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Increased production of cysteinyl leukotrienes in hepatorenal syndrome
263
Increased production
of cysteinyl leukotrienes
in hepatorenal
syndrome
The cyskinyl leukotrimes C, end D, arc potent renal vasoconstrictom which may modulate #omc~Iar hmction in viva, and may tberdore be important in the pathogencsis of hcpctorenal syodmme. Urinmy kokotricm I?,, the major met&mlite of leukotricnes C, and D,, was elevated in patients with hcpatoreoal syodrow (17.8 @I) when compared with oarmel controls (5.1 np ti, of subject;ri zb ~oi 1 fiilure alone (1.9 II.@). Urbmxy lcukotricnc E, was also ckvatcd in subjects with decompcosated liver disease (cirrhosis with escitcs 28.6 nglh, severe hcpatocclhdar dyshrnaioo 57.5 I@), but normal in compensated liver disease (6.7 nglb). In the early stages of hcpatorenal rywkome, leckotdene E, excretion rate wcs up to lW_fold higher (560 og&) then in nontxds. and fell in pamlkl with creatbdne ckamocc, iodicativc of the glomcndar tilt&on rate-dependent renal excretion. Following correction for creatinioe cleemnce, lcokotricnc E,, excretion was mosidcrebly higher in bcpatorcnal syndrome (54.1 p&d creadnine cleemmzo) mmparcd with mmnals (1.0 pghnl meatbdnc ckaranc.c). chronic reoai failure 13.2 anlml crcatbdne clearance). daomocosated liver disease fawites 7.7 disease (1.9 p&l crcadnioe clearance). interpret td, findii, we dct&edreoalhea and cndogcooos metabolism of the cysteioyl leokohicocs by infusion of pH]LukoUicoe C, into a sing!% scbjea with hcpetorenal syndrome end two mntml subjects. Renal dcarencc of leokobiene E,, was rcdoccd ic bepetorenal syndrome (2.4 ml&nio) compercd with controls (>17 ml/mbt) which together with the increased cxwctioo rate of leukotriene E, demonstretes that there is inacescd cysteioyl leckotrienc prodoction in hepatorenal syndrome. This may be one of the facmm involved in its pathogcocsis.
The hepatorenal syndrome may bc defined es renal feilore occurtiog ic patients with liver disease in the absence of say other idcatifiablc cause. It is character&d by vaw constriction of the renal arterioles causing reduced blood flow to the renal cortex, normal or non-specific renal histology and lowurine sodium (1,Z). Vasocoostritiion alone is unlikely to be the only dcterndnant of hepatorend syndrome, however, since many subjects with dcwmpcnsated liver disease, but relatively normal renal function, have renal blood flows comparable to subjects with bepatorenal syndrome (3). suggening that other factors mod-
ulatiog glomerclar function are likely to bc involved in its aetiology. There has been much recent ~pecelation on a role for the cimsaoid5,lculrotr*nes (LT) CJD. end dumnbuxaoe AZ (4-Q, as renal vasoconstrictom and modulatorsof glomender filtration. Info&m of LTC,or LrO, into animals, reduces both renal blood flow cod glomerular tiltration rate (GFR) (7.8). This latter cffcct may be doe, in part, to depression of the glomerular ultrafiltration coefficient, since LTDl is also a potent stimulus for mcsangial cell contraction which decreases the surface area of glomemli
& rapid (ll), with plana Ieve& ofcy&nyl imkMiaa llmmallyklowtbalimitd&~with~as8ny methodology (12). ln man. LTti, is srnetcd 81 a &able mcmbolitc in urine (13.14) and its ntwummetn is beofinviw licvedm9mvideatimeintegratedmeawm pradmc&dcyscdnylknk~(6,15).Tbiaisbt~ tnsttotbe¶ittJatioainMimab.,*t4liuycxrctim prdmtlbuIks,andiawhiibiliwyhwbbavebccnnad to monitor leukottiene pmdwtion ita viva (16,17). In the rmauredurinatyoxaetiond preaatstudywbava Lq in patients with hepatomnal syndmm! and a@atemntro~~todeter&ewheUwrulsrstievi-
265 sepsis (blwd, urine + asciric culture) was carried out in all patients in groups 3-7 at the time of study. On entering patients into the study, those with either determined or previously dmumented septicaemia, bacteraemia, bacterial peritonitis or urinary tract infection were excluded. No subject had had any gastrointestinal hemorrhage in the preceding 10 days. Timed urine collections of between 3 and 14 h (mean 7.5 h) were obtained, kept at 4 ‘C until completion and stored at -70 ‘C until assay. Urine was also collected every 12 h within 1 dav of admission to hooo&al from four subjects with acute alc?halic or fulminant~viml hepatitis, but normal wum cneatmine, and continued as they developed hepatorenal syndrome. Urine from these subjects and those with hepatorenal syndrome was collected via a urinary catheter. The sample collected from these sub. jects and wd for analysis for Figs. 1 and 4 was that obtained when the senun creatinine first exceeded MOpM. Clinical and laboratory data is shown in Table 1. The semm bilimbin and prothm!ttbin times were similar in those with hepatxenal syndrome (group 6) and the control group with severe hepatoeellular dysfunction (group 5) (p > 0.05). Chemicals HPLC grade mnhanol and water WOK purchased fmm Rathbum Chemicals, U.K.; synthetic leukotrienes were obtaiued from Cascade Biochom Ltd, University of Reading, U.K., [14,1S-3H]L’IE4 was England Nuclear (56 Wmmol).
