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

我们研究了在糖尿病肾病过程中起重要作用的Smad1相关基因的信号机制。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，在体内糖尿病肾病的系膜基质扩张过程中发挥关键作用。我们的研究还表明，尿Smad1排泄量可能是比蛋白尿更好的诊断指标，可用于评估糖尿病肾病的系膜基质扩张。我们还证明了AGEs诱导系膜细胞BMP4、Smad1和COL4的mRNA和蛋白表达。转化生长因子-β中和抗体或NOGGIN(骨形态发生蛋白拮抗剂)可抑制这些诱导。此外，通过针对每个基因的siRNA转染来抑制ALK3/6的诱导，阻断了诱导。此外，Smad1和BMP4在糖尿病肾病小鼠的硬化区高表达。这些结果提示，转化生长因子-β/Smad1和BMP4/Smad1信号通路的调节与糖尿病肾病的发生发展密切相关，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在糖尿病肾病的发生发展中起关键作用。然而，Ang II介导的糖尿病肾病的确切作用机制仍不清楚。我们首次发现Ang II在体外和体内都能促进Smad1、磷酸化Smad1和磷酸化Src的表达，这些诱导作用可被奥美沙坦、PP2(一种酪氨酸激酶抑制剂)和显性负性Src的过表达抑制。此外，加入针对Src的siRNA显著降低了Smad1的磷酸化和COL4的合成。这些结果表明，Ang II可能通过调节Src和Smad1在糖尿病肾病早期肾小球系膜基质扩张中发挥作用。

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.