Oxidation of extracellular matrix modulates susceptibility to degradation by the mesangial matrix metalloproteinase-2

Protein oxidation occurs in aging and in various inflammatory conditions. Glomerulosclerosis is characterized by an accumulation of extracellular matrix (ECM) proteins and a paucity of glomerular mesangial cells and can be seen as an end-result of glomerular injury and in aging. ECM accumulation is the net result of the balance between synthesis and degradation. ECM may become oxidized as a part of inflammatory renal injury and with aging. We evaluated the hypothesis that oxidation of mesangial ECM could alter its susceptibility to the action of ECM degrading enzymes. Radiolabeled mesangial ECM was generated by growing cells on tissue culture plastic and incubating with [3H]proline. After removal of cells, leaving behind ECM, selected wells were oxidized using a FeCl3/EDTA/ascorbate system or treated under control conditions. The control and oxidized matrices were then incubated with concentrated supernatants from mesangial cells containing the major mesangial ECM degrading enzyme, the matrix metalloproteinase-2, whose activity was confirmed by gelatin substrate zymography. Counts released corresponding with ECM degraded were measured. ECM oxidized with this system was significantly less susceptible to degradation compared to control ECM. To confirm that this effect was specifically due to oxidative modification of the ECM rather than changes unrelated to oxidation we coincubated ECM with the oxidizing system plus the radical spin trap N-tert-butyl-α-phenylnitrone (PBN). PBN treatment was able to prevent the impaired susceptibility to degradation induced by exposure to the oxidizing system. Exposure of ECM to milder oxidative stress, however, modestly enhanced susceptibility to degradation. These data suggest that oxidation of mesangial ECM can modulate its susceptibility to degradation. This may account for the development of ECM accumulation and glomerulosclerosis in inflammatory renal injury and in aging.