Lemon-Derived Exosomes Mitigate Sepsis-associated acute kidney injury in Mice by Inhibiting Ferroptosis in Proximal Tubule Epithelial Cells

Lemon-Derived Exosomes Mitigate Sepsis-associated acute kidney injury in Mice by Inhibiting Ferroptosis in Proximal Tubule Epithelial Cells | 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 Research Article Lemon-Derived Exosomes Mitigate Sepsis-associated acute kidney injury in Mice by Inhibiting Ferroptosis in Proximal Tubule Epithelial Cells Yuexian He, Henghe Zheng, Jingtong Ou, Guanghong Wu, Xinbei Wen, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8831181/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Sepsis-associated acute kidney injury (SAKI) is a common and life-threatening complication of sepsis. The cellular heterogeneity of SAKI and the molecular mechanisms driving injury to proximal tubular epithelial cells (PTCs) remain incompletely understood, and targeted therapies are currently lacking. Methods Here, using single-cell RNA sequencing (scRNA-seq), network pharmacology, molecular docking, and experimental validation, we investigated the cellular heterogeneity and injury mechanisms of SAKI and explored targets of lemon-derived extracellular vesicles (EVs) for SAKI treatment. Results Single-cell RNA sequencing analysis of 6,353 renal cells revealed pronounced cellular heterogeneity in SAKI, mainly characterized by a marked reduction in proximal tubular epithelial cells (PTCs) and disruption of intercellular communication networks. By cross-analyzing 2,472 lemon-related potential targets with 3,406 PTC injury–related pathogenic targets, 365 common targets were identified; functional enrichment analysis indicated that lipid peroxidation and ferroptosis pathways may be involved. Molecular docking showed strong binding between lemon bioactive compounds and ferroptosis regulators (binding energies up to − 10.7 kcal·mol − 1). Based on these results, we isolated lemon-derived EVs and confirmed their physicochemical properties and biocompatibility. Administration of the EVs inhibited PTCs ferroptosis with ACSL4 acting as an effector, reduced renal edema, proteinuria, and tissue damage in septic mice, ameliorated CLP-induced hypothermia, and improved short-term survival. Conclusion This work delineates ACSL4-mediated ferroptosis in tubular cells as a key pathological driver of SAKI. By leveraging integrative omics and experimental validation, we further demonstrate that lemon extracellular vesicles serve as a natural, multi-targeted nanoplatform capable of suppressing this pathway, attenuating renal injury and enhancing survival in sepsis. These findings support EV-mediated inhibition of ferroptosis as a therapeutic rationale for SAKI and warrant further preclinical development. Sepsis-associated acute kidney injury Single-cell RNA sequencing Network pharmacology Lemon-Derived Exosomes Ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Sepsis is a life‑threatening organ dysfunction caused by a dysregulated host response to infection[ 1 ] and is the leading cause of acute kidney injury (AKI) in critically ill patients[ 2 ]. Septic acute kidney injury (SAKI) has a complex pathogenesis and limited therapeutic options, and is characterized by high incidence, high mortality, and high treatment costs[ 3 ]. It is estimated that about 60% of patients with sepsis develop secondary AKI; the mortality rate of patients with SAKI exceeds 50%, which is 7–9 times that of patients without AKI; survivors have a threefold increased risk of developing chronic kidney disease (CKD)[ 4 ]. Current understanding of the molecular regulatory mechanisms of SAKI is still limited, and clinical treatment for SAKI is restricted to routine supportive care and renal replacement therapies, which prolong hospitalization, increase the risk of adverse outcomes, and impose a substantial socioeconomic burden[ 5 , 6 ]. Therefore, elucidating the pathogenesis of SAKI and identifying potential therapeutic targets will provide a theoretical basis for new clinical treatment strategies. The pathophysiology of SAKI is complex. Current evidence indicates that metabolically active proximal tubules are the primary site of injury. Damage to proximal tubular cells activates multiple cell death pathways, including apoptosis, ferroptosis, pyroptosis and necroptosis[ 7 ]. In recent years, ferroptosis — an iron‑dependent, lipid peroxidation‑driven form of cell death — has been identified as a key mechanism of oxidative stress–induced injury to proximal tubular cells[ 8 ]. Therefore, identifying regulators of ferroptosis in PTCs may provide therapeutic targets beyond conventional supportive care to suppress lipid peroxidation and ferroptotic cell death. Plant-derived bioactive compounds are attractive candidates because of their multifaceted antioxidant and anti-inflammatory properties[ 9 ]. Various plants have been reported to exert antioxidant effects and inhibit ferroptosis, including Panax notoginseng (Sanqi), birch bark, and lemon[ 10 , 11 ]. Lemon (Citrus limon) is rich in bioactive constituents such as flavonoids, limonene and volatile oils, which have been shown to possess significant antioxidant and cytoprotective activities[ 12 , 13 ]. Notably, using lemon‑derived extracellular vesicles (EVs) as natural delivery carriers can effectively enhance the in vivo stability of these bioactive compounds while improving their targeted delivery efficiency to specific tissues, thereby supporting the functional activity of the associated components[ 14 – 16 ]. However, while integrative multi‑omics approaches have been successfully applied to elucidate therapeutic mechanisms in other diseases[ 17 ], to date no studies have systematically employed these combined methods to elucidate ferroptosis‑related injury mechanisms in SAKI, nor have they investigated in depth the specific actions and molecular targets by which lemon‑derived EVs inhibit ferroptosis in this pathological context. Based on this, the present study innovatively integrates the above multidisciplinary techniques with the aim of filling this research gap and providing new insights for mechanism elucidation and targeted therapy of SAKI. We designed and implemented an integrated analytical workflow. First, single-cell RNA sequencing (scRNA-seq) was applied to map cellular heterogeneity in SAKI and to identify molecular pathways associated with PTCs injury. Next, network pharmacology was used to integrate targets of lemon-related bioactive compounds with PTCs injury–associated targets, and molecular docking was performed to prioritize candidate regulators of ferroptosis. Finally, guided by these bioinformatic leads, EVs were isolated and characterized, and their effects on prioritized targets and ferroptosis-related pathways were evaluated in vivo. Through this multi-level strategy—spanning high-resolution data mining to experimental validation—this work aims to elucidate the cellular and molecular mechanisms of SAKI and to investigate how lemon EVs may modulate these pathways, providing a foundation for further mechanistic studies and preclinical development. (Fig. 1 ). 2. Materials and Methods 2.1 scRNA-seq and bioinformatic analysis We retrieved scRNA seq data from GEO (GSE151658)[ 18 , 19 ]. The raw data were used as provided; no additional ethical approval was required because the dataset is publicly available. Quality control and downstream processing were performed using Seurat v5.0.3. Cell cluster annotation was executed through rigorous manual curation based on canonical cell-type-specific molecular markers and supported by existing functional studies[ 20 ]. Differential expression analysis between PTCs populations derived from sepsis versus normal tissues was carried out with the FindMarkers function. Intercellular communication networks were subsequently analyzed using the CellChat platform, leveraging its comprehensive interaction database[ 21 ]. 2.2 Network pharmacological screening of targeted pathways related to lemons The network pharmacology analysis was performed as follows: active compounds of lemon were identified by compiling constituents from BATMAN-TCM and peer-reviewed literature using search terms "Lemon," "Ingredient," or "Compound"[ 22 ]. Molecular structures were retrieved from PubChem, and canonical SMILES were submitted to SwissADME to filter compounds based on gastrointestinal absorption and drug-likeness. Putative protein targets of the filtered compounds were predicted using SwissTargetPrediction, and overlapping targets were visualized with Venny (v2.1.0). A compound–target–disease network and a pathway–target network were then constructed in Cytoscape v3.10.1 to illustrate interactions among lemon compounds, predicted targets, and SAKI-related genes. Functional enrichment analysis was conducted using clusterProfiler (v3.14.3) to perform Gene Ontology (GO) enrichment (including biological processes, molecular functions, and cellular components) and KEGG pathway enrichment via a hypergeometric test (adjusted p < 0.05)[ 23 ]. Molecular docking simulations were executed using AutoDockTools (v1.5.6) and AutoDock Vina (v4.2), with interaction visualization performed in PyMOL[ 24 ]. 2.3 Preparation and characterization of lemon derived EVs EVs were isolated from fresh lemon juice by sequential centrifugation (300 × g, 10 min; 2,000 × g, 20 min; 10,000 × g, 30 min), concentration with 100 kDa MWCO filters, and ultracentrifugation at 100,000 × g for 70 min (Optima XE 90, Beckman Coulter). Pellets were washed and resuspended in sterile PBS. Protein concentration was measured by BCA assay (Pierce™ BCA Kit #23225). For fluorescent labelling, EVs (100 µg) were labeled with PKH26 (2 µM) for 10 min at room temperature; excess dye was removed by ultracentrifugation at 100,000 × g for 70 min. The particle size (diameter, nm) and zeta potential (mV) were determined using a dynamic light scattering analyzer (Zetasizer Nano ZS, Malvern).The morphology of EVs was observed by transmission electron microscopy (TEM; JEM 1400Flash, JEOL) after negative staining with 2% uranyl acetate. In vitro experiments 2.4 RNA extraction and quantitative PCR (qPCR) HK-2 cells were treated and harvested at 24 h post-stimulation. Total RNA from both disease and treatment groups was extracted using an RNA isolation kit (Omega Bio-Tek, USA) and reverse-transcribed into cDNA (Takara, Japan). RT-qPCR was performed with LightCycler 480 SYBR Green Master Mix (Takara). Ferroptosis targets (ACSL4, GCLC, TFRC, VDAC3) were normalized to GAPDH and calculated with the 2 − ΔΔCt method. The qPCR primer pairs were as follows. ACSL4 forward: 5′-TGTGGACAATAAGGCTATCA-3′; ACSL4 reverse: 5′-TGGTCTACTTGGAGGAATG-3′; GCLC forward: 5‘- GGAGGAAACCAAGCGCCAT-3’; GCLC reverse: 5’-CTTGACGGCGTGGTAGATGT-3’; TFRC forward: 5′-TGAGGGAGGAGCCAGGAGAGG-3′; TFRC reverse: 5′-CTTGATGGTGCCGGTGAAGTCTG-3′; VDAC3 forward: 5′-TCAGATGAGTTTTGACACAGCC-3′; VDAC3 reverse: 5′-GAAGTCCGCAGCCTTGTAAC-3′; GAPDH forward: 5′- AATGGGCAGCCGTTAGGAAA-3′; GAPDH reverse: 5′- GCGCCCAATACGACCAAATC-3′. 2.5 EV Uptake Assay in HK-2 Cells To investigate the cellular internalization of EVs labeled with PKH26, HK-2 cells were cultured separately with EVs for 24 h. Following incubation, cells underwent sequential fixation with 4% paraformaldehyde (15 min), permeabilization using 0.1% Triton X-100 (10 min), and blocking with 5% BSA (1 h). Nuclei were counterstained with Hoechst 33342 (Sigma, USA). Subcellular localization of PKH26-labeled EVs was analyzed using a ZEISS LSM 980 confocal laser microscope (Germany), with fluorescence signals quantified via ZEN imaging software to assess endocytic efficiency. 2.6 Cytotoxicity The cytotoxicity of lemon-derived EVs on HK-2 cells was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo). Briefly, HK-2 cells were seeded in 96-well plates at a density of 8 × 10³ cells per well and allowed to adhere overnight. Cells were then treated with a concentration gradient of EVs (0, 10, 20, 40, and 80 µg/mL) for 24 hours. Subsequently, 10 µL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of the formed formazan product was measured at 450 nm using a microplate reader (Multiskan GO, Thermo Fisher). Cell viability was expressed as a percentage relative to the control group (0 µg/mL EVs). 