Fig 2: Expression of CD40 in glomeruli and cultured GECs.Panel A. Detection of CD40 by flow cytometry in cultured human GECs. Panel B. Indirect immunofluorescence staining for CD40 in cultured human GECs (x400). Panel C. Representative PCR gels for CD40 expression in cultured human (upper panel) and mouse (lower panel) GECs. Lane 1: 100 bp molecular weight markers; Lane 2: PCR for CD40, no cDNA control; Lane 3: PCR for CD40. Panel D. Representative PCR gels for CD40 expression in primary cultures of rat GECs and rat glomeruli. Lane 1: 100 bp molecular weight markers; Lane 2: PCR for CD40, no cDNA control; Lane 3: PCR for CD40 on primary cultures of rat GECs. Lane 4: PCR for CD40 on rat glomeruli. Panel E. Indirect immunofluorescence staining for CD40 in frozen mouse kidney sections (x63). Images are representative of at least 3 separate experiments with similar results.

Fig 3: Effect of the partially purified Permeability Factor (PF), prepared from plasmapheresis eluates from patients who presented post-transplant recurrence of FSGS, on nephrin expression in cultured GECs (A) and on Permeability activity of albumin (Palb) in isolated rat glomeruli (B). (A) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with PF (500 ng/ml) for 30 min in the absence (black bar) or presence of different inhibitors of CD40-CD40L interaction. Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to GEC stimulation, whereas the CD40L-muCD8 fusion protein (10 μg/ml) was added to cultured GECs 30 minutes before adding hr-sCD40L (see Methods for details). **P < 0.01 versus unstimulated control and PF + inhibitors of CD40-CD40L interaction. Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (B) Permeability activity of albumin (Palb) induced by PF on isolated rat glomeruli: Palb was determined after the incubation of rat glomeruli with PF (500 ng/ml), as detailed in the Methods section. A significant increase in glomerular permeability is expressed by values of Palb greater than 0.5 (black bar). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to glomerulus stimulation, whereas the CD40L-muCD8 fusion protein (10 μg/ml) was added to the glomeruli 10 minutes before adding PF. At least five animals were studied per each experimental group.

Fig 4: Effect of sCD40L on nephrin expression and cytoskeleton organization in cultured GECs.(A-C) Micrographs representative of immunofluorescence staining for nephrin in non-permeabilized GECs: (A) GECs incubated with vehicle alone for 30 min; (B) GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min; (C) Effect of pretreatment with an inhibitor of CD40-CD40L interaction, CD40-muIg fusion protein (20 ng/ml), on loss of nephrin induced by hr-sCD40L. Original magnification: ×400 (F-H). Bars = 10 μm. Images are representative of at least 5 separate experiments with similar results. (D) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with various concentrations of hr-sCD40L for 30 min (upper graph), and of the time-course effect of incubation of GECs with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer; dashed bars) or vehicle alone (open bars) on nephrin expression, as detected by immunofluorescence staining (lower graph). Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (E-G) Micrographs representative of fluorescein isothiocyanate phalloidin staining of actin microfilaments in permeabilized GECs. (E) GECs incubated with vehicle alone for 30 min; (F) GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min; (G) Effect of pretreatment with an inhibitor of CD40-CD40L interaction, CD40-muIg (20 ng/ml), on reorganization of cytoskeleton induced by hr-sCD40L. Original magnification: ×400 (D-I). Bars = 10 μm. Images are representative of at least 5 separate experiments with similar results. (H) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min in the absence (black bar) or presence of different inhibitors of CD40-CD40L interaction (dashed bars). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to GEC stimulation, whereas the CD40L-muCD8 fusion protein (50 ng/ml) was added to cultured GECs 30 minutes before adding hr-sCD40L (see Methods for details). Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (I) Immunoblot of a representative experiment on the effect of hr-sCD40L and PF on nephrin expression as detected by Western blot analysis. GEC lysates were immunoblotted with antibodies anti-nephrin or Beta-actin after incubation with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) or PF (500 ng/ml), in the presence or absence of CD40-muIg fusion protein (20 ng/ml) to inhibit CD40-CD40L interaction. Blots are representative of three independent experiments with similar results.