Effect of hyperglycemia on P-glycoprotein expression in MDCK II cells

• Main Authors: Szu-Yu Yeh, 葉思妤
• Other Authors: Chun-Jung Lin
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/52400730320477852914

碩士 === 國立臺灣大學 === 藥學研究所 === 99 === Diabetes mellitus is a chronic metabolic disease and is related to various complications including nephropathy. Diabetic nephropathy is the most common cause of end-stage renal disease. P-glycoprotein (P-gp) is abundant in the kidney and is closely related to the excretion of xenobiotics and drugs. In this regard, P-gp represents a protective mechanism to exclude endogenous and exogenous toxins in the kidney. Our previous study has shown that P-gp expression is significantly lower in the kidney of animals with type 1 diabetes (Non-obese diabetic, NOD females) or type 2 diabetes (New Zealand obese, NZO males). The present study is to investigate the underlying mechanisms regulating P-gp expression in the kidney under diabetic condition Madin-Darby canine kidney (MDCK) type II cells were used as an in vitro model to study the regulation mechanisms of P-gp expression in the kidney under diabetic condition. P-gp protein expression was analyzed by Western blotting under the treatment of IL-6、TNF-α、insulin and glucose. Quantitative polymerase chain reaction (qPCR) was used to detect P-gp mRNA expression. In addition, the changes of intracellular ROS、PXR、CAR and ETR-A / B expressions were measured to evaluate the effect of high glucose condition on P-gp expression. In MDCK cells, P-gp protein and mRNA expression were reduced in the presence of high glucose concentration, while superoxide production was increased. After pro-inflammatory cytokines, IL-6 and TNF-α treatment, P-gp expression did not show any significantly change, as well as after insulin treatment. The reduction of P-gp expression was abolished by the treatment of an antioxidant, N-acetylcysteine (NAC). Treatment with hydrogen peroxide (H2O2) or protein kinase C (PKC) agonist, phorbol myristate acetate (PMA)significantly reduced P-gp expression. On the other hand, NAC could not reverse the reduction of P-gp expression caused by PMA treatment. However, the decrease of P-gp expression after high glucose exposure was abolished by the treatment of a PKC inhibitor, staurosporine. Furthermore, the roles of PXR, CAR and ET (Endothelin-1, ET) on P-gp regulation were investigated. The results showed that PXR and CAR mRNA expression were significantly increased under high glucose condition for 24 hours. On the other hand, PXR and CAR mRNA expression were decreased by the treatment of PKC agonist, PMA, for 12 hours. Except for PXR at 48 hours glucose treatment, in both situations, there are no significantly changes of PXR and CAR protein expression. In terms of endothelin receptor A or B (ETR-A / B), the RT-qPCR analysis did not show the mRNA expression of ETR-A / B expression in MDCK II cells. In conclusion, the expression of P-gp was decreased in MDCK II cells under high glucose condition. The regulatory pathway of P-gp expression under high glucose concentration was associated with reactive oxygen species (ROS) production and then through PKC activation. PXR / CAR, ET and ETR-A /B pathway do not play a role in this regulation. Further studies are required to understand whether there are any other factors involved in regulating P-gp.