Platelet-derived microparticles induce CD8+ T cell senescence in end-stage renal disease via the transfer of microRNA-550a-5p

Platelet-derived microparticles induce CD8+ T cell senescence in end-stage renal disease via the transfer of microRNA-550a-5p | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Platelet-derived microparticles induce CD8 + T cell senescence in end-stage renal disease via the transfer of microRNA-550a-5p Jinghong Zhao, Yuewen Sun, Qigang Lan, Yaqin Wang, Minhua Xie, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8468315/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Infection is a leading cause of high mortality in patients with chronic kidney disease (CKD), in which T cell dysfunction is considered as a typical feature in CKD patients. Although platelets are active participants in immune response, their role in regulating T cell function in CKD patients remains unclear. In this study, we demonstrate that CD8 + T cell senescence may be a major contributor to the immunosuppressed state of end-stage renal disease (ESRD) patients, and that platelets derived from ESRD patients can induce premature CD8 + T cell senescence, potentially through mediating mitochondrial dysfunction. Further investigations reveal that platelet-derived microparticles (PMPs) from ESRD patients promote CD8 + T cell senescence via transferring miR-550a-5p, thereby decreasing PMPCB expression and inducing NDUFS8 cleavage failure. Finally, inhibition of miR-550a-5p and pretreatment with Nicotinamide Mononucleotide is capable of preventing platelet-induced CD8 + T cell senescence in ESRD patients. Collectively, these findings suggest that the ectopic transient expression of platelet-derived miR-550a-5p in CD8 + T cells promotes cellular senescence by regulating the PMPCB-NDUFS8 axis, which can be exploited to treat the ESRD-associated immunosenescence. Biological sciences/Cell biology/Senescence Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Immunology/Lymphocytes/T cells/CD8-positive T cells Health sciences/Diseases/Kidney diseases/Chronic kidney disease/End-stage renal disease End-stage renal disease CD8+ T cells cellular senescence platelets miR-550a-5p PMPCB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Chronic kidney disease (CKD) has emerged as a critical global public health concern and is accompanied by a high incidence of multiple complications [ 1 , 2 ] . It has been well recognized that infection is a leading cause of morbidity and mortality in patients with CKD, particularly those with end-stage renal disease (ESRD) [ 3 ] . Notably, the immunosuppressed state of CKD patients is characterized by dysfunction of innate and adaptive immune system, resulting in a proinflammatory milieu and a decreased cellular immunity [ 4 ] . Unfortunately, the understanding of the susceptibility to infection and immune dysfunction in CKD patients is still limited, and more effective measures are still needed in clinical practice. The adaptive immune system possesses highly specific features in response to threats from pathogen and memory function [ 5 ] . T cells are the major cell components and responsible for mediating cell-based immune responses [ 6 ] . In CKD, there is a profound change in the total number and function of circulating T cells [ 7 , 8 ] . Plenty of studies, including our own, have demonstrated a decrease in T cell number in CKD, as well as a concomitant increase in terminal differentiation and proinflammatory T cells [ 9 , 10 ] . Furthermore, in the presence of numerous stimulations, such as uremia and dialysis, T cell replicative capacity is impaired, resulting in functional exhaustion and senescence [ 11 , 12 ] . These two states share overlapping phenotypic and functional characteristics but differ in their distinct molecular and developmental signatures [ 13 ] . T cell exhaustion is marked by increased expression of a suite of inhibitory receptors, including programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) [ 14 ] , while T cell senescence is defined by features such as telomere shortening and loss of CD28, along with reciprocal expression of CD57 [ 15 ] . However, the underlying mechanisms by which T cell exhaustion and senescence contribute to immune dysfunction upon ESRD remain largely unexplored. Platelets, one of the most prevalent blood components, are well-known for their hemostatic and thrombotic functions. Interestingly, accumulating evidence now indicates that platelets also actively modulate immune responses [ 16 , 17 ] . It is becoming increasingly clear that platelets can not only directly interact with immune cells by changing their surface expression of P-selectin or other receptors, such as Toll-like receptors, but also can release a number of granules and microparticles that regulate adaptive immunity [ 18 ] . Our findings have also revealed disrupted platelet homeostasis in the CKD milieu, with enhanced platelet-immune cell interactions and increased platelet-derived microparticles (PMPs) release [ 19 , 20 ] . Given that plasma microparticles can alter T cell function and phenotype, and that PMPs are the most abundant microparticles present in the blood [ 21 ] , it is important to improve our understanding of the role of platelet-T cell interactions in immune dysregulation upon ESRD. In this study, we demonstrate that platelets from ESRD patients transfer miR-550a-5p through the release of PMPs, leading to miR-550a-5p ectopic expression in CD8 + T cells, which induces mitochondrial dysfunction-mediated cellular senescence through negatively regulating peptidase-mitochondrial processing subunit beta (PMPCB) expression, culminating in cleavage failure of NADH: ubiquinone oxidoreductase core subunit S8 (NDUFS8) and a decrease in NAD + level. Importantly, inhibition of miR-550a-5p and supplement with Nicotinamide Mononucleotide (NMN) significantly attenuate mitochondrial dysfunction and cellular senescence of CD8 + T cells. Hence, our findings uncover a novel platelet-CD8 + T cell interaction and provided a promising strategy to prevent CD8 + T cell senescence in ESRD. 2. Materials and Methods 2.1 Study Subjects A total of 75 CKD patients in stages 3–5, who were not on dialysis and aged between 25 and 45 years, were enrolled from the Department of Nephrology at Xinqiao Hospital (Chongqing, China), and 45 healthy volunteers were recruited. All participants provided informed consent to take part in this study (Table S1 ). The study protocol was approved by the ethics committee of Xinqiao Hospital, Army Medical University (Institutional Review Board at No. 2025-267-01). 2.2 Animal Experiments ​ MiR-550a-5p agomir and negative control were synthesized by Biomics (The sequences listed in Table S2). Male C57BL/6J mice (8 weeks old) were purchased from Chongqing Tengxin Bioscience (Chongqing, China). Mice received tail vein intravenous injections of miR-550a-5p agomir or the negative control at a dose of 20 nmol once every four days for four weeks. All mice were euthanized 48 hours after the final injection. All the experimental procedures were approved by the Animal Care and Use Committee of the Army Medical University (No. AMUWEC20255414). 2.3 Isolation of Human CD8 + T cells and Platelets Human whole blood was collected using sodium citrate anticoagulant. CD8 + T cells were isolated using the ImunoSep Human CD8 + T Cell Enrichment Kit (Precision Biomedicals, Shenzhen, China). The purity of the isolated CD8 + T cells, assessed by flow cytometry, exceeded 95%. Purified cells were resuspended in RPMI 1640 medium (Meilunbio, Dalian, China) supplemented with 10% fetal bovine serum (Corning, NY, USA). Platelet isolation was performed as previously described [ 20 ] . Briefly, blood was centrifuged at 160g for 15 minutes at room temperature. The upper two-thirds of the platelet-rich plasma (PRP) was collected and then recentrifuged at 160g for 10 minutes in the presence of 0.1 µg/mL prostacyclin. The resulting PRP was then centrifuged at 1000g for 10 minutes. The platelet pellet was resuspended in Tyrode's-HEPES buffer containing 10 µmol/L indomethacin and 0.1 µg/mL prostacyclin. Flow cytometric analysis of CD45 + events confirmed that leukocyte contamination in the platelet suspension was less than 0.001%. 2.4 PMPs Isolation PMPs were isolated as previously described [ 22 ] . Purified platelets were resuspended in saline with 1 mM calcium and 1 U/mL thrombin at 37℃ for 60 minutes. Activation was terminated by adding EDTA to a final concentration of 20 mM, followed by centrifugation at 2000g for 20 minutes to remove platelets. The supernatants containing PMPs were centrifugated at 20,000g for 90 minutes at 4 o C, the pellet was collected for downstream analysis. 2.5 Co-culture CD8 + T cells co-cultured with platelets as previously described [ 16 ] . Purified CD8 + T cells and platelets were co-cultured at a ratio of 1:450 in prepared RPMI 1640 medium. T cell activation was induced using coated anti-CD3 monoclonal antibody and soluble anti-CD28 monoclonal antibody (all Kxbiotech, beijing, China). Interleukin-2 (IL-2; Beyotime, Shanghai, China) was added to maintain cell expansion. Cells were cultured at 37oC in a humidified atmosphere containing 5% CO 2 for 7 days. According to the experimental design, CD8 + T cells were harvested for subsequent analysis. 2.6 Flow Cytometry and Cell Sorting Platelets were identified were identified based on scatter properties and CD41 + PMPs were detected as particles < 1 µm in size and identified as CD41 + and Annexin V + (Thermo Fisher Scientific) [ 19 ] . Platelet activation was assessed by measuring surface expression of P-selectin and activated integrin GPIIb/IIIa (both Thermo Fisher Scientific). To determine T cell phenotype in peripheral blood, 100 µL of whole blood was processed to isolate peripheral blood mononuclear cells (PBMCs). For human T cells, PBMCs were stained with a lineage antibody cocktail comprising anti-CD3, anti-CD4, anti-CD8, anti-CD57, anti-PD-1, and anti-CD28 antibodies. For mouse T cells, a cocktail containing anti-CD3, anti-CD8, anti-CD44, and anti-CD153 antibodies was used. To determine mitochondrial membrane potential, cells were incubated with 100 nM tetramethylrhodamine methyl ester (TMRM; Thermo Fisher Scientific) at 37 o C for 20 min. To determine reactive oxygen species (ROS) production, cells were incubated with 10 µM dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO, USA) at 37 o C for 30 min. Senescence-associated β-galactosidase (SA-β-Gal) activity was evaluated using the CellEvent™ Senescence Green Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. To determine p21 and γ-H2A.X levels, cells were washed and fixed resuspended with 1 ml Foxp3 Fixation/Permeabilization working solution (Thermo Fisher Scientific) and incubated at room temperature for 30 min. Then, the cells were permeabilized with Permeabilization buffer (Thermo Fisher Scientific) in the presence of anti-phospho-H2A.X (Ser19) and anti-p21 at room temperature for another 30 min. For mouse CD8 + T cells sorting, PMBCs were stained with indicative antibodies and isolated by fluorescence-activated cell sorting. Cells were detected using a BD FACSverse flow cytometer (BD Biosciences) or sorted using a FACSAriaII (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star Inc.). 2.7 Relative Telomere Length Measurement To determine telomere length in CD8 + T cells, genomic DNA was extracted from isolated CD8 + T cells using the Fast DNA Extraction Kit (Bioroyee, Beijing, China) according to the manufacturer's protocol. Relative telomere length was determined using the Relative Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories, San Diego, CA, USA). 2.8 Cell Mito Stress Test Oxygen consumption rate (OCR) was measured using the Seahorse XF HS Mini Analyzer (Agilent Technologies, Santa Clara, CA, USA). CD8 + T cells were seeded in XF8 cell culture microplates and analyzed with the Seahorse XFp Cell Mito Stress Test Kit (Agilent Technologies). Following three baseline respiration measurements without additives, sequential injections of mitochondrial modulators were performed: 1.5 µM oligomycin (ATP synthase inhibitor), by the addition of 2 µm FCCP and Rotenone & antimycin A (0.5 µm Rot/AA) for further measurements. OCR was expressed as units of picomoles (pmol) per minute, and data were normalized against the number of cells. 2.9 NAD+/NADH Assay NAD+/NADH levels were quantified using the NAD+/NADH Assay Kit (LABLEAD, Beijing, China) according to the manufacturer's instructions. Concentrations were normalized to total protein content, expressed as µmol NAD+/NADH per mg protein. Relative NAD+/NADH levels were then analyzed and compared with control groups. 2.10 Cell Transfection CD8⁺ T cells were transfected with PMPCB overexpression plasmids (Youbio Biotechnology, Hunan, China), PMPCB-targeting siRNA or miR-550a-5p mimics/inhibitors (all from Biomics Biotechnologies, Jiangsu, China), using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were harvested 48 hours post-transfection for downstream analysis (listed in Table S4, 5). 2.11 Construction of Reporter Plasmids and Dual Luciferase Reporter Analysis A DNA fragment containing the predicted miR-550a-5p binding site within the 3' untranslated region (3' UTR) of human PMPCB was amplified from genomic DNA by PCR. The PCR product was cloned into the pmirGLO™ dual-luciferase vector (RabbitBio, Chengdu, China) and verified by Sanger sequencing. To assess binding specificity, site-directed mutagenesis was performed on the putative miR-550a-5p binding site (5'-CAGGCAC-3' → 5'-AGUUAGA-3') in the PMPCB 3' UTR. For luciferase assays, CD8 + T cells were co-transfected with either pmirGLO-PMPCB-WT or pmirGLO-PMPCB-Mut, and miR-550a-5p mimic or negative control mimic (mimic-NC). Luciferase activity was measured 24 hours post-transfection using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA). Firefly luciferase signals were normalized to Renilla luciferase activity for data analysis. 