Anti-inflammatory and renal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritis

Abstract

We have previously shown that stanniocalcin-1 (STC1) inhibits the transendothelial migration of macrophages and T cells, suppresses superoxide generation in macrophages, and attenuates macrophage responses to chemoattractants. To study the effects of STC1 on inflammation, in this study we induced a macrophage- and T-cell-mediated model of anti-glomerular basement membrane disease in STC1 transgenic mice, which display elevated serum STC1 levels and preferentially express STC1 in both endothelial cells and macrophages. We examined the following parameters both at baseline and after anti-glomerular basement membrane antibody treatment: blood pressure; C(3a) levels; urine output; proteinuria; blood urea nitrogen; and kidney C(3) deposition, fibrosis, histological changes, cytokine expression, and number of T cells and macrophages. Compared with wild-type mice, after anti-glomerular basement membrane treatment STC1 transgenic mice exhibited: i) diminished infiltration of inflammatory macrophages in the glomeruli; ii) marked reduction in crescent formation and sclerotic glomeruli; iii) decreased interstitial fibrosis; iv) preservation of kidney function and lower blood pressure; v) diminished C(3) deposition in the glomeruli; and vi) reduced expression of macrophage inhibitory protein-2 and transforming growth factor-beta2 in the kidney. Compared with baseline, wild-type mice, but not STC1 transgenic mice, had higher proteinuria and a marked reduction in urine output. STC1 had minimal effects, however, on both T-cell number in the glomeruli and interstitium and on cytokine expression characteristic of either TH1 or TH2 activation. These data suggest that STC1 is a potent anti-inflammatory and renal protective protein.

Figure 1

Inhibition of anti-GBM GN in STC1 Tg mice. A: Periodic acid-Schiff- and Masson’s trichrome-stained kidney tissue. Anti-GBM Ab injection to WT mice produced severe crescentic GN, characterized by extra- and intracapillary mononuclear infiltration, hyaline cast formation (H), glomerulosclerosis, and interstitial fibrosis (see trichrome staining). STC1 Tg mice display reduced inflammation, crescent formation, and fibrosis after anti-GBM. Arrow points to a crescent. G, Glomeruli; H, hyaline casts in dilated tubules. B: STC1 Tg mice display fewer crescents, fewer glomerular endocapillary lesions, fewer sclerotic glomeruli, and less expansion in the tubulointerstitial compartment.

Figure 2

Inhibition of crescentic GN in STC1 Tg mice. WT mice had fourfold rise in BUN, 50% reduction in urine output, and more proteinuria; there were no significant changes in BUN, urine output, or proteinuria in STC1 Tg mice. Although baseline blood pressure was similar in WT and STC1 Tg mice, it was higher in WT mice after anti-GBM GN, consistent with renal protection by STC1.

Figure 3

A: Equal binding of sheep IgG to GBM in WT and STC1 Tg mice. Ten days after anti-GBM Ab injection, frozen sections of kidney tissue were labeled with FITC-tagged anti-sheep IgG Ab. Equal binding of sheep anti-mouse GBM antibody to the GBM in both WT and STC1 Tg mice is illustrated (arrows point to glomeruli). B: STC1 levels in the serum. Western blot demonstrates higher levels of STC1 protein in the serum of STC1 Tg mice compared with WT mice. C and D: STC1 levels in the kidney. Western blot and bar graph demonstrate equal expression of STC1 in the kidneys (representing whole kidney lysates) of WT and STC1 Tg mice after anti-GBM. E: STC1 expression in macrophages. Dual staining for STC1 (red) and F4/80 (brown, a marker of macrophages and dendritic cells) demonstrates high-level expression of STC1 in kidney macrophages/dendritic cells of STC1 Tg mice compared with WT mice.

Figure 4

Expression of STC1 in the endothelium. Immunohistochemistry studies reveal high levels of STC1 expression (brown) in the endothelium of STC1 Tg mice (best appreciated in the glomerular capillaries), compared with WT mice. Also, expression of STC1 in injured tubules of WT mice after anti-GBM GN. G, Glomeruli; T, tubule.

Figure 5

STC1 Tg mice exhibit decreased infiltration of macrophages into the glomeruli after anti-GBM GN. In a model of mouse anti-GBM GN, F4/80⁺ cells (resident macrophages and dendritic cells) were absent from the glomeruli in both WT and STC1 Tg mice. Macrophage infiltration into the glomeruli was observed only in WT mice, and the macrophages were predominantly F4/80⁻/CD68⁺ (inflammatory/exudative). T cells (CD3) were predominantly interstitial and increased to a similar degree in WT and STC1 Tg mice after anti-GBM GN. Image resolution was dictated by available antibodies. Staining for F4/80 was performed on paraformaldehyde-fixed sections, whereas staining for CD68 and CD3 was performed on frozen sections. G, Glomeruli.

Figure 6

STC1 Tg mice exhibit decreased expression of MIP-2 and TGF-β. A–C: Densities of respective bands corresponding to RNase protection assay were normalized to L32, and data represent the means and SE of at least six independent determinations. **P < 0.01. D and E: Western blots and bar graph show MCP-1 and ICAM-1 protein expression in the kidneys of WT and STC1 Tg mice after anti-GBM GN.

Figure 7

A: Decreased deposition of mouse C₃ but not IgG in the glomeruli of STC1 Tg mice after anti-GBM GN. FITC-labeled anti-mouse C₃ or IgG antibodies were used to determine the deposition of mouse C₃ and IgG in the glomeruli of WT and STC1 Tg mice 10 days after the administration of anti-GBM Ab. Top: Representative images for C_3. Bottom: Representative images for mouse IgG. G, Glomerulus. B–D: Equivalent complement activity, anti-sheep IgG isotypes in the sera, and mouse IgG levels in the kidneys of WT and STC1 Tg mice. Bar graphs depict eluted mouse IgG from the kidneys (B), mouse anti-sheep IgG isotypes in the serum at baseline and after priming with sheep IgG (C), and serum C_3a levels at baseline, after priming with sheep IgG and after anti-GBM Ab administration (D). Data represent the means ± SEM of six independent determinations.