Steroid-sensitive nephrotic syndrome candidate gene CLVS1 regulates podocyte oxidative stress and endocytosis
Brandon M. Lane, … , Jameela A. Kari, Rasheed Gbadegesin
Brandon M. Lane, … , Jameela A. Kari, Rasheed Gbadegesin
Published December 7, 2021
Citation Information: JCI Insight. 2022;7(2):e152102. https://doi.org/10.1172/jci.insight.152102.
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Research Article Nephrology

Steroid-sensitive nephrotic syndrome candidate gene CLVS1 regulates podocyte oxidative stress and endocytosis

Abstract

We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1-knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function.

Authors

Brandon M. Lane, Megan Chryst-Stangl, Guanghong Wu, Mohamed Shalaby, Sherif El Desoky, Claire C. Middleton, Kinsie Huggins, Amika Sood, Alejandro Ochoa, Andrew F. Malone, Ricardo Vancini, Sara E. Miller, Gentzon Hall, So Young Kim, David N. Howell, Jameela A. Kari, Rasheed Gbadegesin

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Figure 5

CLVS1 is required for clathrin-mediated endocytosis in human podocytes.

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CLVS1 is required for clathrin-mediated endocytosis in human podocytes....
To examine specific modes of endocytosis in CLVS1 podocytes, we evaluated the internalization of fluorescently labeled transferrin (clathrin-mediated endocytosis), 70,000 MW dextran (macropinocytosis), and albumin molecules (caveolae-mediated endocytosis). (A and B) Quantification of the number of internalized transferrin molecules per cell revealed a decrease in clathrin-mediated endocytosis in CLVS1-KO and homozygous H310Y-KI podocytes compared with controls that was unaffected by pretreatment with 1 μM dexamethasone. (n > 30 for each group, P < 0.0001, 1-way ANOVA.) (C and D) Macropinocytosis, as measured by the internalization of fluorescently labeled 70,000 MW dextran molecules, was unaffected by CLVS1 KO or H310Y KI (P = 0.9572 and P = 0.9604 respectively, 1-way ANOVA). However, treatment with dexamethasone did significantly increase macropinocytosis in all cell lines compared with vehicle-treated controls (P < 0.001 for all). (E and F) Caveolae-mediated endocytosis was comparable among CLVS1-KO, homozygous H310Y-KI podocytes, and controls when internalized albumin molecules were quantified, and this was unaffected by treatment with dexamethasone (n > 30 for each group, P = 0.993 and P = 0.1844 respectively, 1-way ANOVA). Error bars depict SEM in graphs. Scale bars = 12 μm. Each dot in the graphs represents the mean number of molecules per podocyte for a single well.