Center for Quantitative Biology: A focus on "omics", from organisms to single cells Supplement 2

项目概要/摘要 巨噬细胞和粒细胞（例如，中性粒细胞）被认为是 肾小球肾损伤的发病机制，肾小球肾炎（GN）。然而，肾内 这些髓样细胞引起肾小球损伤的机制和亚细胞特异性 不知道。这种巨大的知识差距源于获取方法的局限性， 并分析这些高度异质性的细胞。例如，正常密度（NDN）和 低密度中性粒细胞（LDN），两者都与GN有关，在scRNAseq研究中没有捕获， 肾脏和尿液，因为这些细胞因样品冷冻而损失。考虑到它们的异质性 和疾病的重要性，实施定义GN中骨髓群体的技术 肾脏以及它们如何与肾脏结构细胞相互作用对于了解它们的功能至关重要。 致病作用我们的空间转录组学数据揭示了GN中的巨噬细胞、NDN和LDN 肾脏，具有独特和共享的空间组织。使用DNA甲基化（DNAm）数据 从尿细胞中，我们捕获GN尿中的巨噬细胞和粒细胞。整体 该提案的目的是：（i）定义GN肾脏中的髓样细胞群，（ii）识别 巨噬细胞与粒细胞与肾结构细胞之间的致病性相互作用，以及 (iii)实施尿DNAm测定以分析GN中髓样细胞。核心假设是， 肾小球与LDN的相互作用反映了LN的免疫病理学和更差的肾功能， 尿液DNAm将允许一种侵入性较小的方法来评估致病性骨髓细胞， 人口。将以两个特定目的检验该假设：1）定义巨噬细胞， 肾小球肾炎中粒细胞群及其与结构细胞的通讯途径 和2）使用DNAm定量和确定尿中的肾浸润性骨髓群体。在目标1中， 单细胞空间转录组学将用于确定GN中的原位髓系异质性 通过整合大量RNA-seq衍生的转录组学特征， 亚群与肾小球、间质和肾小管内皮细胞的致病性相互作用， 将定义上皮结构。在目标2中，将对巨噬细胞和粒细胞进行定量 使用与肾功能相关的独特DNA标记。这项研究具有创新性 因为它将提出参与肾小球损伤的分子和细胞途径， 介绍一种通过非侵入性尿液检测致病细胞群新方法 分析。这项研究意义重大，因为它有望为 靶向GN中的特定髓样群体。

Project Summary / Abstract Macrophages and granulocytes (e.g., neutrophils) are considered key players in the pathogenesis of glomerular kidney injury, glomerulonephritis (GN). However, the intra-renal mechanisms and sub-cellular specificity by which these myeloid cells cause glomerular injury are not known. This large gap in knowledge stems from the limitations in approaches to capture and profile these highly heterogeneous cells. For example, neither normal density (NDN) nor low-density neutrophils (LDN), both implicated in GN, are not captured in scRNAseq studies of kidneys and urine due to the loss of these cells by sample freezing. Given their heterogeneity and importance in disease, implementing technologies that define myeloid populations in GN kidneys and how they interact with renal structural cells is critical for understanding their pathogenic role. Our spatial transcriptomic data reveal macrophages, NDNs, and LDNs in GN kidneys, with unique and shared spatial organizations. Using DNA methylation (DNAm) data from urine cells, we capture both macrophages and granulocytes in GN urine. The overall objectives in this proposal are to: (i) define myeloid populations in GN kidneys, (ii) identify pathogenic interactions between macrophages vs. granulocytes with renal structural cells, and (iii) implement urine DNAm assay to profile myeloid cells in GN. The central hypothesis is that glomerular interactions with LDNs reflect LN immunopathology and worse kidney function and that urine DNAm will allow a less invasive approach to evaluate the pathogenic myeloid populations. The hypothesis will be tested with two specific aims: 1) Define macrophage and granulocyte populations and their communication pathways with structural cells in GN kidneys and 2) Quantify and define kidney infiltrating myeloid populations in urine using DNAm. In Aim 1, single-cell spatial transcriptomics will be used to define in situ myeloid heterogeneity in GN kidneys by integrating bulk RNA-seq-derived transcriptomic signatures of sorted subpopulations. Pathogenic interactions with glomerular, interstitial, and tubular endothelial and epithelial structures will be defined. In Aim 2, macrophages and granulocytes will be quantified in urine using unique DNAm signatures in relation to kidney function. This research is innovative because it will propose molecular and cellular pathways involved in glomerular injury as well as introduce a novel approach to detect pathogenic cell populations via non-invasive urine analyses. This research is significant because it is expected to provide a scientific rationale for targeting specific myeloid populations in GN.