Localization of matrix metalloproteinases and their inhibitors in experimental progressive kidney scarring
Localization of matrix metalloproteinases and their inhibitors in experimental progressive kidney scarring
复制标题
DOI：
10.1038/sj.ki.5002108
复制
发表时间：
2007-04-01
影响因子：
19.6
通讯作者：
Johnson, T. S.
中科院分区：
文献类型：
--
作者：
Ahmed, A. K.;Haylor, J. L.;Johnson, T. S.
细胞外基质 (ECM) 处于持续周转状态，通过平衡合成和降解速率来维持稳态。在进行性肾脏疤痕形成过程中，会出现不平衡，导致 ECM 积聚。基质金属蛋白酶（MMP）活性降低被认为是造成这种不平衡的主要原因。然而，大多数与 MMP 及其天然抑制剂（基质金属蛋白酶组织抑制剂 (TIMP)）相关的数据都是基于匀浆研究，其中原位区室化丢失，因此 MMP 活性的变化可能是人为的。为了解决这个问题，我们开发了一种灵敏、高分辨率的原位酶谱技术，并将其与免疫组织化学一起应用于肾脏疤痕的 5/6 肾次全切除模型。肾匀浆中的 ECM 蛋白水解活性在 SNx 后针对明胶 (-82%) 和胶原蛋白 I (-78%) 底物逐渐下降。原位酶谱分析显示，与 SNx 相比，正常肾小管细胞细胞质内的两种底物的活性更高。相比之下，SNx 肾小球的活性比正常情况高出 96%。免疫组织化学证实所有 MMP 和 TIMP 主要位于细胞内肾小管内。肾小管显示 MMP-3 减少和 TIMP-2 升高，而肾小球中的 MMP-1 显着增加，尤其是系膜基质中。通过蛋白质印迹分析，TIMP-1 在残余肾脏中增加了四倍，但无法定位。匀浆中 MMP 活性降低是由于肾小管细胞内活性降低所致，表明 MMP 活性降低可能不会在疤痕形成中肾小管 ECM 扩张中发挥直接作用。然而，肾小球中 MMP-1 活性升高可能在启动肾小球重塑中发挥重要作用。
The extracellular matrix (ECM) is in a continual state of turnover with homeostasis maintained by balancing synthesis and degradation rates. During progressive kidney scarring an imbalance occurs leading to ECM accumulation. Reduced matrix metalloproteinase (MMP) activity is believed to central to this imbalance. However, most of the data relating to MMPs and their natural inhibitors (tissue inhibitors of matrix metalloproteinase (TIMP)) is based on homogenate studies where in situ compartmentalization is lost and thus changes in MMP activity may be artificial. To address this we have developed a sensitive, high-resolution in situ zymography technique and applied it, along with immunohistochemistry, to the 5/6th subtotal nephrectomy model of kidney scarring. ECM proteolytic activity in kidney homogenates progressively declined post-SNx against both gelatin (-82%) and collagen I (-78%) substrates. In situ zymography revealed higher activity with both substrates within the cytoplasm of normal tubular cells compared to the SNx. In contrast, there was 96% greater activity in the SNx glomeruli than normal. Immunohistochemistry confirmed a predominantly intracellular tubular location of all MMPs and TIMPs. Tubules showed reduced MMP-3 and elevated TIMP-2, whereas MMP-1 increased significantly in the glomeruli, especially in the mesangial matrix. TIMP-1 showed a fourfold increase in the remnant kidney by Western blot analysis, but could not be localized. Lowered MMP activity in homogenates results from reduced intracellular activity in the tubules, indicating that reduced MMP activity may not play a direct role in the expansion of the tubular ECM in scarring. However, elevated MMP-1 activity in the glomeruli may play a significant role in initiating glomerular remodelling.