Endotoxin, neutrophil function and albumin in renal insufficiency

Background/state of the art: Chronic kidney disease is widespread in the western world with bacterial infection and sepsis as common complication. It has been shown that innate immune defence, represented by dysfunction of neutrophil granulocytes, is impaired in chronic kidney disease. Another impact of chronic kidney disease on innate immunity is the chronic activation of neutrophils leading to high levels of inflammatory cytokines, thus contributing to protein oxidation. Oxidation of human serum albumin (HSA), the major plasma protein, occurs in chronic kidney disease and leads to further activation of neutrophils. Another important impact of HSA oxidation is the decrease of its binding capacity leading to impaired detoxification ability of albumin. This includes reduced clearance of endotoxin, a major component of the gram negative bacterial cell wall. Circulating endotoxin is recognized by complex formation with lipopolysaccharide binding protein (LBP) followed by binding to CD14 and toll-like receptor (TLR) 4. High systemic endotoxin levels occur in chronic kidney disease and may be the result of decreased clearance ability of HSA and increased gut permeability in combination with intestinal bacterial overgrowth. High systemic endotoxin is associated with worse outcome in several diseases and could be used as predictor for mortality in chronic kidney disease patients. Hypothesis and aims: We hypothesize that endotoxemia in renal insufficiency leads to impaired neutrophil function and to increased albumin oxidation. Oxidized albumin is not able to bind endotoxin adequately any more, which leads to a further increase in oxidative stress and neutrophil dysfunction, resulting in a vicious cycle. Methods: One hundred ninety-five patients with renal dysfunction will be enrolled and divided into 5 groups. Additionally, samples of 25 age and sex-matched healthy controls will be collected. Endotoxin in patients` samples will be detected by an adapted limulus amoebocyte lysate (LAL) assay. In combination with that, lipopolysaccharide binding protein (LBP) and soluble CD14 (sCD14) will be determined by means of ELISA. HPLC will be used to determine nitrate, nitrite, albumin fractions, albumin binding capacity, iNOS expression and energy status of neutrophils. For investigation of oxidation driven by leukocyte derived myeloperoxidase, mass spectrometry analysis will be used. Determination of carbonyl contents of proteins by ELISA will be the completion of determination of oxidative changes of albumin. Neutrophil function will be determined by the Phagoburst and Phagotest kits and determination of TLR2, 4 and 9 expression by flow cytometrical analysis. For cell culture tests, freshly isolated neutrophils or differentiated HL60 cells will be used and incubated with albumin and/or endotoxin. Conclusion: This concept will change the understanding of several aspects of chronic kidney disease and will potentially help to stratify patients into different groups at risk according to their endotoxin status, and their immune and albumin dysfunction. The results of this study will have important implications into the development of novel therapeutic strategies.

Chronic kidney disease is a common disorder, affecting 10% of the population. In Austria around 8700 patients suffer from end-stage renal disease. Among these patients, infections are the second most common cause of death. Prevention of infections is still an unmet clinical need. We hypothesize that end-stage renal disease is associated with profound alterations of gut microbiome and gut permeability, leading to immune dysfunction and therefore an increased risk of developing severe infections. The main finding of this study was an increase in serum endotoxin levels (= contents of the bacterial cell wall) associated with a decrease in neutrophil (= immune cells) function in patients undergoing haemodialysis. These patients were also more prone to die from infections during the follow up period. Gut permeability, oxidative stress, cellular energy charge or inflammation did not completely explain these differences. Oxidative stress however increased platelet activation in chronic kidney disease. We could show in an ancillary study that ultrafiltration during haemodialysis leads to vasoconstriction in gut vessels. Especially in haemodialysis patients, who are also diabetic, this might lead to gut ischemia. To explore potential mechanisms for the observed decrease in neutrophil function, we also studied gut microbiome composition in a subset of patients. Analysis of these data is ongoing. Another part of the study focused on new biomarkers and found that some molecules (microRNAs) are very promising to predict problems in bone metabolism. In conclusion, this cross sectional study could extend the knowledge on immune function, gut permeability and oxidative stress in chronic kidney disease. The most important finding was the strong influence of the mode of dialysis (haemodialysis) on immune function. Defining strategies to improve immune function in this high-risk group and thereby maybe decrease the risk of infection related mortality is an unmet clinical need. One possible strategy is the modulation of the gut microbiome by probiotics. An intervention study is in planning and submitted to the KLIF program from the Austrian Science Foundation.