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IDNEY BIOPSY TEACHING CASE

Diffuse Glomerular Crescents and Peritubular Immune Depositsin a Transplant Kidney

Ramesh Nair, MD, Daniel A. Katz, MD, and Christie P. Thomas, MD

NDEX WORDS: Crescentic glomerulonephritis; tubulointerstitial nephritis; immune deposits; transplant; kidney;

olyoma; virus; simian virus 40.

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RESCENTIC GLOMERULONEPHRITISis an uncommon occurrence in renal trans-

lant recipients.1 Most cases are caused by recur-ent disease, and de novo disease occurs eveness frequently. Differential diagnoses of cres-ents in the allograft are similar to those in theative kidney, which includes such anti–neutro-hil cytoplasmic antibody–associated diseases asegener granulomatosis and microscopic polyar-

eritis,2,3 such immune-complex glomerulone-hritides as systemic lupus erythematosus andmmunoglobulin A (IgA) nephropathy,4,5 andnti–glomerular basement membrane disease.3,6

reexisting crescents from the donor kidneylso can be present in the transplant kidney inarly posttransplantation biopsy specimens.7

e present the case of a kidney transplant recipi-nt with acute renal failure 8 months after hisecond transplantation. Renal biopsy showed dif-use crescents and a pleomorphic interstitial infil-rate associated with diffuse peritubular immuneeposits. A complete workup elucidated theathogenesis of these lesions.

CASE REPORT

linical HistoryA diagnosis of medullary cystic kidney disease was made

n a 56-year-old patient at the age of 27 years based onlinical features and family history. He received his firstenal transplant from a deceased donor in 1992, which lasted

From the Departments of Pathology, Surgery, and Inter-al Medicine, University of Iowa Carver College of Medi-ine and Veterans Affairs Medical Center, Iowa City, IA.

Received March 3, 2006; accepted in revised form April 3,006.Originally published online as doi:10.1053/j.ajkd.2006.04.064

n May 30, 2006.Address reprint requests to Ramesh Nair, MD, Assistant

rofessor, Department of Pathology, RCP 5243, Universityf Iowa, 200 Hawkins Dr, Iowa City, IA 52242. E-mail:amesh-nair@uiowa.edu

© 2006 by the National Kidney Foundation, Inc.0272-6386/06/4801-0023$32.00/0

odoi:10.1053/j.ajkd.2006.04.064

American Journal74

or 9 years. The allograft was thought to have failed becausef chronic allograft nephropathy. He returned to dialysisherapy for 3 years and then received his current renalransplant from a deceased donor in late 2004. Initial immu-osuppression consisted of induction with methylpred-isolone and thymoglobulin. The patient was administered aotal of 12 mg/kg of thymoglobulin divided into 7 doses, andecause of delayed graft function, the patient received hemo-ialysis on days 1, 4, 6, and 8. Maintenance immunosuppres-ion consisted of methylprednisolone and mycophenolateofetil begun on day 2 and tacrolimus begun on day 9. A

ransplant renal biopsy performed on day 5 showed vacu-lated tubular epithelium, confirming acute tubular necrosis.e was discharged from the hospital on day 11. His creati-ine level gradually improved and reached his nadir of 1.8g/dL (159 �mol/L) 5 weeks after surgery. After discharge,

acrolimus levels were maintained between 8 and 12 �g/L. Aerum mycophenolic acid level was checked once, with aevel of 5.2 �g/mL, and the patient was maintained onycophenolate mofetil, 500 mg twice a day.The patient was seen at this transplant center 8 months

fter the second transplant with a serum creatinine level of.2 mg/dL (283 �mol/L). He was asymptomatic and specifi-ally denied hemoptysis, nasal or ear discharge, arthralgias,kin rash, or fatigue. Clinical examination was uninforma-ive except for mild tenderness over the renal allograft andhe presence of a small traumatic ulcer on the right shin.rinalysis was positive for heme; negative for protein,itrites, and leukocyte esterase; and showed 14 to 19 whitelood cells/high-power field and 3 to 4 red blood cells/high-ower field without cellular casts. Complement 3 (C3) and4 levels were 0.89 mg/mL (0.89 g/L) and 0.24 mg/mL

