Involvement of de novo ceramide synthesis in radiocontrast-induced renal tubular cell injury
Involvement of de novo ceramide synthesis in radiocontrast-induced renal tubular cell injury
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DOI：
10.1038/sj.ki.5000057
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发表时间：
2006-01-01
影响因子：
19.6
通讯作者：
Oishi, R
中科院分区：
文献类型：
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作者：
Itoh, Y;Yano, T;Oishi, R
我们以前报道过不同的造影剂可引起猪近端小管(LLC-PK1)细胞的凋亡，其中涉及B细胞淋巴瘤(Bcl2)表达的降低和caspase-3的激活。在本研究中，我们研究了神经酰胺在造影剂诱导的肾小管上皮细胞凋亡中的作用。将LLC-PK1细胞置于造影剂中30min，然后在正常培养液中孵育24 h。2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)5-(2，4-二磺苯基)-2H-四氮唑单钠盐法检测细胞存活率，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法检测细胞凋亡率。用抗磷酸化Akt、cAMP反应元件结合蛋白(PCREB)和神经酰胺的抗体进行免疫荧光染色。用逆转录聚合酶链式反应和酶免疫法分别检测Bcl2的mRNA表达和蛋白含量。建立单侧肾阻塞小鼠造影剂诱导的在体肾损伤模型。抑制伏马菌素B-1(FB1)和L-环丝氨酸的从头合成可逆转非离子放射造影剂碘佛醇所致的细胞损伤，但不能通过D609抑制神经鞘磷脂的降解。FB1逆转异烟肼诱导的Pakt和pCREB免疫反应活性降低、Bcl2表达减少和caspase 3活化。与ioversol类似，C2神经酰胺和Akt抑制剂Src Homology-6通过减少Pakt和pCREB样免疫反应、降低Bcl2表达和增强Caspase-3活性来诱导细胞凋亡。事实上，各种放射造影剂，不包括显示出最小肾毒性的碘二氧醇，都增强了神经酰胺样免疫反应。在放射性造影剂肾病体内模型中也显示了神经酰胺合成的作用。我们在这里第一次证明了神经酰胺合成的增强有助于放射造影剂肾病。
We reported previously that various radiocontrast media cause apoptosis in porcine proximal tubular (LLC-PK1) cells, in which reduction in B-cell lymphoma (Bcl)-2 expression and caspase-3 activation are implicated. In the present study, we investigated a role for ceramide in radiocontrast media-induced apoptosis in renal tubular cells. LLC-PK1 cells were exposed to radiocontrast media for 30 min, followed by incubation for 24 h in normal medium. Cell viability was assessed by 2-(2- methoxy-4-nitrophenyl)-3-(4-nitrophenyl)5-( 2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, while apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling stain. Immunofluorescent stains were performed using antibodies against phosphorylated Akt (pAkt) and cAMP response element binding protein ( CREB) (pCREB), and ceramide. The mRNA expression and protein content of Bcl-2 were determined by reverse transcriptase-polymerase chain reaction and enzyme immunoassay, respectively. In vivo model of contrast-induced renal injury was induced in mice with unilateral renal occlusion. The cell injury induced by the nonionic radiocontrast medium ioversol was reversed by inhibiting de novo ceramide synthesis with fumonisin B-1 (FB1) and L-cycloserine, but not by suppressing sphingomyelin breakdown with D609. FB1 reversed ioversol-induced decrease in the immunoreactivities of pAkt and pCREB, reduction in Bcl-2 expression and caspase-3 activation. Like ioversol, C2 ceramide and the Akt inhibitor Src homology-6 induced apoptosis by reducing pAkt and pCREB-like immunoreactivities, lowering Bcl-2 expression and enhancing caspase-3 activity. Indeed, various radiocontrast media, excluding iodixanol which showed the least nephrotoxicity, enhanced ceramide-like immunoreactivity. The role for de novo ceramide synthesis was also shown in the in vivo model of radiocontrast nephropathy. We demonstrated here for the first time that the enhancement of de novo ceramide synthesis contributes to radiocontrast nephropathy.