TRIPLE REGIMEN WITH RITUXIMAB, AND INTRAVENOUS IMMUNOGLOBULIN IN THE CaseTREATMENT Report OF DIALYSIS DEPENDENT ACUTE HUMORAL-MEDIATED REJECTION IN KIDNEY GRAFTS

Rev Port Nefrol Hipert 2006; 20 (2): 131-135

Triple regimen with rituximab, plasmapheresis and intravenous immunoglobulin in the treatment of dialysis dependent acute humoral-mediated rejection in kidney grafts

Pedro Pessegueiro1, Fernando Nolasco2,3, Sandra Sampaio4, Fernanda Carvalho3 Felicidade Manuel2,3, Engrácia Barber2,3, Helena Viana2,3, João Sousa2,3, Marília Possante2,3, Margarida Domingos2,3, José Reimão Pinto2,3, João Ribeiro Santos2,3, Eduardo Barroso2

1 Unidade de Nefrologia, Hospital Espírito Santo, Évora 2 Unidade de Transplante, Hospital Curry Cabral, Lisboa 3 Serviço de Nefrologia, Hospital Curry Cabral, Lisboa 4 Serviço de Nefrologia, Hospital Distrital de Faro, Faro

ABSTRACT globulin therapy. Treatment with plas- mapheresis/intravenous immunoglobulin/ Introduction: The clinical importance of hu- rituximab resulted in rapid reversal of oliguria, moral-mediated acute rejection has been pro- and recovery of renal function within the 1st week gressively recognised. Early recognition and of treatment in 2/3 patients. Diagnosis was con- treatment with plasmapheresis and intravenous firmed by graft biopsies revealing peritubular immunoglobulin have recently improved short neutrophiles and C4d deposits. term prognosis. Sequential graft biopsies in all three patients Case report: In this report we describe the revealed complete histological recovery within clinical features of three 2nd transplant patients two weeks. One patient never recovered renal developing severe acute humoral rejection dur- function, and one patient lost his graft at three ing the first week post-transplant while on anti- months following hemorrhagic shock. After 2 years follow up, the remaining patient maintains a serum creatinine of 1.1mg/dl. Received for publication: 16/09/2005 Conclusion: The regimen using plasmaphe- Accepted in revised form: 07/02/2006 resis plus intravenous immunoglobulin and

Revista Portuguesa de Nefrologia e Hipertensão 131 Pedro Pessegueiro, Fernando Nolasco, Sandra Sampaio, Fernanda Carvalho, Felicidade Manuel, Engrácia Barber, Helena Viana, João Sousa, Marília Possante, Margarida Domingos, José Reimão Pinto, João Ribeiro Santos, Eduardo Barroso

rituximab was effective in rapidly reversing se- CASE REPORT vere acute humoral rejection. The authors describe the clinical features of Key-Words: Acute humoral-mediated rejection; three kidney transplanted patients all receiving kidney transplant; plasmapheresis; rituximab. a 2nd kidney graft developing severe AHR (Table I). All were presensitized patients, two with >75% panel-reactive . Protocol induction therapy included ATG (2mg/kg/day; 10 days), tacrolimus (0.07mg/Kg/day increasing to 0.2mg/ INTRODUCTION Kg/day by 7th day) plus mycophenolate mophetil (MMF; 500mg/day increasing to 2g/day) plus The importance and clinical significance of prednisolone (20mg/day). humoral alloimmunity in renal transplant (Tx) is Patient A received his second renal transplant currently under re-evaluation. from a cadaver donor (20% glomerular sclero- Acute humoral rejection (AHR) seems more sis on the donor kidney biopsy), and had a pre-Tx prevalent than previously thought, with a preva- weakly positive HLA-II cytometric flux crossmatch lence of 5% to 7%, increasing to 20-30% in cases (FCC), unknown at the time of transplant. He was of biopsy performed for acute rejection1. anuric and required haemodialysis. The criteria for diagnosis of AHR have recently Patient B received a second kidney allograft been established2,3: 1) evidence of clinical graft from a cadaver donor, and although presenting disfunction, 2) histologic evidence of tissue in- a pre-Tx negative FCC he had a historical posi- jury ( margination in the peritubular tive control (Table II). After Tx, a rapid recovery capillaries, acute tubular injury, fibrinoid necro- of renal function was attained, with serum crea- sis), 3) immunopathological evidence for anti- tinine reaching 2.0 mg/dl at the 5th day. 48 hours body (C4d positive staining in the peritubullar after, low grade fever, rapid decrease in diuresis capilaries or Ig/C3 deposition in arteries) 4) and and renal function were observed, leading to ur- serological evidence of anti-donor antibodies at gent haemodialysis. time of biopsy. Patient C, hepatitis C virus positive, received Prognosis for AHR varies, depending on the his second kidney transplant from a cadaver severity of clinical/histological lesions, and timing donor (presenting an anatomical abnormality: of treatment. two small renal arteries). Although pre-Tx In this report we describe the prompt reco- citotoxic crossmatch was negative, FCC turned very of two out of three patients with severe AHR, out slightly positive (Table II). After an initial slight developed under treatment with anti-thymocyte recovery of renal function until the 4th day post- gobulin (ATG)/tacrolimus/micophenolate mofetil, Tx., low grade fever appeared, and a rapid de- and rapidly recovering after treatment with plas- crease in urinary output occurred which lead to mapheresis (PMF), intravenous immunoglobu- induction of dialysis at the 7th day. lin (IVIG) and rituximab. Blood cultures returned negative and allograft ecodoppler was normal in all three patients. All three patients underwent graft biopsy and this showed the presence of neutrophiles in the peritubular capillaries and a imunofluorescence

