declined by 48h post-IR injury (Fig. 1 H, Figure S1 A-B). Conclusively, these data demonstrate that VISTA is constitutively expressed in RM and dynamically induced in IM macrophages during the early phase of renal IR injury, with T cells serving as secondary responders to injury signals. Vsir deficiency exacerbates IR-induced AKI and fibrosis To investigate the functional role of Vsir in renal IR injury, we utilized Vsir knockout ( Vsir ⁻/⁻) mice subjected to unilateral renal pedicle clamping (45 minutes) or sham surgery (Fig. 2 A). Baseline comparisons between wild-type (WT) and Vsir ⁻/⁻ mice in sham-operated groups revealed no significant differences in serum creatinine and blood urea nitrogen (BUN) or histological architecture, as assessed via hematoxylin and eosin (H&E), Masson trichrome, Sirus Red, and Periodic Acid-Schiff (PAS) staining (Figure S2 A). However, following IR injury, Vsir ⁻/⁻ mice exhibited significantly elevated serum creatinine and BUN levels compared to WT mice (Fig. 2 B). Histopathological analysis further demonstrated that Vsir ⁻/⁻ mice displayed more severe tubular necrosis, interstitial inflammation, and glomerular damage, as evidenced by H&E and PAS staining (Fig. 2 C-D). To evaluate long-term fibrotic outcomes, Sirus Red and Masson trichrome staining were performed at 28 days post-IR injury. A marked increase of collagen deposition (fibrotic area) in Vsir ⁻/⁻ mice was observed compared to WT mice (Fig. 2 E). Collectively, these findings demonstrated that Vsir deficiency exacerbated both AKI and subsequent fibrotic progression in the context of IRI, suggesting the protective role of VISTA in mitigating renal damage. Macrophage-specific Vsir deficiency aggravates IR-induced AKI and fibrosis To determine whether Vsir deficiency in macrophage contributes to renal injury, we generated Vsir fl/fl Lyz2 Cre (macrophage-specific Vsir knockout) mice by crossing Vsir fl/fl mice with lyz2-Cre mice (Fig. 2 F). Genotyping confirmed Cre-mediated recombination in Vsir fl/fl Lyz2 Cre mice (Figure S2 B). Baseline comparisons between Vsir fl/fl and Vsir fl/fl Lyz2 Cre mice undergoing sham surgery revealed no differences in serum creatinine and BUN (Fig. 2 G), as well as histology (Figure S2 C). However, following IR, Vsir fl/fl Lyz2 Cre mice exhibited significantly elevated serum creatinine and BUN levels 48h post-injury compared to Vsir fl/fl controls (Fig. 2 G). Histopathological analysis further demonstrated that Vsir fl/fl Lyz2 Cre mice displayed exacerbated tubular necrosis, interstitial inflammation, and glomerular damage, as evidenced by H&E and PAS staining (Fig. 2 H-I). Sirus red and Masson trichrome staining were performed at 28 days post IR injury to evaluate chronic fibrotic outcomes and revealed marked increases of collagen deposition (fibrotic area) in Vsir fl/fl Lyz2 Cre mice, with no differences observed between sham-operated groups (Fig. 2 J, Figure S2 D). Collectively, these findings demonstrate that macrophage-specific Vsir deficiency exacerbates both AKI and fibrotic progression following IR injury, mirroring the effects observed in global Vsir knockout mice. HIF-1α directly induces VISTA expression via transcriptional activation in macrophages IRI triggers hypoxia and stabilizes hypoxia-inducible factor-1α (HIF-1α), which is a key regulator of cellular adaptation to hypoxia 19 . To determine whether HIF-1α regulates VISTA expression in macrophages, we performed chromatin accessibility profiling using H3K27ac CUT&Tag sequencing in macrophages isolated from IR-injured mouse kidneys. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis of H3K27ac-enriched regions identified immune-related pathways, including NF-κB and HIF-1 signaling (Fig. 3 A). Western blot analysis further revealed a positive correlation between HIF-1α and VISTA protein levels in kidneys subjected to IR injury (Fig. 3 B). To validate HIF-1α’s functional role in VISTA regulation, we administered PX-478, a specific HIF-1α inhibitor, to mice undergoing IR injury. PX-478 suppressed IR injury-induced VISTA upregulation (Fig. 3 C) and worsened renal dysfunction, as evidenced by elevated serum creatinine and BUN levels (Fig. 3 D). Histopathological analysis confirmed exacerbated tubular injury in PX-478-treated mice (Fig. 3 E). Mechanistically, bioinformatic prediction of HIF-1α binding sites in the Vsir promoter region identified multiple putative binding motifs. To validate this, we performed H3K27ac and H3K4me1 CUT&Tag sequencing in human macrophages transduced with HIF-1α-overexpressing lentivirus. HIF-1α overexpression significantly increased H3K27ac enrichment at the Vsir promoter (Fig. 3 F). Chromatin immunoprecipitation (ChIP)-qPCR confirmed direct binding of HIF-1α to the Vsir promoter (Fig. 3 G). Finally, a luciferase reporter assay demonstrated that HIF-1α drives Vsir transcription via a conserved HIF-1α binding motif in the promoter region (Fig. 3 H). Collectively, these findings establish that HIF-1α directly upregulates VISTA expression in macrophages through transcriptional activation. Vsir deficiency skews macrophage polarization toward pro-inflammatory phenotypes via NF-κB activation Macrophages exhibit functional heterogeneity, including pro-inflammatory (M1: CD86 + CD206 − ) and anti-inflammatory (M2: CD86 + CD206 + ) subsets 20 . To determine the role of VISTA in regulating this balance, we analyzed macrophage polarization in Vsir fl/fl Lyz2 Cre mice. Compared to Vsir fl/fl controls, Vsir fl/fl Lyz2 Cre mice exhibited increased M1 macrophage frequency and decreased M2 macrophage frequency in the kidney post-IR injury (Figue 4A). Flow cytometric analysis further revealed elevated expression of M1 markers (IL-12 and iNOS) and reduced expression of M2 markers (Arg1 and IL-10) in both RM and IM isolated from Vsir fl/fl Lyz2 Cre mice (Fig. 4 B–E). These findings suggest that VISTA suppresses pro-inflammatory macrophage polarization and promotes M2 reprogramming in both RM and IM subsets. To establish the functional relevance of this polarization shift, we depleted macrophages in Vsir fl/fl Lyz2 Cre mice using clodronate liposomes. This intervention abolished the differences in serum creatinine and BUN levels between Vsir fl/fl and Vsir fl/fl Lyz2 Cre mice (Figure S3 A), as well as histological disparities in tubular injury (H&E/PAS staining; Fig. 4 F). Furthermore, mouse bone marrow-derived macrophage (BMDMs) from CD45.1 + WT and CD45.1 + Vsir −/− mice were isolated and injected into IRI model CD45.2 + mice (Figure S3 B) subjected to IR injury. Injection of Vsir −/− BMDMs exacerbated the renal dysfunction and tubular injury (Figure S3 C, 4G). Mechanistically, chromatin accessibility profiling (H3K27ac CUT&Tag) identified NF-κB signaling as an enriched pathway in IR-injured kidneys (Fig. 3 A). Given that NF-κB is able to promote M1 polarization and IL-12 secretion 21 , 22 , we explored its interaction with VISTA. Immunofluorescence (IF) staining revealed increased phosphorylation of IκBα, a key driver of NF-κB nuclear translocation, in macrophages from Vsir fl/fl Lyz2 Cre mice compared to controls (Figure S3 D). Co-immunoprecipitation (Co-IP) experiments further demonstrated physical interaction between VISTA and NF-κB (Figure S3 E). siRNA-mediated knockdown of Vsir in cultured macrophages enhanced NF-κB nuclear translocation (Figure S3 F), corroborating that VISTA suppresses NF-κB activation. Therefore, the above results establish that VISTA restrains pro-inflammatory macrophage polarization by inhibiting NF-κB signaling. Vsir deficiency alters infiltrating T cell phenotypes and mediates renal injury via T cell-macrophage crosstalk While VISTA is expressed in naive T cells, scRNA-seq demonstrated that macrophages are the primary renal cell type expressing Vsir , suggesting their central role in AKI pathogenesis. To dissect the contribution of T cells to Vsir -deficient macrophage-mediated kidney injury, we employed Rag1 −/− mice (lacking T and B cells) as recipients for adoptive cell transfer experiments. Transfer of Vsir −/− bone marrow-derived macrophages (BMDMs) from Vsir fl/fl Lyz2 Cre mice into Rag1 −/− mice induced only minor renal dysfunction compared to transfers of Vsir fl/fl BMDMs (Fig. 5 A). However, co-transfer of Vsir −/− BMDMs with CD3 + T cells into Rag1 −/− mice significantly increased serum creatinine and BUN levels compared with co-transfer of Vsir fl/fl BMDMs with T cells (Fig. 5 B). These findings indicate that T cells are required for Vsir -deficient macrophages to mediate renal injury, necessitating functional crosstalk between these cell types. To identify the T cell subsets involved, we analyzed kidney-infiltrating T cells 48h post-IR injury. Vsir -deficient macrophages were associated with increased proportions of CD4 + IFNγ + Th1, CD8 + IFNγ + T cells (Fig. 5 C), and decreased CD4 + Foxp3 + regulatory T (Treg) cells (Fig. 5 D), while no significant changes were observed in Th2 (CD4 + IL4 + ) or Th17 (CD4 + IL17A + ) (Fig. 5 D, Figure S4 A). In vitro co-culture experiments further revealed that Vsir −/− BMDMs potently suppressed Treg differentiation and promoted Th1 polarization (Fig. 5 E) of naive CD4 + CD62L + T cells, suggesting a mechanistic link between Vsir deficiency and T cell phenotypic reprogramming. To validate the functional relevance of these T cell subsets in vivo, we employed IFNγ −/− mice and FoxP3-DTR mice (allowing Treg depletion via diphtheria toxin). IFNγ −/− mice exhibited reduced serum creatinine and BUN levels (Fig. 5 F) and ameliorated histopathological injury (Figure S4 B) compared to wild-type mice post-IR injury. Conversely, Treg depletion in FoxP3-DTR mice worsened renal dysfunction (Fig. 5 G) and tubular injury (Figure S4 C). Collectively, these data demonstrate that Vsir deficiency in macrophages disrupts T cell homeostasis by promoting Th1 differentiation and suppressing Treg activity, thereby amplifying renal injury through Th1-dependent mechanisms. Vsir deficiency macrophages disrupt CD4 + T cell balance via IL-12 driven Th1 differentiation IL-12 is a critical cytokine that drives Th1 cell differentiation 23 and suppresses Th2/Treg responses. In Vsir fl/fl Lyz2 Cre mice subjected to IR injury, we observed a significant increase in IL-12-secreting macrophages (Fig. 4 B), accompanied by elevated renal IL-12 positive area as demonstrated by immunohistochemistry (IHC) staining (Fig. 6 A). To determine whether IL-12 mediates the T cell phenotypic shifts observed in Vsir -deficient macrophages, we administered anti-IL-12 neutralizing antibodies to Vsir fl/fl Lyz2 Cre mice undergoing renal IR injury. Anti-IL-12 treatment significantly attenuated renal injury in terms of serum creatinine and BUN (Fig. 6 B) and reduced histopathological damages (Fig. 6 C), mirroring the protective effects of IFNγ knockout. Further analysis revealed that anti-IL-12 administration reduced Th1 cell proportions while increasing Treg cell frequencies (Fig. 6 D). These findings align with IL-12’s established role in promoting Th1 polarization (via IFNγ induction) and suppressing Treg differentiation, thereby skewing the CD4 + T cell balance toward pro-inflammatory phenotypes in Vsir -deficient macrophage-mediated renal injury. Recombinant VISTA exerts renoprotective effects via modulating immune responses in IR injury To evaluate the therapeutic potential of VISTA in renal IR injury, we administered recombinant VISTA protein intravenously at the onset of reperfusion in a murine IR injury model. VISTA treatment significantly reduced serum creatinine and BUN levels compared to vehicle controls (Fig. 7 A), accompanied by histopathological improvements in tubular necrosis and glomerular damage as assessed by H&E and Periodic Acid-Schiff (PAS) staining (Fig. 7 B-C). Collagen deposition, displayed by Sirus red and Masson trichrome staining, was also markedly attenuated in VISTA-treated mice (Fig. 7 D), indicating reduced fibrotic progression. Moreover, Vista administration suppressed pro-inflammatory macrophage polarization, in terms of decreased M1 markers and increased M2 markers (Fig. 7 E-H). Additionally, VISTA treatment reduced the frequency of pathogenic CD4 + IFNγ + Th1 cells and IFNγ-producing CD8 + T cells (Figure S4 D), aligning with its previously demonstrated role in inhibiting Th1 differentiation. Collectively, these findings demonstrate that exogenous VISTA administration mitigates renal IR injury. Discussion Interstitial fibrosis following AKI represents a critical clinical challenge due to its strong association with progression to CKD and poor patient outcomes 24 – 26 . Although molecular mechanisms underlying AKI-to-fibrosis transition remain poorly understood. Our study identifies VISTA as a key immunoregulatory checkpoint molecule in kidney-resident and infiltrating macrophages that critically governs this process. By modulating macrophage polarization and T cell phenotypes, VISTA acts as a sentinel molecule to suppress inflammation and fibrosis, offering a mechanistic framework and therapeutic avenue for AKI-CKD progression. Macrophages exhibit dynamic plasticity between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes during AKI pathogenesis 27 . Following injury, kidney resident macrophages orchestrate inflammatory and reparative processes, while recruited monocytes differentiate into activated macrophages to amplify these responses. Notably, VISTA demonstrates constitutive expression in kidney resident macrophages under homeostasis and inducible expression in infiltrating macrophages and T cells post IR injury 28 . This spatiotemporal expression pattern suggests its regulatory role in balancing immune activation and resolution. Genetic ablation of VISTA exacerbated renal fibrosis, indicating its non-redundant function in mitigating post-AKI fibrogenesis. In our murine renal IR injury model, Vsir −/− mice and myeloid-specific Vsir knockout mice exhibited aggravated kidney injury, mirroring the protective role of HIF-1α in ischemic AKI 29 . Mechanistically, HIF-1α-mediated induction of VISTA in macrophages promoted their anti-inflammatory polarization, thereby establishing a novel HIF-1α/VISTA axis in renal protection 30 . Importantly, exogenous VISTA administration attenuated IRI-induced kidney damage, highlighting its therapeutic potential for preventing AKI progression. The immunomodulatory function of VISTA extends to T cell regulation 31 , 32 . Similar to PD-1 and CTLA-4 immune checkpoints that protect against organ injury 33 , 34 . VISTA deficiency in macrophages elevated IFNγ production from T cells post-IRI. As a Th1 cytokine implicated in post-AKI inflammation and fibrosis 35 – 37 , IFNγ overproduction likely drives fibrotic cascades through paracrine activation of fibroblasts and sustained macrophage pro-inflammatory programming 38 . This aligns with observations in pulmonary fibrosis models where VISTA depletion exacerbates fibroblast activation 39 . Inflammatory modulations targeting macrophages phenotypic regulation represents a promising therapeutic strategy for mitigating tissue injury following renal IR injury. Upon tissue damage, macrophages undergo reprogramming into a pro-inflammatory state, exacerbating inflammatory infiltration and tissue damage 40 . However, the molecular mechanism regulating the reprogramming remains incompletely understood. Emerging evidence suggests that VISTA modulates T cell activity through macrophage reprogramming. In our study, genetic ablation of VISTA in macrophages was associated with elevated IL-12 production and increased iNOS expression, indicating a shift toward a pro-inflammatory phenotype. Previous studies have reported that macrophage reprogramming is regulated by NF-κB signaling pathway, whose activation exacerbates inflammatory responses 41 , 42 . Notably, VISTA deficiency in macrophages enhanced NF-κB pathway activation, thereby sustaining a pro-inflammatory macrophage state post IR injury. Importantly, silencing of NF-κB signaling reversed the upregulation of IL-12 and iNOS observed in VISTA-depleted macrophages. In conclusion, our study elucidates that Vsir deficiency exacerbates AKI-CKD progression by promoting M1 polarization and amplifying IFNγ-driven T cell responses. Injection of VISTA attenuated renal damage, providing preclinical evidence for targeting this checkpoint in AKI management. These discoveries advance our understanding of immune checkpoints in renal fibrosis and offer a translatable strategy to interrupt the AKI-CKD continuum. Materials and Methods Animals Male C57BL/6(B6), B6.CD45.1 + , FoxP3-DTR and B6. Rag1 −/− mice were purchased from Cyagen (Shanghai, China). Vsir -knockout ( Vsir −/− ), Vsir-floxed ( Vsir fl/fl ), and IFNγ-knockout ( IFNγ −/− ) were also obtained from Cyagen. To generate myeloid-specific VISTA conditional knockout mice, Vsir fl/fl mice were crossed with Lyz2 Cre mice (Jackson Laboratory) to generate heterozygous offspring, followed by interbreeding to obtain Vsir fl/fl Lyz2 Cre mice. All animals were housed under standardized conditions (temperature: 18–23℃; humidity: 40–60%). Animal experiments were conducted in compliance with protocols approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University. Renal ischemia reperfusion injury model and interventions The renal IR injury model was established using 6- to 8-week-old male mice following the ARRIVE guidelines. Briefly, unilateral renal pedicle clamping was performed for 45 minutes under anesthesia, as previously described 43 . Sham-operated mice underwent identical procedures without vascular occlusion. Mice were euthanized via isoflurane overdose at 48 hours, 7 days, or 14 days post-ischemia for tissue and serum collection. To deplete macrophages, IRI-induced mice received intraperitoneal injections of clodronate liposomes (Encapsula NanoSciences, CLD-8901) or PBS-containing control liposomes, following the manufacturer’s protocol. Analysis of public scRNA-seq data Public single-cell RNA sequencing data were obtained from the Gene Expression Omnibus (GEO) database (accession: GSE161201). Raw data were processed using Cell Ranger 3.0.1 and analyzed with Seurat 4.1.1 in R (version 4.1.2). Low-quality cells were excluded based on thresholds of 20% mitochondrial UMI content. Datasets were normalized, scaled, and integrated into a harmonized reference using the Harmony algorithm. Clusters were defined by differentially expressed genes (DEGs), and results were visualized via Uniform Manifold Approximation and Projection (UMAP). Enriched gene expression patterns were illustrated using violin plots, dot plots, and feature plots. Preparation of mouse bone marrow-derived macrophages (BMDMs) and human macrophages Mouse BMDMs were isolated from 6- to 8-week-old mice. Bone marrow cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 20% L929-conditioned medium. THP-1 cells (Cell Bank of the Chinese Academy of Sciences) were differentiated into macrophages using 10 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, P8139) 44 . Cells were maintained at 37°C in a humidified 5% CO 2 incubator. For hypoxia studies, cells were exposed to 1%O 2 , 5% CO 2 , and 94% N 2 for 12h, followed by 6h or 12h of reoxygenation. Plasmids, lentiviruses, and dual luciferase reporter assay The VISTA promoter region containing HIF-1α binding sites were cloned into psiCHECK2 vector, with an empty psiCHECK2 vector serving as the control. Lentiviral constructs (lentivirus-NC and lentivirus-HIF-1α) were procured from HanBio (Shanghai, China). For dual-luciferase assays, cells were transfected with plasmids for 48 h, lysed, and analyzed using the Dual-Luciferase Reporter Assay Kit (MCE, Shanghai). Renilla luciferase activity was normalized to firefly luciferase, and the firefly/Renilla ratio quantified promoter activity. Flow cytometry Kidneys from IRI- and sham-operated mice were dissociated in RPMI 1640 medium containing collagenase IV (1 mg/mL; Sigma-Aldrich) and DNase I (50 µg/mL; Sigma-Aldrich) for 30 min at 37°C. Single-cell suspensions were filtered through a 70-µm strainer, resuspended in FACS buffer, and stained with fluorophore-conjugated antibodies against: CD45-FITC (BioLegend, 103108), CD4-PE-Cy7 (BioLegend, 100422), Foxp3-AF647 (BioLegend, 126408), IL-17a-PerCP-Cy5.5 (BioLegend, 506919), IFN-γ-BV421 (BioLegend, 505829), CD8-BV510 (BioLegend, 100752), CD44-BV421 (BioLegend, 103051), CD11b-PE-Cy7 (BioLegend, 101216), F4/80-PerCP-Cy5.5 (BioLegend, 123128), VISTA-PE (BioLegend, 150203), iNOS-PE (BioLegend, 696805), and IL-12-APC (BioLegend, 505205). Intracellular staining was performed using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher). Data were acquired on a BD LSRFortessa™ and analyzed with FlowJo v8.7. Histologic analysis Renal tissues were paraffin-embedded and sectioned (5 µm). For AKI assessment, sections were stained with Periodic Acid-Schiff (PAS) and Hematoxylin & Eosin (H&E). Tubular injury was scored as follows: 0 (none), 1 (0–10%), 2 (11–25%), 3 (26–45%), 4 (46–75%), or 5 (> 75%) based on necrosis and brush border loss. Fibrosis was evaluated using Sirius Red and Masson’s trichrome staining. Collagen-positive areas (blue for Masson’s, red for Sirius Red) were quantified using ImageJ (NIH). Immunofluorescence Immunofluorescence staining was performed as previously reported 45 . Kidney tissues were fixed in 4% paraformaldehyde for 2 h, cryoprotected in 30% sucrose overnight, and embedded in OCT compound. Cryosections (5–10 µm) were blocked in 0.1 M Tris buffer containing 0.1% Triton X-100 and 10% donkey serum. Primary antibodies against phospho-IκBα (Cell Signaling Technology Danvers, MA, USA, 4814T; 1:100) and F4/80 (Abcam, ab111101; 1:100) were incubated overnight at 4°C. After PBS washes, sections were incubated with Alexa Fluor-conjugated secondary antibodies (1:500; Invitrogen, Waltham, MA, USA) for 45 min, counterstained with DAPI (1:10,000; Cell Signaling Technology), and mounted with ProLong Gold Antifade (Invitrogen, P10144). Images were acquired using a confocal microscope (Zeiss LSM 880). Western blot analysis Kidney tissues and cells were lysed in RIPA buffer (supplemented with protease inhibitors) on ice. Lysates were denatured in SDS-PAGE sample buffer, resolved on 10% SDS-PAGE gels, and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST: 10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature (RT) and incubated overnight at 4°C with primary antibodies: rabbit anti-NF-κB (1:1000, Cell Signaling Technology, 8242S), rabbit anti-β-actin (1:5000, Cell Signaling Technology, 8457S), and rabbit anti-VISTA (1:1000, Cell Signaling Technology, 54979). After incubation with HRP-conjugated secondary antibodies (1:5000; Cell Signaling Technology) for 1 h at RT, protein bands were visualized using enhanced chemiluminescence (ECL; Santa Cruz Biotechnology) and quantified via densitometry with ImageJ (NIH). β-actin served as the loading control. CUT&TAG sequencing and ChIP-qPCR assay CUT&TAG sequencing was performed on THP-1-derived macrophages. Cells were immobilized on Concanavalin A-coated beads and incubated with antibodies against H3K27ac (Active Motif, 39133), H3K4me1 (Cell Signaling Technology, 5326S), or IgG control (Cell Signaling Technology, 2729S) in primary antibody buffer for 2 h at RT. After washing, beads were incubated with goat anti-rabbit IgG secondary antibody (1:500; Abcam, ab205718) for 1 h, followed by tagmentation with ChiTag pAG-Tn5 transposase (Vazyme) in ChiTag buffer. Tagmented DNA was purified using MagNA Pure beads (Roche) and amplified via PCR with the following cycling conditions: 72°C for 5 min (gap filling); 98°C for 30 s; 14 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min; final extension at 72°C for 5 min. For ChIP-qPCR, cells were crosslinked with 10% formaldehyde for 15 min, lysed, and sonicated using a ChIP assay kit (Cell Signaling Technology, 9005S). Immunoprecipitated DNA was analyzed by qPCR. Adoptive transfer of T cells CD4 + CD62L + naive T cells and CD8 + naive T cells were isolated from spleens using anti-biotin microbeads (Miltenyi Biotec, 130-106-643 and 130-096-543) with > 95% purity. Cells from wild-type or Vsir −/− mice were adoptively transferred into Rag1 −/− mice 24 h prior to IR injury induction. In vitro Th1, Tregs differentiation CD4 + CD62L + naïve T cells were isolated as above and cultured in 48-well plates pre-coated with anti-CD3 (5 µg/mL; eBioscience, 16-0032-86) and anti-CD28 (2 µg/mL; eBioscience, 16-0281-86). For Th1 cell differentiation, cells were cultured in a medium containing 20 ng/ml IL-2 (PeproTech, 212 − 12), 20 ng/ml IL-12 (R&D system, 419-ML-010), and 10 µg/ml anti-IL4 (eBioscience, 16-7041-81). For Tregs differentiation, cells were cultured in a medium containing 50 ng/ml IL-2 (PeproTech, 212 − 12) and 5 ng/ml TGF-β (R&D system, 240-B-010). Statistical analysis Data are expressed as mean ± standard deviation (SD). Differences between two groups were assessed by unpaired Student’s t -test; multiple comparisons used one-way ANOVA with Tukey’s post hoc test (GraphPad Prism 9). Experiments were repeated ≥ 3 times. Significance was defined as P < 0.05. Declarations Data availability All data generated or analyzed during this study are included in this published article. The single-cell RNA sequencing (scRNA-seq) datasets supporting the findings of this study are publicly available in the Gene Expression Omnibus (GEO) repository under accession number GSE161201. Additional datasets are currently being deposited in appropriate public repositories and will be made accessible upon completion. The authors confirm that any remaining data supporting the conclusions of this work are available from the corresponding authors upon reasonable request. Funding This study was supported by the National Natural Science Foundation of China (82270789 to Ruiming Rong; 82370754, 82170765 to Cheng Yang; 82241213 to Tongyu Zhu), and Shanghai Municipal Key Clinical Specialty (shslczdzk05802). Conflict of interests All the authors declared no competing interests. Contribution Cuidi Xu and Juntao Chen drafted the manuscript. Cheng Yang revised the manuscript. Cuidi Xu and Ruiming Rong conceived and designed the study. Cuidi Xu and Hao Zeng conducted bioinformatics analysis. Cuidi Xu, Pingbao Zhang and Lifei Liang participated in the animal experiments. Siyue Chen, Xuanchuan Wang and Pingbao Zhang provided technical support. Ruiming Rong, Tongyu Zhu and Cheng Yang supervised all the work. References Belcher, J. M. et al. Association of AKI with mortality and complications in hospitalized patients with cirrhosis. Hepatology. 57, 753–762 (2013). Kashif, R. F., Rashad, M. A., Said, A. M. A., Rabie, M. A. & Gomaa, W. A. 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MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function. Immunity. 55, 65–81 (2022). Ji, H. et al. Programmed death-1/B7-H1 negative costimulation protects mouse liver against ischemia and reperfusion injury. Hepatology. 52, 1380–1389 (2010). Khan, M. A. et al. CTLA4-Ig mediated immunosuppression favors immunotolerance and restores graft in mouse airway transplants. Pharmacol. Res. 178, 106147 (2022). Xu, Y. et al. A Role for Tubular Necroptosis in Cisplatin-Induced AKI. J. Am. Soc. Nephrol. 26, 2647–2658 (2015). Bajwa, A. et al. Sphingosine Kinase 2 Deficiency Attenuates Kidney Fibrosis via IFN-gamma. J. Am. Soc. Nephrol. 28, 1145–1161 (2017). Ferhat, M. et al. Endogenous IL-33 Contributes to Kidney Ischemia-Reperfusion Injury as an Alarmin. J. Am. Soc. Nephrol. 29, 1272–1288 (2018). Li, L. et al. IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury. J. Clin. Invest. 120, 331–342 (2010). Kim, S. et al. VISTA (PD-1H) Is a Crucial Immune Regulator to Limit Pulmonary Fibrosis. Am. J. Respir. Cell. Mol. Biol. 69, 22–33 (2023). Li, Z. et al. HIF-1alpha inducing exosomal microRNA-23a expression mediates the cross-talk between tubular epithelial cells and macrophages in tubulointerstitial inflammation. Kidney Int. 95, 388–404 (2019). Morrissey, S. M. et al. Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming. Cell Metab. 33, 2040–2058 (2021). Cheng, Q. J. et al. NF-kappaB dynamics determine the stimulus specificity of epigenomic reprogramming in macrophages. Science. 372, 1349–1353 (2021). Chen, J. et al. Inhibition of ALKBH5 attenuates I/R-induced renal injury in male mice by promoting Ccl28 m6A modification and increasing Treg recruitment. Nat. Commun. 14, 1161 (2023). Mercurio, A. M. & Shaw, L. M. Macrophage interactions with laminin: PMA selectively induces the adherence and spreading of mouse macrophages on a laminin substratum. J. Cell. Biol. 107, 1873–1880 (1988). Conway, B. R. et al. Kidney Single-Cell Atlas Reveals Myeloid Heterogeneity in Progression and Regression of Kidney Disease. J. Am. Soc. Nephrol. 31, 2833–2854 (2020). Additional Declarations (Not answered) Supplementary Files FigureS1.jpg Figure S1 FigureS2.jpg Figure S2 FigureS3.jpg Figure S3 FigureS4.jpg Figure S4 WBrawdata.pdf WB raw data Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6575812","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453371755,"identity":"6a87e54b-6c13-44ae-82fb-2bd02bbbad6b","order_by":0,"name":"Cheng Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYDADfmbmww9I0yLZzpZmQJoWg/M8ChLEuYf/jNmDj3sOyxsf5mEwYKixiSbsnhk55oYznh023HaY98ADhmNpuQ0E3XODx0ya58DhBLPDfAkGjA2HCWuxP3/GTPoPUItxM4+BBFFaDBhyzKQZgFoMmInVInEjrdyw50C64YzDwEBOIMYv/P2Htz34ccBaHsg4/OBDjQ1hLUDABsTNEGYCEcphWuqIVDsKRsEoGAUjEgAA+Wo9jAmHP4AAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4060-2746","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Yang","suffix":""},{"id":453371756,"identity":"0d8420b1-478a-4fde-8b3c-da900a0bf3cd","order_by":1,"name":"Cuidi Xu","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Cuidi","middleName":"","lastName":"Xu","suffix":""},{"id":453371757,"identity":"c5afdbd8-c426-47c9-88e5-e945ce960cec","order_by":2,"name":"Juntao Chen","email":"","orcid":"","institution":"Zhongshan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Juntao","middleName":"","lastName":"Chen","suffix":""},{"id":453371758,"identity":"37ee8bc5-01bb-41fc-ad19-37e7ca1116d8","order_by":3,"name":"Siyue Chen","email":"","orcid":"","institution":"Zhongshan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Siyue","middleName":"","lastName":"Chen","suffix":""},{"id":453371759,"identity":"ec088ada-3ac7-4c29-a75d-e1175ca142b4","order_by":4,"name":"Lifei Liang","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Lifei","middleName":"","lastName":"Liang","suffix":""},{"id":453371760,"identity":"e0ec2972-57d5-4091-ab7b-bcf8a8e986fa","order_by":5,"name":"Hao Zeng","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Zeng","suffix":""},{"id":453371761,"identity":"e003a951-de16-4723-b420-e255344cf0bb","order_by":6,"name":"Pingbao Zhang","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Pingbao","middleName":"","lastName":"Zhang","suffix":""},{"id":453371762,"identity":"1067b542-eb34-4004-9a37-95bb19c4d8c8","order_by":7,"name":"Xuanchuan Wang","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xuanchuan","middleName":"","lastName":"Wang","suffix":""},{"id":453371763,"identity":"c78bf32a-b257-4764-844a-0e992b51a849","order_by":8,"name":"Tongyu Zhu","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Tongyu","middleName":"","lastName":"Zhu","suffix":""},{"id":453371764,"identity":"8b5b7aff-c55d-4a8f-889c-13429494aa93","order_by":9,"name":"Ruiming Rong","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Ruiming","middleName":"","lastName":"Rong","suffix":""}],"badges":[],"createdAt":"2025-05-02 06:20:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6575812/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6575812/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82635321,"identity":"1492f4b6-484c-4ef8-8d47-02272a711a46","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4211376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVISTA expression following renal IR injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B) \u003c/strong\u003eFlow cytometric analysis of resident and recruited macrophage proportions in IRI-induced kidneys. \u003cstrong\u003e(C)\u003c/strong\u003e Uniform Manifold Approximation and Projection (UMAP) plots of renal cells from IRI-treated mice at distinct time points. Abbreviations: PT, proximal tubule; PC, principal cell of collecting duct; RM, resident macrophages; TAL, thick ascending limb of loop of Henle; DCT, distal convoluted tubule; EC, endothelial cells; DL-tAL, descending limb and thin ascending limb of loop of Henle; IC, intercalated cells of collecting duct; IM, infiltrating macrophages; MYO, myofibroblasts; T, T cells; FIB, fibroblasts; PODO, podocyte. \u003cstrong\u003e(D)\u003c/strong\u003eHeatmap depicting parenchymal cell cluster identification. \u003cstrong\u003e(E)\u003c/strong\u003e Dot plot to identify clusters of immune cells. \u003cstrong\u003e(F)\u003c/strong\u003e Feature plot showing \u003cem\u003eVsir\u003c/em\u003e gene expression across all clusters. \u003cstrong\u003e(G)\u003c/strong\u003e Dot plot comparing expression levels of AKI markers and extracellular matrix (ECM) markers at various post-IRI time points. \u003cstrong\u003e(H)\u003c/strong\u003e of VISTA expression in M1 and M2 macrophages at different days post-IRI.