Enhanced Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Renal Ischemic Injury

Enhanced Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Renal Ischemic Injury | 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 Enhanced Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Renal Ischemic Injury Fei Yuan, Jie Liu, Liang Zhong, Pengtao Liu, Ting Li, Kexin Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5266177/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Background Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) provide significant protection against renal ischemia-reperfusion injury (IRI). Hypoxia is considered an important method for enhancing the tissue repair capabilities of MSCs. However, the specific effects of hypoxia on MSCs and MSC-EVs, as well as their therapeutic potential for renal IRI, remain unclear. In this study, we investigated the alterations in MSCs and the production of MSC-EVs following hypoxia pre-treatment, and further explored the key intrinsic mechanisms by which hypoxic MSC-EVs treat renal IRI. Methods Human umbilical cord MSCs were cultured under normoxic and hypoxic conditions. Proliferation and related pathways were measured, and RNA sequencing was used to detect changes in the transcription profile. MSC-EVs from both normoxic and hypoxic conditions were isolated and characterized. In vivo , the localization and therapeutic effects of MSC-EVs were assessed in a rat renal IRI model. Histological examinations were employed to assess the structure, proliferation, and apoptosis of IRI kidney tissue respectively. Renal function was measured by analyzing serum creatinine and blood urea nitrogen levels. In vitro , the therapeutic potential of MSC-EVs were measured in renal tubular epithelial cells injured by antimycin A. Protein sequencing analysis of hypoxic MSC-EVs was conducted, and the depletion of Glutathione S-Transferase Omega 1 (GSTO1) in hypoxic MSC-EVs was performed to verify its key role in alleviating renal injury. Results Hypoxia alters MSCs transcription, promotes their proliferation, and increases the production of EVs. Hypoxia-pretreated MSC-EVs exhibited a superior ability to mitigate renal IRI, enhancing proliferation and reducing apoptosis of renal tubular epithelial cells both in vivo and in vitro . Protein profiling of the EVs revealed an accumulation of numerous anti-oxidative stress proteins, with GSTO1 being particularly prominent. GSTO1 knock down was significantly reduced the antioxidant and therapeutic effects in renal IRI of hypoxic MSC-EVs. Conclusions Hypoxia significantly promotes MSC-EVs generation and enhances the therapeutic effect of EVs on renal IRI. The effect of antioxidant stress induced by GSTO1 is one of the most important underlying mechanisms. Our findings underscore that hypoxia-pretreated MSC-EVs represent a novel and promising therapeutic intervention for renal IRI. hypoxia pretreated mesenchymal stromal cells extracellular vesicles renal ischemia reperfusion injury anti-oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Acute kidney injury (AKI), marked by a rapid decline in renal excretory function, poses a significant threat to patient well-being and survival [ 1 ]. It is estimated that one in five adults and one in three children experience AKI during hospital stays, typically due to sudden kidney failure or damage [ 2 ]. Renal ischemia-reperfusion injury (IRI) is a common cause of AKI, frequently occurring in patients undergoing sepsis or surgical procedures such as kidney transplantation [ 3 ]. Currently, therapeutic options for AKI are limited. While pharmacological interventions like angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have been employed to modulate renal function [ 4 ], they have not been effective in reversing the progression of kidney dysfunction. Therefore, more effective approaches to alleviate kidney tissue damage and promote regeneration are urgently needed. Mesenchymal stromal cells (MSCs) possess remarkable self-renewal capabilities and have demonstrated success in treating a wide range of diseases, including tissue damage repair, inflammation suppression, and immune modulation [ 5 ]. MSCs have proven effective in promoting kidney repair following AKI by mitigating tubulointerstitial damage [ 6 – 8 ]. Furthermore, extracellular vesicles (EVs) secreted by MSCs serve as crucial cellular regulators across numerous biological processes [ 9 – 11 ]. Characterized by a lipid bilayer structure, MSC-EVs facilitate intercellular communication by delivering a diverse array of cargos, including RNAs, proteins, and lipids [ 12 , 13 ]. Notably, MSC-EVs have been reported to ameliorate renal damage induced by IRI and contribute to cellular repair mechanisms [ 14 ]. Compared to their parental MSCs, EVs exhibit lower immunogenicity and enhanced biological safety, positioning them as promising candidates for therapeutic interventions in various diseases. Hypoxia pre-treatment significantly enhances protective capabilities of MSCs for injured tissues and organs. Hypoxia-pretreated MSCs have shown the capacity to alleviate spinal cord injury and improve bone repair. The production of MSC-EVs is also influenced by hypoxic conditions. However, the precise effects of hypoxic preconditioning on MSC-EV production, as well as the underlying mechanisms by which hypoxic EVs facilitate tissue repair, remain not well understood. Therefore, in this study, we aim to investigate the impact of hypoxia on human umbilical cord MSCs and validate the therapeutic efficacy of hypoxic EVs in a rat model of IRI-induced AKI. Furthermore, we seek to elucidate the potential mechanisms driving the therapeutic effects of hypoxic EVs by investigating their proteomics. Materials and methods Cell culture MSCs were prepared and identified as described in our previous study [ 15 ]. In brief, umbilical cord tissues were cut and then attached to culture plates individually. The collection and subsequent use of the umbilical cord were approved by the Institutional Ethical Review Committee of Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiao Tong University. Cells were cultured with media mixed with α-MEM medium (12571063, Gibco) containing 5% UltraGRO™-Advanced cell culture supplement (HPCFDCGL50, Helios) in an incubator with 5% CO 2 at 37°C. When the cell confluency reaches 90%, MSCs were passaged at a 1:5 ratio. The medium was changed every 2 days. The rat renal tubular epithelial cell line NRK-52E (CL-0174, Procell Life Science & Technology Co., Ltd.) was cultured in DMEM (11965092, Gibco) supplemented with 5% fetal bovine serum (A5669701, Gibco). Cells were incubated in an incubator with 5% CO 2 at 37°C. When cells reached 70% confluency, they were passaged at a 1:3 ratio. The medium was changed every 2 days. Hypoxia pre-treatment of MSCs MSCs were plated in 10 cm dishes and cultured until reached approximately 80%-90% confluence for downstream experiments. Hypoxia pre-treatment is conducted by different concentration of oxygen (a humidified atmosphere containing 5% CO 2 with 10%, 5% or 3% O 2 at 37°C) for 24 h. In the normoxic group, cells were incubated in an incubator with 5% CO 2 and 21% O 2 at 37°C. CCK-8 assay The cell proliferation of MSCs and NRK-52E were determined by CCK-8 assay (K1018, APEx BIO) following the instructions. The absorbance at 450 nm wavelength was measured by a microplate reader (Thermo Fisher). Transcriptomic analysis Total RNA was extracted using Fast Pure Cell/Tissue Total RNA Isolation Kit (RC101, Vazyme) according to the manufacturer's instructions. The primary experimental procedures for transcriptome sequencing analysis include RNA quantification and qualification, library preparation for transcriptome sequencing, clustering, and sequencing, and data analysis. HTSeq v0.6.0 was used to count the number of reads mapped to each gene. The FPKM of each gene was calculated based on the length of the gene and the read count mapped to this gene. Differential expression analysis of the two groups was performed using the DESeq2 R package (1.10.1). Adjusted p < 0.05 was considered significantly differential expression. GO enrichment analysis of differentially expressed genes (DEGs) was performed using the clusterProfiler R package. DEGs were correlated with annotations, including GO terms. Significant enrichments by differentially expressed genes were identified with corrected p-values less than 0.05. The transcriptome sequencing analysis in our research was supported by BGIgene Co, Ltd. Isolation and purification of MSC-EVs The extraction method of EVs was performed as described in the previously published literature [ 15 ].MSCs incubated in normoxia (21% O 2 ) and hypoxia (5% O 2 ) conditions, were cultured with UltraGRO™-Advanced cell culture supplement-free media for 24 h, and the supernatants were subsequently collected for MSC-EVs extraction. In brief, the supernatants centrifuged at 2000 g, 4°C, for 20 min to remove cell debris, and then centrifuged again at 100,000 g (Beckman Coulter, Fullerton, CA) for 1 h at 4°C. The concentrate was re-suspended in cold PBS for washing and centrifuged as above to collect to the EVs. Isolated EVs were stored at -80°C. Characterization of EVs The morphology of EVs was observed by transmission electron microscopy (TEM). The particle size was detected by Nanoparticle tracking analysis (NTA). Surface markers including CD9, D63 and CD81 were analyzed by Western Blot analysis. Protein concentration of EVs derived from 1 × 10 7 MSCs was quantified by the bicinchoninic acid assay kit (BCA kit, A55860, Thermo Fisher). Western blot analysis Western blot was performed as previously described [ 16 ]. The following primary antibodies were used: antibodies against CD9 (dilution 1:1000; ab236630, Abcam), CD63 (dilution 1:1000; ab134045, Abcam), CD81(dilution 1:1000; ab79559; Abcam), hypoxia-inducible factor-1α (HIF-1α) (dilution 1:1000; 14179, CST), serine/threonine kinase (AKT) (dilution 1:1000; 9272, CST), phosphorylated Serine/threonine kinase (p-AKT) (dilution 1:1000; 9271, CST), phosphorylated proline-rich AKT substrate of 40 kDa (p-PRAS40) (dilution 1:1000; ab151719, Abcam), RAB22a ( dilution 1:1000; ab137093, Abcam), Glutathione S-Transferase Omega 1 (GSTO1) (dilution 1:1000; 15124-1-AP, Proteintech), Beta Actin (β-actin) (dilution 1:1000; ab8226, Abcam). Labeling and location of MSC-EVs in vivo and in vitro To label MSC-EVs, MSCs were incubated with 10 µM PKH-26 dye (HY-D1451, MCE) in serum-free culture medium at room temperature for 15 min, followed by two washes with sterile PBS to remove excess dye. The cultures were continued for MSCs 2 days and observed using fluorescence microscopy (Stellris 8, Leica). The supernatants were used to isolate PKH-26-labeled EVs in the same procedure as above. In this way, the free dye could be removed to the utmost extent. In vivo , PKH-26 labeled EVs were administered into rats via inferior vena cava. After 48 hours, the kidneys of model rats underwent tissue fixation, dehydration, embedding and sectioning for subsequent observation of EVs’ location under a fluorescent microscope. Renal tissues showed green auto-fluorescence. In vitro , EVs labeled by PKH-26 were added to the medium of NRK-52E for 24h. After the cell crawling slides were fixed with 4% paraformaldehyde, cytoplasm and nucleus were localized with Phalloidin (A12379, Thermo Fisher) and DAPI (62248, Thermo Fisher), respectively. And the intracellular location of EVs observed under a fluorescence microscope. Rats and surgical preparation The ethics Committee of Shanghai Children's Medical Center, School of Medicine, Shanghai Jiao Tong University approved this study. 8 week male Sprague-Dawley rats (180-200g) purchased from Shanghai Jihui Experimental Animal Breeding Co. Ltd was used in this study. All the experimental rats lived in a suitable temperature and humidity environment with a normal diet. An AKI model was established as previously described [ 17 ]. Rats were prepared under 2% isoflurane anesthesia through the respiratory tract. Rats were subjected to random allocation into four groups (n = 6), comprising those undergoing sham surgery (sham), IRI treated with PBS (IRI), IRI treated with normoxic MSC-EVs (IRI/nEVs), or IRI treated with hypoxic MSC-EVs (IRI/hEVs). A mid-abdominal incision was employed for the excision of the right kidney and temporary occlusion of the left renal pedicle for a duration of 45 minutes. Sham-treated subjects underwent an identical surgical intervention, albeit devoid of occlusion of the renal pedicle. 