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Fish oil feeding modulates leukotriene production in murine lupus nephritis
Fish Oil Feeding Modulates Leukotriene Production in Murine Lupus Nephritis Robert F. Spurney, Phillip Ruizl, Christine R. Albrightson2, David S. Pisetsky3 and Thomas M. Coffman Divisions of Nephrology and 3Rheumatology and Immunology, Department of Medicine, Duke University and Durham VA Medical Centers, Durham, N.C., 27710; and ‘Department of Pathology, University of Miami, Miami, FL, 33 10 1, 2Smithkline Beecham Pharmaceuticals, King of Prussia, PA Diets enriched with fish oil (FO) ameliorate kidney disease in the MRL-lpr/lpr murine model of lupus nephritis. Although the mechanisms of this effect are not known, FO is rich in the polyunsaturated fatty acid eicosapentaenoic acid (EPA) which may have profound effects on eicosanoid metabolism. In MRL-lpr/lpr mice, FO feeding reduces renal production of cyclooxygenase metabolites. However, EPA may also affect the metabolism of arachidonate by the 5lipoxygenase (5-LO) pathway and enhanced production of 5-LO metabolites has been implicated in the pathogenesis of kidney disease in MRL-lpr/lpr mice. We therefore investigated the effects of FO feeding on production of 5-LO metabolites in 20 week old MRL-lpr/ lpr mice. After 8 weeks of dietary supplementation with FO, both renal hemodynamic function and glomerular histology were improved compared to safflower oil (SO) controls, Amelioration of kidney disease was associated with alterations in the pattern of leukotriene production by macrophages and kidneys from FO fed mice. There was a significant decrease in the production of leukotriene B, (LTB,) and tetraene peptidoleukotrienes by peritoneal macrophages isolated from mice given FO compared to control animals. Similarly, dietary supplementation with FO decreased renal production of LTB,. Reduced production of tetraene leukotrienes was accompanied by a modest increase in the production of pentaene leukotrienes by macrophages from FO fed mice. We speculate that this modulation of leukotriene production by FO feeding may have beneficial effects on renal disease in autoimmune nephritis.
0 1994 Butterworth-Heinemann
Prostaglandins
1994:48, November
33 1
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Keywords: Leukotriene; eicosapentaenoic acid; fish oil; lupus nephritis; eicosanoid
Introduction
MRL-lpr/Ipr mice spontaneously develop an autoimmune disease with nephritis which is similar to human systemic lupus erythematosus (SLE).' In these animals, the development of renal disease is associated with enhanced production of both cyclooxygenase and 5-1ipoxygenase (5-LO) metabolites by the kidney. 2,3 While an important pathogenic role for cyclooxygenase metabolites has been clearly documented in murine lupus 3,6and in human SLE,4,s recent studies suggest that enhanced production of leukotrienes may also contribute to autoimmune nephritis. 2 In vitro, LTB4 promotes adhesion of leukocytes to endothelial cells, 7 is a potent chemotactic agent, 8 and stimulates aggregation, enzyme release, and generation of reactive oxygen intermediates in neutrophils. 9 The peptidoleukotrienes are potent renal vasoconstrictors/0, H and cause mesangial cell contraction '2 and proliferation. ~3Within the kidney, enhanced leukotriene production could therefore promote renal inflammation and injury by a variety of direct and indirect mechanisms. Dietary supplementation with FO ameliorates renal disease and prolongs survival in murine lupus nephritis. ~4-~8FO is rich in the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which may have profound effects on metabolism of endogenous lipids '8-~ including leukotrienes. 24~ For example, EPA is metabolized by 5-LO to LTBs.24,2s This compound has reduced biologic activity compared to the arachidonic acid (AA) metabolite, LTB4.24,2s Moreover, EPA acts as competitive antagonist of AA for both the cyclooxygenase and 5-LO pathways; ~6-~ thus, EPA reduces the production of AA metabolites in a variety of cells,~9,~°including glomerular mesangial cells. ~ These results suggest that diets enriched with fish oil might alter the profile of lipid mediators produced in murine lupus nephritis. Indeed, when renal disease is prevented in MRL-Ipr/lpr mice by FO feeding, production of cyclooxygenase metabolites by the kidney is reduced. TM However, the effect of FO feeding on leukotriene production in murine lupus has not been previously investigated. To further investigate the effects of FO feeding on eicosanoid production in murine lupus, we examined the effects of dietary supplementation with FO on production of leukotrienes by macrophages and kidneys from MRL-lpr/lpr mice. Our findings suggest that FO feeding reduces production of tetraene leukotrienes and enhances production pentaene leukotrienes in murine lupus. We speculate that these changes in leukotriene production may contribute to the beneficial effects of FO feeding in autoi m m u n e nephritis. 332
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Materials and Methods Animals MRL-lpr/lpr were originally obtained from the Jackson Laboratory (Bar Harbor, ME). These animals were subsequently maintained and bred in the animal facility of the Durham VA Medical Center. All animals were given tap water ad libitum and received standard laboratory chow until 12 weeks of age when mice were randomized to the experimental diets as described below.
Experimental Protocol Beginning at 12 weeks of age, MRL-lpr/Ipr mice were randomized to diets that differed only in the composition of fat. FO fed mice (N = 16) received a diet enriched with 20% menhaden oil and the control group (N = 18) received a 20% SO diet (ICN Biochemical, Cleveland, OH). At 20 weeks of age, mice were placed in metabolic cages and urine collected for 24 hours. The urine was chilled throughout the collection period and then stored at -70°C until urinary eicosanoids and proteinuria were quantitated as described below. Following the urine collections, clearances of inulin (CIN)and PAH (CpAH)were measured. After the renal hemodynamic studies, mice were sacrificed and kidneys were removed for histologic study and for measurement of eicosanoid production as described below.
Renal Hemodynamic Studies C~N and Cp~ were measured as previously described (2) to determine the glomerular filtration rate (GFR) and renal plasma flow (RPF), respectively. On the day of study, animals were anesthetized with 0.04 rag/gram pentobarbital, and a polyethylene catheter (PE 90) was inserted into the trachea to facilitate spontaneous ventilation. The left carotid artery and left jugular vein were cannulated with polyethylene catheters (PE-10) for intravenous infusions, to monitor mean arterial pressure (MAP) using a Gould-Statham strain gauge, and to allow intermittent sampling of arterial blood. Following surgery, normal saline (2.0% of the body weight) was infused intravenously over 20 minutes to replace fluid lost during surgery. A priming dose of carboxyl-14C-inulin (5~Ci/kg) and glycyl-3HPAH (10~Ci/kg) was given, followed by infusion of carboxyl-~4C-inulin (0.8~zCi/ml) and glycyl-3H-PAH (4~Ci/ml) in normal saline at a rate of 25 ~l/minute/100 gram body weight. The bladder was cannulated via a suprapubic incision with a PE-50 catheter. After 30 minutes of equilibration, renal function was measured during two consecutive 30 minute clearance periods. Carboxyl-14C-inulin, and glycyl-3H-PAH in plasma and
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urine were measured in a liquid scintillation counter (Nuclear ChicagoTM Analytical Inc., Elk Grove, IL). Clearances were calculated using standard formulas.
Urinary Protein Excretion Following the 24 hour urine collections, an aliquot of urine was removed for measurement of protein concentration using the Coomassie Brilliant Blue dye-binding assay (Bio-Rad). 33 Results for urinary protein excretion are expressed as rag/24 hours.
Eicosanoid Production by Renal Cortical Suspensions Following the renal h e m o d y n a m i c studies, mice were sacrificed and kidneys prepared for measurement of renal leukotriene and thromboxane production as well as for histopathologic examination as described previously. 2,6 For the eicosanoid measurements, the left kidney was rapidly removed, weighed, and placed in Kreb's buffer at 4°C. The kidney was bisected and a central slice obtained. Cortex was separated from medulla by macrodissection. Portions of cortex were uniformly minced with a razor blade and suspended in 2 ml of iced Kreb's buffer containing 5 ~M A23187. Because the cortical suspensions have significant ~/-glutamyl transpeptidase and dipeptidase activity, C-series leukotrienes were rapidly converted to D-series leukotrienes and then to E-series leukotrienes by ~-glutamyl transpeptidase and dipeptidase, respectively (32 and data not shown). Therefore, in order to measure peptidoleukotrienes, 20 m M Lcysteine was added to the preparation to inhibit enzymatic conversion of D-series leukotrienes to E-series leukotrienes. 32 This concentration of Lcysteine effectively inhibited conversion of LTD4 to LTE4 during the 30 minute incubation (data not shown). After preparation, suspensions were incubated for thirty minutes at 37°C in 95% 02. Samples were then centrifuged at 2000 rpm for 10 rain at 4°C and supernatants were stored at - 70°C until thromboxane and leukotrienes were measured as described below. The tissue pellet was resuspended in 0.5 ml of cold Kreb's buffer by sonication and the protein concentration was measured using the Coomassie Brilliant Blue dye-binding assay (Bio-Rad). 33
Leukotriene Production by Peritoneal Macrophages In some mice (SO: N = 6 and FO: N=6), resident peritoneal cells were harvested by lavage using an exudate pipet (Bellco Inc., Vineland, NJ) as previously described. 34 Cells were pelleted and resuspended in DMEM at a concentration of 1-5 × 106 cells/ml. This suspension was plated into 16 m m diameter culture wells and the cells incubated for 1 hour at 37°C in 95% 02 and 5% CO2. Non-adherent cells were removed by washing 334
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three times with Dulbeccos phosphate buffered saline (D-PBS)(Sigma, St. Louis, MO) and the remaining cells were incubated for 1 hour in DMEM with 30 ixM A231287. After incubation, the supernatant was removed and immediately frozen at -70°C for measurement of leukotrienes as described below. Because murine peritoneal macrophages have little ~/glutamyl transpeptidase activity (35 and data not shown), peptidoleukotriene production by macrophages results primarily in production of Cseries leukotrienes. Therefore, macrophage peptidoleukotriene production was quantitated by measuring either LTC4 or LTCs. The average yield of peritoneal macrophages was not significantly different between groups (5.3 + 0.8 [SO] vs 3.8 + 0.7 [FO] x 106/mouse).
