Modulation of renin secretion by renal cortical interstitial calcium

描述（由申请人提供）：我们的长期目标是阐明肾素分泌的机制。增加的细胞外钙（Ca2+）通过增加肾小球旁（JG）细胞内Ca2+和抑制腺苷酸环化酶V （AC-V）来减少体外肾素释放。细胞外Ca2+如何在体内减少肾素分泌，以及是什么引发了Ca2+的这些变化尚不清楚。我们的假设是，增加远端NaCI递送增加Ca2+在肾皮质间质通过paracellin-1，刺激钙感应受体（CaR），抑制AC-V活性，导致肾素分泌减少。AIM 1：假设：增厚升肢NaCI转运通过paracellin-1增加皮质间质[Ca2+]并减少肾素分泌。我们将使用原位微透析测量肾皮质间质Ca2+的变化，以及肾素分泌率（RSR）随NaCI递送变化的变化。我们将使用RNA干扰降低paracellin-1的表达后测量间质Ca2+和RSR， paracellin-1是一种紧密连接蛋白，负责厚升肢的细胞旁Ca2+重吸收。我们将测量间质Ca2+和RSR的变化，以响应用速尿阻断厚升肢NaCI重吸收。目的2：假设：体内通过皮质间质[Ca2+]刺激CaR可减少肾素分泌。我们将测量RSR，同时使用钙化剂对大鼠进行CaR致敏。我们将测量由于paracellin-1敲低导致间质[Ca2+]减少的情况下，RSR对CaR激活的变化。我们将用解解剂测量CaR脱敏后RSR的变化。我们将在钙溶剂增加间质[Ca2+]和CaR脱敏的情况下测量RSR。AIM 3：假设：在皮质间质[Ca2+]减少的情况下，体内AC-V抑制会降低肾素分泌。我们将测量对AC-V特异性抑制剂NKY80的基础RSR。我们将使用siRNA技术降低大鼠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[Ca2+]和RSR。我们将使用RNA干扰来抑制体内AC-V的表达，并测量间质[Ca2+]和RSR对速尿的反应。

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.