INTER ALPHA TRYPSIN INHIBITOR IN CAOX UROLITHIASIS

间α胰蛋白酶抑制剂治疗 CAOX 尿石症

• 批准号: 2906404
• 负责人: SUSAN RUTH MARENGO
• 金额: $ 16.61万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-29 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906404
• 关键词: affinity chromatography; androgens; clinical research; crystallization; diagnosis design /evaluation; disease /disorder model; enzyme linked immunosorbent assay; gender difference; hormone regulation /control mechanism; laboratory rat; method development; northern blottings; nuclear magnetic resonance spectroscopy; pathologic process; polymers; trypsin inhibitors; urinalysis; urinary calculi; western blottings

Using samples from 101 volunteers and Western blot analysis of spot and 24 hr urine specimens, we have demonstrated that males who are confirmed active calcium oxalate (CaOx) stone formers (SF) typically expressed inter-alpha-trypsin inhibitor-trimer (IaTI-trimer) whereas age and ethnically matched normal (NRM) males expressed detectable levels of IaTI-trimer much less frequently (86 percent versus 23 percent; P is less than 0.0001). It is unlikely that the increased levels of IaTI- trimer in CaOx SF males are actively causing the episode of CaOx disease, because in females, who have a 2-3 fold lower incidence of CaOx disease than do males, the incidence of IaTI-trimer expression is similar in CaOx SF versus NRM females (94 percent versus 77 percent; P= 0.1938). IaTI-trimer has been shown to inhibit CaOx crystallization in vitro and has been recovered from the organic matrix of CaOx stones. It could be hypothesized that the increased expression of IaTI-trimer is in response to the damage caused by CaOx disease. In Specific Aim 1, Western blot analysis will be utilized to determine if the expression of urinary IaTI-trimer is linked to active CaOx stone disease in males. In Experiment 1, the expression of urinary IaTI-trimer will be determined in urine samples from males: NRM, clinically active CaOx SF, and CaOx who have not had an episode in more than two years. Inactive statues will be confirmed by KUB. In Experiment 2, active male CaOx SF will be followed for 3 years from the time of clinical CaOx episode to determine if the incidence of urinary IaTi-trimer decreases during periods of inactive CaOx disease. In Specific Aim 2, IaTI-trimer will be isolated from urine using gel and trypsin affinity chromatography. The isolated protein will be used as a standard to develop an ELISA to allow quantification of urinary IaTI-trimer. In Specific Aim 3, the ELISA will be utilized to determine if IaTI-trimer is negatively regulated by androgens as the preliminary data suggests. Levels of urinary IaTI-trimer in males undergoing androgen deprivation for prostatic cancer, will be compared with those of age and ethnically matched NRM and CaOX SF. Lastly, a rat urolithiasis model (0.75 percent ethylene glycol and AIN-76 diet) will be utilized for formal hormonal deprivation/replacement studies. The rat model will also be utilized to determine if induction of urolithiasis increases the expression of IaTI- trimer systematically (i.e. the liver the primary source of serum IaTI- trimer), or locally (kidney) as judged by Northern blot analysis of RNA message for each of the IaTI-subunits and by Western blot/ELISA of the protein.

使用来自101名志愿者的样品和Western印迹分析斑点和斑点的蛋白质， 24小时尿液标本，我们已经证明，男性谁是确诊 活性草酸钙（CaOx）结石形成者（SF）通常表达 间-α-胰蛋白酶转运蛋白-三聚体（IaTI-三聚体），而年龄和 种族匹配的正常（NRM）男性表达可检测水平的 IaTI三聚体的频率要低得多（86%对23%; P <0.05）。 小于0.0001）。不太可能增加IaTI水平- CaOx SF雄性中的三聚体积极地引起CaOx发作 因为在女性中，CaOx的发病率低2-3倍 疾病比男性，IaTI-三聚体表达的发生率是 CaOx SF与NRM女性相似（94%与77%; P= 0.1938）。 IaTI-三聚体已经显示出抑制CaOx结晶， 体外，并已从CaOx结石的有机基质中回收。 可以假设，IaTI-三聚体的表达增加， 是对CaOx病造成的损害的反应。 具体目标 1、蛋白质印迹分析将用于确定表达是否 尿IaTI-三聚体与男性活动性CaOx结石病有关。 在实验1中，尿IaTI-三聚体的表达将是 在男性尿样中测定：NRM，临床活性CaOx SF， 和CaOx，他们已经两年多没有发作了。 非活动 雕像将由KUB确认。 在实验2中，活性雄性CaOx SF 将从临床CaOx发作至 确定尿IaTi-三聚体的发生率是否在 非活动性CaOx疾病的时期。在具体目标2中，IaTI-三聚体将 使用凝胶和胰蛋白酶亲和层析从尿液中分离。 分离的蛋白质将用作标准品以开发ELISA， 允许定量尿IaTI-三聚体。 在Aim Specific 3中， ELISA将用于确定IaTI-三聚体是否为阴性。 由雄激素调节。 水平 接受雄激素剥夺的男性尿IaTI-三聚体 前列腺癌，将与年龄和种族 匹配NRM和CaOX SF。 最后，大鼠尿石症模型（0.75% 乙二醇和AIN-76饮食）将用于正式的激素 剥夺/替代研究。大鼠模型也将用于 确定尿石症的诱导是否增加IaTI的表达， 三聚体系统性地（即肝脏是血清IaTI的主要来源， 三聚体）或局部（肾），如通过RNA的北方印迹分析所判断的 每个IaTI-亚基的信息，并通过蛋白质印迹/ELISA检测IaTI-亚基的表达。 蛋白