Quantitation of Leukotrienes High Performance Liquid Chromatography Supernatants from incubations of renal cortex and peritoneal macrophages were separated by high performance liquid chromatography (HPLC) as previously described ~ prior to measurement of leukotrienes by RIA. Samples were acidified to pH 3.0 to 3.5, applied to C-18 columns preconditioned with methanol and water and then washed with water followed by a 10% solution of acetonitrile. The samples were eluted with a 70% solution of acetonitrile, evaporated to dryness under nitrogen, and then reconstituted in a 1:1 solution of acetonitrile and 2.5 mM trifluoroacetic acid. Samples and leukotriene standards (Advanced Magnetics Inc., Cambridge, Mass) were injected onto a Pecosphere HS3-C 18 cartridge (PerkinElmer Corp., Norwalk, Conn.) and eluted with a linear gradient from 100% 2.5 mM trifluoroacetic acid to 100% acetonitrile over 8 minutes at a flow rate of 3.0 ml/min. Elution of leukotrienes from the column was monitored by absorbance at wavelength 280 n m with a programmable multiwavelength UV spectrophotometer (Waters Associates, Milford, MA). All separations were performed at ambient temperature. Collected fractions were evaporated to dryness under nitrogen and reconstituted in appropriate buffer for quantitation of leukotrienes by RIA. Using this technique, recoveries ranged from 50-60% for B-series leukotrienes and 40-50% for peptidoleukotrienes.
Radioimmunoassays Concentrations of B-series leukotrienes were measured by radioimmunoassay (RIA)(Amersham, Arlington Hts., Ill.) using a polyclonal antibody that crossreacts with both LTB, and LTBs. Concentrations of peptidoleukotrienes were measured by RIA using a polyclonal antibody that crossreacts extensively with all the peptidoleukotrienes.36 For the LTB4 and LTB5
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assays, samples (measured in duplicate) and standards (measured in triplicate) were incubated with [3H]-LTB4and antisera at 25°C for 2 hours. For the peptidoleukotriene assays, samples or unlabeled standards (Cayman Chemical, Ann Arbor, MI) were incubated with [3H]-LTD4 (New England Nuclear, Boston, MA) and antisera for 4-6 hours at 4°C. After incubation, unbound eicosanoids were removed from the mixture with a suspension of dextran-coated activated charcoal. Sample concentrations were determined by a standard curve in which the logarithm of the concentration was plotted vs the logit of the B/Bo value. Results for renal cortical preparations were expressed as pg/30 min/mg protein and production rates for peritoneal macrophages were expressed as pg/30 min/106 cells. For the LTB4 and LTBs assays, the concentration of unlabeled leukotriene that inhibited 50% binding of leukotriene tracer was: 60_+4 pg/100 ~1 for LTB4, 65_+21 pg/100~l for LTBs. For the peptidoleukotriene assays, the concentration of unlabeled leukotriene that inhibited 50% binding of the leukotriene tracer was: 290 + 50 pg/100~l for LTC4, 254 -+32 pg/100pA for LTCs, 138 -+6 pg/100~l for LTD4, and 181 + 7 pg/100~l for LTDs. The lower limit of detectability was: 1.6 pg/100~l sample for LTB4 and LTBs in the LTB4 RIA, and 8 pg/100pA sample for LTC4, LTCs, LTD4, and LTDs in the peptidoleukotriene RIA. Cross reactivity of the LTB4 antibody with LTC4, LTCs, LTD4, and LTD5 was < 0.1%; and cross reactivity of the peptidoleukotriene antibody with LTB4 and LTBs was < 1%.
Quantitation of Thromboxane High Performance Liquid Chromatography Urinary thromboxane metabolites were extracted and separated by HPLC as described previously, s Samples were acidified to pH 3-3.5 and then applied to Sep-Pak C 18 columns (Waters Associates, Milford, MA) preconditioned with methanol and water. After washing the column with a 1:9 solution of acetonitrile and water, samples were eluted with ethyl acetate. Samples were evaporated to dryness under nitrogen and then reconstituted in a 1:3 solution of acetonitrile and 5.0 m M phosphoric acid. To separate thromboxane metabolites in urine, a Pecosphere HS3-C 18 cartridge (Perkin-Elmer Corp., Norwalk, CT) was used. Samples, unlabeled TxB2 standard (Advanced Magnetics Inc., Cambridge, MA), unlabeled 2,3-dinor-TxB~ standard (Cayman Chemical, Ann Arbor, MI) or [3H]-TxB2 standard (New England Nuclear, Boston, MA) were injected onto the column and eluted by a linear gradient from 100% 2.5 mM trifluoroacetic acid (Aldrich, Milwaukee, WI) to 100 % acetonitrile (Fisher Scientific, Fairlawn, NJ) over 10 minutes at a flow rate of 3.0 ml/min. Elution of thromboxane metabolites from the column was monitored by absorbance at a wavelength of 195 n m with a programmable multiwavelength ultraviolet spectrophotometer (Waters Associates, Milford, MA). 336
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All separations were performed at ambient temperature. Specific eluate fractions were collected based on the retention time of TxB2 and 2,3dinor-TxB2 standards. The TxB2 and 2,3-dinor-TxB2 fractions were dried under a stream of nitrogen and resuspended in radioimmunoassay (RIA) buffer. Concentrations of TxB2 and 2,3-dinor TxB2 were then quantitated by RIA as described below. Recoveries of TxB2 and 2,3-dinor TxB2 ranged from 60-80%.
Radioimmunoassays Immunoreactive thromboxane B~ (TxB2) was measured in cortical suspensions by direct RIA using an antibody that crossreacts extensively with thromboxane metabolites (Seragen Inc., Boston, MA). Urinary TxB2 and urinary 2,3-dinor TxB~ was quantitated by RIA after HPLC separation. For measurement of thromboxane metabolites, either TxB2 standard or 2,3-dinor-TxB2 standard (measured in triplicate) or unknowns (measured in duplicate) were incubated with a mixture of antisera and [3H]-TxB~ overnight at 4°C. After incubation, free eicosanoid was adsorbed with dextran-coated charcoal. Sample concentrations were determined by a standard curve in which the logarithm of the concentration was plotted against the logit of the B/Bo value. The results for urinary TxB2 are expressed as pg/24 hours. For renal cortical homogenates, results are expressed as pg/30 min/mg protein.
Histologic Studies Following the renal hemodynamic studies, the right kidney was removed and placed in 10% buffered formalin. After formalin fixation, kidney sections were stained with hematoxylin and eosin and examined by light microscopy. The slides were reviewed by a pathologist (PR) blinded to treatment group. At least 20 glomeruli were evaluated in each animal. The severity of glomerular crescent formation, hyperlobulation, hypercellularity, and necrosis were each graded separately using a semiquantitative scale where 0 was normal, and 1 +, 2 +, 3 +, and 4 + represented mild, moderate, moderately-severe, and severe abnormalities, respectively. An overall glomerular histopathologic score for each animal was obtained by summing the grades for each individual glomerular abnormality (maxim u m score 16). To investigate the effects of FO feeding on inflammatory cell infiltration in the kidney, the number of polymorphonuclear cells in glomeruli were counted and the severity of interstitial inflammatory cell infiltrates was assessed using a semiquantitative scale from zero to 4 + as described above. To quantitate the number of polymorphonuclear cells in glomeruli, additional sections were stained with PAS. For each animal, the number
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of glomerular polymorphonuclear cells per glomerulus was obtained by averaging the number of cells in 20 randomly selected glomeruli.
Statistical Analysis Data are presented as the mean + standard error of the mean. For the hemodynamic studies, data points for each animal represent the mean of the values measured during two clearance periods. For comparisons between two groups, statistical significance was assessed using an unpaired t-test or Wilcox rank sum as appropriate.
