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 July 8, 2013
Citation Information: J Clin Invest. 2013;123(8):3243-3253. https://doi.org/10.1172/JCI69134.
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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 3

Effects of disease-causing ARHGDIA mutations on protein-protein interaction, RHO GTPase activity, and podocyte migration.

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Effects of disease-causing ARHGDIA mutations on protein-protein interact...
(A) Interaction of wild-type ARHGDIA and 2 mutants (p.R120X and p.G173V) with RHO GTPases. FLAG-tagged ARHGDIA constructs were transfected into podocytes and were coimmunoprecipitated with endogenous RHO GTPases. Note that the R120X and G173V mutants abrogated interaction with RHOA, RAC1, and CDC42. (B) Active GTP-bound forms of RAC1 and CDC42 precipitated from podocytes expressing FLAG-ARHGDIA (wild-type and mutants) using a GST-PAK1 (CRIB, CDC42, and RAC interactive binding domain) pulldown assay. Five percent input represents the controls for equal loading. Note that, compared with mock cells, podocytes expressing ARHGDIA-WT exhibited a substantial decrease in active RAC1 and CDC42. This decrease was abrogated in the null mutant R120X and is diminished in the G173V mutant. (C) Active GTP-bound RHOA precipitated from podocytes expressing FLAG-ARHGDIA (wild-type and mutants) using a GST-rhotekin (RHO-binding domain [RBD]) pulldown assay. Overexpression of either wild-type or mutant ARHGDIA resulted in a substantial decrease in relative RHOA activity compared with mock cells. PD, pulldown. All IPs and PDs are representative of more than 3 experiments. (D) Effect on podocyte migration of wild-type ARHGDIA and 2 mutants found in patients with SRNS. Migration assay was performed using the xCELLigence system (described in Methods). Overexpression of wild-type ARHGDIA in podocytes inhibited serum-induced migration (green). However, the mutants G173V and R120X failed to inhibit migration (red). Error bars are shown in one direction only for clarity and indicate SDs for more than 3 independent experiments (see also Supplemental Figure 5).