Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome
Jia Rao, … , Jose C. Martins, Friedhelm Hildebrandt
Jia Rao, … , Jose C. Martins, Friedhelm Hildebrandt
Published October 23, 2017
Citation Information: J Clin Invest. 2017;127(12):4257-4269. https://doi.org/10.1172/JCI94138.
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Research Article Nephrology

Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome

Abstract

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avil or PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein.

Authors

Jia Rao, Shazia Ashraf, Weizhen Tan, Amelie T. van der Ven, Heon Yung Gee, Daniela A. Braun, Krisztina Fehér, Sudeep P. George, Amin Esmaeilniakooshkghazi, Won-Il Choi, Tilman Jobst-Schwan, Ronen Schneider, Johanna Magdalena Schmidt, Eugen Widmeier, Jillian K. Warejko, Tobias Hermle, David Schapiro, Svjetlana Lovric, Shirlee Shril, Ankana Daga, Ahmet Nayir, Mohan Shenoy, Yincent Tse, Martin Bald, Udo Helmchen, Sevgi Mir, Afig Berdeli, Jameela A. Kari, Sherif El Desoky, Neveen A. Soliman, Arvind Bagga, Shrikant Mane, Mohamad A. Jairajpuri, Richard P. Lifton, Seema Khurana, Jose C. Martins, Friedhelm Hildebrandt

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Figure 5

The ARP complex is an effector for podocyte migration downstream of EGF, AVIL, and PLCE1.

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The ARP complex is an effector for podocyte migration downstream of EGF,...
(A) Human podocytes transfected with WT Avil (green) or PLCE1 (blue) showed a significant increase in the PMR compared with mock (black). Overexpression of Avil mutants failed to increase the PMR. Mutations: Arg135Gln (red triangle dotted line), Leu425Met (red rhombus solid line), and Phe656Valfs*7 (red cross dotted line). (B) Knockdown of AVIL (pink) showed a strongly reduced PMR compared with scrambled shRNA (black), which was partially rescued by overexpression of WT Avil (green) but not by mutants. (C) EGF stimulation increased the PMR in scrambled shRNA (black solid line). The PMR reduction upon AVIL knockdown (pink dotted line) was partially rescued by EGF treatment (pink solid line), but overexpression of PLCE1 (blue dotted line) showed no additional rescue effect over EGF (blue solid line). (D) The PMR reduction upon PLCE1 knockdown (turquoise dotted line) was partially rescued by EGF (turquoise solid line). Overexpression of AVIL (green dotted line) failed to rescue the PMR reduction in PLCE1-knockdown cells (turquoise dotted line). Overexpression of Avil showed no additional rescue effect over EGF treatment (green solid line). (E) Human podocytes transfected with the scrambled control showed a decreased PMR upon treatment with the ARP inhibitor CK666 (black solid line) compared with the scrambled control without CK666 treatment (black dotted line). The PMR increase upon overexpression of WT Avil (green dotted line) or PLCE1 (blue dotted line) was blocked by the addition of CK666 (green or blue solid lines, respectively). (F) Human podocytes were pretreated with CK666 for 45 minutes before the wound scratch was performed. Migration assays with scrambled shRNA as a negative control were conducted with (gray solid line) or without (black dotted line) EGF stimulation following the wound scratch in podocytes. The reduction in PMR upon AVIL knockdown (pink dotted line) was rescued by EGF (green solid line), but was not rescued in the presence of CK666 (brown solid line). The relative migration rate and wound density (percentage) were calculated by IncuCyte assay. Multiple scratch wounds were made in confluent cells. Scratch wounds were allowed to heal for 24 hours, and the PMR was plotted on graphs.