Influence of pretransplant dialysis modality on the change of lymphocyte subset populations and acute rejection rates after renal transplantation
Shigeru Satoh md, Department of Urology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. Email: firstname.lastname@example.org
Background: The influence of dialysis modality on the acute rejection (AR) rate after renal transplantation is controversial. We investigated whether the pretransplant dialysis modality correlated with the lymphocyte subset populations and the incidence of AR after renal transplantation.
Methods: Thirty-eight first living renal transplant recipients, consisting of 22 patients on pretransplant hemodialysis (HD) and 16 patients on pretransplant peritoneal dialysis (PD), were studied. Peripheral blood samples were taken on days -1 through 28 after transplantation, and the lymphocyte fractions were exposed to the monoclonal antibodies anti-CD3, CD19, CD4, CD8 and CD28 for a flow cytometer analysis. Biopsy specimens were obtained at the time of presumed AR episodes and on day 28 after transplantation.
Results: The PD patients had a higher frequency of AR (37.5% in PD vs 9.1% in HD patients, P = 0.034). In contrast, two HD patients showed graft loss at 18 and 30 months after transplantation. The increases of CD3, CD19, CD4 and CD4+ CD28+ cells in the PD patients occurred earlier than in the HD patients and the numbers of these cells in the PD group were higher than those in the HD between days 3–28 after transplantation, most significantly on day 7.
Conclusions: These findings suggest that the PD patients with similar clinical characteristics could potentially have a higher immunocompetence and immune responsiveness associated with a higher rate of AR in the early stage of renal transplantation when compared with the HD patients.
Cell-mediated immunodeficiency appears in an early stage of end-stage renal disease (ESRD) and is associated with a higher incidence of infections when compared with the normal population.1,2 Hemodialysis (HD) has no effect on improving the affected cellular immunity,1 while patients on peritoneal dialysis (PD) are recognized to have a better immune responsiveness than those on HD.2–4 The risk of infectious complications after surgical trauma is generally considered to be higher in HD patients than in PD patients.5
Consequently, the immunodeficiency of HD patients, compared with PD patients, may influence the acute rejection (AR) rate after transplantation. But the effect of pretransplant dialysis modality on the incidence of AR episodes and graft survival rates is controversial.6–14 Most studies reported no significant differences in the incidence of AR episodes and graft survival after renal transplantation between HD and PD patients.6–11 On the other hand, in 1984, Guillou et al. reported that the 1-year graft survival rate of HD patients was higher than that of PD patients.12 Two other studies reported a greater incidence of AR in PD patients after cadaveric renal transplantation, but found no significant difference in graft function.13,14 In these studies, however, the reasons why the AR rate was higher in PD patients were not fully discussed.
The monitoring of the circulating lymphocyte population is a useful measure for evaluating the immunodeficient and immunosuppressive states. In patients with ESRD, the immune state is characterized by a decrease in circulating T cells.15 In this respect, the monitoring of the peripheral lymphocyte subset populations may be useful to clarify the relation between the peri-transplant immunodeficiency by the pretransplant dialysis modality and the clinicopathological events after renal transplantation.
In the present study, we monitored the numbers of peripheral white blood cells (WBC), lymphocytes and lymphocyte subsets, including CD4+ CD28+ T cells, which activate the costimulatory signal of T cells,16,17 in the renal transplant recipients, referring to the pretransplant dialysis modality, the incidence of AR episodes and the pathological findings of allografts.
Patient selection and study design
The present study was carried out in 38 renal transplant recipients (17 men, 21 women) who received dialysis treatment for at least 6 months. The mean age of the recipients at the time of transplantation was 40 years with a range of 8–66 years, the mean dialysis period was 51 months with a range of 6–123 months and the mean post-transplant follow-up period was 23 months with a range of 4–49 months. The criteria for enrollment in the present study were; (i) having pretransplant dialysis for at least 6 months; (ii) first living donor transplantation; (iii) not ABO incompatible; (iv) same tacrolimus-based immunosuppressive regimen; (v) no evidence of delayed graft function or opportunistic infection during the study period; and (vi) patients who gave informed consent for participation in the study. The number of peripheral lymphocyte subsets in the healthy subjects was studied in the pretransplant 38 living donors (21 men, 17 women) with a mean age of 56 years ranging from 33–74 years.
The recipients were classified into two groups according to the method of dialysis before transplantation. Twenty-two patients who received HD alone (n = 21) or HD with a preceding short-term PD (n = 1) were defined as HD patients. Sixteen patients who received PD alone (n = 15) or PD with preceding short-term HD (n = 1) were defined as PD patients. Blood samples for the analysis of the WBC and peripheral lymphocyte population were taken on days −1, 1, 3, 7, 14 and 28 after transplantation. Histological specimens were obtained by an allograft core biopsy at the time of transplantation, presumed AR episodes and on day 28 after transplantation under an echographic guide.
