Simultaneous pancreas/kidney transplant recipients present with late-onset BK polyomavirus-associated nephropathy

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1 16. Paoletti E, Marsano L, Bellino D et al. Effect of everolimus on left ventricular hypertrophy of de novo kidney transplant recipients; a 1 year, randomized, controlled trial. Transplantation 2012; 93: Sahn DJ, DeMaria A, Kisslo J et al. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 1978; 58: Devereux RB, Alonso DR, Lutas EM et al. Echocardiographic assessment of left ventricular hypertrophy: comparison to necroscopy findings. Am J Cardiol 1986; 57: Ferrara LA, Vaccaro O, Cardoni O et al. Indexation criteria of left ventricular mass and predictive role of blood pressure and body composition. Am J Hypertens 2005; 18: Levey AS, Stevens LA, Schmid CH et al. CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration). A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150: Opelz G, Dohler B. Cardiovascular death in kidney recipients treated with renin-angiotensin system blockers. Transplantation 2014; 97: Levy D, Garrison RJ, Savage DD et al. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med 1990; 322: Park M, Hsu CY, Li Y et al. Associations between kidney function and subclinical cardiac abnormalities in CKD. J Am Soc Nephrol 2012; 23: Cai QZ, Lu XZ, Lu Y et al. Longitudinal changes of cardiac structure and function in CKD (CASCADE study). J Am Soc Nephrol 2014; 25: Arnol M, Starc V, Knap B et al. Left ventricular mass is associated with ventricular repolarization heterogeneity one year after renal transplantation. Am J Transplant 2008; 8: Siedlecki A, Foushee M, Curtis JJ et al. The impact of left ventricular systolic dysfunction on survival after renal transplantation. Transplantation 2007; 84: Fernandez-Fresnedo G, Escallada R, Martin de Francisco AL et al. Association between pulse pressure and cardiovascular disease in renal transplant patients. Am J Transplant 2005; 5: Philipp T, Martinez F, Geiger H et al. Candesartan improves blood pressure control and reduces proteinuria in renal transplant recipients: results from SECRET. Nephrol Dial Transplant 2010; 25: Murbraech K, Holdaas H, Massey R et al. Cardiac response to early conversion from calcineurin inhibitor to everolimus in renal transplant recipients: an echocardiographic substudy of the randomized controlled CENTRAL trial. Transplantation 2014; 97: Paoletti E, Bellino D, Gallina AM et al. Is left ventricular hypertrophy a powerful predictor of progression to dialysis in chronic kidney disease? Nephrol Dial Transplant 2011; 26: Chen SC, Su HM, Hung CC et al. Echocardiographic parameters are independently associated with rate of renal function decline and progression to dialysis in patients with chronic kidney disease. Clin J Am Soc Nephrol 2011; 6: Fellstrom B, Jardine AG, Soveri I et al. Renal dysfunction is a strong and independent risk factor for mortality and cardiovascular complications in renal transplantation. Am J Transplant 2005; 5: Weiner DE, Carpenter MA, Levey AS et al. Kidney function and risk of cardiovascular disease and mortality in kidney transplant recipients: the FAVORIT Trial. Am J Transplant 2012; 12: Received for publication: ; Accepted in revised form: Nephrol Dial Transplant (2016) 31: doi: /ndt/gfv441 Advance Access publication 12 January 2016 Simultaneous pancreas/kidney transplant recipients present with late-onset BK polyomavirus-associated nephropathy Thomas Schachtner 1,2, Marina Zaks 1, Andreas Kahl 1,2 and Petra Reinke 1,2 1 Department of Nephrology and Internal Intensive Care, Charité University Medicine Berlin, Campus Virchow Clinic, Berlin, Germany and 2 Berlin-Brandenburg Center of Regenerative Therapies (BCRT), Berlin, Germany Correspondence and offprint requests to: Thomas Schachtner; thomas.schachtner@charite.de ABSTRACT Background. Infections have increased in simultaneous pancreas/kidney transplant recipients (SPKTRs) with BK polyomavirus (BKV)-associated nephropathy (BKVN) being the most important infectious cause of allograft loss. Comparisons of BKVN with kidney transplant recipients (KTRs), however, are lacking. Methods. We studied all SPKTRs and KTRs at our transplant centre between 2003 and Eleven of 106 SPKTs (10.4%) and 21 of 1062 KTRs (2.0%) were diagnosed with BKVN with allograft