Jaffa A A, Miller B S, Rosenzweig S A, Naidu P S, Velarde V, Mayfield R K
Department of Medicine, Medical University of South Carolina, Charleston, USA.
Am J Physiol. 1997 Dec;273(6):F916-24. doi: 10.1152/ajprenal.1997.273.6.F916.
Glomerular hypertension and glomerular hypertrophy act early and synergistically to promote glomerular injury in diabetes. We have previously shown that increased renal kinin production contributes to the glomerular hemodynamic abnormalities associated with diabetes. Glomerulosclerosis, characterized by mesangial cell proliferation and matrix expansion, is the final pathway leading to renal failure. The signal(s) initiating mesangial cell proliferation is ill defined. In the present study, we utilized immunofluorescence, immunoprecipitation, and immunoblotting techniques to identify substrates that are tyrosine phosphorylated in response to bradykinin action in mesangial cells. Immunofluorescence microscopy of mesangial cells stained with anti-phosphotyrosine (anti-PY) antibodies following bradykinin treatment (10(-9)-10(-6) M) revealed a dose-dependent increase in the labeling of cytoplasmic and nuclear proteins. Immunoprecipitation with anti-PY, followed by immunoblot revealed bradykinin-induced tyrosyl phosphorylation of tubulin and mitogen-activated protein kinase (MAPK). Confocal microscopy of mesangial cells stained for MAPK indicated that bradykinin stimulation resulted in translocation of MAPK from the cytoplasm to the nucleus by 2 h. These data demonstrate that bradykinin action results in the tyrosine phosphorylation of cellular proteins in mesangial cells and suggest a role for tubulin and MAPK in the signaling cascade of bradykinin leading to altered mesangial function.
肾小球高血压和肾小球肥大在糖尿病中早期协同作用，促进肾小球损伤。我们之前已经表明，肾脏激肽生成增加会导致与糖尿病相关的肾小球血流动力学异常。以系膜细胞增殖和基质扩张为特征的肾小球硬化是导致肾衰竭的最终途径。启动系膜细胞增殖的信号尚不明确。在本研究中，我们利用免疫荧光、免疫沉淀和免疫印迹技术来鉴定在系膜细胞中对缓激肽作用产生酪氨酸磷酸化反应的底物。用抗磷酸酪氨酸（抗 - PY）抗体对缓激肽处理（10^(-9) - 10^(-6) M）后的系膜细胞进行免疫荧光显微镜检查，结果显示细胞质和核蛋白的标记呈剂量依赖性增加。用抗 - PY进行免疫沉淀，随后进行免疫印迹，结果显示缓激肽诱导微管蛋白和丝裂原活化蛋白激酶（MAPK）的酪氨酸磷酸化。对系膜细胞进行MAPK染色的共聚焦显微镜检查表明，缓激肽刺激导致MAPK在2小时内从细胞质转位到细胞核。这些数据表明，缓激肽作用导致系膜细胞中细胞蛋白的酪氨酸磷酸化，并提示微管蛋白和MAPK在缓激肽信号级联反应中发挥作用，从而导致系膜功能改变。
