Targeted Overexpression and Ablation of Renin in Connecting Tubule

连接小管中肾素的靶向过度表达和消除

• 批准号: 7393119
• 负责人: JEAN-MARC LALOUEL
• 金额: $ 18.31万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7393119
• 关键词: Ablation; Acute; Affect; Age-Years; Aldosterone; Amiloride; Angiotensin II; Angiotensinogen; Animal Model; Animals; Blood Plasma Volume; Blood Pressure; Cells; Chronic; Complex; Condition; Control Animal; Development; Diagnosis; Distal; Electrolytes; Elements; Enzymes; Equilibrium; Excretory function; Experimental Models; Future; Gene Expression; Generations; Genes; High Blood Pressure; Homeostasis; Hypertension; In Situ; Infusion procedures; Intake; Juxtaglomerular Apparatus; Kidney; Knock-in Mouse; Knock-out; Liquid substance; Location; Mediating; Messenger RNA; Modeling; NIH Program Announcements; National Institute of Diabetes and Digestive and Kidney Diseases; Nephrons; Patients; Physiological; Pilot Projects; Plasma; Population; Positioning Attribute; Potassium; Procedures; Protein Overexpression; Proteins; Protocols documentation; RNA; Rate; Regulation; Renin; Renin-Angiotensin System; Research; Research Proposals; Rest; Role; Scientist; Site; Sodium; Sodium Channel; Sodium Chloride; Sorting - Cell Movement; Stem cells; Stress; System; Testing; Time; Tissues; Tubular formation; United States; Urine; Water; Work; blood pressure regulation; epithelial Na+ channel; epithelial amiloride-sensitive sodium channel; genetic manipulation; homologous recombination; hypertension treatment; mouse model; novel; paracrine; pressure; programs; response; vector

DESCRIPTION (provided by applicant): This Exploratory/Developmental Research Proposal (R21) is submitted in response to the NIDDK Program Announcement PA-06-148 "Pilot and Feasibility Program Related to the Kidney". Rationale: This proposal rests on four key elements: (1) Recent evidence stresses the functional significance of the connecting tubule (CNT) in electrolyte and fluid homeostasis; (2) With the observation of inducible renin expression in CNT, we have proposed that a paracrine tubular renin-angiotensin system (RAS) participates in the coordinated regulation of sodium reabsorption at proximal and distal sites along the nephron; (3) CNT renin mRNA and protein, not detected under euvolemic conditions, can be markedly increased in parallel or independently of renin at the juxtaglomerular apparatus (JGA); (4) There is now convincing experimental evidence that luminal angiotensin II can affect sodium reabsorption through the amiloride-sensitive epithelial sodium channel in distal nephron. Hypothesis: Renin expression at the merging points of multiple nephrons is strategically positioned to act on angiotensinogen originating in proximal tubule and thereby to modulate tubular A-II formation and electrolyte transactions in distal nephron. Research Aims. In this Exploratory/Developmental R21, we will perform the following pilot studies: (1) To begin the development of mouse models with targeted alterations of CNT renin expression (1.1) by generating an animal with targeted overexpression of renin in distal nephron (knock-in, KI) by homologous recombination using a Cre/Lox strategy and (1.2) by developing the targeting vectors and the embryonal stem cell clones necessary to ablate expression of renin in CNT (knock-out, KO). (2) To perform preliminary characterization of the KI animal, we will: (2.1) Test and quantitate heterologous and endogenous renin mRNA and protein in KI and control animals in response to a step increase in sodium intake, both in situ and in pools of segments of distal nephron; (2.2) Test the effects of sodium loading on acute and chronic changes in blood pressure, sodium excretion, plasma renin and aldosterone in KI and control animals; (2.3) Test and quantitate gene expression of key regulators and effectors of electrolyte transport in distal nephron using nephron segments isolated in (2.1). Perspective. Achieving these limited and focused objectives will demonstrate and validate experimental models and protocols for a subsequent R01 research proposal involving complementary expertise in the field. Animal models and protocols will be shared with other scientists. The work should help defining the physiological role of CNT renin in sodium balance, plasma volume and arterial pressure regulation. Summary Over 50% of the population of the United States have hypertension, or high blood pressure of unknown cause, by the time they reach 65 years of age, and only a fraction of these patients have their blood pressure under control. The renin-angiotensin system (RAS) is a major controller of blood pressure and of its relationship to dietary salt, but its function is complex because it involves overlapping circulating and tissue systems. We have delineated a tubular RAS operating along the nephron, the filtering unit of the kidney that controls sodium reabsorption, that includes renin expression at a site that controls the final rate of sodium excretion. We propose to investigate the consequences of genetic manipulations of the expression renin, the rate limiting enzyme of the RAS, at this strategic site to probe its significance in the regulation of plasma volume and blood pressure in response to dietary salt. The work should advance diagnosis and treatment of hypertension.

描述（由申请人提供）：本探索性/发展性研究提案（R21）是根据NIDDK计划公告PA-06-148“与肾脏相关的试点和可行性计划”提交的。理论基础：该建议基于四个关键要素：(1)最近的证据强调了连接管（CNT）在电解质和流体稳态中的功能意义；(2)通过观察CNT中可诱导的肾素表达，我们提出旁分泌肾素-血管紧张素系统（RAS）参与沿肾元近端和远端钠重吸收的协调调节；(3) CNT肾素mRNA和蛋白在大容量条件下未被检测到，可在肾小球旁器（JGA）与肾素平行或独立显著增加；(4)目前有令人信服的实验证据表明，管腔血管紧张素II可以通过远端肾元阿米洛利敏感上皮钠通道影响钠的重吸收。假设：肾素在多个肾单位汇合点的表达被策略性地定位于作用于起源于近端小管的血管紧张素原，从而调节远端肾单位的小管A-II的形成和电解质交易。研究目标。在这个探索性/发展性R21中，我们将进行以下试点研究：(1)通过使用Cre/Lox策略的同源重组产生远端肾素靶向过表达的动物（敲入，KI），开始开发具有靶向改变CNT肾素表达的小鼠模型（1.1），（1.2）通过开发靶向载体和胚胎干细胞克隆来消除CNT中肾素的表达（敲出，KO）。(2)为了对KI动物进行初步表征，我们将：（2.1）检测和定量KI动物和对照动物体内的外源和内源肾素mRNA和蛋白，以应对钠摄入量的逐步增加，包括原位和远端肾元段池；（2.2）检测钠负荷对KI和对照动物血压、钠排泄、血浆肾素和醛固酮急慢性变化的影响；（2.3）使用（2.1）中分离的肾元片段检测和定量远端肾元电解质转运关键调控因子和效应因子的基因表达。视角。实现这些有限而集中的目标，将为随后的R01研究计划展示和验证实验模型和协议，其中包括该领域的互补专业知识。动物模型和方案将与其他科学家共享。这项工作将有助于确定碳纳米管肾素在钠平衡、血浆容量和动脉压调节中的生理作用。在美国，超过50%的人在65岁时患有高血压或原因不明的高血压，其中只有一小部分人的血压得到了控制。肾素-血管紧张素系统（RAS）是血压及其与饮食盐关系的主要控制者，但其功能复杂，因为它涉及重叠的循环系统和组织系统。我们已经描绘了一个管状RAS沿着肾元运行，肾元是肾脏的过滤单元，控制钠的重吸收，包括肾素在控制钠的最终排泄速率的部位的表达。我们建议研究基因操纵RAS的限速酶肾素（renin）在这一战略位点的表达的后果，以探讨其在饮食盐对血浆容量和血压的调节中的意义。这项工作将促进高血压的诊断和治疗。