Armağan Hamit Hakan, Nazıroğlu Mustafa
Department of Emergency Medicine, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey.
Department of Biophysics, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey; Drug Discovery Unit, BSN Health, Analyses, Innovation, Consultancy, Organization, Agriculture and Industry Ltd, Göller Bölgesi Teknokenti, Isparta, Turkey.
Chem Biol Interact. 2021 Jan 25;334:109306. doi: 10.1016/j.cbi.2020.109306. Epub 2020 Dec 9.
Oxidative stress (OS)-induced glutathione (GSH) depletion plays an essential role in several kidney diseases such as chronic kidney disease and nephrotoxicity. The OS-dependent activation of TRPM2 cation channel in several neurons and cells were modulated by the concentration of intracellular GSH. However, the effects of GSH alteration on TRPM2 activation, OS, and apoptosis in the cortical collecting duct (mpkCCD) cells still remain elusive. We investigated the effects of GSH supplementation on OS-induced TRPM2 activation, mitochondrial oxidative stress, and apoptosis in the human embryonic kidney 293 (HEK293) and mpkCCD cells treated with buthionine-sulfoximine (BSO), a GSH synthase inhibitor. The HEK293 and mpkCCD cells were divided into five groups as control, GSH (10 mM for 2 h), BSO (0.5 mM for 6 h), BSO + GSH, and BSO + TRPM2 channel blockers. Apoptosis, cell death, mitochondrial OS, caspase -3, caspase -9, cytosolic free Zn, and Ca concentrations were increased in the BSO group of the TRPM2 expressing mpkCCD cells, although they were diminished by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2-APB). The BSO-induced decreases in the levels of cell viability and cytosolic GSH were increased by the treatments of GSH, ACA, and 2-APB. However, the effects of BSO and GSH were not observed in the non-TRPM2 expressing HEK293 cells. Current results show that maintaining GSH homeostasis is not only important for quenching OS in the cortical collecting duct cells but equally critical to modulate TRPM2 activation. Thus, suppressing apoptosis and mitochondrial OS responses elicited by oxidant action of GSH depletion.
氧化应激（OS）诱导的谷胱甘肽（GSH）耗竭在多种肾脏疾病如慢性肾脏病和肾毒性中起重要作用。在多个神经元和细胞中，TRPM2阳离子通道的OS依赖性激活受细胞内GSH浓度调节。然而，GSH改变对皮质集合管（mpkCCD）细胞中TRPM2激活、OS和凋亡的影响仍不清楚。我们研究了补充GSH对用谷胱甘肽合成酶抑制剂丁硫氨酸亚砜胺（BSO）处理的人胚肾293（HEK293）和mpkCCD细胞中OS诱导的TRPM2激活、线粒体氧化应激和凋亡的影响。将HEK293和mpkCCD细胞分为五组：对照组、GSH组（10 mM，处理2小时）、BSO组（0.5 mM，处理6小时）、BSO + GSH组和BSO + TRPM2通道阻滞剂组。在表达TRPM2的mpkCCD细胞的BSO组中，凋亡、细胞死亡、线粒体OS、半胱天冬酶-3、半胱天冬酶-9、胞质游离锌和钙浓度增加，不过GSH、聚（ADP-核糖）聚合酶-1抑制剂（PJ34和DPQ）和TRPM2阻滞剂（ACA和2-APB）处理可使其降低。GSH、ACA和2-APB处理可增加BSO诱导的细胞活力水平降低和胞质GSH降低。然而，在不表达TRPM2的HEK293细胞中未观察到BSO和GSH的作用。目前的结果表明，维持GSH稳态不仅对消除皮质集合管细胞中的OS很重要，而且对调节TRPM2激活同样关键。因此，可抑制GSH耗竭的氧化作用引发的凋亡和线粒体OS反应。
