VISTA Confers Renal Protection in Murine Hemorrhage/Infection-Induced Acute Kidney Injury: Associations with Altered NGAL and HMGB1 Expression

Abstract

Background

Acute kidney injury (AKI) represents a serious complication associated with both short- and long-term morbidity and mortality in critically ill patients. Severe trauma complicated by infection represents a high‑risk clinical trigger for AKI. V‑domain immunoglobulin suppressor of T‑cell activation (VISTA) is an immune‑checkpoint molecule with immunomodulatory functions, yet its role in trauma‑sepsis‑induced AKI remains incompletely defined.

Methods

In this experimental study, male wild‑type (WT) and VISTA‑deficient (VISTA^−/−) C57BL/6 mice were utilized to establish a two‑hit murine AKI model combining hemorrhagic shock and cecal ligation and puncture (Hem/CLP). Blood and kidney tissues were collected 24 hours after hemorrhage/CLP. VISTA expression on neutrophils and monocytes in peripheral blood was assessed by flow cytometry. Serum creatinine and blood urea nitrogen were measured according to manufacturer protocols. Cytokine levels were determined by ELISA, and corresponding mRNA expression was analyzed via RT-PCR. Kidney injury severity was evaluated histologically by pathologists. Protein levels of neutrophil gelatinase-associated lipocalin and high mobility group box 1 were examined by Western blot.

Results

VISTA expression was significantly elevated on circulating neutrophils and monocytes following injury (all p < 0.05). Histopathological assessment revealed aggravated kidney injury in the AKI model (p = 0.0056), which was further exacerbated in VISTA-deficient mice (p = 0.0357). Consistent with this, serum creatinine (p = 0.014) and blood urea nitrogen (p = 0.005) levels were markedly higher in VISTA-deficient mice compared to controls. Pro-inflammatory cytokines IL-6 (p = 0.0335) and TNF-α (p = 0.0283) were increased in AKI mice and further elevated in VISTA-deficient mice (p = 0.0014 and 0.0001, respectively), whereas anti-inflammatory IL-10 showed a converse trend (p = 0.0056 and 0.0019, respectively). RT-PCR confirmed corresponding changes in cytokine mRNA expression. Western blot analysis indicated decreased neutrophil gelatinase-associated lipocalin (p < 0.0001) and increased high mobility group box 1 protein (p = 0.0009) levels in VISTA-deficient mice.

Conclusion

Collectively, our results demonstrate that VISTA‑deficient mice develop more severe early‑phase hemorrhage/sepsis‑induced AKI. Altered NGAL and HMGB1 protein expression occur in parallel with VISTA‑associated renal protection; further functional validation is required to establish causal mechanistic relationships and to evaluate the translational potential of VISTA‑targeted approaches.

Introduction

AKI is a clinical syndrome characterized by a rapid decline in kidney function, typically over hours to days, resulting in the accumulation of waste products (eg, creatinine, urea).¹ It represents a major global health concern, affecting millions of patients worldwide, with high morbidity and mortality, especially in hospitalized and critically ill populations.^1–3 The definition and staging of AKI have evolved over time, with the current consensus based on the KDIGO (Kidney Disease: Improving Global Outcomes) criteria, which use changes in serum creatinine and urine output to classify severity into three stages.^1,4 Despite established consensus criteria for its definition and classification and advances in clinical management strategies that improve early detection and risk stratification, the incidence, severity, and long-term outcomes of AKI continue to present substantial challenges, with prognosis often remaining poor.⁵ As a major public health concern, AKI not only directly contributes to increased short-term mortality but also accelerates the progression of underlying chronic kidney disease (CKD), leading to decreased long-term survival.⁶

The pathogenesis of AKI is multifactorial and includes hemodynamic disturbance, tubular epithelial injury, and robust immune‑mediated inflammation.^7,8 In ischemic AKI, reduced renal perfusion causes tubular epithelial metabolic failure, cell death, cast formation, and reduced glomerular filtration, accompanied by recruitment of neutrophils and monocytes that amplify local tissue damage.^9–11 Additionally, nephrotoxic insults trigger direct tubular injury via reactive oxygen species and cellular stress.^12,13 Accumulating evidence underscores that systemic inflammation and immune dysregulation are central drivers of AKI initiation and progression, especially in settings of major trauma, surgery, and sepsis. Circulating innate immune cells such as neutrophils and monocytes migrate to injured kidneys and propagate tissue harm by releasing pro‑inflammatory mediators and reactive oxygen species.¹⁴

VISTA, is a multifunctional B7‑family immune‑checkpoint molecule that can operate both as a membrane‑bound ligand on myeloid cells and as an inhibitory receptor on lymphocytes to maintain innate‑adaptive immune homeostasis.¹⁵ VISTA exhibits restricted myeloid-dominant expression pattern per bulk and single-cell transcriptomic literature: constitutively abundant on monocytes, tissue macrophages and myeloid-derived suppressor cells (MDSCs), moderately expressed on naïve CD4⁺/CD8⁺ T cells and Foxp3⁺ regulatory T cells (Tregs), while absent from mature B cells and most resting NK cells.^16,17 It has been reported that VISTA is constitutively expressed on various cell types and functions as an independent immune modulator in several inflammatory and autoimmune conditions.¹⁸ Among myeloid populations, circulating neutrophils and monocytes are rapidly mobilized upon hemorrhagic shock and secondary sepsis. These cells are central drivers of systemic inflammation, can infiltrate injured renal tissue, and exhibit high VISTA expression. Accordingly, they represent key cellular candidates mediating crosstalk along the proposed periphery‑kidney inflammatory axis in trauma‑associated AKI, which is why we focused our initial analysis on these two peripheral innate immune cell subsets. Pre-clinical studies have documented VISTA-deficient mice display heightened susceptibility to certain auto-inflammatory phenotypes, consistent with its function as an immune-dampening checkpoint.¹⁹ Despite the established immunomodulatory functions of VISTA in inflammatory and autoimmune disorders, a critical knowledge gap persists: it remains unclear whether and how VISTA modulates the crosstalk between peripheral innate immune activation and renal injury during AKI, particularly in the clinically relevant setting of combined hemorrhage and secondary infection.

