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The effect of the stimulation of alpha or beta adrenergic receptors on the generalized Shwartzman reaction in rabbit with denervated kidney
THE EFFECT OF THE ST154CLATIOX CF ALPHA CR BETA ADRES ERGIC RECEPTORS OS THE GENERr’.LIZED SH:.‘:‘.~.RTZ:;;XI; REACTION IS RABBIT :!;ITH DESERls.TED KIDKEY
Aiaciel
Szczepanski,
Cezary
Department of Biochemistry Diagnostics, ::Iedical
(Received
of alpha-
endotoxin-induced
Elibieta
Staff-Zieliriska
and Department of Pathomorphological Center of Postgraduate Education, C’arsaw, Poland
9.8.1977; Accepr;ed by
The influence
Lucer,
in revised Editor 1.X.
form 11.11.1977. Silsson)
or beta-adrenergic
intraglomerular
agonists
fibrin
deposition
was studied in rabbits with one denervated norepinephrine infused together with the
on the
\ i .f.d.,
kidney. The provocative
endotoxin dose did not influence i. f-d. ted kidney or in the intact one. The
within the denervaisoproterenol
administered
with
endotoxin
also
dose
in a common
had no effect
infusion on
i. f. d.
provocative
in the
intact
kidney
but significantly augmented this deposition within the denervated kidney. The results of the fibrinolytic activity of kidney cortex cannot explain the effects of adrenergic agonists on endotoxin-induced i.f.d. It is supposed that the enhancing effect of isoproterenol on i.f.d. within the denervated kidney can be ascribed to the vasodilating effect of this drug, promoted in the denervated kidney by the absence of endogenous norepinephrine at the nerve endings.
ISTRODUCTION Our previous studies /l/ confirmed the findings of Palmerio et al. !2/ that the sympathetic denervation of the kidney diminished the endotoxin-induced intraglomerular fibrin deposition ( i. f. d. ) although the increase of endotoxin dose significantly reduced this effect.
It has been reported that the i.v. injection of endotoxin resulted in the elevated level of circulating epinephrine /j ,L, 5/ and norepinephrine
236
XDREXERGIC
The
/3,6!.
contribution
AGONISTS
of catecholamines
OS GSR
is believed
vo1.12,so.2
to be essential
for the generalized Shwartzman reaction /7,Si. It is known that the epinephrine is secreted mainly by the adrenal medulla and that the norepinephrine is synthetized for the most part at the adrenergic postganglionic nerve endings /9,10/. The disruption of these nerves is expected to abolish the local s_ynthesis of norepinephrine and it, indeed, has been proved that the denervated tissue is devoid of this substance /10111,12/. If the absence of norepinephrine and, consequently, the lack of alpha-adrenergic receptors stimulation in the denervated kidney was responsible for the diminished endotoxin-induced i.f .d. then the infusion of exogenous norepinephrine should abolish this effect of denervation. The vasomotor response of the denervated organ to the catecholamines is even more expressed than that of the exogenous intact one / 10,13,14/ and, therefore, we expected the common infusion of norepinephrine and endotoxin to result in the enhanced i.f .d. within the denervated kidney but this was not the case. We decided then to stimulate the be ta-adrenergic receptors and the rabbits receiving the endotoxin
and isoproterenol
were
MATERIALS Albino to 2.7
rabbits,
kg were
Popielno
used.
included
AND
investigations.
METHODS
strain,
Sympathetic
in these
of both sexes,
denervation
weighing
from
of the left kidney
1 .j
was
performed as described previously /I/ and after the lapse of 7 days the rabbits were injected i.v. with saline or endotoxin ( Lipopolysacccharide 055: B5, Difco) . The 4-hours infusion of saline or endotoxin B, E.coli or/and adrenergic agonist was begun in unanesthetized animals 24 hours later, according to the protocol illustrated in Fig. 1. Norepinephrine ( Levonor, Polfa) was administered as an alpha-adrenergic agonist and isoproterenol (Isuprel, Winthrop) as a beta-adrenergic agonist. The blood samples were obtained for the baseline determinations from the central ear artery before the saline or endotoxin injection and for the final determinations by the direct heart puncture after the completion of infusion. Both kidneys were removed immediately afterwards and the animals were sacrificed. The blood was obtained through the siliconized needles into the siliconized test tubes, mixed with 4.8% sodium citrate, 9: 1 and kept in melting ice till the beginning of laboratory tests. The 1.
following
Ethanol
Relation
determinations test,
according
were
performed:
to Godal
/15/.
:nlcctions. !nfus ions .
SaCl: SaCl: Nor:
normal normal
saline, saline,
norepinephrine,
2 ml. Ex: endotoxin, 2’3 mcg kg. 8 ml/h, Ex: endotoxin, 12.3 mcg’kg 73 mcggikgih.
