TYPE IV COLLAGEN, LAMININS, AND INTEGRINS IN NORMAL AND DISEASED GLOMERULI

肾小球的发育是由一系列的IV型胶原同种型替代标志的。 GBM。具体而言，IV型胶原a1 a2 a1网络，存在于最早的肾小球基底膜 (GBM)在成熟GBM中，未成熟肾单位被胶原IV a3 a4 a5网络取代。我们的中央 假设是：GBM胶原IV网络转换对于两者的发展和维持至关重要 内皮细胞和足细胞。我们推测：细胞调查其不断发展的IV型胶原蛋白网络 通过整合素; IV型胶原蛋白的合成与其同源整合素受体相关联;并且当 受体参与异常的胶原网络，或者如果受体表达不正确， 传达。这种异常信号然后阻碍正常的基底膜蛋白合成，导致 肾小球结构和功能的进一步缺陷。我们的目标是：目标1）检查可能的缺陷 在Col 4a 3突变小鼠（Alport病模型）中的GBM-整联蛋白信号传导。我们将（a）定义GBM （B）表征Alport进展过程中的整联蛋白表达 疾病;和（c）使用免疫组织化学方法在正常和Alport小鼠之间的肾小球细胞蛋白质的库存变化。 蛋白质组学方法。目的2）研究Col 4a 1杂合子小鼠肾小球的特征， 微量白蛋白尿伴薄GBM。我们将（a）定义来自Col 4a 1突变体的GBM组成;（B） 表征它们的整联蛋白和（c）比较它们的肾小球蛋白质组与野生型。目标3） 确定整合素a1 <$1和a2 <$1对胶原IV网络形成的贡献。整合素a1 1 is 抗纤维化，而A2 <$1是促纤维化的。我们将（a）将整合素a1缺失小鼠与Col 4a 3和Col 4a 1杂交 预期纤维化增加的背景;和（B）将整联蛋白α 2缺失小鼠与预期纤维化增加的背景小鼠杂交 改良我们还将通过体内施用以下物质来测试GBM组装扰动： 含有整合素结合位点的异源三聚体三螺旋胶原肽， 针对IV型胶原整合素结合位点的抗体。目的4）确定GBM蛋白是否 合成和整合素表达是相关联的。这将通过qRT-PCR在肾小球细胞培养物中进行测试， Western印迹、siRNA敲除、共聚焦免疫荧光和免疫电子显微镜 显微镜 总之，这些研究将提供有关动态互惠性的全新信息， 肾小球细胞和IV型胶原网络之间的关系，以及整合素如何介导这一至关重要的 相互关系

Glomerular development is marked by a sequence of type IV collagen isotype replacements in the GBM. Specifically, collagen IV a1a2a1 network, present in the earliest glomerular basement membrane (GBM) of immature nephrons, is replaced in mature GBM by collagen IV a3a4a5 network. Our central hypothesis is: GBM collagen IV network switching is critical for development and maintenance of both endothelial cells and podocytes. We speculate that: cells survey their evolving type IV collagen networks through integrins; synthesis of type IV collagen and its cognate integrin receptors are linked; and when receptors engage abnormal collagen networks, or if receptor expression is incorrect, altered signals are conveyed. This abnormal signaling then thwarts normal basement membrane protein synthesis, leading to further defects in glomerular structure and function. Our aims are: Aim 1) To examine possible defects in GBM-integrin signaling in Col4a3 mutant mice, a model of Alport disease. We will (a) define the GBM composition from Col4a3 mutants; (b) characterize integrin expression during progression of Alport disease; and (c) inventory changes in glomerular cell proteins between normal and Alport mice using a proteomics approach. Aim 2) To characterize glomeruli of Col4a1 heterozygous mice, which are microalbuminuric with thin GBM. We will (a) define the GBM composition from Col4a1 mutants; (b) characterize their integrins and (c) compare their glomerular proteomes with wild type. Aim 3) To determine the contribution of integrins a1 ¿1 and a2¿1 on collagen IV network formation. Integrin a1¿1is anti-fibrotic whereas a2¿1 is pro-fibrotic. We will (a) cross integrin a1-null mice onto Col4a3 and Col4a1 backgrounds expecting increased fibrosis; and (b) cross integrin a2-null mice onto the same, expecting amelioration. We will also test for GBM assembly perturbations through in vivo administration of heterotrimeric triple helical collagen peptides containing integrin binding sites and by injection of antibodies against the type IV collagen integrin binding sites. Aim 4) Determine whether GBM protein synthesis and integrin expression are linked. This will be tested in glomerular cell cultures by qRT-PCR, Western blotting, siRNA knockdowns, and confocal immunofluorescence and immunoelectron microscopy. Together, these studies will provide fundamentally new information on the dynamic reciprocity between glomerular cells and collagen IV networks and how integrins mediate this critically important interrelationship.