足細胞於高糖環境下Rosiglitazone 對乙型變形成長因子及其受器表現的影響

The Influence of Rosiglitazone on Transforming Growth Factor-beta and Receptors Expression of Podocytes in High Glucose Environment

李政學(Cheng-Hsueh Lee)

指導教授 ： 陳鴻鈞

高雄醫學大學/醫學院/醫學研究所/碩士(2008年)

摘要

【背景】腎絲球足細胞在維持腎絲球過濾的結構上與功能上扮演了關鍵性的角色。足細胞的傷害在多種腎絲球疾病，包括糖尿病腎病變，的發病成因上扮演重要的角色。有很多種因子，包括乙型變形成長因子 (TGF-β)，可能會促進足細胞的凋亡。TGF-β家族的配體要發揮它的作用必須經由與第一型及第二型受器(TβR-I, TβR-II)結合，引發Smad 蛋白的磷酸化及活化，並與其他的轉錄因子協同對基因表現進行調控。先前的研究顯示糖尿病動物的腎臟中TGF-β 的訊息核糖核酸及蛋白質表現都增加，且用於結合配體的TβR-II在糖尿病腎臟中也被向上調節。TZDs類藥物(如rosiglitazone)，是PPARγ 的特殊配體，是新一類可以減少胰島素阻抗性並降低血糖，減少尿中白蛋白的排出的藥物。本研究的目的在評估TZDs類藥物是否也能同時對因為高糖而被向上調節TGF-β及其受器(包含TβR-I, 及TβR-II)產生抑制的效果。
【材料及方法】分化的足細胞株分別培養於正常糖(glucose 5.5mM)或高糖(glucose 25mM) 的環境下，並分別加入DMSO 及rosiglitazone 1uM 或10uM培養一及七天。利用反轉錄-聚合脢鏈反應(RT-PCR)、西方墨點法及酵素免疫分析法(ELISA)分別測量PPARγ、TGFβ1、TβR-I及TβR-II在高糖環境及加入rosiglitazone的情形下，其mRNA 及蛋白質的表現。
【結果】於高糖環境下，我們發現足細胞TGF-β1的mRNA表現在1天時是被抑制的(p=0.0471)，而在7天後，TGF-β1的mRNA表現有略為增加的趨勢，但未達顯著意義(p=0.1740)。而在高糖環境下不論是1天或是7天，TGF-β1的蛋白質表現卻是明顯增加的(p=0.0002；p=0.0003)。在TβR-I的部份，在高糖環境下不論是1天或是7天， TβR-I不論是mRNA表現或是蛋白質表現都沒有受到明顯的影響。在TβR-II的部份，在高糖環境下，TβR-II的mRNA表現不受影響；但在培養7天的時候，TβR-II的蛋白質表現有明顯增加(p=0.0011)。經分化後的足細胞確定有PPARγ的表現，且在高糖環境培養7天後並未發現足細胞PPARγ的mRNA表現有受到影響(p=0.4723)。在足細胞於正常糖及高糖環境下使用不同濃度的rosiglitazone對TGF-β1、TβR-I 及TβR-II的表現部分，在高糖環境培養7天的情形下，於加入rosiglitazone 1uM後TGF-β1 之mRNA 的表現明顯被抑制下來(p=0.0009)；於加入rosiglitazone 10uM後TGF-β1 之mRNA 的表現亦明顯被抑制下來 (p=0.0002)但劑量增加並無效果，於蛋白質表現部分加入rosiglitazone 低劑量後TGF-β1的表現無差異，但提高劑量TGF-β1的表現明顯被抑制下來(p=0.0116)。另外於正常糖環境培養7天的情形下，rosiglitazone 10uM 會使TGF-β1的蛋白質表現明顯被抑制下來(p<0.0001)。在高糖環境加入rosiglitazone 10uM培養7天的情形下TβR-I 之mRNA 的表現有明顯增加的情形(p<0.0001)。而在高糖環境下TβR-I蛋白質表現方面並無顯著差異。在高糖環境加入rosiglitazone 10uM培養7天的情形下TβR-II 之mRNA 的表現有明顯增加的情形(p=0.0361)。而在高糖環境下TβR-II蛋白質表現方面並無顯著差異。
【結論】總合上述結果，我們認為較長時間處於高糖環境之下，可能會造成TGF-β1及TβR-II的表現增加，但對TβR-I的表現並無影響。於高糖環境下使用較高濃度的rosiglitazone 劑量對TGF-β1 的表現有明顯的抑制效果。使用較高濃度的rosiglitazone 劑量在較長時間處於高糖環境之下對TβR-I及TβR-II 的mRNA表現有明顯的增加效果。
使用rosiglitazone對TGF-β1 的表現有明顯的抑制效果是我們所期待的；但對TβR-I及TβR-II的影響及後續整個TGF-β訊息傳遞鏈的影響則需要近一步的研究證實。

