New Thoughts on Parietal Epithelial Cells

描述(申请人提供)：虽然肾小球早已被认为由四种居留细胞类型组成，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几乎已经引起了迄今为止所有研究的关注。相比之下，人们对顶叶上皮细胞(PECs)的正常生物学功能以及这些细胞在疾病状态下如何应对损伤知之甚少。尽管成熟的PECs起源于与足细胞相同的间充质细胞，但它们代表着一种独特的细胞类型，因为它们在构成上表达不同的基因，并且在受伤时容易增殖，与通常静止的足细胞形成对比。这项赠款提案的总体目标是为PECs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PECs在处理肾小球超滤液中的白蛋白方面起关键作用的假设。我们认为，与传统的肾小球滤过屏障(包括内皮细胞、基底膜、足细胞)不同，PECS衬里的Bowman基底膜形成了第二个肾小球通透性屏障。我们将验证这样一种假设，即在实验性疾病中，定义明确的紧密连接和相邻细胞之间的连接蛋白是异常的，导致白蛋白通透性增加到肾小球周围间隙。研究将在培养的PECs以及KSP-EYFP小鼠(PECs为绿色)的足细胞和PEC疾病的实验模型中进行，注射不同类型和大小的荧光分子探针作为示踪剂。我们还将测试假设，即PECS通常会将过滤后的白蛋白吸收到空泡中进行溶菌酶降解。然而，当蛋白尿在原发性足细胞疾病中显著时，我们认为摄取增加导致PEC内白蛋白含量过高是有害的。第二个目的是验证这样一种假设，即当足细胞数量减少时，PECs的关键功能是充当转化/分化为足细胞的局部前体细胞。我们已经将KSP-EYFP小鼠(绿色PECs)与neparin CFP小鼠(蓝色足细胞)杂交。在两种小鼠模型(白喉毒素受体在足细胞中的特异性；抗足细胞抗体)中将诱导足细胞的凋亡，从而导致足细胞的丢失。荧光细胞(PECs)的迁移将受到仔细的连续监测，并将基于绿色荧光标记与足细胞特异性蛋白(如WT-1和Synaptopodin)的双重表达而建立的足细胞转分化。同时进行的PEC细胞培养研究将加强体内实验。综上所述，这些研究旨在为PECs的正常功能以及PECs对直接损伤和邻近足细胞损伤的反应提供重要和新的信息。与公共卫生相关：在美国，肾脏疾病正在以流行的速度增加。十分之一的成年人有某种形式的肾脏损伤。这笔赠款的总体目标是勾勒出壁上皮细胞生物学的新范例，以便最终能够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