Clin. exp. Immunol. (1977) 28, 173-179.
Induction of anti-glomerular basement membrane antibodies in the Brown-Norway rat by mercuric chloride C. SAPIN, E. DRUET & P. DRUET Laboratoire de Morphologie et d'Immunopathologie Renale, INSERM U28, CNRS ERA 48, Hdpital Broussais, Paris, France
(Received 9 August 1976)
SUMMARY
Anti-glomerular basement membrane (GBM) antibodies were induced in the Brown-Norway rat by mercuric chloride. The existence of anti-GBM antibodies was suspected because of the immunofluorescent linear pattern. It was proved because eluted antibodies from kidneys and circulating IgG had an anti-glomerular basement membrane activity. A glomerular basement membrane antigen modified by HgCl2 is probably responsible for the appearance of such antibodies. The previous demonstration of the occurrence of an immune complex type glomerulonephritis, in outbred Wistar rats, under the same experimental conditions, suggests the existence of a genetic control for this type of immune response. INTRODUCTION Immune type glomerulonephritides, in man or in animal, are induced, either by anti-glomerular basement membrane (GBM) antibodies or by circulating immune complexes. The agents responsible for such diseases are most often unknown in man. Toxic agents, like mercury compounds, may injure the kidney in several ways. An acute intoxication is responsible for the well-known tubular lesions (Gritzka & Trump, 1968). A chronic intoxication with mercury (Mandema et al., 1963) or with gold salts (Lee et al., 1965) is able to induce in man an immune complex type glomerulonephritis. Finally, it has been recently suggested that other toxic agents, like hydrocarbon solvents, could be responsible for anti-GBM antibody induced glomerulonephritis in man (Zimmerman, Groehler & Bierne, 1975). In a previous study, an immune complex type glomerulonephritis has been described in Wistar rats when injected s.c. for several weeks with mercuric chloride (Bariety et al., 1971). The responsible antigen has not been identified. The aim of this study is to report the induction of anti-GBM antibodies in Brown-Norway (BN) rat submitted to the same experimental procedure. MATERIALS AND METHODS Animals. Males and females BN inbred rats weighing 80-120 g were purchased from the CSEAL (CNRS, Orleans, France). Experimental procedure. The rats studied were divided into three groups. Group A. Five males and five females, were s.c. injected three times a week for 2 weeks with 0-10 ml of a 2 mg/ml solution of mercuric chloride in distilled water per 100 g body weight. The solution was prepared extemporaneously and sterilized by filtration through a 0 45 u millipore filter. Group B. Five females received two injections of the same solution on day 1 and 3. Group C. Five females received only one injection of the same solution on day 1.
Correspondence: Dr C. Sapin, Laboratoire de Morphologie et d'Immunopathologie Renale, INSERM U28, CNRS ERA 48 H6pital Broussais, 96 rue Didot 75674, Paris Cedex 14, France.
