Meprins-Metalloproteinases of the Kidney and Intestine

描述(由申请人提供)：这项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的同工酶受到调控、相互作用、激活和降解细胞表面和细胞外的多肽和蛋白质的机制，并确定这些酶在健康和肾脏和尿路疾病等疾病中的作用。这项工作导致了大量、复杂和独特的哺乳动物金属蛋白酶的发现和鉴定，这些金属蛋白在肾脏和肠道上皮细胞的刷状缘膜上大量表达，在一定条件下在白细胞中也有大量表达。该假说认为meprint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学。下一阶段的具体目标是：(1)确定Meprinα/β在低氧或缺血再灌注时是否对肾上皮细胞中的特定蛋白质造成损伤。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基因敲除小鼠的肾脏和近端小管，以确定刷状缘膜、胞浆和细胞外隔间中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性细胞对缺血肾脏的运动和活性，从而加重损伤。野生型和meprin基因敲除小鼠的骨髓细胞将被辐射破坏，取而代之的是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。供体白细胞在缺血再灌注嵌合小鼠肾脏中的运动，以及梅普林阳性和阴性白细胞所造成的损伤，将有助于深入了解这些细胞所造成的损害。(3)确定肾脏或白细胞分泌的甲肾上腺素是否影响尿路疾病的建立和严重程度。不同梅普林基因型鼠将感染膀胱中的泌尿系病原菌，并将测定其对膀胱和肾脏感染的敏感性。将使用嵌合小鼠评估白细胞和肾小球的作用。将对感染部位meprin的激活、活性β-防御素的浓度以及meprin亚型与细菌的相互作用进行评估，以深入了解保护机制。Meprinα和β基因敲除小鼠的可用性是这些研究的独特资源，这些金属蛋白酶的基础知识将导致肾脏和尿路疾病的新概念、治疗方法和干预措施。

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.