Protective Effects of Cyclosporin H Against Sepsis-Induced Acute Kidney Injury via Modulation of Fpr1 Signaling and Inhibiting Pyroptosis

Introduction

Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection, typically resulting from pathogens such as bacteria, viruses, fungi, or parasites.^1,2 Sepsis represents a common life-threatening condition in intensive care units (ICUs), with approximately 48.9 million cases and 11 million deaths globally each year, approximately 19.7% of all global deaths, imposing a tremendous burden on healthcare systems.^3,4 Sepsis not only affects short-term patient survival but may also lead to long-term organ dysfunction, with kidney injury being one of its common complications.⁵ Sepsis-associated Acute Kidney Injury (SA-AKI) refers to the rapid deterioration of kidney function during sepsis, leading to accumulation of metabolic waste and fluid, sometimes necessitating renal replacement therapy.^6,7 The occurrence of SA-AKI significantly increases patient mortality and hospital stay; therefore, in-depth research on the pathogenesis of SA-AKI and identification of effective therapeutic targets is of substantial clinical significance.

Pyroptosis, a gasdermin-mediated lytic cell death, is recognized in tubular epithelial cells during endotoxemia.⁸ Its activation requires NLRP3/ASC inflammasome assembly, caspase-1 autoproteolysis, cleavage of gasdermin-D (GSDMD), and the subsequent release of mature IL-1β/IL-18 through membrane pores.⁹ However, the upstream danger sensor that licenses NLRP3 in renal tubular epithelial cells under septic conditions remains undefined.

FPR1 (Formyl Peptide Receptor 1) is a G protein-coupled receptor initially described on human neutrophils, primarily involved in inflammatory responses, immune regulation, and phagocytosis.^10,11 FPR1 activates downstream signaling pathways by recognizing ligands such as N-formyl peptides, promoting chemotaxis and activation of inflammatory cells.¹² In various inflammatory diseases, FPR1 expression and activity are closely associated with disease severity.^13–15 For instance, in acute lung injury (ALI) and rheumatoid arthritis (RA), FPR1 activation can exacerbate inflammatory responses, resulting in tissue damage.^13,16 Recent research shows that FPR1 activation amplifies reactive oxygen species (ROS) generation via NOX2 and promotes K⁺ efflux triggers of NLRP3.¹⁷ FPR1 may influence tissue inflammation and injury by regulating inflammatory cell activation and cytokine release.^12,13 However, the direct mechanistic link between FPR1 and tubular epithelial cells pyroptosis during SA-AKI has not been established.

This research aims to explore the mechanism of Fpr1 in SA-AKI based on transcriptomic technology. By establishing a SA-AKI mouse model, we used transcriptomic analysis to identify Fpr1 as a key target and elucidate relevant signaling pathways in SA-AKI. Furthermore, we investigated changes in tissue histopathology, inflammation, and downstream pathways following intervention with the Cyclosporin H. The findings of this study will provide new insights into the molecular mechanisms of SA-AKI and establish a theoretical foundation for developing therapeutic strategies targeting Fpr1.

Materials and Methods

Experimental Animals

Thirty-six male ICR mice, 6–8 weeks old, were purchased from SPEF (Suzhou) Biotechnology Co., Ltd. All animal experimental procedures complied with the regulations of the animal welfare ethics committee. All animals were housed in SPF-level barrier systems with clean laminar flow racks, with daily ultraviolet irradiation of the room. Cages, bedding, drinking water, and feed were all sterilized by autoclaving. The room temperature was maintained at (25±1)°C with relative humidity of 40%–60%. Mice underwent adaptive feeding for 1 week.

Sepsis-Associated Acute Kidney Injury Model and Drug Intervention

Sepsis-Associated Acute Kidney Injury Model

After the adaptive feeding period, 18 ICR mice were randomly divided into control and model groups, with 9 mice per group. Mice in the model group received intraperitoneal injection of 20 mg/kg LPS¹⁸ (Sigma, 916374), while mice in the control group received an equal volume of normal saline intraperitoneally. Samples were collected 72 h later.

