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Entering the Third Decade After Kidney Transplantation: Excellent Graft Function Refers to Superior Graft but Not Patient Survival

Kidney transplant recipients (KTRs) with ultralong-term survival represent a growing, yet insufficiently studied patient cohort. In this single-center retrospective study, we analyzed 248 ultralong-term survivors (≥20 years). KTRs were classified into those with superior graft function (defined as eGFR ≥45 ml/min + proteinuria ≤300 mg/day + eGFR-slope ≤ 2 ml/min/1.73 m2/year) and inferior graft function regarding the risk of CKD progression. 20 years post-transplant, median eGFR was 54 ml/min (11–114), proteinuria 200 mg/24 h (0–7,620), eGFR decline 0.45 ml/min/1.73 m2/year (11.7 6.5) and DSA had been detected in 19.7% of KTRs. We identified 96 KTRs (38.7%) with superior (group 1) and 152 KTRs (61.3%) with inferior graft function (group 2). Donation after cardiac death, female sex, glomerulonephritis as primary disease, and early TCMR were independently associated with inferior graft function. Graft survival was significantly better in group 1 compared to group 2 (LogRank, p < 0.001). Besides group affiliation (HR 20.515, p = 0.003), multivariable analysis identified DSA development (HR 3.081, p = 0.023) and donor age (HR 1.032, p = 0.024) as independent factors. Interestingly, there was no significant difference in patient survival (LogRank, p = 0.350). In ultralong-term survivors, excellent graft function refers to superior graft survival but does not extend ultimate patient survival. DSA-formation should be taken seriously even in the ultralong-term.


INTRODUCTION
Kidney transplantation has become standard procedure in care of patients with end stage kidney disease (ESKD) and by today, is the preferred treatment for most of them (1). Over the past decades, short-and long-term graft survival have improved remarkably (2,3,4,5). For Europe, the Collaborative Transplant Study (CTS) reports an estimated 20-year death-uncensored graft survival rate of 41% for first deceased donor kidney transplant recipients (KTRs) from 1990-2020 and 16.8 years death-uncensored graft half-life (6). According to Coemans et al., who performed a comprehensive analysis of CTS data, deathcensored 20-year graft survival rate even exceeded 50% for the transplant decade 1996-2005 (2). However, the authors reported survival data beyond 20 years to be sparse (2). The latest registry report from Australia and New Zealand (ANZDATA) reveals 30% 20-year death-uncensored graft survival for first deceased donor KTRs (3). Other comprehensive registry reports limit their analysis to a maximum of 10-year death-uncensored graft survival (for deceased donors 49.5% in the US (4), 58.5% in Canada (5).
To optimize ultralong-term aftercare and to overcome the still important challenge of further improving long-term outcome (16), it is crucial to learn more about this particular patient group (9). To address these needs, we studied a large cohort of KTRs who have lived with a functioning graft for ≥20 years and aimed to investigate the following questions:

KTRs and Data Collection
This single-center retrospective study was approved by the local Ethics committee of Zurich, Switzerland (Basec Number: 2019-02082) without informed consent requirement and performed in adherence to the declaration of Helsinki. We considered all adult (age ≥16 years at the date of transplantation) KTRs transplanted at University Hospital Zurich between 1 January 1981 and 31 December 1999. Among a total of 1,180 single-kidney transplantations performed at our institution during this era, we identified 304 KTRs with documented graft survival ≥20 years. 22 KTRs who had denied consent had to be excluded, further 34 KTRs due to insufficient data. This led to a total study cohort of 248 KTRs (Figure 1).
At our center, follow-up care after the first post-transplant year is generally performed quarterly in our outpatient clinic or, in stable conditions, by local nephrologists, complemented by an annual visit at our center. For data collection, we reviewed medical records from the electronic database of the hospital registry. End of follow-up and data collection was 31 January 2021. To evaluate characteristics 20 years post-transplant, we identified the 20-year post-transplant visit for each KTR, defined as the closest and most complete visit to the date of transplantation plus 20 years. For all KTRs, median time from transplantation to the 20-year post-transplant visit was 240 months (range 228-248 months). If the 20-year posttransplant visit did not reveal full data, we checked medical records from 19-21 years post-transplant for completion. Cases with insufficient documentation between 19-21 years post-transplant were excluded, as stated above.

