Endotoxin, Neutrophil Function and Albumin in Renal Insufficiency
Endotoxin, Neutrophil Function and Albumin in Renal Insufficiency
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.

 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.

 195 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.

 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
Laboratory methods Endotoxin will be detected by an adapted limulus amoebocyte lysate assay.
 LBP and 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. Carbonyl contents of proteins will be detected by ELISA.
 Neutrophil function and TLR2, 4 and 9 expression will be studied 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. Stool samples will be used for
 16srDNA sequencing of the gut microbiome.
Inclusion Criteria:

 Age between 18-80 years, informed consent Groups 1a, 1b, 2a, 2b, 3a, 3b Patients with
 chronic kidney disease as defined previously [65] either

 1a) with an eGFR between 30 and 45 (KDIGO 3B)

 1. b) with an eGFR between 15 and 30 (KDIGO 4)

 2. a) undergoing hemodialysis for ESRD

 2b) undergoing hemodiafiltration for ESRD

 3a) undergoing peritoneal dialysis for ESRD without signs of infection

 3b) undergoing peritoneal dialysis for ESRD with peritonitis ≥2 out of the 4 criteria (>100
 leucocytes/50%neutrophils, cloudy peritoneal dialysate, typical clinical presentation with
 fever and abdominal pain, positive culture from the peritoneal dialysate)

 Group 4 Patients with acute kidney injury (AKIN 3 [66] defined as an increase in serum
 creatinine to 300% (3-fold) from baseline or serum creatinine 4.0 mg/dl with an acute rise
 of at least 0.5mg/dl or urine output of < 0.3ml/kg/h 24h or anuria 12h) Initiation of acute
 renal replacement therapy

 Group 5 Stable patients after kidney transplantation with either an eGFR > 45, between 30
 and 45 or < 30

 Group 6: Healthy controls

 Exclusion Criteria:

 -  Malignancy, pregnancy,chronic inflammatory bowel disease, celiac disease, active
 alcohol abuse, any severe organ dysfunction unrelated to renal dysfunction Groups 1a,
 1b, 2a, 2b, 3 Organ transplantation Clinical evidence of active infection (except for
 group 3b) Treatment with antibiotics within the last 2 weeks (except for group 3b)

 Group 4 Preexisting ESRD

 Group 5 Clinical evidence of active infection Treatment with antibiotics within the last 2
 weeks

 Group 6:

 Any evidence of acute or chronic disease