loss in 1 SPKTR (9.1%) and 2 KTRs (9.5%). A control of 95 SPKTRs without BKVN was used for comparison. Results. SPKTRs showed an increased incidence of BKVN compared with KTRs (P < 0.001). Onset of BKVN in SPKTRs was significantly later compared with KTRs (P = 0.033). While 67% of KTRs showed early-onset BKVN, 64% of SPKTRs developed late-onset BKVN. Older recipient age and male gender increased the risk of BKVN in SPKTRs (P < 0.05). No differences were observed for patient and allograft survival (P > 0.05). The Author Published by Oxford University Press Downloaded from onhttps://academic.oup.com/ndt/article-abstract/31/7/1174/ behalf of ERA-EDTA. All rights reserved. 1174

2 However, SPKTRs with BKVN showed inferior estimated glomerular filtration rate and a higher incidence of de novo donor-specific antibodies compared with SPKTRs without BKVN in long-term follow-up (P < 0.05). SPKTRs showed higher peak BKV loads, a need for more intense therapeutic intervention and were more likely not to recover to baseline creatinine after BKVN (P < 0.05). Conclusions. Our results suggest a higher incidence, more severe course and inferior outcome of BKVN in SPKTRs. An increased vulnerability of the allograft kidney due to inferior organ quality may predispose KTRs to early-onset BKVN. In contrast, SPKTRs present with late-onset BKVN in the presence of high-dose immunosuppression. Keywords: BKV-associated nephropathy, donor-specific antibodies, kidney transplantation, simultaneous pancreas/kidney transplantation INTRODUCTION BK polyomavirus (BKV) is still in the case of progression to BKV-associated nephropathy (BKVN) one of the most challenging infectious complications after renal transplantation [1 3]. BKV is known to persist in tubular epithelial cells with intermittent reactivation and low-level viruria in up to 50% of kidney transplant recipients (KTRs) [4, 5]. Reactivation of BKV from the persistent subclinical state is monitored using quantitative urine and plasma PCR, and primarily occurs in KTRs [5 7]. Here, positive screening for BKV in urine or plasma has been shown to have a high positive predictive value for BKVN [8, 9]. Progression to BKVN occurs in 1 10% of KTRs with varying degrees of allograft dysfunction and increasing serum creatinine concentrations [10]. The gold standard for the diagnosis of BKVN is a tissue biopsy of the allograft kidney, not only to identify BKV inclusions [6, 11, 12], but also to identify drug toxicity, recurrence of the underlying renal disease and in particular acute cellular rejection. Previous work on the clinical relevance of BKVN in simultaneous pancreas/kidney transplant recipients (SPKTRs) suggested that BKVN represents the leading cause of kidney allograft loss in simultaneous pancreas/kidney transplantation (SPKT), while the pancreas allograft is not directly vulnerable to BKVN and does not lead to an increased risk of rejection [13 15]. However, these data were obtained in an era of cyclosporinebased maintenance immunosuppression and little is known about the clinical course of BKVN in SPKT under modern immunosuppressive agents. In particular, potent immunosuppressive drug regimes containing tacrolimus and/or mycophenolate mofetil (MMF) have been suggested to stimulate BKV replication and to lead to the current high prevalence of BKVN [12, 16 18]. Since limited therapeutic options in the treatment of BKVN are further restricted in the setting of SPKT by the fear of rejection of the allograft pancreas due to reduction of maintenance immunosuppression, an increased insight into BKVN in SPKTRs is required. Despite a rising number of studies addressing risk factors of BK viraemia and BKVN in large cohorts in single KTRs, a differentiation of BKVN in KTRs and SPKTRs is lacking. Therefore, we attempted to address the following open questions: (i) Are there differences in risk factors between BKVN in SPKTRs and BKVN in KTRs? (ii) Are there differences in outcome between BKVN in SPKTRs and BKVN in KTRs? (iii) Are there differences in severity and viral kinetics between BKVN in SPKTRs and BKVN in KTRs? MATERIALS AND METHODS Patients This study was performed in compliance with the Declaration of Helsinki and Istanbul. We examined 1062 adult solitary KTRs and 106 SPKTRs transplanted at our centre at Charité Campus Virchow Clinic between 2003 