Protective effects of rituximab on puromycin-induced apoptosis, loss of adhesion and cytoskeletal alterations in human podocytes

Abstract

Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes. We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury. A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods. Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data, Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8, -9 and -3, suggesting that rituximab may inhibit apoptosis. In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes.

Cytoskeleton Rearrangement in Podocytopathies: An Update

“…RTX can directly affect the actin cytoskeleton and prevent the reduced expression of nephrin and podocin [ 149 ]. In an in vitro PAN model, treatment with RTX dramatically prevented disruption of the actin cytoskeleton and increased podocyte actin stress fibers, which was related to a substantial enhancement in cell adhesion [ 150 ].…”

Diagnostik und Therapie der Fokal-Segmentalen Glomerulosklerose – 2023

“…Bei der primären FSGS wird als Initialereignis von einer immun-mediierten, direkten Podozytenschädigung ausgegangen. Hierfür spricht vor allem das Ansprechen auf immunsuppressive Maßnahmen, wobei auch direkte podozytenstabilisierende Effekte von GC, CNI und Rituximab beschrieben wurden und der Beweis einer zugrundeliegenden Autoimmunität bis zuletzt ausständig ist [ 2 , 4 , 5 ]. Einige Beobachtungen, wie beispielsweise eine unmittelbare und oft fulminante Rekurrenz der Grunderkrankung direkt nach Nierentransplantation sowie das therapeutische Ansprechen auf Plasmaaustausch, sprechen für das Vorliegen eines zirkulierenden Permeabilitätsfaktors, beispielsweise eines Zytokins [ 6 ].…”