Renal Stroma Derived Endothelial Precursors are Critical for Renal Development

描述（由申请人提供）：肾脏结构异常是儿科慢性肾脏疾病的主要原因之一，导致显著的发病率和死亡率。了解不同的肾脏谱系如何发展和相互作用对于影响结构性肾病至关重要。虽然该领域的大部分都集中在输尿管，肾和基质谱系，很少有人询问的血管系统在肾脏发育过程中的作用，除了它涉及到肾小球。此外，肾血管系统的起源一直存在争议，认为是来自肾源性间充质（肾脏内）还是入侵血管。我们的初步数据有力地表明，肾皮质间质在肾脏中产生了相当大比例的内皮细胞，这对肾脏发育至关重要。在转基因小鼠中，通过荧光激活细胞分选（FACS）和免疫荧光法检测不同胚胎期肾细胞亚群中基质和内皮标志物的共表达。对永久标记的肾皮质基质细胞的流式细胞仪分析显示，在出生后的肾脏中，存活的细胞中有很大一部分是内皮细胞。免疫荧光显示谱系标记的肾间质来源的内皮细胞引起了显着的一部分，但不是肾小球毛细血管的管周毛细血管网络。在功能上，胚胎基质阳性细胞分化成管状网络，表达内皮标记物在体外内皮tubulogenesis测定和内吞乙酰化低密度脂蛋白（功能性内皮细胞测定）。为了检测肾间质是否在体内产生肾内皮，Flk 1 （内皮发育的关键）在肾间质中条件性缺失（FlklST-/-小鼠）。Flk 1ST-/-小鼠有一个显着的先天性肾缺陷和扩张的管周毛细血管，而含有正常的肾小球毛细血管。还观察到输尿管分支和肾单位形成明显减少。因此，假设是肾皮质基质细胞的一个子集是许多肾管周内皮细胞的前体，并且该内皮细胞群体是肾脏中其他谱系的正常模式所必需的。为了验证这一假设，提出了以下目标：目标1：确定肾皮质基质发育成内皮细胞的功能潜力。将通过FACS分离Foxd 1基质，并使其经受体外条件以驱动内皮细胞分化。 目的2：确定Foxd 1/Flk 1细胞的命运以及这些细胞的缺失如何影响血管发育。将对内皮和血管系统进行全面的组织学、结构和功能分析。目的3：探讨FoxD 1源性内皮细胞在肾脏形成中的作用。将对Flk 1ST-/-小鼠的肾脏谱系进行全面的组织学、结构和生理学评估。这些研究将为肾内皮细胞的起源及其对肾脏发育的贡献提供新的见解。这种新的祖细胞池的操作可能会在治疗上影响血管相关的先天性肾脏异常和疾病。

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.