—  SPECIALTY CONFERENCE  —

Renal Pathology

Case 1 - 1) BK-Polyomavirus Nephropathy, Stage A
2) Acute Tubulo-interstitial Cellular Rejection (Banff '97: Type IB, C4d Negative)

Volker Nickeleit
University of North Carolina
Chapel Hill, NC


Click on each slide thumbnail image for an enlarged view
Clinical History
The patient is a 39-year-old African American male who had developed end stage renal disease (presumably secondary to hypertension). He had been on hemodialysis for more than 5 years. In August 2004 the patient received a renal transplant of cadaveric origin. (A zero-hour implantation biopsy revealed only unremarkable medullary parenchyma.) The renal allograft functioned reasonably well post surgery (baseline serum creatinine reading 10 days post transplantation = 1.6 mg/dl). The patient was enrolled into a "blinded drug trial protocol" with baseline immunosuppression consisting of cysclosporine, prednisone and a study drug (either mycophenolate mofetil or FTY720). During the first 2 months after surgery, he experienced a complicated clinical course with wound dehiscence and infection requiring surgical intervention and antibiotic therapy.


Case 1 - Figure 1 - Low power magnification of the renal cortex with unremarkable glomeruli and a patchy inflammatory cell infiltrate in the interstitial compartment. H&E stained section, 4x original magnification.
Case 1 - Figure 2 - Low power magnification of the renal cortex with unremarkable glomeruli and a patchy inflammatory cell infiltrate in the interstitial compartment. H&E stained section, 4x original magnification.
Case 1 - Figure 3 - Renal cortex with unremarkable glomeruli and a patchy infilammatory infiltrate in the interstitial compartment. 20x original magnification; PAS stained sections, 10x original magnification.



Case 1 - Figure 4 - There is interstitial inflammation and some tubulitis. 20x original magnification.
Case 1 - Figure 5 - Interstitial fibrosis is inconspicuous. Trichrome stained section.
Case 1 - Figure 6 - The tubulo-interstitial compartment shows focal edema and inflammatory cell infiltrates composed of mononuclear cell elements and scattered plasma cells. Some non-atrophic tubules show marked tubulitis. 40x original magnification.



Case 1 - Figure 7 - The nuclei of tubular epithelial cells show structural irregularities possibly representing intranuclear polyomavirus inclusion bodies (types 2 and 3). Suboptimal tissue processing resulted in nuclear artifacts precluding a definitive assessment. H&E stained section, 100x original magnification.
Case 1 - Figure 8 - Immunohistochemical incubation to detect the SV-40T antigen (i.e. the "pan-polyomavirus antigen"). The interstitial compartment shows a diffuse inflammatory cell infiltrate and focal tubulitis; only very rare individual tubular epithelial cells reveal a positive (brown) intranuclear staining reaction. 10x original magnification.



Case 1 - Figure 9 - Immunofluorescence microscopy to detect the complement degradation product C4d. C4d is not detected along peritubular capillaries. (C4d is noted in the mesangial region of a glomerulus. This is a non-diagnostic finding which serves as a convenient internal staining control). 20x original magnification.
Case 1 - Figure 10 - Immunofluorescence microscopy to detect tubular epithelial MHC-class II (HLA-DR) expression. Tubular epithelial cells/tubules focally upregulate class II. (Peritubular and glomerular capillaries constantly express class II; this endothelial staining pattern serves as a convenient internal staining control.) 20x original magnification.

Laboratory Results
Serum creatinine:

8/04 1.6 mg/dl
9/04 2.3 mg/dl
10/04 2.6 mg/dl
11/04 2.3 mg/dl

Urinalysis:

No hematuria or proteinuria
Urine cytology 8/04
9/04
10/04 		no decoy cells
no decoy cells
40 - 100 decoy cells / 10 high power fields
Urine PCR 10/04 200.000.000 BK-virus copies/ml

Blood:

Serum PCR 8/04 - 11/04
10/04 		no detection of CMV or EBV
600 BK-virus copies / ml serum

Unsuccessful biopsy attempts end of October; diagnostic graft biopsy 11/18/2004.