All renal transplant recipients received immunosuppressive therapy according to our protocol as previously reported.18 An initial oral dose of 0.15 mg/kg tacrolimus was administered twice per day for 2 days prior to operation. A 24-h continuous intravenous infusion of 0.05 mg/kg/day tacrolimus was administered for 6 days after the operation. At 09.00 hours on the 6th postoperative day, the intravenous administration was discontinued and the same dose as an initial oral administration was administered. The daily dose was adjusted according to the clinical state of the patients, but the whole blood trough target level was maintained at 18–20 ng/mL for 7 days after transplantation and was followed by 14–16 ng/mL for 2 weeks, by 10–12 ng/mL for 4 weeks and by less than 10 ng/mL thereafter. Methylprednisolone (MP) was administered concomitantly: the dose of 500 mg on the operation day was tapered to 40 mg/day during the first week, 30 mg/day of prednisolone (PSL) in the second week, 20 mg/day of PSL in the third week, 15 mg/day in the fourth week and 10 mg/day thereafter. A dose of 1000 mg of mycopherolate mofetil twice per day was also given, starting two days before the operation.
Pathological diagnosis and treatment of acute rejection episodes
Allograft biopsy specimens were fixed in 10% neutral buffered formalin. Sections were stained with hematoxylin-eosin and periodic acid-Schiff's reagent to evaluate the histological rejection according to the Banff classification.19 Patients with presumed AR episodes underwent renal biopsy within 48 h of the initiation of antirejection treatments. Acute rejection crises were treated with three intravenous 500 mg MP administrations. Steroid-resistant rejection was experienced in none of the recipients.
Flow cytometric analysis
All heparinized blood samples remained at room temperature until they were prepared within 2 h after collection. Mononuclear cells were isolated from the peripheral blood by density gradient centrifugation. The cells were exposed to the monoclonal antibodies (mABs) conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). The mABs used were anti-CD3, CD19, CD4, CD8 and CD28. Immunofluorescence staining was performed after washing the cells twice with phosphate-buffered saline plus 0.5% human serum albumin. Cells were incubated for 20 min at 4°C with each mAB (5 mg/mL, 105 cells per test). After washing three times with a FITC and PE conjugated second-step mAb for 20 min at 4°C, a flow cytometer analyzer (FACS-calibur, CELL Quest software; Becton Dickinson, Mountain View, CA) was used for the single- or two-color cell analysis.
Results were expressed as means ± standard error. Group differences for qualitative variables were performed by χ2 analysis. Comparisons of the data between the HD and PD patients and donors were performed by post-hoc test, repeated measure anova or unpaired Student's t-test, where appropriate. Statistical significance was defined as a P-value < 0.05.
Patient characteristics and clinical outcome
Overall, the two groups of dialysis were balanced with regard to the baseline demographic and clinical characteristics (Table 1). No significant differences in the recipient gender, age, dialysis duration, donor gender or donor age were observed between the two groups. The percentage of living unrelated donor transplantations and the average number of human lymphocyte antigen mismatches in the HD patients appeared to be higher than those in the PD patients, but there were no significant differences. The causes of ESRD were chronic glomerulonephritis including IgA nephropathy in 25 patients (15 in HD, 10 in PD), Alport syndrome in 3 patients (1 in HD, 2 in PD), reflux nephropathy in 2 patients (1 in HD, 1 in PD), pregnacy toxicosis in 2 patients (1 in HD, 1 in PD), nephrosclerosis in 2 patients (1 in HD, 1 in PD), diabetes mellitus in 1 patient (HD), systemic lupus erythematodes in 1 patient (HD), Wegener's granuromatosis in 1 patient (PD) and was unknown in 1 patient (HD).
