Incidence of polyomavirus-nephropathy in renal allografts: influence of modern immunosuppressive drugs

Michael Mengel1,, Magali Marwedel1, Jörg Radermacher2, Gabriele Eden2, Anke Schwarz2, Hermann Haller2 and Hans Kreipe1

1 Institut für Pathologie der Medizinischen Hochschule Hannover and 2 Abteilung für Nephrologie der Medizinischen Hochschule Hannover, Hannover, Germany



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. In recent years an increasing number of cases with polyomavirus (PV)-nephropathy after renal transplantation were reported from several transplant centres. New, highly potent immunosuppressive drugs like tacrolimus or mycophenolate mofetil were accused as risk factors for this increase. However, data about the incidence of PV-nephropathy in correlation to different immunosuppressive therapy concepts are lacking.

Methods. All renal transplant biopsies performed at Hannover Medical School between 1999 and 2001 (n=1276) were immunohistochemically screened for the presence of PV-specific proteins. The results were correlated to the different immunosuppressive therapy protocols and patients with PV-nephropathy were compared with a matched control group.

Results. PV-nephropathy was found in <1% of all investigated allograft biopsies (11/1276) and in ~1% of all patients (7/638), respectively. All patients being immunohistochemically positive for PV-specific proteins also showed the typical morphological changes of PV-nephropathy. Four out of seven patients with PV-nephropathy were under triple immunosuppression comprising tacrolimus and mycophenolate mofetil. Under this immunosuppressive therapy protocol an eight times higher incidence and a 13 times higher risk (multivariate odds ratio 12.7) of PV-nephropathy was observed in our patients compared with the control group.

Conclusions. PV-nephropathy is a rare but serious complication after renal transplantation. A small group of patients under intensive immunosuppression comprising tacrolimus in combination with mycophenolate mofetil has a significantly increased risk of acquiring this deleterious complication.

Keywords: polyomavirus; renal transplantation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In 1971 a new virus, a human polyomavirus (PV), was isolated from the urine of a renal transplant recipient and named BK virus according to the initials of this patient [1]. Together with the simian virus 40 (SV40) and the JC virus, these viruses share an ~70% homology with each other [2]. Their genome has an early region encoding the large T and small t protein, a late region encoding viral capsid proteins, and a non-coding regulatory region [3]. More than 80% of adults have serologic evidence of PV-infection with the viruses remaining latent in the urogenital tract [4,5]. Among renal transplant recipients 10–60% have reactivation of PV generally accompanied by shedding into the urine virus-infected urothelial cells, called ‘decoy cells’, which contain characteristic intranuclear viral inclusion [6,7]. This shedding is inconsistently associated with serious graft dysfunction, predicting in ~28% the manifestation of a severe interstitial nephritis in the allograft [8]. In recent studies, the BK virus has been implicated as the cause of interstitial (BK-) nephritis in as many as 3–5% of renal transplant patients [813]. Diffuse interstitial infiltrates with lymphocytes and occasional abundant plasma cells in combination with necrosis of tubular epithelial cells and detachment of inclusion-bearing cells from tubular basement membranes are the morphological hallmarks of BK-virus nephritis [11,12]. The interstitial nephritis is frequently accompanied by focal tubulitis making the differential diagnosis of concomitant acute cellular rejection rather difficult by light microscopy alone [11,12]. Therefore, the definite identification of PV-infected cells relies frequently on the immunohistochemical detection of virus-specific proteins [6,14]. PV-nephropathy is related to a deleterious outcome leading to graft failure in as many as 45% of affected patients and to various degrees of chronic allograft nephropathy in the remaining [9,11].

Recent studies observed a remarkable increase in the incidence of PV-nephropathy [810,12]. A possible explanation for the increased frequency of PV-nephropathy may be found in the nowadays widespread application of modern, highly potent immunosuppressive drugs like tacrolimus and mycophenolate mofetil (MMF) often as rescue therapy after early acute rejection episodes [8,10,13]. Against this background, and as doses and combinations of immunosuppressive drugs vary between different transplant centres, we prospectively screened all renal allograft biopsies performed at Hannover Medical School between 1999 and 2001 (n=1276) for the presence of PV-specific proteins by immunohistochemical analysis.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Between June 1999 and December 2001, 1276 biopsies from 638 renal transplant patients (254 female, 384 male; age 2–77 years, mean age 45.4 years; 1–7 biopsies/patient) were performed at the Hannover Medical School, Germany. Indications for biopsy were: baseline biopsies of donor organs, initially non-functioning graft, clinical suspicion of acute rejection due to creatinine increase, chronic deterioration of graft function, proteinuria and routine protocol biopsies without clinical suspicion of rejection. Biopsies were taken 0–1252 weeks (median 12 weeks, mean 87.6 weeks) after transplantation. For all patients, complete data were available concerning their individual immunosuppressive therapy and clinical outcome.

