B cell-derived IL-4 acts on podocytes to induce proteinuria and foot process effacement

Abstract

The efficacy of B cell depletion therapies in diseases such as nephrotic syndrome and rheumatoid arthritis suggests a broader role in B cells in human disease than previously recognized. In some of these diseases, such as the minimal change disease subtype of nephrotic syndrome, pathogenic antibodies and immune complexes are not involved. We hypothesized that B cells, activated in the kidney, might produce cytokines capable of directly inducing cell injury and proteinuria. To directly test our hypothesis, we targeted a model antigen to the kidney glomerulus and showed that transfer of antigen-specific B cells could induce glomerular injury and proteinuria. This effect was mediated by IL-4, as transfer of IL-4-deficient B cells did not induce proteinuria. Overexpression of IL-4 in mice was sufficient to induce kidney injury and proteinuria and could be attenuated by JAK kinase inhibitors. Since IL-4 is a specific activator of STAT6, we analyzed kidney biopsies and demonstrated STAT6 activation in up to 1 of 3 of minimal change disease patients, suggesting IL-4 or IL-13 exposure in these patients. These data suggest that the role of B cells in nephrotic syndrome could be mediated by cytokines.

Figure 1. Rituximab does not bind SMPDL3B in nonfixed samples.

(A) Western blot of transfected 293T and Raji cells. Whole-cell lysates were subjected to SDS-PAGE, followed by immunoblotting with antibodies as indicated. β-Actin was used as loading control. (B) Rituximab does not bind to SMPDL3B in nonfixed cells. Nonfixed or PFA-fixed 293T cells expressing HA-SMPDL3B were stained with α-HA-AF488 and rituximab-AF647 and analyzed by flow cytometry. Data are representative of 3 independent experiments.

Figure 2. IL-4 induces podocyte membrane ruffling and widespread foot process retraction.

(A) Differential interference contrast live-cell imaging was used to obtain kymographs from individual differentiated cultured murine podocytes. Each cell was imaged individually from separate podocyte cultures. Representative kymographs (time = 20 minutes) before (left column) and after (right column) the addition of IL-4 (10 ng/ml, top row) or IL-4 with anti–IL-4 antibody (5 μg/ml, bottom row) are shown. IL-4 caused pronounced membrane ruffling that was abrogated by anti–IL-4 treatment (also see Supplemental Videos 1 and 2). Original magnification, ×~1,100. (B) Kymograph analysis was used to quantitate actin spike lengths (ruffling index) of 5 locations per cultured podocyte for the cytokines tested (IL-13: 10 ng/ml, IFN-γ: 100 ng/ml, TNF-α: 10 ng/ml). IL-4 and IL-13 have enhanced membrane ruffling dynamics. IL-4 promoted an equivalent level of ruffling as a known activator of Rac, EGF (20 ng/ml), which was reversed with anti–IL-4 antibody. Each symbol represents the average relative cell membrane displacement over time at 5 regions of a single cell. Each cell mean ± SD of 3 experiments, with 3–4 cells/experiment (total of 10 cells analyzed/group). (C) Representative scanning electron microscopy images of glomeruli from minced renal cortices incubated ex vivo for 20 minutes with IL-4 (10 ng/ml), IL-4 with anti–IL-4 antibody (5 μg/ml), TNF-α (10 ng/ml), or positive control EGF (20 ng/ml). IL-4 treatment induced massive foot process retractions similar to that induced by EGF. TNF-α and IL-4 plus anti–IL-4 did not alter foot process morphology, consistent with the membrane ruffling data. Scale bar: 20 μm. *P < 0.001, **P < 0.005, ***P < 0.004, ****P < 0.03 by 1-way ANOVA with Bonferroni correction.

Figure 3. Mice treated with a plasmid encoding IL-4 exhibited proteinuria, foot process abnormalities, and STAT6 activation in glomeruli.

(A) 129X1/SvJ mice were hydrodynamically injected with either IL-4 piggyBac plus transposase vectors to induce IL-4 expression (IL-4 treated) or empty piggyBac plus transposase vectors (control). Representative Coomassie blue–stained SDS-PAGE. (B) Spot albumin/creatinine ratios of urine from control or IL-4–treated mice demonstrated proteinuria with IL-4 expression. Urine was collected 12 hours after plasmid injection. (C) Serum IL-4 ELISA confirmed elevated expression of IL-4 in IL-4 gene–treated mice. Symbols represent individual mice, and bars represent the geographic mean in B and mean in C. Mean ± SD of 3 experiments, with total of 5 mice/group. *P < 0.006, **P < 0.001 by 2-tailed Mann-Whitney. (D) Representative scanning electron microscopy (scale bar: 1 μm) revealed foot process retraction with focal foot process effacement in IL-4–treated mice compared with control. (E) Representative immunohistochemical analysis of glomerular pSTAT6 expression reveals substantial STAT6 phosphorylation and nuclear translocation in IL-4–treated mice (black arrows) compared with control. Original magnification, ×400. Data are representative of 4 independent experiments.