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Controls. Five females and five males were s.c. injected three times a week for 2 weeks with 0-10 ml of sterilized distilled water. This solution was brought to the same pH (3 8) as the mercuric chloride solution with 0-01 N HC1. Kidney biopsies. Kidney open wedge biopsies were performed at different days after the first injection of HgCl2. Rats were anaesthesized by i.p. injection of 2-5 mg per 100 g body weight of Nembutal (Abbott Laboratories). Biopsies were performed in group A on the 15th day after the first injection. Group B and C rats had sequential renal biopsies on day 3, 6 and 15. Control rats had only one biopsy on the 15th day after the beginning of the experiment. Immunohistochemistry and light microscopy. Kidney specimens were all examined by immunohistochemistry. They were immediately snap-frozen and 4 pum-thick cryostat sections were obtained. The sections were studied without any fixation, by immunoperoxidase and/or by immunofluorescence. The sections were incubated with the conjugate for 30 min at laboratory temperature. The slides were then washed with PBS (5 min, three times) and immediately examined by immunofluorescence using a Leitz microscope (SM-Lux). For immunoperoxidase, the peroxidase activity was revealed using the method of Graham & Karnovsky (1966) washed with PBS (5 min, three times) and examined using a Zeiss microscope. When indirect immunofluorescence was used, the kidney sections were first incubated for 30 min with the unlabelled reagent, washed with PBS (5 min, three times) and then stained with the labelled conjugate in the same manner as for direct immunohistochemistry. Kidney samples from group A and control rats were also examined by light microscopy. For that purpose a specimen of the biopsy was fixed in Bouin's fixative and stained with the Masson's trichrome. Preparation of antisera. An anti-rat IgG antiserum was raised in a sheep with rat IgG (Lalibert6 et al., 1975). Rat IgG was isolated from normal rat serum by precipitation at 40% ammonium sulphate saturation and by chromatography on a DEAE (DE 52 Whatmann) cellulose column equilibrated with a 0-017 M phosphate buffer pH 6-9. A rabbit anti-rat C3 antiserum was prepared according to the method of Mardiney & Muller-Eberhard (1965). Another rabbit anti-rat C3 antiserum was purchased from Cappel Laboratories. A sheep anti-rat albumin antiserum was prepared as described elsewhere (Bignon et al., 1976). All the antisera were found to be monospecific when tested by immunoelectrophoretic analysis. A rabbit anti-rat IgM antiserum was obtained following the same procedure as for anti-human jp antiserum (Sapin et al., 1975) because of the well-known antigenic relationship between rat and human IgM. Briefly, rat serum was applied on an anti-human pu polyacrylamide beads immunoadsorbent (Sapin & Druet, 1976). The rat IgM was then eluted using a 0-2 M glycine C1H buffer pH 2-8, neutralized and concentrated. A rabbit was immunized with the eluted rat IgM. The anti-serum obtained was monospecific for rat IgM after adsorption on a glutaraldehyde insolubilized rat IgG immunoadsorbent (Avrameas & Ternyck, 1969). A rabbit anti-whole normal rat serum antiserum was purchased from Behringwerke AG. The gammaglobulin fraction from all of these antisera was obtained by ammonium sulphate precipitation at 40% saturation and fluoresceinated according to Goldstein, Slizys & Chase (1961). The anti-rat IgG and anti-rat albumin antibodies were labelled with peroxidase (Boehringer Grade I) according to Avrameas (1969). Elutions studies. Rats from group A were killed the day following the renal biopsy. These rats were bled out by cardiac puncture. Their serum was frozen. The kidney cortex was diced, homogeneized, and washed in PBS until the OD at 280 nm of the supernatant was equal to 0. The kidney homogenate was then again mixed with PBS for 1 hr at laboratory temperature under constant stirring. The supernatant was concentrated by ultrafiltration and used as a control ('neutral eluate'). The homogenate was then treated with a 0-02 M citrate buffer pH 3-2 during 1 hr at laboratory temperature under constant stirring (Lerner, Glassock & Dixon, 1967). This 'acid eluate' was immediately neutralized with 0-01 N NaOH, dialysed against PBS and concentrated by ultrafiltration up to 1 mg/ml. It was tested by immunoelectrophoretic analysis using a sheep anti-rat IgG antiserum and a rabbit anti-whole normal rat serum antiserum. Both 'neutral' and 'acid eluates' were tested in vitro by indirect immunofluorescence as described above, on a normal BN rat kidney cryostat section. The eluate was revealed using the fluoresceinated sheep anti-rat IgG antiserum. The 'acid eluate' was also tested in vivo: an unilaterally nephrectomized male BN rat, weighing 70 g was i.v. injected with 1 ml of the eluate containing 1 mg proteins. A renal biopsy was performed 24 hr later and the kidney specimen was tested by immunofluorescence using the anti-rat IgG com-
jugate. Circulating anti-glomerular basement membrane antibodies. The sera from group A rats were pooled and the IgG fraction was isolated as described above for the preparation of the sheep anti-rat IgG antiserum. The fraction was tested in vitro (McPhaul & Dixon, 1969) and in vivo as described above (see: elution studies) after concentration up to 6 mg/ml. The IgG fraction from control BN rats, was isolated and tested in the same way. Proteinuria. The 24-hr protein urinary excretion was determined by the Biuret reaction after trichloracetic acid precipitation.
