Duan Shaobin, Zhou Xiaorong, Liu Fuyou, Peng Youming, Chen Yinyin, Pei Yuanyuan, Ling Guanghui, Zhou Letian, Li Ying, Pi Yihua, Tang Ke, Liu Ruihong, Li Guiyan
Institute of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
J Nephrol. 2006 Nov-Dec;19(6):717-24.
Radiocontrast-induced nephropathy is a clinically important complication of intravascularly applied radiocontrast media. A predominant toxic effect of contrast media on renal tubules has been shown in previous clinical trials and animal experiments. Bax and Bcl-2 are members of the Bcl-2 family. Caspases are a family of cell death proteases, caspase-3 is one of the key executioners of apoptosis. In this study, we evaluated the cytotoxicity of high-osmolar contrast media (HOCM; diatrizoate) and low-osmolar contrast media (LOCM; iohexol) on human renal tubular epithelial cells (HKCs), and determined the regulatory roles of Bax/Bcl-2 and caspase-3 on apoptosis induced by contrast media (CM) in HKCs.
An HKC line was used. Experiments were divided into 7 groups: the HOCM group with iodine 111 mg/mL, HOCM group with iodine 74 mg/mL, LOCM group with iodine 111 mg/mL, LOCM group with iodine 74 mg/mL, mannitol high-osmolar control group, mannitol low-osmolar control group and a culture media control group . The cytotoxicity of HOCM and LOCM were evaluated by cell proliferation and viability assay (MTT assay) and lactate dehydrogenase (LDH) release. Apoptosis were assessed by Hochest 33258 fluorescence-stained cytospins, TUNEL staining, DNA agarose gel electrophoresis, electron microscope and flow cytometric DNA analysis. The protein ex-pression of Bax/Bcl-2 was determined by Western blot analysis, and caspase-3 activity was also determined by the fluo-rometric method.
Compared with the control group, LDH levels increased significantly (p<0.05) and cell viability decreased in cells treated with HOCM or LOCM (p<0.05) in an osmotic pressure-, iodinated ion- and time-dependent manner; in the HOCM groups, diatrizoate induced cultured HKC apoptosis. In the LOCM groups, iohexol did not induce apoptosis. Compared with equal osmotic pressure mannitol, apoptosis increased in HKCs incubated with diatrizoate (p<0.05). Bax/Bcl-2 production and caspase-3 activity were up-regulated in cultured HKCs treated with HOCM iodine 74 or 111 mg/mL meglumine diatrizoate.
Both HOCM and LOCM had toxic effects on HKCs, HOCM was more cytotoxic than LOCM; HOCM induced cultured HKC apoptosis while LOCM did not induce cultured HKC apoptosis in the indicated concentrations. The regulation of apoptosis induced by HOCM in HKCs may be regulated by Bax/Bcl-2 and caspase-3.
放射性造影剂诱发的肾病是血管内应用放射性造影剂的一种临床上重要的并发症。先前的临床试验和动物实验已表明造影剂对肾小管具有主要的毒性作用。Bax和Bcl-2是Bcl-2家族的成员。半胱天冬酶是一类细胞死亡蛋白酶，半胱天冬酶-3是细胞凋亡的关键执行者之一。在本研究中，我们评估了高渗造影剂（HOCM；泛影葡胺）和低渗造影剂（LOCM；碘海醇）对人肾小管上皮细胞（HKC）的细胞毒性，并确定了Bax/Bcl-2和半胱天冬酶-3对HKC中造影剂（CM）诱导的细胞凋亡的调节作用。
使用HKC细胞系。实验分为7组：碘含量为111mg/mL的HOCM组、碘含量为74mg/mL的HOCM组、碘含量为111mg/mL的LOCM组、碘含量为74mg/mL的LOCM组、甘露醇高渗对照组、甘露醇低渗对照组和培养基对照组。通过细胞增殖和活力测定（MTT法）以及乳酸脱氢酶（LDH）释放评估HOCM和LOCM的细胞毒性。通过Hochest 33258荧光染色细胞涂片、TUNEL染色、DNA琼脂糖凝胶电泳、电子显微镜和流式细胞术DNA分析评估细胞凋亡。通过蛋白质印迹分析确定Bax/Bcl-2的蛋白表达，并且也通过荧光法测定半胱天冬酶-3的活性。
与对照组相比，用HOCM或LOCM处理的细胞中LDH水平显著升高（p<0.05）且细胞活力降低（p<0.05），呈渗透压、碘离子和时间依赖性；在HOCM组中，泛影葡胺诱导培养的HKC凋亡。在LOCM组中，碘海醇未诱导凋亡。与等渗透压的甘露醇相比，用泛影葡胺孵育的HKC中细胞凋亡增加（p<0.05）。在用碘含量为74或111mg/mL的葡甲胺泛影葡胺处理的培养HKC中，Bax/Bcl-2表达和半胱天冬酶-3活性上调。
HOCM和LOCM对HKC均有毒性作用，HOCM的细胞毒性比LOCM更大；在所示浓度下，HOCM诱导培养的HKC凋亡而LOCM未诱导培养的HKC凋亡。HOCM在HKC中诱导的细胞凋亡调节可能受Bax/Bcl-2和半胱天冬酶-3调控。
