Increased Leukotriene B4 Synthesis in Immune Injured Rat Glomeruli

Increased leukotriene B4 synthesis in immune injured rat glomeruli.

M A Rahman, … , S N Emancipator, M J Dunn

J Clin Invest. 1988;81(6):1945-1952. https://doi.org/10.1172/JCI113542.

Research Article

We examined glomerular synthesis of the 5-lipoxygenase metabolite, LTB4, in normal and immune-injured rat glomeruli. Glomeruli isolated from normal rats and from rats with nephrotoxic serum nephritis (NSN), passive Heymann nephritis (PHN) and cationic bovine gamma globulin (CBGG)-induced glomerulonephritis were incubated with the calcium ionophore A23187 (3 microM). Lipids in the glomeruli and media were extracted with ethyl acetate, and were purified and fractionated by HPLC. Immunoreactive-LTB4 (i-LTB4) was determined by radioimmunoassay on HPLC fractions with a detection limit of 50 pg of i-LTB4. A large peak of i-LTB4 that comigrated with authentic LTB4 was found exclusively in glomeruli isolated from the CBGG-injected rats. Addition of the lipoxygenase inhibitor BW755C (50 micrograms/ml) to glomerular incubation resulted in greater than 90% inhibition of i-LTB4. Synthesis of i-LTB4 by glomeruli from normal, NSN and PHN rats was undetectable. Glomerular LTB4 synthesis by CBGG-injected rats was confirmed by radiometric HPLC and by gas chromatography mass-spectroscopy (GC-MS) analysis. In order to rule out synthesis of LTB4 by neutrophils entrapped in the glomeruli, a group of rats received 1,000 rad total body x irradiation, with shielding of the kidneys before induction of CBGG glomerulonephritis. Despite greater than 95% reduction in total leukocyte count, glomerular synthesis of LTB4 remained enhanced. Augmented glomerular synthesis of the proinflammatory lipid, LTB4, in the CBGG model of glomerular disease could […]

Find the latest version: https://jci.me/113542/pdf Increased Leukotriene B4 Synthesis in Immune Injured Rat Glomeruli Mohamed A. Rahman, M. Nakazawa, Steven N. Emancipator, and Michael J. Dunn Department ofMedicine, Hines Veterans Administration Hospital and Loyola Medical Center, Hines, Illinois and Maywood, Illinois 60141; and Department ofMedicine and Institute ofPathology Case Western Reserve University, and Division ofNephrology, University Hospitals of Cleveland, Cleveland, Ohio 44106

Abstract glomerular synthesis of 12-HETE has been demonstrated in We examined glomerular synthesis of the 5-lipoxygenase me- rats with antibody-mediated glomerular injury, nephrotoxic tabolite, LTB4, in normal and immune-injured rat glomeruli. serum nephritis (NSN)' (3). This enhancement of enzymatic Glomeruli isolated from normal rats and from rats with activity could play a role in the development of glomerular nephrotoxic serum nephritis (NSN), passive Heymann disease, since 12-HETE has a proinflammatory effect on neu- nephritis (PHN) and cationic bovine gamma globulin trophils. (CBGG)-induced glomerulonephritis were incubated with the In recent years much interest has focused on the 5-lipoxy- calcium ionophore A23187 (3 ,M). Lipids in the glomeruli and genase pathway of oxidative arachidonic acid metabolism. media were extracted with ethyl acetate, and were purified and This pathway produces 5-hydroperoxyeicosatetraenoic acid fractionated by HPLC. Immunoreactive-LTB4 (i-LTB4) was (5-HPETE), which is converted to several leukotrienes. Leu- determined by radioimmunoassay on HPLC fractions with a kotriene C4 (LTC4) and leukotriene D4 (LTD4) were previously detection limit of 50 pg of i-LTB4. A large peak of i-LTB4 that known as slow reacting substance of anaphylaxis (4, 5). Leu- comigrated with authentic LTB4 was found exclusively in glo- kotriene B4 (LTB4) is produced by stimulated inflammatory meruli isolated from the CBGG-injected rats. Addition of the cells, mainly neutrophils and macrophages (6), and is present lipoxygenase inhibitor BW755C (50 ;&g/ml) to glomerular in- in perfusate obtained from antigen challenged lung (7). LTB4 cubation resulted in > 90% inhibition of i-LTB4. Synthesis of is a powerful chemotactic and chemokinetic agent for granu- i-LTB4 by glomeruli from normal, NSN and PHN rats was locytes, stimulates the generation of reactive oxygen radicals in undetectable. Glomerular LTB4 synthesis by CBGG-injected neutrophils and induces leukocyte adhesion to the endothelial rats was confirmed by radiometric HPLC and by gas chroma- surface (8-10). When injected intradermally with PGE2, LTB4 tography mass-spectroscopy (GC-MS) analysis. increased permeability of dermal vessels to a degree dependent In order to rule out synthesis of LTB4 by neutrophils en- on its dose, although LTB4 alone had no effect on capillary trapped in the glomeruli, a group of rats received 1,000 rad permeability (1 1-14). Therefore LTB4 is considered to be a total body x irradiation, with shielding of the kidneys before major mediator of inflammation, including enhanced vascular induction of CBGG glomerulonephritis. Despite > 95% reduc- permeability, and could play a part in the pathogenesis of tion in total leukocyte count, glomerular synthesis of LTB4 glomerulonephritis. remained enhanced. Augmented glomerular synthesis of the The aims of the present study were: first, to examine the proinflammatory lipid, LTB4, in the CBGG model of glomeru- ability of normal glomerular cells to metabolize arachidonic lar disease could have an important role in the development of acid through the 5-lipoxygenase pathway and synthesize glomerular injury and proteinuria. LTB4; second, to examine glomerular synthesis of LTB4 in infiltrative and noninfiltrative models of immune glomerular Introduction disease. The infiltrative model we examined was the heterologous phase of NSN, which is characterized by Several lipoxygenase products of arachidonic acid are synthe- heavy glomeru- sized in the in lar infiltration with neutrophils. Two noninfiltrative, nonpro- kidney, especially glomeruli. Murine and liferative models were examined; passive Heymann nephritis human glomeruli have lipoxygenase activity and metabolize (PHN) and cationic bovine arachidonic acid to gamma globulin (CBGG)-induced 12-hydroxyeicosa tetraenoic acid (12- glomerulonephritis, both pathologically similar to human HETE) and 12- and 15-HETE, respectively (1, 2). Enhanced membranous nephropathy. * Parts of this work were presented at the Annual Meeting of the Methods Central Society for Clinical Research, Chicago, IL, in November, 1985, and at the Annual Meeting of the American Society of Nephrol- Materials. Bovine gamma globulin (BGG), and fluoresceinated goat ogy, New Orleans, LA in December, 1985. antisera to BGG, rat IgG, and rat complement C3 were obtained from Address reprint requests to Dr. Rahman, Division of Nephrology, U. S. Biochemicals, Cleveland, OH; l-ethyl-3-[(3-dimethylamino- Hines VA Hospital, Room G335, Hines-Loyola Medical Center, Hines propyl-carbodiimide hydrochloride] (EDC) and complete Freund's IL 60141. adjuvant were from Sigma Chemical Co., St. Louis, MO, and ethylene Received for publication 1I March 1987 and in revised form 8 diamine was from Fisher Scientific, Pittsburgh, PA. Frozen rat kidneys January 1988. were obtained from Pel-Freeze Biological, Inc., Rogers, AR. Indo- methacin was from Merck Sharp and Dohme, West Point, PA. LTB4 1. Abbreviations used in this paper: CBGG, cationic bovine gamma standards were kindly provided by Dr. J. Gleason, SmithKline & globulin; GBM, glomerular basement membrane; LTB4, leukotriene French, Philadelphia, PA, and anti-LTB4 antiserum was graciously B4; 5-HPETE, 5-hydroperoxy-eicosatetraenoic acid; NSN, nephro- provided by Dr. A. Rosenthal of Merck Sharp and Dohme. [3H]- toxic serum nephritis; PHN, passive Heymann nephritis. Leukotrienes B4, C4, and D4, and [3H]arachidonic acid were from New England Nuclear (NEN), Boston, MA. BW755C was generously pro- J. Clin. Invest. vided by Dr. S. Moncada, The Wellcome Research Laboratory, Kent, © The American Society for Clinical Investigation, Inc. England. 0021-9738/88/06/1945/08 $2.00 Preparation of anti-rat glomerular basement membrane (anti- Volume 81, June 1988, 1945-1952 GBM) serum. Particulate GBM was prepared from frozen rat kidneys