2.12 RNA Extraction and Quantitative real-time polymerase chain reaction (qPCR) Analysis Total RNA was extracted from PMPs or CD8⁺ T cells using TRIzol™ reagent (Thermo Fisher Scientific). Reverse transcription was performed using The RevertAid First Strand cDNA Synthesis Kit (MCE, Monmouth Junction, NJ, USA) for mRNA analysis or the microRNA (miRNA) 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) for miRNA analysis. qPCR was carried out using SYBR Green Master Mix (MCE) on a real-time PCR system. Small nuclear RNA U6 (RNU6) and β-actin served as endogenous controls for miRNA and mRNA quantification, respectively. Relative gene expression was calculated using the 2^(−ΔΔCt) method. Primer sequences are provided in Supplementary Table S6, 7. 2.13 MicroRNA Expression Profiling Total RNA was isolated from PMPs obtained from five ESRD patients and five healthy controls using the TaKaRa RNAiso Plus reagent. The integrity and concentration of the extracted RNA were verified prior to library construction. Sequencing libraries were prepared and sequenced by BGI Genomics (Shenzhen, China) to generate miRNA expression profiles. Raw miRNA expression data were subjected to quantile normalization and log₂ transformation. miRNAs with expression levels above the sample-specific median were classified as highly expressed. Differential expression analysis identified miRNAs with > 1.2-fold upregulation or < 0.833-fold downregulation for subsequent bioinformatic investigation. 2.14 Western Blot Mitochondria were isolated using a mitochondrial isolation kit (ACMEC, Shanghai, China). Proteins were extracted from the purified mitochondrial fraction using RIPA lysis (Beyotime) buffer supplemented with protease and phosphatase inhibitors. Protein expression levels were determined using anti-p21, anti-γ-H2A.X, anti-PMPCB, anti-NDUFS8, anti-β-actin, and anti-COX IV. Then, the membranes were incubated with appropriate secondary antibodies (all Abcam) for 1 h at 37°C and imaged by an Odyssey Infrared Imaging System (LI-COR Biosciences). 2.15 Immunofluorescence CD8 + T cells were fixed with 4% PFA, and blocked with 2% BSA. Cells were incubated overnight at 4°C with anti-p21, anti-γ-H2A.X, or anti-PMPCB, followed by FITC-conjugated secondary antibody (37°C, 1 h). Nuclei were stained with DAPI (Beyotime). Visualization was performed using a Zeiss LSM800 NLO confocal microscopy (Carl Zeiss). 2.16 Bioinformatics Analysis The human PBMC single-cell RNA sequencing dataset (GSE233315) was obtained from the NCBI Gene Expression Omnibus (GEO) database. Data processing and analysis were performed using the Seurat R package (version 5.3.0). Quality control filtering was applied as follows: cells with gene counts between 500 and 4,000 were retained, UMI counts > 500 and < 15,000 were included; cells exhibiting high ambient RNA contamination (log10(genes per UMI) ≤ 0.8) were excluded; cells with mitochondrial gene content exceeding 10% were removed. 2.17 Statistical Analysis Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software). Data are presented as mean ± standard deviation (SD). The number of independent biological replicates (N) is specified in respective figure legends. Comparisons between two groups were analyzed using paired or unpaired two-tailed Student's t-tests, as appropriate. Comparisons among three or more groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc tests. Associations between variables were assessed using Spearman's rank correlation coefficient. Statistical significance was defined as p < 0.05 for all analyses. 3. Results 3.1 Peripheral blood CD8⁺ T cells develop premature senescence in ESRD patients Initially, we re-analyzed a published single-cell RNA sequencing (scRNA-seq) data from PBMC obtained from healthy controls and ESRD patients (GSE233315). Interestingly, we found that the markers of T cell exhaustion, PDCD1 and CTLA-4 were comparable between healthy and ESRD patients, while Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed a robust upregulated in cellular senescence score, particularly in CD8 + T effector cells (Fig. 1 a, b and S1a, b). Similarly, t-distributed Stochastic Neighbor Embedding (t-SNE) analysis by flow cytometry identified a mild increase in PD-1 in CD4 + and CD8 + T cells from patients with CKD stage 3–5 compared to healthy controls (Fig. 1 c-e and S1c). Notably, CD57, a recognized marker used to identify terminally differentiated senescent T cells, and the proportion of CD57 + cells within the CD4 + T cell population showed no significant, while it was significantly higher within CD8 + T cells of patients with CKD stage 3–5 (Fig. 1 c-e and S1c). Furthermore, we found that CD57 + CD8 + T cells were progressively developed along with advancing CKD and were most significant in ESRD (CKD stage 5) patients (Figure S1 d). Besides, CD28 is an age-related immune biomarker, and the loss of CD28 coupled with the presence of CD57 (CD28 − CD57 + ) constitutes a key hallmark of immunosenescence. Flow cytometry also demonstrated that the proportion of CD28 − CD57 + cells was significantly increased within the CD8 + T cell compartment of ESRD patients, whereas no significant difference in their proportion within CD4 + T cells (Fig. 1 f, g). Taken together, these data indicate that the T cell immune incompetence develops in the pathophysiological context of ESRD due to multifaceted dysregulation, and that premature CD8 + T cell senescence potentially serving as a central contributor. 3.2 CD8 + T cells exhibit upregulated senescence biomarkers accompanied with mitochondrial dysfunction in ESRD Cellular senescence is defined as the irreversible loss of replicative potential caused by persistent DNA damage, accompanied by increased β-galactosidase activity, cell cycle arrest and telomere shortening [ 15 ] . Indeed, we observed significantly elevated expression of the DNA damage marker γ-H2A.X and the cyclin-dependent kinase inhibitor p21 in CD8⁺ T cells from ESRD patients compared with healthy controls (Fig. 2 a-c and S2a, b), as well as significantly increased SA-β-Gal activity and expression of the senescent proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ) (Fig. 2 d, e). Meanwhile, relative telomere length analysis revealed progressive age-dependent telomere shortening in CD8⁺ T cells in both groups, with significantly shorter telomeres observed in ESRD patients (Fig. 2 f). Furthermore, senescence-associated mitochondrial dysfunction is considered an essential component of the senescent phenotype [ 23 ] . Accordingly, we detected a decrease in OCR, maximal respiratory capacity and Adenosine triphosphate (ATP) production (Fig. 2 g). Mitochondrial dysfunction was further confirmed by the loss of mitochondrial membrane potential (Fig. 2 h). Mitochondria are sensitive to oxidative stress, which is the most prominent characteristic of the CKD milieu, and we also observed ROS overproduction in CD8⁺ T cells of ESRD patients (Fig. 2 i). Collectively, these findings demonstrate that CD8⁺ T cells from ESRD patients exhibit a comprehensive senescent phenotype characterized by well-established senescence markers and severe mitochondrial dysfunction. 3.3 Platelets from ESRD patients induce premature CD8 + T cell senescence of by mediating mitochondrial dysfunction We next sought to identify the role of platelets from ESRD patients in premature CD8⁺ T cell senescence. We first established a co-culture system in which platelets derived from healthy donors or ESRD patients respectively were co-cultured with CD8⁺ T cells derived from healthy donors (Fig. 3 a). As shown, CD8 + T cells cultured alone or co-cultured with platelets derived from healthy individuals did not show a cellular senescence phenotype over time (Fig. 3 b-e). However, CD8 + T cells co-cultured with platelets derived from ESRD patient exhibited a dramatic acceleration of premature CD8⁺ T cell senescence by day 5, as evidenced by the upregulation of γ-H2A.X, p21, SA-β-Gal and CD57 (Fig. 3 b, c). Besides, upregulation of senescent proinflammatory cytokines and shortened telomeres were also detected in CD8⁺ T cells after co-cultured with platelets from ESRD patients (Fig. 3 d, e). Surprisingly, mitochondrial membrane potential decreased, impaired mitochondrial respiratory function and ROS overproduction were observed in CD8⁺ T cells at day 3 after co-cultured with platelets from ESRD patients, preceding the appearance of cellular senescence (Fig. 3 f-h). In contrast, pretreatment with N-acetylcysteine (NAC), an antioxidant agent, effectively prevented mitochondrial dysfunction and cellular senescence in CD8 + T cells induced by platelets from ESRD patients (Figure S3), indicating that platelets from ESRD patients might be involved in the initiation of premature CD8 + T cell senescence through mediating mitochondrial dysfunction. 3.4 PMPs from ESRD patients mediate mitochondrial dysfunction and cellular senescence in CD8 + T cells Previously, we have shown that CKD promotes platelet hyperreactivity, inducing the large-scale release of PMPs, which have been reported to mediate mitochondrial dysfunction [ 19 , 24 ] . Consistently, remarkably increased platelet activity including P-selectin, activated GPIIb/IIIa expression, and PMPs release were verified in ESRD patients (Figure S4a, b). Notably, PMPs release in the peripheral blood of ESRD patients was correlated positively with cellular senescence biomarkers and ROS levels in CD8 + T cells, whereas correlated negatively with mitochondrial membrane potential, hinting that PMPs release may facilitate mitochondrial dysfunction and cellular senescence of CD8 + T cells in ESRD milieu (Fig. 4 a-f). To interrogate whether PMPs directly regulate premature CD8 + T cells senescence, CD8 + T cells were treated with PMPs ex vivo (Fig. 4 g). Accordingly, Dio-labeled PMPs were successfully internalized by CD8 + T cells (Fig. 4 g). PMPs from ESRD patients significantly induced CD8 + T cells to undergo mitochondrial dysfunction and cellular senescence, while NAC dramatically relieved these effects (Fig. 4 h-l and S4c-l). Hence, these results demonstrate that a direct action of PMPs on premature CD8 + T cells senescence by inducing mitochondrial dysfunction. 3.5 PMPs promote premature CD8 + T cell senescence in an miR-550a-5p dependent manner PMPs are enriched in platelet microRNAs, which play an important role in regulating mitochondrial dysfunction and cellular senescence [ 25 ] . PMPs were isolated from healthy controls and ESRD patients for miRNA profile detection. There were profound differences in PMP expression between healthy controls and ESRD patients, with 109 miRNAs upregulated and 57 miRNAs downregulated in ESRD-derived PMPs (Fig. 5 a). Importantly, we identified that the greatest change in miRNA expression was miR-550a-5p, which was also confirmed to be the most remarkably upregulated (Fig. 5 a, b). Unexpectedly, the Starbase, TargetScan and miRDB databases revealed that miR-550a-5p was not expressed in mice (data not shown). Interestingly, the miR-550a-5p content in CD8 + T cells was much lower than in PMPs, while the miR-550a-5p content in in CD8 + T cells of ESRD patients was higher than in healthy controls, suggesting that high levels of miR-550a-5p in ESRD-derived PMPs were transferred to CD8 + T cells (Figure S5a). Similar results were also observed in CD8 + T cells and platelets co-cultured system (Fig. 5 c). To investigate the miR-550a-5p's role in mediating mitochondrial dysfunction and cellular senescence in CD8 + T cells, we transfected the cells with miR-550a-5p mimic, which was verified by qPCR and immunofluorescence (Figure S5b, c). Following 48 hours of transfection, we observed that overexpression of miR-550a-5p induced a markedly elevated levels of cellular senescence and mitochondrial dysfunction biomarkers (Fig. 5 d-j and S5d), as well as increased senescent proinflammatory cytokines expression and shortened telomeres (Fig. 5 k, l). Conversely, treatment CD8 + T cells with miR-550a-5p inhibitor significantly rescued platelets from ESRD patients-induced these effects under co-cultured system (Figure S6). Collectively, these results demonstrate platelets from ESRD patients-derived PMPs induce CD8 + T cell senescence via miR-550a-5p-mediating mitochondrial dysfunction axis. 3.6 MiR-550a-5p induce mitochondrial dysfunction and cellular senescence in CD8 + T cells via targeting PMPCB To investigate the underlying mechanisms by which miR-550a-5p regulates CD8 + T cells mitochondrial dysfunction-induced cellular senescence, bioinformatic analysis predicted 19 potential target mitochondrial genes of miR-550a-5p based on TargetScan, miRDB and MitoCarta 3.0 databases (Fig. 6 a, Table S8). Gene Ontology (GO) enrichment analysis showed that the mitochondrial genes were markedly enriched in mitochondrial protein transport (Fig. 6 b), suggesting that miR-550a-5p may induce mitochondrial dysfunction through mediating mitochondrial transport process disorders. Of note, considering that miRNAs negatively regulate gene expression at the post-transcriptional level [ 26 ] . we observed that PMPCB expression was significantly decreased at both mRNA and protein levels in CD8 + T cells of ESRD patients (Figure S7a-c). PMPCB