RESEARCH Printed in the United
THROMBOSIS
vol. States
6, pp. 399-408, Pergamon Press,
1975 Inc.
SOLUBLE FIBRINMONOMER COMPLEXES ANDFIBRINOLYTIC ACTIVITYIN KIDNEY DURING THEGENERALIZED SHWARTZMAN REACTION IN RABBIT Maciej Szczepanski,
Cezary Lucer
Department of Biochemistry and Department of Pathomorphological Diagnostics, Medical Center of Postgraduate Education, Warsaw, Poland (Received 6.11.1974; in revised form 10.3.1975. Accepted by Editor I.M. Nilsson)
ABSTRACT The generalized Shwartzman reaction (GSR) was induced in rabbits. Blood samples were obtained at intervals and determinations were made of fibrinogen concentration, platelet count, soluble fibrin monomer complexes (SFMC) and the fibrinolytic activity of the euglobulin fraction. In addition, the tissue plasminogen activator activity in kidney sections was measured. An increase in SFMCand decrease in tissue activator activity in the outer medullary zone of the kidney were observed 6 hours after the preparative endotoxin dose as well as 3 and 6 hours after the provocative injection. The maximal fibrin deposition within the kidney glomeruli was observed 6 hours after the provocative endotoxin injection and 18 hours later the amount of intraglomerular fibrin decreased but the secondary, necrotic changes of the kidney cortex progressed. ‘Ihe fibrinolytic activity in the outer medullary zone of kidney returned to the normal values at the same time. The importance of the fibrinolytic activity in kidney as a defensive mechanism in GSR is discussed. INTRODUCTION In previous experiments we have studied the fibrinolytic activity in plasma euglobulins and in kidneys of rabbits after two spaced Salmonella typhi endotoxin injections (1). The determinations were performed within one hour after the second endotoxin injection and, therefore, renal cortical necrosis did not develop in those animals. The aim of this study was to determine the soluble fibrin monomer complexes, the fibrinolytic activity in euglobulins and some Other parameters of coagulation system and to investigate the activity of plasminogen activator in kidney during the generalized Shwartzman reaction (GSR) in rabbit upto 24 hours after the second endotoxin injection.
399
KIDNEY FIDRINOLYSIS DURING GSR
400
v~11.6,~0.5
MATERIALS AND METHODS Male, albino rabbits weighing from 1.5 to 2.5 kg were used. They were given a standard LSK diet (Bacutil, Warsaw) and water ad libitum through the experiments. The animals were anesthetized intravenously with Eunarcon (Riedel de Ha&, Hannover), the kidneys were removed by translumbar route and the blood samples were obtained through siliconized needle and plastic tube by direct cardiac puncture. The animals were then sacrificed. The Escherichia coli endotoxin (Lipopolysaccharide B, 055:85, Difco, USA) was dissolved in isotonic saline and administered through the marginal ear vein. 50 mcg/kg was injected as a preparative dose and 200 mcg/kg was injected as a provocative dose, 24 hours later. The materials (Table 1).
consisted
of the control
group and of 9 test groups
TABLE 1 Intervals between Endotoxin Injections, Blood Samplings and Kidney Removals in the Respective Groups of Rabbits Interval
Group Control
Preparative
Provocative
endotoxin injection: 15 min lh 6h 24 h endotoxin injection: 15 min lh 3h 6h 24 h
Number of rabbits
50 mcg/kg
200
mcg/kg
Platelet counts were done by means of phase microscopy, Nygaard (2) . Fibrinogen concentration of Bidwell (3). Ethanol gelation
8 7 5 5 5
5 6 6 9 6
as described
in plasma was determined by the calorimetric
test was performed according
by method
to Godal (4).
Protamine sulfate paracoagulation test was done according to Niewiarowski The pH of the 1% stock solution of protamine sulfate and Gurewich (5) The 0.2 ml (Wytwornia Surowic i Szczepionek, Warszawa) was adjusted to 7.8. samples of its dilutions were mixed with equal volumes of titrated plateletpoor plasma and incubated at +37’C for 30 min. The results were expressed in mg/ml of protamine sulfate in the last dilution with visible paracoagulation. Fibrinolytic activity in euglobulins was determined on fibrin plates. 1 ml of titrated plasma was diluted with 8 ml of distilled water, acidified to was dissolved in 0.25 ml pH 5.3 with 1% acetic acid (6)) and the precipitate
vo1.6,No.5
KIDNEY FIBRINOLYSIS
DURING GSR
401
0.03 ml of these concentrated of 0.23 M Palitsch borate buffer, pH 7.6. euglobulins was placed in triplicate on the surface of fibrin plate. The fibrin plates were done according to Brakman (7). Tissue activator activity and localization in rabbit kidney were estimated This activity was expressed as the shortest lysis according to Todd (8). time, corresponding to the shortest incubation time in previous investigations (1). Histological examinations of the second kidney were done in each animal. This kidney was immediately fixed in 10% formalin and stained with haematoxylin-eosin for morphologic changes and with phosphotungstic acid The percentage of involved glomeruli haematoxylin for fibrin deposits. was counted and the amount of fibrin deposits was approximately estimated. Statistical analysis of data was done according to the Wilcoxon rank-sum test. The differences among the groups of animals were regarded as signicant on the probability leval of alpha = 0.05. RESULTS The histological examinations revealed the glomerular microthrombi in kidneys obtained 3, 6 and 24 hours after the provocative endotoxin injection. The incidence of glomerular microthrombosis did not differ among these three groups of animals but the intensity of fibrin deposition was quite different The percentage of involved glomeruli and the amount of deposin each group. ited fibrin increased up to the 6th hour after the provocative endotoxin injection and then decreased. This decrease was accompanied, however, by the secondary morphologic changes that could be interpreted as the results of the occlusion of kidney cortex microcirculation by fibrin deposits (Table 2). The platelet count diminished as early as 15 min after the preparative endotoxin injection and the next statistically significant decrease of platelet count was observed between 6th and 24th hours after this injection. The further changes in platelet count were insignificant until 6 hours after the provocative endotoxin injection when statistically significant decline in platelet count was noted in comparison to the last value before the injection (Fig. 1). The fibrinogen level in plasma increased by 60% of the control value 24 hours after the preparative endotoxin injection and the statistically significant decrease of this level was observed 3 hours after the provocative endotoxin injection. The next rise of fibrinogen level was noted 24 hours after this injection. The first statistically significant increase of the protamine sulfate paracoagulable material was observed 6 hours after the first endotoxin injection and the second increase was noted 3 hours after provocative Then, the content of this material diminished 24 hours endotoxin injection. after this last injection. The ethanol gelation test was positive hours after preparative endotoxin injection. animals 3 and 6 hours after the provocative negative in all animals 24 hours after this
in four among five animals 6 This test was positive in all endotoxin injection but was last injection (Fig. 2).