Drug Intervention

Based on transcriptome results, after the adaptive feeding period, 18 ICR mice were randomly divided into control, model, and Fpr1 inhibitor groups, with 6 mice per group. Mice in the model and Fpr1 inhibitor groups received intraperitoneal injection of 20 mg/kg LPS. Mice in the Fpr1 inhibitor group were administered 5 mg/kg Cyclosporin H¹³ (MCE, HY-P1122) intraperitoneally 1 h before LPS injection. Mice in the control group received an equal volume of normal saline intraperitoneally. Samples were collected 72 h later.

Sample Collection

Mice were fasted but allowed water for 12 h before sample collection. Blood was collected from the orbital venous plexus using pro-coagulation tubes. After collection, blood samples were left at room temperature for 30–60 min to allow natural coagulation. After settling, samples were centrifuged at 1000 g for 10–15 min at 4°C, and the supernatant (serum) was collected and stored at −80°C for later use. Mice were immediately euthanized by cervical dislocation. After dissection, intact kidneys were removed and divided into three portions, which were fixed in 4% PFA, fixed in electron microscopy fixative, and stored at −80°C for later use, respectively.

HE Staining

Mouse kidney tissues fixed in 4% PFA from each group were embedded in paraffin and sectioned. Sections were dehydrated through different concentration gradients of xylene and absolute ethanol, stained with hematoxylin (Solarbio, G1140) for 3–5 min, washed with tap water, differentiated with differentiation solution, washed with tap water again, blued, and rinsed with running water. Sections were then stained with eosin (Solarbio, G1100) for 5 min, processed through graded concentrations of ethanol and xylene for clearing, mounted with neutral balsam, and subjected to image collection and analysis.

ELISA

Serum samples from each group of mice were analyzed according to the instructions for creatinine (Nanjing Jiancheng Bioengineering Institute, C011-2-1), urea nitrogen (Nanjing Jiancheng Bioengineering Institute, C013-2-1), Mouse Tumor Necrosis Factor Alpha (TNFα) ELISA Kit (JONLNBIO, JLW10484), and Mouse Interleukin 1 Beta (IL-1β) ELISA Kit (JONLNBIO, JL18442). Changes in creatinine, urea nitrogen, TNF-α, and IL-1β levels in mouse serum were measured using a microplate reader at different wavelengths.

RNA-Seq Analysis

Kidney tissues from control and model groups were collected, with three biological replicates per group. RNA was extracted and assessed for purity and integrity. Libraries were constructed using Illumina’s NEBNext^® UltraTM RNA Library Prep Kit (NEB, E3330S) and sequenced on the Illumina platform to generate 150 bp paired-end reads. The paired-end sequences were aligned to the reference genome using HISAT2. Genes with adjusted P-value <0.05 and |Log2FC| ≥2 were designated as differentially expressed genes (DEGs) using DESeq2. The DEGs were further analyzed using clusterProfiler (3.8.1) software for GO functional enrichment and KEGG pathway enrichment analyses. GO functional enrichment mainly included biological process (BP), cellular component (CC), and molecular function (MF).

RT-qPCR Analysis

Kidney tissues from each group of mice were homogenized with an appropriate amount of RNAiso Plus (Takara, 9109) according to the Trizol RNA extraction procedure, followed by chloroform extraction and isopropanol precipitation to obtain RNA. 1 μg of RNA was used to synthesize cDNA using NovoScript^®Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge) (novoprotein, E047). Real-time quantitative PCR (qPCR) was performed on the ArchimedTM platform (Kunpeng Gene (Beijing) Technology Co., Ltd.) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711) to analyze the mRNA expression of C3, B2m, Itgam, Ubd, Fpr1, and Fpr2 (primers shown in Table 1). The results of fluorescence quantitative PCR were calculated using the relative quantification method, with gene expression F = 2^−ΔΔCT.

Table 1 RT-qPCR Primer Sequence

TUNEL Staining

Paraffin sections of mouse kidney tissues from each group were deparaffinized in xylene, rehydrated through graded ethanol, and rinsed with PBS. The sections were processed according to the TUNEL Cell Apoptosis Detection Kit (Shanghai Yisheng Biotechnology Co., Ltd., 40308ES60). Briefly, sections were treated with 20 μg/mL Proteinase K and rinsed with PBS. DNase I treatment was used as a positive control. After equilibration with 1× Equilibration Buffer, sections were incubated with TdT incubation buffer at 37°C for 60 min in the dark. Sections were washed with PBS, and background was reduced using Triton X-100 and BSA solution. Sections were stained with DAPI, washed with deionized water, and moistened with PBS. Fluorescence microscopy was used to observe red fluorescence at 620 nm and blue DAPI at 460 nm. Slides could be stored overnight at 4°C in the dark.

