Urinary excretion of neutral proteinases in nephrotic rats with a glomerular disease

Abstract

A proliferative glomerulonephritis was induced in rats pre-immunized with rabbit IgG by injecting intravenously a subnephrotoxic dose of rabbit anti-glomerular basement membrane (GBM) IgG (A rats). Most rats (80%) developed a severe proteinuria (>100 mg/24 hr) within two to five days after the injection of anti-GMB IgG. At the same time, microscopic examination of the kidneys revealed a glomerular infiltration of mononuclear phagocytes and a prominent decrease in the intensity of the colloidal iron reaction in glomeruli. A non-proliferative glomerular disease was induced in another group of rats (B rats) by intraperitoneal administration of aminonucleoside of puromycin. A marked proteinuria (>100 mg/24 hr) occurred after six days in 90% of animals. Histochemical studies then revealed a decrease in staining intensity of glomeruli for polyanion. No glomerular hypercellularity was noted. In normal rats and in non-proteinuric A or B rats, the 24 hour urinary excretion of neutral proteinases ranged from 1.4 to 7.8 units (mean value ± SEM, 4.69 ± 0.60, N = 11), that of laminin ranged from 100 to 3,900 ng (mean value ± SEM, 1,154 ± 325, N = 10), and that of type IV collagen ranged from 160 to 240 ng (mean value ± SEM, 306 ± 26.5 ng, N = 8). In proteinuric rats from groups A (N = 11) and B (N = 9), the 24 hour urinary excretion of neutral proteinases significantly increased (mean values ± SEM, 38.55 ± 8.66 U for A rats and 42.17 ± 7.92 U for B rats) and ran parallely with that of proteins, laminin and type IV collagen. Significant direct linear correlations were observed between: (a) the urinary neutral proteinase activity and the proteinuria, the urinary excretion of laminin and that of type IV collagen; and (b) the proteinuria and the urinary excretion of laminin. In A and B rats, as in normal rats, proteinase activity occurred in urine as an active form exhibiting an apparent molecular weight of 30,000 and an isoelectric point of 9.0 was maximal in the neutral range, was eliminated by heating at 60° C for 30 minutes and was found capable of degrading GBM polypeptides (laminin and type IV collagen) in vitro. In normal urine, a significant inhibition of the neutral proteinase activity was seen with phenylmethylsulfonylfluoride, soybean trypsin inhibitor or aprotinin, but not with EDTA or cysteine, which suggests that the enzymatic mainly results from (a) serine proteinase(s). In urine from A or B rats, neutral proteinase activity was significantly reduced by all the inhibitors tested which indicates the presence of both (a) serine proteinase(s) and (a) metallo-proteinase(s). Our in vitro and in vivo findings strongly suggest that in these two experimental models of glomerular disease, neutral metalloproteins may be involved in the damage to the GBM and in the development of proteinuria.