urinary tissue factor in glomerulonephritis: markerof ...immunecomplex(ic) glomerulonephritis (p

3 Clin Pathol 1997;50:336-340

Urinary tissue factor in glomerulonephritis: a

potential marker of glomerular injury?B A Lwaleed, P S Bass, M Chisholm, J L Francis

AbstractAim-To investigate the significance ofurinary tissue factor (uTF) concentra-tions in patients with glomerulonephritis.Methods-Urine samples were collectedfrom normal subjects (n = 57), patientswith uncomplicated renal stones (n = 30),and patients with glomerulonephritis(n = 150). Samples were then centrifugedand the pellets solubilised in n-octyl-p-glucopyranoside. uTF concentrationswere determined using a one stage kineticchromogenic assay.Results-The uTF concentration washigher in patients with glomerulonephri-tis than in normal controls (p < 0.01) or inpatients with renal stones (p < 0.05). uTFactivity correlated with the protein creati-nine index (PCI, r = 0.41, p < 0.001) andseven patients with glomerulonephritisand a PCI < 0.1 g/mmol had raised uTF.Glomerulonephritis patients were subdi-vided into two groups depending on thePCI: < 0.2 g/mmol creatinine (mild tomoderate proteinuria, group I) and¢ 0.2 g/mmol creatinine (heavy proteinu-ria, group II). In group I, uTF concentra-tions were higher in patients with eitherimmune complex (IC) glomerulonephritis(p < 0.01) or non-IC (p < 0.05) glomeru-lonephritis than in normal controls. Ingroup II, the IC glomerulonephritis grouphad higher uTF concentrations than nor-mal controls (p < 0.001) or patients withrenal stones (p < 0.01); and non-ICglomerulonephritis patients had higheruTF than normal controls (p < 0.01).When the glomerulonephritis groups weredivided into broad WHO subtypes, thesignificance level varied with the type ofglomerulonephritis.Conclusions-uTF is increased in patientswith glomerulonephritis, and its concen-tration may reflect the aetiopathogenesisof glomerulonephritis.(7 Clin Pathol 1997;50:336-340)

Keywords: urinary tissue factor; monocyte/macrophagetissue factor; coagulopathy; glomerulonephritis

Activation of blood coagulation and fibrindeposition are associated with various forms ofinflammatory glomerular diseases in humansand laboratory animals. 1-3 Fibrin depositionwithin and around the glomerulus has beenobserved by several investigators4 and isthought to play a major role in the developmentand the progression of some forms of glomeru-lar disease.9 The underlying biological mech-anism of such phenomena remains poorly

understood. However, it has been suggestedthat the source of glomerular procoagulantactivity could either be bloodborne or gener-ated by resident glomerular cells. Glomerularfibrin deposition is dependent on leucocyteaccumulation,'0 and rabbits treated with mus-tine hydrochloride develop severe leucopenia,which prevents glomerular macrophage accu-mulation and glomerular fibrin depositionwithout any functional alteration of the hostcoagulation factors."' Mononuclear cells areknown to be a potent source of procoagulantactivity." 12 Increased expression of procoagu-lant activity by monocytes/macrophages in sev-eral inflammatory conditions, particularly afterstimulation with Escherichia coli endotoxin, iswell documented."' '-` Similarly, isolatedhuman and animal glomeruli with differentforms of glomerular disease express high levelsof procoagulant activity.2 16`1 Further evidenceof the involvement of mononuclear and intrin-sic glomerular cells can be drawn from tissueculture studies of isolated glomeruli whereboth cell types have been shown to expressincreased levels of procoagulant activity.2 10However, fibrin deposition in the kidney mayalso result from impaired activity of thefibrinolytic system.'7

Initially, renal procoagulant activity wasthought to activate the extrinsic pathway of thecoagulation cascade by at least three differentmechanisms.9-2' However, it has recently beenshown that blood coagulation can only proceedthrough a tissue factor dependent pathway.21This is supported by the findings of Hoyer et alwho excluded the involvement of factor VIII inglomerular fibrin deposition,22 and the correla-tion between tissue factor from glomerularsupernatants and thromboxane B2 (TxB2)formation in platelets.23 Tissue factor apopro-tein is a 46 kDA, single chain, integral plasmamembrane glycoprotein with no intrinsic pro-tease activity.24 25 Tissue factor serves as areceptor and essential cofactor for the serineprotease blood coagulation factors VII andVIIa26 in the activation of factors X andIX-thus it is an important initiator of bloodcoagulation.27 Tissue factor is not only foundon mononuclear cells or in lysed isolatedglomeruli but also in a lipid associated form inthe urine (uTF),2' where its concentration maybe of clinical significance, particularly inpatients with inflammatory diseases andneoplasia.29 '5 Indeed, uTF concentrationswere found to be increased in patients withmalignant diseases compared with patientswith corresponding benign diseases and withnormal controls, but not compared withpatients with inflammatory diseases.33-35 Analy-sis of uTF in pathological renal states has until

