Evaluation of Serum sCD30 in Renal Transplantation Patients With and Without Acute Rejection

Evaluation of Serum sCD30 in Renal Transplantation Patients With and Without Acute Rejection

Evaluation of Serum sCD30 in Renal Transplantation Patients With and Without Acute Rejection C. Cervelli, G. Fontecchio, M. Scimitarra, R. Azzarone, A...

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Evaluation of Serum sCD30 in Renal Transplantation Patients With and Without Acute Rejection C. Cervelli, G. Fontecchio, M. Scimitarra, R. Azzarone, A. Famulari, F. Pisani, C. Battistoni, B. Di Iulio, D. Fracassi, M.A. Scarnecchia, and F. Papola ABSTRACT Despite new immunosuppressive approaches, acute rejection episodes (ARE) are still a major cause of early kidney dysfunction with a negative impact on long-term allograft survival. Noninvasive markers able to identify renal ARE earlier than creatinine measurement include sCD30. We sought to establish whether circulating levels of sCD30 in pretransplantation and posttransplantation periods were of clinical relevance to avoid graft damage. Quantitative detection of serum sCD30 was performed using an enzyme-linked immunosorbent assay. Our results demonstrated that the mean concentrations of sCD30 were significantly higher in the sera of renal transplant recipients with ARE (30.04 U/mL) and in uremic patients on the waiting list (37.7 U/mL) compared with healthy controls (HC; 9.44 U/mL), but not nonrejecting patients (12.01 U/mL). Statistical analysis revealed a strong association between high sCD30 levels in posttransplantation sera and ARE risk. This study suggested that sCD30 levels were a reliable predictor of ARE among deceased-donor kidney recipients. KEY factor in renal allograft loss is the incidence of acute rejection episodes (ARE), especially during the first year following transplantation. At present, renal biopsy and serum creatinine levels represent the basic clinical tools for ARE recognition; unfortunately using these techniques, the graft damage is detected too late. For this reason, much effort has been made to identify noninvasive biomarkers that establish an early diagnosis of acute graft rejection.1 sCD30, the soluble form of CD30, released by hydrolytic cleavage, is a 120-kDa transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNF-r) superfamily. It is preferentially localized on CD30⫹ T-cell clones that secrete T helper 2 (Th2)–type cytokines. Greater serum levels of sCD30 have been observed in a variety of pathophysiological conditions, including certain autoimmune diseases, hematopoietic malignancies, viral infections, as well as allosensitization states. Thus, detection of high serum values of sCD30, which are usually low in healthy subjects, reflects increased circulating CD30⫹ cells, thereby indicating immune system activation. A growing amount of evidence suggests that monitoring sCD30 levels in the plasma of patients during kidney pretransplantation and posttransplantation kidney periods may be useful to monitor the immunosuppressive regimen and prevent graft loss.2,3,8

A

MATERIALS AND METHODS Our retrospective study involved a total of 370 subjects divided into the following 4 groups: 49 healthy controls (HC); 185 subjects on the kidney waiting list undergoing dialysis treatment (WL) 32 kidney transplant recipients with rejection (TxR); and 104 kidney transplant recipients without rejection (TxWR). The observation period included the first year after transplantation. The patient demographic details and HLA-A/B/DR mismatching are shown in Table 1. During the period of investigation no patient produced donor-specific antibodies. All 136 recipients (the TxR and TxWR groups) underwent renal transplantation from cadaveric donors, using immunosuppressive therapy based on anti-interleukin (IL)-2R (20 ␮g), Urbason (500 mg), Cyclosporine (100 mg X2), or Tacrolimus (1 mg X2) and mycophenolate mofetil (750 mg X2). The estimates of sCD30 in recipient sera were performed using a quantitative sandwich enzyme immunoassay (sCD30 ELISA, Biotest AG, Dreieich, Germany), according to the manufacturer’s From the Centro Regionale di Immunoematologia e Tipizzazione Tissutale (C.C., G.F., M.S., R.A., C.B., B.D.I., D.F., M.A.S., F.P.), ASL n 4, L’Aquila, and Cattedra di Chirurgia Sostitutiva e dei Trapianti d’organo dell’Università degli Studi di L’Aquila (A.F., F.P.), Italy. Address reprint requests to Franco Papola, Centro Regionale di Immunoematologia e Tipizzazione Tissutale, ASL n 4, L’Aquila, Ospedale San Salvatore, 67010 Coppito, L’Aquila, Italy. E-mail: [email protected]

