Extracellular vesicle miR-93-5p cargo regulates glomerular endothelial cell damage in Alport syndrome
Charmi Dedhia, Valentina Villani, Xiaogang Hou, Paolo Neviani, Geremy Clair, Mohammadreza Kasravi, Cristina Grange, Paolo Cravedi, Paola Aguiari, Velia Alcala, Giuseppe Orlando, Xue-Ying Song, Jonathan E. Zuckerman, Roger E. De Filippo, Stefano Da Sacco, Sargis Sedrakyan, Benedetta Bussolati, Laura Perin
Charmi Dedhia, Valentina Villani, Xiaogang Hou, Paolo Neviani, Geremy Clair, Mohammadreza Kasravi, Cristina Grange, Paolo Cravedi, Paola Aguiari, Velia Alcala, Giuseppe Orlando, Xue-Ying Song, Jonathan E. Zuckerman, Roger E. De Filippo, Stefano Da Sacco, Sargis Sedrakyan, Benedetta Bussolati, Laura Perin
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Research Article Cell biology Nephrology

Extracellular vesicle miR-93-5p cargo regulates glomerular endothelial cell damage in Alport syndrome

Abstract

Modulation of miRNA expression in glomerular cells is associated with renal disease. Here, we investigated the role of miR-93-5p in mitigating glomerular damage in Alport syndrome and whether the disease-modifying activity of extracellular vesicles from human amniotic fluid stem cells (hAFSC-EVs) is mediated by their miR-93-5p cargo. We identified downregulation of miR-93-5p specifically in glomerular endothelial cells in Alport syndrome along disease progression. Silencing of miR-93-5p in hAFSC-EVs changed the transcriptomic and proteomic profile, regulating EV disease-modifying activity. Compared with naive hAFSC-EVs, silenced hAFSC-EVs did not rescue glomerular endothelial function in vitro and did not restore kidney function in vivo. We established that hAFSC-EVs regulate VEGFR1 and VEGFR2 signaling by miR-93-5p cargo transfer, highlighting that miR-93-5p can restore glomerular endothelial cell biology. Spatial transcriptomics analysis of hAFSC-EV–injected kidneys showed that these EVs can reverse pathways altered during disease progression by stimulating proregenerative processes, specifically in the glomerulus, by regulating miR-93-5p targets. Alteration of glomerular endothelial cell transcriptomics and miR-93-5p targets was also confirmed in biopsies of patients with Alport syndrome using spatial molecular imaging. We demonstrated the critical role of miR-93-5p in glomerular endothelial cells and the capability of hAFSC-EVs to regulate miR-93-5p and its targets in Alport syndrome.

Authors

Charmi Dedhia, Valentina Villani, Xiaogang Hou, Paolo Neviani, Geremy Clair, Mohammadreza Kasravi, Cristina Grange, Paolo Cravedi, Paola Aguiari, Velia Alcala, Giuseppe Orlando, Xue-Ying Song, Jonathan E. Zuckerman, Roger E. De Filippo, Stefano Da Sacco, Sargis Sedrakyan, Benedetta Bussolati, Laura Perin

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Figure 3

EVs regulate the miR-93/VEGFR1/VEGFR2 axis in GECs.

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EVs regulate the miR-93/VEGFR1/VEGFR2 axis in GECs. (A) Relative miR-93 ...
(A) Relative miR-93 expression in GECs at baseline, VEGF-induced damage, VEGF-induced damage plus EVs, and VEGF-induced damage plus KD_EVs. Note: P = 0.0.55 between the VEGF-induced damage and the VEGF-induced damage plus EVs group. Small nuclear RNA U6 was used as a reference to calculate relative expression. (B) Densitometric analysis for fibronectin (261 kDa) by Western blot (WB) in GECs under indicated conditions. Quantification was normalized to β-actin (42 kDa). WB bands are shown below. (C) Relative miR-93 expression in GECs under indicated conditions. Small nuclear RNA U6 was used for normalization. (D and E) Densitometric analysis of VEGFR1 (75 kDa) in hAFSCs and KD hAFSCs (D) and in EVs and KD_EVs (E). For hAFSCs in D, quantification was normalized to β-actin (42 kDa). (F) Coimmunoprecipitation assay for VEGF (25 kDa) using an anti-VEGFR1 antibody on EVs and KD_EVs exposed to VEGF. Expression of VEGF by WB analysis as indicated. WB bands are shown below. (G) Densitometric analysis for VEGFR1 (75 kDa) in GECs under indicated conditions, normalized to β-actin (42 kDa). WB bands are shown below. (H) Densitometric analysis for p-VEGFR2/VEGFR2 (250 kDa and 192 kDa, respectively) ratio in GECs under indicated conditions. WB bands are shown below. (I) Left: Graph showing fluorescein absorbance (log scale) in the GEOAC in channel F (“filtrate”) after 60 minutes in GECs under indicated conditions (8–12 chips per group). Right: 3D confocal Z-stack images showing GEC monolayer formation, GECs with VEGF-induced damage, GECs with VEGF-induced damage plus EVs, and GECs with VEGF-induced damage with KD_EVs. GECs were labeled with CellTracker Deep Red. White asterisks represent disruption of the continuous monolayer in the damaged GEOAC and GEOAC treated with KD_EVs or EVs (scale bars: 100 μm). Data are reported as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with uncorrected Fisher’s LSD post hoc test (A–C and G–I) or unpaired, 2-tailed Student’s t test (D–F).