132 Revista Portuguesa de Nefrologia e Hipertensão TRIPLE REGIMEN WITH RITUXIMAB, PLASMAPHERESIS AND INTRAVENOUS IMMUNOGLOBULIN IN THE TREATMENT OF DIALYSIS DEPENDENT ACUTE HUMORAL-MEDIATED REJECTION IN KIDNEY GRAFTS

Table I - Patient’s outlined characteristics Figure 1

Patient A/G/R CRF actiology Loss of previousd Tx Mismatch

A 44/M/C IgAN Chronic rejection 4

B 40/M/C HNAS SHU 3

C 50/M/B HNAS Unknown* 2

* Rejection occurred at 3 months after transplant; A/G/R: age/gender (Male, Fe- male)/race (Caucasian, Black); CRF: chronic renal failure; IgAN: nephropathy; HNAS: Hypertensive nephroangiosclerosis; Tx: transplant

Acute hummoral mediated rejection. Presence of neutrophiles in the peritubular capillaries (arrows) and pericapillaries Table II - Crossmatch and panel-reactive antibodies monitoring imunofluorescence C4d positive stain. Acute tubular necrosis features also observed. IMF: immunofluorescence; H&E: hematoxylin and eosin staining. Historical Pre Tx Pre Tx Post Tx Post Tx Post Tr Post Tr PCr after Patient FCC (UI) FCC (UI) PRA (%) FCC (UI) PRA (%) FCC (UI) PRA (%) 2 years 141 (I) 17% (I) 503 (I) 241 (I) A HD presence of ischemic glomeruli. 260 (II) 63% (II) 425 (II) 83% (II) 2915 (II) 41% (II) Although all patients attained resolution of the 398 (I) 158 (I) 96% (I) 7733 (I) 99% (I) 289 (I) 97% (I) B 1.1mg/dl 130 (II) 135 (II) 0% (II) 2268 (II) 0% (II) 772 (II) 0% (II) AHR, two of them lost permanently their graft

220 (I) 71% (I) 4691 (I) 71% (I) 667 (I) 92% (I) function. Patient A, while partly recovering renal C HD 725 (II) 99% (II) 2736 (II) 99% (II) 3504 (II) 91% (II) function (minimal serum creatinine of 1.9mg/dl Tx: transplant; Tr: treatment; FCC: flux citometry crossmatch; PRA: panel-reac- even after an episode of acute celular rejection tive antibodies; PCr: plasma creatinine; I: HLA class I (normal values <200UI); II: HLA antigens class II (normal values <250UI) BANF IA), eventually had his graft removed as a consequence of multiple surgical complications (infected volumous linfocele, life-threatening haemorrhage after a graft biopsy). Patient C C4d positive result. Severe AHR was diagnosed continued to show ischemic glomerular lesions, (Figure 1). and further investigation revealed a high pres- All patients were treated with 5 daily PMF sure arteriovenous shunt as the cause of de- sessions (one plasma volume exchange), IVIG creased diastolic blood flow. Even after AV shunt (400mg/kg) after each PMF, repeated once af- removal, no renal recovery was observed. ter 21 days, plus one or two (21 days apart) ad- In conclusion, the treatment regimen used ministrations of rituximab (375 mg/m2). allowed 100% resolution of severe AHR and al- Three days after initiating this regimen, 2/3 though only 33% of graft survival was attained, patients presented a rapid increase in diuresis, no graft loss was directly caused by humoral with a steady recovery of renal function (patient A: rejection, but by haemodynamic complications. creatinine 1.9mg/dl at day 50; patient B: creati- Moreover, all patients saw their initial histologi- nine 1.1mg/dl at day 90). Patient C remained cal abnormalities completely resolved. anuric, dialysis-dependent. During follow up of the remaining patient (B) Post-treatment graft biopsies showed a com- with functioning allograft, a cutaneous-born sys- plete resolution of the AHR features in all pa- temic infection by Alternaria spp fungus was dia- tients. However, patient A still showed donor gnosed. It was treated with long term anti-my- severe chronic lesions and patient C showed cotic therapy. No other severe complication was

