BK virus (BKV) replication in renal allograft recipients may progress from asymptomatic viruria to progressive allograft dysfunction leading to allograft failure (1), systemic infection (2), and bladder cancer (3). The diagnosis of BK virus nephritis (BKN) is often encountered unexpectedly on the histologic examination of the allograft biopsy (4). The clinical and functional outcomes often correlate with the duration and histologic progression (stage) of BKN (5).
Because of the lack of specific antiviral therapy, at present, the treatment of choice is to reduce the dose of maintenance immunosuppressive therapy empirically. In the absence of a clinical assay, however, the definition of over- or intense immunosuppression is difficult to define (6). Although nonspecific dose reduction or conversion from cyclosporine A to tacrolimus has been associated with a decrease in plasma BKV load, it does not necessarily lead to improved graft survival (1, 7, 8). This type of intervention is often associated with an increased risk of acute allograft rejection (4, 9), potentially initiating to a vicious cycle of progressive viral and immunologic graft damage (7, 8, 10).
Sirolimus is an evolving immunosuppressive agent that is relatively devoid of nephrotoxicity (11) and that has been demonstrated to have antiproliferative properties (12). On the basis of the assumption that damage to the graft microenvironment is permissive to BK viral activation and replication (13), one might hypothesize that the lack of nephrotoxicity may have salutary effects on the initiation and progression of BKN.
From May 2002 to July 2002, protocol biopsies were performed in 25 recipients of renal allografts with progressive allograft dysfunction as a part of our study protocol to decrease or stop the calcineurin inhibitor therapy. The protocol was approved by the Institutional Review Board. We unexpectedly identified three patients with BKN, who were subsequently withdrawn from the original protocol because presence of BKN was an exclusion criteria.
A 31-year-old white man with end-stage renal failure caused by glomerulonephritis required peritoneal dialysis for 16 months before receiving a two-human leukocyte antigen mismatch living-donor kidney transplant. Baseline serum creatinine (median) was 159 μmol/L (1.8 mg/dL), and median estimated creatinine clearance (eCrCl) (14) was 51.1 mL/min. There were no acute allograft rejections or opportunistic infections in the posttransplant period. The renal allograft protocol biopsy was performed at 36 months posttransplantation. The histologic examination of the allograft biopsy demonstrated BKN.
A 51-year-old African American woman developed end-stage renal disease caused by type 2 diabetes mellitus. She was on hemodialysis for 1 year before receiving a zero-mismatch living-donor kidney transplant. The patient had an episode of acute rejection (Banff type 1A) during the first week after transplantation and was treated with three bolus doses of corticosteroid therapy. Eighteen months later, the baseline serum creatinine (median) was 150 μmol/L (1.7 mg/dL) with a median eCrCl of 54 mL/min. A protocol renal allograft biopsy revealed BKN.
A 51-year-old white man developed end-stage renal disease caused by focal segmental glomerulosclerosis. He required hemodialysis for 13 months before receiving a three-human leukocyte antigen mismatch cadaveric kidney transplant. Anti-CD25 monoclonal antibody (basiliximab) was administered for induction therapy. Sixteen months after kidney transplantation, baseline serum creatinine (median) was 150 μmol/L (1.7 mg/dL) with a median eCrCl of 47 mL/min. A protocol allograft biopsy was performed at that time and showed BKN.
The maintenance immunosuppression in all three patients consisted of tacrolimus (goal 12-hr trough level of 8 to 10 ng/mL), mycophenolate mofetil (1.5 g/day in patients 1 and 3 and 2 g/day in patient 2) and prednisone (5 mg/day).
Histology of Allograft Biopsy
Renal core biopsies were fixed in formalin and routinely processed. Sections were stained with hematoxylin-eosin. After identification of BKN, periodic acid-Schiff, Masson’s trichrome, and periodic acid methenamine silver stains were added. Immunohistochemical stains using SV40 large T-antigen monoclonal antibodies were performed. Paraffin-embedded biopsy specimens were analyzed for BKV-DNA quantification.
Cytologic evaluations of urine samples for the presence of polyomavirus-infected “decoy cells” were performed as described previously (5) at the time of biopsy and every 4 to 6 weeks during the follow-up period.