Results Dietary supplementation with FO improved renal hemodynamic function in MRL-lpr/Ipr mice as shown in Table 1. GFR was significantly higher in mice given FO compared with SO controls. Similarly, RPF tended to be higher in FO fed mice compared to control animals. This improvement in renal hemodynamic function in the FO group was associated with a reduction in the severity of the glomerular histomorphologic abnormalities as reflected by the significant decrease in the glomerular histopathologic score (Table 1). Specifically, glomerular hypercellularity and hyperlobulation tended to be reduced in FO fed mice and glomerular crescents were seen exclusively in mice given SO. This improvement in glomerular histomorphology was accompanied by a significant decrease in the number of polymorphonuclear cells in glomeruli of mice receiving FO compared to SO controls (2.6 + 0.5 [SO] vs 1.3 + 0.2 [FO] polymorphonuclear cells per glomerulus; P<0.025). In contrast, the severity of the interstitial inflammatory cell infiltrates was similar in both groups of MRL-lpr/lpr mice (semiquantitative score 1.6 + 0.6 [FO] vs 1.7 ___0.6 [SO]; P= NS). Urinary protein excretion also tended to be reduced in FO fed mice (Table 1) but this difference in proteinuria did not reach statistical significance. To investigate the effect of FO feeding on leukotriene production in MRL-Ipr/Ipr mice, 5-LO metabolites in supernatants from macrophages TABLE 1.
Effect of FO feeding on nephritis in
MRL-Ipr/Iprmice FO
C,N (ml/min/kg) CpAH (ml/min/kg) Glomerular histopathologic score Proteinuria (mg/24 hours)
11.1 33.6 2.3 9.0
_+ 0.8* _+ 4.2 +_ 0.4t +_ 0.9
SO 6.6 26.9 4.3 22
_+ 2.9 _+ 2.9 _+ 0.4 _+ 8.9
*P < 0.005 or t P < 0.05 vs SO.
338
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and kidney preparations were separated by HPLC and concentrations of immunoreactive leukotrienes in specific eluate fractions were determined by RIA. Elution of leukotrienes from the column was monitored at a wavelength of 280 nm and column eluate fractions were collected based on the appearance of absorbance peaks for leukotriene standards. As shown in panel A of Figure 1, supernatants from macrophages of mice given SO
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Time (min) FIGURE 1. HPLC separation of leukotrienes from peritoneal macrophages of MRL-Ipr/Ipr mice. Peritoneal macrophages were stimulated with the calcium ionophore A2.3187 (30p,M) and leukotrienes in supernatants were separated by HPLC as described in the Methods Section. Elution of leukotrienes from the column was monitored by absorbance at a wavelength of 280 nm. As shown in panel A, an absorbance peak coeluting with LTC4standard was detected in supernatants from macrophages of mice given SO. As shown in panel B, this LTC, peak was reduced in mice given FO, but a second peak coeluting with LTCs standard was detected. This LTCs peak was never seen in supematants from macrophages of SO fed mice. Absorbance peaks coeluting with either LTB4 or LTB5 standards were not detected in either group of MRL-Ipr/Ipr mice.
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Fish oil feeding in murine lupus:
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contained an absorbance peak that eluted at the same time as LTC4 standard. In mice receiving FO, this LTC4 peak was reduced, and a second peak coeluting with LTCs standard was detected (Figure 1, panel B). This LTCs peak was never seen in supernatants from macrophages of mice given SO. Quantitation of immunoreactive leukotrienes in specific eluate fractions revealed differences in the pattern of leukotriene production by macrophages and renal cortices from FO fed mice compared to SO controls. As shown in Figure 2 and Table 2, dietary supplementation with FO significantly reduced macrophage production of both LTC4 and LTB4 as measured by RIA. This decrease in macrophage production of tetraene
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FIGURE2. Leukotriene production by peritoneal macrophages. Production of both B-series leukotrienes (panel A) and C-series leukotrienes (panel B) were measured by RIA after HPLC separation of 5-1ipoxygenase metabolites as described in the Methods Section. FO feeding reduced macrophage production of LTB4 and LTC4 compared to SO controls. Reduced production of tetraene leukotrienes was associated with enhanced production of pentaene leukotrienes. There was a significant increase in LTCs production by peritoneal macrophages from mice fed FO compared to control animals. (*P < 0.025 vs SO, **P < 0.05 vs SO)
340
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TABLE 2. Effect of dietary supplementation with FO on production of leukotrienes by Ipr macrophages
MRL-Ipr/
Leukotrienes (pg/30 min/106 cells)
LTB4 LTC. LTBs LTCs
FO
SO
9.2 -+ 0.5* 345 _+ 48* trace 240 _+ 421-
20.8 _+ 4.6 777 _+ 162 trace 42 _+ 12
*P < 0.025 or I P < 0.005 vs Safflower oil
leukotrienes was accompanied by enhanced production of pentaene peptidoleukotrienes by macrophages from FO fed mice compared to control animals (Figure 2 and Table 2). Renal production of LTB4 followed a similar pattern. As shown in Figure 3, renal production of LTB4 was significantly decreased by dietary supplementation with FO (26_+ 18 [FO] vs 151 +46 [SO] pg/30 m i n / m g protein; P<0.025). In contrast, renal production of peptidoleukotrienes was not affected by FO feeding. Similar amounts of LTD4 were produced by renal cortices from both groups of MRL-lpr/lpr mice (255 + 29 [FO] vs 297_+39 [SO] pg/30 rain/rag protein; P = NS). Production of pentaene peptidoleukotrienes or LTBs were not detected in kidney preparations from either group. To assess the relative magnitude of the effect of FO feeding on production of cyclooxygenase compared with 5-LO metabolites, we also measured thromboxane production in mice receiving FO and SO controls. As reported previously by Kelley et al, ~8 FO feeding reduced production of TxB2 by suspensions of renal cortices (3060 _+300 [SO] to 1230___270 [FO] pg/30 min/mg protein; P<0.005). To investigate the effects of FO feeding on thromboxane production in vivo, we also measured urinary excretion of thromboxane metabolites. In these studies, FO feeding reduced urinary excretion of TxB~ excretion from 708+144 to 390+47 pg/24 hours (P<0.05). Similarly, urinary excretion of 2,3-dinor-TxB2 was reduced from 14,040 to 2,190 pg/24 hours (P<0.005).
Discussion These studies demonstrate that amelioration of nephritis in MRL-lpr/lpr mice by FO feeding is associated with reduced production of tetraene leukotrienes by both macrophages and kidneys from MRL-Ipr/Ipr mice. Several mechanisms may contribute to reduced production of tetraene leukotrienes in mice given FO. For example, EPA inhibits metabolism of
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FIGURE 3. LTB4 production by renal cortical preparations from MRL-Ipr/Ipr mice. Production of LTB4 by renal cortices was measured by RIA after HPLC separation of 5-LO metabolites as described in the Methods Section. There was a significant decrease in renal cortical LTB4 production in mice given FO compared to SO controls. (*P < 0.025 vs SO)
AA by enzymes of both the cyclooxygenase and 5-LO pathways in a variety of cell t y p e s . 19,2°,21,26,~8 Reduced tetraene leukotriene production may result from direct enzyme inhibition as in the case of leukotriene A hydrolase, 3° or competition between EPA and AA as substrates for 5-LO.~9.28Moreover, FO might indirectly inhibit tetraene leukotriene production by reducing production of other inflammatory mediators which, in turn, might affect eicosanoid production rates. In this regard, FO inhibits production of TxA2, 31 platelet activating factor (PAF)7a interleukin-1 (IL-I), 37 and tumor necrosis factor 37 in vitro. Each of these inflammatory mediators has been implicated in the pathogenesis of lupus nephritis, 3~,38,39and both PAF4° and IL-135 directly stimulate leukotriene production by mononuclear inflammatory cells. FO feeding significantly reduced production of LTB4 by both macrophages and kidneys from MRL-lpr/lpr mice. In contrast, while FO feeding reduced macrophage production of peptidoleukotrienes, it did not affect peptidoleukotriene production by the kidney. This difference may reflect 342
