糖基化終末產物經由MEK/ERK訊息傳遞路徑促使神經膠質細胞源神經營養因子誘發大鼠近曲腎小管上皮細胞凋亡

Advanced glycation end-products enhance GDNF induced apoptosis via MEK/ERK signaling pathway in rat proximal tubular epithelial cells

楊承琳(Cheng-Lin Yang)

指導教授 ： 林清淵

長榮大學/健康科學學院/醫學研究所/碩士(2009年)

https://doi.org/10.6833/CJCU.2009.00051

摘要

過去的研究發現糖尿病孕母高血糖會導致胎兒腎臟先天性的結構缺損畸形。組織學上發現糖尿病孕母的胚胎腎臟其細胞有細胞凋亡增加的情況。並發現糖尿病孕母的胎兒其早期發育中的腎臟，神經膠質細胞營養因子 (GDNF)之表現較非糖尿病孕母之胚胎腎臟為低。GDNF在胚胎腎臟發育中扮演著不可或缺的角色。GDNF會特異性的與其受體GFRα-1結合並誘發Ret受體型酪胺酸激酶的高度磷酸化，以誘發訊號傳遞錄經之活化，並使基因進行轉錄。前人研究中發現在高血糖的環境中有糖基化終末產物 (AGE)的存在。因此本研究之目的為探討在大鼠近曲腎小管上皮細胞 (NRK-52E)中，AGE對於GDNF功能之影響。研究中吾人使用Streptozotocin誘導高血糖的糖尿病小鼠作為動物模式。小鼠胚胎腎臟基因的表現量是以即時定量聚合酶鏈鎖反應來進行測定。NRK-52E細胞的凋亡反應是以流式細胞儀來偵測。NRK-52E的RAGE、GFRα-1、及EGR-1基因表現量是使用即時定量聚合酶鏈鎖反應來進行測定；MEK及ERK的磷酸化則是使用西方墨點法。研究結果中發現，在NRK-52E細胞，AGE會增加RAGE及EGR-1 mRNA的表現。RAGE及EGR-1 mRNA 之量都會隨著AGE刺激時間的增加而上升，且分別在24及48小時達到高峰。GDNF與GFRα-1結合後會增加EGR-1之表現、誘發MEK卅ERK訊息傳遞路徑之活化，並且驅使NRK-52E細胞凋亡。此作用是經由活化MEK卅ERK訊息傳遞路徑，並可藉由ERK抑制劑來將其阻斷。因此吾人推論AGE會促進糖尿病性胚胎病變其機制可能是經由增加RAGE的表現，再經由MEK/ERK訊息傳遞路徑來造成NRK-52E細胞的凋亡。

關鍵字

細胞外訊息調節激酶細胞凋亡 ； 神經膠質細胞營養因子 ； 腎小管上皮細胞 ； 糖基化終末產物

並列摘要

The uncontrolled hyperglycemia of maternal diabetes has been found to negatively affect pregnancy and can result renal structure malformations. Histologically, cell apoptosis was significantly increased in fetal kidneys of diabetic group. Glial cell line-derived neurotrophic factor (GDNF) expression was down-regulated at peritubular region of early developing fetal kidney of diabetic group. GDNF binds to GDNF receptor (GFRα-1) and Ret. Phosphorylation of the tyrosine kinase receptor Ret, induced a signal cascade, lead to gene transcription. Advanced glycation end-products (AGE) were found in hyperglycemic condition. The aim of this study was to examine whether AGEs directly affect GDNF function in rat renal tubular epithelial cell line (NRK-52E). Diabetes mellitus mice induced by Streptozotocin as an animal model. Gene expressions of embryonic kidneys were quantified by Q-PCR. NRK-52E was used as target. AGE was prepared as originally described previously. NRK-52E apoptosis was determined by flow cytometer. Expression RAGE, GFRα-1, EGR-1, phosphorylated MEK and ERK of NRK-52E cells were quantified by Q-PCR and Western blot. The results showed that AGE up-regulated RAGE and EGR-1 expression on NRK-52E cells. RAGE and EGR-1 expression were increased at mRNA level in a time-dependent manner and peaked at 24 hrs and 48hr respectively. GDNF bind to GFRα-1 can induce NRK-52E cells apoptosis. AGE co-incubation with GDNF promoted NRK-52E cells apoptosis. This effect was via MEK/ERK pathway and could be significantly inhibited by ERK inhibitor. In conclusion, AGE might be promote diabetic embryopathy by up-regulating NRK-52E cells RAGE expression and enhancing NRK-52E cells apoptosis via MEK/ERK pathway.

並列關鍵字

advanced glycation end-products ； apoptosis ； GDNF ； ERK ； kidney epithelial cells

參考文獻

Lin, H.Y., and Moustakas, A. (1994). TGF-beta receptors: structure and function. Cell Mol Biol (Noisy-le-grand) 40, 337-349.

Farr, A.G., Mannschreck, J.W., and Anderson, S.K. (1987). Expression of Ia antigens by murine kidney epithelium after exposure to streptozotocin. Am J Pathol 126, 561-568.

Barasch, J., Yang, J., Ware, C.B., Taga, T., Yoshida, K., Erdjument-Bromage, H., Tempst, P., Parravicini, E., Malach, S., Aranoff, T., et al. (1999). Mesenchymal to epithelial conversion in rat metanephros is induced by LIF. Cell 99, 377-386.

Airaksinen, M.S., and Saarma, M. (2002). The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 3, 383-394.

Airaksinen, M.S., Titievsky, A., and Saarma, M. (1999). GDNF family neurotrophic factor signaling: four masters, one servant? Mol Cell Neurosci 13, 313-325.