The patient was a 50-year-old Japanese man with end-stage renal disease secondary to chronic glomerulonephritis. After hemodialysis for 10 years, he underwent cadaveric renal allografting on 7 October, 2000. His immunosuppression regimen comprised of Tac, azathioprine (Az), prednisolone (PSL) and antilymphocyte globulin (Fig. 1). Since mild liver dysfunctions occurred on day 3, Az was replaced with MMF. Initially the graft functioned satisfactorily and serum creatinine levels (SCr) decreased from 9.62 mg/dL to 1.51 mg/dL within 35 days. On day 30, screening computed tomography detected a lymphocele with a diameter of 10 cm and mild hydronephrosis of the allograft in the pelvic cavity. On day 39, SCr increased to 2.10 mg/dL. After needle aspiration of the lymphocele with injection of a sclerosant, SCr decreased to 1.78 mg/dL. However, allograft function became worse on day 107, along with hydronephrosis, probably related to recurrence of lymphocele. The patient underwent allograft biopsy followed by needle aspiration with an injection of sclerosant and ureteric stenting. Serum creatinine levels immediately decreased from 2.43 mg/dL to 1.96 mg/dL, but increased again to 2.23 mg/dL after 5 days. Since allograft biopsy revealed hydronephrosis, toxic tubulopathy and no evidence of acute rejection (Fig. 2a), the dose of Tac was reduced from 10 mg to 6 mg. On day 125, the patient underwent laparoscopic fenestration because of the re-recurrence of lymphocele. Serum creatinine levels initially decreased to 1.83 mg/dL, but gradually increased to 2.12 mg/dL with proteinuria and an increase of serum lactic dehydrogenase 10 days after the procedure. Viral inclusion cells (decoy cells) with ground-glass nuclear inclusion bodies were detected by routine urine cytology on day 120 (Fig. 3). Immunostaining with anti-BKV antibody (a mouse monoclonal anti-BK virus large T antigen antibody; Cemicon, Temecula, CA) on postoperative day 108 also showed many positive nuclei in the tubular epithelium (Fig. 2b). A second allograft biopsy (Fig. 2c) was carried out and the specimen was immunostained with anti-BK virus antibody. Acute rejection (type IA, Banff 97) was detected with a much milder toxic tubulopathy than detected at the time of the first allograft biopsy, while BK infected uroepithelial cells decreased in number (Fig. 2d). Although steroid pulse therapy was duly considered, we were afraid of BKV reactivation, as BKV infects a transplanted kidney and the rate of graft loss can reach approximately 50%.1 Shiraki et al. reported that Mizoribine (Mz) suppressed human cytomegalovirus replication depending on increases in concentration.5 Since BKV belongs to the same double-stranded DNA virus category as cytomegalovirus, we expected a suppression of BKV replication. Therefore, we decreased the dose of MMF from 2000 mg to 1500 mg for the suppression of BKV reactivation, increased the dose of Tac from 6 mg to 8 mg for prevention of acute rejection, and added Mz at a dose of 50 mg for suppression of BKV replication or immunosuppression. Five months after transplantation, SCr was stabilized at 1.9–2.2 mg/dL. At 6 months after transplantation, pneumonia developed due to mixed infection with Staphylococcus aureus and Mycobacterium avium. The doses of Tac and MMF were reduced from 8 mg to 6 mg and from 1500 mg to 1000 mg, respectively, and the dose of PSL was also decreased to 10 mg. Serum creatinine levels remained stable at 2.0–2.3 mg/dL, but urinary excretion of protein increased to about 500 mg/day. Decoy cells were still found in the urine, but showed a decreasing tendency. At 8 months after transplantation, the patient suffered from Haemophilus influenzae pneumonia. Since pneumonia was not very severe, we reduced only the dose of prednisolone to 7.5 mg. Serum creatinine levels gradually rose to 3.4 mg/dL and urinary protein loss became higher than 1000 mg/day. Fourteen months after transplantation, renal allograft biopsy (Fig. 2e) revealed tubulointerstitial nephritis and borderline acute rejection (t:1, i:1, g:0, ah:1, v:0). Only a few BK-positive cells were seen (Fig. 2f). At this point, some clinical findings of rejection were seen, so we considered that the development of the renal allograft rejection coincided with that of BKV-ISN. First, methylprednisolone pulse therapy was performed for 3 days. Then, for the treatment of BK nephropathy, maintenance immunosuppression was reduced by gradual decreases in the dose of Tac, a reduction in the dose of MMF to 750 mg and the discontinuance of Mz. Subsequently, SCr decreased from 3.40 to 3.03 mg/dL within 2 weeks, but increased again to 4.03 mg/dL a week later. Decoy cells were still detected in the urine.