AKI is not a single disease but a syndrome with diverse etiologies, unified by rapid decline in renal function. Our understanding of its pathophysiology continues to evolve, yet management remains largely supportive. Ongoing research into biomarkers, novel therapies (eg, anti-inflammatory agents), and precision medicine approaches holds promise for improving outcomes. It has been reported, VISTA pathway has dual-direction therapeutic druggable targets, alongside its binding ligands and intracellular signaling nodes as actionable targets across preclinical and early-clinic development.²⁰ Single‑hit AKI models (eg, isolated ischemia‑reperfusion or cecal ligation and puncture alone) cannot faithfully reproduce the clinical trajectory of trauma‑associated AKI, where hypovolemic shock is frequently complicated by subsequent polymicrobial infection. Therefore, we employed a combined hemorrhage‑then‑CLP “two‑hit” murine model. In this two‑hit hemorrhage‑CLP model, robust systemic innate immune activation and early‑phase renal pathological damage are detectable at 24 h following CLP insult. This time‑point allows us to capture peak early‑systemic inflammation together with established acute kidney functional and histological injury, making 24 h suitable for evaluating VISTA‑dependent immune‑regulatory effects in the early AKI phase.

Materials and Methods

Ethical Statement and Animal Welfare

Male C57BL/6 mice were obtained from the Research Animal Center of Shanghai Pudong Hospital (Shanghai, China). All animals were maintained in a specific pathogen-free (SPF) environment under controlled conditions, including a 12-hour light/dark cycle, ambient temperature of 22 ±2 °C, and relative humidity of 50 ± 10%. Standard laboratory chow and autoclaved water were provided with unlimited access. To ensure the health and welfare of the animals, regular veterinary monitoring was conducted, and all mice were acclimatized for at least one week prior to experimentation. The present study was conducted in strict accordance with the ARRIVE guidelines (Animals in Research: Reporting of in vivo Experiments).²¹ All experimental protocols were reviewed and approved by the Institutional Animal Ethics Committee of Shanghai Pudong Hospital (Approval Protocol Code: 2020-DS-017). All procedures were performed in adherence to the relevant guidelines and regulations under this protocol, following the Chinese Guidelines for Ethical Review of Animal Welfare in Experiments. Briefly, mice were anesthetized with isoflurane during the operation and were given euthanasia under carbon dioxide after the sample was harvested. Of note, male mice were used in this study because it had been previously observed sex-based differences in immune responses to Hem/CLP, with males being significantly more sensitive than pro-estrus females.^22,23

Animals and Experimental Groups

Male C57BL/6 wild-type (WT) mice (aged 8–12 weeks, 25–30 g) were obtained from the Animal Experimentation Center. VISTA-deficient (VISTA^−/−) mice on a C57BL/6 background were generated by Shanghai Model Organisms Co., Ltd. (Shanghai, China) using CRISPR/Cas9-mediated gene-editing technology. These knockout mice were backcrossed for at least six successive generations onto the C57BL/6 background to ensure genetic homogeneity relative to wild-type controls. All mice were housed under specific pathogen-free conditions with free access to standard laboratory chow and water for at least one week prior to experimentation. WT mice were randomly assigned to either the hemorrhage/cecal ligation and puncture (Hem/CLP) group or the sham‑Hem/CLP group as we performed previously.²⁴ Randomization was performed using a sequence number system. Preliminary investigations confirmed no baseline differences in VISTA expression on circulating neutrophils and monocytes or in kidney histology between naive WT groups. VISTA^−/− mice were allocated to the Hem/CLP group only. We acknowledge that inclusion of a VISTA^−/− sham group would be ideal to formally exclude intrinsic baseline renal alterations caused by VISTA deletion independent of injury, and such controls should be incorporated in future investigations.

Experimental Model of Acute Kidney Injury (AKI)

To simulate the clinical progression from hypovolemic shock to secondary infection, a double-hit model of AKI was established by combining hemorrhagic shock with subsequent polymicrobial sepsis.

Hemorrhagic Shock (Hem)

Under isoflurane anesthesia, mice were subjected to pressure-controlled hemorrhagic shock. Mean arterial pressure was maintained at 35 ± 5 mmHg for 90 minutes by withdrawing blood from the femoral artery. Subsequently, resuscitation was performed by intravenous infusion of Ringer’s lactate solution at four times the volume of shed blood, administered via a micro-injection pump.

