INTRODUCTION

Avoidance or early cessation of steroids in kidney transplantation is supported by recent guidelines, however later cessation of steroids was not supported recently.¹ Steroid-free regimens have been tried to avoid steroids’ adverse effects such as diabetes mellitus, hypertension, hyperlipidemia, avascular necrosis and osteopenia; nevertheless, an increase is observed in acute rejection rates.^2-3 On the first few days of posttransplantation, acute rejection incidence was found lower in steroid-free group, on the other hand there are many restrictions due to design of the clinical trials.^4-5 In renal transplantation, acute rejection incidence is higher in those steroid-free immunosuppressive regimens despite the improvement of steroid- free therapies.

In addition, long-term graft survival is not known in patients with acute rejection in a steroid-free immunosuppressive regimen. ⁶ Our knowledge for whether a steroid-free immunosuppressive regimen will become the first line therapy is insufficient. That is why, only selected population can be preferred for this.⁷ In an updated metaanalysis, discontinuation of the steroid after kidney transplantation significantly increased the risk of acute rejection, there was no difference in patient mortality or graft loss up to five years after transplantation, so prospective long-term studies are recommended.⁸

In chronic graft dysfunction, contribution of antibody mediated rejection (AMR) is highly accepted. AMR is triggered by humoral immunity that is mediated by several antibodies, especially donor specific HLAs. These antibodies cause serious problems in renal transplantation and donor specific antibodies (DSA) that occur after renal engraftment cause acute rejection.⁹⁾ Several clinical trials showed that the presence of DSAs are related with poor graft function. ^10-12 Our aim in this study is to emphasize the importance of preventing unnecessary immunosuppression with immunological monitoring. At the same time, it is to increase the graft survival by intervening early in the treatment in the patient who needs it.

METHODS

We evaluated PRA and DSA levels of adult individuals with functioning grafts (25 ml/ min/1,73 m² or more) in our transplantation unit that has been working since 1994. Individuals with inadequate information were excluded. We included 82 steroid free individuals and 52 steroid user individuals. By the way, we noted individuals’ primary kidney diseases, dialysis type, donor type, duration of transplantation, comorbidities such as diabetes mellitus (DM), hypertension (HT) or cardiovascular disease (CVD), DGF ratios, induction therapy, duration of steroid use, cessation of steroid and its etiology. Pretransplant PRA and posttransplant PRA and DSA levels which had been used for immunological monitorization were also noted with pre and posttransplant current graft function. Steroid cessation time, etiology of avoidance and duration of steroid use before cessation were also noted in steroid free group. Acute rejection or BK virus nephropathy history were noted if present, in both groups.

SSO method

Sequence-specific oligonucleotides method was performed according to the manufacturer’s instructions (Lifecodes HLA SSO Typing Kit Immucor, USA). For the first amplification step, 16 pl of mix containing master mix, H₂O, and Taq polymerase was added on four microliters of DNA (15-200 ng) in an Eppendorf tube (200 pl). The total volume of 20 pl samples was placed in the thermal cycle and the program was run. For the second hybridization step, the probe mix was warmed at 56° C for seven minutes. The probe mix was sonicated and vortexed before use. Then, 15 pl of probe mix was added on five microliters of amplicon in 96 well plates, and the samples were placed in the thermal cycler and the hybridization program was run for 20 minutes. During this run, the Luminex fluoro analyzer instrument was prepared for the analysis. When the hybridization program ended at 56° C, 170 pl diluted Streptavidin were added on the samples in the wells, and the wells were placed in the Luminex instrument. The results were analyzed by MatchIt Software Program.

Panel reactive antibody method

Lifecodes LifeScreen Class I and II ID Kits (Immucorgamma, USA) were used for Class I and Class II identification, respectively. After the 96 well plates were moisturized, wash buffer, patient/control sera and HLA Class I or II ID beads were added into the wells. The plate was incubated at room temperature for 30 minutes in the dark. After incubation the wells were washed with 200 pl buffer for three times.

Then, conjugate was prepared in appropriate concentration and added into the wells. After incubation at room temperature for 30 minutes in the dark, 150 pl wash buffers were added into the wells. The plate was gently mixed in the Luminex Fluoroanalyzer instrument, and the results were analyzed by MatchIt Software program.

Statistical analysis

Statistical analyzes were performed using IBM" SPSS" 25 (NY, USA) software. The suitability of variables to normal distribution is examined using analytical methods (Kolmogorov-Smirnov / Shapiro-Wilk tests). Descriptive statistics were done by giving the mean ± standard deviation, median and IQR, minimum-maximum value. In categorical variables, frequency and percentage values were given and Pearson’s or Fisher’s Exact Chi Square test were used for comparison of categorical variables. In comparison of independent groups between continuous variables, t-test was used for variables that conform to normal distribution, and Mann-Whitney U test was used for non- normal distribution. In comparison of more than two groups, Kruskal-Wallis test was used and after post hoc

Bonferroni correction was used. p<0.05 was considered significant.