SouthamptonUniversity Hospitals,Southampton, UnitedKingdom:UniversityDepartment ofHaematologyB A LwaleedM Chisholm

Department ofPathologyP S Bass

Hemostasis andThrombosis ResearchUnit, Walt DisneyMemorial CancerInstitute at FloridaHospital, AltamonteSpring, Florida, USAJ L Francis

Correspondence to:J L Francis, Hemostasis andThrombosis Research Unit,Walt Disney MemorialCancer Institute at FloridaHospital, 616 E AltamonteDrive, Suite 100, AltamonteSprings, FL 32701, USA.

Accepted for publication10 January 1997

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Urinary tissue factor in glomerulonephritis

recently been limited to animal models, withrather conflicting results.'7 Recently, we havedeveloped a highly standardised assay for uTFmeasurement (to be published). In this pre-liminary study, we applied this assay to patientswith glomerulonephritis and examined thehypothesis that uTF concentrations may reflectthe pathogenesis of glomerular damage or itsdegree.

MethodsPATIENTSEthics committee approval was obtained forthe study and informed consent was soughtfrom each patient on arrival at the outpatientclinic at the Royal South Hants Hospital,Southampton, during 1993 to 1994. Allpatients included in the study had biopsyproven renal disease (biopsies performedbetween 1990 and 1994). The biopsy reportswere retrieved and classification of glomeru-lonephritis was made according to the criteriaof the Collaborating Centre for the Histologi-cal Classification of Renal Diseases of theWHO.36 Urine was collected from 237 sub-jects: controls (healthy volunteers (n = 57) andpatients with "uncomplicated" renal stonesand a normal erythrocyte sedimentation rate(n = 30)), and patients with glomerulonephri-tis (n = 150). The glomerulonephritis groupwas made up of minor glomerular abnormali-ties (n = 4); diffuse glomerulopathy (type Imembrano-proliferative glomerulonephritis(n = 1 1), mesangio-proliferative glomerulone-phritis (n = 33), and membranous glomeru-lonephritis (n = 19)); focal segmental prolif-erative glomerulonephritis (n = 26); focalsegmental glomerulosclerosis (n = 20); andmiscellaneous diseases (n = 37). The lattergroup included patients with diabetes, amy-loid, and ischaemic changes. All patients onsteroids at the time of urine collection wereexcluded from the study, as were patients withcrescentic glomerulonephritis.

UTF MEASUREMENTUrine samples were collected from eachsubject into sterile universal containers withoutpreservative. Samples were then sedimented,solubilised, and assayed. Briefly, the extracteduTF, in the presence of recombinant factorVIIa (rVIIa) and Ca"+, forms a complex (tissuefactor/rVIIa/Ca2) which then directly activatesfactor X to factor Xa. Generation of factor Xa,which is proportional to the amount of tissuefactor in the urine sample, was determined bymeasuring its action on a factor Xa specificchromogenic substrate. The rate of the reactionat 405 nM was determined in a BiokineticsEL-312e microplate reader (Bio Tek Instru-ments, Winooski, Vermont, USA), pro-grammed to read the absorbance at 30 secondintervals over a period of 35 minutes. Absorb-ance values were converted to tissue factor (ng/ml) using a calibration curve constructed fromserial dilutions of recombinant relipidatedtissue factor (rrTF, 0.4-83 ng/ml) preparedaccording to Carson and Konigsberg37 in BOG(P-octyl-glucopyranoside, Sigma, Poole, Dor-

set). For maximum accuracy the calibrationstandards were measured in each plate.

EXPRESSION OF UTF RESULTSThe results (ng/ml) were corrected for thedilution of the urine using the creatinineconcentration of the sample. Final results wereexpressed as uTF ng/ml per mg creatinine:uTF (ng/ml)/creatinine (mg/dl) x 100.

PROTEIN CREATININE INDEX MEASUREMENTSTotal protein and creatinine were measuredaccording to the standard CX7 protocol auto-mated analyser (Beckman, Brea, California,USA). The protein creatinine index (PCI) wasthen calculated as: total protein (g/l)/creatinine(g/mmol).38 39

STATISTICAL ANALYSISData were included in a database and analysedby the Statgraphics statistical software system.Data were not normally distributed, and sum-mary statistics were expressed as medians andinterquartile ranges (IQR). Differences be-tween two groups were assessed by the Mann-Whitney U test.

