Presence of Donor- and Recipient-derived DNA in Cell-free Urine Samples of Renal Transplantation Recipients: Urinary DNA Chimerism

Clinical Chemistry 45:10 1741 1746 (1999) Molecular Diagnostics and Genetics Presence of Donor- and Recipient-derived DNA in Cell-free Urine Samples of Renal Transplantation Recipients: Urinary DNA Chimerism Jun Zhang, 1 Kwok-Lung Tong, 2 Philip K.T. Li, 3 Albert Y.W. Chan, 4 Chung-Kwong Yeung, 5 Calvin C.P. Pang, 6 Teresa Y.H. Wong, 3 Kam-Cheong Lee, 4 and Y.M. Dennis Lo 1* Departments of 1 Chemical Pathology, 5 Surgery, and 6 Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR. Departments of 2 Medicine and 4 Pathology, Princess Margaret Hospital, Kowloon, Hong Kong SAR. 3 Department of Medicine and Therapeutics, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR. *Address correspondence to this author at: Department of Chemical Pathology, Prince of Wales Hospital, Room 38023, 1/F Clinical Sciences Bldg., 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong Special Administrative Region. Fax 852-2194-6171; e-mail loym@cuhk.edu.hk. Received June 23, 1999; accepted July 21, 1999. Background: Previous studies have indicated that microchimerism is present in body tissues, peripheral blood, and plasma of recipients after organ transplantation. We hypothesize that donor-derived DNA may also be present in cell-free urine of renal transplant recipients and that the concentrations of urine DNA may be correlated with graft rejection. Methods: Thirty-one female patients who had renal transplantation were enrolled in the study. In women with male organ donors, the SRY gene on the Y chromosome was used as a marker for donor-derived DNA. Real-time quantitative PCR for the SRY and -globin genes was carried out on cell-free urinary DNA from these patients. Serial urine samples from a female renal transplant recipient undergoing an acute rejection episode were also collected and analyzed with the -globin quantitative PCR system. Results: SRY sequences were detected in the urine of 14 of 17 female patients with male organ donors. None of the 14 patients with female organ donors had detectable SRY sequences in urinary DNA. The median fractional concentration of donor-derived DNA was 8.7% (interquartile range, 1.9 26.4%). During the acute rejection episode, urinary concentrations of the -globin gene were markedly increased, with the concentrations returning rapidly to normal following antirejection treatment. Conclusions: Our results demonstrate that urinary DNA chimerism is present following renal transplantation. The measurement of urinary DNA using quantitative PCR may be useful for the diagnosis and monitoring of graft rejection. 1999 American Association for Clinical Chemistry Multiple types of donor-derived cells are transferred to the recipient following transplantation of a solid-organ allograft and can exist in the recipient s tissues or peripheral blood for long periods after transplantation (1 5). This finding has led to the hypothesis that microchimerism may play a role in the acceptance of a foreign organ (1, 5, 6). In patients with short- or long-term surviving organ grafts, however, donor microchimerism is not always detectable (7, 8). On the contrary, it may also be present in patients with rejection (9 11). It thus is still unclear at present whether microchimerism is the cause or the consequence of graft survival or is merely an associated phenomenon of organ transplantation (12, 13). Detection of microchimerism following transplantation has been achieved predominantly by PCR-based techniques (2, 7, 8, 14). Both male-specific Y-chromosomal sequences and the human leukocyte antigen genes on chromosome 6 have been used for this purpose (11, 15). Recently, donor-derived DNA has also been detected in cell-free plasma of liver and kidney transplantation recipients, a phenomenon referred to as plasma DNA chimerism (16). In addition to plasma DNA, DNA extracted from urine is also a potential source of material for molecular diagnosis (17). For example, point mutations and microsatellite alterations have been detected in urine samples of patients with bladder cancer (18 20). A urine specimen is potentially more accessible than blood and can be col- 1741

