Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis

Abstract

Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized by proteinuria, progression to end-stage renal disease, and recurrence of proteinuria after kidney transplantation in about one-third of patients. It has been suggested that rituximab might treat recurrent FSGS through an unknown mechanism. Rituximab not only recognizes CD20 on B lymphocytes, but might also bind sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) protein and regulate acid sphingomyelinase (ASMase) activity. We hypothesized that rituximab prevents recurrent FSGS and preserves podocyte SMPDL-3b expression. We studied 41 patients at high risk for recurrent FSGS, 27 of whom were treated with rituximab at time of kidney transplant. SMPDL-3b protein, ASMase activity, and cytoskeleton remodeling were studied in cultured normal human podocytes that had been exposed to patient sera with or without rituximab. Rituximab treatment was associated with lower incidence of posttransplant proteinuria and stabilization of glomerular filtration rate. The number of SMPDL-3b(+) podocytes in postreperfusion biopsies was reduced in patients who developed recurrent FSGS. Rituximab partially prevented SMPDL-3b and ASMase down-regulation that was observed in podocytes treated with the sera of patients with recurrent FSGS. Overexpression of SMPDL-3b or treatment with rituximab was able to prevent disruption of the actin cytoskeleton and podocyte apoptosis induced by patient sera. This effect was diminished in cultured podocytes where SMPDL-3b was silenced. Our study suggests that treatment of high-risk patients with rituximab at time of kidney transplant might prevent recurrent FSGS by modulating podocyte function in an SMPDL-3b-dependent manner.

Figure 1. CD20 is not expressed in human podocytes, while SMPDL-3b is expressed in podocyte lipid rafts

(A) Immunofluorescence staining for CD20 (top, green) or SMPDL-3b (bottom, green) and synaptopodin (both, red) is shown on frozen tissue sections of a normal human kidney. Human lymph node tissue was used as a positive control for CD20. An irrelevant IgG served as negative control. Images were acquired at 63× magnification. (B)- FITC-rituximab (FITC-RITUX; 10 μg/ml) binds to normal kidney sections. Binding of FITC-RITUX can be blocked by pre-incubation with an SMPDL-3b blocking peptide. FITC-IgG1 and FITC-IF5 anti-CD20 monoclonal antibodies were used as negative controls and did not show any binding to human kidney section at 10 μg/mL. (C) Immunoperoxidase amplification staining of rituximab (10 μg/ml) binding to normal human podocytes (shown in brown) can be prevented by pre-incubation with the SMPDL-3b blocking peptide. Binding of rituximab to Raji cells, which express CD20, was used as positive control (63×1.7× magnification was utilized). (D) Cultured, differentiated human podocytes do not express CD20, as assessed by mRNA expression levels for both transcript variant 1 (v1, 200 bp) and 3 (v3, 240 bp) using standard RT-PCR in cultured differentiated human podocytes (Podo). Raji cell line was the positive control. (E) CD20 protein expression was also analyzed in both cultured differentiated human podocytes and Raji cells by Western blotting. A unique band of 35 kDa was observed in Raji cells, but did not appear in differentiated human podocytes. (F) PCR and Western blot analyses for SMPDL-3b in cultured differentiated human podocytes. Both isoforms 1 (50 kDa) and 2 (40 kDa) could be identified. Actin was utilized as housekeeping gene. (G) Cultured HEK293 cells and differentiated human podocytes were utilized to separate membrane extracts (ME) from cytosolic extracts (CE). ME were identified as EGFR-1+ and CE as AKT+. SMPDL-3b 50 kDa isoform localizes predominantly at the plasma membrane. SMPDL-3b (50 kDa) was only identified in the membrane extract from human podocytes. The 40 kDa isoform was not detected in any fraction, in either podocytes or HEK293 cells. (H) Lipid raft (+) were separated from non-lipid raft (−) fractions to determine if SMPDL-3b localizes specifically to the lipid raft microdomains. Flotillin-1 (48 kDa) was used as a specific raft fraction marker. (I) Lysates of HEK293 cells transfected with GFP–SMPDL-3b were immunoprecipitated with either rituximab (Rit) or with a monoclonal antibody directed against IL8 (αIL8). Both the initial lysates and the immunoprecipitation eluates (IP) were tested for the presence of SMPDL-3b.

Figure 2. Rituximab prevents the downregulation of SMPDL-3b in recurrent FSGS

(A) Low and high power images (at 63× and 63×1.7× magnification) of immunoperoxidase staining for SMPDL-3b (red) and synaptopodin (brown) in post-reperfusion biopsies of patients with recurrent (REC) and non-recurrent (NON-REC) FSGS. Black arrows point to podocytes. (B) Number of SMPDL-3b+ podocytes per glomerulus, as evaluated by SMPDL-3b and synaptopodin labeling in post-reperfusion kidney biopsies from patients that later on developed recurrent (REC) disease (n=8) and patients that did not develop clinical recurrence (NON-REC) (n=12). All kidney biopsies were obtained prior to initiation of treatment with rituximab. An average of 13±4 glomeruli per patient were analyzed. *p<0.05, unpaired Student’s t-test. (C) Regulation of podocyte SMPDL-3b mRNA expression by normal (NHS), non-recurrent (NON-REC) FSGS, and recurrent (REC) FSGS human sera (n=4) and by rituximab. *p<0.05, **p<0.01 by one-way ANOVA. (D) The amount of SMPDL-3b protein is normalized to actin in human podocytes treated with normal (NHS, n=5), REC (n=12), NON-REC (n=10) human sera and exposed to rituximab. *p<0.05, **p<0.01 by one-way ANOVA. (E) Western blot for SMPDL-3b protein of normal podocytes cultured with sera from consecutive non-recurrent (n=4) and recurrent (n=4) FSGS patients in the presence or absence of rituximab. (F) The amount of 52 and 54 kDa ASMase protein is normalized to actin in human podocytes that were exposed to normal (NHS) (n=5), non-recurrent (NON-REC) FSGS (n=10), and recurrent (REC) FSGS (n=12) human sera in the presence or absence of rituximab. *p<0.05, **p<0.01 by one-way ANOVA (G) ASMase activity per μg of total lysate protein as evaluated by enzyme-linked immunosorbent assay. **p<0.01 by one-way ANOVA.

