Background
Circulating neutrophil-derived extracellular vesicles (NEVs) are elevated in critically ill patients and may act as a hidden reservoir of inflammation. We assessed their role in systemic and renal glomerular inflammation and their potential involvement in the development of acute kidney injury during critical illness.

Methods
NEVs were isolated from lipopolysaccharide-treated healthy donor blood or critically ill COVID-19 patients with acute respiratory distress syndrome. NEVs were incubated for 4 hours in our in vitro models comprising human umbilical vein endothelial cells or renal glomerular endothelial cells, co-cultured with peripheral blood mononuclear cells.

Results
NEVs were internalised by monocytes leading to their activation via the p38 pathway (NEVs vs untreated, p38 median fluorescence intensity (MFI), 694 vs 459, p=0.006), causing the release of tumour necrosis factor (NEVs vs untreated, TNF pg ml-¹, 676 vs 27, p=0.003), which subsequently increased cell adhesion molecule expression on human umbilical vein endothelial cells (NEVs vs untreated, E-Selectin MFI, 4120 vs 1438, p=0.008) or human renal glomerular endothelial cells (NEVs vs untreated, E-Selectin MFI, 2960 vs 931, p=0.003). These NEVs contained substantial amounts of matrix-metalloproteinase-8 and 9, inhibitors of which effectively ablated NEV-induced activation of the endothelial cells.

Conclusions
We have demonstrated a comprehensive pathway detailing how circulating NEVs induce significant renal endothelial inflammation in a monocyte-dependent fashion. Our data suggest a mechanistic role for circulatory NEVs in the pathophysiology of acute kidney injury in critically ill patients, including patients with COVID-19.