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the heterogeneous cell population contributing to renal eicosanoid production. In addition to macrophages, a number of other inflammatory cells are capable of leukotriene synthesis 4~and may be differently affected by a diet enriched with FO. In human neutrophils, FO feeding reduces production of LTB4 but has no significant effect on neutrophil production of peptidoleukotrienes. 26 EPA and its metabolites may also have different effects on the enzymes involved in eicosanoid synthesis. EPA is a potent inhibitor of cyclooxygenase3~ and the EPA metabolite leukotriene As (LTAs) inhibits leukotriene A hydrolaseY Inhibition of cyclooxygenase may cause shunting of both AA and EPA through the 5-LO pathway resulting in enhanced synthesis of LTA4 and LTAs. Because LTAs is both a substrate and inhibitor of leukotriene A hydrolase, 3°this shunting mechanism may favor production of peptidoleukotrienes. A diet enriched with FO might therefore inhibit production of LTB4and cyclooxygenase metabolites without significantly altering production of peptidoleukotrienes. Our findings in this study are consistent with this model. In MRL-lpr/Ipr mice, enhanced production of tetraene peptidoleukotrienes contributes to renal hemodynamic impairment. 2 Nevertheless, GFR in mice given FO was significantly higher than SO controls despite similar rates of tetraene peptidoleukotriene production by kidneys from both groups of MRL-Ipr/Ipr mice. There are several potential explanations for this apparent discrepancy. First, multiple factors probably contribute to renal hemodynamic impairment in murine lupus. For example, prevention of glomerular injury by FO feeding may maintain GFR at levels comparable to values for normal controls 6 despite the adverse effects of peptidoleukotrienes on other aspects of renal hemodynamic such as RPF. The inability of dietary supplementation with FO to reduce renal peptidoleukotriene production might therefore account for the relatively modest effects of FO feeding on RPF in the present study. Secondly, measurements of renal leukotriene production in vitro may not reflect the inhibitory effect of FO feeding on peptidoleukotriene production in vivo. Indeed, the highly stimulated conditions in our renal homogenate system may define the capacity for renal eicosanoid synthesis rather than the rate of eicosanoid production in vivo. 6 Thirdly, peptidoleukotrienes may affect renal hemodynamics by enhancing production of other inflammatory mediators which, in turn, may be inhibited by FO feeding. In this regard, LTC4 stimulates production of the potent vasoconstrictor eicosanoid TxA2 in rat peritoneal macrophages, 42and renal vasoconstriction induced by LTC4 can be blocked by specific thromboxane synthase inhibitors. 43 These observations are of particular relevance to renal disease in SLE because TxA2 is an important mediator of renal hemodynamic impairment in both human and murine lupus nephritis. S,6 To determine if renal thromboxane production was affected by FO feeding in the present study, we measured both urinary excretion of throm-
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boxane metabolites and production of TxB2 by renal cortical preparations. As reported previously by Kelley et al, '8 dietary supplementation with FO decreased renal thromboxane production in vitro. In addition, we found that FO feeding significantly reduced urinary excretion of thromboxane metabolites compared to SO controls. Excretion of unmetabolized, native TxB2 in urine is thought to reflect thromboxane production by the kidney whereas, urinary 2,3-dinor-TxB~ may reflect extrarenal thromboxane production largely derived from platelets.44 Taken together, these data suggest that FO feeding inhibits both renal and extrarenal thromboxane production in vivo. Furthermore, because TxA2 mediates renal hemodynamic impairment in murine lupus, s this reduction in renal thromboxane production may contribute to the beneficial effects of FO feeding on renal hemodynamics in MRL-lpr/lpr mice. Essential fatty acid deficiency (EFAD) has also been shown to inhibit renal LTB4 production and ameliorate nephritis in several animal models of immune-mediated kidney disease 4s,46 including murine lupus. 47 It has been suggested that EFAD decreases renal leukotriene production by reducing the number of renal inflammatory cells. 4s In contrast, dietary supplementation with FO may decrease leukotriene production without significantly affecting influx of inflammatory cell to sites of inflammation. 48 To determine if FO feeding affected renal inflammation in the present study, we assessed the number of polymorphonuclear cells in glomeruli and the severity of interstitial inflammatory cell infiltrates. Are found that FO feeding caused a statistically significant decrease in the number of glomerular polymorphonuclear cells. This decrease in glomerular polymorphonuclear cells might be a consequence of reduced renal production of the potent chemotactic agent LTB4as has been suggested in EFAD. 49 Alternatively, polymorphonuclear cells have a great capacity for production of LTB4 and it is possible that this decrease in glomerular polymorphonuclear cells contributed to the reduction in renal production of LTB4 we observed in the present study. In summary, FO feeding was associated with significant alterations in production of 5-LO metabolites by both macrophages and kidneys from MRL-lpr/lpr mice. Dietary supplementation with FO inhibited production of tetraene leukotrienes and promoted the synthesis of pentaene leukotrienes. We speculate that these alterations in 5-LO metabolism may contribute to the beneficial effects of FO feeding on renal disease in MRLlpr/lpr mice. Furthermore, understanding the mechanisms of action of FO in murine lupus may suggest novel treatments for nephritis in patients with SLE.
Acknowledgments A preliminary version of this report has been published in abstract form (J. Am. Soc. Nephrol., 1:449, 1990). 344
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These studies were supported by grants from the Research Service of the Veterans Administration. Dr. Spurney performed these studies as a fellow of the National Kidney Foundation. The authors wish to t h a n k Pat Flannery for his expert technical assistance, and N o r m a Turner for her secretarial assistance in preparing the manuscript.
References 1. Theofilopoulos, A.N., and F.J. Dixon. Murine models of systemic lupus erythematosus. Adv Immunol 37:269, 1985. 2. Spurney, R.F., P. Ruiz, D.S. Pisetsky, and T.M. Coffman. Enhanced renal leukotriene production in murine lupus: role of lipoxygenase metabolites. Kidney Int 39:95, 1991. 3. Kelley, V.E., S. Sneve, and S. Musinski. Increased renal thromboxane production in murine lupus nephritis. J Clin Invest 77:252, 1986. 4. Patrono, C., G. Ciabattoni, G. Remuzzi, E. Gotti, S. Bombardieri, O. DiMunno, G. Tartarelli, G.A. Cinotti, B.M. Simonetti, and A. Pierucci. Functional significance of renal prostacyclin and thromboxane A2 production in patients with systemic lupus erythematosus. J Clin Invest 76:1011, 1985. 5. Pierucci, A., B.M. Simonetti, G. Pecci, G. Mavrikakis, S. Feriozzi, G.A. Cinotti, P. Patrignani, G. Ciabattoni, and C. Patrono. Improvement of renal function with selective thromboxane antagonism in lupus nephritis. N Engl J Med 329:421, 1989. 6. Spurney, R.F., R.J. Bernstein, P. Ruiz, P.E. Klotman, D.S. Pisetsky, and T.M. Coffman. Physiologic role for enhanced renal thromboxane production in murine lupus nephritis. Prostaglandins 42:15, 1991. 7. Dahlen, S.E., J. Bjork, P. Hedqvist, K.E. Afors, S. Hammarstrom, J.A. Lindgren, and B. Samuelsson. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory response. Proc Natl Acad Sci (USA) 78:3887, 1981. 8. Ford-Hutchinson, A.W., M.A. Gray, M.V. Doig, M.E. Shipley, and M.J.H. Smith. Leukotriene B4, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286:264, 1980. 9. Showell, H.J., P.H. Naccache, P. Borgeat, S. Picard, P. Vallerand, E.L. Becker, and R.I. Sha'afi. Characterization of the secretory activity of leukotriene B4 toward rabbit neutrophils. J Immunol 128:811, 1982. 10. Badr, K.F., C. Baylis, J.M. Pfeffer, M.A. Pfeffer, RJ. Soberman, R.A. Lewis, K.F. Austen, E.J. Corey, and B.M. Brenner. Renal and systemic hemodynamic responses to intravenous infusion of leukotriene C4 in the rat. Circ Res 54:492, 1984. 11. Badr, K.F., B.M. Brenner, and I. Ichikawa. Effects of leukotriene D 4 o n glomerular dynamics in the rat. Am J Physiol 253:F239, 1987. 12. Simonson, M.S., and M.J. Dunn. Leukotriene C4 and D4 contract rat glomerular mesangial cells in culture. Kidney Int 30:524, 1986. 13. Badr, K.F., S. Mong, R.L. Hoover, M. Schwartzberg, J. Ebert, H.R. Jacobson, and R.C. Harris. Leukotriene D~ binding and signal transduction in rat glomerular mesangial cells. Am J Physiol 257:F280, 1989.
