Combined Impact of Pre-sensitization and Delayed Graft Function on Allograft Rejection in Deceased Donor Kidney Transplantation: Nationwide Cohort Study

The aim of this study is to investigate whether or not delayed graft function (DGF) and pre-transplant sensitization has a synergistic adverse effect on allograft outcome after deceased donor kidney transplantation (DDKT) using the Korean Organ Transplantation Registry (KOTRY) database, the nationwide prospective cohort. The study included 1,370 cases between May 2014 and June 2019. The cases were divided into 4 subgroups according to pre-sensitization and the development of DGF post-transplant (non-pre-sensitized-DGF(- )(n=1100), non-pre-sensitized-DGF(+)(n=133), pre-sensitized-DGF(-)(n=116), and pre-sensitized-DGF(+)(n=21)). We compared the incidence of biopsy-proven allograft rejection (BPAR), time-related change in allograft function, allograft or patient survival, and post-transplant complications across 4 subgroups. The incidence of overall BPAR and acute antibody-mediated rejection (ABMR) was significantly higher in the pre-sensitized-DGF(+) subgroup than in other 3 subgroups. In addition, multivariable cox regression analysis demonstrated that pre-sensitization combined with DGF is an independent risk factor for both overall BPAR (hazard ratio 3.253, p = 0.005) and acute ABMR (hazard ratio 7.589, p < 0.001). Moreover, DGF and pre-sensitization showed significant interaction. Pre-sensitization combined with DGF did not show significant impact on allograft function, and allograft or patient survival. In conclusion, pre-sensitization and DGF had a synergistic adverse impact on allograft rejection after DDKT.


Introduction
Delayed graft function (DGF) is a manifestation of acute kidney injury (AKI), which is more prevalent in deceased donor kidney transplantation (DDKT).The definition of DGF varies according to the study; however, it is mostly based on the use of dialysis within 1 week from transplant [1][2][3] .The mechanism underlying the development of DGF still needs to be unveiled, but it is suggested that post-ischemic acute tubular necrosis resulting from ischemia and reperfusion injury (IRI) developing during deceased donor management or recovery of organs, and calcineurin inhibitor (CNI) toxicity may be the major contributors 4 .The activation of adaptive immune system induced by DGF also increases the risk of allograft rejection.
Meanwhile, it is well known that the presence of preexisting donor-specific anti-human leukocyte antigen antibody (HLA-DSA), so-called "pre-sensitized state" is an important obstacle preventing successful kidney transplantation (KT) [5][6][7][8][9] .In such patients, HLA-DSA can increase the risk of acute or chronic antibody-mediated rejection (ABMR) resulting in worse allograft outcomes 10,11 .In the setting of DDKT, DGF combined with subclinical rejection resulted in far worse allograft outcomes.In addition, the detrimental impact of DGF on allograft was enhanced by the presence of pre-transplant HLA-DSA in DDKT 12 .
Based on the above background, it is possible that DGF in patients with pre-sensitization has a synergistic adverse impact on the allograft outcomes.However, it has yet to be fully investigated and only a single center study is available 12 .In this regard, the aim of this study is to investigate the combined impact of DGF and pre-sensitization on the development of allograft rejection using the well-established nationwide prospective cohort, Korean Organ Transplantation Registry (KOTRY).

Baseline clinical and immunological patient characteristics
DGF developed in 11.2% (154/1370) out of the total DDKT recipients.Between presensitized and non-pre-sensitized subgroups, no difference was detected in the frequency of DGF (10.8% vs. 15.3%, p=0.110).Table 1 describes baseline characteristics of the donor and recipients of four subgroups.Baseline estimated glomerular filtration rate (eGFR) was significantly lower in donors of DGF (+) subgroups irrespective of pre-sensitization.Cold ischemic time showed a longer tendency in DGF (+) subgroups irrespective of presensitization without statistical significance.However, donor age, gender, body mass index (BMI), underlying disease including DM or hypertension (HTN) and the proportion of donors after cardiac death (DCD) or donors after brain death (DBD) did not differ significantly across 4 subgroups.Among recipient factors, there was a significantly longer dialysis vintage and also additional number of female patients in both pre-sensitized subgroups than in nonpre-sensitized subgroups.In addition, a previous KT history and the proportion of antithymocyte globulin (ATG) use as induction therapy were higher in the pre-sensitized subgroups than in the non-pre-sensitized subgroups.The proportion of DM as the primary renal disease was lower in the pre-sensitized subgroups than in the non-pre-sensitized subgroups.A significantly higher proportion of patients undergoing hemodialysis as the dialysis modality prior to KT were selected from the non-pre-sensitized-DGF(+) subgroup, when compared with the non-pre-sensitized-DGF(-) subgroup.Although the majority of patients received tacrolimus as the main immunosuppressant, more patients in DGF(+) subgroups received sirolimus compared with DGF(-) subgroups.

