Differentiating BK virus nephropathy (BKVN) from acute rejection (AR) is crucial in clinical practice, as both of them have interstitial inflammation in the grafts. The purpose of the study is to describe the inflammatory cellular constituents of BKVN and to determine the clinical utility of immunophenotyping findings in distinguishing BKVN from AR. In addition, the expression of the HLA-DR was investigated. Sixty-five renal allograft recipients were included in this study, including 22 cases of BKVN, 31 cases of AR, and 12 cases of stable allograft. Immunostaining for infiltrating lymphocytes showed that the number of CD20 cells (
BK virus nephropathy (BKVN) has been recognized as a cause of renal dysfunction following kidney transplantation. The incidence of biopsy-proven BKVN can vary between 1% and 10% with subsequent graft loss in more than 50% of cases [
The patients were retrospectively selected from among 356 renal allograft recipients who had recieved renal biopsy between June 2007 and February 2013 at Jinling Hospital, Nanjing University School of Medicine, Nanjing, China. Informed consent was obtained from all patients, and the Human Subjects Committee of Jinling Hospital, Nanjing University School of Medicine, approved all of the study protocols. Among them, 22 recipients were assigned to the BKVN when the biopsy demonstrated viral cytopathic changes with intranuclear inclusion bodies, associated renal tubular epithelial cell injury including tubular epithelial cell necrosis and denudation of basement membranes, and positive immunohistochemical staining for the BK T antigen (Figure
(a) BK virus allograft nephropathy. The histological manifestations are characterized by intranuclear viral inclusions in tubular epithelial cells and epithelial cell necrosis (arrow). Hematoxylin and eosin stained paraffin section. (b) Immunohistochemical staining of a renal biopsy showing positive staining for the BK T antigen.
Biopsies were performed upon clinical indication and according to local standard of practice. Two needle biopsy cores were obtained from each renal allograft for morphologic study: one for formalin fixation and the other for quick-freezing. Hematoxylin and eosin, periodic acid Schiff, methenamine-silver, and Masson stains were routinely used on the formalin-fixed tissue. The residual biopsy tissues were stored for future use. Fresh-frozen tissues were analyzed by immunofluorescence microscopy using a conventional panel of antibodies against IgG, IgM, IgA, C3, C4, C1q, HLA-DR, BKV, and C4d. BKV infection was evaluated using anti-BKV antibody (mouse monoclonal antibody specific for anti-BK virus large T antigen; Millipore Biosciences, Temecula, CA, USA). C4d staining was routinely performed on frozen slides using an indirect immunofluorescence technique with a primary affinity-purified monoclonal antibody (mouse anti-human; Quidel, San Diego, CA) and an FITC-labeled affinity-purified secondary rabbit anti-mouse IgG antibody (Dako, Denmark). The staining was performed using standard procedures. Positive C4d staining was defined as a bright linear stain along the capillary basement membranes that involved over half of the sampled capillaries in accordance with the 2001 Banff Meeting [
Formalin-fixed, paraffin-embedded renal biopsy sections were deparaffinized in xylene and rehydrated in graded ethanol (100%–70%). After deparaffinization, the sections were incubated with 3% H2O2 for 10 min to inactivate endogenous peroxidase. Microwave antigen retrieval was performed with citric acid solution (pH = 6.0) for 10 min. The slides were incubated with antibodies at room temperature for 1 hour. CD3, CD4, CD8, CD68, and CD20 were regularly detected (Figure
Immunohistochemical characterization of the inflammatory infiltrate in BK virus allograft nephropathy.
Statistical analyses were conducted using SPSS (v16.0) software. Pairwise comparisons of variables based on proportions were done by Fisher’s exact test with Bonferroni correction for
Sixty-five renal allograft recipients were included in this study, including 22 cases of BKVN, 31 cases of AR, and 12 cases of stable allograft as controls. The baseline patient characteristics are listed in Table
Clinical characteristics of patients that participated in this study.