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14. Balow, J.E., H.A. Austin III, G.C. Tsokos, T.T. Antonovych, A.D. Steinberg, and J.H. Klippel. Lupus nephritis. Ann Int Med 106:79, 1987. 15. Robinson, D.R., J.D. Prickett, R. Polisson, A.D. Steinberg, and L. Levine. The protective effect of dietary fish oil on murine lupus. Prostaglandins 30:51, 1985. 16. Prickett, J.D., D.R. Robinson, and A.D. Steinberg. Dietary enrichment with the polyunsaturated fatty acid eicosapentaenoic acid prevents proteinuria and prolongs survival in NZB x NZW F, mice. J Clin Invest 68:556, 1981. 17. Prickett, J.D., D.R. Robinson, and A.D. Steinberg. Effects of dietary enrichment with eicosapentaenoic acid upon autoimmune nephritis in female NZB x NZW/G mice. Arthritis Rheum 26:133, 1983. 18. Kelley, V.E., A. Ferretti, S. Izui, and T.B. Strom. A fish oil diet rich in eicosapentaenoic acid reduces cyclooxygenase metabolites, and suppresses lupus in MRL-lpr mice. J Immunol 134:1914, 1985. 19. Spector, A.A., Kaduce, T.L., P.H. Figard, K.C. Norton, J.C. Hoak, and R.L. Czervionke. Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells. J Lipid Res 24:1595, 1983. 20. Levine, L., and N. Worth. Eicosapentaenoic acid: its effects on arachidonic acid metabolism by cells in culture. J Allergy Clin Immunol 74:430, 1984. 21. Scharschmidt, L.A., N.B. Gibbons, and R. Neuwirth. Eicosapentaenoic and dihomo?linolenic acid metabolism by cultured rat mesangial cells. Am J Physiol 25:C101, 1989. 22. Knapp, H.R., I.A.G. Reilly, P. Alessandrini, and G.A. FitzGerald. In vivo indexes of platelet and vascular function during fish-oil administration in patients with atherosclerosis. N Engl J Med 314:937, 1986. 23. Sperling, R.I., J.-L. Robin, K.A. Kylander, T.H. Lee, R.A. Lewis, and K.F. Austen. The effects of N-3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes. J Immuno1139:4186, 1987. 24. Lee, T.H., J.-M. Mencia-Huerta, C. Shih, E.J. Corey, R.A. Lewis, and K.F. Austen. Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene Bs and the double lipoxygenase product. J Biol Chem 259:2383, 1984. 25. Strasser, T., S. Fischer, and P.C. Weber. Leukotriene Bs is formed in human neutrophils after dietary supplementation with eicosapentaenoic acid. Proc Nat1 Acad Sci 82:1540, 1985. 26. Lee, T.H., R.L. Hoover, J.D. Williams, R.I. Sperling, J. Ravalese III, B.W. Spur, D.R. Robinson, E.J. Corey, R.A. Lewis, and K.F. Austen. Effect of dietary enrichment with eicosapentaenoic and docosahexanenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function. N Engl J Med 312:1217, 1985. 27. Leitch, A.G., T.H. Lee, E.W. Ringel, J.D. Prickett, D.R. Robinson, S.G. Pyne, E.J. Corey, J.M. Drazen, K.F. Austen, and R.A. Lewis. Immunologically induced generation of tetraene and pentaene leukotrienes in the peritoneal cavities of menhaden-fed rats. J Immunol 132:2559, 1984. 28. Broughton, K.S., J. Whelan, I. Hardarbottir, and J.E. Kinsella. Effect of increasing the dietary (n-3) to (n-6) polyunsaturated fatty acid ratio on murine liver and peritoneal cell fatty acids and eicosanoid formation. J Nutr 12I :155, 1990.
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Clark, W.E, A. Parbtani, M.W. Huff, B. Reid, B.J. Holub, and P. Falardeau. Omega-3 fatty acid dietary supplementation in systemic lupus erythematosus. Kidney Int 36:653, 1989. Nathaniel, D.J., J.F. Evans, Y. Leblanc, C. Leveille, B.J. Fitzsimmons, and A.W. Ford-Hutchinson. Leukotriene As is a substrate and an inhibitor of rat and human neutrophil LTA4hydrolase. Biochem Biophys Re Comm 13I:827, 1985. Needleman, P., A. Raz, M.S. Minkes, J.A. Ferrendelli, and H. Sprecher. Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties. Proc Natl Acad Sci 76:944, 1979. Sraer, J., M. Bens, J. Oudinet, and R. Ardaillou. Bioconversion of leukotriene C4 by rat glomeruli and papilla. Prostaglandins 5:909, 1986. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem 72:248, 1976. Dan-vu, A.P., D.S. Pisetsky DE, and J.B.Weinberg. Functional alterations of macrophages in autoimmune MRL-lpr/lpr mice. J Immunol 138:1757, 1987. Censini, S., M. Bartalini, A. Tagliabue, and D. Boraschi. Interleukin-1 stimulates production of LTC4 and other eicosanoids by macrophages. Lymphokine Res 8:107, 1989. Spurney, R.F., S. Ibrahim, D. Butterly, F. Sanfilippo, P.E. Klotman, and T.M. Coffman. Leukotrienes in renal transplant rejection: Distinct roles for leukotriene B4 and peptidoleukotrienes in the pathogenesis of allograft injury. J Immunol I52:867, 1994. Endres, S., R. Ghorbani, V.E. Kelley, K. Georgilis, G. Lonnemann, J.W.M. van der Meer, J.G. Cannon, T.S. Rogers, M.S. Klempner, P.C. Weber, E.J. Schaefer, S.M. Wolff, and C.A. Dinarello. The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med 320:265, 1989. Baldi, E., S.N. Emancipator, M.O. Hassan, and M.J. Dunn. Platelet activating factor receptor blockade ameliorates murine systemic lupus erythematosus. Kidney Int 38:1030, 1990. Brennan, D.C., J.A. Yui, R.P. Wulthrich, and V.E. Kelley. Tumor necrosis factor and IL-1 in New Zealand Black/White mice. J Immunol 143:3470, 1989. Fauler, J., B. Sielhorst, and J.C. Frolich. Platelet-activating factor induces the production of leukotrienes by human monocytes. Biochimica Biophysica Acta 1013:80, 1989. Lewis, R.A., and R. Austen. The biologically active leukotrienes. J Clin Invest 73: 889, 1984. Feuerstein, N., M. Foegh, and P.W. Ramwell. Leukotrienes C4 and D4 induce prostaglandin and thromboxane release from rat peritoneal macrophages. Br J Pharm 72:389, 1981. Albrightson, C.R., A.S. Evers, A.C. Griffin, and P. Needleman. Effect of endogenously produced leukotrienes and thromboxane on renal vascular resistance in rabbit hydronephrosis. Circ Res 61:514, 1987. Catella, F., J. Nowak, and G.A. Fitzgerald. Measurement of renal and nonrenal eicosanoid synthesis. Am J Med 81:23, 1986.
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8chreiner, G.F., B. Rovin, and J.B. Lefkowith. The antiinflammatory effects of essential fatty acid deficiency in experimental glomerulonephritis. J Immunol 143:3192, 1989. Harris, K.P.G., J.B. Lefkowith, 8. Klahr, and G.F. Schreiner. Essential fatty acid deficiency ameliorates acute renal dysfunction in the rat after the administration of the aminonucleoside of puromycin. J Clin Invest 86:1115, 1990. Hurd, E.R., J.M. Johnston, J.R. Okita, and P.C. MacDonald. Prevention of glomerulonephritis and prolonged survival in New Zealand black/New Zealand white F hybrid mice fed an essential fatty acid-deficient diet. J Clin Invest 67:476, 1981. Lefkowith, J.B., A. Morrison, V. Lee, and M. Rogers. Manipulation of the acute inflammatory response by dietary polyunsaturated fatty acid modulation. J Immunol 145:1523, 1990. Lefkowith, J.B. Essential fatty acid deficiency inhibits the in vivo generation of leukotriene B4 and suppresses levels of resident and elicited leukocytes in acute inflammation. J Immunol 140:228, 1988.
Editor: W. Lands
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Accepted: 9-19-94
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0 1994 Butterworth-Heinemann
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Keywords: Leukotriene; eicosapentaenoic acid; fish oil; lupus nephritis; eicosanoid
Introduction
MRL-lpr/Ipr mice spontaneously develop an autoimmune disease with nephritis which is similar to human systemic lupus erythematosus (SLE).' In these animals, the development of renal disease is associated with enhanced production of both cyclooxygenase and 5-1ipoxygenase (5-LO) metabolites by the kidney. 2,3 While an important pathogenic role for cyclooxygenase metabolites has been clearly documented in murine lupus 3,6and in human SLE,4,s recent studies suggest that enhanced production of leukotrienes may also contribute to autoimmune nephritis. 2 In vitro, LTB4 promotes adhesion of leukocytes to endothelial cells, 7 is a potent chemotactic agent, 8 and stimulates aggregation, enzyme release, and generation of reactive oxygen intermediates in neutrophils. 9 The peptidoleukotrienes are potent renal vasoconstrictors/0, H and cause mesangial cell contraction '2 and proliferation. ~3Within the kidney, enhanced leukotriene production could therefore promote renal inflammation and injury by a variety of direct and indirect mechanisms. Dietary supplementation with FO ameliorates renal disease and prolongs survival in murine lupus nephritis. ~4-~8FO is rich in the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which may have profound effects on metabolism of endogenous lipids '8-~ including leukotrienes. 24~ For example, EPA is metabolized by 5-LO to LTBs.24,2s This compound has reduced biologic activity compared to the arachidonic acid (AA) metabolite, LTB4.24,2s Moreover, EPA acts as competitive antagonist of AA for both the cyclooxygenase and 5-LO pathways; ~6-~ thus, EPA reduces the production of AA metabolites in a variety of cells,~9,~°including glomerular mesangial cells. ~ These results suggest that diets enriched with fish oil might alter the profile of lipid mediators produced in murine lupus nephritis. Indeed, when renal disease is prevented in MRL-Ipr/lpr mice by FO feeding, production of cyclooxygenase metabolites by the kidney is reduced. TM However, the effect of FO feeding on leukotriene production in murine lupus has not been previously investigated. To further investigate the effects of FO feeding on eicosanoid production in murine lupus, we examined the effects of dietary supplementation with FO on production of leukotrienes by macrophages and kidneys from MRL-lpr/lpr mice. Our findings suggest that FO feeding reduces production of tetraene leukotrienes and enhances production pentaene leukotrienes in murine lupus. We speculate that these changes in leukotriene production may contribute to the beneficial effects of FO feeding in autoi m m u n e nephritis. 332
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Materials and Methods Animals MRL-lpr/lpr were originally obtained from the Jackson Laboratory (Bar Harbor, ME). These animals were subsequently maintained and bred in the animal facility of the Durham VA Medical Center. All animals were given tap water ad libitum and received standard laboratory chow until 12 weeks of age when mice were randomized to the experimental diets as described below.