402
KIDNEY FIDRINOLYSIS DURING GSR
v01.6,~0.5
The fibrinolytic activity in euglobulins increased during the first hour after the preparative endotoxin injection. The decrease of this activity was observed 6 and 24 hours after this injection and the provocative endotoxin injection resulted again in a rise in this activity within next 15 min. There were not any significant changes of this activity in the further groups of animals. The activity of tissue activator in kidney, referred to as the shortest lysis time, decreased significantly for the first time 6 hours after the preparative endotoxin injection. This activity returned then to the normal yalues and a subsequent decrease was observed 3 and 6 hours after the provoThe tissue activator activity was again within cative endotoxin injection. the normal values 24 hours after this injection (Fig. 3). Figures 4 and 5 illustrate the difference in the intensity of lysis of the fibrin films covering the kidney sections. The section of normal kidney revealed the fibrinolytic ‘activity as early as after 30 min of
TABLE2 Histological Changes in Animals after Endotoxin Injection. Histological
changes
Animals with glomerular microthrombi (All animals in the group) Mean percentage of involved glomeruli in animals with microthrombi Amount of deposit; material ? within glomeruli Leukocytic infiltration Necrosis of glomeruli ad/or of tubular epithelium
the Provocative
Lapse of time after endotoxin injection
the provocative
3 hours
6 hours
4 (6)
g (9)
24 hours 5 (6)
31%
74%
39%
+
+++
++
absent _
absent +
present ++
x/ + - a few fibrin strands within glomeruli, ++ - moderate deposition of fibrin within glomeruli, +++ massive deposition of fibrin within glomeruli. ++ large or confluent foci of necrosis. xx/ + - small foci of necrosis,
KIDNEY FIBRINOLYSIS
DURING GSR
FIG. 1 Platelet count. Ordinate: platelet count in thousands lines: standard deviations. C: control group. 1Ex: injection . 2 Ex: provocative endotoxin injection.
403
per mm3. Vertical preparative endotoxin
FIG. 2 Paracoagulation tests. Upper part of the figure: protamine sulfate paracoagulation test. Ordinate: protamine sulfate in mg/ml in last dilutions with paracoagulation. Lower part of the figure: ethanol gelation test. Each square denotes one determination. For further details see legend to Fig. 1.
404
KIDNEY FIBRINOLYSIS
DURING GSR
v01.6,~0.5
(6
14 12 10 6 6 L 2
15rmn. 1
6
2L
15mn
I
3
SLllnmm 10
20 30 LO
50 60 70
I I
FIG. 3 Fibrinolytic activity in euglobulins and in kidney. Upper part of the figure: fibrinolytic activity in euglobulins. Ordinate: diameter of fibrin plate lysis. Lower part of the figure: fibrinolytic activity in kidney. Ordinate: shortest lysis time. For further details see legend to Fig. 1.
incubation and the fibrin film lysis was extensive after 60 min of incubation On the other hand, the section of kidney obtained 3 hours after (Fig. 4). the provocative endotoxin injection had to be incubated for 60 min until the first signs of fibrin film lysis were visible (Fig. 5). The localization of the plasminogen activator in normal kidney was confined mainly to the outer medullary zone. The interlobar arteries and the veins passing through this zone as well as the arcuate vessels, however, did not reveal the activator activity (Fig. 4). It was also possible to observe this activity connected with the medullary rays of the outer medullary zone extending into cortex in section incubated for 50 or 60 min. The localization of plasminogen activator in kidney with decreased fibrinolytic activity, i.e. with the shortest lysis time equal or longer than 40 min, was often restricted to the inner part of the outer medullary zone in the close proximity of the inner medullary zone (Fig. 5).
vo1.6,No.5
KIDNEY FIBRXNOLYSIS
FIG. 4 Fibrinolytic activity in kidney of 60 min. normal rabbit . Incubation:
DURING GSR
405
FIG. 5 Fibrinolytic activity in kidney of endotoxin-treated rabbit. Kidney obtained 3 hours after the provocative endotoxin injection. Incubation : 60 min.