關鍵字

乙型變形成長因子 ； 足細胞 ； Rosiglitazone ； 高糖

並列摘要

【Background】Podocytes play a critical role in maintain the structure and function of the glomerular filter. Podocyte injury plays a role in the pathogenesis of various glomerular diseases, including diabetic nephropathy. Numerous other factors, including transforming growth factor-β (TGF-β), may also promote podocyte apoptosis. The TGF family of ligands mediate the effects by binding specific type I and type II receptors (TβR-I, TβR-II), inducing phosphorylation and the activation of the Smad proteins, and then collaborating with other transcription factors to regulate gene expression. Previous studies have shown that TGF-β1 mRNA and protein are elevated in the kidneys of diabetic animals, and the ligand-binding TβR-II is up-regulated in the diabetic kidney. Thiazolidinediones (TZDs) ,such as rosiglitazone are specific ligands of PPARγ, are a new class of agents that lower insulin resistance and thus blood glucose. Additionally, TZDs lower urinary albumin excretion significantly more than other types of glucose-lowering agents. The aim of this study is to evaluate whether TZDs can suppress the expression of TGF-β1 and TGF-β receptors (TβR-I and TβR-II) that were up-regulated by a hyperglycemic environment.
【Methods】Differential podocyte cell linse were culturde in normal glucose(5.5uM) or high glucose(25uM) environments. DMSO or rosiglitazone 1uM or 10uM were added to the culture medium for 1 or 7 days. In order to evaluate the mRNA and protein expression of PPARγ, TGF-β1 and receptors (TβR-I and TβR-II) in high glucose and rosiglitazone treat condition, reverse transcription-polymerase chain reaction (RT-PCR), immunoassays ELISA assay and western blot method were performed.
【Results】The expression of TGF-β1 mRNA was suppressed after being cultured in a high glucose environment for 1 day (p=0.0471). However, after being cultured for 7 days, the expression of TGF-β1 mRNA has a trend to be enhanced, although the following trend did not achieve statistical significance (p=0.1740). The protein expression of TGF-β1 was enhanced significantly when cultured in high glucose mediums for 1day or 7 days (p=0.0002 and p=0.0003, respectively). With regards to TβR-I, both mRNA and protein expression were not affected by high glucose. In a high glucose environment, the expression of TβR-II mRNA was not affected by high glucose, but the protein expression of TβR-II increased significantly at day 7 (p=0.0011). Differential podocyte cell lines showed the expression of PPARγ, and there was no effect to PPARγ mRNA expression after being cultured in high glucose medium for 7 days (p=0.4723). After treating podocytes with different concentrations of rosiglitazone (1uM or 10uM) in normal or high glucose conditions, we found that the expression of TGF-β1 mRNA was suppressed significantly (p=0.0009) when cultured with high glucose for 7 days and treated with rosiglitazone 1uM. Moreover, our result showed that when treated with rosiglitazone 10uM, the expression of TGF-β1 mRNA was also suppressed (p=0.0002). Consequently, our results showed that treatments with rosiglitazone 1uM or 10uM did not yield different effects on TGF-β1 mRNA expression. The protein expression of TGFβ1 did not change after being treated with low doses of rosiglitazone; however, high dosage could result in the suppression of TGF-β1 protein expression (p=0.0116). In addition, when cultured in normal glucose medium for 7 days, treatments with rosiglitazone 10uM could result to suppress TGF-β1 protein expression (p<0.0001). When cultured with high glucose and treated with rosiglitazone 10uM for 7 days, the expression of TβR-I mRNA was significantly enhanced (p<0.0001), but no significant change to TβR-I protein expression were observed. Similarly, when cultured with high glucose and treated with rosiglitazone 10uM for 7 days, the expression of TβR-II mRNA was significantly enhanced (p=0.0361), but no significant changes were found with regards to TβR-II protein expression.
【Conclusions】Our study shows that when podocytes are cultured in a high glucose condition for a long time, the expression of TGF-β1 and TβR-II may be enhanced, but the expression of TβR-I is not significantly affected. After being treated with high dose rosiglitazone in a high glucose condition, the expression of TGF-β1 is significantly suppressed while the expressions of TβR-I and TβR-II mRNA are significantly enhanced.
The suppression of TGF-β1 expression by rosiglitazone occurred as we originally expected. However, the unforeseen effects that rosiglitazone had on TβR-I and TβR-II and even on the TGF-β signal transduction calls for further evaluation through additional studies.

並列關鍵字

transforming growth factor-beta ； podocyte ； rosiglitazone ； high glucose

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