Fukami Kei, Ueda Seiji, Yamagishi Sho-ichi, Kato Seiya, Inagaki Yosuke, Takeuchi Masayoshi, Motomiya Yoshihiro, Bucala Richard, Iida Shuji, Tamaki Kiyoshi, Imaizumi Tsutomu, Cooper Mark E, Okuda Seiya
Department of Nephrology, Internal Medicine III, the Cardiovascular Research Institute, Department of Pathology, Kurume University School of Medicine, Kurume, Japan.
Kidney Int. 2004 Dec;66(6):2137-47. doi: 10.1111/j.1523-1755.2004.66004.x.
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.
The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. 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 [3H]leucine incorporation.
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 (AT1R) 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.
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.
肾素-血管紧张素系统（RAS）和晚期糖基化终末产物（AGEs）的蓄积与糖尿病肾病的发病机制有关。在糖尿病肾病中，RAS与AGEs之间是否存在功能相互作用尚不清楚。在本研究中，我们调查了AGEs是否能激活自分泌血管紧张素II（Ang II）信号，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。
使用荧光探针CM-H2DCFDA检测细胞内活性氧（ROS）的形成。通过放射免疫分析法测定Ang II。采用酶联免疫吸附测定（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确定Smad2、p27（Kip1）（p27）、纤连蛋白和AGEs受体（RAGE）的蛋白表达。通过荧光素酶测定分析TGF-β诱导的启动子活性。通过5-溴-2'-脱氧尿苷（BrdU）掺入评估DNA合成，并通过[3H]亮氨酸掺入测定新生蛋白合成。
AGEs增加了系膜细胞内ROS的生成，抗RAGE抗血清可显著抑制这种作用。还发现AGEs以时间和剂量依赖性方式刺激Ang II的产生，抗氧化剂N-乙酰半胱氨酸（NAC）可完全阻止这种作用。坎地沙坦（一种Ang II 1型受体（AT1R）拮抗剂）可完全阻断AGE诱导的TGF-β过量产生。坎地沙坦和抗TGF-β中和抗体均完全阻止了AGEs诱导的Smad2磷酸化和TGF-β诱导的启动子活性。此外，发现AGEs抑制DNA合成，并刺激新生蛋白合成和纤连蛋白产生，同时伴有p27上调。坎地沙坦或抗TGF-β多克隆抗体可完全阻止所有这些现象。
本研究表明，AGE-RAGE介导的ROS生成激活TGF-β-Smad信号，随后通过Ang II的自分泌产生诱导系膜细胞肥大和纤连蛋白合成。该途径可能在促进糖尿病肾病的发生和发展中提供代谢和血流动力学因素之间的重要联系。
