{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T16:02:03Z","timestamp":1774454523828,"version":"3.50.1"},"reference-count":33,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T00:00:00Z","timestamp":1774396800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Research Foundation of Korea\n\t\t\t\t\t\t\t\t              https:\/\/ror.org\/013aysd81","award":["RS-2023-00279934"],"award-info":[{"award-number":["RS-2023-00279934"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Chronic kidney disease (CKD) involves uremic toxin-driven tubular injury and systemic vascular dysfunction, in which mitochondrial impairment and apoptotic cell loss contribute to progressive tissue deterioration. Accordingly, a targeted EV platform is required to enable efficient miRNA delivery to the toxin-stressed tubular\u2013endothelial compartment. Based on our previous study showing that melatonin restores miR-4516 levels under CKD-related stress, we directly loaded miR-4516 into engineered extracellular vesicles (EVs) to evaluate its effects on mitochondrial function and cell survival. Here, we engineered EVs with a G3-C12\/RGD surface modification and established a miR-4516 loading strategy to enhance delivery to kidney proximal tubule cells and vascular endothelial cells. miR-4516 loading increased EV-associated miR-4516 levels without major changes in particle size distribution, and EV identity was supported by CD9 and CD81 expression. Confocal microscopy and flow cytometry demonstrated increased cellular uptake of miR-4516-loaded G3-C12\/RGD-EVs compared with control EVs in TH1 proximal tubule cells and HUVECs. Under indoxyl sulfate stress, engineered EV treatment restored intracellular miR-4516 and improved mitochondrial function, as indicated by recovery of respiratory Complex I and Complex IV activities and improved Seahorse bioenergetic parameters (OCR\/ECAR, basal and maximal respiration, ATP-linked respiration, and spare respiratory capacity). Annexin V staining further indicated reduced toxin-induced apoptosis. In an adenine diet-induced CKD mouse model, intravenous administration of miR-4516-loaded G3-C12\/RGD-EVs improved urinary albumin-to-creatinine ratio (UACR), blood urea nitrogen (BUN), and serum creatinine. These findings indicate that miR-4516-loaded, targeting-engineered EVs may mitigate uremic toxin-associated mitochondrial dysfunction and renal impairment in CKD.<\/jats:p>","DOI":"10.3390\/ijms27072997","type":"journal-article","created":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T15:34:27Z","timestamp":1774452867000},"page":"2997","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["miR-4516-Loaded Engineered Milk Extracellular Vesicles Attenuate Indoxyl Sulfate-Induced Mitochondrial Dysfunction and Improve Renal Function in a CKD Mouse Model"],"prefix":"10.3390","volume":"27","author":[{"given":"Jeongkun","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences, College of Medicine, Program in Biomedical Science & Engineering, Inha University, 3-ga, Sinheung-dong, Jung gu, Incheon 22332, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun Young","family":"Yoon","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, College of Medicine, Program in Biomedical Science & Engineering, Inha University, 3-ga, Sinheung-dong, Jung gu, Incheon 22332, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jae Young","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, College of Medicine, Program in Biomedical Science & Engineering, Inha University, 3-ga, Sinheung-dong, Jung gu, Incheon 22332, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sang Hun","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, College of Medicine, Program in Biomedical Science & Engineering, Inha University, 3-ga, Sinheung-dong, Jung gu, Incheon 22332, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"(2024). Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2024 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney Int., 105, S117\u2013S314.","DOI":"10.1016\/j.kint.2023.10.018"},{"key":"ref_2","unstructured":"GBD Chronic Kidney Disease Collaboration (2020). Global, regional, and national burden of chronic kidney disease, 1990\u20132017: A sytematic analysis for the Global Burden of Disease Study 2017. Lancet, 395, 709\u2013733."