Graft Function
From serum-creatinine at the 1-year and 20-year post-transplant visit, we calculated baseline 1-year and 20-year eGFR, using the following formulas: Modification of Diet in Renal Disease (MDRD) (17), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) (18), and . We did not include the race coefficient for MDRD and CKD-EPI (20). For MDRD and CKD-EPI, we additionally calculated body surface area (BSA)-deindexed eGFR, multiplying eGFR by KTR's individual BSA (21), divided by 1.73 m 2 (22,23). To indicate stability or decline of graft function, respectively, eGFR (CKD-EPI) slopes (24) were calculated for the last 5 years of the 20-year period, i.e., 15-20 years post-transplant. Baseline 20-year proteinuria was assessed by multiplying urineto-creatinine ratio (PCR) (mg/mmol) from spot urine at the 20year post-transplant visit by 10 (25). For 31 KTRs (12.5%), PCR was calculated from 24-hour collection urine, as before 2005, measurement of proteinuria was obtained by 24-hour collection urine at our center. PCR below detection limit was included in the analysis with a value of zero.

Maintenance Immunosuppression and DSA-Screening
All donors and recipients were typed for human leukocyte antigen (HLA)-A, -B and -DR. Since approximately 2009, annual HLA antibody-monitoring using Luminex based assay (One Lambda, Canoga Park, CA, United States) became standard procedure in KTR-care at our center. In case of worsening graft function or progression of proteinuria, screening may have been performed more often. If Luminex mix assay was positive and/or clinical suspicion was high, an additional Luminex single antigen bead assay was performed to test for DSA. We classified KTRs with ≥1 DSA-positive Luminex single antigen bead assay up to 21 years post-transplant as DSA-positive, irrespective of the level of mean fluorescence intensity (MFI). KTRs were classified as DSA-negative in case HLA antibody-monitoring (Luminex mix assay only or both, Luminex mix and single bead assay) did not show DSA up to 21 years post-transplant or if the very first screening was performed beyond 21 years post-transplant and negative for DSA. KTRs were excluded from this sub-analysis in case of missing HLA antibody-screening during the observation period (n = 36, 14.5%) or if the very first screening was performed beyond 21 years post-transplant and DSA-positive, thus the date of DSA-occurrence was indeterminable (n = 14, 5.6%).

Group Categorization
According to the KDIGO 2012 Clinical Practice Guideline for the risk of CKD progression, KTRs were stratified into two groups based on graft function 20 years post-transplant. (50) Criteria for superior graft function (Group 1) were: 1) 20-year eGFR ≥45 ml/ min (BSA-deindexed CKD-EPI), 2) 20-year proteinuria ≤300 mg/24 h, and 3) eGFR (CKD-EPI) decline ≤-2 ml/min/1.73 m 2 /year 15-20 years post-transplant. Subjects in group 1 had to meet all 3 criteria. KTRs who did not pass ≥1 criteria were assigned to group 2. Two cases with missing data on proteinuria that fulfilled the other criteria for group 1 were classified as insufficient data and excluded, consequently. Two cases were categorized according to BSAindexed CKD-EPI, because of unknown BSA (missing documentation of patient's height).

Survival
We separately studied patient survival (treating graft loss as a censored event), death-uncensored and death-censored (treating death as a censored event) graft survival, calculated from the date of transplantation to KTR's death or graft loss (return to permanent dialysis or retransplantation), whatever came first. If there was no event, survival dates were censored at the date of last follow-up or end of data collection (31 January 2021). Specific causes of graft loss and results of indication biopsy were evaluated for KTRs with death-censored graft loss in group 1.

