Quantitation of allograft ®brosis and chronicallograft nephropathy

Grimm PC, Nickerson P, Gough J, McKenna R, Jeffery J, Birk P, RushDN. Quantitation of allograft ®brosis and chronic allograftnephropathy.Pediatr Transplantation 1999: 3: 257±270. # Munksgaard, 1999

Abstract: Despite improvements in the prevention and treatment ofacute renal allograft rejection, the long-term survival of renal transplantshas not increased. Immunologic and non-immunologic factorscontribute to the gradual deterioration of graft function and to thehistologic lesion characterized by vascular and interstitial ®brosis(`chronic rejection'). Quantitation of this process has been attemptedusing various invasive and non-invasive methods. These methods,performed at different times post-transplant, are reviewed in this article.In particular, pathology scoring systems and the potential of usingcomputerized image analysis of biopsy material are discussed.

Paul C. Grimm1,2,6, PeterNickerson2,3, Jim Gough4,5, RachelMcKenna2,3,5, John Jeffery3, PatriciaBirk1 and David N. Rush3

Departments of 1Pediatrics and Child Health,2Immunology, 3Internal Medicine and 4Pathology,University of Manitoba, Winnipeg, Manitoba and5University of Calgary, Calgary, Alberta, Canadaand 6University of California at San Diego,California, USA

Key words: ®brosis ± image analysis ± chronic

rejection ± transplantation review ± pathology

Dr Paul C. Grimm, Division of Pediatric Nephrology,

University of California at San Diego, 9500 Gilman

Dr Mail Code 0831, La Jolla, California 92093±0831,

USA

Tel: (619) 543 5218

Fax: (619) 543 3575

E-mail: pgrimm@ucsd.edu

Accepted for publication 22 April 1999

Advances in solid organ transplantation havebeen numerous since its inception into clinical usein the 1950s. In kidney transplantation, improve-ments in the short-term allograft survival ratehave been particularly impressive, to the pointthat 1-yr graft survival exceeds 90% in manycenters (1). Unfortunately, the same success hasnot been re¯ected in long-term allograft survival.The rate of allograft loss following the ®rst yr hasin fact not changed in the last 30 yr (2±4). Themajor causes of late allograft losses are deathwith a functioning graft, and slow deteriorationof allograft function with increasing ®brosis,termed chronic rejection. Current forms ofimmunosuppression are ineffective in the preven-

tion and treatment of chronic rejection.Moreover, the potency of the newer agents hasbeen associated with life-threatening complica-tions (5±7).

Chronic rejection is a complex, multi-factorialprocess in which immune and non-immunefactors have a role in its pathogenesis. Therecognition of non-immune factors has led tothe use of terms such as `chronic transplantnephropathy' and `chronic allograft dysfunction'to describe the entity. Emphasis on the patholo-gical features is embodied in the term `transplantassociated vasculopathy'. There are no univer-sally accepted diagnostic criteria for chronicrejection (8), although diagnostic criteria havebeen suggested (9). The clinical diagnosis ofchronic rejection is characterized by gradualdeterioration of graft function with a slowlyrising serum creatinine, hypertension of increas-ing severity, and the development of proteinuria.

Abbreviations: ESRD, end-stage renal disease; TGF-b, transform-

ing growth factor b; CsA, cyclosporin A; CADI, Chronic Allograft

Damage Index; CGDS, Chronic Graft Damage Score.

Pediatr Transplantation 1999: 3: 257±270

Printed in UK. All rights reserved

Copyright # Munksgaard 1999

Pediatric TransplantationISSN 1397±3142

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The onset of chronic rejection is dif®cult toestablish with certainty. However, protocolbiopsy studies such as our own have shownthat patients whose graft function declines overthe ®rst 24 months can have increased chronichistologic changes as early as 3 months post-transplant (10).

Failure of a renal allograft due to chronicrejection is now a major cause of chronic renalfailure requiring dialysis (11). Many such patientsreturn to the transplant waiting list, once againcompeting for the short supply of cadaverkidneys. However, these patients are oftensensitized and their waiting time is extendeddue to positive cross-matches (12). The burden ofgraft failure due to chronic rejection is especiallyimportant in children with ESRD for whom theexpected healthy life span of 70±80 yr wouldmean multiple transplant procedures, assuming atypical renal allograft survival half-life of8±10 yr. Intensive research is required to studythe cause(s) and develop new prevention andtreatment strategies for chronic rejection.

Unfortunately, this research is hampered bythe time periods over which chronic rejectiondevelops (13, 14). The ultimate end-point is long-term graft survival. However, the cost andlogistical problems of performing an interven-tional study, which may not come to a conclusionfor 10 yr, are prohibitive. In addition, the rapidtempo of development of paradigms and treat-ment modalities might render the original ques-tion obsolete prior to the termination of thestudy. Consequently, attention has been turnedto the identi®cation of surrogate markers ofchronic rejection. Some of the proposed markersstudied to date are the incidence of acuterejection, rate of deterioration of graft functionand ®brosis, among others. These will bediscussed subsequently.

