Molecular function of Klotho protein on vascular walls and trying to identify unknown factors protecting against endothelial dysfunction

在本研究中，我们发现Klotho蛋白在培养的血管内皮细胞中通过ANP增加cGMP的产生，同时在培养的血管平滑肌细胞中通过CNP增加cGMP的产生。PKC抑制剂GF 109203X可抑制Klotho蛋白的增殖。Klotho蛋白还能加速Angⅱ诱导的血管平滑肌细胞的增殖。蛋白激酶A和C的特异性抑制剂（PKA， PKC）抑制Klotho蛋白的加速。此外，我们发现Klotho蛋白增加了体外培养血管内皮细胞中ec-cNOS蛋白的表达，但不增加ec-cNOS基因的表达。我们在人伸长因子的控制下，产生了过表达klotho的转基因大鼠系。转基因大鼠与野生型大鼠尿液中一氧化氮代谢物含量无显著差异。另一方面，我们试图确定在肾小管中表达的内皮功能障碍的未知因素。从球囊介导的主动脉损伤大鼠和对照大鼠中收集肾脏。取肾皮质。总RNA采用硫氰酸胍-苯酚-氯仿法提取。为了获得主动脉球囊损伤大鼠肾皮质的基因表达谱，我们进行了微阵列分析。主动脉球囊损伤大鼠的基因表达谱与对照组无显著差异。

In this study, we found that Klotho protein proliferated the cGMP production by ANP in cultured vascular endothelial cells and also proliferated the cGMP production by CNP in cultured vascular smooth muscle cells. The proliferation by Klotho protein was inhibited by GF 109203X, a PKC inhibitor. Klotho protein also accelerated Ang II - induced proliferation of vascular smooth muscle cells. Specific inhibitors of protein kinases A and C (PKA, PKC) inhibited the acceleration by Klotho protein. Moreover, we showed that Klotho protein increases ec-cNOS protein expression, but dose not increase ec-cNOS gene expression in cultured vascular endothelial cells. We generated transgenic lines of rats that over-express klotho under the control of the human elongation factor. No significant difference was observed in nitric oxide metabolites in urine between the transgenic rats and wild-type rats.On the other hand, we tried to identify the unknown factors protecting against endothelial dysfunction, that express in renal tubules in the kidney. Kidneys were collected both from the rats suffering from balloon-mediated aortic injury and from controlled rats. Renal cortex was collected from the kidneys. Total RNA was extracted by the acid guanidium thiocyanate-phenol-chloroform method. To obtain gene expression profile of the renal cortex prepared form the rats suffering from balloon-mediated aortic injury, a microarray analysis was performed. No significant difference was observed in gene expression profile between the rats suffering from balloon-mediated aortic injury and the controlled rats.