2.7 Live/dead staining HK-2 cells were incubated with a staining solution containing 2 µM Calcein-AM (Invitrogen) and 4 µM propidium iodide (PI, Invitrogen) in PBS for 30 minutes at 37°C in the dark. Cell viability was assessed using laser scanning confocal microscopy (ZEISS LSM 980). Viable cells (green fluorescence) and dead cells (red fluorescence) were counted in three random fields per condition. 2.8 Biosafety assessment Mouse blood was centrifuged at 500 × g for 5 min. Erythrocytes were incubated with EVs (40 µg/mL), PBS (negative control), or 1% Triton X-100 (positive control) for 1 h at 37°C. The appearance of the supernatant was recorded, and absorbance at 540 nm was measured to assess the extent of hemolysis. In vivo experiments 2.9 Animals and ethics All the experimental procedures were approved by the Institutional Animal Care and Use Committee of The Fifth Affilated Hospital Of Zunyi Medical University,Zhuhai (Approval Number: 2024KY0015). Male C57BL/6 mice (8–12 weeks, 20–25 g) were obtained from the Zhuhai BesTest Bio-Tech Co.,Ltd and housed under specific pathogen free conditions (12 h light/dark, 22–24°C, 50–60% humidity) with ad libitum access to food and water. Animals were randomized to groups and procedures were designed to minimize suffering. 2.10 CLP sepsis model and experimental groups Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were anesthetized with 3% isoflurane, a 1.5 cm midline laparotomy exposed the cecum, the distal one third was ligated with 4 0 silk and punctured twice with a 21 gauge needle; a small amount of fecal material was expressed. Sham controls underwent laparotomy without ligation/puncture. Postoperative care included subcutaneous 0.9% saline (1 mL/25 g) and buprenorphine (0.1 mg/kg). Experimental groups:(1) CLP (n = 10), (2) CLP + lemon derived EVs (40 µg/mouse, n = 10). For in vitro validation, LPS (5 mg/kg) was administered intraperitoneally as described in Results. EVs (40 µg/mouse in 100 µL PBS) were injected via tail vein on days 1, 3, and 5 post CLP. Body temperature was recorded by infrared thermography (FLIR E60) at 0, 3, 6, 12, and 24 h post CLP. Survival was monitored for 60 h. 24 h urine was collected in metabolic cages. The Kidney/body weight ratio was determined at the time of sacrifice. Urinary protein was measured by BCA assay. Serum creatinine and BUN were measured using commercial kits (ab65335, ab83362; Abcam). 2.11 Histological Studies: Excised tissue specimens were fixed in 10% neutral buffered formalin and processed for paraffin embedding. Serial sections (4 µm thickness) were prepared, deparaffinized using xylene, and rehydrated through an ethanol gradient. Tissue morphology was evaluated using hematoxylin and eosin (H&E) staining. All stained sections were imaged using brightfield microscopy. 2.12 Immunohistochemical detection Kidney tissues were embedded in paraffin after fixed in 4% formaldehyde and sliced into 4 µm sections for staining. Then, the slices were subjected to heat induced epitope retrieval and cooled with sodium citrate. After being treated with 3% H2O2 to inhibit endogenous peroxidase, the slices were incubated with 10% blocking serum for 30 min and then incubated overnight at 4°C with primary antibody: anti-ACSL4 (1:1000, ab155282), anti-GPX4 (1:1000, ab125066). The slices were treated with HRP-conjugated secondary antibody for 1 h, then, developed with diaminobenzidine and counterstained with hematoxylin. After drying, the slices were photographed with a light microscope (Lycra, Germany). 2.13 Statistical analysis Data are mean ± SD. Normality was assessed by Shapiro–Wilk test. Two group comparisons used Student’s t test; multiple groups were compared by one way ANOVA with Tukey’s post hoc test. Survival was analyzed by log rank test. P < 0.05 was considered significant. Analyses were performed in GraphPad Prism 9.0. 3. Result 3.1 Single-cell transcriptomic atlas of SAKI We analyzed the single-cell transcriptome of mouse SAKI samples to investigate cellular heterogeneity in SAKI and the important role of PTCs injury in SAKI. After quality filtering, the dataset contained 5,323 PTCs together with other renal epithelial and immune cell types. To correct for batch effects, we applied anchor-based integration, followed by normalization, centering, and principal component analysis (PCA); the first 30 principal components were used for clustering and visualized with UMAP (Fig. 2 C). Unsupervised clustering of 6,353 cells yielded 14 clusters (Fig. 2 A). Cell type annotations were assigned using canonical markers from the literature (Fig. 2 B): PTCs (Slc34a1, Slc22a6), PMN (S100a8, Lcn2), Peri/St (Rgs5), NK (Nkg7, Gzmb), Macs (Csf1r, Adgre1), LOH (Slc12a1, Umod), Endo (Eng, Pecam1), DCT (Slc12a3, Pvalb), cDC (H2-Ab1), CD-PC (Aqp2, Aqp3), CD-IC (Aqp6, Avpr1a). Cell-type proportions revealed a marked reduction of PTCs in SAKI samples (Fig. 2 E), underscoring their importance in SAKI pathophysiology. 3.2 Intercellular communication involving PTCs in SAKI Using ligand–receptor analysis of the single-cell data, we evaluated intercellular signaling centered on PTCs. Overall communication involving PTCs was reduced in SAKI compared with normal kidney (Fig. 3 A–B), indicating progressive impairment of PTCs signaling during disease. Pathway-level analysis revealed altered interaction frequency and strength in SAKI (Fig. 3 C), with several pathways—SPP1, ANGPTL, FN1, and Annexin—exhibiting increased activity. Substantial changes in specific ligand–receptor pairs between PTCs and other cell types were observed (Fig. 3 D). These results implicate PTCs damage in disrupted intercellular signaling in SAKI and highlight potential signaling targets for intervention. 3.3 Identification of lemon bioactive compounds and predicted targets Database mining (BATMAN-TCM) and SwissADME filtering yielded 57 lemon-derived bioactive compounds. The Canonical SMILES entries for these compounds were retrieved from PubChem and submitted to SwissTargetPrediction, producing 2,472 unique predicted therapeutic targets after removing duplicates. These targets were intersected with 3,406 PTCs injury–related pathogenic genes in SAKI, identifying 365 overlapping therapeutic targets (Figure. 4B). These 365 intersecting genes may represent potential targets through which lemon treats SAKI. A compound–target–disease network was constructed to visualize these relationships. (Fig. 4 C). 3.4 Functional enrichment and candidate mechanisms of lemon in SAKI GO enrichment of the 365 intersecting targets (BP, CC, MF) showed that lemon-associated targets are enriched in processes including organic anion transport, fatty acid metabolism, lipid transport, and response to oxidative stress; cellular components were enriched for apical cell compartments and focal adhesions; molecular functions included transmembrane transporter and protein kinase activities (Fig. 4 D, Fig. 5 A). These functional signatures emphasize oxidative stress and lipid metabolism as central themes. KEGG enrichment analysis, combined with literature review, revealed that the ferroptosis pathway plays a significant role in oxidative stress in SAKI. A schematic summarizing lemon-compound interactions with ferroptosis-related targets is shown in Fig. 5 B. Chord diagrams (Fig. 5 A) illustrate connections among ferroptosis, arachidonic acid metabolism, glutathione metabolism, and cysteine/methionine metabolism—pathways relevant to lipid peroxidation and ferroptosis. Network visualization (Fig. 5 B) highlights multiple genes linking lemon compounds to ferroptosis in SAKI. Notably, five upregulated ferroptosis-related targets—ACSL4, GCLC, PRNP, TFRC, and VDAC3—emerged as central nodes, suggesting that lemon may modulate SAKI pathogenesis in part via effects on ferroptosis-associated pathways. 3.5 Molecular docking Five protein crystal structures corresponding to the targets were obtained in the PDB database. After importing 90 groups of target proteins and compound molecules into AutoDock Vina, the affinity value of the best binding postures was calculated. The average value of 285 groups of affinity is -6.16 kcal/mol, and there are 10 groups with an affinity ≤ -9.00 kcal/mol. So, it is verified that the therapeutic effect of lemon of SAKI treatment from molecular docking level. Five groups with top of affinity absolute value are: ACSL4-Hesperidin complex(− 10.2), GCLC-Krukovine complex(-9.4), PRNP-Hesperetin complex(-7.2), TFRC-Hesperidin complex(-10.7) and VDAC3-Obamegine complex(-8.5).The specific combination message is shown in Fig. 6 . 3.6 EV characterization, uptake and cytotoxicity To verify the lemon extract–derived exosomes used in subsequent experiments, we performed physicochemical characterization, cellular uptake assays, and tests of their inhibitory effects on ferroptosis-related genes. To enhance the therapeutic efficacy of the lemon material, researchers isolated lemon nanoparticles from lemon homogenate by density gradient centrifugation. During this process, EVs became progressively enriched (Figure. 7A). Nanoparticle tracking analysis indicated that the EVs exhibited a primary size distribution centered at approximately 93 nm. The isolated vesicles displayed a zeta potential of − 18.74 mV; the combined zeta potential and particle size measurements confirm successful extraction of lemon EVs and reveal that the prepared vesicles possess a stable surface charge and a uniform size distribution (Figure. 7C–D). Transmission electron microscopy confirmed the characteristic exosomal morphology (cup-shaped vesicles) (Figure. 7E). Immunofluorescence of HK-2 cells treated with labeled EVs showed red punctate signals in the cytoplasm and around DAPI-counterstained nuclei (scale bar: 20 µm) (Figure. 7F), indicating that HK-2 cells efficiently uptake the lemon-derived EVs. 3.7 EV biocompatibility in vitro and in vivo In CCK-8 assays across concentrations of 0, 10, 20, 40, and 80 µg/mL, cell viability remained at an acceptable level at 40 µg/mL; therefore, 40 µg/mL was selected as the working concentration for subsequent in vitro and in vivo experiments (Fig. 8 A). Biocompatibility of EVs was assessed by live/dead staining, hemolysis assay, and histological examination of major organs. Live/dead staining of HK-2 cells treated with control or 40 µg/mL EVs revealed comparable numbers of viable cells and minimal PI-positive cells (Fig. 8 B). Quantification by counting live and dead cells per field (n = 3) yielded similar live-cell counts for both groups (Fig. 8 C). Representative hemolysis images and quantitative measurement at 540 nm showed hemolysis rates below 2% for control and 40 µg EVs groups, whereas Triton-100 (positive control) produced extensive hemolysis (Fig. 8 D–E). H&E staining of heart, liver, spleen, lung and kidney sections from mice treated with control or EVs revealed no overt histopathological abnormalities (Fig. 8 F). These data indicate favorable in vitro and in vivo biocompatibility of the EVs at the tested dose. Figure 8 G presents the relative expression of four ferroptosis-related genes (ACSL4, GCLC, TFRC, VDAC3) selected from the bioinformatics analysis. There was a significant difference in the expression of ACSL4 between the treatment group and the LPS group (compared to the LPS group, the expression in the treatment group decreased by approximately 74%). However, GCLC, TFRC, and VDAC3 did not show any significant changes. Based on concordance with the transcriptomic screening, ACSL4 was prioritized as a therapeutic target in PTCs for downstream intervention studies. 