Tubular Chimerism Occurs Regularly in Renal Allografts and Is Not Correlated to Outcome
Michael Mengel*,
Danny Jonigk*,
Magali Marwedel*,
Wolfram Kleeberger*,
Martin Bredt*,
Oliver Bock*,
Ulrich Lehmann*,
Wilfried Gwinner,
Hermann Haller and
Hans Kreipe*
*Institut fuer Pathologie der Medizinischen Hochschule Hannover and Abteilung fuer Nephrologie der Medizinischen Hochschule Hannover, Germany.
Correspondence to: Prof. Dr. med. Hans Kreipe, Institut fuer Pathologie, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, 30625 Hannover, Germany. Phone: +49-511-5324500; Fax: +49-511-5325799; E-mail: kreipe.hans{at}mh-hannover.de
ABSTRACT. Recent studies have demonstrated an integration ofrecipient-derived progenitor cells into solid allografts withdifferentiation into parenchymal cells. Whether or to what extentthis phenomenon influences allograft outcome has still to beelucidated. To detect epithelial chimerism tubular cells wereharvested from sequential renal allograft biopsy samples bylaser microdissection in 36 patients. Recipient-derived cellswere detected by short-tandem repeat–based genotyping.In cases with gender-mismatched transplantation, chimerism wassemiquantitatively evaluated by in situ hybridization for theY-chromosome. Findings were correlated to different pathomechanismsof epithelial injury as well as to morphologic and clinicaloutcome. Epithelial chimerism was detectable as early as 8 dafter transplantation and lasted for 8 yr. A total of 88% ofthe patients showed an epithelial chimerism; 72% had a stablechimerism in sequential biopsy samples. Evaluation of Y-chromosomeby in situ hybridization revealed low percentages of chimericaltubular epithelial cells (2.4% to 6.6%). No correlation to morphologywas found. Chimerism was detectable in inconspicuous protocolbiopsy samples, cases of drug toxicity, and rejected allograftswith and without chronic changes. No correlation was found toallograft function. Epithelial microchimerism is an early andpersistent phenomenon after renal transplantation. There isno correlation to morphologic or functional outcome. Probablyrecipient-derived stem cells contribute in a minor fashion totissue homeostasis, and cell turnover in renal allografts ispredominantly enabled by donor cell renewal.
Immediately after transplantation, different nonimmunologicaland immunological mechanisms can damage the tubular epithelialcells of a renal allograft (1–3). From observations innative kidneys, it is assumed that the source of regenerationafter acute tubular damage are the viable epithelial cells atboth ends of the damaged nephron segment, as well as other residualepithelial cells surviving along the tubular basement membrane(4,5). However, the definite origin of regenerating cells andthe sequence of cellular recovery after acute tubular damageare not known exactly. On the basis of the mesenchymal-epithelialcell transdifferentiation during nephrogenesis, it might bealso possible that interstitial multipotent, bone marrow–derivedstem cells transdifferentiate into tubular epithelial cellsunder pathologic conditions (4,6–8).
In transplanted kidneys, recipient-derived pluripotent stemcells are thought to be an additional source for replacementof perished allograft cells. Recently, this could readily bedemonstrated in animal models for tubular epithelial cells,stromal cells, glomerular podocytes, and endothelial cells undergoingdamage from a variety of causes (6,9). In human renal allografts,recipient-derived endothelial cells were detected after vascularrejection (10). Neointimal and interstitial mesenchymal cellsof recipient origin have been described in transplants withchronic allograft nephropathy (11). The chimeric coexistenceof recipient- and donor-derived epithelial cells was recentlydemonstrated in other solid organ transplants (heart, lung,liver), indicating the tremendous plasticity of circulatingrecipient-derived precursor cells (12–15). From theserecent studies, which were all based on relatively low numbersof cases (n < 15), it was assumed that cellular injury isthe preceding event introducing intragraft chimerism after transplantationin humans. How different pathomechanisms and various types ofcell damage induce intragraft microchimerism is not known. Furthermore,it is still a matter of debate whether recipient-derived intragraftmicrochimerism influences the immunresponse to the allograftin the sense of allograft accommodation, as was postulated byMedawar (16) four decades ago. Although the mechanism that leadsto an integration of recipient-derived cells, transdifferentiation,or cell fusion is still under discussion, the effect of resultingchimerism on allograft outcome deserves study (17,18).
