PATTERNS OF MATRIX SYNTHESIS IN GLOMERULAR SCLEROSIS

受最终赞助商热情的影响，申请者 在分子生物学方面产生了越来越大的兴趣 在他两年的临床肾病研究员期间，他的肾小球疾病。 在这次临床培训结束时，申请者开始 研究性培训，希望发展出一门多产的学术 职业生涯。在发起人的指导和监督下，申请人 把他实验室培训的头两年用来开发 一种允许定位RNA的免疫组织化学方法 转录到单个细胞，以前的分辨率级别 得不到。随后，我们使用这项新技术来识别 特定类型的肾小球细胞表达肾小球基质(ECM)和 基底膜(GBM)蛋白mRNAs，从而定义细胞 这些蛋白质的来源和演示，与之前相反 认为肾小球基底膜合成是不对称的 其单个成分的细胞来源是多样化的。这 新的信息为进一步研究比较提供了框架 正常的基底膜代谢到参与疾病的过程 模特们。 大鼠5/6肾切除模型的初步研究 肾小球硬化揭示了细胞合成模式 发生在NTX出现类似于在 肾小球胚胎发生。这些发现为研究提供了依据 以下假设：1)初始肾小球适应性反应 至5/6 NTX代表ECM/GBM协调回归胎儿模式 2)5/6NTX模型的后期或硬化期 代表一种不适应、不协调的ECM/GBM合成模式；以及 3)这涉及ECM/GBM蛋白类型的细胞模式的变化 合成或其相应的降解酶。 申请者在研究期间和之后的长期研究目标 奖励期涉及对以下机制的更好理解 控制肾小球疾病的进展。了解 单个肾小球细胞类型内的代谢变化及其临床意义 因此，与基质变化的关系是一个重要的目标。这个 该提案的具体短期目标涉及以下方面的问题 硬化过程的具体背景和最近的这一结果 申请人及其赞助商完成的工作。具体来说，我们 建议研究降解性肾小球基质的细胞模式 酶和细胞因子在5/6肾切除后的表达 该模型的肥厚期(早期)和硬化期(晚期)。vbl.使用 ISRT和定量RNA转录本的互补技术， 这些蛋白质的数量和细胞位置将被评估， 为每种肾小球细胞类型在肾小球疾病中的作用提供新的见解 肾小球硬化的发病机制。 完成该项目所需的所有设备和资源 已经在申请人的设施中提供。安排已经完成 也被要求为他的受保护的、不间断的研究时间提供保险。 这一点，除了发起人对申请人的持续支持外， 将为申请人的进一步发展提供必要的环境 科学进步和这一项目的圆满完成。

Influenced by the enthusiasm of his eventual sponsors, the applicant developed an increasing interest in the molecular biologic aspects of glomerular disease during his two-year clinical nephrology fellowship. At the conclusion of this clinical training, the applicant started research training with the hope of developing a productive academic career. With the sponsors' guidance and supervision, the applicant devoted the first two years of his laboratory training towards developing an immunohistochemical method that permits the localization of RNA transcripts to a single cell, a level of resolution previously unobtainable. Subsequently, we used this new technique to identify specific glomerular cell types expressing glomerular matrix (ECM) and basement membrane (GBM) protein mRNAs, thus, defining the cellular origins for these proteins and demonstrating, in contrast to previously held concepts, that glomerular basement membrane synthesis is asymmetric and the cellular origins of its individual components are diverse. This new information provides the framework for further studies comparing normal basement membrane metabolism to processes involved in disease models. Our preliminary studies on the rat 5/6 nephrectomy (NTX) model of glomerular sclerosis reveal that the cellular synthetic patterns which occur soon after NTX appear similar to cellular events seen during glomerular embryogenesis. These findings provide the basis for the following hypotheses: 1) that the initial glomerular adaptive response to 5/6 NTX represents a coordinated return to fetal patterns of ECM/GBM synthesis; 2) that the later or sclerotic phase of the 5/6 NTX model represents a maladaptive, discoordinate pattern of ECM/GBM synthesis; and 3) this involves changes in cellular patterns of types of ECM/GBM protein synthesis or their corresponding degradative enzymes. The long-term research objectives of the applicant during and after the award period relate to an improved understanding of the mechanisms that govern the progression of glomerular disease. Understanding the metabolic changes within individual glomerular cell types and their relation to matrix alterations is, therefore, an important goal. The specific short-term objectives of this proposal address these issues in the specific context of the sclerotic process and result from this recent work accomplished by the applicant and his sponsors. Specifically, we propose to study the cellular patterns of glomerular matrix, degradative enzyme, and cytokine expression after 5/6 nephrectomy during both the hypertrophic (early) and sclerotic (late) stages of this model. Using ISRT and complimentary techniques that quantitate RNA transcripts, the amount and cellular location of these proteins will be assessed, providing new insights into the role of each glomerular cell types in the pathogenesis of glomerular sclerosis. All equipment and resources required for the completion of this project are already available at the applicant's facility. Arrangements have also been made to insure his protected, uninterrupted research time. This, in addition to the sponsors' continued support of the applicant, will provide the environment necessary for the applicant's further scientific progress and for the successful completion of this project.