and 2012 for development of BKVN. Eleven of 106 SPKTRs (10.4%) were diagnosed with BKVN and 1 SPKTR lost his allograft due to BKVN (0.9%). Twenty-one of 1062 KTRs (2.0%) were diagnosed with BKVN and 2 KTRs lost their allograft due to BKVN (0.2%). BKVN was classified into histological patterns as reported previously [19]. Patients were divided into different groups: (i) 11 SPKTRs with BKVN, (ii) 95 SPKTRs without BKVN and (iii) 21 KTRs with BKVN. The definition of early-onset BK viraemia as <6 months posttransplantation based on almost linearly increasing incidence during the first 6 months posttransplantation and decline afterwards. For analysis of clinical and virological characteristics, SPKTRs with BKVN were compared with a control group of 95 SPKTRs without BKVN and 21 KTRs with BKVN. SPKTRs with kidney or pancreas allograft retransplants were excluded from the study. Recipients of an ABO-incompatible transplant were excluded from the analysis. Patients were followed until allograft loss, death or their last patient follow-up date in the aftercare plan. Estimated glomerular filtration rate (egfr) was calculated by the abbreviated MDRD equation: 186 (creatinine/88.4) (age) (0.742 if female). Kidney and pancreas transplantation procedures Donor pancreata/kidneys were procured from deceased donors with no evidence of pancreatic or renal dysfunction. Pancreata were placed in the recipient s right iliac fossa with enteric drainage of exocrine secretions using a diverting Roux-en-Y anastomosis and anastomoses to the iliac vasculature. Kidneys were placed in the recipient s left iliac fossa with vascular anastomoses to the iliac vessels. Immunosuppressive therapy and rejection treatment Primary immunosuppression in SPKTRs and KTRs was a triple-drug regimen with a calcineurin inhibitor (tacrolimus or cyclosporine), MMF or mycophenolic acid (MPA), and steroid (Table 1). Tacrolimus was initially dosed as 0.15 mg/kg/day, and trough levels were maintained at 8 10 ng/ml for 6 months and at 5 7 ng/ml afterwards. Initial daily MMF dosage was 2 g. SPKTRs receiving mtor inhibitor according to the EURO SPK BKV in simultaneous pancreas/kidney transplantation 1175

3 1176 T. Schachtner et al. Table 1. Patient characteristics SPKT total (n = 106) BKVN in SPKTRs (n = 11) SPKT control (n = 95) P-value BKVN in SPKTRs (n = 11) BKVN in KTRs (n = 21) P-value Recipient factors Age, years a 43 (18 65) 48 (39 59) 43 (18 65) 0.046* 48 (39 59) 57 (26 70) Male sex, n (%) 64 (60) 11 (100) 53 (56) 0.003* 11 (100) 15 (71) First kidney allograft, n (%) 106 (100) 11 (100) 95 (100) 1 11 (100) 17 (81) Diabetes mellitus, n (%) 106 (100) 11 (100) 95 (100) 1 11 (100) 5 (24) <0.001* Time on dialysis, months a 24 (0 243) 30 (5 132) 23 (0 243) (5 132) 47 (0 121) CMV viraemia, n (%) 23 (22) 2 (18) 21 (22) 1 2 (18) 11 (52) Immunosuppression, n (%) Cyclosporine 0 (0) 0 (0) 0 (0) 1 0 (0) 2 (10) Tacrolimus 106 (100) 11 (100) 95 (100) 1 11 (100) 19 (90) Mycophenolate mofetil 106 (100) 11 (100) 95 (100) 1 11 (100) 21 (100) 1 Steroids 106 (100) 11 (100) 95 (100) 1 11 (100) 21 (100) 1 Induction with IL-2R antagonist, n (%) (0) 0 (0) 0 (0) 1 0 (0) 16 (76) <0.001* Lymphocyte-depleting induction, n (%) 106 (100) 11 (100) 95 (100) 1 11 (100) 5 (21) <0.001* Acute rejection, n (%) Total 52 (49) 6 (54) 46 (48) (54) 13 (62) Borderline 16 (15) 0 (0) 16 (17) (0) 2 (10) IA/IB 28 (26) 6 (54) 22 (23) (54) 8 (38) IIA/IIB/III 8 (8) 0 (0) 8 (8) 1 0 (0) 3 (14) HLA-A mismatch, n (%) 39 (37) 3 (27) 36 (38) (27) 4 (19) HLA-B mismatch, n (%) 70 (66) 5 (45) 65 (68) (45) 9 (43) 1 2 HLA-DR mismatch, n (%) 61 (58) 4 (36) 57 (60) (36) 2 (10) Total HLA mismatch, n (%) 4-6 HLA mismatch 80 (75) 7 (64) 73 (77) (45) 8 (38) PRA, n (%) 0 10% 102 (96) 11 (100) 91 (96) 1 11 (100) 19 (90) >10% 4 (4) 0 (0) 4 (4) 1 0 (0) 2 (10) De novo DSA, n (%) 10 (10) 4 (36) 6 (6) 0.010* 4 (36) 4 (19) Donor and allograft factors Donor age, years a 34 (18 59) 38 (18 45) 34 (10 50) (18 45) 57 (17 74) 0.008* Donor male sex, n (%) 55 (52) 5 (45) 50 (53) (45) 9 (43) 1 Cadaveric donation, n (%) 106 (100) 11 (100) 95 (100) 1 11 (100) 17 (81) Cold ischaemia time, h : min a 9 : 34 (4 : : 05) 10 : 27 (8 : : 35) 9 : 20 (4 : : 05) : 27 (8 : : 35) 09 : 15 (4 : : 10) Delayed graft function, n (%) 13 (12) 0 (0) 13 (14) (0) 6 (29) PRA, panel reactive antibodies. a Median (range). *P < 0.05.