Pathogenesis of chronic rejection

The alloimmune response

As stated above, the pathogenesis of chronicrejection is clearly multi-factorial, and in differentpatient populations different mechanisms may bemore relevant. The most important initiatingfactor is the alloimmune response. Only a smallhandful of patients receive perfectly matchedkidneys or have HLA identical living donors (15).The consequence of mismatching of donor andrecipient HLA is persistent alloreactivity betweenrecipient and donor and the process of acuterejection. A number of studies have indicated thatacute rejection is the single most important

predictor of chronic rejection (16, 17). There isevidence that the alloresponse leading to acuteand chronic rejection may be reduced by higherlevels or dosage of immunosuppression (10, 18).Individual biological variation in immuneresponse may also contribute to differences ingraft outcome. El-Gamel et al. have shown thatpolymorphisms in the promoter region of theTGF-b gene in lung allograft recipients isassociated with the risk of developing lungallograft ®brosis (19).

The issue of non-compliance is now recognizedas a serious cause of graft loss in all ages andgroups. Unrecognized non-compliance may beresponsible for a signi®cant number of acuterejections (20). Certain age groups such as teen-agers may be especially susceptible to this processand warrant extra attention. As pediatric recipi-ents are expected to pass through adolescence the®nding that 60% of adolescent renal transplantrecipients dabble in non-compliance is a cause forgreat concern (21). The prevalence of non-compliance is not well studied but is recognizedas important in patients following liver (22), heart(23), renal (24) and bone marrow transplantation(25). The intermittent failure of the pharmacolo-gic umbrella to protect the graft from immuno-logic injury may be an important reason for lateallograft acute and chronic rejection.

Our group has recently shown that sub-clinicalrejection is present in patients with stable serumcreatinines in the ®rst 6 months post-transplant(26). This has been supported by other investi-gators (27, 28). We have recently published theoutcome of a randomized study where patientswere randomly allotted to one of two groups. Onegroup underwent early protocol biopsies at 1, 2and 3 months post-transplant, and receivedcorticosteroid treatment if sub-clinical rejectionwas present. The other received no biopsies at 1, 2or 3 months and therefore no treatment of sub-clinical rejection was given. The group thatreceived biopsies and treatment of sub-clinicalrejection had a signi®cantly lower serum creati-nine at 24 months than the group receivingstandard therapy. This study suggests that earlysub-clinical rejection episodes may be deleteriousto the long-term function of the allograft in thatthere was a signi®cant advantage to detection andtreatment of early sub-clinical rejections. Thiswas accompanied by minimal increase in side-effects (29).

Non-allogeneic damage

There is evidence that CsA and tacrolimus, thecalcineurin inhibitors that are the cornerstones of

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modern immunosuppression, may actually causechronic allograft damage. Both drugs have beenimplicated in the pathogenesis of graft dysfunc-tion with vasospasm leading to rapid renalallograft dysfunction on an acute basis andinterstitial ®brosis and vasculopathy on a chronicbasis. In therapeutic doses, CsA acutely increasesrenal allograft vascular resistance in humans (30).This appears to be related to speci®c afferentarteriolar vasoconstriction (31). This causes long-term impairment of the true glomerular ®ltrationrate in CsA-treated pediatric allograft recipients(32). CsA has also been shown to directly inducethe expression of TGF-b (33), and downstreamTGF-b-dependent genes related to ®brosis suchas procollagen (34). CsA-induced ®brosis is awell-recognized cause of chronic nephropathy innon-renal transplant recipients (35).

In renal transplantation the shortage ofcadaver donors has required that transplantprograms extend the criteria that de®ne theacceptable donor. As a consequence, organsretrieved from older donors may already beplagued by atherosclerosis, interstitial ®brosisand glomerulosclerosis. This pre-existing damageand consequent reduced renal functional reservemay predispose the organ to accelerated dete-rioration of graft function. In addition, pre-existing donor disease may sensitize the organ toCsA-related nephrotoxicity (36). Placing a smallkidney (female, younger or older) into a largerecipient is associated with a higher rate of graftfailure. The balance between supply and demandwith reference to renal function may induce ahyper®ltration injury leading to chronic allograftloss (37). A number of studies, including our own,have shown an increased risk of chronic graftdeterioration associated with older donors (10,17, 38). Rather than older donors simply havingpre-existing injury at the time of transplant, theymay be also be on the road to senescence andtherefore limited in their ability to respond toinjury (39). Younger organs may respond to agiven injury by repairing without ®brosis (heal),while older organs may respond to the sameinjury with ®brosis (scar) (10, 40).

Hyperlipidemia is present in up to 30% of renaltransplant recipients (41). Hyperlipidemia and,speci®cally, hypercholesterolemia is a clinicalmarker of chronic rejection in heart transplanta-tion (42). This has not been so easily con®rmed inrenal transplant recipients (43). Low-densitylipoproteins may be directly toxic to endotheliumand lead to vascular damage and glomerulo-sclerosis (44). In the presence of macrophages andin¯ammation, low-density lipoproteins maybecome oxidized leading to a reactive product

that may in turn activate the immune system byincreasing MHC class II antigen expression (45).