as the catalytic subunit of mitochondrial processing peptidase, which promotes the maturation of precursor proteins transported to mitochondria [ 27 ] . Besides, we detected that co-cultured with platelets from ESRD patients can reduce PMPCB expression in CD8 + T cells (Fig. 6 c, D and S7d). Similar results were observed in CD8 + T cells when transfected with miR-550a-5p mimic (Figure S7e, f). Furthermore, bioinformatic analysis predicted perfect complementary sequences between miR-550a-5p and the PMPCB 3'-untranslated region (UTR) (Fig. 6 e). Dual-luciferase reporter assays were conducted to identify the targeting efficacy of miR-550a-5p on PMPCB. Overexpression of miR-550a-5p markedly decreased luciferase activity, while no effect was detected on PMPCB 3'-UTR mutant luciferase reporter (Fig. 6 f). Interestingly, interrogation of sequences in the 3’UTR of mouse PMPCB gene also identified a miR-550a-5p binding site (Figure S7g). Moreover, we treated mice with miR-550a-5p modified oligonucleotides (agomir). Analogous to human CD8 + T cells, cellular senescence, mitochondrial dysfunction and PMPCB downregulation were also detected in CD8 + T cells of mice treated with miR-550a-5p agomir (Figure S7h, Figure S8). Above all, these results demonstrate that miR-550a-5p could directly bind and downregulate PMPCB expression. To determine the role of PMPCB in mitochondrial dysfunction and cellular senescence in CD8⁺ T cells, we first transfected siRNA against PMPCB in CD8⁺ T cells ex vivo (Fig. 7 a). As expected, the absent of PMPCB induced decreased mitochondrial membrane potential and impaired mitochondrial respiratory, but increased ROS levels (Fig. 7 b-d). Consistently, upregulation of cellular senescence biomarkers and pro-inflammation cytokines, as well as shortened telomeres were also observed in CD8⁺ T cells with PMPCB knockdown, indicating its important role in maintaining mitochondrial homeostasis (Fig. 7 e-k and S9a, b). On the contrary, overexpression of PMPCB significantly alleviated miR-550a-5p-induced mitochondrial dysfunction, ultimately mitigating cellular senescence in CD8⁺ T cells (Figure S10). Taken together, these data underscore a critical role of miR-550a-5p in mediating premature CD8 + T cell senescence by targeting PMPCB, which is essential for the maintenance of mitochondrial homeostasis. 3.7 NMN counteracts platelets from ESRD patients-induced and NDUFS8-mediated cellular senescence in CD8 + T cells To further elucidate PMPCB-mediated CD8 + T cell senescence mechanisms, we identified six PMPCB-interacting proteins using a protein-protein interaction analysis (Fig. 8 a). Notably, the nuclear-encoded NDUFS8 is a subunit of monitoring NADH dehydrogenase in mitochondrial complex I, which would be cleaved by mitochondrial processing peptides [ 28 ] . We found no significant difference in NDUFS8 expression in CD8 + T cells between ESRD patients and healthy controls, as well as co-cultured with platelets from ESRD patients or treatment with the miR-550a-5p mimic in human and miR-550a-5p agomir in mouse CD8 + T cells (Fig. 8 b, c and S11a-d). Interestingly, we detected the precursor and mature forms of NDUFS8 in CD8 + T cells of ESRD patients, while only mature form of NDUFS8 existed in CD8 + T cells of healthy controls (Fig. 8 b). Moreover, co-cultured with platelets of ESRD patients or treatment with miR-550a-5p mimic in human and miR-550a-5p agomir in mouse CD8 + T cells induced cleavage failure of NDUFS8, while overexpression of PMPCB significantly alleviated these effects (Fig. 8 c and S11c, d). These results indicate the regulatory effect of PMPCB on the cleavage of NDUFS8. As reported, NDUFS8 suppression will impair electron transfer from NADH to ubiquinone, thereby decreasing NAD+/NADH ratio, which has been reported to drive cells into senescence [ 29 , 30 ] . Indeed, ESRD patients exhibited decreased NAD + levels and NAD+/NADH ratios in CD8 + T cells (Fig. 8 d). Meanwhile, co-cultured with platelets of ESRD patients or treatment with miR-550a-5p mimic resulted in a similar reduction in NAD + levels and NAD+/NADH ratios, conversely, overexpression of PMPCB partially ameliorated this phenomenon (Fig. 8 e and S11e). Therefore, we treated CD8 + T cells with NMN, an intermediate in NAD + biosynthesis which plays a vital role in a variety of biological processes, and analyzed their mitochondrial function [ 31 ] . NMN treatment effectively attenuated mitochondrial dysfunction in CD8 + T cells, thereby preventing platelets from ESRD patients-induced cellular senescence (Fig. 8 f-j and S11f-i). Moreover, we performed an intraperitoneal injection of NMN (500 mg/kg per mouse) once every 3 days in miR-550a-5p agomir-treated mice for 4 weeks. Consistently, NMN treatment effectively alleviated mitochondrial dysfunction and cellular senescence in CD8 + T cells of mice (Figure S12). Above all, these findings indicate that NMN may serve as a promising therapeutic drug for alleviating platelets from ESRD patients-mediated mitochondrial dysfunction and cellular senescence. 4. Discussion Infection is a leading cause of morbidity and mortality in patients with CKD [ 3 ] . T cell activation significantly influences an individual's immune function and susceptibility to infection [ 6 ] . Although disturbances in T cell homeostasis in CKD have been extensively studied, significant knowledge gaps remain regarding the underlying mechanisms of T cell dysfunction [ 10 ] . In this study, we identify that premature CD8 + T cell senescence is a key feature of altered T cell homeostasis in ESRD patients. We further demonstrate that platelets accelerate this senescence process by transferring PMPs containing miR-550a-5p into CD8⁺ T cells, thereby downregulating PMPCB expression and inhibiting NDUFS8/NAD + axis. Conversely, inhibiting miR-550a-5p function or overexpressing PMPCB ameliorated platelet-induced premature CD8⁺ T cell senescence in ESRD. Patients with CKD are more prone to develop premature age-related diseases [ 11 ] . Immunosenescence contributes to the development of infections, cancers and autoimmune diseases, as well as accelerating the progression of fatal diseases, including cardiovascular and metabolic disorders, non-alcoholic fatty liver disease, and neurodegenerative diseases [ 32 , 33 ] . However, the characteristics and molecular mechanisms underlying immune cell senescence in ESRD remain incompletely understood. In this study, integrated bioinformatic analysis and clinical sample validation revealed that CD8⁺ T cells exhibit more pronounced senescent change characters compared to CD4⁺ T cells, which is consistent with previous reports [ 9 , 34 ] . Currently, the cause of T cell senescence in CKD is attributed to the chronic inflammation resulting from uremia or maintenance dialysis [ 11 ] . In our study, we enrolled patients who were not on dialysis, and observed that CD8 + T cell senescence progressively developed along with advancing CKD. Interestingly, we demonstrate that another factor, co-cultured with platelets from ESRD patients can induce CD8 + T cell senescence, which cells derived from healthy controls. While the role of platelets in hemostasis is well known, they are also considered a cellular component of the innate immune system [ 17 ] . Based on our findings, we discovered that, in addition to uremia, platelets can serve as immune regulatory cells to mediate CD8 + T cell senescence. As known, platelets can directly interact with immune cells via specific receptors, such as CD41, CD42 and CD154, but our study excluded this effect using a flow cytometry gating strategy [ 17 ] . Besides, platelets can also release granules or microparticles to regulate innate and adaptive immunity. We also confirmed that increased PMP release in ESRD patients, and found that PMP levels correlated positively with CD8⁺ T cell senescence. Of note, recently studies indicate that platelet-releasing particles are closely associated with cellular senescence, but their performance is inconsistent in different milieu [ 35 , 36 ] . The release of platelet factor 4 by platelets in young mice alleviates cognitive impairment in older mice, while the release of miR-146 is upregulated in the hippocampus and frontal cortex of patients with Alzheimer disease. Given platelets have fully functional miRNA machinery, we isolated PMPs and performed a miRNA profiling to identify the leading cause of CD8 + T cell senescence. Here, we detect that miR-550a-5p is the most significantly upregulated miRNA in PMPs of ESRD patients and that it accelerates CD8 + T cell senescence. In addition, the upregulation of miR-550a-5p can also promote the proliferation and migration of cancer cells [ 37 ] . Taken together, it is plausible that miR-550a-5p may be implicated in proliferation and migration of cancer cells via mediating CD8 + T cell senescence and immunosuppression. On the contrary, treating CD8 + T cells with a miR-550a-5p inhibitor effectively attenuates cell senescence when co-cultured with platelets from ESRD patients. Interestingly, the aspirin, an antiplatelet agent, also exhibits anti-aging properties via antioxidant, proteostatic and immunometabolic mechanisms [ 38 – 40 ] . Hence, it is conceivable that antiplatelet may serve a promising strategy for alleviating CD8 + T cell senescence. Mitochondrial dysfunction and associated ROS production have also been found in aging and many aging-associated pathological conditions, whereas the impairment of mitochondrial function and morphology also govern the senescent phenotype [ 23 ] . In our study, we observe that, after co-cultured with platelets of ESRD patients, mitochondrial disorders in CD8 + T cells preceded the appearance of senescence phenotypes, while NAC treatment significantly rescue these effects, indicating that platelets of ESRD may induce CD8 + T cell senescence by mediating mitochondrial dysfunction. In order to characterize the molecular mechanisms by which miR-550a-5p regulates CD8 + T cell senescence, we for the first time identify PMPCB as a target gene of miR-550a-5p using bioinformatics and dual luciferase reporter analyses. PMPCB is the catalytic subunit of the mitochondrial processing peptidase, which identifies and cleaves mitochondrial importing signals necessary for the proper transport of proteins into the mitochondria, and thus facilitates their maturation. A decrease or mutation in PMPCB levels would induce mitochondrial processing peptidase dysfunction, thereby accumulating the processing intermediates such as frataxin [ 41 ] . Meanwhile, PMPCB has been shown to play a key role in regulating oxidant stress and apoptosis mediated by mitochondrial dysfunction [ 42 , 43 ] . Here, knockdown of PMPCB in CD8 + T cells exhibit similar effects, including decreased mitochondrial membrane potential, impaired mitochondrial respiratory function and increased ROS production. However, the specific mechanisms by which PMPCB deficiency induces mitochondrial dysfunction are not fully elucidated. Further investigations reveal that the decreased PMPCB impairs the maturation of the pro-NDUFS8 precursor protein, leading to a reduced NAD+/NADH ratio. NDUFS8 is a nuclear-encoded core subunit of human mitochondrial Complex I, and NDUFS8 deficiency displays an apparent reduction in NAD+/NADH ratio and a decrease in mitochondrial membrane potential [ 44 ] . Crucially, intracellular reduced NAD⁺ levels are intimately linked to aging [ 45 ] , while NMN treatment effectively attenuated mitochondrial dysfunction and cellular senescence in CD8 + T cells. Hence, our findings suggest that therapeutic intervention of PMPCB-NDUFS8 axis opens new opportunities to promote CD8 + T cell senescence and immune function. In summary, these findings demonstrate that platelet-derived miR-550a-5p is responsible for CD8⁺ T cell senescence via the PMPCB-NDUFS8 axis in ESRD. This study not only substantially advances our understanding of the immune-platelet interactions, but also provided a potential strategy to prevent premature CD8⁺ T cell senescence upon ESRD. Declarations Data availability Data from this study are available upon a reasonable request to the corresponding author. Acknowledgments The investigators thank all patients who participated in this study. Funding This study was supported by the Joint Funds of the National Natural Science Foundation of China (No. U22A20279), Key program of the Natural Science Foundation of China (Nos. 82530025 and 82030023), Chongqing Graduate Research and Innovation Project (No. CYB23281). CRediT authorship contribution statement Yuewen Sun : Investigation, Formal analysis, Writing – original draft. Qigang Lan: Conceptualization, Investigation, Formal analysis, Supervision, Writing – original draft, Writing – review & editing. Yaqin Wang : Investigation, Data curation. Minhua Xie: Investigation, Data curation. Xinyi Wu: Investigation, Data curation. Liangjing Lv : Investigation, Data curation. Qin Xin: Investigation, Data curation. Jun Chen: Investigation, Data curation. Xiaoyi Zhong: Investigation, Data curation. Mengying Yao: Investigation, Data curation. Shuiqin Gong: Investigation, Data curation. Siyan Zhou: Investigation, Data curation. Junping Wang: Conceptualization, Supervision, Writing – review & editing. Jinghong Zhao: Conceptualization, Supervision, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary information Supplementary information is available at (journal name)’s website 1. Supplemental Material 2. MicroRNA profile References Foreman KJ, Marquez N, Dolgert A, et al. Forecasting life expectancy, years of life lost, and all-cause and cause-specific mortality for 250 causes of death: reference and alternative scenarios for 2016-40 for 195 countries and territories. Lancet. 