4 trial protocol were excluded from the control group. All KTRs received induction therapy either with an IL-2R antagonist (basiliximab) or with a lymphocyte-depleting agent (OKT 3, anti-thymocyte globulin or alemtuzumab). All SPKTRs received induction therapy with a lymphocyte-depleting agent. If acute rejection was suspected, a kidney biopsy was performed and the rejection classified according to the Banff classification. Rejections were treated with intravenous steroid for 3 5 days. Kidney biopsies grade Banff IIA or higher were treated with a lymphocyte-depleting agent. Pancreas rejections were diagnosed clinically. Suspected pancreas rejection based upon enzyme elevation was treated with intravenous methylprednisolone. Biopsy-proven pancreas rejection was treated with intravenous methylprednisolone for Maryland Grade 0 I rejection, and with a lymphocyte-depleting agent for Grade II rejection or higher. Diagnosis and treatment of BKVN Diagnosis of BKVN was based on biopsy with histological findings that were performed upon detection of renal dysfunction or significant BK viraemia (> copies/ml in two consecutive samples). Therapeutic interventions were applied according to our predefined protocol of switching maintenance immunosuppression and antiviral treatment (Table 2). In a first step, KTRs with BKVN were treated by reduction of immunosuppression. Calcineurin inhibitor dose was reduced according to trough levels (tacrolimus trough level of 3 5 ng/ml and Table 2. Characteristics of BKV infection cyclosporine trough level of ng/ml) and where necessary MMF/MPA dose was reduced to mg/day. In a second step, KTRs with BKVN were treated by changing immunosuppression. Tacrolimus was replaced by cyclosporine with a trough level of ng/ml. MMF/MPA was replaced by azathioprine with a dose of mg/kg body weight dependent on blood count. Methylprednisolone was maintained unchanged. KTRs who did not respond to the modification of immunosuppression were treated in a third step with cidofovir and intravenous immunoglobulins (IVIG) in a fourth step. Treatment steps were initiated if BKV load remained unchanged or was rising after two consecutive measurements over an interval of 2 4weeks. Infection monitoring and prophylaxis Screening for BKV load in serum was performed 3 months during the first posttransplant year and annually thereafter. In addition, screening for BKV load was performed at any unexplained rise in serum creatinine and in case of acute rejection or anti-rejection treatment. All KTRs/SPKTRs with lymphocyte depletion induction received a prophylaxis with valgangciclovir for 3 months posttransplantation. All KTRs with a high-risk CMV constellation (D+ R ) received a prophylaxis with valganciclovir for 3 months posttransplantation. Oral prophylaxis for Pneumocystis jirovecii pneumonia with trimethoprim/sulphamethoxazole was administered 6 months posttransplantation. BKVN in SPKTRs (n = 11) BKVN in KTRs (n = 24) P-value Transplant age at BK viraemia, months a 14 (2 109) 4 (1 50) 0.033* Duration of BK viraemia, months a 11 (2 52) 12 (3 60) month, n (%) 2 3 months, n (%) 1 (9) 1 (5) months, n (%) 5 (45) 10 (48) >12 months, n (%) 5 (45) 10 (48) BKV-load in serum, copies/ml BKV load at diagnosis a ( ) ( ) Peak BKV load a ( ) ( ) 0.023* Concomitant CMV infection CMV load at diagnosis, copies/ml a ( ) Peak CMV load, copies/ml a ( ) Maintenance immunosuppression Tac trough levels at diagnosis, µg/l a 7.3 ( ) 9.3 ( ) CyA trough levels at diagnosis, µg/l a 177 MMF dosing at diagnosis, mg/day a 2000 ( ) 2000 ( ) Therapeutic intervention, n (%) Switch from Tac to CyA 10 (91) 21 (100) Switch from MMF to Aza 9 (82) 17 (81) 1 +Cidofovir 9 (82) 8 (38) 0.028* +IVIG 5 (45) 4 (19) No intervention, n (%) 0 (0) 0 (0) Allograft function Serum creatinine at diagnosis, mg/dl a 1.66 ( ) 1.92 ( ) Peak serum creatinine, mg/dl a 2.75 ( ) 2.23 ( ) Δ serum creatinine, mg/dl a 0.76 ( ) 0.75 ( ) Recovery to baseline creatinine, n (%) 2 (18) 14 (67) 0.023* Graft loss due to BKVN, n (%) 1 (9) 2 (10) 1 Tac, tacrolimus; CyA, cyclosporine A; Aza, azathioprine. a Median (range). *P < BKV in simultaneous pancreas/kidney transplantation 1177