1742 Zhang et al.: Posttransplantation Urinary DNA Chimerism lected noninvasively on multiple occasions, a feature that is highly desirable for the purposes of disease monitoring. In this report, we demonstrate the use of a real-time quantitative PCR for determining whether donor-derived DNA exists in cell-free urine samples of recipients who underwent sex-mismatched renal transplantation. We also provide data indicating that urinary DNA measurement may be useful for monitoring graft rejection. Materials and Methods patients Approval of the study was obtained from the Clinical Research Ethics Committee of The Chinese University of Hong Kong. Thirty-one female renal transplantation recipients were recruited from the Prince of Wales Hospital and Princess Margaret Hospital, Hong Kong. Among them, 17 received grafts from male donors, and the remaining 14 received grafts from female donors. During the study period, one female renal transplant recipient, who had a female organ donor, was admitted with a clinical diagnosis of acute graft rejection, confirmed by histological examination of a renal graft biopsy. Serial urine samples were collected from this individual up to the 34th day of admission. Antirejection treatment was instituted promptly after admission. To elucidate whether renal biopsy might lead to any variation in concentration of urinary DNA, urine samples from six patients who suffered from renal diseases but who had not been transplanted were also studied before and after renal biopsy. collection and processing of urine samples Spontaneous urine samples from the patients were carefully collected into sterile plain bottles. Urine samples were centrifuged at 1560g for 10 min. The supernatant was transferred carefully into plain polypropylene tubes without disturbing the pellet at the bottom of the bottle. The supernatant was recentrifuged at 1560g for 10 min and then transferred into fresh plain tubes. The samples were frozen at 20 C until further use. dna extraction from urine samples DNA from the cell-free urine samples was extracted using a QIAamp Viral RNA Kit (Qiagen) according to the QIAamp viral RNA purification protocol as recommended by the manufacturer. A 560- L cell-free urine sample was used for DNA extraction via spin column. An elution volume of 50 L was used. real-time quantitative pcr Primers and fluorescent probes for real-time quantitative PCR assays for the SRY and -globin genes were as described previously (21); 5 L of extracted urinary DNA was used as the template for the PCR reaction. Each sample was analyzed in duplicate. Real-time quantitative PCR was carried out in a Perkin-Elmer Applied Biosystems 7700 Sequence Detector (Perkin-Elmer). The theoretical and practical aspects of real-time PCR analysis have been described in detail elsewhere (21, 22). The compositions and conditions of the PCR assays for SRY and -globin sequence quantification as well as data computation procedures were as detailed previously for the measurement of fetal DNA in maternal plasma (21). The concentration of urinary DNA was expressed as genomeequivalents/mmol creatinine (Cr). One genome-equivalent was defined as the quantity of a particular DNA sequence present in one diploid male cell. The urinary Cr concentration was measured with a Hitachi 911 analyzer. The fractional concentration of donor-derived sequences in urinary DNA was calculated by: Quantity of SRY sequence Quantity of -globin sequence 100% PCR contamination was strictly controlled as described previously (21). Uracil N-glycosylase was used to further reduce the risk of carryover contamination (21, 23). Multiple water blanks were included in every analysis. Results assay precision The analytical intraassay CVs of the threshold cycle values obtained with real-time TaqMan SRY and -globin assays were 1.3% and 1.2% (mean SD, 33.0 0.45 and 32.39 0.38), respectively, as determined by 10-replicate SRY and -globin PCRs on urinary DNA extracted from a healthy male subject. The total CVs of threshold cycle values for SRY and -globin measurements, including the DNA extraction and quantitative PCR analysis steps, were 1.7% and 1.7% (mean SD, 33.31 0.55 and 32.53 0.55), respectively, by 10-replicate extractions of a urine sample from the same male subject, followed by quantitative PCR analysis. Serial