The effect of pepsin on autologous immune complex glomerulonephritis

Life Sciences, Vol. 33, pp. 671-677 Printed in the U.S.A.

Pergamon Press

THE EFFECT OF PEPSIN ON AUTOLOGOUS IMMUNE COMPLEX GLOMERULONEPHRITIS Haruo Ohnishi Fuji Central Research Laboratory, Mochida Pharmaceutical Co., Ltd., 722 Jimba-aza-Uenohara, Gotemba, Shizuoka 412, Japan (Received in final form June i, 1983) SUMMARY The effects of intravenous administration of pepsin on autologous immune complex 81omerulonephritis, which is an established experimental model of membranous glomerulopathy in human, were investigated. Sensitization of rats with renal tubular antigen induced an increase in urinary protein excretion, decreases in serum levels of total protein, albumin and immunoglobulin G and histopathologieal abnormalities in glomerulus. A significant increase in serum immune complex and glomerular immune complex deposit were also observed. These abnormalities were ameliorated by pepsin. Pepsin may be effective and beneficial in the treatment of immune complex nephritis.

The author has previously reported that pepsin selectively decomposes immune complex at neutral pH (H. Ohnishi, Ryumachi, submitted for publication, 1982) and that immune complex-type glomerulonephritis of MRL/I mice and spontaneous muscular dystrophy of C57BL/6J dy/dy mice are ameliorated by intravenous pepsin (H. Ohnishi, Life Sciences and N. Engl. J. Med., submitted for publication, 1982). These results indicated that pepsin might be effective in the treatment of autoimmune diseases and nephritis. In the present study, the author has investigated the effects of intravenous pepsin on autologous immune complex glomerulonephritis in rats induced by the injection of renal tubular antigen in Freund's complete adjuvant.

MATERIALS & METHODS Animals; Forty 78-day old male Wistar rats weighing 300-349 g were used. Pepsin; Pepsin was extracted and purified from human urine according to Seijffers et al. I. Pepsin activity was assayed by the method of Anson and Mirsky 2. The specific activity of the preparation used in the present study was 1,912 units/mg-protein and the preparation showed a single band on SDS-polyacrylamide gel electrophoresis. Antigen; Renal tubular antigen (FxlA antigen) was prepared from rats according to Edgington et al. 3. Experimental procedure; One milligram of the antigen was emulsified in 0.25 ml of Freund's complete adjuvant (Iatron) according to the method of Edgington et al. 3. The emulsion was subcutaneously injected to hind leg paw of a Wistar rat. Sixty-four days after the injection, the rats, aged 142 days at that 0024-3025/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

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time, were randomly divided into 4 groups, each consisting of 8 animals. Thereafter, 1,000, 3,000 or i0,000 units/kg/day of pepsin was dissolved in 4 ml of physiological saline and intravenously injected twice a day for 80 days. The animals in the control group received only vehicle. Throughout the study, urinary protein excretion in 24-hour urine 4 was measured. Blood was sampled 24 hour after the final injection of pepsin, when the animals were 222-day old, and kidneys were removed and weighed. Serum levels of total protein 4. albumin S , immunoglobulin G (IgG) 6, immune complex 7, 50% complement hemolysis ~ (CH50) and C36 were measured. Another untreated 8 rats which were 222-day old were examined as an untreated control group. The removed kidneys were further subjected to histopathological study. The kidneys were fixed in 10% formalin buffered with phosphate buffer solution (pH 7.4), embedded in paraffin, then PAS-stained and observed with the light microscope. Also, renal tissue was double-fixed with 2.5% glutaraldehyde and i% osmic acid, embedded in epoxy resin then double-stained with uranium acetate and lead citrate, and observed with the electron microscope. Further, renal tissue was cut with a cryostat at 2 to 4 pm. The sections were reacted with fluorescein-isothiocyanate-labelled goat anti-rat IgG antibody (Fuji Zohki) for 1 hour at room temperature and examined under the fluorescence microscope with epi-illumination 9 .

RESULTS As shown in FIG. I, urinary protein excretion of the control was 53 ± 1 mg/day before the injection of renal tubular antigen. At 64 days after the injection of the antigen the protein level was increased to 124 ± 36 mg/day, and at 144 days after the injection, to 479 ± 84 mg/day. At the same point, the urinary protein levels of the animals received I0,000 units/kg/day of pepsin were 55.3 ± 3.0, 118 ± 25 and 179 ± 30 mg/day, respectively, and significant decreases were observed. With 1,000 and 3,000 units/kg/day of pepsin, urinary protein excretion was decreased with statistical significance at 144 days after the injection of the antigen.