ResultsThere was a significant difference in uTF con-centrations between patients with glomerulo-nephritis and normal controls (p < 0.01) andpatients with renal stones (p < 0.05, fig 1);54.9% of patients with glomerulonephritisshowed a uTF level above the upper quartile ofthe normal controls, in contrast to 36.7% in therenal stones group. There was a weak correla-tion between uTF and PCI values (r = 0.41,p < 0.001). The glomerulonephritis group wasthen subdivided according to the PCI level atthe time of the biopsy. Group I had a PCI of< 0.2 g/mmol (that is, less than about 2 g/day,mild to moderate proteinuria), and group II. 0.2 g/mmol (heavy proteinuria). In addition,two broad groups of glomerulonephritis pa-tients were distinguished-namely immunecomplex (IC) glomerulonephritis (type Imembrano-proliferative glomerulonephritis,mesangio-proliferative glomerulonephritis, andmembranous glomerulonephritis) and non-ICglomerulonephritis (minor glomerular abnor-malities and focal segmental glomerulosclero-sis). In group I (low PCI), the IC group(p < 0.01) and the non-IC group (p < 0.05)both had higher uTF values than the normalcontrols, but not than the renal stones group.In group II (high PCI), the IC glomerulone-phritis group (p < 0.001) and the non-ICglomerulonephritis group (p < 0.01) also hadhigher uTF values than the normal controls,and a significant difference was observedbetween the renal stones group and the ICglomerulonephritis group as well (p < 0.01, fig2A and 2B).

In group I, no significant difference wasfound between the IC and the non-IC groups;in group II, the IC glomerulonephritis grouphad a higher, but wider, range ofuTF values (8to 29) compared with the non-IC glomeru-lonephritis group (7 to 16), but the differencewas not significant (fig 3A and 3B). There was

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Figure1 Urinary tissue factor (uTF) concentrations in control groups and patients withglomerulonephritis. Results are shown as median and the interquartile ranges.

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Figure 2 Comparison of urinary tissue factor (uTF) concentrations in controls andpatients with immune complex (IC) glomerulonephritis and non-IC glomerulonephritis(A, PCI < 0. 2; B, PCI 0. 2). Results are shown as median and interquartile ranges. PCI,protein creatinine index.

no correlation between the stage of themembranous glomerulonephritis or the degreeof tubulo-interstitial damage and the uTF con-

centrations in all three forms of IC glomeru-lonephritis studied (r = 0.1, p > 0.05).

Analysis of the uTF concentrations in otherforms of glomerulonephritis showed that forlow PCI there was a significant differencebetween the focal segmental proliferative groupand normal controls only (p < 0.05), and

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Figure 3 Comparison of urinary tissue factor (uTF)concentrations in patients with immune complex (IC)glomerulonephritis and non-IC glomerulonephritis (A, PCI< 0.2; B, PCI 0.2). Results are shown as median andinterquartile ranges. PCI, protein creatinine index.

between the miscellaneous group and normalcontrols only (p < 0.05). With high PCI, therewas a significant difference between themiscellaneous group and both the normal con-trols (p < 0.01) and the renal stones group(p < 0.05).

DiscussionThe fact that several human and experimentalmodels of glomerulonephritis show alterationsin kidney tissue factor levels highlights theimportance of this glycoprotein in glomerularpathology. Tissue factor, the normal route tocoagulation activation, triggers glomerular fi-brin deposition,"6 which has been observed inmany forms of clinical and experimentalglomerulonephritis.6 40 Increased concentra-tions of tissue factor in glomerular diseases arefound not only in the kidney itself, but also inthe urine, where it is associated with lipid.28Assessment of uTF in renal diseases has untilnow been mainly restricted to experimentalmodels, with conflicting results.'7 Thus urinaryprocoagulant activity in rabbits with nephro-toxic nephritis is reported to have disappearedduring the onset of glomerulonephritis,4' whilein a subsequent study using the same animalmodel a marked increase in the urinary proco-agulant activity was observed during the devel-opment of glomerular injury.'8 Recently, how-

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Urinary tissue factor in glomerulonephritis

ever, the relation between the urinaryprocoagulant activity and fibrinolytic activity inhuman glomerulonephritis has been evaluatedand a negative association found.'7 The assaysystem employed in this latter study, however,may not have been completely reliable. Inaddition, the samples were dialysed, thoughuTF is bound to membrane microvesicles2"which provide the lipid for its functional activ-ity. The structure and the orientation of thesevesicles in relation to uTF is still poorly under-stood. This is important since the orientationofthe lipid vesicles has been shown to affect theaccessibility of tissue factor to factor VII/VIIa.42Recently we have developed and validated asimple, clinically applicable kinetic chromo-genic assay for measuring uTF activity whichdoes not use dialysis (to be published).The clinical applications of this new assay to