RESULTS Group A rats were obviously sick at the time of the biopsy (day 15). Control females weighed an average of 150 g and males 190 g. Group A females weighed an average of 125 g and males 160 g. These rats had a moderate proteinuria (Table 1) Control rats were in apparent good health and they had a mean proteinuria of 2 mg/24 hr (from 0-7-7 mg/24 hr); Group B and C rats were also in good health, their weight was similar to that of control rats. Four rats were proteinuric in group B and only one in group C.
HgCl2 induced anti-GBM antibodies
175
TABLE 1. Immunohistochemical results and proteinuria in BN rats injected with mercuric chloride
Kidney immunohistochemistry
Number of rats
Total dose HgCl2 injected (mg)
Day of
Group A
10
1-2
15
10/10*
8/10
10/10
0/10
Group B
5
04
3 6 15
0/5 0/5 5/5
n.d. 0/5 0/5
n.d. 0/5
3/5
n.d. n.d. 0/5
3 6 15
0/5 0/5
3/5
n.d. 0/5 0/5
n.d. 0/5 3/5
n.d. n.d. 0/5
15
0/10
0/10
0/10
0/10
biopsy
Anti-IgG Anti-IgM Anti-C3
Anti-albumin
Proteinuria (mg/24 hr) 20 (4-91)t n.d. n.d. 22-5
(4-39) Group C
Controls
5
10
0-2
0
n.d. n.d. 63 (0-24) 2 (0-7 7)
* Expressed as number of positive rats by number of rats studied. t Extreme values.
Immunohistochemistry Results are summarized in Table 1. Both immunofluorescence and immunoperoxidase studies with anti-rat IgG conjugates of the group A rats showed an intense, linear, continuous smooth staining of all the glomerular capillary walls (Fig. 1). The intensity of the staining was the same for all of the ten rats tested. Bowman's capsule, tubules and vessels were not labelled. A similar localization was observed with the anti-rat C3 antisera (Fig. 2), but the labelling was less intense than with the anti-rat IgG conjugates. When the sections were stained with the anti-rat pu antiserum, a much more discrete but quite distinct staining of some glomerular capillary walls appeared. It was much more difficult to ascertain whether the pattern was linear. Tubules and vessels were negative. No staining of the glomerular capillary walls was observed with the anti-rat albumin conjugates. Occasionally some droplets were observed probably within the epithelial cells of some glomerular tufts. Numerous droplets were observed in the tubular cells.
FIG. 1. Kidney cryostat section from a group A BN rat. Staining with the fluoresceinated sheep anti-rat IgG antiserum. Note the continuous, linear and smooth pattern of the labelling on the basement membrane of the glomerular capillary walls. (Magnification x 392.)
Group B and C rats were all negative when stained with the anti-rat IgG, anti-rat C3 and anti-rat pu antisera on days 3 and 6. However, on day 15 all five rats in group B and three out of the five rats in group C were positive when studied with the anti-rat IgG conjugates. The pattern observed was also a
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C. Sapin, E. Druet & P. Druet
FIG. 2. Same rat as in Fig. 1. Staining with the fluoresceinated rabbit anti-rat C3 antiserum. The same linear pattern of labelling is observed but with a lesser intensity. (Magnification x 392.)
smooth, linear, continuous one, but the fixation was often less intense than in group A. A similar fixation was observed with the anti-rat C3 antisera. No significant fixation was observed with the anti-rat 4 antiserum. Control rats were completely negative when studied with the same conjugates.
Light microscopy In group A, the glomerular tufts were essentially normal. No extra or endocapillary cellular proliferation could be observed, and the glomerular capillary walls _ had a normal thickness. On the other hand, ;rA very important tubular lesions were observed in all ten rats: the tubules were enlarged, tubular cells had desquamated and numerous casts were noted.