0.24 g/L), respectively. Ultrasound with Doppler examina-ion showed a normal-sized kidney without hydronephrosisnd with normal Doppler indices. A renal biopsy was per-ormed to evaluate the acute renal failure.

enal Biopsy FindingsOf 11 glomeruli present, 4 showed global sclerosis and 6

f the remaining glomeruli had parietal epithelial cell cres-ents. These included 3 fibrocellular and 3 cellular crescentsFig 1). The globally sclerotic glomeruli also had fibrousrescents. There was no evidence of endothelial or mesan-ial proliferation, necrosis, or thickening of glomerularapillary loops. There was severe interstitial inflammationith infiltrating lymphocytes, eosinophils, and plasma cells.oderate to severe interstitial fibrosis and tubular atrophyere noted, as well as moderate tubulitis, in relatively intact

ubules. Features typical of polyomavirus consisting ofmudgy basophilic inclusions were present focally in nuclei

f tubular epithelial cells. The nucleus of a cell forming a

of Kidney Diseases, Vol 48, No 1 (July), 2006: pp 174-178

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POLYOMAVIRUS, CRESCENTS, AND IMMUNE DEPOSITS 175

ellular crescent also contained a smudgy basophilic inclu-ion consistent with polyomavirus (Fig 2). Immunohisto-hemical stain for simian virus 40 (SV40) large T-antigenhowed mild to moderate positivity in scattered tubularpithelial cell nuclei. Two glomeruli with cellular crescentshowed positive staining for SV40 in nuclei of cells consti-uting the crescent (Fig 3).

By means of immunofluorescence (IF), there was diffuse,ariable, faint to bright, granular staining of tubular base-ent membranes (TBMs) for IgG, 1 to 3�, and C3, 1 to 3�.4d was present in the TBM in a granular fashion (Fig 4)nd was brighter than IgG and C3 and stained with lessariable intensity of 2 to 3�. Peritubular capillary stainingor C4d was absent. IgM and IgA were negative in TBMs.

Fig 1. Renal cortex showing glomeruli with cellularnd fibrocellular crescents in a background of markednterstitial fibrosis, inflammation, and tubular atrophyJones silver stain; original magnification �100).

Fig 2. Glomerulus with parietal epithelial cell prolif-ration. One epithelial cell nucleus in the crescentontains a smudgy basophilic inclusion typical of poly-

ma virus (arrow) (hematoxylin and eosin stain; origi-al magnification �400).

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ecause the first biopsy sample saved for IF consisted ofenal medulla only, glomeruli could not be examined formmunoglobulin and complement deposition at the time.he follow-up renal biopsy 2 weeks later contained 15lomeruli by light microscopy, 4 globally sclerotic, 4 withrescents, 1 cellular, and 3 fibrocellular. Tissue for IF thisime contained 2 glomeruli, both with crescents. There wasrace nonspecific blotchy staining for C4d in mesangialegions and segmentally in a glomerulus in what was prob-bly an area of sclerosis. Granular staining with IgG, C3,nd C4d was present in Bowman capsule (1�). The glomer-lar mesangium and capillary loops were negative for immu-oglobulins (IgG, IgA, IgM) and complements (C3, C1q),hereas TBMs showed the same staining pattern as in therst biopsy specimen. TBM immune deposits were diffuselyresent in both the cortex and medulla and did not show aarticular pattern of involvement with respect to whichegment of the tubule was involved.

Fig 3. Immunoperoxidase stain for SV40 large T-ntigen shows positive staining in nuclei of parietalpithelial cell crescent (long arrows). An adjacent atro-hic tubule also shows positive nuclear staining (shortrrow) (original magnification �400).

Fig 4. Diffuse granular tubular basement mem-�

rane staining for C4d (variable 2 to 3 ) and in Bow-

an capsule (1�) (original magnification �200).

