Background Acute rejection is a serious and frequent complicationof renal transplantation, and its diagnosis is contingent onthe invasive procedure of allograft biopsy. A noninvasive diagnostictest for rejection could improve the outcome of transplantation.
Methods We obtained 24 urine specimens from 22 renal-allograftrecipients with a biopsy-confirmed episode of acute rejectionand 127 samples from 63 recipients without evidence of acuterejection. RNA was isolated from the urinary cells. MessengerRNA (mRNA) encoding the cytotoxic proteins perforin and granzymeB and a constitutively expressed cyclophilin B gene were measuredwith the use of a competitive, quantitative polymerase-chain-reactionassay, and the level of expression was correlated with allograftstatus.
Results The log-transformed mean (±SE) levels of perforinmRNA and granzyme B mRNA, which encode cytotoxic proteins, butnot the levels of constitutively expressed cyclophilin B mRNA,were higher in the urinary cells from the 22 patients with abiopsy-confirmed episode of acute rejection than in the 63 recipientswithout an episode of acute rejection (perforin, 1.4±0.3vs. –0.6±0.2 fg per microgram of total RNA; P<0.001;and granzyme B, 1.2±0.3 vs. –0.9±0.2 fgper microgram of total RNA; P<0.001). Analysis involvingthe receiver-operating-characteristic curve demonstrated thatacute rejection can be predicted with a sensitivity of 83 percentand a specificity of 83 percent with the use of a cutoff valueof 0.9 fg of perforin mRNA per microgram of total RNA, and witha sensitivity of 79 percent and a specificity of 77 percentwith the use of a cutoff value of 0.4 fg of granzyme B mRNAper microgram of total RNA. Sequential urine samples were obtainedfrom 37 patients during the first nine days after transplantation,and measurements of the levels of mRNA that encoded cytotoxicproteins identified those in whom acute rejection developed.
Conclusions Measurement of mRNA encoding cytotoxic proteinsin urinary cells offers a noninvasive means of diagnosing acuterejection of renal allografts.
Renal transplantation is the treatment of choice for most patientswith end-stage renal disease. However, because of the wide disparitybetween the supply of organs and the demand,1 many patientswait three to four years for a suitable organ. Allograft failureis one of the four most common causes of end-stage renal diseasein the United States2 and is an important factor in the organ-shortageproblem. Indeed, about 20 percent of the patients in the UnitedStates who are on the waiting list are those with a failed graft,1and about 15 percent of the procedures performed are repeatedtransplantations.3
Acute rejection, defined as a sudden deterioration in renal-allograftfunction as a result of the recipient's immune response to thedonor organ, is a major risk factor for allograft failure.3,4,5,6,7,8About 35 percent of allograft recipients have an episode ofacute rejection in the first year after transplantation.4 Acuterejection is associated with a 20 percent reduction in the one-yearsurvival rate of cadaveric grafts, and the projected half-lifeof the allografts is four years shorter in patients who havehad an episode of acute rejection than in patients who havenot had an episode of acute rejection.8
Needle biopsy of allografts is the standard test for the diagnosisof acute rejection. Recent refinements have reduced but noteliminated biopsy-associated complications, such as hematuria,anuria, perirenal hematoma, bleeding and shock, arteriovenousfistulas, and graft loss.9,10,11 Sampling errors pose an additionalproblem, and multiple samples are therefore needed to increasediagnostic accuracy.12,13,14,15 The development of an accurate,noninvasive diagnostic test that also provides insights intothe mechanisms of rejection would be of considerable value.
