Renal Stroma Derived Endothelial Precursors are Critical for Renal Development

描述（由申请人提供）：肾脏结构异常是儿童慢性肾脏疾病的主要原因之一，产生显著的发病率和死亡率。了解不同肾脏谱系如何发展和相互作用是对结构性肾脏疾病产生影响的关键。虽然该领域的大多数研究都集中在输尿管、肾源性和间质谱系上，但很少有人研究血管系统在肾脏发育过程中的作用，除非它与肾小球有关。此外，肾脏血管的起源一直被认为是来自肾源间质（肾内）或入侵血管。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，这对肾脏的发育至关重要。在转基因小鼠中，通过荧光活化细胞分选（FACS）和免疫荧光检测了不同胚胎阶段肾细胞亚群中基质和内皮标记物的共表达。对永久标记的肾皮质间质细胞的FACS分析显示，在出生后的肾脏中，存活的细胞中有很大比例是内皮细胞。免疫荧光显示，谱系标记的肾基质来源的内皮细胞产生了很大一部分小管周围毛细血管网络，但没有肾小球毛细血管。在功能上，胚胎基质阳性细胞分化成表达内皮标志物的管状网络，在体外内皮小管形成试验和内噬乙酰化低密度脂蛋白（功能性内皮细胞试验）中。在体内检测肾间质是否产生肾内皮，Flk1

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.