2018. 392(10159): 2052–2090. Kovesdy CP. Epidemiology of chronic kidney disease: an update 2022. Kidney Int Suppl (2011). 2022. 12(1): 7–11. Cheikh Hassan HI, Tang M, Djurdjev O, Langsford D, Sood MM, Levin A. 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Additional Declarations There is no conflict of interest Supplementary Files GraphicalAbstract.jpg Graphical Abstract MicroRNAprofile.xls MicroRNA profile UneditedWBgel.pdf Unedited WB gel SupplementalMaterial.docx Supplemental Material Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 11 Mar, 2026 Review # 2 received at journal 10 Mar, 2026 Reviewer # 2 agreed at journal 23 Feb, 2026 Review # 1 received at journal 05 Feb, 2026 Reviewer # 1 agreed at journal 22 Jan, 2026 Reviewers invited by journal 22 Jan, 2026 Submission checks completed at journal 29 Dec, 2025 Editor assigned by journal 28 Dec, 2025 First submitted to journal 28 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":9321063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeripheral blood CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells develop premature senescence in CKD patients. \u003c/strong\u003e(a) t-SNE projection of PBMCs from healthy controls and ESRD patients, colored by major cell clusters identified through scRNA-seq analysis. Key T cell subsets and natural killer (NK) cells are indicated. TFH: T follicular helper cells. (b) Comparison of senescence scores across different T cell populations between the ESRD group and healthy group. (c) t-SNE plot of T cells overlaid with expression of indicated cell surface receptors determined by flow cytometry. (d, e) Quantitative analysis of the percentages of PD-1\u003csup\u003e+ \u003c/sup\u003ecells and CD57\u003csup\u003e+ \u003c/sup\u003ecells within CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells between healthy controls and CKD patients (n = 30). (f, g) Representative flow cytometric analysis of the percentage of CD28\u003csup\u003e-\u003c/sup\u003eCD57\u003csup\u003e+ \u003c/sup\u003ecells within CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells between healthy controls and ESRD patients (n = 30). Data are presented as mean ± SD and were analyzed by 2-tailed, unpaired Student's t-test. ns: no significance. *P \u0026lt; 0.05, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/1e0df1d4fe492bfe691e267b.png"},{"id":101207133,"identity":"bfbf5f5a-9a1e-4ae1-a644-9f30018aa530","added_by":"auto","created_at":"2026-01-27 09:57:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4846812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells exhibit upregulated senescence biomarkers accompanied with mitochondrial dysfunction in ESRD. \u003c/strong\u003e(a, b) Representative immunofluorescence staining images showing γ-H2A.X and p21 expression in CD8⁺ T cells of healthy controls and ESRD patients. Scale bar, 20 μm. (c) Western blot analysis of γ-H2A.X and p21 protein levels in CD8⁺ T cells of healthy controls and ESRD patients (n = 3). (d) Flow cytometric analysis of SA-β-GAL activity in CD8⁺ T cells of healthy controls and ESRD patients (n = 30). (e) qPCR analysis of TNF-α and IFN-γ expression in CD8⁺ T cells of healthy controls and ESRD patients (n = 30). (f) Correlation analysis between relative telomere length and age in CD8⁺ T cells of healthy controls and ESRD patients (n = 30). (g) OCR in CD8⁺ T cells of healthy controls and ESRD patients and quantitation of basal respiration, ATP-linked respiration, and maximal respiration (n = 9). (h, i) Flow cytometric analysis of TMRM (h) and ROS (i) levels in CD8⁺ T cells of healthy controls and ESRD patients (n = 30). Data are presented as mean ± SD and were analyzed by 2-tailed, unpaired Student's t-test and Pearson’s correlation analysis (F). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/8b4a91934e0fe001f92ab00f.png"},{"id":101171948,"identity":"7efa9e51-97cc-428c-aee6-3386a905bea2","added_by":"auto","created_at":"2026-01-27 00:11:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4317044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlatelets from ESRD patients induce premature CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell senescence of by mediating mitochondrial dysfunction. \u003c/strong\u003e(a) Schematic diagram illustrating the co-culture system of platelets and CD8⁺ T cells. (b) Flow cytometric analysis of γ-H2AX and p21 expression levels, and SA-β-Gal activity in CD8⁺ T cells co-cultured with platelets from healthy controls or ESRD patients (n=3). (c) Flow cytometric analysis of the proportion of CD57⁺ cells in CD8⁺ T cells co-cultured with platelets from healthy controls or ESRD patients (n = 3). (d) qPCR analysis of TNF-α and IFN-γ expression in CD8⁺ T cells from different groups (n = 6). (e) Relative telomere length in CD8⁺ T cells from different groups (n = 6). (f, g) Flow cytometric analysis of TMRM (f) and ROS (g) levels in CD8⁺ T cells across different groups (n = 3 for each analysis). (h) OCR in CD8⁺ T cells from different groups, with quantitation of basal respiration, ATP production, and maximal respiration (n = 9). Data are presented as mean ± SD and were analyzed by one-way ANOVA. ns: no significance. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/1088b61baa238f3d70e7c49e.png"},{"id":101171949,"identity":"7b3f60fc-f97f-460e-885d-c9a943fb8e33","added_by":"auto","created_at":"2026-01-27 00:11:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4930461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlatelets from ESRD patients-released MPs mediate mitochondrial dysfunction and cellular senescence in CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells. \u003c/strong\u003e(a–f) Correlation analysis between the levels of PMPs in peripheral blood and the proportion of CD57⁺ cells (a), SA-β-Gal activity (b), γ-H2AX level (c), p21 expression (d), TMRM levels (e), and ROS levels (f) in CD8⁺ T cells from ESRD patients (n = 30). (g) Schematic diagram and representative immunofluorescence images illustrating the internalization of PMPs by CD8⁺ T cells. PMPs were labeled with DiO (green), and nuclei of CD8⁺ T cells were stained with Hoechst (blue). Scale bar, 20 μm. (h–l) Flow cytometric analysis of SA-β-Gal activity (h), γ-H2AX level (i), p21 expression (j), TMRM levels (k), and ROS levels (l) in CD8⁺ T cells treated with PMPs from healthy controls or ESRD patients (n = 6). Data are presented as mean ± SD and were analyzed by one-way ANOVA or Pearson’s correlation analysis (a-f). **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/55997e3fa03f4fe5b8b6c1ca.png"},{"id":101206299,"identity":"ea763f0a-7f0b-4c8d-90e1-7bf8db796a85","added_by":"auto","created_at":"2026-01-27 09:55:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4809023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePMPs promote premature CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cell senescence in an miR-550a-5p dependent manner. \u003c/strong\u003e(a) Volcano plot illustrating differentially expressed miRNAs between healthy controls and ESRD patients, with thresholds set at |log₂(fold change)| \u0026gt; 0.263 (equivalent to \u0026gt;1.2 or \u0026lt;0.833-fold change) and p\u0026lt;0.05. (b) qPCR analysis of the expression of selected miRNAs in PMPs of healthy controls and ESRD patients (n = 12). (c) qPCR analysis of miR-550a-5p expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells of healthy controls and ESRD patients (n = 6). (d-l) Flow cytometric analysis of SA-β-Gal activity (d), γ-H2AX level (e), p21 expression (f), TMRM levels (g), and ROS levels (h) in CD8⁺ T cells treated with or without miR-550a-5p mimic (n = 6). (i) OCR in CD8⁺T cells treated with or without miR-550a-5p mimic and quantitation of basal respiration, ATP production, and maximal respiration (n = 9). (j) Flow cytometric analysis the proportion of CD57⁺ cells in CD8⁺ T cells treated with or without miR-550a-5p mimic (n = 6). (k-l) qPCR analysis of TNF-α and IFN-γ expression (k) and relative telomere length (l) in CD8\u003csup\u003e+\u003c/sup\u003e T cells treated with or without miR-550a-5p mimic (n = 6). Data are presented as mean ± SD and were analyzed by 2-tailed, unpaired Student's t-test or one-way ANOVA (b, f). ns: no significance. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/aae521ffe6ff551080d25e8a.png"},{"id":101171938,"identity":"ff19f03d-f487-4968-9bca-8720ce17197b","added_by":"auto","created_at":"2026-01-27 00:11:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6861437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-550a-5p regulates PMPCB expression via directly binding to its 3’UTR region. \u003c/strong\u003e(a) Venn diagram showing the overlap between predicted target genes of miR-550a-5p from TargetScan and miRDB databases and human mitochondrial genes sourced from the MitoCarta database. (b) GO enrichment analysis of the predicted mitochondrial target genes. (c) Representative immunofluorescence staining images showing PMPCB expression (red) in CD8⁺ T cells co-cultured with platelets from healthy controls or ESRD patients. Mitochondria were labeled with Tom20 (green). Scale bar, 10 μm. (d) Western blot analysis of PMPCB protein levels in CD8⁺ T cells co-cultured with platelets from healthy controls or ESRD patients (n = 3). (e) Schematic illustration of the wild-type (WT) and mutant (Mut) putative miR-550a-5p binding sites within the 3′ untranslated region (UTR) of the PMPCB gene cloned into a luciferase reporter vector. (f) Relative luciferase activity in CD8⁺ T cells co-transfected with either the WT or Mut PMPCB 3′ UTR reporter plasmid and miR-550a-5p mimic or negative control (NC) miRNA. Luciferase activity was normalized to Renilla luciferase activity (n = 6). Data are presented as mean ± SD and were analyzed by 2-tailed, unpaired Student's t-test or one-way ANOVA (D). ns: no significance. **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/c452b4a41ea52a303af8b626.png"},{"id":101171950,"identity":"b542e63f-17fc-480a-a7d5-ed5f72be1bbb","added_by":"auto","created_at":"2026-01-27 00:11:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4683795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of PMPCB induces mitochondrial dysfunction and cellular senescence in CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells. \u003c/strong\u003e(a) Western blot analysis of PMPCB protein levels in CD8⁺ T cells treated with PMPCB-specific siRNA or control siRNA (n = 3). (b) Flow cytometric analysis of TMRM levels in CD8⁺ T cells treated with PMPCB-specific siRNA or control siRNA (n = 6). (c) OCR in CD8⁺ T cells treated with PMPCB-specific siRNA or control siRNA, with quantitation of basal respiration, ATP production, and maximal respiration (n = 9). (d–h) Flow cytometric analysis of reactive oxygen species (ROS) levels (d), proportion of CD57⁺ cells (e), SA-β-Gal activity (f), γ-H2AX level (g), and p21 expression (h) in CD8⁺ T cells treated with PMPCB-specific siRNA or control siRNA (n = 6). (i) Western blot analysis of γ-H2AX and p21 protein levels in CD8⁺ T cells treated with PMPCB-specific siRNA or control siRNA (n = 3). (j, k) qPCR analysis of TNF-α and IFN-γ expression (j) and relative telomere length (k) in CD8⁺ T cells treated with PMPCB-specific siRNA or control siRNA (n = 6). Data are presented as mean ± SD and were analyzed by 2-tailed, unpaired Student's t-test. ns: no significance. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/0cd92fb8f1d0c15e4530412a.png"},{"id":101206565,"identity":"0a014aa9-0b69-4c01-bbcd-c3d6b8347901","added_by":"auto","created_at":"2026-01-27 09:56:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5536754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNMN counteracts platelets from ESRD patients-induced and NDUFS8-mediated cellular senescence in CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells. \u003c/strong\u003e(a) Prediction of PMPCB-interacting proteins using the STRING database platform. (b) Western blot analysis of NDUFS8 expression in CD8⁺ T cells of healthy controls and ESRD patients (n = 3). (c) Western blot analysis of NDUFS8 expression in CD8⁺ T cells transfected with or without PMPCB overexpression plasmids under co-cultured system (n = 3). (d) Measurement of NAD⁺ content and NAD⁺/NADH ratio in CD8⁺ T cells of healthy controls and ESRD patients (n = 10). (e) Measurement of NAD⁺ content and NAD⁺/NADH ratio in CD8⁺ T cells transfected with or without PMPCB overexpression plasmids under co-cultured system (n = 6). (f-j) Flow cytometric analysis of TMRM levels (f), ROS levels (g), SA-β-Gal activity (h), γ-H2AX level (i), p21 expression (j) in CD8⁺ T cells pretreated with or without 100 µM NMN under co-cultured system (n = 6). Data are presented as mean ± SD and were analyzed by 2-tailed, unpaired Student's t-test (b, d) or one-way ANOVA. ns: no significance. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.00\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/229c4d96b17f133e7be3db3a.png"},{"id":101209262,"identity":"ea80427a-decb-4aaa-a2a3-c9562aadefbc","added_by":"auto","created_at":"2026-01-27 10:15:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":46480683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/17a28f99-aa80-4c43-875b-9a3f92559a39.pdf"},{"id":101206240,"identity":"258ea30d-45bd-40e8-9c8c-579423365a95","added_by":"auto","created_at":"2026-01-27 09:55:44","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":244440,"visible":true,"origin":"","legend":"Graphical Abstract","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/83367e3658c37deabafce56d.jpg"},{"id":101171944,"identity":"7c255828-d285-4832-bc0b-ae78b33817dc","added_by":"auto","created_at":"2026-01-27 00:11:31","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":427542,"visible":true,"origin":"","legend":"MicroRNA profile","description":"","filename":"MicroRNAprofile.xls","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/ea678d627f732b96dabaa6af.xls"},{"id":101207111,"identity":"5fc8261d-11c5-4b5c-b57f-0284a576a0d9","added_by":"auto","created_at":"2026-01-27 09:57:29","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":406735,"visible":true,"origin":"","legend":"Unedited WB gel","description":"","filename":"UneditedWBgel.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/f98db0d13ad19742947209e6.pdf"},{"id":101171940,"identity":"a45cf458-ccd1-4cb4-83bb-10ad61309b4f","added_by":"auto","created_at":"2026-01-27 00:11:31","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17073857,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8468315/v1/1a43536fca325dc475e270c7.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"\u003cp\u003ePlatelet-derived microparticles induce CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence in end-stage renal disease via the transfer of microRNA-550a-5p\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChronic kidney disease (CKD) has emerged as a critical global public health concern and is accompanied by a high incidence of multiple complications \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. It has been well recognized that infection is a leading cause of morbidity and mortality in patients with CKD, particularly those with end-stage renal disease (ESRD) \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Notably, the immunosuppressed state of CKD patients is characterized by dysfunction of innate and adaptive immune system, resulting in a proinflammatory milieu and a decreased cellular immunity \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Unfortunately, the understanding of the susceptibility to infection and immune dysfunction in CKD patients is still limited, and more effective measures are still needed in clinical practice.\u003c/p\u003e \u003cp\u003eThe adaptive immune system possesses highly specific features in response to threats from pathogen and memory function \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. T cells are the major cell components and responsible for mediating cell-based immune responses \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In CKD, there is a profound change in the total number and function of circulating T cells \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Plenty of studies, including our own, have demonstrated a decrease in T cell number in CKD, as well as a concomitant increase in terminal differentiation and proinflammatory T cells \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Furthermore, in the presence of numerous stimulations, such as uremia and dialysis, T cell replicative capacity is impaired, resulting in functional exhaustion and senescence \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. These two states share overlapping phenotypic and functional characteristics but differ in their distinct molecular and developmental signatures \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. T cell exhaustion is marked by increased expression of a suite of inhibitory receptors, including programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, while T cell senescence is defined by features such as telomere shortening and loss of CD28, along with reciprocal expression of CD57 \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. However, the underlying mechanisms by which T cell exhaustion and senescence contribute to immune dysfunction upon ESRD remain largely unexplored.\u003c/p\u003e \u003cp\u003ePlatelets, one of the most prevalent blood components, are well-known for their hemostatic and thrombotic functions. Interestingly, accumulating evidence now indicates that platelets also actively modulate immune responses \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. It is becoming increasingly clear that platelets can not only directly interact with immune cells by changing their surface expression of P-selectin or other receptors, such as Toll-like receptors, but also can release a number of granules and microparticles that regulate adaptive immunity \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Our findings have also revealed disrupted platelet homeostasis in the CKD milieu, with enhanced platelet-immune cell interactions and increased platelet-derived microparticles (PMPs) release \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Given that plasma microparticles can alter T cell function and phenotype, and that PMPs are the most abundant microparticles present in the blood \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, it is important to improve our understanding of the role of platelet-T cell interactions in immune dysregulation upon ESRD.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that platelets from ESRD patients transfer miR-550a-5p through the release of PMPs, leading to miR-550a-5p ectopic expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells, which induces mitochondrial dysfunction-mediated cellular senescence through negatively regulating peptidase-mitochondrial processing subunit beta (PMPCB) expression, culminating in cleavage failure of NADH: ubiquinone oxidoreductase core subunit S8 (NDUFS8) and a decrease in NAD\u0026thinsp;+\u0026thinsp;level. Importantly, inhibition of miR-550a-5p and supplement with Nicotinamide Mononucleotide (NMN) significantly attenuate mitochondrial dysfunction and cellular senescence of CD8\u003csup\u003e+\u003c/sup\u003e T cells. Hence, our findings uncover a novel platelet-CD8\u003csup\u003e+\u003c/sup\u003e T cell interaction and provided a promising strategy to prevent CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence in ESRD.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Subjects\u003c/h2\u003e \u003cp\u003eA total of 75 CKD patients in stages 3\u0026ndash;5, who were not on dialysis and aged between 25 and 45 years, were enrolled from the Department of Nephrology at Xinqiao Hospital (Chongqing, China), and 45 healthy volunteers were recruited. All participants provided informed consent to take part in this study (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The study protocol was approved by the ethics committee of Xinqiao Hospital, Army Medical University (Institutional Review Board at No. 2025-267-01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2 Animal Experiments\u003c/b\u003e​\u003c/h2\u003e \u003cp\u003eMiR-550a-5p agomir and negative control were synthesized by Biomics (The sequences listed in Table S2). Male C57BL/6J mice (8 weeks old) were purchased from Chongqing Tengxin Bioscience (Chongqing, China). Mice received tail vein intravenous injections of miR-550a-5p agomir or the negative control at a dose of 20 nmol once every four days for four weeks. All mice were euthanized 48 hours after the final injection. All the experimental procedures were approved by the Animal Care and Use Committee of the Army Medical University (No. AMUWEC20255414).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Isolation of Human CD8\u003csup\u003e+\u003c/sup\u003e T cells and Platelets\u003c/h2\u003e \u003cp\u003eHuman whole blood was collected using sodium citrate anticoagulant. CD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated using the ImunoSep Human CD8\u003csup\u003e+\u003c/sup\u003e T Cell Enrichment Kit (Precision Biomedicals, Shenzhen, China). The purity of the isolated CD8\u003csup\u003e+\u003c/sup\u003e T cells, assessed by flow cytometry, exceeded 95%. Purified cells were resuspended in RPMI 1640 medium (Meilunbio, Dalian, China) supplemented with 10% fetal bovine serum (Corning, NY, USA).\u003c/p\u003e \u003cp\u003ePlatelet isolation was performed as previously described \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Briefly, blood was centrifuged at 160g for 15 minutes at room temperature. The upper two-thirds of the platelet-rich plasma (PRP) was collected and then recentrifuged at 160g for 10 minutes in the presence of 0.1 \u0026micro;g/mL prostacyclin. The resulting PRP was then centrifuged at 1000g for 10 minutes. The platelet pellet was resuspended in Tyrode's-HEPES buffer containing 10 \u0026micro;mol/L indomethacin and 0.1 \u0026micro;g/mL prostacyclin. Flow cytometric analysis of CD45\u003csup\u003e+\u003c/sup\u003e events confirmed that leukocyte contamination in the platelet suspension was less than 0.001%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 PMPs Isolation\u003c/h2\u003e \u003cp\u003ePMPs were isolated as previously described \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Purified platelets were resuspended in saline with 1 mM calcium and 1 U/mL thrombin at 37℃ for 60 minutes. Activation was terminated by adding EDTA to a final concentration of 20 mM, followed by centrifugation at 2000g for 20 minutes to remove platelets. The supernatants containing PMPs were centrifugated at 20,000g for 90 minutes at 4\u003csup\u003eo\u003c/sup\u003eC, the pellet was collected for downstream analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Co-culture\u003c/h2\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells co-cultured with platelets as previously described \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Purified CD8\u003csup\u003e+\u003c/sup\u003e T cells and platelets were co-cultured at a ratio of 1:450 in prepared RPMI 1640 medium. T cell activation was induced using coated anti-CD3 monoclonal antibody and soluble anti-CD28 monoclonal antibody (all Kxbiotech, beijing, China). Interleukin-2 (IL-2; Beyotime, Shanghai, China) was added to maintain cell expansion. Cells were cultured at 37oC in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e for 7 days. According to the experimental design, CD8\u003csup\u003e+\u003c/sup\u003e T cells were harvested for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6 Flow Cytometry and Cell Sorting\u003c/b\u003e\u003c/h2\u003e \u003cp\u003ePlatelets were identified were identified based on scatter properties and CD41\u003csup\u003e+\u003c/sup\u003e PMPs were detected as particles\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;m in size and identified as CD41\u003csup\u003e+\u003c/sup\u003e and Annexin V\u003csup\u003e+\u003c/sup\u003e (Thermo Fisher Scientific) \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Platelet activation was assessed by measuring surface expression of P-selectin and activated integrin GPIIb/IIIa (both Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eTo determine T cell phenotype in peripheral blood, 100 \u0026micro;L of whole blood was processed to isolate peripheral blood mononuclear cells (PBMCs). For human T cells, PBMCs were stained with a lineage antibody cocktail comprising anti-CD3, anti-CD4, anti-CD8, anti-CD57, anti-PD-1, and anti-CD28 antibodies. For mouse T cells, a cocktail containing anti-CD3, anti-CD8, anti-CD44, and anti-CD153 antibodies was used.\u003c/p\u003e \u003cp\u003eTo determine mitochondrial membrane potential, cells were incubated with 100 nM tetramethylrhodamine methyl ester (TMRM; Thermo Fisher Scientific) at 37\u003csup\u003eo\u003c/sup\u003eC for 20 min. To determine reactive oxygen species (ROS) production, cells were incubated with 10 \u0026micro;M dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO, USA) at 37\u003csup\u003eo\u003c/sup\u003eC for 30 min. Senescence-associated β-galactosidase (SA-β-Gal) activity was evaluated using the CellEvent\u0026trade; Senescence Green Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.\u003c/p\u003e \u003cp\u003eTo determine p21 and γ-H2A.X levels, cells were washed and fixed resuspended with 1 ml Foxp3 Fixation/Permeabilization working solution (Thermo Fisher Scientific) and incubated at room temperature for 30 min. Then, the cells were permeabilized with Permeabilization buffer (Thermo Fisher Scientific) in the presence of anti-phospho-H2A.X (Ser19) and anti-p21 at room temperature for another 30 min.\u003c/p\u003e \u003cp\u003eFor mouse CD8\u003csup\u003e+\u003c/sup\u003e T cells sorting, PMBCs were stained with indicative antibodies and isolated by fluorescence-activated cell sorting.