檢測腎毒性物質的體外腎細胞評估系統之建立

Establishment of an in vitro cultured renal cell system for evaluating nephrotoxic ingredients

蔡伯政(Po-Cheng Tsai)

指導教授 ： 李志恒

高雄醫學大學/藥學院/藥學系碩士班/碩士(2015年)

https://doi.org/10.6832/KMU.2015.00006

摘要

環境中存在的許多會對腎臟產生毒性的化學物質，包括重金屬、草藥植物以及一些醫療用途的藥品。而為了避免環境中潛在的腎毒性物質對腎臟產生傷害，此研究的目的在於建立一個以體外細胞試驗的方式來評估物質是否會對腎臟造成危害。
實驗方法：選取具特定腎部位毒性之物質，包括valproic acid、mercury(II) chloride等，藉由三株體外腎細胞HEK293、HK-2及MDBK來模擬腎臟的腎絲球、近曲小管及遠曲小管這三個部位，並且用另一株中國倉鼠卵巢細胞CHOK1來做為對照，以瞭解是否只具有腎毒性的物質不會對其他非腎臟細胞產生毒性，透過MTT assay來檢驗物質的細胞毒性，使用具有直接及間接腎毒性的藥物進行測試，觀測是否只有直接毒性的藥物有作用。並使用腎損傷的生物指標kidney injury molecule-1 (KIM-1)更進一步的確認腎臟細胞受到損害，KIM-1為近曲小管細胞膜上的跨膜蛋白，當腎細胞受到傷害時會被metalloproteinase切成兩段，膜外的蛋白會脫離細胞，因此細胞內完整的KIM-1 protein會減少，以此做為評斷的基準。
實驗結果與討論：單獨使用體外細胞毒性試驗並無法準確的檢測出具有腎毒性的化學物質。但是生物指標KIM-1在腎毒性物質的檢測上具有良好的敏感性和專一性，KIM-1只會在近曲小管細胞表現，且只有直接會對腎細胞造成毒性的藥物會完整的KIM-1蛋白表現量減少。因此，體外細胞系統結合腎損傷的生物指標可能夠具有篩選潛在腎毒性的物質。

關鍵字

腎毒性 ； 體外試驗

並列摘要

Objectives: Many chemicals, including heavy metals, herbals, and therapeutic agents, have been shown to be nephrotoxic. To prevent the renal injury from nephrotoxic chemicals, an in vitro cultured renal cell system was established for predicting the potential nephrotoxicity of chemicals.
Methods: Three quasi-normal renal cell lines, HEK293, HK-2, and MDBK, representing the glomerular, proximal tubular, and distal tubular parts of the kidney, respectively, were used for evaluation. The cytotoxicity of nephrotoxic drugs were evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. To compare the specificity of cytotoxicity between nephrotoxic and non-nephrotoxic drugs in renal cells, CHOK1 cells were used as reference. Kidney injury was also assessed by nephrotoxic drugs-affected expression of kidney injury molecule-1 (KIM-1). KIM-1, a membrane protein that is cut by metalloproteinase after renal cell injury, could serve as a kidney injury biomarker.
Results and Discussion: The cytotoxicity of nephrotoxic drugs was not significantly different among the in vitro cultured renal and non-renal cells. However, the expression of complete KIM-1 could serve as a biomarker of kidney injury. In this research, KIM-1 was sensitive and specific for evaluating the chemicals with direct toxicity of proximal tubules Therefore, the biomarkers of kidney injury could be used to predict the potential nephrotoxicity of chemicals in the in vitro cultured renal cell system.

並列關鍵字

nephrotoxicity ； in vitro

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