高濃度葡萄糖誘導腎間質纖維母細胞表現第一型單核球趨化蛋白質之訊息傳遞機轉

Signal Transduction Mechanisms of High Glucose-induced Monocyte Chemoattractant Protein-1 Expression in Renal Interstitial Fibroblasts

洪崇尹(Chung-Ying Hung)

指導教授 ： 陳永銘

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00766

摘要

背景：糖尿病腎病變是糖尿病的重要併發症，也是造成末期腎衰竭最常見的單一病因。臨床上糖尿病腎臟傷害如果併發腎小管間質性發炎，繼而產生腎間質纖維化，常導致較差的預後。第一型單核球趨化蛋白質（MCP-1）是一種 CC 亞族趨化激素，具有強力驅動單核球的作用。過去的研究發現腎臟所製造的 MCP-1 與糖尿病腎病變的嚴重度有密切關係。再者，腎間質內的纖維母細胞過去被發現與腎間質纖維化有關，然而是否與糖尿病腎病變之發炎現象有關，迄今尚無報告。在本研究，吾人假設腎間質纖維母細胞和 MCP-1 參與糖尿病腎病變之發炎反應，因而探討高濃度葡萄糖對於腎間質纖維母細胞表現 MCP-1，及其訊息傳遞路徑的影響。
研究方法：NRK-49F 細胞（大鼠腎間質纖維母細胞）以不同濃度葡萄糖（5.6 - 35 mmol/L）培養最長達五天。MCP-1 蛋白在培養液中的濃度由西方墨點法檢測，MCP-1 mRNA 的表現由北方墨點法檢測。為了探討 nuclear factor-kappa B（NF-kapp B）和 mitogen-activated protein kinase（MAPK）在高濃度葡萄糖引發 MCP-1 表現所扮演的訊息傳遞角色，NRK-49F 細胞在接受高濃度葡萄糖處理之前先給予 NF-kB 抑制劑 MG132（0.025-0.1 μM），extracellular signal-regulated kinases（ERK）1/2 抑制劑 PD98059（10-40 μM），c-Jun amino-terminal kinases（JNK）抑制劑 SP600125（5-20 μM）或 p38 MAPK 抑制劑 SB203580（5-20 μM）。
結果：以高濃度葡萄糖培養 NRK-49F 會隨著時間增加 MCP-1 mRNA 和蛋白的表現。將細胞以高濃度葡萄糖和 MG132 一起培養會減少高濃度葡萄糖所刺激的 MCP-1 mRNA 表現。SP600125、PD98059 以及 SB203580 也分別會降低高濃度葡萄糖所刺激的 MCP-1 mRNA 表現。
結論：這些結果顯示高濃度葡萄糖可以經由 ERK 1/2、JNK-c-Jun/AP-1、p38 MAPK 以及 NF-kB 等訊息傳遞路徑誘發大鼠腎間質纖維母細胞表現 MCP-1。

關鍵字

高濃度葡萄糖 ； 腎間質纖維母細胞 ； 第一型單核球趨化蛋白質 ； 訊息傳遞

並列摘要

Background. Diabetic nephropathy is a major complication of diabetes and the single most common disorder leading to end stage renal disease (ESRD). In diabetic nephropathy the prognosis worsens significantly if interstitial inflammation that leads to interstitial fibrosis develops. Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine well known for its ability in promoting the migration of monocytes. Locally produced MCP-1 is involved in the development of advanced diabetic nephropathy, especially in the formation of tubulointerstitial lesions. Moreover, renal interstitial fibroblasts have been implicated in the development of interstitial fibrosis. However, it is not clear whether renal fibroblasts play a role in renal inflammation in diabetic nephropathy. In this study we hypothesized that renal interstitial fibroblasts and MCP-1 participate in the inflammatory responses of diabetic nephropathy. We thereby investigated the effects of high glucose on the expression of MCP-1 by renal interstitial fibroblasts, and examined the signal transduction mechanisms whereby high glucose acts to affect MCP-1 production.
Methods. NRK-49F cells (rat kidney interstitial fibroblast) were incubated with glucose (5.6 to 35 mmol/L) for up to five days. The MCP-1 concentration in the conditioned media was determined by Western blot analysis and the MCP-1 mRNA expression was examined using Northern blot analysis. To investigate the role of nuclear factor-kappa B (NF-kappa B) and mitogen-activated protein kinase (MAPK) in high glucose induced MCP-1 expression, NRK-49F cells were treated with NF-kappa B inhibitor MG132, extracellular signal-regulated kinases（ERK）1/2 inhibitor PD98059, c-Jun amino-terminal kinases（JNK）inhibitor SP600125, or p38 MAPK inhibitor SB203580 before high glucose treatment.
Result. Incubation of NRK-49F with high glucose (35 mM) resulted in a time-dependent increase in MCP-1 mRNA expression and protein secretion. Coincubation of the cells with high glucose and MG132 blunted high glucose-mediated MCP-1 mRNA expression. PD98059, SP600125 and SB203580 also attenuated high glucose induced MCP-1 mRNA expression.
Conclusion. These results suggest that high glucose induces MCP-1 synthesis in kidney interstitial fibroblasts by ERK 1/2, p38 MAPK, JNK-c-Jun/AP-1 and NF-kappa B signaling pathways.

並列關鍵字

High glucose ； Renal interstitial fibroblasts ； Monocyte chemoattractant protein-1 ； Signal transduction

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