Revista Portuguesa de Nefrologia e Hipertensão 133 Pedro Pessegueiro, Fernando Nolasco, Sandra Sampaio, Fernanda Carvalho, Felicidade Manuel, Engrácia Barber, Helena Viana, João Sousa, Marília Possante, Margarida Domingos, José Reimão Pinto, João Ribeiro Santos, Eduardo Barroso

noticed, allowing, after 24 months, a plasma cre- cific. IVIG efficacy appears to be mainly due to atinine level of 1.1mg/dl (Table II). mechanisms such as an anti-idiotypic effect and inhibition of the activation12,17. Rituximab is a chimeric antibody directed DISCUSSION against CD20, which is only expressed on rest- ing and activated B cells, not on plasma cells. Over the last 40 years, most clinical and re- With the isolated use of this drug, antibody pro- search emphasis has been on the cellular as- duction by plasma cells is maintained, but a de- pects of allograft rejection. It is only recently that crease in de novo antibody production occurs there has been more emphasis on the antibody- and, by clearing B cells, it also clears B cells mediated injury, due to the development of a new -presenting function. Its role seems li- diagnostic technique, C4d staining4,5. This mited to maintaining a low level of production of showed that AHR is more prevalent than previ- donor antibodies after gross removal has been ously considered6, and that these patients seem attained, suggesting a possible role in prevent- to have worse prognosis, with an increased rate ing further AHR episodes. of early graft loss and an early and increased The triple therapeutic regimen using PMF, IVIG progression to chronic graft dysfunction7-10. and rituximab seems effective in treating the se- Crespo et al1 have shown that the presence vere AHR14. Not only was a significant reduction of C4d correlates very well with the presence of in the anti-donor antibodies levels observed, but anti-donor specific antibodies in the serum. also a complete return to normal of the histo- These anti-HLA antibodies can be rather non logical findings on graft biopsy. specific, being directed mostly against class I1. As in all these three patients, a higher histori- The antibodies are mainly IgG, produced by cal and pretransplant sensitization or a previous plasma cells. These cells represent the goal in failed allograft are known risk factors for AHR, terms of inhibiting antibody production. suggesting that a humoral response against do- Controversy stills reigns over how best to treat nor-specific antigens plays a role in the these patients. Several therapies have been tried, pathogenesis of this type of rejection1. but all with limited success, mainly due to their Although the considered triple regimen inability to inhibit the antibody-producers plasma showed a good effect in the acute severe set- cells1,11-16. The therapies currently available are ting, its long-term effect has to be validated. MMF, IVIG, rituximab, and removal of antibodies Moreover, with such an intensive immunosup- by PMF. pressive regimen, the infectious complications Mycophenolate acts by inhibiting B cell proli- have become the major potential problem. feration, mostly by reducing expression of mo- Recommendations published in 200414 con- lecules responsible for B cell activation and sur- cluded that effective treatment exists to reverse vival. It therefore inhibits de novo immune post transplant AHR. While high IVIG pulses responses, having a negligible effect in terms of (1-2g/kg) or plasmapheresis plus low dose IVIG inhibiting antibodies that already exist. modalities have proven efficacy for antibody re- IVIG is effective in reducing anti-HLA antibo- moval14, they have yet to be tested in rigorous dies in sensitized patients12. It is also effective prospective, multicentre studies. Rituximab or in treating mild AHR12,17. However, it is expen- splenectomy was also reported to increase the sive, has significant side effects, and is non-spe- treatment potency in the more severe cases14.