Characteristics | BKVN ( |
AR ( |
SF ( |
|
---|---|---|---|---|
Gender, male (%) | 16 (72.73) | 19 (61.29) | 8 (66.67) | 0.686 |
Age (years) |
|
|
|
0.527 |
Donor age (years) |
|
|
|
0.349 |
Banff 97 (IA : IB : IIA : IIB) | — | 4 : 6 : 11 : 10 | — | — |
Positive pretransplant PRA ( |
0 | 0 | 0 | — |
Previous transplant | 0 | 0 | 0 | — |
Cold ischemic time (h) |
|
|
|
0.473 |
Warm ischemic time (min) |
|
|
|
0.724 |
Induction with IL-2R antibody, |
22 (100) | 31 (100) | 12 (100) | — |
Baseline immunosuppressants | 0.118 | |||
MMF + Tac + Pred | 21 | 23 | 9 | |
MMF + CsA + Pred | 1 | 8 | 3 | |
MPA |
|
|
|
<0.001 |
Tac level (ng/mL) |
|
|
|
0.410 |
Time of biopsy after Tx (month) | 14 (10–21) | 15 (2–25) | 6 (2–16) | 0.10 |
BKVN: BK virus nephropathy; AR: acute rejection; SF: stable allograft function; PRA: panel-reactive antibody; IL: interleukin; MMF: mycophenolate mofetil; Pred: prednisolone; Tac: Tacrolimus; CsA: cyclosporine A; MPA: mycophenolic acid;
The morphological findings were quite similar between BKVN and AR. Tubulointerstitial nephritis with varying degrees of inflammatory infiltrates was a major histologic changes in both entities. A spectrum of viral inclusions could be identified in the tubules in 16 of 22 BKVN patients. We used immunohistochemistry to detect CD3, CD4, CD8, CD68, and CD20 expression. The number of lymphocytes positive by immunostaining for CD3, CD4, CD8, CD20, and CD68 for patients with BKVN and AR is shown in Table
Inflammatory cellular constituents in different groups.
BKVN ( |
AR ( |
SF ( |
Post hoc | |||
---|---|---|---|---|---|---|
|
|
| ||||
CD3 (cells/mm2) | 502 (402–753) | 624 (414–773) | 180 (122–267) | 0.958 | <0.001 | <0.001 |
CD4 (cells/mm2) | 272 (177–389) | 294 (207–388) | 98 (69–147) | 0.743 | <0.001 | <0.001 |
CD8 (cells/mm2) | 276 (184–400) | 278 (196–436) | 96 (45–129) | 0.770 | <0.001 | <0.001 |
CD20 (cells/mm2) | 322 (108–636) | 76 (30–300) | 14 (11–23) | <0.001 | <0.001 | 0.003 |
CD68 (cells/mm2) | 646 (250–858) | 480 (259–617) | 85 (25–138) | 0.055 | <0.001 | <0.001 |
BKVN: BK virus nephropathy; AR: acute rejection; SF: stable allograft function.
The percentage of lymphocytes positive by immunostaining for CD3, CD4, CD8, CD20, and CD68 for patients with BKVN and AR is shown in Table
Proportions of infiltrating lymphocytes and HLA-DR expression in BKVN and AR.
BKVN ( |
AR ( |
|
|
---|---|---|---|
CD3/total (%) |
|
|
<0.001 |
CD4/total (%) |
|
|
0.004 |
CD8/total (%) |
|
|
0.005 |
CD20/total (%) |
|
|
0.002 |
CD68/total (%) |
|
4 |
0.324 |
CD3/CD68 |
|
|
0.059 |
CD3/CD20 |
|
|
<0.001 |
CD3/CD20 > 5 | 5 | 25 | <0.001 |
CD3/CD20 > 10 | 2 | 15 | 0.003 |
HLA-DR ≥ 10% | 11 | 22 | 0.156 |
BKVN: BK virus nephropathy; AR: acute rejection; SF: stable allograft function; HLA: human lymphocyte antigen.
Increased HLA-DR expression was observed in the tubule cells of 33 of the 53 renal transplants. HLA-DR expression was noted in 11/22 (50.0%) of the BKVN cases and 22/31 (71.0%) of the AR cases (Table
Proportions of infiltrating lymphocytes in HLA-DR-positive and HLA-DR-negative BKVN patients.