3.8 EVs mitigate CLP-induced sepsis and renal injury To further validate the therapeutic effect of EVs on SAKI, we conducted a series of in vivo experiments. A murine CLP sepsis model was established, with treatment and control groups. After treatment we recorded body temperature, assessed kidney-to-body weight ratio, 24-hour urine protein, serum creatinine (Scr), and blood urea nitrogen (BUN) as pathological indicators, and monitored mouse survival (Figure. 9A). We evaluated the efficacy of EVs in the CLP sepsis model. Infrared thermography and rectal temperature measurements were taken at 0, 3, 6, 12, and 24 hours after surgery (Figure. 9B–C). The average temperatures of CLP mice at 0, 3, 6, 12, and 24 hours were 36.57 ± 0.10°C, 33.23 ± 0.15°C, 30.47 ± 0.15°C, 27.93 ± 0.25°C, and 24.87 ± 0.85°C, respectively, showing a progressive decline. By contrast, mice treated with 40 µg EVs had average temperatures at the same time points of 36.60 ± 0.10°C, 36.10 ± 0.26°C, 36.23 ± 0.15°C, 36.33 ± 0.15°C, and 36.33 ± 0.25°C, remaining close to baseline, indicating that EVs can ameliorate the hypothermia associated with SAKI. At 24 hours after CLP, we measured renal injury-related parameters. Compared with the EV-treated group, CLP model mice showed a significantly increased kidney/body weight ratio (8.86 vs. 7.04), indicative of renal edema or enlargement. Renal function markers were also significantly elevated: 24-hour urine protein excretion roughly doubled (232.00 mg vs. 113.67 mg), serum creatinine (Scr) increased by 43.9% (178.00 µmol/L vs. 123.67 µmol/L), and blood urea nitrogen (BUN) approximately doubled (13.80 mmol/L vs. 6.70 mmol/L). These data consistently indicate that CLP surgery successfully induced acute kidney injury and that EV treatment effectively attenuated renal damage and improved the above pathophysiological indicators (Figure. 9D). The histological assessment (H&E staining) results showed that in the renal tubular area, the EV intervention significantly improved the vacuolar degeneration and necrotic shedding of renal tubular epithelial cells, and simultaneously reduced the incidence of casts formation and renal tubular dilation. These morphological improvements indicated that the administration of EVs significantly alleviated the kidney tissue damage induced by CLP (Fig. 9 E). Immunohistochemistry revealed that, compared with the control group, the lemon-derived EV group exhibited lower ACSL4 expression and higher GPX4 expression in PTCs. In the 40 µg EV group, ferroptosis-associated inflammation was markedly reduced and tissue reparative capacity was enhanced. This indicates that EVs derived from lemons can inhibit ferroptosis in PTCs. (Figure. 9F). Survival monitoring further confirmed the survival benefit of EV treatment (Figure. 9G). In the CLP model group (n = 10), survival decreased sharply within 60 hours post‑surgery, with a mortality rate of 80% (2/10 survived at 60 hours). In contrast, mice receiving 40 µg EVs (n = 10) showed a markedly slower decline in survival, with mortality reduced to 20% (8/10 survived at 60 hours). These results indicate that EV treatment significantly improves short‑term survival in septic mice. Overall, administration of 40 µg lemon-derived extracellular vesicles effectively alleviated CLP-induced progressive hypothermia and significantly improved renal function indicators (including kidney/body weight ratio, 24-hour urine protein, serum creatinine, and BUN). In addition, EV treatment increased short-term survival. Histological analyses demonstrated that EVs inhibited renal ferroptosis and associated inflammation—downregulating ACSL4 and upregulating GPX4—thereby reducing glomerular and tubular pathological damage. Collectively, these findings across systemic stability, renal function, tissue pathology, and survival measures support the potential of lemon-derived EVs as a therapeutic strategy for SAKI. 4. Discussion and Conclusion SAKI is a critical and frequently fatal complication in intensive care, characterized by complex cellular pathophysiology and a pronounced lack of mechanism-based therapies[ 25 ]. Single-cell transcriptomic analysis reveals that PTCs depletion and disruption of intercellular communication are hallmarks of SAKI. Our scRNA-seq profiling of septic kidneys identified 14 distinct cell populations and demonstrated that PTCs are the most vulnerable compartment, as evidenced by their significant numerical reduction. Beyond cellular loss, intercellular communication analysis revealed profound disruption of ligand–receptor signaling networks centered on PTCs. Key pathways involved in cell adhesion, survival, and tissue repair—such as SPP1, ANGPTL, and FN1—were substantially altered. This breakdown in communication indicates that SAKI involves not only autonomous PTCs death but also a collapse of the paracrine and juxtacrine signaling essential for renal tissue coordination and repair[ 26 , 27 ]. PTCs are prone to injury in SAKI, and the extent of PTCs injury serves as a critical determinant of clinical outcome. This vulnerability stems from the proximal tubule’s physiological traits: its high mitochondrial content and dependence on oxidative phosphorylation for ATP production render it particularly sensitive to hypoxic and septic insults[ 28 ]. Furthermore, its major role in solute reabsorption, mediated by numerous transporters, predisposes it to toxic metabolite accumulation and severe ATP depletion under stress[ 29 ]. Consequently, the proximal tubule is the primary site of injury in acute kidney injury. Importantly, PTCs dysfunction is not a passive bystander event but a pivotal driver of maladaptive signaling and fibrotic reprogramming; the severity and persistence of PTCs injury directly influence the transition from acute kidney impairment to progressive chronic kidney disease[ 30 ]. Given their high metabolic activity and susceptibility to injury, PTCs represent a logical and critical target for therapeutic intervention. In light of the central role of PTCs dysfunction in SAKI pathogenesis, we employed network pharmacology and molecular docking to identify targetable pathways. These approaches converged on ferroptosis as a key targetable pathway for lemon-derived bioactives. Intersection analysis between putative targets of lemon compounds and SAKI-associated genes highlighted pathways linked to oxidative stress and lipid metabolism, with ferroptosis among the most enriched. Ferroptosis has been established to contribute significantly to SAKI pathology, primarily through iron accumulation and excessive mitochondrial ROS production, which trigger oxidative stress and amplify renal cellular injury[ 31 ]. Among the candidate targets, ACSL4 was prioritized as a critical mediator of ferroptosis in PTCs. Experimental validation confirmed its significant upregulation in LPS-stimulated tubular cells. ACSL4 promotes ferroptosis by activating polyunsaturated fatty acids such as arachidonic acid, thereby driving phospholipid peroxidation and cell death. Knockdown of ACSL4 reduces cellular sensitivity to ferroptosis, underscoring its critical role in various forms of kidney injury, including ischemia-reperfusion injury[ 32 – 34 ]. Molecular docking revealed strong binding of lemon flavonoids, particularly hesperidin, to ACSL4 (affinity: −10.2 kcal/mol), suggesting a mechanism through which EVs could directly inhibit this enzymatic step, reduce peroxidizable lipid bioavailability, and preserve tubular integrity. Lemons are rich in antioxidant components such as vitamin C and flavonoids, which neutralize free radicals and mitigate oxidative cellular damage[ 13 ]. Additionally, lemon exhibits anti-inflammatory properties that can suppress inflammatory factor production[ 35 ]. Hesperetin, a key lemon bioactive, has been shown to downregulate oxidative stress, inflammation, apoptosis, and ferroptosis[ 36 ]. Previous studies have utilized lemon-derived exosomes as natural nanocarriers to encapsulate and protect bioactive compounds like hesperidin, enhancing their stability and targeted delivery. Unlike traditional botanical extracts, plant-derived EVs possess nanoscale dimensions and improved skin-penetrating ability, facilitating deep transport of active constituents[ 15 ]. Compared to mammalian-derived EVs, plant-derived EVs not only exhibit comparable therapeutic properties such as anti-fibrotic, anti-viral, and anti-tumor effects but also offer distinct advantages including higher yield, easier accessibility, and reduced immunogenicity[ 37 ]. Our study, through multi-omics integration and experimental validation, further elucidates that lemon-derived exosomes exert therapeutic effects in SAKI by targeting and regulating ACSL4, a key ferroptosis regulator. However, the specific bioactive compounds in lemons and the precise mechanisms of their exosomes in modulating ferroptosis, as well as antioxidant and anti-inflammatory pathways, remain incompletely understood. Future studies will further elucidate these synergistic mechanisms, thereby facilitating the targeted application of plant-derived extracellular vesicles. The therapeutic efficacy of bioactives depends on effective and biocompatible delivery[ 38 ]. Our lemon-derived EVs exhibited favorable nanoscale properties that facilitated uptake by renal tubular cells, enabling functional delivery. Comprehensive safety assessments confirmed excellent biocompatibility, with no significant cytotoxicity, hemolytic activity, or organ toxicity at therapeutic doses. In a CLP-induced sepsis model, EVs treatment significantly improved systemic stability in mice, increased short-term survival rates, reduced renal edema, proteinuria, and histopathological damage. While EVs suppressed proximal tubular cell ferroptosis by modulating ACSL4 and preserving GPX4 expression, thereby curbing lipid peroxidation and promoting tissue repair. These findings establish lemon EVs as a safe and effective delivery platform that translates computationally predicted bioactivity into targeted renal protection through ferroptosis inhibition. In summary, this integrative study employs multi-omics profiling and functional validation to establish ACSL4-driven ferroptosis in proximal tubular cells as a central pathogenic mechanism in SAKI. Furthermore, it identifies lemon-derived extracellular vesicles as a biocompatible, multi-target nanotherapeutic that effectively blocks this deleterious pathway, thereby preserving renal function and improving survival in a preclinical sepsis model. Collectively, our work provides a mechanism-based rationale for advancing plant-derived EV therapies targeting ferroptosis in SAKI and related disorders. Abbreviations ACSL4 Acyl-CoA synthetase long-chain family member 4 AKI Acute kidney injury ANGPTL Angiopoietin-like protein ANNEXIN Annexin BCA Bicinchoninic acid BP Biological process BUN Blood urea nitrogen CC Cellular component CCK-8 Cell Counting Kit-8 cDC Conventional dendritic cell CLP Cecal ligation and puncture CRRT Continuous renal replacement therapy DAPI 4′,6-Diamidino-2-phenylindole DCT Distal convoluted tubule DEG Differentially expressed gene DMEM Dulbecco’s Modified Eagle Medium EVs Extracellular vesicles FN1 Fibronectin 1 GEO Gene Expression Omnibus GO Gene Ontology GPX4 Glutathione peroxidase 4 GSH Glutathione GSSG Oxidized glutathione H&E Hematoxylin and eosin HK-2 Human kidney-2 proximal tubular epithelial cell line IHC Immunohistochemistry KEGG Kyoto Encyclopedia of Genes and Genomes LCN2 Lipocalin-2 LPS Lipopolysaccharide MDA Malondialdehyde MF Molecular function MWCO Molecular weight cut-off NK Natural killer cell PCA Principal component analysis PBS Phosphate-buffered saline PDB Protein Data Bank PTCs Proximal tubular epithelial cells PUFA Polyunsaturated fatty acid qPCR Quantitative polymerase chain reaction ROS Reactive oxygen species SAKI Sepsis-associated acute kidney injury scRNA-seq Single-cell RNA sequencing Scr Serum creatinine SPP1 Secreted phosphoprotein 1 TBARS Thiobarbituric acid reactive substances TEM Transmission electron microscopy TFRC Transferrin receptor UMAP Uniform Manifold Approximation and Projection VDAC3 Voltage-dependent anion channel 3 Declarations The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical Approval The Fifth Affilated Hospital Of Zunyi Medical University,Zhuhai (Approval Number: 2024KY0015) Consent for publication All authors unanimously consent to publication. Funding This work was supported by grants from the Science and Technology Key Program for Social Development of Zhuhai (2420004000180) to Yuexian He, the Science and Technology Planning Project of Zunyi ( HZ 2025 − 266) to Yuexian He, the Guizhou Provincial Health Commission Science and Technology Fund Project (gzwkj2026-General 483) to Yuexian He, the National Science Foundation of China (82460375 and 81960361) to Xiaoyue Li, the Science and Technology Key Program for Social Development of Zhuhai (2420004000300) to Xiaoyue Li, the Guizhou Provincial Health Commission Science and Technology Fund Project (gzwkj2025-011) to Xiaoyue Li. Author contributions YXH: Writing – original draft, Visualization, Software, Methodology, Funding acquisition, Data curation, Conceptualization. 