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Inhibition of leukotriene biosynthesis improves renal function in experimental glomerulonephritis
J. Lipid Mediators Cell Signalling 110995) 231-240
ELSEVIER
Inhibition of leukotriene biosynthesis improves renal function in experimental glomerulonephritis Rosemary Petric a,b,* , Anthony Ford-Hutchinson
ayb
aDepartment of Pharmacology, Merck Frost Centre for Therapeutic Research, P.O. Box 1005, Pointe Claire-Dorual, Quebec H9R 4P8, Canada b Department of Pharmacology, McGill University, 3655 Drummond St., Montreal, Quebec, H3G lY6 Canada
Received 29 July 1994; revised 14 November 1994; accepted 22 November 1994
Abstract The development of renal dysfunction in experimental glomerulonephritis (GN) is mediated in part by enhanced leukotriene (LT) formation. In our studies the pathophysiological role of LTs was investigated through pharmacological inhibition of LT biosynthesis in a rat model of nephrotoxic serum nephritis. MK-0591, an indirect inhibitor of Slipoxygenase activity, was co-administered to rats injected with nephrotoxic rabbit serum, followed by assessment of renal function, morphology and microsomal LTC, synthase activity on day 7. A significant improvement in glomerular function was noted (p < 0.051, together with a 50% reduction in proteinuria (p < 0.01) in animals receiving MK-0591 (60 mg kg-’ day-‘). In addition, the fall in renal LTC, synthase activity which occurred in nephritic rats (to 74% of control values, p < 0.01) was prevented in drug-treated animals. Based on these results, it appears that inhibition of LT biosynthesis protects against both renal impairment and alterations in LTC, synthase activity during the development of experimental GN, and may provide a useful therapeutic adjunct in the treatment of this disease. Keywords:
Leukotrienes;
Experimental
glomerulonephritis;
Leukotriene
C, synthase activity
1. Introduction The pathophysiologic effects of leukotrienes (LT) which have been described in a number of disease states (Samuelsson et al. 1987; Ford-Hutchinson, 1989; Huber
* Corresponding
author. Tel. (514) 428 3187; Fax (514) 428 3921.
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et al. 1989) appear to be exerted predominantly during the inflammatory stage. Indeed, the renal vascular, hemodynamic and morphological alterations which are manifest in experimental glomerulonephritis (GN), have been attributed to cysteinyl LTs early in the course of disease development (Badr et al., 1988; Wu and Lianos, 1993). Inhibition of LTs, therefore, may provide a therapeutic approach to limiting or preventing progression of nephritis. Oxygenation of arachidonic acid, liberated from membrane phospholipids through the action of phospholipase A,, results in the formation of leukotrienes (Lewis and Austen, 1984). LTA,, derived from arachidonic acid by 5lipoxygenase in conjunction with Uipoxygenase activating protein (FLAP) (Miller et al., 1990), is then metabolized enzymatically in diverging pathways by two distinct enzymes, LTA, hydrolase and LTC, synthase. The former catalyzes the formation of LTB,, a potent chemotactic mediator, while the latter enzyme initiates the formation of cysteinyl LTs. Conjugation of LTA, with the tripeptide glutathione (GSH), by LTC, synthase results in the formation of LTC,, which is subsequently metabolized to LTD, by y-glutamyl transpeptidase and to LTE, by cytosolic dipeptidases. These LTs, also known as the slow reacting substances of anaphylaxis (SRS-A), are potent mediators of smooth muscle contraction in bronchial and vascular tissue. In the rat, N-acetylation of LTE, produces NAcLTE,, a major metabolite excreted primarily in the bile (Hagmann et al., 1986). Renal metabolism of cysteinyl LTs and their excretion in the urine has also been demonstrated (Fauler et al., 19911, with enhanced urinary elimination of LTC, occurring in a rat model of nephrotoxic serum nephritis (NSN) (Petric and Ford-Hutchinson, 1994). Inhibition of LTs has resulted in improved functional and histological parameters in several disease models, including obstructive nephropathy (Reyes et al., 19921, renal transplant rejection (Spurney et al., 1994) and nephritis (Katoh et al., 1993; Wu and Lianos, 19931, in which indirect inhibitors of LT biosynthesis (MK-886) or specific LTD, receptor antagonists (SK&F 106203 and SK&F 104353) were used. In a preliminary study we reported a significant reduction in urinary protein excretion in nephritic rats co-administered MK-0591 (Petric et al., 19951, a more potent second generation inhibitor of FLAP (Brideau et al., 1991). We now report the results of our further investigations of MK-0591 treatment on renal function, LTC, synthase activity and urinary LT excretion in a rat model of NSN.
