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 7

Defective ROS regulation contributes to decreased viability in CLVS1 KO and H310Y KI podocytes.

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Defective ROS regulation contributes to decreased viability in CLVS1 KO ...
(A and B) Automated live-cell imaging and quantification of ROS levels in CLVS1-KO podocytes as well as controls using a fluorescent reporter of multiple ROS types, including hydrogen peroxide, peroxynitrite, and hydroxl radicals (A) (n > 20 for each group, P < 0.0001 for all time points for KO, P < 0.05 for all time points, and after 20 minutes for KI, 2-way ANOVA), as well as a second independent fluorescent reporter that detects superoxide generation (B) (n > 20 for each group, P < 0.001 for all time points after 10 hours, 2-way ANOVA), revealed an increase in ROS and superoxide accumulation in CLVS1-KO and homozygous KI podocytes that could be rescued with pretreatment with 1 μM dexamethasone. (C and D) The increased susceptibility to apoptosis in CLVS1-KO and homozygous KI podocytes (P < 0.05 for all time points after 36 hours) could be rescued by treatment with a superoxide scavenger or an ROS inhibitor (5 nM MitoTEMPO and 500 nM elamipretide) (n > 10 for all conditions, 2-way ANOVA). The ROS inducer pyocyanin (300 μM) and ROS inhibitor N-acetyl-l-cysteine (5 mM) were used as positive and negative controls, respectively, for all experiments. Error bars depict SEM.