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NAIR, KATZ, AND THOMAS176

Ultrastructural evaluation showed a glomerulus with abrocellular crescent. Capillary loops were of normal thick-ess. Electron-dense and fibrillary deposits were absent inlomeruli. Multiple electron-dense deposits were detectedasily in basement membranes of approximately 25% ofubules, which showed varying degrees of atrophy. Thextent of TBM deposits were less than that seen with IF anday represent a sampling bias. Tightly packed hexagonal

iral particles averaging 40 nm and typical of polyomavirusere present in rare (2) tubular cell nuclei. The basementembrane of 1 of these tubules showed a few tiny electron-

ense deposits.

iagnosisThe diagnosis was polyomavirus tubulointerstitial nephri-

is associated with TBM immune complex deposits andiffuse crescent formation in glomeruli.

ollow-Up HistoryLaboratory workup was performed to rule out immuno-

ogic causes for crescentic glomerulonephritis, a concernspecially in the presence of diffuse TBM immune deposits.ntinuclear antibody, anti–double-stranded DNA antibody,

ytoplasmic and perinuclear antineutrophil cytoplasmic anti-odies, and anti–glomerular basement membrane antibodyere negative. Urine cytological examination showed numer-us urothelial cells with viral cytopathic changes consistentith polyomavirus infection. Polyomavirus quantitative poly-erase chain reaction in blood showed a high viral load at

41,000 copies/mL.The patient was administered 3 doses of methylpred-

isolone for the severe crescentic nephritis, after whichreatment with tacrolimus and mycophenolate mofetil wasiscontinued. He was started on treatment with leflunomide,0 mg/d, and sirolimus, 4 �g/d. Despite these measures,erum creatinine levels continued to increase, and the patientubsequently underwent another biopsy 2 weeks later thathowed ongoing polyomavirus nephropathy and crescenticesions with persistent tubulointerstitial inflammation. Le-unomide dosing was increased to 30 and then 40 mg/d toaintain a leflunomide level greater than 15 mg/L. The

irolimus dose was adjusted to maintain levels of 4 to 8 g/L.e was administered an infusion of low-dose cidofovir (0.25g/kg intravenously after a 500-mL normal saline bolus) on

ay 23 after the initial biopsy because of a continuedncrease in serum creatinine levels and persistently higholyomavirus copy numbers. A second dose of cidofoviras administered 3 months after the first dose. Polyomavi-

us polymerase chain reaction in blood became negative, buterum creatinine levels continued to increase despite appar-nt response to antiviral therapy, probably as a consequencef the severe tubulointerstitial fibrosis that already hadccurred. The patient returned to dialysis therapy 6 monthsfter the original diagnosis was made.

DISCUSSION

The definition of a glomerular crescent asndorsed by the World Health Organization is 2

r more layers of parietal epithelial cells that c

artially or completely fill Bowman space.8 Theerm crescentic glomerulonephritis implies in-olvement of more than 50% of glomeruli byrescents. Polyomavirus can cause infection ofarietal epithelial cells of Bowman capsule with-ut inducing crescent formation.9,10 Althoughsolated crescents were reported previously inssociation with polyomavirus infection,9 theccurrence of active crescent-type lesions in theajority of glomeruli is distinctly unusual and

as not been reported previously.The accepted prerequisite for crescent forma-

ion is a breach of capillary loop integrity withbrin leakage into Bowman space, leading toarietal epithelial cell proliferation.11,12 Thisechanism of crescent formation usually is

econdary to such immunologic phenomena asnti–glomerular basement membrane disease,ntineutrophil cytoplasmic antibody–associatediseases, and immune-complex glomerulone-hritides. Immune deposits were absent in glo-eruli by both IF and electron microscopy. Focal

rescents also can occur from glomerular capil-ary loop breakage in other conditions, such asiabetic nephropathy, because of microaneurysmormation.13 There was no evidence of capillaryoop breakages, such as fibrin in Bowman spacer necrosis. It is most likely that crescent forma-ion here represents an exuberant proliferation ofarietal epithelial cells caused by direct infectiony polyomavirus through contiguous spread fromhe adjacent proximal tubule. The positive SV40taining in glomerular crescents and proximalubule present a strong argument that the poly-mavirus tubulointerstitial nephritis and crescen-ic glomerulonephritis are causally related.

