Department of Pathology, Τhe Third Hospital of Hebei Medical University, Shijiazhuang, Hebei 050051, P.R. China.
Department of Nephrology, Τhe Third Hospital of Hebei Medical University, Shijiazhuang, Hebei 050051, P.R. China.
Int J Mol Med. 2015 Jul;36(1):294-300. doi: 10.3892/ijmm.2015.2193. Epub 2015 Apr 22.
The Notch pathway is known to contribute to the development of glomerular disease. Angiotensin II (Ang II), an important member of the renin-angiotensin system, stimulates the accumulation of extracellular matrix components in glomerular disease; however, the exact mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of the Notch pathway on the synthesis of extracellular matrix components in Ang II-stimulated podocytes. Mouse podocytes were stimulated with Ang II (10-6 mol/l). The activation of the Notch pathway was inhibited by a vector carrying short hairpin RNA (shRNA) targeting Notch1 (sh-Notch1) or by γ-secretase inhibitor (GSI). The protein levels of Notch1, Notch intracellular domain 1 (NICD1), hairy and enhancer of split-1 (Hes1), matrix metalloproteinase (MMP)-2, MMP-9, transforming growth factor-β1 (TGF-β1), type IV collagen and laminin were determined by western blot analysis. The Notch1, Hes1, MMP-2, MMP-9, TGF-β1, type IV collagen and laminin mRNA levels were detected by RT-PCR. The MMP-2 and MMP-9 activity was measured using a cell active fluorescence assay kit. The levels of TGF-β1, type IV collagen and laminin were determined in the culture medium of the podocytes by enzyme-linked immunosorbent assay (ELISA). Our results revealed that Ang II upregulated Notch1, NICD1, Hes1, TGF-β1, type IV collagen and laminin expression and downregulated MMP-2 and MMP-9 expression in the cultured podocytes. The inhibition of the Notch pathway by sh-Notch1 or GSI increased MMP-2 and MMP-9 expression, decreased the TGF-β1 level and suppressed type IV collagen and laminin expression. The inhibition of the Notch pathway by sh-Notch1 or GSI also increased MMP-2 and MMP-9 activity, and decreased TGF-β1 levels, type IV collagen levels and laminin secretion. These findings indicate that the Notch pathway potentially mediates the Ang II-induced synthesis of extracellular matrix components in podocytes through the regulation of MMPs and TGF-β1.
已知 Notch 通路有助于肾小球疾病的发展。血管紧张素 II（Ang II）是肾素-血管紧张素系统的重要成员，可刺激肾小球疾病中外基质成分的积累；然而，具体的机制仍有待阐明。本研究旨在探讨 Notch 通路对 Ang II 刺激足细胞中外基质成分合成的影响。用 Ang II（10-6mol/l）刺激小鼠足细胞。用靶向 Notch1 的短发夹 RNA（shRNA）载体（sh-Notch1）或 γ-分泌酶抑制剂（GSI）抑制 Notch 通路的激活。通过 Western blot 分析测定 Notch1、Notch 细胞内结构域 1（NICD1）、毛状和分裂增强子 1（Hes1）、基质金属蛋白酶（MMP）-2、MMP-9、转化生长因子-β1（TGF-β1）、IV 型胶原和层粘连蛋白的蛋白水平。通过 RT-PCR 检测 Notch1、Hes1、MMP-2、MMP-9、TGF-β1、IV 型胶原和层粘连蛋白的 mRNA 水平。用细胞活性荧光测定试剂盒测定 MMP-2 和 MMP-9 的活性。用酶联免疫吸附试验（ELISA）测定足细胞培养液中 TGF-β1、IV 型胶原和层粘连蛋白的水平。结果表明，Ang II 上调了培养的足细胞中 Notch1、NICD1、Hes1、TGF-β1、IV 型胶原和层粘连蛋白的表达，下调了 MMP-2 和 MMP-9 的表达。用 sh-Notch1 或 GSI 抑制 Notch 通路增加了 MMP-2 和 MMP-9 的表达，降低了 TGF-β1 水平，并抑制了 IV 型胶原和层粘连蛋白的表达。用 sh-Notch1 或 GSI 抑制 Notch 通路也增加了 MMP-2 和 MMP-9 的活性，降低了 TGF-β1、IV 型胶原和层粘连蛋白的水平。这些发现表明，Notch 通路可能通过调节 MMP 和 TGF-β1 介导 Ang II 诱导的足细胞中外基质成分的合成。