Because tubules are the major target of most mechanisms of acutecell damage in renal allografts, leading to tubular atrophyand chronic allograft nephropathy, we investigated chimerismof tubular epithelial cells. For this purpose, laser microdissectionof tubular epithelial cells was combined with subsequent highlysensitive genotyping (DNA fingerprinting) by short-tandem repeat(STR)–PCR (12,13). This technique enables study of allallografts independent of gender mismatch. To further analyzethe influence of different mechanisms of tubular injury on theinduction of epithelial chimerism, and to assess whether epithelialchimerism has any effect on tubular regeneration after acuteinjury, we investigated sequential renal allograft biopsy samplesfrom 36 patients with variable morphologic and functional outcome.
Cases
Sequential renal allograft biopsy samples from 36 patients wereinvestigated. The first biopsy was performed within 3 mo aftertransplantation (8 to 91 d), and the second follow-up biopsywas performed at least 6 mo after transplantation (166 to 2910d). All biopsy samples were histologically evaluated followingthe Banff ’97 classification (1). The patients were dividedinto seven subgroups according to different pathomechanismsthat cause tubular epithelial damage (Table 1). Depending onthe diagnosis made on the basis of the first biopsy sample andon whether, in the second biopsy sample, signs of chronic allograftnephropathy (tubular atrophy, interstitial fibrosis) were found,a patient was assigned to one of the following groups: (1) histopathologicallyunremarkable in first and second biopsy samples; (2) calcineurininhibitor (CNI) toxicity in the first biopsy sample and chronicallograft nephropathy (CAN) in the follow-up sample; (3) CNItoxicity in the first biopsy sample and no CAN in the follow-upsample; (4) acute rejection in the first biopsy sample and CANin the second sample; (5) acute rejection in the first biopsysample and no CAN in the second sample; (6) Banff ’97borderline changes in the first biopsy sample and CAN in thesecond sample; and (7) Banff ’97 borderline changes inthe first biopsy sample and no CAN in the second sample. Additionally,in four patients, a baseline biopsy performed before implantationof the allograft was investigated as the first biopsy. Follow-updata concerning allograft function and immunosuppression protocolswere retrieved from patient’s files.
Table 1. Time point and morphological classification of investigated biopsiesa
Laser Capture Microdissection and DNA Preparation
Sections (3 µm) from formalin-fixed and paraffin-embeddedallograft biopsy samples were mounted on glass slides coatedwith polyethylene foil. To exclude any contamination by infiltratingrecipient leukocytes, an immunohistochemical stain for leukocytecommon antigen (LCA/CD45; Dako, Hamburg, Germany) followinga standard avidin-biotin complex (ABC) technique was performed(Figure 1). Laser-based microdissection of cortical tubularepithelial cells was performed with the PALM Laser MicroBeamsystem (PALM, Wolfratshausen, Germany) (19). At least 700 tubularepithelial cells were dissected from the renal cortex area inan immunohistochemically stained (anti CD45) section of eachbiopsy sample (n = 72) (Figure 2). After collecting the cellsinto the lid of a 0.5-ml reaction tube, the DNA was isolatedby applying 25 µl of proteinase K digestion buffer (50mmol/L Tris, pH 8.1, 1 mmol/L EDTA, 0.5% Tween 20, 40 µg/mlproteinase K) into the lid. The reaction tubes were incubatedovernight at 55°C, and after denaturation at 94°C for8 min, the lysate was used for subsequent PCR analysis.
Figure 1. Immunohistochemical staining for CD45 before laser microdissection of an allograft biopsy sample. Allograft-infiltrating leukocytes (arrows) are readily identifiable; hence, erroneous harvesting of recipient-derived infiltrating cells representing pseudo-microchimerism can be prevented.
Figure 2. Sequence of pictures (A through D) demonstrating harvest of pure tubular epithelial cells by laser microdissection.
