Ludwig Thomas, Riethmüller Christoph, Gekle Michael, Schwerdt Gerald, Oberleithner Hans
Institut für Physiologie II, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Kidney Int. 2004 Jul;66(1):196-202. doi: 10.1111/j.1523-1755.2004.00720.x.
Cisplatin and its analogs oxaliplatin and carboplatin are widely used antitumor drugs. Nephrotoxicity is a common and relevant adverse effect that occurs especially in cisplatin therapy. Cellular and molecular mechanisms of cisplatin-induced nephrotoxicity are not completely understood. The nephrotoxicity of platinum complexes was evaluated by a new in vitro system that utilizes the high Trans Epithelial Electrical Resistance (TEER) of the C7 clone of the MDCK (Madin-Darby canine kidney) cells. By means of this assay system we addressed the question whether the side of application of renal epithelia influences platinum complex toxicity.
C7 cells were grown in membrane filter cups, and the apical or basolateral membranes were exposed to 100-micromol/L cis-, oxali-, or carboplatin. TEER and caspase-3 activity were determined. Cimetidine was used as an inhibitor of organic cation transporters (OCTs). C7 cell lysates were analyzed for OCT-1 and -2 by Western blot analysis.
TEER dropped by 89.5 +/- 9.3% (mean +/- SEM; N= 6) within 24 hours after addition of cisplatin to the basolateral side of C7 cells, while caspase activity increased up to 840.6 +/- 17.4% (mean +/- SEM; N= 6) compared to control cells. Exposure of the apical membrane to cisplatin reduced TEER by only 13.4 +/- 8.7% (mean +/- SEM; N= 6), and increased caspase-3 activity up to 213.9 +/- 7.6% (mean +/- SEM; N= 6). Oxaliplatin and carboplatin reduced TEER to a lesser extent than cisplatin. Oxaliplatin lowered TEER stronger than carboplatin. In general, basolateral application led to higher caspase activities and lower TEERs. The OCT-inhibitor cimetidine inhibited the TEER decrease induced by platinum complexes. Immunoblotting confirmed the presence of OCT-2 in C7 cells.
Toxic effects of platinum complexes on renal epithelia depend on the platinum complex used and the site of application. We conclude that cell polarity and basolateral transport mechanisms are essential in nephrotoxicity of platinum drugs.
顺铂及其类似物奥沙利铂和卡铂是广泛使用的抗肿瘤药物。肾毒性是一种常见且相关的不良反应，尤其在顺铂治疗中会出现。顺铂诱导肾毒性的细胞和分子机制尚未完全明确。通过一种新的体外系统评估铂类复合物的肾毒性，该系统利用了MDCK（Madin-Darby犬肾）细胞C7克隆的高跨上皮电阻（TEER）。借助这个检测系统，我们探讨了肾上皮细胞给药侧是否会影响铂类复合物的毒性这一问题。
将C7细胞培养在膜滤器小室中，使顶膜或基底外侧膜暴露于100 μmol/L的顺铂、奥沙利铂或卡铂。测定TEER和半胱天冬酶-3活性。西咪替丁用作有机阳离子转运体（OCTs）的抑制剂。通过蛋白质印迹分析检测C7细胞裂解物中的OCT-1和OCT-2。
在C7细胞基底外侧加入顺铂后24小时内，TEER下降了89.5±9.3%（平均值±标准误；N = 6），而与对照细胞相比，半胱天冬酶活性增加至840.6±17.4%（平均值±标准误；N = 6）。顶膜暴露于顺铂仅使TEER降低了13.4±8.7%（平均值±标准误；N = 6），并使半胱天冬酶-3活性增加至213.9±7.6%（平均值±标准误；N = 6）。奥沙利铂和卡铂使TEER降低的程度小于顺铂。奥沙利铂比卡铂更能降低TEER。总体而言，基底外侧给药导致更高的半胱天冬酶活性和更低的TEER。OCT抑制剂西咪替丁抑制了铂类复合物诱导的TEER降低。免疫印迹证实C7细胞中存在OCT-2。
铂类复合物对肾上皮细胞的毒性作用取决于所用的铂类复合物和给药部位。我们得出结论，细胞极性和基底外侧转运机制在铂类药物的肾毒性中至关重要。