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Nephron 146 Melanie PH, Craig RB, Ewout JH, Andrew MH (2024) Biology of the proximal tubule in body homeostasis and kidney disease. Nephrol Dial Transpl 40 Brent RS, Xuejun J, Wei G (2020) Emerging Mechanisms and Disease Relevance of Ferroptosis. Trends Cell Biol 30 Kaiyue D, Chongbin L, Li L, Ming Y, Na J, Shilu L, Lin S (2023) Acyl-CoA synthase ACSL4: an essential target in ferroptosis and fatty acid metabolism. Chin Med J (Engl) 136 Boyi G (2022) ACSL4, PUFA, and ferroptosis: new arsenal in anti-tumor immunity. Signal Transduct Target Ther 7 Yue W, Menghan Z, Ran B, Yali S, Fei Q, Yanting L, Chongxiu Y, Xinmeng C, Qixiang Z, Siliang L et al (2022) ACSL4 deficiency confers protection against ferroptosis-mediated acute kidney injury. Redox Biol 51 Hamideh P, Ali R, Fatemeh S, Ramin R, Mehrdad I (2014) Antioxidant and anti-inflammatory properties of the citrus flavonoids hesperidin and hesperetin: an updated review of their molecular mechanisms and experimental models. Phytother Res 29 Jinzhi W, Yuanyuan Y, Ting Y, Qingmiao S, Yifan Z, Lanjuan L (2024) Hesperetin Alleviated Experimental Colitis via Regulating Ferroptosis and Gut Microbiota. Nutrients 16 Nai M, Jie L, Li Z, Juan Y, Rong L, Anquan Q, Xueping L, Fang Y, Zheng Z (2023) Plant-Derived Exosome-Like Nanovesicles: Current Progress and Prospects. Int J Nanomed 18 Sheva N, Mousa J, Faramarz E, Kevin R, Ali K, P C (2012) Biocompatibility of engineered nanoparticles for drug delivery. J Control Release 166 Supplementary Files CRediTStatement.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 25 Feb, 2026 Reviewers invited by journal 19 Feb, 2026 Editor assigned by journal 16 Feb, 2026 First submitted to journal 09 Feb, 2026 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. 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13:46:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8831181/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8831181/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103306530,"identity":"7853a188-0298-47fe-aa16-9faea8ef9ee1","added_by":"auto","created_at":"2026-02-24 09:12:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2201544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall experimental procedure\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/e1513b1e6eb1715b8c5854c9.png"},{"id":103306521,"identity":"633b58d6-4094-4156-b6f2-c79c0635c6a6","added_by":"auto","created_at":"2026-02-24 09:12:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4458589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of single-cell profiles of normal samples and acute kidney injury (AKI) samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The UMAP plot shows that the single-cell samples are clustered into 14 different clusters.\u003c/p\u003e\n\u003cp\u003e(B) 11 cell types were determined based on the expression of specific marker genes.\u003c/p\u003e\n\u003cp\u003e(C) UMAP clustering plot for comparing the differences between normal tissues and AKI tissues.\u003c/p\u003e\n\u003cp\u003e(D) The expression of selected marker genes in each of the identified cell types is shown in a dot plot.\u003c/p\u003e\n\u003cp\u003e(E) The proportion distribution of different cell types in each sample.\u003c/p\u003e\n\u003cp\u003e(F) The UMAP map clearly shows the expression patterns of marker genes in the seven identified cell types.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/e41bf56466638e72292d8295.png"},{"id":103306553,"identity":"695382a9-3cbf-4d72-9a5a-438cdbfd57c3","added_by":"auto","created_at":"2026-02-24 09:12:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7306074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell-cell communication analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Compare the frequency and intensity of cell communication between AKI tissues and normal tissues.\u003c/p\u003e\n\u003cp\u003e(B) Compare the interaction network diagrams of normal tissues and AKI tissues, with thicker lines indicating more frequent interactions between cell types.\u003c/p\u003e\n\u003cp\u003e(C) Compare the differences in the frequency and intensity of cell communication related to signal pathways between AKI tissues and normal tissues.\u003c/p\u003e\n\u003cp\u003e(D) Show the key ligand-receptor pairs with significant expression changes between proximal renal tubular epithelial cells and other cell types.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/64a8006f793f1551fcaa0ca3.png"},{"id":103306531,"identity":"341282e7-0887-44fb-b9c6-fb584be2cb56","added_by":"auto","created_at":"2026-02-24 09:12:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5627936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the targets associated with lemon and SAKI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A).Volcano map executing to show the distribution and expression of renal tubular epithelial cells DEGs.\u003c/p\u003e\n\u003cp\u003e(B) Venn diagram visualizing the common targets between lemon’s targets and SAKI-lesioned renal tubular epithelial cells-related targets.\u003c/p\u003e\n\u003cp\u003e(C) The “Drug-Active Component-Intersecting Gene-Disease” network. The light blue triangle represents lemon, the blue hexagon denote target genes, and the deep green rhombus indicate active components, and the light blue arrow represents SAKI.\u003c/p\u003e\n\u003cp\u003e(D) GO enrichment analysis results, displaying the top 6 representative biological processes (BPs), molecular functions (MFs), and cellular components (CCs) with the lowest p-values (P \u0026lt; 0.05). Pathways highlighted in red are associated with oxidative stress.\u003c/p\u003e\n\u003cp\u003e(E) KEGG pathway enrichment analysis identified 10 significant pathways associated with crossover genes (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/83bea45931c600d0d7e0d6b9.png"},{"id":103306563,"identity":"ab9be2c7-f4a1-4171-9cb0-165a7b791a24","added_by":"auto","created_at":"2026-02-24 09:12:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4428579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the targets associated with Ferroptosis pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Chord diagram analysis of the 4 key pathways involved in signal transduction.\u003c/p\u003e\n\u003cp\u003e(B) Gene counts of cell death-related GO pathways.\u003c/p\u003e\n\u003cp\u003e(C) The “Drug-Pathway-Crossover Gene-Disease” network. The blue rhombus represents lemon, the deep blue oval denote target genes, the green hexagon represents Ferroptosis pathway,and the light blue arrow represents SAKI.\u003c/p\u003e\n\u003cp\u003e(D) Ferroptosis pathway, the up-regulated genes were marked in blue.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/cc64865e7fa3fa07b628653c.png"},{"id":103306568,"identity":"3ae33ea3-135f-4e91-ad85-4dd57c106f8a","added_by":"auto","created_at":"2026-02-24 09:12:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5458458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe results of molecular docking between the upregulated targets and active components of lemon.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Heat map of molecular docking results.\u003c/p\u003e\n\u003cp\u003e(B) Molecular docking results of ACSL4 and Hesperidin(affinity -10.20).\u003c/p\u003e\n\u003cp\u003e(C) Molecular docking results of GCLC and Krukovine (affinity -9.40).\u003c/p\u003e\n\u003cp\u003e(D) Molecular docking results of VDAC3 and Obamegine (affinity -8.50).\u003c/p\u003e\n\u003cp\u003e(E) Molecular docking results of PRNP and Hesperetin (affinity -7.20).\u003c/p\u003e\n\u003cp\u003e(F) Molecular docking results of TFRC and Hesperidin (affinity -10.70).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/9d0c33d4a17045a08de156a0.png"},{"id":103306559,"identity":"9e142658-cad5-455f-9f8e-94567069ca9b","added_by":"auto","created_at":"2026-02-24 09:12:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6983735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEV characterization, gene validation and HK‑2 uptake.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Flowchart for extracting EVs from lemons.\u003c/p\u003e\n\u003cp\u003e(B) Particle size distribution of EVs.\u003c/p\u003e\n\u003cp\u003e(C) Zeta potential of isolated EVs.\u003c/p\u003e\n\u003cp\u003e(D) TEM image of lemon EVs.\u003c/p\u003e\n\u003cp\u003e(E) TEM image of EVs (left) showing typical vesicular morphology; immunofluorescence images (right) show uptake of labeled EVs by HK‑2 cells (red, EVs; blue, DAPI nuclear stain). Scale bar: 20 µm.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/e2900c7abf0768c5f130514a.png"},{"id":103306607,"identity":"fccea99a-8ea0-4135-a489-db72bb85828a","added_by":"auto","created_at":"2026-02-24 09:13:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6195808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiocompatibility assessment of EVs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Relative gene expression of ferroptosis‑related genes (ACSL4, GCLC, TFRC, VDAC3) measured in Control and LPS groups (n indicated in Methods). ACSL4 shows marked upregulation in the LPS group; other genes show no notable change.\u003c/p\u003e\n\u003cp\u003e(B) Live/dead staining images of HK‑2 cells after treatment with control or 40 µg/mL EVs. First row: Control; second row: 40 µg EVs. Columns: Live (Calcein AM), Dead (PI), Merge. Scale bar: 50 µm.\u003c/p\u003e\n\u003cp\u003e(C) Quantitative analysis by counting live and dead cells per field (n = 3 fields per sample; values presented as mean ± SD).\u003c/p\u003e\n\u003cp\u003e(D) Representative images from in vitro hemolysis assays.\u003c/p\u003e\n\u003cp\u003e(E) Quantification of hemoglobin release measured at 540 nm (n = 3).\u003c/p\u003e\n\u003cp\u003e(F) Representative H\u0026amp;E staining images of major organs (heart, liver, spleen, lungs, kidneys) from mice treated with control or EVs; no overt histopathological abnormalities observed. Scale bar: 50 µm.\u003c/p\u003e\n\u003cp\u003e(G) CCK‑8 cytotoxicity assay for EVs at 0, 10, 20, 40 and 80 µg/mL; 40 µg/mL was selected for downstream experiments. Data shown as mean ± SD (n indicated in Methods).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/ff6c1e2b5e89bf797d598059.png"},{"id":103306564,"identity":"e89c69f3-ae35-4474-bbfd-8fa27b5a198d","added_by":"auto","created_at":"2026-02-24 09:12:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4325599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic effects of EVs in CLP sepsis model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Flowchart of in vitro experiment.\u003c/p\u003e\n\u003cp\u003e(B) Infrared thermal images of septic mice following indicated treatments (n indicated in Methods) at 0, 3, 6, 12 and 24 h.\u003c/p\u003e\n\u003cp\u003e(C) Rectal temperature measurements across time points (n indicated in Methods).