New Thoughts on Parietal Epithelial Cells

描述（由申请人提供）：虽然肾小球早已被认为是四种常驻细胞类型的损害，但内皮细胞、系膜细胞和足细胞对肾小球结构和功能的贡献迄今为止已经获得了几乎所有的研究关注。相比之下，对壁上皮细胞（佩奇）的正常生物学功能以及这些细胞如何在疾病状态下对损伤做出反应知之甚少。尽管与足细胞起源于相同的间充质细胞，但成熟佩奇代表了一种明显独特的细胞类型，因为它们组成性地表达不同的基因，并且在损伤时容易增殖，与通常静止的足细胞形成对比。这项拨款提案的总体目标是为佩奇在健康和疾病中的生物学和功能提供新的见解。在第一个目标中，我们将检验佩奇在肾小球超滤液中处理白蛋白方面具有关键作用的假设。我们认为，佩奇内衬鲍曼氏基底膜形成第二肾小球渗透性屏障，不同于传统的肾小球滤过屏障（包括内皮细胞，GBM，足细胞）。我们将检验这样的假设，即在实验性疾病中，相邻细胞之间的明确的紧密连接和连接蛋白是异常的，导致白蛋白渗透到肾小球周围空间。将在培养的佩奇中以及Ksp-EYFP小鼠（佩奇为绿色）的足细胞和PEC疾病实验模型中进行研究，这些小鼠注射了不同类型和大小的荧光分子探针作为示踪剂。我们还将检验佩奇通常将过滤的白蛋白吸收到空泡中进行溶菌酶降解的假设。然而，当蛋白尿是显着的原发性足细胞疾病，我们建议，增加摄取导致过多的内PEC白蛋白含量是有害的。第二个目的是检验佩奇的关键功能是充当当足细胞数量减少时转化/分化成足细胞的局部前体细胞的假设。我们将Ksp-EYFP小鼠（绿色佩奇）与nephrin CFP小鼠（蓝色足细胞）杂交。在两种小鼠模型（足细胞中特异性白喉毒素受体;抗足细胞抗体）中将诱导足细胞凋亡并因此导致丧失。将以连续方式仔细监测荧光细胞（佩奇）的迁移，并基于绿色荧光标签与足细胞特异性蛋白如WT-1和突触足蛋白的同时双重表达建立向足细胞的转分化。同时进行的PEC细胞培养研究将增加体内实验。总之，这些研究旨在为佩奇的正常功能提供重要和新颖的信息，以及佩奇如何对直接损伤及其邻近足细胞的损伤做出反应。公共卫生相关性：在美国，肾脏疾病的流行率正在上升，十分之一的成年人患有某种形式的肾损伤。这项资助的总体目标是描绘壁上皮细胞生物学的新范例，以便最终开发新的策略来减轻肾脏疾病的负担。

DESCRIPTION (provided by applicant): Although the glomerulus has long since been known to be compromised of four resident cell types, the contributions of the endothelial cell, mesangial cell, and podocyte to glomerular architecture and function have garnered virtually all of the research attention to date. In contrast, very little is known about the normal biological function of parietal epithelial cells (PECs), and how these cells respond to injury in disease states. Despite originating from the same mesenchymal cell as podocytes, mature PECs represent a distinctly unique cell type as they constitutively express different genes, and readily proliferate when injured, contrasting from the typically quiescent podocyte. The overall goals of this grant proposal are to provide novel insights into the biology and function of PECs in health and disease. In the first aim, we will test the hypothesis that PECs have a critical role in the handling of albumin in the glomerular ultrafiltrate. We propose that PECs lining Bowman's basement membrane form a second glomerular permeability barrier, distinct from the conventional glomerular filtration barrier (which comprises the endothelium, GBM, podocytes). We will test the hypothesis that the well-defined tight junctions and junctional proteins between neighboring cells are abnormal in experimental disease causing increased albumin permeability into the peri-glomerular space. Studies will be conducted in cultured PECs and in experimental models of podocyte and PEC disease in Ksp-EYFP mice (PECs are green) injected with different types and sizes of fluorescent molecular probes as tracers. We will also test the hypothesis that PECs normally take up filtered albumin into vacuoles for lysozymal degradation. However, when proteinuria is marked in primary podocyte disease, we propose that increased uptake leading to excess intra-PEC albumin content is then injurious. The second aim tests the hypothesis that a critical function of PECs is to serve as local precursor cells that transform/differentiate into podocytes when podocyte number decreases. We have crossed Ksp-EYFP mice (green PECs) with nephrin CFP mice (blue podocytes). Podocyte apoptosis and thus loss will be induced in two mouse models (diphtheria-toxin receptor specifically in podocytes; anti-podocyte antibody). The migration of fluorescent cells (PECs) will be carefully monitored in a serial fashion and trans-differentiation into podocytes established based on dual expression of green fluorescent tag concurrently with podocyte-specific proteins such as WT-1 and synaptopodin. Concurrent PEC cell culture studies will augment the in vivo experiments. In summary, these studies are aimed at providing important and novel information into the normal function of PECs, and how PECs respond to direct injury and to injury of their neighboring podocytes. PUBLIC HEALTH RELEVANCE: Kidney disease is increasing at epidemic rates in the U.S. One out of ten adults has some form of kidney damage. The overall goal of this grant is to delineate new paradigms in the biology of parietal epithelial cells, so that ultimately new strategies can be developed to reduce the burden of kidney disease