New Thoughts on Parietal Epithelial Cells

描述（由申请人提供）：尽管人们早就知道肾小球有四种驻留细胞类型，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几乎获得了所有研究的关注。相比之下，人们对壁上皮细胞（PECs）的正常生物学功能以及这些细胞在疾病状态下如何对损伤作出反应知之甚少。尽管与足细胞起源于相同的间充质细胞，但成熟的PECs是一种独特的细胞类型，因为它们在组成上表达不同的基因，并且与典型的静止足细胞相比，受伤时容易增殖。这项拨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中起关键作用的假设。我们认为，与传统的肾小球滤过屏障（包括内皮细胞、GBM和足细胞）不同，内衬鲍曼基底膜的PECs形成了第二个肾小球渗透性屏障。我们将验证这样的假设：在实验性疾病中，相邻细胞之间定义明确的紧密连接和连接蛋白异常，导致白蛋白渗透到肾小球周围间隙增加。在培养的PECs和Ksp-EYFP小鼠（PECs为绿色）足细胞和PEC疾病的实验模型中注射不同类型和大小的荧光分子探针作为示踪剂进行研究。我们还将验证PECs通常将过滤后的白蛋白带入液泡以进行溶菌降解的假设。然而，当原发性足细胞疾病中出现蛋白尿时，我们认为摄取增加导致pec内白蛋白含量过量是有害的。第二个目的是验证PECs的一个关键功能是充当足细胞数量减少时转化/分化为足细胞的局部前体细胞的假设。我们将Ksp-EYFP小鼠（绿色PECs）与nephrin CFP小鼠（蓝色足细胞）杂交。两种小鼠模型（足细胞特异性白喉毒素受体；抗足细胞抗体）将诱导足细胞凋亡并因此丢失。将仔细监测荧光细胞（PECs）的连续迁移，并根据绿色荧光标签与足细胞特异性蛋白（如WT-1和synaptopodin）的双重表达，将其转化为足细胞。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PECs的正常功能，以及PECs如何对直接损伤及其邻近足细胞的损伤作出反应提供重要的新信息。公共卫生相关性：肾脏疾病在美国以流行病的速度增长，十分之一的成年人有某种形式的肾脏损害。这项资助的总体目标是描述壁上皮细胞生物学的新范式，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