A Uroguanylin Based ELISA for Salt Sensitivity

基于尿鸟苷蛋白的盐敏感性 ELISA

• 批准号: 6882465
• 负责人: STEPHEN L CARRITHERS
• 金额: $ 21.34万
• 依托单位: SEQUELA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6882465
• 关键词: analytical chemistry; clearance rate; diagnosis design /evaluation; diagnostic tests; dietary sodium; enzyme linked immunosorbent assay; gastrointestinal hormones; guanylate cyclase; high performance liquid chromatography; intravenous administration; kidney function; laboratory mouse; monoclonal antibody; oral administration; peptide hormone; sodium chloride; technology /technique development; western blottings

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.

描述：本提案是为了响应国家糖尿病、消化和肾脏疾病研究所的计划公告 PA-03-123“盐敏感性诊断筛查测试 (SBIR/STTR) 的开发”而提交。我们实验室的总体假设是，部分通过肠肾内分泌轴维持 NaCl 稳态，其中尿鸟苷素 (UGN) 和鸟苷素 (GN) 快速调节肾脏 NaCl 排泄，以改变 NaCl 摄入量，并且这些肠源性肽可以调节急性肾功能以响应口服氯化钠负荷。在人类和实验动物中都已证实，口服给予的氯化钠负荷比静脉内给予的等量盐负荷可以更快地排泄（在四小时内），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 参与并促成了这一应对措施。然而，定量和常规测定生物体液（即血清、尿液）中 UGN、GN 及其激素原水平的方法尚未开发出来。因此，缺乏定量评估这些肽所需的试剂限制了我们对其在盐敏感性高血压、充血性心力衰竭和水肿等病理状况中的作用的了解。为了建立原理证明，我们将在目标 1 中开发 UGN 单克隆抗体，用于鼠 UGN 的定量捕获 ELISA。在目标 2 中，我们将在我们实验室开发的小鼠模型中验证该 ELISA，其中将在（1）静脉注射 UGN 和（2）注射之前和之后测量肾反应以及 UGN 的血清和尿液水平。胃（口服）盐负荷； UGN 将通过 ELISA 进行测量并通过 HPLC 和蛋白质印迹进行确认。我们预测，这种定量 ELISA 的开发将使我们能够确定鸟苷酸肽在对口服 NaCl 负荷的急性肾脏反应中的作用。