Inhibition of Podocyte FAK Protects against Proteinuria and Foot Process Effacement

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays a critical role in cell motility. Movement and retraction of podocyte foot processes, which accompany podocyte injury, suggest focal adhesion disassembly. To understand better the mechanisms by which podocyte foot process effacement leads to proteinuria and kidney failure, we studied the function of FAK in podocytes. In murine models, glomerular injury led to activation of podocyte FAK, followed by proteinuria and foot process effacement. Both podocyte-specific deletion of FAK and pharmacologic inactivation of FAK abrogated the proteinuria and foot process effacement induced by glomerular injury. In vitro, podocytes isolated from conditional FAK knockout mice demonstrated reduced spreading and migration; pharmacologic inactivation of FAK had similar effects on wild-type podocytes. In conclusion, FAK activation regulates podocyte foot process effacement, suggesting that pharmacologic inhibition of this signaling cascade may have therapeutic potential in the setting of glomerular injury.The glomerulus forms the filtration barrier of the kidney and is composed of a fenestrated endothelium, glomerular basement membrane (GBM), and the podocytes that interdigitate to form slit diaphragms.^1,2 When the podocytes are damaged, foot process fusion occurs. This process involves the rearrangement of the actin cytoskeleton and retraction of the foot processes toward the cell body, allowing mechanical forces and signaling events to be transmitted into the cell. Since the identification that mutations of the podocyte slit diaphragm specific NPHS1 gene cause congenital nephrotic syndrome,^3–5 podocytes have been recognized as critical regulators of glomerular injury. Other podocyte slit diaphragm proteins such as podocin, synaptopodin, and CD2AP have generated further interest in the regulation of the kidney filtration barrier^6–8; however, little is still known about cell–matrix interactions in podocytes. Mice lacking the focal adhesion protein integrin-linked kinase (ILK), specifically in the podocytes, also develop proteinuria, resulting in renal failure and death.⁹ Moreover, mice lacking α3β1 integrin have demonstrated inability to form mature foot processes.¹⁰ These cell–matrix interactions, which seem important in podocyte development, may also play a critical role after podocyte injury, because the process of podocyte effacement requires cell process retraction and movement, processes that suggest focal adhesion disassembly.Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase, in which integrin- or growth factor–induced autophosphorylation at tyrosine 397 results in activation of critical signaling pathways required for focal adhesion turnover.^11–16 It has been demonstrated that cell spreading and migration are significantly diminished in cells lacking FAK.¹⁷ This inhibition in motility has brought excitement in cancer therapeutics, resulting in the development and use of FAK inhibitors.^18–21 In a recent study, inhibition of urokinase plasminogen activator (uPAR), a glycosylphosphatidylinositol-anchored protein that is important for cell invasion and metastasis, has been demonstrated to reduce proteinuria and podocyte effacement significantly, suggesting that this dynamic podocyte cell movement may mimic the molecular signaling events observed in cancer cell invasion.²²In this study, we demonstrated that after podocyte injury in vivo and in vitro, FAK activation was significantly increased in wild-type (WT) mice, prompting us to address whether inhibition or loss of FAK activation would reduce podocyte cell motility by inhibiting focal adhesion turnover, thereby preventing proteinuria and effacement. Because complete FAK gene deletion results in lethality at embryonic day 8.5, a time point before glomerular development has been initiated, the ability to study this protein''s role in podocyte development as well as repair after injury has been limited.¹⁷ Hence, selective loss of FAK expression in the podocytes of the kidney was achieved using a Cre-loxP approach.^23,24 These mice were born without evidence of podocyte/glomerular developmental defects but were resistant to the foot process fusion and subsequent proteinuria that typically accompany LPS and rabbit anti-mouse GBM-induced podocyte damage. We postulate this inhibition of foot process effacement is due to diminished podocyte spreading and motility, supported by our in vitro data. In addition, pharmacologic treatment of WT mice using the FAK inhibitor TAE-226 significantly reduced proteinuria and podocyte effacement, raising the possibility for therapeutic use in glomerular diseases.