Neutrophil extracellular* traps in the blood of patients with chronic kidney disease of various stages Текст научной статьи по специальности «Фундаментальная медицина»

УДК 612.112.91

I. V.Beinikova1*, A.S.Nurgaliyeva1, L.A.Demidchik1, V.Hendrikson1

'Karaganda State Medical University, Almaty, Republic Kazakhstan

NEUTROPHIL EXTRACELLULAR TRAPS IN THE BLOOD OF PATIENTS WITH CHRONIC KIDNEY DISEASE OF VARIOUS STAGES

SUMMARY

We studied the possibility of the formation of neutrophil extracellular traps in patients with chronic kidney disease different stages. The object of the study: whole blood of 63 patients with chronic kidney disease distributed in five groups depending on the degree of destruction of renal parenchyma. Formation of net-like structures during NETosis of neutrophil, called neutrophil extracellular traps, having in its composition of DNA, histones, defensins and enzymes {elastase, myeloperoxidase, gelatinase, lactoferrin and lysozyme), was triggered by interaction with lipopolysaccharide substances of exogenous (bacteria) and autologous origin (products of oxidative metabolism), which act through the FcY-receptors and activate NADPH-oxidase complex. The results of our study revealed the following picture of the detection of neutrophil extracellular traps: neutrophil extracellular traps were detected in 33 % of patients with chronic kidney disease 1 stage; amount was in the range from 4 to 10 in 100 neutrophils, in the group with 2 stage - in 5% of patients; amount - 4 in 100 neutrophils, with 3 stage - in 25% of patients; amount was in the range from 1 to 7 in 100 neutrophils, with 4 stage - in 33% of patients in an amount from 2 to 12 in 100 neutrophils, in the group with 5 stage - in 35% of patients in an amount from 2 to 14 in 100 neutrophils. Thus, the highest frequency of detection and the maximum amount of neutrophil extracellular traps was noted in the groups of patients with chronic kidney disease 1, 4 and 5 stages. Some features in morphology of neutrophil extracellular traps different types have been found. Distinctive feature of the first type of neutrophil extracellular traps was hypertrophy of core segments with the concentration of the pellets inside the net-like DNA. Distinctive feature of the second type of neutrophil extracellular traps was rupture of membrane structures of organelles and plasmolemma of neutrophils. The appearance of neutrophil extracellular traps is an unfavorable factor that provokes the activation of procoagulant mechanism of the hemostatic system.

Key words: neutrophil granulocytes, neutrophil extracellular traps, chronic kidney disease.

Introduction. Neutrophil granulocytes, after contact with microorganisms and exogenous cytotoxic agents autogenic origin, throw out into the extracellular space net-like structures, which include DNA, histones, defensins and enzymes such as elastase, myeloperoxidase, gelatinase, lactoferrin and lysozyme. These structures have been named as «neutrophil extracellular traps» (NETs). Formation of NETs - is an alternative neutrophil death, called NETosis. Morphological differences of NETosis compared with apoptosis and necrosis are the decay of the nucleus membrane before disintegration of the cytoplasmic membrane, mixing of nuclear and cytoplasmic material, reorganization of cell cytosk-eleton and disappearance of cytoplasmic organelles [2,4,7].

NETs formation is studied in various pathological conditions including acute renal lesions. There are studies related to the assessment of the role of extracellular histones and NETs in acute renal lesions [3]. Study of NETs in patients with CKD is not conducted.

The aim of study: identify the possibility of the formation of neutrophil extracellular traps in the blood of patients with chronic kidney disease (CKD) of various stages.

Materials and methods: Object of study is whole blood of 63 patients with CKD. All patients were divided into five groups according to the degree of destruction of renal parenchyma. A group of patients with CKD 1 consisted of 12 persons, with CKD 2-5 persons, with CKD 3-20 persons, with CKD 4-12 persons, with CKD 5-14 persons. The control group was represented by 13 healthy donors. Each of the examined gave informed consent to participate in the study.

Detection of neutrophil extracellular traps was performed by the method of Dolgushin et al [1]. Staining of blood smears was performed by the method of May-Grunwald. Chi-square test and Fisher criterion were used for statistical analysis.

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Results and discussion. According to obtained data, NETs were detected in 33 % of patients with CKD I; amount of NETs was in the range from 4 to 10 in 100 neutrophils. In the group with CKD 2 NETs were dctcctcd in 5 % of patients; amount - 4 in 100 neutrophils. In the group with CKD 3 NETs were detected in 25 % of patients; amount was in the range from 1 to 7 in 100 neutrophils. In the group with CKD 4 NETs were detected in 33 % of patients in an amount from 2 to 12 in 100 neutrophils. Finally, in the group with CKD 5 NETs were detected in 35 % of patients in an amount from 2 to 14 in 100 neutrophils. In the control group, NETs were not found.

