NOVEL GROWTH FACTOR RELEASED BY KIDNEY EPITHELIAL CELLS

这个项目的目标是定义一个人的生理意义 刺激肾上皮细胞生长的新蛋白质 细胞。 我们之前发现了一种蛋白质，这种蛋白质在未转化的猴子身上出现 肾上皮细胞(BSC-1系)暴露3-5分钟 降低了培养介质中的钠浓度；它被称为 低钠生长因子(LSGF)。这种蛋白质有一个明显的分子 体重约6500，刺激猴子和狗肾脏的生长 在培养中的上皮细胞，而不是成纤维细胞，通过 二硫苏糖醇，似乎与已知的类似的生长因子不同 与生物活性和氨基酸组成有关的大小；以及 驻留在细胞外基质中，当细胞外基质 降低了介质中的钠浓度。它的氨基末端 LSGF蛋白被阻断。一种反相提纯LSGF的方法 高效液相色谱仪是在03年度拨款年度开发的。 本修订申请描述了一种新的具有生物活性的亚型 反相高效液相色谱法检测氨基酸组成不同的LSGF 先前描述的蛋白质。此外，一种兔抗血清 LSGF已经准备好，可以识别两种LSGF亚型的ELISA，以及 还中和了每种植物的促生长活性。要定义 LSGF的结构及其发挥生理作用的机制 对体外和体内肾细胞的影响，需要新的试剂 这将用于实现以下具体目标： 1.对LSGF蛋白进行部分测序； 2.用寡核苷酸探针或免疫筛选法克隆 编码该蛋白，并研究其在细胞内表达的调节 增长； 3.建立LSGF免疫分析方法，研究蛋白质的生理学； 4.确定LSGF是否改变了肾上皮细胞的营养物质和离子转运 细胞。 实现这些目标可以定义这种自分泌增长因子的作用 肾脏生理学和病理生理学。确定机制，通过这些机制 LSGF法案可能是朝着我们的长期目标迈出的重要一步 配制一种含有特定成分的治疗性溶液，如 能加速肾脏再生的营养物质和生长因子 受伤后。

The goal of this project is to define the physiological significance of an apparently novel protein that stimulates growth of kidney epithelial cells. We previously identified a protein that appears when nontransformed monkey renal epithelial cells (BSC-1 line) are exposed for 3-5 minutes to a reduced concentration of sodium in the culture medium; it is called the low sodium growth factor (LSGF). This protein has an apparent molecular weight about 6,500, stimulates growth of both monkey and canine kidney epithelial cells in culture but not fibroblasts, resists denaturation by dithiothreitol, appears to differ from known growth factors of similar size with respect to biological activity and amino acid composition, and resides in the extracellular matrix from which it is released when the sodium concentration of the medium is reduced. The amino terminus of the LSGF protein is blocked. A protocol to purify LSGF by reversed-phase (RP) HPLC was developed during the 03 grant year. This revised application describes a new biologically active isoform of LSGF detected by RP HPLC that has a different amino acid composition than the previously described protein. In addition, a rabbit antiserum against LSGF has been prepared that recognizes both LSGF isoforms of ELISA, and also neutralizes the growth-promoting activity of each. To define the structure of LSGF and mechanisms by which it exerts its physiological effects on renal cells in vitro and in vivo, new reagents are required that will be used to achieve the following specific aims: 1. partially sequence the LSGF protein; 2. use oligonucleotide probes or immunoscreening to clone the gene that encodes the protein, and study regulation of its expression during cell growth; 3. develop LSGF immunoassays to study the physiology the protein; 4. determine if LSGF alters nutrient and ion transport in renal epithelial cells. Achieving these aims could define the role of this autocrine growth factor in renal physiology and pathophysiology. Identifying mechanisms by which the LSGF acts may be an important step towards our long-term goal of formulating a therapeutic solution containing specific components such as nutrients and growth factors that could speed regeneration of the kidney after injury.