5 Quantitative PCR for BKV-DNA detection BKV load was measured by TaqMan Real Time PCR as described previously [7]. Briefly, DNA was isolated from serum using a QIAamp DNA Mini Kit (Qiagen Corp., Hilden, Germany) according to the manufacturer s instructions. PCR was based on the TaqMan platform (ABI). PCR amplifications were set up in a reaction volume of 25 U/µL using primer and probe at final concentrations of 900 nm and 5 µm, respectively. Primers and probe were designed to amplify the VP1 region of BKV, respectively. A plasmid standard containing the VP1 coding region of the respective virus was used to determine the copy number per milliliter. Thermal cycling was begun with an initial denaturation step at 95 C for 10 min that was followed by 40 cycles at 95 C for 15 s (denaturation) and 60 C for 1 min (reannealing and extension). The detection level is the lowest viral load measured within the range of linearity, 3000 copies/ml serum. Statistical methods Statistical tests were performed using SPSS Version 22 (SPSS, Chicago, IL, USA). For comparisons of study groups, two-sided Mann Whitney U test for nonparametric independent samples was used. For comparisons between paired samples, two-sided Wilcoxon signed-rank test for nonparametric dependent samples was used. Outcomes were measured with Kaplan Meier models and overall strata comparisons measured by log-rank tests. Cox regression with time-dependent covariate analysis was used to compare patient survival and deathcensored allograft survival between those with BKVN and those without BKVN. Clinical characteristics were compared across groups using Fisher s exact test. Two-sided P-values <0.05 were considered statistically significant. RESULTS Clinical characteristics Altogether 11 SPKTRs and 21 KTRs with BKVN were analysed. Median follow-up of SPKTRs posttransplantation was 98 months (range months), during which two KTRs died (18.2%) and one returned to dialysis (9.1%). Median followup of KTRs posttransplantation was 104 months (range months), during which two KTRs died (9.5%) and two returned to dialysis (9.5%). One of 11 SPKTRs (9.1%) and 2 of 21 KTRs (9.5%) with BKVN showed allograft loss due to BKVN. The control group consisted of 95 SPKTRs with a median follow-up posttransplantation of 101 months (range months). Eighteen SPKTRs died (18.9%), 5 returned to dialysis (5.2%) and 14 lost their pancreas allograft function (14.7%). Among those with pancreas allograft loss within the first 6 months posttransplantation, 0 of 10 SPKTRs (0%) developed BKVN in follow-up. Risk factors associated with BKVN in SPKTRs and KTRs Analysis of clinical characteristics of SPKTRs with BKVN, SPKTRs without BKVN and KTRs with BKVN is shown in- Table 1. Risk factors associated with development of BKVN in SPKTRs included recipient age and recipient male gender. SPKTRs developing BKVN were significantly older compared with SPKTRs not developing BKVN (P = 0.033). In addition, SPKTRs developing BKVN were more likely to be male compared with SPKTRs not developing BKVN (P = 0.003). KTRs with BKVN showed a tendency for a higher incidence of CMV replication (52 versus 18%; P = 0.128). Outcomes in SPKTRs and KTRs with BKVN Overall no differences for patient survival [hazard ratio (HR) 1.156, confidence interval (CI) , P = 0.848] and death-censored kidney (HR 2.142, CI , P = 0.483) and pancreas allograft survival (HR 1.785, CI , P = 0.599) were observed between SPKTRs with BKVN and SPKTRs without BKVN (Figures 1 and 2). SPKTRs developing BKVN showed significantly worse renal function compared with SPKTRs without BKVN starting at +36 months posttransplantation (Figure 3; P < 0.05). SPKTRs developing BKVN showed significantly worse renal function compared with KTRs with BKVN at any time posttransplantation (Figure 3; P < 0.05). SPKTRs with BKVN were more likely not to recover to baseline creatinine after BKV clearance compared with KTRs with BKVN (P < 0.001). Despite a lowering of immunosuppression in SPKTRs and KTRs with BKVN, there was no difference in acute rejection episodes compared with the control group (P > 0.05). Two of 28 SPKTRs with BKVN (18%) and 7 of 21 KTRs with BKVN (33%) developed acute cellular rejection within 6 months after reduction of maintenance immunosuppression for treatment of BKVN. While 4 of 11 SPKTRs with BKVN (36.4%) developed de novo donor-specific antibodies after BKVN, only 6 of 95 SPKTRs without BKVN (6.3%) developed de novo donorspecific antibodies in follow-up (P = 0.01) and only 3 of 21 KTRs with BKVN (14.3%) developed donor-specific antibodies after BKVN (P = 0.197). FIGURE 1: Kaplan Meier plot of patient survival by BKVN after SPKT and single kidney transplantation. No differences were observed between SPKTRs with BKVN, KTRs with BKVN and SPKTRs without BKVN (log-rank, P = 0.778) T. Schachtner et al.

