Kidney International, Vol. 41(1992), pp. 1543—1548
Synthesis and metabolism of cysteinyl leukotrienes by the
isolated pig kidney
KEVIN P. MOORE, GRAHAM W. TAYLOR, CHRISTOPHER GOVE, JOHN WooD, KAI-CHAH TAN,
JOHN EASON, and ROGER WILLIAMS
The Institute of Liver Studies, Kings College School of Medicine and Dentistry, Denmark Hill; and Department of Clinical Pharmacology,
Royal Postgraduate Medical School, Du Cane Road, London, England, United Kingdom
Synthesis and metabolism of cysteinyl leukotrienes by the isolated pig
kidney. The metabolism and synthesis of cysteinyl leukotrienes by the
isolated perfused pig kidney has been investigated. Kidneys were
maintained for up to six hours in a recirculating perfusion system by
kidney has the capacity to generate leukotnenes. Assem and
Abduilah demonstrated increased LTC4 immunoreactivity following immunologic challenge in the isolated guinea pig kidney
[11], and more recently Merab and colleagues demonstrated
increased renal venous effluent levels of LTC4 and LTD4
following challenge with calcium ionophore (A23 187) in the
resulting in a urine output of between 20—180 mi/hr. Infusion of isolated rabbit kidney [12]. In this study we have demonstrated
3H-LTC4 into the renal artery resulted in rapid and complete metabothat the isolated pig kidney can both synthesize and metabolize
lism, with the major urinary metabolites comprising LTE4, io-hydroxyLTE4, w-carboxy-LTE4 and N-acetyl-w-hydroxy-LTE4. The capacity cysteinyl leukotrienes in the absence of circulating cells.
using an oxygenated Krebs-Henseleit buffer containing albumin and the
perfluorinated oxygen carrier, FC-43. Perfusion pressure was maintained at 12—13.5 kPa, with perfusion flow rates of 150—250 mllmin
of the isolated kidney to synthesize cysteinyl leukotrienes was monitored by measuring urinary LTE4 excretion; there was a basal urinary
excretion of LTE4 (median 43 pg/mm, range 8—470 pg/mm). Neither
lipopolysaccharide or human recombinant tumor necrosis factor alpha
had any effect on basal excretion. Treatment with the calcium ionophore A23 187, however, resulted in a 38.1 9.6-fold increase in urinary
LTE4 excretion. We conclude that the isolated pig kidney, in the
absence of circulating cells, can synthesize cysteinyl leukotrienes in the
absence of circulating cells, which can then undergo extensive oxidative metabolism.
Methods
Authentic standards of LTC4, D4 and E4 and their metabo-
lites were obtained from Cascade Biochem (University of
Reading, Berks, UK). Solvents for high performance liquid
chromatography (HPLC) were obtained from Rathburn (Peebleshire, UK). Calcium ionophore (A23 187, Novabiochem,
Notts, UK) was prepared as a 10 m solution in dimethyl
suiphoxide. Lipopolysaccharide (LPS, E.Coli 055.B5) was obtained from Sigma (Poole, UK) and human recombinant tumor
necrosis factor alpha (TNF) was a gift from Eurocetus, Amsterdam.
The cysteinyi leukotrienes (LTC4, D4 and E4) are potent
modulators of renal function, and when infused in vivo may
Isolated pig kidney model
reduce both renal blood flow and glomerular filtration [1—3].