\u003c/p\u003e\n\u003cp\u003e(D) Renal injury endpoints at 24 h post‑CLP: kidney/body weight (g/kg), 24 h urine protein (µg/24 h), serum creatinine (Scr, µmol/L) and BUN (mmol/L). Bars represent mean ± SD; significance indicated in-figure where applicable (* p \u0026lt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e(E) Representative HE staining of kidney sections showing histopathological changes across groups. Scale bar: as indicated in figure panels.\u003c/p\u003e\n\u003cp\u003e(F) Immunohistochemical detection of ACSL4 and GPX4 in different groups.\u003c/p\u003e\n\u003cp\u003e(G) Kaplan–Meier survival curves for treatment groups (n indicated in Methods).\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/d04422b7bedf7b50b794ea45.png"},{"id":103306677,"identity":"ad99da28-843c-4b45-b92c-0be1499a7f46","added_by":"auto","created_at":"2026-02-24 09:13:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45718524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/5df18a5b-e80c-4bba-b010-38e05abeb8c9.pdf"},{"id":103306565,"identity":"5229323d-90ce-4c23-b25b-42c14e5daaf7","added_by":"auto","created_at":"2026-02-24 09:12:41","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10722,"visible":true,"origin":"","legend":"","description":"","filename":"CRediTStatement.docx","url":"https://assets-eu.researchsquare.com/files/rs-8831181/v1/24ac895145cd045a5f9d4347.docx"}],"financialInterests":"","formattedTitle":"Lemon-Derived Exosomes Mitigate Sepsis-associated acute kidney injury in Mice by Inhibiting Ferroptosis in Proximal Tubule Epithelial Cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSepsis is a life‑threatening organ dysfunction caused by a dysregulated host response to infection[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and is the leading cause of acute kidney injury (AKI) in critically ill patients[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Septic acute kidney injury (SAKI) has a complex pathogenesis and limited therapeutic options, and is characterized by high incidence, high mortality, and high treatment costs[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is estimated that about 60% of patients with sepsis develop secondary AKI; the mortality rate of patients with SAKI exceeds 50%, which is 7\u0026ndash;9 times that of patients without AKI; survivors have a threefold increased risk of developing chronic kidney disease (CKD)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Current understanding of the molecular regulatory mechanisms of SAKI is still limited, and clinical treatment for SAKI is restricted to routine supportive care and renal replacement therapies, which prolong hospitalization, increase the risk of adverse outcomes, and impose a substantial socioeconomic burden[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, elucidating the pathogenesis of SAKI and identifying potential therapeutic targets will provide a theoretical basis for new clinical treatment strategies.\u003c/p\u003e \u003cp\u003eThe pathophysiology of SAKI is complex. Current evidence indicates that metabolically active proximal tubules are the primary site of injury. Damage to proximal tubular cells activates multiple cell death pathways, including apoptosis, ferroptosis, pyroptosis and necroptosis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In recent years, ferroptosis \u0026mdash; an iron‑dependent, lipid peroxidation‑driven form of cell death \u0026mdash; has been identified as a key mechanism of oxidative stress\u0026ndash;induced injury to proximal tubular cells[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, identifying regulators of ferroptosis in PTCs may provide therapeutic targets beyond conventional supportive care to suppress lipid peroxidation and ferroptotic cell death.\u003c/p\u003e \u003cp\u003ePlant-derived bioactive compounds are attractive candidates because of their multifaceted antioxidant and anti-inflammatory properties[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Various plants have been reported to exert antioxidant effects and inhibit ferroptosis, including Panax notoginseng (Sanqi), birch bark, and lemon[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Lemon (Citrus limon) is rich in bioactive constituents such as flavonoids, limonene and volatile oils, which have been shown to possess significant antioxidant and cytoprotective activities[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notably, using lemon‑derived extracellular vesicles (EVs) as natural delivery carriers can effectively enhance the in vivo stability of these bioactive compounds while improving their targeted delivery efficiency to specific tissues, thereby supporting the functional activity of the associated components[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, while integrative multi‑omics approaches have been successfully applied to elucidate therapeutic mechanisms in other diseases[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], to date no studies have systematically employed these combined methods to elucidate ferroptosis‑related injury mechanisms in SAKI, nor have they investigated in depth the specific actions and molecular targets by which lemon‑derived EVs inhibit ferroptosis in this pathological context. Based on this, the present study innovatively integrates the above multidisciplinary techniques with the aim of filling this research gap and providing new insights for mechanism elucidation and targeted therapy of SAKI.\u003c/p\u003e \u003cp\u003eWe designed and implemented an integrated analytical workflow. First, single-cell RNA sequencing (scRNA-seq) was applied to map cellular heterogeneity in SAKI and to identify molecular pathways associated with PTCs injury. Next, network pharmacology was used to integrate targets of lemon-related bioactive compounds with PTCs injury\u0026ndash;associated targets, and molecular docking was performed to prioritize candidate regulators of ferroptosis. Finally, guided by these bioinformatic leads, EVs were isolated and characterized, and their effects on prioritized targets and ferroptosis-related pathways were evaluated in vivo. Through this multi-level strategy\u0026mdash;spanning high-resolution data mining to experimental validation\u0026mdash;this work aims to elucidate the cellular and molecular mechanisms of SAKI and to investigate how lemon EVs may modulate these pathways, providing a foundation for further mechanistic studies and preclinical development. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 scRNA-seq and bioinformatic analysis\u003c/h2\u003e \u003cp\u003eWe retrieved scRNA seq data from GEO (GSE151658)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The raw data were used as provided; no additional ethical approval was required because the dataset is publicly available. Quality control and downstream processing were performed using Seurat v5.0.3. Cell cluster annotation was executed through rigorous manual curation based on canonical cell-type-specific molecular markers and supported by existing functional studies[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Differential expression analysis between PTCs populations derived from sepsis versus normal tissues was carried out with the FindMarkers function. Intercellular communication networks were subsequently analyzed using the CellChat platform, leveraging its comprehensive interaction database[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Network pharmacological screening of targeted pathways related to lemons\u003c/h2\u003e \u003cp\u003eThe network pharmacology analysis was performed as follows: active compounds of lemon were identified by compiling constituents from BATMAN-TCM and peer-reviewed literature using search terms \"Lemon,\" \"Ingredient,\" or \"Compound\"[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Molecular structures were retrieved from PubChem, and canonical SMILES were submitted to SwissADME to filter compounds based on gastrointestinal absorption and drug-likeness. Putative protein targets of the filtered compounds were predicted using SwissTargetPrediction, and overlapping targets were visualized with Venny (v2.1.0). A compound\u0026ndash;target\u0026ndash;disease network and a pathway\u0026ndash;target network were then constructed in Cytoscape v3.10.1 to illustrate interactions among lemon compounds, predicted targets, and SAKI-related genes. Functional enrichment analysis was conducted using clusterProfiler (v3.14.3) to perform Gene Ontology (GO) enrichment (including biological processes, molecular functions, and cellular components) and KEGG pathway enrichment via a hypergeometric test (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Molecular docking simulations were executed using AutoDockTools (v1.5.6) and AutoDock Vina (v4.2), with interaction visualization performed in PyMOL[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation and characterization of lemon derived EVs\u003c/h2\u003e \u003cp\u003eEVs were isolated from fresh lemon juice by sequential centrifugation (300 \u0026times; g, 10 min; 2,000 \u0026times; g, 20 min; 10,000 \u0026times; g, 30 min), concentration with 100 kDa MWCO filters, and ultracentrifugation at 100,000 \u0026times; g for 70 min (Optima XE 90, Beckman Coulter). Pellets were washed and resuspended in sterile PBS. Protein concentration was measured by BCA assay (Pierce\u0026trade; BCA Kit #23225). For fluorescent labelling, EVs (100 \u0026micro;g) were labeled with PKH26 (2 \u0026micro;M) for 10 min at room temperature; excess dye was removed by ultracentrifugation at 100,000 \u0026times; g for 70 min. The particle size (diameter, nm) and zeta potential (mV) were determined using a dynamic light scattering analyzer (Zetasizer Nano ZS, Malvern).The morphology of EVs was observed by transmission electron microscopy (TEM; JEM 1400Flash, JEOL) after negative staining with 2% uranyl acetate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro experiments\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RNA extraction and quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eHK-2 cells were treated and harvested at 24 h post-stimulation. Total RNA from both disease and treatment groups was extracted using an RNA isolation kit (Omega Bio-Tek, USA) and reverse-transcribed into cDNA (Takara, Japan). RT-qPCR was performed with LightCycler 480 SYBR Green Master Mix (Takara). Ferroptosis targets (ACSL4, GCLC, TFRC, VDAC3) were normalized to GAPDH and calculated with the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method. The qPCR primer pairs were as follows. ACSL4 forward: 5\u0026prime;-TGTGGACAATAAGGCTATCA-3\u0026prime;; ACSL4 reverse: 5\u0026prime;-TGGTCTACTTGGAGGAATG-3\u0026prime;; GCLC forward: 5\u0026lsquo;- GGAGGAAACCAAGCGCCAT-3\u0026rsquo;; GCLC reverse: 5\u0026rsquo;-CTTGACGGCGTGGTAGATGT-3\u0026rsquo;; TFRC forward: 5\u0026prime;-TGAGGGAGGAGCCAGGAGAGG-3\u0026prime;; TFRC reverse: 5\u0026prime;-CTTGATGGTGCCGGTGAAGTCTG-3\u0026prime;; VDAC3 forward: 5\u0026prime;-TCAGATGAGTTTTGACACAGCC-3\u0026prime;; VDAC3 reverse: 5\u0026prime;-GAAGTCCGCAGCCTTGTAAC-3\u0026prime;; GAPDH forward: 5\u0026prime;- AATGGGCAGCCGTTAGGAAA-3\u0026prime;; GAPDH reverse: 5\u0026prime;- GCGCCCAATACGACCAAATC-3\u0026prime;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 EV Uptake Assay in HK-2 Cells\u003c/h2\u003e \u003cp\u003eTo investigate the cellular internalization of EVs labeled with PKH26, HK-2 cells were cultured separately with EVs for 24 h. Following incubation, cells underwent sequential fixation with 4% paraformaldehyde (15 min), permeabilization using 0.1% Triton X-100 (10 min), and blocking with 5% BSA (1 h). Nuclei were counterstained with Hoechst 33342 (Sigma, USA). Subcellular localization of PKH26-labeled EVs was analyzed using a ZEISS LSM 980 confocal laser microscope (Germany), with fluorescence signals quantified via ZEN imaging software to assess endocytic efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cytotoxicity\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of lemon-derived EVs on