Meprins-Metalloproteinases of the Kidney and Intestine

描述（由申请人提供）：本研究的总体长期目标是阐明细胞表面金属蛋白酶及其分泌对应物在细胞表面和细胞外被调节、相互作用、激活和降解肽和蛋白质的机制，并确定这些蛋白酶在健康和疾病（如肾脏和泌尿道疾病）中的作用。meprins是一种复杂而独特的哺乳动物金属蛋白酶，在一定条件下大量表达于肾脏和肠上皮细胞的刷状边界膜以及白细胞中。假设meprins在尿路感染的宿主防御中具有保护作用，但α / β形式积极参与急性肾损伤的病理生理。在接下来的阶段，具体目标是：(1)确定meprin α / β是否在缺氧或缺血再灌注时损害肾上皮细胞中的特定蛋白质。免疫组织化学、免疫沉淀和蛋白质组学技术将用于野生型和meprin敲除小鼠的肾脏和近端小管，以确定刷状边界膜、细胞质和细胞外区室中meprin相互作用和水解的特定靶点。(2)确定白细胞是否影响这些炎症细胞对缺血肾的运动和活性，并增强损伤。来自野生型和meprin基因敲除小鼠的骨髓细胞将被辐射破坏，并被来自携带绿色荧光蛋白的野生型或meprin基因敲除小鼠的供体白细胞所取代。通过观察缺血再灌注嵌合小鼠供体白细胞向肾脏的运动，以及meprin阳性和阴性白细胞对肾脏的损伤，可以了解这些细胞的损伤情况。(3)确定肾脏分泌的meprina或白细胞分泌的meprina是否影响尿路疾病的建立和严重程度。将不同meprin基因型小鼠感染膀胱内尿病菌，测定其对膀胱和肾脏感染的易感性。白细胞蛋白和肾蛋白的作用将在嵌合小鼠中进行评估。将评估meprins在感染部位的激活、活性β -防御素的浓度以及meprins同型体与细菌的相互作用，以深入了解保护机制。meprin α和β敲除小鼠的可用性为这些研究提供了独特的资源，对这些金属蛋白酶的基础知识将为肾脏和尿路疾病带来新的概念、治疗和干预。

DESCRIPTION (provided by applicant): The overall long-term objectives of this research are to elucidate mechanisms by which cell surface metalloproteinases and their secreted counterparts are regulated and interact, activate and degrade peptides and proteins at the cell surface and extracellularly, as well as to define the roles of these proteases in health and diseases such as kidney and urinary tract disease. The work has led to the discovery and characterization of meprins, complex and unique mammalian metalloproteinases abundantly expressed at the brush border membrane of kidney and intestinal epithelial cells, and in leukocytes under certain conditions. The hypothesis is that meprins have a protective role in host defense in urinary tract infections, but that the alpha/beta form is actively involved in the pathophysiology of acute renal injury. In the next period, the Specific Aims are to: (1) Determine whether meprin alpha/beta is damaging to specific proteins in kidney epithelial cells in response to hypoxia or ischemia-reperfusion. Immunohistochemical, immunoprecipitation, and proteomic techniques will be used with kidneys and proximal tubules from wild-type and meprin knockout mice to identify specific targets of meprin interaction and hydrolysis in brush border membrane, cytosol and extracellular compartments. (2) Determine whether leukocytic meprins affect movement and activity of these inflammatory cells to ischemic kidney and enhance damage. Bone marrow cells from wild-type and meprin knockout mice will be destroyed by radiation, and replaced by donor leukocytes from wild-type or meprin knockout mice carrying a green- fluorescent protein. The movement of the donor leukocytes to kidneys in the chimeric mice subjected to ischemia-reperfusion, and the injury caused by meprin positive and negative leukocytes will give insight into the damage caused by these cells. (3) Determine whether meprin alpha secreted from the kidney, or leukocytic meprins, affect the establishment and severity of urinary tract disease. Mice of different meprin genotypes will be infected with uropathic bacteria in the bladder, and the susceptibility to bladder and kidney infections will be determined. The roles of leukocytic and kidney meprins will be assessed using the chimeric mice. The activation of meprins at sites of infection, concentrations of active beta-defensins, and interactions of meprin isoforms with bacteria will be assessed to gain insights into mechanisms of protection. The availability of the meprin alpha and beta knockout mice are unique resources for these studies, and fundamental knowledge of these metalloproteinases will lead to new concepts, treatments, and interventions for kidney and urinary tract diseases.