Surrogate markers of chronic rejection

A major current goal of renal transplantation isthe improvement of long-term graft survival.However, as the success rates at 1 yr are so good,it will require many patients and many years offollow-up to determine the impact of anyprotocol on the outcome of interest (13, 14).There is therefore a need to develop surrogatemarkers for long-term graft outcomes.

Proteinuria

As in native renal disease, proteinuria tends todevelop as renal function deteriorates andquantitative estimates can be used as a surrogateof chronic rejection. Proteinuria at 1 yr followingtransplant has been shown to predict laterdeterioration of renal function. In one study,patients with stable renal function had a meanprotein of 180 mg/d whereas patients withsubsequent deterioration of renal function hada urinary protein excretion of 400 mg/d (46). Inanother study 67% of post-transplant proteinuriawas due to chronic rejection. Conversely, thismeans one-third of post-transplant proteinuria inadults is not due to chronic rejection. Proteinurianot due to chronic rejection may be even morefrequent in children who have been transplantedfor diseases that recur, such as FSGS (47).

Acute rejection

One of the most frequently used surrogatemarkers of chronic rejection is the ®rst episodeof acute rejection. This has been shown to be astrong predictor of long-term chronic rejection(48, 49). Moreover, speci®c characteristics of anacute rejection episode may be predictive ofoutcome. For example, vascular rejection is moredetrimental to long-term function than interstitiallimited rejection (50), particularly if ®brinoidnecrosis or interstitial hemorrhage are present(51). Different post-transplant time periods inwhich an acute rejection episode is recognized areassociated with different impacts on long-termgraft function. Most studies suggest that rejectionepisodes occurring at later times post-transplantare better markers of subsequent chronic rejec-tion than episodes occurring early, i.e. less than3 months, post-transplant (10, 52±54).

Using the UNOS renal transplant registry,Hunsicker and Bennett explored the impact ofacute rejection episodes observed in different

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epochs on the rate of later graft loss. Theseauthors found that changes in the rate ofrejections that occurred in different epochs werenot predictive of the rate of late graft loss (14).This ®nding has thrown the use of acute rejectionas a surrogate marker for chronic rejection intodoubt. Moreover, recent studies of newer immu-nosuppressive medications such as mycophenolicacid and tacrolimus have shown that in spite ofreducing the 1-yr rate of acute rejection thereappears to be no improvement in long-termallograft dysfunction (6, 7).

Serum creatinine

An alternative and relatively non-invasive surro-gate marker for long-term graft outcomes may bethe serum creatinine measured at de®ned timepoints following transplantation. Kasiske et al.reported that higher serum creatinines at a latepost-transplant period predict a stepwise dete-rioration in graft function. For example, patientswith a serum creatinine greater than 2 mg/dLhad, on average, 4 yr to graft loss. A serumcreatinine greater than 3 mg/dL was associatedwith an average of slightly more than 1 yr to graftloss and a serum creatinine greater than 4 mg/dLwas associated with 6 months to graft loss in 50%of the patients. These authors counseled againstusing a relative rise of the serum creatinine as apercentage of the initial value because they feltthis would not gain much in sensitivity and wouldlose substantial speci®city (13).

Reciprocal of the serum creatinine

An alternative method of analysis is the utiliza-tion of the reciprocal of the serum creatinine vs.time. These graphs typically initiate at the nadirof the post-transplant serum creatinine andimprove their accuracy with longer periods ofobservation (55). In fact, the change in the slopeof the 1/serum creatinine graph has been used toindicate changes in renal function in response tochanges in immunosuppressive therapy (56). Animportant note of caution has been raised bysome authors that the rate of decline of GFR maybe variable and therefore short-term observationsmay provide false information (46). A study byShah and Levey indicated that spontaneouschanges in the rate of decline of the reciprocalserum creatinine graph may occur in 30±50% ofpatients with native kidney disease progressing toend-stage renal failure. This typically occurred ata serum creatinine of about 5 mg/dL (57). Leveyet al. raised the possibility that the actual rate ofdeterioration of GFR is slow, but felt that otherissues, such as the tubular secretion of creatinine

and the generation of creatinine, may be moreimportant (58). Levey et al. recommend thatchanges in the rate of progression using the 1/serum creatinine method should not be used inintervention studies.

An important issue is the duration of follow-upand data gathering in studies using the 1/serumcreatinine method. In transplant recipients whodevelop chronic rejection, the slope of the 1/serum creatinine graph frequently changes withinthe ®rst 3 yr post-transplant. The direction,magnitude and onset of change falls into one ofa small number of patterns. Twelve per cent ofpatients have an initial deterioration and thenstabilize, the in¯ection point in the graphoccurring at 2.9 6 1.2 yr post-transplant.Another 26% of patients show initial stablegraft function followed by the onset of deteriora-tion occurring at 2.2 6 1.2 yr post-transplant(46). The Modi®cation of Diet in Renal DiseaseStudy Group reported that `regression to themean' tends to slow the measured rate ofdeterioration of renal function after entry intostudies. They reported that the correlationbetween the slope of the 1/serum creatinine andGFR improves with time to an R-value of 0.76with 15 months of follow-up. Similarly, theprecision of the 1/serum creatinine methodimproves with duration of follow-up (59). Apediatric twist on the reciprocal of the serumcreatinine is using it to predict renal function atage 10 yr in patients with a progressive congenitalrenal disease such as cystinosis (60). The authorsnoted that when using the slope of the reciprocalof the serum creatinine to extrapolate the renalfunction at later time points required that thevariation of the renal function at baseline betaken into account.