Table 1: Clinical, biochemical and immunological analysis features of the study groups. 

RESULTS

Age, sex, number of transplantation, mismatch, current GFR, acute rejection, posttransplant malignancy, HT presence were similar in both groups. Clinical, biochemical and immunological analysis features of the study groups were shown in Table 1 . Posttransplant creatinine (mg/dl), actual creatinine (mg/dl), posttransplant eGFR (ml/min per/1.73 m²), posttransplant spot urine protein/creatinine, actual spot urine protein/creatinine, DM, BKVN presence and induction therapy were significantly different. Immunological analysis showed no marked difference.

Steroid free individuals’ initial therapy was CNI based regimen and 61% of individuals received induction therapy. After cessation of steroid, most patients had continued CNI based regimen. 95% of individuals under steroid treatment were stopped after one year of transplantation. Etiology of avoidance was not specified but most common causes were osteoporosis, avascular necrosis, uncontrolled DM and HT.

Transplant time and steroid use duration were compared with other parameters in Table 2. Transplant time and steroid use duration were significantly different in those who had received induction therapy. It was observed that those who received induction therapy had shorter transplant and steroid use times. Posttransplant PRA I positivity was found to be more significant for those with shorter steroid use duration

Table 2: Tx time, steroid usage time compare with other parameters. 

DISCUSSION

DSAs that occur against renal graft causes antibody mediated rejection (AMR) which is a reason for graft dysfunction.¹³ It has become essential to detect antibodies. Solid-phase immunity analysis, especially LUMINEX®, is more sensitive than previous complement dependent lymphocytotoxicity (CDC) analysis, that is why it is recommended for high risk patients.¹⁴ We monitorized these parameters periodically and if needed, we perform pathological analysis for direct treatment.

Clinicians should be careful in case of development of AMR as it once happens, no efficient treatment is available. Posttransplant DSA occurrence risk is determined by the intensity of immunosuppressive therapy and patients’ compliance. Thus, immunological tests may be helpful in the follow-up for those patients who have quitted steroid therapy.

Our experiment revealed that spot urine/ creatinine ratio and serum creatinine levels are lower in contrast to higher eGFR. This may be related to the selection of patients with low immunological risk. This also may be the sign that we defined patients’ risk accurately.

Patients’ history of DM, occurence of new onset diabetes mellitus after transplantation (NODAT) or poorly controlled DM under steroid therapy might cause switching therapy to steroid-free regimens. A clinical trial reported that switching therapy to steroid-free regimen was related to lower risk of NODAT occurrence in 3 years of follow- up.¹⁵ This is possible if there is an early cessation of steroid.

In our work, we found that the suspension of steroids was done too late. Also the fact that BKVN was detected more in this group may be the reason why the clinicians decided to use reduced doses of immunosuppressive agents. The high induction therapy rates of the steroid-free group may have led the clinician to cease steroids more comfortably. The fact that those with less steroid use in the steroid-free group are among those who received more induction therapy supports this view. In a clinical trial it was reported that duration of steroid use is shorter in the group who had taken effective induction therapy, and this was related to lower steroid side effects and better graft function.¹⁶

This study is a retrospective study and tried to compare a steroid-free group with a control group.

The steroid was discontinued for two reasons. In the first place, when a steroid related side effect was present and second, when the patient had poor kidney function. Therefore, the results we have obtained may seem in favor of steroids. For this reason, it is stated that following this study in the long term, conducting a randomised controlled trial will give much better results.

This might be a warning for clinicians that posttransplant PRA I levels are higher in the group that used steroids for a shorter time, but it must be supported by other parameters and more clinical trials. In a clinical trial it was reported that steroid might be quitted early and showed that it could potentially be useful in elderly patients as well as sensitized recipients with PRA <60%, regardless of the degree of HLA sensitivity. However, it seems to be beneficial to continue steroids in young and highly sensitized patients.¹⁷

In this study, choice of the steroid-free regimen was determined by the clinician according to each patient’s condition, and the patients’ discontinuation was one year after the transplant. Despite this, acute rejection rates were not found to be higher in contrast to literature.

Complement based DSAs/ IgG subgroups might be more useful in immunological monitorization.^18,19 Immunologic responses are important factors for renal transplantation and anti-HLA antibodies may affect long term graft function.²⁰ That’s why more innovative approaches should be performed to prevent critical sensitization, occurence of anti-HLA antibodies, posttransplant non immunologic complications and optimal treatment of chronic active ABMR.²¹

CONCLUSIONS

Randomized controlled trials are needed to assess the reliability of steroid-free regimens. Until then, it would be safer to choose steroid-free regimens only in a selected group of patients.