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 2

Knockdown of the clavesin gene in zebrafish (clvs2) results in edema phenotypes.

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Knockdown of the clavesin gene in zebrafish (clvs2) results in edema phe...
(A and B) Translation blocking and splice block morpholinos were used to knock down clvs2 expression in zebrafish. Morpholino efficacy was confirmed through Western blot (A) and reverse transcription PCR (RT-PCR) (B), showing that both morpholinos were able to knock down clvs2 expression. (C) Larval phenotypes were evaluated at 4 days postfertilization (dpf) as having edema (arrows for periorbital and pericardial) or as unaffected (no edema). (D and E) Analysis revealed significantly increased edema phenotypes with clvs2 knockdown compared with controls, and this edema could be rescued by coinjection of wild-type human CLVS1 mRNA but not the p.H310Y variant (E) (*P < 0.05, n > 60 for all groups, 1-way ANOVA). (F) Quantification of excreted GFP-labeled vitamin D in the Tg(lfabp:VDBP-GFP) reporter line revealed a loss of GFP in clvs2 morpholino-injected fish compared with controls (n = 10 for each group, P < 0.0001, 2-tailed t test), demonstrating that GFB integrity was affected by clvs2 knockdown. (G and H) Transmission electron microscopy images show healthy podocyte foot processes with intact slit diaphragms (white arrowheads) around a capillary loop in 5 dpf control morphant larvae and podocyte effacement (red arrowhead) in clvs2 morphants (scale bars = 800 nm and 1 μm, respectively), confirming that edema phenotypes are due to reduced GFB integrity.