We have developed a competitive, quantitative polymerase-chain-reaction(PCR) assay that permits the noninvasive diagnosis of allograftrejection at the molecular level. We assessed the diagnosticaccuracy of measuring the levels of perforin and granzyme Bmessenger RNA (mRNA) in urinary cells from renal-allograft recipients.We measured the mRNA of perforin, a pore-forming protein,16and the mRNA of granzyme B, a serine peptidase,17 because theseproteins are integral components of the lytic machinery of cytotoxiccells,18,19,20,21,22,23 and cytotoxic cells are often presentin allografts that are undergoing acute rejection.24
Methods
Collection of Urine Samples and Renal-Biopsy Specimens
We collected 151 urine specimens (110 in the first month aftertransplantation, 24 one to six months after transplantation,and 17 more than six months after transplantation) from 85 renal-allograftrecipients. Thirty-eight of these patients underwent needlebiopsy (yielding 44 specimens) to identify the basis for graftdysfunction; urine was collected before each biopsy. The biopsyspecimens were fixed in formalin, embedded in paraffin, andstained with hematoxylin and eosin, periodic acid–Schiff,or Masson's trichrome. The specimens were reviewed and classifiedwith use of the Banff 97 classification12 by a single pathologistwho did not know the results of the molecular studies. On thebasis of histologic findings, 24 renal-biopsy specimens from22 recipients (mean [±SD] age, 44±15 years; 10women and 12 men) were classified as showing acute rejection,5 specimens from 5 recipients (mean age, 52±17 years;3 women and 2 men) were classified as showing chronic allograftnephropathy, and 15 specimens from 11 recipients (mean age,50±13 years; 2 women and 9 men) were classified as showingother findings according to the Banff 97 classification. Sevenof the 15 samples showed toxic tubulopathy, 4 had nonspecificchanges, 3 showed acute tubular necrosis, and 1 had signs ofrenal-vein thrombosis.
The remaining 107 urine specimens were from 47 patients whowere classified as having stable allograft function after transplantation(mean age, 47±12 years; 12 women and 35 men). In thesepatients, the serum creatinine levels either had decreased orhad not changed by more than 0.2 mg per deciliter (18 µmolper liter) during the seven days before and the seven days afterurine collection. Sequential urine samples were obtained from37 patients (mean age, 46±12 years; 14 women and 23 men)during the first nine days after transplantation.
Eleven of the 85 renal-allograft recipients (mean age, 52±13years; 1 woman and 10 men) had impaired graft function in thefirst week after transplantation and required dialysis therapyat that time. This group was classified as having delayed graftfunction.
Immunosuppression consisted of a cyclosporine-based or tacrolimus-basedregimen, with antilymphocyte antibodies (muromonab-CD3 [OKT3]or antithymocyte globulin) given for episodes of glucocorticoid-resistantacute rejection.25 The study was approved by the institutionalreview board at the Weill Medical College of Cornell University,and each patient gave written (or oral, if only urine sampleswere involved) informed consent.
Isolation of RNA
Urine was centrifuged at 10,000xg for 30 minutes at 4°C.RNA was extracted from the pellet with use of a commercial kit(RNeasy minikit, Qiagen, Chatsworth, Calif.). More than 95 percentof urine specimens yielded RNA suitable for PCR. For each sample,1 µg of RNA was reverse-transcribed to complementary DNA(cDNA).26
Construction of Gene-Specific DNA Competitors and Quantitative PCR
The design and construction of gene-specific DNA competitorsare shown in Figure 1. The cDNA of granzyme B, perforin, orcyclophilin B was amplified with different concentrations ofDNA competitors. The PCR products were resolved by electrophoresis,stained with ethidium bromide, and scanned by laser densitometry.26We quantified the concentrations of naturally occurring genetranscripts by measuring the ratio of the cDNA band to the bandof the specific competitor. Transcript levels were expressedin femtograms of specific mRNA per microgram of total RNA.
Figure 1. Design and Construction of DNA Competitors.
A DNA competitor of granzyme B cDNA was prepared by digestion of the 180-bp naturally occurring product of PCR (GenBank accession number M28879) with MseI and ligation of the subfragments with a 44-bp DNA insert with appropriate cohesive ends at the 5' and 3' ends. A DNA competitor of perforin cDNA was prepared by digestion of the 176-bp naturally occurring product of PCR (GenBank accession number M28393) with NlaIII and ligation of the subfragments with a 36-bp DNA insert. The 274-bp cDNA competitor of cyclophilin was amplified with use of a modified sense primer that contains the external sense primer at its 5' end and a 16-bp subfragment internal sense primer at its 3' end corresponding to sequences 302 to 317 within the naturally occurring product of PCR (GenBank accession number M60857).