Transmission Electron Microscopy for Pyroptosis Observation

Several 1 mm³ kidney tissue blocks were immediately fixed in 2.5% glutaraldehyde (TED PELLA INC, 18426) for 24 h. The fixative was then replaced with PBS buffer for 6 h, followed by post-fixation in 1% osmium tetroxide (TED PELLA INC, 18456) for 2 h. Tissues were dehydrated through graded ethanol, infiltrated with epoxy propane:epoxy resin (1:1) for 2 h and pure epoxy resin for 3 h. After embedding in pure epoxy resin, samples were placed in a 45°C oven for 12 h and then in a 72°C oven for 24 h. The embedded blocks were trimmed and sectioned at 70 nm thickness using a Leica UC-7 ultramicrotome. Sections were collected on copper grids, stained with lead, and photographed using a transmission electron microscope (Japan).

Western Blot Analysis

Kidney tissues from each group of mice were lysed with an appropriate amount of RIPA lysis buffer (Beyotime, P0013B) to extract total protein. 5× SDS loading buffer (Sangon Biotech, C516031) was added and samples were denatured at 100°C. Proteins were separated by SDS-PAGE and transferred to NC membranes (Merck, HATF00010). Membranes were blocked with 5% BSA (Solarbio, SW3015) for 2 h and then incubated overnight at 4°C with primary antibodies: anti-Caspase-1 (1:1000) (Proteintech, 81482-1-RR), anti-NLRP3 (1:2000) (Proteintech, 68102-1-Ig), anti-ASC (1:500) (Boster Biological Technology Co. Ltd., A00362-4), anti-FPR1 (1:500) (Abcam, ab113531), anti-IL-1β (1:1000) (CST, 12426S), and anti-GAPDH (1:5000) (Proteintech, 10494-1-AP) as internal reference. Membranes were then incubated with secondary antibodies (1:10,000) (Proteintech, SA00001-1, SA00001-2) at 37°C for 2 h, washed three times, and visualized using an ECL chemiluminescence imaging system (Savior, SCG-W2000). Image J software was used to analyze the optical density values of target proteins and internal reference.

Statistical Analysis

All data analyses were performed using GraphPad Prism 8 software. All experiments were repeated at least three times, and all experimental data were expressed as mean ± standard deviation (). All data satisfied the normal-distribution. Two-group comparisons were performed with paired Student’s t-tests; multi-group comparisons were analysed by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.

Results

Establishment of Sepsis-Associated Acute Kidney Injury Mouse Model

To verify the successful establishment of the SA-AKI mouse model, we assessed the degree of kidney injury through renal histopathology, kidney function, and inflammatory marker evaluation. HE staining results showed that the control group had intact kidney tissue structure without degeneration, swelling, or inflammatory cell infiltration, while the SA-AKI group exhibited disrupted kidney tissue structure, glomerular atrophy, vacuolar degeneration of renal tubular epithelial cells, inflammatory cell aggregation, and interstitial hemorrhage (Figure 1A). Compared with control, urea nitrogen (BUN) and creatinine levels were significantly increased in the SA-AKI group (Figures 1B and C; Table S1) (P<0.001). The levels of TNF-α and IL-1β in both kidney tissue and serum of the SA-AKI group were significantly higher than those in the control group (P<0.001). The serum level of TNF-α was approximately 3 times higher than that of the control group, and the serum level of IL-1β was approximately 5 times higher than the control group. In kidney tissue, the levels of TNF-α and IL-1β were both approximately 2 times higher than those in the control group (Figures 1D–G; Table S1).

Figure 1 Pathology, renal function, and inflammatory status of the SA-AKI mouse model. ICR mice were intraperitoneally injected with 20 mg/kg LPS and humanely euthanized after 72 h. Serum and kidney tissues were collected. (A) HE staining of kidney tissue sections (red arrows indicate normal glomeruli, green arrows indicate normal renal tubules, blue arrows indicate atrophic glomeruli, purple arrows also indicate vacuolar degeneration of glomeruli, black arrows indicate inflammatory cell infiltration, yellow arrows indicate interstitial hemorrhage), bar=100 μm; (B and C) Levels of blood urea nitrogen and creatinine in serum; (D–G) Levels of TNF-α and IL-1β in serum and kidney tissue, value see Table S1. N=9, comparisons between groups were performed using t-test, significant differences are indicated by asterisks, ***P<0.001.