from
Du
Pant-New
L TE, away Urinary LTE, was assayed by high-pressure liquid cbmmatography (HPLC)-radioimmunoassay (RIA) essentially as previously described (15.19). Urine samples were adjusted to pH 3.8 folkwing the addition of (=4tXHl dpm) [WLTE, as an internal standard and centrifuged
(15 min. 1500 x 8 at 4 “c); between 2 and 10 ml were loaded onto a Brownlee prewlumn containing MCHlO packing, washed with 20 ml of buffer, and switched online intotheHPLCs@em. HPLCwssperfom~edonaH~ersil column (5.0 x 250 mm, Spm particles) elutingwith a35 min linear gradient of methanollwater (5743 to 83:17) buffered to pH 3.8 with acetic acid (0.04%). ammonia (0.02%) and containing oxalic acid (0.5 mM) at a flow rate of 2 mUmin. The HPLC retention times of LTC,, LTD,, LTE, and &cetyl-LTE, were 16, 27, 22.5 and 18.5 nin, respectively. One ml fmctiom were ootlected and the solvent was removed under vacuum: the VBCUU~was hraken with argon and resuspended in 3CQ@ of assay buffer of which duplicate 95 /II samples were used for radioimmunoassay (Amersham leukotriene Q/D&, ‘H-assay system) and 95 PI used to determine recovery by scintillation counting. Mean recovery of LTE4 from 82 samples foIlowing HPLC and resuspension in assay buffer was 40.5% + 1.5 (SE.). Radioimmunoassay was carried out in duplicate in 6-8 fractions around the LTE, elution position and the presence of LTE4 ‘YBSaccepted only when there was a rise and fall of immtmoreactivity coeluting with the internal standard. Urinary LTE, was calculated followbtg correction for background immunoreactivity, recovery of the 3H-labelled internal standard and 64% cross-reactivity with respect tea the LTC, standard pm vided in the kit. Inter- and intra-away variations were 16% and 12% in normal urine, but was somewhat higher in a sample from a deeply jaundiced patient at 25.1% and 23.6%. There was no decrease in the urinary LTQ concentration in one sample stored at -70 OC over a Cmonth period (131-126 ngWIO ml). Urine from a severely jaundiced patient wa9 subjected to HPLC and radioimmunoassay was carried out at different dilutions (l:l, 1x2 and 1:4): LTEl was determined as 389, 316 and 336 pg per sample, demonstrating that there is unlikely to be a meluting species pat-HPLC which could affect ligand bind-
75.0 + 11.5 51.9 + 4.8 76.5 f 4.3 60.3 e 6.1 81.3 * 60.8 f
*ct.*
x&0*31.7 413.llf65.9
N.D. 0.09 f Cl.05 0.20
11.1 0.56 f
9.1 f 3.3 15.2 * 3.1
40.0- L?o.ll 75-120
0.24 + 0.06 N.D. -
K.P. MGCIRE et al.
266 ing to the antibody. The identity of immunoreactive LTE4 was confirmed by derivatisatio”. A sampleof urine from a patient with hepatorenal syndrome was extracted and chromatographedon HPLC. The fractions containing immunoreactive LTEl were pooled and N-acetylated (20). The sample was recbromatographedon HPLC: over 90% of immunoreactivity now coeluted with authentic N-acety,-LiE,. LTB, was expressedeither as excretion rate (“g/h) or adjus:rd to take account of creatinine clearance (Cr.Ci.) using the formula below:
Biochenkwy This was determined by a Technicon autoanalyser, urine creatinine by a modified Jaffe reaction (Signta kit) on a” LKR Ultraspec 4050, and urine sodium by flame photometry. staristics All results are reported as mea” or median values where indicated. Intergroup mmptiso” was carried out with a Mann-Whitney U-test unlessotherwise stated. Cm. relations were carried out by the Speanna” rank test. Differenceswere consideredto be significant if p < 0.05.
LTE~(pg/min)lCr.Cl. (mUmin) The units are expressed as pglml creaiinine clearance. Correction for urinary creatinine alone in subjects with venalimpairment is not valid.
Ethical approval for these studies was obtained from the Ethics committee at King’s College Hospital.
LTC, infusion study A single female subject (A) with hepatorenal syndrome and two mate subjects(B,C) with biliary obstruction due to a gall stone and a T tube in situ placed 4-6 weeks previously were studied. Subject A (aged 53 years) had altoholic hepatitis (bilirubi” 556 FM, prathrombin time 25 s) and hepatorenal syndrome (serum creatinine 274 &f, urine sodium i3 mM). Subject B (aged 27 years) had pmtal vein thrombosis, but normal liver and renal function tests, and subject C (aged 63 years) was otherwise normal. All gaveinfomted eonsent for an infusion with 3.5-7 #Ci of [‘HILTC, over 1 h. Urine was collected at O-l h, I-4 h, 4-S h, 8-24 hand then daily up to72 hforsubjects B and C. Complete hourly collections were obtained for the patient with hepatorenal syndrome (A) through a urinary catheter for the first 4 h, and then at 8 h, 16 h, 24 h, 48 h and 72 h. Bile was collected from subjectsB and C a?
Results
the same time points as urine samples. Samples were stored at -70 ‘Cc. Blood was obtained through a” indwelling arterial cannula in subject A, and collected into a serinemorate-L-cyrteine-heparin (19) mixture an ice. Blood sampleswere collected at 30, MI and 90 min. centrifuged at 4 ‘C within 15 min and plasma deproteinized with 4 vols. of ice-cold aqueousmethanol @I%, containing 0.5 mM EDTA, 1 mM hydroxyTEMP0, pH 7.4) (11). Sampies were stored at -70 “C under argon, and solventswere removed under vacuum before assav.Total recovetv of infused radioactivity was determined by scintillation counting following quench correction; samplesof bile and urine were also freeze dried to remove v&tiles (e.g.,
: water)
tri-
tiate and the rccovery of non-volatile radioactitity was determined. Plasma and urine were extracted on line as described above and the HPLC radio-profile was compared with authenticatedstandards.