c: cortex. OMZ: outer medullary zone. IMZ: inner medullary zone. AV: arcuate vessels. ILV: interlobar vessels. MR: fibrinolytic activity extending into cortex along medullary rays. FA: fibrinolytic activity confined to the inner part of outer medullary zone in the close proximity of inner medul lazy zone. Magnification: x 5. DISCUSSION It is well known that a single endotoxin dose does not elicit fibrin deposition within kidney glomeruli and that a lapse of 3 to 6 hours after the second provocative endotoxin injection is necessary for the appearance of these deposits. The mechanism by which fibrin deposition occurs in this animal model is not well understood. Few measurements have been performed 24 hours after the second dose of endotoxin. Surprisingly we found that the percentage of glomeruli with fibrin deposits decreased 24 hours after the second dose of endotoxin although this did not prevent development of the secondary changes, i.e., necrosis of glomeruli and/or tubular epithelium with surrounding leukocyte infiltration. The early decrease of platelet count after the first endotoxin dose was observed by others (9,10,11). We found another drop in platelet count between the 6th and 24th hours after the first endotoxin injection. This decrease could not be related to the direct influence of endotoxin since it is known that endotoxin is completely eliminated from the circulation within the first hour after intravenous injection (12).
406
KIDNEY FIBRINOLYSIS
DURING GSR
vo1.6,No.5
We have noted the rise in fibrinogen concentration in plasma 24 hours after each endotoxin injection and a decrease of this concentration 3 and 6 hours after the provocative injection only.’ This decrease corresponded to the time during which fibrin deposition occurred within kidney glomeruli. Similar patterns of fibrinogen level changes during the GSRwere observed by other authors (9,11,13). In vitro studies have demonstrated that the paracoagulation of plasma with protamine sulfate occurs in the presence of either Fragment X0 or of fibrin monomers (14,lS). Accordi.ng to Latallo et al (16) the mechanism of protamine sulfate action on the fibrin-FDP complexes consists primarily in binding FDP into readily soluble PS-FDP complexes, thus allowing the liberated fibrin to polymerize spontaneously. Findings of Musumeci (17) and of Konttinen et al(14) suggested the value of ethanol gelation test in demonstration of fibrin monomer complexes with early FDP. The recent studies of Gurewich et al (18) proved, however, that the ethanol gelation test is sensitive to fibrin monomers but not to fdp and that this gelation might be non-specific. The investigations performed by Hedner and Nilsson (19) and by Jacobsen and Southers (20) revealed that the ethanol gelation test was more often positive than protamine sulfate test in patients with such disorders where chronic or acute intravascular coagulation was present. The increased content of soluble fibrin monomer complexes (SFMC), as expressed by the positive results of both paracoagulation tests in our experiments, was observed simultaneously with time of intraglomerular fibrin deposition, i.e., 3 and 6 hours after the provocative endotoxin injection. On the other hand, a similar increase of SFMCwas also noted 6 hours after the preparative ‘endotoxin injection but without any accompanying histologic changes of kidney. The decrease of fibrinolytic activity in the euglobulin fraction after the Salmonella typhi endotoxin injections in rabbits was noted by Lipinski et a1(13), but the first determination in their experiments was done 6 hours after the preparative endotoxin dose. Sutton et al(21) investigated the fibrinolytic activity in euglobulins in baboons after one endotoxin injection. They observed a marked shortening of the euglobulin clot lysis time as early returning to the normal value one as 10 min after the endotoxin injection, hour after injection. A rise of fibrinolytic activity in the euglobulin fraction was observed The increase of immediately after each endotoxin dose in our experiments. this activity after the first endotoxin injection was followed by a decrease The second endotoxin injection was followed 6 and 24 hours after injection. also by the rise in the fibrinolytic activity as early as within 15 min. A great dispersion of the further results does ,qti-allow to conclude whether the fibrinolysis was activated or inhibitedtill the end of experiments. The fibrinolytic activity of rabbit kidney after endotoxin injections was investigated by Graeff et al (22) and by Bergstein and Michael Jr. (23)) using the Todd method. Graeff et al (22) infused E. coli endotoxin by means of a continuous intravenous drip and found the disappearance of fibrinolytic activity in the niedullary zone of the kidney in animals with intraglomerular fibrin deposits. They did not observe fibrinolytic activity in the kidney cortex even in normal rabbits. Bergstein and Michael Jr. (23) studied the
Vo1.6,No.5
KIDNEY FIFEUNOLYSIS
DURING GSR
407
fibrinolytic activity only in the kidney cortex after one or two spaced enThis activity disappeared after the first endotoxin dotoxin injection. injection and this disappearance was not accompanied by any histological or activity of the immunofluorescent changes in the kidney. The fibrinolytic kidney cortex was measured again 24 hours after this injection. The second endotoxin injection resulted in intraglomerular fibrin deposition and in the persistent disappearance of fibrinolytic activity in kidney cortex. The tissue activator activity in kidney in our experiments was decreased 6 hours after each endotoxin injection and also in the animals that were The decrease investigated 3 hours after the provocative endotoxin injection. of this activity after the provocative injection was accompanied by the fibrin deposition within the kidney glomeruli but we have not seen any glomerular changes when the similar decrease was observed after the preparative injection. If the fibrinolytic activity in the outer medullary zone of the kidney might be related to the changes in the kidney cortex, one would suppose that there is another mechanism besides this activity that prevents the fibrin deposition within glomeruli after the first endotoxin injection but fails after the second one. This supposition is in agreement with the opinion of Lee and coworkers (24) on the role of reticuloendothelial system in disseminated intravascular coagulation. In spite of the fact that the fibrinolytic activity in the outer medullary zone of the kidney returned to the normal values 18 hours after the observed maximal intraglomerular fibrin deposition and that the amount fibrin within glomeruli decreased at the same time, the secondary changes in the kidney cortex progressed. It seems reasonable to conclude that the return of the fibrinolytic activity in rabbit kidney to the normal values will not prevent the kidney cortex necrosis once the massive intraglomerular fibrin deposition is initiated, at least during the Generalized Shwartzman Reaction. REFERENCES 1. 2. 2. 3. 4. 5. 6. 7. 8. 9.