One other instance of viral infection leading toiffuse crescentic glomerulonephritis in a trans-lant was reported previously by Detwiler etl.14 This patient had crescentic and necrotizinglomerulonephritis associated with cytomegalo-irus infection of the allograft. Epithelial cellsithin crescents showed characteristic viral inclu-

ions. Unlike our patient, this biopsy also showedecrotizing glomerulitis, which was the likelyntecedent to crescent formation.

A second notable finding in the biopsy was theresence of diffuse TBM immune deposits byoth IF and electron microscopy. This is the firstime we encountered this finding. This may oc-

ur in part because we do not routinely perform

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POLYOMAVIRUS, CRESCENTS, AND IMMUNE DEPOSITS 177

F studies in transplant biopsies, except for C4d.he diffuse and granular staining pattern for C4d

n TBMs prompted us to perform a full IF panel,esulting in detection of IgG and C3 deposits.wo very recent studies in abstract form reported

hat immune deposits may be seen in as many as3% of specimens of polyomavirus tubulointer-titial nephritis.15,16 In 1 of these studies, TBMeposits were focal, and the investigators sug-ested that localization of TBM deposits immedi-tely adjacent to infected tubular cells may beresumptive evidence of an in situ antibodyesponse, directed to either shed viral antigens orltered cellular antigens shed from infectedells.15 Conversely, our biopsy showed diffuse,ather than patchy, TBM staining in nearly allubules without a particular propensity for obvi-usly infected tubules. In the second study, posi-ive staining for SV40 was noted in these depos-ts, lending support to the view that this mayepresent viral antigen-antibody complexes, al-hough we could not confirm these findings.16

hese studies, along with ours, raise the possibil-ty that TBM deposits or a granular TBM stain-ng pattern for C4d may serve as a marker forolyomavirus infection.Clinically significant polyomavirus nephropa-

hy can be diagnosed with certainty only bydentification of characteristic features of tubulo-nterstitial nephritis on renal biopsy. Urine cyto-ogical examination and quantitative blood poly-erase chain reaction for viral copy number are

xcellent screening tests to monitor for diseasend can be used to follow up therapy.17 When theiagnosis is established, the initial goal of therapys to reduce overall immunosuppression, unlesshere is concomitant evidence for acute rejection,or which judicious use of antirejection therapylso may be tried.18 Evidence from several smallonrandomized studies showed that decreasingmmunosuppression alone is effective in someatients in improving renal function and clearingiral load, although up to 45% of patients experi-nce accelerated graft loss.19

The light microscopic evidence for polyomavi-us inclusions in tubular epithelial cell nuclei inur biopsy specimen was not overwhelming.ight microscopic features suspicious for poly-mavirus inclusion were noted in only 1 epithe-ial cell within a crescent and in only a few

ubular epithelial cells, even by using immunohis- J

ochemistry. Conversely, immunohistochemicaltaining was strongly positive and easily de-ected in cellular crescents. Therefore, polyoma-irus infection could be overlooked in the pres-nce of severe crescentic glomerular disease ifmmunohistochemical and electron microscopictudies are not performed. The mechanism of cres-ent formation appears to be different, although itimics classic crescent formation. The recognition

hat the crescents are of infectious origin can avoidnnecessary immunosuppression.

This is the first reported instance of polyoma-irus infection that caused diffuse parietal epi-helial crescentic proliferation and diffuse tubu-ointerstitial immune complex deposition. Therescentic process and diffuse TBM immuneeposits seen here probably are a correlate ofeavy viral infection, and in cases like this, therognosis for recovery is likely to be poor. Al-hough the occurrence of diffuse crescent forma-ion is rare, we suggest that polyomavirus infectione included in the differential diagnosis of crescen-ic glomerulonephritis in transplant recipients anderhaps in patients with other conditions in whicholyomavirus infection may be seen, such as inative kidneys after nonrenal solid-organ trans-lants or after stem cell transplantation.