6 FIGURE 2: (A) Kaplan Meier plot of death-censored kidney allograft survival by BKVN after SPKT and single kidney transplantation. No differences were observed between SPKTRs with BKVN, KTRs with BKVN and SPKTRs without BKVN (log-rank, P = 0.716). (B) Kaplan Meier plot of death-censored pancreas allograft survival by BKVN after SPKT. No differences were observed between SPKTRs with BKVN and SPKTRs without BKVN (log-rank, P = 0.298). FIGURE 3: Decreased median egfr in SPKTRs and KTRs with BKVN (P < 0.05). SPKTRs developing BKVN showed significantly worse renal function compared with SPKTRs without BKVN starting at +36 months posttransplantation (P < 0.05). No differences were observed for serum lipase levels and haemoglobin A1c levels at any time between SPKTRs with BKVN and SPKTRs without BKVN (P > 0.05). Virological characteristics in SPKTRs and KTRs with BKVN Analysis of virological characteristics of SPKTRs with BKVN and KTRs with BKVN is shown in Table 2. The primary onset of BK viraemia was significantly earlier in KTRs developing BKVN compared with SPKTRs developing BKVN with a median of 4 months (range 1 50 months) compared with 14 months (range months; P = 0.033). Four of 11 SPKTRs (36%) and 15 of 21 KTRs (71%) showed early-onset BKVN within the first 6 months posttransplantation (Figure 4). FIGURE 4: Onset of BK viraemia after SPKT and single kidney transplantation. Onset of BKVN in SPKTRs was significantly later compared with KTRs (P = 0.033). While 71% of KTRs showed early-onset BKVN, 64% of SPKTRs developed late-onset BKVN. SPKTRs with BKVN showed significantly higher peak BKV loads compared with KTRs with BKVN (P = 0.023). SPKTRs with BKVN were more likely to require additional therapeutic interventions over modification and reduction of maintenance immunosuppression compared with KTRs with BKVN (P = 0.028). SPKTRs with BKVN were less likely to recover to baseline serum creatinine after BKVN compared with KTRs with BKVN (P = 0.023). DISCUSSION In contrast to available data on SPKTRs with BKVN, to the best of our knowledge,this is the first study focusing solely on BKV in simultaneous pancreas/kidney transplantation 1179

7 SPKTRs with biopsy-proven BKVN, all transplanted in the modern era of immunosuppression and with a clear comparison of SPKTRs and KTRs with BKVN. Our results suggest a significantly higher incidence of biopsyproven BKVN in SPKTRs compared with that in KTRs. This observation is in line with previous works suggesting an increased incidence of 7.5 and 6.2% for BKVN in SPKT [13, 20]. These studies, however, describe patients in the era of cyclosporine-based maintenance immunosuppression and do not reflect the impact of modern immunosuppression with tacrolimus and MMF. Firstly, our data suggest a predominantly late-onset of BKVN in SPKTRs compared with an early-onset of BKVN in KTRs. The use of lymphocyte-depleting induction has been associated with an increased incidence of BKV replication most likely due to an elimination of protective BKV-specific cellular immunity [21 23]. However, since BKVN rarely develops in recipients of non-renal solid organ allografts, additional risk factors in the allograft kidney seem to be decisive for the development of BKV-replication and progression to BKVN [24, 25]. Since SPKTRs present with late-onset BKVN despite the use of lymphocyte-depleting induction, our data suggest additional factors contributing to the increased risk of early-onset BKVN in KTRs and late-onset BKVN in SPKTRs. Risk factors that have been identified to predispose KTRs to early-onset BKVN include acute cellular rejection episodes and systemic CMV reactivation [21, 26, 27]. In addition, a delay in initial kidney allograft function that may reflect a potential permissive effect of ischaemic allograft injury may contribute to an increased risk of BKVN. An increased vulnerability of the allograft kidney due to inferior organ quality due to older donor age and comorbidities, which may potentiate the adverse impact of cold ischaemia time, may represent the decisive factor for earlyonset BKV replication. In contrast, the excellent organ quality in SPKT with minimum initial ischaemic allograft injury and no delayed graft function may play the decisive role for the lower incidence of early-onset BKVN despite lymphocyte-depleting induction. SPKTRs may be at an increased risk for the development of late-onset BKVN because of a greater risk of overimmunosuppression compared with KTRs alone [20]. Immunosuppressive protocols have been designed around the greater risk of rejection presented by the pancreas allograft, and these patients may have a greater rate of exposure to T-cell-depleting antibodies and pulsed steroids compared with KTRs alone. In addition, pre-existing diabetes itself has been suggested to be a possible risk factor for BKVN and at least in part explain the increased incidence of BKVN in SPKTRs. In this context, a previous study on transplant recipients of pancreas only were found to have BK viruria in 10% of cases, which supports the concept that diabetes may predispose to BKVN [28, 29]. Our results suggest recipient age and recipient male gender as risk factors for the development of BKVN in SPKTRs, confirming several other studies that have also shown a male predominance in BKVN in KTRs alone [5, 6, 28, 30]. To what extent differences in seroprevalence, methods of exposure or different immune responses between males and females contribute to this finding remains unclear. Secondly, our data suggest a more severe course of BKVN in SPKTRs compared with that in KTRs, with higher peak BKV loads, a need for more intense therapeutic intervention and less likely recovery to baseline serum creatinine. Here, the more severe course in SPKT may be attributed to the later onset of BKVN with a less frequent BKV screening density and