M. Fibrinolytic activity in rabbit kidney after the endotoxin injections . Thrombosis Res. : 4_, 587, 1974 NYGAARD, K. Direct method of counting platelets in oxalated blood. Proc. Mayo Clin.: 8, 365, 1933. of humanplasma. Biochem. J. : 55, 497, 1953. BIDWELL,E. Fibrinolysins GODAL,H.C. and ABILDGAARD, U. Gelation of soluble fibrin z plasma by ethanol. Stand. J.Haemat: z, 342, 1966. NIEWIAROWSKI, S. and GUREWICH, V. Laboratory identification of intravascular coagulation. J. Lab. Clin. Med.: 77, 665, 1971. KOWALSKI, E . , KOPEC.M. and NIEWIAROWSKI. S. An evaluation of the J. Clin. Path.: euglobulin method for the determination of fibrinolysis. 12, 215, 1959. BRAMAN, P. Fibrinolysis. A standarized fibrin plate method and a fibrinolytic assay of plasminogen. Amsterdam,Scheltema and Holkema, N.V., 1967. TODD,A.S. The tissue activator of plasminogen and thrombosis. In: Thrombosis and Anticoagulant Therapy. W. Walker (Ed.). University of St. Andrews. 1961. CORRIGAN,J.J.-JR., ABILDGAARD, C.F . , VANDERHEIDEN, J.R. and SCHUIMN,L. Quantitative aspects of blood coagulation in the Generalized Shwartzman Reaction. Pediat. Res.: 1, 39, 1967. SZCZEPANSKI ,
408
10. 11. 12.
13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
KIDNEY FIBRINOLYSIS
DURING GSR
Vo1.6,No.5
GAYNOR,E., BOUVIER,C. and SPAET, T.D. Vascular lesions: Possible pathogenetic base of the Generalized Shwartzman Reaction. Science: 170, 986, 1970. MCKAY,D.G. and SHAPIRO,S.S. Alterations in the blood coagulation system induced by bacterial endotoxin. In vivo. (Generalized Shwartzman Reaction). J. Exp. Med.: 107, 353, 1958. CAREY,F.J., BRAUDE,A.I. and ZALESKY,M. Studies with radioactive endotoxin. II. The effect of tolerance on the distribution after intravenous injection of Escherichia coli endotoxin labelled with Cr51. J. Clin. Invest.: 37, 441, 1958. LIPINSKI, B., WOROWSKI, K., JELJASZEWICZ, J., NIEWIAROWSKI, S. and REJNIAK,L. Participation of soluble fibrin monomer complexes and platelet factor 4 in the Generalized Shwartzman Reaction. Thromb. Diath. haemorrh: 20, 285, 1968. KONTTINEN, Y . P . , KEMPAINEN, L. and TURUNEN, 0. Comparison of ethanol and protamine tests in demonstration of soluble fibrin and early products of fibrin degradation. Thromb. Diath. haemorrh.: 28, 342, 1972. NIEWIAROWSKI, S. and GLJRBWICH, V. Laboratory identificationof intraJ. Lab. Clin.Med.: 77, 665, 1971. vascular coagulation. LATALLO,Z.S., WEGRZYNOWICZ, Z., BUDZYNSK1,A.S. and KOPEC,M. Effect of protamine sulphate on the solubility of fibrinogen, its derivatives and other plasma proteins. Stand. J. Haemat.: Suppl. 13, 151, 1971. MUSUMECI, V. Ethanol gelation test and protamine sulphax test in diagnosis of intravascular coagulation. Stand. J. Haemat.: Suppl. 13, 97, 1971. GUREWICH, V., LIPINSKI, B., and LIPINSKA, I. Comparative study of precipitation and paracoagulation by protamine sulfate and ethanol gelation tests. Thromb. Res.: 2, 539, 1973. HEDNER,U. and NILSSON,I.M. Parallel determinations of FDP and fibrin monomers with various methods. Thromb. Diath. haemorrh: 28, 268, 1972. JACOBSEN,C.D., and SOUTHERS,N.J. Ethanol gelation and protamine sulfate Comparison and critique. Thromb. Diath. haemorrh.: 29, 130, 1973. test. F. Coagulation, fibrinolysis and renal SUTTON,D., RAO, P. and BACHMAN, flow changes following endotoxin injection in baboons. Fed. Proc.: 28, 510, 1969. enzyme system GPXFF, H. , MITCHELL,P.S. and BELLER,F.K. Fibrinolytic of the kidney related to renal function after infusion of endotoxin in rabbits. Lab. Invest. : 19, 169, 1968. BERGSTEIN,J.M. and MICE, A.F. Jr. Renal cortical fibrinolytic Thromb. activity in the rabbit following one or two doses of endotoxin. Diath. haemorrh.: 2, 27, 1973. LEE, E., PROSE, P.M. and COHEN,M.A. The role of reticuloendothelial system in diffuse, low grade intravascular coagulation. Thromb. Diath. haemorrh.: Suppl. 20, 88,1966.
