대한신장학회

논문분류	춘계학술대회 초록집
제목	Impact of P-glycoprotein Induction on Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells
저자	Hyo Wook Gil, Jong Oh Yang, Eun Young Lee, Sae Yong Hong
출판정보	2014; 2014(1):
키워드	콜리스틴, 활성산소, 아포프토시스
초록	Background: Intravenous colistin is used to treat resistant Gram-negative infections and is associated with nephrotoxicity. The pathogenesis of colistin-associated nephropathy has not been clarified, and there is currently no effective therapeutic or prophylactic agent available. This study aimed to examine the mechanism of colistin induced nephrotoxicity and p-glycoprotein (p-gp) induction could prevent nephrotoxicity. Methods: We examined the cytotoxicity of colistin in cultured human proximal tubular cells (HK-2) by MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay and LDH measuring. To evaluate colistin induced reactive oxygen species (ROS), ROS was measured by 5-(and-6)-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). To investigate colistin induced apoptosis, Tunnel assay and caspase 3/7 activity was done. Autophagy was assessed by measuring of LC3 and acridine orange. To evaluate that p-glycoprotein was involved efflux of colistin in HK2 cell, the expression of p-gp and the cell toxicity was determined in colistin with or without dexamethanson (p-gp inducer) and verapamil (selective P-gp inhibitor) by real time PCR and MTT assay. Results: Colistin showed cell toxicity at dose and time dependent pattern in HK2 cell line (6.25, 12.5, 25, 50 ug/mL at 12, 24, 48 hrs). IS provoked the reactive oxygen species at dose dependent pattern on DCF-DA. Caspase 3/7 activity increased in dose dependent pattern at 6 hours (25, 50,100 ug/mL) and was prevented by N-acetylcystein. Apoptosis was confirmed by Tunnel assay at 6 hr (50 ug/mL). The ratio of LC3 II to LC3 I increased at 2hr (25 ug/mL). Colistin itself suppress the expression of p-gp. P-gp expression and activity in the colistin-treated HK2 cell line were enhanced by dexamethasone treatment and cytotoxicity by colistin was decreased. In addition, induced P- gp transporter was shown to improve the efflux effect on colistin treated HK2 cell lines as was demonstrated using the Calcein-AM fluorescence accumulation assay. Conclusion: Colistin induce ROS related apoptosis and autophagy. P-gp is involved with efflux of colistin in HK2 cell. This study suggested the protective effect of p-glycoprotein induction against colistin-induced nephrotoxicity.
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