Arachidonic Acid Infusion in Rabbits

Path. Res. Pract. 176, 297-305 (1983)

Department of Pedlatncs (Nephrology DlVlslOn) and Department of Pathology, UnIversIty of Montreal Hopltal Ste-Justme, Centre de Recherche Pedlatnque, Montreal, Quebec, Canada.

Arachidonic Acid Infusion in Rabbits Renal Pathologic Alterations S. O'REGAN and CLAIRE TURGEON

Summary ArachIdonIc aCId mfuslon m r abbIts IS hIghly lethal, death occunng m greater than 90% of anImals and most wlthm 1 to 2 mmutes. ThIs effect may be inhIbIted by pnor admmlstratlon of vltamm E. PathologIc exammatlOn of renal tIssue from anImals kIlled m thIS manner demonstrated ImmunoglobulIn and ftbrm deposItIOn m the glomerulI and mterstltlUm of the kIdney. No pathologIc alteratIons were detected by hght mIcroscopy. Electromlcroscopy revealed some endothehal cell swelhng with no eVIdence of electron dense deposIts, mdICatmg the absence of Immune complexes. PathologIc exammatlon of the kIdneys suggest the deposItIon of Immunoglobuhn m a non complexed form.

Introduction A major role ill the evolutlOn of both glomerulonephritlc and illterstitlal nephritic lesions has been ascribed to the 10calizatlOn of immune complexes in the kidneys with the consequent cascade of complement activation, neutrophil mflltration, etc. and subsequent damage to the kidney (21). With the utilization of Immunofluorescence mICroscopy, immune depOSits have been localized ill the kidney. In general, though antigen detectlOn may be achieved utilizing these methods ill ammal models of disease in whom a known specIfic illducing antigen has been admimstrated (22) this has not been generally possible in human studies (13). Studies m humans and animals have generally conSidered the presence of immunoglobulm and complement depOSits in the kidney as indicative of the presence of immune complexes. Up to recently, these immune complexes were considered to be deposited from Circulating Immune com-

* ) Supported by a grant from the MedICal Research CounCIl of Canada (MA-6122 ).

298 . S. O'Regan and Claire Turgeon

plexes (21). More recent m vItro studIes indicate that in anImal models of membranous glomerulopathy such as Heymann's nephritIs (10) and also possibly in bovine serum albumin (B. S. A.) mduced rabbbit nephritIs (6), Immune complex formation may evolve locally due to the prior deposition of antigen in the kidney. Ascribing of immune deposits m the kidney to be due solely to circulating immune complexes contams certain contractions. In humans with bIlateral cortIcal necrosis, Immune deposIts are observed (9). In addItIOn, Immune deposits may be observed m apparently normal kidney at autopsy in the absence of renal dIsease (8). In cystmosis (2), an mborn error of metabolism, immunoglobulms may be deposited m the kidney. Also complement may be observed deposited on the vessels in normal renal tissue (20). Because of the failure to reproduce consistantly Immune complex deposit disease in animals with pre-formed Immune complexes (5, 7, 14), It has been suggested that immunoglobulins may be deposited m non-complexed form in renal tissue (3). Thus m the Shwartzman reaction m whIch a rabbit IS primed WIth endotoxin, a succeeding dose of endotoxin results, in many anImals, in a pathologic cascade resulting m a shock like state assoCIated with fIbrin, IgM and complement depOSItiOn in renal glomeruh (3). Though m this anImal model, the immunoglobulm depoSition may be due to the filtratIOn and the deposition m a non-complexed form, endotoxm IS immunogenIc so the possibility that the IgM is m immune complex form cannot be outruled. InfUSIOn of arachidOnIC aCId, a precursor of prostaglandins, into rabbits in an excess of 1.1 mg/kg mtravenously is lethal, death occunng in a period of seconds to mmutes m 90% of anImals (18). In this animal model death is attributed to productIOn of various prostaglandin fractIOns which result in bronchospasm and IS accompanied by platelet aggregation (4,17). While mvestlgatmg the role of a-tocopherol m blockmg these effects and mhlbiting the lethality of arachldoOiC aCId mfusIOn m thiS animal model (1), we noted the Similarity to the Shwartzman reactIOn and m additIOn varymg deposition of immunoglobulin and complement in associatIOn with fibrin depositIOn in the kidneys of these aOimals infused with this non-antigeOic substance.