Cecal Ligation and Puncture (CLP)

At 24 hours post-resuscitation, polymicrobial sepsis was induced via CLP. Briefly, the cecum was exposed, ligated below the ileocecal valve, and punctured once with a 21-gauge needle. A small amount of fecal content was extruded to ensure patency before the cecum was returned to the abdominal cavity. The sham Hem/CLP group underwent identical surgical procedures, including femoral artery exposure and laparotomy, but without blood withdrawal or cecal ligation/puncture.

Sample Collection and Processing

At 24 hours after the CLP procedure (or corresponding time for sham groups), mice were deeply anesthetized with isoflurane. Blood was collected via cardiac puncture using heparinized syringes. Plasma was separated by centrifugation at 3000 × g for 5 minutes at 4°C and stored at −80°C for subsequent analysis. Both kidneys were promptly harvested. One kidney was fixed in 10% neutral buffered formalin for histology, while the contralateral kidney was snap-frozen in liquid nitrogen and stored at −80°C for molecular analyses.

Biochemical and Cytokine Measurements

Plasma levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were quantified using an automatic dry biochemical analyzer (Hitachi Auto Analyzer 7170, Japan). The concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10) in plasma and kidney homogenates were determined using specific commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, USA), strictly following the manufacturer’s protocols. Optical density was measured at 450 nm with a correction wavelength of 550 nm. A four-parameter logistic curve fit was applied to the standard values for sample concentration calculation.

Flow Cytometry

Peripheral blood leukocytes were analyzed 24 hours post-Hem/CLP. Single-cell suspensions were stained with the following fluorochrome-conjugated antibodies: anti-CD11b-APC (clone GK-1.5, eBioscience), anti-F4/80-Brilliant Violet (clone BM8, BioLegend), anti-Ly6G-VioBlue (clone 53–6.7, eBioscience), and anti-VISTA-PE (clone OX-90, eBioscience). Appropriate unstained controls and isotype-matched antibody were used for gate setup. Doublet events were excluded using forward‑scatter area versus forward‑scatter height (FSC‑A vs FSC‑H) gating within the total leukocyte population prior to further subset identification. Neutrophils were identified as CD11b⁺Ly6G⁺ cells and monocytes/macrophages as CD11b⁺F4/80⁺ cells. For VISTA expression analysis, MFI was calculated exclusively within pre-gated single-positive target cell populations on a per-mouse basis. Each individual MFI value represents the averaged fluorescence intensity of thousands of single cells acquired from one independent animal. Representative fluorescence histograms were generated to show the single-cell distribution of VISTA expression on a logarithmic scale.

Histopathological Assessment

Formalin-fixed kidney tissues were embedded in paraffin, sectioned at 4–5 μm thickness, and stained with hematoxylin and eosin (H&E). For blinded assessment, all tissue slides were de‑identified and assigned random unique codes with no group information. Three independent pathologists performed semi‑quantitative tubular injury scoring in a blinded fashion using a scoring system:²⁵ 0 (no injury), 1 (1–10% cell necrosis), 2 (11–25% lobular necrosis), 3 (26–45% lobular necrosis), and 4 (>46% lobular necrosis). This scoring system comprehensively evaluates pathological manifestations including tubular necrosis, karyopyknosis, tubular degeneration, interstitial edema, and inflammatory cell infiltration. Group identities were unblinded only after completion of all scoring.

Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from kidney tissues using TRIzol reagent (Invitrogen). After DNase I treatment to eliminate genomic DNA, complementary DNA (cDNA) was synthesized using a reverse transcription system. qRT-PCR was performed using SYBR Green PCR Master Mix on a Chromo Real-Time PCR Detection System (Bio-Rad). Gene expression levels of IL-6, TNF-α, and IL-10 were normalized to the housekeeping gene GAPDH and calculated using the 2^(-ΔΔCt) method.²⁶ Primer sequences were as follows: GAPDH: Forward 5′-CCC CCA ATG TGT CCG TCG-3′, Reverse 5′-CGG CAT CGA AGG TGG AAG A-3′. IL-6: Forward 5′-GGA GCC CAC CAA GAA CGA T-3′, Reverse 5′-TCA CCA GCA TCA GTC CCA AGA-3′. TNF-α: Forward 5′-TCT TCT CAT TCC TGC TTG TGG-3′, Reverse 5′-TTG GTG GTT TGC TAC GAC G-3′. IL-10: Forward 5′-CCA GTT TTA CCT GGT AGA AGT GAT G-3′ Reverse 5′-TGT CTA GGT CCT GGA GTC CAG CAG ACT C-3′.

Western Blot Analysis

Kidney tissues were homogenized in RIPA lysis buffer (Thermo Fisher Scientific). Protein concentrations were determined using a Bradford protein assay kit (Thermo Fisher Scientific) with bovine serum albumin as the standard. Equal amounts of protein were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight at 4°C with primary antibodies against NGAL, HMGB1 (both from Cell Signaling Technology), or GAPDH (as a loading control). Membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence detection system (Amersham) and quantified by densitometry using ImageJ software (NIH).