\u003c/p\u003e \u003cp\u003eCells were detected using a BD FACSverse flow cytometer (BD Biosciences) or sorted using a FACSAriaII (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Relative Telomere Length Measurement\u003c/h2\u003e \u003cp\u003eTo determine telomere length in CD8\u003csup\u003e+\u003c/sup\u003e T cells, genomic DNA was extracted from isolated CD8\u003csup\u003e+\u003c/sup\u003e T cells using the Fast DNA Extraction Kit (Bioroyee, Beijing, China) according to the manufacturer's protocol. Relative telomere length was determined using the Relative Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell Mito Stress Test\u003c/h2\u003e \u003cp\u003eOxygen consumption rate (OCR) was measured using the Seahorse XF HS Mini Analyzer (Agilent Technologies, Santa Clara, CA, USA). CD8\u003csup\u003e+\u003c/sup\u003e T cells were seeded in XF8 cell culture microplates and analyzed with the Seahorse XFp Cell Mito Stress Test Kit (Agilent Technologies). Following three baseline respiration measurements without additives, sequential injections of mitochondrial modulators were performed: 1.5 \u0026micro;M oligomycin (ATP synthase inhibitor), by the addition of 2 \u0026micro;m FCCP and Rotenone \u0026amp; antimycin A (0.5 \u0026micro;m Rot/AA) for further measurements. OCR was expressed as units of picomoles (pmol) per minute, and data were normalized against the number of cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 NAD+/NADH Assay\u003c/h2\u003e \u003cp\u003eNAD+/NADH levels were quantified using the NAD+/NADH Assay Kit (LABLEAD, Beijing, China) according to the manufacturer's instructions. Concentrations were normalized to total protein content, expressed as \u0026micro;mol NAD+/NADH per mg protein. Relative NAD+/NADH levels were then analyzed and compared with control groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Cell Transfection\u003c/h2\u003e \u003cp\u003eCD8⁺ T cells were transfected with PMPCB overexpression plasmids (Youbio Biotechnology, Hunan, China), PMPCB-targeting siRNA or miR-550a-5p mimics/inhibitors (all from Biomics Biotechnologies, Jiangsu, China), using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were harvested 48 hours post-transfection for downstream analysis (listed in Table S4, 5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Construction of Reporter Plasmids and Dual Luciferase Reporter Analysis\u003c/h2\u003e \u003cp\u003eA DNA fragment containing the predicted miR-550a-5p binding site within the 3' untranslated region (3' UTR) of human PMPCB was amplified from genomic DNA by PCR. The PCR product was cloned into the pmirGLO\u0026trade; dual-luciferase vector (RabbitBio, Chengdu, China) and verified by Sanger sequencing. To assess binding specificity, site-directed mutagenesis was performed on the putative miR-550a-5p binding site (5'-CAGGCAC-3' \u0026rarr; 5'-AGUUAGA-3') in the PMPCB 3' UTR. For luciferase assays, CD8\u003csup\u003e+\u003c/sup\u003e T cells were co-transfected with either pmirGLO-PMPCB-WT or pmirGLO-PMPCB-Mut, and miR-550a-5p mimic or negative control mimic (mimic-NC). Luciferase activity was measured 24 hours post-transfection using the Dual-Luciferase\u0026reg; Reporter Assay System (Promega, Madison, WI, USA). Firefly luciferase signals were normalized to Renilla luciferase activity for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 RNA Extraction and Quantitative real-time polymerase chain reaction (qPCR) Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from PMPs or CD8⁺ T cells using TRIzol\u0026trade; reagent (Thermo Fisher Scientific). Reverse transcription was performed using The RevertAid First Strand cDNA Synthesis Kit (MCE, Monmouth Junction, NJ, USA) for mRNA analysis or the microRNA (miRNA) 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) for miRNA analysis. qPCR was carried out using SYBR Green Master Mix (MCE) on a real-time PCR system. Small nuclear RNA U6 (RNU6) and β-actin served as endogenous controls for miRNA and mRNA quantification, respectively. Relative gene expression was calculated using the 2^(\u0026minus;ΔΔCt) method. Primer sequences are provided in Supplementary Table S6, 7.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 MicroRNA Expression Profiling\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from PMPs obtained from five ESRD patients and five healthy controls using the TaKaRa RNAiso Plus reagent. The integrity and concentration of the extracted RNA were verified prior to library construction. Sequencing libraries were prepared and sequenced by BGI Genomics (Shenzhen, China) to generate miRNA expression profiles. Raw miRNA expression data were subjected to quantile normalization and log₂ transformation. miRNAs with expression levels above the sample-specific median were classified as highly expressed. Differential expression analysis identified miRNAs with \u0026gt;\u0026thinsp;1.2-fold upregulation or \u0026lt;\u0026thinsp;0.833-fold downregulation for subsequent bioinformatic investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Western Blot\u003c/h2\u003e \u003cp\u003eMitochondria were isolated using a mitochondrial isolation kit (ACMEC, Shanghai, China). Proteins were extracted from the purified mitochondrial fraction using RIPA lysis (Beyotime) buffer supplemented with protease and phosphatase inhibitors. Protein expression levels were determined using anti-p21, anti-γ-H2A.X, anti-PMPCB, anti-NDUFS8, anti-β-actin, and anti-COX IV. Then, the membranes were incubated with appropriate secondary antibodies (all Abcam) for 1 h at 37\u0026deg;C and imaged by an Odyssey Infrared Imaging System (LI-COR Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Immunofluorescence\u003c/h2\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells were fixed with 4% PFA, and blocked with 2% BSA. Cells were incubated overnight at 4\u0026deg;C with anti-p21, anti-γ-H2A.X, or anti-PMPCB, followed by FITC-conjugated secondary antibody (37\u0026deg;C, 1 h). Nuclei were stained with DAPI (Beyotime). Visualization was performed using a Zeiss LSM800 NLO confocal microscopy (Carl Zeiss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Bioinformatics Analysis\u003c/h2\u003e \u003cp\u003eThe human PBMC single-cell RNA sequencing dataset (GSE233315) was obtained from the NCBI Gene Expression Omnibus (GEO) database. Data processing and analysis were performed using the Seurat R package (version 5.3.0). Quality control filtering was applied as follows: cells with gene counts between 500 and 4,000 were retained, UMI counts\u0026thinsp;\u0026gt;\u0026thinsp;500 and \u0026lt;\u0026thinsp;15,000 were included; cells exhibiting high ambient RNA contamination (log10(genes per UMI)\u0026thinsp;\u0026le;\u0026thinsp;0.8) were excluded; cells with mitochondrial gene content exceeding 10% were removed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The number of independent biological replicates (N) is specified in respective figure legends. Comparisons between two groups were analyzed using paired or unpaired two-tailed Student's t-tests, as appropriate. Comparisons among three or more groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc tests. Associations between variables were assessed using Spearman's rank correlation coefficient. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Peripheral blood CD8⁺ T cells develop premature senescence in ESRD patients\u003c/h2\u003e \u003cp\u003eInitially, we re-analyzed a published single-cell RNA sequencing (scRNA-seq) data from PBMC obtained from healthy controls and ESRD patients (GSE233315). Interestingly, we found that the markers of T cell exhaustion, PDCD1 and CTLA-4 were comparable between healthy and ESRD patients, while Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed a robust upregulated in cellular senescence score, particularly in CD8\u003csup\u003e+\u003c/sup\u003e T effector cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b and S1a, b). Similarly, t-distributed Stochastic Neighbor Embedding (t-SNE) analysis by flow cytometry identified a mild increase in PD-1 in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells from patients with CKD stage 3\u0026ndash;5 compared to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e and S1c). Notably, CD57, a recognized marker used to identify terminally differentiated senescent T cells, and the proportion of CD57\u003csup\u003e+\u003c/sup\u003e cells within the CD4\u003csup\u003e+\u003c/sup\u003e T cell population showed no significant, while it was significantly higher within CD8\u003csup\u003e+\u003c/sup\u003e T cells of patients with CKD stage 3\u0026ndash;5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e and S1c). Furthermore, we found that CD57\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells were progressively developed along with advancing CKD and were most significant in ESRD (CKD stage 5) patients (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed). Besides, CD28 is an age-related immune biomarker, and the loss of CD28 coupled with the presence of CD57 (CD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD57\u003csup\u003e+\u003c/sup\u003e) constitutes a key hallmark of immunosenescence. Flow cytometry also demonstrated that the proportion of CD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD57\u003csup\u003e+\u003c/sup\u003e cells was significantly increased within the CD8\u003csup\u003e+\u003c/sup\u003e T cell compartment of ESRD patients, whereas no significant difference in their proportion within CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, g). Taken together, these data indicate that the T cell immune incompetence develops in the pathophysiological context of ESRD due to multifaceted dysregulation, and that premature CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence potentially serving as a central contributor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 CD8\u003csup\u003e+\u003c/sup\u003e T cells exhibit upregulated senescence biomarkers accompanied with mitochondrial dysfunction in ESRD\u003c/h2\u003e \u003cp\u003eCellular senescence is defined as the irreversible loss of replicative potential caused by persistent DNA damage, accompanied by increased β-galactosidase activity, cell cycle arrest and telomere shortening \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Indeed, we observed significantly elevated expression of the DNA damage marker γ-H2A.X and the cyclin-dependent kinase inhibitor p21 in CD8⁺ T cells from ESRD patients compared with healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c and S2a, b), as well as significantly increased SA-β-Gal activity and expression of the senescent proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Meanwhile, relative telomere length analysis revealed progressive age-dependent telomere shortening in CD8⁺ T cells in both groups, with significantly shorter telomeres observed in ESRD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Furthermore, senescence-associated mitochondrial dysfunction is considered an essential component of the senescent phenotype \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Accordingly, we detected a decrease in OCR, maximal respiratory capacity and Adenosine triphosphate (ATP) production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Mitochondrial dysfunction was further confirmed by the loss of mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Mitochondria are sensitive to oxidative stress, which is the most prominent characteristic of the CKD milieu, and we also observed ROS overproduction in CD8⁺ T cells of ESRD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Collectively, these findings demonstrate that CD8⁺ T cells from ESRD patients exhibit a comprehensive senescent phenotype characterized by well-established senescence markers and severe mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Platelets from ESRD patients induce premature CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence of by mediating mitochondrial dysfunction\u003c/h2\u003e \u003cp\u003eWe next sought to identify the role of platelets from ESRD patients in premature CD8⁺ T cell senescence. We first established a co-culture system in which platelets derived from healthy donors or ESRD patients respectively were co-cultured with CD8⁺ T cells derived from healthy donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). As shown, CD8\u003csup\u003e+\u003c/sup\u003e T cells cultured alone or co-cultured with platelets derived from healthy individuals did not show a cellular senescence phenotype over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-e). However, CD8\u003csup\u003e+\u003c/sup\u003e T cells co-cultured with platelets derived from ESRD patient exhibited a dramatic acceleration of premature CD8⁺ T cell senescence by day 5, as evidenced by the upregulation of γ-H2A.X, p21, SA-β-Gal and CD57 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). Besides, upregulation of senescent proinflammatory cytokines and shortened telomeres were also detected in CD8⁺ T cells after co-cultured with platelets from ESRD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). Surprisingly, mitochondrial membrane potential decreased, impaired mitochondrial respiratory function and ROS overproduction were observed in CD8⁺ T cells at day 3 after co-cultured with platelets from ESRD patients, preceding the appearance of cellular senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-h). In contrast, pretreatment with N-acetylcysteine (NAC), an antioxidant agent, effectively prevented mitochondrial dysfunction and cellular senescence in CD8\u003csup\u003e+\u003c/sup\u003e T cells induced by platelets from ESRD patients (Figure S3), indicating that platelets from ESRD patients might be involved in the initiation of premature CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence through mediating mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 PMPs from ESRD patients mediate mitochondrial dysfunction and cellular senescence in CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003c/h2\u003e \u003cp\u003ePreviously, we have shown that CKD promotes platelet hyperreactivity, inducing the large-scale release of PMPs, which have been reported to mediate mitochondrial dysfunction \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Consistently, remarkably increased platelet activity including P-selectin, activated GPIIb/IIIa expression, and PMPs release were verified in ESRD patients (Figure S4a, b). Notably, PMPs release in the peripheral blood of ESRD patients was correlated positively with cellular senescence biomarkers and ROS levels in CD8\u003csup\u003e+\u003c/sup\u003e T cells, whereas correlated negatively with mitochondrial membrane potential, hinting that PMPs release may