Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxide Production

Abstract

Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O₂^•-) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O₂^•- formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O₂^•- formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O₂^•- formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O₂^•- formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.

Keywords: acellular therapy; anion superoxide; extracellular vesicles; mesenchymal cells; mitochondria; proximal tubular cells; regenerative medicine; renal ischemia/reperfusion.

Figure 1

Characterization of extracellular vesicles (EVs) secreted by adipose mesenchymal cells. (A) Morphological characterization. Transmission electron microscopy (TEM) at the two indicated magnifications. (B) Characterization by using vesicular surface antigens. The immunodetections show that the EVs, whose images are presented in (A), are CD9⁺, CD63⁺, TSG101⁺, and CD81⁺, as indicated.

Figure 2

Evaluation of structural mitochondria damage after AKI and EVs treatment. (A) Transmission electron microscopy images of mitochondria from HK-2 cells in CTR, HPX, and HPX + EVs conditions, as indicated above the panels. Arrowhead in HPX points to a typical myelin figure. (B) Quantification of the mitochondrial areas. Bars represent mean ± SEM (n = 34, 37, 30 areas for CTR, HPX, and HPX + EVs groups, respectively). (C) Representative fluorescence images of MitoTracker™ Orange (red areas) to evaluate mitochondrial Δψ in CTR, HPX, and HPX + EVs. Nuclei are stained in blue (DAPI). Magnifications are indicated on the left side of the panels. The insets in the 100× magnification pictures are presented just below the panels. (D) The percentage of events in cytometry analyses (normalized to modal value) at the arbitrary fluorescence intensity units indicated on the abscissa. The small colored squares indicate the experimental conditions: orange, CTR; blue, HPX; red, HPX + EVs. The histograms of CTR, HPX, and HPX+EVs overlap. (E) The mitochondrial mean fluorescence intensities (MFI) of MitoTracker™ Orange were quantified from the groups indicated on the abscissa. Scatter plots represent determinations in which median values of fluorescence were recorded. In (B,E), differences were assessed using one-way ANOVA followed by Tukey’s test. (B,E) *** p < 0.001; **** p < 0.0001; NS: not significant.

Figure 3

Generation of O₂^•− in renal mitochondria 24 h after ischemia/reperfusion (I/R). Representative time courses of H₂O₂ formation from the O₂^•− generated in renal cortex corticis mitochondria during different respiratory states, which were elicited by successive additions of substrates/inhibitors. (A) SHAM rats. (B) I/R conditions. (C) I/R + EVs; EVs were injected at the beginning of reperfusion. The numbers (spikes) indicate additions to the medium. 1:10 mM succinate; 2:0.1 mM ADP; 3:1 mM ADP; 4:0.2 μg/mL oligomycin; 5:1.5 μM FCCP; 6:2.5 μM Antimycin A. The insets allow a better comparison of the rate of formation of H₂O₂ over 2 min after the addition of succinate. The dismutation reaction 2O₂^•− + 2H⁺ →H₂O₂ + O₂ was non-limiting because an excess of superoxide dismutase (SOD) (60 U/mL) was added to the reaction medium. Standard curves were obtained by successive additions of 10 nM H₂O₂ pulses at 2 min time intervals, thereby allowing calculation of rates in pmol H₂O₂ × mg⁻¹ × min⁻¹ (see the following figure). (D) Quantification of the H₂O₂ formation after addition of succinate (insets). Bars are mean ± SEM; n = 7 (SHAM); n = 7 (I/R); n = 5 (I/R+EVs). Means were compared using one-way ANOVA followed by Tukey′s test. **** p < 0.0001; NS: not significant.

Figure 4

Quantification of H₂O₂ formation (energization with succinate) 24 h after I/R. Assays were carried out in the different respiratory states indicated on the abscissae. (A) Phosphorylating (1 mM ADP). (B) Non-phosphorylating (oligomycin). (C) Uncoupled (FCCP). (D) Residual (Antimycin A). Experimental groups are also indicated on the abscissae. Bars indicate mean ± SEM of the number of determinations indicated in the legend to Figure 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; NS: not significant (one-way ANOVA followed by Tukey´s test).

Figure 5

Proton leak (QO₂ measured in the presence of oligomycin). The reaction medium was that used to determine the production of reactive O₂ species in Figure 3, without addition of the components required for the formation of H₂O₂. The bars correspond to mean ± SEM of 6 (SHAM and I/R) and 5 (I/R + EVs) determinations with different mitochondrial preparations. Groups are those indicated on the abscissae. Not significant differences (NS) were found among the 3 groups (one-way ANOVA followed by Tukey´s test).

Figure 6

The expression (relative quantification) of the renal injury biomarkers 24 h after I/R are not modified by EVs. (A) KIM-1. (B) NGAL. The expression of the genes was investigated from total RNA extracted from cortex corticis. The bars represent mean ± SEM that were compared using one-way ANOVA followed by Tukey’s test. The experimental groups are indicated on the abscissae; n = 4 (SHAM), n = 7 (I/R), n = 5 (I/R + EVs), for both biomarkers. **** p < 0.0001; NS: not significant.

Figure 7

Expression of pro-inflammatory cytokines and antioxidant enzymes, of which expression was investigated from the total RNA extracted from cortex corticis. (A) IL-6. (B) TNF-α. (C) Heme oxygenase-1. (D) Catalase. (E) Superoxide dismutase-1. Bars represent mean ± SEM that were compared using one-way ANOVA followed by Tukey’s test. Groups are indicated on the abscissae; n = 4 (SHAM), n = 7 (I/R), n = 5 (I/R+EVs). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; NS: not significant.

Figure 8

Co-culture with EVs decreases apoptosis of a lineage of proximal tubule cells (HK-2 line) subjected to hypoxia. Representative cytometry analysis of 3 × 10⁵ HK-2 cells cultivated in 1.5 mL of DMEM without serum for 24 h. (A) Cultures in normoxia (CTR). (B) Hypoxia (1% O₂) (HPX) in the absence of EVs. (C) HPX in the presence of 2 × 10⁹ EVs (HPX+EVs). Then the cells were incubated for 24 h under 21% O₂ in the same medium. (D–F) Quantification of cell death is expressed as a percent of total cells. (D) Early apoptosis, corresponding to ANX⁺PI⁻ cells. (E) Late apoptosis, corresponding to ANX⁺PI⁺ cells. (F) Sum of the cells that suffered early and late death: ANX⁺PI⁻ cells + ANX⁺PI⁺ cells. Bars represent mean ± SEM of different cultures in the conditions CTR (n = 16), HPX (n = 14), and HPX+EVs (n = 9). * p < 0.05; ** p < 0.01; *** p < 0.001; NS: not significant (one-way ANOVA followed by Tukey´s test).

Figure 9

Suppressing the excess of O₂^•−: Proposed mechanisms for the rapid effects of EVs (24 h of reperfusion after renal ischemia). Mesenchymal cells (MSCs) secrete EVs that, after subcapsular administration and diffusion into the renal parenchyma [10], reach the tubular segments injured by I/R. By releasing several factors, including the catalase they carry [58], they contribute to maintaining the normal local redox state existing in the absence of injury. With mitochondrial and cytoplasmic redox homeostasis restored, the mitochondrial processes required for ATP synthesis are preserved and, therefore, the appropriate ATP supply is preserved for the transport demands and maintenance of tubular structures.