[so:
‘1.
isoproterenol,
5 mcg/kg,‘h.
2. c?
.
/ C.
5.
Fibrinogen concentration in plasma by Swair: and Feder method ~‘16 . i:ibrinolytic activity in concentrated euglobulins i 17,‘. Fibrinolytic activity of kidney cortexaccording to the Astrup and Albrechtsen assay for tissue stable activator of plasminoger. ‘1s’. The potassium thiocyanate extracts of kidney cortex specimens were incubated on bovine fibrin plates for 22 hours. Intraglomerular fibrin deposition was estimated as the percentage C? involved glomeruli in the kidney specimens stained with phosphotungt--t: acid haematoxylin ,l PTAHj / 17 /.
Statistical sum test.
analysis
of data was done according
to the V’:..ilcoxon rsnk-
RESULTS The results samples and were and 9 , Fig.
2, .
cf ethanol gelation test were negative in all baseline positive in final samples cnly in animals of groups 7.5
The fibrin ..L3en 717 concentration in plasma of animals of groups 1) 2. and 3 decreased throughout the experiments and no differences were The inrease of this concentration was observed among these groups. between observed in animals of groups i, 5, and 6 without the differences
r
Ia
‘aJ;rilVE
RESULT
cl FIG.
The
results
of ethanol
NEGATIVE
RESULT
2. gelation
test
in final
samples.
The animals receiving both endotoxin doses, i. e. these groups, either. of groups 7,8 and 9 revealed the decline of plasma fibrinogen concentration during the experiments and this decline was significantly greater in animals of group 9 than in animals of group 7 or of group 8 (alpha = Y 0.01). There were not any significant in euglobulins between the various All
animals
of group
differences in the fibrinolytic groups of rabbits.
3 had the undetectable
fibrinolytic
activity
activity
of
both kidney cortices at the end of experiments and it was in contrast to the results obtained in animals of groups 1 and 2 (Table 1) .There were of not any noteworthy differences in this activity between the animals groups L,S and 6. The fibrinolytic activity of both kidney cortices was undetectable in most of the animals of groups 7 and 8. The 70% of animals of group 9 revealed fibrinolytic activity in at least one kidney control cortex and the pattern of this activity resembled that of the animals
of group
1.
The intraglomerular fibrin deposition was not seen in any of the animals of groups 1 to 6 and Table 2 illustrates the numbers of animals of groups 7,s anil 9 that developed these changes.
T .
-i-
/
c
t
i
L
c
!
I
_:
i
2
?
!
I
-
I
7
’ ! -1
f.a.
in both kidney
Ii
cortices
;
‘?
i
I-
/-
!
,
‘7 :
.i i/
1
/
I
T
I
I
s
, i
1‘he Numbers of Animals with intra
Groups
Animals
with
i.f.d.
in both kidneys
Animals
with
i.f.d.
in the intact
ml> Animals
without
i.f.d.
3 kidney
2 \:
j I
i
I / I
, -
t; I
2
1 I I 1 1
fi
-/
There were not any significant differences in the percentage of lnvalved glomeruli in intact kidney cortex among the ani.mals of groups 7.5 and 9. This percentage in denervated kidney cortex did not differ, too, ‘between the animals of groups 7 and 8. T:he percentage of
/
involved
glomeruli
m denervated
kidnev
was significar,tl_i- increased in comparison corresponding cortex in animals of group .alphn = 0.03, *Fig. 3;.
tort ex in animals
of
group
9
with this percentage in the 7 as well as of group d
Group
la
INTbCT
FIG.
KIDNEY
cl
DENEZVATED
KIDNEY
3.
in The mean percentage of glomeruli with fibrin deposition animals of groups 7,s and 9. Vertical lines: standard errors of means.
DISCUSSION None of the adrenergic agonists preceded by saline or by endotoxin injection elicited the generation of soluble fibrin monomer complexes as was
revealed
by the negative
complexes
appeared,
endotoxin
doses
however,
combined
results
of the ethanol
gelation
in most of the animals
or not combined
test.
receiving
with adrenergic
These both
agonists.
.The infusion of saline or of adrenergic agonists preceded by saline injection resulted in the decreased fibrinogen concentration; this phenomenon can be explained by the hemodiluting effect of infused solutions. The rise in fibrinogen concentration observed after the infusion of corresponding solutions but preceded by the preparatory endotoxin injection was due to the well known stimulatory effect of this injection on the fibrinogen synthesis /19,20/. Both endotoxin doses decreased fibrinogen concentration in plasma and this decrease was augmented by isoproterenol but not influenced by norepinephrine. These but findings suggest that stimulation of beta-adrenergic receptors
r_or of alpha-sdr2riergzc fibrinogen
receptors
Increases
tk
2nd~tcxLn-rnducsd
cmsumpr~m.