STR-PCR
For detection of recipient-derived cells within the microdissectedtubular epithelial cells, a PCR assay was applied that analyzesa highly polymorphic STR marker located within the human -actin–relatedpseudogene, H-beta-Ac-psi2. This marker is also known as SE33and contains a tetranucleotide repeat that displays considerablepolymorphism and a high heterozygosity rate of up to 95% (20,21).To increase sensitivity for partially degraded DNA isolatedfrom formalin-fixed, paraffin-embedded tissue, we used modifiedprimers that reduce the length of the PCR products to fragmentsbetween 140 and 236 bp (13). The amplification reaction wasperformed in a final volume of 25 µl containing 22 nMof each primer, 0.5 units Hot Start Taq polymerase (Quiagen,Hilden, Germany), 1.5 mM MgCl2, 250 nM of dNTP, and up to 10µl DNA lysate. The reaction mixture was preheated at 95°Cfor 15 min, followed by 35 cycles at 94°C for 30 s, 56°Cfor 30 s, and 72°C for 1 min, with a final elongation stepat 72°C for 10 min. The PCR products were analyzed witha capillary electrophoresis device (ABI310; Applied Biosystems,Darmstadt, Germany). One microliter of the PCR product was mixedwith 0.5-µl size standard (GeneScan350, Applied Biosystems,Darmstadt, Germany) and 12 µl formamide. This mixturewas heated for 2 min at 91°C and then immediately chilledon ice. Electrophoresis was performed following the manufacturer’sinstructions. Electropherograms displaying size and intensityof the PCR products were created by the software package suppliedwith the instrument (GeneScan310 analysis).
To identify recipient-derived cells within the microdissectedtubular epithelia cells, first the allelotype of the recipienthad to be revealed. This was performed by analogously analyzinga tissue specimen from the recipient, which had been archivedbefore the renal transplantation. Analyzing an allograft biopsysample without microdissection (total DNA extraction) supplieda mixture of recipient and donor allelotype. By subtractingthe already known recipient allelotype from the mixed allelotype,the donor-specific alleles could readily be identified. Thefour preimplantation baseline biopsy samples served as negativecontrols. In 2 of the 72 investigated biopsy samples, insufficientDNA amounts were extracted from the microdissected cells.
Y-chromosome Hybridization Combined with Immunohistochemistry
From eight male patients who received a female renal allograft,sufficient biopsy material was available after STR analysisfor additional Y-chromosome hybridization. For the detectionof the Y-chromosome in the formalin-fixed and paraffin-embeddedtissue, we applied a modified version of a previously describedchromogenic in situ hybridization protocol (22). The Y-chromosomehybridization with a specific centromere probe (Appligene-Oncor,Heidelberg, Germany) was combined with immunohistochemistryfor a pan-cytokeratin antibody (clone KL-1; Beckman-CoulterImmunotech, Krefeld, Germany). In brief, paraffin was removedfrom sections of allograft biopsy samples and rehydrated. Afterincubation of primary antibody, the detection was done by astandard ABC-technique with BCIP/NBT/INT (Dako) as chromogenproducing a deep blue membrane stain of epithelial cells. Subsequentlya DNA probe for the centromeric region of the Y-chromosome wasincubated overnight (37°C) and detected by an optimizedABC technique (NenLifeSience). ACE (Dako) served as substratedeveloping a red to brown color.
For evaluation, the number of tubular epithelial cells showinga nuclear, spotlike brown signal per 500 tubular epithelialcells was recorded. Cells without a blue membrane stain (cytokeratinnegative) were not counted, to exclude any contamination bygraft infiltrating recipient-derived leukocytes. Female allograftsin female recipients served as negative controls. Male nontransplanted,native kidneys served as positive controls.
Statistical Analyses
SPSS statistical software version 10.0 (SPSS, Chicago, IL) wasused for statistical analyses. Correlation between a persistentor discontinuously present/absent epithelial chimerism and allograftfunction was done by Wilcoxon test for paired data and the Mann-Whitneytest for unpaired data. Allograft function was determined bycalculation of quotient of the maximal calculated creatinineclearance within the first 6 mo after transplantation and thecalculated creatinine clearance at 18 mo after transplantation.The creatinine clearance was calculated from the respectiveserum creatinine levels applying the formula provided by Cockcroftand Gault (23). Results were considered significant when theP value was less than 0.05.
