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

肾小球的发育以IV型胶原同型替换序列为标志。 GBM。具体地说，IV型胶原a1a2a1网络，存在于最早的肾小球基底膜 未成熟肾单位(GBM)中，成熟的GBM被IV型胶原a3a4a5网络取代。我们的中央 假说是：GBM IV型胶原网络切换对两者的发育和维护至关重要 内皮细胞和足细胞。我们推测：细胞调查它们正在进化的IV型胶原网络 通过整合素；IV型胶原的合成与其同源整合素受体相连； 受体参与异常的胶原蛋白网络，或者如果受体表达不正确，信号改变 传达出去了。这种异常的信号然后会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Alport病模型Col4A3突变小鼠的GBM-整合素信号转导中。我们将(A)定义GBM 来自Col4A3突变体的组合物；(B)表征Alport进展过程中整合素的表达 疾病；和(C)正常和Alport小鼠之间肾小球细胞蛋白的库存变化 蛋白质组学方法。目的2)研究COL4a1杂合子小鼠肾小球的特征。 微量白蛋白尿伴肾小球基底膜变薄。我们将(A)定义Col4a1突变体的GBM组成；(B) 鉴定它们的整合素和(C)将它们的肾小球蛋白质组与野生型进行比较。目标3) 确定整合素a1？1和a2？1在IV型胶原网络形成中的作用。整合素A1？1是 抗肝纤维化，而a2？1促肝纤维化。我们将(A)将整合素A1缺失的小鼠与Col4a3和Col4a1杂交 预期纤维化增加的背景；以及(B)将整合素a2缺失的小鼠杂交到同样的小鼠上，预期 改进。我们还将通过体内给药来测试GBM组装的扰动 含整合素结合部位的异三聚体三螺旋胶原肽 抗IV型胶原整合素结合部位的抗体。目的4)检测GBM蛋白 整合素的合成和表达是相关联的。这将通过qRT-PCR在肾小球细胞培养中进行测试， Western blotting、siRNA敲除、共聚焦免疫荧光和免疫电子 显微镜。 总之，这些研究将从根本上提供关于动态互惠的新信息 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作用 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.