Increased Leukotriene B4 Synthesis in Immune Injured Glomeruli 1945 as previously described (15, 16). Female New Zealand white rabbits later, as described above. On days 11-13, treated rats were given 100 received intradermal injections of rat GBM, 5 mg of GBM per rabbit, mg/kg per d of BW755C as a solution in normal saline per os in two in complete Freund's adjuvant as we have previously described (3). divided doses; the dose, route, and timing were adapted from a pre- The injections were repeated after 2 wk. 1 mo after the initial injection vious report of the in vivo use of BW755C in rats (22). Six control rats the nephrotoxicity of the rabbit serum was evaluated by the develop- were given normal saline per os. Rats were maintained on drug or ment of proteinuria after injections of 1 ml serum i.v. in 300 g male vehicle during a 24-h urine collection, and a blood sample was taken at Sprague-Dawley rats. Immunofluorescent examination of glomeruli the midpoint of the urine collection. Protein and creatinine content of from injected rats confirmed linear staining of GBM for rabbit IgG and urine and serum creatinine were assessed, and glomeruli isolated from rat C3. 1 ml of rabbit serum contained 14.4 mg of IgG by radial kidneys after sacrifice were incubated to determine LTB4 production. immunodiffusion (3). Measurement ofproteinuria. Urine was collected from individual Preparation ofanti-FxJA antibody. Fx1A tubular antigen was pre- rats over the 24 h immediately before death. Protein excretion was pared from Sprague-Dawley renal cortex (17). New Zealand white determined by multiplying the urine volume by the concentration of rabbits were immunized with 10 mg Fx IA in complete Freund's adju- protein in the urine measured as the turbidity induced by 3% sulfosali- vant in the rear foot pads. 2 wk later, a booster immunization with the cylic acid. Bovine serum albumin solutions at various concentrations same preparation was given in multiple subcutaneous sites and re- were used as protein standards. peated biweekly. Rabbits were periodically bled and antibody against Immunofluorescence microscopy (IF). A small sample of cortical tubular antigen assessed by indirect immunofluorescence. Antiserum tissue was snap frozen in cold 2-methyl butane immersed in liquid against FxlA was heat-inactivated (560C, 30 min) and absorbed with nitrogen. Frozen tissue was sectioned at 4 ,um in a Spencer Cryostat 0.1 ml normal rat serum and subsequently with 1 ml packed rat blood (American Optical, Buffalo, NY) and stored at -70'C. Staining was cells per ml antiserum. The IgG fraction of the rabbit antiserum was done as previously described (3, 18, 20). Sections were layered with prepared by precipitation with half-saturated (NH4)2SO4 followed by goat antiserum to rat IgG (rhodamine conjugated), bovine IgG, rabbit DEAE cellulose column chromatography in 0.0175 M potassium IgG or rat complement C3 (fluorescein conjugated), all diluted at 1:20. phosphate buffer, pH 6.30. This preparation is referred to as Heymann There was no cross-reactivity between any of the antisera among antibody; its specificity was confirmed by indirect immunofluores- themselves or with rat IgG, bovine IgG, or rat serum (for C3) in double cence (18). immunodiffusion. Coded sections (to prevent observer bias) were ex- Preparation of the CBGG. BGG was cationized as previously de- amined in a microscope (Ernst Leitz Inc., Wetzlar, West Germany). scribed (19, 20). 2 g BGG was dissolved in 20 ml 0.01 M NaCl, mixed Electron microscopy (EM). Small (1 mm in each dimension) por- with 40 ml ethylenediamine, and the pH adjusted to 7.0 with 2 N HCI. tions of renal cortex were immersion fixed in 2.5% glutaraldehyde in 4 g EDC was added and the pH was maintained at 7.0. The mixture 0.1 M sodium cacodylate adjusted to pH 7.3. The tissue was postfixed was left overnight at room temperature, then dialyzed against 0.15 M in (Os 04) and embedded in Spurr's epoxy. Ultrathin (50 nm) sections NaCl for 24 h. Cationic BGG prepared by this procedure has a pl were cut, picked up on uncoated nickel grids, and stained with uranyl ranging from 9.5 to 11.5. acetate and lead citrate. Specimens (coded to prevent observer bias) Experimental models. Sprague-Dawley rats, 200-300 g were di- were evaluated in an electron microscope (201 C; Philips Medical vided into four groups: a control group, a NSN group, a PHN group, Electronics Div., Piscataway, NJ) for the number and size of electron and a group with CBGG-induced membranous nephropathy. In each dense deposits in subendothelial, intramembranous, subepithelial, and of the four groups kidney tissues were examined by light, immunofluo- mesangial glomerular sites, in a manner similar to that previously rescence and electron microscopy, and glomeruli were isolated for described ( 18, 20). LTB4 measurement. Light microscopy (LM). Tissue was immersion fixed in 10% for- NSN was induced in rats by injecting 1 ml of anti-GBM serum i.v.; malin buffered to pH 7.5 with 0.01 M sodium phosphate for 24 h. The the rats were sacrificed 48 h later (3). To induce PHN, 60 mg of IgG tissue was then processed through graded alcohols and xylenes and fraction of anti-Fx I A was injected into rats in three divided doses given infiltrated in molten Paraplast (Fisher Scientific). The tissue was em- 1 h apart, via tail vein ( 18). Rats were sacrificed 18 d later, during the bedded, sectioned at 2 um and stained with periodic acid silver-meth- peak of the autologous phase. For induction of the CBGG model, rats enamine, and with hematoxylin and eosin. Coded sections were exam- were immunized in the foot pads with 1 mg CBGG in complete ined in a light microscope (American Optical, Buffalo, NY). Freund's adjuvant. 7 d later the rats received daily intravenous antigen Glomerular isolation and incubation. After small portions of the challenge with increasing doses of CBGG for 5 d (day 1: 2 mg, day 2: 4 cortex were taken for electron, light and immunofluorescence micros- mg, day 3: 6 mg, day 4: 8 mg, day 5: 10 mg) and were killed 3-4 d after copy, the remainder of the kidney tissue was placed in ice-cold PBS the last intravenous injection of antigen. The pathologic features ofthe (0.15 M NaCl, 0.01 M sodium phosphate, pH 7.6) and used for glo- CBGG model have been described recently (20). merular isolation. Glomeruli were isolated by a differential sieving X-Irradiation to the CBGG model. To exclude the possibility that technique (20). The cortex was separated from the medulla and circulating or infiltrating neutrophils were the source of measured minced to a pastelike consistency. The minced cortical paste was LTB4, leukocytes were depleted by total body x-irradiation (21). 5 d passed through a 106-,sm metal sieve to remove large tubular frag- after foot pad immunization with CBGG each rat received a total dose ments and suspended in PBS. This suspension was triturated and of 1,000 rad (Theratron model 780; Atomic Energy of Canada, Ltd., poured over a 75-um sieve. The glomeruli trapped on the 75-Mm sieve Ottawa, Canada); an 8 cm thick cerrobend shield was placed around were collected and suspended in PBS. The purity of the glomerular the abdomen and the lumbar spine region shielding the kidneys. 4 days preparation was assessed by light microscopy and varied between 92 later, blood leukocytes were ennumerated and rats received four intra- and 98%. The isolated glomeruli were transferred to a siliconized glass venous injections of CBGG; 2, 4, 6, and 8 mg, 12 h apart. This protocol tube and were suspended in Earle's balanced salt solution with 20 mM was adapted to develop the disease in a shorter period of time due to Hepes, 3.0 AM calcium ionophore (A23 187) and 2 mM reduced gluta- the decreased life span of the irradiated rats. One day after the last thione, at a total volume of 1 ml. In some experiments we added the CBGG injection proteinuria was examined, a blood sample was drawn cyclo- and lipoxygenase inhibitor BW755C (50 Atg/ml) or [3H1- for measurement of total white blood cell and neutrophil count, and arachidonic acid, specific activity 80 Ci/mmol (5 X 105 cpm/ml of the rats were killed. media yielding 1.8 ng arachidonate/ml). The glomerular suspension In vivo inhibition ofLTB4 synthesis with BW75SC. To determine was incubated for 30 min at 370C with mild agitation. the effects of 5-lipoxygenase inhibition in the CBGG model of glomer- Extraction and purification of lipids. Incubation was terminated by ulonephritis we administered the cyclo- and lipoxygenase inhibitor acidification to pH 3.5 with 0.1 N HCI and lipids were extracted three BW755C to rats for 3 d after the last antigenic challenge. Six rats were times with 3-4 vol of ethyl acetate. The extracted lipids were dried preimmunized and challenged for four successive days starting a week under nitrogen and reconstituted in 100% methanol and then in 5%