Calculation of Predicted Indirectly ReCognizable HLA-Epitopes Scores
The HLA-derived mismatched peptide epitopes presented by KTRs HLA-molecules were calculated using the PIRCHE-II algorithm. Presentation of both HLA class I (HLA-A, B) and HLA class II derived peptides (HLA-DR, DQ) were calculated for each HLA locus. Detection of HLA antigens was performed by DNA-based HLA-typing technology using blood samples. Either sequence-specific oligonucleotide (SSO) or sequence-specific primer (SSP) technologies were used to generate lowresolution HLA typing results. The imputation of probable allele resolution results needed for the PIRCHE-II calculation was achieved by the use of the imputation algorithm included in the PIRCHE-II calculation. The PIRCHE-II algorithm is available online (https://www.PIRCHE-II.org).

Statistical Analysis
Statistical analysis was performed using SPSS (Version 26, IBM, Armonk, NY, United States). Continuous variables are expressed as median (range minimum-maximum) and compared using Mann Whitney-U Test. Categorical data are expressed as number (%) and compared using Chi (2) test, corrected for Yates in 2x2 tables. If expected cell count was ≤5, we used Fisher's Exact test instead. Missing values were not imputed. Survival was analysed using the Kaplan-Meier method and compared with LogRank test. Univariable and multivariable Cox proportional hazards models with enter method were used to investigate factors associated with survival. Variables with a p-value ≤0.05 in the univariable analysis were included in the multivariable model. For categorical variables in the multivariable model, assumption of proportional hazards was assessed visually by Kaplan Meier curves (26). For all tests, statistical significance was assumed for a two-tailed p-value <0.05. Table 1 shows basic characteristics, Table 2 post-transplant complications and 1-year graft function, and Table 3 multivariable Cox regression analysis for group categorization of the 248 KTRs included in this study. Median KTR-age at the date of transplantation was 39.9 years, 92/248 (37.1%) KTRs were female. Table 4 shows detailed information on graft function 20 years post-transplant. Median serum-creatinine was 124 μmol/L, median eGFR 54 ml/min (BSA-deindexed CKD-EPI), median proteinuria 200 mg/24 h, and median eGFR decline −0.45 ml/ min/1.73 m 2 /year. CKD-related laboratory findings are shown in the Supplementary Table S1.

Immunosuppression and DSA-formation
Maintenance immunosuppression is shown in Table 5, results of HLA antibody-screenings in Table 6. Within the

KTR-Categorization and Group Comparison
Subdivision of the cohort is shown in Figure 1. 96/248 (38.7%) KTRs fulfilled the criteria for superior graft function (group 1). The remaining 152/248 (61.3%) KTRs were classified to group 2. Figure 2 displays distribution of all KTRs according to baseline 20-year eGFR and proteinuria, group subdivision is marked by color. Multivariable Cox regression analysis is shown in  .5%) due to DC-GF. These latter 3 KTRs were analyzed more closely: 1 KTR was DSA-negative 20 years post-transplant but developed de novo DSA (dnDSA) during the third post-transplant decade. Graft loss resulted from biopsy-proven chronic antibody mediated rejection (ABMR). The other two KTRs both had their first HLA antibodyscreening performed during the 28th year post-transplant and were DSA-positive by then (which, due to indeterminable date of DSA-development, led to exclusion from DSA-sub-analysis, as stated above). Indication biopsy showed glomerulopathy and low level glomerulitis in one, and glomerulopathy and vasculopathy with signs of de novo IgA nephropathy in the other case. In group 2, 67/152 (44.1%) grafts failed during follow-up: 30/67 (44.8%) due to DWFG, 37/67 (55.2%) due to DC-GF. Death-censored and death-uncensored graft survival was significantly superior in group 1 (LogRank, both p < 0.001, Figures 4A,C). In contrast, there was no significant difference in patient survival (LogRank, p = 0.35, Figure 4B).
Univariable and multivariable Cox regression analysis are shown in Tables 7,8. For DC-GF (