A _~~~~ :
Induction of anti-glomerular basement membrane antibodies in the Brown-Norway rat by mercuric chloride C. SAPIN, E. DRUET & P. DRUET Laboratoire de Morphologie et d'Immunopathologie Renale, INSERM U28, CNRS ERA 48, Hdpital Broussais, Paris, France
(Received 9 August 1976)
SUMMARY
Anti-glomerular basement membrane (GBM) antibodies were induced in the Brown-Norway rat by mercuric chloride. The existence of anti-GBM antibodies was suspected because of the immunofluorescent linear pattern. It was proved because eluted antibodies from kidneys and circulating IgG had an anti-glomerular basement membrane activity. A glomerular basement membrane antigen modified by HgCl2 is probably responsible for the appearance of such antibodies. The previous demonstration of the occurrence of an immune complex type glomerulonephritis, in outbred Wistar rats, under the same experimental conditions, suggests the existence of a genetic control for this type of immune response. INTRODUCTION Immune type glomerulonephritides, in man or in animal, are induced, either by anti-glomerular basement membrane (GBM) antibodies or by circulating immune complexes. The agents responsible for such diseases are most often unknown in man. Toxic agents, like mercury compounds, may injure the kidney in several ways. An acute intoxication is responsible for the well-known tubular lesions (Gritzka & Trump, 1968). A chronic intoxication with mercury (Mandema et al., 1963) or with gold salts (Lee et al., 1965) is able to induce in man an immune complex type glomerulonephritis. Finally, it has been recently suggested that other toxic agents, like hydrocarbon solvents, could be responsible for anti-GBM antibody induced glomerulonephritis in man (Zimmerman, Groehler & Bierne, 1975). In a previous study, an immune complex type glomerulonephritis has been described in Wistar rats when injected s.c. for several weeks with mercuric chloride (Bariety et al., 1971). The responsible antigen has not been identified. The aim of this study is to report the induction of anti-GBM antibodies in Brown-Norway (BN) rat submitted to the same experimental procedure. MATERIALS AND METHODS Animals. Males and females BN inbred rats weighing 80-120 g were purchased from the CSEAL (CNRS, Orleans, France). Experimental procedure. The rats studied were divided into three groups. Group A. Five males and five females, were s.c. injected three times a week for 2 weeks with 0-10 ml of a 2 mg/ml solution of mercuric chloride in distilled water per 100 g body weight. The solution was prepared extemporaneously and sterilized by filtration through a 0 45 u millipore filter. Group B. Five females received two injections of the same solution on day 1 and 3. Group C. Five females received only one injection of the same solution on day 1.
Correspondence: Dr C. Sapin, Laboratoire de Morphologie et d'Immunopathologie Renale, INSERM U28, CNRS ERA 48 H6pital Broussais, 96 rue Didot 75674, Paris Cedex 14, France.
173
174
C. Sapin, E. Druet & P. Druet
Controls. Five females and five males were s.c. injected three times a week for 2 weeks with 0-10 ml of sterilized distilled water. This solution was brought to the same pH (3 8) as the mercuric chloride solution with 0-01 N HC1. Kidney biopsies. Kidney open wedge biopsies were performed at different days after the first injection of HgCl2. Rats were anaesthesized by i.p. injection of 2-5 mg per 100 g body weight of Nembutal (Abbott Laboratories). Biopsies were performed in group A on the 15th day after the first injection. Group B and C rats had sequential renal biopsies on day 3, 6 and 15. Control rats had only one biopsy on the 15th day after the beginning of the experiment. Immunohistochemistry and light microscopy. Kidney specimens were all examined by immunohistochemistry. They were immediately snap-frozen and 4 pum-thick cryostat sections were obtained. The sections were studied without any fixation, by immunoperoxidase and/or by immunofluorescence. The sections were incubated with the conjugate for 30 min at laboratory temperature. The slides were then washed with PBS (5 min, three times) and immediately examined by immunofluorescence using a Leitz microscope (SM-Lux). For immunoperoxidase, the peroxidase activity was revealed using the