Shift of C3 deposition from localization in the glomerulus into the tubulo-interstitial compartment in the absence of secreted IgM in immune complex glomerulonephritis

Abstract

The role of secretory IgM in protecting kidney tissue from immune complex glomerulonephritis induced by 4 mg horse spleen apoferritin and 0.05 mg lipopolysaccharide has been investigated in mutant mice in which B cells do not secrete IgM, but are capable of expressing surface IgM and IgD and secreting other Ig isotypes. Glomerular size, number of glomeruli per cross-section, glomerular cellularity and urine content of protein and creatinine was comparable in treated secreted IgM (sIgM)-deficient and wild-type mice. Assessment of urinary proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a 30 kDa low molecular weight protein in treated sIgM-deficient animals only, reflecting dysfunction of proximal tubules. A shift of bound C3 from glomeruli to the tubulo-interstitial compartment in sIgM-deficient mice also suggests tubulo-interstitial damage. In contrast, local C3 synthesis within the kidney tissue did not differ between the two treated groups. Apoptosis physiologically present to maintain kidney cell homeostasis was increased slightly in treated wild-type mice. These results indicate that secretory IgM can protect the tubulo-interstitial compartment from immune complex-induced damage without having an effect on the glomerulus.

Fig. 1

Histochemical analysis of kidney sections from lipopolysaccharide/horse spleen apoferritin (LPS/HSA)-treated sIgM-deficient and wild-type animals. Secreted IgM (sIgM)-deficient and wild-type mice were treated with intraperitoneal injection of 0·05 mg LPS three times a week and 4 mg HSA five times a week. After 6 weeks renal tissue was fixed in paraformaldehyde and 4 μm paraffin sections were stained with haematoxylin and eosin (H&E), Masson's Trichrome (MT), periodic acid Schiff (PAS) and methinamine silver (Silver) according to standard protocols. Ongoing mesangial and endocapillary hypercellularity occurs in both experimental groups alike and is indicated by arrows. Magnification × 250.

Fig. 2

Immunohistochemical determination of C3 deposits on proximal tubules and CD11b expression on macrophages in kidney sections of lipopolysaccharide/horse spleen apoferritin (LPS/HSA)-treated secreted IgM (sIgM)-deficient and wild-type littermates. Only mesangial deposits of C3 (red) were found in treated wild-type mice (a), while C3 also bound to the Bowman's capsule and to proximal tubules in treated sIgM-deficient mice (b). C3 (blue) does not bind to distal tubules of LPS/HSA-treated sIgM-deficient (d) and wild-type mice (c) stained with PNA (red). Infiltrating CD11b⁺ macrophages (red) were found scattered intra- and periglomerular in LPS/HSA-treated wild-type mice (e) and more in the interstitial compartment in LPS/HSA-treated sIgM-deficient (f); sections were counterstained with C3 (blue). No signs of myofibroblast transformation indicated by alpha smooth muscle actin binding was observed (g, h). Glomerular FasL (red) expression was found to be stronger in LPS/HSA-treated wild-type mice (i) than in treated sIgM-deficient mice (j). Magnification × 250.

Fig. 3

Deposition of C3, IgM and IgG in renal tissue of lipopolysaccharide/horse spleen apoferritin (LPS/HSA)-treated sIgM-deficient and wild-type littermates detected by confocal laser scanning microscopy. Mesangial deposits of C3 (red), IgM (green) and IgG (blue) were found in treated wild-type mice (a–d). C3 (red) was found along the basement membrane of tubules and Bowman's capsule (e, f) with little mesangial staining in treated secreted IgM (sIgM)-deficient mice, while trace amounts of IgG (blue) could be observed on the mesangium (h). Strong caspase 3 (i, k) and only trace Fas (i, j) expression in glomeruli of treated wild-type mice, while no caspase 3 (l, n) or Fas (l, m) could be found in glomeruli of treated sIgM-deficient mice. Magnification × 630.

Fig. 4

(a) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 24-h urine samples of secreted IgM (sIgM)-deficient and wild-type animals after 6 weeks of lipopolysaccharide/horse spleen apoferritin (LPS/HSA) treatment. Α 30 kDa low molecular weight protein was found in urine samples of LPS/HSA-treated sIgM-deficient mice (lanes 4, 5, 6), but not in the urine of wild-type animals (lanes 1, 2, 3). (b) Quantitative fluorogenic reverse transcription–polymerase chain reaction of mC3. C3 expression is increased significantly in LPS/HSA treated animals (box plots) compared to untreated animals of each experimental group. C3 expression was calculated by the 2^–ΔΔCT-method normalized to the highly expressed housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, relative to C3 expression of untreated animals of the corresponding genotype. Significant increase of C3 expression was found between untreated and treated animals, whereas no statistically significant difference in C3 expression could be detected between both LPS/HAS-treated groups. Statistical significance P is based on the Mann–Whitney U-test.