Gene-regulatory networks in podocytes in health and disease

足细胞是维持肾小球滤过的关键，它们的损伤总是导致蛋白尿和局灶性和节段性肾小球硬化（FSGS）的发展。在这种情况下，许多信号通路和足细胞细胞器以及其他肾小球细胞类型的突变和调节已被证明可以作为足细胞损伤和疾病进展的触发、繁殖或衰减者。此外，许多遗传性足细胞疾病的发病是不同步的，集中在单个细胞水平上，导致患病和健康足细胞之间的细胞间接触，以及患病和未患病的肾小球细胞之间的空间相关反应。许多触发足细胞损伤或形成足细胞对损伤反应的途径要么向肾小球细胞核发出信号，要么本身就是基因调节因子。因此，通过单细胞转录组学和表观遗传学，它们的信号集中在基因调控反应上，从而可以全面而公正地了解机制。在我们之前的工作中，我们已经证明这种基因调控反应是动态调节的，作为两种功能，足细胞疾病进展状态以及引发疾病的初始触发。然而，这些基因调控轨迹沿着疾病进展，以及触发特异性基因调控反应的性质仍然是尚未表征。因此，目的1提出定义足细胞和其他肾小球细胞在疾病进展中的序列基因调控特征。为此，我们将采用整合的基于基因调控网络的肾小球单核RNA测序数据（snRNAseq，用于RNA表达）和肾小球细胞类型特异性ATACseq数据（“转座酶可及染色质测序测定”，以揭示染色质可及性）分析。通过这种联合方法，我们将在两种小鼠FSGS模型中确定信号通路和细胞成分调制的触发特异性序列。这将允许对与FSGS疾病进展相关的机制进行公正和全面的见解。在目标2中，我们将讨论局灶性足细胞损伤对空间相关但间接影响的足细胞以及肾小球非足细胞的基因调控作用。为此，我们将通过条件性和他莫昔芬诱导的关键足细胞转录因子Lmx1b的敲除，利用马赛克诱导足细胞损伤。肾小球snRNAseq将用于在单个细胞水平上进行基因分型，然后结合肾小球细胞类型特异性ATACseq进行基于网络的单细胞转录组分析。这些分析将为足细胞维持机制、肾小球内疾病触发因素的空间传播以及肾小球非足细胞对局灶性足细胞疾病的继发性反应提供前所未有的见解。

Podocytes are key to maintaining glomerular filtration and their damage invariably results in albuminuria and the development of focal and segmental glomerulosclerosis (FSGS). In this context, mutations within and modulations of many of signaling pathways and cellular organelles of podocytes as well as other glomerular cell types have been shown to function as either triggers and propagators or attenuators of podocyte damage and disease progression. Furthermore, the onset of many hereditary types of podocyte disease is asynchronous and focal on the level of individual cells, resulting in intercellular contacts shared between diseased and healthy podocytes as well as in spatially correlated responses between disease affected and non-affected glomerular cells. Many of the pathways triggering podocyte damage or shaping their response to injury either signal to glomerular cell nuclei or are gene-regulatory modulators themselves. Hence, their signals converge on gene-regulatory responses amenable to comprehensive and unbiased mechanistic insight by single-cell transcriptomics and epigenetics. In our previous work, we have shown that such gene-regulatory responses are dynamically regulated as a function of both, the state of podocyte disease progression as well as the initial trigger eliciting the disease. However, these gene-regulatory trajectories along disease progression as well as the nature of trigger-specific gene-regulatory responses remain uncharacterized as yet. Therefore, aim 1 proposes to define sequential gene-regulatory signatures of podocytes and other glomerular cells upon disease progression. To this end, we will employ integrated gene-regulatory network-based analyses of glomerular single-nucleus RNA-sequencing data (snRNAseq, for RNA expression) and glomerular cell-type specific ATACseq data (‘assays for transposase-accessible chromatin sequencing’ to reveal chromatin accessibility). With this combined approach we will determine trigger-specific sequences of signaling pathway and cell constituent modulation in two mouse models of FSGS. This will allow for unbiased and comprehensive insights into mechanisms pertinent to disease progression in FSGS. In aim 2, we will address the gene-regulatory effects of focal podocyte damage on spatially correlated yet indirectly affected podocytes as well as glomerular non-podocyte cells. To this end, we will use mosaic induction of podocyte damage by conditional and tamoxifen-induced knock-out of the key podocyte transcription factor Lmx1b. Glomerular snRNAseq will be used for genotyping on an individual cell level followed by network-based analysis of single-cell transcriptomes integrated with glomerular cell-type specific ATACseq. These analyses will provide unprecedented insight into mechanisms of podocyte maintenance, spatial propagation of disease triggers within a glomerulus, and secondary responses of glomerular non-podocyte cells to focal onset of podocyte disease.