Targeted Overexpression and Ablation of Renin in Connecting Tubule

描述(由申请人提供)：本试探性/发展性研究建议书(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”而提交的。理论基础：这一建议基于四个关键因素：(1)最近的证据强调了连接小管(CNT)在电解质和液体动态平衡中的功能意义；(2)通过观察CNT中可诱导的肾素表达，我们提出，旁分泌小管肾素-血管紧张素系统(RAS)参与了沿肾单位近端和远端钠重吸收的协调调节；(3)在正常血容量条件下未检测到的CNT肾素mRNA和蛋白可以平行或独立于肾素在肾小球旁小球装置(JGA)显著增加；(4)现在有令人信服的实验证据表明，腔血管紧张素II可以通过远端肾单位对阿米洛利敏感的上皮钠通道影响钠的重吸收。假设：肾素在多个肾单位的汇合点的表达具有战略意义，可以作用于起源于近端肾小管的血管紧张素原，从而调节肾小管A-II的形成和远端肾单位的电解质转运。研究目的。在这个试探性/发展性R21中，我们将进行以下先导研究：(1)开始建立靶向改变CNT肾素表达的小鼠模型(1.1)，通过使用CRE/Lox策略进行同源重组来获得远端肾单位(KING-in，KI)中肾素的靶向过表达的动物，(1.2)通过开发靶向载体和去除CNT肾素表达所需的胚胎干细胞克隆(KINK-OUT，KO)。(2)为了初步确定KI动物的特征，我们将：(2.1)测试和定量KI和对照动物在原位和远端肾单位节段池中对钠摄入量逐步增加的异源和内源性肾素mRNA和蛋白；(2.2)测试钠负荷对KI和对照动物血压、钠排泄、血浆肾素和醛固酮急性和慢性变化的影响；(2.3)使用(2.1)中分离的肾单位节段来测试和定量KI和远端肾单位电解质转运的关键调节因子和影响因子的基因表达。透视。实现这些有限和集中的目标将展示和验证后续R01研究提案的实验模型和协议，该提案涉及该领域的补充专业知识。动物模型和方案将与其他科学家共享。这项工作应该有助于确定CNT肾素在钠平衡、血浆容量和动脉压调节中的生理作用。超过50%的美国人到65岁时患有高血压，或不明原因的高血压，而这些患者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器，但其功能复杂，因为它涉及重叠的循环系统和组织系统。我们已经描绘了沿肾单位工作的管状RAS，肾单位是控制钠重吸收的过滤单位，它包括肾素在控制最终钠排出速率的部位的表达。我们建议研究RAS的限速酶肾素在这一关键位置的表达的遗传操作的后果，以探讨其在调节血浆容量和血压对饮食盐的反应中的意义。这项工作将促进高血压的诊断和治疗。

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.