dilutions of DNA extracted from the male individual indicated that the SRY and -globin quantitative PCR systems were able to detect the DNA equivalent of a single cell. quantitative analysis of cell-free dna in urine samples The values of the urinary SRY and -globin sequences in the renal transplant recipients without evidence of acute graft rejection are shown in Table 1. Fourteen of 17 (82.4%) female transplant recipients with male donors had detectable SRY DNA sequences in their cell-free urine samples. The median concentration of donor-derived SRY sequences was 14 428 genome-equivalents/mmol Cr (interquartile range, 10 382 to 17 560 genome-equivalents/ mmol Cr). No Y-chromosome signal was detected in the 14 female recipients with female donors. As an amplification control, -globin sequences were detectable in urinary DNA of all 31 subjects. The median urinary -globin DNA concentration was 115 773 genome-equivalents/mmol Cr (interquartile range, 40 380 to 346 207 genome-equivalents/mmol Cr). The median fractional concentration of

Clinical Chemistry 45, No. 10, 1999 1743 Case Table 1. Urinary concentrations of SRY and -globin sequences in sex-mismatched and sex-matched female renal transplant recipients. Donor sex Urinary SRY sequence, genomeequivalents/mmol Cr Urinary -globin sequence, genome-equivalents/mmol Cr Fractional concentration, % T1 M 9616 500 039 1.9 T2 M 17 560 324 184 5.4 T3 M 11 384 94 327 12.1 T4 M 13 498 728 863 1.9 T5 M 25 781 128 905 20.0 T6 M 13 263 521 031 2.5 T7 M 0 122 827 0 T8 M 16 835 93 194 18.1 T9 M 22 695 38 820 58.5 T10 M 10 382 35 299 29.4 T11 M 25 069 2 352 669 1.1 T12 M 0 192 876 0 T13 M 0 36 337 0 T14 M 17 525 66 473 26.4 T15 M 15 358 1 431 695 1.1 T16 M 1990 54 168 3.7 T17 M 4863 9306 52.3 T18 F 0 1 135 425 0 T19 F 0 240 385 0 T20 F 0 50 152 0 T21 F 0 13 686 0 T22 F 0 30 538 0 T23 F 0 353 548 0 T24 F 0 14 797 0 T25 F 0 223 214 0 T26 F 0 939 850 0 T27 F 0 45 060 0 T28 F 0 192 780 0 T29 F 0 101 718 0 T30 F 0 115 773 0 T31 F 0 27 411 0 donor-derived sequences in urinary DNA was 8.7% (interquartile range, 1.9 26.4%). urinary cell-free dna measurement during a graft rejection episode During the study period, a female renal transplant recipient whose graft was obtained from a female organ donor was admitted with a clinical diagnosis of acute graft rejection. Renal biopsy was carried out after the first urine sample was collected. No postbiopsy complication was observed. Sequential urine samples were collected before and after antirejection treatment. Because SRY PCR analysis was not possible in this case of female-to-female transplantation, we investigated the potential use of the -globin gene as a marker for monitoring renal graft rejection. The variation in the concentration of -globin sequences in the recipient s urine is shown in Fig. 1 for the period from 2 days before treatment to 1 month posttreatment. A dramatic increase in the concentration of urinary -globin sequences was observed during the rejection episode. Following antirejection treatment, the concentration of urinary -globin sequences declined rapidly. To exclude the possibility that renal biopsy might lead to a significant increase in the concentration of urinary DNA, six patients who suffered from miscellaneous renal diseases but who had not undergone transplantation and who were undergoing routine renal biopsy were studied. Prebiopsy and 2-h and 1-day postbiopsy urine samples were collected from these patients and analyzed by -globin quantitative PCR. The quantitative PCR results for the -globin sequences at various time points and clinical information of the patients are summarized in Table 2. The median prebiopsy and 2-h and 1-day postbiopsy -globin gene concentrations were 88 584 genome-equivalents/mmol Cr (interquartile range, 55 899 to 257 242 genome-equivalents/mmol Cr), 77 636 genome-equivalents/mmol Cr (interquartile range, 31 201 to 296 302 genome-equivalents/mmol Cr) and 83 847 genomeequivalents/mmol Cr (interquartile range, 6739 to 275 482 genome-equivalents/mmol Cr), respectively. Statistical analysis reveal no significant difference between these three time points (Friedman test, P 0.846), indicating that renal biopsy did not produce significant changes in the concentration of urinary cell-free DNA.

