The Role of Aminopeptidase-A in GBM Thickening in Diabetic Nephropathy

描述(由申请人提供)： 糖尿病是慢性肾脏疾病最常见的原因。大约40%的糖尿病患者发展为糖尿病肾病(DN)；在美国，所有终末期肾病病例中近一半是由糖尿病肾病引起的。尽管广泛使用靶向治疗来降低血糖和拮抗肾素-血管紧张素系统，但糖尿病肾病的患病率一直保持稳定。因此，需要对糖尿病肾病进行有效的、针对疾病的治疗。申请人的近期目标是研究糖尿病肾病患者肾小球基底膜(GBM)增厚的发生机制。申请人的长期目标是充分详细地了解糖尿病肾病的发病机制，以开发基于机制的治疗糖尿病肾病的方法。申请者和指导团队已经设计了一个明确和重点突出的职业发展计划，以提供关于细胞-细胞外基质相互作用的新的和增强的培训，包括糖尿病肾病、胰岛素受体信号转导、沟通和演示技能、赠款和手稿写作以及负责任的研究行为。在CDA-2奖的过程中，有一个逐步提高独立性的计划。具体目标的时间表、实验室空间分配、申请人和指导团队之间的预定会议、申请人的期望以及每个导师的具体角色(S)都在职业发展计划中进行了详细的阐述。此外，预计申请者将在前几年提交较小的赠款申请，并在CDA-2奖的最后两年提交独立资助申请(VA Merit奖，NIH R01)。申请人目前正在进行的研究表明，氨基肽酶-A(APA)是一种表达于内脏肾小球上皮细胞(足细胞)的II型跨膜蛋白，可能在糖尿病肾病肾小球基底膜增厚的发病机制中发挥重要作用。初步结果显示，APA基因敲除小鼠的基底膜显著增厚。此外，APA在糖尿病肾病患者和2型糖尿病大鼠模型中表达下调。此外，敲除培养的肾小球上皮细胞中的胰岛素受体会减少APA的表达。在这项提案中，申请者将研究APA下调在调节参与GBM重塑的基因表达中的作用。总体假设是，糖尿病状态下APA下调导致转录因子(ZHX蛋白)移位，这些转录因子迁移到足细胞核，并诱导基质相关基因表达的变化，从而导致基底膜增厚。建议研究的目的是增加我们对导致基底膜增厚和糖尿病肾病发生的关键分子变化的了解。在具体目标1中，我们将研究APA下调对基质相关基因表达的影响。在具体目标2中，将研究APA-ZHX相互作用在GBM增厚发展中的重要性。在特定的目标3中，将研究胰岛素受体缺陷对APA和ZHX蛋白的影响。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Diabetes mellitus is the most common cause of chronic kidney disease. Approximately 40% of diabetic patients develop diabetic nephropathy (DN); and nearly half of all incident cases of end-stage renal disease in the United States are due to DN. Despite widespread use of targeted therapies to lower glucose and to antagonize the renin-angiotensin system, the prevalence of DN has remained stable. Thus, effective and disease-specific therapies for DN are needed. The immediate goal of the applicant is to study the mechanisms underlying the development of glomerular basement membrane (GBM) thickening in DN. The applicant's long- term goal is to understand the pathogenesis of diabetic kidney disease in sufficient detail to develop mechanism-based therapies for DN. A clear and focused career development plan has been designed by the applicant and the mentoring team to provide new and enhanced training in cell-extracellular matrix interactions in DN, insulin receptor signaling, communication and presentation skills, grant and manuscript writing, and responsible conduct in research. There is a plan for progressive increase in independence over the course of the CDA-2 Award. Timelines for the specific aims, laboratory space allocation, scheduled meetings between the applicant and the mentoring team, expectations of the applicant, and the specific role(s) of each mentor are addressed in detail in the career development plan. In addition, the applicant is expected to submit smaller grant proposals in the earlier years and an application for independent funding (VA Merit Award, NIH R01) in the last two years of the CDA-2 Award. Studies currently being conducted by the applicant have demonstrated that aminopeptidase-A (APA), a type II transmembrane protein expressed in visceral glomerular epithelial cells (podocytes), may play an important role in the pathogenesis of GBM thickening in DN. Initial results show that APA knockout mice have significantly thickened GBMs. Moreover, APA is down-regulated in patients with DN and in rat models of type 2 DN. Also, knockdown of the insulin receptor in cultured glomerular epithelial cells reduces the expression of APA. In this proposal, the applicant will study the role of APA downregulation in mediating the expression of genes involved in GBM remodeling. The overall hypothesis is that APA down-regulation in the diabetic state causes displacement of transcriptional factors (ZHX proteins) that migrate into the podocyte nucleus and induce changes in the expression of matrix- related genes, thereby leading to GBM thickening. The goal of the proposed studies is to increase our understanding of key molecular changes that result in the development of GBM thickening and DN. In Specific Aim 1, the effects of APA down-regulation on matrix-related gene expression will be studied. In Specific Aim 2, the importance of APA-ZHX interaction in the development of GBM thickening will be examined. In Specific Aim 3, the effects of insulin receptor deficiency on APA and ZHX proteins will be investigated.