Glomerular endothelial derived vesicles mediate podocyte dysfunction: A potential role for miRNA.

N Hill,M Ramirez-Solano,D L Michell,K J Woollard,Q Sheng,K C Vickers,C Pusey,Jean-Claude Dussaule

doi:10.1371/journal.pone.0224852

N Hill, M Ramirez-Solano + Show 6 more

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https://doi.org/10.1371/journal.pone.0224852

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Abstract

MicroRNAs (miRNA) are shown to be involved in the progression of several types of kidney diseases. Podocytes maintain the integrity of the glomerular basement membrane. Extracellular vesicles (EV) are important in cell-to-cell communication as they can transfer cellular content between cells, including miRNA. However, little is known about how extracellular signals from the glomerular microenvironment regulate podocyte activity. Using a non-contact transwell system, communication between glomerular endothelial cells (GEnC) and podocytes was characterised in-vitro. Identification of transferred EV-miRNAs from GEnC to podocytes was performed using fluorescence cell tracking and miRNA mimetics. To represent kidney disease, podocyte molecular profiling and functions were analysed after EV treatments derived from steady state or activated GEnC. Our data shows activation of GEnC alters EV-miRNA loading, but activation was not found to alter EV secretion. EV delivery of miRNA to recipient podocytes altered cellular miRNA abundance and effector functions in podocytes, including decreased secretion of VEGF and increased mitochondrial stress which lead to altered cellular metabolism and cytoskeletal rearrangement. Finally, results support our hypothesis that miRNA-200c-3p is transfered by EVs from GEnC to podocytes in response to activation, ultimately leading to podocyte dysfunction.

Highlights

In the kidney, glomerular endothelial cells (GEnC) in direct contact with the bloodstream bear fenestrations which allow the passage of certain molecules through the glomerular filtration barrier
Heatmaps of the most upregulated miRNAs from podocytes treated with GLU and puromycin aminonucleoside (PAN)-stimulated GEnC-derived Extracellular vesicles (EV), as compared to podocytes treated with EV from resting GEnC (SS), are presented in S6 Fig. Pathway and data enrichment analysis of predicted target genes of the significantly altered miRNAs was completed and we identified many of the upregulated miRNAs to be associated
We found a significant decrease in VEGF secretion in podocytes treated with EVs from glucose, LPS or PAN treated GEnCs compared to podocytes treated with steady state GEnC-derived EVs (Fig 5A)

Summary

Introduction

Glomerular endothelial cells (GEnC) in direct contact with the bloodstream bear fenestrations which allow the passage of certain molecules through the glomerular filtration barrier. The capillary endothelial cell layer of the glomerulus is characterised by circular, transcellular pores, around 60–80 nm in diameter that occupy 30–50% of the endothelial surface [1, 2]. This makes them ideal opportunists to transport proteins, biochemicals and vesicles from the blood stream to the underlying glomerular basement membrane (GBM) and podocytes. The glomerular endothelial glycocalyx is another important layer to consider, which may regulate vascular permeability, controlling both size and charge exclusion properties [3].

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