1 ml EVs at a concentration of 100 µg/ml was administered to the respective groups via the inferior vena cava. Rats were euthanized at 48 h after injury, and kidneys and serum samples were collected for the following examinations. Rats were euthanized by excessive carbon dioxide under anesthesia conditions. Rats were housed in the same animal facility and underwent relevant procedures performed by the same surgical personnel. All animal experiments were reported in line with the ARRIVE guidelines 2.0. Histological examination and renal function assessment Renal histological examination and renal function assessment were performed as previously described [ 17 ]. Parts of the left kidney were fixed in 4% paraformaldehyde, then dehydrated in ethanol and embedded in paraffin. Kidney tissue blocks were cut into 4 µm sections and subjected to hematoxylin–eosin (H&E) staining. Subsequently, the stained sections were viewed using light microscopy. A score was given based on the grade of tubular necrosis, brush border loss, cast formation and tubular dilatation in ten randomly chosen areas. The histological scoring was determined in a blind manner based on the following criteria refer to previous literature: (0) none; (1) 0–10%; (2) 11–25%; (3) 26–45%; (4) 46–75% and (5) 76–100% [ 17 ]. Renal cell apoptosis was assessed by using TUNEL staining (C1091, Beyotime). Renal tissues and cultured cell slides underwent fixation, permeabilization with 0.1% Triton X-100 (A110694, Sangon Biotech) for 10 minutes followed by wash with PBS. TUNEL staining was performed following manufacturer's guidelines. The numbers of TUNEL-positive tubular cells and average fluorescence intensity were quantified by counting the cells in ten randomly chosen non-overlapping fields per slide. Renal cell proliferation was assessed immunohistochemistry staining of Ki67. The kidney tissue paraffin sections were permeabilized with 0.3% Triton X-100, followed by blocking with 10% donkey serum and incubation with the primary antibodies against Ki67 (dilution 1:200; ab16667, Abcam). Then, the kidney tissue sections were sequentially incubated with secondary anti-rabbit IgG horseradish peroxide (dilution 1:200; 7074S, CST). Positive staining was detected via a 3,3’-diaminobenzidine (D8001, Sigma-Aldrich) reaction. Tissue images were captured under a microscope (DMI4000 B, Leica). The blood urea nitrogen (BUN) and serum creatinine (SCr) levels were determined by the Urea Nitrogen Assay Kit (D799849, Sangon Biotech) and Creatinine Assay kit (D799853, Sangon Biotech) according to the manufacturers’ protocol, respectively. Blood samples of rats were collected after injecting EVs for 48 h. Centrifuge the blood at 2500 rpm for 10 min, collect the supernatant and store at -80°C for further analysis. Proteomic Analysis of MSC-EVs The normoxic and hypoxic EVs samples were prepared and analyzed in triplicates (n = 3). The primary experimental procedures for proteomics analysis include protein extraction, trypsin digestion, liquid chromatograph mass spectrometer (LC-MS/MS) analysis and data analysis. The resulting MS/MS data were processed using the DIA-NN search engine (Version 1.8). Tandem mass spectra were searched against the Human UniProt database (20376 entries) concatenated with a reverse decoy database. Subcellular localization analysis of the differentially expressed protein in MSC-EV was identified by PSORTbsoftware (v3.0). Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for KEGG pathway enrichment analysis. Reactome pathway annotation and WikiPathways pathway annotations were identified by Reactome database and WikiPathways database. The proteomics analysis in our research is supported by Jingjie PTM BioLabs. Assessment the levels of reactive oxygen species (ROS) in vivo and in vitro The fluorescent probe Dihydroethidium (DHE) (S0063, Beyotime) was used to detect the ROS levels of fresh-frozen kidney tissue. On the other hand, 10 mM 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA, S0033S, Beyotime) was used to assess the ROS production of NRK-52E in situ visualization. Nuclei were stained with DAPI before captured the image under microscopy. Fluorescence intensity of staining was measured by Image J (Version 1.54h 15, Wisconsin, USA). Six representative visual fields of each group were counted. Assessment the levels of mitochondrial membrane potential (Δψm) NRK-52E were plated in confocal petri dishes. Cells were treated with drugs and then loaded with the potentiometric dye 500 nM TMRE (C2001S, Beyotime) at 37°C in cell culture chamber for 20 min and Hoechst 33342(C1027, Beyotime) for 5 min. The staining was viewed by a confocal scanning microscope after washing 3 times with PBS. Assessment of GSH and GSSG levels The concentration of GSH and GSSG of both NRK-52E and fresh rat kidney tissue were quantified by the GSH content detection kit (colorimetric method) (D799613, Sangon Biotech) and the GSSG content detection kit (colorimetric method) (D799615, Sangon Biotech). For tissue, weigh 0.1g of fresh kidney tissue, and then add 1mL of Reagent One. Use a homogenizer gently grind tissue samples on ice. Centrifuge samples at 8000g and 4°C for 10 minutes and save the supernatant for testing. Then, proceed with the tests as the manufacturer's instructions. The absorbance at 412-nm wavelength was measured by a microplate reader (Aligent). Cell transfection of siRNA MSCs were seeded in a 10-cm dish at 70% confluence one day prior to transfection. Nucleotides formed transfection complexes with Lipofectamine 2000 (11668027, Thermo Fisher), and were added to cells and incubated for 6–8 h prior to refreshing the medium. Small interfering RNAs (siRNAs) were synthesized by Sangon Biotech Co., Ltd. based on the following sequences. siGSTO1-214: sense 5'-GCCUGAGUGGUUCUUUAAGAATT-3', antisense 5'-UUCUUAAAGAACCACUCAGGCTT-3'. hGSTO1-411: sense 5'-CCUUGGUAGGAAGCUUUAUUATT-3', antisense 5'-UAAUAAAGCUUCCUACCAAGGTT-3'. hGSTO1-411: sense 5'-GUUAAAUGAGUGUGUAGACCATT-3', antisense 5'-UGGUCUACACACUUUAACTT-3'. FAM labeled siRNA was used to assess the efficiency of transfection. Cells which expressed green fluorescence stably were considered to successful transfection. Quantitative real-time PCR (qPCR) Total RNA was initially extracted from both treated and control cell samples using a TRIzol™ Reagent (15596026, Thermal Fisher), following the manufacturer's protocol. Subsequently, RNA samples were reverse transcribed into complementary DNA (cDNA) using a HiScript IV RT SuperMix for qPCR (+ gDNA wiper) kit (R423-01, Vazyme). Specific primers designed against the sequences of interest and a reference gene were utilized for qPCR amplification. The qPCR reactions were carried out in triplicate using a ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme) to monitor DNA synthesis in real time. Fluorescence data collected during the annealing phase were used to calculate the threshold cycle values for each target gene. Expression levels were normalized to β-actin. Here is the sequence of primers: β-actin sense 5’-CACCATTGGCAATGAGCGGTTC-3’ and antisense 5’-AGGTCTTTGCGGATGTCCACGT-3’; GSTO1 sense 5’-GAAGACGACCTTCTTTGGTGGC-3' and antisense 5’-CTTCATGGCTGCCATCCACAGT-3’. Statistical analysis Statistical analysis was conducted using GraphPad Prism 10.0. We used the Shapiro-Wilk test to assess the normality of the data distribution. Data that were normally distributed are presented as mean ± standard deviation (SD). For comparing two groups, an unpaired t-test was used to determine statistical significance. For multiple group comparisons, one-way analysis of variance (ANOVA) was performed. A P-value of less than 0.05 (P < 0.05) was considered statistically significant. Results Hypoxia promotes proliferation and alters transcriptome of MSCs After 24 h of hypoxia pre-treatment, MSCs retained characteristic spindle-shaped morphology with a radial distribution but exhibited a significantly increased degree of cell fusion compared to those cells in normoxic conditions (Fig. 1 A). The CCK-8 assay validated a marked increase in the hypoxic group, indicating that hypoxia substantially promotes cell proliferation (Fig. 1 B). Western blot analysis further demonstrated a significant up-regulation of HIF-1α expression in MSCs under hypoxic conditions. Additionally, activation of the AKT signaling pathway, known to be associated with cell proliferation, was observed, with significant increases in the levels of AKT, phosphorylated AKT (p-AKT), and the p-AKT/AKT ratio in the hypoxic group (Figs. 1 C, D). Transcriptomic analysis of hypoxia-pretreated MSCs revealed profound alterations in gene expression profiles, as illustrated in the heatmap of DEGs (Fig. 1 E). GO analysis unveiled a significant enrichment of these DEGs in cellular membrane components and transporter activities, both of which are closely linked to the production and secretion of extracellular vesicles (Fig. 1 F). Alterations in the MSC characteristics and yield of EVs following hypoxia preconditioning TEM revealed that EVs from both normoxic and hypoxic conditions displayed similar morphologies, appearing as round or elliptical vesicles with intact structures (Fig. 2 A). The expression levels of specific membrane protein markers associated with EVs, such as CD9, CD63, and CD81 were determined by Western blot. The results showed increased expression of CD9 and CD63 in hypoxic EVs, although the alteration in CD81 was not statistically significant (Figs. 2 B, C). Measurement of protein concentration in EVs produced by 1x10 7 MSCs revealed that the EVs from hypoxia pre-treated MSCs had a significantly higher concentration compared to those from the normoxic group (Fig. 2 D). NTA demonstrated that the size of EVs ranged from 50–500 nm in both groups, suggesting no significant difference (Figs. 2 E, F). However, the particle concentration in the hypoxic group was significantly elevated compared to the normoxic group (Fig. 2 G). Furthermore, we examined the expression of proteins involved in EV secretion in MSCs, specifically PRAS40 and RAB22a. PRAS40 exhibited a marked rise under 5% oxygen with significantly increased expression of HIF-1α under 10% and 5% oxygen conditions (Figs. 2 H-J). Although RAB22a did not increase under 10% oxygen conditions, it was significantly up-regulated under 5% and 3% oxygen conditions (Figures S1 A-B). Hypoxic EVs exhibited superior efficacy in promoting recovery from renal IRI PKH26-labeled EVs were systemically administered to rats undergoing IRI. After 48 hours, PKH26 fluorescence was clearly detectable within the kidney tissue (Fig. 3 A). H&E staining revealed numerous necrotic areas in the proximal epithelium and abundant tubular protein casts in the IRI -affected kidneys. In contrast, treatment with both normoxic and hypoxic EVs resulted in a reduction of tubular lesions (Fig. 3 B). Renal injury scoring, based on structural alterations, indicated that hypoxic EVs were more effective at alleviating morphological changes associated with IRI compared to their normoxic counterparts (Fig. 3 E). Ki67, a marker of proliferation, demonstrated that enhanced tissue proliferation following IRI, interestingly, further augmented by both normoxic and hypoxic EVs treatments (Figs. 3 C, F). TUNEL staining unveiled IRI-induced apoptosis, markedly attenuated by both normoxic and hypoxic EVs administrations (Figs. 3 D, G). Renal function was assessed by measuring SCr and BUN. Rats with renal IRI that received PBS showed a rapid increase in SCr and BUN levels. In contrast, treatment with normoxic and hypoxic EVs significantly attenuated these increases (Figs. 3 H, I). Notably hypoxic EV-treated kidneys exhibited lower renal injury scores, higher Ki67 expression, fewer apoptotic cell counts, and decreased SCr levels compared to those treated with normoxic EVs. MSC-EVs promote anti-apoptosis of renal tubular epithelial cells in vitro To model hypoxic injury to renal tubular epithelial cells, rat tubular epithelial cell line NRK-52E were exposed to Antimycin (AMA), an inhibitor of mitochondrial electron transport chain. As depicted in Fig. 4 A, AMA induced a dose-dependent reduction in NRK-52E viability, a concentration of 100 µM leading to approximately a 50% reduction in cell viability. Treatment with both normoxic EVs and hypoxic EVs restored cell viability (Fig. 4 B). To investigate the uptake of MSC-EVs by renal tubular epithelial cells in vitro , MSCs were labeled with PKH-26, allowing the secreted EVs to carry red fluorescence (Fig. 4 C). Internalization of PKH26-labeled EVs was confirmed in NRK-52E cells. Quantitative analysis revealed that hypoxic EVs exhibited enhanced cellular uptake compared to those from normoxic conditions (Figs. 4 D, F). TUNEL staining revealed that AMA induced apoptosis in renal tubular epithelial cells. Both normoxic EVs and hypoxic EVs effectively attenuated cell apoptosis. Particularly, hypoxic EVs demonstrating superior efficacy in protecting