Statistical Analysis

All statistical analyses were performed using SPSS software (version 19.0). Data are presented as the mean ± standard error of the mean (SEM). For comparisons between two groups, an unpaired Student’s t-test was used. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was applied after confirming homogeneity of variance. Two‑way ANOVA was applied for datasets with two factors: genotype (WT vs VISTA^−/−) and surgical intervention (Sham vs Hem/CLP). This test evaluates the main effects of genotype, surgical injury, and their genotype × injury interaction term. One‑way ANOVA was only used when comparing three or more experimental groups under a single factor condition. If variances were homogeneous, Tukey’s HSD post‑hoc test was used to control type‑I error for multiple pairwise comparisons. If the assumption of homogeneity of variances was violated, Welch’s ANOVA followed by the Dunnett T3 post-hoc test was employed. A p-value of less than 0.05 was considered statistically significant. All experiments were repeated at least once to ensure reproducibility. Per-group animal numbers (n = 4–13 mice per experimental group) are reported in respective figure legends. Animals that died prior to the experimental endpoint or reached humane euthanasia criteria were excluded from analyses. No data imputation was performed; only complete biological samples were included in the final dataset. A-priori power calculation was not performed for this study.

Results

The Development of AKI

To investigate the pathophysiology of AKI, a double-hit model combining hemorrhagic shock (Hem) and subsequent polymicrobial sepsis (via CLP) was established (Figure 1). This model recapitulates the clinical scenario of AKI following severe trauma/hemorrhage complicated by sepsis, a common and devastating clinical pathway to renal failure.

Figure 1.

Double-hit induction model of AKI in mice via sequential hemorrhagic shock and polymicrobial sepsis. This schematic depicts a clinically relevant two-hit murine model of AKI. In the first hit, under isoflurane anesthesia, mice undergo controlled hemorrhagic shock: blood is withdrawn via a femoral artery catheter to reduce mean arterial pressure to 35 ± 5 mmHg for 90 minutes, inducing renal hypoperfusion/ischemia and initial tubular injury. Animals are then resuscitated with four times the shed blood volume of lactated Ringer’s solution, triggering reperfusion injury. Twenty-four hours later, the second hit is delivered via CLP under isoflurane anesthesia. The cecum is ligated and punctured with a 21G needle to induce polymicrobial sepsis, releasing bacteria and inflammatory mediators into the circulation. The combination of ischemia/reperfusion injury and systemic inflammation drives synergistic renal damage, culminating in acute kidney injury characterized by tubular cell damage, dysfunction, and renal failure.

VISTA Expression Is Upregulated on Circulating Neutrophils and Monocytes Following Hem/CLP

To investigate the role of VISTA in the systemic immune response, we first assessed its expression on key innate immune cells. Neutrophils and monocytes were identified in peripheral blood as CD11b⁺Ly6G⁺ and CD11b⁺F4/80⁺ populations, respectively. Flow cytometry analysis revealed a significant increase in VISTA expression on both cell types 24 hours after Hem/CLP injury. Compared to the WT-sham group, VISTA‑positive neutrophils were elevated significantly (95% CI: −50.12 to −6.418; p=0.016; Figure 2A) and mean fluorescence intensity (MFI) was markedly elevated (95% CI: −4.549 to −2.224; p=0.002; Figure 2B). VISTA‑positive monocytes (95% CI: −69.59 to −31.00; p<0.001; Figure 2C) and MFI were markedly elevated (95% CI: −15.19 to −10.00; p<0.001; Figure 2D). This result indicates that VISTA is activated during the early innate immune response triggered by hemorrhagic shock followed by polymicrobial sepsis.

Figure 2.

Vista expression is upregulated on circulating neutrophils and monocytes following Hem/CLP injury. Flow cytometry analysis was performed on peripheral blood leukocytes collected 24 hours after Hem/CLP or sham surgery. Representative histogram and quantitative analysis of VISTA expression on CD11b⁺Ly6G⁺ neutrophils, presented as both (A) the percentage of VISTA-positive cells and (B) MFI. Representative histogram and quantitative analysis of VISTA expression on CD11b⁺F4/80⁺ monocytes, presented as both (C) the percentage of VISTA-positive cells and (D) MFI. Both the proportion of VISTA-expressing cells and VISTA MFI were significantly elevated in the WT-Hem/CLP group compared to WT-sham controls for both cell populations. Each dot represents the MFI value calculated from one individual mouse, which reflects the overall VISTA expression level averaged across single cells within the gated population of that animal. Bar graphs indicate the mean ± SEM of each group. Statistical differences were determined by two-way ANOVA with Tukey’s post-hoc test. *p < 0.05, ***p < 0.001 vs WT-sham group. n = 6 mice per group.

VISTA Deficiency Exacerbates Hem/CLP-Induced Renal Dysfunction

Consistent with the establishment of AKI, Scr and BUN levels were significantly elevated in WT mice subjected to Hem/CLP compared to WT-sham controls (Scr: 95% CI: 0.2853 to 20.51, p=0.045, Figure 3A; BUN: 95% CI: 4.065 to 55.79, p=0.025, Figure 3B), confirming the successful establishment of the AKI model. Notably, this renal dysfunction was further aggravated in VISTA^−/− -Hem/CLP mice. Compared to the WT- Hem/CLP group, VISTA^−/−-Hem/CLP mice exhibited significantly higher levels of both Scr (95% CI: −23.40 to −3.181, p=0.014, Figure 3A) and BUN (95% CI: −65.85 to −14.12, p=0.005, Figure 3B). These data strongly suggest that VISTA plays a protective role in mitigating renal functional impairment induced by hemorrhage/sepsis.

Figure 3.

