高血糖影響腎臟上皮 MDCK II細胞中 P-glycoprotein 表現之機轉探討

Abstract

糖尿病 (diabetes) 是一種慢性代謝性疾病，會造成全身微循環的障礙，進而引起腎臟方面的病變。P-glycoprotein (P-gp) 大量存在於腎臟中，主要功能是排除其受質及藥物等異質體，與腎臟組織的保護有關。由本實驗室先前西方墨點法與組織免疫染色的結果顯示，不論在第一型糖尿病Non-obese diabetic (NOD) 母鼠或第二型糖尿病New Zealand obese (NZO) 公鼠的腎臟中，P-gp表現量皆明顯下降；因此本實驗將接續先前的實驗結果，探討糖尿病腎臟中P-gp表現下降之調控機制。 在本研究中，使用犬腎小管上皮細胞MDCK II，作為研究腎臟P-gp表現量之體外試驗模型。為探討糖尿病造成腎臟P-gp表現下降之可能的調控機制，MDCK II分別以IL-6、TNF-α、insulin及高濃度葡萄糖處理後，萃取細胞膜蛋白做西方墨點法試驗，觀察P-gp表現量變化的情形。並以定量即時聚合酶鏈鎖反應 (qRT-PCR) 測定P-gp mRNA 表現量。結果顯示，高濃度葡萄糖會誘導活性氧化物 (ROS) 之生成，同時造成P-gp表現顯著下降；而在細胞激素 (IL-6, TNF-α) 和胰島素環境下則未呈現顯著影響。另一方面，ROS也可以造成P-gp表現下降, 並可被抗氧化劑 N-acetylcysteine (NAC) 所抵消。在高濃度葡萄糖環境下給予細胞PKC inhibitor staurosporine (STS)，可抑制高濃度葡萄糖所造成P-gp 表現下降之現象，顯示PKC 的活化參與高濃度葡萄糖對於P-gp的調控。以PKC agonist PMA處理後P-gp 表現下降，然而共同處理NAC 與 PMA後，PMA誘導之P-gp 表現下降並未被NAC反轉。此外，也藉由觀察細胞內細胞核受體PXR、CAR及內皮素 (Endothelin-1, ET) 與P-gp 調控之關連性。實驗結果發現在高濃度葡萄糖處理24小時後，細胞中 PXR及CAR的mRNA 表現量皆顯著上升。另一方面，在PKC agonist PMA環境下處理12小時後，細胞中 PXR及CAR的mRNA 表現量則下降，這些處理皆未造成蛋白質表現量的改變；RT-qPCR分析並未發現MDCK II 細胞上有內皮素受體 ETR-A / B mRNA 的表現 總結上述研究結果，在高濃度葡萄糖環境下P-gp的表現量是顯著下降的。顯示由葡萄糖調節異常所引起的高血糖 (hyperglycemia) 為糖尿病造成腎臟P-gp 表現下降之主要機轉；且其調控P-gp表現量的機轉與ROS之生成以及活化PKC路徑有關。PXR、CAR、ET以及ETR-A / B路徑並未在此調控中扮演角色。然而是否有其他調控因子參與其中仍需做更進一步的探討。

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.