ines. Methods We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2, encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. Results All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. Conclusions Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. Podcast This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3...

中文翻译：

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使用 NPHS2 点变体等位基因系列生成的肾脏类器官揭示了独特的细胞内 Podocin 错误运输

伊内斯。方法我们生成了一系列带有 NPHS2 致病性错义变异的人类诱导多能干细胞 (iPSC) 系，编码蛋白质变化 p.G92C、p.P118L、p.R138Q、p.R168H 和 p.R291W，以及对照系。iPSC 系也是从一名患有类固醇抵抗性肾病综合征的患者（p.R168H 纯合子）和一个健康的杂合子父母中产生的。所有细胞系均分化为肾类器官。免疫荧光评估了 PODOCIN 表达和亚细胞定位。对足细胞进行转录分析并研究 PODOCIN-NEPHRIN 相互作用。结果 所有变体系均显示在转录减少的情况下 PODOCIN 蛋白水平降低。尽管野生型 PODOCIN 定位于膜，但不同的变异蛋白显示出独特的亚细胞蛋白运输模式，其中一些尚未报道。P118L和R138Q优先保留在内质网（ER）中；R168H和R291W积聚在高尔基体中。足细胞分析显示与疾病相关的转录变化最小。所有变体均表现出足细胞特异性凋亡，这与内质网应激无关。NEPHRIN-PODOCIN 共定位阐明了对 NEPHRIN 关联以及 NEPHRIN 运输的变体特异性影响。结论 内源性 NPHS2 的特定变体导致类器官足细胞内不同的亚细胞 PODOCIN 定位。了解每种变体对蛋白质水平和定位的影响以及对 NEPHRIN 的影响，可以为了解 NPHS2 变体的病理学提供更多见解。播客 本文包含一个播客，网址为 https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3...