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/e112d8106bef1a8596fc816d.jpg"},{"id":82635320,"identity":"09451ade-6715-47b3-8905-e9f7dfec9f4a","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4386581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eVsir\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deficiency exacerbates IR-induced AKI and fibrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic of experimental groups: WT and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. \u003cstrong\u003e(B)\u003c/strong\u003e Serum creatinine and blood urea nitrogen (BUN) levels in IR-injured WT and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. \u003cstrong\u003e(C-D) \u003c/strong\u003eRepresentative H\u0026amp;E and PAS staining images (original magnification: 20×; scale bar: 50 μm) and tubular injury scores in IR-injured \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. \u003cstrong\u003e(E)\u003c/strong\u003e Representative Masson’s trichrome and Sirius red staining (original magnification: 20×; scale bar: 50 μm) of renal fibrosis in WT and\u0026nbsp;\u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;mice 28 days post-IR injury. \u003cstrong\u003e(F)\u003c/strong\u003e Schematic of experimental groups: \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice. \u003cstrong\u003e(G)\u003c/strong\u003e Serum creatinine and BUN levels in IR-injured\u003cem\u003e Vsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice. \u003cstrong\u003e(H-I)\u003c/strong\u003e Representative H\u0026amp;E and PAS staining (original magnification: 20×; scale bar: 50 μm) and tubular injury scores in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice 28 days post-IR injury. \u003cstrong\u003e(J)\u003c/strong\u003e Representative Masson’s trichrome and Sirius red staining (original magnification: 20×; scale bar: 50 μm) of renal fibrosis in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice 28 days post-IR injury.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/444de2031ed8910879604d11.jpg"},{"id":82635324,"identity":"c486d2bf-c72c-4e35-9fe3-3de14587a94e","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3834013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIF signaling induces VISTA expression following renal IR injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eKEGG pathway analysis of motifs enriched in H3K27ac chromatin immunoprecipitation. \u003cstrong\u003e(B)\u003c/strong\u003e Expression protein levels of VISTA and HIF-1α in kidneys following IR injury. \u003cstrong\u003e(C)\u003c/strong\u003e VISTA expression after pharmacological inhibition of HIF-1α. \u003cstrong\u003e(D)\u003c/strong\u003e Representative H\u0026amp;E and PAS staining of renal tissue sections from IR injury-subjected mice treated with PX-478.\u003cstrong\u003e (E) \u003c/strong\u003eSerum creatinine and BUN levels in IR injury-subjected mice treated with PX478. \u003cstrong\u003e(F)\u003c/strong\u003e H3K27ac and H3K4me1 histone modification enrichment at the\u003cem\u003e Vsir\u003c/em\u003e gene locus following HIF-1α overexpression. \u003cstrong\u003e(G)\u003c/strong\u003eChromatin immunoprecipitation quantitative PCR (ChIP-qPCR) demonstrating HIF-1α binding to the \u003cem\u003eVsir\u003c/em\u003e promoter region. \u003cstrong\u003e(H)\u003c/strong\u003e Luciferase reporter assay confirming HIF-1α-dependent transcriptional activation of \u003cem\u003eVsir\u003c/em\u003e through direct binding to its regulatory region.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/8f645eb6640f36622d581691.jpg"},{"id":82635343,"identity":"be5e910d-8b5d-4bdb-a629-864a47805130","added_by":"auto","created_at":"2025-05-13 14:25:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5243930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eVsir\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deficiency modulates macrophage phenotype.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Proportions of M1 and M2 macrophages in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice following sham or IR injury. \u003cstrong\u003e(B-E)\u003c/strong\u003e Flow cytometric analysis of IL-12, iNOS, Arg-1, and IL-10 expressions in RM and IM post IR injury in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice. \u003cstrong\u003e(F)\u003c/strong\u003e H\u0026amp;E and PAS staining of renal tissue sections from IR injury-subjected \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003emice injected with PBS- or clodronate-loaded liposomes. \u003cstrong\u003e(G)\u003c/strong\u003e H\u0026amp;E and PAS staining of renal tissue sections from IR injury-subjected mice injected with bone marrow-derived macrophages (BMDMs) isolated from CD45.1\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eVsir\u003c/em\u003e \u003csup\u003e-/-\u003c/sup\u003e mice or CD45.1\u003csup\u003e+\u003c/sup\u003e WT mice.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/ad568a2300a144529ed04a30.jpg"},{"id":82635329,"identity":"f540ef67-7719-4d0f-9edb-43d15c5f447b","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3770384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVsir deficiency alters the functional profile of infiltrating T cells following renal injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Serum creatinine and BUN levels in \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice injected with BMDMs from \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e or \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003emice. \u003cstrong\u003e(B)\u003c/strong\u003e Serum creatinine and BUN levels in \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice injected with CD3\u003csup\u003e+\u003c/sup\u003eT cells and BMDMs from \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e or \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003emice. \u003cstrong\u003e(C)\u003c/strong\u003e Frequency of IFNγ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and IFNγ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice 48 hours post-IR injury. \u003cstrong\u003e(D)\u003c/strong\u003e Frequency of Tregs and Th17 cells in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice 48 hours post-IR injury. \u003cstrong\u003e(E)\u003c/strong\u003e \u0026nbsp;Treg frequency in CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells co-cultured with WT or\u0026nbsp;\u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;BMDMs. \u003cstrong\u003e(F)\u003c/strong\u003e Serum creatinine and BUN levels in WT and \u003cem\u003eIFNγ\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice post-IR injury. \u003cstrong\u003e(G)\u003c/strong\u003e Serum creatinine and BUN levels in Tregs depleted mice post-IR injury.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/5982d5deeed8cb116d9a284a.jpg"},{"id":82635954,"identity":"cabbf15f-4ff2-474b-b10d-fc86684c024e","added_by":"auto","created_at":"2025-05-13 14:33:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4793877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVsir deficiency alters the phenotype of infiltrating T cells by IL-12.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eImmunohistochemical (IHC) staining showing IL-12-positive areas in renal tissues of \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice post IR injury. \u003cstrong\u003e(B) \u003c/strong\u003eSerum creatinine and BUN levels in IR injury-subjected\u003cem\u003e Vsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice treated with or without anti-IL-12 neutralizing antibody. \u003cstrong\u003e(C)\u003c/strong\u003e Representative H\u0026amp;E and Masson’s trichrome staining of renal tissue sections (original magnification: 20×; scale bar: 50 μm) from\u003cem\u003e Vsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice with or without anti-IL-12 intervention. \u003cstrong\u003e(D) \u003c/strong\u003eFlow cytometry analysis of Th1 (IFNγ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e), IFNγ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e and Treg cells frequencies in IRI-injured mice following anti-IL-12 treatment.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/67630f9d43976380c610394a.jpg"},{"id":82637252,"identity":"a6dd6c0d-66aa-4be5-afde-3106a440b4d5","added_by":"auto","created_at":"2025-05-13 14:41:54","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3706412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVISTA confers renoprotective effects in vivo following renal IR injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eSerum creatinine and BUN levels inmice treated with VISTA or vehicle control after sham surgery or IR induction. \u003cstrong\u003e(B-C)\u003c/strong\u003e Representative H\u0026amp;E and PAS staining of renal tissue sections (original magnification: 20×; scale bar: 50 μm) and tubular injury scores in mice treated with or without VISTA 48 hours post-IR injury. \u003cstrong\u003e(D)\u003c/strong\u003e Representative Masson’s trichrome and Sirius red staining (original magnification: 20×; scale bar: 50 μm) of renal fibrosis in mice treated with or without VISTA 14 days post-IR injury. \u003cstrong\u003e(E)\u003c/strong\u003eFlow cytometry analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage frequencies in mice treated with or without VISTA 48 hours post-IR injury.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/e3f18eda49010c81223e0cb6.jpg"},{"id":88725286,"identity":"c751c491-dd77-4f35-9ba1-32ec7f0439d8","added_by":"auto","created_at":"2025-08-10 15:03:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31271120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/36aadd31-d351-4348-92a1-412483aceb1f.pdf"},{"id":82635323,"identity":"90e760e3-0370-4833-92c4-c2ead7b8235a","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3164410,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/3d71aaeea9ca9601a174a8e2.jpg"},{"id":82635330,"identity":"2f247dc5-8001-4167-ba62-cacd934d79b0","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3653363,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/0f0984e15dc383b73e487b6e.jpg"},{"id":82635326,"identity":"d630dc47-0335-46eb-9027-25ea22dfc6df","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3540711,"visible":true,"origin":"","legend":"Figure S3","description":"","filename":"FigureS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/8a91f3885015017f7e79d6ba.jpg"},{"id":82635332,"identity":"d801cecd-ffc3-4cda-9bc5-da36d5b5ef5c","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3582748,"visible":true,"origin":"","legend":"Figure S4","description":"","filename":"FigureS4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/d57cd8f7de9781d6c652e190.jpg"},{"id":82635328,"identity":"bdf5906a-e0f6-4203-b14a-645a431ad4f5","added_by":"auto","created_at":"2025-05-13 14:25:53","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1260869,"visible":true,"origin":"","legend":"WB raw data","description":"","filename":"WBrawdata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6575812/v1/7bd9d77f171d65f3fd2b3ee4.