N-ethylmaleimide Sensitive Factor (NSF) Is Essential for Maintaining Podocyte Focal Adhesion

Background: Focal adhesion proteins in podocytes are critical for attaching to the glomerular basement membrane. Reduction in these proteins causes podocyturia, frequently observed in focal segmental glomerulosclerosis patients. We aimed to explore the regulation of focal adhesion molecules in podocytes. Methods: We used a mouse model in which the Talin1 gene (Tln1) can be specifically knocked out in podocytes by administering doxycycline (Dox). These mice, referred to as Tln1 cKO mice, develop progressive proteinuria and eventually experience renal failure (J Clin Invest. 2019 1;129(3):1295-13). We focused on the early phase of podocyte injury by examining Tln1 cKO mice 10 days after Dox treatment (Tln1 cKO 10d), as they showed proteinuria without elevated serum creatinine at this stage. We performed single-cell RNA sequencing (scRNAseq) on kidney samples from Tln1 cKO 10d mice and analyzed the differentially expressed genes (DEGs). Based on the DEG analysis results, we generated another mouse model with podocyte-specific knockout of the Nsf gene (Nsf cKO mice). Using primary cultured podocytes from these Nsf cKO mice, we investigated the role of NSF in focal adhesion. Results: Nsf was significantly down-regulated in Tln1 cKO 10d mice podocytes. Notably, Nsf cKO mice developed proteinuria as early as 2 weeks of age and succumbed to end-stage kidney disease by 9 weeks. Nsf cKO podocytes showed a marked reduction in cell spreading, decreased mature Integrin α3β1, and increased immature Integrin α3. Immunofluorescence staining indicated expansion of the ER and ER-Golgi intermediate compartment (ERGIC) area in Nsf cKO podocytes. Additionally, COP2 and COP2 associated SNARE protein increased in Nsf cKO podocytes. These findings suggested that the deletion of Nsf in podocytes leads to a disruption in ER-to-Golgi transport, thereby impairing the trafficking of focal adhesion proteins such as Integrin α3. Conclusion: NSF plays a crucial role in maintaining podocyte focal adhesions by regulating ER-to-Golgi transport and the trafficking of proteins such as Integrin α3β1. This study provides new insights into the molecular mechanisms of podocyte injury and kidney disease progression.