MO009NEUTRALIZATION OF UPAR WITH AN ANTI-UPAR ANTIBODY AMELIORATES RECURRENT FSGS SERA INDUCED PODOCYTE INJURY
Authors: Vincenti Flavio , Rashmi Priyanka , Da Silva Andrea Alice , Shoji Jun , Craik Charles , Sarwal Minnie 
Published: June 2020

Abstract

Background and Aims
The role of suPAR as a biomarker and/or causative factor in the pathogenesis of recurrent FSGS remains unclear (Harel E., et al. Transplantation 2020; 104:54-60). While anti-suPAR antibodies have been shown to block suPAR induced podocyte injury in mouse models of FSGS, this beneficial effect has not yet been demonstrated in FSGS patients.
We report here the inhibitor effects of 2G10, a fully human anti-suPAR antibody that blocks the interaction of suPAR with the B3 integrin on podocytes.

Method
The immortalized podocyte cell line was developed by transfection with the temperature sensitive SV40 T gene. These cells proliferate at the “permissive” temperature (33°C) and are considered undifferentiated. After transferring to the “nonpermissive” temperature (37°C), they enter growth arrest and by day 10-14 express markers of differentiated podocytes in vivo, such as nephrin, podocin, CD2 associated protein (CD2AP), synaptopodin, and known molecules of the slit diaphragm ZO-1, α, β, and γ-catenin, and P-cadherin. The podocytes were cultured in RPMI medium supplemented with insulin, transferrin, selenium, sodium pyruvate (ITS-A, Gibco #513000), 10% FBS and penicillin/streptomycin. After differentiation for 14 days, cells were serum starved for 1h. Serum from rFSGS patients or from a control patient was added (4% final) and cells were cultured for an additional 24h. After fixation in PFA/sucrose. actin cytoskeleton was visualized by labeling with rhodamine-conjugated phalloidin. DAPI was used for nuclei staining. Cells were imaged by confocal microscopy at 40X magnification and the number of cells with intact stress fibers were counted. For rescue of stress fibers, podocytes were cultured in the presence of a fully humanized anti uPAR antibody (2G10, 1 ug/ml; Duriseti S, J Biol Chem, 2010, 285:26878-88) or an isotype IgG control antibody (1 ug/ml).

Results
Sera from three patients with recurrence of FSGS after transplant were used in the study. Podocyte culture in the presence of sera from all three patients caused significant depolarization of stress fibers as determined by number of stress fiber positive cells (30%, 59% and 49% reduction with respect to untreated podocytes respectively). Treatment of podocytes with control sera did not cause any significant changes (data not shown). Culture of podocytes with patient sera in the presence of 2G10 antibody against uPAR rescued stress fibers (Fig 1A and 1B). On the other hand, a control human IgG was unable to rescue the loss of stress fibers induced by sera from recurrent FSGS patients.

Conclusion
The therapeutic potential of a human anti-suPAR antibody in samples from patients with recurrent FSGS has not been previously demonstrated. The in vitro findings of 2G10 antibody on preserving the stress fibers in human podocytes from the disrupting effect of the sera of patients with recurrent FSGS suggest that antibodies that block suPAR could be effective in preventing recurrence of FSGS.