Steroid-sensitive nephrotic syndrome candidate gene CLVS1 regulates podocyte oxidative stress and endocytosis
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
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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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 3

Loss of CLVS1 expression increases podocyte susceptibility to apoptosis that can be restored by corticosteroid treatment.

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Loss of CLVS1 expression increases podocyte susceptibility to apoptosis ...
(A and B) Western blot showing significantly reduced expression of CLVS1 in CRISPR/Cas9-mediated knockout (KO) podocyte cell lines compared with control cell lines (n = 4, P < 0.001, 2-tailed t test). (C) Still images depicting cleaved caspase-3 activity (green) as reporter for early apoptosis and propidium iodide staining (red) for late apoptosis and necrosis in human podocytes 72 hours after serum starvation (scale bars = 1 mm). (D) Quantification of these images over time revealed an increase in podocyte susceptibility to serum starvation–induced apoptosis in CLVS1-KO and homozygous H310Y-knockin (KI) podocytes compared with controls (P < 0.05 for all time points after 16 hours for KO and after 34 hours for KI, n > 25 for each group, 2-way ANOVA). Heterozygous H310Y-KI podocytes displayed similar apoptotic phenotypes to controls. The elevated apoptosis in CLVS1-KO and homozygous H310Y-KI podocytes was rescued by treatment with 1 μM dexamethasone.