Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion

Abstract

Mesangial cells maintain normal glomerular function by mediating ECM remodeling and immune complex disposal. We have recently identified megsin, a novel member of the serine protease inhibitor (serpin) superfamily predominantly expressed in the mesangium. While our previous studies suggested a role for megsin in the pathogenesis of human glomerular diseases, its exact biological significance remained unknown. Here we produced two lines of megsin transgenic mice. Overexpression of megsin led to progressive mesangial matrix expansion and an increase in the number of mesangial cells. These glomerular lesions were accompanied by an augmented immune complex deposition, together with Ig's and complement. Binding and functional assays in vitro identified plasmin as one biological substrate of megsin and confirmed its activity as a proteinase inhibitor. Transgenic animals exhibiting nephritis as a result of treatment with anti--glomerular basement membrane antiserum showed significantly more persistent expansion of the mesangial ECM than was seen in parental mice. Megsin therefore exerts a biologically relevant influence on mesangial function, and on the mesangial microenvironment, such that simple overexpression of this endogenous serpin engenders elementary mesangial lesions.

Figure 1

Generation and characterization of human megsin transgenic mice. (a) Megsin transgene construct. Full-length human megsin cDNA was subcloned in the rabbit β-globin gene including a part of the second intron, the third exon, and the 3′ untranslated region. The positions of primers for PCR analysis are indicated above the construct. (b) Identification of human megsin transgene by PCR of genomic DNA. Lane 1, a wild-type mouse DNA; lane 2, a wild-type mouse DNA with one copy of megsin transgene added; lane 3, F₀ megsin transgenic DNA (line A); lane 4, F₀ megsin transgenic DNA (line B). (c) Identification of human megsin transgene by genomic Southern blot analysis. Southern blot analysis after EcoRV digestion of genomic DNA. Lane 1, a wild-type mouse DNA; lane 2, F₀ megsin transgenic DNA (line A); lane 3, F₀ megsin transgenic DNA (line B). Approximately 9.0 kb and 2.6 kb of fragments in line A and 10.0 kb and 1.5 kb of fragments in line B, but not endogenous murine megsin genome, are detected with human megsin transgene probe.

Figure 2

Northern blot analysis of megsin transgene. Upper panels: total RNAs of kidneys, hearts, livers, or lungs from a wild-type mouse (lane 1), an F₁ megsin transgenic mouse (line A) (lane 2), and an F₁ megsin transgenic mouse (line B) (lane 3). Lower panels: the ethidium bromide staining pattern of 18S rRNA in each total RNA sample.

Figure 3

Western blot analysis of megsin gene product. (a) Characterization of anti-human megsin antibody. Lane 1, human megsin fusion protein with MBP expressed in E. coli (86 kDa); lanes 2 and 4, kidneys from wild-type mice; lane 3, kidney from F₁ megsin transgenic mouse (line A); lane 5, kidney from F₁ megsin transgenic mouse (line B). Arrow indicates megsin product with an approximate molecular weight of 47 kDa. (b) Immunohistochemical detection of human megsin gene product in the kidneys of a wild-type mouse (left) and an F₁ megsin transgenic mouse (line A) (right). ×100.

Figure 4

Histopathological analysis of murine kidneys. PAS staining. As compared with 40-week-old wild-type mice (left, ×200), F₁ megsin transgenic mice (line A) of the same age developed mesangial matrix expansion and an increase in the number of mesangial cells (middle, ×200; right, ×50).

Figure 5

Computer-assisted morphometry of glomerular abnormalities in megsin transgenic mice. Glomerular tuft areas (a) and numbers of mesangial cells (b) were measured in 25 wild-type mice and 19 megsin transgenic mice (line A). Normal limits are considered as a range of mean + 2 SD in wild-type mice. Filled circles represent animals above the normal limits, while open circles demonstrate mice within normal limits. The mean ± SD is indicated.

Figure 6

Immunohistochemical analysis of ECM. Marked deposition of type IV collagen and laminin was observed in the glomeruli of transgenic mice, while the amount of fibronectin was much less in the transgenic animals than in wild-type mice. Accumulation of type I collagen was not observed in either wild-type or transgenic animals. Upper row, a wild-type mouse; lower row, a megsin transgenic mouse. ×200.

Figure 7

Immunofluorescent studies of Ig’s. Diffuse granular deposits of IgG and complement were augmented in the mesangial area of 40-week-old F₁ transgenic mice (line A) (right) as compared with wild-type mice of the same age (left). ×50.

Figure 8

Electron microscopic analysis. Severe matrix expansion with numerous electron-dense deposits was detected in the mesangial matrix of F₁ megsin transgenic mouse (line A) (right). Glomerular endothelial cells and epithelial cells remained intact. Left, a wild-type mouse. ×2500.

Figure 9

Anti-GBM nephritis experiments. (a) PAS staining of anti-GBM nephritis mouse kidneys. While glomerular injury in transgenic mice and wild-type mice was essentially the same at day 7, only wild-type mice showed significant improvement at day 28. Upper panels, a wild-type mouse; lower panels, a megsin transgenic mouse. ×200. (b) Semiquantitative analysis of mesangial matrix expansion of anti-GBM nephritis mice. Note that mesangial matrix expansion persisted at day 28 in the transgenic mice, whereas it improved in wild-type mice. Squares, wild-type mice; circles, megsin transgenic mice.

Figure 10

Functional assays of megsin. (a) SDS-PAGE under a nonreduced condition, followed by CBB staining (left and middle panels) or immunoblotting using anti–t-PA antibody (right panel), revealed functional binding of megsin to plasmin, but not to other serine proteases such as t-PA and thrombin. Lane 1, plasmin; lane 2, antiplasmin; lane 3, plasmin plus antiplasmin; lane 4, megsin; lane 5, plasmin plus megsin; lane 6, thrombin; lane 7, antithrombin; lane 8, thrombin plus antithrombin; lane 9, megsin; lane 10, thrombin plus megsin; lane 11, t-PA; lane 12, PAI-1; lane 13, t-PA plus PAI-1; lane 14, megsin; lane 15, t-PA plus megsin. The complex of serine protease and serpin is indicated by asterisks. (b) The inhibitory effect of megsin to plasmin activity. Diamonds, substrate alone; open squares, plasmin; filled squares, plasmin and antiplasmin (1:2); open circles, plasmin and megsin (1:10); filled circles, plasmin and inactivated megsin (1:10); open triangles, ovalbumin; filled triangles, albumin.