1946 Rahman, Nakazawa, Emancipator, and Dunn methanol in water. The reconstituted lipids were further purified on GBM serum showed tuft necrosis, hypercellularity, and neu- ODS silica cartridges, pretreated with 1 ml methanol followed by 3-5 trophilic infiltration. Rats given anti-Fx IA had normocellular ml of water immediately before use. After application of the sample, without the cartridge was washed with 2-3 ml of water then the sample glomeruli crescents, infiltration or necrosis, but capil- was walls were eluted with I ml of methanol. Samples were stored in methanol under lary thickened and focal epimembranous spikes N2 at -60'C until separation by reverse phase HPLC and LTB4 mea- were evident. Immunofluorescence (Fig. 1) demonstrated surement by RIA. characteristic linear staining of rabbit IgG and rat C3 on the HPLC and RIA for LTB4. Samples obtained from glomerular in- GBM in rats given anti-GBM serum (A), intense granular glo- cubation in the presence of calcium ionophore (A23187) were ana- merular capillary wall deposits of rabbit IgG, rat IgG and C3 in lyzed by HPLC-RIA. Reverse phase HPLC was carried out on an glomeruli from rats given anti-FxlA (B), and granular capil- instrument model 5000; Varian Instruments, Palo Alto, CA fitted with lary wall deposition of BGG, rat IgG and C3 in rats a given Nucleosil C- 18 column. The column was eluted at 0.9 ml/min, with a CBGG (C); mesangial were not in rat. linear gradient deposits prominent any from 93.4% 0.01 M phosphate buffer (pH 7.4), 6% Control rats were negative for all immune reactants. methanol, and 0.6% t-amyl alcohol, to 99.4% methanol with 0.6% By EM, rats with NSN had areas of and mononu- t-amyl alcohol (% by volume) over 100 min (23). Fractions were col- polymorphonuclear lected at 1-min intervals, clear cell infiltration, and detachment of endothelial lining dried under nitrogen and reconstituted in 0.5 2 ml of PBS containing 1% gelatin. Immunoreactive LTB4 was deter- (Fig. A). Electron dense deposits were not evident, but a few mined in each fraction using LTB4 antibodies. The sensitivity and the foci of tuft necrosis were observed. All rats given anti-Fx1A cross-reactivity of the antibodies were previously described (24). antibody had numerous electron dense deposits in the subepi- Radiometric-HPLC. Samples obtained from glomerular incuba- thelial layer of the capillary basement membrane, and foot tion with A23 187 and [3H]arachidonic acid were analyzed by HPLC as processes were diffusely broadened (Fig. 2 B). Intramem- described above. The radioactivity in each fraction was determined by branous deposits were seen rarely, and subendothelial and liquid scintillation counting and was expressed as counts per minute mesangial deposits were absent. EM of kidney tissues removed per milligram glomerular protein. from rats given CBGG revealed of the foot GC-MS. HPLC fractions that with broadening pro- comigrated authentic LTB4 and cesses and numerous electron dense were shown to contain i-LTB4 peaks, and fractions eluted 2 min before regularly spaced subepithelial as well as a few and 2 min after the LTB4 retention time, were further studied by deposits, mesangial deposits (Fig. 2 C). There was no GC-MS by Dr. Ian Blair, Vanderbilt University, Nashville, TN (25). thickening of capillary basement membrane and Briefly, after esterification of the fatty acid esters in the HPLC fractions no neutrophil or monocyte infiltration. with pentafluorophenol, the bis-trimethylsilyl ether was generated by Glormerular LTB4 production. Immunoreactive LTB4 heating with bis(trimethylsilyl)trifluoroacetamide in pyridine (1:1) at (i-LTB4) was measured by RIA on HPLC fractions with a 60°C for I h. Derivatized samples were chromatographed on a 6 m detection limit of 50 pg of i-LTB4. The elution profile of au- DB- I capillary column (0.25 mm i.d.) with an injection temperature of thentic leukotrienes measured by flow photometry at 280 nm 100°C and a 5°C/min thermal column program; elution was with indicated retention times of 53, 57, and 61 min for LTC4, helium as carrier gas at an inlet pressure of 90 kPa. Ions were detected LTD4, and LTB4, respectively (Fig. 3 A). The amount of in a Nermag RIOIOC mass spectrometer operating in negative ion i-LTB4 in the HPLC fraction that comigrates with authentic mode with an ion-source temperature of 2 10°C at 89 eV, at a source pressure of LTB4 from both control rats (n = 9 rats in three experiments) 30 Pa. Synthetic [2H]LTB4 served as an internal standard. 3 The capillary column chromatography separates the various isomers of (Fig. B) and those with NSN (n = 10 rats in three experi- 5-, 12-dihydroxyeicosatetraenoic acids (26). ments) (Fig. 3 C), did not exceed the background noise of the assay, which resulted from cross reactivity of LTB4 antibodies Results with other lipid fractions. Likewise, glomeruli isolated from Proteinuria. Table I shows the 24-h protein excretions in the rats with PHN did not synthesize measurable amounts of three models of glomerular injury and in control rats. All rats i-LTB4 (n = 11 rats in four experiments, data not shown). In that received NTS and CBGG developed significant protein- contrast to control, NSN, and PHN rats, the synthesis of uria in excess of 60 mg/24 h. Rats with PHN developed mild i-LTB4 was greatly enhanced in glomeruli isolated from rats proteinuria, 37±13.8 mg/24 h, and control rats excreted 8±0.7 immunized and challenged with CBGG (n = 11 rats in four mg/24 h. experiments) (Fig. 3 D) in which we detected 12.4±2.3 ng Glomerular morphologic changes. LM examination of glo- i-LTB4/mg glomerular protein, that comigrated with authen- meruli from rats given CBGG was not different from normal tic LTB4. controls; no mesangial or endocapillary proliferation, base- In different experiments glomeruli isolated from CBGG ment membrane changes, crescents or cellular infiltration injected rats were incubated with [3H] C 20:4. Lipids extracted were seen. On the other hand, glomeruli from rats given anti- from glomerular incubations were chromatographed, and the counts per minute per milligram glomerular protein in each Table L Results ofProteinuria in Control Rats and Rats fraction plotted against retention time. A large radioactive with Experimental Models ofGlomerular Disease peak, 2803 (cpm/mg glomerular protein) ml was detected, comigrating with authentic LTB4, while no radioactive peaks Experimental model No. of rats Proteinuria comigrating with authentic LTC4 or LTD4 were seen (Fig. 4). mg/24 h Glomeruli from normal rats did not reveal any leukotriene peak (data not shown). Control 9 8±0.7 Glomerular incubation with NSN 10 106±11.6* lipoxygenase inhibitor. Glo- meruli isolated from rats injected with CBGG were incubated PHN 11 37±13.8* with the CBGG cyclo- and lipoxygenase inhibitor BW755C, 50 gg/ml. 11 112±12.7* In two separate experiments, glomerular LTB4 synthesis decreased from 11.8±4.4 without * P < 0.01 vs. control. ng/mg glomerular protein inhibitor to 0.71±0.30 ng/mg in the presence of BW755C.