Table 1. Characteristics of the study groups according to the dialysis methods and the percentage of patients with acute rejection episodes, pathological borderline changes and graft loss
|Number of patients||22||16|| |
|Gender (% male)||50.0||37.5||0.442|
|Age (years)||41.7 ± 2.8||36.4 ± 3.5||0.242|
|Dialysis duration (months)||49.5 ± 8.3||54.1 ± 13.2||0.762|
|Donor gender (% male)||63.6||43.8||0.224|
|Donor age (years)||57.3 ± 1.7||55.1 ± 2.6||0.475|
|Living unrelated (% patients)||18.2|| 6.3||0.287|
|HLA mismatch number|| 2.5 ± 1.6|| 1.8 ± 0.9||0.102|
|Acute rejection (% patients)|| 9.1||37.5||0.034|
|Border line changes (% patients)||36.4||50.0||0.401|
|Graft loss (% patients)|| 9.1|| 0.0||0.215|
Eight of the 38 recipients (21.1%) experienced steroid-dependent clinical AR episodes in the early stage of the post-transplant state. These included six PD patients, in whom AR occurred in the second through to the fourth week after transplantation, and two HD patients, in whom AR occurred in the fourth through the sixth week. Compared with the HD patients, the PD patients showed a significantly higher frequency of AR (9.1% in HD patients vs 37.5% in PD patients, P = 0.034) and an earlier onset of AR. Episode biopsy specimens in these eight patients showed borderline (BL) or type Ia changes according to the Banff classification. Protocol biopsy specimens on day 28 after transplantation showed BL changes in 16 (42.1%) of the 38 recipients, including the eight patients with AR episodes. The frequency of BL changes was not significantly different between the HD and PD patients (50.0%vs 36.4%, P = 0.401). Two HD patients had graft losses due to chronic rejection at 18 and 30 months after transplantation during the follow-up period.
Peripheral lymphocyte subsets of the hemodialysis and peritoneal dialysis patients and donors
As shown in Table 2, the WBC count of PD patients in the preoperative state was higher than that of HD patients and donors. The number of lymphocytes, CD19, CD4 and CD4+ CD28+ cells were significantly lower in the HD patients than the donors.
Table 2. Absolute number of WBC, lymphocytes and lymphocyte subsets of 22 hemodialysis patients, 16 peritonieal dialysis patients and 38 donors in the pre-transplant state
|WBC||4855 ± 401*||6238 ± 521**||5055 ± 185|
|Lym||1184 ± 107**||1278 ± 108**||1680 ± 89|
|CD3|| 858 ± 82|| 885 ± 88||1003 ± 55|
|CD19|| 118 ± 18*,**|| 180 ± 52|| 250 ± 17|
|CD4|| 499 ± 46**|| 538 ± 54|| 631 ± 32|
|CD8|| 403 ± 42|| 371 ± 35|| 459 ± 33|
|4/8|| 1.39 ± 0.10|| 1.48 ± 0.11|| 1.52 ± 0.09|
|4+ 28+|| 447 ± 44**|| 576 ± 68|| 678 ± 38|
As shown in Table 3, the WBC counts in the HD and PD patients in the peri-operative state increased immediately after transplantation and reverted to the baseline level on day 28 in the PD patients, but not in the HD patients. In contrast, the numbers of lymphocytes, CD3, CD4 and CD4+ CD28+ T cells decreased immediately after the operation and continuously increased between days 3–28 in the PD patients. However, the numbers of CD4 and CD4+ CD28+ cells in the HD patients only increased significantly on day 28. The PD patients showed an earlier increase of lymphocyte subset population after transplantation.
Table 3. Absolute number of WBC, lymphocytes and lymphocyte subsets of 22 hemodialysis and 16 peritoneal dialysis patients in the peri-operative period
| HD||4855 ± 401*||10 477 ± 861**|| 9718 ± 631**||8384 ± 606**||7886 ± 494**||6027 ± 485**|
| PD||6238 ± 521||12 131 ± 814**||10 225 ± 877**||9631 ± 776**||9575 ± 796**||7238 ± 708|
| HD||1184 ± 107|| 758 ± 109***|| 1175 ± 144||1396 ± 141||1665 ± 189||2000 ± 251**|
| PD||1278 ± 108|| 820 ± 88***|| 1493 ± 181||1918 ± 317**||1886 ± 237**||1795 ± 222**|
| HD|| 858 ± 82|| 538 ± 88***|| 856 ± 108|| 932 ± 107*||1112 ± 124||1234 ± 164|
| PD|| 885 ± 88|| 492 ± 59***|| 985 ± 128||1268 ± 206||1353 ± 177**||1360 ± 173**|
| HD|| 118 ± 18*|| 113 ± 17*|| 191 ± 27**|| 230 ± 32*,**|| 264 ± 44**|| 266 ± 45**|
| PD|| 180 ± 52|| 191 ± 29|| 380 ± 71**|| 465 ± 110**|| 451 ± 99**|| 356 ± 62|
| HD|| 499 46|| 302 ± 43***|| 489 ± 57|| 589 ± 59*|| 722 ± 80|| 834 ± 103**|
| PD|| 538 ± 54|| 280 ± 39***|| 619 ± 88|| 796 ± 118**|| 842 ± 100**|| 826 ± 94**|
| HD|| 403 ± 42|| 258 ± 49***|| 348 ± 56|| 403 ± 53|| 410 ± 48|| 499 ± 75|
| PD|| 371 ± 35|| 234 ± 30***|| 369 ± 48|| 495 ± 96|| 472 ± 75|| 513 ± 75**|
| HD|| 1.39 ± 0.10|| 1.18 ± 0.09***|| 1.69 ± 0.14|| 1.73 ± 0.15**|| 2.01 ± 0.18**|| 1.80 ± 0.14**|
| PD|| 1.48 ± 0.11|| 1.31 ± 0.17|| 1.82 ± 0.23|| 1.85 ± 0.21**|| 2.07 ± 0.22**|| 1.91 ± 0.19**|
| HD|| 447 ± 44|| 272 ± 46***|| 460 ± 54*|| 601 ± 62*|| 684 ± 85|| 744 ± 115**|
| PD|| 576 ± 68|| 257 ± 38***|| 659 ± 88**|| 830 ± 129**|| 840 ± 97**|| 911 ± 101**|
In comparison between HD and PD patients, the numbers of CD3, CD19, CD4 and CD4+ CD28+ cells were significantly higher in the PD patients than in the HD patients on day 7. On days 14 and 28, the numbers of these cells were still higher in the PD patients than in the HD patients, however, there were no significant differences due to the late occurrence of an increase in the numbers of these cells in the HD patients.
