Extracellular Vesicle Transplantation Is Beneficial for Acute Kidney Injury
<p><b>Characterization of RACs.</b> (<b>A</b>) Total stem and progenitor levels increased after vasculogeneic conditioning. (<b>B</b>) The EPCs were quantitatively and (<b>C</b>,<b>D</b>) qualitatively enhanced in post-vasculogenic culture (the majority by definitive EPC expansion). (<b>E</b>,<b>F</b>) Vasculogenic conditioning dramatically accelerated M1 macrophage phenotype conversion to regenerative macrophage type 2 (<b>G</b>). The level of regulatory T cells. * <span class="html-italic">p</span> < 0.05; *** <span class="html-italic">p</span> < 0.01; **** <span class="html-italic">p</span> < 0.0001 vs. the control group; Statistical significance was determined using a Mann–Whitney test. n = 10 per group. The results are presented as mean ± SEM. * <span class="html-italic">p</span> < 0.05; *** <span class="html-italic">p</span> < 0.01; **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 2
<p><b>Characterization of RAC-derived extracellular vesicles.</b> (<b>A</b>) EV-specific anti-CD63 and anti-CD9 biomarker expression in RACev. (<b>B</b>) Representative transmission-electron microscopy figures showed the lipid bilayer structure in RACev. (<b>C</b>) (<b>a</b>) Quantification of one million RAC-derived EVs, (<b>b</b>) average size, and (<b>c</b>) protein amount.</p> "> Figure 3
<p><b>RACev transplantation restored kidney function.</b> (<b>A</b>) Serum creatinine level at day three significantly decreased in RACev vs. control. (<b>B</b>) Similarly, serum BUN level was dramatically diminished in the RACev transplanted group compared to the Control group. * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; ns is not significant vs. the control group; statistical significance was determined using a 2-way ANOVA followed by Tukey’s multiple comparison test. The results are presented as mean ± SEM.</p> "> Figure 4
<p><b>RACev transplantation preserved renal interstitial fibrosis.</b> (<b>A</b>) Representative Masson trichrome staining depicts reduced or preserved fibrosis area in RACev-transplanted group in comparison to control groups. (<b>B</b>) Fibrosis area quantification in cortex area and (<b>C</b>) medullary area. (<b>D</b>) Anti-fibrosis miRs were significantly upregulated in RACev. (<b>E</b>) Fibrosis-related genes markedly upregulated RACev vs. control four days after the onset of R-IRI. * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; ns is not significant vs. the control group. Transcriptome analysis at day four after the onset of AKI demonstrated fibrosis-related gene upregulation in control group vs. RACev. Statistical significance was determined using a one-way ANOVA followed by Dunn’s multiple comparison test. The results are presented as mean ± SEM (n = 8–10 per group).</p> "> Figure 5
<p><b>Regulation of inflammatory and apoptosis pathways.</b> (<b>A</b>) Gene co-expression network analysis of control group revealed (<b>B</b>) inflammation, EMT, and hypoxia pathway upregulation. (<b>C</b>) RACev-transplanted group demonstrated regeneration-associated pathway upregulation. (<b>D</b>) Anti-inflammatory miRs are abundantly expressed in RACev. (<b>E</b>) Anti-apoptotic and proliferation-associated miR expression in RACev. Differentially expressed miRs were determined using a threshold of absolute values of fold change ≥ 2.</p> "> Figure 6
<p><b>Enhanced angiogenesis in infarcted tissues.</b> (<b>A</b>) Microvascular density was enhanced in ischemic injured kidney tissue in the RACev-transplanted group. (<b>B</b>) CD31 positive capillary count at day four and (<b>C</b>) day 28 after onset of R-IRI. (<b>D</b>) Angiogenic miRs, also known as angiomiRs, are markedly expressed in RACev. (<b>E</b>) Angiogenesis-related gene expression of RACev vs. control groups’ kidney tissues. ** <span class="html-italic">p</span> < 0.01; ns is not significant vs. control group. Statistical significance was determined using one-way ANOVA with Dunn’s multiple comparisons test. Results are presented as the mean ± SEM (n = 8–10 per group). Differentially expressed miRs were determined using a threshold of absolute values of fold change ≥ 2.</p> "> Figure 7
<p><b>Selective accumulation of RACev.</b> (<b>A</b>) Schematic design of an in vivo study. (<b>B</b>) Systemic transplantation of labeled RACev preferentially accumulated into the ischemia-injured kidneys. (<b>C</b>,<b>D</b>) Transcriptome cell annotation and mapping showed possible RACev accumulation in the kidney.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Cell Phenotype Characterization
2.3. Endothelial Progenitor Cell Colony Formation Assay
2.4. Extracellular Vesicle Isolation and Characterizations
2.5. EV Labeling and Tracking
2.6. Flow Cytometry Analysis of EVs
2.7. Animals
2.8. Renal Ischemia-Reperfusion Injury Induction
2.9. Immunohistochemistry Analysis
2.10. Library Preparation, Sequencing, and Bioinformatics Analysis
2.11. Statistical Analysis
3. Results
3.1. Characterization of Regeneration-Associated Cells
3.2. Characterization of EVs
3.3. RACev Transplantation Improved Kidney Function
3.4. RACev Therapy Preserved from Kidney Fibrosis
3.5. Regulatory Role of RACev on Epithelial Mesenchymal Transition, Inflammation, and Hypoxia after Ischemic Injury
3.6. RACev Restored Capillary Densities
3.7. Preferential RACev Accumulation in the Injured Kidney
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Salybekov, A.A.; Okamura, S.; Ohtake, T.; Hidaka, S.; Asahara, T.; Kobayashi, S. Extracellular Vesicle Transplantation Is Beneficial for Acute Kidney Injury. Cells 2024, 13, 1335. https://doi.org/10.3390/cells13161335
Salybekov AA, Okamura S, Ohtake T, Hidaka S, Asahara T, Kobayashi S. Extracellular Vesicle Transplantation Is Beneficial for Acute Kidney Injury. Cells. 2024; 13(16):1335. https://doi.org/10.3390/cells13161335
Chicago/Turabian StyleSalybekov, Amankeldi A., Shigeaki Okamura, Takayasu Ohtake, Sumi Hidaka, Takayuki Asahara, and Shuzo Kobayashi. 2024. "Extracellular Vesicle Transplantation Is Beneficial for Acute Kidney Injury" Cells 13, no. 16: 1335. https://doi.org/10.3390/cells13161335