2. Materials and methods 2.1. Induction of nephritis and renal function studies Nephritis was induced in male Sprague Dawley rats (Charles River, QC) weighing 300 + 15 g, by injection of rabbit anti-rat glomerular membrane serum as described previously (Petric and Ford-Hutchinson, brief, rabbits immunized with rat cortical basement membrane were serum decomplemented by heating and adsorbed against washed rat
Montreal, basement 1994). In bled, the red blood
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233
cells to remove non-specific reactivity. Rats were then injected, via the tail vein, with approx. 1 ml of rabbit serum (120 mg protein/ml) and progression of nephritis was assessed on days 1 and 7, as described below. Control rats received an equal volume of pre-immunized rabbit serum, which had been prepared in a similar fashion. Animals were maintained on standard rodent chow (PM1 Feeds Inc., Richmond, IN) containing a minimum of 22.5% protein and tap water ad libitum. All procedures were conducted in accordance with the Canadian Council of Animal Care (CCAC) guidelines. Renal function studies were performed to determine a biochemical profile for each animal on either day 1 or 7. Rats were placed in individual metabolic cages without food, but with access to tap water, for a 24 h urine collection. Following collection of urine, animals were anaesthetized with ethyl ether (Fisher Scientific) and blood obtained via cardiac puncture. Plasma and urine were assayed by conventional methods to determine creatinine clearance (Cc,), blood urea nitrogen (BUN), sodium and potassium clearance (C,, and C,) and their respective fractional excretions (Fe,, and Fe,). Animals were killed by cervical dislocation while still under anaesthesia and the kidneys removed for histological assessment. The left kidney was bisected, decapsulated and fixed in either 10% formalin (Fisher Scientific) or 5% glutaraldehyde (Sigma, St. Louis, MO) for haematoxylin and eosin staining or electron microscopy and the results evaluated by a pathologist blinded to the treatment groups. Measurement of urinary protein concentration utilized BioRad reagents (BioRad Laboratories, Richmond, CA) and followed the method of Bradford (1976). 2.2. LTC,
synthase activity and urinary leukotriene excretion
Renal LTC, synthase activity was measured in rat cortical microsomes as described previously (Petric et al., 1995). Immediately following death, the kidneys were bisected, decapsulated, placed in ice-cold 1.15% KC1 and the medulo-papillary tissue discarded. Cortical microsomes were prepared by polytron homogenization and differential centrifugation at 4°C and the microsomal pellet fraction resuspended in phosphate-buffered saline to a final concentration of 20 mg protein/ml. Microsomes were stored at -80°C. To measure microsomal enzyme activity, 5 mg of microsomal protein/ml were diluted with 0.1 M potassium phosphate buffer (pH 7.4) in a final incubation volume of 150 ~1 containing 50 mM serine-borate complex (an inhibitor of y-glutamyl transpeptidase), 10 mM GSH and 40 PM LTA,. The reaction mixture was incubated for 10 min at 25°C and the reaction stopped with an equal volume of acetonitrile-MeOH-acetic acid (50:50:1 v/v>. Following centrifugation of the mixture, LTC, formation was measured in the resulting supernatant by isocratic reverse-phase HPLC (Waters Associates) and expressed as pmol LTC, formed min- ’ mg protein- ‘. Excretion of cysteinyl leukotrienes in the urine was assayed in separate studies as described previously (Petric and Ford-Hutchinson, 1994). An aliquot of urine was extracted through Sep Pak C-18 cartridges (Waters, Milford, MA) which had been conditioned with equal volumes of MeOH and water. Samples were washed
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with 1% (w/v) ammonium acetate buffer containing 1 mM EDTA (pH 5.4) and the leukotrienes eluted with MeOH. Leukotrienes were resolved isocratically by HPLC and 1 ml fractions were collected for subsequent RIA. Quantification of LTs in each fraction was achieved by RIA using a rat monoclonal antibody to LTE, (Rehovot University, Israel). Urinary LT excretion was expressed as pg/h. 2.3. Leukotriene inhibition studies To determine the effects of LT biosynthesis inhibition on the development of nephritis, rats were administered MK-0591 (60 mg kg-l day-‘) in divided doses throughout the study interval, with the first dose given 2 h prior to the injection of nephrotoxic rabbit serum. MK-0591, a quindole inhibitor of FLAP (Prasit et al., 1993) was dissolved in 0.05% EtOH and suspended in 0.1% methocel at a concentration of 10 mg/ml. The suspension was prepared fresh and a volume of 3 ml/kg was administered orally by gavage every 12 h. Control rats received 0.1% methocel containing 0.05% EtOH in the same dosing regimen. To assess the effects of MK-0591 on normal renal function, control rats were treated with MK-0591 in a similar fashion. The four treatment groups, containing six to seven rats per group, were as follows: group 1, control; group 2, GN; group 3, GN + MK, group 4, control + MK. A kinetic profile of MK-0591 metabolism was determined from a single 30 mg/kg oral dose in control and nephritic rats (n = 3). Blood was collected from the tail vein over a 24 h interval into heparinized vacutainers, centrifuged at 300 x g for 15 min at 4°C and the plasma stored at - 20°C. At the time of assay samples were thawed, extracted with an equal volume of acetonitrile and centrifuged at 14 000 x g for 10 min, and the resulting supernatant injected onto a reverse-phase HPLC column (Nova-Pak C-18, 3.9 X 150 mm). MK-0591 was resolved isocratically at 230 nm with a mobile phase consisting of acetonitrile: 0.1% (w/v) phosphoric acid (55:45 v/v) at a flow rate of 1 ml/min. Plasma concentrations were determined by integration of the MK-0591 peak relative to a spiked sample of known concentration. 2.4. StatMcal analysis Comparison between treatment groups utilized a one-way analysis of variance, followed by the t-test using the Bonferroni correction for multiple comparisons. Data are expressed as mean + SE.
3. Results 3.1. Renal function and hivFtology Renal function studies in nephritic rats (GN) demonstrated a significant drop in glomerular filtration on day 1. C,, fell from 458 f 16 to 404 f 14 ~1 min-’ 100 g
R. Petric, A. Ford-Hutchinson /J. Lipid Mediators Cell SignaIling I1 (1995) 231-240 Table 1 Protective lonephritis
effect
of MK-0591
C,, (~1 min-’ Control GN MK+ GN
46Ok9 386 + 16” 428 + 103b
on renal
100 g body wt-‘)
function
parameters
in a rat model
of experimental
235
glomeru-
BUN (mmol/l)
c ($/mitt)
Fe,, (%)
Cx (PI/mitt)
Fe, (%o)
5.8 + 0.3 8.2+0.5a 6.1 +0.4b
4.4OkO.72 4.29 f 0.38 3.12kO.35
0.311+0.045 0.375 f 0.044 0.266+0.030
488+41 423 + 63 514+26
35+3 40+4 40+2
C,,, creatinine clearance; BUN, blood urea nitrogen; CNa, clearance of sodium; FeNa, fractional fractional excretion of potassium. Renal excretion of sodium; C,, clearance of potassium; Fe,, function was measured on day 7 of the study. Values are expressed as the mean + SE with 6-7 rats per group. Staistical significance is expressed as “p < 0.01 for GN vs control; and “p < 0.05 for MK+ GN vs GN.
