PI3K/mTOR/S6K1訊息傳遞路徑在糖尿病腎病變的角色

Role of PI3K/mTOR/S6K1 signaling pathway in Diabetic Nephropathy

鄭竹芸(Chu-Yun Cheng)

指導教授 ： 莊麗月

高雄醫學大學/醫學院/生物化學研究所/碩士(2011年)

摘要

糖尿病腎病變(diabetic nephropathy, DN)為糖尿病患者常見的嚴重併發症，其病理特徵包括早期腎臟細胞增生、後期細胞肥大和細胞外間質堆積，進而造成腎臟纖維化，最終演變為末期腎病(end-stage renal disease, ESRD)。高糖與乙型轉型生長因子(transforming growth factor-β, TGF-β)是誘發腎臟肥大和纖維化的重要因子，而表皮生長因子(epidermal growth factor, EGF)也與糖尿病腎病變的致病機轉相關。高糖會活化Phosphatidy-linositol 3-kinase (PI3K)來導致腎膈細胞肥大，Mammalian target of rapamycin (mTOR)則在腎臟疾病中會被活化，而且與細胞增生、肥大及纖維化有關，但高糖與DN其他致病因子是否經由PI3K/mTOR/S6 kinase 1 (S6K1)訊息傳遞路徑導致DN仍不清楚。因此本研究以近端腎小管上皮細胞LLC-PK1為對象，首先加入高糖及DN各種致病因子，發現高糖、TGF-β1及EGF會增加S6K1磷酸化(Thr 389)。以高糖處理12小時之下，觀察到S6K1活化最為明顯。此外，加入各種訊息傳遞抑制劑後，其中PI3K抑制劑LY294002最為顯著減弱S6K1的活性，因此高糖確實會透過PI3K來活化mTOR/ S6K1。利用EGF receptor (EGFR)抑制劑Iressa發現會降低S6K1磷酸化，表示高糖也會經由活化EGFR來提高S6K1之活性。已知FKBP38為mTOR之內生性抑制因子，實驗發現高糖不會影響FKBP38的表現，因此高糖並非藉由抑制FKBP38來活化mTOR訊息傳遞路徑。PI3K抑制劑LY294002、mTOR抑制劑Rapamycin和S6K1抑制劑PF4708671皆會降低p21waf1/cip1 promoter活性以及蛋白質表現，並改善高糖所造成的細胞肥大。抑制PI3K/mTOR/S6K1訊息傳遞路徑，則會弱化Fibronectin以及加強Matrix metalloprotease 9 (MMP-9)的表現來減少細胞外間質堆積，也會弱化α-smooth muscle actin (α-SMA)表現來逆轉高糖所引起的EMT。但是高糖並不會經由PI3K/mTOR/ S6K1路徑影響Cyclin D1和Cyclin-dependent kinase 4 (Cdk4)蛋白質表現而抑制細胞增生。利用免疫組織化學染色法觀察Streptozotocin (STZ)誘導DN之老鼠腎臟組織，發現p-mTOR (Ser 2448)及p-S6K1蛋白質表現量較正常鼠增加。因此，活化PI3K/mTOR/S6K1訊息傳遞路徑會誘發其下游分子機制，促使近端腎小管上皮細胞肥大及纖維化，最後導致糖尿病腎病變。

關鍵字

糖尿病腎病變 ； 磷脂醯肌醇3-激酉每 ； 哺乳類雷帕霉素靶蛋白 ； 核醣體蛋白S6激酉每1

並列摘要

Diabetic nephropathy (DN) is a common complication of diabetes mellitus and a leading cause of end-stage renal disease (ESRD). It is characterized by early cell proliferation, late hypertrophy, accumulation of extracellular matrix (ECM), and epithelial-to-mesenchymal transition (EMT), resulting in renal fibrosis and ESRD. Hyperglycemia and transforming growth factor-β (TGF-β) are important factors in renal hypertrophy and fibrosis. Epidermal growth factor (EGF) is also associated with the pathogenesis of DN. High glucose-induced hypertrophy is dependent on phosphatidylinositol 3-kinase (PI3K) in kidney mesangial cells. Activation of mammalian target of rapamycin (mTOR) in renal disease plays a major role in mediating the glomerular hypertrophy and interstitial fibrosis. However, it’s not known whether high glucose and other pathologic factors (TGF-β1, EGF) contribute to DN via the PI3K/mTOR/S6 kinase 1 (S6K1) pathway. In this study, we found that high glucose, TGF-β1 and EGF induced S6K1 phosphorylation (Thr 389) in porcine proximal tubular epithelial (LLC-PK1) cells. High glucose increased S6K1 activity via the PI3K/mTOR pathway. In addition, inhibition of EGF receptor by Iressa decreased S6K1 phosphorylation. High glucose didn’t reduce FKBP38 (a endogenous mTOR inhibitor) protein expression to activate the mTOR/S6K1 pathway. Furthermore, we found that high glucose increased p21waf1/cip1 promoter activity and protein expression via the PI3K/mTOR/S6K1 pathway, leading to cell hypertrophy. Inhibition of the PI3K/mTOR/S6K1 pathway not only attenuated high glucose-induced fibronectin protein expression but also restored matrix metalloprotease 9 (MMP-9) protein expression in high glucose-treated cells. Moreover, downregulation of the PI3K/mTOR/ S6K1 pathway attenuated high glucose-induced α-smooth muscle actin (α-SMA) protein expression, a biomarker of EMT. In the kidneys of streptozotocin (STZ)-induced diabetic rats, we found that phospho-mTOR (Ser 2448) and phospho-S6K1 protein expression were increased. In conclusion, PI3K/mTOR/S6K1 pathway plays key roles in the pathogenesis of DN, including renal hypertrophy and fibrosis.

並列關鍵字

Diabetic nephropathy ； Phosphatidylinositol 3-kinase ； Mammalian target of rapamycin ； S6 kinase 1

參考文獻

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