New Thoughts on Parietal Epithelial Cells

描述（由申请人提供）：尽管早已知道肾小球由四种驻留细胞类型所损害，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几乎吸引了迄今为止所有的研究注意力。相比之下，人们对壁上皮细胞（PEC）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。尽管与足细胞起源于相同的间充质细胞，但成熟的 PEC 代表了一种明显独特的细胞类型，因为它们组成型表达不同的基因，并且在受伤时容易增殖，这与典型的静止足细胞形成鲜明对比。该拨款提案的总体目标是为 PEC 在健康和疾病中的生物学和功能提供新的见解。第一个目标是检验 PEC 在处理肾小球超滤液中白蛋白方面发挥关键作用的假设。我们认为鲍曼基底膜内衬的 PEC 形成第二个肾小球通透性屏障，与传统的肾小球滤过屏障（包括内皮、GBM、足细胞）不同。我们将检验以下假设：在实验疾病中，相邻细胞之间明确的紧密连接和连接蛋白是异常的，导致白蛋白进入肾小球周围空间的通透性增加。研究将在培养的 PEC 以及注射了不同类型和大小的荧光分子探针作为示踪剂的 Ksp-EYFP 小鼠（PEC 为绿色）中的足细胞和 PEC 疾病实验模型中进行。我们还将测试以下假设：PEC 通常会将过滤后的白蛋白吸收到液泡中进行溶菌酶降解。然而，当原发性足细胞疾病中蛋白尿明显时，我们认为，摄入增加导致 PEC 内白蛋白含量过多是有害的。第二个目标测试以下假设：PEC 的关键功能是充当局部前体细胞，当足细胞数量减少时，其转化/分化为足细胞。我们将 Ksp-EYFP 小鼠（绿色 PEC）与去氧肾上腺素 CFP 小鼠（蓝色足细胞）杂交。在两种小鼠模型中将诱导足细胞凋亡并因此损失（白喉毒素受体，特别是足细胞中的白喉毒素受体；抗足细胞抗体）。将以连续方式仔细监测荧光细胞 (PEC) 的迁移，并转分化为基于绿色荧光标签与足细胞特异性蛋白（如 WT-1 和突触蛋白）双重表达而建立的足细胞。并行的 PEC 细胞培养研究将增强体内实验。总之，这些研究旨在为 PEC 的正常功能以及 PEC 如何响应直接损伤及其邻近足细胞的损伤提供重要且新颖的信息。公共卫生相关性：在美国，肾脏疾病正在以流行率增加。十分之一的成年人患有某种形式的肾脏损伤。这笔赠款的总体目标是描绘壁上皮细胞生物学的新范例，以便最终可以开发新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