Itgam and Fpr1 as a Key Target in the Sepsis-Associated Acute Kidney Injury Model

SA-AKI is a common and severe complication in sepsis patients, with complex pathogenesis. Early identification of the mechanisms and biomarkers of sepsis-induced injury can provide a basis for early diagnosis and intervention of sepsis-associated kidney injury. Therefore, this study used transcriptome sequencing to analyze the mechanisms and biomarkers in the sepsis kidney injury model. The heatmap showed clear differentially expressed genes between the SA-AKI group and the control group, with a total of 209 differentially expressed genes (Figure 2A). A volcano plot of these 209 differentially expressed genes showed that 194 genes were upregulated and 15 genes were downregulated (Figure 2B). When ranked by logFC, the top 3 upregulated genes were Ubd, Fpr1, and Fpr2; when ranked by P-value, the top 3 upregulated genes were C3, B2m, and Itgam.

Figure 2 Transcriptome sequencing analysis of pathways in the SA-AKI mouse model. Eight mice each from the control and SA-AKI groups had their kidney tissues subjected to transcriptome sequencing on the Illumina platform for differential gene expression analysis. (A) Heatmap of differentially expressed genes comparing SA-AKI and control groups. The left side represents the SA-AKI group, the right side represents the control group, red indicates upregulated expression, and blue indicates downregulated expression. (B) Volcano plot. Compared to the control group, red nodes represent upregulated DEGs, blue nodes represent downregulated DEGs, and gray nodes represent genes without significant differential expression. (C) GO functional annotation analysis. DEGs participated in 50 biological processes (BP), 50 molecular functions (MF), and 48 cellular components (CC); the figure shows only the top 8 most significant functions in each category. (D) KEGG pathway enrichment analysis. The deeper the red color in the bar chart, the smaller the p.adjust value, indicating greater relevance of that entry.

GO indicated that DEGs were involved in 50 biological processes (BP), mainly including leukocyte migration, phagocytosis, leukocyte chemotaxis, myeloid leukocyte migration, and cell chemotaxis; 50 molecular functions, primarily carbohydrate binding, immune receptor activity, MHC class I protein binding, pattern recognition receptor activity, and chemokine activity; and 48 cellular components, mainly phagocytic cup, endocytic vesicle membrane, phagocytic vesicle membrane, MHC class I peptide loading complex, and phagocytic vesicle (Figure 2C). KEGG pathway enrichment analysis revealed the main pathways involved were chemokine signaling pathway, viral protein interaction with cytokine and cytokine receptor, natural killer cell mediated cytotoxicity, phagosome, and cytokine–cytokine receptor interaction (Figure 2D).

We performed qPCR validation of the top 3 genes with the highest fold change (Ubd, Fpr1, Fpr2) and the top 3 genes with the most significant P-values (C3, B2m, Itgam). The results showed that the expression levels of all 6 genes were significantly higher in the SA-AKI group compared to the control group (P<0.01) (Figure 3A). PPI protein interaction network analysis of these 6 genes showed that, except for Ubd, the remaining 5 proteins had interaction relationships, forming 6 PPI networks. These 6 genes were visualized using Cytoscape software (Figure 3B). Hub gene identification using the cytohubba plugin confirmed that Itgam and Fpr1 as the top 2 hub genes. In conclusion, Itgam and Fpr1 serve as a key target in the SA-AKI model.

Figure 3 Validation of key genes in SA-AKI. (A) RT-qPCR analysis of mRNA expression levels of Ubd, Fpr1, Fpr2, C3, B2m, and Itgam genes in kidney tissues of control and SA-AKI group mice; (B) STRING analysis of PPI protein interaction network and screening of key genes using Cytoscape software. Data are from 6 mice, comparisons between groups were performed using t-test, significant differences are indicated by asterisks, **P<0.01, ***P<0.001.