body wt- ’ (p < 0.01) in control and GN groups, respectively. BUN was not affected (data not shown). On day 7, a further decline in C,, occurred to 386 k 13 ~1 min-’ 100 g body wt-’ (p < 0.05) in the nephritic group, with a 50% elevation in BUN in the nephritic animals (p < 0.01) (Table 1). Co-administration of MK-0591 ameliorated this decline in filtration, (442 k 24 ~1 min-’ 100 g body wt - ‘) on day 1 (ns) and by improving C,, by 10% on day 7 (p < 0.03, and reducing BUN by 25% (p < 0.05) compared to the GN group. No significant changes in electrolyte excretion were evident in the GN or MK-0591 treated groups on day 7 compared to controls. MK-0591 treatment in control rats (administered pre-immune rabbit serum) produced no significant effects on renal function during the study interval (data not shown). Proteinuria was present in both nephritic and drug-treated groups compared to controls throughout the study interval. On day 7 urinary protein excretion rose from control values of 6.4 k 0.4 mg 24 h-i 100 g body wt- ’ to 301 f 24 mg 24 h- ’ 100 g body wt-’ in the GN group (p < 0.001) (Fig. 1). Inhibition of LT biosynthesis with MK-0591, however, resulted in a 50% reduction in proteinuria (p < 0.01). No histological improvement was evident with inhibition of LT biosynthesis in rats injected with nephrotoxic rabbit serum by light or electron microscopy. Both treated and untreated nephritic rats demonstrated basement membrane thickening, sub-endothelial electron-dense deposition and fusion of foot processes (data not shown). 3.2. LTC,
synthase activity and leukotriene excretion
Measurement of urinary LT excretion, however, indicated incomplete inhibition of LT biosynthesis, presumably in the kidney. Detectable levels of LTD, and NAcLTE,, but not LTC, were observed in the urine of 3/7 nephritic rats treated with MK-0591 on day 1. This group of animals also demonstrated detectable levels of NAcLTE, only, in the urine of 3/6 animals on day 7 (data not shown). Measurement of renal LTC, synthase activity in cortical microsomes showed a significant reduction in enzyme activity in GN rats, which was prevented by
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/J. Lipid Mediators Cell SignaIling 11 (1995) 231-240
CONTROL
GN
MK+GN
Fig. 1. Inhibition of leukotriene biosynthesis with MK-0.591 (60 mg kg-’ day-‘) reduced urinary protein excretion in nephritic rats. The significant proteinuria evident in the GN rats compared to controls (* * p < O.Ol), was reduced in MK+GN animals (#p < 0.01) compared to the GN group on day 7 of the study. Data are expressed as the mean f SE of 6-7 rats per group. ??
co-administration of MK-0591 (Fig. 2). Enzyme activity in the GN group dropped to 2.41 f 0.50 pmol LTC, formed min-’ mg protein-’ from control levels of 9.14 f 1.50 pmol LTC, formed min-’ mg protein-’ (p < 0.01). LTC, synthase activity in the MK-591 treated rats, however, rose to 6.41 f 1.10 pmol LTC, formed min-’ mg protein-’ (p < O.Ol>, a significant improvement compared to GN animals and not statistically different from control values.
CONTROL
ON
MK+GN
Fig. 2. Renal LTC, synthase activity measured in rat cortical microsomes. A significant reduction in enzyme activity was evident in the GN rats compared to controls ( * * p < 0.01) on day 7 of the study. LTC, synthase activity in the MK+ GN group, however, improved significantly ($p < 0.01) and was not statistically different from control values. Data represent the mean f SE of 6-7 assays each performed in triplicate.
R. Petric, A. Ford-Hutchinson /.I. Lipid Mediators Cell Signalling 11 (1995) 231-240
231
3.3. MK-0591 plasma concentrations
Kinetic studies to determine plasma concentration of MK-0591 were conducted. Blood levels of MK-0591 were achieved which have been previously shown to inhibit LT formation in the rat ex vivo and in vitro (Brideau et al., 1991). No difference was found in the kinetic profile of MK-0591 in nephritic and control rats over 24 h (data not shown), and is consistent with the known biliary excretion of MK-0591 and its metabolites (unpublished data).
4. Discussion Published data support a pathophysiological effect of cysteinyl LTs on renal function in models of experimental GN. Badr et al. (1988) have demonstrated an LTD,-mediated decrease in renal blood flow and a reduction in glomerular filtration (which was independent of perfusion) while LTC,/D,-dependent glomerular cell proliferation was demonstrated in vitro by Baud et al. (1985, 1989) and in vivo by Wu and Lianos (1993). In addition, both the hemodynamic and proliferative changes occurring in the nephritic rat were reduced or prevented by LT inhibition using either LTD, receptor antagonists or LT biosynthesis inhibitors. MK-0591, a quindole derivative of MK-886, was investigated for its renal protective effect during the development of nephrotoxic serum nephritis. This compound is an extremely potent inhibitor of LT biosynthesis in the rat as assessed by whole blood and pleurisy assays (Brideau et al., 19911, urinary excretion of LTE, and measurement of LTB, in rectal dialysis bags (Hilling@ et al., 1994). Co-administration of MK-0591 to rats injected with nephrotoxic rabbit serum resulted in a significant improvement in renal function. Glomerular filtration (as measured by C,, and BUN) was elevated and urinary protein excretion reduced by nearly 50%. Failure to achieve a greater degree of renal protection with MK-0591 treatment may be a consequence of several factors. The most probable explanation is that mediators other than 5-lipoxygenase-derived products contribute to the pathology. However, it can not be excluded that a greater degree of inhibition of LT biosynthesis locally in the kidney might have resulted in superior efficacy, particularly as incomplete inhibition of urinary cysteinyl LT excretion was observed in several animals on days 1 and 7, as described in the results section. In addition, inhibition of LT biosynthesis may have caused a shift in arachidonic acid metabolism toward the cycle-oxygenase pathway, resulting in enhanced prostanoid and thromboxane formation. Lianos et al. (1983) and Stork and Dunn (1985) have demonstrated elevated prostanoid synthesis in the early stage of disease development, therefore any additional increase of these eicosanoids could further exacerbate the derangement of renal hemodynamics already present. For example, the decrease in Fe Na present in the MK + GN group could have resulted from enhanced thromboxane synthesis, since thromboxane has been suggested to augment proximal tubular sodium reabsorption (Papanicolaou et al., 1987; Petric et al., 1990). Morphologically, no alleviation of the structural injury caused by
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injection of nephrotoxic rabbit serum was evident in MK-0591 treated rats, a finding consistent with other reports also demonstrating no alteration in immune deposition (Rahman et al., 1991) or leukocytic infiltration (Wu et al., 1993) in nephritic models. Of particular interest were the changes in renal microsomal LTC, synthase activity in the MK-0591 treated group. Whereas nephritic rats demonstrated a significant reduction in enzymic activity, no drop in enzyme activity was found in the MK-0591 group. LTC, synthase activity is negatively phosphorylated through a protein kinase C (PKC)-mediated event (Kargman et al., 1994; Ali et al., 1994). It has been suggested that such a mechanism may explain the alterations in LTC, synthase activity in nephritic kidneys (Petric and Ford-Hutchinson, 1994). Baud et al. (1989) have demonstrated an LTD,-mediated increase in PKC activity in glomerular epithelial cells while investigating the proliferative effects of LTD,. Given that renal LTC, synthase activity in rats treated with an LT biosynthesis inhibitor was not statistically different from controls, it is interesting to speculate whether LT autoregulation of enzyme activity is occurring. The early rise in LTC, synthase activity which we have described during the initial 24 h following injection of nephrotoxic rabbit serum, gives way to a decline in enzymic activity by day 7, and a gradual recovery thereafter (Petric et al., 1995). Whether the increase in enzyme activity evident on day 1 is also reversed by MK-0591 treatment remains to be determined. In summary, we have shown that LT biosynthesis inhibition provides a protective effect on renal function in a rat model of nephrotoxic serum nephritis. In addition, normalization of renal LTC, synthase activity in nephritic animals co-administered MK-0591 suggests a level of enzyme autoregulation, and provides a possible explanation for the rapid rise and fall in cysteinyl LT formation which has been described in nephritis.