‘71 ‘72, Contrarv to the results obtained in man and in rat b:: others _i, receptors 1~ rabbit dir! c7f both types of adrenergc I37' 3 : , the stirwlatian not Influence the fibrL?olq-tic acti>yity- in euglobu!xs ~1 our experiment 5. Thus can be esplair.ed by the fact that rabbit is kn~,u-n as can an~nal with with other s?aael; a particularly low fibrinolytic potential as ccm?ared agonists w’:; 5 .‘2’,23 ,2&,?’and it is FossiSle that none of t he ndT,inistered able to induce
systezrc
activation
of its fibrlnoi:tic
activitTU*.
The infusion of isoproterenol, preceded b>- saline injoctio:?, in the decline of the flbrinolytic activity of both kidne?- cortices
resulted below
the sensitivity threshold of this particular method and it was ln contrast with the results obtained in animals infused with saline or norepinephrine It can be supposed that the isoproterenol-induced vasodilation brought about the considerable release of tissue plasminogen activator and that the observed disappearance of this activator from the kidney cortex corresponded to the exhaustion stage at the end of infusion. This is 111accordance with the opinion of Gader et al. 27.’ that the plasminogen activator response appears to be a beta-adrenergic mediated phenomenz no such phenomenon was observed when. i soproterenol followed I iowever, the preparatory endotoxin injection. Even more intriguing was the fact that the pattern of the fibrinolytic activity of kidney cortex in animals infused with endotoxin and isoproterenol resembled that in animals given saline only. This finding evidently contrasted with the lack of fibrinolytic activity of both kidney cortices in most of the animals receiving provocative endotoxin infuslon alone or in combination with norepinephrine. This seemingly protective influence of beta-adrenergic agonist on the kidney cortex fibrinolytic activity is difficult to explain, the more so because the animals infused with endotoxin and isoproterenol revealed the most pronounced i.f.d. of all animals in our materials. The
animals
not infused
with the
provocative
endotoxin
dose
were
free of i. f.d. and these findings are consistent with the opinion of Whitaker /28/ that even massive doses of epinephrine or norepinephrine did not induce renal capillary thrombi. The i.f.d. was significantly less pronounced in the denervated kidney than in the intact one in animals receiving both endotoxin doses and this difference was mantained even when the norepinephrine was added to the endotoxin infusion. The former findings confirmed the results of our previous studies :li on and those of Fine /29/. The lack of any effect of norepinephrine endotoxin induced i.f.d. was rather suprising because it has been found by several authors that norepinephrine increases the renal vascular resistance and decreases renal blood flow ‘3O,j1,32,3j,3~,35,36, 3;,28,39:’ and these vasomotor changes are believed to promote
intravascular fibrin deposition. In spite of the fact t!lat the sympathetic denervation of the kidney is supposed to increase the sensitivit>of its vessels to norepineahrine ;‘13,L0.‘Y the infusion of exogenous norepinephrine did not influence the endotoxin-induced i. f.d. in the It car: be supposed that th e infused exosenous denervated kidney , either. norepinephrine did not compensate efficiently the lack of endogenous norepinephrine at the nerve endings in this kidney. The stimulation of beta-adrenergic receptors with isoproterenol did not influence the endotosin-induced i.f .d. in the intact kidney but It glomeruli in the dene-ated almost doubled the percentage o_f involved kidney as compared with that in the corresponding kidney in animals receiving endotoxin alone. As for the intact kidney, ?.!oriau et al. .‘Ll ’ have not found the enhancing effect of isoproterenol on thrombininduced i . f. d. in rabbit kidney. Our findings are different from the results of Collins et al. who found that norepinephrine 14.2; but not isoproterenol !L3/, augmented the endotoxin-induced renal lesions in rabbit. This discrepancy can be explained by the different experimental schedule applied in their investigations, The changes of the fibrinolytic activity of kidney cortex resulting from the infusion of isoproterenol cannot explain the effect of this drug on the endotoxin-induced i.f.d. in denervated kidney and we suppose that this effect is related to the influence of isoprotercnol on renal vessels. Aviado et al. /31/ and Johnson et al. /44/ found that isoproterenol injected into the renal artery increased renal blood flow. Carriere /L5/ observed that small doses of isoproterenol given into the renal artery did not result in any changes of renal blood flow. The administrat’ion of this agonist together with phenoxybenzamine, however, caused a marked vasodilation in kidney cortex. According to this author, alpha-adrenergic blocking action of phenoxybenzamine enabled isoprotcrenol to exert its stimulating effect on beta-adrenergic receptors in kidney cortex. It is possible that the disruption of the postganglionic adrenergic renal nerves in our experiments simulated to some extent
the action
of phenoxybenzamine
in Carriere’s
studies.
