高糖經由PI3K-UCHL5訊息傳遞路徑誘發腎膈細胞肥大與纖維化

High glucose induces hypertrophy and fibrosis via the PI3K-UCHL5 pathway in mesangial cells

張純瑛(Chun-Ying Chang)

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(2010年)

摘要

糖尿病腎病變(diabetic nephropathy，DN)是糖尿病患者嚴重的併發症，也是國內近年來十大死因之一，其病理變化包括早期腎臟細胞增生、後期細胞肥大和細胞外基質堆積，終而導致腎臟纖維化，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)是糖尿病腎病變最重要的病理因素，且TGF-β在誘發糖尿病患者腎臟肥大與胞外基質堆積上更是扮演舉足輕重之角色。泛素(ubiquitin)所引起的蛋白質降解参與訊息傳遞、細胞週期以及基因表現等調控。L5型泛素羧端水解酶(ubiquitin carboxyl-terminal hydrolase L5，UCHL5)為去泛素化酶(deubiquitinating enzyme，DUB)，可移除標定於乙型轉型生長因子第一型受器(TGF-β type Ι receptor，TGFβRI)上的泛素鏈(polyubiquitin chain)，使其無法被降解，因而穩定TGFβRI，以持續下游TGF-β/Smad訊息傳遞路徑的進行。有關高糖與UCHL5間的關係及UCHL5在糖尿病腎病變所扮演的角色尚無文獻探討，故實驗以腎膈細胞(MES 13)為對象，外加高糖作為模擬糖尿病的誘發因子，探討UCHL5蛋白質之表現及其對高糖誘發下游生理效應之調控。本論文發現高糖刺激腎膈細胞(MES 13)會增加UCHL5與TGFβRI蛋白質之表現，外加PI3K (LY294002)與TGFβRI (SB431542)抑制劑則可逆轉高糖增加UCHL5蛋白質之表現，利用轉染PI3K與Smad2 dominant-negative plasmids再次確認高糖經由PI3K與TGFβRI訊息傳遞路徑增加UCHL5蛋白質之表現。分別轉染UCHL5 shRNA與PI3K dominant-negative plasmids弱化高糖增加UCHL5與TGFβRI蛋白質之表現，抑制下游TGF-β/Smad訊息傳遞，且進一步經由弱化p21waf1/cip1蛋白質表現來逆轉高糖所引起的細胞肥大。轉染UCHL5 shRNA與PI3K dominant-negative plasmids也會經由弱化NF-κB與COX-2蛋白質表現來逆轉高糖所引起的發炎反應。轉染UCHL5 shRNA與PI3K dominant-negative plasmids也可經由弱化fibronectin與collagen type I蛋白質表現來逆轉高糖所引起的胞外基質堆積，免於腎膈細胞(MES 13)走向纖維化之命運。利用免疫組織化學染色觀察發現Streptozotocin (STZ)誘導糖尿病腎病變的大鼠腎臟組織中UCHL5及TGFβRI表現量較正常鼠增加，且p21waf1/cip1、NF-κB、COX-2、fibronectin、collagen type I等下游相關分子表現亦增加。綜合上述，高糖經由PI3K-UCHL5訊息傳遞路徑誘發腎膈細胞(MES 13)肥大與纖維化，導致糖尿病腎病變的發生，可知UCHL5在高糖誘發糖尿病腎病變的機制中扮演重要之角色。

關鍵字

糖尿病腎病變 ； L5型泛素羧端水解酶

並列摘要

Diabetic nephropathy is a major cause of end-stage renal disease and an important cause of morbidity and mortality in diabetic patients. The pathology of diabetic nephropathy consists of cell proliferation, hypertrophy and accumulation of extracellular matrix, leading to renal fibrosis and finally end-stage renal disease. High glucose-induced transforming growth factor-beta (TGF-β) production causes glomerular mesangial cell hypertrophy and accumulation of extracellular matrix, contributing to diabetic nephropathy. Ubiquitin-proteasome degradation pathway plays a key role in a number of biological processes including signal transduction, cell cycle, and gene expression. Ubiquitin C-terminal hydrolase L5 (UCHL5) is a deubiquitinating enzyme (DUB), that could promote TGF-β signalling and upregulate TGF-β-dependent gene expression by deubiquitinating and stabilizing the type I TGF-β receptor (TGFβRI). Thus, we studied the role of UCHL5 in high glucose-induced effects in mouse kidney mesangial cells. We found that high glucose-induced UCHL5 protein expression via the phosphoinositide-3 kinase (PI3K) pathway. High glucose increased TGFβRI protein expression and TGF-β bioactivity via the PI3K-UCHL5 pathway. High glucose-induced upregulation of p21waf1/cip1 protein expression, via the PI3K-UCHL5 pathway, which contributed to cell hypertrophy. High glucose-induced upregulation of NF-κB and COX-2 protein expression, via the PI3K-UCHL5 pathway, which contributed to inflammatory responses. High glucose-induced upregulation of fibronectin and collagen type I protein expression, via the PI3K-UCHL5 pathway, which contributed to extracellular matrix accumulation and renal fibrosis. Upregulation of UCHL5 protein expression caused upregulation of TGFβRI, p21waf1/cip1, NF-κB, COX-2, fibronectin and type I collagen protein expression in the kidney of streptozotocin (STZ)-induced diabetic rats. Thus, high glucose-induced UCHL5 protein expression, via the PI3K pathway, contributed to the development of diabetic nephropathy in vitro and in vivo.

並列關鍵字

diabetic nephropathy ； ubiquitin carboxyl-terminal hydrolase L5

參考文獻

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