STR-PCR after Laser Microdissection
Thirty-two (88%) of 36 investigated patients showed an intragraftepithelial microchimerism in at least one of their biopsy samples(Figure 3, Table 2). In 26 of these patients (72%), a stablechimerism (i.e., first and second biopsy chimeric) of tubularepithelial cells was found. In four patients (11.1%), no microchimerismwas detectable in any of their sequential biopsy samples (Figure 4).In three cases, the first biopsy showed no microchimerism,whereas the second did. Just in one patient, the opposite constellationwas found, with chimerism in the first biopsy sample only.
Figure 3. Short-tandem repeat–PCR detecting epithelial microchimerism in an allograft. (A) Allelotype of recipient, determined on tissue taken before transplantation. (B) Chimeric allelotype in tubular epithelial cells of the allograft (pale arrows pointing to alleles of recipient, dark arrows pointing to alleles of donor). Corresponding individual peak size is given in the table at the bottom.
Figure 4. Negative short-tandem repeat–PCR without detection of epithelial microchimerism in an allograft. (A) Allelotype of recipient, determined on tissue taken before transplantation. (B) Allelotype of tubular epithelial cells of the allograft. Corresponding individual peak size is given in the table at the bottom.
An epithelial microchimerism was detectable as early as 8 dafter transplantation. Chimeric cells were found, too, in abiopsy sample taken 8 yr after transplantation. In our cohort,this was the longest time period between two sequential biopsiesavailable and demonstrating a stable (i.e., first and secondbiopsy chimeric) microchimerism. As expected, none of the preimplantationbiopsy samples revealed an epithelial microchimerism.
Correlation of Epithelial Microchimerism with Biopsy Pathology
For correlation of epithelial microchimerism, the patients withbaseline biopsy findings at first biopsy were grouped accordingto the diagnosis of their second biopsy (Table 3). All fourpatients from the first group (histopathologically unremarkablein first and second biopsy samples) and all three patients fromthe second group (CNI toxicity in the first biopsy and CAN inthe follow-up biopsy samples) developed an intragraft microchimerism.Within the third group (CNI toxicity in first biopsy sampleand no CAN in second biopsy sample), one of two patients waschimeric. In patients with acute rejection (group 4) in thefirst and CAN in the second biopsy samples, 93% (13 of 14) ofthe patients showed an epithelial microchimerism. In 80% ofthe patients (four of five) of the fifth group (acute rejectionin first and no CAN in second), a microchimerism was found.All patients from group 6 (Banff ’97 borderline changesin first and CAN in second), and four of five patients fromgroup 7 (Banff ’97 borderline changes in first and noCAN in second) were chimeric.
Table 3. Correlation of microchimerism to biopsy findings
Correlation of Epithelial Microchimerism with Immunosuppression Protocols
Most of the patients (n = 15) received tacrolimus and corticosteroidsas a maintenance immunosuppressive therapy. Analysis of samplestaken via biopsy revealed 11 of these patients to be chimeric,1 only in the second biopsy sample; 3 had no chimerism at all.Only two patients received cyclosporine A and corticosteroids,one being stably chimeric and the other chimeric only in thesecond biopsy sample. One patient with mycophenolatmofetil whowas receiving corticosteroid therapy was chimeric in both biopsysamples. The rest of the investigated patients (n = 16) hada triple immunosuppression comprising variable combinationsof tacrolimus or cyclosporine A with corticosteroids and mycophenolatmofetilor azathioprine. The majority of these patients (n = 11) hada stable chimerism in both biopsy samples (Table 4).
Table 4. Correlation of microchimerism to immunosuppression
Y-chromosome In Situ Hybridization
After laser microdissection and STR-PCR analysis from eightmale patients (11 samples) who received a female allograft,sufficient material was available for Y-chromosome in situ hybridization.Without exception, these samples all showed an epithelial microchimerismin STR-PCR analysis, as well as in the Y-chromosome in situhybridization approach (Figure 5). All samples with suitableY-chromosome in situ hybridization showed morphologic signsof acute cellular rejection (Table 5).