Increased Leukotriene B4 Synthesis in Immune Injured Glomeruli 1947 Figure 1. Immunofluorescence micrographs. Representative staining for rat C3 in glomeruli from rats with NSN (A), PHN (B), and CBGG- induced glomerulonephritis (C). Linear staining as seen for C3 in A was also observed for rat IgG and rabbit IgG in rats with NSN. Rats with PHN manifest granular staining for rat and rabbit IgG similar to the pattern present for C3 depicted in B. Similarly, rats with CBGG glomerulonephritis had granular deposits of BGG and rat IgG in capillary walls, in a distribution similar to the pattern for C3 depicted in C. (x 400).

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Nk,, - <.;',i

Figure 2. Electron micrographs. Ultrastructurally, rats with NSN reduced glomerulonephritis are also in epimembranous sites, but are veal leukocytic infiltration of glomeruli with abutment of leukocytes smaller and more discrete than those in PHN, without new basement on basement membranes denuded of endothelium, and foci of fibrin membrane formation (C). An electron micrograph of a section of (A). Rats with PHN have large, coalescent epimembranous electron capillary wall from a normal rat (D) is included for comparison. (a-c dense deposits, and a few smaller scattered intramembranous de- x 20,000; d x 30,000.) posits (B). The deposits present in glomeruli of rats with CBGG-in-

1948 Rahman, Nakazawa, Emancipator, and Dunn Leukotriene standards 2500- LTB4 . Inm A *o 2000-. 0 CL I, 0 4 Ej 1500-. 0 .0