THROMBOSIS
vol. States
6, pp. 399-408, Pergamon Press,
1975 Inc.
SOLUBLE FIBRINMONOMER COMPLEXES ANDFIBRINOLYTIC ACTIVITYIN KIDNEY DURING THEGENERALIZED SHWARTZMAN REACTION IN RABBIT Maciej Szczepanski,
Cezary Lucer
Department of Biochemistry and Department of Pathomorphological Diagnostics, Medical Center of Postgraduate Education, Warsaw, Poland (Received 6.11.1974; in revised form 10.3.1975. Accepted by Editor I.M. Nilsson)
ABSTRACT The generalized Shwartzman reaction (GSR) was induced in rabbits. Blood samples were obtained at intervals and determinations were made of fibrinogen concentration, platelet count, soluble fibrin monomer complexes (SFMC) and the fibrinolytic activity of the euglobulin fraction. In addition, the tissue plasminogen activator activity in kidney sections was measured. An increase in SFMCand decrease in tissue activator activity in the outer medullary zone of the kidney were observed 6 hours after the preparative endotoxin dose as well as 3 and 6 hours after the provocative injection. The maximal fibrin deposition within the kidney glomeruli was observed 6 hours after the provocative endotoxin injection and 18 hours later the amount of intraglomerular fibrin decreased but the secondary, necrotic changes of the kidney cortex progressed. ‘Ihe fibrinolytic activity in the outer medullary zone of kidney returned to the normal values at the same time. The importance of the fibrinolytic activity in kidney as a defensive mechanism in GSR is discussed. INTRODUCTION In previous experiments we have studied the fibrinolytic activity in plasma euglobulins and in kidneys of rabbits after two spaced Salmonella typhi endotoxin injections (1). The determinations were performed within one hour after the second endotoxin injection and, therefore, renal cortical necrosis did not develop in those animals. The aim of this study was to determine the soluble fibrin monomer complexes, the fibrinolytic activity in euglobulins and some Other parameters of coagulation system and to investigate the activity of plasminogen activator in kidney during the generalized Shwartzman reaction (GSR) in rabbit upto 24 hours after the second endotoxin injection.
399
KIDNEY FIDRINOLYSIS DURING GSR
400
v~11.6,~0.5
MATERIALS AND METHODS Male, albino rabbits weighing from 1.5 to 2.5 kg were used. They were given a standard LSK diet (Bacutil, Warsaw) and water ad libitum through the experiments. The animals were anesthetized intravenously with Eunarcon (Riedel de Ha&, Hannover), the kidneys were removed by translumbar route and the blood samples were obtained through siliconized needle and plastic tube by direct cardiac puncture. The animals were then sacrificed. The Escherichia coli endotoxin (Lipopolysaccharide B, 055:85, Difco, USA) was dissolved in isotonic saline and administered through the marginal ear vein. 50 mcg/kg was injected as a preparative dose and 200 mcg/kg was injected as a provocative dose, 24 hours later. The materials (Table 1).
consisted
of the control
group and of 9 test groups
TABLE 1 Intervals between Endotoxin Injections, Blood Samplings and Kidney Removals in the Respective Groups of Rabbits Interval
Group Control
Preparative
Provocative
endotoxin injection: 15 min lh 6h 24 h endotoxin injection: 15 min lh 3h 6h 24 h
Number of rabbits
50 mcg/kg
200
mcg/kg
Platelet counts were done by means of phase microscopy, Nygaard (2) . Fibrinogen concentration of Bidwell (3). Ethanol gelation
8 7 5 5 5
5 6 6 9 6
as described
in plasma was determined by the calorimetric
test was performed according
by method
to Godal (4).
Protamine sulfate paracoagulation test was done according to Niewiarowski The pH of the 1% stock solution of protamine sulfate and Gurewich (5) The 0.2 ml (Wytwornia Surowic i Szczepionek, Warszawa) was adjusted to 7.8. samples of its dilutions were mixed with equal volumes of titrated plateletpoor plasma and incubated at +37’C for 30 min. The results were expressed in mg/ml of protamine sulfate in the last dilution with visible paracoagulation. Fibrinolytic activity in euglobulins was determined on fibrin plates. 1 ml of titrated plasma was diluted with 8 ml of distilled water, acidified to was dissolved in 0.25 ml pH 5.3 with 1% acetic acid (6)) and the precipitate
vo1.6,No.5
KIDNEY FIBRINOLYSIS
DURING GSR
401
0.03 ml of these concentrated of 0.23 M Palitsch borate buffer, pH 7.6. euglobulins was placed in triplicate on the surface of fibrin plate. The fibrin plates were done according to Brakman (7). Tissue activator activity and localization in rabbit kidney were estimated This activity was expressed as the shortest lysis according to Todd (8). time, corresponding to the shortest incubation time in previous investigations (1). Histological examinations of the second kidney were done in each animal. This kidney was immediately fixed in 10% formalin and stained with haematoxylin-eosin for morphologic changes and with phosphotungstic acid The percentage of involved glomeruli haematoxylin for fibrin deposits. was counted and the amount of fibrin deposits was approximately estimated. Statistical analysis of data was done according to the Wilcoxon rank-sum test. The differences among the groups of animals were regarded as signicant on the probability leval of alpha = 0.05. RESULTS The histological examinations revealed the glomerular microthrombi in kidneys obtained 3, 6 and 24 hours after the provocative endotoxin injection. The incidence of glomerular microthrombosis did not differ among these three groups of animals but the intensity of fibrin deposition was quite different The percentage of involved glomeruli and the amount of deposin each group. ited fibrin increased up to the 6th hour after the provocative endotoxin injection and then decreased. This decrease was accompanied, however, by the secondary morphologic changes that could be interpreted as the results of the occlusion of kidney cortex microcirculation by fibrin deposits (Table 2). The platelet count diminished as early as 15 min after the preparative endotoxin injection and the next statistically significant decrease of platelet count was observed between 6th and 24th hours after this injection. The further changes in platelet count were insignificant until 6 hours after the provocative endotoxin injection when statistically significant decline in platelet count was noted in comparison to the last value before the injection (Fig. 1). The fibrinogen level in plasma increased by 60% of the control value 24 hours after the preparative endotoxin injection and the statistically significant decrease of this level was observed 3 hours after the provocative endotoxin injection. The next rise of fibrinogen level was noted 24 hours after this injection. The first statistically significant increase of the protamine sulfate paracoagulable material was observed 6 hours after the first endotoxin injection and the second increase was noted 3 hours after provocative Then, the content of this material diminished 24 hours endotoxin injection. after this last injection. The ethanol gelation test was positive hours after preparative endotoxin injection. animals 3 and 6 hours after the provocative negative in all animals 24 hours after this
in four among five animals 6 This test was positive in all endotoxin injection but was last injection (Fig. 2).