renal tubular epithelial cells from AMA- induced injury (Figs. 4 E, G). Hypoxia preconditioning up-regulated antioxidant stress pathway revealed by the proteomics of EVs To elucidate the underlying mechanism by which hypoxic EVs exhibit superior efficacy for renal IRI, we conducted mass spectrometry analysis of the proteomic cargo of EVs. PCA clearly distinguished the proteomic signatures of hypoxic EVs from those of normoxic EVs, indicating that hypoxia alters the protein profiles of MSC-EVs (Fig. 5 A). We identified significant differences in protein expression levels between the two groups, with 264 proteins up-regulated and 288 proteins down-regulated in hypoxic EVs (P < 0.05) (Fig. 5 B). Among these significant regulated proteins, GSTO1, an enzyme involved in the redox reaction of GSH, exhibited the most pronounced difference (Fig. 5 C). Unsupervised hierarchical clustering analysis confirmed the distinct protein patterns between normoxic and hypoxic EVs, as shown in the heat map (Fig. 5 D). Subcellular localization classification of the differentially expressed proteins indicated that the predominant proteins were extracellular (38.59%) and cytoplasmic (19.75%) (Fig. 5 E). Functional enrichment analysis of the up-regulated proteins revealed significant enrichment in GSH synthesis metabolism pathways, such as cysteine and methionine metabolism and glutathione metabolism pathways, as indicated by KEGG analysis (Figs. 5 F). Moreover, highlighted significant enrichment of pathways associated with nuclear factor erythroid 2–related factor 2 (NRF2), a key regulator of cellular resistance to oxidative stress. This includes pathways related to nuclear events mediated by NFE2L2, the KEAP1-NFE2L2 pathway, photodynamic therapy-induced NRF2 survival signaling, and the NRF2 pathway itself (Figs. 5 G, H). Collectively, these findings suggest that hypoxia preconditioning enhances the expression of antioxidant stress-related proteins in MSC-EVs. Validation of hypoxic EVs in alleviating renal IRI via antioxidant mechanisms We first evaluated the differential effects of hypoxic and normoxic EVs on oxidative stress in rat kidney tissue. The results demonstrated that IRI significantly elevated ROS levels. Notably, hypoxic EVs markedly reduced ROS levels in IRI-affected kidneys compared to normoxic EVs (Figs. 6 A, C). In vitro assays with NRK-52E cells exposed to AMA corroborated this trend, showing that ROS production and mitochondrial membrane potential changes mirrored those observed in rat kidney tissue (Figures B, D, and E). Furthermore, we assessed levels of GSH, the foremost substance involved in antioxidant. Renal IRI markedly diminished GSH levels and increased GSSG levels, thereby reducing the GSH/GSSG ratio. However, hypoxic EVs treatment significantly restored GSH and GSSG levels in the IRI kidney, surpassing the restorative effects of normoxic EVs. Ultimately, the GSH/GSSG ratio in hypoxic EV group saw a significant increase, even reaching levels comparable to the sham group (Figs. 6 F-H). We further validated the protein level of GSTO1, identified as the most differentially expressed protein in the hypoxic EVs. The results confirmed that GSTO1 was up-regulated indeed in both hypoxia pre-treated MSCs and their daughter EVs (Figs. 6 I-K). To investigate the role of GSTO1 in mitigating oxidative stress in hypoxic EVs, we knock down GSTO1 using three different siRNAs. Except for siGSTO1-214, both siGSTO1-411 and siGSTO1-601 effectively silenced the mRNA and protein expression of GSTO1 in MSCs (Figs. 6 L, M). Hypoxic EVs from MSCs transfected with both siGSTO1-411 and siGSTO1-601 didn’t reduce the ROS levels of renal tubular epithelial cells as effectively as those transfected with a negative control siRNA (Figs. 6 N, O). Furthermore, GSTO1 knockdown diminished the ability of the hypoxic EVs to restore the mitochondrial membrane potential in the renal tubular epithelium (Figures S2 A, C) and reduced the protective effect against AMA-induced apoptosis (Figures S2 B, D). Upon treating IRI model rats with hypoxic EVs in which GSTO1 was knocked down, we observed notable changes. ROS level significantly increased compared to the control group receiving hypoxic EVs (Figures S3 A, D). Additionally, a noticeable dip in Ki67 expression and an increment in apoptotic were spotted (Figures S3 B, C, E, F). Further analysis of kidney function revealed that hypoxic EVs with GSTO1 knockdown was less effective in mitigating elevated SCr and BUN levels resulting from IRI compared to the hypoxic EVs group (Figures S4 A, B). Taken together, these findings indicate that GSTO1 in hypoxic EVs is essential for alleviating renal IRI. Discussion IRI is the leading cause of AKI, resulting in damage to renal tubules and rapid deterioration of renal function. The current study demonstrates that hypoxia not only stimulates the proliferation of MSCs but also enhances the secretion of EVs. Additionally, MSC-EVs pre-treated with hypoxia show a pronounced ability to protect against renal IRI. Importantly, we have presented novel evidence that hypoxia pre-treated MSC-EVs mitigate acute renal IRI through an anti-oxidative stress mechanism, with the antioxidant protein GSTO1 playing a crucial role in this protective process. Conventional in vitro cell culture typically occurs under ambient oxygen conditions (21% O 2 ), referred to as ‘normoxia’. In contrast, in vivo , MSCs are often situated in niches with lower oxygen tensions [ 18 ]. Culturing MSCs under hypoxic conditions aims to mimic this natural microenvironment. MSCs residing in hypoxic niches, with oxygen tensions ranging from 3–9%, are capable of self-renewal, proliferation, migration, and differentiation [ 19 , 20 ]. While previous studies have demonstrated that hypoxia preconditioning can enhance MSC proliferation and differentiation [ 21 ], the precise mechanisms remained elusive. Our study identified that hypoxia could activate the HIF-1α/AKT pathway to promote MSCs proliferation. Extensive cellular responses to hypoxic stress are typically mediated by HIF-1α [ 22 ]. And, overexpression of HIF-1α has been shown to enhance MSCs proliferation and osteogenic capacity [ 23 ]. The AKT signaling pathway plays a key role in cell proliferation, and previous research has shown that HIF-1α is involved in the up-regulation of p-AKT protein expression [ 24 , 25 ]. Hence, this evidence supports that the HIF-1α/AKT pathway involve in the proliferation of MSC under hypoxic conditions. MSCs participate in tissue repair through endocrine or paracrine mechanisms, with EVs being one of their main means of communication [ 26 ]. These EVs play a crucial role in tissue repair by delivering proteins and genetic materials. Notably, the generation of EVs is influenced by the microenvironment. The impact of MSC-EVs can be amplified under certain physical and biological stimuli, such as hypoxia, lipopolysaccharide, and TNF-α. These conditions not only boost EV production but may also modify their contents, ultimately enhancing their beneficial effects [ 27 – 30 ]. The RAB family comprises key signaling molecules involved in EVs production, while PRAS40 is involved in the release of EVs following cellular stress [ 31 , 32 ]. The production of MSC-EVs significantly increased under hypoxic culture conditions, with the activation of RAB22a and PRAS40 observed in MSCs in this study. Furthermore, the plasma membrane plays a crucial role in shaping and facilitating the functions of EVs during their generation. It is actively involved in cell signaling, membrane adaptability, and uptake by recipient cells. Interestingly, our transcriptome analysis of MSCs revealed that under hypoxic conditions, signaling pathways related to cell membrane components and transporter activities associated with EV production are activated. Certain researchers have documented that hypoxic EVs are effective in mitigating organ ischemia injury, such as in the brain and limbs, by transporting microRNAs or proteins [ 33 – 35 ]. Zhang et al reported that hypoxic preconditioning of MSCs can enhance the repair of injured kidneys, with increased angiogenesis and antioxidant effects playing a role [ 36 ]. Consequently, the therapeutic effects of hypoxic EVs on renal IRI were explored in this study. The results indicated that hypoxia preconditioning enhances the ability of MSC-EVs to repair IRI kidneys, improving both renal morphology and function. During AKI, tubular cell apoptosis and proliferation occur. Both in vivo and in vitro experiments showed that hypoxia preconditioning of MSC-EVs significantly reduced tubular cell apoptosis and promoted their proliferation, demonstrating a stronger protective effect against injury. EVs rely on their biologically active internal components, including proteins, lipids, and nucleic acids, to carry out their functions. However, it remains unclear which of these components plays the central role. While substantial research has focused on alterations in miRNAs within EVs, less attention has been paid to their protein content, which can directly impact recipient cells [ 37 ]. Recently, EVs have emerged as a novel mechanism for delivering proteins [ 38 ]. Eirin et al highlighted in their study on MSC-EV-mediated kidney injury repair that the delivery of IL-10 protein is a key factor in the protective effects of MSC-EVs [ 39 ]. All of this underscores the potential significance of proteins in the cellular communication of MSC-EVs. To further elucidate the mechanisms by which hypoxic EVs alleviate renal injury, a proteomic analysis of the protein cargo of MSC-EVs was performed. Compared to normal MSC-EVs, hypoxic EVs exhibited a substantial accumulation of anti-oxidative stress proteins, with GSTO1 being the most significantly elevated. Oxidative stress is a crucial factor in the pathogenesis of ischemic kidney injury, and it has been confirmed that MSC-EVs reduce oxidative reactions and protect kidney function [ 40 , 41 ]. In this study, it was also found that MSC-EVs significantly reduced oxidative stress levels in injured kidneys, with hypoxic EVs showing an even greater effect, demonstrating their potent antioxidant properties. GSTO1 is an important enzyme that promotes the reduction of ROS through redox reactions involving GSH. It has been shown to play a significant role in protecting the function of kidneys in end-stage renal disease [ 42 ]. It has been reported that GSTO1 is involved in the regulation of oxidative stress and protective effects in various diseases [ 43 ]. To further verify the role of GSTO1 in hypoxic EVs, GSTO1-knockdown hypoxic EVs were obtained and used to treat ischemic renal injury. The data show that in the absence of GSTO1, the antioxidant effects of hypoxic EVs were significantly reduced, along with their protective effects on tubular cells, resulting in diminished kidney function protection. Thus, this finding suggests that the antioxidant protein GSTO1 plays a pivotal role in the protective effects of hypoxic EVs against renal IRI. Conclusions This study demonstrates that EVs derived from hypoxia pre-treated MSCs offer renal protection against IRI. The cargo of antioxidant proteins, particularly GSTO1, plays a pivotal role in alleviating acute renal IRI. Consequently, our findings present a promising avenue for the clinical treatment of renal IRI. Abbreviations ACEIs Angiotensin-converting enzyme inhibitors AKI Acute kidney injury AKT Serine/threonine kinase AMA Antimycin A ARBs Angiotensin receptor blockers BUN Blood urea nitrogen DEGs Differential express genes ETC mitochondrial electron transport chain EVs Extracellular vesicles GO Gene ontology GSTO1 Glutathione S-Transferase Omega 1 H&E hematoxylin–eosin HIF-1α Hypoxia-inducible factor-1α huMSCs Human umbilical cord MSCs IRI Renal ischemia-reperfusion injury KEGG Kyoto Encyclopedia of Genes and Genomes MSCs Mesenchymal stromal cells NRF2 Nuclear factor erythroid 2–related factor 2 NTA Nanoparticle tracking analysis p-AKT Phosphorylated Serine/threonine kinase PBS Phosphate-buffered saline PCA Principal component analysis p-PRAS40 Phosphorylated proline-rich AKT substrate of 40 kDa RAB22a RAB22a, Member RAS Oncogene Family ROS Reactive oxygen species SCr serum creatinine TEM Transmission electron microscopy Declarations Acknowledgments The authors declare that they have not use AI-generated work in this manuscript. Author contributions FY, JL, LZ and PT L: experiments conducting, data acquirement and original manuscript preparation; FY, JL, LZ , PT L and XY Z: manuscript editing/validation and data analysis; WG , TL, KX Y and XY Z: technical support and experimental assistance; GY Z, JS and XY Z: study design guidance, manuscript reviewing and editing; JS and XY Z: project supervision and funding acquisition. All authors read and approved the final manuscript. Funding This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81900618); the Tai-Shan Scholar Program from Shandong Province, China (Grant No. tsqn202103116); the Pudong New Area Science and Technology Development Fund (Grant No. PKJ2020-Y04; the Natural Science Foundation of Fujian Province (Grant No. 2023J01183); the Program of Scientific and Technological Development of Weifang (Grant No. 2023GX026). Declaration of competing interest The authors declare that they have no competing interests. Availability of data and materials All datasets to support current study are available from the corresponding author on reasonable request. The sequence data in the current study will be available in NCBI’s BioProject and can be accessed by the public (ID: PRJNA1064232).