Evaluation the performance of serum neutrophil gelatinase associated lipocalin as a biomarker of allograft dysfunction in kidney recipients from living donors

Implication for health policy/practice/research/medical education: Evidence signifies that serum neutrophil gelatinase-associated lipocalin (sNGAL) is a reliable predictor of early graft function organ recipients from deceased donors. We aimed to evaluate the diagnostic performance of sNGAL for the prediction of earlyand long-term graft restoration in kidney recipients from living donors. Organ recipients (n=39) were consecutively enrolled. Serial measurements were performed before transplantation and at 2, 16, 24, 36, 48 hours after surgery. Immediate graft function (IGF) was defined as an estimated glomerular filtration rate (eGFR) >60 mL/min per 1.73 m2 on day 5 post-transplantation. We have uncovered that the value of sNGAL at 2hours post-surgery correlates allograft function at 6, 9 and 12 months posttransplantation. Multicenter study with large samples size is required to ascertain the usefulness of sNGAL as a diagnostic tool for the prediction of allograft function in renal transplant patients from living donors. Please cite this paper as: Adlravan E, Javandoust Gharehbagh F, Taghizadeh Afshari A, Baradaran B, Nourooz-Zadeh J. Evaluation the performance of serum neutrophil gelatinase associated lipocalin as a biomarker of allograft dysfunction in kidney recipients from living donors. J Renal Inj Prev. 2021; 10(4): e30. doi: 10.34172/jrip.2021.30. O rig in al Introduction: Serum neutrophil gelatinase-associated lipocalin (sNGAL) has been proposed as an early biomarker for the prediction of delayed graft function (DGF) from cadaveric donors. Objectives: The purposes of this investigation were to explore the time-based trend for sNGAL in kidney recipients from living donors and to evaluate its correlation with graft function recovery during a one-year follow-up. Patients and Methods: Kidney recipients (n = 39) were consecutively enrolled. Sample collection was performed before transplantation and at 2, 16, 24, 36, 48 hours after surgery. Kidney recipients were split into immediate graft function (IGF) and DGF based on estimated glomerular filtration rate (eGFR) on day 5 post-surgery. eGFRs >60 mL/min/1.73 m2 on day 5 post-transplantation were considered as IGF. sNGAL was assessed by ELISA. Serum creatinine (sCr) was measured by the Jaffe method. Results: Rates of participants with IGF or DGF were 25 and 14, respectively. Pre-surgery, sNGAL levels in the DGF subset were 21% higher than that of the IGF group. At 2-hours checkpoint, area under curve, sensitivity, specificity and cut-off (ng/mL) for sNGAL were 0.73, 100%, 52% and 151.8. sNGAL levels correlated with allograft function at 6, 9 and 12 months post-transplantation (r = 0.66; P = 0.007; r = 0.836; P = 0.031 and r = 0.93; P = 0.016). Conclusion: We have uncovered that monitoring sNGAL in kidney recipients is a useful biomarker for the evaluation of shortand long postoperative outcome in renal transplant patients from living donors. However, multicenter study with large samples-size is required to ascertain the usefulness of sNGAL as diagnostic tool for the evaluation of allograft dysfunction in renal transplant patients from living donors. A R T I C L E I N F O