402
KIDNEY FIDRINOLYSIS DURING GSR
v01.6,~0.5
The fibrinolytic activity in euglobulins increased during the first hour after the preparative endotoxin injection. The decrease of this activity was observed 6 and 24 hours after this injection and the provocative endotoxin injection resulted again in a rise in this activity within next 15 min. There were not any significant changes of this activity in the further groups of animals. The activity of tissue activator in kidney, referred to as the shortest lysis time, decreased significantly for the first time 6 hours after the preparative endotoxin injection. This activity returned then to the normal yalues and a subsequent decrease was observed 3 and 6 hours after the provoThe tissue activator activity was again within cative endotoxin injection. the normal values 24 hours after this injection (Fig. 3). Figures 4 and 5 illustrate the difference in the intensity of lysis of the fibrin films covering the kidney sections. The section of normal kidney revealed the fibrinolytic ‘activity as early as after 30 min of
TABLE2 Histological Changes in Animals after Endotoxin Injection. Histological
changes
Animals with glomerular microthrombi (All animals in the group) Mean percentage of involved glomeruli in animals with microthrombi Amount of deposit; material ? within glomeruli Leukocytic infiltration Necrosis of glomeruli ad/or of tubular epithelium
the Provocative
Lapse of time after endotoxin injection
the provocative
3 hours
6 hours
4 (6)
g (9)
24 hours 5 (6)
31%
74%
39%
+
+++
++
absent _
absent +
present ++
x/ + - a few fibrin strands within glomeruli, ++ - moderate deposition of fibrin within glomeruli, +++ massive deposition of fibrin within glomeruli. ++ large or confluent foci of necrosis. xx/ + - small foci of necrosis,
KIDNEY FIBRINOLYSIS
DURING GSR
FIG. 1 Platelet count. Ordinate: platelet count in thousands lines: standard deviations. C: control group. 1Ex: injection . 2 Ex: provocative endotoxin injection.
403
per mm3. Vertical preparative endotoxin
FIG. 2 Paracoagulation tests. Upper part of the figure: protamine sulfate paracoagulation test. Ordinate: protamine sulfate in mg/ml in last dilutions with paracoagulation. Lower part of the figure: ethanol gelation test. Each square denotes one determination. For further details see legend to Fig. 1.
404
KIDNEY FIBRINOLYSIS
DURING GSR
v01.6,~0.5
(6
14 12 10 6 6 L 2
15rmn. 1
6
2L
15mn
I
3
SLllnmm 10
20 30 LO
50 60 70
I I
FIG. 3 Fibrinolytic activity in euglobulins and in kidney. Upper part of the figure: fibrinolytic activity in euglobulins. Ordinate: diameter of fibrin plate lysis. Lower part of the figure: fibrinolytic activity in kidney. Ordinate: shortest lysis time. For further details see legend to Fig. 1.
incubation and the fibrin film lysis was extensive after 60 min of incubation On the other hand, the section of kidney obtained 3 hours after (Fig. 4). the provocative endotoxin injection had to be incubated for 60 min until the first signs of fibrin film lysis were visible (Fig. 5). The localization of the plasminogen activator in normal kidney was confined mainly to the outer medullary zone. The interlobar arteries and the veins passing through this zone as well as the arcuate vessels, however, did not reveal the activator activity (Fig. 4). It was also possible to observe this activity connected with the medullary rays of the outer medullary zone extending into cortex in section incubated for 50 or 60 min. The localization of plasminogen activator in kidney with decreased fibrinolytic activity, i.e. with the shortest lysis time equal or longer than 40 min, was often restricted to the inner part of the outer medullary zone in the close proximity of the inner medullary zone (Fig. 5).
vo1.6,No.5
KIDNEY FIBRXNOLYSIS
FIG. 4 Fibrinolytic activity in kidney of 60 min. normal rabbit . Incubation:
DURING GSR
405
FIG. 5 Fibrinolytic activity in kidney of endotoxin-treated rabbit. Kidney obtained 3 hours after the provocative endotoxin injection. Incubation : 60 min.