Statistical Analysis
We used SAS software (SAS, version 7.0, SAS Institute, Cary,N.C.) for data analysis. Before we compared the steady-statelevels of mRNA in the various groups, we examined the normalityof the distributions of transcript levels. The levels of perforinmRNA, granzyme B mRNA, and cyclophilin B mRNA deviated significantlyfrom the normal distribution (P<0.001), and the extent ofthe deviation was substantially reduced through the use of alog transformation. We used the natural logarithm (ln) of mRNAlevels as the dependent variable in a one-way mixed-level analysisof variance27 to identify any differences among the four groups.We then used Dunnett's test for multiple comparisons to controlfor the risk of a type I error while comparing the mRNA levelsin the acute-rejection group with those in the group with otherfindings, the group with chronic allograft nephropathy, andthe group with a stable course after transplantation. We useda conventional receiver-operating-characteristic (ROC) curveto analyze mRNA levels in order to determine the cutoff pointsthat yielded the highest combined sensitivity and specificitywith respect to distinguishing patients with an episode of acuterejection from those without such an episode. We calculatedthe area under the curve and used generalized estimating equations28to reestimate the sensitivity and specificity at the selectedcutoff point after adjustment for the lack of independence resultingfrom the inclusion of multiple urine specimens from some patients.
Results
Histologic Classification of Renal-Allograft–Biopsy Specimens
The Banff 97 classification12 was used to categorize the biopsyspecimens as showing acute rejection in 24 specimens from 22patients, chronic allograft nephropathy in 5 specimens from5 patients, and other findings in 15 specimens from 11 patients.Of the 24 biopsy specimens that showed acute rejection, 2 weregraded as borderline, 6 as grade IA (focal moderate tubulitis),8 as grade IB (severe tubulitis), 5 as grade IIA (mild-to-moderateintimal arteritis), 2 as grade IIB (severe intimal arteritis),and 1 as grade III (transmural arteritis). Among the 22 patientswith biopsy evidence of acute rejection, the clinical diagnosis,as assessed by the response to antirejection therapy with glucocorticoidsor antilymphocyte antibodies in 20 patients and by histologicanalysis of nephrectomy specimens in 2 patients, was consistentwith the biopsy diagnosis. Two of the biopsy specimens showingacute rejection had features of chronic allograft nephropathy:one had severe interstitial fibrosis, tubular atrophy, and tubularloss (grade III chronic allograft nephropathy), and the otherhad moderate (grade II) changes. Among the five biopsy specimensclassified as showing chronic allograft nephropathy, three hadgrade II chronic allograft nephropathy and two had grade I changes.
Levels of mRNA in Urinary Cells
The levels of perforin and granzyme B mRNA, but not those ofconstitutively expressed cyclophilin B mRNA, were higher inurinary cells from patients with an episode of acute rejectionthan in those without such an episode (Figure 2 and Table 1).The log-transformed mean (±SE) level of perforin mRNAwas 1.4±0.3 fg per microgram of total RNA in the patientswith an episode of acute rejection (24 samples from 22 patients)and –0.6±0.2 fg per microgram of total RNA in thepatients without an episode of acute rejection (127 samplesfrom 63 patients) (P<0.001). (A negative value for the log-transformeddata corresponds to an untransformed value of mRNA that is lessthan 1 fg per microgram of total RNA but greater than zero.)The levels of perforin mRNA in urinary cells obtained from patientswith an episode of acute rejection were significantly higherthan the levels in patients with stable graft function aftertransplantation (P<0.001), patients with other findings (P<0.001), or patients with chronic allograft nephropathy (P=0.03).
Box and whisker plots show the 10th, 25th, 50th (median), 75th, and 90th percentile values for perforin mRNA, granzyme B mRNA, and cyclophilin B mRNA in urine samples from patients classified as having an episode of acute rejection, other findings on allograft biopsy (acute tubular necrosis, toxic tubulopathy, or nonspecific changes), chronic allograft nephropathy, or a stable course after transplantation. The levels of perforin and granzyme B mRNA, but not those of cyclophilin B, were significantly higher in the patients with an episode of acute rejection than in the patients in the other groups (P=0.001 by one-way mixed-level analysis of variance). Values in parentheses are the numbers of urine samples. In all cases log-transformed values are shown.