Figure 5. Photomicrograph of a proximal tubular cross section with nuclear Y-chromosome signals (pale arrow) in epithelial cells (arrowheads, membrane stain for pan-cytokeratin) of a female allograft in a male recipient. In the adjacent interstitium, recipient-derived leukocytes with nuclear Y-chromosome signals (dark arrow) can be seen.
Table 5. Results of Y-chromosome in situ hybridization
Quantitative evaluation of Y-chromosome signals revealed 0.8%to 2.2% of recipient-derived tubular epithelial cells in theinvestigated allografts. No Y-chromosome signals were seen innegative controls (female allografts in female recipients).Because the Y-chromosome is not visible in every nucleus in5-µm sections, male native kidneys were examined as positivecontrols and for comparison. These showed Y-chromosome signalsin approximately one-third of tubular epithelial cells. Therefore,we adjusted the cell count from the allografts by a factor ofthree, to 2.4% to 6.6% chimeric tubular epithelial cells (6).
From three patients (patients 7, 23, and 24), Y-chromosome insitu hybridization results for both sequential samples wereavailable. In one patient (patient 23), the number of chimericcells increased, whereas it decreased in two (patients 7 and24) over time. In the same slides, frequently nuclei with aY-chromosome signal were detectable within glomerular tufts,but only in single cells at the endothelial site of small- andmedium-size arteries. Without performing simultaneous immunohistochemicalphenotyping, it was impossible to clearly discriminate thesecells on a purely morphologic basis from invading monocytesor leukocytes.
Chimerism and Outcome
For 33 patients, follow-up data concerning the calculated creatinineclearance 18 mo after transplantation were available. Twenty-nineof these patients showed a decrease in their creatinine clearance,two an increase, and two returned to dialysis. A total of 21of the 33 patients had a persistent epithelial chimerism. Thequotient of best creatinine clearance in the first 6 mo aftertransplantation and creatinine clearance at 18 mo ranged withinthese patients with persistent chimerism from 0.94 and 4.6 (mean1.61, SD 0.84). Ten patients had no (n = 4) or a discontinuous(n = 6) epithelial chimerism with a creatinine clearance quotientbetween 0.71 and 1.65 (mean 1.21, SD 0.33). No statistical significantcorrelation between chimerism and allograft function at 18 moafter transplantation was found (P > 0.05).
Currently, the classic paradigm of stem cell differentiationrestricted to its organ-specific lineage is challenged by intriguingfindings suggesting that adult stem cells, including hematopoieticstem cells, retain a previously unrecognized degree of developmentalplasticity that allows them to differentiate across boundariesof lineage, tissue, and germ layer (24–27). Numerous studiessuggest that tissue injury is the preceding and stimulatingevent recruiting circulating stem cells into various solid organs,subsequently transdifferentiating into tissue-specific cells(reviewed in (24)). However, the involved mechanisms are notfully understood. Solid organ transplants in particular suffersevere tissue damage, at least during their harvest, as wellas quite frequent numerous additional insults during their earlyposttransplant period. Therefore, the finding that recipient-derivedstem cells settle into the graft and transdifferentiate intoorgan-specific cells created tremendous hope that this can bethe source for organ restitution and unlimited long-term graftfunction.
According to the results presented here from a large seriesof study of renal allograft biopsy samples, it is unlikely thatthe presence of recipient-derived epithelial cells (i.e., intragraftmicrochimerism) has any influence on graft outcome, either withregard to morphology (onset of chronic tubulointerstitial damage)or to allograft function (decrease in creatinine clearance).Although recipient-derived epithelial cells were found veryearly after transplantation in 88% of the patients, which persistedin the majority of these patients for a long time, no obviouscorrelation to a specific mechanism of epithelial injury wasfound. Moreover, even cases lacking morphologically visiblesigns of early cell damage became rapidly chimeric after transplantation.A possible explanation might be that all allografts will suffersome diffuse epithelial damage during organ harvest and reperfusion.However, we looked in our collective for allografts after livingdonation (n = 2). Both showed a stable chimerism in their sequentialbiopsies (data not shown). Although others were able to showa correlation between the extent of tissue damage and an increasedchimerism in various solid organ transplants (10,12,13,28,29),we were not able to demonstrate this for tubular epithelialcells in renal allografts.
