Expression data from cultured WT and NCK1/2 knockout mouse podocyte cells

Background: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins to trigger actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 in podocytes remain to be fully elucidated. Methods: We generated podocytes deficient in Nck1 and Nck2, and used transcriptomic approaches to profile expression differences. We further applied proteomic techniques to identify specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2 along with podocyte injury models to comprehensively verify our findings. Results: Compound loss of Nck1/2 resulted in altered expression of genes involved in actin binding, cell adhesion and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2, and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling and integrin activation at focal adhesions via α-actinin-4. Furthermore, we found that loss of either Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2 deficient mice, consistent with enhanced susceptibility to podocyte injury. Conclusion: These findings highlight distinct yet complementary roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition and sustaining filtration barrier integrity. We generated conditionally immortalized Widtype and NCK1/2 knockout mouse podocyte cell lines. Podocytes were differentiated for 10 days, after which RNA was isolated for hybridization on Affymetrix microarrays. There were three independent replicates for each line.