134 Revista Portuguesa de Nefrologia e Hipertensão TRIPLE REGIMEN WITH RITUXIMAB, PLASMAPHERESIS AND INTRAVENOUS IMMUNOGLOBULIN IN THE TREATMENT OF DIALYSIS DEPENDENT ACUTE HUMORAL-MEDIATED REJECTION IN KIDNEY GRAFTS

In conclusion, although the diagnosis of se- 7. Worthington JE, Martin S, Al-Husseini DM, Dyer PA, Johnson vere AHR requiring dialysis in patients already RW. Posttransplantation production of donor HLA-specific under ATG is usually associated with renal graft antibodies as a predictor of renal transplant outcome. Trans- plantation 2003;75:1034-40 loss, it is possible to obtain complete histologi- 8. Herzenberg AM, Gill JS, Djurdjev O, Magil AB. C4d deposi- cal recovery, and clinical improvement, by em- tion in acute rejection: an independent long-term prognostic ploying triple therapy with PMF, IVIG and factor. J Am Soc Nephrol 2002;13:234-41 Rituximab. Prompt diagnosis and treatment is 9. Gloor JM, DeGoey S, Ploeger N, et al. Persistence of low essential for such a result. levels of alloantibody after desensitization in crossmatch- positive living-donor kidney transplantation. Transplantation 2004;78:221-7 Correspondence and offprint requests to: 10. Regele H, Böhmig G, Habicht A, et al. Capillary deposition of Dr. Pedro Pessegueiro complement split product C4d in renal allografts is associ- Unidade de Nefrologia, Hospital Espírito Santo ated with basement membrane injury in peritubular and Largo Sr. da Pobreza, 7000-811 Évora, Portugal glmerular capillaries: a contribution of humoral to E-mail: [email protected] chronic allograft rejection. J Am Soc Nephrol 2002;13:2371- -80 11. Becker Y, Becker B, Pirsch J, Sollinger H. Rituximab as REFERENCES treatment for refractory kidney . Am J Transplantation 2004;4:996-1001 1. Crespo M, Pascual M, Tolkoff-Rubin N, Mauiyyedi S, Collins 12. Rocha PN, Butterly DW, Greenberg A, Reddan DN, Tuttle- AB, Fitzpatrick D, et al. Acute humoral rejection in renal Newhall J, Collins BH, et al. Beneficial effect of plasmaphe- allograft recipients: I. Incidence, serology and clinical cha- resis and intravenous immunoglobulin on renal allograft sur- racteristics. Transplantation 2001;71:652-8 vival of patients with acute humoral rejection. Transplantation 2. Racusen LC, Colvin RB, Solez K, Mihatsch MJ, Halloran PF, 2003;75:1490-5 Campbell PM, et al. Antibody-mediated rejection criteria - an 13. Zachary A, Montgomery R, Ratner L, et al. Specific and addition to the Banff 97 classification of renal allograft re- durable elimination of antibody to donor HLA antigens in jection. Am J Transplant 2003;3:708-14 renal-transplant patients. Transplantation 2003;76:1519-25 3. Mauiyyedi S, Crespo M, Collins AB, Schneeberger EE, 14. Takemoto S, Zeevi A, Feng S, et al. National conference to Pascual MA, Saidman SL, et al. Acute humoral rejection in assess antibody-mediated rejection in solid organ trans- kidney transplantation: II. Morphology, immunopathology, and plantation. Am J Transplantation 2004;4:1033-41 pathologic classification. J Am Soc Nephrol 2002;13:779- 15. Shah A, Nadasdy T, Arend L, et al. Treatment of C4d-posi- -87 tive acute humoral rejection with plasmapheresis and rabbit 4. Nickeleit V, Zeiler M, Gudat F, Thiel G, Mihatsch MJ. Detec- polyclonal antithymocyte globulin. Transplantation tion of the complement degradation product C4d in renal 2004;77:1399-405 allografts: diagnostic and therapeutic implications. J Am Soc 16. Luke P, Scantlebury V, Jordan M, et al. Reversal of steroid- Nephrol 2002;13:242-51 and anti- antibody-resistant rejection using in- 5. Nickeleit V, Mihatsch MJ. Kidney transplants, antibodies and travenous immunoglobulin (IVIG) in renal transplant reci- rejection: is C4d a magic marker? Nephrol Dial Transplant pients. Transplantation 2001;72:419-22 2003;:2232-9 17. Casadel D, Rial M, Opelz G, et al. A randomized and 6. Mauiyedi S, Pelle PD, Saidman S, et al. Chronic humoral prospective study comparing treatment with high-dose in- rejection: identification of antibody-mediated chronic renal travenous immunoglobulin with monoclonal antibodies for allograft rejection by C4d deposits in peritubular capillaries. rescue of kidney grafts with steroid-resistant rejection. J Am Soc Nephrol 2001;12:574-82 Transplantation 2001;71:53-8

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