The renal biopsy in chronic rejection

In the absence of reliable non-invasive methodsof quantitating chronic rejection, renal biopsyremains the gold standard for this diagnosis in1998. There are a number of changes detectableon biopsy which have been correlated withchronic allograft dysfunction. These changesoccur in the four primary compartments of thekidney, the vessels, glomeruli, tubules and inter-stitium.

Pathologic changes of chronic rejection havebeen documented for many years (61). It is widelybelieved that the primary lesion is an obliterativeendarteritis sometimes associated with oblitera-tive capillaritis of the glomeruli (62). Graftatherosclerosis is a major manifestation of

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chronic rejection in cardiac transplantation and isdetected sporadically in biopsies of kidneys withchronic rejection. Transplant atherosclerosis ischaracterized by narrowing of arterial andarteriolar vessels. These frequently show evidenceof an `onion-skin' lesion suggesting recurringwaves of intimal injury as well as diffuselyproliferative smooth muscle cell expansion,drop-out of medial myocytes and increase inthe intimal surface area (61). The typical arterialchange of chronic rejection is ®brous intimalthickening with occasional elastica breaks(Fig. 1). This lesion is patchy, at least early on,and its detection is therefore prone to samplingerror. This change typically affects arteries largerthan those regularly seen on renal biopsy (63).Transplant glomerulopathy is highly speci®c forchronic rejection, but is only present in 5% ofdocumented cases (63). A detailed discussion ofthe diagnosis and differential diagnosis of trans-plant vasculopathy and glomerulopathy has beenpublished recently (64). Interstitial ®brosis andtubular atrophy are more readily apparent inrenal biopsies. These lesions are less prone tosampling error than vascular or glomerularabnormalities but are thought to be non-speci®cchanges induced by glomerular or vasculardamage to the kidney (63).

A recently described lesion has been suggestedto be relatively speci®c for chronic rejection. Thelesion of basement membrane splitting andlaminations was identi®ed by Monga et al. atthe EM level (65). These authors subsequentlystudied 61 biopsies from patients averaging 2 yrpost-transplant. They found peritubular capillarychanges in all patients with transplant glomer-ulopathy as well as in nine additional patients.The lesion increased in severity with timefollowing transplant (66). The signi®cance ofthe peritubular capillary changes in the diagnosis

of chronic rejection has been con®rmed by others(67); this lesion will probably become importantin diagnosis and quantitation of chronic allograftnephropathy and speci®cally chronic rejection inthe future (63). The precursors to the peritubularcapillary changes found at approximately 2 yrpost-transplant may be the post-capillary venule-like changes in peritubular capillaries detected byelectron microscopy within the ®rst 3 monthspost-transplantation described by Ivany et al.(68). These authors showed that there was a2-fold increase in endothelial thickness and cross-sectional area in acutely rejecting as compared tonormal grafts. In addition, there was increasedadherence and passage of lymphocytes andmonocytes through the endothelium. If recurrent,these may be the forerunners of the changesdescribed by Monga et al. (65). Recently, ourgroup has shown positive staining for PCNA(`proliferating cell nuclear antigen') in endothe-lium of peritubular capillaries in biopsies ofpatients with more severe forms of acute renaltransplant rejection (69). This lesion could alsorepresent a precursor to the lesion described byMonga et al. and might provide a link betweenacute and chronic rejection (65).

Abrass et al. (70) have attempted to distinguish®brosis related to rejection from that due to CsAtoxicity by studying the distribution of moleculesinvolved in ®brosis by immuno¯uorescent stain-ing. These authors suggest that CsA toxicity ischaracterized by an increase in interstitial col-lagen types I and III whereas corticosteroidresponsive rejection was characterized insteadby de novo expression of collagen IV alpha chain3 and laminin-b2 in proximal tubular basementmembrane. It is possible, therefore, that thistechnique may differentiate between ®brosis thatis due to chronic rejection from that due to CsAtoxicity, but this is as yet unproven.

The renal biopsy in chronic rejection shouldnot be interpreted in isolation of clinical para-meters. At a recent consensus conference it wasrecommended that a diagnosis of chronic rejec-tion in addition to graft histology requires that`the reciprocal of plasma-creatinine over time issigni®cantly different from zero. At least 10consecutive values of plasma-creatinine or theplasma-creatinine over a 3-month interval shouldbe used' (9).

The renal biopsy: quantitation of chronic rejection

A number of groups have attempted to develop ascoring schema that would be predictive of long-term graft failure in chronic rejection. In

Fig. 1. Renal allograft biopsy from patient with chronicrejection. Notice neointima in vessel.