Absence of significant differences in an amount of NETs between the groups of patients with CKD of various stages may be due to insufficient sample size and requires further research with

increasing the number of patients in these groups.

Analysis of morphological features of NETs, detected in the blood of patients with CKD different stages, showed the presence of 2 types of NETs (Figure I and Figure 2).

Distinctive features of the first type of NETs: the conservation the nuclear apparatus of the cell with hypertrophy of its segments, neutrophil granules are concentrated inside of the formed trap. Distinctive features of the second type of NETs: rupture of membrane structures of neutrophil organelles with formation of retilorm DNA.

Conclusion. According to obtained data, NETs were detected in all groups of patients with CKD, but not at all patients in each group, the amount of NETs also was varied. No significant correlation between the amount of NETs and stages of CKD was observed.

Figure 1 Blood smear of patient with CKD with image of formation the first type of NETs (magnification x 1400, painting with hematoxylin-eosin)

neutrophil extracellular trap

neutrophil

Figure 2 Blood smear of patient with CKD with image of formation the second type of NETs (magnification x 1400, painting with hematoxylin-eosin)

The appearance of NETs in the blood of patients, in our opinion, is an unfavorable factor. Fir st of all, the formation of NETs in the blood may also render the procoagulant action. As mentioned before, the formation of extracellular net-like structures consisting of DNA and histones takes place during formation of NETs [4, 5]. Extracellular histones can cause endothelial damage also participate in the inflammatory response and provoke the thrombin formation [7, 8], that can undoubtedly aggravate CKD.

Conclusions:

- The highest frequency of detection of NETs was observed in the groups of patients with CKD 1, 4 and 5 stages (from 33 - 35%).

- The largest number of NETs in the blood was also observed in the groups of patients with CKD 1, 4 and 5 stages (from 10 - to 14 pieces).

- NETs with different morphological characteristics can be found in the blood smear* of one patient.

LITERATURE

1 Dolgushin 1.1., Andreeva Y.S., Savochkina A.Y. Neutrophil extracellular* traps and methods of assessing the functional status of neutrophils. - Moscow: Russian Academy of Medical Sciences, 2009. -208 p.

2 Pinegin B.V., Mayansky A.N. Neutrophils: structure and function // Immunology. - 2007. - Vol. 28, № 6. - P. 374-382.

3 Rosin D.L., Okusa M.D. Dying cells and extracellular histones in AKI: beyond a NET effect? // J. Am. Soc. Nephrol. - 2012. - № 23 (8). - P. 1275-1277.

4 Brmkmann V., Zychlinsky A. Beneficial suicide: why neutrophils die to, make NETs // Nature Rev. - 2007. - № 5. - P. 577-582.

5 Fttchs T.A., Abed U., Goosmann C. Novel cells death program leads to neutrophil extracelllular traps // J. Cell Biol. - 2007. - № 176 (2). - P. 231-241.

6 Palmer L.J. Neutrophil extracellular traps in periodontitis // A thesis submitted to The University of Birmingham for the degree of doctor of philosophy. - 2010. - P. 307.

7 All am R., Kumar S.V.R., Darisipudi M.N., Anders H.-J. Extracel lular histones in tissue injury and inflammation // Journal of Molecular Medicine. - 2014. - Vol. 92, Is. 5. - P. 465-472.

8 Semeraro F.. Ammollo C.T., Morrissey J.H., Dale G.L. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4 // Blood. -2011.-Vol. 118, №7.-P. 1952-1961.

