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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-specific 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 to 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-EVs injected kidneys showed that EVs can reverse pathways altered during disease progression by stimulating pro-regenerative 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 human Alport patients 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE318476 | GEO | 2026/02/11

REPOSITORIES: GEO

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Project description:Purpose: MicroRNA-21 contributes to the pathogenesis of fibrogenic diseases in multiple organs including the kidney. To evaluate the therapeutic utility of antimiR-21 oligonucleotides in chronic kidney disease, we silenced miR-21 in mice that develop Alport Nephropathy due to a defect in the Col4a3 gene. The goals of this study to assess the effect of inhibiting miR-21 in the Col4a3-/- Alport Syndrome mouse model at 9 weeks of age. Methods: Col4a3-/-, Col4a3+/-, and Col4a3+/+ mice in the 129X1/SvJ genetic background were obtained. Mice received anti–miR-21 (25 mg/kg) or control anti-miR (25mg/kg) in phosphate-buffered saline (PBS) by inter-scapular subcutaneous injection twice per week. In some experiments mice received a range of doses from 12.5mg/kg once a week to 50mg/kg once a week. Anti–miR-21 is a high-affinity oligonucleotide complementary to the active site of miR-21. Mice received injections starting at 24 days (3.5 weeks) after birth and ending at 5, 7, 9 or 16 weeks after birth depending on the study objectives. Total RNA from kidney tissue was extracted as per manufacturer’s instructions (miREASY kit, Qiagen). RNA quality was assessed using BioAnalyzer (Agilent). mRNA expression profiles were determined using next-generation sequencing (NGS) on the Illumina HiSeq 2000 platform producing 50bp paired-end reads. Bowtie/TopHat suites were used to align the reads to mouse genome or transcriptome and RSEM were used to quantify gene abundances. Gene level counts were then normalized with the R/Bioconductor package limma using the voom/variance stabilization method. Results: Anti-miR-21 enhanced PPAR?/RXR activity and associated downstream signaling pathways in glomerular, tubular and interstitial cells, enhanced mitochondrial function, which reduced mitochondrial ROS production and preserved tubular cell functions. In addition, inhibition of miR-21 reduced fibrogenic and inflammatory signaling in glomerular and interstitial cells, likely as a consequence of enhanced PPAR?/RXR activity and mitochondrial function. Inhibition of miR-21 represents a novel therapeutic strategy for chronic kidney diseases including Alport Nephropathy. Whole kidney mRNA profiles of wild type and Col4a3-/- mice treated with either PBS or antimiR-21, ending at 9 weeks of age, were generated by Next Generation Sequencing in triplicate using Illumina HiSeq 2000

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

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