MICROVESICLES (MVs) DERIVED FROM ENDOTHELIAL PROGENITOR CELLS (EPCs) PROTECTS KIDNEYS FROM ISCHEMIA-REPERFUSION INJURY (IRI) THROUGH THE microRNA-DEPENDENT REPROGRAMMING OF RENAL RESIDENT CELLS

INTRODUCTION AND AIMS: EPCs are bone marrow-derived stem cells able to trigger tissue regeneration by paracrine mechanisms including the secretion of growth factors and MVs. MVs are small membrane fragments carrying genetic information that play a key role in cell-to-cell communication. MVs released from EPCs activate an angiogenic program in endothelial cells through the horizontal transfer of RNAs. The aim of this study was to evaluate whether EPC-derived MVs prevent IRI-associated AKI, identifying the role of microRNAs (miRNAs) shuttled by MVs in the reprogramming of renal resident cells. METHODS: EPCs were isolated from peripheral blood of healthy volunteers. MVs were isolated by ultracentrifugation and characterized for protein and RNA content. Wistar rats were treated as follows: 1) Sham; 2) IRI (renal pedicle clamping for 45 min); 3) IRI + 30 µg/ml EPC MVs; 4) IRI + 30 µg/ml EPC MVs pre-treated with 1U/ml RNase; 5) IRI + 30 µg/ml MVs from control fibroblasts. Renal function and histology were evaluated. In vitro, we studied the effects of MVs on isolated kidney endothelial, tubular epithelial and CD133+ progenitor cells. In selected experiments, MVs were isolated from EPCs engineered by siRNA to knock-down Dicer, the intracellular enzyme essential for miRNA production. RESULTS: MVs expressed markers of stem and endothelial cells and molecules of the integrin/selectin families essential for their internalization into target cells. MVs carried different mRNAs and 146 miRNAs such as miR-126 and miR-296 able to modulate proliferation, angiogenesis and apoptosis. After injection in rats, EPC MVs localized within peritubular capillaries and tubular cells and protected rats from IRI-associated AKI (decrease of serum creatinine/BUN and of histologic tubular injury) MVs significantly reduced apoptosis, sustained tubular proliferation and inhibited leukocyte infiltration. All these protective effects were not observed using fibroblast-derived MVs or when EPC MVs were pre-treated with RNase or produced by EPCs subjected to Dicer knock-down. In vitro, MVs inhibited hypoxia-induced apoptosis and functional alterations of kidney endothelial and epithelial cells by up-regulating genes involved in cell proliferation, differentiation, angiogenesis and inhibition of inflammation. In addition, MVs induced the differentiation of CD133+ renal progenitors towards an endothelial phenotype. CONCLUSIONS: EPC MVs induced a protective effect on IRI-associateed AKI. This effect may be ascribed to the reprogramming of renal resident cells induced by MV-shuttled miRNAs. Our results suggest that EPC-derived MVs could be exploited as a potential new therapeutic approach for AKI and other ischemic diseases without the potential risks of stem cell therapy such as maldifferentiation and tumorigenesis.