Renal Biopsy Findings

  1. Light Microscopy
    The biopsy consisted of 1 tissue core showing renal cortex with 30 unremarkable glomeruli. The interstitial compartment revealed focal mild edema and a patchy (35%) inflammatory cell infiltrate composed of mononuclear cell elements, plasma cells and scattered rare eosinophilic leukocytes. Some tubules showed marked lymphocytic tubulitis or mild atrophy with thickening of the TBM. Interstitial fibrosis was inconspicuous. Tubular epithelial cells demonstrated reactive nuclear atypia and very focal irregularities suggestive of intranuclear viral inclusion bodies. Frank tubular epithelial cell necrosis with denudation of tubular basement membranes were not present. Arterioles and small arteries demonstrated enlarged endothelial cells but no evidence of transplant endarteritis.
  2. Immunofluorescence Microscopy/Immunohistochemistry
    1. Immunofluorescence microscopy: No diagnostically significant deposition of immunoglobulins, complement factors C1q and C3, and fibrin were found in glomeruli. The complement degradation product C4d was not detected along peritubular capillaries. Tubules/tubular epithelial cells focally expressed MHC-class II (HLA-DR).
    2. Immunohistochemistry: Incubations with a monoclonal antibody directed against the SV40T antigen ("pan-polyomavirus antigen") revealed a positive nuclear staining reaction in tubular epithelial cells (involving approximately 15% -20% of all tubules).
  3. Electron Microscopy
    Not performed.
Final Diagnosis:
  1. BK-polyomavirus nephropathy, stage A
  2. Acute tubulo-interstitial cellular rejection (Banff '97: type IB, C4d negative)
Discussion:
Polyomavirus nephropathy, commonly referred to as BK-virus nephropathy (BKN) after the main causative agent, the BK-polyomavirus strain, was first described as a single case report by the pathologist Mackenzie in 1978 [1]. In subsequent years, however, during the era of cyclosporine and azathioprine based immunosuppression, BKN was largely 'forgotten' [2, 3] . The clinical scenario changed dramatically in the mid 1990s when new third generation immunosuppressive drugs, specifically, high dose tacrolimus and mycophenolate-mofetil were introduced into the routine management of kidney transplant recipients in many centers worldwide [2, 3, 4, 5, 6, 7] . Interestingly, one of the largest initial series of patients suffering from BKN was reported from the University of Pittsburgh one of the first transplant centers that had largely replaced cyclosporine with tacrolimus [8]. Risk factors for BKN, however, are still not fully understood, and "high dose immunosuppression" with new drugs is likely only one component in a multi-factorial risk profile promoting viral disease [9, 10, 11, 12, 13] .

Currently, BKN is reported with a prevalence of 1% to up to 10% with rising incidence rates [7, 11, 14, 15, 16, 17] . BKN is by far the most important infectious complication affecting kidney transplants. It exceeds productive CMV infections of renal allografts by approximately 30 times. Since effective anti-viral treatment strategies are poorly defined, BKN often leads to severe allograft dysfunction and graft loss [3, 11, 16, 18] . Graft failure rates, especially when BKN is diagnosed late (stages B and C) and/or treatment strategies fail, can reach 50% to 100% within 24 months following the initial diagnosis [7, 13, 19] . Improved graft survival has recently been reported from centers with vigorous patient screening programs which facilitate an early diagnosis of BKN and a better outcome [9, 11, 20] .

BKN in renal allograft recipients is typically limited to the transplanted kidney. Depending on the extent of virally induced tubular injury, patients clinically present with varying degrees of allograft dysfunction. Serum creatinine levels vary from normal (BKN stage A) to markedly increased (BKN stages B and C) [9, 11, 14] . The native kidneys are free of disease and systemic signs of an infection (fatigue, fever) are generally absent. BK-virus associated hemorrhagic cystitis, often seen after bone marrow transplantation, is not found in the setting of BKN in renal allograft recipients. Early observations linking productive infections of BK viruses with ureteral stenosis could not be confirmed in recent reports [11, 15, 21] . The definitive diagnosis of BKN requires a kidney biopsy and the detection of characteristic histologic changes [3, 4, 5, 9, 11, 13, 20, 22, 23, 24, 25] .