Comparison of overall biopsy-proven allograft rejection (BPAR) and acute ABMR
The incidence of overall BPAR was the highest in the pre-sensitized-DGF(+) subgroup (33.3%, 7/21).The incidence rate of acute ABMR was higher in the pre-sensitized-DGF(+) subgroup (23.8%, 5/21) than in other 3 subgroups.The incidence of chronic ABMR was higher in pre-sensitized subgroups compared to non-pre-sensitized subgroups.In contrast, acute and chronic T-cell mediated rejection (TCMR) rate showed no statistically significant difference across four subgroups (Table 2).

Comparison of the change in allograft function and death-censored allograft survival
At the end of the 3-year follow-up, allograft function measured by eGFR using chronic kidney disease-epidemiology collaboration (CKD-EPI) equation was the lowest in non-presensitized-DGF(+) subgroup.The change in time-related allograft function of non-presensitized-DGF(+) subgroup was significantly different from that of other subgroups in linear mixed model (Figure 3) (p<0.001 vs. non-pre-sensitized-DGF(-), p<0.001 vs. pre-sensitized-DGF(-), p=0.034 vs. pre-sensitized-DGF(+)).

Discussion
Pre-sensitization to HLA is a well-known pre-transplant factor, which can increase the risk for allograft rejection and allograft failure.Meanwhile, DGF is a well-known post-transplant factor, which also induces adverse allograft outcomes.This study demonstrated that the combination of post-transplant factor (DGF) and pre-transplant risk factor (pre-sensitization) had a synergistic adverse effect on allograft outcomes, especially higher incidence of allograft rejection.
First, we compared baseline characteristics of donors and recipients across 4 clinical subgroups.In terms of donor factors, baseline renal function was significantly lower in patients who showed DGF, which was consistent with previous studies, which reported that low baseline kidney function is a risk factor for DGF 13 .In contrast, there was no significant difference in the frequency of DGF between pre-sensitized and non-pre-sensitized subgroups, which suggests that pre-sensitization may not have a significant effect on the development of DGF.Among recipient factors, the dialysis was significantly prolonged in pre-sensitized subgroups, which suggested that sensitized subjects need longer wait time for DDKT allocation [14][15][16] .As expected, the proportion of female recipients was higher in both presensitized subgroups 15 and the proportion of recipients with previous KT history was higher and tended to be high in both pre-sensitized subgroups than in non-pre-sensitized subgroups.
In addition, although a majority of patients received primary maintenance immunosuppression with tacrolimus, a significantly higher number of patients received sirolimus in the non-pre-sensitized-DGF(+) subgroup.This finding suggested that physicians decided a switch from CNI to mammalian target of rapamycin (mTOR) inhibitor, given that CNI may contribute to delayed recovery of allograft function 17 .Second, we compared the incidence of overall BPAR according to pre-sensitization or the development of DGF.As a result, the incidence of overall BPAR was the highest in the presensitized-DGF(+) subgroup and was mainly attributed to the increase in acute ABMR rather than acute and chronic TCMR as expected.Interestingly, pre-sensitization and DGF showed significant interaction with each other, which suggests their synergistic impact on the development of acute ABMR.This finding can be explained by two factors.First, DGF per se can increase the immunogenicity of allograft, and thereby increase the vulnerability to immune reaction of pre-formed HLA-DSA.Indeed, IRI in DGF can up-regulate the major histocompatibility complex (MHC) class I and II antigens, and enhance the expression of adhesion and costimulatory molecules of allograft tissue [18][19][20][21] .Moreover, the IRI induces ligands of toll-like receptors (TLRs) and activate cells of the innate immune system, inducing activation and maturation of dendritic cells, followed by adaptive immune response 21 .