a more advanced stage of BKVN at diagnosis. In addition, the more severe course of BKVN may be attributed to a less effective reduction of maintenance immunosuppression at the time of diagnosis due to a higher fear of acute cellular rejection in the presence of the allograft pancreas. This may in particular contribute to the need for more therapeutic interventions including cidofovir and IVIG and the more severe injury of the kidney allograft with inferior kidney allograft function after BKV clearance. According to our results, with a higher incidence of late-onset BKVN in SPKT and associated inferior kidney allograft outcome, a more intense screening of BKV replication needs to be implemented in SPKTRs. Here, we suggest 3-month intervals during the first 2 years and annual screening thereafter. It is more than likely that earlier detection of BK viraemia may have a significant impact on severity and outcomes of BKVN in SPKTRs. The practicability of guidance of immunosuppression reduction using BKVspecific T-cell monitoring in SPKTRs needs to be addressed in upcoming studies [22, 23]. Thirdly, SPKTRs with BKVN showed severely impaired kidney allograft function in long-term follow-up. This finding can be attributed to the late-onset of BKVN with delayed diagnosis and a more pronounced injury of the allograft kidney due to BKVN. The fear of pancreatic allograft rejection may in addition contribute to inadequate treatment of BKVN in SPKTRs that can lead to a more severe and prolonged course of BKVN. Our findings may be at least in part explained by the very recently suggested association of BKV replication with allosensitization in terms of development of de novo donor-specific antibodies, which we are able to confirm here in the setting of SPKT [31]. Our own data very recently showed that late-onset BKV replication in KTRs is highly associated with allosensitization in retransplant KTRs. Here, inflammation within the graft as a result of previous episodes of acute rejections predisposing to allosensitization and a chronic inflammatory state in the presence of donor-specific antibodies can be considered a decisive risk factor for BKV replication and the development of late-onset BKVN [24, 25]. Previous work in SPKTRs suggested that reducing immunosuppression in an attempt to salvage the kidney allograft did not result in subsequent pancreas allograft rejection or dysfunction [13]. Very little is currently known about the clinical course of BKVN in SPKTRs with regards to the outcome of the pancreas allograft. Although our study shows that a careful, stepwise approach in immunosuppression reduction is safe and reliable as we did not observe any pancreatic allograft loss during immunosuppression reduction for BKVN treatment, our data raise fears of inferior kidney and pancreas allograft survival and function in long-term follow-up due to the high incidence of de novo donor-specific antibodies in SPKTRs with BKVN. Our study has several strengths. We describe the incidence of BKVN in a cohort of SPKTRs and KTRs almost exclusively 1180 T. Schachtner et al.

8 maintained on tacrolimus, MMF and methylprednisolone with a standardized immunosuppression reduction approach on BKVN. We are able to show for the first time a predominantly late onset of BKVN in SPKTRs compared with KTRs and suggest differences in the pathogenesis, risk factors and outcomes. Our study further has some limitations. Firstly, although the BK virus screening protocol was instituted prospectively, this study is a single-centre retrospective study. Secondly, screening for BK viruria was not performed routinely in our study population, so BK viruria only cannot be addressed among our control group. In summary, our results suggest a predominantly late onset of BKVN in SPKTRs compared with that in KTRs. An increased vulnerability of the allograft kidney due to inferior organ quality in KTRs compared with SPKTRs may predispose KTRs to early-onset BKVN. In contrast, SPKTRs present with late-onset BKVN, which may be attributed to a greater risk of overimmunosuppression due to the higher rejection risk presented by the pancreas allograft. AUTHORS CONTRIBUTIONS T.S. participated in data collection, writing of the paper, the performance of the research and data analysis. M.Z. participated in data collection and data analysis. A.K. participated in research design and data analysis. P.R. participated in research design, writing of the paper, the performance of the research and data analysis. ACKNOWLEDGEMENTS The authors gratefully thank Anett Sefrin, Cordula Giesler and all the members of the Department of Immunology at Charité Berlin and BCRT who participated in the research of this study. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1. Gardner SD, Field AM, Coleman DV et al. New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1971; 1: Nickeleit V, Hirsch HH, Binet IF et al. Polyomavirus infection of renal allograft recipients: from latent infection to manifest disease. J Am Soc Nephrol 1999; 10: Hirsch HH, Steiger J. Polyomavirus BK. Lancet Infect Dis 2003; 3: Huang G, Chen LZ, Qiu J et al. Prospective study of polyomavirus BK replication and nephropathy in renal transplant recipients in China: a singlecenter analysis of incidence, reduction in immunosuppression and clinical course. Clin Transplant 2009; 5: Hirsch HH, Knowles W, Dickenmann M et al. Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med 2002; 347: Hirsch HH, Brennan DC, Drachenberg CB et al. Polyomavirus associated nephropathy in renal transplantation. Interdisciplinary analyses and recommendations. Transplantation 2005; 79: Babel N, Fendt J, Karaivanov S et al. Sustained BK viruria as an early marker for the development of BKV-associated nephropathy: analysis of 4128 urine and serum samples. 