IgM exacerbates glomerular disease progression in complement-induced glomerulopathy

Abstract

Although glomerular immunoglobulin M (IgM) deposition occurs in a variety of glomerular diseases, the mechanism of deposition and its clinical significance remain controversial. Some have theorized IgM becomes passively trapped in areas of glomerulosclerosis. However, recent studies found that IgM specifically binds damaged glomeruli. Therefore, we tested whether natural IgM binds to neo-epitopes exposed after insults to the glomerulus and exacerbates disease in mice deficient in the complement regulatory protein factor H; a model of non-sclerotic and nonimmune-complex glomerular disease. Immunofluorescence microscopy demonstrated mesangial and capillary loop deposition of IgM, whereas ultrastructural analysis found IgM deposition on endothelial cells and subendothelial areas. Factor H-deficient mice lacking B cells were protected from renal damage, as evidenced by milder histologic lesions on light and electron microscopy. IgM, but not IgG, from wild-type mice bound to cultured murine mesangial cells. Furthermore, injection of purified IgM into mice lacking B cells bound within the glomeruli and induced proteinuria. A monoclonal natural IgM-recognizing phospholipids also bound to glomeruli in vivo and induced albuminuria. Thus, our results indicate specific IgM antibodies bind to glomerular epitopes and that IgM contributes to the progression of glomerular damage in this mouse model of non-sclerotic glomerular disease.

Figure 1. Factor H deficient mice demonstrate progressive IgM deposition within the glomerulus over time

Glomerular IgM and C3 deposition were assessed by immunofluorescence microscopy of kidney sections from fH^−/− mice. (a and b) IgM is seen in the mesangium of young mice (3 months old). IgM deposition expands into capillary loops in older mice (9 months old). (c and d) Glomerular deposition of C3 appears in glomerular capillary loops at both ages. (e and f) Dual staining with IgM and C3 demonstrate distinct locations of IgM and C3 in fH^−/− mice at 3 months of age and diffuse deposition of IgM at 9 months of age. Representative glomeruli from mice in each group are shown. Original magnification × 200. Bar = 100 μm.

Figure 2. Complement components are deposited within glomeruli of factor H deficient mice

Glomerular deposition of C4 (a) and C1q (c) are seen in fH^−/− mice. However, fH^−/−/μMT mice did not demonstrate glomerular C4 (b) or C1q (d) deposition. Neither fH^−/− or fH^−/−/μMT mice demonstrated glomerular IgG deposition (e and f). Arrows show location of glomeruli. Original magnification × 400 and bar = 100 μm.

Figure 3. IgM deposits within glomerular capillary loops of diseased glomeruli

Kidney sections from 9 month-old fH^−/− mice were assessed by immunofluorescence for distribution of IgM within the glomerular capillary loops. (a) Synaptopodin, a marker for podocytes is shown. (b) Glomerular IgM staining is shown. (c) Dual staining of synaptopodin and IgM demonstrates distribution within separate compartments of the capillary loops. (d) Immunogold electron microscopy staining for IgM demonstrates IgM deposition occurring on endothelial cells and in the subendothelial area (arrows show locations of gold beads and IgM). (e) Kidney sections from fH^−/−/μMT animals lacked IgM deposition. Representative glomeruli are shown. Original magnification × 400 and bar = 100 μm for panels a, b, and c. Bar = 200 nm for panels d and e.

Figure 4. IgM binds to mesangial cells in vitro

Cultured murine mesangial cells were incubated with normal mouse serum at 37°C. Cells were analyzed by flow cytometry for the presence of IgM, IgG, and C3. (a) IgM binds to mesangial cells (MES-13) (positive staining demonstrated by grey shaded histogram and isotype control represented by black outline). (b) IgG does not bind to mesangial cells. (c) IgM binding was specific to mesangial cells, as a pancreatic endothelial cell line (MS-1) did not bind IgM. (d) Mesangial cells that bound IgM also bound C3 when incubated with mouse serum.

Figure 5. Purified polyclonal and monoclonal IgM binds glomerular cells in vitro

Cultured murine mesangial cells were incubated with polyclonal IgM or seven different monoclonal IgM clones at 37°C. Following incubation cells were analyzed by flow cytometry to determine the degree of IgM binding. (a) Purified polyclonal IgM bound to cultured primary mesangial cells (Primary MC) and bound to a mesangial cell line (MES-13, panel b). (b) Two of the monoclonal IgM clones exhibited positive binding to mesangial cells (the monoclonal IgM clone C2 in green and the monoclonal IgM clone F632 in blue). (c) The remaining five monoclonal IgM clones did not demonstrate binding to mesangial cells. Isotype control is represented by the purple shaded histogram.

