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We have previously documented amelioration of rat autologous anti-GBM nephritis with the antiproteolytic drugs epsilon-aminocaproic acid (EACA) and aprotinin, given from the day of induction or later in the course of disease. In the present study we investigated potential mechanisms of this effect by assessing interactions of the drugs with proteinase-dependent generation of superoxide anion in glomeruli, and their influence on both GBM degradation in vitro and activity of glomerular proteolytic enzymes. Release of O2- by enzymatically disrupted glomeruli, isolated from nephritic control or EACA/aprotinin-treated rats, was measured with the ferricytochrome reduction method and its activity was correlated with proteinuria and glomerular cellularity at the early phase of the disease. The hydroxyproline release assay was used to quantitate degradation of rat GBM in vitro by leukocyte proteinases stimulated by phorbol myristate acetate (PMA), in the presence or absence of EACA and aprotinin. Finally, the activities of elastase, cathepsins B and L, and plasmin, together with collagenase-like activity, were assessed fluorimetrically in homogenates of glomeruli isolated from control and antiproteolytic-drug-treated nephritic rats. EACA and aprotinin notably inhibited production of superoxide by nephritic glomeruli (by 47% and 66%, respectively), and this effect was not significantly correlated with proteinuria or glomerular hypercellularity at the early stage of disease. On the other hand, generation of O2- by glomeruli of untreated nephritic rats was notably correlated with total glomerular cell counts and numbers of macrophages infiltrating glomeruli. PMA-stimulated neutrophils and macrophages caused degradation of isolated rat GBM in vitro, markedly attenuated in the presence of EACA (P<0.0005) and, to a lesser extent, by addition of aprotinin (P<0.01). The activity of elastase was significantly reduced in glomeruli of nephritic rats treated with EACA or aprotinin (both P<0.001), while activities of remaining proteinases were not appreciably affected. The beneficial influence of proteinase inhibitors on rat anti-GBM disease may be due, at least in part, to abrogation of superoxide generation in nephritic glomeruli. EACA and aprotinin also have potential to interfere with digestion of GBM, and both these effects may be related to suppression of glomerular elastase.

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BACKGROUND: Some preliminary observations suggest that predisposition to a particular type of glomerulonephritis (GN) may be connected with the genetically determined charge of the glomerular capillary wall. A correlation between erythrocyte surface and the glomerular capillary wall charges has also been observed. The purpose of this study was to verify and extend previous investigations. Therefore we measured erythrocyte and platelet surface charge from patients with idiopathic membranous and mesangial GN as well as idiopathic membranoproliferative GN and lupus nephritis. METHODS: The erythrocyte and platelet surface charge was determined by the binding of the cationic dye, alcian blue (AB). A fresh alcoholic AB solution was made for each experiment, which were run in batches of four, each including cells from a healthy person and from patients each with a different type of GN. RESULTS: In patients with idiopathic membranous and membranoproliferative GN, a significant decrease in the erythrocyte and platelet charges was observed irrespective of their clinical state (remission or nephrotic syndrome). Erythrocyte charge was decreased despite the normal amount of membranous sialic acid. In contrast, patients with idiopathic mesangial GN, in complete or partial remission, exhibited normal erythrocyte and platelet surface charges. Exclusively in this type of GN, the appearance of nephrotic proteinuria was associated with a slight decrease, the erythrocyte charge, which was not statistically significant (P > 0.1). A reduction in the negative erythrocyte charge in lupus nephritis was less in magnitude than in idiopathic membranous or membranoproliferative GN, and occurred independently of the level of daily proteinuria, whereas the platelet charge was normal. CONCLUSION: The decrease of the erythrocyte and platelet charge in idiopathic membranous and mebranoproliferative GN seems to be a pre-morbid feature.

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BACKGROUND: Given the evidence accrued by other authors on beneficial effect of protease inhibitors on experimental immune nephritis, and following our preliminary report on abrogation of immune glomerulopathy in the rat by antifibrinolytic and antiproteolytic drug, epsilon-aminocaproic acid (EACA), we investigated the effect of this drug on the rat autologous anti-GBM nephritis. Along with the EACA we evaluated another protease inhibitor, aprotinin, an antagonist of serine proteases. METHODS: EACA (0.3g/kg) or aprotinin (5000 kallkrein inhibition units, KIU/kg) was administered intraperitoneally (t.i.d.) from day 0 (preventive protocol) or day 3 (therapeutic protocol) of autologous anti-GBM nephritis induced in Wistar rats. Proteinuria, creatinine clearance and renal histopathology were assessed as markers of disease activity, while glomerular fibrin deposits (immunoperoxidase staining) and standard parameters of coagulation/fibrinolysis of peripheral blood enabled insight into local and systemic haemostatic mechanisms. Glomerular binding of anti-GBM antibodies (immunofluorescence) and serum titres of autologus nephrotoxic antibodies (haemagglutination assay) represented conditions of immune induction of glomerulopathy. RESULTS: Our experiments indicated that EACA, and to a lesser extent also aprotinin, are capable of preventing proteinuria (EACA, reduction by 57.6%; aprotinin, reduction by 26.8%, compared to untreated nephritic rats, day 3 post-induction) and glomerular histopathological changes, without affecting endogenous creatinine clearance, otherwise depressed in this model of glomerulonephritis. More importantly, both drugs significantly ameliorated glomerular lesions and proteinuria, even when the treatment was initiated on day 3 post-induction, after the injury has begun (EACA reduced proteinuria by 32.0%, and aprotinin reduced it by 20.9% day 7). Administration of EACA and aprotinin at doses reducing glomerular injury did not cause appreciable fibrin deposition in glomeruli of nephritic rats, nor did it modify parameters of systemic coagulation and fibrinolysis in these animals, EACA and aprotinin did not interfere with serum titres of nephrotoxic antibody, nor with the intensity of its binding to the glomerular basement membrane in vivo. CONCLUSIONS: Antiproteolytic drugs utilized in our studies exert their beneficial effect on autologous anti-GBM nephritis through interference with inflammatory phase of the disease, while sparing its immune induction and mechanisms of coagulation/fibrinolysis.

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