A vesicular trafficking pathway mediated by N-ethylmaleimide-sensitive factor governs integrin maturation and podocyte integrity.

Abstract

Podocyte adhesion to the glomerular basement membrane is essential for kidney function, and adhesion failure leads to podocyte loss and glomerulosclerosis. However, how initial adhesion impairment progresses to irreversible podocyte dysfunction remains poorly understood. Here, we investigated early molecular changes following adhesion impairment to identify new therapeutic targets. We performed single-cell RNA sequencing on kidneys from doxycycline-inducible podocyte-specific Tln1 knockout (Tln1-cKO) mice, an established model of impaired focal adhesion, before overt kidney dysfunction. To test the function of a key downregulated gene, we generated podocyte-specific Nsf knockout (Nsf-cKO) mice and analyzed them using biochemical, cell biological, and live-cell imaging approaches. We also examined publicly available human kidney single-nucleus RNA sequencing datasets. Nsf was among the most significantly downregulated genes in Tln1-cKO podocytes. NSF (N-ethylmaleimide-sensitive factor) is an AAA+ ATPase essential for vesicular trafficking through disassembly of SNARE complexes. Nuclear Yes-associated protein (YAP) was reduced in Tln1-cKO podocytes, and pharmacological inhibition of TEA domain transcription factor (TEAD) or soft-substrate culture lowered NSF expression, indicating that YAP/TEAD signaling links mechanotransduction to Nsf expression. Podocyte-specific Nsf deletion caused massive proteinuria, podocyte loss, glomerulosclerosis, and death by three weeks. NsfcKO podocytes showed impaired spreading and defective integrin regulation: integrin α3 maturation was blocked, while integrin β1 underwent ER stress-induced proteasomal degradation. Trafficking of nephrin and podoplanin was also affected. Live-cell imaging showed markedly delayed ER-to-Golgi trafficking in Nsf-cKO podocytes (90-120 minutes versus 15 minutes in controls). In human datasets, NSF expression was reduced in podocytes from patients with diabetic and hypertensive chronic kidney disease. Adhesion impairment suppresses NSF through reduced YAP/TEAD activity, disrupting ER-to-Golgi trafficking and integrin maturation, and generating a feed-forward cascade of progressive adhesion failure. NSF-mediated trafficking is essential for podocyte homeostasis and a potential therapeutic target in proteinuric kidney diseases.