Fundamental Analysis of Regulation of Smadl-related Genes in Diabetic Nephropathy

我们研究了smad1相关基因在糖尿病肾病过程中发挥重要作用的信号传导机制。我们证明Smad1是一个关键的转录因子，通过调节IV型胶原（Co14）和α-平滑肌肌动蛋白的表达，在体内糖尿病肾病系膜基质扩张的进展中起关键作用。我们的研究还表明，尿Smad1的排泄量将是一个比蛋白尿更好的诊断标志物，以评估糖尿病肾病的肾小球系膜基质扩张。我们还证实了AGEs诱导系膜细胞中BMP4、Smad1和Col4 mRNA和蛋白的表达。TGF-β中和抗体或Noggin（一种BMP拮抗剂）抑制这些诱导。此外，用siRNA转染每个基因敲低ALK3/6可阻断诱导。此外，Smad1和BMP4在糖尿病肾病小鼠的硬化区高表达。这些结果表明，TGF-β/Smad1和BMP4/Smad1信号通路的调节与糖尿病肾病的发生和发展密切相关，阻断Smad1信号通路可能有利于糖尿病并发症的发生。血管紧张素II （Ang II）在糖尿病肾病的发展中起着关键作用。然而，angii介导的糖尿病肾病作用的确切机制尚不清楚。我们首先发现Ang II在体外和体内增加Smad1、phospho-Smad1和phospho-Src的表达，而这些诱导作用被奥美沙坦、PP2（一种Src酪氨酸激酶抑制剂）和显性阴性Src的过表达所抑制。此外，针对Src添加siRNA可显著降低Smad1的磷酸化和Col4的合成。这些结果表明，Ang II可以通过Src和Smad1调控糖尿病肾病早期系膜基质扩张的发展。

We studied the signaling mechanisms of Smad1-related genes, which play important roles in the process of diabetic nephropathy. We demonstrated that Smad1 is a key transcriptional factor by regulating the expression of type IV collagen (Co14) and α-smooth muscle actin, and plays a critical role for the progression of mesangial matrix expansion in diabetic nephropathy in vivo. Our study also indicates that the amount of urinary Smad1 excretion would be a better diagnostic marker than albuminuria to assess mesangial matrix expansion in diabetic nephropathy. We also demonstrated that AGEs induced BMP4, Smad1, and Col4 mRNA and protein expressions in mesangial cells. TGF-β neutralizing antibody or Noggin (a BMP antagonist) inhibited these inductions. Moreover knockdown of ALK3/6 by using siRNA transfection against each gene blocked the induction. Furthermore, Smad1 and BMP4 were highly expressed in sclerotic regions of diabetic nephropathy mice. These results suggest that the modulation of both TGF-β/Smad1 and BMP4/Smad1 signaling is deeply responsible for initiation and progression of diabetic nephropathy and that blocking of Smad1 signaling pathway may be beneficial for diabetic complications.Angiotensin II (Ang II) is known to play a pivotal role in the development of diabetic nephropathy. However, the precise mechanism of Ang II-mediated effects on diabetic nephropathy is still unknown. We first showed that Ang II increased expression of Smad1, phospho-Smad1, and phospho-Src in vitro and in vivo, and these inductions were inhibited by olmesartan, PP2, a Src tyrosine kinase inhibitor, and overexpression of dominant negative Src. Moreover, addition of siRNA against Src significantly reduced the phosphorylation of Smad1 and synthesis of Col4. These results indicate that Ang II can regulate the development of mesangial matrix expansion in the early phase of diabetic nephropathy through Src and Smad1.