UREMIC TOXIN GUANIDINE ACETIC ACID INHIBITS THE OXIDATIVE METABOLISM OF NEUTROPHILS IN DOGS

Abstract Among the uremic toxins proven to affect the neutrophil function in humans with chronic kidney disease (CKD), guanidine compounds stand out. To achieve a clearer understanding of the mechanisms that affect the immunity of uremic patients, the hypothesis that guanidine acetic acid (GAA) contributes to the inhibition of oxidative metabolism and an increase in neutrophil apoptosis in healthy dogs was investigated in vitro. To this end, neutrophils isolated from ten healthy dogs were incubated in pure RPMI 1640 (control) and enriched with 5 mg/L of GAA. Capillary flow cytometry was used to quantify superoxide production in neutrophils with the probe (hydroethidine), in the presence and absence of phorbol-12-myristate-13-acetate (PMA), in order to assess oxidative metabolism. Apoptotic indices were quantified using the Annexin V-PE system, with and without the inductive effect of camptothecin. Neutrophils isolated and incubated in a GAA-enriched medium produced smaller amounts of superoxide (p<0.001) when activated with PMA, however, this inhibition of oxidative metabolism occurred without significantly altering their viability or rate of apoptosis. Thus, the results show guanidine compounds contribute to immunosuppression in dogs with CKD.

摘要 已被证实可影响慢性肾脏病（chronic kidney disease, CKD）患者中性粒细胞功能的尿毒症毒素中，胍类化合物尤为突出。为更清晰地阐明影响尿毒症患者免疫功能的潜在机制，本研究针对胍乙酸（guanidine acetic acid, GAA）可抑制健康犬中性粒细胞氧化代谢并促进其凋亡这一假说开展体外实验。为此，本研究将从10只健康犬体内分离得到的中性粒细胞，分别置于单纯RPMI 1640培养基（对照组）以及添加了5 mg/L GAA的培养基中进行孵育。为评估氧化代谢水平，本研究采用毛细流式细胞术，以氢乙啶（hydroethidine）为荧光探针，在佛波醇-12-肉豆蔻酸酯-13-乙酸酯（phorbol-12-myristate-13-acetate, PMA）存在与缺失两种条件下，对中性粒细胞的超氧化物生成量进行定量检测。同时，本研究采用Annexin V-PE检测体系，在喜树碱（camptothecin）诱导与非诱导两种条件下，对细胞凋亡指数进行定量分析。经PMA激活后，在添加GAA的培养基中孵育的中性粒细胞所产生的超氧化物含量极显著降低（p<0.001）；但该氧化代谢抑制作用并未显著改变细胞存活率或凋亡速率。综上，本研究结果表明，胍类化合物可导致慢性肾脏病犬出现免疫抑制。