Table 1. Levels of mRNA in Urinary Cells from Patients with an Episode of Acute Rejection and Patients with Other Findings on Allograft Biopsy, Patients with Biopsy Evidence of Chronic Allograft Nephropathy, and Patients with Stable Graft Function after Transplantation.
The levels of granzyme B mRNA were 1.2±0.3 fg per microgramof total RNA in the patients with an episode of acute rejectionand –0.9±0.2 fg per microgram of RNA in the patientswithout an episode of acute rejection (P<0.001). The levelsof granzyme B mRNA in urinary cells obtained from patients withan episode of acute rejection were significantly higher thanthose in patients with stable graft function after transplantation(P<0.001) and patients with other findings (P=0.001), butnot in patients with chronic allograft nephropathy (P=0.12).The levels of cyclophilin B mRNA did not vary significantlyamong the four groups of patients (P=0.90) (Table 1 and Figure 2).
Sixteen of the 24 biopsy specimens showing acute rejection wereobtained within three months after transplantation. The mRNAlevels of cytotoxic genes in the urinary cells from these 16biopsy specimens were similar to the levels in the urinary cellsfrom the 8 biopsy specimens that were obtained more than threemonths after transplantation (perforin, 1.4±0.4 vs. 1.5±0.5fg per microgram of total RNA, P=0.76; and granzyme B, 1.0±0.3vs. 1.6±0.4 fg per microgram of total RNA, P=0.23).
Sixteen of 24 biopsy specimens showing acute rejection had hadhistologic changes of grade IB or less. The mean level of perforinmRNA was 1.6±0.3 fg per microgram of total RNA in urinesamples from these 15 patients, and it was 1.0±0.5 fgper microgram of total RNA in the urine samples from the 7 patientswith changes of grade II or grade III (P=0.29). The mean levelof granzyme B mRNA was 1.6±0.3 fg per microgram of totalRNA in the patients with changes of grade IB or less and 0.6±0.4fg per microgram of total RNA in the patients with changes ofgrade II or grade III (P=0.05).
ROC-Curve Analysis of mRNA Levels
The ROC curves (Figure 3) show the fraction of true positiveresults (sensitivity) and false positive results (1 –specificity) for various cutoff levels of perforin mRNA, granzymeB mRNA, and cyclophilin B mRNA. The log-transformed thresholdthat gave the maximal sensitivity and specificity for perforinmRNA was 0.9 fg per microgram of total RNA; at this threshold,the sensitivity was 83 percent and the specificity was 83 percent(P<0.001) (Figure 3). The log-transformed threshold was 0.4fg per microgram of total RNA for granzyme B mRNA; at this threshold,the sensitivity was 79 percent and the specificity was 77 percent(P<0.001) (Figure 3). The levels of cyclophilin B mRNA werenot useful in identifying allografts that would show acute rejection.
Figure 3. Receiver-Operating-Characteristic Curve of mRNA Levels.
The fraction of true positive results (sensitivity) and false positive results (1 – specificity) for perforin mRNA levels, granzyme B mRNA levels, and cyclophilin B mRNA levels as markers of acute rejection are shown. The calculated area under the curve was 0.86 for perforin mRNA levels, 0.86 for granzyme B mRNA levels, and 0.58 for cyclophilin B mRNA levels. A value of 0.5 is no better than expected by chance, and a value of 1.0 reflects a perfect indicator.
The ROC-curve analysis included all 151 urine specimens in whichtranscript levels were measured. Forty-four samples were frompatients who had undergone renal-allograft biopsy, and 107 werefrom patients classified on the basis of clinical criteria ashaving a stable course after transplantation. Whereas the presenceor absence of acute rejection is known with a high degree ofcertainty in patients who have undergone allograft biopsy, somepatients classified on the basis of clinical criteria as havinga stable course after transplantation might have histologicchanges of acute rejection.29 In order to eliminate this possibility,we repeated the ROC-curve analysis using only the results fromthe patients who had undergone allograft biopsy. This evaluationrevealed that the log-transformed perforin mRNA level of 0.9fg per microgram of total RNA had a sensitivity of 83 percentand a specificity of 83 percent (P<0.001) and that a granzymeB mRNA level of 0.4 fg per microgram of total RNA had a sensitivityof 79 percent and a specificity of 64 percent (P=0.006) forthe diagnosis of acute rejection (Table 2).