Materials and Methods Male New Zealand white rabbits welghmg 2.5 to 3 kg were uttllzed for these studies. Expenments were performed accordmg to the method of Silver et al. by mtravenous mJectlOn of arachldomc aCid mto the margmal ear vem (18). Arachldomc aCid (Sigma Product no. A-6382) was solublhzed m salme With the aid of a few drops ot Tween 80 and diluted to make a 4 mtlhmolar solutIOn. A 30 umts per ml solutIOn of vltamm E (D/L-a-tocopherol acetate, USV New York 10707) was prepared by mlxmg 30 umts per 0 6 ml With 0.4 ml of 10 mg/ml of Tween 80

ArachidOnIC ACid InfusIOn m Rabbits . 299' (Fisher SCientific Canada) m salme. A slmtiar concentratIOn of Tween 80 was prepared m physiologIc sahne. Group 1. A group (n = 10) of rabbits were mJected mtravenously with a lethal dose of arachidonIc aCid I.e. 6 mg/kg. This high dose was chosen to ensure maximum potentlallethahty. Group 2. This group of anImals (n = 8) were mJected via margmal ear vem With 150 unttslkg of D.L.-a-tocopherol mixed m Tween 80 5 mmutes pnor to the admmlstratlOn of a lethal dose of arachidonIc aCid. Group 3. This group of antmals (n = 5) were mJected via margmal ear vem With 10 mg/ml of Tween 80 m salIne, m a slmtiar dose to that given antmals m Group 2. For Immunofluorescent studies, tissue was snap frozen m pre-cooled Isopentane m dry Ice Cryostat seCtIons 4 It m thickness were stamed with fluorescem Isothlocyanate (F.I.T.C) monospecifiC goat antisera to rabbit anti IgM, IgG, C 1 and flbrm. Total protem concentrations of antisera were 10 to 25 mg/ml With antibody protem concentrations of 10 mg/ml and fluorescem protem ratios of 2.75 to 5.5 mg/gr. All sectIOns were washed three times m phosphate buffer Ph 7.2 pnor to and after stammg With antisera as prevIOusly descnbed (11). For lIght microscopy kidney tissue was fixed m 10% formalm sal me and paraffm sectIOns 2 & 3 It thick were stamed With hemelum phloxme safron (H.P.S.) penodlc aCid Schiff (P.A.S.), penodlc aCid Schiff methenamme (P.A.S.M ) and Masson's tnchrome stams. Pieces of kidney 1 mm 3 m dimensIOn were fixed for 3 hours m glutaraldehyde fixative diluted 2.5% m phosphate buffer, at Ph 7.3. The tissues were nnsed several times m a solutIOn of 1 % sucrose m phosphate buffer. They were transfered m 2% phosphate buffered osmIUm tetroXide. After several washmgs m distilled water, the tissues were stamed m uranyl acetate 2% m dlsttlled water, and then the dehydratIOn was started Epon 812 was used m the Impregnation and embedding, and polymensatlon took place m an over at 60°C. Sections were cut With a Sorvall MT-1 microtome The thick sectIOns (1 micron) were stamed m 0.25% toluidme blue and the ultrathm sectIOns were stamed m lead citrate for 15 mmutes and exammed on the electron mICroscope (Phlitps 201).

Results InfUSIOn of arachIdOnIC In a dose of 6 mg/kg resulted In death WIthIn 1 to 2 mInutes in all animals. As prevIOusly decribed (1) prior admInistration of vitamin E resulted In 100% survival rate of the anImals In Group 2. InfUSIOn WIth Tween 80 did not result In any discernIble illness in animals in Group 3.

Group 1 Pathology. StainIng WIth F.I.T.C labelled goat antI-sera to rabbIt fibrIn demonstrated 4 + heavy depOSItIOn of fibnn WIthIn all glomerulI IncludIng the capillary lumen a as well as diffuse deposition of fibrin within the interstitium of the kidneys (Fig. 1). ThIS was uniform In all anImals, involved all glomeruli, and was observed in both cortical and medullary regions. Staining with F.I.T.C. labelled goat antI-rabbit IgM demonstrated depOSIts of IgM along capillary walls (Fig. 2). In four anImals In addItIOn, fine deposits of IgG were

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S. O'Regan and Claire Turgeon

Fig. 1. Cryostat sectIOn of arachidonic aCid Injected animal staIned with F.I.T.C. conjugated goat anti rabbit fibrIn demonstratIng diffuse fIbIn depOSits In renal InterstitIUm (x 100).

Arachldomc ACid InfusIOn m Rabbits . 301

Fig. 2. Cryostat sectIOn of arachldomc aCid mJected ammal stamed with F I.T.C. conjugated goat anti rabbit IgM demonstratmg glomerular IgM deposits (x 250)

also present in a similar distribution. Glomerular localization along capillary walls and in the mesangium of C3 was also visible in four animals. Light mtcroscopy. There was no evidence of pathologic abnormality in animals who had expired immediately after bemg mfused with arachidonIc acid. There was no eVIdence of any inflammatory cell infiltration and all glomeruli and the interstitium were normal. Electromtcroscopy. On electromicroscopy granular matenal was VISIble within the capIllary lumina of glomerulI (Fig. 3). It dId not have the ftbnllar appearance of fibrin but appeared protemaceous m nature. There was some swelling of endothelial cells. The basement membrane, the foot processes and mesangium appeared normal.