Histology:
Two morphologic features define BKN: i) intranuclear viral inclusion bodies in tubular and parietal (glomerular) epithelial cells, and ii) virally induced tubular epithelial cell injury and necrosis [3, 4, 6, 8, 11, 23] . BK viruses use the proliferative "machinery" of the host cells for replication. The formation of intranuclear viral inclusion bodies is a characteristic histologic sign of a productive polyomavirus infection, i.e. BKN. Four distinct variants of viral intranuclear inclusion bodies as well as "hybrid forms" exist which can often be seen side by side:

Type 1 (the most frequent form) - an amorphous basophilic ground-glass inclusion body;
Type 2 – a central, eosinophilic, granular type surrounded by a (mostly incomplete) clear halo;
Type 3 – an eosinophilic finely granular form without a halo; and
Type 4 - a vesicular variant with clumped, irregular chromatin and occasional nucleoli [3, 11, 23]

It is currently undetermined whether the different phenotypes of inclusion bodies represent various stages of intranuclear viral replication and maturation and/or potential fixation artifacts. A productive infection with polyomaviruses does not induce cytoplasmic inclusion bodies. Ultimately, the intranuclear viral replication and assembly results in the lysis of the host cell and the release of mature daughter virions.

The most important histologic changes are seen in the tubules since virally induced tubular epithelial cell injury and necrosis with denudation of basement membranes are the morphologic correlates for graft dysfunction in stages B and C [3, 9, 11, 23] . Of special note: Despite the marked virally induced epithelial cell damage, the tubular basement membranes remain intact. They can serve as the structural skeleton for tubular regeneration and healing once the viral replication ceases. Therefore, BKN, especially stages A and B, can heal with morphologic and functional restitution.

BKN typically involves renal tubules and collecting ducts in a focal fashion. Often, severely injured tubules containing many inclusion bearing epithelial cells are located adjacent to normal tubules. This observation may potentially reflect the ascending route of viral spread within "infected" nephrons.

The replication of polyomaviruses is limited to epithelial cell elements. A productive infection is generally not seen in stromal, mesangial, endothelial, and inflammatory cells or in podocytes. Occasionally, inclusion bodies are observed in the parietal epithelial cell layer lining Bowman's capsule and within small "pseudo-crescents" [3] . Signs of a productive BK virus infection can also be detected in the transitional cell layer lining the renal pelvis, the ureters and/or the urinary bladder [3] . Viral inclusion bodies in transitional cells, however, are not part of the histologic features that define BKN.

Diagnostic confirmation of BKN is generally achieved by immunohistochemistry, in-situ hybridization and/or electron microscopy. These techniques are well suited to identify various viral families and potentially viral strains if a productive viral infection is already suspected by light microscopy. However, their routine diagnostic use as generalized screening tools to "hunt" for a productive polyomavirus infection is neither helpful nor cost effective [3, 7] .

Histologic stages/patterns of BKN:
BKN can present with different histologic patterns and progress through various stages [3, 9, 11, 13, 22, 23, 26] . Three patterns/stages have recently been defined at the "Polyomavirus Allograft Nephropathy Consensus Conference" (Basel, Switzerland, October 2003, [13]). They are listed here with slight modifications:

  1. Pattern/stage A (limited/early stage): Signs of viral replication in less than 25% of cortical and medullary tubular cross sections with only minimal evidence of epithelial cell necrosis and denudation of tubular basement membranes. Interstitial inflammation, fibrosis and tubular atrophy are inconspicuous. Changes classified as pattern/stage A are often most pronounced in the renal medulla.
  2. Pattern/stage B (florid, fully developed stage): Signs of viral replication in more than 10% of cortical and medullary tubular cross sections with conspicuous epithelial cell necrosis, denudation of tubular basement membranes, and interstitial edema. Inflammation, sometimes rich in plasma cells and polymorphonuclear leukocytes, is common, whereas interstitial fibrosis and tubular atrophy are minimal. Changes classified as pattern B can be found in the renal cortex and medulla.
  3. Pattern/stage C (late, sclerosing phase): Signs of viral replication in generally less than 25% of cortical and medullary tubular cross sections associated with tubular epithelial cell injury. Interstitial inflammation can vary from minimal to marked. Fibrosis and tubular atrophy are typically seen in more than 25% of the tissue sample. Changes classified as pattern C are frequently most pronounced in the renal cortex.
Pattern/stage A represents the earliest stage of BKN with only scattered intranuclear viral inclusion bodies likely located in re-activated intra-renal foci of latent BK virus infections. BKN-stage A, in contrast to stages B and C, responds to therapy more frequently with favorable graft survival and cessation of viral replication in up to 50% of patients [11, 14, 20, 23, 27, 28] . Since tubular injury in pattern A is very limited, (often confined to the renal medulla) graft function typically remains stable, and the optimal timing of a diagnostic graft biopsy, which is generally triggered by allograft dysfunction, becomes a clinical challenge. BKN- stage A can progress to stages B or C if productive viral replication spreads and virally induced tubular injury persists over weeks to months. In one series, progression was observed in 70% of follow-up graft biopsies [22]. The therapeutic goal of BKN in stages A and B is to limit viral replication and tubular injury, to promote tubular epithelial cell regeneration and to prevent disease progression to stage C with irreversible fibrosis and tubular atrophy. BKN-stage C is typically associated with severe allograft dysfunction or loss [3, 22] .

BKN, interstitial nephritis and rejection:
The correct diagnostic interpretation of "inflammatory changes" in the setting of BKN can be difficult and has led to considerable controversy. Often, BKN is simply referred to as "BK-Virus interstitial nephritis". Although, this descriptive term accurately describes the findings, it implies that the inflammatory changes observed are secondary only to the viral replication. Using this terminology, one major differential diagnosis of "inflammation" in a renal allograft – rejection – remains without further consideration. In my opinion, it is crucial to establish a diagnosis of rejection in the setting of BKN since patients with BKN are generally treated with lowering the immunosuppression. A prerequisite for such an approach is the absence of rejection. This is the reason why I preferentially use the term "BK-Virus nephropathy" to clearly distinguish viral disease from potential concurrent acute allograft rejection.

BKN, stage A is typically not associated with significant inflammation [3, 23] . BKN, stages B and C can be associated with varying degrees of inflammatory cell infiltrates. Tubular injury results in back-leak of urine into the interstitial compartment, edema and a mixed inflammatory cell infiltrate including polymorphonuclear leukocytes [23]. The inflammatory reaction is often rich in plasma cells including a plasma cell tubulitis likely promoted by tubular expression of cytokines (e.g. IL-1, IL-6, IL-11, RANTES) and cytokine receptors (IL-4R, IL-13R) [3, 26, 29] . An inflammatory reaction rich in lymphocytes and lymphocytic tubulitis should raise the level of suspicion of concurrent rejection.

We diagnose acute rejection in the setting of BKN if we detect the following changes:

  1. transplant endarteritis or fibrinoid arterial wall necrosis,
  2. transplant glomerulitis,
  3. the deposition of the complement degradation product C4d along peritubular capillaries,
  4. significant lymphocytic inflammation and tubulitis in regions/tubules free of virally induced cytopathic changes (by LM and IHC), or
  5. the expression of MHC-class II (HLA-DR) in tubules [9, 11, 23, 28, 30]
All of the changes are typical for acute rejection and they are not defining features of BKN. The immunohistochemical detection of C4d and/or MHC-class II appear to be of specific diagnostic value to support a diagnosis of concurrent acute rejection. C4d is not detected along peritubular capillaries in "uncomplicated" cases of BKN [31, 32] . Productive polyomavirus infections seem to be associated with tubular upregulation of MHC-class I molecules (i.e. HLA-B, HLA-C, HLA-F) but not with the expression of MHC-class II (i.e. HLA-DR) [23, 29, 30] . The outlined approach to diagnose acute rejection in the setting of BKN has largely been adopted by the "Polyomavirus Allograft Nephropathy Consensus Conference" (Basel, Switzerland, October 2003, [13]).

