Renin-expressing Cell Lines for Hypertension Research

描述（由申请人提供）：在正常成人肾脏中，肾素表达的主要部位是肾小球细胞，那里的肾素表达与血压和电解质平衡的调节有关。在肾器官发生过程中，发现大量的肾素表达与发育中的肾动脉系统短暂相关，并且肾素-血管紧张素系统（RAS）信号传导的遗传或药理学破坏与脉管系统的畸形相关。为了研究调节肾素基因表达模式的转录调控机制，已通过转基因靶向肿瘤发生产生了一种表达肾素的细胞系（As4.1），并用于瞬时转染试验，以确定表达所需的启动子中的顺式作用位点。已经开发了在肾素启动子控制下表达绿色荧光蛋白（GFP）的转基因报告细胞系，并用于验证体内鉴定位点的功能。GFP报告细胞系也被用于通过FACS从肾个体发育的不同阶段的肾中纯化天然的表达肾素的细胞，用于利用Affytek和MPSS平台进行表达谱分析。分析研究已经允许识别转录因子家族的特定成员，这些转录因子家族构成在肾素启动子中的顺式调节位点相互作用的反式作用因子，并且已经暗示肾素是Notch调节的直接下游靶点。分析还允许鉴定一系列其他基因，其产物在分离的细胞中特异性地与肾素共富集。表达模式表明，早期肾发育的表达肾素的细胞对应于活化的血管周细胞，其主动参与与内皮细胞和其他细胞的通讯以影响动脉血管系统的发育。周细胞是具有相当发育可塑性的壁细胞，被认为在血管生成、平滑肌分化和血管重塑中起重要作用。它们的失调与糖尿病微血管病、高血压和血管组织的不适当钙化有关。在目标1和2下，提出了通过开发完成肾素启动子调控的主要顺式和反式作用组分的描述和体内验证的优先计划。在目标3下，将开发和分析一组转基因报告菌株，其将允许在正常发育期间的不同阶段和响应于病理生理学扰动的肾素表达细胞的谱系追踪和肾素表达细胞或其后代的表达谱。这些研究将允许评估如何在转录因子库的变化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.