Introduction
Kidney transplantation is the most gainful therapeutic option for patients with end-stage renal disease (ESRD) (1). However, the incidence of early delayed graft function (DGF) is still much inferior than desired. Occurrence of DGF varies from 5% to 50% depending on organ source (living donors versus cadaveric donors) (2,3). Ischemiareperfusion injuries, immunological responses and donor-related organ quality are the major recognized causes of DGF (4)(5)(6). Current clinical indicators of DGF are serum creatinine (sCr) and urine output (UO). The major inherited problem with the sCr measurement is that the alteration emerges several days after a tubular insult. UO test is a simple and non-invasive index of successful reperfusion but lacks the required functional specificity.
Thus, efforts have been directed towards the search for innovative biomarkers that will facilitate site-specific DGF detection. Pioneering studied have shown that measurement of urine or blood biomarkers such as neutrophil gelatinase associated lipocalin (NGAL), cystatin C, interleukin 18 (IL-18) and kidney injury molecule 1 outperform sCr in spotting renal dysfunction (7,8). In kidney recipients from cadaveric donors with more than one co-morbid disorder, the marked elevation of sera-and urine NGAL precedes that of sCr (9,10). Additionally, there are correlations between serum neutrophil gelatinase associated lipocalin (sNGAL) and biomarkers of ischemia-reperfusion related kidney injury (9,10). A multicenter investigation involving 225 kidney recipients from brain death donors (The CONTEXT study) revealed that plasma NGAL (pNGAL) predicted early graft dysfunction at day one post-surgery (11). Buemi et al (12) explored the diagnostic performance of pNGAL from kidney recipients involving deceased donors (n = 80) and living donors (n = 17). In the organ recipients from the deceased donors, pNGAL at 24-and 48-hours post-surgery were predictor of initial recovery allograft function. With respect to the organ recipient from the living donors, no conclusion was reached since all kidney recipients were non-DGF.

Objectives
In this study, we aimed to: 1) revisit the temporal trend of sNGAL of kidney recipients from living donors withand without postoperative complication and 2) evaluate its diagnostic performance to predict allograft recovery at 3-, 6-, 9-and 12-months post-surgery.

Study design
In this cross-sectional analysis, 39 renal transplant candidates were enrolled through Imam-Khomeini hospital complex affiliated with Urmia university of medical sciences. Inclusion criteria were age >12 years, resident of West Azerbaijan province. Relevant demographic and clinical data (recipient-and donor age on the day of the surgery, cold ischemia time, type and duration of preoperative dialysis, end-stage renal failure etiology, postoperative daily sCr and UO) were collected from the patients' hospital records. Organ recipients received a triple-drug-based immunosuppressive regimen combining tacrolimus, mycophenolate mofetil and methylprednisolone.

Serum preparation
Blood (4 mL) was drawn into a Vacutainer by venipuncture. Sampling was performed at pre-operation day and at 2, 16, 24, 36 and 48 hours after surgery. The blood samples were allowed to clot at room temperature for 30 minutes. The samples were then centrifuged at 1500 rpm for 10 minutes. The sera (0.5 mL aliquots) were transferred into Eppendorf tubes and subsequently stored at -70°C until further analysis.

Biomarker measurements
Serum NGAL levels were measured using an ELISA Kit (Shanghai Crystal Day Biotech, China) according to the manufacturer's instructions. Creatinine measurement was performed according to the Jaffe method using a commercial kit (Pars Azmoon Inc., Tehran) on a BT-auto analyzer (13).

Statistical analysis
The data were analyzed using SPSS version 17. Normal distribution continuous variables are presented as mean ± standard deviation. Non-normal distribution continuous variables are presented as median inter-quartile range variables. Categorical variables were displayed by number and percentage. The Kolmogorov-Smirnov test was used to determine each continuous variable's normal distribution .The student t test or Mann-Whitney U test measured the difference in continuous variables between two groups and the chi-square test or the Fisher's exact test assessed categorical variables between two groups. To assess the diagnostic significance of the indicators, receiver operating characteristic (ROC) analysis was performed. Optimum cut-off value was determined as described elsewhere (14).
Pre-surgery, median eGFR (mean; range) in the kidney recipients as a whole was 7.3 mL/min per 1.73 m 2 (7.77 ± 3.30; 3.30-16.85). In the IGF group, the eGFR progressively increased after surgery, reaching 60 at day 3 post-surgery. A maximum eGFR was obtained at day 14 post-surgery 70.92 (62.45 ± 19.30; 23.18 to 110.13). In the case of the DGF group, an improvement in the eGFR was also improved but at slower rate. At day 14 post-surgery, median eGFR was 46 (48.34 ± 19.31; 23.18 to 81.28).
Trends for eGFR in the IGF-and DGF groups are shown in Figure 1. Comparison of eGFR between IGF and DGF groups revealed that statistical differences were seen at checkpoint 16 hours post-surgery and onwards.
Retrospective classification of the organ recipients according to post-operative outcome showed that median sNGAL (mean; range) in the DGF subset was higher than that of the IGF group 202.35 ng/mL (330.80 ± 341.18 ng/mL) versus 124.30 ng/mL (294.60 ± 324.70 ng/mL). Interestingly, a statistical difference was seen at 2 hours post-surgery. Figure 2 displays the trends for sNGAL in the organ recipient during the follow-up.
At 2 hours post-surgery, AUC, sensitivity and specificity for sNGAL were 0.73, 100% and 52%. Figure 3 displays the receiver operating characteristics for sNGAL and eGFR at two hours surgery.