Excretion rate of LTEd was significantly higher in hepatorenal syndrome (group 6, median 17.8 “g/h, range 3.2-104 ngih) compared with “ommls (group 1.5.8 ngih,
‘“I
0.8-16.7 n&) and patients with renal failure alone (group 7. 1.9 ngih, O-7.7 ttgl$ (~2< 0.001). Patients with
tion. Data relating to this were obtained in four patients followed serially throughout the development of hepato-
chronic renal failure had a significantly lower LTE, excxetion rate compared with alI other groups (p < 0.05). Patients with hepatorenal syndrome also had a higher excretion rate compared with compensated liver disease (group 2, 6.7 tt@t, 2.1-17.9 rig/h)) @ < O.OS), but were ttot significantly different from obstructive jaundice (group 3, 26.4 n#h, 14.4-41.3 no), or those with ascites (group 4, 28.6 npm, 16.3-44.6 @I) @ > 0.05). The excretion rate of LTEa was, however, significantiy higher in those with severe hepatocelhdar dysfunction fgrovp 5, 57.5 ngh, 27.5-90 nglh). eanpared with all other groups @ < O.U2) (Fig. 1). Ir. normal stibjects, there was a weak positive cnt’rel~tion between urinary flow rate and LTE, excretion
renal syndrome (Fig. Za-d). Urinary LTE, excretion was markedly elevated (45-560 rig/h)) in the first samples collected and at a” early stage during the development of renal failure (creatinine clearances of 39-80 mumin), and fell approximately in parallel with creatinbte clearance indicative of GFR-dependent exaeticm. One patient subse-
rate (I, = 0.55,~ < iN.tSj such that the urinary LTEa excretion rate increased with urine flow suggesting that tw bttiar reabsor~ticm occurs.
was plotted against LTE, excretion for all patients with developing or established hepatorenal syndrome (Fig. 3). Tbe data for normal cmnrols and tbae with severe hep atwellular dystimctkm are also &own in Fis. 3. After adjustment cd LTE, excxetkm for creatinine clearance (Fig. 4). patients with hepatorenal syndrome bad the highest ratio of LTE.#Zr.Cl (median 41.1 pg/ml Cr.‘& rfmp 11.5-242.1) and this was significantly different from all other groups: normals (1.0 pghnl Cr.Cl,
Expression of the results as absolute urinary excretion rates @gilt) does not take into account the possible effects of varying degrees of renal impairment on LTEd exert-
quently recovered: folkming a 2-&y period of aria, creatinine clearance inereascd to 49 mlltttin simultaneous with recovery from Liver failure. Urinary LTEa excretion increased to 54 ng!h at this time (Fig. 2~). and was below that o&wed at an early stage of renal failvre (150 “g/h. crearininc cleara~lcr 33 minnin). A linear correlation (r, = 0.86, p < O.Wl) was observed when ereatinine clearance
serum bilirubin and LTE,excr&m
(r, = 0.6L.p < 0.0X).
LTC, in$i&onr Following infusion of [3H]LTC, into the three subjects between 27.5 and 43% of total radioactivity appeared in urine within 72 h. Tritiated water (determined as total volatile radioactivity and formed through o.oxidati”n 01 the (14,15-8) radiolabel) was present in the urine of all three subjects, accounting for 3%. 4.5% and 8.5% of the infused dose ior s$bj:cts A, B and C, respeaively. The major metabolite present in urine was LTE,, xcountiug for 3.5-4.4% of total radioactivity infused wer 24 h (Fig.
0.13-X86), conpensated liver disease (1.9 pg/ml Cr.CI, OS-6.67), obstructive jaundice (8.5 pg/ml Cr.CI, 5.0-15.0). ascites (7.7 pglml C.Cr, 3.8-15.2), severe hepatocellulat dysfunction (11.0 pg!ml C&I, 6.5-42.4), and chronic renal failure (3.2 pglml Cr.CI, O-6.9) (p c: 0.005). Following this correction for renal function, the group with renal failure (resulting in low excretion rates) now became similar to nomml controls. Effectof liverfunction To assess the effects of liver dysfunction
per se on cys-
teinyl leukotriene production, all subjects with liver disease but without hepatorenal syndrome (groups 2,4 and 5) were grouped together; following correction for renal function there was a significant correlation between LTEI excretion and plasma bilirubin (I, = 0.61, p < O.Wl). In order to take into account the contribution of liver dysfunction, and BSSOSSthe additive effect of hepatorensl syndrome on cysteinyl leukatriene production, the values for group 6 “we also plotted on the same graph (Fig. 5). This gave a higher parallel line of regression indicative of higher LTE, excretion for a given plasma bilirubin.
6). LTE, excretion was considerably delayed in the subject with hepatorenal syndrome, suggesting reduced renal clearance. In the two subjects (B,C) with biliary T-tuber in situ, large volumes of bile (lWO-lZCN3 ml) were mllected over 72 h; 21% and 27.5% of the total tadiaactivity inhrsed was recovered. In the patient with hepatorenal syndrome. HPLC analysis of arterial bled during infusion of [‘H]LTC, showed that LT& wan the major radiolabelled species at all time points (>%I%), with fess than 5% fwxe”t as LTC, or LTD,,. For~oe~~~ed~o~~to IMmin, the antouot of [%jLl’& excreted was 84 @IO dpm. Using the midpoint arledzd pla~rna wncentration of [‘H]LTE, at this time (615 dpmM) and conventional cleaxsnce formulae, the renal clearame of LT& is detotmiwd as 2.3 mUmin. This was lower than the measured creatinine clearance of 10.1 mUmin, in keep+ with tubular reabsorption of LT&. The measured excretion rate of LT& in this subject was 54 @It. Awmting that the contribution of the kidney to urinary LTB, is minimal, Uwt by using the detemtined clearance of radiolabelM LTEd and measured mt~enous f.TE& excretion one can back-catcutate the maximal art&al ptatma fevef. i.e., 3% p$tnL fn the two normaf &jects iltfuwd with [%i]LTC* tile plasam (IHjLTE, was mo low for detetmination, therefore the renal clearance lyas determined using the excredon rate over the l-h infusion period (1.4 and 3.5% as [“HjLTF$, and assuming a renal plasma flow of 600 mt/min with 50% of arterial radiowtivily ii LTE,. The minimum renal clearao~s in these two subjects were eslimated as 17 and 42 mlhnin (renal ckaranm = renal plasma flow x fraction extraeted bv the kidnw’). With a nor-
accurate
