Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers 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 adaptors in podocytes remain to be fully elucidated.

We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified 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.

Compound loss of Nck1/2 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 at focal adhesions via α actinin-4. Furthermore, loss of 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.

These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.

肾脏过滤屏障的维持需要足细胞和下面的肾小球基底膜（GBM）之间的协调相互作用。GBM 配体结合足细胞整合素，从而触发对粘附至关重要的基于肌动蛋白的信号传导事件。Nck1/2 接头已成为足细胞细胞骨架动力学的重要调节因子。然而，足细胞中 Nck1/2 接头介导的精确信号传导机制仍有待充分阐明。

我们生成了 Nck1 和 Nck2 缺陷的足细胞，并使用转录组学方法来分析表达差异。蛋白质组学技术鉴定了足细胞中 Nck1 和 Nck2 的特异性结合伙伴。我们使用培养的足细胞和 Nck1 和/或 Nck2 缺陷的小鼠以及足细胞损伤模型来全面验证我们的发现。

Nck1/2 的复合缺失改变了参与肌动蛋白结合、细胞粘附和细胞外基质组成的基因表达。因此，Nck1/2缺陷的足细胞在体外表现出肌动蛋白组织和细胞粘附的缺陷，在体内存在足细胞脱离和GBM形态改变。我们鉴定了 Nck1 和 Nck2 的不同相互作用组，并揭示了 Nck1 和 Nck2通过 α actinin-4合作调节粘着斑处肌动蛋白成束的机制。此外，Nck1或Nck2的缺失导致体内基质沉积增加，Nck2缺陷小鼠的缺陷更明显，这与足细胞损伤的易感性增强一致。

这些发现揭示了 Nck 蛋白在调节足细胞粘附、控制 GBM 组成和维持过滤屏障完整性方面的独特但互补的作用。