BACKGROUND Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. METHODS We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. RESULTS Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of purified podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homolog Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. CONCLUSIONS We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype-to-protein correlation.

中文翻译：

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分离的足细胞的蛋白质组分析揭示了肾小球硬化的应激反应。

背景技术在系统水平上了解足细胞对损伤的特异性反应是困难的，因为损伤导致足细胞损失或细胞外基质增加，从而改变肾小球细胞组成。寻找早期足细胞损伤的窗口可能有助于识别参与足细胞应激反应的分子途径。方法我们开发了一种将蛋白质组学分析应用于极少量纯化足细胞部分样品的方法。为了检查FSGS小鼠模型中处于早期疾病状态的足细胞，我们使用了在化学诱导肾小球疾病（用阿霉素或LPS）后从个别小鼠中分离的足细胞部分。我们还将单肾小球蛋白质组分析应用于FSGS患者的组织。结果FSGS患者肾小球的转录组和蛋白质组分析表明，晚期疾病中足细胞特异性基因和蛋白质的表达不足。从FSGS小鼠模型中纯化的足细胞部分的蛋白质组分析表明，早期的应激反应包括对代谢，机械和蛋白稳定蛋白的干扰。进一步的分析显示蛋白尿量与机械传感器蛋白Filamin-B的表达水平之间存在高度相关性。FSGS患者的活检样品，蛋白尿大鼠的单肾小球以及经受机械应激的足细胞中足细胞中Filamin-B的表达增加表明该蛋白在有害的应激反应中起作用。在果蝇中，纤维蛋白同系物Cher减少的肾细胞显示出改变的滤过能力，但缝隙隔膜结构没有变化。结论我们通过对人肾小球FSGS组织和疾病早期早期纯化的天然小鼠足细胞进行超灵敏蛋白质组分析，确定了足细胞应激反应的保守机制。这种方法可以对大型蛋白质组学数据和表型与蛋白质的相关性进行系统的比较。