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"931","DOI":"10.1159\/000512263","article-title":"Assessment of endothelial and microvascular function in CKD: Older and newer techniques, associated risk factors, and relations with outcomes","volume":"51","author":"Theodorakopoulou","year":"2020","journal-title":"Am. J. Nephrol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Fontecha-Barriuso, M., Lopez-Diaz, A.M., Guerrero-Mauvecin, J., Miguel, V., Ramos, A.M., Sanchez-Ni\u00f1o, M.D., Ruiz-Ortega, M., Ortiz, A., and Sanz, A.B. (2022). Tubular mitochondrial dysfunction, oxidative stress, and progression of chronic kidney disease. Antioxidants, 11.","DOI":"10.3390\/antiox11071356"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1478","DOI":"10.1093\/ndt\/gfz055","article-title":"Most exposed: The endothelium in chronic kidney disease","volume":"35","author":"Hordijk","year":"2020","journal-title":"Nephrol. Dial. Transplant."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Harlacher, E., Wollenhaupt, J., Baaten, C.C.F.M.J., and Noels, H. (2022). Impact of uremic toxins on endothelial dysfunction in chronic kidney disease: A systematic review. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23010531"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1038\/labinvest.2011.194","article-title":"Indoxyl sulfate-induced epithelial-to-mesenchymal transition and apoptosis of renal tubular cells as novel mechanisms of progression of renal disease","volume":"92","author":"Kim","year":"2012","journal-title":"Lab. Investig."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"620826","DOI":"10.1155\/2015\/620826","article-title":"Indoxyl sulfate-induced oxidative stress, mitochondrial dysfunction, and impaired biogenesis are partly protected by vitamin C and N-acetylcysteine","volume":"2015","author":"Lee","year":"2015","journal-title":"Sci. World J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5317","DOI":"10.1038\/s41598-018-23663-1","article-title":"Chronic kidney disease induces a systemic microangiopathy, tissue hypoxia and dysfunctional angiogenesis","volume":"8","author":"Prommer","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Yoon, Y.M., Go, G., Yun, C.W., Lim, J.H., Lee, J.H., and Lee, S.H. (2020). Melatonin suppresses renal cortical fibrosis by inhibiting cytoskeleton reorganization and mitochondrial dysfunction through regulation of miR-4516. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21155323"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/s43556-024-00230-x","article-title":"Targeted delivery of extracellular vesicles: The mechanisms, techniques and therapeutic applications","volume":"5","author":"Zhao","year":"2024","journal-title":"Mol. Biomed."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1038\/s12276-024-01201-6","article-title":"Recent advances in extracellular vesicles for therapeutic cargo delivery","volume":"56","author":"Kim","year":"2024","journal-title":"Exp. Mol. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e12404","DOI":"10.1002\/jev2.12404","article-title":"Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches","volume":"13","author":"Welsh","year":"2024","journal-title":"J. Extracell. Vesicles"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Ludwig, B.S., Kessler, H., Kossatz, S., and Reuning, U. (2021). RGD-binding integrins revisited: How recently discovered functions and novel synthetic ligands (Re-)shape an ever-evolving field. Cancers, 13.","DOI":"10.3390\/cancers13071711"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1038\/s41573-021-00284-4","article-title":"Emerging therapeutic opportunities for integrin inhibitors","volume":"21","author":"Slack","year":"2022","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1038\/kisup.2014.20","article-title":"Hypoxia and fibrosis in chronic kidney disease: Crossing at pericytes","volume":"4","author":"Kawakami","year":"2014","journal-title":"Kidney Int. Suppl."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1007\/s00467-013-2430-y","article-title":"Peritubular capillary rarefaction: A new therapeutic target in chronic kidney disease","volume":"29","author":"Kida","year":"2014","journal-title":"Pediatr. Nephrol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1080\/15476286.2019.1667215","article-title":"Emerging role of miRNAs in renal fibrosis","volume":"17","author":"Fan","year":"2020","journal-title":"RNA Biol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Yoon, Y.M., Go, G., Yoon, S., Lim, J.H., Lee, G., Lee, J.H., and Lee, S.H. (2021). Melatonin treatment improves renal fibrosis via miR-4516\/SIAH3\/PINK1 axis. Cells, 10.","DOI":"10.3390\/cells10071682"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Timofeeva, A.M., Paramonik, A.P., Sedykh, S.S., and Nevinsky, G.A. (2023). Milk exosomes: Next-generation agents for delivery of anticancer drugs and therapeutic nucleic acids. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241210194"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"102577","DOI":"10.1016\/j.jddst.2021.102577","article-title":"Emerging roles and biopharmaceutical applications of milk derived exosomes","volume":"64","author":"Desai","year":"2021","journal-title":"J. Drug Deliv. Sci. Technol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.canlet.2015.10.020","article-title":"Bovine milk-derived exosomes for drug delivery","volume":"371","author":"Munagala","year":"2016","journal-title":"Cancer Lett."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"857","DOI":"10.21873\/anticanres.15543","article-title":"Inhibitory effect of oxaliplatin-loaded engineered milk extracellular vesicles on tumor progression","volume":"42","author":"Go","year":"2022","journal-title":"Anticancer Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41392-022-01259-6","article-title":"Targeting integrin pathways: Mechanisms and advances in therapy","volume":"8","author":"Pang","year":"2023","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Johnson, V., Vasu, S., Kumar, U.S., and Kumar, M. (2023). Surface-engineered extracellular vesicles in cancer immunotherapy. Cancers, 15.","DOI":"10.3390\/cancers15102838"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1777","DOI":"10.1021\/bc500291r","article-title":"Surface functionalization of exosomes using click chemistry","volume":"25","author":"Smyth","year":"2014","journal-title":"Bioconjug. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Cheng, T.-H., Ma, M.-C., Liao, M.-T., Zheng, C.-M., Lu, K.-C., Liao, C.-H., Hou, Y.-C., Liu, W.-C., and Lu, C.-L. (2020). Indoxyl sulfate, a tubular toxin, contributes to the development of chronic kidney disease. Toxins, 12.","DOI":"10.3390\/toxins12110684"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"77722","DOI":"10.18632\/oncotarget.20773","article-title":"Protein-bound uremic toxins impaired mitochondrial dynamics and functions","volume":"8","author":"Sun","year":"2017","journal-title":"Oncotarget"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ho, H.-J., and Shirakawa, H. (2022). Oxidative stress and mitochondrial dysfunction in chronic kidney disease. Cells, 12.","DOI":"10.3390\/cells12010088"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Babaker, M.A., Aljoud, F.A., Alkhilaiwi, F., Algarni, A., Ahmed, A., Khan, M.I., Saadeldin, I.M., and Alzahrani, F.A. (2022). The therapeutic potential of milk extracellular vesicles on colorectal cancer. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23126812"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Kong, C., Huang, L., Yang, M., Yue, N., Zhang, Y., Tian, C., Wang, Y., Wei, D., Shi, R., and Liang, Y. (2025). Milk-derived extracellular vesicles: Nature\u2019s nanocarriers for drug delivery and therapeutics. Front. Pharmacol., 16.","DOI":"10.3389\/fphar.2025.1595891"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s13036-024-00470-z","article-title":"Stabilizing milk-derived extracellular vesicles (mEVs) through lyophilization: A novel trehalose and tryptophan formulation for maintaining structure and bioactivity during long-term storage","volume":"19","author":"Dogan","year":"2025","journal-title":"J. Biol. Eng."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"11","DOI":"10.7150\/ntno.129284","article-title":"Rapid method for the high purity isolation of bovine milk-derived extracellular vesicles via polyester (PET) capillary-channeled polymer (C-CP) fiber columns","volume":"10","author":"Mata","year":"2026","journal-title":"Nanotheranostics"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/7\/2997\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T15:34:41Z","timestamp":1774452881000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/7\/2997"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,25]]},"references-count":33,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ijms27072997"],"URL":"https:\/\/doi.org\/10.3390\/ijms27072997","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,25]]}}}