This effect may be due to their vasoconstrictor action and
Pigs (20—25 kg) were anesthetized with halothane, ketamine
stimulation of mesangial cell contraction [4, 5]. The leuko- and fentanyl. The left or right kidney was exposed, and the
trienes have been implicated in the pathogenesis of a number of renal artery cannulated with a clamp time of less than 90
renal disorders, including nephrotoxic serum nephritis in the seconds. The kidney was perfused in situ at 37°C at approxirat, murine lupus nephritis, and hepatorenal syndrome in hu- mately 150 mllmin with oxygenated Gelofusine: Krebs-Hense-
mans [6—9]. It is unlikely that circulating leukotrienes have any leit buffer (4:1 vol/vol) until complete excision of the kidney and
direct renal effects. LTC4 and LTD4 are rapidly metabolized in clearing of red cells (less than 2 mm). The perfusate was then
blood to the weak spasmogen, LTE4, with the maximum changed to comprise Krebs-Henseleit buffer (2 parts) and FC 43
circulating levels of the LTE4 less than 7 pg/mi [8, 10]; even in (a perfluorinated oxygen carrier, Merck UK) (1 part), contain-
patients with hepatorenal syndrome, where substantial leukotnene synthesis occurs, maximal plasma levels of LTE4 are still
too low to have a direct effect on the kidney [8]. In order for
leukotrienes to influence renal hemodynamics, synthesis must
occur within the kidney, and there is now evidence that the
ing a final concentration of bovine serum albumin (3.2%),
glucose (10 mM), and Synthamin 19 (1%). The final concentra-
tion of glucose takes into account the glucose present in the
Synthamin. The perfusate was continuously gassed (95% 02,
5% C02) using a pediatric hollow fiber oxygenator (Dideco,
Masterfiow). The kidney was removed and immediately placed
in an organ bath at 37°C, suspended in netting in the perfusate.
Received for publication November 22, 1991
and in revised form January 16, 1992
Accepted for publication January 16, 1992
© 1992 by the International Society of Nephrology
Perfusion pressure was monitored with a pressure transducer
and maintained at 12 to 13.5 kPa. Perfusion was continued for
up to five hours; the effluent from the renal vein was allowed to
pass directly back into the organ bath from where it passed into
1543
1544
Moore et al: Renal synthesis and metabolism of leukotrienes
LTE4 0.18%; LTB4 <0.2%; arachidonic acid <0.1%. The
Ureter
Vein
Renal artery
antibody used in the assay has a useable range from 5—160 pg,
with 50% binding at —30 pg. The antibody is available from
Cascade Biochem (Reading, Berks, UK).
Immunoaffinity columns
Immunoafflaity columns were prepared according to the
method of Chiabrando et al [13]. Briefly, 2.4 g cyanogen
bromide activated sepharose 4B (Pierce Warmer) was washed
in 500 ml hydrochloric acid (1 mM). Two ml of the immunoglobulin fraction in 10 ml of sodium bicarbonate buffer (pH 8.3)
was shaken with activated sepharose for four hours at room
temperature. This was then washed with acetate buffer (pH
95% 02/5% CO2
4.0), followed by borate buffer (pH 8.0) and the remaining
÷[Mernbrane_oxygenator
active groups inactivated with 6% ethanolamine. The slurry was
Fig. 1. Schematic of the isolated pig kidney perfusion system. The washed with acetone:water (9:1) and stored in 25 ml 0.1 M
kidney is immersed in an organ bath at 37°C and perfused through the phosphate buffer before pouring into 2 ml Analytichem columns
renal artery at approximately 150 to 200 ml/min with oxygenated for use.
Urine
Krebs-Henseleit buffer containing bovine serum albumin, amino acids,
glucose and the perfluorinated oxygen carrier, FC43. Perfusion pressure was monitored and maintained at 12 to 13.5 kPa.
the oxygenator and recirculated into the artery (Fig. 1). The
recirculating system had a total volume of 1 liter, and after
each urine collection, the volume was replaced with an equal
volume of Krebs-Henseleit buffer diluted 1:2 with water. Approximately 30 minutes were allowed for the kidney to equili-
LTE4 extraction and assay
Renal production of cysteinyl-leukotrienes was assessed by
measuring urinary LTE4 excretion using HPLC-RIA. A known
volume of urine containing —8000 dpm [3H]-LTE4 (58 Ci!