Vista deficiency exacerbates Hem/CLP-induced renal dysfunction. Plasma levels of (A) Scr and (B) BUN were measured 24 hours after WT-Hem/CLP or sham surgery in WT and VISTA^−/− -Hem/CLP mice. Data are presented as mean ± SEM. Statistical differences were determined by two-way ANOVA with Tukey’s post-hoc test. *p < 0.05 vs WT-sham group; ##p < 0.01, ###p < 0.001 vs WT-Hem/CLP group. n = 4–5 mice per group.

VISTA Deficiency Amplifies Histopathological Kidney Injury

Blinded histopathological assessment of kidney sections by three independent pathologists confirmed that, compared with the WT-sham group (Figure 4A), severe tubular injury in the WT-Hem/CLP group (Figure 4B) was characterized by karyopyknosis, apoptosis, edema, and tubular degeneration. Strikingly, the injury was markedly more severe in VISTA^−/− -Hem/CLP mice (Figure 4C), with pathologists noting extensive necrotic areas. Semi-quantitative injury scores were significantly higher in WT‑Hem/CLP mice compared to sham controls (95% CI: 0.6391 to 3.028, p=0.0056, Figure 4D). Furthermore, the injury score was significantly worse in VISTA^−/−-Hem/CLP mice compared to WT littermates subjected to the same insult (95% CI: −2.664 to −0.1027, p=0.0357, Figure 4D). These histological findings further confirm that VISTA deficiency worsens pathological kidney damage.

Figure 4.

Histopathological kidney injury is amplified in the absence of Vista. Representative hematoxylin and eosin (H&E) staining of kidney sections from (A) WT-sham and (B) WT-Hem/CLP groups in WT and (C) VISTA^−/−-Hem/CLP mice. Pathological features including tubular necrosis, karyopyknosis, tubular degeneration, interstitial edema and inflammatory infiltration are annotated in representative images. (D) Semi-quantitative tubular injury scores assessed by blinded pathologists. Data are presented as mean ± SEM. Statistical differences were determined by two-way ANOVA with Tukey’s post-hoc test. **p < 0.01 vs WT-sham group; ###p < 0.001 vs WT-Hem/CLP group. n = 5–6 mice per group.

Pro-Inflammatory Cytokine Cascade Is Enhanced, While Anti-Inflammatory IL-10 Is Suppressed in VISTA^−/−-Hem/CLP Mice

The renal inflammatory milieu was profoundly altered by Hem/CLP and further modulated by VISTA status. ELISA analysis of kidney homogenates showed that pro-inflammatory cytokines IL-6 (95% CI: 2.010 to 45.14, p=0.0335, Figure 5A) and TNF-α (95% CI: 2.358 to 39.87, p=0.0283, Figure 5B) were elevated, while the anti-inflammatory cytokine IL-10 was decreased (95% CI: −71.19 to −15.00, p=0.0056, Figure 5C) in WT‑Hem/CLP mice versus WT-sham controls. This inflammatory imbalance was more severe in VISTA^−/−-Hem/CLP mice. Compared to the WT-Hem/CLP group, VISTA^−/− -Hem/CLP mice exhibited higher levels of IL-6 (95% CI: −51.74 to −18.61, p=0.0014; Figure 5A) and TNF-α (95% CI: −63.18 to −25.66, p=0.0001; Figure 5B), and lower levels of IL-10 (95% CI: 21.64 to 74.04, p=0.0019; Figure 5C).

Figure 5.

Pro-inflammatory cytokine cascade is enhanced, while anti-inflammatory IL-10 is suppressed in Vista^−/−-Hem/CLP mice. Protein levels of (A) IL-6, (B) TNF-α, and (C) IL-10 in kidney homogenates were measured by ELISA 24 hours after WT-Hem/CLP or WT-sham surgery. Gene expression of inflammatory cytokines is upregulated in VISTA^−/−-Hem/CLP mice. mRNA levels of (D) IL-6, (E) TNF-α and (F) IL-10 in kidney tissues were determined by quantitative real-time PCR (qRT-PCR) and normalized to GAPDH. Data are presented as mean ± SEM. Statistical differences were determined by two-way ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p<0.001 vs WT-sham group; #p < 0.05, ###p < 0.001 vs WT- Hem/CLP group. n = 5–6 mice per group.

Further gene expression analysis (via RT-PCR) results were largely consistent with the protein-level data. Compared to the WT-sham group, mRNA expression levels of IL-6 (95% CI: 3.330 to 51.17, p=0.0274; Figure 5D) and TNF-α (95% CI: 20.14 to 90.54, p=0.0073; Figure 5E) were upregulated in the kidneys of WT‑Hem/CLP mice. In VISTA^−/−-Hem/CLP mice, this upregulation was more pronounced for IL-6 (95% CI: −88.80 to −40.97, p<0.0001, Figure 5D) and TNF-α (95% CI: −88.65 to −18.26, p=0.0087, Figure 5E). Contrary to its protein level, IL-10 mRNA was increased in WT- Hem/CLP kidneys (95% CI: 34.61 to 71.52, p=0.0001; Figure 5F) but was significantly lower in VISTA^−/−-Hem/CLP mice compared to WT (95% CI: 17.41 to 54.32, p=0.0015; Figure 5F), suggesting potential post-transcriptional regulation. These results collectively indicate that VISTA may play a key immunomodulatory role in the kidney, potentially by suppressing the pro-inflammatory response and maintaining the level of the anti-inflammatory cytokine IL-10.