that the urinary emtion rate of LTE, is increased in patients witb hepatomnai syndrome aswell as thosewith obstructive jaundice and dewmpensated liver disease. This confirms the recent report by Huber and colleagues (6). and extendstheir obwvations to include controls with severe hepatic dysfunction or rend Failure alone.. Funher, the effects of renal failure on elimination kinetics of the cysteinyl leukotrienes have also been characterised. Tois study was designed to control for most variables which reflect the different circmnstawes oaxrring in hepatoreoai syndrome. Renal failure atone leads to a reduction in the LTf5 excretion rate, which Norma when emrated for creatiaioe cleamnce. Reduced bit&y clearance and bepatic metabolism would lead to an increased plasma LT!& umcentmtion and thus increased excretion rate. This is clearly seen in the diseased liver mntrol groups, particularly in scvere hepatoc&dar dysfunction. Each of these groups have relatively normal renal function, and presumably normal renal cleamoce of LTE* When renal ftmction is taken into acmunt, however, by correcting for creatittiw clearance, LTE, excretion was elevated in decompensated liver disease and hepatorettai syndnnne. This suggests that increased syntLxe& of Ll& occurs in demmpensated liver disease sod hepatoretrai syndrome, but when as+es& by excretion rate aloae, tI& is masked in the lanergroup bytbeeffeaof renal faiiua. Front the intitsioo data on the patient with hefWwntl syndrome, we have &own that renal clearance of LTE,4 is reduced compared with normals. The number of subjects studied by infwion of [WLTC, was small, hut further sopportiq evidence wa the observed Parallel fall of eb oogenous LTE, excretion and creatinine clearawe as subjects developed hepatorenal syndrome (Fig. 4). A reduction in renal clearance w.Md reduee the rate of o&tary LTE, exuetion. In xmtrast, we have obsenwl an inaeased exation rate of LTE, in kepatorena! syndrome (17.8 o#tt) campared with normals (5.1 II&). Thus, we eon&de that there is itweaed qsteinyl ieok&me production in hepatozenal syndrome compared with ttomtal subjects and, assoming Lm eiiiation is proportional to GFR, is also incremed with respect to disease cimtrol
Urinary LTB, excretion has been used to monilor cysteittyl leukotriene production since it is rapidly produced from LX, in viva, and is a stable identifiable wbwy metaboliie providing a time-integzated t&w produetioion (6,13-15). Our
approach to fettkoresutrs demonstrate
Poops. The site of incmaxd eysteinyi leukottiene production in patients with hepatorenal syndrome is not known, altboogh a variety of circulating and tiwe resident inflammatory ceus inckJding IelKaywS, fer o&s will produca leokotrienes have recently demo&rated
maaopbages and Kopfin vitro (4). Two sh&?s
cystcinyt fetikatiene
tion by the isolated kidney (22X%
pmdttc-
bat it is itot known
270
whether the glomeruli are capable of synthesisingcysteinyl leukotrienes. The presenceof 5.lipoxygenase (24) in glomeruli, as well as the known action of LTD, on the mesangiumand the effect of leukotrieee antagonistson ret serum nephritis make it likely that synthetic capacity exists (9.10.25). Further, recent studies have shown that human arterial rings can synrhesisecysteinyl leukotrienes when stimulated by the calcium ionophore A23187 (26).
,.
We have estimated tbe maximum circulating level of LTJZI in one reprerentative subject with hepatorenal syndrome at 390 pglod (0.9 nM). This calculation is basedon the unlikely assumptionthat the kidney does no wntribute to urinary LTEI, and therefore representsthe highest possible plasma levels that may be oixswd. Such concentrations of circulating Ll& would correspond with less than 50 pM of the more biologically active species (LTC, or LTD& Such concentrationsare too low to have an effect on the kidney, 1s severa! studies have demonstrated that concentrations of hetwen 1 and 10 nM of LTD+ are required to affect rena! Iunction (27,28). Forther, hepatorenal syndrome and decompensatedliver dir ease are character&d by systemicvasodilatation; it is unlikely therefore that circulating leukotrienes have any significant systemichemadynamic effects, Clearly, the local concentrationsof LTC, and LTD, are the final arbiters of whether or not there is a pharmacological action. If urinary LTEn is, at least partly, representative of renal leukotriene production, then thesemediatorscould be one of the important modulators of glomerular hemodynamics and function in viva in hepatorenal syndmme. Tbe mechanismsby which increased svnthesisof cvsteinyl kokootrieneso&r in hepatorenal syndmme are not known. We have recently shown that urinary LTE, is increased in patients with asthma, whrre kokotriene synthesisis stimulated in an lgB-dependent manner (15). In hepatorenal syndrome other primary mechanismsprobably operate, and activate kukotriene formation io other sites such as the kidney. One potential factor that mold increase leukotriene formation includes endotoxins, which are frequently present in the systemiccirculation of
patients with hepatorenal syndrome and decompensated liver diseaseeven in the absenceof demonstrable sepsis (‘26-28). Infusion of endotoxins into the rat increasesbiliary cysteinyl lcukotriene excretion arising presumably from increased kukotriene production (17,32). Endotoxin infusion into the rat reducesthe renal blood flow and the glomendar filtration rate, the effects of which are ameliorated by antagonistsof the cysteinyl leukotrienes C, and D,, and thmmboxane A, (33). Recent studieshave also demonstrated that chronic endotoxemia in rats inaeest5 the free arachidonic acid pool, thus providing a further mechanismby which general eicosanoid synthesis is increased (34). Other potential agents include tomoor necrosis factor, whose formation by peripheral monocytes is increased in certain liver diseases (35.36). and may act through activating phospholipw AZ. We have demonstratedthat there is marked inerase in cysteinyl leukotriene production in bepatorenal syw drome, although circulatbq levels of these mediators are too low to have significant renal 01 hemodyxmmiceffects. Local production within the kidney, however, could resolt in significant renal effects. Without the use of suitable antagonists,it is not possibleto demonstrate a causalrole of cysteinyl leukotrienes in the patbogenesisof hepatorenal syodmme, although the availability of leukotriene antagonists opens a new area of pmsible therapeutic intervenlion.
ACk”OUtCdgC~“b
We are grateful to Mr. 1. Dawon and Drs. V. Pamoos and D. Tauhe for allowing us to study their patients, Mr. M. Shipley (London School of Hygiene and Tropical Medicine) for advice on statistical analysis, and Dr. Alan Boohis end Professor D. Davies for helpful advice on the pharmacokinetics. Tlds work was supported by the Medical Research Council and the Nuffield Foundation. K.M. is io rueipt of an MUC Training Fellowship.