The diagnoses of BKN and concurrent acute rejection are not purely academic but rather carry direct therapeutic consequences. Of note: Productive polyomavirus infections, i.e. persistent cases of BKN, are not associated with an increased incidence of acute rejection episodes. Thus, BKN does not trigger rejection [15, 23] .

Patient management:
Both the detection of "decoy-cells" in the urine and quantitative PCR analyses on plasma (and urine samples) are useful adjunct tools to assess the risk for BKN, to indicate the necessity for a graft biopsy and to monitor the response to therapy during persistent BKN [9, 11, 13, 20, 22, 23, 24, 25, 33] . Figure A illustrates our algorithmic diagnostic and therapeutic approach.

Highlighted below are some key aspects of acute rejection episodes occurring in the setting of BKN as many clinicians still hesitate to "treat" rejection:

  1. If the therapy of BKN includes low dose immunosuppression, acute rejection should either be absent or treated adequately.

  2. Anti rejection therapy follows a "two-step-approach":
    1. the transient specific increase of the immunosuppression is subsequently followed by
    2. therapeutic attempts to treat the BKN (e.g. low dose maintenance immunosuppression, leflunomide, cidofovir) [9, 11, 20, 23, 28]

  3. Anecdotal reports demonstrate that transient anti rejection treatment (followed by therapeutic attempts to treat BKN) can result in long-term functional improvement, resolution of BKN, and even return of kidney function to baseline levels [20, 28, 30] .

  4. BK-viruses are "slow-actors" (unlike EBV or adenoviruses). Brief increases of the immunosuppression do not seem to stimulate viral replication. Anecdotal reports indicate that transient anti-rejection therapy (with bolus steroids or even anti-lymphocytic preparations) does not result in explosive viral replication, i.e. plasma viral load levels and the number of decoy cells in the urine remain unchanged [20].

  5. BKN is limited to the allograft; transient anti-rejection therapy has not resulted in systemic viral spread or mortality (unlike CMV, adenoviruses or EBV).

  6. Transient anti-rejection treatment may not result in an immediate improvement of renal function and "therapeutic unresponsiveness" should not be regarded as evidence for a histologic "misdiagnosis of rejection". If BKN concurs with acute rejection, then allograft dysfunction is caused to different degrees by
    1. virally induced tubular injury, and
    2. rejection.
    If virally induced tubular injury predominates (i.e. BKN stage B), then allograft function will only improve after BKN has additionally been treated.

  7. In general BKN carries an unfavorable long-term prognosis; specific attempts to treat concurrent acute rejection cannot worsen graft outcome significantly.
Thus, it seems that attempts are clinically justified to diagnose and treat acute rejection episodes in the setting of BKN.

Histopathologist's role:
The definitive diagnosis of BKN is made in a graft biopsy. The diagnosis should indicate the pattern/stage of disease (A-C) and comment upon the presence or absence of rejection. Crucial clinical data required at the time of histologic work-up include information on the activation of polyomaviruses, i.e. the shedding of decoy cells in the urine and plasma viral load levels. Clinical evidence of "polyomavirus activation" at time of biopsy should automatically trigger an intensive histologic search for cytopathic signs (including immunohistochemistry or in-situ hybridization).

Clinical follow-up:
In order to treat the acute cellular tubulo-interstitial rejection episode, the patient under discussion received bolus steroid therapy in mid November 2004. His baseline immunosuppression was subsequently lowered/changed to cyclosporine (2x125 mg/day), prednisone (5 mg/day) and leflunomide (40 mg/day). In early December 2004 (less than 4 weeks after the initial diagnosis) renal function had improved and returned to "baseline" levels (S-Cr 1.7 mg/dl), i.e. the rejection episode had been adequately treated. Not surprisingly BK-virus load levels remained largely unchanged (urine cytology: 8/decoy cells per 10 HPF; urine PCR: 200.000.000 BK virus copies per ml urine; serum PCR: 8500 BK virus copies per ml serum); long-term follow-up will be needed to assess the clinical response to leflunomide.

References

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