MMP Expression in Kidneys of <i>α3(IV) ^−/−</i> Mutant and Wild-Type Mice

<div><p>Frozen sections of control C57/Bl6 mice and <i>α3(IV)^−/−</i> mice were stained with primary antibodies for MMP-2, MMP-3, and MMP-9 (FITC-green), and sections were counterstained with Rhodamine-labeled antibodies for entactin (red). </p> <p>(A) In control C57/Bl6 mice, MMP-2 is present sporadically in the glomeruli and in some proximal tubules (arrows).</p> <p>(B) After 4 wk of age, MMP-2 is increased in the glomeruli (arrows) in <i>α3(IV)^−/−</i> mice compared to age-matched control kidneys, even before manifestation of proteinuria. </p> <p>(C) After 8 wk of age (after manifestation of GBM-splitting and initiation of interstitial fibrosis), MMP-2 is increased in the interstitium (arrowheads) and in the glomeruli (arrows) in kidneys from <i>α3(IV)^−/−</i> mice. </p> <p>(D) MMP-2–staining was quantified using NIH ImageJ software in the glomeruli (left panels) and in total kidney cortex. Glomerular MMP-2–staining was significantly increased at 4 wk in <i>α3(IV)^−/−</i> as compared with control. *** <i>p</i> < 0.001 versus control. </p> <p>(E) MMP-2 expression was analyzed by immunoblot using total protein from the kidney cortex of single mice. A representative blot is shown, confirming increased expression of MMP-2 in <i>α3(IV)^−/</i>⁻. The lower blot displays the control blot for actin to confirm equal loading. </p> <p>(F) Immunofluorescence staining revealed that MMP-3–staining is present in the glomeruli (arrows) of control C57/Bl6 mice.</p> <p>(G) MMP-3 is significantly increased in the glomeruli of <i>α3(IV)^−/−</i> mice after 4 wk of age. </p> <p>(H) In 8-wk-old <i>α3(IV)^−/−</i> mice, MMP-3 is up-regulated in the interstitium (arrowheads) and MMP-3 is also present in the glomeruli (arrows). </p> <p>(I) Quantification of MMP-3–staining revealed a significant increase of MMP-3–staining in the glomeruli and total cortex of <i>α3(IV)^−/−</i> mice at 4 wk and 8 wk of age. *** <i>p</i> < 0.001 versus control. </p> <p>(J) Immunoblot analysis revealed increased renal MMP-3 protein.</p> <p>(K) MMP-9 is present in the glomeruli of C57/Bl6 control mice (arrows).</p> <p>(L) The onset of proteinuria is preceded by a significant increase of MMP-9 in the glomeruli of <i>α3(IV)^−/−</i> mice (arrows). </p> <p>(M) MMP-9 is significantly up-regulated in the interstitium (arrowheads) and in the glomeruli (arrows) of 8-wk-old <i>α3(IV)^−/−</i> mice compared to control. </p> <p>(N) Quantification of immunofluorescence staining using ImageJ software confirmed a significant increase of MMP-9 expression in the glomeruli (left panels) and total cortex region (right panels) of <i>α3(IV)^−/−</i>mice as compared with control. *** <i>p</i> < 0.001 versus control. </p> <p>(O) Increased MMP-9 expression was confirmed by immunoblot.</p>