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Pepsin and Membranous Glomerulonephritis

673

At 144 days after the injection of the antigen, serum levels of total protein, albumin, IgG, CH50 and C3 were decreased, and serum level of immune complex was higher than those of normal animals. These changes were evidently improved by the administration of pepsin, as shown in Table I. Kidney weight of the control was 1.52 i 0.II g and increased from 1.14 ± 0.02 g of the untreated control. In the antigen-treated animals that received 1,000, 3,000 and i0,000 units/kg/day of pepsin, kidney weights were 1.32 ± 0.05, 1.29 ± 0.07 and 1.24 ± 0.02 (p < 0.05) g, respectively. Granular deposit of rat IgG was observed along the glomerular capillary wall in the kidney of the animals sensitized with renal tubular antigen, as shown in FIG. 2, and I0,000 units/kg/day of pepsin diminished this IgG deposit, as shown in FIG. 3.

Table I

Effect of pepsin on autologous immune complex glomerulonephritis

Total protein (g/dl) Albumin (g/dl) IgG (mg/dl) CH50 (units/ml) C3 (%) Immune complext

Control

Treated with I000 units/kg of pepsin

Treated with 3000 units/kg of pepsin

Treated Untreated with I0000 control units/kg of pepsin

6.25±0.18 3.11±0.06 130±20 17.6±2.7 83.6±2.9 542±89

6.53±0.08 3.22±0.05 199±24" 24.0±2.7 89.4±1.6 392±57

6.54±0.23 3.38±0.10" 222±29* 26.0±3.1 88.1±1.9 411±107

6.64±0.05 7.25±0.07 3.36±0.05* 3.96±0.03 289±20** 328±6 30.0±2.2** 43.0±0.9 93.5±1.6"* 100±I 291±43" 63±11

?; ~g-aggregated IgG equivalent/ml.

FIG. 2 Granular deposit of fluorescencestained rat IgG is evident along the glomerular basement membrane of renal tubular antigen-sensitized rat.

*; p<0.05, **; p<0.01, from control.

FIG. 3 Faint granular deposit of rat IgG is observed in the glomerular basement membrane of renal tubular antigensensitized rat given 10000 units/kg/ day of pepsin.

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In the kidneys of the animals sensitized with renal tubular antigen, thickening of the basement membrane of glomerulus and Bowman's sac, dilation and hyaline cast formation in renal tubuli, development of cell infiltration into interstitium were observed as shown in FIG. 4 and 6; in electron microscopy, irregular deposit of highly electron-dense materials in the subepithelial layer of the basement membrane of glomerulus and simplification of the visceral epithelial processes were observed, as shown in FIG. 8. These histopathological changes were ameliorated or abolished by pepsin administration. FIG. 5, 7 and 9 show renal tissue of the rats given 10,000 units/kg/day.

FIG. 4 Thickening of basement membrane of glomerulus and Bowman's sac, dilation and hyaline cast formation in renal tubuli and cell infiltration in interstitium are observed in renal tubular antigen-sensitized rat.

FIG. 6 Thickening of the glomerular basement membrane is observed in renal tubular antigen-sensitized rat.

FIG.

5

Thickening of basement membrane, tubular changes and cell infiltration are slightly observed in renal tubular antigen-sensitized rat given i0000 units/kg/day of pepsin.

FIG. 7 Thickening of the glomerular basement membrane is slightly observed in reanl antigen-sensitized rat given 10000 units/kg/day of pepsin.

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Pepsin and ~ m b r a n o u s Glomerulonephritis

FIG. 9

FIG. 8 Simplification of visceral epithelial processes and highly electron-dense materials in subepithelial layer are observed in renal tubular antigensensitized rat. Bar = 1 ~m.

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Simplification of visceral epithelial processess and electrondense materials in subepithelial layer are slightly observed in renal tubular antigen-sensitized rat given 10000 units/kg/day of pepsin. Bar = 1 B m .

DISCUSSION The author has previously reported that pepsin selectively decomposes immune complex at neutral pH (H. Ohnishi, Ryumachi, submitted for publication, 1982) and that spontaneous glomerulonephritis of MRL/I mice and spontaneous muscular dystrophy are ameliorated by intravenous administration of pepsin (H. Ohnishi, Life Sciences and N. Engl. J. Med., submitted for publication, 1982), indicating that pepsin may be useful for the treatment of autoimmune diseases and nephritis. In the present report, the effects of intravenous pepsin on the autologous immune complex glomerulonephritis of rats, in which immune complex plays an important role in its pathogenesis and development, have been studied. Autologous immune complex glomerulonephritis of rats was first reported to be induced by sensitizing animals with renal homogenate of rats and Freund's complete adjuvant I0, then with renal tubular antigen (FxlA antigen) and Freund's complete adjuvant 3 . The mechanisms of the pathogenesis and development of this autologous immune complex glomerulonephrits are generally accepted that a marked increase in capillary permeability, resulting in proteinuria and nephrotic syndrome, is caused by glomerular deposit of immune complex derived from (i) the circulation where they were formed through the autologous tubular antigen in the serum and the antibody against the tubular antigen II,12 or from (2) in situ binding of the antibody to the substance with the same antigen determinant as tubular antigen present in the basement membranel3, I~ The complements are activated and consumed by these immune complexes. In the present experiments, sensitization of rats with renal tubular antigen induced proteinuria and nephrotic syndromes; increase in urinary protein excretion and decreases in serum levels of total protein, albumin and IgG and histopathological abnormalities in glomerulus such as thickening of basement membrane and simplification of the visceral epithelial processes. Furthermore, a significant increase in serum level of immune complex and immune