HK-2 cells was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo). Briefly, HK-2 cells were seeded in 96-well plates at a density of 8 \u0026times; 10\u0026sup3; cells per well and allowed to adhere overnight. Cells were then treated with a concentration gradient of EVs (0, 10, 20, 40, and 80 \u0026micro;g/mL) for 24 hours. Subsequently, 10 \u0026micro;L of CCK-8 reagent was added to each well and incubated at 37\u0026deg;C for 2 hours. The absorbance of the formed formazan product was measured at 450 nm using a microplate reader (Multiskan GO, Thermo Fisher). Cell viability was expressed as a percentage relative to the control group (0 \u0026micro;g/mL EVs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Live/dead staining\u003c/h2\u003e \u003cp\u003eHK-2 cells were incubated with a staining solution containing 2 \u0026micro;M Calcein-AM (Invitrogen) and 4 \u0026micro;M propidium iodide (PI, Invitrogen) in PBS for 30 minutes at 37\u0026deg;C in the dark. Cell viability was assessed using laser scanning confocal microscopy (ZEISS LSM 980). Viable cells (green fluorescence) and dead cells (red fluorescence) were counted in three random fields per condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Biosafety assessment\u003c/h2\u003e \u003cp\u003eMouse blood was centrifuged at 500 \u0026times; g for 5 min. Erythrocytes were incubated with EVs (40 \u0026micro;g/mL), PBS (negative control), or 1% Triton X-100 (positive control) for 1 h at 37\u0026deg;C. The appearance of the supernatant was recorded, and absorbance at 540 nm was measured to assess the extent of hemolysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo experiments\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Animals and ethics\u003c/h2\u003e \u003cp\u003e All the experimental procedures were approved by the Institutional Animal Care and Use Committee of The Fifth Affilated Hospital Of Zunyi Medical University,Zhuhai (Approval Number: 2024KY0015). Male C57BL/6 mice (8\u0026ndash;12 weeks, 20\u0026ndash;25 g) were obtained from the Zhuhai BesTest Bio-Tech Co.,Ltd and housed under specific pathogen free conditions (12 h light/dark, 22\u0026ndash;24\u0026deg;C, 50\u0026ndash;60% humidity) with ad libitum access to food and water. Animals were randomized to groups and procedures were designed to minimize suffering.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 CLP sepsis model and experimental groups\u003c/h2\u003e \u003cp\u003ePolymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were anesthetized with 3% isoflurane, a 1.5 cm midline laparotomy exposed the cecum, the distal one third was ligated with 4 0 silk and punctured twice with a 21 gauge needle; a small amount of fecal material was expressed. Sham controls underwent laparotomy without ligation/puncture. Postoperative care included subcutaneous 0.9% saline (1 mL/25 g) and buprenorphine (0.1 mg/kg). Experimental groups:(1) CLP (n\u0026thinsp;=\u0026thinsp;10), (2) CLP\u0026thinsp;+\u0026thinsp;lemon derived EVs (40 \u0026micro;g/mouse, n\u0026thinsp;=\u0026thinsp;10). For in vitro validation, LPS (5 mg/kg) was administered intraperitoneally as described in Results. EVs (40 \u0026micro;g/mouse in 100 \u0026micro;L PBS) were injected via tail vein on days 1, 3, and 5 post CLP. Body temperature was recorded by infrared thermography (FLIR E60) at 0, 3, 6, 12, and 24 h post CLP. Survival was monitored for 60 h. 24 h urine was collected in metabolic cages. The Kidney/body weight ratio was determined at the time of sacrifice. Urinary protein was measured by BCA assay. Serum creatinine and BUN were measured using commercial kits (ab65335, ab83362; Abcam).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Histological Studies:\u003c/h2\u003e \u003cp\u003eExcised tissue specimens were fixed in 10% neutral buffered formalin and processed for paraffin embedding. Serial sections (4 \u0026micro;m thickness) were prepared, deparaffinized using xylene, and rehydrated through an ethanol gradient. Tissue morphology was evaluated using hematoxylin and eosin (H\u0026amp;E) staining. All stained sections were imaged using brightfield microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Immunohistochemical detection\u003c/h2\u003e \u003cp\u003eKidney tissues were embedded in paraffin after fixed in 4% formaldehyde and sliced into 4 \u0026micro;m sections for staining. Then, the slices were subjected to heat induced epitope retrieval and cooled with sodium citrate. After being treated with 3% H2O2 to inhibit endogenous peroxidase, the slices were incubated with 10% blocking serum for 30 min and then incubated overnight at 4\u0026deg;C with primary antibody: anti-ACSL4 (1:1000, ab155282), anti-GPX4 (1:1000, ab125066). The slices were treated with HRP-conjugated secondary antibody for 1 h, then, developed with diaminobenzidine and counterstained with hematoxylin. After drying, the slices were photographed with a light microscope (Lycra, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Statistical analysis\u003c/h2\u003e \u003cp\u003eData are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Normality was assessed by Shapiro\u0026ndash;Wilk test. Two group comparisons used Student\u0026rsquo;s t test; multiple groups were compared by one way ANOVA with Tukey\u0026rsquo;s post hoc test. Survival was analyzed by log rank test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. Analyses were performed in GraphPad Prism 9.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Single-cell transcriptomic atlas of SAKI\u003c/h2\u003e \u003cp\u003eWe analyzed the single-cell transcriptome of mouse SAKI samples to investigate cellular heterogeneity in SAKI and the important role of PTCs injury in SAKI. After quality filtering, the dataset contained 5,323 PTCs together with other renal epithelial and immune cell types. To correct for batch effects, we applied anchor-based integration, followed by normalization, centering, and principal component analysis (PCA); the first 30 principal components were used for clustering and visualized with UMAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Unsupervised clustering of 6,353 cells yielded 14 clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cell type annotations were assigned using canonical markers from the literature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB): PTCs (Slc34a1, Slc22a6), PMN (S100a8, Lcn2), Peri/St (Rgs5), NK (Nkg7, Gzmb), Macs (Csf1r, Adgre1), LOH (Slc12a1, Umod), Endo (Eng, Pecam1), DCT (Slc12a3, Pvalb), cDC (H2-Ab1), CD-PC (Aqp2, Aqp3), CD-IC (Aqp6, Avpr1a). Cell-type proportions revealed a marked reduction of PTCs in SAKI samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), underscoring their importance in SAKI pathophysiology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Intercellular communication involving PTCs in SAKI\u003c/h2\u003e \u003cp\u003eUsing ligand\u0026ndash;receptor analysis of the single-cell data, we evaluated intercellular signaling centered on PTCs. Overall communication involving PTCs was reduced in SAKI compared with normal kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B), indicating progressive impairment of PTCs signaling during disease. Pathway-level analysis revealed altered interaction frequency and strength in SAKI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with several pathways\u0026mdash;SPP1, ANGPTL, FN1, and Annexin\u0026mdash;exhibiting increased activity. Substantial changes in specific ligand\u0026ndash;receptor pairs between PTCs and other cell types were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results implicate PTCs damage in disrupted intercellular signaling in SAKI and highlight potential signaling targets for intervention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Identification of lemon bioactive compounds and predicted targets\u003c/h2\u003e \u003cp\u003eDatabase mining (BATMAN-TCM) and SwissADME filtering yielded 57 lemon-derived bioactive compounds. The Canonical SMILES entries for these compounds were retrieved from PubChem and submitted to SwissTargetPrediction, producing 2,472 unique predicted therapeutic targets after removing duplicates. These targets were intersected with 3,406 PTCs injury\u0026ndash;related pathogenic genes in SAKI, identifying 365 overlapping therapeutic targets (Figure. 4B). These 365 intersecting genes may represent potential targets through which lemon treats SAKI. A compound\u0026ndash;target\u0026ndash;disease network was constructed to visualize these relationships. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Functional enrichment and candidate mechanisms of lemon in SAKI\u003c/h2\u003e \u003cp\u003eGO enrichment of the 365 intersecting targets (BP, CC, MF) showed that lemon-associated targets are enriched in processes including organic anion transport, fatty acid metabolism, lipid transport, and response to oxidative stress; cellular components were enriched for apical cell compartments and focal adhesions; molecular functions included transmembrane transporter and protein kinase activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These functional signatures emphasize oxidative stress and lipid metabolism as central themes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG enrichment analysis, combined with literature review, revealed that the ferroptosis pathway plays a significant role in oxidative stress in SAKI. A schematic summarizing lemon-compound interactions with ferroptosis-related targets is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. Chord diagrams (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) illustrate connections among ferroptosis, arachidonic acid metabolism, glutathione metabolism, and cysteine/methionine metabolism\u0026mdash;pathways relevant to lipid peroxidation and ferroptosis. Network visualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) highlights multiple genes linking lemon compounds to ferroptosis in SAKI. Notably, five upregulated ferroptosis-related targets\u0026mdash;ACSL4, GCLC, PRNP, TFRC, and VDAC3\u0026mdash;emerged as central nodes, suggesting that lemon may modulate SAKI pathogenesis in part via effects on ferroptosis-associated pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Molecular docking\u003c/h2\u003e \u003cp\u003eFive protein crystal structures corresponding to the targets were obtained in the PDB database. After importing 90 groups of target proteins and compound molecules into AutoDock Vina, the affinity value of the best binding postures was calculated. The average value of 285 groups of affinity is -6.16 kcal/mol, and there are 10 groups with an affinity \u0026le; -9.00 kcal/mol. So, it is verified that the therapeutic effect of lemon of SAKI treatment from molecular docking level. Five groups with top of affinity absolute value are: ACSL4-Hesperidin complex(\u0026minus;\u0026thinsp;10.2), GCLC-Krukovine complex(-9.4), PRNP-Hesperetin complex(-7.2), TFRC-Hesperidin complex(-10.7) and VDAC3-Obamegine complex(-8.5).The specific combination message is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6 EV characterization, uptake and cytotoxicity\u003c/h2\u003e \u003cp\u003eTo verify the lemon extract\u0026ndash;derived exosomes used in subsequent experiments, we performed physicochemical characterization, cellular uptake assays, and tests of their inhibitory effects on ferroptosis-related genes. To enhance the therapeutic efficacy of the lemon material, researchers isolated lemon nanoparticles from lemon homogenate by density gradient centrifugation. During this process, EVs became progressively enriched (Figure. 7A).\u003c/p\u003e \u003cp\u003eNanoparticle tracking analysis indicated that the EVs exhibited a primary size distribution centered at approximately 93 nm. The isolated vesicles displayed a zeta potential of \u0026minus;\u0026thinsp;18.74 mV; the combined zeta potential and particle size measurements confirm successful extraction of lemon EVs and reveal that the prepared vesicles possess a stable surface charge and a uniform size distribution (Figure. 