End-stage renal disease induces a clinical state of immunodeficiency.1,2,20 A decrease in relative or absolute lymphocyte numbers was observed in the peripheral blood from uremic, HD and PD patients compared with healthy controls.15 In the pretransplant state of the present study, the lymphocyte numbers of HD and PD patients were lower than those in the donors. The numbers of CD19, CD4 and CD4+ CD28+ cells in HD patients, but not in PD patients, were also significantly lower than those in the donors. Vanholder et al. suggested that PD patients have high immunocompetence immediately before and after renal transplantation.14 The influence of pretransplant dialysis on the AR rate early after kidney transplantation should be considered, because there would be potential differences in immune function between each pretransplant dialysis modality.4,21
The changes of lymphocyte subsets have been shown to correlate with the clinical events following liver and renal transplantation.22 An increase in CD3 and CD4 T cell counts was documented in acute cellular rejection, but the change of the B lymphocyte (CD19) population after transplantation is controversial.23,24 Furthermore, the CD4+ CD28+ T cell is a mediator for the costimulatory signal on T cells.25 The CD4CD28 expression significantly increased in AR recipients, whereas the CD4CD28 expression was significantly down-regulated in the non-acute rejection recipients.16 The CD28 signaling pathway activates CD3 and CD4 T cells and cellular interactions mediated by CD28 may represent an important component of the functional interactions between T and B lymphocytes.25 In the present study, the numbers of CD3, CD4, CD4+ CD28+ and CD19 cells in PD patients were higher than those in HD patients between days 3–28, significantly on day 7, after transplantation. The results suggest that PD patients have a higher immunocompetence mediated by both the T and B lymphocytes and the T-cell costimulatory pathway pre- and immediately post-transplant states. A different primary immunosuppressive strategy between PD and HD patients might be considered in the early stage of renal transplantation in the near future.
The onset of AR in the PD patients in whom AR occurred was earlier than that in the HD patients in whom AR occurred; between 2 and 4 weeks after transplantation in PD patients and between 4 and 6 weeks in HD patients. The onset of significantly increasing numbers of CD4 and CD4+ CD28+ cells compared with the baseline levels in PD patients occurred between days 3–7 after transplantation. However, this onset in HD patients occurred only on day 28. These results suggest that the patients on PD have a better immune responsiveness after transplantation than those on HD, leading to the early onsets of AR. Furthermore, several studies have suggested that the changes of peripheral lymphocyte subsets have previously been related to clinical events, before clinical signs of rejection, and are a predictive marker for the later occurrence of AR episode.16,22–24 Based on these studies, the onsets of increasing numbers of peripheral lymphocyte subsets may be predictive for the later occurrence of clinical AR episodes in the present study.
Many factors, including tacrolimus trough levels, may influence the occurrence of AR. The two groups in the present study showed a good balance in regard to clinical characteristics and no difference in tacrolimus pharmacokinetics, not only trough levels, but also for other parameters (data not shown).
In conclusion, the present preliminary study suggested that PD patients with similar clinical characteristics could potentially be affected by an enhanced immune responsiveness, compared with HD patients, that appears to be associated with a higher rate of AR episodes in the early stage of renal transplantation. However, the pretransplant dialysis modality may not influence the later graft survival, because of the graft loss in two HD patients in the current study. Since our findings came from a small number of patients, a large-scale study is needed to confirm the influence of pretransplantation dialysis modality on post-transplantation immune response and to establish a different primary immunosuppressive strategy between HD and PD patients.