Multidimensional cellular interrogation of the kidney in AKI and CKD

AKI 和 CKD 中肾脏的多维细胞检查

• 批准号: 9910956
• 负责人: Zoltan Laszik
• 金额: $ 44.04万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910956
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Address; Algorithmic Analysis; Atlases; Biological Assay; Biological Markers; Biology; Biopsy; Cells; Chronic Kidney Failure; Cohort Studies; Communities; Computer-Assisted Image Analysis; Coupled; Cytometry; Data; Data Analyses; Databases; Diagnosis; Diagnostic; Disease; Electron Microscopy; Ensure; Fluorescence; Formalin; Gene Expression Profiling; Gene Proteins; Generations; Goals; Health; Image; Image Analysis; Immunofluorescence Immunologic; Immunohistochemistry; In Situ; In Situ Hybridization; Inflammatory; Kidney; Kidney Diseases; Knowledge; Laboratories; Literature; Maps; Mass Spectrum Analysis; Messenger RNA; Methods; Microscopy; Modality; Molecular; Molecular Abnormality; Paraffin Embedding; Pathologic; Patients; Phase; Population; Preparation; Proteins; Proteomics; Protocols documentation; Reproducibility; Research Design; Resolution; Role; Running; Sampling; Site; Source; Stains; Standardization; Structure; Technology; Time; Tissue Embedding; Tissue Viability; Tissues; Transplantation; Work; base; cohort; data hub; data integration; data visualization; kidney biopsy; light microscopy; multidimensional data; new technology; next generation; novel; novel diagnostics; prototype; standard of care; tool; transcriptome sequencing; transcriptomics; whole slide imaging

Project Summary Current standard routine pathologic work-up of kidney biopsies has been in place for about 50 years and involves light microscopy (LM), immunofluorescent microscopy (IF)/immunohistochemistry (IHC), and electron microscopy (EM). Current practice of using kidney biopsies as one of the main diagnostic tools in the management of kidney has significant limitations. First, interpretation is founded on classic pathologic principles and approaches, which are by definition, descriptive, or semi-quantitative at most. Therefore, the interpretation has an inherent component of subjectivity and reproducibility and can be suboptimal which has been well documented both in the transplant and native kidney biopsy literature. Second, immunophenotypical characterization in the kidney is not part of the diagnostic work-up. Furthermore, in spite of advances in a number of various novel technologies potentially applicable to tissue interrogation of kidney biopsies, there have been no new technologies added to the diagnostic armamentarium with the sole exception of mass spectrometry for some limited applications. Therefore, it is evident that diagnostic potential of kidney biopsies is underutilized. This, coupled with the unmet need of better understanding the biology of acute kidney injury (AKI) and chronic kidney disease (CKD) makes kidney biopsy a potential high value source to acquire new knowledge through the application of novel tissue interrogation technologies. The ideal next generation tissue- based assay need to be highly reproducible, quantitative, fluorescence-based, and multiplexed to visualize multiple mRNAs and proteins at single cell resolution level. In this carefully designed study proposal we are going to serve as a Tissue Interrogation Site of the KPMP project. We will utilize two multiplex assays (1) multiplex Immunofluorescence and In Situ Hybridization (mIFISH) and (2) CODEX that have been developed by Dr. Nolan (Stanford) and by Dr. Laszik (UCSF) that fulfill the criteria listed above. The assays will use formalin-fixed paraffin-embedded (FFPE) tissue which has the best potential for standardization and to be representative due to its relatively large size. The new generation (in situ) assays that retain the structural integrity of tissues will be supplemented by additional ancillary tissue-based assays of proteomics and RNAseq on homogenized tissues which will be led by Drs. Sarwal and Sigdel at UCSF. Data generated by proteomics and RNAseq will be correlated with the conventional pathologic findings, and also with data generated on the in situ platforms of mIFISH and CODEX (i.e., tissue atlases). Cytometry by Time of Flight (CyTOF) assay will help interrogate the inflammatory compartment of AKI and CKD on fresh/cyoprotected samples. Data generated by proteomics, RNAseq, and CyTOF will also be used to identify bio-markers that will be incorporated into the in situ mIFISH and CODEX assays which are the “epicenter” of this proposal.

项目摘要 目前标准的肾脏活检常规病理检查已经存在了大约50年， 涉及光学显微镜(LM)、免疫荧光显微镜(IF)/免疫组织化学(IHC)和电子 显微镜(EM)。目前使用肾脏活检作为主要诊断工具之一的做法 肾脏的管理有很大的局限性。首先，解释是建立在经典的病理学基础上的 原则和方法，从定义上讲，这些原则和方法至多是描述性的或半定量的。因此， 口译具有固有的主观性和再现性，并可能是次优的，这具有 在移植和自然肾活检文献中都有很好的记录。第二，免疫表型 肾脏的特征不是诊断工作的一部分。此外，尽管在 许多可能适用于肾活检组织询问的各种新技术，在那里 没有新的技术添加到诊断医疗设备中，唯一的例外是质量 用于某些有限应用的光谱分析。因此，很明显，肾脏活检的诊断潜力 没有得到充分利用。这一点，加上更好地了解急性肾损伤的生物学方面尚未得到满足的需要。 (AKI)和慢性肾脏疾病(CKD)使肾活检成为潜在的高价值来源，以获得新的 通过应用新的组织讯问技术获得知识。理想的下一代组织- 基于分析的分析需要具有高度的重复性、定量、基于荧光和多路传输才能可视化 单个细胞分辨率水平的多个mRNAs和蛋白质。在这份精心设计的研究提案中，我们 将作为KPMP项目的组织审问现场。我们将使用两种多重检测方法(1) 已开发的多重免疫荧光和原位杂交(MIFISH)和(2)法典 符合上述标准的Nolan博士(斯坦福大学)和Laszik博士(加州大学旧金山分校)。化验结果将使用 福尔马林固定石蜡包埋(FFPE)组织，最有可能标准化和 由于它的体型相对较大，所以具有代表性。新一代(原位)分析保留了结构 组织完整性的补充将由额外的基于组织的辅助蛋白质组学分析和 由加州大学旧金山分校的Sarwal和Sigdel博士领导的均质组织的RNAseq。数据生成者 蛋白质组学和RNAseq将与传统的病理结果和数据相关联 在MIFISH和CODEX的原位平台(即组织地图集)上生成。按飞行时间进行的细胞测定法 (CyTOF)检测将有助于询问新鲜/细胞保护的AKI和CKD的炎性间隔 样本。蛋白质组学、RNAseq和CyTOF产生的数据也将被用于识别将 纳入本提案的“中心”--MIFISH和CODEX原位分析。