Morpho-functional characterisation of protective mechanisms of neutrophils in the patients at the terminal stage of chronic kidney disease

Abstract

Patients with chronic kidney disease (CKD) are a unique clinical “model” for studying the functions of immunocompetent cells under the conditions of severe endogenous intoxication, inflammation and immunosuppression. The aim of our study was to perform a comprehensive assessment of morphological and functional activity of neutrophils in patients at the terminal stage of chronic renal failure. We examined 49 patients (16 women, 33 men, 22 to 63 years old) with CKD 5 D (according to the K/DOQI classification, 2006), and 60 healthy individuals (controls). Leukoconcentrates of venous blood were used as biological samples for the study. The following functional properties of neutrophils were evaluated: the formation of extracellular networks (netosis, NETs) determined microscopically with Romanovsky-Giemsa staining during cell cultivation for 30 and 150 minutes; production of reactive oxygen species (ROS) using cytochemical method with nitro blue tetrazolium, apoptotic activity (luminescence microscopy after staining with a mixture of acridine orange/ethidium bromide), as well as absorption capacity in the reaction of phagocytosis. The tests were performed in spontaneous and stimulated versions. The ATCC 25923 S. aureus strain killed by heating was used as a stimulator. An increase in netosis parameters was revealed in 30-min (but not 150-min) cell cultures in both spontaneous and stimulated versions (p < 0.001), as well as apoptosis (p = 0.02). ROS production by neutrophils and their absorption activity did not significantly change. The parameters of netosis and apoptosis directly correlated with each other (rs = 0.34; p = 0.03), being also dependent on the level of azotemia (correlation coefficient for the indices of netosis and urea level was: rs = 0.41; p = 0.01; for apoptosis and urea, rs = 0.34; p = 0.02). A relationship was found between the level of apoptosis and ROS production by neutrophils (r s = -0.51; p = 0.03). These studies have shown that the cells from patients with terminal-stage CKD are prone for a suicidal program – apoptosis and netosis. Considering the increased netosis just upon shorter cultivation of leukocytes (30 minutes), it can be assumed that NADPH-independent mechanisms are primarily involved. The direct dependence of netosis and apoptosis parameters on the level of azotemia allows us to consider accumulation of endogenous intoxication products, in particular, oxidized proteins, in the patient’s body as one of the primary reasons for increasing the suicidal neutrophil program.

Keywords

• neutrophils
• functional activity
• chronic kidney disease