Acknowledgements
This work was supported in part by the Medical Research Council Industrial Studentship award no. 1550. We wish to thank Richard Frenette and Serge Leger for the measurement of MK-0591 levels and Dr Donald Nicholson and Philip Tagari for advice on leukotriene measurements. We are also grateful to Dr Madeleine Chagnon, Bio Research Laboratories Ltd, Senneville, QC, for the pathological evaluation of histological samples, and to MS Faith Carr for assistance in the preparation of this manuscript.
References Ali, A., Nicholson, D.W. and Ford-Hutchinson, A.W. (1994) Activation of protein kinase C dependent down-regulation of sulfidopeptide leukotriene biosynthesis and leukotrine C, synthase in neutrophilic HL-60 cells. Mol. Pharmacol., in press.
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Badr, K.F., Schreiner, G.F., Wassermann, M. and Ichikawa, I. (1988) Preservation of the glomerular capillary ultrafiltration coefficient during rat nephrotoxic serum nephritis by a specific leukotriene D4 receptor antagonist. J. Clin. Invest. 81, 1702-179. Baud, L., Sraer, J., Perez, J., Nives, M-P. and Ardaillou, R. (1985) Leukotriene C, binds to human glomerular epithelial cells and promotes their proliferation in vitro. J. Clin. Invest. 76, 374-377. Baud, L., Perez, J., Cherqui, G., Cragoe, E.J. and Ardaillou R. (1989) Leukotriene D,-induced proliferation of glomerular epithelial cells: PKC- and Na+-Ht exchanger-mediated response. Am. J. Physiol. 257, C232-C239. Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Chem. 72, 248-254. Brideau, C., Chan, C., Charleson, S., Denis, D., Evans, J.F., Ford-Hutchinson, A.W., Fortin, R., Gillard, J.W., Guay J., Guevremont, D., Hutchinson, J.H., Jones, T.R., tiger, S., Mancini, J.A., McFarlane, C., Pickett, C.B., Piechuta, H., Prasit, P., Reindeau, D., Rouzer, C.A., Tagari, P., Vickers, P.J. and Young, R.N. (1991) Pharmacology of MK-0591 (3-(l-(4-chlorobenzyl)-3-(rbutylthio)-5-(quinolin-2-yl-methoxy)-indol-2-yl)-2,2-dimethylpropanoic acid), a potent orally active leukotriene inhibitor. Can. J. Physiol. Pharmacol. 70, 799-807. Fauler, J., Wiemeyer, A., Yoshizawa, M., Schurek, H.J. and Frolich, J.C. (1991) Metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney. Prostaglandins 42, 239-249. Ford-Hutchinson, A.W. Evidence for the involvement of leukotrienes and other lipoxygenase products in disease states. In Rokach, J. (Ed.), Leukotrienes and Lipoxygenases, Chemical, Biological and Clinical Aspects, Vol. II, Elsevier, Amsterdam, 1989, pp. 406-425. Hagmann, W., Denzlinger, C., Rapp, S., Weckbecker, G. and Keppler, D. (1986) Identification of the major endogenous leukotriene metabolites in the bile of rats as N-acetyl leukotriene E,. Prostaglandins 31, 239-251. Hillingss, J., Kjeldsen, J., Laursen, L.S., Lauritsen, K., von Spreckelsen, S., DeprC, M., Friedman, B.S., Malmstrijm, K., Shingo, S., Bukhave, K. and Rask-Madsen, J. (1994) Selective blockade of rectal leukotriene B, (LTB,) and systemic leukotriene production by a single, oral dose of MK-591 in patients with active ulcerative colitis. Gastroenterogy 106, A698. Huber, M. Kastner, S., Scholmerich, J., Gerok, W. and Keppler, D. (1989) Analysis of cysteinyl leukotrienes in human urine: enhanced excretion in patients with liver cirrhosis and hepatorenal syndrome. Eur. J. Clin. Invest. 19, 53-60. Kargman, S., Ali, A., Vaillancourt, J.P., Evans, J.F. and Nicholson, D.W. (1994) Protein kinase C dependent regulation of sulfidopeptide leukotriene biosynthesis and leukotriene C4 synthase in neutrophilic HL-60 cells. Mol. Pharmacol. 45, 1043-1049. Katoh, T., Lianos, E.A., Fukunagta, M., Takahashi, K. and Badr, K.F. (1993) Leukotriene D4 is a mediator of proteinuria and glomerular hemodynamic abnormalities in passive Heymann nephritis. J. Clin. Invest. 91, 1507-1515. Lewis, R.A. and Austen, F.K. (1984) The biologically active leukotrienes. Biosynthesis, metabolism, receptors, functions and pharmacology. J. Clin. Invest. 73, 889-897. Lianos, E.A., Andres, G.A. and Dunn, M.J. (1983) Glomerular prostaglandin and thromboxane synthesis in rat nephrotoxic serum nephritis. J. Clin. Invest. 72, 1439-1448. Miller, D.K., Gillard, J.W., Vickers, P.J., Saowski, S., Leveille, C., Mancini, J.A., Charleson, P., Dixon, R.A.F., Ford-Hutchinson, A.W., Fortin, R., Gauthier, J.-Y., Rodkey, J., Rosen, R., Rouzer, C., Sigal, I.S., Strader, C.D. and Evans, J.F. (1990) Identification and isolation of a membrane protein necessary for leukotriene production. Nature 343, 278-281. Papanicolaou, N., Hatziantoniou, C., Dontas, A., Gkikas, E-L., Paris, M., Gkikas, G. and Bariety, J. (1987) Is thromboxane a potent antinatriuretic factor and is it involved in the development of acute renal failure? Nephron 45, 277-282. Petric, R. and Ford-Hutchinson, A.W. (1994) Elevated cysteinyl leukotriene excretion in experimental glomerulonephritis. Kidney Int. 46, 1322-1329. Petric, R., Freeman, D., Wallace, C., McDonald, J., Stiller, C. and Keown, P. (1990) Modulation of experimental cyclosporine nephrotoxicity by inhibition of thromboxane synthesis. Transplant 50, 558-563.