DISCUSSION
ULS represent a growing, yet insufficiently studied patient population (8,9). To address this new challenge in transplant long-term aftercare (7,9), we herein analyzed 248 KTRs with a functioning graft ≥20 years. In line with earlier ULS-reports (7,8,9,11,12,14,15) graft function was remarkably good: 20 years post-transplant, the majority (64.5%) of the KTRs was in stage 1-3a of CKD classification (25). 38.7% fulfilled the criteria for superior graft function, i.e., had high and stable 20-year eGFR and low proteinuria (group 1). Group comparison revealed a significant difference in DCD, early TCMR, recipient gender, and glomerulonephritis as primary disease. For the first, previous studies suggest comparable survival rates for kidneys from DCD. (27) For the second, although in general associated with reduced graft survival, the impact of successfully treated early TCMR on ultralong-term survival has not been well studied. (51) However, our data suggest that initial acute kidney injury and associated nephron loss either due to DCD or early TCMR may have an impact in the ultralong-term, and predispose these KTRs to decreased graft function and proteinuria through chronic hyperfiltration and chronic histologic lesions of interstitial fibrosis/tubular atrophy. Regarding the effect of KTR-gender on ultralong-term survival, results are conflicting (8,9,10,13). However, the predominance of men in group 1 surprises, given their higher risk of chronic graft failure (28). Our finding could result from potential underestimation of GFR in women by the applied equations and stresses the need for further, gender-specific studies (29). Although the time of onset and severity of recurrence of the underlying disease vary widely, our data suggest that glomerulonephritis recurrence strongly influences the risk of impaired renal function in the ultralongterm. However, in our analysis, no factor had an independent impact on further survival.
Data on long-term maintenance immunosuppression is extremely limited (30). In our center, standard  (8,12). Given the prolonged exposure to immunosuppression, determining optimal long-term immunosuppression is especially important for ULS (9). But this urgent question still remains unanswered (33).
To target therapeutic interventions and optimize ultralongterm aftercare, we need to improve our understanding of late graft loss (16,31), which includes patient's death (DWFG) and loss of graft function while still alive (DC-GF) (16). In this study, we drew a detailed picture of graft and patient survival of 248 ULS. While overall graft survival was already remarkably good (median death-uncensored graft survival 29.9 years), for KTRs with superior graft function, it was outstanding. In fact, group 1 only involved 3 events of DC-GF. In contrast, graft survival in group 2 was clearly inferior. Corresponding with the fact that graft failure is preceded by graft dysfunction (16,34), 92.5% of all events of DC-GF in this study occurred in group 2.
In their comprehensive study of 177 ULS, McCaughan et al. observed that DC-GF after 20 years is uncommon (9). Our study shows that this is particularly true for ULS with preserved graft function, while in group 2, DC-GF accounted for the majority (55.2%) of graft losses. A multivariable Cox regression model for DC-GF confirmed a strong influence of group affiliation.
Donor age profoundly impacts graft quality (35) and is an important risk factor in graft outcome (2,35). In this study, comparable to earlier ULS-reports (7,8,9,10,12,13), donors were young (median 32 years), a clear difference to more recently transplanted KTRs (2). Very interestingly, donors were significantly younger in group 1, and donor age had significant impact on DC-GF beyond 20 years post-transplant. This phenomenon might be attributed to the loss of functional nephrons with aging and consecutive decreased functional reserve (36) and increased vulnerability to transplant-related injury (37,38). In previous studies, univariable analyses revealed significant association of donor age with ultralongterm survival, however, in multivariable models the effect showed only a trend and missed statistical significance (9,10).
Late DC-GF is profoundly driven by alloimmune mechanisms (31,39,51). DSA are associated with increased risk of late graft   failure (40,41) and provide a well-established biomarker predicting ABMR and graft loss (40,42). However, little is known about the role of DSA in the context of ultralong-term survival (12,43). Analysis of DSA-screenings revealed several interesting findings: First, in our cohort of ULS only, cumulative incidence of DSA-formation during the first two post-transplant decades was 19.7% and thus within the range reported from general KTR-population (42,44). Secondly, we could not find any significant group difference in cumulative incidence of DSA-positive KTRs, duration to first DSA-detection, HLA-class, HLA-mismatches, PIRCHE-II socres, MFI, and number of detected DSA. These results surprise, as KTRs who develop dnDSA have been shown to have higher rates of eGFR decline (41). Thirdly, however, DSA-formation was