Renal Stroma Derived Endothelial Precursors are Critical for Renal Development

肾基质衍生的内皮前体对肾脏发育至关重要

• 批准号: 8880199
• 负责人: Sunder Sims-Lucas
• 金额: $ 11.14万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8880199
• 关键词: Affect; Biological Assay; Blood Vessels; Blood capillaries; Blood flow; Breeding; Cell Differentiation process; Cell Separation; Cells; Cessation of life; Child; Childhood; Chronic Kidney Failure; Congenital Abnormality; Data; Defect; Development; Disease; Embryo; Embryonic Development; Endocytosis; Endothelial Cells; Endothelium; Epithelium; Fetal Kidney; Glomerular Capillary; Green Fluorescent Proteins; Health; Immunofluorescence Immunologic; In Vitro; Invaded; Kidney; Kidney Diseases; Label; Lead; Lipoprotein (a); Mesenchyme; Morbidity - disease rate; Mus; Mutant Strains Mice; Nephrons; Pattern; Physiological; Plastics; Population; Process; Reporter; Role; Rosa; Signal Transduction; Sorting - Cell Movement; Staging; Stromal Cells; Structural defect; Structure; Testing; Time; Transgenic Mice; Tubular formation; acetyl-LDL; capillary; in vivo; insight; kidney cell; mortality; mutant; nephrogenesis; novel; progenitor; reconstruction; red fluorescent protein

DESCRIPTION (provided by applicant): Kidney structural abnormalities are amongst the leading causes of pediatric chronic kidney disease, producing significant morbidity and mortality. Understanding how different kidney lineages develop and interact is critical for making an impact on structural kidney disease. While most of the field has focused on ureteric, nephrogenic and stromal lineages, few have interrogated the role of the vasculature in the process of kidney development, except as it relates to the glomerulus. Moreover, the origin of the kidney vasculature has been debated to come from either the nephrogenic mesenchyme (within the kidney) or invading vessels. Our preliminary data strongly suggests that the renal cortical stroma gives rise to a significant percentage of the endothelium in the kidney that is critical for kidney development. In transgenic mice, co-expression of stromal and endothelial markers was detected in subsets of kidney cells at different embryonic stages by fluorescent activated cell sorting (FACS) and immunofluorescence. FACS analysis of permanently tagged renal cortical stromal cells showed a significant proportion of the surviving cells were now endothelial cells in post-natal kidneys. Immunofluorescence revealed lineage tagged renal stromal-derived endothelial cells gave rise to a significant portion of the peritubular capillary network, but not glomerular capillaries. Functionally, embryonic stroma-positive cells differentiated into tubular networks that expressed endothelial markers in an in vitro endothelial tubulogenesis assay and endocytosed Acetylated Low density lipoprotein (a functional endothelial cell assay). To test whether renal stroma gives rise to renal endothelium in vivo, Flk1 (critical for endothelial development) was conditionally deleted in the renal stroma (Flk1ST-/- mice). Flk1ST-/- mice had a dramatic congenital kidney defect and dilated peritubular capillaries while containing normal-appearing glomerular capillaries. An apparent reduction in ureteric branching and nephron formation was also observed. Thus, the hypothesis is that a subset of renal cortical stromal cells are precursors to many of the renal peritubular endothelial cells and that this endothelial cell population is necessary for normal patterning of other lineages in the kidney. To test this hypothesis, the following aims are proposed: Aim 1: To determine the functional potential of the renal cortical stroma to develop into endothelium. Foxd1 stroma will be isolated by FACS and subjected to in vitro conditions to drive endothelial cell differentiation. Aim 2: Determine the fate of Foxd1/Flk1 cells and how deletion of these cells affects vascular development. A comprehensive histological, structural, and functional analysis of the endothelium and vasculature will be performed. Aim 3: To investigate the role of the FoxD1- derived endothelium in formation of the kidney. A thorough histological, structural and physiological assessment of the renal lineages of the Flk1ST-/- mice will be performed. These studies will provide new insights into the origins of renal endothelium and their contribution to renal development. Manipulation of this novel progenitor pool may therapeutically impact vascular related congenital kidney abnormalities and diseases.

描述（由申请人提供）：肾脏结构异常是小儿慢性肾脏疾病的主要原因之一，产生了明显的发病率和死亡率。了解不同的肾脏谱系如何发展和相互作用对于对结构性肾脏疾病产生影响至关重要。尽管该领域的大多数集中在输尿管，肾脏基和基质谱系上，但很少有人询问脉管系统在肾脏发育过程中的作用，除非与肾小球相关。此外，肾脏脉管系统的起源是辩论的。我们的初步数据强烈表明，肾皮质基质在肾脏中占据着肾脏发育至关重要的内皮的很大比例。在转基因小鼠中，通过荧光活化的细胞分选（FACS）和免疫荧光在不同胚胎阶段的肾细胞子集中检测到基质和内皮标记的共表达。永久标记的肾皮质基质细胞的FACS分析显示，在产后肾脏中，存活的细胞中很大一部分是内皮细胞。免疫荧光揭示了谱系标记为肾质基质衍生的内皮细胞的谱系会导致周围毛细管网络的很大一部分，但没有肾小球毛细血管。在功能上，胚胎基质阳性细胞分化为管状网络，这些细胞在体外内皮细胞生成测定法中表达内皮标记和内吞乙酰化的乙酰化低密度脂蛋白（一种功能性内皮细胞测定）。测试肾脏基质是否引起体内肾脏内皮，FLK1 （对于内皮发育至关重要）在肾脏基质（FLK1ST - / - 小鼠）中有条件地删除。 FLK1ST - / - 小鼠具有明显的先天性肾脏缺陷，并且含有正常表现的肾小球毛细血管的周围毛细血管扩张。还观察到了输尿管分支和肾单位形成的明显减少。因此，假设是肾皮质基质细胞的一部分是许多肾周周内皮细胞的前体，并且该内皮细胞群对于肾脏中其他谱系的正常构图是必需的。为了检验该假设，提出了以下目的：目标1：确定肾皮质基质的功能潜力以发展成内皮。 FOX1基质将通过FACS分离，并经过体外条件，以驱动内皮细胞分化。 目标2：确定FOXD1/FLK1细胞的命运以及这些细胞的缺失如何影响血管发育。将对内皮和脉管系统进行全面的组织学，结构和功能分析。目标3：研究FOXD1衍生的内皮在形成肾脏中的作用。将对FLK1ST的肾脏谱系进行彻底的组织学，结构和生理评估。这些研究将为肾脏内皮的起源及其对肾脏发育的贡献提供新的见解。对这个新型祖细胞池的操纵可能会在治疗上影响与血管相关的先天性肾脏异常和疾病。