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 1

miR-93 expression decreases along disease progression in mice and humans.

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miR-93 expression decreases along disease progression in mice and humans...
(A) Graph showing miR-93 expression in glomeruli isolated from WT mice (C57BL/6J) at 4 months old (4m) and AS (Col4a5–/–; B6.Cg-Col4a5tm1Yseg/J) mice at 2m, 3.5m, and 5.5m. (B) Graph showing proteinuria levels, measured as albumin-to-creatinine ratio in WT mice and AS mice at 2m, 3.5m, and 5.5m. (C) Graph showing miR-93 expression in GECs, podocytes, and mesangial cells isolated from WT (4m), and AS mice at 2m, 3.5m, and 5.5m. No significant difference in miR-93 expression was noted in podocytes and mesangial cells. (D) Graph showing miR-93-5p (left) and miR-93-3p (right) expression in the kidney cortex of a human biopsy of AS patient (no. 2; male, 17 years old) compared to healthy tissue of a partial nephrectomy (used as reference; reference no. 1; male, 66 years old), showing that miR-93-5p is less expressed in AS and that miR-93-3p is much less expressed compared with miR-93-5p. (E) Representative images of human glomeruli, healthy tissue (reference no. 3) of a partial nephrectomy (male, 7 years old) showing miR-93 expression by in situ hybridization. Upper left corner: DAPI in blue. Upper right corner: EHD3 (green), identifying GECs. Lower left corner: miR-93 probe (red). Lower right corner: merged panels. White arrows: GECs showing coexpression of the miR-93 probe and EHD3. Yellow arrows: RBCs (autofluorescence in red). Scale bars: 20 μm. For miR-93 expression, small nuclear RNA U6 was used as a reference to calculate relative expression. All experiments were run in triplicate. All values are reported as mean ± SEM. Statistical significance was assessed using 1-way ANOVA with post hoc uncorrected Fisher’s LSD test. *P < 0.05; **P < 0.01; ****P < 0.0001.