BK Virus-DNA in Blood
BKV load was determined in serum samples as described previously by Hirsch et al. (15). Briefly, 200 μL of the specimens were processed for DNA extraction using protease K digestion and silica spin columns. Quantification was achieved by a BKV-specific real-time polymerase chain reaction method (TaqMan ABI Prism 7700; Applied Biosystems, Rotkreuz, Switzerland) using the primer pair 5′-AGCAGGCAAGGGTTCTATTACTAAAT-3′ and 5′-GAAGCAACA-GCAGATTCTCAACA-3′ and the FAM/TAMRA-labeled probe 5′-AAGACCCTAAAGACTTTCCCTCTGATCTACACCAGT-TT-3′. Briefly, a reaction mix of 20 μL consisting of 12.5 μL Master Mix, 3.25 μL (20 pmol) of either primer, 0.75 μL (300 nmol) of the probe, and 0.25 μL double-distilled water was added to 5 μL of sample DNA preparation, the negative and positive template controls. Calibration curves were generated in the same run using triplicates of template controls of known copy number ranging from 102 to 106. The threshold cycle number CT was plotted against the known copy number yielding correlation coefficients r2 of 0.97 to 0.99. The patient samples were analyzed in duplicates, and the viral load per milliliter plasma was calculated from the arithmetic mean of both measurements.
BK Virus-DNA in Biopsy Tissue
The BKV load in paraffin-embedded biopsy material was determined after extraction of total DNA by normalizing the BKV load to the aspartoacylase gene number by using the primers 5′-CCCTGCTACGTTTATCTGATTGAG-3′ and 5′-CCCACAGGATACTTTGGCTATGG- 3′ and the TaqMan Probe 5′CCTTCCCTCAAATATGCGACCACTCG-3′ as described previously (15, 16) and a titrated plasmid copy numbers of the cloned aspartoacylase target.
Changes in Maintenance Immunosuppression Therapy
After the histologic diagnosis of BKN, the dosage of baseline immunosuppression medications was reduced by at least 50%. During the following 12 weeks the number of decoy cells and plasma viral load remained unchanged with a further increase in serum creatinine levels. At this stage, tacrolimus and mycophenolate mofetil were replaced with sirolimus with continuation of low-dose prednisone therapy (2.5 mg every other day). After a sirolimus loading dose of 7 mg, the maintenance dose was adjusted to achieve the concentration-controlled goal trough level of 10 to 12 ng/mL. During the first 6 months of sirolimus therapy, patients received primary prophylaxis against Pneumocystis carinii with cotrimoxazole/trimethoprim in two patients or dapsone in one patient with G6PD deficiency.
Three of twenty-five recipients (8.3%) of kidney allografts with gradual deterioration of renal function showed a histologic diagnosis of BKN. The median time to the diagnosis of BKN was 18 months (range 16–36 months) after transplantation.
Kidney Allograft Biopsy Findings
All three biopsy specimens stained positive for SV40 large T antigen. Quantification of the BKV load in the biopsy specimen from two patients (patients 1 and 3) showed 3,269 and 33 copies of BK viral genome/cell, respectively, and BKV-DNA was undetectable in the other specimen.
Table 1 describes the number of decoy cells and plasma BKV-DNA copies per milliliter from the time of diagnosis through the follow-up period. After a switch to combination of sirolimus and prednisone therapy, urine cytology results became negative for decoy cells after an additional 16 weeks in one patient and 20 weeks in the other two patients urine cytology remained negative during the follow-up period. Similarly, the median plasma BKV load was 60,303 copies per milliliter (range 12,481–326,117) at the time of BKN diagnosis. The plasma viral load decreased by more than 50% during the first 2 months after sirolimus therapy and was undetectable in all three patients at the time of last follow-up. The disappearance of decoy cells from the urine preceded the clearance of BKV viremia (Fig. 1). In addition, repeat allograft biopsies in patients 1 and 2 demonstrated the disappearance of cytopathic effects of BKV and negative staining by SV40 large T-antigen antibody.
Allograft function was evaluated by eCrCl from 24 weeks before the diagnosis of BKN and through the follow-up period (Table 2). Overall, the combination of sirolimus and prednisone was well tolerated. Allograft function improved in all three patients. None of these patients developed acute rejection during the follow-up period. At the time of the last follow-up, 18 to 19 months after switching to sirolimus- and prednisone-based therapy, the median serum creatinine decreased from 159 μmol/L (1.8 mg/dL) to 132 μmol/L (1.5 mg/dL), and the median eCrCl increased from 52 mL/min (range 51–54 mL/min) to 67 mL/min (range 62–75 mL/min) (Table 2, Fig. 1).