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Petric, R., Nicholson, D.W. and Ford-Hutchinson, A.W. (1995) Renal LTC, synthase: Characterization, partial purification and alterations in experimental glomerulonephritis. Biochim. Biophys. Acta 1254, 207-215. Prasit, P., Belley, M., Blouin, M., Brideau, C., Chan, C., Charleson, S., Evans, J.F., Frenette, R., Gauthier, J-Y., Guay, J., Gillard, J.W., Grimm. E., Ford-Hutchinson, A.W., Hutchinson, J.H., Fortin, R., Jones, T.R., Mancini, J., LRger, S., McFarlane, C.S., Pietchuta, H., Reindeau, D.E., Roy, P., Tagari, P., Vickers, P.J., Young, R.N. and Zamboni, R. (1993) A new class of leukotriene biosynthesis inhibitor: the development of MK-0591. J. Lipid Mediators 6, 239-244. Rahman, M.A., Sauter, D.C. and Young, M.R. (1991) Effects of dietary fish oil on the induction of experimental membranous nephropathy in the rat. Lab. Invest. 64, 371-376. Reyes, A.A., Letkowith, J., Pippin, J. and Klahr, S. (1992) Role of the 5-lipooxygenase pathway in obstructive nephropathy. Kidney Int. 41, 100-106. Samuelsson, B., Dahlen, SE., Lindren, J.A., Rouzer, CA. and Serhan, C.N. (1987) Leukotrienes and lipoxins: Structures, biosynthesis, and biological effects. Science 237, 1171-1176. ‘Spurney, R.F., Ibrahim, S., Butterly, D., Klotman, P.E., Sanfilippo, F. and Hoffman, M. (1994) Leukotrienes in renal transplant rejection in rats. J. Immunol. 152, 867-876. Stork, J.E. and Dunn, M.J. (1985) Hemodynamic roles of thromboxane A2 and prostaglandin E, in glomerulonephritis. J. Pharmacol Exp Ther. 233, 672-678. Wu, S.H. and Lianos, E.A. (1993) Modulatory effect of arachidonate 5-lipoxygenation on glomerular cell proliferation in nephrotoxic serum nephritis. J. Lab. Clin. Med. 122, 703-710. Wu, S.-H., Bresnahan, B.A. and Lianos, E.I. (1993). Hemodynamic role of arachidonate 12- and 5-lipoxygenases in nephrotoxic serum nephritis. Kidney Int. 43, 1280-1285.
ELSEVIER
Inhibition of leukotriene biosynthesis improves renal function in experimental glomerulonephritis Rosemary Petric a,b,* , Anthony Ford-Hutchinson
ayb
aDepartment of Pharmacology, Merck Frost Centre for Therapeutic Research, P.O. Box 1005, Pointe Claire-Dorual, Quebec H9R 4P8, Canada b Department of Pharmacology, McGill University, 3655 Drummond St., Montreal, Quebec, H3G lY6 Canada
Received 29 July 1994; revised 14 November 1994; accepted 22 November 1994
Abstract The development of renal dysfunction in experimental glomerulonephritis (GN) is mediated in part by enhanced leukotriene (LT) formation. In our studies the pathophysiological role of LTs was investigated through pharmacological inhibition of LT biosynthesis in a rat model of nephrotoxic serum nephritis. MK-0591, an indirect inhibitor of Slipoxygenase activity, was co-administered to rats injected with nephrotoxic rabbit serum, followed by assessment of renal function, morphology and microsomal LTC, synthase activity on day 7. A significant improvement in glomerular function was noted (p < 0.051, together with a 50% reduction in proteinuria (p < 0.01) in animals receiving MK-0591 (60 mg kg-’ day-‘). In addition, the fall in renal LTC, synthase activity which occurred in nephritic rats (to 74% of control values, p < 0.01) was prevented in drug-treated animals. Based on these results, it appears that inhibition of LT biosynthesis protects against both renal impairment and alterations in LTC, synthase activity during the development of experimental GN, and may provide a useful therapeutic adjunct in the treatment of this disease. Keywords:
Leukotrienes;
Experimental
glomerulonephritis;
Leukotriene
C, synthase activity
1. Introduction The pathophysiologic effects of leukotrienes (LT) which have been described in a number of disease states (Samuelsson et al. 1987; Ford-Hutchinson, 1989; Huber
* Corresponding
author. Tel. (514) 428 3187; Fax (514) 428 3921.