All biopsies investigated were routinely fixed in buffered formalin and embedded in paraffin following standard protocols. Serial sections for haematoxylin and eosin staining and periodic acid–Schiff (PAS) staining were performed for each biopsy. Morphological classification by light microscopy was carried out according to the Banff ‘97 working classification [15]. Prospectively, all allograft biopsies were immunohistochemically stained using a monoclonal antibody against the SV40 large-T antigen (Callbiochem/Oncogene Research Products, Cambridge, MA), which is expressed by cells infected with replicating primate (SV40) or human (BK and JC) PV [10]. In brief, after deparaffination and heat-induced epitope retrieval the primary antibody was incubated at a dilution of 1:50 overnight at 4°C. A biotinylated secondary rabbit-anti-mouse antibody was detected by a sensitive Streptavidin–alkaline-phosphatase complex. A distinct nuclear staining in epithelial cells was regarded as positive.

At the Hannover renal transplantation centre the initial immunosuppression is chosen according to risk profiles.

Protocol 1 includes all patients with a normal immunologic risk. Basal immunosuppression consists of Basiliximab (days 1 and 4) and cyclosporine A [trough levels (TL) of 150–200 ng/ml]. Additionally steroids are given at doses of 500, 250 and 125 mg on the first 3 days after transplantation and are subsequently tapered from 1 mg/kg to 5–7.5 mg over the following 3 months.

Protocol 2 is applied to patients with an increased immunologic risk, i.e. patients with panel reactive antibodies (PRA) >30%, patients with prior allograft loss, high urgency patients and recipients of living kidney donation. In these patients, Basiliximab, steroids and cyclosporine A are applied as shown above. Additionally MMF is given at 2 g daily.

Protocol 3 includes patients with an increased risk of calcineurine-inhibitor toxicity, i.e. donor >65 years or recipient with haemolytic–uraemic syndrome as primary disease. Those patients initially receive a calcineurine-inhibitor-free protocol comprising steroids at the dose shown above, MMF at 3 g daily and ATG (Fresenius HemoCare Immune Therapy GmbH, Gräfelfing, Germany) 5 mg/kg i.v. given prior to perfusion and once daily for the first 7 days post-transplant.

Switch to tacrolimus generally serves as rescue therapy after repeated acute rejection or after acute rejection >3 months after transplantation, these patients are adjusted to TL of 8 ng/ml. Some patients receive sirolimus as part of a new immunosuppressive protocol (TL 4–12 ng/ml). Azathioprine (AZA) 0.5–2 mg/kg was routinely given to patients as part of a protocol prior to the introduction of modern immunosuppressive drugs like cyclosporine A and as part of a triple therapy before the introduction of MMF.