patients with glomerulonephritis have not beenexplored before, although the assay has beensuccessfully used to distinguish normal sub-jects from patients with malignant disease inthe absence of inflammation.33-35Using this assay system, uTF was found to

be increased in patients with glomerulonephri-tis compared to normal controls and to patientswith uncomplicated renal stones: 54.9% of theglomerulonephritis group had results above theupper quartile of the healthy control group,while only 36.7% of the renal stones group hadabnormal values. Although the glomerulone-phritis group had increased uTF concentra-tions, there was an overlap with the normal andrenal stones groups (fig 1). This may have beendue to the heterogeneity of the various groups,including the stage of evolution of the diseaseor treatment schedules (some patients were ontreatment at the time of sample collection,though all patients on steroids were excludedfrom the study).Although it could be argued that circulating

tissue factor may enter the urinary filtrate inglomerulonephritis, tissue factor is constitu-tively expressed only by cells outside thevasculature,43 though its expression could beinduced in endothelial cells and monocytes byendotoxin or cytokines." 44 However, the pro-coagulant activity of blood monocytes indifferent forms of glomerulonephritis was notsignificantly different from that in a normalcontrol group,'7 neither were plasma tissue fac-tor concentrations in patients with chronicglomerulonephritis.45

In our study, we found that uTF showed onlya weak correlation with urinary protein excre-tion (expressed as protein creatinine index).We then divided the glomerulonephritis studygroups into group I, with a PCI of < 0.2 g/mmol, and group II, with PCI values . 0.2 g/mmol. When the types of glomerulonephritiswere divided into immune complex andnon-immune complex subtypes, both groupsshowed a significant increase in the uTFconcentrations compared with the controls,even when the PCI was only moderately raised.Although the immune complex subtypes ingroup II showed a wider range ofuTF (8 to 29)compared with the non-immune complexgroup (7 to 16), the differences between the

groups failed to reach statistical significance.The high levels of uTF observed in theimmune complex subgroup could be related tovarious factors, including immunoglobulin andcomplement deposition, mononuclear cell in-filtration, fibrin formation, and glomerular cellproliferation. This may cause direct or indirectactivation of the resident glomerular cells orinfiltrating inflammatory cells to express in-creased amounts of tissue factor. The presenceof fibrin or fibrin related products acts as amacrophage aggregating agent"' and stimu-lates glomerular mesangial cells directlythrough cytotoxic effects.49 In addition,changes in membrane structure cause tissuefactor de-encryption which would result inenhanced tissue factor procoagulant activity.An obvious example of this is cell apoptosis,which has been shown to be associated with anincreased cell surface tissue factor procoagu-lant activity.50 Whatever the mechanisms in-volved, this may lead to local activation ofblood coagulation or of the fibrinolytic system.The extent of glomerular damage may there-fore be reflected in the amount of uTFproduced. This is supported by the finding ofWiggins et al, who showed, using an animalmodel, that procoagulant activity in rabbitswith nephrotoxic nephritis not only increasedin glomeruli but also appeared in urine duringthe progression of glomerular injury.'8 Thissuggests that haemostatic alterations mayrepresent changes of pathological importancein the development and progression of the dis-eases. However, in patients with diffuseglomerulonephritis we found no significantassociation between uTF activity and eithertuft morphological changes or interstitial cellinfiltration. The apparent lack of correlationfound in this study could be a time factor, sincethe period between sample collection andbiopsy exceeded three years in some cases.However, uTF may be a useful tool in assessingglomerular damage, particularly in immunecomplex glomerulonephritis, and perhaps inmonitoring response to treatment. Furtherstudies are required to evaluate its use as amarker of glomerular injury.

In conclusion, the uTF concentration isincreased in patients with glomerulonephritiscompared with controls, and is increased evenwhen the protein creatinine index is only mod-erately raised. Our immune complex glomeru-lonephritis group showed wider ranges thanthe non-immune complex glomerulonephritisgroup and the results suggested that uTF pro-duction may be more important in immunecomplex glomerulonephritis. Further studiesto evaluate uTF level in patients with glomeru-lar abnormalities are required, especially toevaluate the influence of disease activity, stage,and the influence of treatment.

We thank Dr Rod Dathan and Dr Mary Rogerson, ConsultantPhysicians at Southampton University Hospitals for allowing usto study patients under their care. We thank Dr Mary Rogersonfor commenting on the text. We also acknowledge the coopera-tion ofMrs Natasha Wilson, Renal Failure Monitoring Unit, forher help with the retrieval and collation of medical records.

1 Winter H, Majid NH. Glomerulonephritis-an emergingdisease? Vet Bull 1984;54:327-35.

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