Figure 4. Inhibition of IL-4 signaling with JAK1/3 inhibitor abrogated ruffling and proteinuria in IL-4–treated mice.

(A) Representative kymographs obtained by membrane ruffling and (B) quantification, as performed in Figure 1, from cultured podocytes treated with the JAK1/3 inhibitor tofacitinib (100 nM, bottom row), which significantly attenuated IL-4–induced membrane ruffling compared with negative control (DMSO). Each symbol represents the average relative cell membrane displacement over time at 5 regions of a single cell. Mean ± SD of 2 experiments, with 2–3 cells/experiment (total of 5 cells analyzed/group). (C) Coomassie blue–stained SDS-PAGE and (D) spot albumin/creatinine ratios of urine from IL-4–treated mice treated with JAK1/3 inhibition, which significantly reduced IL-4–induced proteinuria. Urine was collected 24 hours after plasmid administration. Symbols represent individual mice, and bars represent the mean. Mean ± SEM of 2 experiments, with total of 5 mice/group. *P < 0.008 by 2-tailed Mann-Whitney.

Figure 5. Multimerized hen egg lysozyme embedded within the glomerular basement membrane following i.v. injection.

(A) The production of multimerized hen egg lysozyme (HEL). Monomeric HEL was biotinylated and then complexed with avidin to form multimers. (B) Representative Coomassie blue–stained SDS-PAGE of monomeric and multimerized HEL demonstrated multimerized complexes of at least 120 kDa were formed. (C) Immunofluorescence microscopy images (green = HEL complex, blue = DAPI) of frozen sections from kidneys of mice injected with multimerized HEL (left: 30 minutes after injection, center: 7 days after injection) or monomeric HEL (right: 30 minutes after injection) and stained with anti-HEL antibody. Monomeric HEL was not visible in glomeruli, while multimerized HEL was found in the glomeruli up to 7 days after injection. Glomeruli are outlined in circles. Original magnification, ×20. Data are representative of 3 independent experiments.

Figure 6. HEL-specific IL-4–secreting B cells generated proteinuria in mice treated with multimerized HEL.

(A) HEL-specific B cells were either polarized in vitro into B effector 2 (Be2) cells to produce IL-4, or left unpolarized, and then stimulated through the B cell receptor with anti-μ antibody overnight. Only Be2-polarized B cells secreted IL-4 into the supernatant, as measured by ELISA. (B) Coomassie blue–stained SDS-PAGE and (C) spot albumin/creatinine ratios of urine from mice treated with multimerized HEL, followed by transfer of either Be2-polarized HEL-specific B cells or IL-4–deficient, Be2-polarized HEL-specific B cells. Proteinuria was observed only when B cells were capable of producing IL-4. Urine was collected 24 hours after B cell transfer. Symbols represent individual mice, and bars represent the mean. (D) Representative still images obtained from intravital 2-photon microscopy movies (Supplemental Videos 3 and 4) of mice injected with PBS (top) or multimerized HEL (bottom), followed by the transfer of fluorescently labeled HEL-specific B cells. When glomerular HEL was present, HEL-specific B cells arrested within glomeruli. In the absence of HEL, B cell trafficking through glomeruli was not changed. Quantum dots were injected i.v. to identify glomeruli and vessels. Glomeruli are outlined in circles. Red: glomeruli and vessels; blue: renal tubules; yellow: HEL-specific B cells. Original magnification, ×20. Mean ± SD of 2 experiments, with a total of 5 mice/group. *P < 0.001, **P < 0.008 by 2-tailed Mann-Whitney.

Figure 7. B cell–induced proteinuria generated foot process effacement.

(A) Representative light microscopy of H&E-stained kidney sections from mice with B cell–induced proteinuria revealed no pathologic changes. Original magnification, ×40. (B) Representative immunofluorescence microscopy images of kidneys from mice with B cell–induced proteinuria (left) did not demonstrate immunoglobulin or complement component C3 in glomeruli compared to positive control nephrotoxic serum (right). (C) Representative scanning electron microscopy from kidneys showed focal foot process effacement in mice with B cell–induced proteinuria (right) compared with control mice (left). Scale bar: 10 μm. Data are representative of 3 independent experiments.

Figure 8. A subset of minimal change disease patients has activated glomerular STAT6.

Representative immunohistochemistry of glomerular pSTAT6 expression in selected MCD patients (described in Supplemental Table 1) and a control. Ten of twenty-nine MCD patients screened demonstrated pSTAT6 staining (untreated MCD, arrows point to stained nuclei). Twenty-two of twenty-three healthy controls had no detectable pSTAT6 staining. Of 29 MCD patient samples stained for pSTAT6, 2 were strongly positive, 8 were weakly positive, and 19 were negative; of 22 controls samples stained for pSTAT6, 1 was weakly positive and 22 were negative (P = 0.019). Original magnification, ×400.I