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD057088. Ethics approval and consent to participate The project has been approved by the ethics committee of Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine. All human subjects gave informed consent for tissue donation. Project title: “The study on the mechanism by which hypoxia promotes mesenchymal stem cell-derived extracellular vesicle production and enhances the repair capacity of ischemia-reperfusion kidney injury”, protocol number SCMCIRB-Y2019005, approved 2019/2/25. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5266177","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373550416,"identity":"85ea7fa1-8ee4-48c4-9569-e297af8a367e","order_by":0,"name":"Fei Yuan","email":"","orcid":"","institution":"Shanghai Children's Medical Center Affiliated to Shanghai Jiaotong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Yuan","suffix":""},{"id":373550417,"identity":"e884d46f-5184-4151-b7fb-98dfb48216d7","order_by":1,"name":"Jie Liu","email":"","orcid":"","institution":"Shanghai 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hypoxic (n=3) and normoxic groups(n=3) (*P \u0026lt; 0.05). \u003cstrong\u003eC:\u003c/strong\u003e Western blot analysis of HIF-1α, AKT, p-AKT, and β-actin levels in hypoxic (n=4) and normoxic (n=4) conditions. \u003cstrong\u003eD:\u003c/strong\u003eStatistical analysis of Western blot results, showing protein expression levels of HIF-1α, AKT, and p-AKT (*P \u0026lt; 0.05 ). \u003cstrong\u003eE:\u003c/strong\u003e Heatmap clustering analysis of DEGs in hypoxic (n=3) and normoxic (n=3) conditions. \u003cstrong\u003eF:\u003c/strong\u003e GO enrichment analysis of the DEGs.\u003c/p\u003e","description":"","filename":"Figure161.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/673cde91936263c857ab0247.jpg"},{"id":69001394,"identity":"95d65d44-1dbc-4f16-bba0-289be4ed5b51","added_by":"auto","created_at":"2024-11-14 11:46:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":670684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in EVs production and characterization following hypoxia pre-treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Morphological observation of EVs in normoxic and hypoxic conditions using TEM (scale bar = 200 nm). \u003cstrong\u003eB:\u003c/strong\u003e Western blot analysis of EV markers, including CD9, CD63, and CD81 in hypoxic (n=3) and normoxic (n=3) conditions. \u003cstrong\u003eC:\u003c/strong\u003e Relative quantification and statistical analysis of CD9, CD63, and CD81 expression levels (*P \u0026lt; 0.05). \u003cstrong\u003eD:\u003c/strong\u003e Protein concentration of EVs derived from 1 × 10\u003csup\u003e7\u003c/sup\u003e MSCs in hypoxic (n=5) and normoxic (n=5) conditions (*P \u0026lt; 0.05). \u003cstrong\u003eE:\u003c/strong\u003e Size distribution and diameter of EVs analyzed by NTA. \u003cstrong\u003eF:\u003c/strong\u003e Statistical analysis of EV diameter in hypoxic (n=5) and normoxic (n=5) conditions.\u003cstrong\u003e G:\u003c/strong\u003e Statistical analysis of EV particle concentration in hypoxic (n=5) and normoxic (n=5) conditions (****P \u0026lt; 0.0001). \u003cstrong\u003eH:\u003c/strong\u003e Western blot analysis of HIF-1α, PRAS40, and β-actin levels at 21% (normoxic, n=3), 10% (n=3), 5% (n=3), and 3% O2 conditions (n=3). \u003cstrong\u003eI:\u003c/strong\u003e Statistical analysis of HIF-1α protein expression levels normalized to β-actin (*P \u0026lt; 0.05). \u003cstrong\u003eJ:\u003c/strong\u003e Western blot analysis of PRAS40 protein levels normalized to β-actin (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure162.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/8328108cf49b8c10798a45e5.jpg"},{"id":69002284,"identity":"7a7ae709-5d8e-47a4-84e2-684399a2fd08","added_by":"auto","created_at":"2024-11-14 11:54:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1038062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxia EVs attenuate renal IRI in rat.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Localization of MSC-EVs labeled with PKH-26 in IRI kidneys, with DAPI staining for nuclei; background shows spontaneous green fluorescence of kidney (scale bar = 100 µm). \u003cstrong\u003eB:\u003c/strong\u003eH\u0026amp;E staining of rat kidneys across different groups (scale bar = 100 µm): sham surgery (Sham), renal IRI injected with PBS (IRI), with normoxic EVs (IRI/nEVs), or with hypoxic EVs (IRI/hEVs).\u003cbr\u003e\n \u003cstrong\u003eC:\u003c/strong\u003e Representative images of Ki67-positive cells in the tubulointerstitial area (scale bar = 100 µm). \u003cstrong\u003eD:\u003c/strong\u003e Representative images of apoptotic cells identified by TUNEL staining (scale bar = 100 µm). \u003cstrong\u003eE:\u003c/strong\u003eStatistical analysis of kidney injury scores assessed from histological slides (n=6). \u003cstrong\u003eF:\u003c/strong\u003e Quantification of Ki67-positive cells per high-power field (HPF) (n=6). \u003cstrong\u003eG:\u003c/strong\u003e Quantification of apoptotic cells per HPF (n=6). \u003cstrong\u003eH:\u003c/strong\u003eSCr levels across different groups (µmol/L) (n=6). \u003cstrong\u003eI: \u003c/strong\u003eBUN levels in different groups (µg/mL) (n=6). (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure163.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/cc6e11b786990dce1ad6aa14.jpg"},{"id":69002283,"identity":"22cce195-98a9-493e-92b6-cb848a46676a","added_by":"auto","created_at":"2024-11-14 11:54:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":496795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of hypoxic EVs on NRK-52E cells under AMA-induced injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e CCK-8 assay demonstrating the toxicity of various concentrations of AMA on NRK-52E cells (n=5). \u003cstrong\u003eB:\u003c/strong\u003eCell viability of NRK-52E stimulated with 100 µM AMA for 24 hours across different treatment groups (n=5). \u003cstrong\u003eC:\u003c/strong\u003e MSCs labeled with PKH-26 (scale bar = 100 µm). \u003cstrong\u003eD:\u003c/strong\u003e Internalization of labeled EVs in the cytoplasm of NRK-52E (scale bar = 50 µm; blue: DAPI-stained nuclei, green: Phalloidin-stained cytoskeleton, red: PKH-26). \u003cstrong\u003eE:\u003c/strong\u003e Representative images of TUNEL staining in NRK-52E (scale bar = 200 µm). \u003cstrong\u003eF:\u003c/strong\u003e Quantification of internalized PKH-26-labeled EVs derived from normoxic (n=3) and hypoxic MSCs (n=3) in NRK-52E (*P \u0026lt; 0.05). \u003cstrong\u003eG:\u003c/strong\u003e Quantification of apoptotic cells HPF (n=5) (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure164.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/8e9d84a7d610880231e7561f.jpg"},{"id":69003020,"identity":"e6edc500-8afb-4cb5-8755-c17329b219c4","added_by":"auto","created_at":"2024-11-14 12:02:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1002290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxia up-regulated antioxidant proteins in MSC-EVs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e PCA distinguishing normoxic (n=3) and hypoxic EVs (n=3). \u003cstrong\u003eB:\u003c/strong\u003e Summary of protein types in EVs, with red indicating up-regulated and blue indicating down-regulated proteins. \u003cstrong\u003eC:\u003c/strong\u003eTop 30 proteins showing the most significant differences in expression. \u003cstrong\u003eD:\u003c/strong\u003eHeatmap clustering analysis of differentially expressed proteins. \u003cstrong\u003eE:\u003c/strong\u003eSubcellular localization analysis of up-regulated and down-regulated proteins. \u003cstrong\u003eF:\u003c/strong\u003eKEGG functional enrichment analysis of up-regulated differential proteins. \u003cstrong\u003eG:\u003c/strong\u003eWikiPathways enrichment analysis of up-regulated differential proteins. \u003cstrong\u003eH:\u003c/strong\u003eReactome pathway enrichment analysis of up-regulated differential proteins.\u003c/p\u003e","description":"","filename":"Figure165.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/a9f53caf381de390be5a97c8.jpg"},{"id":69001398,"identity":"19ebee88-5263-4b26-82d1-eea39352faa3","added_by":"auto","created_at":"2024-11-14 11:46:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":642266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxic EVs alleviate acute renal IRI through the cargo of antioxidant proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003eRepresentative images of reactive oxygen species (ROS) fluorescence in rat kidneys (scale bars = 100 µm). \u003cstrong\u003eB:\u003c/strong\u003e Representative images of mitochondrial membrane potential injury in NRK-52E cells (scale bars = 50 µm). \u003cstrong\u003eC:\u003c/strong\u003eQuantitative analysis of ROS fluorescence intensity in rat kidneys (n=5).\u003cstrong\u003e D:\u003c/strong\u003eROS fluorescence quantified in NRK-52E following 24-hour exposure to 100 µM AMA (n=5). \u003cstrong\u003eE:\u003c/strong\u003e Quantitative analysis of mitochondrial membrane potential fluorescence intensity in NRK-52E cells (n=5). \u003cstrong\u003eF:\u003c/strong\u003e GSH levels across different experimental groups (n=5). \u003cstrong\u003eG:\u003c/strong\u003e GSSG levels across different experimental groups (n=5). \u003cstrong\u003eH:\u003c/strong\u003e GSH/GSSG ratio across different experimental groups (n=5). \u003cstrong\u003eI:\u003c/strong\u003e Western blot analysis of GSTO1 expression levels in MSCs and EVs following hypoxic treatment. \u003cstrong\u003eJ:\u003c/strong\u003e Statistical analysis of GSTO1 protein expression in MSCs, normalized to β-actin, as determined by Western blot (n=3). \u003cstrong\u003eK:\u003c/strong\u003e Statistical analysis of GSTO1 protein expression in EVs, normalized by total protein concentration and volume (n=3). \u003cstrong\u003eL:\u003c/strong\u003e mRNA expression levels of GSTO1 post-transfection with GSTO1-targeting siRNA, assessed by qPCR (n=3). \u003cstrong\u003eM:\u003c/strong\u003e Protein expression levels of GSTO1 post-transfection with GSTO1-targeting siRNA, evaluated by Western blot (n=2). \u003cstrong\u003eN:\u003c/strong\u003eRepresentative images of ROS fluorescence in NRK-52E (scale bars = 50 µm). \u003cstrong\u003eO:\u003c/strong\u003eQuantitative analysis of ROS fluorescence in NRK-52E (n=5) (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure166.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/4d191e125f69e9dfed4ed98a.jpg"},{"id":75930474,"identity":"53870df3-bba6-4fa9-a6e5-d9516b0b9b7b","added_by":"auto","created_at":"2025-02-10 16:12:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6020863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/20cc57ef-16e0-4752-b046-a0435cf86ceb.pdf"},{"id":69002286,"identity":"6e7acfed-13a7-4693-a5d3-993f5de5c9ee","added_by":"auto","created_at":"2024-11-14 11:54:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":566472,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial0801.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/3e04116f352ca074fc9f19e8.pdf"},{"id":69001401,"identity":"a0527e23-30cd-4e13-bef5-91ae11d7a1c6","added_by":"auto","created_at":"2024-11-14 11:46:37","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":454077,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5266177/v1/ea0f3f0f1fc41af3cfb1b168.jpeg"}],"financialInterests":"","formattedTitle":"Enhanced Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Renal Ischemic Injury","fulltext":[{"header":"Background","content":"\u003cp\u003eAcute kidney injury (AKI), marked by a rapid decline in renal excretory function, poses a significant threat to patient well-being and survival [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is estimated that one in five adults and one in three children experience AKI during hospital stays, typically due to sudden kidney failure or damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Renal ischemia-reperfusion injury (IRI) is a common cause of AKI, frequently occurring in patients undergoing sepsis or surgical procedures such as kidney transplantation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, therapeutic options for AKI are limited. While pharmacological interventions like angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have been employed to modulate renal function [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], they have not been effective in reversing the progression of kidney dysfunction. Therefore, more effective approaches to alleviate kidney tissue damage and promote regeneration are urgently needed.