method of Graham & Karnovsky (1966) washed with PBS (5 min, three times) and examined using a Zeiss microscope. When indirect immunofluorescence was used, the kidney sections were first incubated for 30 min with the unlabelled reagent, washed with PBS (5 min, three times) and then stained with the labelled conjugate in the same manner as for direct immunohistochemistry. Kidney samples from group A and control rats were also examined by light microscopy. For that purpose a specimen of the biopsy was fixed in Bouin's fixative and stained with the Masson's trichrome. Preparation of antisera. An anti-rat IgG antiserum was raised in a sheep with rat IgG (Lalibert6 et al., 1975). Rat IgG was isolated from normal rat serum by precipitation at 40% ammonium sulphate saturation and by chromatography on a DEAE (DE 52 Whatmann) cellulose column equilibrated with a 0-017 M phosphate buffer pH 6-9. A rabbit anti-rat C3 antiserum was prepared according to the method of Mardiney & Muller-Eberhard (1965). Another rabbit anti-rat C3 antiserum was purchased from Cappel Laboratories. A sheep anti-rat albumin antiserum was prepared as described elsewhere (Bignon et al., 1976). All the antisera were found to be monospecific when tested by immunoelectrophoretic analysis. A rabbit anti-rat IgM antiserum was obtained following the same procedure as for anti-human jp antiserum (Sapin et al., 1975) because of the well-known antigenic relationship between rat and human IgM. Briefly, rat serum was applied on an anti-human pu polyacrylamide beads immunoadsorbent (Sapin & Druet, 1976). The rat IgM was then eluted using a 0-2 M glycine C1H buffer pH 2-8, neutralized and concentrated. A rabbit was immunized with the eluted rat IgM. The anti-serum obtained was monospecific for rat IgM after adsorption on a glutaraldehyde insolubilized rat IgG immunoadsorbent (Avrameas & Ternyck, 1969). A rabbit anti-whole normal rat serum antiserum was purchased from Behringwerke AG. The gammaglobulin fraction from all of these antisera was obtained by ammonium sulphate precipitation at 40% saturation and fluoresceinated according to Goldstein, Slizys & Chase (1961). The anti-rat IgG and anti-rat albumin antibodies were labelled with peroxidase (Boehringer Grade I) according to Avrameas (1969). Elutions studies. Rats from group A were killed the day following the renal biopsy. These rats were bled out by cardiac puncture. Their serum was frozen. The kidney cortex was diced, homogeneized, and washed in PBS until the OD at 280 nm of the supernatant was equal to 0. The kidney homogenate was then again mixed with PBS for 1 hr at laboratory temperature under constant stirring. The supernatant was concentrated by ultrafiltration and used as a control ('neutral eluate'). The homogenate was then treated with a 0-02 M citrate buffer pH 3-2 during 1 hr at laboratory temperature under constant stirring (Lerner, Glassock & Dixon, 1967). This 'acid eluate' was immediately neutralized with 0-01 N NaOH, dialysed against PBS and concentrated by ultrafiltration up to 1 mg/ml. It was tested by immunoelectrophoretic analysis using a sheep anti-rat IgG antiserum and a rabbit anti-whole normal rat serum antiserum. Both 'neutral' and 'acid eluates' were tested in vitro by indirect immunofluorescence as described above, on a normal BN rat kidney cryostat section. The eluate was revealed using the fluoresceinated sheep anti-rat IgG antiserum. The 'acid eluate' was also tested in vivo: an unilaterally nephrectomized male BN rat, weighing 70 g was i.v. injected with 1 ml of the eluate containing 1 mg proteins. A renal biopsy was performed 24 hr later and the kidney specimen was tested by immunofluorescence using the anti-rat IgG com-
jugate. Circulating anti-glomerular basement membrane antibodies. The sera from group A rats were pooled and the IgG fraction was isolated as described above for the preparation of the sheep anti-rat IgG antiserum. The fraction was tested in vitro (McPhaul & Dixon, 1969) and in vivo as described above (see: elution studies) after concentration up to 6 mg/ml. The IgG fraction from control BN rats, was isolated and tested in the same way. Proteinuria. The 24-hr protein urinary excretion was determined by the Biuret reaction after trichloracetic acid precipitation.
