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: 6381496
• 负责人: SUSAN RUTH MARENGO
• 金额: $ 15.25万
• 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/6381496
• 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.

利用101名志愿者的样本和斑点和蛋白质印迹分析 24小时尿液样本，我们已经证明了被确认的男性 活性草酸钙(CaOx)结石形成物(SF)通常表达 α-胰蛋白酶抑制因子-三聚体(IATI-Trimer)而AGE和 种族匹配正常(NRM)男性表达可检测到的水平 IATI-Trimer频率要低得多(86%对23%；P 不到0.0001)。IATI水平的增加不太可能- CaOx SF男性中的三聚体是导致CaOx发病的积极因素 疾病，因为在女性中，CaOx的发病率降低了2-3倍 疾病的发病率高于男性，IATI-三聚体表达的发生率为 CaOx SF与NRM女性相似(94%对77%；P= 0.1938)。IATI-三聚体已被证明抑制CaOx在 在体外，已从CaOx结石的有机基质中恢复。 可以推测，IATI-Trimer的表达增加 是为了应对CaOx病造成的损害。以特定的目标 1，利用Western印迹分析来确定是否表达 在男性中，尿液IATI-三聚体的增加与活动性CaOx结石有关。 在实验1中，尿IATI-三聚体的表达将是 从男性尿样中检测出：NRM，临床上活跃的CaOx SF， 和CaOx，他们已经两年多没有发作过一次了。非活动 雕像将由KUB确认。在实验2中，活跃的雄性CaOx SF 将从临床CaOx发作时起随访3年至 确定尿液IATI-三聚体的发生率是否在 不活跃的CaOx病时期。在具体目标2中，IATI-Trimer将 用凝胶和胰酶亲和层析从尿液中分离得到。 分离出的蛋白将作为标准建立一种用于检测 允许定量尿液IATI-三聚体。在具体目标3中， 将使用ELISA法来确定IATI-Trimer是否为阴性 正如初步数据显示的那样，受雄激素的调节。级别 男性去雄激素治疗后尿IATI-三聚体的变化 前列腺癌，将与年龄和种族的前列腺癌进行比较 匹配NRM和CaOX SF。最后，建立大鼠尿石症模型(0.75% 乙二醇和AIN-76饮食)将用于正式激素 剥夺/替代研究。大鼠模型也将被用来 确定诱导尿石症是否会增加IATI-1的表达 三聚体系统(即肝脏是血清IATI的主要来源- 三聚体)，或通过RNA的Northern印迹分析判断为局部(肾脏) 每个IATI亚基的信息和通过Western印迹/ELISA法检测 蛋白。