\u003c/p\u003e \u003cp\u003eMesenchymal stromal cells (MSCs) possess remarkable self-renewal capabilities and have demonstrated success in treating a wide range of diseases, including tissue damage repair, inflammation suppression, and immune modulation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. MSCs have proven effective in promoting kidney repair following AKI by mitigating tubulointerstitial damage [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, extracellular vesicles (EVs) secreted by MSCs serve as crucial cellular regulators across numerous biological processes [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Characterized by a lipid bilayer structure, MSC-EVs facilitate intercellular communication by delivering a diverse array of cargos, including RNAs, proteins, and lipids [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notably, MSC-EVs have been reported to ameliorate renal damage induced by IRI and contribute to cellular repair mechanisms [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Compared to their parental MSCs, EVs exhibit lower immunogenicity and enhanced biological safety, positioning them as promising candidates for therapeutic interventions in various diseases.\u003c/p\u003e \u003cp\u003eHypoxia pre-treatment significantly enhances protective capabilities of MSCs for injured tissues and organs. Hypoxia-pretreated MSCs have shown the capacity to alleviate spinal cord injury and improve bone repair. The production of MSC-EVs is also influenced by hypoxic conditions. However, the precise effects of hypoxic preconditioning on MSC-EV production, as well as the underlying mechanisms by which hypoxic EVs facilitate tissue repair, remain not well understood. Therefore, in this study, we aim to investigate the impact of hypoxia on human umbilical cord MSCs and validate the therapeutic efficacy of hypoxic EVs in a rat model of IRI-induced AKI. Furthermore, we seek to elucidate the potential mechanisms driving the therapeutic effects of hypoxic EVs by investigating their proteomics.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eMSCs were prepared and identified as described in our previous study [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In brief, umbilical cord tissues were cut and then attached to culture plates individually. The collection and subsequent use of the umbilical cord were approved by the Institutional Ethical Review Committee of Shanghai Children\u0026rsquo;s Medical Center, School of Medicine, Shanghai Jiao Tong University. Cells were cultured with media mixed with α-MEM medium (12571063, Gibco) containing 5% UltraGRO\u0026trade;-Advanced cell culture supplement (HPCFDCGL50, Helios) in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. When the cell confluency reaches 90%, MSCs were passaged at a 1:5 ratio. The medium was changed every 2 days.\u003c/p\u003e \u003cp\u003eThe rat renal tubular epithelial cell line NRK-52E (CL-0174, Procell Life Science \u0026amp; Technology Co., Ltd.) was cultured in DMEM (11965092, Gibco) supplemented with 5% fetal bovine serum (A5669701, Gibco). Cells were incubated in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. When cells reached 70% confluency, they were passaged at a 1:3 ratio. The medium was changed every 2 days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHypoxia pre-treatment of MSCs\u003c/h3\u003e\n\u003cp\u003eMSCs were plated in 10 cm dishes and cultured until reached approximately 80%-90% confluence for downstream experiments. Hypoxia pre-treatment is conducted by different concentration of oxygen (a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e with 10%, 5% or 3% O\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C) for 24 h. In the normoxic group, cells were incubated in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e and 21% O\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eCCK-8 assay\u003c/h3\u003e\n\u003cp\u003eThe cell proliferation of MSCs and NRK-52E were determined by CCK-8 assay (K1018, APEx BIO) following the instructions. The absorbance at 450 nm wavelength was measured by a microplate reader (Thermo Fisher).\u003c/p\u003e\n\u003ch3\u003eTranscriptomic analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using Fast Pure Cell/Tissue Total RNA Isolation Kit (RC101, Vazyme) according to the manufacturer's instructions. The primary experimental procedures for transcriptome sequencing analysis include RNA quantification and qualification, library preparation for transcriptome sequencing, clustering, and sequencing, and data analysis. HTSeq v0.6.0 was used to count the number of reads mapped to each gene. The FPKM of each gene was calculated based on the length of the gene and the read count mapped to this gene. Differential expression analysis of the two groups was performed using the DESeq2 R package (1.10.1). Adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significantly differential expression. GO enrichment analysis of differentially expressed genes (DEGs) was performed using the clusterProfiler R package. DEGs were correlated with annotations, including GO terms. Significant enrichments by differentially expressed genes were identified with corrected p-values less than 0.05. The transcriptome sequencing analysis in our research was supported by BGIgene Co, Ltd.\u003c/p\u003e\n\u003ch3\u003eIsolation and purification of MSC-EVs\u003c/h3\u003e\n\u003cp\u003eThe extraction method of EVs was performed as described in the previously published literature [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].MSCs incubated in normoxia (21% O\u003csub\u003e2\u003c/sub\u003e) and hypoxia (5% O\u003csub\u003e2\u003c/sub\u003e) conditions, were cultured with UltraGRO\u0026trade;-Advanced cell culture supplement-free media for 24 h, and the supernatants were subsequently collected for MSC-EVs extraction. In brief, the supernatants centrifuged at 2000 g, 4\u0026deg;C, for 20 min to remove cell debris, and then centrifuged again at 100,000 g (Beckman Coulter, Fullerton, CA) for 1 h at 4\u0026deg;C. The concentrate was re-suspended in cold PBS for washing and centrifuged as above to collect to the EVs. Isolated EVs were stored at -80\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of EVs\u003c/h2\u003e \u003cp\u003eThe morphology of EVs was observed by transmission electron microscopy (TEM). The particle size was detected by Nanoparticle tracking analysis (NTA). Surface markers including CD9, D63 and CD81 were analyzed by Western Blot analysis. Protein concentration of EVs derived from 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e MSCs was quantified by the bicinchoninic acid assay kit (BCA kit, A55860, Thermo Fisher).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eWestern blot was performed as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The following primary antibodies were used: antibodies against CD9 (dilution 1:1000; ab236630, Abcam), CD63 (dilution 1:1000; ab134045, Abcam), CD81(dilution 1:1000; ab79559; Abcam), hypoxia-inducible factor-1α (HIF-1α) (dilution 1:1000; 14179, CST), serine/threonine kinase (AKT) (dilution 1:1000; 9272, CST), phosphorylated Serine/threonine kinase (p-AKT) (dilution 1:1000; 9271, CST), phosphorylated proline-rich AKT substrate of 40 kDa (p-PRAS40) (dilution 1:1000; ab151719, Abcam), RAB22a ( dilution 1:1000; ab137093, Abcam), Glutathione S-Transferase Omega 1 (GSTO1) (dilution 1:1000; 15124-1-AP, Proteintech), Beta Actin (β-actin) (dilution 1:1000; ab8226, Abcam).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLabeling and location of MSC-EVs\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo label MSC-EVs, MSCs were incubated with 10 \u0026micro;M PKH-26 dye (HY-D1451, MCE) in serum-free culture medium at room temperature for 15 min, followed by two washes with sterile PBS to remove excess dye. The cultures were continued for MSCs 2 days and observed using fluorescence microscopy (Stellris 8, Leica). The supernatants were used to isolate PKH-26-labeled EVs in the same procedure as above. In this way, the free dye could be removed to the utmost extent. \u003cem\u003eIn vivo\u003c/em\u003e, PKH-26 labeled EVs were administered into rats via inferior vena cava. After 48 hours, the kidneys of model rats underwent tissue fixation, dehydration, embedding and sectioning for subsequent observation of EVs\u0026rsquo; location under a fluorescent microscope. Renal tissues showed green auto-fluorescence. \u003cem\u003eIn vitro\u003c/em\u003e, EVs labeled by PKH-26 were added to the medium of NRK-52E for 24h. After the cell crawling slides were fixed with 4% paraformaldehyde, cytoplasm and nucleus were localized with Phalloidin (A12379, Thermo Fisher) and DAPI (62248, Thermo Fisher), respectively. And the intracellular location of EVs observed under a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eRats and surgical preparation\u003c/h3\u003e\n\u003cp\u003e The ethics Committee of Shanghai Children's Medical Center, School of Medicine, Shanghai Jiao Tong University approved this study. 8 week male Sprague-Dawley rats (180-200g) purchased from Shanghai Jihui Experimental Animal Breeding Co. Ltd was used in this study. All the experimental rats lived in a suitable temperature and humidity environment with a normal diet. An AKI model was established as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Rats were prepared under 2% isoflurane anesthesia through the respiratory tract. Rats were subjected to random allocation into four groups (n\u0026thinsp;=\u0026thinsp;6), comprising those undergoing sham surgery (sham), IRI treated with PBS (IRI), IRI treated with normoxic MSC-EVs (IRI/nEVs), or IRI treated with hypoxic MSC-EVs (IRI/hEVs). A mid-abdominal incision was employed for the excision of the right kidney and temporary occlusion of the left renal pedicle for a duration of 45 minutes. Sham-treated subjects underwent an identical surgical intervention, albeit devoid of occlusion of the renal pedicle. 1 ml EVs at a concentration of 100 \u0026micro;g/ml was administered to the respective groups via the inferior vena cava. Rats were euthanized at 48 h after injury, and kidneys and serum samples were collected for the following examinations. Rats were euthanized by excessive carbon dioxide under anesthesia conditions. Rats were housed in the same animal facility and underwent relevant procedures performed by the same surgical personnel. All animal experiments were reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistological examination and renal function assessment\u003c/h2\u003e \u003cp\u003eRenal histological examination and renal function assessment were performed as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Parts of the left kidney were fixed in 4% paraformaldehyde, then dehydrated in ethanol and embedded in paraffin. Kidney tissue blocks were cut into 4 \u0026micro;m sections and subjected to hematoxylin\u0026ndash;eosin (H\u0026amp;E) staining. Subsequently, the stained sections were viewed using light microscopy. A score was given based on the grade of tubular necrosis, brush border loss, cast formation and tubular dilatation in ten randomly chosen areas. The histological scoring was determined in a blind manner based on the following criteria refer to previous literature: (0) none; (1) 0\u0026ndash;10%; (2) 11\u0026ndash;25%; (3) 26\u0026ndash;45%; (4) 46\u0026ndash;75% and (5) 76\u0026ndash;100% [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRenal cell apoptosis was assessed by using TUNEL staining (C1091, Beyotime). Renal tissues and cultured cell slides underwent fixation, permeabilization with 0.1% Triton X-100 (A110694, Sangon Biotech) for 10 minutes followed by wash with PBS. TUNEL staining was performed following manufacturer's guidelines. The numbers of TUNEL-positive tubular cells and average fluorescence intensity were quantified by counting the cells in ten randomly chosen non-overlapping fields per slide.