According to Egdahl /4/, the intact sympathetic neural pathways are essential for the endotoxin-induced release of catecholamines from adrenal medulla and it is probable that the same is valid for the norepinephrine release from the postganglionic adrenergic nerve endings. The local norepinephrine release in the intact kidney, elicited with the by the provocative endotoxin dose, could have interfered vasodilatory action of isoproterenol on renal vessels and this might explain the lack of this drug effect on i. f. d. in the intact kidney. This interference probably did not occur in the kidney devoid of sympathetic innervation and isoproterenol was able to display effectively its vasodilatory influence on the vessels of this kidney.
‘:t
?? 7Fc,r-aFpe c.
fZA
be conclnded
L..._)
that:
1. The alpha-adrenergic
stimulation with norepinephrine or_ the endotoxin-induced i.f. d. IX the kidne)- devoid ir.nervat;on or in rhe intact kidney.
has no effec: of s_empathetic
2.
The beta-adrenergic
stimulation with isoprot erenol does not influence :.f.d. within t:he intact kidney but significantly augments this doposyiion in the denervated kidney and this latter outcome seems to resu!: :rci:1 II?:2 rcca! vasodilation.
; L.
‘Phe lsogroterenol-induced 3c_ the concomitant changes cortex.
aggravation of i. f. d. cannot be exploiiled In the fibrinolytic activit;j of kidney
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PhLh!ERIO,C., MIXG,S .C., FRAXK,E. and FINE, J. The role of s_ympat’netic nervous system in the generalized Shwartzman reaction. J . Exp. hIed .:&,
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1962.
BOUQUET,P. and IZhRD,Y. response of rabbit to typhoid
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EGDAHL ,R .FI. medulla
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ROSENBERG, J.C., LILLEHE1,R.C.) LONGERBEAM, J. and ZIXIMERILIAN ,B. Studies on the hemorrhagic and endotoxin shock in relation to vasomotor changes and endogeneous circulating epinephriand serotonin. Ann. Surg: 1% 611, 1961. ne, norgpinephrine A
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CAVANAGH, D., RAO,P.S., BACXhl Aiv, F. Pathophysiology Amer.
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SUTTON, M.C, BHAGAT,B. D. and of endotoxin shock in the primate. 705,
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:vIUELLER-BERGHAUS ,C. and MCKAY, D. G. Prevention of generalized Shwartzman reaction in pregnant rats by alpha-adrenergic blocking agents. Lab.Invest. : 2, 276, 1967.
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MCKhY,D.G., LhTOUR,J.G. and LOPEZ ,A. hi. Production of the generalized Shwartzman reaction by activated Hageman factor and alpha-adrenergic stimulation. Thromb.Diath.haemorrh.: 26,71,1971.
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actions
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EELL,C.H,, of Phvsioloq 106S.
DAiJIDSOXtj.X. and Biochemistrv.
ami SC~+RB~ROU
Textbook Livingst0r.e)
and London:
HERT1SG.G.. ASELROD,J., KOPiS,I.J. and \l!HITBY-L.G. of uptake of catecholamines after chronic denervation of synpathetic nerves. -Xature: -183. 66, 1961. S1CKENSA,O. of the canine
Lack
C. and ANGELAKOS t E .T. i\drsnergrc innemation kidney. Circulation Res. : 22, 313,19&S.
13.
BERNE,R.hf.? HOF‘F>\IAX ,I’?. K. f Jr. , K;:AGi?S , ;‘i. and LEVY. X1.X, Response of the normal and denervated kidnev to 1’ epinephrine and l’nor-epinephrine. Amer. J.Phvsiol. : 12, &, 1912.
li.
SURTSHIX ,A. and HOELTZENBEIS !J. Effect ~71‘ chronic denervation on response of renal vessels to epinephrine. Amer. J.Phvsiol. :
1;1, 15.
163, 1953.
GODAL,H.C. plasma
16.
SWAIK,W.R. Chemistrv:
17.
and ABILDGAAR
by ethanol.
and FEDERS,M.B. 13,
D, V. Gelation
S cand. J. Haemat . : _, 342,
1026,
Fibrinogen
of soluble
fibrin
in
1906. assay.
z.
1967.
SZCZEPA;US KI, h$. and LUCER ,C. Soluble fibrin monomer complexes and t’ibrinolytic activity in kidney during the generalized Shwartzman reaction in rabbit. Thromb .Res. : 4, 339, 1975.
18.