Table 2. Levels of Perforin mRNA and Granzyme B mRNA in Patients with Acute Rejection and in Those without Acute Rejection.
Renal-Allograft Recipients with Delayed Graft Function
Of 11 biopsy specimens from patients with delayed graft function,2 showed acute tubular necrosis, 7 had evidence of toxic tubulopathy,1 had nonspecific changes, and 1 had evidence of both acutetubular necrosis and acute rejection. The levels of perforinmRNA and granzyme B mRNA were significantly lower in the 19urine samples from the 10 patients with delayed graft functionfrom nonimmunologic causes than in the 24 samples from the 22patients with an episode of acute rejection (perforin mRNA,–0.8±0.5 vs. 1.4±0.3 fg per microgram oftotal RNA, P<0.001; and granzyme B mRNA, –0.4±0.5vs. 1.2±0.3 fg per microgram of total RNA, P= 0.004).The levels of perforin mRNA and granzyme B mRNA in the onlypatient with a clinical diagnosis of delayed graft functionand a histologic diagnosis of acute rejection were 1.0 and 1.2fg per microgram of total RNA, respectively, and were similarto those in the patients with an episode of acute rejection.
Serial Studies in the Early Post-Transplantation Period
Sequential urine samples were obtained from 37 patients duringthe first nine days after transplantation. A mixed-level two-wayanalysis of variance was used to estimate and compare the meanlevels of perfo-rin mRNA, granzyme B mRNA, and cyclophilin BmRNA during days 1 through 3, 4 through 6, and 7 through 9 in8 patients in whom acute rejection developed within 10 daysafter transplantation and in 29 patients in whom acute rejectiondid not develop within the first 10 days. The levels of perforinmRNA and granzyme B mRNA, but not those of cyclophilin B mRNA,were higher in urine samples obtained on days 4 through 6 and7 through 9 from patients in whom acute rejection developedthan in samples from those without acute rejection (Figure 4).
Figure 4. Mean (±SE) Levels of Perforin mRNA, Granzyme B mRNA, and Cyclophilin B mRNA in Sequential Urine Samples.
Perforin mRNA, granzyme B mRNA, and cyclophilin B mRNA were measured in urine samples obtained during the first nine days after transplantation. The levels of perforin mRNA and granzyme B mRNA but not those of cyclophilin B mRNA were higher in the 8 patients in whom acute rejection developed within the first 10 days after transplantation than in the 29 patients in whom acute rejection did not develop within the first 10 days after transplantation. The respective numbers of urine samples obtained from the patients with an episode of acute rejection and those without such an episode were as follows: 6 and 43 on day 1, 2, or 3 after transplantation; 5 and 26 on day 4, 5, or 6; and 6 and 14 on day 7, 8, or 9. Means, standard errors, and P values were estimated with use of a mixed-level two-way analysis of variance. In all cases, log-transformed values are shown.
Discussion
We found that an episode of acute rejection of a renal allograft,an important and treatable risk factor for allograft failure,can be diagnosed accurately and noninvasively by measurementsof perforin and granzyme B mRNA in urinary cells. Perforin,which is stored in and secreted by the granules of cytotoxiceffector cells, forms pores in target-cell membranes and causescell death.16 Granzyme B, which is expressed primarily by activatedcytotoxic cells, is an integral member of the lytic machineryof cytotoxic cells.17 In the granule–exocytosis modelof cytotoxicity, perforin creates holes in the membrane of thetarget cell and facilitates the entry of granzyme B into thecell.18,19,20,21,22,23 Granzyme B then induces DNA fragmentationand cell death through the activation of caspase 3.30
Studies in animals and patients have implicated perforin andgranzyme B in allograft rejection. Perforin-deficient mice haveimpaired cytotoxic effector cells and weakly reject cardiacallografts.31 Granzyme B–deficient mice have reduced cytolyticactivity.20 Clinical studies suggest that acute rejection ischaracterized by the heightened expression of cytotoxic geneswithin the allograft.32,33,34,35,36,37,38 The functional attributesof perforin and granzyme B provided the rationale for the evaluationof levels of perforin mRNA and granzyme B mRNA in urinary cellsas markers of acute rejection.