7C\u0026ndash;D). Transmission electron microscopy confirmed the characteristic exosomal morphology (cup-shaped vesicles) (Figure. 7E). Immunofluorescence of HK-2 cells treated with labeled EVs showed red punctate signals in the cytoplasm and around DAPI-counterstained nuclei (scale bar: 20 \u0026micro;m) (Figure. 7F), indicating that HK-2 cells efficiently uptake the lemon-derived EVs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7 EV biocompatibility in vitro and in vivo\u003c/h2\u003e \u003cp\u003eIn CCK-8 assays across concentrations of 0, 10, 20, 40, and 80 \u0026micro;g/mL, cell viability remained at an acceptable level at 40 \u0026micro;g/mL; therefore, 40 \u0026micro;g/mL was selected as the working concentration for subsequent in vitro and in vivo experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBiocompatibility of EVs was assessed by live/dead staining, hemolysis assay, and histological examination of major organs. Live/dead staining of HK-2 cells treated with control or 40 \u0026micro;g/mL EVs revealed comparable numbers of viable cells and minimal PI-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Quantification by counting live and dead cells per field (n\u0026thinsp;=\u0026thinsp;3) yielded similar live-cell counts for both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eRepresentative hemolysis images and quantitative measurement at 540 nm showed hemolysis rates below 2% for control and 40 \u0026micro;g EVs groups, whereas Triton-100 (positive control) produced extensive hemolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD\u0026ndash;E). H\u0026amp;E staining of heart, liver, spleen, lung and kidney sections from mice treated with control or EVs revealed no overt histopathological abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). These data indicate favorable in vitro and in vivo biocompatibility of the EVs at the tested dose.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG presents the relative expression of four ferroptosis-related genes (ACSL4, GCLC, TFRC, VDAC3) selected from the bioinformatics analysis. There was a significant difference in the expression of ACSL4 between the treatment group and the LPS group (compared to the LPS group, the expression in the treatment group decreased by approximately 74%). However, GCLC, TFRC, and VDAC3 did not show any significant changes. Based on concordance with the transcriptomic screening, ACSL4 was prioritized as a therapeutic target in PTCs for downstream intervention studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8 EVs mitigate CLP-induced sepsis and renal injury\u003c/h2\u003e \u003cp\u003eTo further validate the therapeutic effect of EVs on SAKI, we conducted a series of in vivo experiments. A murine CLP sepsis model was established, with treatment and control groups. After treatment we recorded body temperature, assessed kidney-to-body weight ratio, 24-hour urine protein, serum creatinine (Scr), and blood urea nitrogen (BUN) as pathological indicators, and monitored mouse survival (Figure. 9A).\u003c/p\u003e \u003cp\u003eWe evaluated the efficacy of EVs in the CLP sepsis model. Infrared thermography and rectal temperature measurements were taken at 0, 3, 6, 12, and 24 hours after surgery (Figure. 9B\u0026ndash;C). The average temperatures of CLP mice at 0, 3, 6, 12, and 24 hours were 36.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u0026deg;C, 33.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u0026deg;C, 30.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u0026deg;C, 27.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u0026deg;C, and 24.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u0026deg;C, respectively, showing a progressive decline. By contrast, mice treated with 40 \u0026micro;g EVs had average temperatures at the same time points of 36.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u0026deg;C, 36.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u0026deg;C, 36.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u0026deg;C, 36.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u0026deg;C, and 36.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u0026deg;C, remaining close to baseline, indicating that EVs can ameliorate the hypothermia associated with SAKI.\u003c/p\u003e \u003cp\u003eAt 24 hours after CLP, we measured renal injury-related parameters. Compared with the EV-treated group, CLP model mice showed a significantly increased kidney/body weight ratio (8.86 vs. 7.04), indicative of renal edema or enlargement. Renal function markers were also significantly elevated: 24-hour urine protein excretion roughly doubled (232.00 mg vs. 113.67 mg), serum creatinine (Scr) increased by 43.9% (178.00 \u0026micro;mol/L vs. 123.67 \u0026micro;mol/L), and blood urea nitrogen (BUN) approximately doubled (13.80 mmol/L vs. 6.70 mmol/L). These data consistently indicate that CLP surgery successfully induced acute kidney injury and that EV treatment effectively attenuated renal damage and improved the above pathophysiological indicators (Figure. 9D).\u003c/p\u003e \u003cp\u003eThe histological assessment (H\u0026amp;E staining) results showed that in the renal tubular area, the EV intervention significantly improved the vacuolar degeneration and necrotic shedding of renal tubular epithelial cells, and simultaneously reduced the incidence of casts formation and renal tubular dilation. These morphological improvements indicated that the administration of EVs significantly alleviated the kidney tissue damage induced by CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). Immunohistochemistry revealed that, compared with the control group, the lemon-derived EV group exhibited lower ACSL4 expression and higher GPX4 expression in PTCs. In the 40 \u0026micro;g EV group, ferroptosis-associated inflammation was markedly reduced and tissue reparative capacity was enhanced. This indicates that EVs derived from lemons can inhibit ferroptosis in PTCs. (Figure. 9F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSurvival monitoring further confirmed the survival benefit of EV treatment (Figure. 9G). In the CLP model group (n\u0026thinsp;=\u0026thinsp;10), survival decreased sharply within 60 hours post‑surgery, with a mortality rate of 80% (2/10 survived at 60 hours). In contrast, mice receiving 40 \u0026micro;g EVs (n\u0026thinsp;=\u0026thinsp;10) showed a markedly slower decline in survival, with mortality reduced to 20% (8/10 survived at 60 hours). These results indicate that EV treatment significantly improves short‑term survival in septic mice.\u003c/p\u003e \u003cp\u003eOverall, administration of 40 \u0026micro;g lemon-derived extracellular vesicles effectively alleviated CLP-induced progressive hypothermia and significantly improved renal function indicators (including kidney/body weight ratio, 24-hour urine protein, serum creatinine, and BUN). In addition, EV treatment increased short-term survival. Histological analyses demonstrated that EVs inhibited renal ferroptosis and associated inflammation\u0026mdash;downregulating ACSL4 and upregulating GPX4\u0026mdash;thereby reducing glomerular and tubular pathological damage. Collectively, these findings across systemic stability, renal function, tissue pathology, and survival measures support the potential of lemon-derived EVs as a therapeutic strategy for SAKI.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion and Conclusion","content":"\u003cp\u003eSAKI is a critical and frequently fatal complication in intensive care, characterized by complex cellular pathophysiology and a pronounced lack of mechanism-based therapies[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Single-cell transcriptomic analysis reveals that PTCs depletion and disruption of intercellular communication are hallmarks of SAKI. Our scRNA-seq profiling of septic kidneys identified 14 distinct cell populations and demonstrated that PTCs are the most vulnerable compartment, as evidenced by their significant numerical reduction. Beyond cellular loss, intercellular communication analysis revealed profound disruption of ligand\u0026ndash;receptor signaling networks centered on PTCs. Key pathways involved in cell adhesion, survival, and tissue repair\u0026mdash;such as SPP1, ANGPTL, and FN1\u0026mdash;were substantially altered. This breakdown in communication indicates that SAKI involves not only autonomous PTCs death but also a collapse of the paracrine and juxtacrine signaling essential for renal tissue coordination and repair[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePTCs are prone to injury in SAKI, and the extent of PTCs injury serves as a critical determinant of clinical outcome. This vulnerability stems from the proximal tubule\u0026rsquo;s physiological traits: its high mitochondrial content and dependence on oxidative phosphorylation for ATP production render it particularly sensitive to hypoxic and septic insults[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, its major role in solute reabsorption, mediated by numerous transporters, predisposes it to toxic metabolite accumulation and severe ATP depletion under stress[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Consequently, the proximal tubule is the primary site of injury in acute kidney injury. Importantly, PTCs dysfunction is not a passive bystander event but a pivotal driver of maladaptive signaling and fibrotic reprogramming; the severity and persistence of PTCs injury directly influence the transition from acute kidney impairment to progressive chronic kidney disease[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Given their high metabolic activity and susceptibility to injury, PTCs represent a logical and critical target for therapeutic intervention.\u003c/p\u003e \u003cp\u003eIn light of the central role of PTCs dysfunction in SAKI pathogenesis, we employed network pharmacology and molecular docking to identify targetable pathways. These approaches converged on ferroptosis as a key targetable pathway for lemon-derived bioactives. Intersection analysis between putative targets of lemon compounds and SAKI-associated genes highlighted pathways linked to oxidative stress and lipid metabolism, with ferroptosis among the most enriched. Ferroptosis has been established to contribute significantly to SAKI pathology, primarily through iron accumulation and excessive mitochondrial ROS production, which trigger oxidative stress and amplify renal cellular injury[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among the candidate targets, ACSL4 was prioritized as a critical mediator of ferroptosis in PTCs. Experimental validation confirmed its significant upregulation in LPS-stimulated tubular cells. ACSL4 promotes ferroptosis by activating polyunsaturated fatty acids such as arachidonic acid, thereby driving phospholipid peroxidation and cell death. Knockdown of ACSL4 reduces cellular sensitivity to ferroptosis, underscoring its critical role in various forms of kidney injury, including ischemia-reperfusion injury[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Molecular docking revealed strong binding of lemon flavonoids, particularly hesperidin, to ACSL4 (affinity: \u0026minus;10.2 kcal/mol), suggesting a mechanism through which EVs could directly inhibit this enzymatic step, reduce peroxidizable lipid bioavailability, and preserve tubular integrity.