Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury.

Davide Medica,Vincenzo Cantaluppi,Alessandra Stasi,Giuseppe Castellano,Loreto Gesualdo,Massimiliano Migliori,Federico Figliolini,Vincenzo Panichi,Giovanni Camussi,Rossana Franzin

doi:10.3390/cells10071675

Abstract

Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis.

Highlights

The glomerulus is a crew of capillaries implicated in the ultrafiltration processes of the kidney
We studied in vitro the protective effects of Endothelial Progenitor Cells (EPC)-derived extracellular vesicles (EVs) on Glomerular endothelial cells (GEC) and podocytes cultured with Tumor Necrosis Factor α (TNF-α), IL-6, and C5a, in an inflammatory microenvironment resembling that observed in glomerulonephritis
As shown by confocal microscopy studies (Figure 1A), we observed that PKH26 red-labeled EVs were efficiently internalized in vitro in GECs as well as in podocytes

Summary

Introduction

The glomerulus is a crew of capillaries implicated in the ultrafiltration processes of the kidney. The glomerular capillary wall is composed of three layers: a fenestrated endothelium of glomerular endothelial cells, a glycocalyx with a complex mesh of proteins called glomerular basement membrane (GBM), and a layer of specialized visceral epithelial cells called podocytes [1]. Glomerular Filtration Barrier (GFB) has a very high hydraulic permeability combined with a marked selective permeability that excludes macromolecules such as albumin. Podocytes constitute the slit diaphragms between their inter-digitating foot processes that prevent large molecules from reaching the urinary space [3]. The expression of nephrin in the podocyte slit diaphragm is crucial for maintaining GFB selectivity [4]. The glomerular microenvironment maintains GEC function stimulating expression of endothelial receptors such as Platelet Endothelial Cell

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