Figure 1. Clinical course after kidney transplantation. †, POD 125–Laparoscopic unroofing; ‡, POD 180–pneumonia (Staphylococcus aureus and Mycobacterium avium); §, POD 355 pneumonia (Haemophilus influenzae); ¶, methylprednisolone 500 mg. Cr, serum creatinine; MMF, mycophenolate mofetil; Mz, mizoribine; POD, postoperative day; PSL, prednisolone; Tac, tacrolimus; U-Pro, excretion of urinary protein.
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Figure 2. Light microscopy after transplantation on POD 108, 135 and 454. BKV antigen-positive tubular cell nuclei appear in accordance with the disappearance of lymphocytic infiltration from the stroma. (A) Hydronephrosis and drug-induced toxic tubulopathy. HE staining. (B) Many tubular cells have nuclei that are positive for BK virus large T antigen. Immunostaining for BK virus large T antigen. (C) Numerous lymphocytes infiltrating the interstitium with wide-spread tubulitis. HE staining. (D) Few BK virus-infected cells are seen. Immunostaining for BK virus large T antigen. (E) Borderline acute rejection with marked tubular damage is seen. The infiltrating inflammatory cells consist of heterogeneous lymphocytes and occasional plasma cells. HE staining. (F) Some tubular cells show positive nuclear staining. Immunostaining for BK virus large T antigen. MMF, mycophenolate mofetil; Mz, mizoribine; POD, postoperative day; PSL, prednisolonine; Tac, tacrolimus.
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Two years after transplantation, decoy cells have disappeared from the urine, but SCr has increased to 4.58 mg/dL and the urinary protein loss is over 2500 mg/day. Proteinuria is very severe, but there was no evidence of glomerulonephritis in any of the three allograft biopsy specimens and the other laboratory findings. Cytomegalovirus, herpes simplex virus and Epstein-Barr virus have not been detected.
Polymerase chain reaction amplification
Equivalent amounts of DNA (1.5 µg), as determined by spectrophotometry at 260 nm, were placed into Eppendorf tubes. Fifty nanograms of DNA was subjected to PCR amplification in a reaction mixture with a total volume of 25 µL; containing 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, each primer at 0.5 µm, dNTPs at 200 µm and 0.625 U of Taq Polymerase. Denaturation was carried out for 2 min at 94°C, followed by 35 cycles of denaturation at 91°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min. A final extension cycle of 1 min at 55°C and 4 min at 72°C was added. Polymerase chain reaction was performed using an automated DNA thermal cycler (Hybaid, Teddington, Middlesex). Two pairs of primers were used to detect VP-1 (VP1-327–1: CAAGTGC CAAAACTACTAAT and VP1-327–2r: TGCATGAAG GTTAAGCATGC) and non-coding region (NCR; NCR-1: TCCATGAGCTCCATGGATTCTTC and NCR-2r: CTAGGTCCCCCAAAAGTGCTAGA).6 Polymerase chain reaction products were subjected to electrophoresis on 2.5% agarose gel in tris-borate-EDTA buffer and stained with ethidium bromide. The PCR product was expected to be 327 bp in size for the subtype-specific region of VP-1 and 600–800 bp for the entire NCR. To confirm the PCR product, the 327 bp band of VP-1 was cut out from the gel, purified, and directly sequenced in the forward and reverse directions using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Results of PCR amplification
Urine samples showed PCR products corresponding to the VP-1 region (327 bp) and the NCR (600–800 bp). In Figure 4a, lanes 1–3 and lanes 5–7 are from patient urine, while lanes 4 and 8 are negative controls. When the 327 bp band was cut from the gel, purified and directly sequenced in the forward and reverse directions, the VP-1 sequence was detected. The PCR product of VP-1 was cleaved by Alu I into two pieces of 186 and 141 bp (Fig. 4b, lane 1). When the PCR products were further digested with XmnI, no additional band appeared (Fig. 4b, lane 3). To confirm the results of restriction enzyme analysis, we also digested the PCR product with RsaI. The cleaved product (281 bp) is shown in Figure 4b, lane 5, while lanes 2 and 4 are the original VP-1 products. These findings indicated that the virus belonged to BKV genotype I.
Figure 4. (a) Polymerase chain reaction (PCR) amplification of BKV from urine samples. Postive PCR products corresponding to the VP-1 region (327 bp) and NCR (600–800 bp) are seen. Lanes 1–3 and 5–7 are from the patient's urine. Lanes 4 and 8 are negative controls. (b) BKV typing by restriction enzyme analysis of PCR products. The PCR product of VP-1 was digested with Alu I into two smaller products of 186 and 141 bp (lane 1). When the PCR products were further digested with XmnI, no additional band appeared (lane 3). To confirm this restriction enzyme analysis, we digested the PCR product with RsaI. The cleaved product (281 bp) is shown in lane 5. Lanes 2 and 4 are original VP-1 products.
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