Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
Division of Nephrology and Hypertension, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
Am J Physiol Renal Physiol. 2019 Dec 1;317(6):F1593-F1604. doi: 10.1152/ajprenal.00195.2019. Epub 2019 Sep 30.
Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for , , or , which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.
足细胞的功能与其细胞骨架的复杂组织以及与肾小球基底膜的黏附紧密相关。有报道称，培养的足细胞黏附于各种基质与足细胞的健康状况相关。为了鉴定对足细胞功能重要的新基因，我们设计了一种基于足细胞黏附于纤连蛋白或可溶性 Fms 样酪氨酸激酶-1（sFLT1）/Fc 包被的平板的体外遗传筛选。对永生化的人足细胞进行全基因组规模的 RNA 干扰筛选，鉴定出 77 个可增加对纤连蛋白黏附的基因、101 个可增加对 sFLT1/Fc 黏附的基因，以及 44 个可同时增加对这两种底物黏附的基因。针对二氢喋呤生物合成蛋白 1-4（DPH1-DPH4）的多个 shRNA 是增加黏附的主要靶点。稳定表达这些发夹的永生化人足细胞显示出对这两种底物的黏附增加。然后，我们使用 CRISPR-Cas9 生成足细胞 knockout 细胞，分别缺失 、 或 ，这些细胞也显示出对纤连蛋白和 sFLT1/Fc 的黏附增加以及铺展缺陷。最后，我们表明 足细胞特异性敲低 Dph1、Dph2 和 Dph4 导致了足细胞功能的改变。总之，我们在这里报告了一种新的鉴定足细胞功能重要基因的高通量方法。鉴于足细胞黏附作为足细胞健康的标志物的核心作用，这些数据是肾小球疾病候选调节因子的丰富来源。