© 2009 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.03.077

Transplantation Proceedings, 41, 1159 –1161 (2009)

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CERVELLI, FONTECCHIO, SCIMITARRA ET AL Table 1. Demographics of the Studied Groups

Male No. Age (mean) Female No. Age (mean) HLA mismatching 0 1 2 3 ⬎3

Male No. Age (mean) Female No. Age (mean)

TxR (%)

TxWR (%)

22 (68.7) 46

55 (52.9) 48

10 (31.3) 48

49 (47.1) 49

1 (3.2) 3 (9.4) 5 (15.6) 10 (31.2) 13 (40.6)

0 (0.0) 5 (4.8) 26 (25.0) 34 (32.7) 39 (37.5)

HC (%)

WL (%)

33 (67.3) 49

102 (55.1) 45

16 (32.7) 45

83 (44.9) 50

instructions. A total of 70 ␮L of each serum sample was added to a microtitre plate that had been precoated with mouse anti-human sCD30. The captured sCD30 molecules were then revealed after binding to a HRP-conjugated anti-human sCD30 mAb with a colorimetric reaction using tetramethyl-benzidine (TMB). The reaction was stopped by the addition of phosphoric acid, and OD values were read at 450 nm. The sCD30 concentrations were calculated from a standard curve (OD vs concentration) established for every run using 7 human sCD30 standard dilutions (range, 1.6 –100 U/mL). All samples were run in duplicate. Fisher exact test, ␹2 test, and two-taled Student t test were used for the statistical analyses to compare sCD30 levels between patient groups. The online software Vassar Stats was used for the statistical calculations. The sCD30 cut-off value, represented by sCD30 mean concentration ⫾ 2 SD, was calculated using HC sera, which were chosen with a similar age distribution to WL and the kidney recipients. Statistical significance was defined at P ⬍ .05.

RESULTS

The sCD30 mean concentration ⫾ 2 SD established as the cut-off value from HC sera samples was 22.0 U/mL. The mean value of sCD30 concentrations in the HC group (9.44 U/mL) were lower than the cut-off value and similar to that of the TxWR (12.01 U/mL). Higher values, on the other hand, were observed for the TxR (30.04 U/mL) and WL groups (37.7 U/mL). Student t test indicated statistically significant differences in sCD30 levels between TxR and HC groups (t ⫽ 4.01; P ⫽ 10⫺4), between TxR and TxWR groups (t ⫽ 4.42; P ⬍ 10⫺4), between HC and WL groups (t ⫽ 5.27; P ⫽ 10⫺4), and, finally, between TxWR and WL groups (t ⫽ 10; P ⬍ 10⫺4). In contrast, no significant correlation was observed between HC and TxWR group (t ⫽ 1.29; P ⫽ not significant [NS]) or between TxR group and HC (t ⫽ 1.59; P ⫽ NS). Fisher exact test revealed elevated values of ␹2 and odds ration (OR; ␹2 range,

10.7– 48.3; OR range, 4.22–26.38, P range, 7 ⫻ 10⫺4– 6.0 ⫻ 10⫺14) among all groups except for TxWR and HC (␹2 ⫽ 4.5; OR ⫽ 4.11; P ⫽ .01) as well as for TxR and WL patients (␹2 ⫽ 0.8; OR ⫽ 0.65; P ⫽ NS; Table 2). The TxR who received bolus steroid therapy went into complete remission. DISCUSSION