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complex deposit in the subepithelial layer of glomerulus were observed. Decreases in serum levels of CH50 and C3 was attributable to consumption by the increased immune complex 15. These results were in good agreement with the mechanisms responsible for the pathogenesis and development of autologous immune complex nephritis. Pepsin administration initiated at 64 days after the sensitization with renal tubular antigen continued for 80 days, at which period urinary protein excretion began to rise, suppressed the tubular antigen-induced nephritis in a dose-dependent manner. Pepsin decreased an increased urinary protein excretion, suppressed the decrease in serum proteins and ameliorated the histopathological changes. The mechanism of anti-nephritic action of pepsin may be decomposition of pathogeneic immune complex, formed with tubular antigen or tubular antigen-like substances and antibody against tubular antigen, based on the decomposing action of pepsin on immune complexes at neutral pH (H. Ohnishi, Ryumachi, submitted for publication, 1982). This assumption is supported by the result that the serum level and glomerular deposit of immune complex, which were increased after the sensitization with tubular antigen, were decreased by the administration of pepsin. Suppression of the decreases in CH50 and C3 observed admnistration of pepsin agreed with the decrease of immune complex.

after

the

On the other hand, there are some reports evaluating the effect of drugs on autolougus immune complex glomerulonephritis which was an established experimental model of membranous glomerulopathy in human. However, immunosuppressants or anti-inflammatory drugs have been reported to have "little or no effect °'16 on this model 19. Fleuren and Hoedmaeker demonstrated that triple-drug treatment of cyclophosphamide, azathiopurine and predonisolone was more effective than single-drug treatment of autologous immune complex glomerulonephritis through a decrease in the level of the free-circulating antibody 16. The mechanism of action of the triple-drug treatment is different from that of pepsin. Since pepsinogen exists in circulating blood, this antinephritic action of pepsin may be related to physiological role of serum pepsinogen. These therapeutic effect of pepsin on autologous immune complex glomerulonephritis in rats may, together with the fact that pepsin showed therapeutic effects on spontaneous glomerulonephritis of MRL/I mice, indicate that pepsin may be effective and beneficial in the treatment of immune complex nephritis.

REFERENCES I. M.J. SEIJFFERS, H.L. SEGAL and L.L. MILLER, Am. J. Physiol., 206 1106-1110 (1964) 2. M.L. ANSON and A.E. MIRSKY, J. Gen. Physiol., 1 6 59-63 (1932) 3. T.S. EDGINGTON, R.J. GLASSOCK and F.J. DIXON, J. Immunol., 99 1199-1210 (1967). 4. A.G. GORNALL, C.J. BARDAWILL and M.M. DAVID, J. Biol. Chem., 177 751-766 (1948) 5. B.T. DOUMAS, W.A. WATSON and H.G. BIGGS, Clin. Chim. Acta, 31 87-96 (1971) 6. G. MANCINI~ A.O. CARBONARA and J.F. HEREMANS, Immunochemistry, 2 235-254 (1965) 7. K. ARAI and A. SHIMIZU, Clin. Exp. Immunol., 46 541-546 (1981) 8. M.M. MAYER, Experimental Immunochemistry, pp. 133-240 Thomas, Springfield, (1961) 9. S.P. MAKKER and B. MOORTHY, Lab. Invest., 44 1-5 (1981)

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I0. II. 12. 13. 14. 15. 16. 17.

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W. HEYMANN, D.B. HACKEL, S. HARWOOD, S.G.F. WILSON and J.L.P. HUNTER, Proc. Soc. Exp. Biol. Med., I00 660-664 (1959) R.J. GLASSOCK, T.S. EDGINGTON, J,l. WATSON and F.J. DIXON, J. Exp. Med., 127 573-588 (1968) C.K. ABRASS, W.A. BORDER and R.J. GLASSOCK, Kidney Int., 12 508 (1977) W.G. COUSER, D.R. STEINMULLER, M.M. STILM_ANT, D.J. SALANT and L.M. LOWENSTEIN, J. Clin. Invest., 62 1275-1287 (1978) B.J.C. Van Damme, G.J. FLEUREN, W.W. BAKKER, R.L. VERNIER and Ph.J. HOEDMAEKER, Lab. Invest., 38 502-510 (1978) U. E. NYDEGGER, M.D. KAZATCHKINE and P.H. LAMBERT, Progress in Immunology IV, (Vol. 3) pp. 1025-1043 Academic Press, London (1980) G.J. FLEUREN and Ph.J. HOEDMAEKER, Clin. Exp. Immunol., 41 218-224 (1980) L.R. KUPOR, D.C. LOWANCE and J.J. McPHAUL, Jr., J. Lab. Clin. Med., 87 2736 (1976)