Studies on endogenous glomerular proteinases potentially involved in glomerular diseases
Description
Several lines of evidence have documented the potential of proteolytic enzymes to produce many of the pathological changes associated with glomerulonephritis. However the specific proteinases responsible for such changes, their site of synthesis, and their mechanism(s) of action remain to be identified. Although several mechanisms of proteinase-mediated glomerular injury have been suggested, most of the experimental evidence to date indicate that proteinase-mediated glomerular basement membrane (GBM) damage is a major mechanism of glomerular injury leading to proteinuria. The neutral pH environment of the GBM suggests that such GBM-degrading proteinases should have signficiant activity at neutral pH values, that is they must be 'neutral' proteinases. Furthermore, since many types of experimental and human glomerulonephritis lack signficiant leukocyte involvement, the glomerulus itself may be an important source of proteinases contributing to its own damage. Glomerular neutral proteinase activity was markedly inhibited by EDTA (10mM: $-$65 to $-$89%) and 1,10-phenanthroline (2mM: $-$73%), inhibitors of metalloproteinases. No signficiant inhibition of the glomerular neutral proteinase activity was observed with inhibitors of serine or cysteine proteinases (PMSF, SBTI, TLCK, aprotinin, alpha-1-PI, E-64, Ep-475, and leupeptin). The glomerular metalloproteinase activity was associated with the membrane fraction obtained following centrifugation of sonicated glomeruli at 100,000 x g for 90 min. The glomerular neutral metalloproteinase degraded both gelatin and GBM at the neutral pH of 7.5. SDS-substrate (gelatin) polyacrylamide gel electrophoresis revealed a single major band of EDTA-inhibitable gelatin-degrading activity with a molecular weight of approximately 120-125 kDa. The glomerular neutral metalloproteinase (GLOMP) activity was not inhibited by tissue inhibitors of metalloproteinases (TIMP-1 or TIMP-2), was not latent, and was slightly enhanced in activity by aminophenyl mercuric acetate, 4-chloromercuribenzoate, or p-chloromercuriphenyl sulphonic acid (each 500 $\le$ M), activators of several latent metalloproteinases. GLOMP activity was increased 3-fold following incubation with trypsin (20 $\le$ g/ml, 25 min, 22$\vert$C). However, this increase in activity appeared to be due to solubilization of the membrane-bound proteinase. These data documented the presence of a neutral metalloproteinase in freshly isolated glomeruli and indicated that this glomerular metalloproteinase was distinct from the previously described matrix metalloproteinases as well as other well-characterized metalloproteinases present in the renal glomerular cells including the gelatinase secreted by cultured mesangial cells, and the recently described GBM-degrading neutral metalloproteinase secreted by cultured glomerular epithelial cells