Pulmonary endothelial cells endocytosis of HMGB1 promotes lung injury after renal ischemia-reperfusion injury

Dysfunction of other organs is a significant contributor to the poor outcomes associated with acute kidney injury (AKI). HMGB1 is proposed to play a crucial role in the development of common extra-renal complications: acute lung injury (ALI). However, the mechanisms underlying these connections are not fully understood. Here, we established mice models of renal ischemia-reperfusion injury (IRI). HMGB1 was observed to be released from renal cell nucleus 1 hour after reperfusion. Based on mass spectrometry screening, clathrin was identified and the released HMGB1 entered pulmonary endothelial cells via clathrin-mediated endocytosis after binding to TLR4. Further investigations revealed that blocking HMGB1 with neutralizing antibodies or knocking down clathrin in lungs reversed pulmonary injury induced by renal IRI through inhibiting neutrophil recruitment and the formation and release of neutrophil extracellular traps (NETs) in lungs. Moreover, knocking down HMGB1 in TCMK-1 cells or clathrin in MPVECs in the co-culture systems in vitro obtained similar results. These findings demonstrate the effect of HMGB1 released from kidney in promoting ALI following renal IRI, disclose pulmonary endothelial cell endocytosis of HMGB1 mediated by clathrin as the potential mechanism, and identify neutralizing antibody to HMGB1 as a promising ALI mitigation strategy by inhibiting NETs formation in lungs. Co-cultured untreated TCMK-1 cells or the ATP repleted TCMK-1 cells after ATP depletion with untreated MPVECs for 12 h. Then the MPVECs in the lower chamber underwent gene sequencing.