Metabolic Syndrome Alters the Cargo of Mitochondria-Related microRNAs in Swine Mesenchymal Stem Cell-Derived Extracellular Vesicles, Impairing Their Capacity to Repair the Stenotic Kidney

Figures

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  Figure 1. Schematic of the experimental protocol. MetS: metabolic syndrome, RAS: renal artery stenosis, EVs: mesenchymal stem/stromal cell-derived extracellular vesicles, MDCT: multidetector computed tomography. Pigs were studied after 16 weeks of diet-induced MetS and RAS (MetS+RAS), untreated or 4 weeks after intrarenal delivery of EVs isolated from adipose tissue-derived Lean- or MetS-MSCs (n = 7 each). Lean and MetS sham served as controls.

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  Figure 2. MetS alters the miRNA cargo of MSC-derived EVs. (a) heat map of 11 miRNAs upregulated and 8 miRNAs downregulated in MetS-EVs versus Lean-EVs. (b) Expression of selected differentially expressed candidate miRNAs demonstrated agreement with the miRNA-sequencing analysis, where miR-196a and miR-132 were upregulated and miR-192 and miR-320 downregulated in MetS-EVs. (c) Heat map of 15 out of 19 miRNAs differentially expressed in MetS-EVs versus Lean-EVs that were enriched in MetS-EVs compared to MetS-MSCs. ∗p < 0.05 vs. Lean-EVs.

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  Figure 3. miRNAs dysregulated in MetS-EVs are capable of targeting mitochondria-related genes. Predicted mitochondria target genes of differentially expressed miRNAs were identified (MitoCarta2.0) and classified by their cellular component (a), molecular function (b), and biological process (c). miRNAs dysregulated in MetS-EVs are capable of targeting several genes implicated in mitochondrial energy pathways, including the electron transport chain complexes I, II, III, IV, and V and the tricarboxylic acid (TCA) cycle.

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  Figure 4. MetS altered the expression of mitochondria-related genes and proteins of EVs. (a) Heat map of 43 mRNAs upregulated and 29 mRNAs downregulated in MetS-EVs versus Lean-EVs. (b) Heat map of 14 proteins upregulated and 10 proteins downregulated in MetS-EVs versus Lean-EVs. (c) Functional analysis of genes and proteins dysregulated in MetS-EVs revealed that they are implicated in important mitochondrial functions, such as organization, transport, and oxidation reduction processes.

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  Figure 5. MSC-derived EVs were retained in the poststenotic kidney. Fragments of red immunofluorescent stained EVs (PKH26, arrows; original magnification ×40) were detected in the stenotic kidney of MetS+RAS+Lean-EVs and MetS+RAS+MetS-EVs 4 weeks after administration, but not in untreated MetS+RAS stenotic kidneys. Immunofluorescent costaining with cytokeratin identified EV fragments in the vicinity of renal tubular cells, whereas some of them colocalized with renal tubular cells.

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  Figure 6. MetS-EVs failed to improve the renal mitochondrial density and structure in MetS+RAS. (a) Representative immunofluorescence staining (original magnification ×40) for the mitochondrial outer membrane marker preprotein translocases of the outer membrane (TOM)-20 (green) and transmission electron microscopy of renal tubular cell mitochondria in study groups. (b) Renal mitochondrial density that decreased in MetS+RAS compared to Lean and MetS, improved in MetS+RAS+Lean-EVs, but failed to improve in MetS+RAS+MetS-EVs, as were the mitochondrial area and matrix density. ∗p < 0.05 vs. Lean; †p < 0.05 vs. MetS; ‡p < 0.05 vs. MetS+RAS; #p < 0.05 vs. MetS+RAS+Lean-EVs.

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  Figure 7. MetS-EVs did not improve the renal mitochondrial function in MetS+RAS. (a) Mitochondrial hydrogen peroxide (H202) that increased in MetS compared to Lean and further increased in MetS+RAS, decreased in MetS+RAS+Lean-EVs, but not in MetS+RAS+MetS-EVs. (b) The cytochrome-c oxidase (COX)-IV activity and ATPADP ratio that decreased in MetS compared to Lean and further decreased in MetS+RAS, increased in MetS+RAS+Lean-EVs, but not in MetS+RAS+MetS-EVs. ∗p < 0.05 vs. Lean; †p < 0.05 vs. MetS; ‡p < 0.05 vs. MetS+RAS; #p < 0.05 vs. MetS+RAS+Lean-EVs.

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  Figure 8. MetS-EVs failed to improve poststenotic kidney injury. (a) Representative kidney dihydroethidium (DHE, red), periodic acid-Schiff (PAS), and trichrome staining in study groups. (b) Renal production of superoxide anion, tubular injury score, and tubulointerstitial fibrosis that increased in MetS+RAS compared to Lean and MetS, decreased in MetS+RAS+Lean-EVs, but not in MetS+RAS+MetS-EVs. ∗p < 0.05 vs. Lean; †p < 0.05 vs. MetS; ‡p < 0.05 vs. MetS+RAS; #p < 0.05 vs. MetS+RAS+Lean-EVs.

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  Figure 9. Renal mitochondrial damage correlated with renal injury. (a) The renal mitochondrial density correlated inversely with poststenotic tubular injury. (b) The mitochondrial matrix density correlated inversely with tubulointerstitial fibrosis. (c) Mitochondrial ATP generation correlated inversely with renal production of superoxide anion (DHE). (d) Mitochondrial H202 production correlated directly with tubulointerstitial fibrosis.