c: cortex. OMZ: outer medullary zone. IMZ: inner medullary zone. AV: arcuate vessels. ILV: interlobar vessels. MR: fibrinolytic activity extending into cortex along medullary rays. FA: fibrinolytic activity confined to the inner part of outer medullary zone in the close proximity of inner medul lazy zone. Magnification: x 5. DISCUSSION It is well known that a single endotoxin dose does not elicit fibrin deposition within kidney glomeruli and that a lapse of 3 to 6 hours after the second provocative endotoxin injection is necessary for the appearance of these deposits. The mechanism by which fibrin deposition occurs in this animal model is not well understood. Few measurements have been performed 24 hours after the second dose of endotoxin. Surprisingly we found that the percentage of glomeruli with fibrin deposits decreased 24 hours after the second dose of endotoxin although this did not prevent development of the secondary changes, i.e., necrosis of glomeruli and/or tubular epithelium with surrounding leukocyte infiltration. The early decrease of platelet count after the first endotoxin dose was observed by others (9,10,11). We found another drop in platelet count between the 6th and 24th hours after the first endotoxin injection. This decrease could not be related to the direct influence of endotoxin since it is known that endotoxin is completely eliminated from the circulation within the first hour after intravenous injection (12).
406
KIDNEY FIBRINOLYSIS
DURING GSR
vo1.6,No.5
We have noted the rise in fibrinogen concentration in plasma 24 hours after each endotoxin injection and a decrease of this concentration 3 and 6 hours after the provocative injection only.’ This decrease corresponded to the time during which fibrin deposition occurred within kidney glomeruli. Similar patterns of fibrinogen level changes during the GSRwere observed by other authors (9,11,13). In vitro studies have demonstrated that the paracoagulation of plasma with protamine sulfate occurs in the presence of either Fragment X0 or of fibrin monomers (14,lS). Accordi.ng to Latallo et al (16) the mechanism of protamine sulfate action on the fibrin-FDP complexes consists primarily in binding FDP into readily soluble PS-FDP complexes, thus allowing the liberated fibrin to polymerize spontaneously. Findings of Musumeci (17) and of Konttinen et al(14) suggested the value of ethanol gelation test in demonstration of fibrin monomer complexes with early FDP. The recent studies of Gurewich et al (18) proved, however, that the ethanol gelation test is sensitive to fibrin monomers but not to fdp and that this gelation might be non-specific. The investigations performed by Hedner and Nilsson (19) and by Jacobsen and Southers (20) revealed that the ethanol gelation test was more often positive than protamine sulfate test in patients with such disorders where chronic or acute intravascular coagulation was present. The increased content of soluble fibrin monomer complexes (SFMC), as expressed by the positive results of both paracoagulation tests in our experiments, was observed simultaneously with time of intraglomerular fibrin deposition, i.e., 3 and 6 hours after the provocative endotoxin injection. On the other hand, a similar increase of SFMCwas also noted 6 hours after the preparative ‘endotoxin injection but without any accompanying histologic changes of kidney. The decrease of fibrinolytic activity in the euglobulin fraction after the Salmonella typhi endotoxin injections in rabbits was noted by Lipinski et a1(13), but the first determination in their experiments was done 6 hours after the preparative endotoxin dose. Sutton et al(21) investigated the fibrinolytic activity in euglobulins in baboons after one endotoxin injection. They observed a marked shortening of the euglobulin clot lysis time as early returning to the normal value one as 10 min after the endotoxin injection, hour after injection. A rise of fibrinolytic activity in the euglobulin fraction was observed The increase of immediately after each endotoxin dose in our experiments. this activity after the first endotoxin injection was followed by a decrease The second endotoxin injection was followed 6 and 24 hours after injection. also by the rise in the fibrinolytic activity as early as within 15 min. A great dispersion of the further results does ,qti-allow to conclude whether the fibrinolysis was activated or inhibitedtill the end of experiments. The fibrinolytic activity of rabbit kidney after endotoxin injections was investigated by Graeff et al (22) and by Bergstein and Michael Jr. (23)) using the Todd method. Graeff et al (22) infused E. coli endotoxin by means of a continuous intravenous drip and found the disappearance of fibrinolytic activity in the niedullary zone of the kidney in animals with intraglomerular fibrin deposits. They did not observe fibrinolytic activity in the kidney cortex even in normal rabbits. Bergstein and Michael Jr. (23) studied the
Vo1.6,No.5
KIDNEY FIFEUNOLYSIS
DURING GSR
407
fibrinolytic activity only in the kidney cortex after one or two spaced enThis activity disappeared after the first endotoxin dotoxin injection. injection and this disappearance was not accompanied by any histological or activity of the immunofluorescent changes in the kidney. The fibrinolytic kidney cortex was measured again 24 hours after this injection. The second endotoxin injection resulted in intraglomerular fibrin deposition and in the persistent disappearance of fibrinolytic activity in kidney cortex. The tissue activator activity in kidney in our experiments was decreased 6 hours after each endotoxin injection and also in the animals that were The decrease investigated 3 hours after the provocative endotoxin injection. of this activity after the provocative injection was accompanied by the fibrin deposition within the kidney glomeruli but we have not seen any glomerular changes when the similar decrease was observed after the preparative injection. If the fibrinolytic activity in the outer medullary zone of the kidney might be related to the changes in the kidney cortex, one would suppose that there is another mechanism besides this activity that prevents the fibrin deposition within glomeruli after the first endotoxin injection but fails after the second one. This supposition is in agreement with the opinion of Lee and coworkers (24) on the role of reticuloendothelial system in disseminated intravascular coagulation. In spite of the fact that the fibrinolytic activity in the outer medullary zone of the kidney returned to the normal values 18 hours after the observed maximal intraglomerular fibrin deposition and that the amount fibrin within glomeruli decreased at the same time, the secondary changes in the kidney cortex progressed. It seems reasonable to conclude that the return of the fibrinolytic activity in rabbit kidney to the normal values will not prevent the kidney cortex necrosis once the massive intraglomerular fibrin deposition is initiated, at least during the Generalized Shwartzman Reaction. REFERENCES 1. 2. 2. 3. 4. 5. 6. 7. 8. 9.