VISTA Regulates the Expression of Kidney Injury Markers NGAL and HMGB1

We investigated the expression of two key proteins implicated in AKI pathogenesis: NGAL, a biomarker of tubular injury, and HMGB1, a damage-associated molecular pattern (DAMP) molecule. Western blot analysis of kidney tissue revealed that NGAL protein levels were significantly induced following Hem/CLP in WT mice (95% CI: 79.10 to 141.8, p<0.0001, Figure 6A). Intriguingly, this induction was attenuated in VISTA^−/−-Hem/CLP mice (95% CI: 34.59 to 97.29, p=0.0009, Figure 6A). Conversely, HMGB1 expression was also increased in WT‑Hem/CLP mice (95% CI: 14.61 to 52.61, p=0.0027, Figure 6B) and was further elevated in the absence of VISTA (95% CI: −58.83 to −20.83, p=0.0009, Figure 6B).

Figure 6.

Vista regulates the expression of kidney injury markers NGAL and HMGB1. Protein levels of (A) NGAL) and (B) HMGB1 in kidney tissues from WT-sham and WT-Hem/CLP groups in WT and VISTA^−/−-Hem/CLP mice were determined by Western blot analysis 24 hours after injury. Representative blot images are displayed; densitometric quantification was performed on all independent biological replicates. Representative blots and densitometric quantification are shown. Data are presented as mean ± SEM. Statistical differences were determined by two-way ANOVA with Tukey’s post-hoc test. *p < 0.05, ***p < 0.001 vs WT-sham group; #p < 0.05 vs WT-Hem/CLP group. n = 5–13 mice per group.

Discussion

Despite considerable research efforts, the precise pathogenesis of hemorrhage/sepsis-associated AKI remains incompletely understood. A dysregulated inflammatory response is widely recognized as a pivotal driver in the pathophysiology of such AKI, making the development of targeted anti-inflammatory strategies a promising therapeutic avenue.²⁷ In this study, we provide novel evidence demonstrating a protective role for the immunomodulatory molecule VISTA in a murine model of Hem/CLP-induced AKI.

During our double-hit model, initial hemorrhage induces tubular cell death via ischemia/reperfusion injury, triggering an innate immune response. The subsequent polymicrobial sepsis from CLP imposes a further immune burden, significantly amplifying renal damage. At the molecular level, necrotic cells release DAMPs, including HMGB1, that engage pattern recognition receptors on resident and infiltrating immune cells,^28,29 prompting the secretion of pro-inflammatory cytokines such as TNF-α and IL-6, which may recruit additional leukocytes to the kidney. Simultaneously, circulating neutrophils and monocytes are primed by systemic signals from the septic focus, predisposing them to an exaggerated inflammatory phenotype upon renal infiltration.³⁰ This bidirectional communication constitutes a “periphery-kidney inflammatory axis” that amplifies tubular damage, reduces glomerular filtration, and ultimately impairs renal recovery.^31,32 Our data, showing elevated pro-inflammatory cytokines alongside worsened renal function parameters (Scr, BUN) and histopathological scores in this combined model, are fully consistent with this mechanistic framework.

Using VISTA-deficient mice alongside wild-type controls, we demonstrate that VISTA expression is markedly upregulated on circulating neutrophils and monocytes in response to the combined ischemic and septic insult, and that genetic ablation of VISTA significantly aggravates renal dysfunction, histopathological injury, and intra-renal cytokine imbalance. Collectively, our findings identify VISTA as an endogenous immune checkpoint that restrains the injurious inflammatory cascade in the setting of trauma-associated AKI, and suggest that harnessing the VISTA pathway may represent a novel therapeutic strategy for this devastating condition.

A central finding of this study is the significant upregulation of VISTA on circulating CD11b⁺Ly6G⁺ neutrophils and CD11b⁺F4/80⁺ monocytes at 24 hours after Hem/CLP, as evidenced by both the percentage of VISTA-positive cells and MFI. The upregulation of VISTA on neutrophils and monocytes in this setting may function as a negative feedback signal to calibrate innate immune activation, analogous to the phenomenon of “endotoxin tolerance” in sepsis-surviving monocytes.

Of note, the magnitude of VISTA upregulation differed between neutrophils and monocytes. This is consistent with the known biology of VISTA, which is most abundantly expressed on monocytes and macrophages compared with other leukocyte subsets. While macrophages contribute to early tissue injury,³³ they are essential for later debris clearance, inflammation resolution, and repair.^34,35 Neutrophils, which are the most rapidly mobilized cells in response to both ischemia and bacterial infection, also upregulated VISTA, suggesting that this checkpoint plays a role across different phases of the innate immune response to Hem/CLP. This finding is also consistent with a parallel observation from our group showing that VISTA expression is upregulated on peripheral myeloid cells in a murine model of hemorrhage/sepsis-induced acute respiratory distress syndrome (ARDS),^24,26 suggesting that VISTA-mediated immune modulation may be a conserved response to systemic inflammatory organ dysfunction rather than a kidney-specific phenomenon.