Cyclosporin H Intervention Alleviates Sepsis-Associated Kidney Injury and Inflammation

Although ITGAM is a key inflammation-related gene, in many inflammatory diseases, FPR1 and ITGAM jointly participate in inflammatory responses.¹⁹ FPR1 is a G protein-coupled receptor primarily involved in innate immune responses, activating neutrophils by recognizing formylated peptides from bacterial and host sources.²⁰ Studies have shown that FPR1 and ITGAM are co-expressed in neutrophils and macrophages and act synergistically in various inflammatory diseases.^21,22 Therefore, using FPR1 inhibitors can simultaneously regulate the activity of multiple inflammation-related genes, achieving a more comprehensive therapeutic effect. We investigated the effects of the Fpr1 inhibitor Cyclosporin H on kidney pathology, renal function, and inflammatory factor levels in the SA-AKI mouse model to provide a theoretical basis for early treatment of sepsis. HE staining results showed that the control group had intact kidney tissue structure without degeneration, swelling, or inflammatory cell infiltration; the SA-AKI group exhibited disrupted kidney tissue structure, glomerular atrophy, vacuolar degeneration of renal tubular epithelial cells, and interstitial hemorrhage, while the Cyclosporin H intervention group showed significantly improved kidney tissue damage in SA-AKI mice (Figure 4A). Serum BUN and creatinine levels of were significantly elevated in the SA-AKI group compared with control, whereas in the Cyclosporin H intervention group they were significantly lower than in the SA-AKI group (P<0.001) (Figure 4B and C; Table S2). ELISA showed that TNF-α and IL-1β levels in kidney tissue and serum were markedly higher in SA-AKI group than controls and were reduced by Cyclosporin H treatment versus SA-AKI group (P<0.001; Figure 4D–G; Table S2). These results confirm that Cyclosporin H can improve kidney damage and reduce inflammation in the SA-AKI model.

Figure 4 Effects of Cyclosporin H intervention on kidney injury and inflammation levels in the SA-AKI mouse model. (A) Representative images of HE-stained kidney tissue (red arrows indicate normal glomeruli, green arrows indicate normal renal tubules, blue arrows indicate atrophic glomeruli, purple arrows also indicate vacuolar degeneration of glomeruli, yellow arrows indicate interstitial hemorrhage), bar=100 μm; (B and C) Levels of blood urea nitrogen and creatinine in serum; (D–G) Levels of TNF-α and IL-1β in serum and kidney tissue, value see Table S2. N=6, comparisons between groups were performed using t-test, significant differences are indicated by asterisks, **P<0.01, ***P<0.001.

Cyclosporin H Inhibits Pyroptosis in the Sepsis-Associated Kidney Injury Model

We used TUNEL staining to detect kidney damage in each group. TUNEL staining results showed that the number of TUNEL-positive renal tubular epithelial cells in the kidney tissue of SA-AKI mice was significantly higher than that in the control group, while after Cyclosporin H intervention, the number of TUNEL-positive cells was significantly reduced compared to the SA-AKI group (Figure 5A). We further observed morphological changes in kidney tissue cells through transmission electron microscopy. The results showed that the control group exhibited intact mitochondrial and cell membrane structures with well-formed cristae; the SA-AKI group displayed disrupted mitochondrial cristae structure with numerous vacuoles, localized damage to cell membranes, and slight chromatin condensation in the nucleus, which are markers of pyroptosis, while the Cyclosporin H intervention group showed less severe mitochondrial damage, intact cell membranes, reduced number of vacuoles, and decreased pyroptotic characteristics compared to the SA-AKI group (Figure 5B). We further detected pyroptosis-related markers through Western blot. The results showed that compared with the control group, the levels of ASC, Caspase-1, NLRP3, and IL-1β proteins in the kidney tissue of the SA-AKI group were significantly increased, while after Cyclosporin H intervention, the levels of ASC, Caspase-1, NLRP3, and IL-1β proteins were decreased compared to the SA-AKI group (Figure 5C and D). These results indicate that Cyclosporin H can improve kidney injury in SA-AKI by inhibiting pyroptosis.

