Nephrin Trafficking in the Mouse Model of Experimental Autoimmune Nephrotic Syndrome

Background: Nephrin is an essential protein expressed at the slit diaphragm of podocytes and plays a crucial role in maintaining the integrity of the glomerular filtration barrier. We previously developed a novel experimental autoimmune mouse model resembling idiopathic nephrotic syndrome in humans, induced by immunization with Crb2, a slit diaphragm protein that interacts with nephrin. While proper trafficking of nephrin to the podocyte surface is critical for its function, and impaired nephrin turnover contributes to podocyte injury, the mechanisms underlying nephrin trafficking in this model remain unknown. Methods: Cell surface nephrin expression following anti-Crb2 antibody stimulation was studied by biotinylation and western blotting using immortalized human podocytes. Nephrin expression and localization in mouse kidney were evaluated by western blotting and immunofluorescence staining. Markers of endocytosis and autophagy were used to identify the trafficking pathways involved. Results: Cell surface expression of nephrin was significantly reduced following anti-Crb2 antibody stimulation. In Crb2-immunized mouse kidney, nephrin staining intensity decreased, and its reduced expression was confirmed by western blotting. Nephrin showed increased colocalization with the endocytosis markers Rab5 and Rab7, indicating enhanced endocytosis. Furthermore, nephrin colocalized with the autophagy marker LC3. Triple staining for nephrin, LC3 and Rab7 was also increased, suggesting nephrin processing via the amphisomal pathway. Additionally, colocalization of nephrin with the lysosomal marker LAMP1 was increased, indicating lysosomal degradation of nephrin. Conclusion: Nephrin undergoes endocytosis and is degraded via the amphisome-lysosome pathway in the Crb2-induced nephrotic syndrome mouse model, providing new insights into the pathogenesis of podocyte injury in nephrotic syndrome.