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1991 Kasiske et al. (71) published results from100 biopsies obtained from 74 patients trans-planted between 1978 and 1982. All allograftsfunctioned for at least 1 yr. Biopsies wereperformed either for diagnosis of acute deteriora-tion of renal function that did not reverse inresponse to empiric treatment with corticoster-oids or for progressive declines in renal function.In addition, patients were biopsied to diagnoseproteinuria of greater than 1.0 gm per 24h. Forty-four biopsies were from patients with a chronicprogressive decline in allograft function(CPDAF). They evaluated 16 different para-meters using a 0±100 scale and devised a chronicrejection score that quantitated glomerularmesangial expansion, chronic arterial intimalocclusion, interstitial ®brosis, tubular atrophy,glomerulosclerosis and basement membranereduplication. The chronic rejection score inpatients who were biopsied within the ®rst9 months post-transplant was not differentbetween patients with or without a progressivedecline in function. In patients biopsied late(mean of 44 months post-transplant) the chronicrejection score was signi®cantly higher in patientswith a progressive decline in function whencompared to the score of those patients whosefunction did not decline progressively (116 vs. 23;p , 0.001). Kasiske et al. (71) also showed acorrelation between proteinuria and progressivedecline in renal function. This paper was the ®rstto show a correlation between functional dete-rioration and a scoring system using multipleparameters of the kidney biopsy. The authorssuggested that `in patients with CPDAF, a biopsymay be useful in con®rming the diagnosis ofchronic rejection, and excluding other causes ofallograft dysfunction. Moreover, the severity ofthe chronic histopathologic changes may predictthe duration of subsequent allograft survival.'

The Chronic Allograft Damage Index (CADI)

Isoniemi et al. (18) published their studies oflong-term renal follow-up in 128 patients with a®rst cadaver renal transplant randomized to fourdifferent immunosuppressive regimens. Protocolrenal biopsies were performed in all patients witha functioning graft 2 yr post-transplant and readblindly. The fact that biopsies were performed at2 yr on a protocol, regardless of renal function,distinguishes this study from that of Kasiske et al.(71), where biopsies were from a wide variety oftime points. Histopathologic parameters werescored on a semiquantitative 0±3 scale. Aftercorrelation with clinical parameters the investi-gators developed a `chronic allograft damage

index (CADI)'. This index was the sum of thescores of diffuse in¯ammation and ®brosis in theinterstitium, mesangial matrix increase, sclerosisof glomeruli, intimal proliferation of vessels andtubular atrophy. The CADI was shown to belowest in the triple immunosuppressive therapygroup (CsA, azathioprine, prednisone) comparedwith any double immunotherapy group (18).Subsequently, Isoniemi et al. (72) published theresults of the CADI in 2-yr protocol biopsies in89 patients. A signi®cant correlation was foundbetween the CADI score and transplant function6 yr later (R = 0.717, p = 0.0001) (Fig. 2). Theyalso showed that the correlation coef®cient of theCADI improved with increasing follow-up time.The correlation coef®cient at 3 yr being 0.59, 4 yrbeing 0.57, 5 yr being 0.57 and 6 yr being 0.72(72). They state that `the CADI score is to ourknowledge the earliest predictor of chronicallograft rejection. Thus, a protocol core biopsyand the CADI may be used as an end point inprophylactic studies, before the necessary 5±20year follow-up is completed'.

The Chronic Graft Damage Score (CGDS)

The group from Uppsala (73) have obtainedprotocol biopsies at time points earlier than the2-yr point published by Isoniemi et al. (18) andcorrelated these with long-term graft dysfunction.They developed a pathologic scoring systementitled the `Chronic Graft Damage Score'(CGDS), which is calculated by adding thescores of the degree of vascular intimal hyper-plasia, glomerular mesangial changes, focallymphocytic in®ltration, focal and diffuse inter-stitial ®brosis and tubular atrophy evaluated in ablinded fashion. This score is very similar to the

Fig. 2. Scatter diagram relating the 2-yr CADI to the 6-yrserum creatinine level (mmol/L). The correspondingregression line and correlation coef®cient are also shown(n = 65). Reproduced from (72).

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CADI score of Isoniemi et al. (72). These authors(73) subsequently reported 99 biopsies performedat 6 months. The CGDS score was stronglyassociated with the risk of graft loss between 2and 3 yr following transplant. Patients with aCGDS greater than 6 had a higher graft loss rateof 17% at 2 yr, whereas patients with a CGDS ofless than 6 with a 2-yr graft loss rate of 4%(p = 0.037). The CGDS was also associated with:a higher serum creatinine, more proteinuria anda lower GFR at 2 yr (p = 0.003, p = 0.01,p = 0.008, respectively). At 3 yr post-transplant,patients with a 6-month protocol biopsy CGDSgreater than or equal to 6 had 29% graft loss,compared with only 4% in those with a CGDS ofless than 6. Similarly, for patients with afunctioning graft there was a direct correlationwith CGDS at 6 months and serum creatinine at3 yr (p = 0.0003).

