REN2: Circulating Plasma Extracellular Vesicles (EV) Trigger Sepsis-Associated Acute Kidney Injury (AKI) and are reduced by Regional Citrate Anticoagulation (RCA)

Background and aims: During sepsis, extracellular vesicles (EV) are released from activated leukocytes, platelets and injured cells playing a key role in immune alterations, microvascular thrombosis and organ damage by transferring proteins and RNAs to target cells. Aims of this study were to evaluate: 1) EV from plasma of septic patients defining their potential role in Acute Kidney Injury (AKI); 2) the effect of Regional Citrate Anticoagulation (RCA) to limit EV release from activated cells in vitro and in vivo in an experimental model of sepsis in pigs. Methods: Plasma samples were collected from 30 septic AKI patients treated by CVVH or CVVHD with heparin or citrate to analyze EV (Nanosight and protein/RNA profiling). The effects of EV isolated from plasma were studied on human kidney-derived endothelial and tubular epithelial cells in vitro. Moreover, EV release was evaluated ex vivo using LPS-activated neutrophils, monocytes and platelets in presence of citrate or heparin. The same determinations were also performed in an experimental model of sepsis in pigs treated by a Selective Cytophoretic Device (SCD). Results: Plasma EV concentrations were higher in septic than non-septic patients correlating with organ failure and mortality. Isolated septic EV carried inflammatory proteins/RNA and induced functional alterations and apoptosis of kidney endothelial and tubular epithelial cells (e.g. adhesion of leukocytes, endothelial-to-mesenchymal transition, tubular senescence and injury). RCA but not heparin significantly decreased plasma EV levels during CVVH/CVVHD and their detrimental effects on isolated renal cells in vitro. Using LPS-activated leukocytes and platelets ex vivo, we observed that citrate but not heparin reduced intracellular calcium and consequently the detrimental EV release. These data were confirmed by Nanosight analysis and cell biology studies that revealed a significant decrease of EV concentration, modulation of EV phenotype and of EV-induced endothelial/tubular injury in septic pigs treated by SCD in presence of citrate but not heparin. Conclusion: During sepsis, EV are released by activated leukocytes/platelets and by injured cells playing a key role in the development of multiple organ dysfunction including AKI. Our in vivo and in vitro results strongly suggest that RCA may limit EV release and their detrimental biological activities on endothelial and tubular epithelial cells.