孤立的足细胞的蛋白质组分析揭示了肾小球硬化中的应激反应。
Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis.
机构信息
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom.
出版信息
J Am Soc Nephrol. 2020 Mar;31(3):544-559. doi: 10.1681/ASN.2019030312. Epub 2020 Feb 11.
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 , 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.
背景
由于损伤会导致足细胞丢失或细胞外基质增加，改变肾小球细胞组成，因此很难从系统水平上了解足细胞对损伤的特异性反应。寻找早期足细胞损伤的窗口可能有助于确定参与足细胞应激反应的分子途径。
方法
我们开发了一种方法，将蛋白质组分析应用于纯化的足细胞分数的非常小的样本。为了研究 FSGS 小鼠模型中早期疾病状态的足细胞，我们使用化学诱导肾小球疾病（阿霉素或 LPS）后从单个小鼠中分离的足细胞分数。我们还应用了单肾小球蛋白质组分析来研究 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显示，在晚期疾病中，足细胞特异性基因和蛋白质的表达减少。FSGS 小鼠模型纯化的足细胞分数的蛋白质组分析显示，早期应激反应包括代谢、机械和蛋白质稳态蛋白的扰动。进一步分析显示，蛋白尿的量与机械传感器蛋白 Filamin-B 的表达水平高度相关。FSGS 患者活检样本中的足细胞、蛋白尿大鼠的单个肾小球和机械应激下的足细胞中 Filamin-B 的表达增加表明，该蛋白在有害应激反应中具有作用。在，具有减少的 Filamin 同源物 Cher 的肾细胞显示出改变的过滤能力，但在裂孔隔膜结构中没有变化。
结论
我们通过对人类肾小球 FSGS 组织和早期疾病阶段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。这种方法可以对大规模蛋白质组数据和表型-蛋白质相关性进行系统比较。
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