Gene-regulatory networks in podocytes in health and disease

点击翻译按钮获取中文摘要

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.