Increased production
of cysteinyl leukotrienes
in hepatorenal
syndrome
The cyskinyl leukotrimes C, end D, arc potent renal vasoconstrictom which may modulate #omc~Iar hmction in viva, and may tberdore be important in the pathogencsis of hcpctorenal syodmme. Urinmy kokotricm I?,, the major met&mlite of leukotricnes C, and D,, was elevated in patients with hcpatoreoal syodrow (17.8 @I) when compared with oarmel controls (5.1 np ti, of subject;ri zb ~oi 1 fiilure alone (1.9 II.@). Urbmxy lcukotricnc E, was also ckvatcd in subjects with decompcosated liver disease (cirrhosis with escitcs 28.6 nglh, severe hcpatocclhdar dyshrnaioo 57.5 I@), but normal in compensated liver disease (6.7 nglb). In the early stages of hcpatorenal rywkome, leckotdene E, excretion rate wcs up to lW_fold higher (560 og&) then in nontxds. and fell in pamlkl with creatbdne ckamocc, iodicativc of the glomcndar tilt&on rate-dependent renal excretion. Following correction for creatinioe cleemnce, lcokotricnc E,, excretion was mosidcrebly higher in bcpatorcnal syndrome (54.1 p&d creadnine cleemmzo) mmparcd with mmnals (1.0 pghnl meatbdnc ckaranc.c). chronic reoai failure 13.2 anlml crcatbdne clearance). daomocosated liver disease fawites 7.7 disease (1.9 p&l crcadnioe clearance). interpret td, findii, we dct&edreoalhea and cndogcooos metabolism of the cysteioyl leokohicocs by infusion of pH]LukoUicoe C, into a sing!% scbjea with hcpetorenal syndrome end two mntml subjects. Renal dcarencc of leokobiene E,, was rcdoccd ic bepetorenal syndrome (2.4 ml&nio) compercd with controls (>17 ml/mbt) which together with the increased cxwctioo rate of leukotriene E, demonstretes that there is inacescd cysteioyl leckotrienc prodoction in hepatorenal syndrome. This may be one of the facmm involved in its pathogcocsis.
The hepatorenal syndrome may bc defined es renal feilore occurtiog ic patients with liver disease in the absence of say other idcatifiablc cause. It is character&d by vaw constriction of the renal arterioles causing reduced blood flow to the renal cortex, normal or non-specific renal histology and lowurine sodium (1,Z). Vasocoostritiion alone is unlikely to be the only dcterndnant of hepatorend syndrome, however, since many subjects with dcwmpcnsated liver disease, but relatively normal renal function, have renal blood flows comparable to subjects with bepatorenal syndrome (3). suggening that other factors mod-
ulatiog glomerclar function are likely to bc involved in its aetiology. There has been much recent ~pecelation on a role for the cimsaoid5,lculrotr*nes (LT) CJD. end dumnbuxaoe AZ (4-Q, as renal vasoconstrictom and modulatorsof glomender filtration. Info&m of LTC,or LrO, into animals, reduces both renal blood flow cod glomerular tiltration rate (GFR) (7.8). This latter cffcct may be doe, in part, to depression of the glomerular ultrafiltration coefficient, since LTDl is also a potent stimulus for mcsangial cell contraction which decreases the surface area of glomemli
& rapid (ll), with plana Ieve& ofcy&nyl imkMiaa llmmallyklowtbalimitd&~with~as8ny methodology (12). ln man. LTti, is srnetcd 81 a &able mcmbolitc in urine (13.14) and its ntwummetn is beofinviw licvedm9mvideatimeintegratedmeawm pradmc&dcyscdnylknk~(6,15).Tbiaisbt~ tnsttotbe¶ittJatioainMimab.,*t4liuycxrctim prdmtlbuIks,andiawhiibiliwyhwbbavebccnnad to monitor leukottiene pmdwtion ita viva (16,17). In the rmauredurinatyoxaetiond preaatstudywbava Lq in patients with hepatomnal syndmm! and a@atemntro~~todeter&ewheUwrulsrstievi-
265 sepsis (blwd, urine + asciric culture) was carried out in all patients in groups 3-7 at the time of study. On entering patients into the study, those with either determined or previously dmumented septicaemia, bacteraemia, bacterial peritonitis or urinary tract infection were excluded. No subject had had any gastrointestinal hemorrhage in the preceding 10 days. Timed urine collections of between 3 and 14 h (mean 7.5 h) were obtained, kept at 4 ‘C until completion and stored at -70 ‘C until assay. Urine was also collected every 12 h within 1 dav of admission to hooo&al from four subjects with acute alc?halic or fulminant~viml hepatitis, but normal wum cneatmine, and continued as they developed hepatorenal syndrome. Urine from these subjects and those with hepatorenal syndrome was collected via a urinary catheter. The sample collected from these sub. jects and wd for analysis for Figs. 1 and 4 was that obtained when the senun creatinine first exceeded MOpM. Clinical and laboratory data is shown in Table 1. The semm bilimbin and prothm!ttbin times were similar in those with hepatxenal syndrome (group 6) and the control group with severe hepatoeellular dysfunction (group 5) (p > 0.05). Chemicals HPLC grade mnhanol and water WOK purchased fmm Rathbum Chemicals, U.K.; synthetic leukotrienes were obtaiued from Cascade Biochom Ltd, University of Reading, U.K., [14,1S-3H]L’IE4 was England Nuclear (56 Wmmol).