40 1000

ei 500 2 4 323640 44 485256 6064 68

R n i v;. *7.I c5- Control 5 - 40 45 50 55 60 65 70 Minutes E ° 10- Figure 4. Radiometric HPLC obtained after incubation c c 5- of glomeruli 0 from CBGG rats. Counts per minute per milligram glomerular protein in each fraction is 2 4 32 36 40 44 48 52 56 60 64 68 plotted against retention time in minutes. The retention time of leukotriene standards are indicated by arrows. A large peak of radioactivity (integral under the curve is 2803 (cpm/mg c 15 - NSN : glom. protein). ml), comigrated with standard LTB4. No LTC4 or LTD4 peaks were seen. E 10 cE Lo co c 5 0 at J-4 2 4 32 36 40 44 48 52 56 60 64 68 0.12±0.04 X 103 neutrophils/,l and 26±3.0 X 103 platelets/ A) compared to nonirradiated controls (13.6± 1.0 X 103 WBC/Ml, 6.86±1.7 X 103 neutrophils/jsl, and 318±28 X 103 plate- c 0 lets/gl). E °._ In vivo inhibition ofLTB4 synthesis with BW755C. Treat- c E ment with BW755C resulted in microhematuria, and the urine 0o of these rats contained up to 7 mg/24 h hemoglobin, measured from the optical density ofthe urine at 410 nm versus a turbid- Minutes ity blank at 700 nm. The hemoglobin values were subtracted from the total protein determined by sulfosalicylic acid. Figure 3. Determination of LTB4 by HPLC-RIA. The retention CBGG rats had 77±8.3 mg protein in the 24-h urine collec- times of LTC4, LTD4, and LTB4 standards are 53, 57, and 61 min, respectively (A). The results of RIA of LTB4 on HPLC fractions are tion, whereas rats given BW755C excreted only 28.4±11.8 shown by hatched bars in control (B), NSN (C), and CBGG-injected mg/24 h (P < 0.01). However, the endogenous creatinine rats (D). Data are the means±SE of three or four separate experi- clearance in the treated rats was 0.24±0.07 ml/min, compared ments, expressed in ng/mg glomerular protein and plotted against re- with 0.55±0.05 ml/min in the saline-treated group (P < 0.01). tention time in minutes. Control and NSN synthesized undetectable There was no correlation between proteinuria and endogenous amounts of i-LTB4. Glomeruli from CBGG injected rats synthesized creatinine clearance between the treated and control groups. 12.4±2.3 of i-LTB4. Moreover, while the creatinine clearance was reduced to 44% ofthe control group, the proteinuria was reduced to 37% ofthe control value in treated rats. LTB4 synthesis in the treated rats GC-MS. To verify the structural characteristics of the was 1.06 ng/mg glomerular protein, versus 2.96 ng/mg in un- i-LTB4 in glomeruli isolated from CBGG injected rats, selected treated controls. samples of extracted glomeruli and incubation medium were subjected to GC-MS analysis after HPLC purification. Frac- tions that comigrated with standard LTB4 and contain peaks of i-LTB4 were identified as authentic LTB4 based on an in- Table II. Comparison ofLTB4 Determined by HPLC-RIA tense negative ion at m/z 479, identical to the spectrum of and GC/MS standard LTB4. There was close agreement in the quantifica- Retention LTB4 extracted from medium tion of LTB4 by HPLC-RIA and by GC/MS (Table II). Rats with CBGG- on X-irradiation to the CBGG model Some rats were X-irra- glomerulonephritis column HPLC-RIA GC/MS diated before intravenous injection of CBGG to minimize the min ng/ml possibility that glomerular LTB4 was derived from circulating leukocytes. Irradiated rats developed 118±14 mg proteinuria/ Normal 6 1 117.2 97.7 24 h, comparable to nonirradiated rats. Glomerular i-LTB4 Normal 59 1.6 1.1 production was 8.2 ng/mg glomerular protein (pooled from Normal 63 0.2 0.2 four rats). There was more than a 95% reduction in circulating Leukocyte depleted* 61 37.8 38.8 leukocytes prior to induction of glomerulonephritis on day 4 Leukocyte depleted* 59 17.2 0.8 postirradiation (0.67±0.23 X 103 WBC/Al blood, 0.15±0.02 X 103 neutrophils/gl and 66.0±20.6 X 103 platelets/Ml) and at * These rats received 1000 R total body x-irradiation before induc- sacrifice on day 7 postirradiation (0.05±0.28 X 103 WBC/Ml, tion of glomerulonephritis.