Figure 5 Effects of Cyclosporin H intervention on cell pyroptosis in the SA-AKI mouse model. (A) Representative images of TUNEL-stained kidney tissue (green indicates apoptotic renal epithelial cells, DAPI stains cell nuclei, Merge shows the combination of TUNEL and DAPI staining), bar=100 μm; (B) Morphology of renal tubular epithelial cells observed under TEM in control, SA-AKI, and Cyclosporin H intervention groups (10000×), red arrows indicate damaged cell membranes, purple arrows indicate mitochondria with disappeared cristae, yellow arrows indicate slight chromatin condensation in the nucleus, bar=2 μm; (C) Western blot analysis of ASC, Caspase-1, NLRP3, and IL-1β protein expression changes in kidney tissues from control, SA-AKI, and Cyclosporin H intervention groups. (D) Quantification of the Caspase-1, NLRP3, and IL-1β protein, mean gray values of target protein and GAPDH were measured in ImageJ software and the ratio (Caspase-1/NLRP3/IL-1β)/GAPDH was calculated and expressed as fold change to control. N=3, comparisons between groups were performed using one-way analysis of variance (ANOVA), significant differences are indicated by asterisks, *P<0.05, **P<0.01, ***P<0.001.

Discussion

Sepsis-associated acute kidney injury (SA-AKI) is a common and severe complication in sepsis patients, with complex pathogenesis involving multiple inflammatory mediators and cytokines.^23,24 Clinically, SA-AKI has a 90-day mortality rate of 20%–70%. Furthermore, SA-AKI patients face long-term mortality risks and various complications, which not only increase the consumption of medical resources but also negatively impact patients’ long-term rehabilitation and quality of life.^25–27 Therefore, early identification and intervention are crucial for improving the prognosis of SA-AKI patients.

Lipopolysaccharide (LPS) is a key component of Gram-negative bacterial cell walls that can activate the Toll-like receptor 4 (TLR4) signaling pathway, triggering systemic inflammatory responses.²⁸ The LPS-induced sepsis model is one of the commonly used methods for studying the pathophysiological mechanisms and therapeutic strategies of sepsis.²⁹ In this study, we successfully established an SA-AKI model by intraperitoneal injection of 20 mg/kg LPS in ICR mice for 72 h. Histopathological examination results showed that in LPS-injected mice, kidney tissue structure was disrupted, with glomerular atrophy, vacuolar degeneration of renal tubular epithelial cells, inflammatory cell aggregation, and interstitial hemorrhage (Figure 1A). Additionally, we detected significantly elevated serum creatinine and blood urea nitrogen levels in LPS-injected mice, and ELISA tests revealed that TNF-α and IL-1β levels in both serum and kidney tissue were significantly higher than those in the control group (Figure 1B–G). These results confirm the successful establishment of the SA-AKI model through intraperitoneal LPS injection.

In recent years, transcriptomic sequencing analysis has become a powerful tool for studying the molecular mechanisms of various diseases and has been widely applied in sepsis research.^30,31 To further clarify the mechanisms and biomarkers of sepsis-induced kidney injury, this study analyzed potential targets and their mechanisms in the SA-AKI model through transcriptome sequencing. The results showed 209 differentially expressed genes between the AKI group and the control group, with 194 genes upregulated and 15 genes downregulated. These genes were primarily related to biological processes such as leukocyte migration, phagocytosis, and chemotaxis, and involved in pathways including chemokine signaling, viral protein interaction with cytokines, and others. This indicates that inflammatory responses play a central role in the pathogenesis of SA-AKI, supporting the view that “cytokine storm” is an important mechanism in SA-AKI.³²

Through further analysis using PPI network and Cytoscape software, we identified Fpr1 as a hub gene, which is a significant finding. Fpr1 (formyl peptide receptor 1) belongs to the G protein-coupled receptor superfamily, primarily expressed in neutrophils and macrophages, and can be activated by various inflammation-related molecules.^10–12 Previous studies have shown that Fpr1 plays a key role in multiple inflammatory diseases, such as arthritis, atherosclerosis, and cardiovascular diseases.^33–35 However, the role of Fpr1 in SA-AKI has not been fully elucidated. This study is the first to identify the central role of Fpr1 in SA-AKI through transcriptomic methods and verified its significant upregulation through qPCR. As a pattern recognition receptor, Fpr1 recognizes N-formylated peptides from bacteria and danger signal molecules from the host, activating downstream signaling pathways including MAPK and NF-κB, leading to amplified inflammatory responses.^20,36 In SA-AKI, hyperactivated Fpr1 may promote kidney injury through the following mechanisms: (1) promoting chemotaxis and infiltration of neutrophils and macrophages; (2) enhancing the production of pro-inflammatory cytokines such as TNF-α and IL-1β; (3) activating oxidative stress responses; and (4) promoting pyroptosis and other forms of programmed cell death. These effects collectively lead to expanded inflammatory responses in kidney tissue and impaired renal function.

