Bosco A M, Almeida B F M, Pereira P P, Dos Santos D B, Neto Á J S, Ferreira W L, Ciarlini P C
Department of Clinical Practice, Surgery and Animal Reproduction, Araçatuba College of Veterinary Medicine, São Paulo State University (UNESP), Araçatuba, SP, Brazil.
Department of Clinical Practice, Surgery and Animal Reproduction, Araçatuba College of Veterinary Medicine, São Paulo State University (UNESP), Araçatuba, SP, Brazil.
Vet Immunol Immunopathol. 2017 Mar;185:14-19. doi: 10.1016/j.vetimm.2017.01.006. Epub 2017 Jan 28.
We investigated the hypothesis that the increased concentration of plasma methylguanidine (MG) increases oxidative metabolism and accelerates apoptosis of neutrophils from dogs with chronic kidney disease (CKD). To achieve this, the levels of MG were quantified in healthy (n=16) and uremic dogs with CKD stage 4 of according to the guidelines of the International Renal Interest Society (IRIS, 2015) (n=16) using high performance liquid chromatography (HPLC). To evaluate the isolated effect of MG on neutrophil oxidative metabolism and apoptosis, neutrophils isolated from 12 healthy dogs were incubated with the highest concentration of plasma MG (0.005g/L) observed in dogs with CKD. Neutrophil oxidative metabolism was assessed by flow cytometry, using the probes hydroethidine for superoxide production and 2',7'-dichlorofluorescein diacetate for hydrogen peroxide production, with or without phorbol myristate acetate (PMA) stimulus. Neutrophil apoptosis and viability were also evaluated in flow cytometer using the Annexin V-PE system, with or without the apoptosis-inducing effect of camptothecin. Uremic dogs presented higher concentrations of MG (p<0.0001), increased oxidative stress and primed neutrophils with higher apoptosis rate. The neutrophil abnormalities observed in vivo were also reproduced in vitro, using cells isolated from healthy dogs and incubated with MG. We obtained strong evidence that in dogs with CKD, increased MG levels contributed to oxidative stress and potentially compromised the non-specific immune response by altering the oxidative metabolism and viability of canine neutrophils.
血浆甲基胍（MG）浓度升高会增加氧化代谢并加速慢性肾脏病（CKD）犬中性粒细胞的凋亡。为此，根据国际肾脏利益协会（IRIS，2015）的指南，使用高效液相色谱法（HPLC）对16只健康犬和16只处于CKD 4期的尿毒症犬的MG水平进行了定量。为了评估MG对中性粒细胞氧化代谢和凋亡的单独作用，将从12只健康犬分离的中性粒细胞与CKD犬中观察到的最高血浆MG浓度（0.005g/L）一起孵育。使用用于超氧化物产生的氢乙锭和用于过氧化氢产生的2'，7'-二氯荧光素二乙酸酯探针，通过流式细胞术评估中性粒细胞的氧化代谢，无论是否有佛波醇肉豆蔻酸酯乙酸酯（PMA）刺激。还使用膜联蛋白V-PE系统，在流式细胞仪中评估中性粒细胞的凋亡和活力，无论是否有喜树碱的凋亡诱导作用。尿毒症犬的MG浓度更高（p<0.0001），氧化应激增加，中性粒细胞预激活，凋亡率更高。使用从健康犬分离的细胞并与MG一起孵育，在体外也重现了体内观察到的中性粒细胞异常。我们获得了强有力的证据，即在患有CKD的犬中，MG水平升高导致氧化应激，并可能通过改变犬中性粒细胞的氧化代谢和活力而损害非特异性免疫反应。
