A Uroguanylin Based ELISA for Salt Sensitivity

描述：本提案是针对国家糖尿病、消化和肾脏疾病研究所的PA-03-123“盐敏感性诊断筛查试验(SBIR/STTR)的发展”的计划公告而提出的。我们实验室的总体假设是，部分地通过肠-肾内分泌轴维持氯化钠的动态平衡，在该轴上，尿鸟苷(UGN)和鸟苷(GN)快速调节肾脏NaCI的排泄，以改变NaCI的摄入量，并且这些肠源性多肽可以调节急性肾功能，以响应口服NaCI负荷。在人类和实验动物中，口服的NaCI负荷比静脉注射同等的盐负荷排泄更快(在4小时内)，这一点在人类和实验动物中都得到了很好的证实，这表明肠道因素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参与并促进了这一反应。然而，定量和常规测定生物液(即血清、尿液)中UGN、GN及其前激素水平的方法尚未开发出来。因此，缺乏定量评估这些多肽所需的试剂限制了我们对它们在盐敏性高血压、充血性心力衰竭和水肿等病理条件中的作用的了解。为了建立原理证明，在目标1中，我们将开发抗UGN的单抗，用于定量捕获小鼠UGN的ELISA。在目标2中，我们将在我们实验室建立的小鼠模型上验证该ELISA，在该模型中，将在(1)静脉注射UGN和(2)胃(口服)盐负荷前后测量肾脏反应以及血清和尿液UGN水平；UGN将通过ELISA法测定并通过高效液相和Western blotting确认。我们预测，这种定量ELISA法的发展将使我们能够确定鸟苷肽在口服NaCI负荷的急性肾脏反应中的比例。

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.