A Uroguanylin Based ELISA for Salt Sensitivity

产品说明：本提案是根据国家糖尿病、消化和肾脏疾病研究所PA-03-123“盐敏感性诊断筛查试验（SBIR/STTR）的开发”项目公告提交的。我们实验室的总体假设是这样的原则：氯化钠稳态部分由肠-肾内分泌轴维持，其中肾脏氯化钠排泄受到尿鸟苷素（UGN）和鸟苷肽（GN）的快速调节，以改变氯化钠摄入量，并且这些肠来源的肽可以响应口服氯化钠负荷而调节急性肾功能。在人类和实验动物中均已充分确立，口服施用的NaCl负荷可以比静脉内施用的等效盐负荷更快地（在4小时内）排出，这表明肠因子可能能够急性调节肾功能。来自该实验室和其他实验室的证据表明，UGN和GN参与并促成了这种反应。然而，定量和常规测定生物体液中UGN、GN及其激素原水平的方法（即，血清、尿液）尚未开发。因此，缺乏定量评估这些肽所需的试剂限制了我们对它们在病理条件如盐敏感性高血压、充血性心力衰竭和水肿中的作用的了解。为了建立原理证明，我们将在目标1中开发UGN的单克隆抗体，用于鼠UGN的定量捕获ELISA。在目的2中，我们将在我们实验室开发的小鼠模型中验证该ELISA，其中在（1）静脉内给予UGN和（2）胃内（口服）盐负荷之前和之后测量肾反应以及UGN的血清和尿液水平;通过ELISA测量UGN，并通过HPLC和蛋白质印迹法确认。我们预测，这种定量ELISA的发展将使我们能够确定鸟苷素肽在口服NaCl负荷的急性肾反应中的作用。

DESCRIPTION: This proposal is submitted in response to the program announcement PA-03-123 "Development of diagnostic screening test for salt sensitivity (SBIR/STTR)" from the National Institute of Diabetes and Digestive and Kidney Diseases. The overall hypothesis of our laboratory is the principle that NaCl homeostasis is maintained, in part, by an entero-renal endocrine axis in which renal NaCI excretion is rapidly regulated by uroguanylin (UGN) and guanytin (GN) to altered NaCI intake AND that these intestinally-derived peptides can fhodulate acute renal function in response to oral NaCi loads. It is well established in both humans and in experimental animals that orally-administered NaCI loads can be excreted more rapidly (within four hours) than equivalent salt loads administered intravenously, suggesting that an enteric factor may be capable of acute-regulation of renal function. Evidence from this and other laboratories suggests that UGN and GN are involved in and contribute to this response. However, methods to quantitatively and routinely determine the levels of UGN, GN, and their pro-hormones in biological fluids (i.e., serum, urine) have not been developed. Thus, lack of the reagents required for the quantitative assessment of these peptides has limited our knowledge of their role in pathologic conditions such as saltsensitive hypertension, congestive heart failure, and edema. To establish proof of principle, we will, in Aim 1, develop monoclonal antibodies to UGN to be used in a quantitative capture ELISA for murine UGN. In Aim 2, we will validate this ELISA in a mouse model developed in our laboratory in which the renal response and the serum and urinary levels of UGN will be measured prior to and after (1) intravenous doses of UGN and (2) intra-gastric (oral) salt loading; UGN wiH be measured by ELISA and confirmed by HPLC and Western blotting. We predict that the development of this quantitative ELISA will allow us to determme the rote of guanylin peptides in the acute renal response to oral NaCI loading.