Figure 6. Purified polyclonal and monoclonal IgM binds glomerular cells in vivo

fH^−/−/μMT and μMT mice injected intravenously with either purified polyclonal IgM or monoclonal IgM clones. Kidney sections were assessed for the presence of IgM by immunofluorescence microscopy. Glomeruli from fH^−/−/μMT animals demonstrated deposition of polyclonal IgM (a), the C2 IgM clone (c), and the D5 IgM clone (e). The kidney sections from μMT animals demonstrated trace IgM staining following injection of the C2 IgM clone (d) and lacked IgM deposition following injection of polyclonal IgM and the D5 IgM clone (b and f). Representative glomeruli are shown. Original magnification × 400. Bar = 100 μm.

Figure 7. Purified polyclonal IgM binds the glomeruli of wild type mice

Seven month-old wild type (WT) mice were intravenously injected with purified polyclonal IgM conjugated with biotin (Figure 7, Biotin-polyclonal IgM injected). The kidneys were harvested at 24 hours and were assessed for polyclonal IgM deposition by immunofluorescence microscopy. Evidence of extensive polyclonal IgM deposition (detected using streptavidin-Alexa488) was observed in the glomeruli of injected mice (a), but not in those of un-injected mice (d). Staining specific for the IgM μ chain indicated the presence of mesangial IgM (both purified polyclonal and endogenous forms) within the glomeruli of injected (b) and un-injected (e) mice. Confirmation of significant polyclonal IgM deposition in the glomeruli of injected (c), but not in the un-injected (f) mice is shown in overlaid images. Representative glomeruli from each group are shown and indicated by arrowheads. Original magnification × 400. Bar = 100 μm.

Figure 8. Purified polyclonal and monoclonal IgM induce albuminuria in vivo

(a) Intravenous injection of purified IgM into B cell deficient (μMT mice) resulted in a significant increase in albuminuria at one day after injection (* P < 0.05 by ANOVA for Day 0 versus Day 1 and for Day 1 versus Day 3). Individual data points are presented. Lines represent means. (b) Injection of the C2 IgM clone significantly increased the degree of albuminuria in fH^−/−/μMT animals at Day 1 but did not induce albuminuria in wild type (WT) animals. * P < 0.05. (c) Injection of the D5 IgM clone failed to induce albuminuria in either fH^−/−/μMT or WT animals.

Figure 9. Factor H deficient animals lacking B cells develop fewer pathologic lesions than factor H deficient animals by light microscopy

(a and b) PAS stained kidney sections from 9 month-old wild type (WT) and a μMT mouse show two normal appearing glomeruli with only mild focal mesangial hypercellularity (arrowheads). (c) The kidney section from a fH^−/− mouse demonstrates three glomeruli with global endocapillary proliferation, mesangial hypercellularity, and double contours (double contours marked with arrows, also shown in magnified inset). (d) In fH^−/−/μMT mice these pathologic lesions were attenuated. Two glomeruli from a fH^−/−/μMT kidney section are present with moderate mesangial hypercellularity and no double contours. Representative glomeruli from mice in each group are shown, original magnification × 400. Bar = 100 μm. (e and f) Quantitative assessment demonstrated a significantly higher degree of hypercellularity and double contours in kidney sections from fH^−/− mice compared to wild type or μMT mice. The kidney sections from fH^−/−/μMT mice had significantly attenuated pathologic lesions (both hypercellularity and double contours) compared to fH^−/− mice. * P < 0.05 by ANOVA. Individual data points are presented. Lines represent means.

Figure 10. Factor H deficient animals lacking B cells demonstrate milder pathologic lesions than factor H deficient animals by electron microscopy

(a) Electron microscopy of kidney sections from 9 month-old fH^−/− mice demonstrate numerous sub-endothelial and intramembranous deposits (arrowheads), mesangial interposition (arrow), areas of glomerular basement membrane duplications forming double contours (double-headed arrows), and increased cellularity (asterisk shows location of monocytes and/or endothelial cells). (b) These pathologic lesions were less prominent in 9 month-old fH^−/−/μMT mice. There are relatively small sub-endothelial deposits (arrowheads), otherwise the glomerulus has normal ultra-structure. Original magnification × 3000. Bar = 2 μm.

Figure 11. B cells contribute to the development of albuminuria in factor H deficient mice

Urine albumin/creatinine levels were determined in 9 month-old animals. There was a trend towards greater albuminuria in fH^−/− over wild-type (WT) mice. Albuminuria was attenuated in the fH^−/−/μMT group but did not achieve statistical significance. Individual data points are presented. Lines represent means.