0929-7855/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZO929-7855(94)00040-9
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et al. 1989) appear to be exerted predominantly during the inflammatory stage. Indeed, the renal vascular, hemodynamic and morphological alterations which are manifest in experimental glomerulonephritis (GN), have been attributed to cysteinyl LTs early in the course of disease development (Badr et al., 1988; Wu and Lianos, 1993). Inhibition of LTs, therefore, may provide a therapeutic approach to limiting or preventing progression of nephritis. Oxygenation of arachidonic acid, liberated from membrane phospholipids through the action of phospholipase A,, results in the formation of leukotrienes (Lewis and Austen, 1984). LTA,, derived from arachidonic acid by 5lipoxygenase in conjunction with Uipoxygenase activating protein (FLAP) (Miller et al., 1990), is then metabolized enzymatically in diverging pathways by two distinct enzymes, LTA, hydrolase and LTC, synthase. The former catalyzes the formation of LTB,, a potent chemotactic mediator, while the latter enzyme initiates the formation of cysteinyl LTs. Conjugation of LTA, with the tripeptide glutathione (GSH), by LTC, synthase results in the formation of LTC,, which is subsequently metabolized to LTD, by y-glutamyl transpeptidase and to LTE, by cytosolic dipeptidases. These LTs, also known as the slow reacting substances of anaphylaxis (SRS-A), are potent mediators of smooth muscle contraction in bronchial and vascular tissue. In the rat, N-acetylation of LTE, produces NAcLTE,, a major metabolite excreted primarily in the bile (Hagmann et al., 1986). Renal metabolism of cysteinyl LTs and their excretion in the urine has also been demonstrated (Fauler et al., 19911, with enhanced urinary elimination of LTC, occurring in a rat model of nephrotoxic serum nephritis (NSN) (Petric and Ford-Hutchinson, 1994). Inhibition of LTs has resulted in improved functional and histological parameters in several disease models, including obstructive nephropathy (Reyes et al., 19921, renal transplant rejection (Spurney et al., 1994) and nephritis (Katoh et al., 1993; Wu and Lianos, 19931, in which indirect inhibitors of LT biosynthesis (MK-886) or specific LTD, receptor antagonists (SK&F 106203 and SK&F 104353) were used. In a preliminary study we reported a significant reduction in urinary protein excretion in nephritic rats co-administered MK-0591 (Petric et al., 19951, a more potent second generation inhibitor of FLAP (Brideau et al., 1991). We now report the results of our further investigations of MK-0591 treatment on renal function, LTC, synthase activity and urinary LT excretion in a rat model of NSN.
2. Materials and methods 2.1. Induction of nephritis and renal function studies Nephritis was induced in male Sprague Dawley rats (Charles River, QC) weighing 300 + 15 g, by injection of rabbit anti-rat glomerular membrane serum as described previously (Petric and Ford-Hutchinson, brief, rabbits immunized with rat cortical basement membrane were serum decomplemented by heating and adsorbed against washed rat
Montreal, basement 1994). In bled, the red blood
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233
cells to remove non-specific reactivity. Rats were then injected, via the tail vein, with approx. 1 ml of rabbit serum (120 mg protein/ml) and progression of nephritis was assessed on days 1 and 7, as described below. Control rats received an equal volume of pre-immunized rabbit serum, which had been prepared in a similar fashion. Animals were maintained on standard rodent chow (PM1 Feeds Inc., Richmond, IN) containing a minimum of 22.5% protein and tap water ad libitum. All procedures were conducted in accordance with the Canadian Council of Animal Care (CCAC) guidelines. Renal function studies were performed to determine a biochemical profile for each animal on either day 1 or 7. Rats were placed in individual metabolic cages without food, but with access to tap water, for a 24 h urine collection. Following collection of urine, animals were anaesthetized with ethyl ether (Fisher Scientific) and blood obtained via cardiac puncture. Plasma and urine were assayed by conventional methods to determine creatinine clearance (Cc,), blood urea nitrogen (BUN), sodium and potassium clearance (C,, and C,) and their respective fractional excretions (Fe,, and Fe,). Animals were killed by cervical dislocation while still under anaesthesia and the kidneys removed for histological assessment. The left kidney was bisected, decapsulated and fixed in either 10% formalin (Fisher Scientific) or 5% glutaraldehyde (Sigma, St. Louis, MO) for haematoxylin and eosin staining or electron microscopy and the results evaluated by a pathologist blinded to the treatment groups. Measurement of urinary protein concentration utilized BioRad reagents (BioRad Laboratories, Richmond, CA) and followed the method of Bradford (1976). 2.2. LTC,
synthase activity and urinary leukotriene excretion
Renal LTC, synthase activity was measured in rat cortical microsomes as described previously (Petric et al., 1995). Immediately following death, the kidneys were bisected, decapsulated, placed in ice-cold 1.15% KC1 and the medulo-papillary tissue discarded. Cortical microsomes were prepared by polytron homogenization and differential centrifugation at 4°C and the microsomal pellet fraction resuspended in phosphate-buffered saline to a final concentration of 20 mg protein/ml. Microsomes were stored at -80°C. To measure microsomal enzyme activity, 5 mg of microsomal protein/ml were diluted with 0.1 M potassium phosphate buffer (pH 7.4) in a final incubation volume of 150 ~1 containing 50 mM serine-borate complex (an inhibitor of y-glutamyl transpeptidase), 10 mM GSH and 40 PM LTA,. The reaction mixture was incubated for 10 min at 25°C and the reaction stopped with an equal volume of acetonitrile-MeOH-acetic acid (50:50:1 v/v>. Following centrifugation of the mixture, LTC, formation was measured in the resulting supernatant by isocratic reverse-phase HPLC (Waters Associates) and expressed as pmol LTC, formed min- ’ mg protein- ‘. Excretion of cysteinyl leukotrienes in the urine was assayed in separate studies as described previously (Petric and Ford-Hutchinson, 1994). An aliquot of urine was extracted through Sep Pak C-18 cartridges (Waters, Milford, MA) which had been conditioned with equal volumes of MeOH and water. Samples were washed