Discussion
In the current report, frequencies of DGF was 36% when immediate recovery of renal function was defined as an eGFR >60 mL/min per 1.73 m 2 on day 5 post-surgery (15). On the other hand, the frequency of DGF was 17% when the need for dialysis was defined as an index of postoperative outcome. Our finding is in contrast with the nil percent recently by Buemi et al (12). Of note is that Buemi et al DGF was defined as the requirement for at least one dialysis session during the first week after surgery. In their study, the recipients received anti-CD25 monoclonal antibody on days 1 and 4 as induction therapy. The difference in the frequency of postoperative outcome between the aforementioned investigation(s) is possible due to implementation of induction therapy. Taken together, further studies are required to elucidate the cause(s) of the higher rate of DGF our center.
We have also found that preoperative median sNGAL in our cohort by ELISA was 50% lower than that reported by the Triage NGAL test (12). The latter is a rapid fluorescence immunoassay and is a point-of-care assay. The disparity between our value and that reported by Buemi et al is likely to reflect difference in cold-ischemia time (70 minutes versus 240 minutes).
Thirdly, retrospective segregation of the studied population according to early postoperative outcome  revealed that median sNGAL in the DGF group, prior to surgery, was 21% higher than that of the IGF group. Interestingly, this finding is in line with the results of reported by Nielsen et al (11) evaluating diagnostic performance pNGAL in a cohort of renal transplant patients from brain death donors. A similar phenomenon has been noticed in patients receiving dialysis prior to transplantation (11). Furthermore, higher sNGAL levels has been detected in kidney recipients with underlying risk factors compared to those transplanted preemptive (11). Many organs are capable of expressing NGAL, especially during periods of aggression. Accordingly, NGAL values can be significantly modified by pre-existing conditions in organ donors and organ recipients. Indeed, Portilla et al (16) attributed the paradox to disruption of megalindependent endocytosis in the renal proximal tubule and thus leading to manifestation of higher sNGAL. Taken together these observations imply that donors /recipient dependent factors may be responsible for the elevated sNGAL in the DGF patients. The clinical significance of the elevated sNGAL among the patients with DGF prior to surgery merits further investigation.

Conclusion
This study is the first to uncover that pre-transplant sNGAL levels in DGF were higher compared to IGF and that sNGAL at 2-hours post-surgery correlates allograft function at 6-, 9-and 12-months. Multicenter study with large samples-size is required to ascertain the usefulness of sNGAL as a diagnostic tool for the prediction of allograft function in renal transplant patients from living donors.

Limitations of the study
These were; 1) small sample size; 2) a single-center study that needs further validation at a multi-center level or larger sample size is required; and 3) lack of a standardized measure of glomerular filtration rate.

Acknowledgments
We thank so much for the work and support of all the physicians, nurses and transplant workers of Imam-Khomeini hospital complex affiliated with Urmia university of medical sciences.
Authors' contribution JNZ and ATF were the principal investigators of the study.