Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease
Britta George, … , Maria Pia Rastaldi, Lawrence B. Holzman
Britta George, … , Maria Pia Rastaldi, Lawrence B. Holzman
Published February 1, 2012; First published January 17, 2012
Citation Information: J Clin Invest. 2012;122(2):674-692. https://doi.org/10.1172/JCI60070.
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Categories: Research Article Nephrology

Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

Abstract

The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation — markers of focal adhesion complex–mediated Crk-dependent signaling — was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2–dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases.

Authors

Britta George, Rakesh Verma, Abdulsalam A. Soofi, Puneet Garg, Jidong Zhang, Tae-Ju Park, Laura Giardino, Larisa Ryzhova, Duncan B. Johnstone, Hetty Wong, Deepak Nihalani, David J. Salant, Steven K. Hanks, Tom Curran, Maria Pia Rastaldi, Lawrence B. Holzman

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Figure 1

Crk1/2 interacts with nephrin in a tyrosine phosphorylation–dependent fashion.

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Crk1/2 interacts with nephrin in a tyrosine phosphorylation–dependent fa...
(A) Purified recombinant GST-nephrinCD expressed in BL21 or TKB1 E. coli (to produce nonphosphorylated or tyrosine-phosphorylated nephrin, respectively) or purified GST alone was mixed with rat glomerular lysate, pulled down with glutathione agarose, and immunoblotted with monoclonal anti-Crk1/2 antibody. (B) Glomerular lysates from rats injected with PBS (control) or PAN were immunoprecipitated and/or immunoblotted using the indicated antibodies. (C) Cultured human podocytes expressing CD16/7-nephrinCD (CD16NCD) or CD16/7-HA (CD16HA) and Crk2-myc were activated by clustering: namely, addition of monoclonal anti-CD16 primary antibody (1°) and/or goat anti-mouse IgG Texas Red–conjugated secondary antibody (2°), as indicated, to the media of live cells. Crk2-myc was detected with rabbit polyclonal anti-myc primary antibody and Alexa Fluor 488–labeled secondary antibody. Cells were analyzed by confocal microscopy. CD16/7-nephrinCD (red) and Crk2-myc (green) colocalized in the plane of the plasma membrane. (D) SYF MEFs transiently expressing CD16/7-nephrinCD and Crk-GFP were activated by clustering. Although colocalization of nephrin and Crk was not observed in SYF MEFs, nephrin-Crk association was rescued by reexpressing Fyn, but not by expressing a kinase-dead variant of Fyn (FynKD). (C and D) Original magnification, ×630. yz plane reconstructions are shown at far right.