Cell-matrix interactions of renal tubular epithelial cells: implications of cortical actin structures and the tubular basement

慢性肾脏疾病在世界范围内的发病率持续上升。由于因果治疗选择有限，CKD通常需要透析，这与高社会经济成本负担有关，也导致生活质量严重受限。CKD的进行性器官功能障碍以组织形态学特征为特征，如间质纤维化和肾小管萎缩。肾小管萎缩的主要特征是肾上皮细胞的表型改变和肾小管基底膜（TBM）的大量增厚和多层。基于这些观察和我们的初步工作，我们假设TBM结构和组成的改变可能调节管状萎缩的进展，并且影响细胞-基质相互作用，最终导致信号传导和细胞串扰的改变。除了特定的基质受体（如整合素受体）外，细胞骨架机制也参与平衡细胞-基质相互作用。尽管在了解肾纤维化过程的调节机制方面取得了重大进展，但tbm改变和细胞-基质相互作用失衡之间的相互作用尚不清楚。因此，我们在本项目中旨在解决以下问题：1)肾小管基底膜是如何组成的？病理条件下TBM的成分是如何改变的？是否存在特定的ECM特征（在慢性肾脏疾病模型中）？2)皮质肌动蛋白网络在调节肾小管上皮细胞的细胞-基质相互作用中的确切作用是什么？这些肌动蛋白网络是否调节ECM合成或机械转导等过程？TBM结构改变如何影响肾上皮生物学（如机械信号传导）？这些过程可以通过药物干预来改变吗？第一个问题将采用创新的定量蛋白质组学方法、ecm富集协议和体内小鼠模型来解决。此外，我们已经建立了新的条件小鼠模型，可以分析皮质肌动蛋白网络对肾小管上皮细胞的作用和贡献，以及细胞-基质相互作用的影响。利用广泛的原代细胞系统平台，以及CRISPR/Cas9修饰的细胞系，生物物理方法，各种ecm方案和共培养分析，我们旨在阐明改变的TBM结构如何影响肾小管细胞的上皮表型，以及这些影响如何转化为CKD背景下的进行性肾小管萎缩。

Chronic kidney disease shows a persistent increase in incidence worldwide. Due to limited causal therapy options CKD often requires dialysis, which is related to high socio-economic cost burdens and also results in massively limited quality of life. Progressive organ dysfunction in the context of CKD is characterized by histomorphological features such as interstitial fibrosis and renal tubular atrophy. Hallmark features of renal tubular atrophy are phenotypical alterations of renal epithelial cells and massive thickening and multi-lamellation of the tubular basement membrane (TBM). Based on these observations and our preliminary work we hypothesize that alterations of TBM structure and composition might modulate the progress of tubular atrophy and moreover influences cell-matrix interactions ultimately leading to altered signaling and cellular crosstalk. Aside from specific matrix-receptors (e.g. integrin receptors) also cytoskeletal machineries are involved in balanced cell-matrix interactions. Although there is significant progress in the understanding of mechanisms modulating the process of renal fibrosis, the reciprocal interplay between altered TBMs and dysbalanced cell-matrix interactions are less understood. Therefore, we aim to address following questions in this project: 1) How is the renal tubular basement membrane composed? How is the composition of the TBM altered under pathological conditions? Are there specific ECM signatures present (in models of chronic kidney disease)? 2) What is the exact role of cortical actin networks in modulating cell-matrix interactions of renal tubular epithelial cells? Do these actin networks modulate ECM synthesis or processes such as mechano-transduction? 3) How do altered TBM structures influence renal epithelial biology (e.g. mechano-signaling)? Can these processes be modified by pharmacological interventions? The first question will be addressed employing innovative quantitative proteomics methods, ECM-enrichment protocols and in vivo mice models. Moreover, we´ve already established new conditional mice models allowing to analyze the role and contribution of cortical actin networks for renal tubular epithelial cells and the implications for cell-matrix interactions. Using a broad platform of primary cell systems, as well as CRISPR/Cas9 modified cell lines, biophysical approaches, a variety of ECM-protocols and co-culture assays we aim to elucidate how altered TBM structures influence epithelial phenotypes of renal tubular cells and how these effects translate into progressive renal tubular atrophy in the context of CKD.