AGEs activate mesangial TGF-β-Smad signaling via an angiotensin II type I receptor interaction
AGEs activate mesangial TGF-β-Smad signaling via an angiotensin II type I receptor interaction
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DOI：
10.1111/j.1523-1755.2004.66004.x
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发表时间：
2004-12-01
影响因子：
19.6
通讯作者：
Okuda, S
中科院分区：
文献类型：
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作者：
Fukami, K;Ueda, S;Okuda, S
背景。肾素-血管紧张素系统（RAS）和晚期糖基化终产物（AGE）的积累与糖尿病肾病的发病机制有关。糖尿病肾病中 RAS 和 AGE 之间是否存在功能性相互作用尚不清楚。在本研究中，我们研究了 AGE 是否可以激活自分泌血管紧张素 II (Ang II) 信号传导，并随后在培养的大鼠系膜细胞中诱导转化生长因子-β (TGF-β)-Smad 信号传导。方法。使用荧光探针CM-H(2)DCFDA检测细胞内活性氧(ROS)的形成。通过放射免疫测定法测量Ang II。通过酶联免疫吸附测定 (ELISA) 对释放到培养基中的 TGF-β 进行定量分析。通过蛋白质印迹分析测定 Smad2、p27(Kip1) (p27)、纤连蛋白和 AGE 受体 (RAGE) 蛋白表达。通过荧光素酶测定分析TGF-β诱导型启动子活性。通过 5-bomo-2'-脱氧尿苷 (BrdU) 掺入评估 DNA 合成，并通过 [H-3] 亮氨酸掺入测定从头蛋白质合成。结果。 AGEs 增加了系膜细胞中细胞内 ROS 的产生，而这种效应可被 RAGE 抗血清显着抑制。 AGEs 还被发现以时间和剂量依赖性方式刺激 Ang II 的产生，而抗氧化剂 N-乙酰半胱氨酸 (NAC) 可以完全阻止这种情况。 AGE 诱导的 TGF-β 过量产生可被 Ang II 1 型受体 (AT(1)R) 拮抗剂坎地沙坦完全阻断。坎地沙坦和 TGF-β 中和抗体均完全阻止 AGE 诱导的 Smad2 磷酸化和 TGF-β 诱导型启动子活性。此外，AGEs 被发现可抑制 DNA 合成并刺激蛋白质从头合成和纤连蛋白产生，与 p27 上调相关。所有这些现象都被坎地沙坦或多克隆抗体抗TGF-β完全阻止。结论。本研究表明，AGE-RAGE 介导的 ROS 生成激活 TGF-β-Smad 信号传导，随后通过自分泌 Ang II 诱导系膜细胞肥大和纤连蛋白合成。该通路可能在促进糖尿病肾病发生和进展中提供代谢和血流动力学因素之间的重要联系。
Background. The renin-angiotensin system (RAS) and the accumulation of advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy. Whether there is a functional interaction between the RAS and AGEs in diabetic nephropathy is not known. In this study, we investigated whether AGEs could activate autocrine angiotensin II (Ang II) signaling and subsequently induce transforming growth factor-beta (TGF-beta)-Smad signaling in cultured rat mesangial cells.Methods. The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H(2)DCFDA. Ang II was measured by radioimmunoassay. TGF-beta released into media was quantitatively analyzed in an enzyme-linked immunosorbent assay (ELISA). Smad2, p27(Kip1) (p27), fibronectin, and receptor for AGEs (RAGE) protein expression were determined by Western blot analysis. TGF-beta-inducible promoter activity was analyzed by a luciferase assay. DNA synthesis was evaluated by 5-bomo-2'-deoxyuridine (BrdU) incorporation and de novo protein synthesis was determined by [H-3] leucine incorporation.Results. AGEs increased intracellular ROS generation in mesangial cells, and this effect was significantly inhibited by an antiserum against RAGE. AGEs also were found to stimulate Ang II production in a time- and dose-dependent manner, which was completely prevented by an antioxidant, N-acetylcysteine (NAC). AGE-induced TGF-beta overproduction was completely blocked by candesartan, an Ang II type 1 receptor (AT(1)R) antagonist. Both candesartan and neutralizing antibody against TGF-beta completely prevented AGEs-induced Smad2 phosphorylation and TGF-beta-inducible promoter activity. Furthermore, AGEs were found to inhibit DNA synthesis and to stimulate de novo protein synthesis and fibronectin production in association with up-regulation of p27. All of these phenomena were completely prevented by candesartan or a polyclonal antibody against TGF-beta.Conclusion. The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.