Analysis of haemolysis-based injury in immortalised proximal tubule epithelial cells is impaired by haemoglobin interference with bioluminescence assays.

Acute kidney injury (AKI) is a serious complication which is common amongst cardiac and aortic/vascular surgery patients. Although the cause of AKI following cardiac surgery is multifactorial, haemolysis is an increasingly recognised contributing factor caused by the use of cardiopulmonary bypass machines. Haemolysis, the lysis of red blood cells, releases free haemoglobin (fHb) into the bloodstream which is known to cause kidney injury. Currently, the mechanisms of haemolysis-induced kidney injury are not fully understood. In this study, we investigated the injury response to fHb in a human-derived conditionally immortalised proximal tubule epithelial cell line (ciPTECs). Results showed that fHb interference reduces bioluminescence signal output which is considerably mitigated by removing fHb solutions and haemolysed plasma prior to the beginning of the assay. Interestingly, we found that the effect of fHb on ciPTECs was different with haemolysed plasma compared to fHb solutions, potentially due to factors other than fHb within the plasma. In contrast to expectations, haemolysed plasma induced only a marginal increase in NF-ĸB activity and had minimal effect on mitochondrial function. Together, these findings suggest that caution should be applied when using bioluminescent assays with fHb, both in solution and haemolysed plasma, and that fHb may not inflict severe injury on ciPTECs.