facilitate mitochondrial dysfunction and cellular senescence of CD8\u003csup\u003e+\u003c/sup\u003e T cells in ESRD milieu (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f). To interrogate whether PMPs directly regulate premature CD8\u003csup\u003e+\u003c/sup\u003e T cells senescence, CD8\u003csup\u003e+\u003c/sup\u003e T cells were treated with PMPs \u003cem\u003eex vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Accordingly, Dio-labeled PMPs were successfully internalized by CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). PMPs from ESRD patients significantly induced CD8\u003csup\u003e+\u003c/sup\u003e T cells to undergo mitochondrial dysfunction and cellular senescence, while NAC dramatically relieved these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh-l and S4c-l). Hence, these results demonstrate that a direct action of PMPs on premature CD8\u003csup\u003e+\u003c/sup\u003e T cells senescence by inducing mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 PMPs promote premature CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence in an miR-550a-5p dependent manner\u003c/h2\u003e \u003cp\u003ePMPs are enriched in platelet microRNAs, which play an important role in regulating mitochondrial dysfunction and cellular senescence \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. PMPs were isolated from healthy controls and ESRD patients for miRNA profile detection. There were profound differences in PMP expression between healthy controls and ESRD patients, with 109 miRNAs upregulated and 57 miRNAs downregulated in ESRD-derived PMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Importantly, we identified that the greatest change in miRNA expression was miR-550a-5p, which was also confirmed to be the most remarkably upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Unexpectedly, the Starbase, TargetScan and miRDB databases revealed that miR-550a-5p was not expressed in mice (data not shown). Interestingly, the miR-550a-5p content in CD8\u003csup\u003e+\u003c/sup\u003e T cells was much lower than in PMPs, while the miR-550a-5p content in in CD8\u003csup\u003e+\u003c/sup\u003e T cells of ESRD patients was higher than in healthy controls, suggesting that high levels of miR-550a-5p in ESRD-derived PMPs were transferred to CD8\u003csup\u003e+\u003c/sup\u003e T cells (Figure S5a). Similar results were also observed in CD8\u003csup\u003e+\u003c/sup\u003e T cells and platelets co-cultured system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). To investigate the miR-550a-5p's role in mediating mitochondrial dysfunction and cellular senescence in CD8\u003csup\u003e+\u003c/sup\u003e T cells, we transfected the cells with miR-550a-5p mimic, which was verified by qPCR and immunofluorescence (Figure S5b, c). Following 48 hours of transfection, we observed that overexpression of miR-550a-5p induced a markedly elevated levels of cellular senescence and mitochondrial dysfunction biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-j and S5d), as well as increased senescent proinflammatory cytokines expression and shortened telomeres (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek, l). Conversely, treatment CD8\u003csup\u003e+\u003c/sup\u003e T cells with miR-550a-5p inhibitor significantly rescued platelets from ESRD patients-induced these effects under co-cultured system (Figure S6). Collectively, these results demonstrate platelets from ESRD patients-derived PMPs induce CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence via miR-550a-5p-mediating mitochondrial dysfunction axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 MiR-550a-5p induce mitochondrial dysfunction and cellular senescence in CD8\u003csup\u003e+\u003c/sup\u003e T cells via targeting PMPCB\u003c/h2\u003e \u003cp\u003eTo investigate the underlying mechanisms by which miR-550a-5p regulates CD8\u003csup\u003e+\u003c/sup\u003e T cells mitochondrial dysfunction-induced cellular senescence, bioinformatic analysis predicted 19 potential target mitochondrial genes of miR-550a-5p based on TargetScan, miRDB and MitoCarta 3.0 databases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, Table S8). Gene Ontology (GO) enrichment analysis showed that the mitochondrial genes were markedly enriched in mitochondrial protein transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), suggesting that miR-550a-5p may induce mitochondrial dysfunction through mediating mitochondrial transport process disorders. Of note, considering that miRNAs negatively regulate gene expression at the post-transcriptional level \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. we observed that PMPCB expression was significantly decreased at both mRNA and protein levels in CD8\u003csup\u003e+\u003c/sup\u003e T cells of ESRD patients (Figure S7a-c). PMPCB as the catalytic subunit of mitochondrial processing peptidase, which promotes the maturation of precursor proteins transported to mitochondria \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Besides, we detected that co-cultured with platelets from ESRD patients can reduce PMPCB expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, D and S7d). Similar results were observed in CD8\u003csup\u003e+\u003c/sup\u003e T cells when transfected with miR-550a-5p mimic (Figure S7e, f). Furthermore, bioinformatic analysis predicted perfect complementary sequences between miR-550a-5p and the PMPCB 3'-untranslated region (UTR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Dual-luciferase reporter assays were conducted to identify the targeting efficacy of miR-550a-5p on PMPCB. Overexpression of miR-550a-5p markedly decreased luciferase activity, while no effect was detected on PMPCB 3'-UTR mutant luciferase reporter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Interestingly, interrogation of sequences in the 3\u0026rsquo;UTR of mouse PMPCB gene also identified a miR-550a-5p binding site (Figure S7g). Moreover, we treated mice with miR-550a-5p modified oligonucleotides (agomir). Analogous to human CD8\u003csup\u003e+\u003c/sup\u003e T cells, cellular senescence, mitochondrial dysfunction and PMPCB downregulation were also detected in CD8\u003csup\u003e+\u003c/sup\u003e T cells of mice treated with miR-550a-5p agomir (Figure S7h, Figure S8). Above all, these results demonstrate that miR-550a-5p could directly bind and downregulate PMPCB expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the role of PMPCB in mitochondrial dysfunction and cellular senescence in CD8⁺ T cells, we first transfected siRNA against PMPCB in CD8⁺ T cells \u003cem\u003eex vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). As expected, the absent of PMPCB induced decreased mitochondrial membrane potential and impaired mitochondrial respiratory, but increased ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-d). Consistently, upregulation of cellular senescence biomarkers and pro-inflammation cytokines, as well as shortened telomeres were also observed in CD8⁺ T cells with PMPCB knockdown, indicating its important role in maintaining mitochondrial homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-k and S9a, b). On the contrary, overexpression of PMPCB significantly alleviated miR-550a-5p-induced mitochondrial dysfunction, ultimately mitigating cellular senescence in CD8⁺ T cells (Figure S10). Taken together, these data underscore a critical role of miR-550a-5p in mediating premature CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence by targeting PMPCB, which is essential for the maintenance of mitochondrial homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7 NMN counteracts platelets from ESRD patients-induced and NDUFS8-mediated cellular senescence in CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003c/h2\u003e \u003cp\u003eTo further elucidate PMPCB-mediated CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence mechanisms, we identified six PMPCB-interacting proteins using a protein-protein interaction analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Notably, the nuclear-encoded NDUFS8 is a subunit of monitoring NADH dehydrogenase in mitochondrial complex I, which would be cleaved by mitochondrial processing peptides \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. We found no significant difference in NDUFS8 expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells between ESRD patients and healthy controls, as well as co-cultured with platelets from ESRD patients or treatment with the miR-550a-5p mimic in human and miR-550a-5p agomir in mouse CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c and S11a-d). Interestingly, we detected the precursor and mature forms of NDUFS8 in CD8\u003csup\u003e+\u003c/sup\u003e T cells of ESRD patients, while only mature form of NDUFS8 existed in CD8\u003csup\u003e+\u003c/sup\u003e T cells of healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Moreover, co-cultured with platelets of ESRD patients or treatment with miR-550a-5p mimic in human and miR-550a-5p agomir in mouse CD8\u003csup\u003e+\u003c/sup\u003e T cells induced cleavage failure of NDUFS8, while overexpression of PMPCB significantly alleviated these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and S11c, d). These results indicate the regulatory effect of PMPCB on the cleavage of NDUFS8. As reported, NDUFS8 suppression will impair electron transfer from NADH to ubiquinone, thereby decreasing NAD+/NADH ratio, which has been reported to drive cells into senescence \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Indeed, ESRD patients exhibited decreased NAD\u0026thinsp;+\u0026thinsp;levels and NAD+/NADH ratios in CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Meanwhile, co-cultured with platelets of ESRD patients or treatment with miR-550a-5p mimic resulted in a similar reduction in NAD\u0026thinsp;+\u0026thinsp;levels and NAD+/NADH ratios, conversely, overexpression of PMPCB partially ameliorated this phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee and S11e). Therefore, we treated CD8\u003csup\u003e+\u003c/sup\u003e T cells with NMN, an intermediate in NAD\u0026thinsp;+\u0026thinsp;biosynthesis which plays a vital role in a variety of biological processes, and analyzed their mitochondrial function \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. NMN treatment effectively attenuated mitochondrial dysfunction in CD8\u003csup\u003e+\u003c/sup\u003e T cells, thereby preventing platelets from ESRD patients-induced cellular senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef-j and S11f-i). Moreover, we performed an intraperitoneal injection of NMN (500 mg/kg per mouse) once every 3 days in miR-550a-5p agomir-treated mice for 4 weeks. Consistently, NMN treatment effectively alleviated mitochondrial dysfunction and cellular senescence in CD8\u003csup\u003e+\u003c/sup\u003e T cells of mice (Figure S12). Above all, these findings indicate that NMN may serve as a promising therapeutic drug for alleviating platelets from ESRD patients-mediated mitochondrial dysfunction and cellular senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eInfection is a leading cause of morbidity and mortality in patients with CKD \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. T cell activation significantly influences an individual's immune function and susceptibility to infection \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Although disturbances in T cell homeostasis in CKD have been extensively studied, significant knowledge gaps remain regarding the underlying mechanisms of T cell dysfunction \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In this study, we identify that premature CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence is a key feature of altered T cell homeostasis in ESRD patients. We further demonstrate that platelets accelerate this senescence process by transferring PMPs containing miR-550a-5p into CD8⁺ T cells, thereby downregulating PMPCB expression and inhibiting NDUFS8/NAD\u0026thinsp;+\u0026thinsp;axis. Conversely, inhibiting miR-550a-5p function or overexpressing PMPCB ameliorated platelet-induced premature CD8⁺ T cell senescence in ESRD.\u003c/p\u003e \u003cp\u003ePatients with CKD are more prone to develop premature age-related diseases \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Immunosenescence contributes to the development of infections, cancers and autoimmune diseases, as well as accelerating the progression of fatal diseases, including cardiovascular and metabolic disorders, non-alcoholic fatty liver disease, and neurodegenerative diseases \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. However, the characteristics and molecular mechanisms underlying immune cell senescence in ESRD remain incompletely understood. In this study, integrated bioinformatic analysis and clinical sample validation revealed that CD8⁺ T cells exhibit more pronounced senescent change characters compared to CD4⁺ T cells, which is consistent with previous reports \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Currently, the cause of T cell senescence in CKD is attributed to the chronic inflammation resulting from uremia or maintenance dialysis \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In our study, we enrolled patients who were not on dialysis, and observed that CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence progressively developed along with advancing CKD. Interestingly, we demonstrate that another factor, co-cultured with platelets from ESRD patients can induce CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence, which cells derived from healthy controls. While the role of platelets in hemostasis is well known, they are also considered a cellular component of the innate immune system \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Based on our findings, we discovered that, in addition to uremia, platelets can serve as immune regulatory cells to mediate CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence.\u003c/p\u003e \u003cp\u003eAs known, platelets can directly interact with immune cells via specific receptors, such as CD41, CD42 and CD154, but our study excluded this effect using a flow cytometry gating strategy \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Besides, platelets can also release granules or microparticles to regulate innate and adaptive immunity. We also confirmed that increased PMP release in ESRD patients, and found that PMP levels correlated positively with CD8⁺ T cell senescence. Of note, recently studies indicate that platelet-releasing particles are closely associated with cellular senescence, but their performance is inconsistent in different milieu \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The release of platelet factor 4 by platelets in young mice alleviates cognitive impairment in older mice, while the release of miR-146 is upregulated in the hippocampus and frontal cortex of patients with Alzheimer disease. Given platelets have fully functional miRNA machinery, we isolated PMPs and performed a miRNA profiling to identify the leading cause of CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence. Here, we detect that miR-550a-5p is the most significantly upregulated miRNA in PMPs of ESRD patients and that it accelerates CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence. In addition, the upregulation of miR-550a-5p can also promote the proliferation and migration of cancer cells \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Taken together, it is plausible that miR-550a-5p may be implicated in proliferation and migration of cancer cells via mediating CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence and immunosuppression. On the contrary, treating CD8\u003csup\u003e+\u003c/sup\u003e T cells with a miR-550a-5p inhibitor effectively attenuates cell senescence when co-cultured with platelets from ESRD patients. Interestingly, the aspirin, an antiplatelet agent, also exhibits anti-aging properties via antioxidant, proteostatic and immunometabolic mechanisms \u003csup\u003e[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Hence, it is conceivable that antiplatelet may serve a promising strategy for alleviating CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence.\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction and associated ROS production have also been found in aging and many aging-associated pathological conditions, whereas the impairment of mitochondrial function and morphology also govern the senescent phenotype \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In our study, we observe that, after co-cultured with platelets of ESRD patients, mitochondrial disorders in CD8\u003csup\u003e+\u003c/sup\u003e T cells preceded the appearance of senescence phenotypes, while NAC treatment significantly rescue these effects, indicating that platelets of ESRD may induce CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence by mediating mitochondrial dysfunction. In order to characterize the molecular mechanisms by which miR-550a-5p regulates CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence, we for the first time identify PMPCB as a target gene of miR-550a-5p using bioinformatics and dual luciferase reporter analyses. PMPCB is the catalytic subunit of the mitochondrial processing peptidase, which identifies and cleaves mitochondrial importing signals necessary for the proper transport of proteins into the mitochondria, and thus facilitates their maturation. A decrease or mutation in PMPCB levels would induce mitochondrial processing peptidase dysfunction, thereby accumulating the processing intermediates such as frataxin \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, PMPCB has been shown to play a key role in regulating oxidant stress and apoptosis mediated by mitochondrial dysfunction \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Here, knockdown of PMPCB in CD8\u003csup\u003e+\u003c/sup\u003e T cells exhibit similar effects, including decreased mitochondrial membrane potential, impaired mitochondrial respiratory function and increased ROS production. However, the specific mechanisms by which PMPCB deficiency induces mitochondrial dysfunction are not fully elucidated. Further investigations reveal that the decreased PMPCB impairs the maturation of the pro-NDUFS8 precursor protein, leading to a reduced NAD+/NADH ratio. NDUFS8 is a nuclear-encoded core subunit of human mitochondrial Complex I, and NDUFS8 deficiency displays an apparent reduction in NAD+/NADH ratio and a decrease in mitochondrial membrane potential \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Crucially, intracellular reduced NAD⁺ levels are intimately linked to aging \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, while NMN treatment effectively attenuated mitochondrial dysfunction and cellular senescence in CD8\u003csup\u003e+\u003c/sup\u003e T cells. Hence, our findings suggest that therapeutic intervention of PMPCB-NDUFS8 axis opens new opportunities to promote CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence and immune function.\u003c/p\u003e \u003cp\u003eIn summary, these findings demonstrate that platelet-derived miR-550a-5p is responsible for CD8⁺ T cell senescence via the PMPCB-NDUFS8 axis in ESRD. This study not only substantially advances our understanding of the immune-platelet interactions, but also provided a potential strategy to prevent premature CD8⁺ T cell senescence upon ESRD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from this study are available upon a reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe investigators thank all patients who participated in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Joint Funds of the National Natural Science Foundation of China (No.\u0026nbsp;U22A20279), Key program of the Natural Science Foundation of China (Nos.\u0026nbsp;82530025 and 82030023), Chongqing Graduate Research and Innovation Project (No.\u0026nbsp;CYB23281).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYuewen Sun\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Investigation, Formal analysis, Writing \u0026ndash; original draft. \u003cstrong\u003eQigang Lan:\u0026nbsp;\u003c/strong\u003eConceptualization, Investigation, Formal analysis, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eYaqin Wang\u003c/strong\u003e: Investigation, Data curation. \u003cstrong\u003eMinhua Xie:\u0026nbsp;\u003c/strong\u003eInvestigation, Data curation. \u003cstrong\u003eXinyi Wu:\u0026nbsp;\u003c/strong\u003eInvestigation, Data curation. \u003cstrong\u003eLiangjing Lv\u003c/strong\u003e: Investigation, Data curation. \u003cstrong\u003eQin Xin:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eJun Chen:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eXiaoyi Zhong:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eMengying Yao:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eShuiqin Gong:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eSiyan Zhou:\u0026nbsp;\u003c/strong\u003eInvestigation, Data curation. \u003cstrong\u003eJunping Wang:\u003c/strong\u003e Conceptualization, Supervision, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJinghong Zhao:\u0026nbsp;\u003c/strong\u003eConceptualization, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information is available at (journal name)\u0026rsquo;s website\u003c/p\u003e\n\u003cp\u003e1. Supplemental Material\u003c/p\u003e\n\u003cp\u003e2. MicroRNA profile\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eForeman KJ, Marquez N, Dolgert A, et al. Forecasting life expectancy, years of life lost, and all-cause and cause-specific mortality for 250 causes of death: reference and alternative scenarios for 2016-40 for 195 countries and territories. Lancet. 2018. 392(10159): 2052\u0026ndash;2090.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovesdy CP. Epidemiology of chronic kidney disease: an update 2022. Kidney Int Suppl (2011). 2022. 12(1): 7\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheikh Hassan HI, Tang M, Djurdjev O, Langsford D, Sood MM, Levin A. Infection in advanced chronic kidney disease leads to increased risk of cardiovascular events, end-stage kidney disease and mortality. 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The prophylactic anti-aging effect of aspirin (acetylsalicylic acid) on oxidative stress-induced damage in the buccal mucosa of D-galactose-induced aged rats. Sci Rep. 2025. 15(1): 13053.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi W, Lin H, Di W, He C, Shen Y. Granulosa cell RNA-Seq insights into senescence and sphingolipid metabolism disorder in PCOS: aspirin as a potential therapeutic drug. Reprod Biol Endocrinol. 2025. 23(1): 61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV\u0026ouml;gtle FN, Br\u0026auml;ndl B, Larson A, et al. Mutations in PMPCB Encoding the Catalytic Subunit of the Mitochondrial Presequence Protease Cause Neurodegeneration in Early Childhood. Am J Hum Genet. 2018. 102(4): 557\u0026ndash;573.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakai A, Dang H, Oishi N, et al. Genome-Wide RNAi Screen Identifies PMPCB as a Therapeutic Vulnerability in EpCAM\u0026thinsp;+\u0026thinsp;Hepatocellular Carcinoma. Cancer Res. 2019. 79(9): 2379\u0026ndash;2391.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng JF, He S, Zeng Z, Gu X, Cai L, Qi G. PMPCB Silencing Sensitizes HCC Tumor Cells to Sorafenib Therapy. Mol Ther. 2019. 27(10): 1784\u0026ndash;1795.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIannetti EF, Smeitink J, Willems P, Beyrath J, Koopman W. Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD. Cell Death Dis. 2018. 9(11): 1135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChini C, Cordeiro HS, Tran N, Chini EN. NAD metabolism: Role in senescence regulation and aging. Aging Cell. 2024. 23(1): e13920.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"End-stage renal disease, CD8+ T cells, cellular senescence, platelets, miR-550a-5p, PMPCB","lastPublishedDoi":"10.21203/rs.3.rs-8468315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8468315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInfection is a leading cause of high mortality in patients with chronic kidney disease (CKD), in which T cell dysfunction is considered as a typical feature in CKD patients. Although platelets are active participants in immune response, their role in regulating T cell function in CKD patients remains unclear. In this study, we demonstrate that CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence may be a major contributor to the immunosuppressed state of end-stage renal disease (ESRD) patients, and that platelets derived from ESRD patients can induce premature CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence, potentially through mediating mitochondrial dysfunction. Further investigations reveal that platelet-derived microparticles (PMPs) from ESRD patients promote CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence via transferring miR-550a-5p, thereby decreasing PMPCB expression and inducing NDUFS8 cleavage failure. Finally, inhibition of miR-550a-5p and pretreatment with Nicotinamide Mononucleotide is capable of preventing platelet-induced CD8\u003csup\u003e+\u003c/sup\u003e T cell senescence in ESRD patients. Collectively, these findings suggest that the ectopic transient expression of platelet-derived miR-550a-5p in CD8\u003csup\u003e+\u003c/sup\u003e T cells promotes cellular senescence by regulating the PMPCB-NDUFS8 axis, which can be exploited to treat the ESRD-associated immunosenescence.\u003c/p\u003e","manuscriptTitle":"Platelet-derived microparticles induce CD8+ T cell senescence in end-stage renal disease via the transfer of microRNA-550a-5p","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 00:11:26","doi":"10.21203/rs.3.rs-8468315/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-11T06:56:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-10T04:46:01+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-23T11:32:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-05T09:49:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-22T10:06:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-22T05:36:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-29T23:45:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-29T01:39:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-12-29T01:39:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"009afc43-767c-41b3-9dea-393b8c1b3b81","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":61672123,"name":"Biological sciences/Cell biology/Senescence"},{"id":61672124,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":61672125,"name":"Biological sciences/Immunology/Lymphocytes/T cells/CD8-positive T cells"},{"id":61672126,"name":"Health sciences/Diseases/Kidney diseases/Chronic kidney disease/End-stage renal disease"}],"tags":[],"updatedAt":"2026-03-11T07:00:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 00:11:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8468315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8468315","identity":"rs-8468315","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}