identified as an independent risk factor for DC-GF. The association of steroid use with DC-GF must be interpreted here with the restart of steroids after the onset of DSA. In our cohort, DSA were detected surprisingly late (median 211 months post-transplant), a finding probably biased by transplant era and available techniques. However, it is known that DSA-formation can appear anytime, even several years post-transplant (42,43,44) and that time from dnDSA-onset to graft dysfunction ranges from months to years (44). So nevertheless, it is suggestive that ultralong-term survival of our cohort was favored by substantially late DSAdevelopment and that their deleterious impact on graft survival manifested not until the third post-transplant decade. Given the close relationship between dnDSA, ABMR and ultimate graft loss, this result points towards a potential target of intervention in order to further improve long-term graft survival (45). However, further studies are needed to address this question (45).
Hence, DC-GF is predominantly seen in ULS with inferior graft function. However, despite the known link of declining graft function with increased mortality (46), there was no significant group difference in patient survival. The risk of DWFG increases with time since transplantation (47) and in ULS, it represents the leading cause of graft loss (8,9,14,15). Our results correspond with the findings from Gaston et al. who stated that mortality risk is largely independent of graft function (16). Beyond 20 years posttransplant, leading causes of death are cardiovascular disease and malignancy (8,9), both highly prevalent in ULS (7,8,9,14,15). For example, McCaughan et al. reported cancer in 37% and cardiovascular disease in 27% of 20-year survivors and therefore stated that, in management of ULS, focus should shift on prevention and optimal therapy of these comorbidities (9).
Our results allow us to specify this statement and lead to further clinical implications. Indeed, in case of good, stable graft function up to 20 years post-transplant, risk of ultimate DC-GF is very low, and focus should be on controlling the medical comorbidities (9). In contrast, in KTRs with inferior ultralongterm graft function, risk of DC-GF may not be neglected, and aftercare should equally concentrate on preventing ultimate loss of graft function. Given that DSA and ABMR, respectively, are potentially treatable conditions (45). Our findings argue for continuing DSA-monitoring even in the setting of ultralongterm survival. Additionally, KTRs with inferior ultralong-term graft function might be considered for biopsy, not only to evaluate immune injury, but also to detect evidence of CNItoxicity and to adjust immunosuppression, accordingly (48,49). Our study has several limitations. First, it is a retrospective and single-center analysis with the intrinsic limitations and potential biases. Secondly, our cohort differs in several aspects from more recently transplanted KTRs. Thirdly, DSA-subanalysis is limited by transplant era and available techniques: it affected time of first and frequency of subsequent screenings, 50 cases with missing or insufficient data had to be excluded, and KTRs were mostly not typed for HLA-DP and -DQ.
However, most of these limitations are inevitably associated with the retrospective design of research on ULS and the according necessity of lengthy follow-up (8). In addition, our study cohort is not dominated by living donor transplantations, which would suggest better organ quality and possibly better HLA matching, so our results translate well to the general transplant cohort. The reason for this is that only about 50 living donations were performed in the observation period from 1981 to 1999 at our center. Our study provides an important contribution in improving understanding of this unique, increasingly important patient population (7,8,9). Comprehensive follow-up enables us to give extensive overview of ultralong-term graft function, alloimmunization, and ultimate outcome beyond 20 years post-transplant and to identify corresponding risk factors and potential therapeutic targets required to improve ULS-aftercare.

CONCLUSION
Overall, KTRs with ultralong-term survival ≥20 years do extremely well. Particularly KTRs with stable and high eGFR and low proteinuria likely keep their graft function and ultimately die of medical comorbidities. The risk of graft failure is predominantly seen in KTRs with inferior graft function. This graft function-related risk profile could augment long-term monitoring and treatment.

DATA AVAILABILITY STATEMENT
The data underlying this article will be shared on reasonable request to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Cantonal Ethics Committee Zurich. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
AR participated in data collection, data analysis, and writing of the paper. JN participated in data collection and writing of the paper. RW participated in writing of the paper. TM participated in research design and writing of the paper. TS participated in research design, data collection, data analysis and writing of the paper.