The SPSS statistical package (Version 11.0, SPSS Inc., Chicago, IL) was used for all statistical analyses. Unpaired t-tests, {chi}2 analysis or Kaplan–Meier analysis with log rank test were used as appropriate to assess the differences between groups. Odds ratios for the risk of developing PV-nephropathy were calculated from two-by-two contingency tables (Fisher's exact test). For multivariate analysis the effect of multiple parameters on the development of PV-nephropathy were analysed in the cases and control group, in all patients on tacrolimus, MMF and prednisolone treatment and in the whole group of 638 patients for whom renal biopsies with testing for PV were available. The analysis was performed with stepwise forward logistic regression (significance level for removing the variable from analysis=0.1, for entering the variable=0.05). Parameters investigated before biopsy proven occurrence of PV-nephropathy were: immunosuppressive regime, tacrolimus dose and trough level, prednisolone dose, MMF dose, cytomegalovirus viraemia, donor age, total number of mismatches at the HLA-A, HLA-B and HLA-DR locus, number of acute interstitial rejections within and after the first month after renal transplantation, delayed graft function, living donation or brain death donor, sex, patient age, height, weight, serum creatinine and creatinine clearance. All data are expressed as means±SD unless stated otherwise.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Morphological findings
From 1276 investigated biopsies, 390 (30.6%) had unspecific changes not sufficient for the diagnosis of acute or chronic rejection following the Banff ‘97 classification. 186 biopsies (14.6%) showed an interstitial infiltrate and focal mild tubulitis consistent with borderline changes according to the Banff ‘97 classification. In 203 biopsies (15.9%) an acute rejection was found, mostly as acute tubular-interstitial rejection. No acute Banff ‘97 grade III rejection with fibrinoid necrosis or transmural arteriitis appeared in our collective. 370 biopsies (29%) showed varying degrees of chronic allograft nephropathy. In 46 biopsies (3.6%), signs of acute and chronic rejection were observed simultaneously. Morphological changes consistent with cyclosporine A/tacrolimus toxicity were present in 93 biopsies (7.3%). Eighteen biopsies did not contain sufficient allograft tissue for classification.

Immunosuppressive therapy (Table 1Go)
More than half (61.8%) of our patients were temporarily under a double immunosuppression. Fewer patients had a more intensive triple immunosuppression (47.2%) either from the beginning or as rescue therapy after a late acute rejection episode. In both groups the therapy concept always comprised steroids. The most frequent immunosuppressive combination was steroids and cyclosporine A (45.3%) followed by steroids and tacrolimus instead of cyclosporine A (11.1%). Thirty per cent of the patients received a triple immunosuppressive therapy with steroids, cyclosporine A and MMF. Much less frequent the combination of steroids, tacrolimus and MMF (7.8%) or of steroids, cyclosporine A and AZA (4.7%) or more recently of steroids, cyclosporine A and sirolimus (2.8%) was applied. Taken together, 11.9% of the patients received a double or triple immunosuppressive therapy comprising tacrolimus, 33.8% comprising MMF and 7.8% both, tacrolimus and MMF.


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Table 1.  Maintenance immunosuppressive therapy concepts

 
The rest of the patients were under individually adapted immunosuppressive therapy protocols, modified during their post-transplant course.

Patients with PV-nephropathy (Table 2Go)
Only seven patients were found with tubular epithelial cells expressing the SV40 large T-antigen in at least one biopsy confirming a replicative PV-infection (Figure 1Go). All immunohistochemically positive biopsies (n=11) simultaneously showed the typical morphological features of PV-nephropathy with diffuse interstitial infiltrates in combination with necrosis of tubular and collecting duct epithelial cells and detachment of inclusion-bearing cells from the denuded tubular basement membranes (Figure 2Go). PV was detected 1.5–138 weeks (mean 35.2 weeks) after transplantation. All patients had biopsies before the detection of PV, i.e. biopsies that were immunohistochemically negative for PV. In all except one patient these biopsies showed changes of acute rejection (Banff grade IA to IIA, for details see Table 2Go), preceding PV detection 0–80 weeks (mean 20 weeks). Six out of the seven patients had one to four biopsies after the first detection of PV, revealing in three patients a clearance of PV (mean 76 days after first detection of PV) and in the other three patients a persistence of PV.


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Table 2.  Characteristics of patients with PV-nephropathy

 


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Fig. 1.  Immunohistochemical staining for SV40-large-T antigen with tubular epithelial cells showing strong nuclear expression indicating PV infection. ABC-technique, magnification 630x.

 


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Fig. 2.  PV-nephropathy with diffuse interstitial infiltrate and focal tubulitis (arrow head) and numerous enlarged, inclusion-bearing nuclei of tubular epithelial cells (arrows). PAS-staining, magnification 400x.