Increased Leukotriene B4 Synthesis in Immune Injured Glomeruli 1949 Discussion LTB4; however, their capacity to synthesize 5-lipoxygenase products has not been established. A bone marrow-derived Arachidonic acid is the substrate for cyclo- and lipoxygenase macrophage, recently described as resident in the mesangium, enzymes. Glomerular synthesis of the cyclooxygenase metabo- increases in immune glomerular injury (32), and could be a lites, prostaglandins, and thromboxane, has been studied ex- source of LTB4. These cells may metabolize arachidonic acid tensively. These eicosanoids modulate renal hemodynamics, through the 5-lipoxygenase pathway similar to monocytes and and the inflammatory response in glomerular disease (20, macrophages. Endothelial cells in the glomeruli may also be a 27-29). On the other hand, the data on the role of the lipoxy- source of the LTB4 we observed, but this is very unlikely since genase metabolites in glomerular inflammation are few and endothelial cells are relatively poor producers of LTB4, gener- the capacity of the glomeruli to synthesize 5-lipoxygenase ating i-LTB4 in the pg range/2 X 106 cells/h, even with 10 ,M products has not been established. ionophore (34). In this study, we demonstrate glomerular synthesis of the Regardless of the cellular source of LTB4, we consider de- 5-lipoxygenase metabolite LTB4 in the CBGG-induced model posited immune complexes to be the likely stimulus. Lianos of immune glomerular disease. Our studies were always con- recently reported transient increases in i-LTB4 synthesis within ducted with calcium ionophore added to isolated glomeruli, in hours after induction of the heterologous phase of NSN, but order to stimulate 5-lipoxygenase. This model, pathologically noted that LTB4 synthesis did not persist until the onset of similar to human membranous nephropathy, is a nonprolifer- proteinuria (35). Lianos and Noble also reported similar tran- ative and noninfiltrative model, and injury is independent of sient i-LTB4 synthesis in the heterologous phase of PHN and serum complement and circulating leukocytes (30). The en- active Heymann nephritis, again not associated with protein- hanced glomerular synthesis of LTB4 was demonstrated by uria either temporally or in degree (36). Although PHN is an HPLC/RIA, by radiometric HPLC after incubation of isolated immune complex disease pathologically similar to the CBGG glomeruli with tritiated arachidonic acid, and by GC/MS, model, we did not observe an increment in glomerular LTB4 which unequivocally demonstrated the presence of authentic synthesis in proteinuric rats with PHN or with NSN, in agree- LTB4 in the HPLC fractions shown to contain i-LTB4. Fur- ment with Lianos et al. (35, 36). In contrast, in CBGG-induced thermore, synthesis of immunoreactive LTB4, measured by glomerulonephritis there is more 5-lipoxygenation of arachi- HPLC-RIA, was inhibited by the cyclo- and lipoxygenase in- donic acid than in the other models and heightened LTB4 hibitor BW755C. Although we included reduced glutathione synthesis is sustained for at least a few days after onset of in the incubation medium, we did not observe any sulfidopep- proteinuria. Hence, ongoing formation or deposition of im- tide leukotriene products or their major metabolite, LTE4. We mune complexes which persists in active models of glomerulo- cannot exclude the possibility that some sulfidopeptide leuko- nephritis but occurs only transiently in the passive models triene is produced by glomeruli, but it is clear that the major might be a key factor in stimulating glomerular 5-lipoxy- product of 5-lipoxygenase activity in glomeruli is LTB4. We genase. could not detect LTB4 production by normal glomeruli, even Data on the effects of LTB4 on glomerular function are with addition of ionophore. This is not incompatible with the scarce. Physiologic effects of LTB4 are most extensively stud- recent report by Cattell and her colleagues, since they pooled ied in leukocytes. LTB4 is a potent chemotactic lipid, equipo- glomeruli from many more rats, totaling 5 mg glomerular tent on molar basis to formyl-methionyl-leucyl-phenylalanine protein to obtain 120 pg/mg LTB4 produced by normal glo- and complement component C5a, the two most potent che- meruli without addition of ionophore (31). We detected as motactic agents known (37, 38). In vivo, LTB4 shows chemo- much as 12 ng/mg LTB4 produced by glomeruli from rats with tactic activity towards polymorphonuclear leukocytes, mono- CBGG nephritis containing only 0.5 mg protein; this is clearly cytes, and eosinophils. Also, LTB4 can cause secretion of lyso- different from the synthetic rate of normal glomeruli, whether somal enzymes from neutrophils (10, 39), enhance or not stimulated with ionophore. complement receptor expression (40) and induce expansion of The