АННОТАЦИЯ

Изучена возможность образования нейтрофильных внеклеточных ловушек у больных с хронической болезнью почек различной стадии. Объектом исследования служила цельная кровь 63 пациентов с хронической болезнью почек, распределенных в 5 групп в зависимости от степени поражения почечной паренхимы. Образование сетеподобных структур в процессе нетоза нейтрофила, называемых нейтро-фильными внеклеточными ловушками, имеющими в своем составе ДНК, гистоны, дефензины и ферменты гранул (эластаза, миелопероксидаза, желатиназа, лактоферрин и лизоцим), было спровоцировано взаимодействием с липополисахаридными веществами экзогенного (микроорганизмы) и аутогенного происхождения (продукты окислительного метаболизма), действующими через Fcy-рецепторы и активирующими NADPH-оксидазный комплекс. Результаты исследования определили следующую картину обнаружения нейтрофильных внеклеточных ловушек: у больных с хронической болезнью почек 1 стадии - 33 %, количество ловушек варьировало от 4 до 10 на 100 нейтрофилов, 2 стадии - 5%, количество ловушек составило 4 на 100 нейтрофилов, 3 стадии - 25 %, количество ловушек варьировало от 1 - до 7 на 100 нейтрофилов, 4 стадии - 33%, количество ловушек варьировало от 2 - до 12 на 100 нейтрофилов, 5 стадии - 35 %, количество ловушек варьировало от 2 - до 14 на 100 нейтрофилов. Таким образом, была отмечена наибольшая частота обнаружения и максимальное количество нейтрофильных внеклеточных ловушек в группах больных с хронической болезнью почек 1, 4 и 5 стадий. Были обнаружены некоторые особенности в морфологии нейтрофильных внеклеточных ловушек разных типов. Отличительной особенностью нейтрофильных ловушек первого типа была гипертрофия сегментов ядра с сосредоточением гранул внутри сетеподобной ДНК. Отличительным признаком нейтрофильных ловушек второго типа был разрыв всех мембранных структур органелл и плазмолеммы нейтрофила. Появление нейтрофильных внеклеточных

/ КАЗАХСКИЙ МЕДИЦИНСКИЙ IУНИВЕРСИТЕТ НЕПРЕРЫВНОГО ОБРАЗОВАНИЯ

Вестник АГИУВ № 3, 2016

ловушек является неблагоприятных фактором, провоцирующим активацию прокоагулянтного механизма системы гемостаза.

Ключевые слова: нейтрофильные гранулоциты, нейтрофильные внеклеточные ловушки, хроническая болезнь почек.

Эртурл1 сатыдагы созылмалы буйрек ауруына шалдыккан наукастарда нейтрофильд1 жасушадан тыс туткыштардьщ пайда болу мумкЫштттерш окыту. Зерттеу нысаны ретшде дэрежесше карай 5 топ*а белЫген буйрек улпасыньщ закымдануы, созылмалы буйрек ауруы бар 63 науастьщ тутас каны ы-пал егп. ЫАОРН-оксидт1, Рсу-рецепторлары аркылы колданыстагы экзогенд1 (микроорганизмдер) жэне шыгу теп аутогенд1 колданыстагы (тотыктыргыш зат алмасу ежмдерО липополисахаридл заттармен езара эре-кеттесуше акелген, дефензиндер мен гранула ферменттер1мен (эластаз, миелопероксидаз, желатиназ, лактоферрин жэне лизоцим), гистондар, сондай-ак ез!н1н курамында ДНК бар, нейтрофил нетроз проце-сшде жел1ге уксас курылымньщ курылуын нейтрофилд1 жасушадан тыс какпан деп атаган. Зерттеу коры-тындысымен нейтрофилд1 жасушадан тыс туткышы бар келесщей керЫю аныкталды: 1 сатыда созылмалы буйрек ауруымен ауыратын наукаста р - 33%, 100 нейтрофильге туткыш саны 4-тен 10 дейтд1 курайды, 2 сатыда - 5 %, 100 нейтрофильге туткыш саны 4 — Т( курайды, 3 сатыда - 25 %, 100 нейтрофильге туткыш саны 1-ден 7-ге дейЫд1 курайды, 4 сатыда - 33%, 100 нейтрофильге туткыш саны 2-ден 12-ге дейжд1 курайды, 5 сатыда - 35%, 100 нейтрофильге туткыш саны 2-ден 14-ке дейтд1 курайды. Осылайша, созылмалы буйрек ауруына шалдыккан наукастар тобынан жи1л!г1 1, 4 жэне 5 сатыларында нейтрофильд1 жасушадан тыс туткыш сандарыньщ ей кеп мелшер1 мен жиШ айтарлыктай атап еттдк Морфологияда жасушадан тыс туткыштардьщ эр турл1 типтеп кейбф ерекшел1ктер1 аныкталды.

Нейтрофильд1 туткыштьщ брнип типшщ айрыкша ерекшелИ ДНК ¡шЫдеп жуйе ¡спеттес гранул топта-масымен ядро сегменттершщ гипертрофиясы болды. Нейтрофильд1 туткыштьщ екЫил титнщ айрыкша ерекшелМ нейтрофил плазмолеммалары мен органелла курылымыньщ барлык мембраналык алшамыгы болды. Кан токтату жуйесшде прокоагулянт тетИн белсенд1ру ушш нейтрофильд1 жасушадан тыс туткыштардьщ пайда болуы колайсыз фатор болып табылады.

Туйшд1 сездер: нейтрофильд1 гранулоциттер, нейтрофильд1 жасушадан тыс туткыштар, созылмалы буйрек ауруы.

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