Extracellular Vesicle Transplantation Is Beneficial for Acute Kidney Injury

Abstract

Under vasculogenic conditioning, certain pro-inflammatory subsets within peripheral blood mononuclear cells (PBMCs) undergo phenotypic transformation into pro-regenerative types, such as vasculogenic endothelial progenitor cells, M2 macrophages, and regulatory T cells. These transformed cells are collectively termed regeneration-associated cells (RACs). In this study, we aimed to investigate the therapeutic efficacy of RAC-derived extracellular vesicles (RACev) compared with a vehicle-treated group in the context of renal ischemia-reperfusion injury (R-IRI). Human PBMCs were cultured with defined growth factor cocktails for seven days to harvest RACs. EV quantity and size were characterized by nanoparticle tracking analysis. Notably, the systemic injection of RACev significantly decreased serum creatinine and blood urine nitrogen at day three compared to the control group. Histologically, the treatment group showed less fibrosis in the cortex and medullary areas (p < 0.04 and p < 0.01) compared to the control group. The CD31 staining confirmed enhanced capillary densities in the treatment group compared to the control group (p < 0.003). These beneficial effects were accompanied by angiogenesis, anti-fibrosis, anti-inflammation, and anti-apoptosis RACev miR delivery to ischemic injury to control inflammatory, endothelial mesenchymal transition, and hypoxia pathways. In vivo bioluminescence analysis demonstrated a preferential accumulation of RACev in the IR-injured kidney. The systemic transplantation of RACev beneficially restored kidney function by protecting from tissue fibrosis and through anti-inflammation, angiogenesis, and anti-apoptosis miR delivery to the ischemic tissue.

Keywords: angiogenesis; extracellular vesicles; miR; regeneration-associated cells; renal ischemia-reperfusion injury.

Figure 1

Characterization of RACs. (A) Total stem and progenitor levels increased after vasculogeneic conditioning. (B) The EPCs were quantitatively and (C,D) qualitatively enhanced in post-vasculogenic culture (the majority by definitive EPC expansion). (E,F) Vasculogenic conditioning dramatically accelerated M1 macrophage phenotype conversion to regenerative macrophage type 2 (G). The level of regulatory T cells. * p < 0.05; *** p < 0.01; **** p < 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. * p < 0.05; *** p < 0.01; **** p < 0.0001.

Figure 2

Characterization of RAC-derived extracellular vesicles. (A) EV-specific anti-CD63 and anti-CD9 biomarker expression in RACev. (B) Representative transmission-electron microscopy figures showed the lipid bilayer structure in RACev. (C) (a) Quantification of one million RAC-derived EVs, (b) average size, and (c) protein amount.

Figure 3

RACev transplantation restored kidney function. (A) Serum creatinine level at day three significantly decreased in RACev vs. control. (B) Similarly, serum BUN level was dramatically diminished in the RACev transplanted group compared to the Control group. * p < 0.05; ** p < 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.

Figure 4

RACev transplantation preserved renal interstitial fibrosis. (A) Representative Masson trichrome staining depicts reduced or preserved fibrosis area in RACev-transplanted group in comparison to control groups. (B) Fibrosis area quantification in cortex area and (C) medullary area. (D) Anti-fibrosis miRs were significantly upregulated in RACev. (E) Fibrosis-related genes markedly upregulated RACev vs. control four days after the onset of R-IRI. * p < 0.05; ** p < 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).

Figure 5

Regulation of inflammatory and apoptosis pathways. (A) Gene co-expression network analysis of control group revealed (B) inflammation, EMT, and hypoxia pathway upregulation. (C) RACev-transplanted group demonstrated regeneration-associated pathway upregulation. (D) Anti-inflammatory miRs are abundantly expressed in RACev. (E) Anti-apoptotic and proliferation-associated miR expression in RACev. Differentially expressed miRs were determined using a threshold of absolute values of fold change ≥ 2.

Figure 6

Enhanced angiogenesis in infarcted tissues. (A) Microvascular density was enhanced in ischemic injured kidney tissue in the RACev-transplanted group. (B) CD31 positive capillary count at day four and (C) day 28 after onset of R-IRI. (D) Angiogenic miRs, also known as angiomiRs, are markedly expressed in RACev. (E) Angiogenesis-related gene expression of RACev vs. control groups’ kidney tissues. ** p < 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.

Figure 7

Selective accumulation of RACev. (A) Schematic design of an in vivo study. (B) Systemic transplantation of labeled RACev preferentially accumulated into the ischemia-injured kidneys. (C,D) Transcriptome cell annotation and mapping showed possible RACev accumulation in the kidney.