The Banff Score in chronic rejection

The Banff schema (74) has been used extensivelyto quantitate acute graft rejection and has beeninstrumental in putting numerous studies of acuterejection on a ®rm scienti®c foundation. Its use inquantitation of chronic rejection is less frequent.The most recent iteration of the Banff criteria (seeref 101) includes quantitation of allograft glo-merulopathy, ®brosis, tubular atrophy, vascularintimal thickening and mesangial matrix in thescore. An adequate sample is de®ned as having 10or more glomeruli with at least two arteries. Onegroup performing protocol biopsies at 3 monthsand 2 yr showed that deterioration in the Banffchronic rejection score between these two timepoints is associated with proteinuria, hyperten-sion and donor age (28). Another group (75)reported 157 biopsies performed to investigate anunexplained elevation of serum creatinine orproteinuria greater than 1 gm per 24h. Theseinvestigators found 97 cases of chronic rejectionand showed that increasing severity of chronicrejection was associated with deteriorating graftsurvival following biopsy. Grade I chronicrejection was associated with a 90% graft survivalat 1 yr whereas grade II chronic rejection wasassociated with a 50% graft survival at 1 yrfollowing the biopsy (Fig. 3). Unfortunately,there was no speci®c time point at which thesebiopsies were performed (75). A recent paperused the Banff schema to generate a chronicrejection score in 2-yr protocol biopsies obtainedin patients randomized to either tacrolimus orCsA-based immunosuppression. The Banffchronic score and renal function at 2 yr was

not different between the two treatment groups(76).

Our own group (10) has shown that the chronicrejection score at 3 and 6 months is an indepen-dent risk factor for an elevated serum creatinineat 24 months. In addition, patients who have adeterioration in their serum creatinine between 6and 24 months have a higher total Banff chronicscore (simply summing the individual chronicBanff scores) at 3, 6 and 12 months post-transplant (Fig. 4) (10). Similarly, SeroÂn andcolleagues (77) performed protocol biopsiesbetween 2 and 5 months post-transplant inpatients who were otherwise stable. Theyshowed that the diagnosis of chronic transplantnephropathy as made using the Banff criteria wasassociated with a signi®cantly decreased serumcreatinine at 1 or 2 yr of follow-up (p = 0.0001 at1 yr; p = 0.024 at 2 yr). In addition, graft survivalat 4 yr was 90% in patients without and only 75%in patients with chronic transplant nephropathy(p = 0.024). These authors found that `thepredictive value of protocol biopsies is indepen-dent of the renal functional impairment, at leastwhen patients with a serum creatinine lower than200 mM/L (mg/dl) are considered' (77).

The above information demonstrates that thepresence of chronic graft pathology at any pointpost-transplant has the potential to predict poorlong-term graft outcome. Unfortunately, thecorrelation and predictive value of these estimatesmay not be outstanding in that negative chronicscores may underestimate the true situation dueto sampling error in a patchy process. This meansthat to use these techniques as surrogate markersin intervention studies, an extremely large samplesize (number of patients) may be necessary for anaccurate prediction. The next section discussessome techniques under investigation that mayhold promise for further development.

Fig. 3. Graft survival and the histologic grading in chronicrejection (n = 97). Reproduced from (75).

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Assessment of vasculopathy

In animal models, quantitation of the amount ofvascular neointimal formation has been useful(78). These studies have utilized image analysis todetermine the fraction of the lumen that ispreserved. These studies usually require samplingof the entire transplanted organ (usually heart oraorta) to obtain an adequate sample size toprovide a reasonable value. Unfortunately, threeissues limit the usefulness of this process inhuman renal transplant biopsies. The ®rst is thepatchy nature of the involvement in vessels.Armstrong et al. (79) have performed detailedserial sections of murine cardiac allograftsshowing that the epicardial coronary arterieshave a highly variable expression of intimalthickening at different positions in the sameartery (79). Next, percutaneous renal biopsiestypically show few arterial cross-sections. TheBanff criteria of an adequate renal biopsy samplespecify a minimum of only two vascular cross-sections. This leads to a rather small sample size.Finally, allograft vasculopathy tends to occur inlarger vessels than are sampled typically inpercutaneous renal allograft biopsies. All con-spire to make quantitation of allograft vasculardamage unlikely to provide dependable prognos-tic data.

Assessment of interstitial ®brosis

Assessment of interstitial ®brosis is one of themost reliable indicators of graft damage. The riskof sampling error is low because the interstitiumand tubules of many nephrons are sampled. Thedisadvantage of using interstitial ®brosis as amarker of chronic rejection is its lack ofspeci®city for that diagnosis. Whatever itscause, however, the quantitation of interstitial

®brosis has been shown to be useful as a predictorof long-term outcome. Rasdon et al. published in1968 that the presence of interstitial ®brosis inkidney biopsies was associated with more severerenal functional impairment in patients withglomerulonephritis (80). Bohl et al. used amorphometric technique with point counting toshow a signi®cant correlation between the relativeinterstitial volume fraction and the log of theserum creatinine in membranous glomerulone-phritis (81). Finally, in a study of 96 diabeticpatients, the correlation between interstitialcortical volume and creatinine clearance andlength of time of insulin-dependent diabetesmellitus was recognized (82).