Experimental Protocol Beginning at 12 weeks of age, MRL-lpr/Ipr mice were randomized to diets that differed only in the composition of fat. FO fed mice (N = 16) received a diet enriched with 20% menhaden oil and the control group (N = 18) received a 20% SO diet (ICN Biochemical, Cleveland, OH). At 20 weeks of age, mice were placed in metabolic cages and urine collected for 24 hours. The urine was chilled throughout the collection period and then stored at -70°C until urinary eicosanoids and proteinuria were quantitated as described below. Following the urine collections, clearances of inulin (CIN)and PAH (CpAH)were measured. After the renal hemodynamic studies, mice were sacrificed and kidneys were removed for histologic study and for measurement of eicosanoid production as described below.
Renal Hemodynamic Studies C~N and Cp~ were measured as previously described (2) to determine the glomerular filtration rate (GFR) and renal plasma flow (RPF), respectively. On the day of study, animals were anesthetized with 0.04 rag/gram pentobarbital, and a polyethylene catheter (PE 90) was inserted into the trachea to facilitate spontaneous ventilation. The left carotid artery and left jugular vein were cannulated with polyethylene catheters (PE-10) for intravenous infusions, to monitor mean arterial pressure (MAP) using a Gould-Statham strain gauge, and to allow intermittent sampling of arterial blood. Following surgery, normal saline (2.0% of the body weight) was infused intravenously over 20 minutes to replace fluid lost during surgery. A priming dose of carboxyl-14C-inulin (5~Ci/kg) and glycyl-3HPAH (10~Ci/kg) was given, followed by infusion of carboxyl-~4C-inulin (0.8~zCi/ml) and glycyl-3H-PAH (4~Ci/ml) in normal saline at a rate of 25 ~l/minute/100 gram body weight. The bladder was cannulated via a suprapubic incision with a PE-50 catheter. After 30 minutes of equilibration, renal function was measured during two consecutive 30 minute clearance periods. Carboxyl-14C-inulin, and glycyl-3H-PAH in plasma and
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urine were measured in a liquid scintillation counter (Nuclear ChicagoTM Analytical Inc., Elk Grove, IL). Clearances were calculated using standard formulas.
Urinary Protein Excretion Following the 24 hour urine collections, an aliquot of urine was removed for measurement of protein concentration using the Coomassie Brilliant Blue dye-binding assay (Bio-Rad). 33 Results for urinary protein excretion are expressed as rag/24 hours.
Eicosanoid Production by Renal Cortical Suspensions Following the renal h e m o d y n a m i c studies, mice were sacrificed and kidneys prepared for measurement of renal leukotriene and thromboxane production as well as for histopathologic examination as described previously. 2,6 For the eicosanoid measurements, the left kidney was rapidly removed, weighed, and placed in Kreb's buffer at 4°C. The kidney was bisected and a central slice obtained. Cortex was separated from medulla by macrodissection. Portions of cortex were uniformly minced with a razor blade and suspended in 2 ml of iced Kreb's buffer containing 5 ~M A23187. Because the cortical suspensions have significant ~/-glutamyl transpeptidase and dipeptidase activity, C-series leukotrienes were rapidly converted to D-series leukotrienes and then to E-series leukotrienes by ~-glutamyl transpeptidase and dipeptidase, respectively (32 and data not shown). Therefore, in order to measure peptidoleukotrienes, 20 m M Lcysteine was added to the preparation to inhibit enzymatic conversion of D-series leukotrienes to E-series leukotrienes. 32 This concentration of Lcysteine effectively inhibited conversion of LTD4 to LTE4 during the 30 minute incubation (data not shown). After preparation, suspensions were incubated for thirty minutes at 37°C in 95% 02. Samples were then centrifuged at 2000 rpm for 10 rain at 4°C and supernatants were stored at - 70°C until thromboxane and leukotrienes were measured as described below. The tissue pellet was resuspended in 0.5 ml of cold Kreb's buffer by sonication and the protein concentration was measured using the Coomassie Brilliant Blue dye-binding assay (Bio-Rad). 33
Leukotriene Production by Peritoneal Macrophages In some mice (SO: N = 6 and FO: N=6), resident peritoneal cells were harvested by lavage using an exudate pipet (Bellco Inc., Vineland, NJ) as previously described. 34 Cells were pelleted and resuspended in DMEM at a concentration of 1-5 × 106 cells/ml. This suspension was plated into 16 m m diameter culture wells and the cells incubated for 1 hour at 37°C in 95% 02 and 5% CO2. Non-adherent cells were removed by washing 334
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three times with Dulbeccos phosphate buffered saline (D-PBS)(Sigma, St. Louis, MO) and the remaining cells were incubated for 1 hour in DMEM with 30 ixM A231287. After incubation, the supernatant was removed and immediately frozen at -70°C for measurement of leukotrienes as described below. Because murine peritoneal macrophages have little ~/glutamyl transpeptidase activity (35 and data not shown), peptidoleukotriene production by macrophages results primarily in production of Cseries leukotrienes. Therefore, macrophage peptidoleukotriene production was quantitated by measuring either LTC4 or LTCs. The average yield of peritoneal macrophages was not significantly different between groups (5.3 + 0.8 [SO] vs 3.8 + 0.7 [FO] x 106/mouse).
Quantitation of Leukotrienes High Performance Liquid Chromatography Supernatants from incubations of renal cortex and peritoneal macrophages were separated by high performance liquid chromatography (HPLC) as previously described ~ prior to measurement of leukotrienes by RIA. Samples were acidified to pH 3.0 to 3.5, applied to C-18 columns preconditioned with methanol and water and then washed with water followed by a 10% solution of acetonitrile. The samples were eluted with a 70% solution of acetonitrile, evaporated to dryness under nitrogen, and then reconstituted in a 1:1 solution of acetonitrile and 2.5 mM trifluoroacetic acid. Samples and leukotriene standards (Advanced Magnetics Inc., Cambridge, Mass) were injected onto a Pecosphere HS3-C 18 cartridge (PerkinElmer Corp., Norwalk, Conn.) and eluted with a linear gradient from 100% 2.5 mM trifluoroacetic acid to 100% acetonitrile over 8 minutes at a flow rate of 3.0 ml/min. Elution of leukotrienes from the column was monitored by absorbance at wavelength 280 n m with a programmable multiwavelength UV spectrophotometer (Waters Associates, Milford, MA). All separations were performed at ambient temperature. Collected fractions were evaporated to dryness under nitrogen and reconstituted in appropriate buffer for quantitation of leukotrienes by RIA. Using this technique, recoveries ranged from 50-60% for B-series leukotrienes and 40-50% for peptidoleukotrienes.
Radioimmunoassays Concentrations of B-series leukotrienes were measured by radioimmunoassay (RIA)(Amersham, Arlington Hts., Ill.) using a polyclonal antibody that crossreacts with both LTB, and LTBs. Concentrations of peptidoleukotrienes were measured by RIA using a polyclonal antibody that crossreacts extensively with all the peptidoleukotrienes.36 For the LTB4 and LTB5
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assays, samples (measured in duplicate) and standards (measured in triplicate) were incubated with [3H]-LTB4and antisera at 25°C for 2 hours. For the peptidoleukotriene assays, samples or unlabeled standards (Cayman Chemical, Ann Arbor, MI) were incubated with [3H]-LTD4 (New England Nuclear, Boston, MA) and antisera for 4-6 hours at 4°C. After incubation, unbound eicosanoids were removed from the mixture with a suspension of dextran-coated activated charcoal. Sample concentrations were determined by a standard curve in which the logarithm of the concentration was plotted vs the logit of the B/Bo value. Results for renal cortical preparations were expressed as pg/30 min/mg protein and production rates for peritoneal macrophages were expressed as pg/30 min/106 cells. For the LTB4 and LTBs assays, the concentration of unlabeled leukotriene that inhibited 50% binding of leukotriene tracer was: 60_+4 pg/100 ~1 for LTB4, 65_+21 pg/100~l for LTBs. For the peptidoleukotriene assays, the concentration of unlabeled leukotriene that inhibited 50% binding of the leukotriene tracer was: 290 + 50 pg/100~l for LTC4, 254 -+32 pg/100pA for LTCs, 138 -+6 pg/100~l for LTD4, and 181 + 7 pg/100~l for LTDs. The lower limit of detectability was: 1.6 pg/100~l sample for LTB4 and LTBs in the LTB4 RIA, and 8 pg/100pA sample for LTC4, LTCs, LTD4, and LTDs in the peptidoleukotriene RIA. Cross reactivity of the LTB4 antibody with LTC4, LTCs, LTD4, and LTD5 was < 0.1%; and cross reactivity of the peptidoleukotriene antibody with LTB4 and LTBs was < 1%.
