Kidney pericytes in vascular regeneration after injury

描述（由申请人提供）：本申请涉及广泛的挑战领域（11）：再生医学，以及特定的挑战主题11-DK- 102：工程化或再生组织中的血管网络。慢性肾脏疾病是一种影响数百万人的公共卫生流行病，对卫生保健，我们的社区和个人造成了深刻的损失。慢性肾脏疾病的特征是慢性炎症、实质细胞损失和纤维化以及肾功能丧失。新出现的证据表明，肾小管周围毛细血管损失通过在肾脏中建立导致持续性炎症的缺血环境来介导这些过程。与其他器官相比，肾损伤具体导致毛细血管损失的原因尚不清楚。在最近发表的研究中，我们已经描述了周细胞作为一个组成部分的管周毛细血管。周细胞是间充质来源的细胞，其位于内皮细胞附近并与内皮细胞形成功能性连接。在眼睛中，周细胞是毛细血管稳定和血管完整性所必需的。到目前为止，由于缺乏标记物，周细胞在肾脏中的描述很差。我们已经开发出独特的工具来研究肾脏中的周细胞。我们已经明确表明，在损伤过程中，周细胞脱离毛细血管，增殖并分化成瘢痕形成的肾成纤维细胞。我们现在假设肾纤维化是一种功能性周细胞缺乏的状态，我们进一步假设周细胞丢失解释了纤维化中肾小管周围毛细血管稀疏。在AIM 1中，我们将通过选择性地删除它们来建立肾周细胞在体内的功能。我们将验证一种新的基于细胞的治疗方法，试图通过过继转移补充肾损伤后的周细胞。我们将使用新发现的周细胞特异性标记物来询问肾损伤和纤维化是否以周细胞缺乏为特征。我们将研究周细胞的生物学作用是否是提供促血管生成信号的内皮细胞功能，血管稳定性试验，并通过选择性敲除三个候选因素，可能是不可或缺的肾周细胞功能在本试验中。在AIM 2中，我们将定义周细胞在健康和疾病中的翻译谱，并验证我们假设调节周细胞分化为肌成纤维细胞的候选转录因子。在稳态和纤维化疾病中的周细胞基因谱将使用新的高通量多核糖体免疫亲和纯化技术来完成。使用候选基因的方法，我们将测试三个周细胞特异性转录因子在调节周细胞分化成肌成纤维细胞的潜在作用。我们将使用RNA沉默方法敲低周细胞中的这些因子，并在血管稳定性测定中测试敲低的影响。这些建议代表了第一次详细研究周细胞功能在肾脏和一个全新的方法来了解血管稳定和再生的肾脏。 公共卫生相关性：慢性肾脏病是一种影响数百万人的公共卫生流行病，尽管肾脏具有再生能力，但许多慢性肾脏病患者仍遭受功能逐渐丧失。来自不同学科的新证据表明，将氧气和营养物质运送到肾小管的小血管（称为毛细血管）的损失可能是慢性肾脏疾病的核心问题。我们最近发现，包括内皮细胞的肾毛细血管也被另一种称为周细胞的细胞包裹。到目前为止，对肾脏中的周细胞的描述很少，也没有研究过它们在肾脏中的功能。我们已经开发了独特的工具来研究小鼠的周细胞，在本提案中，我们将在细胞和分子水平上研究周细胞在血管发育、维持健康血管和肾损伤后血管再生中的作用。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (11): Regenerative Medicine, and specific Challenge Topic, 11-DK- 102: Vascular Network in engineered or regenerated tissues. Chronic kidney disease is a public health epidemic affecting millions that exacts a profound toll to health care, our communities and to individuals. Chronic kidney disease and is characterized by chronic inflammation, parenchymal cell loss and fibrosis and loss of kidney functions. Emerging evidence implicates peritubular capillary loss in mediating these processes by setting up an ischemic environment in the kidney that results in persistent inflammation. The reason why kidney injury, in contrast to other organs, specifically results in capillary loss is unclear. In recently published studies, we have described pericytes as an integral component of peritubular capillaries. Pericytes are cells of mesenchymal origin that lie adjacent to and make functional connections with endothelial cells. In the eye, pericytes are necessary for capillary stabilization and vascular integrity. Until now pericytes have been poorly described in the kidney due to lack of markers. We have developed unique tools to study pericytes in the kidney. We have definitively shown that during injury, pericytes detach from capillaries, proliferate and differentiate into scar-forming kidney fibroblasts. We now hypothesize that kidney fibrosis is a state of functional pericyte deficiency and we further hypothesize that pericyte loss explains peritubular capillary rarefaction in fibrosis. In AIM 1 we will establish the function of kidney pericytes in vivo by selectively deleting them. We will validate a novel cell-based therapy by attempting to replenish pericytes following kidney injury by adoptive transfer. We will ask whether kidney injury and fibrosis is characterized by pericyte deficiency using newly identified pericyte specific markers. We will examine whether the biologic role of pericytes is to provide pro-angiogenic signals to endothelium using an in vitro assay of pericyte function, the vascular stabilization assay, and and by selective knockdown of three candidate factors that may be integral to kidney pericyte function in this assay. In AIM 2 we will define the translational profile of pericytes in health and disease and validate candidate transcription factors that we hypothesize regulate differentiation of pericytes into myofibroblasts. Pericyte gene profiling in homeostasis and fibrotic disease will be accomplished using a novel, high throughput polysome immunoaffinity purification technique. Using a candidate gene approach we will test the potential role of three pericyte-specific transcription factors in regulation of differentiation of pericytes to myofibroblasts. We will knock-down these factors in pericytes using RNA silencing methods and test the effects of knock-down in the vascular stabilization assay. These proposals represent the first detailed study of pericyte function in the kidney and a completely novel approach to understanding vascular stabilization and regeneration in the kidney. PUBLIC HEALTH RELEVANCE: Chronic kidney disease is a public health epidemic that affects millions yet despite the regenerative capacity of the kidney, many people with chronic kidney disease suffer progressive loss of function. Emerging evidence from different disciplines indicates that loss of the small blood vessels, known as capillaries that carry oxygen and nutrients to the tubules of the kidney, may be a central problem in chronic kidney disease. We have recently discovered that kidney capillaries comprising endothelial cells are also wrapped in another type of cell called pericytes. Until now pericytes in the kidney have been poorly described and their function in the kidney has not been studied. We have developed unique tools to study pericytes in mice, and in this proposal we will investigate at a cellular and molecular level the role pericytes play in the developing vasculature, maintenance of healthy vasculature and in vascular regeneration following injury of the kidney.