REFERENCES

1. Hariharan S, Adams MB, Brennan DC, et al: Recurrentnd de novo glomerular disease after renal transplantation: Aeport from Renal Allograft Disease Registry (RADR). Trans-lantation 68:635-641, 19992. Nachman PH, Segelmark M, Westman K, et al: Recur-

ent ANCA-associated small vessel vasculitis after transplan-ation: A pooled analysis. Kidney Int 56:1544-1550, 1999

3. Deegens JK, Artz MA, Hoitsma AJ, Wetzels JF: Out-ome of renal transplantation in patients with pauciimmunemall vessel vasculitis or anti-GBM disease. Clin Nephrol9:1-9, 20034. Kowalewska J, Yuan S, Sustento-Reodica N, et al: IgA

ephropathy with crescents in kidney transplant recipients.m J Kidney Dis 45:167-175, 20055. Stone JH, Millward CL, Olson JL, Amend WJ, Cri-

well LA: Frequency of recurrent lupus nephritis amonginety-seven renal transplant patients during the cyclospor-ne era. Arthritis Rheum 41:678-686, 1998

6. Khandelwal M, McCormick BB, Lajoie G, et al: Recur-ence of anti-GBM disease 8 years after renal transplanta-ion. Nephrol Dial Transplant 19:491-494, 2004

7. Kiser RL, Thomas DB, Andreoni K, Klemmer PJ:reexisting crescentic glomerulonephritis in the renal allo-raft. Am J Kidney Dis 42:E20-E26, 20038. Jennette JC: Crescentic glomerulonephritis, in Jennette

C, Olsen JL, Schwartz MS, Silva FG: Heptinstall’s Pathol-

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NAIR, KATZ, AND THOMAS178

gy of the Kidney (ed 5). Philadelphia, PA, Lippincott-Raven,998, pp 625-6569. Celik B, Randhawa PS: Glomerular changes in BK

irus nephropathy. Hum Pathol 35:367-370, 200410. Nickeleit V, Hirsch HH, Binet IF, et al: Polyomavirus

nfection of renal allograft recipients: From latent infectiono manifest disease. J Am Soc Nephrol 10:1080-1089, 1999

11. Min KW, Gyorkey F, Gyorkey P, Yium JJ, Eknoyan: The morphogenesis of glomerular crescents in rapidlyrogressive glomerulonephritis. Kidney Int 5:47-56, 197412. Stejskal J, Pirani CL, Okada M, Mandelanakis N,

ollak VE: Discontinuities (gaps) of the glomerular capil-ary wall and basement membrane in renal diseases. Labnvest 28:149-169, 1973

13. Elfenbein IB, Reyes JW: Crescents in diabetic glo-erulopathy. Incidence and clinical significance. Lab Invest

3:687-695, 197514. Detwiler RK, Singh HK, Bolin P Jr, Jennette JC:

ytomegalovirus-induced necrotizing and crescentic glomer- p

lonephritis in a renal transplant patient. Am J Kidney Dis2:820-824, 199815. Bracamonte E, Furmanczyk P, Smith K, Alpers CE,

owalewska J: Tubular basement membrane deposits asso-iated with polyoma virus nephropathy in renal allografts.od Pathol 19:259A, 2006 (abstr)16. Hever A, Nast C: Polyoma virus antigen-associated

ubular basement membrane immune complex deposition.od Pathol 19:262A, 2006 (abstr)17. Hirsch HH, Brennan DC, Drachenberg CB, et al:

olyomavirus-associated nephropathy in renal transplanta-ion: Interdisciplinary analyses and recommendations. Trans-lantation 79:1277-1286, 200518. Nickeleit V, Mihatsch MJ: Polyomavirus allograft

ephropathy and concurrent acute rejection: A diagnosticnd therapeutic challenge. Am J Transplant 4:838-839, 2004

19. Vasudev B, Hariharan S, Hussain SA, et al: BK virusephritis: Risk factors, timing, and outcome in renal trans-

lant recipients. Kidney Int 68:1834-1839, 2005