Quantitation of Thromboxane High Performance Liquid Chromatography Urinary thromboxane metabolites were extracted and separated by HPLC as described previously, s Samples were acidified to pH 3-3.5 and then applied to Sep-Pak C 18 columns (Waters Associates, Milford, MA) preconditioned with methanol and water. After washing the column with a 1:9 solution of acetonitrile and water, samples were eluted with ethyl acetate. Samples were evaporated to dryness under nitrogen and then reconstituted in a 1:3 solution of acetonitrile and 5.0 m M phosphoric acid. To separate thromboxane metabolites in urine, a Pecosphere HS3-C 18 cartridge (Perkin-Elmer Corp., Norwalk, CT) was used. Samples, unlabeled TxB2 standard (Advanced Magnetics Inc., Cambridge, MA), unlabeled 2,3-dinor-TxB~ standard (Cayman Chemical, Ann Arbor, MI) or [3H]-TxB2 standard (New England Nuclear, Boston, MA) were injected onto the column and eluted by a linear gradient from 100% 2.5 mM trifluoroacetic acid (Aldrich, Milwaukee, WI) to 100 % acetonitrile (Fisher Scientific, Fairlawn, NJ) over 10 minutes at a flow rate of 3.0 ml/min. Elution of thromboxane metabolites from the column was monitored by absorbance at a wavelength of 195 n m with a programmable multiwavelength ultraviolet spectrophotometer (Waters Associates, Milford, MA). 336
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All separations were performed at ambient temperature. Specific eluate fractions were collected based on the retention time of TxB2 and 2,3dinor-TxB2 standards. The TxB2 and 2,3-dinor-TxB2 fractions were dried under a stream of nitrogen and resuspended in radioimmunoassay (RIA) buffer. Concentrations of TxB2 and 2,3-dinor TxB2 were then quantitated by RIA as described below. Recoveries of TxB2 and 2,3-dinor TxB2 ranged from 60-80%.
Radioimmunoassays Immunoreactive thromboxane B~ (TxB2) was measured in cortical suspensions by direct RIA using an antibody that crossreacts extensively with thromboxane metabolites (Seragen Inc., Boston, MA). Urinary TxB2 and urinary 2,3-dinor TxB~ was quantitated by RIA after HPLC separation. For measurement of thromboxane metabolites, either TxB2 standard or 2,3-dinor-TxB2 standard (measured in triplicate) or unknowns (measured in duplicate) were incubated with a mixture of antisera and [3H]-TxB~ overnight at 4°C. After incubation, free eicosanoid was adsorbed with dextran-coated charcoal. Sample concentrations were determined by a standard curve in which the logarithm of the concentration was plotted against the logit of the B/Bo value. The results for urinary TxB2 are expressed as pg/24 hours. For renal cortical homogenates, results are expressed as pg/30 min/mg protein.
Histologic Studies Following the renal hemodynamic studies, the right kidney was removed and placed in 10% buffered formalin. After formalin fixation, kidney sections were stained with hematoxylin and eosin and examined by light microscopy. The slides were reviewed by a pathologist (PR) blinded to treatment group. At least 20 glomeruli were evaluated in each animal. The severity of glomerular crescent formation, hyperlobulation, hypercellularity, and necrosis were each graded separately using a semiquantitative scale where 0 was normal, and 1 +, 2 +, 3 +, and 4 + represented mild, moderate, moderately-severe, and severe abnormalities, respectively. An overall glomerular histopathologic score for each animal was obtained by summing the grades for each individual glomerular abnormality (maxim u m score 16). To investigate the effects of FO feeding on inflammatory cell infiltration in the kidney, the number of polymorphonuclear cells in glomeruli were counted and the severity of interstitial inflammatory cell infiltrates was assessed using a semiquantitative scale from zero to 4 + as described above. To quantitate the number of polymorphonuclear cells in glomeruli, additional sections were stained with PAS. For each animal, the number
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of glomerular polymorphonuclear cells per glomerulus was obtained by averaging the number of cells in 20 randomly selected glomeruli.
Statistical Analysis Data are presented as the mean + standard error of the mean. For the hemodynamic studies, data points for each animal represent the mean of the values measured during two clearance periods. For comparisons between two groups, statistical significance was assessed using an unpaired t-test or Wilcox rank sum as appropriate.
Results Dietary supplementation with FO improved renal hemodynamic function in MRL-lpr/Ipr mice as shown in Table 1. GFR was significantly higher in mice given FO compared with SO controls. Similarly, RPF tended to be higher in FO fed mice compared to control animals. This improvement in renal hemodynamic function in the FO group was associated with a reduction in the severity of the glomerular histomorphologic abnormalities as reflected by the significant decrease in the glomerular histopathologic score (Table 1). Specifically, glomerular hypercellularity and hyperlobulation tended to be reduced in FO fed mice and glomerular crescents were seen exclusively in mice given SO. This improvement in glomerular histomorphology was accompanied by a significant decrease in the number of polymorphonuclear cells in glomeruli of mice receiving FO compared to SO controls (2.6 + 0.5 [SO] vs 1.3 + 0.2 [FO] polymorphonuclear cells per glomerulus; P<0.025). In contrast, the severity of the interstitial inflammatory cell infiltrates was similar in both groups of MRL-lpr/lpr mice (semiquantitative score 1.6 + 0.6 [FO] vs 1.7 ___0.6 [SO]; P= NS). Urinary protein excretion also tended to be reduced in FO fed mice (Table 1) but this difference in proteinuria did not reach statistical significance. To investigate the effect of FO feeding on leukotriene production in MRL-Ipr/Ipr mice, 5-LO metabolites in supernatants from macrophages TABLE 1.
Effect of FO feeding on nephritis in
MRL-Ipr/Iprmice FO
C,N (ml/min/kg) CpAH (ml/min/kg) Glomerular histopathologic score Proteinuria (mg/24 hours)
11.1 33.6 2.3 9.0
_+ 0.8* _+ 4.2 +_ 0.4t +_ 0.9
SO 6.6 26.9 4.3 22
_+ 2.9 _+ 2.9 _+ 0.4 _+ 8.9
*P < 0.005 or t P < 0.05 vs SO.
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and kidney preparations were separated by HPLC and concentrations of immunoreactive leukotrienes in specific eluate fractions were determined by RIA. Elution of leukotrienes from the column was monitored at a wavelength of 280 nm and column eluate fractions were collected based on the appearance of absorbance peaks for leukotriene standards. As shown in panel A of Figure 1, supernatants from macrophages of mice given SO
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Time (min) FIGURE 1. HPLC separation of leukotrienes from peritoneal macrophages of MRL-Ipr/Ipr mice. Peritoneal macrophages were stimulated with the calcium ionophore A2.3187 (30p,M) and leukotrienes in supernatants were separated by HPLC as described in the Methods Section. Elution of leukotrienes from the column was monitored by absorbance at a wavelength of 280 nm. As shown in panel A, an absorbance peak coeluting with LTC4standard was detected in supernatants from macrophages of mice given SO. As shown in panel B, this LTC, peak was reduced in mice given FO, but a second peak coeluting with LTCs standard was detected. This LTCs peak was never seen in supematants from macrophages of SO fed mice. Absorbance peaks coeluting with either LTB4 or LTB5 standards were not detected in either group of MRL-Ipr/Ipr mice.
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contained an absorbance peak that eluted at the same time as LTC4 standard. In mice receiving FO, this LTC4 peak was reduced, and a second peak coeluting with LTCs standard was detected (Figure 1, panel B). This LTCs peak was never seen in supernatants from macrophages of mice given SO. Quantitation of immunoreactive leukotrienes in specific eluate fractions revealed differences in the pattern of leukotriene production by macrophages and renal cortices from FO fed mice compared to SO controls. As shown in Figure 2 and Table 2, dietary supplementation with FO significantly reduced macrophage production of both LTC4 and LTB4 as measured by RIA. This decrease in macrophage production of tetraene
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FIGURE2. Leukotriene production by peritoneal macrophages. Production of both B-series leukotrienes (panel A) and C-series leukotrienes (panel B) were measured by RIA after HPLC separation of 5-1ipoxygenase metabolites as described in the Methods Section. FO feeding reduced macrophage production of LTB4 and LTC4 compared to SO controls. Reduced production of tetraene leukotrienes was associated with enhanced production of pentaene leukotrienes. There was a significant increase in LTCs production by peritoneal macrophages from mice fed FO compared to control animals. (*P < 0.025 vs SO, **P < 0.05 vs SO)
340
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Fish oil feeding in murine lupus:
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TABLE 2. Effect of dietary supplementation with FO on production of leukotrienes by Ipr macrophages
MRL-Ipr/
Leukotrienes (pg/30 min/106 cells)
LTB4 LTC. LTBs LTCs
FO
SO
9.2 -+ 0.5* 345 _+ 48* trace 240 _+ 421-
20.8 _+ 4.6 777 _+ 162 trace 42 _+ 12
*P < 0.025 or I P < 0.005 vs Safflower oil
leukotrienes was accompanied by enhanced production of pentaene peptidoleukotrienes by macrophages from FO fed mice compared to control animals (Figure 2 and Table 2). Renal production of LTB4 followed a similar pattern. As shown in Figure 3, renal production of LTB4 was significantly decreased by dietary supplementation with FO (26_+ 18 [FO] vs 151 +46 [SO] pg/30 m i n / m g protein; P<0.025). In contrast, renal production of peptidoleukotrienes was not affected by FO feeding. Similar amounts of LTD4 were produced by renal cortices from both groups of MRL-lpr/lpr mice (255 + 29 [FO] vs 297_+39 [SO] pg/30 rain/rag protein; P = NS). Production of pentaene peptidoleukotrienes or LTBs were not detected in kidney preparations from either group. To assess the relative magnitude of the effect of FO feeding on production of cyclooxygenase compared with 5-LO metabolites, we also measured thromboxane production in mice receiving FO and SO controls. As reported previously by Kelley et al, ~8 FO feeding reduced production of TxB2 by suspensions of renal cortices (3060 _+300 [SO] to 1230___270 [FO] pg/30 min/mg protein; P<0.005). To investigate the effects of FO feeding on thromboxane production in vivo, we also measured urinary excretion of thromboxane metabolites. In these studies, FO feeding reduced urinary excretion of TxB~ excretion from 708+144 to 390+47 pg/24 hours (P<0.05). Similarly, urinary excretion of 2,3-dinor-TxB2 was reduced from 14,040 to 2,190 pg/24 hours (P<0.005).