\u003c/p\u003e \u003cp\u003eRenal cell proliferation was assessed immunohistochemistry staining of Ki67. The kidney tissue paraffin sections were permeabilized with 0.3% Triton X-100, followed by blocking with 10% donkey serum and incubation with the primary antibodies against Ki67 (dilution 1:200; ab16667, Abcam). Then, the kidney tissue sections were sequentially incubated with secondary anti-rabbit IgG horseradish peroxide (dilution 1:200; 7074S, CST). Positive staining was detected via a 3,3\u0026rsquo;-diaminobenzidine (D8001, Sigma-Aldrich) reaction. Tissue images were captured under a microscope (DMI4000 B, Leica).\u003c/p\u003e \u003cp\u003eThe blood urea nitrogen (BUN) and serum creatinine (SCr) levels were determined by the Urea Nitrogen Assay Kit (D799849, Sangon Biotech) and Creatinine Assay kit (D799853, Sangon Biotech) according to the manufacturers\u0026rsquo; protocol, respectively. Blood samples of rats were collected after injecting EVs for 48 h. Centrifuge the blood at 2500 rpm for 10 min, collect the supernatant and store at -80\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProteomic Analysis of MSC-EVs\u003c/h2\u003e \u003cp\u003eThe normoxic and hypoxic EVs samples were prepared and analyzed in triplicates (n\u0026thinsp;=\u0026thinsp;3). The primary experimental procedures for proteomics analysis include protein extraction, trypsin digestion, liquid chromatograph mass spectrometer (LC-MS/MS) analysis and data analysis. The resulting MS/MS data were processed using the DIA-NN search engine (Version 1.8). Tandem mass spectra were searched against the Human UniProt database (20376 entries) concatenated with a reverse decoy database. Subcellular localization analysis of the differentially expressed protein in MSC-EV was identified by PSORTbsoftware (v3.0). Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for KEGG pathway enrichment analysis. Reactome pathway annotation and WikiPathways pathway annotations were identified by Reactome database and WikiPathways database. The proteomics analysis in our research is supported by Jingjie PTM BioLabs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment the levels of reactive oxygen species (ROS)\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe fluorescent probe Dihydroethidium (DHE) (S0063, Beyotime) was used to detect the ROS levels of fresh-frozen kidney tissue. On the other hand, 10 mM 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA, S0033S, Beyotime) was used to assess the ROS production of NRK-52E in situ visualization. Nuclei were stained with DAPI before captured the image under microscopy. Fluorescence intensity of staining was measured by Image J (Version 1.54h 15, Wisconsin, USA). Six representative visual fields of each group were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssessment the levels of mitochondrial membrane potential (Δψm)\u003c/h2\u003e \u003cp\u003eNRK-52E were plated in confocal petri dishes. Cells were treated with drugs and then loaded with the potentiometric dye 500 nM TMRE (C2001S, Beyotime) at 37\u0026deg;C in cell culture chamber for 20 min and Hoechst 33342(C1027, Beyotime) for 5 min. The staining was viewed by a confocal scanning microscope after washing 3 times with PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of GSH and GSSG levels\u003c/h2\u003e \u003cp\u003eThe concentration of GSH and GSSG of both NRK-52E and fresh rat kidney tissue were quantified by the GSH content detection kit (colorimetric method) (D799613, Sangon Biotech) and the GSSG content detection kit (colorimetric method) (D799615, Sangon Biotech). For tissue, weigh 0.1g of fresh kidney tissue, and then add 1mL of Reagent One. Use a homogenizer gently grind tissue samples on ice. Centrifuge samples at 8000g and 4\u0026deg;C for 10 minutes and save the supernatant for testing. Then, proceed with the tests as the manufacturer's instructions. The absorbance at 412-nm wavelength was measured by a microplate reader (Aligent).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection of siRNA\u003c/h2\u003e \u003cp\u003eMSCs were seeded in a 10-cm dish at 70% confluence one day prior to transfection. Nucleotides formed transfection complexes with Lipofectamine 2000 (11668027, Thermo Fisher), and were added to cells and incubated for 6\u0026ndash;8 h prior to refreshing the medium. Small interfering RNAs (siRNAs) were synthesized by Sangon Biotech Co., Ltd. based on the following sequences. siGSTO1-214: sense 5'-GCCUGAGUGGUUCUUUAAGAATT-3', antisense 5'-UUCUUAAAGAACCACUCAGGCTT-3'. hGSTO1-411: sense 5'-CCUUGGUAGGAAGCUUUAUUATT-3', antisense 5'-UAAUAAAGCUUCCUACCAAGGTT-3'. hGSTO1-411: sense 5'-GUUAAAUGAGUGUGUAGACCATT-3', antisense 5'-UGGUCUACACACUUUAACTT-3'. FAM labeled siRNA was used to assess the efficiency of transfection. Cells which expressed green fluorescence stably were considered to successful transfection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was initially extracted from both treated and control cell samples using a TRIzol\u0026trade; Reagent (15596026, Thermal Fisher), following the manufacturer's protocol. Subsequently, RNA samples were reverse transcribed into complementary DNA (cDNA) using a HiScript IV RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) kit (R423-01, Vazyme). Specific primers designed against the sequences of interest and a reference gene were utilized for qPCR amplification. The qPCR reactions were carried out in triplicate using a ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme) to monitor DNA synthesis in real time. Fluorescence data collected during the annealing phase were used to calculate the threshold cycle values for each target gene. Expression levels were normalized to β-actin. Here is the sequence of primers: β-actin sense 5\u0026rsquo;-CACCATTGGCAATGAGCGGTTC-3\u0026rsquo; and antisense 5\u0026rsquo;-AGGTCTTTGCGGATGTCCACGT-3\u0026rsquo;; GSTO1 sense 5\u0026rsquo;-GAAGACGACCTTCTTTGGTGGC-3' and antisense 5\u0026rsquo;-CTTCATGGCTGCCATCCACAGT-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using GraphPad Prism 10.0. We used the Shapiro-Wilk test to assess the normality of the data distribution. Data that were normally distributed are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). For comparing two groups, an unpaired t-test was used to determine statistical significance. For multiple group comparisons, one-way analysis of variance (ANOVA) was performed. A P-value of less than 0.05 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHypoxia promotes proliferation and alters transcriptome of MSCs\u003c/h2\u003e \u003cp\u003eAfter 24 h of hypoxia pre-treatment, MSCs retained characteristic spindle-shaped morphology with a radial distribution but exhibited a significantly increased degree of cell fusion compared to those cells in normoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The CCK-8 assay validated a marked increase in the hypoxic group, indicating that hypoxia substantially promotes cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Western blot analysis further demonstrated a significant up-regulation of HIF-1α expression in MSCs under hypoxic conditions. Additionally, activation of the AKT signaling pathway, known to be associated with cell proliferation, was observed, with significant increases in the levels of AKT, phosphorylated AKT (p-AKT), and the p-AKT/AKT ratio in the hypoxic group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTranscriptomic analysis of hypoxia-pretreated MSCs revealed profound alterations in gene expression profiles, as illustrated in the heatmap of DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). GO analysis unveiled a significant enrichment of these DEGs in cellular membrane components and transporter activities, both of which are closely linked to the production and secretion of extracellular vesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAlterations in the MSC characteristics and yield of EVs following hypoxia preconditioning\u003c/h2\u003e \u003cp\u003eTEM revealed that EVs from both normoxic and hypoxic conditions displayed similar morphologies, appearing as round or elliptical vesicles with intact structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The expression levels of specific membrane protein markers associated with EVs, such as CD9, CD63, and CD81 were determined by Western blot. The results showed increased expression of CD9 and CD63 in hypoxic EVs, although the alteration in CD81 was not statistically significant (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMeasurement of protein concentration in EVs produced by 1x10\u003csup\u003e7\u003c/sup\u003e MSCs revealed that the EVs from hypoxia pre-treated MSCs had a significantly higher concentration compared to those from the normoxic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). NTA demonstrated that the size of EVs ranged from 50\u0026ndash;500 nm in both groups, suggesting no significant difference (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). However, the particle concentration in the hypoxic group was significantly elevated compared to the normoxic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eFurthermore, we examined the expression of proteins involved in EV secretion in MSCs, specifically PRAS40 and RAB22a. PRAS40 exhibited a marked rise under 5% oxygen with significantly increased expression of HIF-1α under 10% and 5% oxygen conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-J). Although RAB22a did not increase under 10% oxygen conditions, it was significantly up-regulated under 5% and 3% oxygen conditions (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHypoxic EVs exhibited superior efficacy in promoting recovery from renal IRI\u003c/h2\u003e \u003cp\u003ePKH26-labeled EVs were systemically administered to rats undergoing IRI. After 48 hours, PKH26 fluorescence was clearly detectable within the kidney tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). H\u0026amp;E staining revealed numerous necrotic areas in the proximal epithelium and abundant tubular protein casts in the IRI -affected kidneys. In contrast, treatment with both normoxic and hypoxic EVs resulted in a reduction of tubular lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Renal injury scoring, based on structural alterations, indicated that hypoxic EVs were more effective at alleviating morphological changes associated with IRI compared to their normoxic counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Ki67, a marker of proliferation, demonstrated that enhanced tissue proliferation following IRI, interestingly, further augmented by both normoxic and hypoxic EVs treatments (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, F). TUNEL staining unveiled IRI-induced apoptosis, markedly attenuated by both normoxic and hypoxic EVs administrations (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRenal function was assessed by measuring SCr and BUN. Rats with renal IRI that received PBS showed a rapid increase in SCr and BUN levels. In contrast, treatment with normoxic and hypoxic EVs significantly attenuated these increases (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I). Notably hypoxic EV-treated kidneys exhibited lower renal injury scores, higher Ki67 expression, fewer apoptotic cell counts, and decreased SCr levels compared to those treated with normoxic EVs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC-EVs promote anti-apoptosis of renal tubular epithelial cells\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo model hypoxic injury to renal tubular epithelial cells, rat tubular epithelial cell line NRK-52E were exposed to Antimycin (AMA), an inhibitor of mitochondrial electron transport chain. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, AMA induced a dose-dependent reduction in NRK-52E viability, a concentration of 100 \u0026micro;M leading to approximately a 50% reduction in cell viability. Treatment with both normoxic EVs and hypoxic EVs restored cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To investigate the uptake of MSC-EVs by renal tubular epithelial cells \u003cem\u003ein vitro\u003c/em\u003e, MSCs were labeled with PKH-26, allowing the secreted EVs to carry red fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Internalization of PKH26-labeled EVs was confirmed in NRK-52E cells. Quantitative analysis revealed that hypoxic EVs exhibited enhanced cellular uptake compared to those from normoxic conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTUNEL staining revealed that AMA induced apoptosis in renal tubular epithelial cells. Both normoxic EVs and hypoxic EVs effectively attenuated cell apoptosis. Particularly, hypoxic EVs demonstrating superior efficacy in protecting renal tubular epithelial cells from AMA- induced injury (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eHypoxia preconditioning up-regulated antioxidant stress pathway revealed by the proteomics of EVs\u003c/h2\u003e \u003cp\u003eTo elucidate the underlying mechanism by which hypoxic EVs exhibit superior efficacy for renal IRI, we conducted mass spectrometry analysis of the proteomic cargo of EVs. PCA clearly distinguished the proteomic signatures of hypoxic EVs from those of normoxic EVs, indicating that hypoxia alters the protein profiles of MSC-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We identified significant differences in protein expression levels between the two groups, with 264 proteins up-regulated and 288 proteins down-regulated in hypoxic EVs (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Among these significant regulated proteins, GSTO1, an enzyme involved in the redox reaction of GSH, exhibited the most pronounced difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Unsupervised hierarchical clustering analysis confirmed the distinct protein patterns between normoxic and hypoxic EVs, as shown in the heat map (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Subcellular localization classification of the differentially expressed proteins indicated that the predominant proteins were extracellular (38.59%) and cytoplasmic (19.75%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Functional enrichment analysis of the up-regulated proteins revealed significant enrichment in GSH synthesis metabolism pathways, such as cysteine and methionine metabolism and glutathione metabolism pathways, as indicated by KEGG analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Moreover, highlighted significant enrichment of pathways associated with nuclear factor erythroid 2\u0026ndash;related factor 2 (NRF2), a key regulator of cellular resistance to oxidative stress. This includes pathways related to nuclear events mediated by NFE2L2, the KEAP1-NFE2L2 pathway, photodynamic therapy-induced NRF2 survival signaling, and the NRF2 pathway itself (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). Collectively, these findings suggest that hypoxia preconditioning enhances the expression of antioxidant stress-related proteins in MSC-EVs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eValidation of hypoxic EVs in alleviating renal IRI via antioxidant mechanisms\u003c/h2\u003e \u003cp\u003eWe first evaluated the differential effects of hypoxic and normoxic EVs on oxidative stress in rat kidney tissue. The results demonstrated that IRI significantly elevated ROS levels. Notably, hypoxic EVs markedly reduced ROS levels in IRI-affected kidneys compared to normoxic EVs (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, C). \u003cem\u003eIn vitro\u003c/em\u003e assays with NRK-52E cells exposed to AMA corroborated this trend, showing that ROS production and mitochondrial membrane potential changes mirrored those observed in rat kidney tissue (Figures B, D, and E). Furthermore, we assessed levels of GSH, the foremost substance involved in antioxidant. Renal IRI markedly diminished GSH levels and increased GSSG levels, thereby reducing the GSH/GSSG ratio. However, hypoxic EVs treatment significantly restored GSH and GSSG levels in the IRI kidney, surpassing the restorative effects of normoxic EVs. Ultimately, the GSH/GSSG ratio in hypoxic EV group saw a significant increase, even reaching levels comparable to the sham group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further validated the protein level of GSTO1, identified as the most differentially expressed protein in the hypoxic EVs. The results confirmed that GSTO1 was up-regulated indeed in both hypoxia pre-treated MSCs and their daughter EVs (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-K). To investigate the role of GSTO1 in mitigating oxidative stress in hypoxic EVs, we knock down GSTO1 using three different siRNAs. Except for siGSTO1-214, both siGSTO1-411 and siGSTO1-601 effectively silenced the mRNA and protein expression of GSTO1 in MSCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL, M). Hypoxic EVs from MSCs transfected with both siGSTO1-411 and siGSTO1-601 didn\u0026rsquo;t reduce the ROS levels of renal tubular epithelial cells as effectively as those transfected with a negative control siRNA (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN, O). Furthermore, GSTO1 knockdown diminished the ability of the hypoxic EVs to restore the mitochondrial membrane potential in the renal tubular epithelium (Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, C) and reduced the protective effect against AMA-induced apoptosis (Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB, D).\u003c/p\u003e \u003cp\u003eUpon treating IRI model rats with hypoxic EVs in which GSTO1 was knocked down, we observed notable changes. ROS level significantly increased compared to the control group receiving hypoxic EVs (Figures \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA, D). Additionally, a noticeable dip in Ki67 expression and an increment in apoptotic were spotted (Figures \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB, C, E, F). Further analysis of kidney function revealed that hypoxic EVs with GSTO1 knockdown was less effective in mitigating elevated SCr and BUN levels resulting from IRI compared to the hypoxic EVs group (Figures \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA, B). Taken together, these findings indicate that GSTO1 in hypoxic EVs is essential for alleviating renal IRI.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIRI is the leading cause of AKI, resulting in damage to renal tubules and rapid deterioration of renal function. The current study demonstrates that hypoxia not only stimulates the proliferation of MSCs but also enhances the secretion of EVs. Additionally, MSC-EVs pre-treated with hypoxia show a pronounced ability to protect against renal IRI. Importantly, we have presented novel evidence that hypoxia pre-treated MSC-EVs mitigate acute renal IRI through an anti-oxidative stress mechanism, with the antioxidant protein GSTO1 playing a crucial role in this protective process.\u003c/p\u003e \u003cp\u003eConventional \u003cem\u003ein vitro\u003c/em\u003e cell culture typically occurs under ambient oxygen conditions (21% O\u003csub\u003e2\u003c/sub\u003e), referred to as \u0026lsquo;normoxia\u0026rsquo;. In contrast, \u003cem\u003ein vivo\u003c/em\u003e, MSCs are often situated in niches with lower oxygen tensions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Culturing MSCs under hypoxic conditions aims to mimic this natural microenvironment. MSCs residing in hypoxic niches, with oxygen tensions ranging from 3\u0026ndash;9%, are capable of self-renewal, proliferation, migration, and differentiation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While previous studies have demonstrated that hypoxia preconditioning can enhance MSC proliferation and differentiation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the precise mechanisms remained elusive. Our study identified that hypoxia could activate the HIF-1α/AKT pathway to promote MSCs proliferation. Extensive cellular responses to hypoxic stress are typically mediated by HIF-1α [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. And, overexpression of HIF-1α has been shown to enhance MSCs proliferation and osteogenic capacity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The AKT signaling pathway plays a key role in cell proliferation, and previous research has shown that HIF-1α is involved in the up-regulation of p-AKT protein expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Hence, this evidence supports that the HIF-1α/AKT pathway involve in the proliferation of MSC under hypoxic conditions.\u003c/p\u003e \u003cp\u003eMSCs participate in tissue repair through endocrine or paracrine mechanisms, with EVs being one of their main means of communication [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These EVs play a crucial role in tissue repair by delivering proteins and genetic materials. Notably, the generation of EVs is influenced by the microenvironment. The impact of MSC-EVs can be amplified under certain physical and biological stimuli, such as hypoxia, lipopolysaccharide, and TNF-α. These conditions not only boost EV production but may also modify their contents, ultimately enhancing their beneficial effects [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The RAB family comprises key signaling molecules involved in EVs production, while PRAS40 is involved in the release of EVs following cellular stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The production of MSC-EVs significantly increased under hypoxic culture conditions, with the activation of RAB22a and PRAS40 observed in MSCs in this study. Furthermore, the plasma membrane plays a crucial role in shaping and facilitating the functions of EVs during their generation. It is actively involved in cell signaling, membrane adaptability, and uptake by recipient cells. Interestingly, our transcriptome analysis of MSCs revealed that under hypoxic conditions, signaling pathways related to cell membrane components and transporter activities associated with EV production are activated.\u003c/p\u003e \u003cp\u003eCertain researchers have documented that hypoxic EVs are effective in mitigating organ ischemia injury, such as in the brain and limbs, by transporting microRNAs or proteins [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Zhang et al reported that hypoxic preconditioning of MSCs can enhance the repair of injured kidneys, with increased angiogenesis and antioxidant effects playing a role [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Consequently, the therapeutic effects of hypoxic EVs on renal IRI were explored in this study. The results indicated that hypoxia preconditioning enhances the ability of MSC-EVs to repair IRI kidneys, improving both renal morphology and function. During AKI, tubular cell apoptosis and proliferation occur. Both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments showed that hypoxia preconditioning of MSC-EVs significantly reduced tubular cell apoptosis and promoted their proliferation, demonstrating a stronger protective effect against injury.\u003c/p\u003e \u003cp\u003eEVs rely on their biologically active internal components, including proteins, lipids, and nucleic acids, to carry out their functions. However, it remains unclear which of these components plays the central role. While substantial research has focused on alterations in miRNAs within EVs, less attention has been paid to their protein content, which can directly impact recipient cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Recently, EVs have emerged as a novel mechanism for delivering proteins [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Eirin et al highlighted in their study on MSC-EV-mediated kidney injury repair that the delivery of IL-10 protein is a key factor in the protective effects of MSC-EVs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. All of this underscores the potential significance of proteins in the cellular communication of MSC-EVs. To further elucidate the mechanisms by which hypoxic EVs alleviate renal injury, a proteomic analysis of the protein cargo of MSC-EVs was performed. Compared to normal MSC-EVs, hypoxic EVs exhibited a substantial accumulation of anti-oxidative stress proteins, with GSTO1 being the most significantly elevated.\u003c/p\u003e \u003cp\u003eOxidative stress is a crucial factor in the pathogenesis of ischemic kidney injury, and it has been confirmed that MSC-EVs reduce oxidative reactions and protect kidney function [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, it was also found that MSC-EVs significantly reduced oxidative stress levels in injured kidneys, with hypoxic EVs showing an even greater effect, demonstrating their potent antioxidant properties. GSTO1 is an important enzyme that promotes the reduction of ROS through redox reactions involving GSH. It has been shown to play a significant role in protecting the function of kidneys in end-stage renal disease [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It has been reported that GSTO1 is involved in the regulation of oxidative stress and protective effects in various diseases [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. To further verify the role of GSTO1 in hypoxic EVs, GSTO1-knockdown hypoxic EVs were obtained and used to treat ischemic renal injury. The data show that in the absence of GSTO1, the antioxidant effects of hypoxic EVs were significantly reduced, along with their protective effects on tubular cells, resulting in diminished kidney function protection. Thus, this finding suggests that the antioxidant protein GSTO1 plays a pivotal role in the protective effects of hypoxic EVs against renal IRI.