Polymyxin B Hemoadsorption efficiently removes Gram-Negative-derived Quorum Sensing molecules responsible for acute kidney tubular epithelial cell injury

Background. Lipopolysaccharide (LPS) is the main driver of sepsis-associated Acute Kidney Injury (SA-AKI) in Gram-Negative infections and its removal by Polymyxin-B Hemoadsorption (PMX-HA) has been evaluated in clinical trials. Quorum Sensing (QS), diffusible signal molecules used by Gram-Positive and Gram-Negative bacteria for intercellular communication, can interact with eukaryotic cells. Study aims were to evaluate: 1) presence of QS molecules in blood and urine of septic patients and their clinical significance; 2) biological effects of QS on human tubular epithelial cells (TEC); 3) role of PMX-HA in reducing QS blood levels and consequently TEC injury. Methods. Thirty-one patients with endotoxemic shock treated by PMX-HA were enrolled. LC-MS was used to detect QS molecules in blood and urine and their concentrations were correlated with clinical data. The effects of QS on TEC in presence or absence of LPS were assessed by studying cytotoxicity (XTT), apoptosis (TUNEL), stress/injury biomarkers (TIMP-2, IGFBP-7, NGAL), ROS generation, mitochondrial dysfunction and cell polarity (TEER, ZO-1/megalin expression). Results. We found different QS molecules belonging to Acyl homoserine lactones (C4- and 3-oxo-C12-AHL) and Hydroxyquinolones (C7 HQ) in blood and urine of septic patients: C4-AHL was the most abundant QS family member. A correlation between QS concentrations and endotoxin activity, inflammatory biomarkers and organ dysfunction parameters (PaO ₂ /FiO ₂ , serum creatinine) was observed. PMX-HA decreased QS molecules in biofluids: these results were confirmed by ex-vivo hemoadsorption using a PMX-B-loaded minicartridge. In vitro studies on TEC showed that C4-AHL QS induced cytotoxicity, apoptosis, up-regulation of stress and damage biomarkers, ROS production and mitochondrial dysfunction. C4-AHL QS also induced earlier functional alterations of TEC such as loss of polarity and down-regulation of ZO-1 and megalin. The detrimental effects of C4-AHL QS on TEC were enhanced by the presence of LPS. The specificity of C4-AHL QS-induced TEC damage was confirmed by using supernatants derived from isogenic mutants of Pseudomonas aeruginosa not able to produce this molecule. Conclusions. The results of the present study suggested that PMX-HA could remove from blood QS molecules able to synergize with LPS in the triggering of functional alterations and apoptosis of TEC, key pathogenic mechanisms of SA-AKI.