In membranous glomerulonephritis, Riemen-schneider et al. (83) showed a correlation be-tween the relative interstitial volume of the renalcortex and the creatinine clearance, even inpatients whose serum creatinine was in thenormal range. This suggests that quantitatinginterstitial ®brosis can be more sensitive than theserum creatinine in the early stages of renaldisease (83). When interpreting quantitative datafrom transplant biopsies it may be important totake into account the time that the biopsy isobtained. SeroÂn et al. (84) showed that in renaltransplant recipients with excellent renal functionthere is an increase in interstitial ®brosis between3 and 12 months in spite of no change in serumcreatinine. This suggests that sub-clinical damagemay be occurring that may not become clinicallyapparent for many years and this natural historyunder current immunosuppressive protocols mayneed to be accounted for when assessing renalallograft biopsies.

Trichrome staining

Masson's trichrome stain has been used in theassessment of interstitial ®brosis, as it imparts adeep blue color to the interstitium which can thenbe quantitated by computerized image analysis(85). In renal transplantation quantitation ofinterstitial ®brosis was performed on protocolrenal biopsies by Nicholson et al. (86). Theyshowed no correlation between interstitial ®brosisas assessed using a histomorphometric pointcounting technique and subsequent 1-monthgraft survival. However, the 6-month biopsywas predictive of long-term outcome. In patientswhose 6-month biopsy showed an interstitialvolume fraction of . 25%, the subsequent graftsurvival was relatively poor at 80%, whereas if theinterstitial volume fraction was , 25% the sub-sequent 5-yr survival was 100% (p = 0.04). Theseinvestigators recommended that further studies

Fig. 4. Development of chronic allograft pathology overtime in subgroups with stable (u), improving (O) ordeclining (D) allograft function. *ANOVA, p , 0.005 stablevs. improving subgroup or stable vs. declining subgroup.Reproduced from (10).

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be carried out including use of automated imageanalysis to improve the speci®city and turnaround time of this technique. They also ques-tioned whether biopsies earlier than 6 monthswould be just as helpful as 6-month biopsies.

In our studies of quantitation of renal allograft®brosis we also attempted to use trichromestained renal biopsy specimens and computerizedimage analysis. Unfortunately, we encountered anumber of technical problems. The ®rst is that theblue color of the trichrome frequently stains theproximal tubular brush border. This is easy todiscount by a well-trained pathologist or clin-ician. However, the computer would incorporateproximal tubular brush border staining aspositive (Fig. 5). A second problem is thatquantitating the trichrome blue-stained intersti-tium does not separate interstitial expansion dueto cellular in®ltrate from interstitial expansiondue to pure ®brosis. Our initial attempts to usetrichrome stains were disappointing. We thenturned to using a stain speci®c for collagen ®berscalled Sirius Red.

Sirius Red staining

Sirius Red (also known as Sirius Red F3BA orPicro Sirius Red) staining was initially describedin 1964 (87), and was subsequently shown to be aspeci®c method for collagen detection in tissuesections including kidney (88). Sirius Red is along dye molecule that intercalates into thegroove in the collagen molecule. It is birefringentwhen observed under polarized light. In animalmodels Sirius Red staining using digitizedmethodology has been shown to be able toquantitate accurately and also date the age of®brotic lesions (89, 90). Sirius red staining hasbeen used to quantitate radiation-induced ®brosisin lung tissue (91), murine cardiac ®brosis (92)and the collagen content of infarcted myocar-dium following treatment with captopril (93). Inthe rat 5/6 nephrectomy model of renal disease,Sirius Red staining has been used to quantitatethe reduction of renal ®brosis by pharmacologictherapy (94).

In human studies, Sirius Red staining has beenused to quantitate ®brotic changes in pediatricliver transplant recipients (95). Response tointerferon alpha 2B therapy in liver ®brosis inchronic non-A, non-B hepatitis has also beenquantitated using this technique (96). In addition,this study showed that Sirius Red staining washighly signi®cantly correlated with total livercollagen as determined by collagen hydroxypro-line assay (96). It has also been used in humancardiac transplantation and to show that ®brosis

in the transplanted heart is correlated with totalischemic time (p . 0.001) and that donor age(range 10±51 yr) did not correlate with subse-quent ®brosis (97). In human renal biopsies SiriusRed staining has been quantitated using auto-mated image analysis and has been shown tocorrelate with the interstitial volume fraction ofthe cortex, as measured by the more laboriouspoint counting method (98). In addition, SiriusRed staining of collagen and analyzed withmathematical morphometry has been shown tobe highly correlated with the glomerular ®ltrationrate at the time of biopsy (99).

Due to the potential of Sirius Red staining as atool in analysis of ®brosis we have initiated aproject using Sirius Red staining in our bank ofprotocol human renal allograft biopsies (Fig. 6).We are attempting to derive a risk score todetermine: 1, what is the earliest time point (3, 6or 12 months?) in which a biopsy can be highly

(a)

(b)

Fig. 5. (a) Trichrome stained allograft biopsy. (b)Intermediate stage of computerized image analysisshowing that areas of proximal tubular brush border areinappropriately assigned to the blue `®brosis' area (arrows).

