Yasuda T, Kondo S, Homma T, Harris R C
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Clin Invest. 1996 Nov 1;98(9):1991-2000. doi: 10.1172/JCI119003.
Increases in intraglomerular pressure are known to predispose to the development of glomerular sclerosis, which is characterized by accumulation of extracellular matrix within the glomerulus. Glomerular mesangial cells are exposed to pulsatile capillary pressures and are a potential target for mechanical stress. In the present studies, we subjected cultured rat mesangial cells to continuous cycles of stretching and relaxation (stretch/relaxation) and examined alterations in extracellular matrix gene expression. After 48 h of stretch/relaxation, immunofluorescent localization of matrix accumulation indicated increases in types I, III, and IV collagens, fibronectin, and laminin, with the greatest increases seen at the periphery of the culture dish, at the point of the greatest deformation. Northern blot analysis of total RNA revealed time-dependent induction of alpha1(I) collagen, alpha1(III) collagen, alpha1(IV) collagen, fibronectin, and laminin by stretch/relaxation, with maximal increases occurring between 12 and 24 h. Transient transfection of reporter gene constructs of the 5' flanking region of alpha1(I) collagen gene indicated that stimulation of gene transcription was involved in the increased expression of matrix mRNA. Gelatinolytic activity in conditioned media was decreased at 24 and 48 h of stretch/relaxation, in association with a significant decrease in levels of mRNA for matrix metalloproteinase-2 (68-72 kD type IV collagenase) occurring within 6 h of stretch/relaxation. In contrast, expression of tissue inhibitor of metalloproteinase-2 was increased within 12 h of stretch/relaxation. Stretch/relaxation increased immunoreactive TGF-beta at 48 but not 12 h. TGF-beta1 mRNA levels remained unchanged during the initial 12 h of stretch/relaxation, but were significantly elevated at 48 h, and no differences in TGF-beta bioactivity could be detected in conditioned media for up to 12 h of stretch/relaxation. These findings demonstrate that in glomerular mesangial cells, repeated cycles of stretching and relaxation lead to matrix accumulation by stimulating production of extracellular matrix and decreasing activity of degradative enzymes. The observed induction of TGF-beta1 suggests a role in matrix accumulation occurring in response to continued mechanical deformation.
已知肾小球内压力升高易引发肾小球硬化，其特征是肾小球内细胞外基质的积累。肾小球系膜细胞暴露于搏动性毛细血管压力下，是机械应力的潜在靶点。在本研究中，我们使培养的大鼠系膜细胞经历连续的拉伸和松弛循环（拉伸/松弛），并检测细胞外基质基因表达的变化。在拉伸/松弛48小时后，基质积累的免疫荧光定位显示I型、III型和IV型胶原蛋白、纤连蛋白和层粘连蛋白增加，在培养皿周边变形最大的点增加最为明显。对总RNA的Northern印迹分析显示，拉伸/松弛可使α1(I)胶原蛋白、α1(III)胶原蛋白、α1(IV)胶原蛋白、纤连蛋白和层粘连蛋白呈时间依赖性诱导，在12至24小时之间增加最为显著。对α1(I)胶原蛋白基因5'侧翼区域的报告基因构建体进行瞬时转染表明，基因转录的刺激参与了基质mRNA表达的增加。在拉伸/松弛24小时和48小时时，条件培养基中的明胶分解活性降低，与拉伸/松弛6小时内基质金属蛋白酶-2（68 - 72 kD IV型胶原酶）的mRNA水平显著降低相关。相反，金属蛋白酶组织抑制剂-2的表达在拉伸/松弛12小时内增加。拉伸/松弛在48小时而非12小时时增加了免疫反应性TGF-β。在拉伸/松弛的最初12小时内，TGF-β1 mRNA水平保持不变，但在48小时时显著升高，在拉伸/松弛长达12小时的条件培养基中未检测到TGF-β生物活性的差异。这些发现表明，在肾小球系膜细胞中，反复的拉伸和松弛循环通过刺激细胞外基质的产生和降低降解酶的活性导致基质积累。观察到的TGF-β1的诱导表明其在响应持续机械变形而发生的基质积累中起作用。
