Endothelial extracellular vesicle miR-423-5p regulates microvascular homeostasis and renal function after ischemia-reperfusion injury
Francis Migneault, Hyunyun Kim, Alice Doreille, Shanshan Lan, Alexis Gendron, Marie-Hélène Normand, Annie Karakeussian Rimbaud, Martin Dupont, Isabelle Bourdeau, Éric Bonneil, Julie Turgeon, Sylvie Dussault, Pierre Thibault, Mélanie Dieudé, Éric Boilard, Alain Rivard, Héloïse Cardinal, Marie-Josée Hébert
Francis Migneault, Hyunyun Kim, Alice Doreille, Shanshan Lan, Alexis Gendron, Marie-Hélène Normand, Annie Karakeussian Rimbaud, Martin Dupont, Isabelle Bourdeau, Éric Bonneil, Julie Turgeon, Sylvie Dussault, Pierre Thibault, Mélanie Dieudé, Éric Boilard, Alain Rivard, Héloïse Cardinal, Marie-Josée Hébert
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Research Article Cell biology Nephrology Vascular biology

Endothelial extracellular vesicle miR-423-5p regulates microvascular homeostasis and renal function after ischemia-reperfusion injury

Abstract

Microvascular rarefaction substantially contributes to renal dysfunction following ischemia-reperfusion injury (IRI). We characterized the microRNA signature of extracellular vesicles (EVs) released during endothelial apoptosis to identify biomarkers and regulators of microvascular rarefaction and renal dysfunction. Using in vitro models and RNA-Seq, we found miR-423-5p, let-7b-5p, and let-7c-5p enriched in small EVs from apoptotic endothelial cells. In mouse models of renal IRI and a cohort of 51 patients who have undergone renal transplant with delayed graft function, serum miR-423-5p correlated with circulating EVs, while let-7b-5p and let-7c-5p were also present in free form. Early acute kidney injury saw increased serum miR-423-5p levels linked to small EVs with endothelial markers. Over time, higher serum miR-423-5p levels were associated with large EVs and correlated with greater renal microvascular density and reduced fibrosis. Microvascular density and fibrosis predicted renal function 3 years after transplantation. We explored miR-423-5p’s role in renal homeostasis, finding that its injection during renal IRI preserved microvascular density and inhibited fibrosis. Endothelial cells transfected with miR-423-5p showed enhanced resistance to apoptosis, increased migration, and angiogenesis. Localized miR-423-5p injection in hindlimb ischemia model accelerated revascularization. These findings position miR-423-5p as a predictor of renal microvascular rarefaction and fibrosis, highlighting potential strategies for preserving renal function.

Authors

Francis Migneault, Hyunyun Kim, Alice Doreille, Shanshan Lan, Alexis Gendron, Marie-Hélène Normand, Annie Karakeussian Rimbaud, Martin Dupont, Isabelle Bourdeau, Éric Bonneil, Julie Turgeon, Sylvie Dussault, Pierre Thibault, Mélanie Dieudé, Éric Boilard, Alain Rivard, Héloïse Cardinal, Marie-Josée Hébert

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Figure 3

At a distance from IRI, lower miR-423-5p serum levels correlate with more severe microvascular rarefaction and renal fibrosis.

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At a distance from IRI, lower miR-423-5p serum levels correlate with mor...
(A) PLVAP IHC (left) and Sirius red staining (right) in corticomedullary junction in 21 days sham mice or 21 days after 30-minute and 60-minute IRI. Quantification of PLVAP+ PTC (left) and Sirius red+ area within PTC (right) in corticomedullary junction in 21 days sham mice or 21 days after 30-minute and 60-minute IRI; n = 3–5. (B) miR-423-5p serum levels at baseline or 1, 2, 7, and 21 days after 30-minute and 60-minute IRI or in 21-day sham mice. Expression of miR-423-5p measured by qPCR and presented as relative copies of miR-423-5p per μL of total serum ± SEM after normalization with cel-miR-39; n = 3–23 (Supplemental Figure 3E). (C) miR-423-5p serum levels at 21 days after sham surgery (black) or 30-minute (blue) and 60-minute (yellow) IRI correlated with microvascular density (ρ = 0.7802; P = 0.0025) and inversely correlated with collagen deposition (ρ = –0.8626; P = 0.0003). (D) Immunoblots and quantification of different protein markers in large (50,000g) and small (200,000g) extracellular vesicles (EVs) from mouse serum at baseline and 1 and 21 days after IRI. PLVAP for endothelial-derived EVs, PSMA3, and LG3 for apoptotic exosome-like vesicles, ACTB for EV marker, and CD82 for exosome marker; n = 3–10. (E) Distribution of miR-423-5p expression in large (50,000g) and small EVs (200,000g) from the serum of mice at baseline and 2 and 21 days after 30 minutes of IRI; n = 5 for each fraction. Data are shown as mean ± SEM. P values obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Scale bars: 50 µm.