 
Five out of the seven patients with PV had MMF as a component of their initial immunosuppressive therapy and six out of these seven had MMF at the time of diagnosis of PV. Four of these six MMF treated patients had additionally been switched to tacrolimus treatment. In the first three patients after the diagnosis of PV the decline in renal function and the presence of tubulointerstitial infiltrate was considered as a sign of simultaneous rejection and no change (Patients 1 and 2) or an intensification (Patient 3) in the immunosuppressive therapy was made, respectively. In the following four patients immunosuppression was reduced (omitting MMF and/or tacrolimus) after PV diagnosis. Despite this, five of these seven patients lost their graft (21–53 weeks) or rapidly developed signs of severe chronic allograft nephropathy (12–38 weeks) after PV detection. Therefore, neither an intensification nor a decrease in the immunosuppression improved the allograft outcome after the diagnosis of the PV-nephropathy. One patient died with a functioning graft soon after the diagnosis of PV from heart failure without any link to the PV-nephropathy. Only one patient (Patient 2) overcame the PV infection and is still alive with a functioning graft 132 weeks post-transplant.

In none of the 89 baseline biopsies PV was detectable.

Comparison of patients with PV-nephropathy to a matched control group (Table 3Go)
The seven patients with PV-nephropathy were compared with a matched control group that consisted of 42 patients biopsied for allograft dysfunction or as protocol biopsies during the same time period being negative for PV by histology and immunohistochemistry. The two groups were matched (one PV positive case to six PV negative cases) for transplant date and baseline immunosuppression.


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Table 3.  Comparison of patients with PV-nephropathy to a negative control group

 
The PV-patients showed a significant higher serum creatinine, more acute rejection episodes in the first month after transplantation, and a significant higher total number of rejection episodes, than the patients in the control group. The difference between both groups in the immunosuppression was highly significant. More patients from the PV-group were under a triple immunosuppression comprising MMF and tacrolimus. The corresponding calculation of risk factors for the manifestation of a PV-nephropathy (Table 4Go and Figure 3Go) revealed for patients under prednisolone, MMF and tacrolimus a 13 times higher multivariate odds ratio in comparison with control patients and a 17 times higher odds ratio in comparison with all other investigated patients. Further important risk factors were elevated TL for tacrolimus (>8 ng/ml), early acute rejection episodes, increased doses of MMF and tacrolimus, and HLA missmatches >4. Accordingly, graft survival in PV-patients was significant worse in comparison with the control group (P<0.0002).


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Table 4.  Risk factors for PV-nephropathy by stepwise regression

 


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Fig. 3.  Univariate odds ratios with 95% confidence intervals for the occurence of PV-nephropathy.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
PV-nephropathy is a rare but deleterious complication after renal transplantation. At the Hannover renal transplant centre, PV-nephropathy was detected at an incidence of <1% in all allograft biopsies (11/1276) and ~1% (7/638) of all patients, respectively. Potential risk factors for the manifestation of a PV-nephropathy are well described in the literature and also obvious in our patients [8,10,13]. Nearly all (6/7) of our PV-patients had highly potent immunosuppressive therapy comprising tacrolimus and/or MMF at the time of acquiring PV-nephropathy. Furthermore, again six out of seven patients had episodes of acute rejection prior to PV manifestation as a significant risk factor, leading in four patients to an intensification of their basic immunosuppression.

Although the comparison with a matched control group revealed significant differences in the immunosuppression concerning the combination of two highly potent drugs, not all renal transplant patients with a high immunologic risk profile receiving MMF and/or tacrolimus develop PV-nephropathy. In our collective, from 20 patients receiving MMF in their post-transplant course as a part of a double immunosuppressive therapy (steroids+MMF) none acquired PV. From 197 patients under a triple immunosuppression comprising MMF only two (1.02 %) developed PV-nephropathy. There were no cases of PV-nephropathy among the patients receiving tacrolimus alone (n=77) as a part of their immunosuppressive therapy, but four cases among the 50 patients triple treated with tacrolimus plus steroids in combination with MMF (8%). No PV-nephropathy was observed in patients with the Hannover standard immunosuppressive concept (steroids+cyclosporine A), but one case when sirolimus was additionally applied. Therefore, from our results it seems that only the combination of two highly potent immunosuppressive drugs like MMF and tacrolimus is associated with a significant risk of acquiring a PV-nephropathy (multivariate odds ratio=12.7). From other centres large series of PV-nephropathy have been described under tacrolimus and steroids without MMF as rescue therapy after acute rejection [810]. Different dosage regimes of tacrolimus may be a potential explanation for this discrepancy between the individual centres. In our study the TL of tacrolimus was in all PV-patients higher than the generally aspired TL (8 ng/ml) in our center, mostly as the result of a rescue therapy after rejection, but similar to published TL of PV-patients in other centers [8]. Nevertheless, we found no PV-manifestation under tacrolimus alone and within normal dose ranges, and in our hands the combination with MMF was an obvious risk factor for the deleterious PV-nephropathy. Probably differences between transplant centres in the choice of baseline immunosuppression or in the treatment of early acute rejection episodes might explain the different incidence of PV-nephropathy under tacrolimus, comparable with the dose-dependent incidence of the nephrotoxicity of calcineurin inhibitors in general [16,17]. Furthermore, the recent description of a case with PV-nephropathy in the native kidneys of a solitary pancreas transplant recipient suggests also that the immunosuppressive therapy plays an important role in the viral reactivation process [18]. In contrast, in a recently published larger single-centre series of PV-patients (n=67) no influence of the immunosuppression in comparison with a matched control group was found. However, the majority of those patients were on triple baseline immunosuppression with tacrolimus, MMF and steroids, and the baseline TL (12–15 ng/ml) of tacrolimus was higher than in our patients [19]. In this study the PV-group showed a predominance among males and elderly patients; therefore, also other factors might influence the manifestation of PV-nephropathy. However, our collective of PV patients was probably too small to reveal any significant predominance in gender or age.