Side Effects of Sirolimus
Patient 1 developed transient leukopenia with a white cell count decreasing to 2,400 per mm3, which recovered spontaneously. Patient 3 developed small aphthous mouth ulcers in the first week of therapy, which responded to local treatment and after the dose of sirolimus was reduced to maintain a trough level of 8 to 10 ng/mL. Screening test results for cytomegalovirus and other herpetic viruses were negative. Cholesterol and triglyceride levels increased in all three patients during the first months of sirolimus therapy. The median total cholesterol and median triglyceride levels increased from 4 mmol/L (159 mg/dL) at baseline to 6 mmol/L (224 mg/dL) and 2.5 mmol/L (224 mg/dL) to 3.2 mmol/L (289 mg/dL), respectively. All three patients were treated with atorvastatin (20 mg daily), which was tolerated without side effects.
BKN was discovered unexpectedly on protocol biopsies in 3 of 25 renal transplant patients (8.3%) with progressive allograft dysfunction more than 18 months after transplantation. This suggests that BKN might be more common than previously recognized even at times beyond the first year posttransplantation (7). It is conceivable that smoldering BKN could be one of the underdiagnosed and potentially reversible causes of chronic allograft nephropathy.
All three of our patients were not only shedding decoy cells but also had varying degrees of detectable plasma BKV loads greater than 10/mL (4) at the time of histologic diagnosis of BKN. Tissue viral quantification in our patients did not correlate with other parameters of viral replication (16). We assume that inadequate representation because of sampling problems or focal BKN may account for the low or negative results in our patients.
Our most interesting observation was that switching from a combination of tacrolimus and mycophenolate mofetil therapy to a combination of sirolimus and low-dose prednisone was associated with (1) clearance of decoy cell shedding and disappearance of plasma BKV viremia, (2) steady improvement in allograft function, and (3) lack of acute allograft rejection during the follow-up period.
These observations are important because optimal strategy for intervention after the onset of BKN remains undefined. There is a broad consensus that in the absence of specific and well-tolerated antiviral agents, BKN should be treated by judicious reduction in maintenance immunosuppression. However, these strategies vary considerably among studies (1, 4, 7, 8, 10) and with variable outcomes (1, 4, 5, 8).
In this context our observation that treatment with a sirolimus-based regimen was associated with an excellent virologic and immunologic outcome is very promising and may overcome the problem of increased risk of acute rejection associated with nonspecific reduction in immunosuppression (4). Combination therapy with sirolimus and calcineurin inhibitor (either cyclosporine or tacrolimus) has been shown to be equally detrimental for the progression of BKN (4, 17). Other treatment strategies such as the use of cidofovir (18) and leflunomide (either alone or in combination with cidofovir) (19) have been demonstrated to have some antipolyoma activity but need to be added to the immunosuppression therapy, thus as an adjunctive therapy.
We hypothesize that the disappearance of BK viremia after conversion to sirolimus- and prednisone-based therapy could be the result of several factors. The immunologic clearance of BKV could be caused by restitution of T-cell–mediated immunity in the absence of tacrolimus and mycophenolate mofetil (20); this may allow the cells to disfavor viral replication (21). There is decreased permissiveness of the virus in the absence of ongoing graft microenvironment injury secondary to chronic calcineurin inhibitor toxicity. In addition, sirolimus inhibits neointimal and smooth muscle cell proliferation and could decrease fibrogenesis after the onset of graft damage (12). However, sirolimus use has been associated with increased expression of transforming growth factor-β without histologic changes during a short follow-up of 28 days (22). On the basis of the observation that sirolimus has some in vitro anti-human immunodeficiency virus activity (23), it may similarly have antipolyomaviral activity.
Notably, neither rejection episodes nor relapsing BKV replication was observed during the follow-up period. Repeat biopsies in two of our patients revealed an absence of subclinical acute rejection, an absence of active replication of BKV, and no progression in baseline interstitial fibrosis or tubular atrophy. Decoy cells representing high-level polyomavirus replication disappeared earlier than plasma BKV DNA, as has been observed previously (15). However, it is possible that BKV genomes may remain detectable by polymerase chain reaction for extended periods of time after BKV viremia cleared, as documented recently in cases treated with cidofovir therapy (18, 24).
Conversion to sirolimus- and prednisone-based therapy after the diagnosis of BKN is associated with clearance of decoy cells in the urine, disappearance of plasma viremia, improvement in graft function, and without provoking acute rejection. We suggest that sirolimus-based therapy promotes the milieu, the graft microenvironment and immunologic milieu, which may prevent infected cells from viral replication and promote viral latency.
We are indebted to William L. Henrich, M.D., for his continuous support and mentorship, to Eva K. Ritzl, M.D., for her invaluable help with the preparation of the article, and to Geetha Stachowiak for her administrative help.
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Keywords:© 2004 Lippincott Williams & Wilkins, Inc.
Kidney transplantation; Polyoma virus; BK virus nephropathy; Immunosuppression; Decoy cells; Acute rejection