Indeed, the previous studies demonstrated that DGF is associated with an increased risk of allograft loss and acute rejection 22,23 .Second, the conversion of CNI to mTOR inhibitor was more frequently detected in patients who suffered from DGF in this study, perhaps because CNI might be considered as a contributor to DGF.Lower suppressive potency of mTOR inhibitor for humoral immunity in comparison with tacrolimus is another possible cause of higher rate of acute ABMR in pre-sensitized-DGF(+) subgroup 24 .Surprisingly, pre-sensitization or DGF per se had no significant effect on the development of overall BPAR.The reason is unclear, but it may be attributed to the limited definition of both pre-sensitization and DGF in the study using a nationwide cohort.In case of presensitization, since data of DSA were collected from 2017, the results of HLA-DSA were not available in some recipients.Therefore, in such recipients, we defined sensitization to HLA by the presence of panel reactive antibodies (PRA), together with positive crossmatch test results.Even though this definition is used for "pre-sensitization", we cannot assess the degree of sensitization clearly.In case of DGF, the definition of DGF is varies among previous studies 25 .Indeed, the definition of DGF merely depends on the performance of dialysis after KT, and the decision whether or not to perform dialysis can differ according to the transplant centers.In addition, due to the absence of detail data, individualized immunosuppression regimen according to immunologic risk stratification and the serum level of immunosuppressant in each recipient did not be considered in our analysis.Therefore, the aforementioned factors can induce bias that can affect the result of this study.
Interestingly, non-pre-sensitized-DGF(+) group showed worst allograft function at 36 months post-transplant follow-up and worst allograft survival.One of the possible reasons is the baseline status of the corresponding donor kidney (Suppl.Table 1).The donor of this group showed the least renal function at baseline and a relatively higher kidney donor profile index (KDPI) score.In addition, in 5 out of 14 patients with allograft loss in this group, primary non-function was the reason for allograft failure.All of the foregoing findings suggest that the baseline status of donor kidney was the worst in this group, which may result in a higher rate of allograft loss and also sustained low allograft function.In regard to allograft survival, the impact of the baseline kidney function can be more significant than allograft rejection during the limited follow-up duration.Hence, the allograft survival was not the lowest in the pre-sensitized-DGF(+) subgroup in spite of the highest rate of BPAR.Lastly, we compared the post-transplant complications among the 4 subgroups.Non-presensitized-DGF(+) subgroup showed the higher patient death rate.However, only 57 cases out of 1,370 kidney transplantation recipients (KTRs) were found and there was no patient death in the pre-sensitized-DGF(+) subgroup.Therefore, longer observations may be required to arrive at any conclusion.Compared with the post-transplant infection, no difference was detected across 4 subgroups in most infection types, except that the rate of PJP infection was the highest in the pre-sensitized-DGF(+) subgroup.However, there was only 1 case of PJP infection, hence further investigation may be required to clarify this issue as well 26 .
This study has some limitations.First, this nationwide registry analysis reflects similar limitations found in similar large registry analyses as shown in our previous studies 27 .While patient numbers are enhanced, important details for the endpoints are missing, thereby reducing the clinical utility of the findings.For example, the HLA-DSA was not available for analysis in some patients, and we cannot use the class and the strength of DSA in the analysis, which has been reported as an important risk factor for allograft rejection and failure 6,10,[28][29][30] .
Second, the follow-up duration of this registry is limited as mentioned previously.Therefore, traditional risk factors for allograft failure such as DGF and pre-sensitization did not significantly affect allograft outcome.Third, we could not determine the specified protocols at each center in DDKT for highly sensitized recipients.Despite pre-transplant desensitization was performed in 37 recipients, including those whom with positive B-cell crossmatch, no data were available on the protocol.Some centers used rituximab to treat such patients, and others did not, but unfortunately, it was not considered in this analysis.
Nevertheless, our study is the first prospective, multi-centered cohort study to investigate the association of DGF and pre-sensitization in allograft outcomes.
In conclusion, we have shown that combination of DGF and pre-sensitization to HLA had a detrimental impact on allograft outcome in terms of rejection.Therefore, we suggest that more careful monitoring or surveillance of allograft rejection is required.Further, we need to use more intensified immunosuppression protocol to prevent allograft rejection when DGF occurred in DDKT with pre-sensitization.