ASTRUP ,T., CLAS ,P. and KOK ,P. Assay of plasminogen Disorders. - activator in tissues. In: T hrornbos is and Bleeding Bang, F.K.Beller, E. Deutsch, E. F.iMammen ( Ed.). Georg Academic Press, New York, London, 1971, p.370.
19.
20.
21.
N .U , Thieme,
MCKAY, D.G. and SHAPIRO,S .S. Alterations in the blood, coagulation system induced by bacterial endotoxin. I. in vivo. Med. : 107, 353, 1958. _
L Exp.
LERNER,G.R., RAPAPORT,S.I., SIEAISEN, J.K. and SPITZER, J. M. Disappearance of fibrinogen13lI after endotoxin: effects of a first and second injection. Amer. J.Physiol. : a, 532, 1968.
GEXTON induced
, E., KERN, F. , Jr. and VOX KAULLA,K. Fibrinolysis 564, 1961. by pressor amines. Amer. J. Med. : 31,
22.
MATSUMURA,T. dnd SHI MAMOTO,T. Effects of ISHIOKA,T., adrenaline and noradrenaline on euglobulin lysis time and effects of adrenergic receptor blocking agents on shortening of lysis time by adrenaline. Angiologica: z, 104, 1970.
23.
DESNOYERS ,P., ANSTETT,M., LABOUME,J. and SCHMITT,H. Effect of alpha- and beta-adrenoreceptor stimulating and blocking agents on fibrinolytic activity in rats. Pro,gr. Chem. Fibrinolvs . Thrombolys. : l_, 367, 1975.
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’
:.? ::I
.:
37. SCHRIER,R
.\x.‘. Effects of adrenergic on systemic and renal and renin secretion.
catecholamincs water excretion
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BOMZON , L . C . and ROS EN DCRF : C . Renovascular noradrenaline. Amer. J.Phvsiol. : 229, leii7, 1973. -
39.
AHLQUIST, R.P. I. The adrenergic
LO.
CANNON
, W. R.
and
hemodynamics . sodium and Kidnev Int. : -6, 291, 1974. resistance
Present state of alpha- and beta-adrencrgic receptors. Amer. Heart J. : z, 661, 1976. A law of denervation.
Amer.
J. h:ed. S ci. : @,
and
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1939. 41.
MORIAU , h4. , ic’OEL,H. and hlASURE,R. Effects of alpha and beta receptor stimulating and blocking agents on experimental disseminated intravascular coagulation. T hromb, Diath. haemorrh. : 32, 157,
1974. 42.
COLLISS
,A. D.,
HENSON,
E.C.,
IZARD,S.
R.,
BRU’SSON,
J.G.
and JACKSON, M. D. Norepinephrine, cndotoxin shock and the generalized Shwartzman reaction. Arch.Path. : YJ, $2, 1972. L-j.
IZARD,S.R. and BRUNSON,J.G. COLLIXS ,A. D., HENSON ,E.C., endotoxin shock and generalized Shwartzman Isoprotercnol, reaction. Arch.Psth.Lab.Med.: 100, 315, 1976.
LL.
J.A., DAVIS, J.O., GOTSHALL, R.W . , LOHMEIER ,T. E. BRAVERMAN, B. and T EMPEL , G. E . Evidence for an DAVIS,J.L., intrarenal beta receptor in control of renin release. Amer. J, Physiol. : 230, L19, 1976.
45.
CARRIERE
, S . Effect
adrenergic
blockers
JOHNSON,
of norepinephrine, upon the intrarenal
Canad. J.Physiol.Pharmacol.
: 2,
isoproterenol and distribution of blood
199, 1969.
flow.
Aiaciel
Szczepanski,
Cezary
Department of Biochemistry Diagnostics, ::Iedical
(Received
of alpha-
endotoxin-induced
Elibieta
Staff-Zieliriska
and Department of Pathomorphological Center of Postgraduate Education, C’arsaw, Poland
9.8.1977; Accepr;ed by
The influence
Lucer,
in revised Editor 1.X.
form 11.11.1977. Silsson)
or beta-adrenergic
intraglomerular
agonists
fibrin
deposition
was studied in rabbits with one denervated norepinephrine infused together with the
on the
\ i .f.d.,
kidney. The provocative
endotoxin dose did not influence i. f-d. ted kidney or in the intact one. The
within the denervaisoproterenol
administered
with
endotoxin
also
dose
in a common
had no effect
infusion on
i. f. d.
provocative
in the
intact
kidney
but significantly augmented this deposition within the denervated kidney. The results of the fibrinolytic activity of kidney cortex cannot explain the effects of adrenergic agonists on endotoxin-induced i.f.d. It is supposed that the enhancing effect of isoproterenol on i.f.d. within the denervated kidney can be ascribed to the vasodilating effect of this drug, promoted in the denervated kidney by the absence of endogenous norepinephrine at the nerve endings.
