Gelatinase B (MMP-9) is not essential in the normal kidney and does not influence progression of renal disease in a mouse model of Alport syndrome

Abstract

Matrix metalloproteinases are matrix degrading enzymes implicated in many biological processes, including development and inflammation. Gelatinase B (gelB; also known as MMP-9) is expressed in the kidney and is hypothesized to be involved in basement membrane remodeling and in preventing pathogenic accumulation of extracellular matrix in the kidney. Inhibition of gelB activity in metanephric organ culture disrupts branching morphogenesis of the ureteric bud, suggesting that gelB plays a role in kidney development in vivo. We studied kidneys of gelB-deficient mice to search for developmental, histological, molecular, ultrastructural, and functional defects. Surprisingly, no differences between gelB-/- and control kidneys were detected, and renal function was normal in gelB mutants. In addition, gelB-/- embryonic kidneys developed normally in organ culture. Gelatinase B-deficient mice were bred with Col4a3-/- mice, a model for Alport syndrome, to determine whether gelB influences the progression of glomerulonephritis. This is an important question, as it has been hypothesized that proteases are involved in damaging Alport glomerular basement membrane. However, the presence or absence of gelB did not affect the rate of progression of renal disease. Thus, gelB does not have a discernible role in the normal kidney and gelB is not involved in the progression of glomerulonephritis in a mouse model of Alport syndrome.

Figure 1.

Expression of gelA and gelB by embryonic kidney. A: Zymographic analysis of expression of gelA and gelB by gelB+/− (C) and gelB−/− (M) cultured embryonic kidneys. Mutants did not express gelB. A gelB +/+ neutrophil extract (N) was rich in gelB. B: RT-PCR assay for gelB mRNA in E13.5 and E15.5 control kidneys. Both were positive for the appropriate-sized RT-dependent product. The lowest bands represent primer background.

Figure 2.

Histological analyses of gelB+/− and gelB−/− kidneys. No significant differences between gelB+/− (control) and gelB−/− (mutant) kidneys at 5 months of age were detected in H&E-stained paraffin sections (A and B) or by electron microscopy (C and D).

Figure 3.

Immunohistochemical analysis of control and mutant adult kidney basement membranes. No significant differences were detected in deposition of several basement membrane components, including: collagen α1 and α2(IV) (A and B); collagen α4(IV) (C and D); laminin α1 (E and F).

Figure 4.

Analysis of development in control and mutant kidneys. A and B: H&E stained paraffin sections of E13.5 kidneys. Robust ureteric bud (u) branching was apparent in control and mutant kidneys, and many primitive nephron structures were present. C–F: Immunohistochemical analysis of postnatal day 2 kidney basement membranes. Both control and mutant kidneys were undergoing appropriate GBM and other developmental transitions in deposition of laminin α1 (C and D) and laminin α5 (E and F). For example, note the low levels of α1 and high levels of α5 in the maturing GBM (g).

Figure 5.

Kidney development in organ culture. GelB+/− and −/− metanephroi were removed at E12.5, cultured on a filter for several days, and photographed daily. Representative photos after 0 and 2 days in culture are shown. No differences in the extent of growth or branching were observed.

Figure 6.

RT-PCR analysis of gelB, gelA, and GAPDH expression in Col4a3+/− and Col4a3−/− kidneys at various postnatal (P) ages. Higher steady-state levels of gelB mRNA in Col4a3−/− kidneys (M) compared to Col4a3+/− kidneys (C) was apparent at all ages except P42. Levels of gelA mRNA were not obviously higher at most ages in mutant kidneys.

Figure 7.

Physiological analysis of renal function at various ages in gelB+/−; Col4a3−/− and gelB−/−; Col4a3−/− mice. Serum creatinine (A), blood urea nitrogen (B) and urinary protein/creatinine (C) concentrations were measured at the time of sacrifice and plotted versus age. There was no significant effect of the gelB mutation on the progression of renal disease (P > 0.1). Symbols are: (▪) gelB+/−, Col4a3−/− mice and (○) gelB−/−, Col4a3−/− mice.

Figure 8.

Ultrastructural analysis of 8-week-old GBM from gelB+/−; Col4a3−/− (A) and gelB−/−; Col4a3−/− (B) kidney. Both show the characteristic splitting and thickening seen in Alport syndrome.