BACKGROUND Mutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology. METHODS We used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene in vitro and in podocyte-like Drosophila nephrocytes. RESULTS We identified hemizygous missense mutations in the gene TBC1D8B in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine Tbc1d8b with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the Drosophila ortholog of nephrin. Expression of Rab11 RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas RAB11 overexpression revealed a partial phenotypic overlap to Tbc1d8b loss of function. CONCLUSIONS Novel mutations in TBC1D8B are monogenic causes of SRNS. This gene inhibits RAB11. Our findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.

中文翻译：

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TBC1D8B突变牵涉肾病综合征发病机制中依赖RAB11的囊泡贩运。

背景技术已经鉴定出约50个基因的突变是肾病综合征的单基因原因，这是CKD的常见原因。这些基因描绘了致病途径，并为足细胞生物学提供了重要的见识。方法我们使用全外显子组测序来确定类固醇抵抗性肾病综合征（SRNS）的新的单基因原因。我们分析了SRNS相关基因在体外和足细胞样果蝇肾细胞中的功能意义。结果我们在五个肾病综合征家庭中鉴定了基因TBC1D8B的半合错义突变。免疫共沉淀测定表明TBC1D8B与活性形式的RAB11之间存在相互作用。使HEK293T细胞中的TBC1D8B沉默会增加基础自噬和胞吐作用，这是RAB11独立调节的两种细胞功能。这表明TBC1D8B通过抑制内源性RAB11发挥调节作用。免疫共沉淀试验表明，TBC1D8B也与缝隙隔膜蛋白nephrin相互作用，并在永生化细胞系中共定位。与野生型Tbc1d8b蛋白相比，具有患者衍生突变的过表达鼠Tbc1d8b对内源RAB11和nephrin的亲和力较低。击倒果蝇中的Tbc1d8b会破坏足细胞样肾细胞的功能，并导致Sns的错配，这是果蝇的肾上腺素直系同源物。Rab11 RNAi在肾细胞中的表达导致缝隙隔膜蛋白向膜的传递有缺陷，而RAB11的过表达显示Tbc1d8b功能丧失的部分表型重叠。结论TBC1D8B中的新突变是SRNS的单基因原因。该基因抑制RAB11。