HLA-DR < 10% ( |
HLA-DR ≥ 10% ( |
|
|
---|---|---|---|
CD3/total (%) | 36.8 (30.7–43.1) | 35.2 (30.5–44.8) | 0.847 |
CD4/total (%) | 19.9 (17.7–22.4) | 19.9 (16.4–24.9) | 0.652 |
CD8/total (%) | 17.3 (13.8–23.1) | 18.6 (13.7–22.4) | 0.699 |
CD20/total (%) | 13.1 (4.2–17.9) | 16.2 (11.5–25.8) | 0.217 |
CD68/total (%) | 52.4 (35.5–55.8) | 45.2 (39.6–49.0) | 0.309 |
CD3/CD68 | 0.76 (0.60–1.09) | 0.75 (0.63–1.00) | 0.844 |
CD3/CD20 | 3.23 (1.61–9.70) | 2.16 (1.48–3.56) | 0.178 |
BKVN: BK virus nephropathy; HLA: human lymphocyte antigen.
This study revealed that immunophenotyping would aid in differentiating BKVN from acute rejection. We for the first time found that the percentages of CD3, CD4, CD8, and CD20 cells were all significantly different between BKVN and AR. In contrast, the presence of HLA-DR upregulation, however, may not be specific for acute rejection, since it may also be a response to BKVN. Furthermore, our study also found that the high MPA level appears to promote the development of BKV disease.
BKV infection is common after renal transplant, leading to BKVN, which is increasingly an important cause of graft failure. BKVN is a marker for an overimmunosuppressive state, and a reduction in immunosuppressive agents alone is a safe and effective therapy [
In a small sample research, Ahuja et al. characterized the type of infiltrating lymphocytes in renal histology by immunophenotyping and found that the predominance of CD20-positive lymphocytes is suggestive of BKV infection [
The proportions of infiltrating cells of BKVN have not been characterized previously. In comparison to patients with acute rejection, the percentages of CD3, CD4, and CD8 were all significantly fewer in BKVN, while CD20 took a much higher percentage. Furthermore, we compared the value of CD3/CD20 between the two groups and found that the value of CD3/CD20 in BKVN group is significantly lower than in AR group. And interestingly, we found that CD3/CD20 <5 and CD3/CD20 <10 are strongly correlated with BKVN compared with acute rejection. Differentiating BKVN from acute rejection is crucial in clinical practice, but, unfortunately, there is currently no test pathognomonic for BKVN. Histological diagnosis represents the gold diagnostic standard until now; however, it can be mistaken for allograft rejection, that is, tubulointerstitial nephritis with varying degrees of inflammatory infiltrates, tubulitis and tubular atrophy, and fibrosis. Immunohistochemistry with SV40 staining is now routinely used to document the presence of BKV in renal tissue; however, while renal involvement can be focal in earlier stages and could have predominant fibrotic changes with minimal inflammatory changes in the later stages of the disease [
In this study, we found no statistically significant difference in tubule cell HLA-DR expression between BKVN and acute rejection. It is generally believed that tubular epithelial cells may show markedly increased HLA-DR expression during allograft rejection [
In addition, we found that patients with BKVN had significantly higher MPA AUC0–12 levels at the time of diagnosis compared with matched controls, while TAC levels showed no significant differences among the three groups. Nearly all experts believe that the degree of immunosuppression is the primary risk factor for development of BKVN and that the reduction of immunosuppression is the principal treatment of BKVN [
In summary, the findings in this study indicate that due to the focal nature of BKVN, a negative biopsy cannot rule out the disease. However, evaluation of a renal biopsy in combination with the immunophenotyping is necessary for the accurate determination of BKVN. On the other hand, the presence of HLA-DR upregulation may not only be specific for acute rejection but also be a response to BKVN. Moreover, this observation suggests that BKVN is associated with MPA AUC0–12 level, and early detection of MPA level may be of value in avoiding the development of BKVN.
BK virus nephropathy
Acute rejection
Stable allograft function
Human lymphocyte antigen.
All the authors declare that they have no conflict of interests.
This study was supported by Grants from the General Program of National Natural Science Foundation of China (nos. 81070593 and 81270834), a grant from Fund for Distinguished Young of Jiangsu Province, and a grant from 333 Talent Training Program of Jiangsu Province.