enhanced glomerular LTB4 synthesis in the CBGG human suppressor cells (41). model could be derived from endogenous glomerular cells, or The lack of glomerular cellular infiltration in the CBGG circulating neutrophils or monocytes trapped in the glomeruli model, despite the generation of the powerful chemotactic during glomerular isolation. Although there were no infiltrat- lipid LTB4, is consistent with the findings of Kreisle et al. (42), ing monocytes or neutrophils in glomeruli demonstrable by who found that rat PMN do not exhibit chemotaxis to LTB4 at light and electron microscopy in this model, it is conceivable concentrations from 10-10 to 10-6 M. At these concentrations that there were some circulating cells present in the glomeruli LTB4 can elicit a powerful chemotactic response in human not demonstrated by routine morphologic examination (21, PMNs. Thus, any proinflammatory effects of LTB4 in rat glo- 32). The contribution of these cells to the glomerular LTB4 merular disease are likely mediated by mechanisms other than should be minimal, if any. This is supported by our finding of recruitment of inflammatory cells into the glomeruli. increased glomerular LTB4 synthesis associated with CBGG- We do not expect any effect of LTB4 on glomerular hemo- induced glomerulonephritis even when circulating leukocytes dynamics, since LTB4 has no demonstrable spasmogenic activ- are reduced by > 95% through bone marrow irradiation. Fur- ity on rat mesangial cells in culture (43), guinea pig ileum (44), thermore, glomerular synthesis of LTB4 in rats with NSN was and rat and rabbit aorta (45). This is in contrast to the well below the detection limit of our assay, despite heavy glomeru- known contractile effects of the sulfidopeptide leukotrienes, lar infiltration with neutrophils. Therefore, the most likely LTC4 and LTD4 on glomeruli (46) and mesangial cells in cul- source of enhanced LTB4 synthesis in the CBGG model is ture (43, 46), and on the renal vasculature (47, 48). endogenous glomerular cells. Glomerular epithelial and mes- Although LTB4 has no effect on vascular permeability it- angial cells are capable of synthesizing prostaglandins, throm- self, simultaneous injection of LTB4 with vasodilators such as boxane, and 12-HETE (33). They are a possible source of PGE2 or bradykinin increases vascular permeability (1 1-14). 1950 Rahman, Nakazawa, Emancipator, and Dunn The vasodilators alone did not increase vascular permeability mass spectroscopy studies. We are also grateful to Edith Hanzmann, even at relatively high doses, and the increase in permeability Nancy Urankar, and Chen-Lung Hsieh for excellent technical assis- was dependent on the concentration of LTB4 injected. Al- tance. though one group found no increase in vascular permeability This work was supported by a grant from the National Kidney in the kidney after infusion of both eicosanoids into the renal Foundation of Illinois, grant from the Veterans Administration, and artery at doses that did induce increased permeability in skin by National Institutes of Health (NIH) grants HL-22563 and DK-38544. Dr. Rahman was an NIH postdoctoral trainee, grant and other sites, these investigators studied whole kidney rather AM-07470. than alterations in glomerular capillary permeability specifi- cally (49). These experiments were, moreover, quite acute, References being terminated only 8 min after infusion of eicosanoids. Since increased glomerular synthesis of PGE2 is a feature ofthe 1. Jim, K., A. Hassid, F. Sun, and M. J. Dunn. 1982. Lipoxygenase CBGG-induced model of glomerulonephritis (20), synergism activity in rat kidney glomeruli, glomerular epithelial cells and cortical between increased PGE2 and LTB4 in promoting increased tubules. J. Biot. Chem. 257:10294-10299. vascular permeability may well apply to glomerular perme- 2. Sraer, J., M. Rigaud, M. Bens, H. Rabinovitch, and R. Ardaillou. ability to macromolecules in this model. We administered 1983. Metabolism of arachidonic acid via the lipoxygenase pathway in BW755C, a combined lipoxygenase and cyclooxygenase inhib- human and murine glomeruli. J. Biot. Chem. 258:4325-4330. 3. Lianos, E. A., M. A. Rahman, and M. J. Dunn. 1985. Glomeru- itor to a group of CBGG rats and decreased glomerular LTB4 lar arachidonate lipoxygenation in rat nephrotoxic serum nephritis. J. synthesis by 65%, proteinuria by 63%, and GFR by 56%. Clin. Invest. 76:1355-1359. Hence, the reduction of proteinuria after inhibition of glomer- 4. Murphy, R. C., S. Hammarstrom, and B. Samuelsson. 1979. ular LTB4 could also be attributed to a reduced filtered protein Leukotriene C: A slow reacting substance from murine mastocytoma load. Furthermore, BW755C is a cyclooxygenase inhibitor and cells. Proc. Nati. Acad. Sci. USA. 76:4275-4279. undoubtedly reduced glomerular PGE2 production and re- 5. Morris, H. R., G. W. Taylor, P. J. Piper, and J. R. Tippins. 