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Mod Pathol 1996; 9: Drachenberg CB, Papadimitriou JC, Hirsch HH et al. Histological patterns of polyomavirus nephropathy: correlation with graft outcome and viral load. Am J Transplant 2004; 4: Al-Jedai AH, Honaker MR, Trofe J et al. Renal allograft loss as the result of polyomavirus interstitial nephritis after simultaneous kidney-pancreas transplantation: results with kidney retransplantation. Transplantation 2003; 75: Schachtner T, Babel N, Reinke P. Different risk factor profiles distinguish early-onset from late-onset BKV-replication. Transpl Int 2015; 28: Schachtner T, Stein M, Babel N et al. The loss of BKV-specific immunity from pretransplantation to posttransplantation. Am J Transplant 2015; 15: Schachtner T, Müller K, Stein M et al. BK virus-specific immunity kinetics: a predictor of recovery from polyomavirus BK-associated nephropathy. Am J Transplant 2011; 11: Atencio IA, Shadan FF, Zhou XJ et al. Adult mouse kidneys become permissive to acute polyomavirus infection and reactivate persistent infections in response to cellular damage and regeneration. J Virol 1993; 67: Limaye AP, Smith KD, Cook L et al. Polyomavirus nephropathy in native kidneys of non-renal transplant recipients. Am J Transplant 2005; 5: Schachtner T, Stein M, Sefrin A et al. Inflammatory activation and recovering BKV-specific immunity correlate with self-limited BKV replication after renal transplantation. Transpl Int 2014; 27: Schachtner T, Stein M, Reinke P. ABO desensitization affects cellular immunity and infection control after renal transplantation. Transpl Int 2015; 28: Trofe J, Gaber LW, Stratta RJ et al. Polyomavirus in kidney and kidney pancreas transplant recipients. Transpl Infect Dis 2003; 5: Ramos E, Drachenberg CB, Portocarrero M et al. BK virus nephropathy diagnosis and treatment: experience at the University of Maryland Renal Transplant Program. 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9 30. Dadhania D, Snopkowski C, Ding R et al. Epidemiology of BK virus in renal allograft recipients: independent risk factors for bk virus replication. Transplantation 2008; 86: Sawinski D, Forde KA, Trofe-Clark J et al. Persistent BK viremia does not increase intermediate-term graft loss but is associated with de novo donorspecific antibodies. J Am Soc Nephrol 2015; 26: Received for publication: ; Accepted in revised form: Nephrol Dial Transplant (2016) 31: doi: /ndt/gfv338 Advance Access publication 26 September 2015 Measured and not estimated glomerular filtration rate should be used to assess renal function in heart transplant recipients Oscar Kolsrud 1,2, Sven-Erik Ricksten 3, Erik Holmberg 4, Marie Felldin 5, Kristjan Karason 6, Ola Hammarsten 7, Ola Samuelsson 8 and Göran Dellgren 1,2,5 1 Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden, 2 Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 3 Department of Cardiothoracic Anaesthesia and Intensive Care, Sahlgrenska University Hospital, Gothenburg, Sweden, 4 Department of Oncology, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 5 Department of Transplant Institute, Sahlgrenska University Hospital, Gothenburg, Sweden, 6 Department of Cardiology, Sahlgrenska University Hospital, Gothenburg, Sweden, 7 Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden and 8 Department of Nephrology, Sahlgrenska University Hospital, Gothenburg, Sweden Correspondence and offprint requests to: Göran Dellgren; goran.dellgren@vgregion.se ABSTRACT Background. In organ transplanted patients, impaired renal function is of major prognostic importance and influences therapeutic decisions. Therefore, monitoring of renal function with glomerular filtration rate (GFR) is of importance, both before and after heart transplantation (HTx). The GFR can be measured directly (mgfr) or estimated (egfr) with equations based on circulating creatinine or cystatin C levels. However, these equations have not been thoroughly validated in the HTx population. Methods. We investigated the correlation, agreement and accuracy between mgfr (using 51 Cr-ethylenediaminetetraacetic acid or iohexol clearance) and three commonly used egfr equations (Modification of Diet in Renal Disease, Cockcroft Gault and Chronic Kidney Disease Epidemiology Collaboration) in a retrospective analysis of 416 HTx recipients followed between 1988 and Comparisons were performed prior to transplantation and at 1, 5 and 10 years of follow-up. Results. The correlations between egfr and mgfr were only moderate, with r-values ranging from 0.55 preoperatively to 0.82 during follow-up. Most importantly, the level of agreement between egfr and mgfr was very low for all three estimates, with percentage errors ranging from 93.3 to 157.3%. Also, the percentage of patients with egfr within 30% of mgfr (P30) rarely reached the National Kidney Foundation recommended minimum level of 75%. Conclusion. We argue that the accuracy and the precision of the most commonly used estimation equations for assessment of kidney function are unacceptably low and we believe that mgfr should be used liberally as the basis for clinical decision-making both before and after HTx when egfr is subnormal. Keywords: heart transplantation, kidney function, measured and estimated glomerular filtration rate INTRODUCTION Heart transplantation (HTx) is an established treatment with excellent outcome in patients with end-stage heart failure [1]. For several reasons, assessment of renal function with glomerular filtration rate (GFR) is of major importance, both before and after transplantation. All solid-organ transplanted patients need lifelong treatment with immunosuppressant drugs to avoid rejection. These drugs have an effect on and are The Author Published by Oxford University Press Downloaded from onhttps://academic.oup.com/ndt/article-abstract/31/7/1174/ behalf of ERA-EDTA. All rights reserved. 1182