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Page 40 Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10

labeling did not affect the physiological characteristics of the cells, and the MR detectability of labeled-EVs
were confirmed by magnetometer and in vitro MRI phantoms. To demonstrate the utility of MRI- assisted
EV tracking, a proof of concept in vivo biodistribution study was conducted by injecting labeled AFSC EVs
into WT and Alport mice (a model of chronic kidney disease) via different routes of administration, and
tracking them via MRI at 10 min and 3 h post injection. MRI studies showed that homing of AFSC EVs to
the kidney injected intra-cardiacally into Alport mice were more efficient versus the retro-orbital route, and
Prussian blue staining of kidney sections confirmed the MR findings. In summary, we have developed a
clinically applicable novel magnetic nanoparticle agent that can be used to label and track the biodistribution
of EVs in living subjects using non- invasive, safe, and effective MRI technology that’s widely available. This
technology is highly adaptable and can be deployed in both preclinical and clinical settings.

26. Glomerular heterogeneity and modulation of miR-93-5p: the role of extracellular vesicles

Authors: Charmi Dedhia, Paola Aguiari, Hasmik Soloyan, Roger E De Filippo, Sargis Sedrakyan, Laura
Perin
E-mail: lperin@chla.usc.edu
Affiliations: Children’s Hospital Los Angeles/University of Southern California, Los Angeles, CA, USA.
Abstracts: miRNA play important roles in the pathogenesis of renal diseases. Modulation of miRNA
in podocytes and glomerular endothelial cells (GEC) has been associated with cellular damage and
development of renal diseases. miR-93-5 is a potent regulator of various genes and pathways responsible
of glomerular damage during pathological conditions, like VEGF, TGF and Msk2. We have evidence that
miR- 93-5p is altered in the glomeruli of mice affected with X-linked Alport syndrome (AS), as well as in
human AS glomeruli. In this study, we investigated the role of miR- 93-5p in glomerular cells from healthy
and AS mice. We also used extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSC),
which are naturally enriched with mir-93-5p to asses their therapeutic potential to rescue glomerular
damage in vitro and in vivo by regulating miR-93-5p target genes. Mesangial cells, podocytes and GEC were
sorted from glomeruli of male and female WT and AS mice at different stages of disease and miR-93-5p
expression was evaluated by qRT-PCR. We assessed renal cortices from patients affected by AS and disease-
modifying activity of human AFSC EVs by 1) applying EVs to damaged GEC and podocytes in vitro
followed by analysis of mir-93-5p targets and 2) injecting EVs into AS mice followed by RNAseq analysis of
isolated glomeruli and survival. Glomerular miR-93-5p expression differed between male and female mice
and in glomerular cells throughout the progression of disease vs. WT. In glomerular cells from AS male
mice, miR-93-5p levels were significantly lower in GEC, but not in podocytes or mesangial cells, relative
to WT. Consistently, decreased miR-93-5p expression was detected in human samples from AS patients.
Expression of WT1 and miR-93 in puromycin aminonucleoside damaged podocytes and expression of
fibronectin and miR-93 in VEGF damaged GEC was restored to basal level in the presence of hEVs. In vivo,
single injection of hEVs showed therapeutic effect by ameliorating the level of proteinuria and increasing
life span, as shown for mouse EVs (Sedrakyan, 2017). Differential gene expression and pathway enrichment
analysis showed stark differences between male and female glomeruli in WT, involving respiratory and
metabolic pathways, extracellular matrix and cell adhesion molecules. In AS males, genes with functional
role in lipid metabolism and angiogenic pathways were most highly regulated; AS males injected with
EVs showed improved gene modulations in metabolic function and genes with functional role in the
development of vasculature and angiogenesis were most highly enriched. Gender specific variation in miR-
93-5p expression in glomerular cells might indicate important differences in their biology and potential
response to injury during development of kidney diseases. EVs from AFSC demonstrate great potential
to restore lost miR-93-5p expression in glomeruli of AS and therefore can present powerful therapeutic
approach for treatment of CKD.