Figure 3. Both rituximab and SMPDL-3b partially prevent the effect of recurrent FSGS sera on podocyte stress fibers

(A) Representative stress fiber confocal images (40× magnification) of normal human podocytes exposed to normal (NHS) (n=5), non-recurrent (NON-REC) FSGS (n=10), and recurrent (REC) FSGS (n=12) human sera. (B) The percentage of cells with disruption of stress fibers observed after exposure to NHS (n=5), NON-REC sera (n=10), and REC human sera (n=12) (***p<0.001 by one-way ANOVA). (C) Linear correlation between the percentage of cells with loss of stress fibers and the urine protein/creatinine ratio obtained from REC (red squares) and NON-REC (black squares) patients (n=22) in the first 30 days after transplantation (R² = 0.59; p<0.001). (D) Confocal images of stress fiber 40× magnification and bar graph analysis of normal human podocytes exposed to recurrent FSGS sera in the presence (REC+RITUX) or absence (REC) of rituximab. Rituximab protected the loss of stress fibers observed in stressed podocytes exposed to REC sera, but not NON-REC sera (**p<0.01 by one-way ANOVA). (E) Confocal images of stress fibers (40× magnification) and bar graph analysis of normal human podocytes exposed to REC sera transfected with an empty GFP vector (REC) or with a SMPDL-3b-GFP vector (REC + SMPDL-3b). SMPDL-3b overexpression protected the loss of stress fibers observed in podocytes exposed to REC sera (*p<0.05; ***p<0.001 by one-way ANOVA).

Figure 4. The renal-protective effect of rituximab in podocytes is mediated by SMPDL-3b

(A) Confocal images (40× magnification) of normal human podocytes exposed to either non-recurrent FSGS (NON-REC, N=10) sera or recurrent FSGS (REC, N=12) sera in non-targeting (NT) control and siSMPDL-3b (siSMP) cell lines in the absence or presence of rituximab (+Ritux). The protective effect of rituximab was markedly reduced in siSMP podocytes. (B) Bar graph analysis of the disruption of stress fibers observed in NT and siSMP podocytes exposed to REC (n=12) or NON-REC (n=10) sera in the absence or presence of rituximab (+RITUX) (*p<0.05 by one-way ANOVA). Rituximab did not prevent loss of stress fibers in siSMP podocytes (p=0.8). (C) Disruption of stress fibers over time observed in NT control cells exposed to normal human sera (NHS) (closed triangles), REC sera (closed squares), or NON-REC sera (closed circles), and in siSMP cells exposed to NHS (open triangles), REC sera (open squares), and NON-REC sera (open circles) (*p<0.05 when comparing by one-way ANOVA different time points to time 0; #p<0.05 when comparing NT to siSMP cells exposed to REC sera). Error bars represent SD from four independent experiments performed with pooled sera from NHS (n=5), NON-REC (n=10), and REC (n=12) patients (D) Percentage of Annexin V+ cells in NT control cells exposed to NHS (solid triangles), REC sera (solid squares), or NON-REC sera (solid circles), and in siSMP cells exposed to NHS (open triangles), REC sera (open squares) and NON-REC sera (open circles) (*p<0.05 when comparing by one-way ANOVA REC and NON-REC sera treated cells to NHS treated cells; #p<0.05 when comparing NT to siSMP cells exposed to REC sera by one-way ANOVA). Error bars represent SD from four independent experiments performed with pooled sera from NHS (n=5), NON-REC (n=10), and REC (n=12) patients. (E and F) Bar graph analysis for Annexin V staining of four independent experiments performed at 24 hours with pooled sera from NHS (n=5), NON-REC (n=10), and REC (n=12) patients. Apoptosis determination was performed in both (E) NT control cells and in (F) siSMP cells. An increased cell death was observed in NON-REC sera treated podocytes (p<0.05 by one-way ANOVA), but more so in REC sera treated podocytes (p<0.01 by one-way ANOVA) when compared to NHS. Rituximab prevented podocyte apoptosis in NT cells, but not in siSMP cells (*p<0.05, **p<0.01 by one-way ANOVA). (H) Disease model. Although SMPDL-3b deficiency is not sufficient to cause actin remodeling and proteinuria in podocytes, SMPDL-3b downregulation after exposure to FSGS patient sera renders podocytes more susceptible to actin remodeling caused by a variety of permeability factors. Rituximab partially preserves disruption of stress fibers through stabilization of SMPDL-3b.