An increase in the serum creatinine level is often the firstclinical indicator of acute rejection and is currently the bestsurrogate marker of it. However, it lacks sensitivity and specificity.The limitations associated with monitoring allograft rejectionby measurements of serum creatinine have been forcefully broughtto light by the observation that 30 percent of allograft biopsiesperformed in patients with stable renal function or in patientswho were considered to have been successfully treated for rejectionreveal histologic features of acute rejection.29,39 These occultrejections appear biologically relevant, since treatment betterpreserves the structure and function of renal allografts.40
The accurate diagnosis of acute rejection is contingent on theinvasive procedure of needle biopsy of allografts. Repetitivebiopsies, although ideal from a diagnostic perspective, areconstrained by several practical considerations, including thecomplications associated with the procedure and sampling errors.9,10,11,12,13,14,15Thus, the availability of a noninvasive indication of rejectionwould be clinically useful.
We have demonstrated the feasibility of accurate noninvasivediagnosis of acute rejection by measurements of perforin mRNAand granzyme B mRNA in urinary cells, and we think that thisdiagnostic test also has the potential to predict the developmentof acute rejection. In this regard, the diagnostic accuracyand mechanistic insights might be further improved by quantificationof additional genes such as the fas ligand gene.33
Allograft recipients with delayed graft function have low ratesof graft survival and are at higher risk for acute rejectionthan are patients with immediate graft function.3,8,41 Delayedgraft function can result from nonimmunologic causes, immunologiccauses, or a combination of both. Serum creatinine values areuninformative, and biopsy is currently mandatory to establishthe cause. Our data showing that patients with delayed graftfunction owing to nonimmunologic causes can be distinguishedfrom patients with acute rejection offer a method for clarifyingthe mechanism and providing a specific therapy for patientswith impaired renal function in the period immediately aftertransplantation.
Our studies using gene-specific constructs of competitor DNAin quantitative PCR demonstrate that acute rejection of renalallografts can be diagnosed accurately and noninvasively byquantification of perforin mRNA and granzyme B mRNA in urinarycells. In addition to functioning as surrogates for allograftbiopsy, mRNA phenotyping of urinary cells may lead to the molecularclassification of rejection and to the identification of suitabletherapeutic targets.
Supported in part by an award (AI-37206) from the National Institutesof Health. Dr. Hartono is a recipient of the American Societyof Transplantation Fellowship award, and Dr. Ramaswamy is arecipient of the National Kidney Foundation of New York–NewJersey Fellowship award.
A U.S. patent entitled "Methods of Evaluating Transplant Rejection"(6187543) was issued on February 13, 2001; Dr. Suthanthiranis one of the inventors. The patent is owned jointly by Harvard,Cornell, and the Beth Israel Deaconess Medical Center.
We are indebted to Dr. Phyllis August for careful review andto Ms. Linda Stackhouse for assistance in the preparation ofthe manuscript.
Source Information
From the Division of Nephrology, Department of Medicine (B.L., C.H., R.D., V.K.S., R.R., B.Q., D.S., M.S.), and the Department of Pathology (J.M.), Weill Medical College of Cornell University, New York; the Department of Transplantation Medicine, New York–Presbyterian Hospital, New York (C.H., D.S., M.S.); and the Department of Psychiatry, State University of New York at Stony Brook, Stony Brook (J.E.S.).
Address reprint requests to Dr. Suthanthiran at the Division of Nephrology and Department of Transplantation Medicine, 525 E. 68th St., Box 3, New York, NY 10021, or at msuthan{at}med.cornell.edu.
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