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"VISTA attenuates ischemia reperfusion-induced renal injury and fibrosis by macrophage polarization reprogramming","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute kidney injury (AKI) is a clinical complex disorder characterized by multifactorial pathogenesis and high morbidity, with significant global health burdens\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Due to the lack of effective therapeutic interventions, AKI often progresses to irreversible tubular dysfunction and fibrotic matrix deposition in the renal interstitium, linking to the transition toward chronic kidney diseases (CKD)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Despite its clinical prevalence, the precise immunopathological mechanisms underlying AKI remain incompletely defined. Renal ischemia reperfusion (IR), which involves hypoxia, inflammation, apoptosis, and oxidative stress, serves as a widely utilized experimental model to investigate the mechanism bridging AKI and CKD progression\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The dynamic interplay of infiltrating immune cells, including macrophages and T cells, is increasingly recognized as a critical driver of this transition, as these cells orchestrate both the acute inflammatory response and the subsequent fibrotic remodeling observed in the kidney\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notably, while immune cells are known to persist throughout the AKI-to-CKD trajectory, the specific mechanisms by which they mediate this pathological shift, particularly their cytokine-driven polarization, remain poorly understood.\u003c/p\u003e \u003cp\u003eRabb et al. demonstrated that the early phase of renal IR injury is marked by the activation of mononuclear phagocytes, which amplify T cell infiltration and modulate T cell immunometabolic profiles\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Macrophages play a central role in the inflammatory response to AKI and persist throughout the transition to CKD, significantly influencing the outcome of renal injury\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Notably, Mami et al. revealed that macrophages could be trained toward a pro-inflammatory phenotype and exacerbated AKI, and depletion of macrophages by clodronate liposome attenuated renal injury and fibrosis post IR injury\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eV-domain Ig suppressor of T cell activation (VISTA),, also known as PD-1H, DD1α, Gi24, and Dies-1, is a type I transmembrane protein that acts as an immune-regulatory checkpoint on hematopoietic cells and shares homology with both the B7 family ligand programmed death-ligand 1 (PD-L1) and the CD28 family receptor PD-1\u003csup\u003e13\u003c/sup\u003e. As a protein encoded by a member of the B7/CD28 immune modulatory gene family, VISTA expressed on APCs exhibits an inhibitory effect on T cell function\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Blocking VISTA aggravated the progression of T-cell-mediated autoimmune diseases\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In a study that utilized mice bearing CT26 colon carcinoma tumors with complete resistance to PD-1/CTLA-4 blockade, the addition of VISTA blockade led to the rejection of more than half of the tumors\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Among all subsets of hematopoietic cells, myeloid cells, particularly monocytes, granulocytes, and dendritic cells (DCs), display the highest expression of VISTA. It is acknowledged that VISTA is highly constitutively expressed in resident macrophages. Park et al. reported that VISTA-expressing kidney-resident macrophages promote repair during ischemia injury\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In contrast to most other immune checkpoints, which are upregulated during different stages of stimulation or activation, VISTA is expressed by immune cells at steady state. This constitutive expression across a wide range of immune cells is unique and suggests that VISTA plays a role in maintaining immune system homeostasis. Although the expression of VISTA is modest, T cells are sensitive to respond to VISTA. VISTA\u0026ndash;Fc fusion protein and cellular overexpression of VISTA are suppressive to T-cell activation, proliferation, and cytokine production\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, we speculate that VISTA may serve as a potential target to attenuate acute inflammation and chronic fibrosis in renal IR injury.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVISTA expression dynamics in renal IR injury\u003c/h2\u003e \u003cp\u003eMacrophages, including resident macrophages (RM: CD45\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003eint\u003c/sup\u003eF4/80\u003csup\u003ehi\u003c/sup\u003e) and infiltrating macrophages (IM: CD45\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003ehi\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003e), are critical mediators of AKI and CKD pathogenesis. In the renal IR injury model\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, we characterized immune cell populations at key timepoints. Flow cytometric analysis revealed that macrophages (both RM and IM) constituted a significant proportion of CD45\u003csup\u003e+\u003c/sup\u003e immune cells at 6h, 1d, and 7d post-IR injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Notably, macrophages exhibited the highest expression VISTA among all cell types in the kidney. To validate this observation, we performed single-cell RNA sequencing (scRNA-seq) using public datasets in the GEO database (GSE161201) from four groups: control kidneys (Con), 6h post-IR injury (IRI 6h), 1d post-IR injury (IRI 1d), and 7d post-IR injury (IRI 7d). Analysis confirmed robust \u003cem\u003eVsir\u003c/em\u003e (VISTA) transcription in both RM and IM, whereas T cells showed low expression and other cells showed no expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-F). Gene expression profiling further revealed dynamic changes in renal injury markers: \u003cem\u003eHavcr1\u003c/em\u003e and \u003cem\u003eVcam1\u003c/em\u003e (markers of acute injury) peaked at 1d post-IR injury, while collagen genes (\u003cem\u003eCol1a1\u003c/em\u003e and \u003cem\u003eCol2a1\u003c/em\u003e, markers of fibrosis) increased by 7d post-IR injury. The expression of \u003cem\u003eVsir\u003c/em\u003e followed a biphasic pattern, rising sharply at 6h and 1d post-IRI before declining at 7d post-IR injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the VISTA expression in specific macrophage subsets, flow cytometry was performed. Baseline analysis showed that 35% of RM expressed VISTA, while only 10% of IM exhibited detectable VISTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Post-IR injury, RM maintained consistent VISTA expression at 48h, whereas IM macrophages, CD4\u003csup\u003e+\u003c/sup\u003e T cells, and CD8\u003csup\u003e+\u003c/sup\u003e T cells exhibited transient upregulation of VISTA. Notably, VISTA expression across all cell types declined by 48h post-IR injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B). Conclusively, these data demonstrate that VISTA is constitutively expressed in RM and dynamically induced in IM macrophages during the early phase of renal IR injury, with T cells serving as secondary responders to injury signals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVsir\u003c/b\u003e \u003cb\u003edeficiency exacerbates IR-induced AKI and fibrosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the functional role of \u003cem\u003eVsir\u003c/em\u003e in renal IR injury, we utilized \u003cem\u003eVsir\u003c/em\u003e knockout (\u003cem\u003eVsir\u003c/em\u003e⁻/⁻) mice subjected to unilateral renal pedicle clamping (45 minutes) or sham surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Baseline comparisons between wild-type (WT) and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e⁻/⁻\u003c/sup\u003e mice in sham-operated groups revealed no significant differences in serum creatinine and blood urea nitrogen (BUN) or histological architecture, as assessed via hematoxylin and eosin (H\u0026amp;E), Masson trichrome, Sirus Red, and Periodic Acid-Schiff (PAS) staining (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). However, following IR injury, \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e⁻/⁻\u003c/sup\u003e mice exhibited significantly elevated serum creatinine and BUN levels compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Histopathological analysis further demonstrated that \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e⁻/⁻\u003c/sup\u003e mice displayed more severe tubular necrosis, interstitial inflammation, and glomerular damage, as evidenced by H\u0026amp;E and PAS staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). To evaluate long-term fibrotic outcomes, Sirus Red and Masson trichrome staining were performed at 28 days post-IR injury. A marked increase of collagen deposition (fibrotic area) in \u003cem\u003eVsir\u003c/em\u003e⁻/⁻ mice was observed compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these findings demonstrated that \u003cem\u003eVsir\u003c/em\u003e deficiency exacerbated both AKI and subsequent fibrotic progression in the context of IRI, suggesting the protective role of VISTA in mitigating renal damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophage-specific\u003c/b\u003e \u003cb\u003eVsir\u003c/b\u003e \u003cb\u003edeficiency aggravates IR-induced AKI and fibrosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether \u003cem\u003eVsir\u003c/em\u003e deficiency in macrophage contributes to renal injury, we generated \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e (macrophage-specific \u003cem\u003eVsir\u003c/em\u003e knockout) mice by crossing \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e mice with lyz2-Cre mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Genotyping confirmed Cre-mediated recombination in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Baseline comparisons between \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice undergoing sham surgery revealed no differences in serum creatinine and BUN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), as well as histology (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). However, following IR, \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice exhibited significantly elevated serum creatinine and BUN levels 48h post-injury compared to \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Histopathological analysis further demonstrated that \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice displayed exacerbated tubular necrosis, interstitial inflammation, and glomerular damage, as evidenced by H\u0026amp;E and PAS staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I). Sirus red and Masson trichrome staining were performed at 28 days post IR injury to evaluate chronic fibrotic outcomes and revealed marked increases of collagen deposition (fibrotic area) in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice, with no differences observed between sham-operated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD). Collectively, these findings demonstrate that macrophage-specific \u003cem\u003eVsir\u003c/em\u003e deficiency exacerbates both AKI and fibrotic progression following IR injury, mirroring the effects observed in global \u003cem\u003eVsir\u003c/em\u003e knockout mice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHIF-1α directly induces VISTA expression via transcriptional activation in macrophages\u003c/h3\u003e\n\u003cp\u003eIRI triggers hypoxia and stabilizes hypoxia-inducible factor-1α (HIF-1α), which is a key regulator of cellular adaptation to hypoxia\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To determine whether HIF-1α regulates VISTA expression in macrophages, we performed chromatin accessibility profiling using H3K27ac CUT\u0026amp;Tag sequencing in macrophages isolated from IR-injured mouse kidneys. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis of H3K27ac-enriched regions identified immune-related pathways, including NF-κB and HIF-1 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Western blot analysis further revealed a positive correlation between HIF-1α and VISTA protein levels in kidneys subjected to IR injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate HIF-1α\u0026rsquo;s functional role in VISTA regulation, we administered PX-478, a specific HIF-1α inhibitor, to mice undergoing IR injury. PX-478 suppressed IR injury-induced VISTA upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and worsened renal dysfunction, as evidenced by elevated serum creatinine and BUN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Histopathological analysis confirmed exacerbated tubular injury in PX-478-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Mechanistically, bioinformatic prediction of HIF-1α binding sites in the \u003cem\u003eVsir\u003c/em\u003e promoter region identified multiple putative binding motifs. To validate this, we performed H3K27ac and H3K4me1 CUT\u0026amp;Tag sequencing in human macrophages transduced with HIF-1α-overexpressing lentivirus. HIF-1α overexpression significantly increased H3K27ac enrichment at the \u003cem\u003eVsir\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Chromatin immunoprecipitation (ChIP)-qPCR confirmed direct binding of HIF-1α to the \u003cem\u003eVsir\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Finally, a luciferase reporter assay demonstrated that HIF-1α drives \u003cem\u003eVsir\u003c/em\u003e transcription via a conserved HIF-1α binding motif in the promoter region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Collectively, these findings establish that HIF-1α directly upregulates VISTA expression in macrophages through transcriptional activation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVsir\u003c/b\u003e \u003cb\u003edeficiency skews macrophage polarization toward pro-inflammatory phenotypes via NF-κB activation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMacrophages exhibit functional heterogeneity, including pro-inflammatory (M1: CD86\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e\u0026minus;\u003c/sup\u003e) and anti-inflammatory (M2: CD86\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e) subsets\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To determine the role of VISTA in regulating this balance, we analyzed macrophage polarization in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice. Compared to \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e controls, \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice exhibited increased M1 macrophage frequency and decreased M2 macrophage frequency in the kidney post-IR injury (Figue 4A). Flow cytometric analysis further revealed elevated expression of M1 markers (IL-12 and iNOS) and reduced expression of M2 markers (Arg1 and IL-10) in both RM and IM isolated from \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;E). These findings suggest that VISTA suppresses pro-inflammatory macrophage polarization and promotes M2 reprogramming in both RM and IM subsets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo establish the functional relevance of this polarization shift, we depleted macrophages in \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice using clodronate liposomes. This intervention abolished the differences in serum creatinine and BUN levels between \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA), as well as histological disparities in tubular injury (H\u0026amp;E/PAS staining; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Furthermore, mouse bone marrow-derived macrophage (BMDMs) from CD45.1\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eWT\u003c/em\u003e and CD45.1\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were isolated and injected into IRI model CD45.2\u003csup\u003e+\u003c/sup\u003emice (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB) subjected to IR injury. Injection of \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e BMDMs exacerbated the renal dysfunction and tubular injury (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC, 4G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMechanistically, chromatin accessibility profiling (H3K27ac CUT\u0026amp;Tag) identified NF-κB signaling as an enriched pathway in IR-injured kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Given that NF-κB is able to promote M1 polarization and IL-12 secretion\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, we explored its interaction with VISTA. Immunofluorescence (IF) staining revealed increased phosphorylation of IκBα, a key driver of NF-κB nuclear translocation, in macrophages from \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice compared to controls (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD). Co-immunoprecipitation (Co-IP) experiments further demonstrated physical interaction between VISTA and NF-κB (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE). siRNA-mediated knockdown of \u003cem\u003eVsir\u003c/em\u003e in cultured macrophages enhanced NF-κB nuclear translocation (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF), corroborating that VISTA suppresses NF-κB activation. Therefore, the above results establish that VISTA restrains pro-inflammatory macrophage polarization by inhibiting NF-κB signaling.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVsir\u003c/b\u003e \u003cb\u003edeficiency alters infiltrating T cell phenotypes and mediates renal injury via T cell-macrophage crosstalk\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhile VISTA is expressed in naive T cells, scRNA-seq demonstrated that macrophages are the primary renal cell type expressing \u003cem\u003eVsir\u003c/em\u003e, suggesting their central role in AKI pathogenesis. To dissect the contribution of T cells to \u003cem\u003eVsir\u003c/em\u003e-deficient macrophage-mediated kidney injury, we employed \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (lacking T and B cells) as recipients for adoptive cell transfer experiments. Transfer of \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e bone marrow-derived macrophages (BMDMs) from \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003eLyz2\u003csup\u003eCre\u003c/sup\u003e mice into \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice induced only minor renal dysfunction compared to transfers of \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e BMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, co-transfer of \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003eBMDMs with CD3\u003csup\u003e+\u003c/sup\u003eT cells into \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice significantly increased serum creatinine and BUN levels compared with co-transfer of \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e BMDMs with T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These findings indicate that T cells are required for \u003cem\u003eVsir\u003c/em\u003e-deficient macrophages to mediate renal injury, necessitating functional crosstalk between these cell types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the T cell subsets involved, we analyzed kidney-infiltrating T cells 48h post-IR injury. \u003cem\u003eVsir\u003c/em\u003e-deficient macrophages were associated with increased proportions of CD4\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+\u003c/sup\u003e Th1, CD8\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and decreased CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e regulatory T (Treg) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), while no significant changes were observed in Th2 (CD4\u003csup\u003e+\u003c/sup\u003eIL4\u003csup\u003e+\u003c/sup\u003e) or Th17 (CD4\u003csup\u003e+\u003c/sup\u003eIL17A\u003csup\u003e+\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). In vitro co-culture experiments further revealed that \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMDMs potently suppressed Treg differentiation and promoted Th1 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) of naive CD4\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e T cells, suggesting a mechanistic link between \u003cem\u003eVsir\u003c/em\u003e deficiency and T cell phenotypic reprogramming.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the functional relevance of these T cell subsets in vivo, we employed \u003cem\u003eIFNγ\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003eFoxP3-DTR\u003c/em\u003e mice (allowing Treg depletion via diphtheria toxin). \u003cem\u003eIFNγ\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited reduced serum creatinine and BUN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and ameliorated histopathological injury (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB) compared to wild-type mice post-IR injury. Conversely, Treg depletion in \u003cem\u003eFoxP3-DTR\u003c/em\u003e mice worsened renal dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and tubular injury (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC). Collectively, these data demonstrate that \u003cem\u003eVsir\u003c/em\u003e deficiency in macrophages disrupts T cell homeostasis by promoting Th1 differentiation and suppressing Treg activity, thereby amplifying renal injury through Th1-dependent mechanisms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVsir\u003c/b\u003e \u003cb\u003edeficiency macrophages disrupt CD4\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eT cell balance via IL-12 driven Th1 differentiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIL-12 is a critical cytokine that drives Th1 cell differentiation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and suppresses Th2/Treg responses. In \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice subjected to IR injury, we observed a significant increase in IL-12-secreting macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), accompanied by elevated renal IL-12 positive area as demonstrated by immunohistochemistry (IHC) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To determine whether IL-12 mediates the T cell phenotypic shifts observed in \u003cem\u003eVsir\u003c/em\u003e-deficient macrophages, we administered anti-IL-12 neutralizing antibodies to \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice undergoing renal IR injury. Anti-IL-12 treatment significantly attenuated renal injury in terms of serum creatinine and BUN (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and reduced histopathological damages (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), mirroring the protective effects of \u003cem\u003eIFNγ\u003c/em\u003e knockout. Further analysis revealed that anti-IL-12 administration reduced Th1 cell proportions while increasing Treg cell frequencies (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings align with IL-12\u0026rsquo;s established role in promoting Th1 polarization (via IFNγ induction) and suppressing Treg differentiation, thereby skewing the CD4\u0026thinsp;+\u0026thinsp;T cell balance toward pro-inflammatory phenotypes in \u003cem\u003eVsir\u003c/em\u003e-deficient macrophage-mediated renal injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRecombinant VISTA exerts renoprotective effects via modulating immune responses in IR injury\u003c/h3\u003e\n\u003cp\u003eTo evaluate the therapeutic potential of VISTA in renal IR injury, we administered recombinant VISTA protein intravenously at the onset of reperfusion in a murine IR injury model. VISTA treatment significantly reduced serum creatinine and BUN levels compared to vehicle controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), accompanied by histopathological improvements in tubular necrosis and glomerular damage as assessed by H\u0026amp;E and Periodic Acid-Schiff (PAS) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). Collagen deposition, displayed by Sirus red and Masson trichrome staining, was also markedly attenuated in VISTA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), indicating reduced fibrotic progression. Moreover, Vista administration suppressed pro-inflammatory macrophage polarization, in terms of decreased M1 markers and increased M2 markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H). Additionally, VISTA treatment reduced the frequency of pathogenic CD4\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+\u003c/sup\u003e Th1 cells and IFNγ-producing CD8\u003csup\u003e+\u003c/sup\u003e T cells (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eD), aligning with its previously demonstrated role in inhibiting Th1 differentiation. Collectively, these findings demonstrate that exogenous VISTA administration mitigates renal IR injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInterstitial fibrosis following AKI represents a critical clinical challenge due to its strong association with progression to CKD and poor patient outcomes\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Although molecular mechanisms underlying AKI-to-fibrosis transition remain poorly understood. Our study identifies VISTA as a key immunoregulatory checkpoint molecule in kidney-resident and infiltrating macrophages that critically governs this process. By modulating macrophage polarization and T cell phenotypes, VISTA acts as a sentinel molecule to suppress inflammation and fibrosis, offering a mechanistic framework and therapeutic avenue for AKI-CKD progression.\u003c/p\u003e \u003cp\u003eMacrophages exhibit dynamic plasticity between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes during AKI pathogenesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Following injury, kidney resident macrophages orchestrate inflammatory and reparative processes, while recruited monocytes differentiate into activated macrophages to amplify these responses. Notably, VISTA demonstrates constitutive expression in kidney resident macrophages under homeostasis and inducible expression in infiltrating macrophages and T cells post IR injury\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This spatiotemporal expression pattern suggests its regulatory role in balancing immune activation and resolution. Genetic ablation of VISTA exacerbated renal fibrosis, indicating its non-redundant function in mitigating post-AKI fibrogenesis.\u003c/p\u003e \u003cp\u003eIn our murine renal IR injury model, \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and myeloid-specific \u003cem\u003eVsir\u003c/em\u003e knockout mice exhibited aggravated kidney injury, mirroring the protective role of HIF-1α in ischemic AKI\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Mechanistically, HIF-1α-mediated induction of VISTA in macrophages promoted their anti-inflammatory polarization, thereby establishing a novel HIF-1α/VISTA axis in renal protection\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Importantly, exogenous VISTA administration attenuated IRI-induced kidney damage, highlighting its therapeutic potential for preventing AKI progression.\u003c/p\u003e \u003cp\u003eThe immunomodulatory function of VISTA extends to T cell regulation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Similar to PD-1 and CTLA-4 immune checkpoints that protect against organ injury\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. VISTA deficiency in macrophages elevated IFNγ production from T cells post-IRI. As a Th1 cytokine implicated in post-AKI inflammation and fibrosis\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, IFNγ overproduction likely drives fibrotic cascades through paracrine activation of fibroblasts and sustained macrophage pro-inflammatory programming\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This aligns with observations in pulmonary fibrosis models where VISTA depletion exacerbates fibroblast activation\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInflammatory modulations targeting macrophages phenotypic regulation represents a promising therapeutic strategy for mitigating tissue injury following renal IR injury. Upon tissue damage, macrophages undergo reprogramming into a pro-inflammatory state, exacerbating inflammatory infiltration and tissue damage\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanism regulating the reprogramming remains incompletely understood. Emerging evidence suggests that VISTA modulates T cell activity through macrophage reprogramming. In our study, genetic ablation of VISTA in macrophages was associated with elevated IL-12 production and increased iNOS expression, indicating a shift toward a pro-inflammatory phenotype. Previous studies have reported that macrophage reprogramming is regulated by NF-κB signaling pathway, whose activation exacerbates inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Notably, VISTA deficiency in macrophages enhanced NF-κB pathway activation, thereby sustaining a pro-inflammatory macrophage state post IR injury. Importantly, silencing of NF-κB signaling reversed the upregulation of IL-12 and iNOS observed in VISTA-depleted macrophages.\u003c/p\u003e \u003cp\u003eIn conclusion, our study elucidates that \u003cem\u003eVsir\u003c/em\u003e deficiency exacerbates AKI-CKD progression by promoting M1 polarization and amplifying IFNγ-driven T cell responses. Injection of VISTA attenuated renal damage, providing preclinical evidence for targeting this checkpoint in AKI management. These discoveries advance our understanding of immune checkpoints in renal fibrosis and offer a translatable strategy to interrupt the AKI-CKD continuum.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale C57BL/6(B6), B6.CD45.1\u003csup\u003e+\u003c/sup\u003e, FoxP3-DTR and B6.\u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003emice were purchased from Cyagen (Shanghai, China). \u003cem\u003eVsir\u003c/em\u003e-knockout (\u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), Vsir-floxed (\u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e), and IFNγ-knockout (\u003cem\u003eIFNγ\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) were also obtained from Cyagen. To generate myeloid-specific VISTA conditional knockout mice, \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e mice were crossed with \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (Jackson Laboratory) to generate heterozygous offspring, followed by interbreeding to obtain \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice. All animals were housed under standardized conditions (temperature: 18\u0026ndash;23℃; humidity: 40\u0026ndash;60%). Animal experiments were conducted in compliance with protocols approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRenal ischemia reperfusion injury model and interventions\u003c/h3\u003e\n\u003cp\u003eThe renal IR injury model was established using 6- to 8-week-old male mice following the ARRIVE guidelines. Briefly, unilateral renal pedicle clamping was performed for 45 minutes under anesthesia, as previously described\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Sham-operated mice underwent identical procedures without vascular occlusion. Mice were euthanized via isoflurane overdose at 48 hours, 7 days, or 14 days post-ischemia for tissue and serum collection. To deplete macrophages, IRI-induced mice received intraperitoneal injections of clodronate liposomes (Encapsula NanoSciences, CLD-8901) or PBS-containing control liposomes, following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eAnalysis of public scRNA-seq data\u003c/h3\u003e\n\u003cp\u003ePublic single-cell RNA sequencing data were obtained from the Gene Expression Omnibus (GEO) database (accession: GSE161201). Raw data were processed using Cell Ranger 3.0.1 and analyzed with Seurat 4.1.1 in R (version 4.1.2). Low-quality cells were excluded based on thresholds of \u0026lt;\u0026thinsp;200 unique molecular identifiers (UMIs) per cell or \u0026gt;\u0026thinsp;20% mitochondrial UMI content. Datasets were normalized, scaled, and integrated into a harmonized reference using the Harmony algorithm. Clusters were defined by differentially expressed genes (DEGs), and results were visualized via Uniform Manifold Approximation and Projection (UMAP). Enriched gene expression patterns were illustrated using violin plots, dot plots, and feature plots.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of mouse bone marrow-derived macrophages (BMDMs) and human macrophages\u003c/h2\u003e \u003cp\u003eMouse BMDMs were isolated from 6- to 8-week-old mice. Bone marrow cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 20% L929-conditioned medium. THP-1 cells (Cell Bank of the Chinese Academy of Sciences) were differentiated into macrophages using 10 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, P8139)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Cells were maintained at 37\u0026deg;C in a humidified 5% CO\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;2\u0026lt;/sub\u0026thinsp;\u0026gt;\u0026thinsp;incubator. For hypoxia studies, cells were exposed to 1%O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 94% N\u003csub\u003e2\u003c/sub\u003e for 12h, followed by 6h or 12h of reoxygenation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids, lentiviruses, and dual luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe VISTA promoter region containing HIF-1α binding sites were cloned into psiCHECK2 vector, with an empty psiCHECK2 vector serving as the control. Lentiviral constructs (lentivirus-NC and lentivirus-HIF-1α) were procured from HanBio (Shanghai, China). For dual-luciferase assays, cells were transfected with plasmids for 48 h, lysed, and analyzed using the Dual-Luciferase Reporter Assay Kit (MCE, Shanghai). Renilla luciferase activity was normalized to firefly luciferase, and the firefly/Renilla ratio quantified promoter activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eKidneys from IRI- and sham-operated mice were dissociated in RPMI 1640 medium containing collagenase IV (1 mg/mL; Sigma-Aldrich) and DNase I (50 \u0026micro;g/mL; Sigma-Aldrich) for 30 min at 37\u0026deg;C. Single-cell suspensions were filtered through a 70-\u0026micro;m strainer, resuspended in FACS buffer, and stained with fluorophore-conjugated antibodies against: CD45-FITC (BioLegend, 103108), CD4-PE-Cy7 (BioLegend, 100422), Foxp3-AF647 (BioLegend, 126408), IL-17a-PerCP-Cy5.5 (BioLegend, 506919), IFN-γ-BV421 (BioLegend, 505829), CD8-BV510 (BioLegend, 100752), CD44-BV421 (BioLegend, 103051), CD11b-PE-Cy7 (BioLegend, 101216), F4/80-PerCP-Cy5.5 (BioLegend, 123128), VISTA-PE (BioLegend, 150203), iNOS-PE (BioLegend, 696805), and IL-12-APC (BioLegend, 505205). Intracellular staining was performed using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher). Data were acquired on a BD LSRFortessa\u0026trade; and analyzed with FlowJo v8.7.