from
Du
Pant-New
L TE, away Urinary LTE, was assayed by high-pressure liquid cbmmatography (HPLC)-radioimmunoassay (RIA) essentially as previously described (15.19). Urine samples were adjusted to pH 3.8 folkwing the addition of (=4tXHl dpm) [WLTE, as an internal standard and centrifuged
(15 min. 1500 x 8 at 4 “c); between 2 and 10 ml were loaded onto a Brownlee prewlumn containing MCHlO packing, washed with 20 ml of buffer, and switched online intotheHPLCs@em. HPLCwssperfom~edonaH~ersil column (5.0 x 250 mm, Spm particles) elutingwith a35 min linear gradient of methanollwater (5743 to 83:17) buffered to pH 3.8 with acetic acid (0.04%). ammonia (0.02%) and containing oxalic acid (0.5 mM) at a flow rate of 2 mUmin. The HPLC retention times of LTC,, LTD,, LTE, and &cetyl-LTE, were 16, 27, 22.5 and 18.5 nin, respectively. One ml fmctiom were ootlected and the solvent was removed under vacuum: the VBCUU~was hraken with argon and resuspended in 3CQ@ of assay buffer of which duplicate 95 /II samples were used for radioimmunoassay (Amersham leukotriene Q/D&, ‘H-assay system) and 95 PI used to determine recovery by scintillation counting. Mean recovery of LTE4 from 82 samples foIlowing HPLC and resuspension in assay buffer was 40.5% + 1.5 (SE.). Radioimmunoassay was carried out in duplicate in 6-8 fractions around the LTE, elution position and the presence of LTE4 ‘YBSaccepted only when there was a rise and fall of immtmoreactivity coeluting with the internal standard. Urinary LTE, was calculated followbtg correction for background immunoreactivity, recovery of the 3H-labelled internal standard and 64% cross-reactivity with respect tea the LTC, standard pm vided in the kit. Inter- and intra-away variations were 16% and 12% in normal urine, but was somewhat higher in a sample from a deeply jaundiced patient at 25.1% and 23.6%. There was no decrease in the urinary LTQ concentration in one sample stored at -70 OC over a Cmonth period (131-126 ngWIO ml). Urine from a severely jaundiced patient wa9 subjected to HPLC and radioimmunoassay was carried out at different dilutions (l:l, 1x2 and 1:4): LTEl was determined as 389, 316 and 336 pg per sample, demonstrating that there is unlikely to be a meluting species pat-HPLC which could affect ligand bind-
75.0 + 11.5 51.9 + 4.8 76.5 f 4.3 60.3 e 6.1 81.3 * 60.8 f
*ct.*
x&0*31.7 413.llf65.9
N.D. 0.09 f Cl.05 0.20
11.1 0.56 f
9.1 f 3.3 15.2 * 3.1
40.0- L?o.ll 75-120
0.24 + 0.06 N.D. -
K.P. MGCIRE et al.
266 ing to the antibody. The identity of immunoreactive LTE4 was confirmed by derivatisatio”. A sampleof urine from a patient with hepatorenal syndrome was extracted and chromatographedon HPLC. The fractions containing immunoreactive LTEl were pooled and N-acetylated (20). The sample was recbromatographedon HPLC: over 90% of immunoreactivity now coeluted with authentic N-acety,-LiE,. LTB, was expressedeither as excretion rate (“g/h) or adjus:rd to take account of creatinine clearance (Cr.Ci.) using the formula below:
Biochenkwy This was determined by a Technicon autoanalyser, urine creatinine by a modified Jaffe reaction (Signta kit) on a” LKR Ultraspec 4050, and urine sodium by flame photometry. staristics All results are reported as mea” or median values where indicated. Intergroup mmptiso” was carried out with a Mann-Whitney U-test unlessotherwise stated. Cm. relations were carried out by the Speanna” rank test. Differenceswere consideredto be significant if p < 0.05.
LTE~(pg/min)lCr.Cl. (mUmin) The units are expressed as pglml creaiinine clearance. Correction for urinary creatinine alone in subjects with venalimpairment is not valid.
Ethical approval for these studies was obtained from the Ethics committee at King’s College Hospital.
LTC, infusion study A single female subject (A) with hepatorenal syndrome and two mate subjects(B,C) with biliary obstruction due to a gall stone and a T tube in situ placed 4-6 weeks previously were studied. Subject A (aged 53 years) had altoholic hepatitis (bilirubi” 556 FM, prathrombin time 25 s) and hepatorenal syndrome (serum creatinine 274 &f, urine sodium i3 mM). Subject B (aged 27 years) had pmtal vein thrombosis, but normal liver and renal function tests, and subject C (aged 63 years) was otherwise normal. All gaveinfomted eonsent for an infusion with 3.5-7 #Ci of [‘HILTC, over 1 h. Urine was collected at O-l h, I-4 h, 4-S h, 8-24 hand then daily up to72 hforsubjects B and C. Complete hourly collections were obtained for the patient with hepatorenal syndrome (A) through a urinary catheter for the first 4 h, and then at 8 h, 16 h, 24 h, 48 h and 72 h. Bile was collected from subjectsB and C a?
Results
the same time points as urine samples. Samples were stored at -70 ‘Cc. Blood was obtained through a” indwelling arterial cannula in subject A, and collected into a serinemorate-L-cyrteine-heparin (19) mixture an ice. Blood sampleswere collected at 30, MI and 90 min. centrifuged at 4 ‘C within 15 min and plasma deproteinized with 4 vols. of ice-cold aqueousmethanol @I%, containing 0.5 mM EDTA, 1 mM hydroxyTEMP0, pH 7.4) (11). Sampies were stored at -70 “C under argon, and solventswere removed under vacuum before assav.Total recovetv of infused radioactivity was determined by scintillation counting following quench correction; samplesof bile and urine were also freeze dried to remove v&tiles (e.g.,
: water)
tri-
tiate and the rccovery of non-volatile radioactitity was determined. Plasma and urine were extracted on line as described above and the HPLC radio-profile was compared with authenticatedstandards.