From this, and from our results it might be assumed that pre-transplant immunologic risk factors (HLA-match, PRA, etc.) lead to an intensified basic immunosuppression or higher risk for early acute rejection with consecutive rescue therapy and intensification of immunosuppression using drugs like tacrolimus and MMF. Probably, this constellation can then give together with patient-specific factors (gender, age) the background for a PV-manifestation. However, in general and especially in our collective the incidence of the PV-nephropathy is too low to find any definite associations to single risk or pathogenetic factors.

The outcome after the clinical and morphological manifestation of PV-nephritis remains poor. From our seven patients, only one survived the PV-nephropathy with a functioning graft. Although recently a lower rate of graft loss (16.4%) was described in a larger series of PV-patients, specific treatment would be urgently needed [19]. No specific antiviral therapy for PV-nephropathy is currently recommended, although attempts with cidofovir seem to be promising [20]. Nevertheless, reduction of the doses of immunosuppressive drugs should decrease the viral replication [9,12,13]. However, patients with interstitial PV-nephropathy can then enter a vicious cycle, altering between viral interstitial nephritis and rejection precipitated by lowered doses of immunosuppressive drugs [6]. In contrast, a recent large study was able to show in only 16% of their patient's acute rejection episodes after reduction of immunosuppression as a therapeutic approach in PV-nephropathy, suggesting that the risk of acute rejection as a complication is lower than generally expected. Nevertheless, in this study any beneficial effect of a reduced immunosuppression on PV patient's outcome, similar to the findings in our patients, was not demonstrable [19].

From the results reported here a general immunohistochemical PV-screening for all renal transplant biopsies seems not necessary. We found no PV-case by immunohistochemistry alone, which was not already suspected by the typical virus-induced nuclear changes in conventional stains. Thus, a PV-nephropathy can already be assumed by light microscopy, and only for confirmation and identification of the virus type immunohistochemistry is indicated. For a PV-screening, non-invasive techniques using urine (detection of decoy cells) or blood (virus-specific PCR) seem to be more suitable and also sensitive enough to give the indication for an allograft biopsy [10,21,22]. Detection of the viral load in the plasma offers the basis for identifying patients at risk for PV-nephropathy who may be candidates for preemptive therapy. Patients without an impaired renal function but increased viral load may benefit from controlled reduction of their immunosuppression [22,23].

In conclusion, PV-nephropathy is a rare but serious complication after renal transplantation. Currently no PV-specific therapy is available and therefore, the prevention of PV-manifestation seems to be the only effective approach. Especially the group of renal transplant patients receiving a triple immunosuppressive therapy comprising two highly potent drugs like tacrolimus and MMF in addition to steroids has an increased risk of developing a generally deleterious PV-nephropathy.



   Notes
 
Correspondence and offprint requests to: Dr Michael Mengel, Institut fuer Pathologie, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, D-30659 Hannover, Germany. Email: mengel.michael{at}mh-hannover.de Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 3. 7.02
Accepted in revised form: 20.12.02