Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
J Am Soc Nephrol. 2021 Sep;32(9):2175-2193. doi: 10.1681/ASN.2020091346. Epub 2021 Jun 1.
The glomerulus comprises podocytes, mesangial cells, and endothelial cells, which jointly determine glomerular filtration. Understanding this intricate functional unit beyond the transcriptome requires bulk isolation of these cell types for biochemical investigations. We developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP).
We separated glomerular cell types from wild-type or mT/mG mice a novel FACS approach, and validated their purity. Cell type proteomes were compared between strains, ages, and sex. We applied timMEP to the podocyte-targeting, immunologic, THSD7A-associated, model of membranous nephropathy.
timMEP enabled protein-biochemical analyses of podocytes, mesangial cells, and endothelial cells derived from reporter-free mice, and allowed for the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. We identified marker proteins for mesangial and endothelial proteins, and outlined protein-based, potential communication networks and phosphorylation patterns. The analysis detected cell type-specific proteome differences between mouse strains and alterations depending on sex, age, and transgene. After exposure to anti-THSD7A antibodies, timMEP resolved a fine-tuned initial stress response, chiefly in podocytes, that could not be detected by bulk glomerular analyses. The combination of proteomics with super-resolution imaging revealed a specific loss of slit diaphragm, but not of other foot process proteins, unraveling a protein-based mechanism of podocyte injury in this animal model.
timMEP enables glomerular cell type-resolved investigations at the transcriptional and protein-biochemical level in health and disease, while avoiding reporter-based artifacts, paving the way toward the comprehensive and systematic characterization of glomerular cell biology.
肾小球由足细胞、系膜细胞和内皮细胞组成，它们共同决定肾小球滤过功能。要深入了解这一复杂的功能单位，除了转录组学研究外，还需要对这些细胞类型进行批量分离，以进行生化研究。我们开发了一种适用于鼠类系膜细胞和内皮细胞及足细胞的通用三组分分离方法（timMEP）。
我们采用一种新颖的流式细胞分选方法，从野生型或 mT/mG 小鼠中分离肾小球细胞类型，并验证其纯度。比较了不同品系、年龄和性别的细胞类型蛋白质组。我们将 timMEP 应用于足细胞靶向、免疫、THSD7A 相关的膜性肾病模型。
timMEP 使我们能够对无报告基因小鼠来源的足细胞、系膜细胞和内皮细胞进行蛋白质生化分析，并对单个小鼠的足细胞、内皮细胞和系膜细胞蛋白质组进行了特征描述。我们鉴定了系膜细胞和内皮细胞的标记蛋白，并概述了基于蛋白质的潜在通讯网络和磷酸化模式。分析检测到了不同品系小鼠之间细胞类型特异性蛋白质组差异，以及性别、年龄和转基因的影响。在暴露于抗 THSD7A 抗体后，timMEP 解析了一个精细的初始应激反应，主要发生在足细胞中，而这在批量肾小球分析中是无法检测到的。蛋白质组学与超分辨率成像的结合揭示了特定的裂孔隔膜丢失，但其他足突蛋白没有丢失，揭示了该动物模型中足细胞损伤的基于蛋白质的机制。
timMEP 使我们能够在健康和疾病状态下，在转录和蛋白质生化水平上进行肾小球细胞类型分辨研究，同时避免了基于报告基因的人为假象，为全面系统地描述肾小球细胞生物学铺平了道路。