At the functional level, plasma Scr and BUN were significantly higher in VISTA^−/− -Hem/CLP mice. Scr and BUN are the cornerstone biomarkers used to diagnose and stage AKI in both clinical and experimental settings.^36,37 At the histological level, blinded pathological assessment revealed significantly higher tubular injury scores in VISTA^−/− -Hem/CLP mice, with extensive necrotic areas, karyopyknosis, apoptosis, edema, and tubular degeneration. These features are hallmarks of ischemia/reperfusion-mediated tubular injury, in which of the proximal tubule and the medullary thick ascending limb are particularly vulnerable due to their high metabolic demand and limited oxygen supply. The exacerbation of this histological phenotype by VISTA deficiency suggests that VISTA not only mitigates functional impairment but also directly attenuates structural tissue damage, likely by restraining leukocyte-mediated cytotoxicity. This structural protection is likely mediated, at least in part, by the modulation of tubular epithelial cell survival. IL-6 promotes tubular inflammation and fibrotic transitions, while excessive reactive oxygen species (ROS) generated by activated neutrophils and macrophages overwhelm tubular antioxidant defenses.³⁸ By constraining these effector mechanisms, VISTA signaling may protect tubular cell viability, preserve cellular polarity, and facilitate epithelial regeneration. This is consistent with prior reports demonstrating that immune checkpoint molecules can limit collateral tissue damage in sterile and non-sterile inflammatory conditions.³⁹

A key mechanistic insight from this study is that VISTA deficiency profoundly disrupts the intra-renal cytokine balance toward a pro-inflammatory state. In the kidneys of VISTA^−/− mice subjected to Hem/CLP, we observed significantly elevated protein and mRNA levels of TNF-α and IL-6, alongside markedly reduced IL-10 protein levels compared to WT‑Hem/CLP mice. This “enhanced pro-inflammation / weakened anti-inflammation” imbalance appears to be a central mechanism by which VISTA deficiency amplifies AKI severity. TNF-α is primarily produced by activated macrophages and is rapidly detectable in the kidney following ischemic or septic insults. In the absence of VISTA, the unchecked amplification of both TNF-α and IL-6 likely contributes to a self-perpetuating inflammatory cycle that overwhelms the regenerative capacity of the renal epithelium. IL-10 is a major anti-inflammatory and immunoregulatory cytokine that has been shown to be renoprotective in multiple AKI models. It suppresses the production of TNF-α and IL-6 from macrophages, limits neutrophil-mediated oxidative burst, and promotes the resolution of inflammation.⁴⁰ The significant reduction in intra-renal IL-10 protein in VISTA^−/− -Hem/CLP mice, compounded by the pre-existing pro-inflammatory milieu, likely creates conditions of sustained, unresolved inflammation that perpetuates tissue injury.

Notably, we observed an intriguing discordance between IL-10 mRNA and protein levels in our data. While IL-10 mRNA was elevated in both WT and VISTA^−/− -Hem/CLP kidneys (albeit to a lesser extent in the latter), IL-10 protein was significantly decreased in VISTA^−/− -Hem/CLP mice. This mRNA-protein discordance may reflect post-transcriptional regulation, including microRNA-mediated translational repression, accelerated proteolytic degradation of IL‑10 protein within the inflamed renal microenvironment, dysregulated cytokine secretion, or receptor‑mediated internalization.⁴¹ These cytokine findings align with clinical observations linking VISTA signaling to cytokine regulation in inflammatory and autoimmune conditions,⁴² and reinforce the concept that VISTA functions as a “rheostat” for the inflammatory response rather than a simple on/off switch, calibrating the amplitude of both pro- and anti-inflammatory signals to maintain tissue homeostasis.

NGAL is both a highly sensitive early biomarker of tubular injury and a functionally active participant in AKI pathophysiology.^43,44 Consistent with its established role as an AKI biomarker, NGAL protein was markedly and significantly induced in the kidneys of WT -Hem/CLP mice compared to sham controls.

Unexpectedly, this induction was significantly attenuated in VISTA^−/− -Hem/CLP mice. This result appears paradoxical at first glance, since VISTA deficiency worsens AKI. One might expect an even higher NGAL expression in a more severely injured kidney. One plausible but highly speculative interpretation is that reduced NGAL may reflect a “failed repair” phenotype: under overwhelming inflammatory stress, severely injured tubular epithelial cells may lose the transcriptional capacity to mount an adequate injury‑repair‑associated NGAL response. This interpretation is derived solely from correlative protein observations and published literature, and requires dedicated experimental validation. Alternative explanations are also possible and cannot be ruled out with our current dataset.

In contrast to NGAL, HMGB1 protein levels were significantly elevated in VISTA^−/− -Hem/CLP mice compared to WT mice following Hem/CLP. As a prototypical pro‑inflammatory DAMP, HMGB1 can be passively released from necrotic parenchymal cells or actively secreted by activated myeloid cells. In our model, higher renal HMGB1 in VISTA‑deficient animals most likely represents a marker of aggravated tissue necrosis and amplified local inflammation, rather than a protective downstream signal of VISTA activity. Whether VISTA may also directly modulate HMGB1 production or release independent of tissue injury severity remains unproven and would require additional functional experiments.

Collectively, our data demonstrate that genetic VISTA deficiency exacerbates early‑phase AKI in the murine hemorrhage‑CLP two‑hit model, consistent with a protective role for VISTA in this trauma‑associated AKI setting. Associated changes in renal NGAL and HMGB1 protein levels are observed alongside VISTA‑dependent renal injury, yet causal mechanistic links remain unproven. Our work establishes foundational phenotypic observations and generates multiple testable mechanistic hypotheses. Further mechanistic validation across pre‑clinical models and clinical cohorts will be required to evaluate whether VISTA can be pursued as a therapeutic target for AKI.