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistologic analysis\u003c/h2\u003e \u003cp\u003eRenal tissues were paraffin-embedded and sectioned (5 \u0026micro;m). For AKI assessment, sections were stained with Periodic Acid-Schiff (PAS) and Hematoxylin \u0026amp; Eosin (H\u0026amp;E). Tubular injury was scored as follows: 0 (none), 1 (0\u0026ndash;10%), 2 (11\u0026ndash;25%), 3 (26\u0026ndash;45%), 4 (46\u0026ndash;75%), or 5 (\u0026gt;\u0026thinsp;75%) based on necrosis and brush border loss. Fibrosis was evaluated using Sirius Red and Masson\u0026rsquo;s trichrome staining. Collagen-positive areas (blue for Masson\u0026rsquo;s, red for Sirius Red) were quantified using ImageJ (NIH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed as previously reported\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Kidney tissues were fixed in 4% paraformaldehyde for 2 h, cryoprotected in 30% sucrose overnight, and embedded in OCT compound. Cryosections (5\u0026ndash;10 \u0026micro;m) were blocked in 0.1 M Tris buffer containing 0.1% Triton X-100 and 10% donkey serum. Primary antibodies against phospho-IκBα (Cell Signaling Technology Danvers, MA, USA, 4814T; 1:100) and F4/80 (Abcam, ab111101; 1:100) were incubated overnight at 4\u0026deg;C. After PBS washes, sections were incubated with Alexa Fluor-conjugated secondary antibodies (1:500; Invitrogen, Waltham, MA, USA) for 45 min, counterstained with DAPI (1:10,000; Cell Signaling Technology), and mounted with ProLong Gold Antifade (Invitrogen, P10144). Images were acquired using a confocal microscope (Zeiss LSM 880).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eKidney tissues and cells were lysed in RIPA buffer (supplemented with protease inhibitors) on ice. Lysates were denatured in SDS-PAGE sample buffer, resolved on 10% SDS-PAGE gels, and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST: 10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature (RT) and incubated overnight at 4\u0026deg;C with primary antibodies: rabbit anti-NF-κB (1:1000, Cell Signaling Technology, 8242S), rabbit anti-β-actin (1:5000, Cell Signaling Technology, 8457S), and rabbit anti-VISTA (1:1000, Cell Signaling Technology, 54979). After incubation with HRP-conjugated secondary antibodies (1:5000; Cell Signaling Technology) for 1 h at RT, protein bands were visualized using enhanced chemiluminescence (ECL; Santa Cruz Biotechnology) and quantified via densitometry with ImageJ (NIH). β-actin served as the loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCUT\u0026amp;TAG sequencing and ChIP-qPCR assay\u003c/h2\u003e \u003cp\u003eCUT\u0026amp;TAG sequencing was performed on THP-1-derived macrophages. Cells were immobilized on Concanavalin A-coated beads and incubated with antibodies against H3K27ac (Active Motif, 39133), H3K4me1 (Cell Signaling Technology, 5326S), or IgG control (Cell Signaling Technology, 2729S) in primary antibody buffer for 2 h at RT. After washing, beads were incubated with goat anti-rabbit IgG secondary antibody (1:500; Abcam, ab205718) for 1 h, followed by tagmentation with ChiTag pAG-Tn5 transposase (Vazyme) in ChiTag buffer. Tagmented DNA was purified using MagNA Pure beads (Roche) and amplified via PCR with the following cycling conditions: 72\u0026deg;C for 5 min (gap filling); 98\u0026deg;C for 30 s; 14 cycles of 98\u0026deg;C for 10 s, 63\u0026deg;C for 30 s, and 72\u0026deg;C for 1 min; final extension at 72\u0026deg;C for 5 min. For ChIP-qPCR, cells were crosslinked with 10% formaldehyde for 15 min, lysed, and sonicated using a ChIP assay kit (Cell Signaling Technology, 9005S). Immunoprecipitated DNA was analyzed by qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAdoptive transfer of T cells\u003c/h2\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e naive T cells and CD8\u003csup\u003e+\u003c/sup\u003e naive T cells were isolated from spleens using anti-biotin microbeads (Miltenyi Biotec, 130-106-643 and 130-096-543) with \u0026gt;\u0026thinsp;95% purity. Cells from wild-type or \u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were adoptively transferred into \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice 24 h prior to IR injury induction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro Th1, Tregs differentiation\u003c/h2\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e na\u0026iuml;ve T cells were isolated as above and cultured in 48-well plates pre-coated with anti-CD3 (5 \u0026micro;g/mL; eBioscience, 16-0032-86) and anti-CD28 (2 \u0026micro;g/mL; eBioscience, 16-0281-86). For Th1 cell differentiation, cells were cultured in a medium containing 20 ng/ml IL-2 (PeproTech, 212\u0026thinsp;\u0026minus;\u0026thinsp;12), 20 ng/ml IL-12 (R\u0026amp;D system, 419-ML-010), and 10 \u0026micro;g/ml anti-IL4 (eBioscience, 16-7041-81). For Tregs differentiation, cells were cultured in a medium containing 50 ng/ml IL-2 (PeproTech, 212\u0026thinsp;\u0026minus;\u0026thinsp;12) and 5 ng/ml TGF-β (R\u0026amp;D system, 240-B-010).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between two groups were assessed by unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test; multiple comparisons used one-way ANOVA with Tukey\u0026rsquo;s post hoc test (GraphPad Prism 9). Experiments were repeated\u0026thinsp;\u0026ge;\u0026thinsp;3 times. Significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. The single-cell RNA sequencing (scRNA-seq) datasets supporting the findings of this study are publicly available in the Gene Expression Omnibus (GEO) repository under accession number\u0026nbsp;GSE161201. Additional datasets are currently being deposited in appropriate public repositories and will be made accessible upon completion. The authors confirm that any remaining data supporting the conclusions of this work are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (82270789 to Ruiming Rong; 82370754,\u0026nbsp;82170765\u0026nbsp;to Cheng Yang; 82241213 to Tongyu Zhu), and Shanghai Municipal Key Clinical Specialty (shslczdzk05802).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declared no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCuidi Xu and Juntao Chen drafted the manuscript. Cheng Yang revised the manuscript. Cuidi Xu and Ruiming Rong conceived and designed the study. Cuidi Xu and Hao Zeng conducted bioinformatics analysis. Cuidi Xu, Pingbao Zhang and Lifei Liang participated in the animal experiments. Siyue Chen, Xuanchuan Wang and Pingbao Zhang provided technical support. Ruiming Rong, Tongyu Zhu and Cheng Yang supervised all the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBelcher, J. M. et al. Association of AKI with mortality and complications in hospitalized patients with cirrhosis. Hepatology. 57, 753\u0026ndash;762 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKashif, R. F., Rashad, M. A., Said, A. M. A., Rabie, M. A. \u0026amp; Gomaa, W. A. Ultrasound biomicroscopy study of accommodative state in Smartphone abusers. Bmc Ophthalmol. 22, 330 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, C. et al. LTBP4 Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts. Circ. Res. 133, 71\u0026ndash;85 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaguchi, K. et al. 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Nephrol. 31, 2833\u0026ndash;2854 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6575812/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6575812/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRenal ischemia reperfusion (IR) injury is one of the major causes of acute kidney injury (AKI) and may contribute to the development of chronic kidney disease. However, the precise immunological mediators orchestrating these pathophysiological processes remain poorly defined. In the present study, we investigate the protective role of the V-domain Ig suppressor of T cell activation (VISTA), which is and immune checkpoint molecule and highly expressed in macrophages, in attenuating IR-induced renal injury. Using genetic deficiency models, we demonstrate that miace lacking VISTA (\u003cem\u003eVsir\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) and macrophages-specific VISTA knockout (\u003cem\u003eVsir\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e) exhibited significantly exacerbated renal dysfunction and histopathological damage post-IR injury. Mechanistically, hypoxia-inducible factor-1α, as a transcriptional regulator, induced VISTA expression in macrophages post IR injury. VISTA deficiency in macrophages reprogramed these cells toward a pro-inflammatory phenotype via NF-κB nuclear translocation, which amplified Th1 differentiation through IL-12 upregulation while simultaneously suppressing regulatory T cell expansion. Notably, neutralizing IL-12 activity rescued renal injury in VISTA-deficient mice, underscoring its role as a key effector in this pathway. Therapeutically, exogenous VISTA administration attenuated renal inflammation, fibrosis, and functional impairment, highlighting its direct renoprotective capacity. Therefore, VISTA emerges as a sentinel checkpoint protein that balances macrophage polarization and T cell immunity during AKI.\u003c/p\u003e","manuscriptTitle":"VISTA attenuates ischemia reperfusion-induced renal injury and fibrosis by macrophage polarization reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 14:25:48","doi":"10.21203/rs.3.rs-6575812/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d4f614f-0036-4460-8035-8e49a884bfdf","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48214597,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages"},{"id":48214598,"name":"Biological sciences/Immunology/Translational immunology"}],"tags":[],"updatedAt":"2025-08-26T02:38:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 14:25:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6575812","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6575812","identity":"rs-6575812","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}