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with 1% (w/v) ammonium acetate buffer containing 1 mM EDTA (pH 5.4) and the leukotrienes eluted with MeOH. Leukotrienes were resolved isocratically by HPLC and 1 ml fractions were collected for subsequent RIA. Quantification of LTs in each fraction was achieved by RIA using a rat monoclonal antibody to LTE, (Rehovot University, Israel). Urinary LT excretion was expressed as pg/h. 2.3. Leukotriene inhibition studies To determine the effects of LT biosynthesis inhibition on the development of nephritis, rats were administered MK-0591 (60 mg kg-l day-‘) in divided doses throughout the study interval, with the first dose given 2 h prior to the injection of nephrotoxic rabbit serum. MK-0591, a quindole inhibitor of FLAP (Prasit et al., 1993) was dissolved in 0.05% EtOH and suspended in 0.1% methocel at a concentration of 10 mg/ml. The suspension was prepared fresh and a volume of 3 ml/kg was administered orally by gavage every 12 h. Control rats received 0.1% methocel containing 0.05% EtOH in the same dosing regimen. To assess the effects of MK-0591 on normal renal function, control rats were treated with MK-0591 in a similar fashion. The four treatment groups, containing six to seven rats per group, were as follows: group 1, control; group 2, GN; group 3, GN + MK, group 4, control + MK. A kinetic profile of MK-0591 metabolism was determined from a single 30 mg/kg oral dose in control and nephritic rats (n = 3). Blood was collected from the tail vein over a 24 h interval into heparinized vacutainers, centrifuged at 300 x g for 15 min at 4°C and the plasma stored at - 20°C. At the time of assay samples were thawed, extracted with an equal volume of acetonitrile and centrifuged at 14 000 x g for 10 min, and the resulting supernatant injected onto a reverse-phase HPLC column (Nova-Pak C-18, 3.9 X 150 mm). MK-0591 was resolved isocratically at 230 nm with a mobile phase consisting of acetonitrile: 0.1% (w/v) phosphoric acid (55:45 v/v) at a flow rate of 1 ml/min. Plasma concentrations were determined by integration of the MK-0591 peak relative to a spiked sample of known concentration. 2.4. StatMcal analysis Comparison between treatment groups utilized a one-way analysis of variance, followed by the t-test using the Bonferroni correction for multiple comparisons. Data are expressed as mean + SE.
3. Results 3.1. Renal function and hivFtology Renal function studies in nephritic rats (GN) demonstrated a significant drop in glomerular filtration on day 1. C,, fell from 458 f 16 to 404 f 14 ~1 min-’ 100 g
R. Petric, A. Ford-Hutchinson /J. Lipid Mediators Cell SignaIling I1 (1995) 231-240 Table 1 Protective lonephritis
effect
of MK-0591
C,, (~1 min-’ Control GN MK+ GN
46Ok9 386 + 16” 428 + 103b
on renal
100 g body wt-‘)
function
parameters
in a rat model
of experimental
235
glomeru-
BUN (mmol/l)
c ($/mitt)
Fe,, (%)
Cx (PI/mitt)
Fe, (%o)
5.8 + 0.3 8.2+0.5a 6.1 +0.4b
4.4OkO.72 4.29 f 0.38 3.12kO.35
0.311+0.045 0.375 f 0.044 0.266+0.030
488+41 423 + 63 514+26
35+3 40+4 40+2
C,,, creatinine clearance; BUN, blood urea nitrogen; CNa, clearance of sodium; FeNa, fractional fractional excretion of potassium. Renal excretion of sodium; C,, clearance of potassium; Fe,, function was measured on day 7 of the study. Values are expressed as the mean + SE with 6-7 rats per group. Staistical significance is expressed as “p < 0.01 for GN vs control; and “p < 0.05 for MK+ GN vs GN.
body wt- ’ (p < 0.01) in control and GN groups, respectively. BUN was not affected (data not shown). On day 7, a further decline in C,, occurred to 386 k 13 ~1 min-’ 100 g body wt-’ (p < 0.05) in the nephritic group, with a 50% elevation in BUN in the nephritic animals (p < 0.01) (Table 1). Co-administration of MK-0591 ameliorated this decline in filtration, (442 k 24 ~1 min-’ 100 g body wt - ‘) on day 1 (ns) and by improving C,, by 10% on day 7 (p < 0.03, and reducing BUN by 25% (p < 0.05) compared to the GN group. No significant changes in electrolyte excretion were evident in the GN or MK-0591 treated groups on day 7 compared to controls. MK-0591 treatment in control rats (administered pre-immune rabbit serum) produced no significant effects on renal function during the study interval (data not shown). Proteinuria was present in both nephritic and drug-treated groups compared to controls throughout the study interval. On day 7 urinary protein excretion rose from control values of 6.4 k 0.4 mg 24 h-i 100 g body wt- ’ to 301 f 24 mg 24 h- ’ 100 g body wt-’ in the GN group (p < 0.001) (Fig. 1). Inhibition of LT biosynthesis with MK-0591, however, resulted in a 50% reduction in proteinuria (p < 0.01). No histological improvement was evident with inhibition of LT biosynthesis in rats injected with nephrotoxic rabbit serum by light or electron microscopy. Both treated and untreated nephritic rats demonstrated basement membrane thickening, sub-endothelial electron-dense deposition and fusion of foot processes (data not shown). 3.2. LTC,
synthase activity and leukotriene excretion
Measurement of urinary LT excretion, however, indicated incomplete inhibition of LT biosynthesis, presumably in the kidney. Detectable levels of LTD, and NAcLTE,, but not LTC, were observed in the urine of 3/7 nephritic rats treated with MK-0591 on day 1. This group of animals also demonstrated detectable levels of NAcLTE, only, in the urine of 3/6 animals on day 7 (data not shown). Measurement of renal LTC, synthase activity in cortical microsomes showed a significant reduction in enzyme activity in GN rats, which was prevented by
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/J. Lipid Mediators Cell SignaIling 11 (1995) 231-240
CONTROL
GN
MK+GN
Fig. 1. Inhibition of leukotriene biosynthesis with MK-0.591 (60 mg kg-’ day-‘) reduced urinary protein excretion in nephritic rats. The significant proteinuria evident in the GN rats compared to controls (* * p < O.Ol), was reduced in MK+GN animals (#p < 0.01) compared to the GN group on day 7 of the study. Data are expressed as the mean f SE of 6-7 rats per group. ??
co-administration of MK-0591 (Fig. 2). Enzyme activity in the GN group dropped to 2.41 f 0.50 pmol LTC, formed min-’ mg protein-’ from control levels of 9.14 f 1.50 pmol LTC, formed min-’ mg protein-’ (p < 0.01). LTC, synthase activity in the MK-591 treated rats, however, rose to 6.41 f 1.10 pmol LTC, formed min-’ mg protein-’ (p < O.Ol>, a significant improvement compared to GN animals and not statistically different from control values.
