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

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 (r_s = 0.34; p = 0.03), being also dependent on the level of azotemia (correlation coefficient for the indices of netosis and urea level was: r_s = 0.41; p = 0.01; for apoptosis and urea, r_s = 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.

neutrophils, functional activity, chronic kidney disease

1. Vorobyeva N.V., Pinegin B.V. Neutrophil extracellular traps: mechanisms of formation and their role in health and disease. Biokhimiya = Biochemistry, 2014, Vol. 79, no. 12, pp. 1580-1591. (In Russ.)

2. Gusakova N.V., Novikova I.A. Formation of neutrophil extracellular traps in peripheral blood. Problemy zdorovya i ekologii = Problems of Health and Ecology, 2011, Vol. 9, no. 3, pp. 27-31. (In Russ.)

3. Аzzouz D., Palaniyar N. ApoNETosis: discovery of a novel form of neutrophil death with concomitant apoptosis and NETosis. Cell Death Dis., 2018, Vol. 9, pp. 839-841.

4. Carmona-Rivera C., Kaplan M.J. Low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity. Semin. Immunopathol., 2013, Vol. 35, no. 4, pp. 455-463.

5. Cohen G., Rudnicki M., Hörl W. Uremic toxins modulate the spontaneous apoptotic cell death and essential functions of neutrophils. Kidney Int. Suppl., 2001, Vol. 78, pp. S48-S52.

6. Gorman A., McCarthy J., Finucane D., Reville W., Cotter T.G. Morphological assessment of apoptosis. In: Techniques in Apoptosis, A User’s Guide, 1 st Ed., edited by Cotter T.G., Martin S.J., London, UK, Portland Press Ltd, 1996, pp. 1-20.

7. Hakkim A., Fuchs T.A., Martinez N.E., Hess S., Prinz H., Zychlinsky W.H. Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nat. Chem. Biol., 2011, Vol. 7, no. 2, pp. 75-77.

8. Li P., Li M., Lindberg M.R., Kennett M.J., Xiong N., Wang Y. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J. Exp. Med., 2010, Vol. 207, pp. 1853-1862.

9. Muravlyova L.Y., Molotov-Luchanki V.B, Bakirova R.Y., Turmukhambetova A.A., Klyuyev D.A., Demidchik L.A., Kolesnikova Ye.A. The alteration in peripheral neutrophils of patients with chronic kidney disease. Curr. Issues Pharm. Med. Sci., 2015, Vol. 28, no. 1, pp. 17-20.

10. Nicolas-Avila J.A., Adrover J.M., Hidalgo А. Neutrophils in homeostasis, immunity, and cancer. Immunity, 2017, Vol. 46, no. 1, pp. 15-28.

11. Pedersen H.L., Horvei K.D., Thiyagarajan D., Norby G.E., Seredkina N., Moroni G., Eilertsen G.Ø., Holdaas H., Strøm E.H., Bakland G., Meroni P.L., Rekvig O.P. Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression. J. Pathol. Clin. Res., 2018, Vol. 4, no. 3, pp. 193-203.

12. Pindjakova J., Griffin M.D. Defective neutrophil rolling and transmigration in acute uremia. Kidney Int., 2011, Vol. 80, no. 5, pp. 447-450.

13. Silvestre-Roig C., Hidalgo A., Soehnlein O. Neutrophil heterogeneity: implications for homeostasis and pathogenesis. Blood, 2016, Vol. 127, no. 18, pp. 2173-2181.

14. Yipp B.G., Kubes P. NETosis: how vital is it? Blood, 2013, Vol. 122, no. 16, pp. 2784-2794.

15. Zahran N., Sayed A., William I. Mahmoud O., Sabry O., Rafaat M. Neutrophil apoptosis: impact of granulocyte macrophage colony stimulating factor on cell survival and viability in chronic kidney disease and hemodialysis patients. Arch. Med. Sci., 2013, Vol. 9, no. 6, pp. 985-989.