Excretion rate of LTEd was significantly higher in hepatorenal syndrome (group 6, median 17.8 “g/h, range 3.2-104 ngih) compared with “ommls (group 1.5.8 ngih,
‘“I
0.8-16.7 n&) and patients with renal failure alone (group 7. 1.9 ngih, O-7.7 ttgl$ (~2< 0.001). Patients with
tion. Data relating to this were obtained in four patients followed serially throughout the development of hepato-
chronic renal failure had a significantly lower LTE, excxetion rate compared with alI other groups (p < 0.05). Patients with hepatorenal syndrome also had a higher excretion rate compared with compensated liver disease (group 2, 6.7 tt@t, 2.1-17.9 rig/h)) @ < O.OS), but were ttot significantly different from obstructive jaundice (group 3, 26.4 n#h, 14.4-41.3 no), or those with ascites (group 4, 28.6 npm, 16.3-44.6 @I) @ > 0.05). The excretion rate of LTEa was, however, significantiy higher in those with severe hepatocelhdar dysfunction fgrovp 5, 57.5 ngh, 27.5-90 nglh). eanpared with all other groups @ < O.U2) (Fig. 1). Ir. normal stibjects, there was a weak positive cnt’rel~tion between urinary flow rate and LTE, excretion
renal syndrome (Fig. Za-d). Urinary LTE, excretion was markedly elevated (45-560 rig/h)) in the first samples collected and at a” early stage during the development of renal failure (creatinine clearances of 39-80 mumin), and fell approximately in parallel with creatinbte clearance indicative of GFR-dependent exaeticm. One patient subse-
rate (I, = 0.55,~ < iN.tSj such that the urinary LTEa excretion rate increased with urine flow suggesting that tw bttiar reabsor~ticm occurs.
was plotted against LTE, excretion for all patients with developing or established hepatorenal syndrome (Fig. 3). Tbe data for normal cmnrols and tbae with severe hep atwellular dystimctkm are also &own in Fis. 3. After adjustment cd LTE, excxetkm for creatinine clearance (Fig. 4). patients with hepatorenal syndrome bad the highest ratio of LTE.#Zr.Cl (median 41.1 pg/ml Cr.‘& rfmp 11.5-242.1) and this was significantly different from all other groups: normals (1.0 pghnl Cr.Cl,
Expression of the results as absolute urinary excretion rates @gilt) does not take into account the possible effects of varying degrees of renal impairment on LTEd exert-
quently recovered: folkming a 2-&y period of aria, creatinine clearance inereascd to 49 mlltttin simultaneous with recovery from Liver failure. Urinary LTEa excretion increased to 54 ng!h at this time (Fig. 2~). and was below that o&wed at an early stage of renal failvre (150 “g/h. crearininc cleara~lcr 33 minnin). A linear correlation (r, = 0.86, p < O.Wl) was observed when ereatinine clearance
serum bilirubin and LTE,excr&m
(r, = 0.6L.p < 0.0X).
LTC, in$i&onr Following infusion of [3H]LTC, into the three subjects between 27.5 and 43% of total radioactivity appeared in urine within 72 h. Tritiated water (determined as total volatile radioactivity and formed through o.oxidati”n 01 the (14,15-8) radiolabel) was present in the urine of all three subjects, accounting for 3%. 4.5% and 8.5% of the infused dose ior s$bj:cts A, B and C, respeaively. The major metabolite present in urine was LTE,, xcountiug for 3.5-4.4% of total radioactivity infused wer 24 h (Fig.
0.13-X86), conpensated liver disease (1.9 pg/ml Cr.CI, OS-6.67), obstructive jaundice (8.5 pg/ml Cr.CI, 5.0-15.0). ascites (7.7 pglml C.Cr, 3.8-15.2), severe hepatocellulat dysfunction (11.0 pg!ml C&I, 6.5-42.4), and chronic renal failure (3.2 pglml Cr.CI, O-6.9) (p c: 0.005). Following this correction for renal function, the group with renal failure (resulting in low excretion rates) now became similar to nomml controls. Effectof liverfunction To assess the effects of liver dysfunction
per se on cys-
teinyl leukotriene production, all subjects with liver disease but without hepatorenal syndrome (groups 2,4 and 5) were grouped together; following correction for renal function there was a significant correlation between LTEI excretion and plasma bilirubin (I, = 0.61, p < O.Wl). In order to take into account the contribution of liver dysfunction, and BSSOSSthe additive effect of hepatorensl syndrome on cysteinyl leukatriene production, the values for group 6 “we also plotted on the same graph (Fig. 5). This gave a higher parallel line of regression indicative of higher LTE, excretion for a given plasma bilirubin.
6). LTE, excretion was considerably delayed in the subject with hepatorenal syndrome, suggesting reduced renal clearance. In the two subjects (B,C) with biliary T-tuber in situ, large volumes of bile (lWO-lZCN3 ml) were mllected over 72 h; 21% and 27.5% of the total tadiaactivity inhrsed was recovered. In the patient with hepatorenal syndrome. HPLC analysis of arterial bled during infusion of [‘H]LTC, showed that LT& wan the major radiolabelled species at all time points (>%I%), with fess than 5% fwxe”t as LTC, or LTD,,. For~oe~~~ed~o~~to IMmin, the antouot of [%jLl’& excreted was 84 @IO dpm. Using the midpoint arledzd pla~rna wncentration of [‘H]LTE, at this time (615 dpmM) and conventional cleaxsnce formulae, the renal clearame of LT& is detotmiwd as 2.3 mUmin. This was lower than the measured creatinine clearance of 10.1 mUmin, in keep+ with tubular reabsorption of LT&. The measured excretion rate of LT& in this subject was 54 @It. Awmting that the contribution of the kidney to urinary LTB, is minimal, Uwt by using the detemtined clearance of radiolabelM LTEd and measured mt~enous f.TE& excretion one can back-catcutate the maximal art&al ptatma fevef. i.e., 3% p$tnL fn the two normaf &jects iltfuwd with [%i]LTC* tile plasam (IHjLTE, was mo low for detetmination, therefore the renal clearance lyas determined using the excredon rate over the l-h infusion period (1.4 and 3.5% as [“HjLTF$, and assuming a renal plasma flow of 600 mt/min with 50% of arterial radiowtivily ii LTE,. The minimum renal clearao~s in these two subjects were eslimated as 17 and 42 mlhnin (renal ckaranm = renal plasma flow x fraction extraeted bv the kidnw’). With a nor-
accurate