Group 2 and 3 Minimal deposits of fibrin were present in 3 animals given a-tocopherol prior to arachidonic acid infusion. Stammg of renal tissue from the remaining animals was negative as was stammg of tissue from animals in Group 3. There were no pathologic alterations viSIble by lIght microscopy in renal tissue in animals from Group 2 and 3.

302 . S. O'Regan and Claire Turgeon

Fig. 3. Glomerular capillary showmg protemaceous matenal (arrow) m capillary lumen (x 7000).

Discussion Arachidonic acid is a precursor of protaglandins and thIS non antIgenic substance is present in mammalIan cells (12). It IS a potent platelet aggregating agent and admimstratlOn of arachidomc aCid mtravenously m rabbits results

Arachldomc ACid InfusIOn

In

Rabbits

303

in rapid death with associated bronchospasm and Circulatory collapse (18). Prior infusion of vitamm E mhiblts this phenomenon whiCh is probably due to productlOn of vanous prostaglandms (1). A previous preliminary study indicated that immunoglobulins were deposited m the kidney mammals who had been killed by arachldomc aCid mfuslOn. Control ammals killed by nembutal infusion did not exhibit this Immunoglobulin IgM depOSitIOn in assoCIatIOn with fibrin. This evolutIOn of Immunoglobulin deposition in this animal model occurs in many animals in less than 20 seconds m the absence of pnor renal disease. Its rapidity of evolution and ItS mduction by a non-antigemc substance mdicates that circulatory phenomena occur which allow for the depOSItion or trapping of Immunoglobulins m the presence of possible alteratIOns of intrarenal perfusion and coagulatIOn. Routme exammation of renal bIOpsy matenals m humans with nephritiC diseases involves examination for the detection of immunoglobulin deposits as an indicator of immune complex depOSit disease. Studies mammals have indicated that the presence of such Immunoglobulin deposits indicates the presence of immune complexes (22). However, in many human diseases such as cystinosis and in autopsy tissue as well as m animals immune deposits may be observed m the absence of detectable antigen. Similarly Immune depOSits may be observed m some human diseases such as that associated with leukemia (19) and S.S.P.E (15) with mimmal or undetectable morphologic abnormalities by light microscopy. The pathologic abnormalIties we have observed by immunofluorescence microscopy are similar to those m the Shwartzman reactIOn in rabbits as detailed by Bergstein and Michael (3). Admmistration of endotoxin, a highly antigenic substance as a sensitizmg dose followed by a second dose causes a pathologic cascade whiCh results m fibrmogen depOSitIOn With mtravascular coagulation in the kidneys of these ammals m association with Immunoglobu1m and complement deposition. Though the pOSSibility eXists that these Immune deposits are due to complex formatIOn With depOSitIOn, these authors postulated that these abnormalities may be due to or conslstant With alterations in intrarenal hemoperfuslOn. We have utilized a non-antigemc substance, arachidomc acid which when infused results m immunoglobulm deposition with fibrin and complement formation m glomeruli. Immune depOSits were extremely variable from ammal to ammal and m each kidney. No uniformly distinct pattern has been observed apart from fibnn depositlon which was observed withm glomeruli and in the mterstltium m all ammals examined. In these studies IgM deposition was a phenomenon that evolved wlthm a matter of seconds and thus was unlikely to be due to Immune complex formatIOn in the circulation. The absence of abnormalities identifiable by light microscopy and the absence of electron dense depOSits by electronmlcroscopy indiCates the

304 . S. O'Regan and Claire Turgeon

absence of nephritogenic immune complex formation. In addition, since arachidomc aCId IS a non-antIgenic substance this substance would not have mitIated antIbody-antIgen complex formatIOn. Complement depositIOn may have been due to activation secondary to coagulation and plasmin activation. Thus we suggest that this animal model produces Immunoglobulin depoSition in the kidney whICh is secondary to alteratIOns m renal perfUSIOn and not due to CIrculating complex formatIOn.

