Development of new methods for the evaluation and protection of drug induced nephrotoxicity using kidney epithelial cell line (LLC-PK_1)

肾脏是排泄外源性药物的主要器官，容易受到药物性肾毒性的影响。我们试图利用培养的肾上皮细胞系LLC-PK_1建立药物肾毒性评价和保护的新方法。本研究采用氨基糖苷类抗生素、环孢素（免疫抑制剂）和顺铂（抗肿瘤药）。这些药物在临床上被广泛使用，但它们的使用有时会因急性肾毒性而复杂化。氨基糖苷、环孢素和顺铂对LLC-PK_1细胞的影响，发现活细胞数量、漂浮细胞（死亡细胞）数量、形成圆顶的能力和顶端标记酶（氨基肽酶、碱性磷酸酶和γ -谷氨酰转移酶）的活性是药物诱导的细胞毒性的标志。不同氨基糖苷类药物对LLC-PK、细胞顶端酶的抑制作用与体内肾毒性之间存在良好的关系。此外，氨基糖苷和环孢素诱导胞质游离钙浓度升高，这可能是药物性肾毒性的重要决定因素。庆大霉素对根尖酶活性的抑制作用具有剂量依赖性，并与其在细胞中的积累有关。从胞质游离钙水平和活细胞数量判断，胞内环孢素的积累显然也与其细胞毒性有关。顺铂对指数生长细胞的细胞毒性比对融合细胞的细胞毒性更强。谷胱甘肽能减轻顺铂的毒性。环孢素对生长和融合细胞有毒性，而谷胱甘肽对其细胞毒性无影响，说明不同药物肾毒性的生化机制不同。综上所述，体外培养肾上皮细胞系（LLC-PKi）可作为评价药物肾毒性的有效模型系统，为药物肾毒性的保护提供新的途径。少

The kidney is a major organ for the excretion of xenobiotics, and is susceptible to drug-induced nephrotoxicity. We attempted to establish new methods for the evaluation and protection of nephrotoxicity of drugs by using a cultured kidney epithelial cell line, LLC-PK_1. Aminoglycoside antibiotics, cyclosporin (immunosuppressive agent), and cisplatin (antitumor agent) were employed in this study. These drugs are widely used clinically, but their use is sometimes complicated by acute nephrotoxicity.1. The effect of aminoglycoside, cyclosporin, and cisplatin was tested in LLC-PK_1 cells, and it was found that the number of viable cells, the number of floating cells (dead cells), the ability to form domes, and the activities of apical marker enzymes (aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase) are useful as markers of drug-induced cytotoxicity. There was a good relationship between the potencies of various aminoglycosides to inhibit apical enzymes in LLC-PK, cells … More and the nephrotoxic potencies of these drugs in vivo. In addition, aminoglycoside and cyclosporin induced the elevation of cytosolic free calcium concentration, which may be a important determinant of drug-induced nephrotoxicity.2. The inhibitory effect of gentamicin on the apical enzyme activities was dosedependent, and was related to its accumulation in the cells. Apparently, intracellular accumulation of cyclosporin was also related to its cytotoxicity, when judged by the level of cytosolic free calcium and the number of viable cells.3. Cytotoxicity of cisplatin was more potent on exponentially growing cells than on confluent cells. Glutathione attenuated the toxicity of cisplatin. In contrast, cyclosporin was toxic on growing and confluent cells, and glutathione had no effect on its cytotoxicity, indicating that biochemical mechanisms of nephrotoxicity are different among different drugs.In conclusion, cultured kidney epithelial cell line (LLC-PKi) should be a useful model system to evaluate the nephrotoxicity of drugs, and to develop a new method to protect the drug-induced nephrotoxicity. Less