that the urinary emtion rate of LTE, is increased in patients witb hepatomnai syndrome aswell as thosewith obstructive jaundice and dewmpensated liver disease. This confirms the recent report by Huber and colleagues (6). and extendstheir obwvations to include controls with severe hepatic dysfunction or rend Failure alone.. Funher, the effects of renal failure on elimination kinetics of the cysteinyl leukotrienes have also been characterised. Tois study was designed to control for most variables which reflect the different circmnstawes oaxrring in hepatoreoai syndrome. Renal failure atone leads to a reduction in the LTf5 excretion rate, which Norma when emrated for creatiaioe cleamnce. Reduced bit&y clearance and bepatic metabolism would lead to an increased plasma LT!& umcentmtion and thus increased excretion rate. This is clearly seen in the diseased liver mntrol groups, particularly in scvere hepatoc&dar dysfunction. Each of these groups have relatively normal renal function, and presumably normal renal cleamoce of LTE* When renal ftmction is taken into acmunt, however, by correcting for creatittiw clearance, LTE, excretion was elevated in decompensated liver disease and hepatorettai syndnnne. This suggests that increased syntLxe& of Ll& occurs in demmpensated liver disease sod hepatoretrai syndrome, but when as+es& by excretion rate aloae, tI& is masked in the lanergroup bytbeeffeaof renal faiiua. Front the intitsioo data on the patient with hefWwntl syndrome, we have &own that renal clearance of LTE,4 is reduced compared with normals. The number of subjects studied by infwion of [WLTC, was small, hut further sopportiq evidence wa the observed Parallel fall of eb oogenous LTE, excretion and creatinine clearawe as subjects developed hepatorenal syndrome (Fig. 4). A reduction in renal clearance w.Md reduee the rate of o&tary LTE, exuetion. In xmtrast, we have obsenwl an inaeased exation rate of LTE, in kepatorena! syndrome (17.8 o#tt) campared with normals (5.1 II&). Thus, we eon&de that there is itweaed qsteinyl ieok&me production in hepatozenal syndrome compared with ttomtal subjects and, assoming Lm eiiiation is proportional to GFR, is also incremed with respect to disease cimtrol
Urinary LTB, excretion has been used to monilor cysteittyl leukotriene production since it is rapidly produced from LX, in viva, and is a stable identifiable wbwy metaboliie providing a time-integzated t&w produetioion (6,13-15). Our
approach to fettkoresutrs demonstrate
Poops. The site of incmaxd eysteinyi leukottiene production in patients with hepatorenal syndrome is not known, altboogh a variety of circulating and tiwe resident inflammatory ceus inckJding IelKaywS, fer o&s will produca leokotrienes have recently demo&rated
maaopbages and Kopfin vitro (4). Two sh&?s
cystcinyt fetikatiene
tion by the isolated kidney (22X%
pmdttc-
bat it is itot known
270
whether the glomeruli are capable of synthesisingcysteinyl leukotrienes. The presenceof 5.lipoxygenase (24) in glomeruli, as well as the known action of LTD, on the mesangiumand the effect of leukotrieee antagonistson ret serum nephritis make it likely that synthetic capacity exists (9.10.25). Further, recent studies have shown that human arterial rings can synrhesisecysteinyl leukotrienes when stimulated by the calcium ionophore A23187 (26).
,.
We have estimated tbe maximum circulating level of LTJZI in one reprerentative subject with hepatorenal syndrome at 390 pglod (0.9 nM). This calculation is basedon the unlikely assumptionthat the kidney does no wntribute to urinary LTEI, and therefore representsthe highest possible plasma levels that may be oixswd. Such concentrations of circulating Ll& would correspond with less than 50 pM of the more biologically active species (LTC, or LTD& Such concentrationsare too low to have an effect on the kidney, 1s severa! studies have demonstrated that concentrations of hetwen 1 and 10 nM of LTD+ are required to affect rena! Iunction (27,28). Forther, hepatorenal syndrome and decompensatedliver dir ease are character&d by systemicvasodilatation; it is unlikely therefore that circulating leukotrienes have any significant systemichemadynamic effects, Clearly, the local concentrationsof LTC, and LTD, are the final arbiters of whether or not there is a pharmacological action. If urinary LTEn is, at least partly, representative of renal leukotriene production, then thesemediatorscould be one of the important modulators of glomerular hemodynamics and function in viva in hepatorenal syndmme. Tbe mechanismsby which increased svnthesisof cvsteinyl kokootrieneso&r in hepatorenal syndmme are not known. We have recently shown that urinary LTE, is increased in patients with asthma, whrre kokotriene synthesisis stimulated in an lgB-dependent manner (15). In hepatorenal syndrome other primary mechanismsprobably operate, and activate kukotriene formation io other sites such as the kidney. One potential factor that mold increase leukotriene formation includes endotoxins, which are frequently present in the systemiccirculation of
patients with hepatorenal syndrome and decompensated liver diseaseeven in the absenceof demonstrable sepsis (‘26-28). Infusion of endotoxins into the rat increasesbiliary cysteinyl lcukotriene excretion arising presumably from increased kukotriene production (17,32). Endotoxin infusion into the rat reducesthe renal blood flow and the glomendar filtration rate, the effects of which are ameliorated by antagonistsof the cysteinyl leukotrienes C, and D,, and thmmboxane A, (33). Recent studieshave also demonstrated that chronic endotoxemia in rats inaeest5 the free arachidonic acid pool, thus providing a further mechanismby which general eicosanoid synthesis is increased (34). Other potential agents include tomoor necrosis factor, whose formation by peripheral monocytes is increased in certain liver diseases (35.36). and may act through activating phospholipw AZ. We have demonstratedthat there is marked inerase in cysteinyl leukotriene production in bepatorenal syw drome, although circulatbq levels of these mediators are too low to have significant renal 01 hemodyxmmiceffects. Local production within the kidney, however, could resolt in significant renal effects. Without the use of suitable antagonists,it is not possibleto demonstrate a causalrole of cysteinyl leukotrienes in the patbogenesisof hepatorenal syodmme, although the availability of leukotriene antagonists opens a new area of pmsible therapeutic intervenlion.
ACk”OUtCdgC~“b
We are grateful to Mr. 1. Dawon and Drs. V. Pamoos and D. Tauhe for allowing us to study their patients, Mr. M. Shipley (London School of Hygiene and Tropical Medicine) for advice on statistical analysis, and Dr. Alan Boohis end Professor D. Davies for helpful advice on the pharmacokinetics. Tlds work was supported by the Medical Research Council and the Nuffield Foundation. K.M. is io rueipt of an MUC Training Fellowship.