CONTROL
ON
MK+GN
Fig. 2. Renal LTC, synthase activity measured in rat cortical microsomes. A significant reduction in enzyme activity was evident in the GN rats compared to controls ( * * p < 0.01) on day 7 of the study. LTC, synthase activity in the MK+ GN group, however, improved significantly ($p < 0.01) and was not statistically different from control values. Data represent the mean f SE of 6-7 assays each performed in triplicate.
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231
3.3. MK-0591 plasma concentrations
Kinetic studies to determine plasma concentration of MK-0591 were conducted. Blood levels of MK-0591 were achieved which have been previously shown to inhibit LT formation in the rat ex vivo and in vitro (Brideau et al., 1991). No difference was found in the kinetic profile of MK-0591 in nephritic and control rats over 24 h (data not shown), and is consistent with the known biliary excretion of MK-0591 and its metabolites (unpublished data).
4. Discussion Published data support a pathophysiological effect of cysteinyl LTs on renal function in models of experimental GN. Badr et al. (1988) have demonstrated an LTD,-mediated decrease in renal blood flow and a reduction in glomerular filtration (which was independent of perfusion) while LTC,/D,-dependent glomerular cell proliferation was demonstrated in vitro by Baud et al. (1985, 1989) and in vivo by Wu and Lianos (1993). In addition, both the hemodynamic and proliferative changes occurring in the nephritic rat were reduced or prevented by LT inhibition using either LTD, receptor antagonists or LT biosynthesis inhibitors. MK-0591, a quindole derivative of MK-886, was investigated for its renal protective effect during the development of nephrotoxic serum nephritis. This compound is an extremely potent inhibitor of LT biosynthesis in the rat as assessed by whole blood and pleurisy assays (Brideau et al., 19911, urinary excretion of LTE, and measurement of LTB, in rectal dialysis bags (Hilling@ et al., 1994). Co-administration of MK-0591 to rats injected with nephrotoxic rabbit serum resulted in a significant improvement in renal function. Glomerular filtration (as measured by C,, and BUN) was elevated and urinary protein excretion reduced by nearly 50%. Failure to achieve a greater degree of renal protection with MK-0591 treatment may be a consequence of several factors. The most probable explanation is that mediators other than 5-lipoxygenase-derived products contribute to the pathology. However, it can not be excluded that a greater degree of inhibition of LT biosynthesis locally in the kidney might have resulted in superior efficacy, particularly as incomplete inhibition of urinary cysteinyl LT excretion was observed in several animals on days 1 and 7, as described in the results section. In addition, inhibition of LT biosynthesis may have caused a shift in arachidonic acid metabolism toward the cycle-oxygenase pathway, resulting in enhanced prostanoid and thromboxane formation. Lianos et al. (1983) and Stork and Dunn (1985) have demonstrated elevated prostanoid synthesis in the early stage of disease development, therefore any additional increase of these eicosanoids could further exacerbate the derangement of renal hemodynamics already present. For example, the decrease in Fe Na present in the MK + GN group could have resulted from enhanced thromboxane synthesis, since thromboxane has been suggested to augment proximal tubular sodium reabsorption (Papanicolaou et al., 1987; Petric et al., 1990). Morphologically, no alleviation of the structural injury caused by
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injection of nephrotoxic rabbit serum was evident in MK-0591 treated rats, a finding consistent with other reports also demonstrating no alteration in immune deposition (Rahman et al., 1991) or leukocytic infiltration (Wu et al., 1993) in nephritic models. Of particular interest were the changes in renal microsomal LTC, synthase activity in the MK-0591 treated group. Whereas nephritic rats demonstrated a significant reduction in enzymic activity, no drop in enzyme activity was found in the MK-0591 group. LTC, synthase activity is negatively phosphorylated through a protein kinase C (PKC)-mediated event (Kargman et al., 1994; Ali et al., 1994). It has been suggested that such a mechanism may explain the alterations in LTC, synthase activity in nephritic kidneys (Petric and Ford-Hutchinson, 1994). Baud et al. (1989) have demonstrated an LTD,-mediated increase in PKC activity in glomerular epithelial cells while investigating the proliferative effects of LTD,. Given that renal LTC, synthase activity in rats treated with an LT biosynthesis inhibitor was not statistically different from controls, it is interesting to speculate whether LT autoregulation of enzyme activity is occurring. The early rise in LTC, synthase activity which we have described during the initial 24 h following injection of nephrotoxic rabbit serum, gives way to a decline in enzymic activity by day 7, and a gradual recovery thereafter (Petric et al., 1995). Whether the increase in enzyme activity evident on day 1 is also reversed by MK-0591 treatment remains to be determined. In summary, we have shown that LT biosynthesis inhibition provides a protective effect on renal function in a rat model of nephrotoxic serum nephritis. In addition, normalization of renal LTC, synthase activity in nephritic animals co-administered MK-0591 suggests a level of enzyme autoregulation, and provides a possible explanation for the rapid rise and fall in cysteinyl LT formation which has been described in nephritis.
Acknowledgements
This work was supported in part by the Medical Research Council Industrial Studentship award no. 1550. We wish to thank Richard Frenette and Serge Leger for the measurement of MK-0591 levels and Dr Donald Nicholson and Philip Tagari for advice on leukotriene measurements. We are also grateful to Dr Madeleine Chagnon, Bio Research Laboratories Ltd, Senneville, QC, for the pathological evaluation of histological samples, and to MS Faith Carr for assistance in the preparation of this manuscript.
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