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. IntroductionWhen functioning properly in health, the kidney filtration barrier selectively prevents the passage of macromolecules from the blood compartment into the urinary space. Differentiated podocytes form a remarkable octopus-like morphology, extending numerous interdigitating foot processes defined by a unique 3-dimensional actin cytoskeletal architecture and requiring formation of a specialized intercellular junction. These foot processes adhere to and cover an extracellular matrix interposed between podocytes and an endothelium that creates the glomerular capillary wall. Podocytes undergo cytoskeletal remodeling to alter their morphology in nearly all forms of human glomerular disease, exhibiting what has been described as foot process spreading and retraction or as foot process effacement. This process by which podocytes change their cytoskeletal architecture appears to be a component of a common response of the podocyte to cellular injury, correlating with loss of normal filtration barrier selectivity and predicting the development of proteinuria in human disease and in experimental models (1, 2).

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture

“…53 In concordance with our findings, a number of recent works has established a nephrinindependent pathway resulting in Rac activation as an important pathomechanism to drive effacement. 13,54,55 Instead, we find that foot process recovery is impaired in nephrin Y3F/Y3F animals in both injury models, similar to that observed in mice lacking the actin regulators synaptopodin and cofilin. 56,57 Taken together, our data support a model wherein nephrin tyrosine phosphorylation is required to counteract normal podocyte stress and loss of this phosphorylation, as seen in nephrin Y3F/Y3F mice and some human diseases, leads to irreversible injury and chronic disease.…”

“…Within this segment, there exists a number of highly conserved tyrosine (Y) residues ( Table 1) that, on phosphorylation by Fyn kinase, [7][8][9] serve as docking sites for intracellular signaling proteins ( Figure 1A). 10 Through recruitment of actin adaptors, such as p85/PI3K, 11,12 the Cas/Crk complex, 13 and Nck1/2, 8,9,14 nephrin phosphorylation is postulated to facilitate direct and dynamic connection to the podocyte cytoskeleton. Moreover, Nck enhances nephrin phosphorylation via activation of Fyn, 15 and loss of Nck within podocytes leads to reduced nephrin tyrosine phosphorylation and widespread foot process effacement, 15,16 inferring a reciprocal relationship between Nck and nephrin in the maintenance of podocyte structure.…”

Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance

“…Another conceivable explanation is that the loss of talin1 in podocytes results in a modest reduction in cell spreading, thus preventing the podocytes from tightly covering the glomerular capillaries. However, previous results in mice specifically lacking focal adhesion proteins, FAK, or Crk in podocytes revealed no evidence of proteinuria, even though cell spreading was compromised (40,52).…”

“…We next sought to determine the role of talin1 following podocyte injury in an intact organism. Therefore, we perfused mice with protamine sulfate, an agent that has been shown to induce podocyte foot process effacement (40). Compared with Tln1 fl/fl mice glomeruli perfused with HBSS, glomeruli isolated from mice treated with protamine sulfate consistently demonstrated an unexpectedly striking talin1 cleavage product ( Figure 8A; quantified in Supplemental Figure 4A).…”

Involvement of nephrin in human placental trophoblast syncytialization

“…Nephrin is a central component of the slit diaphragm in glomerular podocytes (Ruotsalainen et al 1999). The cell adhesion activities of nephrin have been confirmed in kidney podocytes and HEK-293 cells (Khoshnoodi et al 2003, George et al 2012. It is well established that nephrin plays a critical role in the renal ultrafilter.…”