mmole, Amersham International) as internal standard was
corrected to pH 7.4, centrifuged, and passed through an immunoaffinity column with an equal volume of phosphate buffer at
room temperature over 10 minutes. After loading, the column
was washed with 25 ml water. Elution was carried out by adding
0.75 ml acetonitrile:water (1:3) containing 0.6% concentrated
ammonia, left for 10 minutes, eluted with 0.75 ml acetone:water
(9:1), freeze dried, and resuspended in HPLC buffer. Samples
were then loaded for on-line extraction, and further purification
by HPLC in methanol:water on a Hypersil ODS column (Shan-
brate, after which time basal urine collections were made for up
to one hour before pharmacological intervention (LPS, TNF, or
A23187) or infusion of radiolabelled LTC4. Drugs were infused
into the outflow tract of the organ bath leading to the oxygenator and thence directly into the renal artery. These gave a final
concentration of approximately 10 ILM for calcium ionophore
don Assoc.) using a flow rate of 2 ml/minute as previously
A23 187 (0.1% DM50), 10 tg/ml for lipopolysaccharide and 10
ng!ml for TNF. Urine was collected at suitable intervals for reported [8, 14]. The elution position of LTE4 was identified
assay of LTE4 or identification of cysteinyl-leukotriene metab- from the radiolabeled internal standard and six 1 ml fractions
eluting around this time were collected for radioimmunoassay.
olites.
Immunoassay was carried out in duplicate using the antibody as
LTE4 antibody
Antibodies to LTE4 were prepared by immunization of New
Zealand White rabbits (pre-immunized with BCG) with a con-
jugate of LTE4-PPD (purified protein derivative, Ministry of
Agriculture, Food, and Fisheries, UK) synthesized using the
glutaraldehyde method, and in one rabbit resulted in a final
working titer of 1:64,000. The immunoglobulin fraction was
obtained by ammonium sulphate precipitation and dialysis
against buffer and water at 4°C. Any precipitate formed (mainly
lipoproteins) was discarded and the final solution diluted with a
one-third volume of 0.1 M phosphate buffer. An antiserum
described above. The presence of LTE4 was accepted only
when LTE4 immunoreactivity coeluted with the 5H-LTE4 inter-
nal standard. In each sample, urinary LTE4 was determined
following correction for background immunoreactivity, and
also for recovery of the 3H-labelled internal standard. Background immunoreactivity using this method is low (around 2
pg/tube).
Metabolite analysis
A 10 ml bolus of 3H-LTC4 in saline (1—2 tCi) was infused as
above and urine collected at 15 minute intervals up to 90
minutes. Urine samples were extracted on-line and purified by
munoassay (RIA) standard curves were generated using an HPLC on a Hypersil ODS column using an extended methanol:
antiserum dilution sufficient to give 30 to 50% binding. All water HPLC gradient as described previously for LTE4 metabassays were carried out in phosphate buffered saline with olite analysis [10]. Radioactivity in each fraction (0.75 ml) was
charcoal phase separation. A final dilution of the purified determined by scintillation counting in Optiphase safe (5 ml) on
antiserum giving a 40% bind with a B/B0 of 80%, and 20% for a 2000CA liquid scintillation counter (Packard Technology).
the S pg and 80 pg standards used in all subsequent assays. The
Results
cross reactivity of the antibody was as follows: LTC4 16%;
Renal synthesis and metabolism of the cysteinyl leukotrienes
LTD4 25%; LTE4 100%; 11-trans LTE4 32%; LTE4 sulphoxide
24%; N-acetyl LTE4 72%; w-hydroxy-LTE4 0.35%;
has been investigated in an isolated pig kidney system (Fig. 1);
dilution curve for the final solution was determined. Radioim-
1545
Moore et a!: Renal synthesis and metabolism of leukotrienes
a blood-free system was chosen to prevent non-renal leukotriene production or catabolism which is known to occur with
circulating cells. Sufficient oxygen delivery to the tissues was
achieved at a normal flow rate (150—200 ml/min) and perfusion
pressure (12—13.5 kPa) by the addition of the perfluorinated
oxygen carrier, FC43, to the perfusate. In the absence of FC43,
preliminary studies demonstrated that it was necessary to
increase flow rates to >500 ml/min to prevent tissue hypoxia,
and this was accompanied by an unacceptably high increase in
perfusion pressure. The isolated perfused pig kidney was stable
at 37°C for three to six hours and produced large volumes of
urine (20—180 ml/hr), which tended to increase with time.
Perfusion pressure was maintained at the above level, with a
gradual decrement in perfusion rate, indicating a progressive
increase in vascular resistance.