References 1. Barrette, M., and O'Regan, S.: Vltamm E mhlblts arachldomc aCid mduced mortalIty and renal IgM depositIOn m rabbits. Prostaglandms & MedlCme 5, 337-341 (1980) 2. Berger, ]., Yaneva, M., and Hmlglas, N.: Immunohistochemistry of glomerulonephntls. Adv. Nephro!. 1, 11-30 (1971) 3. Bergstem, J. M., and Michael, A. F.: GeneralIzed Schwartzmann reactIOn m the rabbit. Arch. Path. 97,230-251 (1974) 4. Cerskus, A. L., Alt, M .. Zamlcmk, ] , and McDonald,]. W. D Effects of mdomethacm and sulphmpyrazone on m VIVO formation of thromboxane Bz and prostaglandm D z dunng arachldonate mfuslOn m rabbits. Thromb. Res. 12, 549-553 (1978) 5. Cochrane, C. G : Mechanisms mvolved m the deposltlon of Immune complexes m tissues. J. Exp. Med 134, 79s-89s (1971) 6. Fleuren, G., Grond, J., and Hoedemaeker, P. J.. In Situ formatIOn of subepithelIal glomerular Immune complexes m passive serum SICkness. Kidney Int. 17, 631-637 (1980) 7. Ford, P. M.: Passive serum SICkness m the mouse effect of mter stram differences on glomerular deposltlon of Immune complexes. Bnt.] Exp Path. 56, 199-204 (1975) 8. Larsen, S.: Glomerular Immune depOSits m kidneys from patients With no clImcal and lIght microscopIC eVidence ot glomerulonephntls Acta Path. MlcroblOl Scand. (A) 87,313-319 (1979) 9. Mahleu, P., Dardenne, M., and Bach, J. F. DetectIOn of humoral and cell-mediated ImmUnIty to kidney basement membranes m human renal diseases. Amer ]. Med. 53,185-192 (1972) 10. Makker, S. P., and Moorthy, B.' In Situ Immune complex formatIOn m Isolated perfused kidney usmg homologous antibody. Lab. Invest. 44, 1-5 (1981) 11. Michael, A. F., Drummond, K. N., Good, R. A., and Vermer, R. L.: Acute poststreptococcal glomerulonephntls, Immune depOSit disease. ]. Clm. Invest. 45, 237-248 (1966) 12. Moncada, S., and Vane, J. R.· Arachldomc aCid metabolttes and the mteractlOns between platelets and blood vessel walls. N. Eng!.]. Med. 300, 1142-1147 (1979) 13. O'Regan, S., Smith, M., and Drummond, K. N. Antigens m human Immune complex nephritis. Clm. Nephro!. 6,417-421 (1976) 14. O'Regan,S., Smith, M., and Drummond, K. M : Immune complex mfuslOn m the rat· Renal functIOnal and morphologICal changes. Clm. Exp. Immunol. 24, 110-115 (1976) 15. O'Regan, S., Turgeon-Knaack, E., Mongeau, J. G., Lapomte, N , Nolm, L., and Robitaille, P. O. Subacute sclerosmg panencephalltls associated glomerulopathy. Nephron 23, 304-307 (1979) 16. Rail, L., Keane, W. F, and Michael, A. F.: Umlateral Shwartzmann reactIOn; Cortical neCroSIS m one kidney followmg m VIVO perfUSIOn With endotoxm. Kidney Int. 12, 91-95 (1977)

Arachldomc ACId InfusIOn m Rabbits . 305 17. Seuter, F., and Busse, W. D .. Arachidonic mduced mortalIty mammals - an appropnate model for the evaluation of anti thrombotic drugs? Agents ActIOns (Supp!.) 4, 175-183 (1979) 18. SIlver, M. J., Hoch, W., KOCSIS, J ]., Ingerman, C. M., and Smith, ]. B.: Arachldomc aCid causes sudden death m rabbits. SCience 183, 1085-1087 (1974) 19. Sunderland, J. c., and Mardmey, M. R.: Immune complex disease m the kidney of lymphoma-leukemia patients. The presence of an oncornavlrus related antigen. ]. Nat!' Cancer Inst. 50, 633-639 (1975) 20. Velosa, J., MIller, K., and Michael, A. F.: Immunopathology of the end stage kidney. Amer. J. Path. 84, 149-160 (1976) 21. Wilson, C. B., and Dixon, F. J. The renal response to ImmunologiC mJury. Eds. Brenner, B. M., Rector, F. C. The Kidney. pp 1237-1350, W B. Saunder's Company, London 1981 22. Wilson, C. B., and Dixon, F. J .. QuantatlOn of acute and chromc serum Sickness m the rabbit. J. Exp. Med. 136, 7-18 (1971)

Received m reVised form August 26, 1982 . Accepted August 26, 1982

Key words: Arachidontc acid - Rabbtts - Immunoglobulin deposition Dr. Sean O'Regan, ASSistant Professor, Department of Pediatrics, Nephrology Service, Hopltal Ste-Justme, 3175, Chemm Cote Samte-Catherme, Montreal, Quebec, Canada H3T 1C5

20 Path. Res Pract Vol 176