ISTRODUCTION Our previous studies /l/ confirmed the findings of Palmerio et al. !2/ that the sympathetic denervation of the kidney diminished the endotoxin-induced intraglomerular fibrin deposition ( i. f. d. ) although the increase of endotoxin dose significantly reduced this effect.
It has been reported that the i.v. injection of endotoxin resulted in the elevated level of circulating epinephrine /j ,L, 5/ and norepinephrine
236
XDREXERGIC
The
/3,6!.
contribution
AGONISTS
of catecholamines
OS GSR
is believed
vo1.12,so.2
to be essential
for the generalized Shwartzman reaction /7,Si. It is known that the epinephrine is secreted mainly by the adrenal medulla and that the norepinephrine is synthetized for the most part at the adrenergic postganglionic nerve endings /9,10/. The disruption of these nerves is expected to abolish the local s_ynthesis of norepinephrine and it, indeed, has been proved that the denervated tissue is devoid of this substance /10111,12/. If the absence of norepinephrine and, consequently, the lack of alpha-adrenergic receptors stimulation in the denervated kidney was responsible for the diminished endotoxin-induced i.f .d. then the infusion of exogenous norepinephrine should abolish this effect of denervation. The vasomotor response of the denervated organ to the catecholamines is even more expressed than that of the exogenous intact one / 10,13,14/ and, therefore, we expected the common infusion of norepinephrine and endotoxin to result in the enhanced i.f .d. within the denervated kidney but this was not the case. We decided then to stimulate the be ta-adrenergic receptors and the rabbits receiving the endotoxin
and isoproterenol
were
MATERIALS Albino to 2.7
rabbits,
kg were
Popielno
used.
included
AND
investigations.
METHODS
strain,
Sympathetic
in these
of both sexes,
denervation
weighing
from
of the left kidney
1 .j
was
performed as described previously /I/ and after the lapse of 7 days the rabbits were injected i.v. with saline or endotoxin ( Lipopolysacccharide 055: B5, Difco) . The 4-hours infusion of saline or endotoxin B, E.coli or/and adrenergic agonist was begun in unanesthetized animals 24 hours later, according to the protocol illustrated in Fig. 1. Norepinephrine ( Levonor, Polfa) was administered as an alpha-adrenergic agonist and isoproterenol (Isuprel, Winthrop) as a beta-adrenergic agonist. The blood samples were obtained for the baseline determinations from the central ear artery before the saline or endotoxin injection and for the final determinations by the direct heart puncture after the completion of infusion. Both kidneys were removed immediately afterwards and the animals were sacrificed. The blood was obtained through the siliconized needles into the siliconized test tubes, mixed with 4.8% sodium citrate, 9: 1 and kept in melting ice till the beginning of laboratory tests. The 1.
following
Ethanol
Relation
determinations test,
according
were
performed:
to Godal
/15/.
:nlcctions. !nfus ions .
SaCl: SaCl: Nor:
normal normal
saline, saline,
norepinephrine,
2 ml. Ex: endotoxin, 2’3 mcg kg. 8 ml/h, Ex: endotoxin, 12.3 mcg’kg 73 mcggikgih.
[so:
‘1.
isoproterenol,
5 mcg/kg,‘h.
2. c?
.
/ C.
5.
Fibrinogen concentration in plasma by Swair: and Feder method ~‘16 . i:ibrinolytic activity in concentrated euglobulins i 17,‘. Fibrinolytic activity of kidney cortexaccording to the Astrup and Albrechtsen assay for tissue stable activator of plasminoger. ‘1s’. The potassium thiocyanate extracts of kidney cortex specimens were incubated on bovine fibrin plates for 22 hours. Intraglomerular fibrin deposition was estimated as the percentage C? involved glomeruli in the kidney specimens stained with phosphotungt--t: acid haematoxylin ,l PTAHj / 17 /.
Statistical sum test.
analysis
of data was done according
to the V’:..ilcoxon rsnk-
RESULTS The results samples and were and 9 , Fig.
2, .
cf ethanol gelation test were negative in all baseline positive in final samples cnly in animals of groups 7.5
The fibrin ..L3en 717 concentration in plasma of animals of groups 1) 2. and 3 decreased throughout the experiments and no differences were The inrease of this concentration was observed among these groups. between observed in animals of groups i, 5, and 6 without the differences
r
Ia
‘aJ;rilVE
RESULT
cl FIG.
The
results
of ethanol
NEGATIVE
RESULT
2. gelation
test
in final
samples.