To determine the metabolic capacity of the kidney, [3H]LTC4 was infused into the renal artery in four separate preparations. Radioactivity was slowly excreted into urine, with 19 to
29% of the infused dose recovered within 90 minutes. Following
an initial screening using our standard HPLC system [141, it was
clear that extensive metabolism had occurred with little LTC4
or LTD4 found in urine. LTE4 was present together with a
number of polar and unresolved radiolabelled species. To
separate these metabolites, an extended HPLC gradient was
Table 1. LTC4 metabolite profile in four isolated pig kidneys
Pig I
LTC4
LTD4
LTE4
LTE4-sulphoxide
N-acetyl-LTE4
w-hydroxy-LTE4
w-carboxy-LTE4
N-acetyl-co-hydroxy-LTE4
N-acetyl-o-carboxy-LTE4
w-dinor-LTE4
w-tetranor-dihydro-LTE4
N-acetyl-&-dinor-LTE4
N-acetyl-u-tetranordihydro-LTE4
Unaccounted radiolabela
Total
Pig 2
Pig 3
Pig 4
0
0
0.1
0.1
10.6
3.9
2.9
2.3
50.0
0.5
0.2
7.5
1.0
0
1.4
1.8
0.4
0.9
26.0
0.2
0.8
4.2
10.0
14.3
13.2
18.2
3.1
0
5.8
6.5
2.4
7.6
5.9
4.1
5.4
27.0
35.7
38.5
29.3
100
100
100
100
2.8
9.3
6.8
3.9
13.0
3.8
3.2
3.6
0
3.2
2.1
1.2
1.0
1.3
1.1
1.2
0.8
All values in the table represent the percentage of each metabolite
excreted over 90 minutes. The relative amount of each metabolite
varied depending on the time following administration, with a gradual
decrease in urinary LTE4 which was accompanied by an increase in the
amount of o-oxidation products (Fig. 1).
a The unaccounted radioactivity constituted a generalized increase of
background counts which was not associated with any particular peak
developed [10] and the elution times of authentic LTC4 metabolites were determined: LTC4 47 minutes; LTD4 63 minutes;
radioimmunoassay. There was a basal urinary excretion of
LTE4 57 minutes; LTE4 sulphoxide 52.5 minutes; N-acetyl- LTE4, (median 43 pg/mm) and although there was a wide
LTE4 50 minutes; -hydroxy-LTE4 37.2 minutes; o-Carboxy- difference between the kidney preparations (range 8—470 pg/
LTE4 35.5 minutes; N-acetyl-w-hydroxy-LTE4 33.5 minutes; mm), this basal production was essentially constant for each
N-acetyl-w-Carboxy-LTE4 32.5 minutes; w-dinor-LTE4 29.5 tissue (Fig. 3a). Urinary LTE4 was not affected by including
minutes; N-acetyl-dinor-LTE4 28 minutes; w-tetranor-dihydroLTE4 26 minutes; N-acetyl-o-tetranor-dihydro-LTE4 25 minutes. All metabolite analyses were carried out on this improved
HPLC system and the retention times of the radioactive metabolites compared with those of standards, run at the beginning
and end of every four samples. There was considerable varia-
either lipopolysaccharide or TNF in the superfusate (Fig. 3b, c)
however, treatment with the calcium ionophore A23 187 caused
lites included w-carboxy-LTE4 (5.8—14.3%) and its dinor (3.6—
leukotrienes are extremely low [8, 14], and only when produced
9.6 fold) in urinary LTE4 in 4/5
a marked increase (38.1
kidneys tested (Fig. 4), and this was accompanied by a transient
increase in perfusion pressure (of between 36 and 53 mm Fig).
