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 8

miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis.

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miR-423-5p protects endothelial cells from apoptosis and promotes endoth...
(A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. TUBA1B was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).