Renal Stroma Derived Endothelial Precursors are Critical for Renal Development

描述(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是如何发展和相互作用的，对于对结构性肾脏疾病产生影响至关重要。虽然该领域的大部分研究集中在输尿管、肾源性和间质谱系上，但很少有人探讨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，肾血管的起源一直被争论为来自肾源性间质(在肾脏内)或侵袭的血管。我们的初步数据强烈表明，肾皮质间质在肾脏中产生了相当大比例的内皮细胞，这对肾脏的发育至关重要。在转基因小鼠中，通过荧光激活细胞分选(FACS)和免疫荧光检测到不同胚胎阶段的肾细胞亚群中基质和内皮标志的共同表达。对永久标记的肾皮质基质细胞的FACS分析表明，在出生后的肾脏中，存活的细胞中有相当大一部分是内皮细胞。免疫荧光显示系谱标记的肾间质来源的内皮细胞形成了肾小管周围毛细血管网络的重要部分，但不是肾小球毛细血管。在功能上，胚胎基质阳性细胞分化成管状网络，在体外内皮管形成试验和内吞的乙酰化低密度脂蛋白(一种功能性内皮细胞试验)中表达内皮标记物。为了测试肾间质是否在体内促进了肾内皮细胞的形成，Flk1 (对内皮发育至关重要)在肾间质(Flk1ST-/-小鼠)中有条件地被删除。Flk1ST-/-小鼠有戏剧性的先天性肾缺陷和扩张的管周毛细血管，同时含有看起来正常的肾小球毛细血管。输尿管分支和肾单位的形成也明显减少。因此，假设肾皮质基质细胞的一部分是许多肾小管周内皮细胞的前体细胞，并且该内皮细胞群对于肾脏其他谱系的正常构型是必要的。为了验证这一假设，提出了以下目标：目标1：确定肾皮质间质发育为内皮细胞的功能潜力。Foxd1基质将通过流式细胞仪分离，并在体外条件下诱导内皮细胞分化。 目的2：确定Foxd1/Flk1细胞的命运以及这些细胞的缺失对血管发育的影响。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：探讨FoxD1来源的内皮细胞在肾脏形成中的作用。将对Flk1ST-/-小鼠的肾脏谱系进行彻底的组织学、结构和生理评估。这些研究将为肾内皮细胞的起源及其对肾脏发育的贡献提供新的见解。操作这一新的祖细胞池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.