M. Fibrinolytic activity in rabbit kidney after the endotoxin injections . Thrombosis Res. : 4_, 587, 1974 NYGAARD, K. Direct method of counting platelets in oxalated blood. Proc. Mayo Clin.: 8, 365, 1933. of humanplasma. Biochem. J. : 55, 497, 1953. BIDWELL,E. Fibrinolysins GODAL,H.C. and ABILDGAARD, U. Gelation of soluble fibrin z plasma by ethanol. Stand. J.Haemat: z, 342, 1966. NIEWIAROWSKI, S. and GUREWICH, V. Laboratory identification of intravascular coagulation. J. Lab. Clin. Med.: 77, 665, 1971. KOWALSKI, E . , KOPEC.M. and NIEWIAROWSKI. S. An evaluation of the J. Clin. Path.: euglobulin method for the determination of fibrinolysis. 12, 215, 1959. BRAMAN, P. Fibrinolysis. A standarized fibrin plate method and a fibrinolytic assay of plasminogen. Amsterdam,Scheltema and Holkema, N.V., 1967. TODD,A.S. The tissue activator of plasminogen and thrombosis. In: Thrombosis and Anticoagulant Therapy. W. Walker (Ed.). University of St. Andrews. 1961. CORRIGAN,J.J.-JR., ABILDGAARD, C.F . , VANDERHEIDEN, J.R. and SCHUIMN,L. Quantitative aspects of blood coagulation in the Generalized Shwartzman Reaction. Pediat. Res.: 1, 39, 1967. SZCZEPANSKI ,
408
10. 11. 12.
13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
KIDNEY FIBRINOLYSIS
DURING GSR
Vo1.6,No.5
GAYNOR,E., BOUVIER,C. and SPAET, T.D. Vascular lesions: Possible pathogenetic base of the Generalized Shwartzman Reaction. Science: 170, 986, 1970. MCKAY,D.G. and SHAPIRO,S.S. Alterations in the blood coagulation system induced by bacterial endotoxin. In vivo. (Generalized Shwartzman Reaction). J. Exp. Med.: 107, 353, 1958. CAREY,F.J., BRAUDE,A.I. and ZALESKY,M. Studies with radioactive endotoxin. II. The effect of tolerance on the distribution after intravenous injection of Escherichia coli endotoxin labelled with Cr51. J. Clin. Invest.: 37, 441, 1958. LIPINSKI, B., WOROWSKI, K., JELJASZEWICZ, J., NIEWIAROWSKI, S. and REJNIAK,L. Participation of soluble fibrin monomer complexes and platelet factor 4 in the Generalized Shwartzman Reaction. Thromb. Diath. haemorrh: 20, 285, 1968. KONTTINEN, Y . P . , KEMPAINEN, L. and TURUNEN, 0. Comparison of ethanol and protamine tests in demonstration of soluble fibrin and early products of fibrin degradation. Thromb. Diath. haemorrh.: 28, 342, 1972. NIEWIAROWSKI, S. and GLJRBWICH, V. Laboratory identificationof intraJ. Lab. Clin.Med.: 77, 665, 1971. vascular coagulation. LATALLO,Z.S., WEGRZYNOWICZ, Z., BUDZYNSK1,A.S. and KOPEC,M. Effect of protamine sulphate on the solubility of fibrinogen, its derivatives and other plasma proteins. Stand. J. Haemat.: Suppl. 13, 151, 1971. MUSUMECI, V. Ethanol gelation test and protamine sulphax test in diagnosis of intravascular coagulation. Stand. J. Haemat.: Suppl. 13, 97, 1971. GUREWICH, V., LIPINSKI, B., and LIPINSKA, I. Comparative study of precipitation and paracoagulation by protamine sulfate and ethanol gelation tests. Thromb. Res.: 2, 539, 1973. HEDNER,U. and NILSSON,I.M. Parallel determinations of FDP and fibrin monomers with various methods. Thromb. Diath. haemorrh: 28, 268, 1972. JACOBSEN,C.D., and SOUTHERS,N.J. Ethanol gelation and protamine sulfate Comparison and critique. Thromb. Diath. haemorrh.: 29, 130, 1973. test. F. Coagulation, fibrinolysis and renal SUTTON,D., RAO, P. and BACHMAN, flow changes following endotoxin injection in baboons. Fed. Proc.: 28, 510, 1969. enzyme system GPXFF, H. , MITCHELL,P.S. and BELLER,F.K. Fibrinolytic of the kidney related to renal function after infusion of endotoxin in rabbits. Lab. Invest. : 19, 169, 1968. BERGSTEIN,J.M. and MICE, A.F. Jr. Renal cortical fibrinolytic Thromb. activity in the rabbit following one or two doses of endotoxin. Diath. haemorrh.: 2, 27, 1973. LEE, E., PROSE, P.M. and COHEN,M.A. The role of reticuloendothelial system in diffuse, low grade intravascular coagulation. Thromb. Diath. haemorrh.: Suppl. 20, 88,1966.
