Nephrotic Syndrome Gene <i>TBC1D8B</i> Is Required for Endosomal Maturation and Nephrin Endocytosis in <i>Drosophila</i>.

Abstract

Variants in <i>TBC1D8B</i> cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. We generated a stable deletion in <i>Tbc1d8b</i> and used microhomology-mediated end-joining for genome editing. <i>Ex vivo</i> functional assays utilized slit diaphragms in podocyte-like <i>Drosophila</i> nephrocytes. Manipulation of endocytic regulators and transgenesis of murine <i>Tbc1d8b</i> provided a comprehensive functional analysis of Tbc1d8b. A null allele of <i>Drosophila TBC1D8B</i> exhibited a nephrocyte-restricted phenotype of nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from the <i>Tbc1d8b</i> locus bearing the edited tag predominantly localized to mature early and late endosomes. Tbc1d8b was required for endocytic cargo processing and degradation. Silencing <i>Hrs</i>, a regulator of endosomal maturation, phenocopied loss of <i>Tbc1d8b</i>. Low-level expression of murine <i>TBC1D8B</i> rescued loss of the <i>Drosophila</i> gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel <i>TBC1D8B</i> variants within a cohort of 363 patients with FSGS and validated a functional effect of two variants in <i>Drosophila,</i> suggesting a personalized platform for <i>TBC1D8B</i>-associated FSGS. Variants in <i>TBC1D8B</i> are not infrequent among patients with FSGS. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.