The animals receiving both endotoxin doses, i. e. these groups, either. of groups 7,8 and 9 revealed the decline of plasma fibrinogen concentration during the experiments and this decline was significantly greater in animals of group 9 than in animals of group 7 or of group 8 (alpha = Y 0.01). There were not any significant in euglobulins between the various All
animals
of group
differences in the fibrinolytic groups of rabbits.
3 had the undetectable
fibrinolytic
activity
activity
of
both kidney cortices at the end of experiments and it was in contrast to the results obtained in animals of groups 1 and 2 (Table 1) .There were of not any noteworthy differences in this activity between the animals groups L,S and 6. The fibrinolytic activity of both kidney cortices was undetectable in most of the animals of groups 7 and 8. The 70% of animals of group 9 revealed fibrinolytic activity in at least one kidney control cortex and the pattern of this activity resembled that of the animals
of group
1.
The intraglomerular fibrin deposition was not seen in any of the animals of groups 1 to 6 and Table 2 illustrates the numbers of animals of groups 7,s anil 9 that developed these changes.
T .
-i-
/
c
t
i
L
c
!
I
_:
i
2
?
!
I
-
I
7
’ ! -1
f.a.
in both kidney
Ii
cortices
;
‘?
i
I-
/-
!
,
‘7 :
.i i/
1
/
I
T
I
I
s
, i
1‘he Numbers of Animals with intra
Groups
Animals
with
i.f.d.
in both kidneys
Animals
with
i.f.d.
in the intact
ml> Animals
without
i.f.d.
3 kidney
2 \:
j I
i
I / I
, -
t; I
2
1 I I 1 1
fi
-/
There were not any significant differences in the percentage of lnvalved glomeruli in intact kidney cortex among the ani.mals of groups 7.5 and 9. This percentage in denervated kidney cortex did not differ, too, ‘between the animals of groups 7 and 8. T:he percentage of
/
involved
glomeruli
m denervated
kidnev
was significar,tl_i- increased in comparison corresponding cortex in animals of group .alphn = 0.03, *Fig. 3;.
tort ex in animals
of
group
9
with this percentage in the 7 as well as of group d
Group
la
INTbCT
FIG.
KIDNEY
cl
DENEZVATED
KIDNEY
3.
in The mean percentage of glomeruli with fibrin deposition animals of groups 7,s and 9. Vertical lines: standard errors of means.
DISCUSSION None of the adrenergic agonists preceded by saline or by endotoxin injection elicited the generation of soluble fibrin monomer complexes as was
revealed
by the negative
complexes
appeared,
endotoxin
doses
however,
combined
results
of the ethanol
gelation
in most of the animals
or not combined
test.
receiving
with adrenergic
These both
agonists.
.The infusion of saline or of adrenergic agonists preceded by saline injection resulted in the decreased fibrinogen concentration; this phenomenon can be explained by the hemodiluting effect of infused solutions. The rise in fibrinogen concentration observed after the infusion of corresponding solutions but preceded by the preparatory endotoxin injection was due to the well known stimulatory effect of this injection on the fibrinogen synthesis /19,20/. Both endotoxin doses decreased fibrinogen concentration in plasma and this decrease was augmented by isoproterenol but not influenced by norepinephrine. These but findings suggest that stimulation of beta-adrenergic receptors
r_or of alpha-sdr2riergzc fibrinogen
receptors
Increases
tk
2nd~tcxLn-rnducsd
cmsumpr~m.
‘71 ‘72, Contrarv to the results obtained in man and in rat b:: others _i, receptors 1~ rabbit dir! c7f both types of adrenergc I37' 3 : , the stirwlatian not Influence the fibrL?olq-tic acti>yity- in euglobu!xs ~1 our experiment 5. Thus can be esplair.ed by the fact that rabbit is kn~,u-n as can an~nal with with other s?aael; a particularly low fibrinolytic potential as ccm?ared agonists w’:; 5 .‘2’,23 ,2&,?’and it is FossiSle that none of t he ndT,inistered able to induce
systezrc
activation
of its fibrlnoi:tic
activitTU*.
