Renin-expressing Cell Lines for Hypertension Research

描述(申请人提供)：在正常成人肾脏中，肾素表达的主要部位是肾小球旁细胞，肾素表达与血压和电解质平衡的调节有关。在肾脏器官发生过程中，肾素的大量表达与肾动脉系统的发育有关，肾素-血管紧张素系统(RAS)信号的遗传或药物干扰与血管系统的畸形有关。为了研究肾素基因表达模式的转录调控机制，通过转基因靶向致瘤获得了肾素表达细胞系(As4.1)，并将其用于瞬时转染实验，以确定表达所需的启动子中的顺式作用位点。在肾素启动子的控制下表达绿色荧光蛋白(GFP)的转基因报告系已经被开发出来，并被用于验证体内已识别的位点的功能。GFP报告系也被FACS用于纯化肾脏个体发育不同阶段的天然肾素表达细胞，以利用Affymetrix和MPSS平台进行表达谱分析。这些研究已经鉴定了转录因子家族的特定成员，这些转录因子家族构成了肾素启动子顺式调控位点上相互作用的反式作用因子，并暗示肾素是Notch调控的直接下游靶点。该图谱还允许识别其他基因的光谱，这些基因的产物在分离的细胞中与肾素特异地共富集。这种表达模式表明，早期肾脏发育的肾素表达细胞对应于激活的血管周细胞，该细胞参与与内皮细胞和其他细胞的沟通，从而影响动脉血管的发育。周细胞是一种具有相当发育可塑性的壁细胞，在血管生成、平滑肌分化和血管重塑中发挥重要作用。它们的失调与糖尿病微血管病变、高血压和血管组织的不适当钙化有关。在目标1和目标2下，提出了一个优先计划，通过开发完成肾素启动子调节的主要顺式和反式作用成分的描述和体内验证。在目标3下，将开发和分析一组转基因报告菌株，这将使肾素表达细胞的谱系追踪和肾素表达细胞或其后代在正常发育过程中的不同阶段和对病理生理扰动的反应的表达谱。这些研究将评估转录因子谱系的变化如何与肾素表达的变化相关。

DESCRIPTION (provided by applicant): In normal adult kidney the primary site of renin expression is the juxtaglomerular cell and renin expression there is associated with regulation of blood pressure and electrolyte balance. During renal organogenesis abundant renin expression is found transiently in association with the developing renal arterial system, and genetic or pharmacological disruption of renin-angiotensin system (RAS) signaling is associated with malformations of the vasculature. To study the mechanisms of transcriptional regulation governing the expression pattern of the renin gene, a renin-expressing cell line (As4.1) has been produced by transgene-targeted tumorigenesis, and is being used in transient transfection assays to define the cis-acting sites in the promoter that are required for expression. Transgenic reporter lines expressing green fluorescent protein (GFP) under control of the renin promoter have been developed and are being used to verify the functionality of the identified sites in vivo. The GFP reporter lines are also being used to purify native renin-expressing cells from kidneys at different stages of renal ontogeny by FACS for expression-profiling utilizing the Affymetrix and MPSS platforms. The profiling studies have allowed identification of specific members of transcription factor families that constitute the trans-acting factors interacting at cis regulatory sites in the renin promoter, and have implicated renin as the immediate downstream target of Notch regulation. The profiling has also permitted identification of a spectrum of other genes whose products specifically co-enrich with renin in the isolated cells. The pattern of expression suggests that the renin-expressing cell of early renal development corresponds to an activated vascular pericyte which is instructively engaged in communicating with endothelial and other cells to effect development of the arterial vasculature. Pericytes are mural cells that exhibit considerable developmental plasticity and are recognized to play an important role in angiogenesis, smooth muscle differentiation, and vascular remodeling. Their dysregulation has been implicated in diabetic microangiopathies, hypertension and inappropriate calcification of vascular tissue. Under Aims 1 and 2, a prioritized plan to complete delineation and in vivo verification of major cis and trans- acting components of renin promoter regulation through development is proposed. Under Aim 3 a set of transgenic reporter strains will be developed and analyzed which will permit lineage tracing of the renin-expressing cell and expression-profiling of renin-expressing cells or their descendents at various stages during normal development and in response to pathophysiological perturbations. These studies will permit assessment of how changes in the transcription factor repertoire correlate with changes in renin expression.