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that EVs derived from hypoxia pre-treated MSCs offer renal protection against IRI. The cargo of antioxidant proteins, particularly GSTO1, plays a pivotal role in alleviating acute renal IRI. Consequently, our findings present a promising avenue for the clinical treatment of renal IRI.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACEIs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Angiotensin-converting enzyme inhibitors\u003c/p\u003e\n\u003cp\u003eAKI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Acute kidney injury\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAKT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Serine/threonine kinase\u003c/p\u003e\n\u003cp\u003eAMA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Antimycin A\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eARBs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Angiotensin receptor blockers\u003c/p\u003e\n\u003cp\u003eBUN\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Blood urea nitrogen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDEGs\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Differential express genes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eETC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;mitochondrial electron transport chain\u003c/p\u003e\n\u003cp\u003eEVs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Extracellular vesicles\u003c/p\u003e\n\u003cp\u003eGO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gene ontology\u003c/p\u003e\n\u003cp\u003eGSTO1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Glutathione S-Transferase Omega 1\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;hematoxylin\u0026ndash;eosin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026alpha; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hypoxia-inducible factor-1\u0026alpha;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehuMSCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Human umbilical cord MSCs\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIRI\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Renal ischemia-reperfusion injury\u003c/p\u003e\n\u003cp\u003eKEGG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Kyoto Encyclopedia of Genes and Genomes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMSCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mesenchymal stromal cells\u003c/p\u003e\n\u003cp\u003eNRF2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Nuclear factor erythroid 2\u0026ndash;related factor 2\u003c/p\u003e\n\u003cp\u003eNTA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Nanoparticle tracking analysis\u003c/p\u003e\n\u003cp\u003ep-AKT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphorylated Serine/threonine kinase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003ePCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Principal component analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ep-PRAS40\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Phosphorylated proline-rich AKT substrate of 40 kDa\u003c/p\u003e\n\u003cp\u003eRAB22a\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RAB22a, Member RAS Oncogene Family\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive oxygen species\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSCr \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;serum creatinine\u003c/p\u003e\n\u003cp\u003eTEM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Transmission electron microscopy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not use AI-generated work in this\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emanuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFY,\u0026nbsp;JL,\u0026nbsp;LZ\u0026nbsp;and\u0026nbsp;PT L: experiments conducting, data acquirement and original manuscript preparation;\u0026nbsp;FY, JL, LZ , PT L and XY Z: manuscript editing/validation and data analysis;\u0026nbsp;WG\u0026nbsp;, TL, KX Y\u0026nbsp;and\u0026nbsp;XY Z: technical support and experimental assistance;\u0026nbsp;GY Z, JS\u0026nbsp;and\u0026nbsp;XY Z: study design guidance, manuscript reviewing and editing;\u0026nbsp;JS and XY Z: project supervision and funding acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (Grant No. 81900618); the Tai-Shan Scholar Program from Shandong Province, China (Grant No. tsqn202103116); the Pudong New Area Science and Technology Development Fund\u0026nbsp;(Grant No. PKJ2020-Y04; the Natural Science Foundation of Fujian Province (Grant No. 2023J01183); the Program of Scientific and Technological Development of Weifang (Grant No. 2023GX026).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets to support current study are available from the corresponding author on reasonable request. The sequence data in the current study will be available in NCBI\u0026rsquo;s BioProject and can be accessed by the public (ID: PRJNA1064232).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE \u0026nbsp;partner repository with the dataset identifier PXD057088.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project has been approved by the ethics committee of Shanghai Children\u0026apos;s Medical Center, Shanghai Jiao Tong University School of Medicine. All human subjects gave informed consent for tissue donation. Project title: \u0026ldquo;The study on the mechanism by which hypoxia promotes mesenchymal stem cell-derived extracellular vesicle production and enhances the repair capacity of ischemia-reperfusion kidney injury\u0026rdquo;, protocol number SCMCIRB-Y2019005, approved 2019/2/25. For animal experiments, the study was approved by the Shanghai Children\u0026apos;s Medical Center, Shanghai Jiao Tong University School of Medicine, protocol number SCMCIACUC-K2019042, approved 2019/2/15.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to submission of the manuscript and agree to publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKellum JA, Romagnani P, Ashuntantang G, Ronco C, Zarbock A, Anders HJ. Acute kidney injury. Nat Rev Dis Primers. 2021;7:52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAghajani Nargesi A, Lerman LO, Eirin A. Mesenchymal stem cell-derived extracellular vesicles for kidney repair: current status and looming challenges. Stem Cell Res Ther. 2017;8:273.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonventre JV, Yang L. 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Mol Cell Biochem. 2023;478:1645\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Liu L, Huo Y, Yang Y, Wang Y. Hypoxia-pretreated human MSCs attenuate acute kidney injury through enhanced angiogenic and antioxidative capacities. Biomed Res Int. 2014;2014:462472.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu G, Zheng G, Ge M, Wang J, Huang R, Shu Q, et al. Functional proteins of mesenchymal stem cell-derived extracellular vesicles. Stem Cell Res Ther. 2019;10:359.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeldolesi J. Unconventional Protein Secretion Dependent on Two Extracellular Vesicles: Exosomes and Ectosomes. Front Cell Dev Biol. 2022;10:877344.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEirin A, Zhu XY, Puranik AS, Tang H, McGurren KA, van Wijnen AJ, et al. Mesenchymal stem cell-derived extracellular vesicles attenuate kidney inflammation. Kidney Int. 2017;92:114\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAparicio-Trejo OE, Aranda-Rivera AK, Osorio-Alonso H, Martinez-Klimova E, Sanchez-Lozada LG, Pedraza-Chaverri J et al. Extracellular Vesicles in Redox Signaling and Metabolic Regulation in Chronic Kidney Disease. Antioxid (Basel). 2022;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuevas-Lopez B, Romero-Ramirez EI, Garcia-Arroyo FE, Tapia E, Leon-Contreras JC, Silva-Palacios A et al. NAC Pre-Administration Prevents Cardiac Mitochondrial Bioenergetics, Dynamics, Biogenesis, and Redox Alteration in Folic Acid-AKI-Induced Cardio-Renal Syndrome Type 3. Antioxid (Basel). 2023;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCimbaljevic S, Suvakov S, Matic M, Pljesa-Ercegovac M, Pekmezovic T, Radic T, et al. Association of GSTO1 and GSTO2 Polymorphism with Risk of End-Stage Renal Disease Development and Patient Survival. J Med Biochem. 2016;35:302\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolsch H, Linnebank M, Lutjohann D, Jessen F, Wullner U, Harbrecht U, et al. Polymorphisms in glutathione S-transferase omega-1 and AD, vascular dementia, and stroke. Neurology. 2004;63:2255\u0026ndash;60.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"hypoxia pretreated, mesenchymal stromal cells, extracellular vesicles, renal ischemia reperfusion injury, anti-oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-5266177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5266177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) provide significant protection against renal ischemia-reperfusion injury (IRI). Hypoxia is considered an important method for enhancing the tissue repair capabilities of MSCs. However, the specific effects of hypoxia on MSCs and MSC-EVs, as well as their therapeutic potential for renal IRI, remain unclear. In this study, we investigated the alterations in MSCs and the production of MSC-EVs following hypoxia pre-treatment, and further explored the key intrinsic mechanisms by which hypoxic MSC-EVs treat renal IRI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman umbilical cord MSCs were cultured under normoxic and hypoxic conditions. Proliferation and related pathways were measured, and RNA sequencing was used to detect changes in the transcription profile. MSC-EVs from both normoxic and hypoxic conditions were isolated and characterized. \u003cem\u003eIn vivo\u003c/em\u003e, the localization and therapeutic effects of MSC-EVs were assessed in a rat renal IRI model. Histological examinations were employed to assess the structure, proliferation, and apoptosis of IRI kidney tissue respectively. Renal function was measured by analyzing serum creatinine and blood urea nitrogen levels. \u003cem\u003eIn vitro\u003c/em\u003e, the therapeutic potential of MSC-EVs were measured in renal tubular epithelial cells injured by antimycin A. Protein sequencing analysis of hypoxic MSC-EVs was conducted, and the depletion of Glutathione S-Transferase Omega 1 (GSTO1) in hypoxic MSC-EVs was performed to verify its key role in alleviating renal injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHypoxia alters MSCs transcription, promotes their proliferation, and increases the production of EVs. Hypoxia-pretreated MSC-EVs exhibited a superior ability to mitigate renal IRI, enhancing proliferation and reducing apoptosis of renal tubular epithelial cells both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Protein profiling of the EVs revealed an accumulation of numerous anti-oxidative stress proteins, with GSTO1 being particularly prominent. GSTO1 knock down was significantly reduced the antioxidant and therapeutic effects in renal IRI of hypoxic MSC-EVs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHypoxia significantly promotes MSC-EVs generation and enhances the therapeutic effect of EVs on renal IRI. The effect of antioxidant stress induced by GSTO1 is one of the most important underlying mechanisms. Our findings underscore that hypoxia-pretreated MSC-EVs represent a novel and promising therapeutic intervention for renal IRI.\u003c/p\u003e","manuscriptTitle":"Enhanced Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Renal Ischemic Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 11:46:32","doi":"10.21203/rs.3.rs-5266177/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-11-04T01:03:23+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-04T01:00:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-30T00:05:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2024-10-28T02:59:13+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2024-10-19T06:43:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f0d32ff-a5f7-4afb-ae46-0f9ccccc8fa5","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:03:29+00:00","versionOfRecord":{"articleIdentity":"rs-5266177","link":"https://doi.org/10.1186/s13287-025-04166-z","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-02-04 15:57:49","publishedOnDateReadable":"February 4th, 2025"},"versionCreatedAt":"2024-11-14 11:46:32","video":"","vorDoi":"10.1186/s13287-025-04166-z","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04166-z","workflowStages":[]},"version":"v1","identity":"rs-5266177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5266177","identity":"rs-5266177","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}