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

项目摘要/摘要 巨噬细胞和粒细胞(例如，中性粒细胞)被认为是 肾小球损伤的发病机制，肾小球肾炎(GN)。然而，肾内 这些髓系细胞导致肾小球损伤的机制和亚细胞特异性 都是未知的。这一巨大的知识差距源于捕获方法的局限性 并描述这些高度异质的细胞。例如，无论是正常密度(NDN)还是 低密度中性粒细胞(LDN)，两者都与肾小球肾炎有关，在scRNAseq研究中没有捕获到 肾脏和尿液，因为这些细胞在样品冷冻过程中丢失。鉴于它们的异质性 在疾病中的重要性，实施定义GN髓系人群的技术 肾脏及其与肾脏结构细胞的相互作用对于理解它们的 致病作用。我们的空间转录数据揭示了肾小球肾炎的巨噬细胞、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尿液中的巨噬细胞和粒细胞。整体而言 这项建议的目标是：(I)确定肾小球肾炎肾脏中的髓样细胞群，(Ii)确定 巨噬细胞、粒细胞与肾脏结构细胞的致病相互作用 (3)对肾小球肾炎的髓系细胞进行尿液dNaM测定。中心假设是 肾小球与低密度脂蛋白的相互作用反映了LN的免疫病理和肾功能的恶化 尿dNaM将允许一种侵入性较小的方法来评估致病髓系 人口。这一假设将以两个特定的目标进行检验：1)定义巨噬细胞和 肾小球肾炎肾组织中粒细胞群及其与结构细胞的通讯途径 2)用dNaM定量和确定尿液中肾脏髓样细胞的数量。在目标1中， 单细胞空间转录将用于确定肾小球肾炎的原位髓系异质性 肾脏通过整合批量RNA-seq衍生的转录转录签名进行排序 亚群。与肾小球、间质和肾小管内皮细胞的致病相互作用 上皮性结构将被定义。在目标2中，巨噬细胞和粒细胞将被量化。 尿液中使用与肾功能有关的独特的dNaM信号。本研究具有创新性。 因为它将提出参与肾小球损伤的分子和细胞途径以及 介绍一种通过非侵入性尿液检测致病细胞群的新方法 分析。这项研究具有重要意义，因为它有望为 针对GN中特定的髓系人群。

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.