The infusion of isoproterenol, preceded b>- saline injoctio:?, in the decline of the flbrinolytic activity of both kidne?- cortices
resulted below
the sensitivity threshold of this particular method and it was ln contrast with the results obtained in animals infused with saline or norepinephrine It can be supposed that the isoproterenol-induced vasodilation brought about the considerable release of tissue plasminogen activator and that the observed disappearance of this activator from the kidney cortex corresponded to the exhaustion stage at the end of infusion. This is 111accordance with the opinion of Gader et al. 27.’ that the plasminogen activator response appears to be a beta-adrenergic mediated phenomenz no such phenomenon was observed when. i soproterenol followed I iowever, the preparatory endotoxin injection. Even more intriguing was the fact that the pattern of the fibrinolytic activity of kidney cortex in animals infused with endotoxin and isoproterenol resembled that in animals given saline only. This finding evidently contrasted with the lack of fibrinolytic activity of both kidney cortices in most of the animals receiving provocative endotoxin infuslon alone or in combination with norepinephrine. This seemingly protective influence of beta-adrenergic agonist on the kidney cortex fibrinolytic activity is difficult to explain, the more so because the animals infused with endotoxin and isoproterenol revealed the most pronounced i.f.d. of all animals in our materials. The
animals
not infused
with the
provocative
endotoxin
dose
were
free of i. f.d. and these findings are consistent with the opinion of Whitaker /28/ that even massive doses of epinephrine or norepinephrine did not induce renal capillary thrombi. The i.f.d. was significantly less pronounced in the denervated kidney than in the intact one in animals receiving both endotoxin doses and this difference was mantained even when the norepinephrine was added to the endotoxin infusion. The former findings confirmed the results of our previous studies :li on and those of Fine /29/. The lack of any effect of norepinephrine endotoxin induced i.f.d. was rather suprising because it has been found by several authors that norepinephrine increases the renal vascular resistance and decreases renal blood flow ‘3O,j1,32,3j,3~,35,36, 3;,28,39:’ and these vasomotor changes are believed to promote
intravascular fibrin deposition. In spite of the fact t!lat the sympathetic denervation of the kidney is supposed to increase the sensitivit>of its vessels to norepineahrine ;‘13,L0.‘Y the infusion of exogenous norepinephrine did not influence the endotoxin-induced i. f.d. in the It car: be supposed that th e infused exosenous denervated kidney , either. norepinephrine did not compensate efficiently the lack of endogenous norepinephrine at the nerve endings in this kidney. The stimulation of beta-adrenergic receptors with isoproterenol did not influence the endotosin-induced i.f .d. in the intact kidney but It glomeruli in the dene-ated almost doubled the percentage o_f involved kidney as compared with that in the corresponding kidney in animals receiving endotoxin alone. As for the intact kidney, ?.!oriau et al. .‘Ll ’ have not found the enhancing effect of isoproterenol on thrombininduced i . f. d. in rabbit kidney. Our findings are different from the results of Collins et al. who found that norepinephrine 14.2; but not isoproterenol !L3/, augmented the endotoxin-induced renal lesions in rabbit. This discrepancy can be explained by the different experimental schedule applied in their investigations, The changes of the fibrinolytic activity of kidney cortex resulting from the infusion of isoproterenol cannot explain the effect of this drug on the endotoxin-induced i.f.d. in denervated kidney and we suppose that this effect is related to the influence of isoprotercnol on renal vessels. Aviado et al. /31/ and Johnson et al. /44/ found that isoproterenol injected into the renal artery increased renal blood flow. Carriere /L5/ observed that small doses of isoproterenol given into the renal artery did not result in any changes of renal blood flow. The administrat’ion of this agonist together with phenoxybenzamine, however, caused a marked vasodilation in kidney cortex. According to this author, alpha-adrenergic blocking action of phenoxybenzamine enabled isoprotcrenol to exert its stimulating effect on beta-adrenergic receptors in kidney cortex. It is possible that the disruption of the postganglionic adrenergic renal nerves in our experiments simulated to some extent
the action
of phenoxybenzamine
in Carriere’s
studies.
According to Egdahl /4/, the intact sympathetic neural pathways are essential for the endotoxin-induced release of catecholamines from adrenal medulla and it is probable that the same is valid for the norepinephrine release from the postganglionic adrenergic nerve endings. The local norepinephrine release in the intact kidney, elicited with the by the provocative endotoxin dose, could have interfered vasodilatory action of isoproterenol on renal vessels and this might explain the lack of this drug effect on i. f. d. in the intact kidney. This interference probably did not occur in the kidney devoid of sympathetic innervation and isoproterenol was able to display effectively its vasodilatory influence on the vessels of this kidney.
‘:t
?? 7Fc,r-aFpe c.
fZA
be conclnded
L..._)
that:
1. The alpha-adrenergic
stimulation with norepinephrine or_ the endotoxin-induced i.f. d. IX the kidne)- devoid ir.nervat;on or in rhe intact kidney.
has no effec: of s_empathetic
2.
The beta-adrenergic
stimulation with isoprot erenol does not influence :.f.d. within t:he intact kidney but significantly augments this doposyiion in the denervated kidney and this latter outcome seems to resu!: :rci:1 II?:2 rcca! vasodilation.
; L.
‘Phe lsogroterenol-induced 3c_ the concomitant changes cortex.
aggravation of i. f. d. cannot be exploiiled In the fibrinolytic activit;j of kidney
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