One kidney did not respond to ionophore, and both urinary
tion of oxidative metabolism in each of the four kidneys LTE4 and perfusion pressure remained constant.
studied. co-hydroxy-LTE4 was a major urinary metabolite exDiscussion
creted in three out of the four kidneys (representing 10.0%,
There is now compelling evidence to implicate the cysteinyl
18.2%, and 26.0% of radiolabel excreted over 90 mm), compared with LTE4 which accounted for 7.5%, 3.9%, and 10.6% of leukotrienes as causal agents in the pathogenesis of certain
total radioactivity in this time (Table 1). Other major metabo- forms of renal failure [1—3]. The circulating levels of the
9.3%) and dihydro-tetranor metabolites (0—6.8%). Trace locally within the kidney are these substances likely to modify
amounts of LTE4 suiphoxide were also present. N-acetyl LTE4 renal function. The biological effects of the cysteinyl leuko-
trienes will be controlled not only by the rate of synthesis, but
its co-oxidation products, N-acetyl-w-hydroxy LTE4 (3.8— also by the rate of metabolism and excretion. To investigate
these factors, we have developed an isolated perfused pig
13.2%) and N-acetyl-co-carboxy LTE4 (2.4—3 .2%). In the fourth
pig there was relatively little oxidative metabolism and LTE4 kidney model and examined the synthesis and metabolism of
represented 50% of radioactivity excreted over 90 minutes, and LTC4. The perfiuorinated oxygen carrier, FC43, was used to
this was the only pig to demonstrate any, albeit minor amounts, improve oxygen delivery, and enabled perfusion at physiologiof radiolabeled LTC4 or LTD4 in the urine. Over the perfusion cal flow rates and pressure in the absence of circulating cells.
period there was a gradual loss of LTE4 in all cases, and this The pig kidney was chosen for study (instead of the more
was accompanied by an increase in the proportion of polar readily available rat tissue) since porcine renal physiology and
systemic leukotriene metabolism more closely resemble those
metabolites in the urine (Fig. 2).
The appearance of LTE4 in urine following infusion of LTC4 in humans.
There is a basal production of the cysteinyl leukotrienes by
into the renal artery allows us to monitor renal production of
leukotrienes by immunoaffinity adsorption followed by HPLC- the kidney in the absence of circulating cells. lonophore chalwas also generated, (0.2—0.9%) together with larger amounts of
RIA of LTE4. We chose LTE4 since a radiolabel of this lenge leads to a mean 38-fold increase in LTE4 excretion. It was
compound was readily available and enabled measurement by
not clear why the single kidney did not respond to ionophore
1546
Moore et a!: Renal synthesis and metabolism of leukotrienes
9
3
i
864
10
:
A
75
'V
0.5-
1
2
!
A
0:
0.5
1
LPS
TNF
0.5-
C
0-
1
I
I
0
—-j
I
I
60
B
I
I
I
I
180
120
Time, minutes
Fig. 3. Basal urinary excretion of LTE4 remains stable in the absence
of (A) is not altered by infusion of either tumor necrosis factor (TNF)
(B) or lipopolysaccharide (LPS) (C) into the renal artery.
>.
>
0
0
4-
r
16 -
14
---
.1
I
C
-
4.
lonophore
ai
-l
0
20
40
1
0
0
120
180
240
Time, minutes
60
ml
Fig.
60
Fig. 4. Following stimulation with calcium ionophore A23187, there is
a marked increase in urinary LTE4 excretion by 4/5 isolated pig kidneys
(closed circles). No change in urinary LTE4 was observed in a single
kidney (open circles). Two kidneys had received either LPS or TNF
before ionophore challenge.
2. HPLC-radiochromatogram of urine (from pig 1) collected at
time 0 to 15 minutes (a), 30 to 45 minutes (b) and at 75 to 90 minutes (c)
following infusion of 3H-LTC4. There is a progressive decrease in the
proportion of LTE4 in urine with time formation of co-oxidized metabolites. The major metabolites are marked: (I) LTE4, (2) LTE4 sulphoxide, (3) N-acetyl-LTE4, (4) co-hydroxy-LTE4, (5) co-Carboxy-LTE4, (6)
N-acetyl-w-hydroxy-LTE4, (7) N-acetyl-w-Carboxy-LTE4, (8) co-dinorLTE4, (9) N-acetyl-dinor-LTE4: (10) w-tetranor-dihydro-LTE4: 26 minutes. LTC4 and LTD4 elute at 47 minutes and 63 minutes, respectively.
