ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling
Heon Yung Gee, … , Edgar A. Otto, Friedhelm Hildebrandt
Heon Yung Gee, … , Edgar A. Otto, Friedhelm Hildebrandt
Published August 1, 2013; First published July 8, 2013
Citation Information: J Clin Invest. 2013;123(8):3243-3253. https://doi.org/10.1172/JCI69134.
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Category: Research Article

ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling

Abstract

Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.

Authors

Heon Yung Gee, Pawaree Saisawat, Shazia Ashraf, Toby W. Hurd, Virginia Vega-Warner, Humphrey Fang, Bodo B. Beck, Olivier Gribouval, Weibin Zhou, Katrina A. Diaz, Sivakumar Natarajan, Roger C. Wiggins, Svjetlana Lovric, Gil Chernin, Dominik S. Schoeb, Bugsu Ovunc, Yaacov Frishberg, Neveen A. Soliman, Hanan M. Fathy, Heike Goebel, Julia Hoefele, Lutz T. Weber, Jeffrey W. Innis, Christian Faul, Zhe Han, Joseph Washburn, Corinne Antignac, Shawn Levy, Edgar A. Otto, Friedhelm Hildebrandt

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Figure 5

Functional analysis of arhgdia knockdown in zebrafish.

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Functional analysis of arhgdia knockdown in zebrafish. (A) Control zeb...
(A) Control zebrafish injected with p53 MO (0.2 mM). p53 MO did not produce any phenotype until 168 hpf (n >100). Scale bars: 1 mm (×3-fold for insets) (A and B). (B) Zebrafish coinjected with an arhgdia MO (0.2 mM) targeting the translation initiation site of zebrafish arhgdia and a p53 MO. At 120 hpf, the arhgdia morphants displayed the nephrosis phenotype of periorbital edema (“bug-eye”; arrows) and total body edema in 70% of embryos (255 of 360). (C) Effects of various RAC1 and RHOA inhibitors on the zebrafish nephrosis phenotype. Drugs were applied at 48 hpf in fish water and replenished every day. The number of arhgdia morphants that showed periorbital edema was counted at 144 hpf and is represented as a percentage. Two RAC1 inhibitors and eplerenone were partially effective in reducing edema, whereas RHOA inhibitors were not. Error bars indicate SDs for greater than 3 independent experiments in C and D. (D) Dose-response curve for the RAC1 inhibitor, which shows the greatest efficacy in C. The IC50 value of the RAC1 inhibitor for reducing periorbital edema was 8.85 μM. conc., concentration. (E) Proteinuria assay by ELISA against a fusion protein of vitamin D–binding protein and GFP in /-fabp::VDBP-GFP transgenic zebrafish. Note that knockdown of arhgdia caused significant proteinuria compared with the control. The RAC1 inhibitor was partially effective in reducing proteinuria in arhgdia morphants, whereas the RHO inhibitor Y-27632 was not. Error bars indicate the SEM for more than 3 independent experiments.