Recent reports have proposed sCD30 as a noninvasive serological marker to predict immunological risk and graft failure among kidney transplant recipients. Our study showed that serum levels of sCD30 measured in TxR patients were significantly higher than those in HC and TxWR patients. Moreover, sCD30 values in pretransplantation sera samples of WL subjects were similar to those observed in TxR patients. This finding agrees with that of other authors who reported increased amounts of sCD30 before renal transplantation among patients undergoing dialysis treatments.4 To date, the panel-reactive antibody (PRA) test for donor-specific human leukocyte antigen (HLA) antibody detection is the routine method to identify WL or transplant recipients at risk of ARE. However, Langan et al5 observed that PRA positivity and elevated sCD30 levels behave as additional as well as independent factors for ARE risk and subsequent graft failure. Because all patients involved in this investigation did not develop anti-HLA antibodies, our finding only confirmed that increased plasma sCD30 indicated development of allograft rejection. Furthermore, these results underlined the probability of ARE when sCD30 levels remained high even after administration of immunosuppressive drugs to kidney recipients; vice versa, the ARE rate was considerably reduced following a decrease in sCD30 due to immunosuppression. Some studies have indicated that sCD30 plasma concentrations in humans are characterized by variability over time depending on genetic background, hormone production, hemodialysis, and immunosuppressive therapy.6,7 This variability can determine different individual responses to the same combination of immunosuppressants. On the basis of this observation, we hypothesized that our immunosuppressive regimen provided adequate depression of the immune system in TxWR patients, whereas the same therapy did not induce a down-regulation of the Th2-cytokine pathway among rejecting patients with high sCD30 levels. We believe that the early prediction of ARE using sCD30 measurements allows pretransplantation and posttransplantaTable 2. Fisher Exact Test Results for Statistical Significance of sCD30 Values in the 4 Patient Populations Examined

WL (185)/HC (49) TxWR (104)/HC (49) TxR (32)/HC (49) TxWR (104)/WL (185) TxR (32)/WL (185) TxR (32)/TxWR (104)

␹2

OR

P

48.3 4.5 20.5 45.6 0.8 10.7

26.38 4.11 17.37 0.15 0.65 4.22

6.0 ⫻ 10⫺14 .01 2.0 ⫻ 10⫺6 2.5 ⫻ 10⫺12 NS 7 ⫻ 10⫺4

EVALUATION OF SERUM SCD30

tion patients with enhanced levels of this marker to be treated with either stronger or individually adjusted immunosuppressive therapy, thus avoiding a poor kidney graft survival. Our data highlighted the feasibility of pretransplantation and posttransplantation sCD30 monitoring in the serum of cadaveric kidney recipients to predict ARE, the occurrence of which hampers long-term allograft survival.

REFERENCES 1. Gwinner W: Renal transplant rejection markers. World J Urol 25:445, 2007 2. Ayed K, Abdallah TB, Bardi R, et al: Plasma levels of soluble CD30 in kidney graft recipients as predictors of acute allograft rejection. Transplant Proc 38:200, 2006

1161 3. Schlaf G, Altermann WW, Rothhoff A, et al: Soluble CD30 serum level – an adequate marker for allograft of solid organs? Histol Histopathol 22:1269, 2007 4. Süsal C, Pelzl S, Döhler B, et al: Identification of highly responsive kidney transplant recipients using pretransplant soluble CD30. J Am Soc Nephrol 13:1650, 2002 5. Lagan LL, Park LP, Hughes TL, et al: Post-transplant HLA class II antibodies and high soluble CD30 levels are independently associated with poor kidney graft survival. Am J Transplant 7:847, 2007 6. Frisaldi E, Conca R, Magistroni P: Prognostic values of soluble CD30 and CD30 gene polymorphisms in heart transplantation. Transplantation 81:1153, 2006 7. Altermann W, Schlaf G, Rothhoff A, et al: High variation of individual soluble serum CD30 levels of pre-transplantation patients:sCD30 a feasible marker for prediction of kidney allograft rejection? Nephrol Dial Transplant 22:2795, 2007 8. Vaidya S, Partlow D, Barnes T, et al: Soluble CD30 concentrations in ESRD patients with and without panel reactive HLA antibodies. Clin Transplant 20:461, 2006