challenge, however, given that there are considerable differences in leukotriene metabolism between kidneys (Table 1), it is
bed [17]. There is evidence that the renal failure which accompanies endotoxemia can be ameliorated with leukotriene antag-
onists [18]. Many of the actions of endotoxins are mediated
through TNF, and it has been demonstrated that infusion of
TNF into rats [19] and humans [20] leads to an increase in
cysteinyl leukotriene production. Neither TNF nor lipopolysaccharide had any stimulatory effect on leukotriene production by
the kidney. These data are not consistent with a direct effect of
these substances on renal leukotriene production in vivo,
although we cannot exclude synergism of these agents with
possible that extensive and rapid metabolism of LTE4 has other cytokines or time-dependent effects through secondary
occurred in this case. The cellular source of leukotriene syn- mediators.
thesis is not known although there is evidence for 5-lipoxygenThe conversion of LTC4 through to LTE4 has been docuase activity within the glomerulus [15]. Other sources could mented by others in both rat renal cells 1211 and large vessels in
include tissue resident inflammatory cells [161 and the vascular
the pig [221 and is catalyzed by y-glutamyl transferase and renal
1547
Moore et a!: Renal synthesis and metabolism of leukotrienes
dipeptidase. In our perfused kidney preparation, renal metabolism of LTC4 was observed in all tissues with LTE4 appearing in
the urine. It is likely that LTE4 would also be the predominating
species in the perfusate, as indeed it is in the circulation in
humans [101. Although a proportion of LTE4 was cleared
unchanged, it was further converted to a number of metabolites, with w-oxidation, presumably through the action of cytochromes P450, as a major metabolic pathway in 3/4 kidneys.
The cellular site of this w-oxidation within the kidney is not
defined although it is known that the mammalian kidney possesses cytochromes P450 and is capable of catalyzing w and w-l
oxidations of fatty acids [23]. Only small amounts of N-acetyl
LTE4 were present in urine, although larger amounts of its
o-oxidation products, particularly N-acetyl-w-hydroxy LTE4
3. ALLEN DE GELLAI M: Hemodynarnic responses to leukotriene
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8.
were detected. This may be due to kinetic reasons, with
N-acetylation followed by rapid w-oxidation or through a differential effect of renal clearance on N-acetyl LTE4 and its
oxidative metabolites, In the fourth kidney, although perfusion
pressure and urine output were normal and there was no
evidence of hypoxia, a-oxidation was greatly reduced and the
major urinary metabolite was LTE4. As the enzyme activities of
y-glutamyl transferase and renal dipeptidase were unaffected,
there appears to have been some specific reduction of the
1990
4. GULBINS E, PAREKH N, RAUTERBERG EW, SCHLOTTMANN K,
MOORE KP, TAYLOR GW, MALTBY NH, SIEGERS D, FULLER RW,
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cytochrome P450 activity in this tissue. Metabolism by the pig
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cytochrome P450-mediated oxidation [25] and these differences
12.
may relate to a lack of a specific isozyme in the rat. KrebsHenseleit buffer has a poor oxygen carrying capacity, and it
may be that perfusion of the rat kidney with this buffer has
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13. CHIABRANDO C, BEGNINI A, PICCINELLI A, CARMINATI C, CozzI
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In man, intravenous infusion of LTC4 also results in the
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14.
TAYLOR GW, TAYLOR 1K, BLACK P, MALTBY NH, TURNER N,
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following infusion of 3H-LTC4 in humans, the proportion of
Metabolism of arachidonic acid via the lipoxygenase pathway in
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lower than that produced in the isolated kidney preparation. It 16. ASSEM ES, ABDULLAH NA, COWIE AG: Kidney mast cells, IgE
and release of inflammatory mediators capable of altering renal
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Acknowledgments
A preliminary account of this work was given at the Florence
Prostaglandins meeting in June 1990 127]. We are grateful to the Medical
Research Council, UK for supporting this work. KM was in receipt of
an MRC Training Fellowship. We would like to thank Dr. Brian Ross
for advice and encouragement in the establishment of this model.
Reprint requests to Dr. K. Moore, Department of Clinical Pharmacology, Royal Postgraduate Medical School, Du Cane Road, London
W12 ONN, England, United Kingdom.
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