Nephron Sub-segmental Omics and Quantitative 3D Imaging of Human Kidney.

在理解决定人类肾脏进展的机制方面存在着根本的差距。 疾病。长期的目标是确定关键的细胞和分子通路的特征，这些通路调控着 急性和慢性肾脏疾病(AKI和CKD)，以确定评估疾病进展的新标志物， 并针对这些途径开发特定的治疗干预措施。管状之间的串扰 肾脏中的亚段和免疫细胞是纤维化和疾病进展的重要决定因素。 因此，这项应用的目的是选择性地检查肾小管的转录组 以确定免疫细胞亚型与肾小管亚段和肾脏的关系 AKI和CKD患者的结构。这一应用的中心假设是， 肾小管亚段和免疫细胞亚型的丰度和分布是独一无二的 人类肾脏疾病疾病进展的免费标识。建议的理由是 研究表明，一旦与疾病进展和疾病进展相关的唯一分子和细胞识别符 长期结果是确定的，它们可以用来监测药物干预的有效性， 确定最能代表人类疾病的动物模型进行翻译研究，并揭示新的 治疗这些疾病的方法。核心假设将通过追求三个具体的 目的：1)明确AKI患者活检组织中肾小管亚片段所表达的转录组 和慢性肾脏病，并将分子特征与临床结果相关联。2)确定丰度和 免疫细胞亚型在同一套患者活检组织中的分布。3)反向映射关键分子通路 以确定分子、细胞和结构的相关性。在第一个目标下，激光 来自病例匹配的生物库肾活检将进行管状亚节段的显微解剖 进展迅速的AKI和CKD患者以及进展不迅速的患者。基因表达 将对从管状亚段中分离出来的RNA进行分析，以识别转录 管状亚节段的签名。在第二个目标下，先进的三维(3-D)组织细胞术 将对来自第一个描述的相同病例匹配的患者的生物库肾脏活检进行 目的量化免疫细胞组成，观察空间细胞结构，并详细描绘 进展迅速的患者和进展不迅速的患者之间的形态差异。在第三节 目的，我们将对关键的分子通路进行染色，以创建大规模的人体活检数字模型 这突出了分子、细胞和结构的相关性对疾病病理生理学和 进步。这项拟议的研究具有重要意义，因为它是一系列研究的下一步，即 期望确定疾病进展的迫切需要的生物标志物，优化临床前研究，以及 针对AKI和CKD庞大的临床问题，制定具体和有针对性的治疗干预措施。

There is a fundamental gap in understanding the mechanisms that determine progression in human kidney disease. The long term goal is to characterize key cellular and molecular pathways regulating progression of acute and chronic kidney diseases (AKI and CKD), to identify novel markers that assess disease progression, and to develop specific therapeutic interventions targeting these pathways. The cross-talk between tubular subsegments and immune cells in the kidney is an important determinant of fibrosis and disease progression. Consequently, the objective of this application is to selectively examine the transcriptome of tubular subsegments and to quantify and localize immune cell subtypes in relation to tubular subsegments and renal structures in patients with AKI and CKD. The central hypothesis of this application is that the transcriptome of kidney tubular subsegments and the abundance and distribution of immune cell subtypes are unique and complimentary identifiers of disease progression in human kidney diseases. The rationale for the proposed research is that once the unique molecular and cellular identifiers that correlate with disease progression and long term outcomes are determined, they can be used to monitor efficacy of pharmacologic interventions, identify animal models that best represent human disease for translational research, and reveal novel approaches towards treating these conditions. The central hypothesis will be tested by pursuing three specific aims: 1) Define the transcriptome expressed by the tubular subsegments from biopsies of patients with AKI and CKD and correlate molecular signatures with clinical outcome. 2) Determine the abundance and distribution of immune cell subtypes in the same set of patient biopsies. 3) Backmap key molecular pathways to the renal biopsy in order to define molecular, cellular and structural correlations. Under the first aim, laser microdissection of tubular subsegments will be performed on biobanked kidney biopsies from case-matched patients with AKI and CKD that rapidly progressed and those that did not rapidly progress. Gene expression analysis will be performed on RNA isolated from the tubular subsegments to discern the transcriptomic signature of tubular subsegments. Under the second aim, advanced three-dimensional (3-D) tissue cytometry will be performed on the biobanked kidney biopsies from the same case-matched patients described in the first aim to quantify the immune cell composition, examine spatial cellular organization, and delineate detailed morphologic differences in patients who rapidly progressed and those who did not rapidly progress. In the third aim, we will stain for key molecular pathways in order to create a large-scale digital model of the human biopsy that highlights molecular, cellular and structural correlations important for disease pathophysiology and progression. The proposed research is significant, because it is the next step in a continuum of research that is expected to identify critically needed biomarkers of disease progression, optimize preclinical studies, and develop specific and targeted therapeutic interventions in the vast clinical problem of AKI and CKD.