Pyroptosis, as a form of inflammatory programmed cell death, has been a research hotspot in recent years. It is characterized by Caspase-1 activation, cell swelling, loss of cell membrane integrity, and the release of pro-inflammatory cytokines such as IL-1β and IL-18.^37–39 The NLRP3 inflammasome plays a central role in pyroptosis and consists of NLRP3, ASC, and pro-Caspase-1.⁴⁰ This study found that in the SA-AKI model, NLRP3, ASC, and Caspase-1 expressions were upregulated, and cells displayed typical pyroptotic morphological characteristics. This is consistent with previous research findings on the role of pyroptosis in kidney injury.⁴¹ Therefore, this study further investigated whether the Fpr1 inhibitor Cyclosporin H could improve SA-AKI by inhibiting pyroptosis. Notably, we found that inhibition of Fpr1 significantly reduced pyroptosis. This result suggests that Fpr1 may regulate NLRP3 inflammasome activation directly or indirectly. Possible mechanisms include the following: (1) calcium influx and reactive oxygen species production caused by Fpr1 activation may serve as upstream signals for NLRP3 activation; (2) the NF-κB signaling pathway downstream of Fpr1 may upregulate the expression of NLRP3 and pro-IL-1β; (3) the inflammatory microenvironment mediated by Fpr1 may enhance NLRP3 inflammasome assembly (Figure 6).

Figure 6 Mechanism of FPR1 in SA-AKI. FPR1 promotes the production of reactive oxygen species in SA-AKI, activates the NLRP3 inflammasome, and further activates pro-caspase-1 cleavage to produce caspase-1 which promotes apoptosis. The release of pro-inflammatory factor IL-1β further aggravates kidney injury. Red arrows indicate Red arrows indicate elevated levels of FPR1, NLRP3, ASC proteins and ROS, while black arrows denote the release of intracellular mature inflammatory factors IL-1β into the extracellular space, triggering an inflammatory response.

This study identified the key role of Fpr1 in SA-AKI through transcriptomic methods and demonstrated for the first time that Cyclosporin H improves sepsis-induced kidney injury by inhibiting pyroptosis. First, Fpr1 could serve as a potential diagnostic marker and therapeutic target for SA-AKI; second, Cyclosporin H or other Fpr1 antagonists may become novel therapeutic drugs for SA-AKI. However, this study still has some limitations. First, we used an LPS-induced SA-AKI model, which differs somewhat from clinical sepsis, the LPS-only model fails to replicate the complex hemodynamic, metabolic, and immune phenomena seen in clinical sepsis; thus further models are required to confirm this result; second, the direct molecular connection between Fpr1 and pyroptosis has not been clearly defined, and future research needs to further elucidate the molecular connection between Fpr1 and the NLRP3 inflammasome; additionally, the long-term safety and effective dosage range of Cyclosporin H require further evaluation.

In conclusion, this study reveals that the Fpr1 may serve as a key mediator in the SA-AKI. Intervention strategies targeting the Fpr1-pyroptosis axis may become effective means to improve the prognosis of SA-AKI, offering new solutions for urgent challenges.

Conclusion

This study clarifies that the Fpr1 may serve as a key mediator in the SA-AKI. Through animal models and transcriptome sequencing, we found that Fpr1 expression was upregulated in kidney tissue of the SA-AKI model, while simultaneously increasing the expression of NLRP3, ASC, Caspase-1, and IL-1β, thereby promoting pyroptosis. Treatment with the Fpr1 inhibitor Cyclosporin H significantly improved kidney injury, inflammation, and pyroptosis in the SA-AKI model, suggesting that Cyclosporin H improves the LPS-induced SA-AKI model, but the discussion of FPR1 and pyroptosis mechanisms requires further clarification. And these findings remain exploratory; definitive demonstration of clinical benefit will require rigorous validation in larger, resuscitated-animal models and subsequent Phase I safety and dose-finding studies.