Discussion These studies demonstrate that amelioration of nephritis in MRL-lpr/lpr mice by FO feeding is associated with reduced production of tetraene leukotrienes by both macrophages and kidneys from MRL-Ipr/Ipr mice. Several mechanisms may contribute to reduced production of tetraene leukotrienes in mice given FO. For example, EPA inhibits metabolism of
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Fish oil feeding in murine lupus:
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200A i m
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FIGURE 3. LTB4 production by renal cortical preparations from MRL-Ipr/Ipr mice. Production of LTB4 by renal cortices was measured by RIA after HPLC separation of 5-LO metabolites as described in the Methods Section. There was a significant decrease in renal cortical LTB4 production in mice given FO compared to SO controls. (*P < 0.025 vs SO)
AA by enzymes of both the cyclooxygenase and 5-LO pathways in a variety of cell t y p e s . 19,2°,21,26,~8 Reduced tetraene leukotriene production may result from direct enzyme inhibition as in the case of leukotriene A hydrolase, 3° or competition between EPA and AA as substrates for 5-LO.~9.28Moreover, FO might indirectly inhibit tetraene leukotriene production by reducing production of other inflammatory mediators which, in turn, might affect eicosanoid production rates. In this regard, FO inhibits production of TxA2, 31 platelet activating factor (PAF)7a interleukin-1 (IL-I), 37 and tumor necrosis factor 37 in vitro. Each of these inflammatory mediators has been implicated in the pathogenesis of lupus nephritis, 3~,38,39and both PAF4° and IL-135 directly stimulate leukotriene production by mononuclear inflammatory cells. FO feeding significantly reduced production of LTB4 by both macrophages and kidneys from MRL-lpr/lpr mice. In contrast, while FO feeding reduced macrophage production of peptidoleukotrienes, it did not affect peptidoleukotriene production by the kidney. This difference may reflect 342
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the heterogeneous cell population contributing to renal eicosanoid production. In addition to macrophages, a number of other inflammatory cells are capable of leukotriene synthesis 4~and may be differently affected by a diet enriched with FO. In human neutrophils, FO feeding reduces production of LTB4 but has no significant effect on neutrophil production of peptidoleukotrienes. 26 EPA and its metabolites may also have different effects on the enzymes involved in eicosanoid synthesis. EPA is a potent inhibitor of cyclooxygenase3~ and the EPA metabolite leukotriene As (LTAs) inhibits leukotriene A hydrolaseY Inhibition of cyclooxygenase may cause shunting of both AA and EPA through the 5-LO pathway resulting in enhanced synthesis of LTA4 and LTAs. Because LTAs is both a substrate and inhibitor of leukotriene A hydrolase, 3°this shunting mechanism may favor production of peptidoleukotrienes. A diet enriched with FO might therefore inhibit production of LTB4and cyclooxygenase metabolites without significantly altering production of peptidoleukotrienes. Our findings in this study are consistent with this model. In MRL-lpr/Ipr mice, enhanced production of tetraene peptidoleukotrienes contributes to renal hemodynamic impairment. 2 Nevertheless, GFR in mice given FO was significantly higher than SO controls despite similar rates of tetraene peptidoleukotriene production by kidneys from both groups of MRL-Ipr/Ipr mice. There are several potential explanations for this apparent discrepancy. First, multiple factors probably contribute to renal hemodynamic impairment in murine lupus. For example, prevention of glomerular injury by FO feeding may maintain GFR at levels comparable to values for normal controls 6 despite the adverse effects of peptidoleukotrienes on other aspects of renal hemodynamic such as RPF. The inability of dietary supplementation with FO to reduce renal peptidoleukotriene production might therefore account for the relatively modest effects of FO feeding on RPF in the present study. Secondly, measurements of renal leukotriene production in vitro may not reflect the inhibitory effect of FO feeding on peptidoleukotriene production in vivo. Indeed, the highly stimulated conditions in our renal homogenate system may define the capacity for renal eicosanoid synthesis rather than the rate of eicosanoid production in vivo. 6 Thirdly, peptidoleukotrienes may affect renal hemodynamics by enhancing production of other inflammatory mediators which, in turn, may be inhibited by FO feeding. In this regard, LTC4 stimulates production of the potent vasoconstrictor eicosanoid TxA2 in rat peritoneal macrophages, 42and renal vasoconstriction induced by LTC4 can be blocked by specific thromboxane synthase inhibitors. 43 These observations are of particular relevance to renal disease in SLE because TxA2 is an important mediator of renal hemodynamic impairment in both human and murine lupus nephritis. S,6 To determine if renal thromboxane production was affected by FO feeding in the present study, we measured both urinary excretion of throm-
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boxane metabolites and production of TxB2 by renal cortical preparations. As reported previously by Kelley et al, '8 dietary supplementation with FO decreased renal thromboxane production in vitro. In addition, we found that FO feeding significantly reduced urinary excretion of thromboxane metabolites compared to SO controls. Excretion of unmetabolized, native TxB2 in urine is thought to reflect thromboxane production by the kidney whereas, urinary 2,3-dinor-TxB~ may reflect extrarenal thromboxane production largely derived from platelets.44 Taken together, these data suggest that FO feeding inhibits both renal and extrarenal thromboxane production in vivo. Furthermore, because TxA2 mediates renal hemodynamic impairment in murine lupus, s this reduction in renal thromboxane production may contribute to the beneficial effects of FO feeding on renal hemodynamics in MRL-lpr/lpr mice. Essential fatty acid deficiency (EFAD) has also been shown to inhibit renal LTB4 production and ameliorate nephritis in several animal models of immune-mediated kidney disease 4s,46 including murine lupus. 47 It has been suggested that EFAD decreases renal leukotriene production by reducing the number of renal inflammatory cells. 4s In contrast, dietary supplementation with FO may decrease leukotriene production without significantly affecting influx of inflammatory cell to sites of inflammation. 48 To determine if FO feeding affected renal inflammation in the present study, we assessed the number of polymorphonuclear cells in glomeruli and the severity of interstitial inflammatory cell infiltrates. Are found that FO feeding caused a statistically significant decrease in the number of glomerular polymorphonuclear cells. This decrease in glomerular polymorphonuclear cells might be a consequence of reduced renal production of the potent chemotactic agent LTB4as has been suggested in EFAD. 49 Alternatively, polymorphonuclear cells have a great capacity for production of LTB4 and it is possible that this decrease in glomerular polymorphonuclear cells contributed to the reduction in renal production of LTB4 we observed in the present study. In summary, FO feeding was associated with significant alterations in production of 5-LO metabolites by both macrophages and kidneys from MRL-lpr/lpr mice. Dietary supplementation with FO inhibited production of tetraene leukotrienes and promoted the synthesis of pentaene leukotrienes. We speculate that these alterations in 5-LO metabolism may contribute to the beneficial effects of FO feeding on renal disease in MRLlpr/lpr mice. Furthermore, understanding the mechanisms of action of FO in murine lupus may suggest novel treatments for nephritis in patients with SLE.
Acknowledgments A preliminary version of this report has been published in abstract form (J. Am. Soc. Nephrol., 1:449, 1990). 344
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These studies were supported by grants from the Research Service of the Veterans Administration. Dr. Spurney performed these studies as a fellow of the National Kidney Foundation. The authors wish to t h a n k Pat Flannery for his expert technical assistance, and N o r m a Turner for her secretarial assistance in preparing the manuscript.
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Editor: W. Lands
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Received: 5-2-94
Accepted: 9-19-94
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