Study population
We analysed KOTRY data from the Korean Society for Transplantation 31 , compiling data from 30 kidney transplantation centers in Korea 32 .The KOTRY data includes 1,945 DDKT cases between May 2014 and June 2019, from which we excluded 575 DDKT recipients with unavailable data regarding PRA, HLA-DSA, crossmatch tests or DGF development.
We defined pre-sensitization to HLA by the presence of (i) HLA-DSA (by Luminex single antigen assay) or (ii) PRA (by solid-phase HLA antibody screening), combined with positive crossmatch test results.HLA-DSA data were available in 1,060 recipients (77.3%).Therefore, the sensitization to HLA was defined by the detection of HLA-DSA in those patients.In another 310 DDKT recipients for whom HLA-DSA data were not available, we defined sensitization to HLA based on the positive results of PRA and crossmatch test.Among 310 DDKT recipients, 5 recipients were sensitization to HLA with the presence of PRA and positive B-cell crossmatch.DGF was defined as the need for dialysis within 1 week of transplantation.The medical records were reviwed after receiving informed consent 32 .This study was performed in accordance with the Declaration of Helsinki and the Declaration of Istanbul.The study was approved by the Institutional Review Board of Seoul St. Mary's Hospital (KC14ONMI0460).

Definition of clinical outcomes
The clinical outcomes investigated in this study included the incidence of overall BPAR, acute ABMR, time-related changes in allograft function measured as eGFR, death-censored allograft survival rates, and post-transplant complications such as BKVAN, cardiovascular disease, cerebrovascular disease, infection and malignancy.BPAR was diagnosed according to the Banff 2013 classification 33 .Rejection-free allograft survival was defined as the time elapsed from transplantation to the first episode of BPAR.Serum creatinine levels were collected at six months and later at one-year intervals post-transplant.The eGFR for each concordant time was assessed using the CKD-EPI equation 34 .Allograft survival was defined as the time from transplantation to initiation with alternative renal replacement therapy.
Cardiovascular disease is defined as cardiovascular death, myocardial infarction, ischemic heart disease with relevant clinical evidence (accompanied by therapeutic intervention or objective findings), new-onset congestive heart failure requiring hospitalization, and arrhythmia.Cerebrovascular disease included non-traumatic hemorrhagic or ischemic brain disease confirmed by computed tomography or magnetic resonance imaging 32 .BKVAN was diagnosed by allograft biopsy.All clinical parameters were compared across the four patient subgroups.

Supplementary Files
This is a list of supplementary les associated with this preprint.Click to download. LeeetalDGFandpresensitizationKOTRYSciRsuppl.doc

Figure 1 .
Figure 1.Distribution of the patient population according to DGF or pre-sensitization to

Figure 3 .
Figure 3.Comparison of the time-related changes in allograft function based on eGFR

Figure 4 .
Figure 4. Kaplan-Meier estimates of death-censored allograft survival according to DGF

Figures Figure 1
Figures

Figure 3 Comparison
Figure 3

Table 4 . (a) Causes of death and (b) clinical outcomes among the 4 subgroups according to DGF and pre-sensitization status.
Continuous variables are shown as mean ± standard deviation or median with interquartile range.Categorical variables are shown as number (proportions).DGF, delayed graft function; CNI, calcineurin inhibitor; BKVAN, BK virus associated nephropathy; CMV, cytomegalovirus; PJP, Pneumocystis jirovecii pneumonia.Others: liver disease, cerebral infarction, acute CNI toxicity, gastrointestinal bleeding, intestinal obstruction, acute rejection etc.