Although our phenotypic findings raise the hypothetical possibility of VISTA agonism as an immunomodulatory strategy for trauma‑associated AKI, multiple translational challenges must be considered before therapeutic development. Systemic VISTA agonism carries a theoretical risk of broad immunosuppression, which could increase susceptibility to concurrent or secondary infection, a major concern in trauma‑sepsis patient populations. Tissue‑specific or kidney‑targeted delivery approaches might mitigate off‑target systemic immune effects, but such strategies remain pre‑clinical. Therefore, VISTA‑directed therapy remains a conceptual research direction and cannot be justified for clinical use based solely on the current murine phenotypic data.

However, this study has several important limitations that should be acknowledged. First, it is based solely on a mouse model, and its translation to clinical settings requires cautious validation. Examine the correlation between VISTA expression and disease severity in samples from clinical AKI patients could validate the clinical relevance in the future. Second, mechanistic evidence remains largely associative; further experiments including IHC, additional Western blotting, qPCR and downstream signaling analysis are required to dissect molecular pathways downstream of VISTA. Third, although Scr and BUN are the cornerstones for clinical AKI diagnosis, they have limitations as dynamic markers of glomerular filtration rate. Future studies should incorporate more sensitive early biomarkers. Fourth, we evaluated VISTA expression only on circulating blood neutrophils and monocytes, but we did not quantify renal‑infiltrating neutrophils, macrophages or intrarenal VISTA‑expressing immune cells. Fifth, our data are restricted to a 24 hours early phase time point in a murine two‑hit hemorrhage CLP model; formal a priori power calculation was not performed, and findings cannot be directly extrapolated to human AKI. Therefore, we cannot infer the role of VISTA in later‑stage renal repair or progression from AKI to chronic kidney disease; future studies with prolonged observation periods are warranted. Sixth, although we observe coordinated changes in NGAL and HMGB1 expression linked to VISTA deficiency, our current dataset only demonstrates associative relationships. Direct causal evidence would require additional functional intervention experiments such as genetic or pharmacological modulation of NGAL or HMGB1 in VISTA‑deficient animals. The present study remains largely phenotypic and descriptive, and further mechanistic studies are required to dissect downstream molecular pathways. Seventh, we did not include a VISTA^−/−- sham group. Although preliminary pilot data suggested comparable baseline renal status between WT and VISTA^−/− naive mice, we cannot fully exclude intrinsic renal effects of VISTA deficiency independent of Hem/CLP‑induced injury. Finally, the observed discrepancy between IL‑10 mRNA upregulation and reduced IL‑10 protein in VISTA‑deficient injured kidneys suggests post‑transcriptional regulatory mechanisms, yet the exact underlying mechanism remains unvalidated experimentally and requires further investigation.

Conclusion

In summary, the present study shows that genetic VISTA deficiency exacerbates early-phase acute kidney injury in a murine hemorrhage-and-sepsis two-hit model, supporting a protective phenotype for VISTA in this experimental setting. This worsening of kidney injury is accompanied by deteriorated renal function (elevated Scr and BUN) and a shift toward a more pro-inflammatory intrarenal cytokine environment. We also observe concomitant alterations in renal NGAL and HMGB1 protein abundance in VISTA-deficient injured kidneys. Our findings generate testable hypotheses regarding VISTA in trauma‑associated AKI. Whether pharmacological activation of the VISTA signaling pathway can suppress excessive renal inflammatory responses remains unvalidated and requires further targeted agonist intervention experiments.

Funding Statement

The author(s) declare that financial support was received for the research and/or publication of this article. This work was funded in part by the Research Grant from Health Commission of Pudong New Area Shanghai (PW2025A-23) and the Priority Discipline of Shanghai Pudong Hospital (Yjzdzk2025-17).

Generative AI Statement

The author(s) declare that Generative AI was used in the creation of this manuscript. Generative AI and AI-assisted technology (HelixLife, 1.0) were used solely in the writing process to improve the readability and language of the manuscript. HelixLife was also used to assist in the Figure 1. Further, the AI tool was not used to analyze and/or draw insights from data for any part of the research process. The technology was also applied with human oversight and control, with the authors’ carefully review and editing of the text results, to mitigate incorrect, in complete and/or biased presentation of data here, for which the authors take ultimate responsibility.

Abbreviation

AKI, acute kidney injury; VISTA, V-domain immunoglobulin suppressor of T-cell activation; Hem, hemorrhage; CLP, cecal ligation and puncture; WT, wild-type; Scr, serum creatinine; BUN, blood urea nitrogen; NGAL, Neutrophil gelatinase-associated lipocalin; HMGB1, high mobility group box 1; DAMP, damage-associated molecular pattern; IL-6/10, interleukin-6/10; TNF-α, tumor necrosis factor-alpha; ICAM-1, Intercellular adhesion molecule-1; SHP2, Src homology 2 domain-containing phosphatase 2; ROS, reactive oxygen species; TLRs, Toll-like receptors; RAGE, receptor for advanced glycation endproducts.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

Dr Baoji Hu reports Support for the manuscript from this work, which was funded in part by the Research Grant from the Health Commission of Pudong New Area Shanghai (PW2025A-23) and the Priority Discipline of Shanghai Pudong Hospital (Yjzdzk2025-17) during the conduct of the study. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.