Pawluczyk Izabella Z A, Tan Eddie K C, Lodwick David, Harris Kevin P G
Department of Infection, Immunity and Inflammation, University of Leicester, UK.
J Hypertens. 2008 Jan;26(1):93-101. doi: 10.1097/HJH.0b013e3282f0ca68.
Emerging evidence suggests that kallikrein exerts renoprotective effects independent of its haemodynamic actions. The aim of the current investigation was to delineate the role of kallikrein in the regulation of fibrosis, by 'knocking down' its expression using specific small interfering RNAs (siRNA).
Rat mesangial cells were treated with 12, 60, 120 nmol/l kallikrein-specific siRNAs. The consequent cellular genotypes and phenotypes were analysed.
Western blotting demonstrated that mesangial cells produced a kallikrein protein, which was of a different molecular weight to urinary kallikrein from rats of the same species. Treatment of cells with siRNA resulted in a dose-dependent decrease in kallikrein mRNA levels, which impacted on other components of the kallikrein-kinin system, dose-dependently reducing bradykinin B2 receptor mRNA expression. Kallikrein suppression resulted in significant increases in fibronectin and transforming growth factor-beta protein levels in culture supernatants over control levels. Gelatin zymography demonstrated a siRNA dose-dependent decrease in active MMP-2 enzyme levels. Bradykinin, an effector molecule of the kallikrein system, is known to stimulate tissue plasminogen activator production. Paradoxically, however, tissue plasminogen activator protein levels were augmented with increasing kallikrein mRNA silencing. This was accompanied by a dose-dependent decrease in low-density lipoprotein receptor-related protein mRNA levels, indicating that increased tissue plasminogen activator levels were due to an attenuation of receptor-mediated protease clearance.
These data lend strong support to the hypothesis that kallikrein exerts antifibrotic, renoprotective effects that are independent of its classical haemodynamic actions.
新出现的证据表明，激肽释放酶发挥肾脏保护作用，与其血流动力学作用无关。本研究的目的是通过使用特异性小干扰RNA（siRNA）“敲低”激肽释放酶的表达，来阐明其在纤维化调节中的作用。
用12、60、120 nmol/L的激肽释放酶特异性siRNA处理大鼠系膜细胞。分析由此产生的细胞基因型和表型。
蛋白质印迹法表明，系膜细胞产生一种激肽释放酶蛋白，其分子量与同一物种大鼠的尿激肽释放酶不同。用siRNA处理细胞导致激肽释放酶mRNA水平呈剂量依赖性下降，这影响了激肽释放酶-激肽系统的其他成分，剂量依赖性地降低了缓激肽B2受体mRNA的表达。与对照水平相比，激肽释放酶的抑制导致培养上清液中纤连蛋白和转化生长因子-β蛋白水平显著增加。明胶酶谱法表明，活性MMP-2酶水平呈siRNA剂量依赖性下降。缓激肽是激肽释放酶系统的效应分子，已知可刺激组织纤溶酶原激活物的产生。然而，矛盾的是，随着激肽释放酶mRNA沉默的增加，组织纤溶酶原激活物蛋白水平升高。这伴随着低密度脂蛋白受体相关蛋白mRNA水平的剂量依赖性下降，表明组织纤溶酶原激活物水平的增加是由于受体介导的蛋白酶清除减弱所致。
这些数据有力支持了以下假说：激肽释放酶发挥抗纤维化、肾脏保护作用，与其经典的血流动力学作用无关。