1744 Zhang et al.: Posttransplantation Urinary DNA Chimerism Fig. 1. Changes in the concentration of -globin sequences in urinary DNA during an acute renal graft rejection episode. The x-axis represents the collection time of urine samples. Day 1 was the day of presentation. Urine samples at days 1 and 2 were collected from the patient before antirejection treatment. Urine samples at day 3 and beyond were collected from the patient posttreatment. Renal biopsy was carried out right after the urine sample on day 1 was collected. The y-axis represents urinary -globin DNA concentration expressed as genome-equivalents/mmol Cr. Discussion We have used a real-time quantitative PCR assay to investigate whether donor-derived DNA could be detected in cell-free urine samples of renal graft recipients after renal transplantation. Male donor-derived SRY sequences were found in urine samples from 14 of 17 (82.4%) female transplant recipients with male donors. This is the first demonstration of urinary DNA chimerism following organ transplantation. As an important control, no SRY sequences were detected in urine from female patients with female donors. Urine thus represents an alternative source of material for the detection of posttransplant chimerism. In three female recipients with male kidney donors, no SRY sequences were detected in the urine, indicating that the extent of urinary DNA chimerism, if present, might be below the detection limit of the PCR assay. To evaluate the fractional concentration of donorderived DNA, we also measured the concentration of -globin gene sequences (as a marker of donor- plus recipient-derived DNA) in the urine samples of the recipients. Donor-derived DNA constituted only a minor proportion (median, 8.7%) of total urinary DNA, suggesting that the main bulk of urinary DNA might originate from the circulation or from other organs of the recipients urinary tract, e.g., the bladder. The success or failure of renal transplantation is mainly dependent on whether graft rejection occurs. Several studies have suggested that the expression of genes related to apoptosis might be used as an indicator of renal graft rejection (24 26). These studies, however, required the use of renal biopsy, which is invasive and carries substantial risk. To evaluate whether urinary DNA may be used as a noninvasive marker of renal graft rejection, we studied the variation of urinary DNA during a rejection episode. Our results indicated that the concentration of urinary DNA of the recipient was markedly increased during the rejection episode and returned rapidly to a lower concentration after antirejection treatment. These data provided the first evidence that urinary DNA might be a new marker for monitoring renal graft rejection. The generality of these results would need to be confirmed in a larger series of patients undergoing graft rejection. Because renal biopsy was performed in this individual to confirm the diagnosis of graft rejection, we took special precautions to investigate whether the procedure of renal biopsy might cause an increase in urinary DNA. Thus, six patients undergoing routine renal biopsy were recruited, and urinary DNA was measured both before the biopsy and at two time points after the biopsy. Because no significant systematic difference in urinary DNA concentrations before and after biopsy was observed, the possibility that the biopsy procedure per se may contribute to the changes in urinary DNA concentrations in the subject who had rejection was excluded. Further corroborative evidence that the increase in urinary DNA in the latter subject was not secondary to the biopsy procedure could be seen in Fig. 1, where a substantial increase in urinary DNA concentration was already observed at day 1, prior to the performance of the renal biopsy. The mechanisms whereby increased cell-free DNA is liberated into the transplant recipient