1980. moved the synergistic actions of PGE2 and LTB4 to enhance Structure of slow-reacting substance of anaphylaxis from guinea pig capillary permeability. The precise definition of the role of lung. Nature (Lond.). 285:104-105. LTB4 in the CBGG model must await the availability of spe- 6. Borgeat, P., and B. Samuelsson. 1979. Arachidonic acid metabolism in polymorphonuclear leukocytes. Effects of ionophore A23187. cific 5-lipoxygenase inhibitors and/or LTB4 receptor antago- Proc. Natl. Acad. Sci. USA. 76:2148-2152. nists. 7. Morris, H. R., G. W. Taylor, P. J. Piper, and J. R. Tippins. 1979. Reports of synergy between LTB4 and vasodilators in in- Slow reacting substance of anaphylaxis: Studies on purification and ducing vascular permeability ascribe the actions of the eicosa- characterization. In Prostaglandins and Inflammation. K. D. Rains- noids to an effect on neutrophils (11, 12, 50). Yet, in the ford and A. W. Ford-Hutchinson, editors. Birkhauser-Verlag, Basel. CBGG-induced model of glomerulonephritis, we do not ob- 27-36. serve a cellular infiltrate (20) and in separate studies reported 8. Goetzel, E. J., and W. C. Pickett, 1980. The human PMN leu- elsewhere, we have found that enhanced glomerular prosta- kocyte chemotactic activity of complex hydroxy-eicostatetraenoic glandin production and proteinuria develop independently of acids (HETEs). J. Immunol. 125:1780-1791. circulating leukocytes (30). This does not exclude synergistic 9. Ford-Hutchinson, A. W., M. A. Bray, M. V. Doig, M. E. Shipley, in and M. J. H. Smith. 1980. Leukotriene B: A potent chemokinetic and interaction between PGE2 and LTB4 CBGG-induced glo- aggregating substance released from polymorphonuclear leukocytes. merulonephritis; rather, it indicates that the functions sub- Nature (Lond.). 286:264-265. served by neutrophils in the enhancement of cutaneous vascu- 10. Serhan, C. N., A. Radin, J. E. Smolen, H. Korchak, B. Samu- lar permeability may be provided by cells other than circulat- elsson, and G. Weissman. 1982. Leukotriene B4 is a complete secreta- ing or infiltrating leukocytes in glomerulonephritis, perhaps gogue in human neutrophils: A kinetic analysis. Biochem. Biophys. endogenous glomerular cells. Glomerular mesangial cells in Res. Commun. 107:1006-1012. culture manifest several functions traditionally associated with 11. Wedmore, C. V., and T. J. Williams. 1981. Evidence for two granulocytes, including expression of receptors for immuno- types of vascular permeability-increasing mediators: the direct action globulin Fc region and for activated complement components, of histamine and bradykinin: the polymorph-dependent action ofC5a, generation of reactive oxygen intermediates and leukotriene B4 and formyl tripeptide. Br. J. Pharmacol. 73:209P. inflammatory 12. Bray, M. A., F. M. Cuningham, A. W. Ford-Hutchinson, and eicosanoids, elaboration of neutral proteases, and even phago- M-. J. H. Smith. 1981. Leukotriene B4 a mediator of vascular perme- cytosis and secretion of interleukin 1 (51, 52). Thus, some ability. Br. J. Pharmacol. 72:483-486. mesangial effect of LTB4 in conjunction with PGE2 may ulti- 13. Dahlen, S.-E., J. Bjork, P. Hedqvist, K.-E. Arfors, S. Hammar- mately cause proteinuria. strom, J.-A. Lindgren, and B. Samuelsson. 1981. Leukotrienes pro- In summary, we examined glomerular synthetic capacity mote vascular leakage and leukocyte adhesion in postcapillary venules: of LTB4 by normal rat glomeruli and by glomeruli isolated in vivo effects with relevance to the acute inflammatory response. Proc. from the heterologous phase of NSN, PHN, and CBGG-in- NatL. Acad. Sci. USA. 78:3887-3891. duced membranous nephropathy. Glomerular synthesis of 14. Higgs, G. A., J. A. Salmon, and J. A. Spayne. 1981. The inflam- in matory effects of hydroperoxy and hydroxy acid products of arachi- LTB4 was exclusively enhanced the CBGG model indepen- 74:429-433. dent of or This increment of donate lipoxygenase in rabbit skin. Br. J. Pharmacol. infiltrating leukocytes platelets. 15. Krakower, C. A., and S. A. Greenspon. 1951. Localization of LTB4 may play an important role in the pathophysiology of nephrotoxic antigen within the isolated renal glomerulus. Arch. Pathot. glomerular disease. 51:629-639. 16. Spiro, R. G. 1967. Studies on the renal glomerular basement Acknowledgments membrane: Preparation and chemical composition. J. Biol. Chem. 242:1915-1918. We wish to thank Dr. Ian Blair and Brian Noven, Vanderbilt Univer- 17. Edgington, J. S., R. J. Glassock, and F. J. Dixon. 1968. Autolo- sity, Nashville, Tennessee, for performing the gas chromatography/ gous immune complex nephritis induced with renal tubular antigen. I.

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1952 Rahman, Nakazawa, Emancipator, and Dunn