In two of our cases, investigated semiquantitatively by Y-chromosomein situ hybridization, we observed a decrease of chimeric cellsafter initial acute cellular rejection, whereas in a third case,microchimerism increased in the subsequent biopsy. In the lung,a positive correlation between chimerism and degree of tissuedamage was demonstrated for the epithelial cells in bronchiand peribronchial glands, while it was lacking for chimerictype II pneumocytes (13). These findings might indicate thatin different types of allografts, different cell types are variablyreplaced by recipient-derived stem cells after initial cellulardamage. According to our findings and those of others, a basalmicrochimerism promptly established after transplantation remainsstable, qualitatively and more or less quantitatively, in thelong-term course of the majority of allografts (6,13,14,30,31).
This observation is in contrast to the hypothesis of Medawar(16) that adaptation and tolerance in allografts may be theresult of gradual replacement of the cells of the donor by thoseof the recipient in the posttransplant course. Furthermore,a recent study in liver allografts, which more frequently showpermanent tolerance than kidney allografts, revealed similarendothelial cell chimerism in both tolerant and nontolerantpatients, leading to the conclusion that chimerism has nothingto do with the induction of clinical tolerance (32). From our26 constantly chimeric patients, 62% (16 of 26) developed morphologicsigns of chronic rejection, and 4 even returned to dialysis18 mo after transplantation—events one would not expectfrom accommodated allografts.
Obviously, the majority of solid organ allografts incorporaterecipient-derived stem cells very early after transplantation,but without inducing tolerance (6,13,14,30,31). Furthermore,recipient-derived precursor cells do not represent an inexhaustiblesource for tissue regeneration, and they appear unable to preventlong-term deterioration of an allograft. In the kidney, thesecells might even contribute to chronic irreversible interstitialand vascular scarring (11). Otherwise, in our collective, patientsshowing any chimerism had a favorable morphologic outcome, i.e.,no chronic allograft nephropathy in the follow-up biopsy.
Although our study contributes to the evidence that adult stemcells can transdifferentiate to mature, organ-specific cells,it is not able to demonstrate any clinical relevance for renalallografts at present. To exploit the therapeutic capacity ofrecruitment of pluripotent stem cells to damaged organs (24),more must be known about the biology of these enigmatic cells.
Acknowledgments
Results of this study have been previously published in abstractform (Nephrol Dial Transplant 18[Suppl 4], 2003, abstract M702).Supported by DFG SFB265/C11.
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Received for publication October 27, 2003.
Accepted for publication January 22, 2004.
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Abstract
Introduction
-actin–related pseudogene, H-beta-Ac-psi2. This marker is also known as SE33 and contains a tetranucleotide repeat that displays considerable polymorphism and a high heterozygosity rate of up to 95% (20,21). To increase sensitivity for partially degraded DNA isolated from formalin-fixed, paraffin-embedded tissue, we used modified primers that reduce the length of the PCR products to fragments between 140 and 236 bp (13). The amplification reaction was performed in a final volume of 25 µl containing 22 nM of each primer, 0.5 units Hot Start Taq polymerase (Quiagen, Hilden, Germany), 1.5 mM MgCl2, 250 nM of dNTP, and up to 10 µl DNA lysate. The reaction mixture was preheated at 95°C for 15 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, with a final elongation step at 72°C for 10 min. The PCR products were analyzed with a capillary electrophoresis device (ABI310; Applied Biosystems, Darmstadt, Germany). One microliter of the PCR product was mixed with 0.5-µl size standard (GeneScan350, Applied Biosystems, Darmstadt, Germany) and 12 µl formamide. This mixture was heated for 2 min at 91°C and then immediately chilled on ice. Electrophoresis was performed following the manufacturer’s instructions. Electropherograms displaying size and intensity of the PCR products were created by the software package supplied with the instrument (GeneScan310 analysis). 