RESULTS Group A rats were obviously sick at the time of the biopsy (day 15). Control females weighed an average of 150 g and males 190 g. Group A females weighed an average of 125 g and males 160 g. These rats had a moderate proteinuria (Table 1) Control rats were in apparent good health and they had a mean proteinuria of 2 mg/24 hr (from 0-7-7 mg/24 hr); Group B and C rats were also in good health, their weight was similar to that of control rats. Four rats were proteinuric in group B and only one in group C.
HgCl2 induced anti-GBM antibodies
175
TABLE 1. Immunohistochemical results and proteinuria in BN rats injected with mercuric chloride
Kidney immunohistochemistry
Number of rats
Total dose HgCl2 injected (mg)
Day of
Group A
10
1-2
15
10/10*
8/10
10/10
0/10
Group B
5
04
3 6 15
0/5 0/5 5/5
n.d. 0/5 0/5
n.d. 0/5
3/5
n.d. n.d. 0/5
3 6 15
0/5 0/5
3/5
n.d. 0/5 0/5
n.d. 0/5 3/5
n.d. n.d. 0/5
15
0/10
0/10
0/10
0/10
biopsy
Anti-IgG Anti-IgM Anti-C3
Anti-albumin
Proteinuria (mg/24 hr) 20 (4-91)t n.d. n.d. 22-5
(4-39) Group C
Controls
5
10
0-2
0
n.d. n.d. 63 (0-24) 2 (0-7 7)
* Expressed as number of positive rats by number of rats studied. t Extreme values.
Immunohistochemistry Results are summarized in Table 1. Both immunofluorescence and immunoperoxidase studies with anti-rat IgG conjugates of the group A rats showed an intense, linear, continuous smooth staining of all the glomerular capillary walls (Fig. 1). The intensity of the staining was the same for all of the ten rats tested. Bowman's capsule, tubules and vessels were not labelled. A similar localization was observed with the anti-rat C3 antisera (Fig. 2), but the labelling was less intense than with the anti-rat IgG conjugates. When the sections were stained with the anti-rat pu antiserum, a much more discrete but quite distinct staining of some glomerular capillary walls appeared. It was much more difficult to ascertain whether the pattern was linear. Tubules and vessels were negative. No staining of the glomerular capillary walls was observed with the anti-rat albumin conjugates. Occasionally some droplets were observed probably within the epithelial cells of some glomerular tufts. Numerous droplets were observed in the tubular cells.
FIG. 1. Kidney cryostat section from a group A BN rat. Staining with the fluoresceinated sheep anti-rat IgG antiserum. Note the continuous, linear and smooth pattern of the labelling on the basement membrane of the glomerular capillary walls. (Magnification x 392.)
Group B and C rats were all negative when stained with the anti-rat IgG, anti-rat C3 and anti-rat pu antisera on days 3 and 6. However, on day 15 all five rats in group B and three out of the five rats in group C were positive when studied with the anti-rat IgG conjugates. The pattern observed was also a
176
C. Sapin, E. Druet & P. Druet
FIG. 2. Same rat as in Fig. 1. Staining with the fluoresceinated rabbit anti-rat C3 antiserum. The same linear pattern of labelling is observed but with a lesser intensity. (Magnification x 392.)
smooth, linear, continuous one, but the fixation was often less intense than in group A. A similar fixation was observed with the anti-rat C3 antisera. No significant fixation was observed with the anti-rat 4 antiserum. Control rats were completely negative when studied with the same conjugates.
Light microscopy In group A, the glomerular tufts were essentially normal. No extra or endocapillary cellular proliferation could be observed, and the glomerular capillary walls _ had a normal thickness. On the other hand, ;rA very important tubular lesions were observed in all ten rats: the tubules were enlarged, tubular cells had desquamated and numerous casts were noted.
A _~~~~ :