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265

correlated with long-term graft function (good orpoor), and 2, how strong and consistent is thepredictive value of this tool for risk assessment oflong-term graft outcome? The ®nding of an earlyand highly predictive marker of long-term out-come would allow us to stratify patients intohigh- or low-risk prognostic groups which wouldhave implications for therapy, as well as serve as asurrogate marker which will allow earlier assess-ment of treatment regimens without waiting the10±20 yr necessary for graft failure to occur.

Conclusions

Several non-invasive techniques have been usedin an attempt to quantitate the loss of graftfunction in chronic rejection. The rate of changeof serum creatinine or GFR, and the quantitationof proteinuria are the most prevalent of these.Unfortunately, neither of these methods isreliable in individual patients and over short

periods of time. Some of the problems associatedwith utilizing the 1/serum creatinine methodinclude (a) changes in creatinine due to changesin body weight or composition, (b) strikingchanges in the slope of the curve that mayoccur after various periods of observation, and(c) a lack of sensitivity of the serum creatinine tore¯ect graft damage due to sub-clinical rejection.Quantitation of proteinuria lacks speci®city forchronic rejection as it may result from otherconditions such as recurrent disease and de novoglomerulonephritis. Ultimately, the non-invasiveprediction of allograft outcome may requireaccounting for the complex interrelationships offactors of the donor (age, cold ischemia time, etc.)recipient (cytokine gene expression status, bodyhabitus, etc.) and response to therapy (renalfunction, acute rejection episodes) in a form ofsystems analysis, or neural network decisionanalysis (100).

The percutaneous renal biopsy remains the`gold standard' in the diagnosis of chronicallograft rejection. Recently, a number of inves-tigators have developed scoring systems forvarious chronic pathological changes involvingglomeruli, tubules, vessels and interstitium thatmay be predictive of long-term graft function.The Banff schema is the most frequently used atpresent for this purpose; others include the CADIand CGD methods. The various scoring methodshave not been subject to direct comparison witheach other on the same set of biopsies. Somemethods have been used by only one or a fewcenters or pathologists, and therefore have notyet been proved to be robust enough to be used ina multicenter fashion. Furthermore, some havebeen studied only in older (2 yr) biopsies, othersin earlier (6-month) biopsies. The earliest timepoint from which reliable prognostic informationcan be obtained is not known. None have beensubmitted to formal sensitivity and speci®cityanalysis of their predictive potential.

The impetus for this paper was the need toreview the techniques used to quantitate theseverity of chronic renal allograft injury that isknown as chronic rejection or chronic allograftnephropathy as this information may havetherapeutic and prognostic value (10, 77). Thispaper is not focused on the diagnosis of cause of®brosis, i.e. CsA nephrotoxicity vs. chronicimmunologic injury. Perhaps in this era ofcalcineurin inhibitor-based immunosuppressionboth processes will coexist in most patients to agreater or lesser extent. In the allograft biopsiedwell after the initial events of procurement injury,cold and warm ischemia, clinical and sub-clinicalacute rejection and the pro®brotic effects of high

(a)

(b)

Fig. 6. Sirius Red-stained human renal allograft biopsies. (a)demonstrates normal tubulo-interstitium showing delicatelacy collagen staining (black arrowhead) and a ®brousseptum (gray arrowhead); (b) is a biopsy from a patient withchronic rejection showing coarsening, thickening and multi-lamination of the intertubular collagen (illumination by leftto right gradient of visible light to polarized light).

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initial doses of calcineurin inhibitors, the primaryissue is to quantitate the aggregate of theseindividual injuries which will be re¯ected in long-term outcome. It will be necessary, however, thata formal comparison of the various techniquesused for the quantitation of chronic renalallograft nephropathy be undertaken, and thatsuch important issues as their speci®city/sensitiv-ity, predictive value and reproducibility beestablished. To settle these issues rapidly, oncea technique is developed it should be studied byhaving the same slides assessed in a blindedfashion by another pathologist, preferably atanother institution (inter-observer variation).Assessment of re-cut specimens and reassessmentof slides in a blinded fashion would serve to de®neintra and inter-assay variability. Finally, theresults should be subjected to formal sensitivityand speci®city analysis in the way that otherclinical tests are evaluated. To determine theseissues rapidly the analysis should utilize biopsiesfor which a long period of follow-up is available.De®nitive and quantitative techniques, such ascounting the layers of peritubular basementmembrane and the use of computerized imageanalysis techniques to quantitate interstitial®brosis, are areas requiring further investigation.Similarly, analysis of the constituent proteins inthe ®brous scar in the tubulo-interstitial compart-ment should be further pursued. We are hopefulthat use of some or all of the above-mentionedapproaches will lead to improvement in thediagnostic and prognostic capability of theallograft biopsy in chronic rejection.

Acknowledgments

This study was funded by grants from the BaxterExtramural Grant Program, NIH (NIAID R01-AI43655-02) and The Children's Hospital of WinnipegResearch Foundation. Dr Grimm was supported by theChildren's Hospital of Winnipeg Foundation and theManitoba Medical Services Foundation. Dr Nickerson issupported by a Medical Research Council of CanadaScholarship.

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