\u003c/p\u003e \u003cp\u003eLemons are rich in antioxidant components such as vitamin C and flavonoids, which neutralize free radicals and mitigate oxidative cellular damage[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, lemon exhibits anti-inflammatory properties that can suppress inflammatory factor production[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Hesperetin, a key lemon bioactive, has been shown to downregulate oxidative stress, inflammation, apoptosis, and ferroptosis[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Previous studies have utilized lemon-derived exosomes as natural nanocarriers to encapsulate and protect bioactive compounds like hesperidin, enhancing their stability and targeted delivery. Unlike traditional botanical extracts, plant-derived EVs possess nanoscale dimensions and improved skin-penetrating ability, facilitating deep transport of active constituents[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Compared to mammalian-derived EVs, plant-derived EVs not only exhibit comparable therapeutic properties such as anti-fibrotic, anti-viral, and anti-tumor effects but also offer distinct advantages including higher yield, easier accessibility, and reduced immunogenicity[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our study, through multi-omics integration and experimental validation, further elucidates that lemon-derived exosomes exert therapeutic effects in SAKI by targeting and regulating ACSL4, a key ferroptosis regulator. However, the specific bioactive compounds in lemons and the precise mechanisms of their exosomes in modulating ferroptosis, as well as antioxidant and anti-inflammatory pathways, remain incompletely understood. Future studies will further elucidate these synergistic mechanisms, thereby facilitating the targeted application of plant-derived extracellular vesicles.\u003c/p\u003e \u003cp\u003eThe therapeutic efficacy of bioactives depends on effective and biocompatible delivery[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our lemon-derived EVs exhibited favorable nanoscale properties that facilitated uptake by renal tubular cells, enabling functional delivery. Comprehensive safety assessments confirmed excellent biocompatibility, with no significant cytotoxicity, hemolytic activity, or organ toxicity at therapeutic doses. In a CLP-induced sepsis model, EVs treatment significantly improved systemic stability in mice, increased short-term survival rates, reduced renal edema, proteinuria, and histopathological damage. While EVs suppressed proximal tubular cell ferroptosis by modulating ACSL4 and preserving GPX4 expression, thereby curbing lipid peroxidation and promoting tissue repair. These findings establish lemon EVs as a safe and effective delivery platform that translates computationally predicted bioactivity into targeted renal protection through ferroptosis inhibition.\u003c/p\u003e \u003cp\u003eIn summary, this integrative study employs multi-omics profiling and functional validation to establish ACSL4-driven ferroptosis in proximal tubular cells as a central pathogenic mechanism in SAKI. Furthermore, it identifies lemon-derived extracellular vesicles as a biocompatible, multi-target nanotherapeutic that effectively blocks this deleterious pathway, thereby preserving renal function and improving survival in a preclinical sepsis model. Collectively, our work provides a mechanism-based rationale for advancing plant-derived EV therapies targeting ferroptosis in SAKI and related disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACSL4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcyl-CoA synthetase long-chain family member 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAKI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcute kidney injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANGPTL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAngiopoietin-like protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANNEXIN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnnexin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBicinchoninic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBiological process\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCellular component\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCK-8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCell Counting Kit-8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConventional dendritic cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCecal ligation and puncture\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eContinuous renal replacement therapy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4\u0026prime;,6-Diamidino-2-phenylindole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDistal convoluted tubule\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially expressed gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEVs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular vesicles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFN1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibronectin 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGEO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Expression Omnibus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPX4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione peroxidase 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSSG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxidized glutathione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHK-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman kidney-2 proximal tubular epithelial cell line\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCN2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipocalin-2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolecular function\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMWCO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolecular weight cut-off\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNatural killer cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal component analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein Data Bank\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePTCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProximal tubular epithelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePUFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyunsaturated fatty acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSAKI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSepsis-associated acute kidney injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003escRNA-seq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle-cell RNA sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eScr\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSerum creatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSecreted phosphoprotein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBARS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThiobarbituric acid reactive substances\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransferrin receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUMAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUniform Manifold Approximation and Projection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVDAC3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVoltage-dependent anion channel 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003eThe Fifth Affilated Hospital Of Zunyi Medical University,Zhuhai (Approval Number: 2024KY0015)\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors unanimously consent to publication.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the Science and Technology Key Program for Social Development of Zhuhai (2420004000180) to Yuexian He, the Science and Technology Planning Project of Zunyi ( HZ 2025\u0026thinsp;\u0026minus;\u0026thinsp;266) to Yuexian He, the Guizhou Provincial Health Commission Science and Technology Fund Project (gzwkj2026-General 483) to Yuexian He, the National Science Foundation of China (82460375 and 81960361) to Xiaoyue Li, the Science and Technology Key Program for Social Development of Zhuhai (2420004000300) to Xiaoyue Li, the Guizhou Provincial Health Commission Science and Technology Fund Project (gzwkj2025-011) to Xiaoyue Li.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eYXH: Writing \u0026ndash; original draft, Visualization, Software, Methodology, Funding acquisition, Data curation, Conceptualization.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData and materials will be available on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMervyn S, Clifford SD, Christopher Warren S, Manu S-H, Djillali A, Michael B, Rinaldo B, Gordon RB, Jean-Daniel C, Craig MC et al (2016) The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). 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J Control Release 166\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sepsis-associated acute kidney injury, Single-cell RNA sequencing, Network pharmacology, Lemon-Derived Exosomes, Ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-8831181/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8831181/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSepsis-associated acute kidney injury (SAKI) is a common and life-threatening complication of sepsis. The cellular heterogeneity of SAKI and the molecular mechanisms driving injury to proximal tubular epithelial cells (PTCs) remain incompletely understood, and targeted therapies are currently lacking.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHere, using single-cell RNA sequencing (scRNA-seq), network pharmacology, molecular docking, and experimental validation, we investigated the cellular heterogeneity and injury mechanisms of SAKI and explored targets of lemon-derived extracellular vesicles (EVs) for SAKI treatment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSingle-cell RNA sequencing analysis of 6,353 renal cells revealed pronounced cellular heterogeneity in SAKI, mainly characterized by a marked reduction in proximal tubular epithelial cells (PTCs) and disruption of intercellular communication networks. By cross-analyzing 2,472 lemon-related potential targets with 3,406 PTC injury\u0026ndash;related pathogenic targets, 365 common targets were identified; functional enrichment analysis indicated that lipid peroxidation and ferroptosis pathways may be involved. Molecular docking showed strong binding between lemon bioactive compounds and ferroptosis regulators (binding energies up to \u0026minus;\u0026thinsp;10.7 kcal\u0026middot;mol\u0026thinsp;\u0026minus;\u0026thinsp;1). Based on these results, we isolated lemon-derived EVs and confirmed their physicochemical properties and biocompatibility. Administration of the EVs inhibited PTCs ferroptosis with ACSL4 acting as an effector, reduced renal edema, proteinuria, and tissue damage in septic mice, ameliorated CLP-induced hypothermia, and improved short-term survival.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis work delineates ACSL4-mediated ferroptosis in tubular cells as a key pathological driver of SAKI. By leveraging integrative omics and experimental validation, we further demonstrate that lemon extracellular vesicles serve as a natural, multi-targeted nanoplatform capable of suppressing this pathway, attenuating renal injury and enhancing survival in sepsis. These findings support EV-mediated inhibition of ferroptosis as a therapeutic rationale for SAKI and warrant further preclinical development.\u003c/p\u003e","manuscriptTitle":"Lemon-Derived Exosomes Mitigate Sepsis-associated acute kidney injury in Mice by Inhibiting Ferroptosis in Proximal Tubule Epithelial Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 09:10:31","doi":"10.21203/rs.3.rs-8831181/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-26T04:58:13+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-20T03:28:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T07:03:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2026-02-09T08:46:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"05f2bc8c-8901-4468-95b9-d6be3071f2a7","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T03:07:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 09:10:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8831181","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8831181","identity":"rs-8831181","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}