ABCB1 Protects Kidney Proximal Tubule Cells Against Cadmium-Induced Apoptosis: Roles of Cadmium and Ceramide Transport
ABCB1 Protects Kidney Proximal Tubule Cells Against Cadmium-Induced Apoptosis: Roles of Cadmium and Ceramide Transport
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DOI：
10.1093/toxsci/kfr071
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发表时间：
2011-06-01
影响因子：
3.8
通讯作者：
Thevenod, Frank
中科院分区：
文献类型：
--
作者：
Lee, Wing-Kee;Torchalski, Blazej;Thevenod, Frank
镉（Cd（2+））通过神经酰胺依赖性细胞凋亡损害肾脏近端小管（PT），也是1类致癌物。多药耐药P-糖蛋白（MDR 1，ABCB 1）可抵抗Cd（2+）诱导的细胞凋亡，并推测ABCB 1可直接转运Cd（2+）作为细胞保护的一种模式。我们的目的是研究ABCB 1在Cd（2+）转运和神经酰胺凋亡中的作用。在过表达ABCB 1的大鼠PT或Madin-Darby犬肾（MDCK）细胞中，测定罗丹明123（+）（Rh 123（+））或（109）Cd（2+）的ABCB 1依赖性外排，并通过MTT、H-33342核染色和阻抗传感（电细胞基质阻抗传感[ECIS]）单层完整性测定细胞死亡。ABCB 1抑制剂（PSC 833，UIC-2抗体）对PT细胞的（109）Cd（2+）外排无影响，但对Rh 123（+）转运有阻断作用。此外，增加ABCB 1表达并不增加（109）Cd（2+）流出，但减弱10-50 μ M Cd（2+）或5-25 μ M C（6）-神经酰胺引起的凋亡，这被PSC 833（1 μ M）消除。ABCB 1-MDCK单层的ECIS测量显示出类似的效果。此外，在ABCB 1-MDCK细胞中，通过二酰基甘油激酶测定，Cd（2+）诱导的神经酰胺形成被消除，并且与MDCK细胞相比，观察到硝基-2-1，3-苯并恶二唑-4-基（NBD）-C（6）-神经酰胺和NBD-C（6）-葡萄糖神经酰胺的分泌增加。而药理学阻断鞘磷脂合酶（0.1 mM D 609）或鞘氨醇激酶（1 μ M二甲基鞘氨醇），增加神经酰胺及其代谢产物的水平，增强Cd（2+）诱导的细胞凋亡，Cd（2+）不仅通过阻止神经酰胺的重新合成而显著降低细胞凋亡（0.1 μ M伏马菌素B（1）），但也通过抑制葡糖神经酰胺合酶（2 μ M C（9）DGJ）。因此，我们得出结论，镉（2+）流出是ABCB 1介导的保护镉（2+）凋亡背后的机制。相反，鞘脂葡糖神经酰胺可能是ABCB 1挤出的促凋亡底物。
Cadmium (Cd(2+)) damages the kidney proximal tubule (PT) by ceramide-dependent apoptosis and is also a class 1 carcinogen. Multidrug resistance P-glycoprotein (MDR1, ABCB1) confers resistance to Cd(2+) apoptosis, and it has been hypothesized that ABCB1 can directly transport Cd(2+) as a mode of cellular protection. Our aim was to investigate the role of ABCB1 in Cd(2+) transport and ceramide apoptosis. In rat PT or Madin-Darby canine kidney (MDCK) cells overexpressing ABCB1, ABCB1-dependent efflux of rhodamine 123(+) (Rh123(+)) or (109)Cd(2+) were determined, and cell death was assayed with MTT, H-33342 nuclear staining, and monolayer integrity by impedance sensing (Electric cell-substrate impedance sensing [ECIS]). ABCB1 inhibitors (PSC833, UIC-2 antibody) did not affect (109)Cd(2+) efflux in PT cells though Rh123(+) transport was blocked. Furthermore, increased ABCB1 expression did not augment (109)Cd(2+) efflux but attenuated apoptosis by 10-50 mu M Cd(2+) or 5-25 mu M C(6)-ceramide, which was abolished by PSC833 (1 mu M). ECIS measurements of ABCB1-MDCK monolayers exhibited similar effects. Moreover, in ABCB1-MDCK cells, Cd(2+)-induced ceramide formation, determined by a diacylglycerol kinase assay, was abolished and increased extrusion of nitro-2-1,3-benzoxadiazol-4-yl (NBD)-C(6)-ceramide, and NBD-C(6)-glucosylceramide was observed compared with MDCK cells. Whereas pharmacological block of sphingomyelin synthase (0.1mM D609) or sphingosine kinase (1 mu M dimethylsphingosine), which increase the levels of ceramide and its metabolites, augmented Cd(2+)-induced apoptosis, Cd(2+) apoptosis was significantly decreased not only by prevention of de novo ceramide synthesis (0.1 mu M fumonisin B(1)) but also by inhibition of glucosylceramide synthase (2 mu M C(9)DGJ). We therefore conclude that Cd(2+) efflux is not the mechanism behind ABCB1-mediated protection from Cd(2+) apoptosis. Rather, the sphingolipid glucosylceramide may be the proapoptotic substrate extruded by ABCB1.