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
View: Text | PDF
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

×

Figure 6

Spatial maps of AS biopsies.

Options: View larger image (or click on image) Download as PowerPoint
Spatial maps of AS biopsies. (A) Histology micrographs from serially sec...
(A) Histology micrographs from serially sectioned kidney biopsies from a 3-year-old patient with AS (no. 1), a 36-year-old patient with AS (no. 2), a 42-year-old patient with AS (no. 6), and a 19-year-old patient used as reference were stained for H&E followed by FOV section and CosMx SMI analysis. (B and C) UMAP visualization of cell classification based on the integration of the 4 biopsies (B) and for individual biopsy (C). A total of 29 FOVs were included in the final analysis following QC, distributed among 21 cell types as indicated by color codes. (D) Heatmap showing expression of top 5 markers identifying the renal cell populations classified in B. (E) Cell deconvolution analysis showing the abundance of different cell types in the 29 FOVs from biopsies shown in A. Analysis was based on publicly available RNA-seq experiments compiled into profile matrices by aggregating gene-wise counts of all annotated cell types. (F) Representative glomerulus from nondiseased patient showing cell segmentation and identification of podocytes (orange), GECs (blue), mesangial cells (light blue), and parietal epithelial cells (green). mRNA probe for VEGFA (white dots), KDR (red dots), and FLT1 (green dots) are spatially localized in the podocytes (VEGFA) and in GECs (KDR and FLT1). Cells that failed to pass the QC are represented in black. Scale bar: 25 μm. Images were visualized and captured using Napari 4.0.17 software. (G) Volcano plot showing the DEGs in GECs from all biopsies combined vs. reference (|log2FC| > 0.2; adj. P < 0.05). Statistical analysis was done to determine differential expression by DESeq2 (adj. P < 0.05). ATL, ascending thin limb; TAL, thick ascending limb; B, B lymphocytes; cDC, classical dendritic cells; CNT, connecting tubules; cycMNP, cycling mononuclear phagocytes; DCT, distal convoluted tubules; DTL, descending thin limb; EC, endothelial cells; FIB/aFIB/MyoF, fibroblasts/adaptive fibroblasts/myofibroblasts; IC, intercalated cells; MAC-M2, type 2 macrophages; MAST, mast cells; MC, mesangial cells; MDC, monocyte-derived cells; PC, principal cells; PEC, parietal epithelial cells; PL, plasma cell; POD, podocytes; PT, proximal tubules; T, T lymphocytes.