#718 N-Ethylmaleimide Sensitive Factor (NSF) is essential for maintaining podocyte focal adhesion
Authors: Imai Atsuhiro , Inoue Kazunori , Okushima Hiroki , Katsuma Yusuke , Matsumoto Ayumi , Matsui Isao , Isaka Yoshitaka 
Published: May 2024

Abstract

Background and Aims
Cell-matrix adhesion molecules in podocytes play a critical role in attaching these cells to the glomerular basement membrane, ensuring the proper functioning of the glomerular filtration barrier. Previous studies have shown that patients with proteinuric glomerular diseases often have higher levels of podocyturia. This increase is notably linked to a reduction in focal adhesion proteins, especially in cases of focal segmental glomerulosclerosis. Our study aims to explore the regulation of these focal adhesion molecules in podocytes.

Method
Doxycycline (Dox) inducible podocyte-specific Tln1 knockout mice (Tln1fl/fl Nphs2-rtTA TetO-Cre (Tln1 KO)) were used as the model for podocyte injury. These mice are genetically modified to lack Talin 1, a key protein in focal adhesion. The absence of Talin 1 in these mice leads to the development of pathological changes akin to focal segmental glomerulosclerosis, characterized by extensive proteinuria, thereby providing a valuable model for understanding this kidney disease (J Clin Invest. 2019 1;129(3):1295-13). As Tln1 KO mice 10 days after Dox induction (Tln1 KO 10 d) showed proteinuria without elevated serum creatinine, these mice were considered as the early phase of podocyte injury. Single-cell RNA sequencing (scRNAseq) was performed using these mice kidney samples, followed by differentially expressed genes (DEG) analyses. Podocyte-specific Nsf KO (Nphs2-Cre Rosa-DTRflox Nsf fl/fl (Nsf cKO)) mice were generated based on the result of the DEG analyses and the effect of NSF on focal adhesion was examined using primary podocyte culture.

Results
Nsf was identified as one of the most significantly down-regulated DEGs in the podocytes ofTln1 KO 10d mice. Further analysis of public scRNAseq data revealed a decrease in Nsf mRNA expression levels also in lipopolysaccharide-treated mouse podocytes. Notably, proteinuria was observed in Nsf conditional knockout (cKO) mice as early as 2 weeks of age, with all Nsf cKO mice succumbing to end-stage kidney disease by 6 weeks post-birth. Examination of isolated mutant podocytes showed a marked reduction in cell spreading. Additionally, a significant decrease in Integrin β1 expression was observed in primary podocytes of Nsf cKO mice, as evidenced by immunoblot analysis. In vivo studies using transmission electron microscopy revealed endoplasmic reticulum (ER) expansion in the podocytes of Nsf cKO mice. Immunofluorescence staining indicated a substantial increase in the ER and ER-Golgi intermediate compartment (ERGIC) in isolated Nsf cKO podocytes. These findings suggest that the deletion of Nsf in podocytes leads to a disruption in ER-to-Golgi protein transport, subsequently impairing the trafficking of focal adhesion proteins.

Conclusion
This study provides new insights into the molecular mechanisms underlying podocyte injury and the progression of kidney diseases. NSF plays a pivotal role in maintaining podocyte focal adhesion.