KANK deficiency leads to podocyte dysfunction and nephrotic syndrome
Heon Yung Gee, … , Zhe Han, Friedhelm Hildebrandt
Heon Yung Gee, … , Zhe Han, Friedhelm Hildebrandt
Published May 11, 2015
Citation Information: J Clin Invest. 2015;125(6):2375-2384. https://doi.org/10.1172/JCI79504.
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Research Article Nephrology

KANK deficiency leads to podocyte dysfunction and nephrotic syndrome

Abstract

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. In a recent study, 30% of SRNS cases evaluated were the result of monogenic mutations in 1 of 27 different genes. Here, using homozygosity mapping and whole-exome sequencing, we identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, we determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling.

Authors

Heon Yung Gee, Fujian Zhang, Shazia Ashraf, Stefan Kohl, Carolin E. Sadowski, Virginia Vega-Warner, Weibin Zhou, Svjetlana Lovric, Humphrey Fang, Margaret Nettleton, Jun-yi Zhu, Julia Hoefele, Lutz T. Weber, Ludmila Podracka, Andrej Boor, Henry Fehrenbach, Jeffrey W. Innis, Joseph Washburn, Shawn Levy, Richard P. Lifton, Edgar A. Otto, Zhe Han, Friedhelm Hildebrandt

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Figure 6

Effects of Kank2 knockdown on RHO GTPase activity and podocyte migration in cultured podocytes.

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Effects of Kank2 knockdown on RHO GTPase activity and podocyte migration...
(A) Active GTP-bound RHOA precipitated from cultured human podocytes transfected with scrambled (Scr) or KANK2 siRNA using a GST-rhotekin (RBD) pull-down (PD) assay. Ponceau red staining shows the GST proteins used. Compared with control podocytes, podocytes transfected with KANK2 siRNA exhibit a relative increase in RHOA. The efficiency of knockdown by siRNA was confirmed by immunoblotting with an anti-KANK2 antibody. (B) Active GTP-bound forms of RAC1 and CDC42 precipitated from podocytes transfected with scrambled or KANK2 siRNA using a GST-PAK1 (CRIB) pull-down assay. Cells transfected with scrambled control siRNA versus KANK2 siRNA exhibited no significant difference in relative RAC1 and CDC42 activity. Pull-down experiments are representative of more than 3 experiments. (C) Effect of Kank2 knockdown on podocyte migration. Cultured murine podocytes transfected with Kank2 siRNA (red) exhibited less active migration compared with those transfected with scrambled siRNA (black). Decrease in podocyte migration by Kank2 knockdown was rescued by overexpression of human wild-type KANK2 in podocytes (green) but not by overexpression of the KANK2 p.S181G variant (blue). Overexpression of p.S684F resulted in partial rescue, indicating that this mutation is potentially a hypomorphic mutation. Error bars are shown in only one direction for clarity, and the result shown is representative of 3 experiments.