Modulation of renin secretion by renal cortical interstitial calcium

描述(申请人提供)：我们的长期目标是阐明肾素分泌的机制。在体外，细胞外钙(Ca~(2+))的增加通过增加肾小球旁(JG)细胞内的Ca~(2+)和抑制腺苷环化酶V(AC-V)来减少肾素的释放。细胞外钙离子如何减少体内肾素的分泌，以及是什么启动了这些钙离子的变化，目前尚不清楚。我们的假设是，远端NaCI递送增加通过Paracellin-1刺激钙敏感受体(CAR)，抑制AC-V活性，导致肾素分泌减少，从而增加肾皮质间质中的钙离子。目的1：假设：增厚的上肢NaCI转运通过Paracellin-1增加皮质间质[Ca~(2+)]，减少肾素的分泌。我们将使用原位微透析法测量肾皮质间质钙离子的变化，以及肾素分泌率(RSR)的变化，以响应NaCI递送的变化。我们将利用RNA干扰技术，在降低Paracellin-1的表达后，检测间质中的钙离子和RSR。Paracellin-1是一种紧密连接蛋白，负责粗大升支的细胞旁钙重吸收。我们将测量间质钙离子和RSR的变化，以响应用速尿阻断粗大上肢NACI重吸收的反应。目的2：假设：在体内通过皮质间质[Ca~(2+)]刺激CAR可减少肾素的分泌。我们将在使用仿钙剂致敏大鼠或CAR的同时测量RSR。我们将测量由于Paracellin-1基因敲除而导致间质[Ca~(2+)]降低的情况下，RSR到CAR激活的变化。我们将测量使用溶钙剂对CAR脱敏的RSR的变化。我们将在间质[Ca~(2+)]升高和CAR脱敏的情况下测量RSR。目的3：假设：在体内，在皮质间质[Ca~(2+)]降低的情况下，AC-V抑制可减少肾素的分泌。我们将测量基础RSR对AC-V特异性抑制剂NKY80的反应。我们将使用siRNA技术抑制大鼠Paracellin-1的表达，并在AC-V特异性抑制剂NKY80存在的情况下测量间质[Ca~(2+)]和RSR。我们将利用RNA干扰技术在体内抑制AC-V的表达，并检测间质[Ca~(2+)]和RSR对速尿的反应。 肾素-血管紧张素系统(RAS)参与了几乎所有形式的高血压。 抑制RAS是抗高血压治疗的主要手段。了解RAS的功能和调节将有助于开发新的治疗方法来减轻高血压的影响。

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate the mechanisms of renin secretion. Increased extracellular calcium (Ca2+) decreases renin release in vitro by increasing juxtaglomerular (JG) cell intracellular Ca2+ and inhibiting adenylyl cyclase V (AC-V). How extracellular Ca2+ decreases rennin secretion in vivo and what initiates these changes in Ca2+ is unknown. Our hypothesis is that increased distal NaCI delivery increases Ca2+ in the renal cortical interstitium via paracellin-1 which stimulates the calcium-sensing receptor (CaR), inhibiting AC-V activity, leading to a decrease in renin secretion. AIM 1: Hypothesis: Increasing thick ascending limb NaCI transport increases cortical interstitial [Ca2+] via paracellin-1 and decreases renin secretion. We will measure changes in renal cortical interstitial Ca2+ using in situ microdialysis and changes in the renin secretory rate (RSR) in response to changes in NaCI delivery. We will measure interstitial Ca2+ and RSR after decreasing the expression of paracellin-1, a tight-junction protein responsible for paracellular Ca2+ reabsorption in the thick ascending limb, using RNA interference. We will measure changes in interstitial Ca2+ and RSR in response to the blockade of thick ascending limb NaCI reabsorption with furosemide. AIM 2: Hypothesis: In vivo stimulation of the CaR via cortical interstitial [Ca2+] decreases renin secretion. We will measure RSR while using calcimimetics to sensitize or the CaR in rats. We will measure changes in RSR to CaR activation in the presence of decreased interstitial [Ca2+] due to paracellin-1 knockdown. We will measure changes in RSR to CaR desensitization with calcilytics. We will measure RSR in the presence of increased interstitial [Ca2+] and CaR desensitization with calcilytics. AIM 3: Hypothesis: In vivo AC-V inhibition decreases renin secretion in the presence of decreased cortical interstitial [Ca2+]. We will measure basal RSR in response to the AC-V specific inhibitor NKY80. We will knockdown paracellin-1 expression in rats using siRNA technology and will measure interstitial [Ca2+] and RSR in the presence of the AC-V specific inhibitor, NKY80. We will knockdown AC-V expression in vivo using RNA interference, and measure interstitial [Ca2+] and RSR in response to furosemide. The renin-angiotensin system (RAS) is involved in almost every form of hypertension. Inhibition of the RAS is a mainstay of anti-hypertensive therapy. Understanding the function and regulation of the RAS will allow for the development of novel therapeutics to lessen the impact of high blood pressure.