s urine during graft rejection are unclear at present. One possible mechanism is the increased liberation of donor-derived DNA as a result of cellular destruction secondary to the rejection Case Table 2. Urinary concentrations of -globin sequences for patients undergoing renal biopsy. -globin sequences, genome-equivalents/mmol Cr Prebiopsy 2 h post biopsy 1 day post biopsy Clinical information B1 257 242 12 214 19 849 IgA nephropathy B2 55 899 122 199 275 482 Proteinuria, histologically normal B3 102 103 621 380 6739 Membranous nephropathy B4 49 533 33 073 3538 Lupus nephritis B5 75 064 296 302 450 234 IgA nephropathy B6 287 436 31 201 147 845 Mesangiocapillary glomerulonephritis type III

Clinical Chemistry 45, No. 10, 1999 1745 process. A second mechanism is the liberation of DNA from the recipient s immune effector cells that have been recruited to the rejection site. In this regard, it is interesting to note that the liberation of DNA from lymphocytes has been observed in experimental systems (27). Furthermore, it is intriguing to note that this phenomenon is inhibited by treatment with glucocorticoids (28), an effect that may be related to our observation that urinary DNA is rapidly reduced following antirejection treatment. A third mechanism whereby increased urinary cell-free DNA is observed in association with graft rejection may be related to alteration in glomerular permselectivity (29), thus allowing plasma DNA to be filtered into the glomerular filtrate. The future recruitment and analysis of urinary cell-free DNA from female renal transplant recipients who have received organs from male donors and who are undergoing acute rejection may yield valuable information on the origin of the rise in urinary DNA during a rejection episode. Our data highlight the existence of a previously unknown type of chimerism, namely urinary DNA chimerism, following renal transplantation and opens up a new field of investigation. However, much remains to be learned regarding the biologic factors governing this phenomenon. For example, additional work will be needed to document the intra- and interindividual variations in cell-free urinary DNA to define reference intervals for distinguishing normality and pathology. In this report, we have presented one example of a quantitative aberration in cell-free urinary DNA, namely, during an acute rejection episode. It is possible that other disorders may also produce an increase in urinary cell-free DNA, e.g., graft infection following renal transplantation. It remains to be demonstrated whether quantitative or temporal differences may be seen in the urinary DNA profiles between the various disorders that may affect renal transplant patients. Future prospective studies will be necessary to answer these questions and may ultimately allow us to use urinary DNA analysis as a powerful noninvasive tool for clinical monitoring and research. This project is supported by Direct Grant 2040681 from The Chinese University of Hong Kong. We thank N.M. Hjelm and C.W. Lam for helpful discussion during this project. References 1. Starzl TE, Demetris AJ, Murase N, Ildstad S, Ricordi C, Trucco M. Cell migration, chimerism, and graft acceptance [Review]. Lancet 1992;339:1579 82. 2. Starzl TE, Demetris AJ, Trucco M, Ramos H, Zeevi A, Rudert WA, et al. Systemic chimerism in human female recipients of male livers. Lancet 1992;340:876 7. 3. Starzl TE, Demetris AJ, Trucco M, Ricordi C, Ildstad S, Terasaki PI, et al. Chimerism after liver transplantation for type IV glycogen storage disease and type 1 Gaucher s disease. N Engl J Med 1993;328:745 9. 4. Starzl TE, Demetris AJ, Trucco M, Zeevi A, Ramos H, Terasaki P, et al. Chimerism and donor-specific nonreactivity 27 to 29 years after kidney allotransplantation. Transplantation 1993;55:1272 7. 5. Starzl TE, Demetris AJ, Trucco M, Murase N, Ricordi C, Ildstad S, et al. Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance. Hepatology 1993;17:1127 52. 6. Starzl TE, Demetris AJ, Murase N, Thomson AW, Trucco M, Ricordi C. Donor cell chimerism permitted by immunosuppressive drugs: a new view of organ transplantation. Immunol Today 1993;14: 326 32. 7. Schlitt HJ, Hundrieser J, Hisanaga M, Uthoff K, Karck M, Wahlers T, et al. Patterns of donor-type microchimerism after heart transplantation. Lancet 1994;343:1469 71. 8. Suberbielle C, Caillat-Zucman S, Legendre C, Bodemer C, Noel LH, Kreis H, et al. Peripheral microchimerism in long-term cadaverickidney allograft recipients. Lancet 1994;343:1468 9. 9. Schlitt HJ, Hundrieser J, Ringe B, Pichlmayr R. Donor-type microchimerism associated with graft rejection eight years after liver transplantation [Letter]. N Engl J Med 1994;330:646 7. 10. Chen Y, Baltzan M, George D, Bohm C, Okasha K, Shoker A. Fate of recurrent acute interstitial cellular rejection in an HLA identical kidney transplant recipient: impact of donor microchimerism. Clin Nephrol 1997;48:300 6. 11. Elwood ET, Larsen CP, Maurer DH, Routenberg KL, Neylan JF, Whelchel JD, et al. Microchimerism and rejection in clinical transplantation. Lancet 1997;349:1358 60. 12. Adams DH, Hutchinson IV. Microchimerism and graft tolerance: cause or effect? [Editorial]. Lancet 1997;349:1336 7. 13. Wood K, Sachs DH. Chimerism and transplantation tolerance: cause and effect. Immunol Today 1996;17:584 7. 14. Sahota A, Yang M, McDaniel HB, Sidner RA, Book B, Barr R, et al. Evaluation of seven PCR-based assays for the analysis of microchimerism. Clin Biochem 1998;31:641 5. 15. McDaniel HB, Yang M, Sidner RA, Jindal RM, Sahota A. Prospective study of microchimerism in transplant recipients. Clin Transplant 1999;13:187 92. 16. Lo YMD, Tein MSC, Pang CCP, Yeung CK, Tong KL, Hjelm NM. Presence of donor-specific DNA in plasma of kidney and livertransplant recipients [Letter]. Lancet 1998;351:1329 30. 17. Yokota M, Tatsumi N, Tsuda I, Takubo T, Hiyoshi M. DNA extraction from human urinary sediment. J Clin Lab Anal 1998; 12:88 91. 18. Steiner G, Schoenberg MP, Linn JF, Mao L, Sidransky D. Detection of bladder cancer recurrence by microsatellite analysis of urine. Nat Med 1997;3:621 4. 19. Fitzgerald JM, Ramchurren N, Rieger K, Levesque P, Silverman M, Libertino JA, et al. Identification of H-ras mutations in urine sediments complements cytology in the detection of bladder tumors. J Natl Cancer Inst 1995;87:129 33. 20. Saito S, Hata M, Fukuyama R, Sakai K, Kudoh J, Tazaki H, et al. Screening of H-ras gene point mutations in 50 cases of bladder carcinoma. Int J Urol 1997;4:178 85. 21. Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN, Poon PMK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768 75. 22. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986 94. 23. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 1990;93:125 8. 24. Sharma VK, Ding R, Li B, Bologa RM, Lagman M, Eduafo A, et al. Molecular correlates of human renal allograft rejection. Transplant Proc 1998;30:2364 6.

1746 Zhang et al.: Posttransplantation Urinary DNA Chimerism 25. Matsuno T, Nakagawa K, Sasaki H, Ishine N, Inagaki M, Yagi T, et al. Apoptosis in acute tubular necrosis and acute renal allograft rejection. Transplant Proc 1994;26:2170 3. 26. Matsuno T, Sasaki H, Ishido N, Nakagawa K, Ishikawa T, Oishi A, et al. Apoptosis in human kidney allografts. Transplant Proc 1996;28:1226 7. 27. Rogers JC, Boldt D, Kornfeld S, Skinner A, Valeri CR. Excretion of deoxyribonucleic acid by lymphocytes stimulated with phytohemagglutinin or antigen. Proc Natl Acad Sci U S A 1972;69: 1685 9. 28. Distelhorst CW, Rogers JC. Glucocorticoids inhibit trypsin-induced DNA release from phytohemagglutinin-stimulated blood lymphocytes. J Immunol 1979;123:487 95. 29. Barnas U, Mayer G. Glomerular proteinuria in renal transplant patients: mechanisms and treatment. Kidney Int 1997;52(Suppl 63):S78 80.