Soluble urokinase-type plasminogen activator receptor (suPAR) is a risk indicator for eGFR loss in kidney transplant recipients

The prognostic significance of suPAR in various kidney diseases has recently been demonstrated. Its role in transplantation-specific outcomes is still largely unknown. Therefore, we prospectively investigated the prognostic relevance of suPAR in patients before and one year after kidney transplantation (KTx). We included 100 patients who had received a kidney transplantation between 2013 and 2015. The plasma concentration of suPAR was measured by ELISA assay. In recipients of living donations (LD), pre-transplant suPAR levels were significantly lower than those of recipients of deceased donations (DD). After KTx, suPAR levels significantly declined in LD and DD recipients, without a detectable difference between both groups any more. Higher suPAR levels in recipients one year after KTx were associated with a more severe eGFR loss in the following three years in multivariable cox-regression (n = 82, p = 0.021). suPAR-levels above 6212 pg/ml one year after KTx are associated with eGFR loss > 30%, which occurred almost twice as fast as in patients with suPAR ≤ 6212 pg/ml (p < 0.001). Hence, suPAR level at one year mark might be a risk indicator of increased eGFR loss.

www.nature.com/scientificreports/ The elevation of suPAR is not solely associated with kidney diseases, but was also linked to inflammatory and diverse pathologic conditions such as rheumatologic diseases 13,14 , acute respiratory distress syndrome (ARDS) 15 , or different types of cancer 16 .
It is still unclear whether suPAR has a pathophysiologic or prognostic role in kidney transplant (KTx) patients. At present, only a few KTx studies have tried to clarify its biomarker value for recurrent FSGS after KTx [17][18][19] . Regarding contrary results of different studies, Winnicki et al. showed that suPAR can serve as valuable biomarker for FSGS, but as a pitfall values must be interpreted in the context of population and test methods used considering test-specific cut-offs 17 . However, its role in kidney function or transplant specific outcomes needs further clarification. Therefore, we herein first prospectively investigated the prognostic relevance of suPAR in patients immediately before and one year after KTx and the influence of KTx on its values.

Materials and methods
Study population. In this study, we prospectively included 100 consented patients (age ≥ 18 years) in the final analysis who had received a kidney transplant at our transplant center between April 2013 and October 2015 and were able to provide both serum samples (at KTx, and 1-year after KTx) for suPAR analysis. We originally enrolled a total of 160 patients, but had to exclude 60 patients who did not present to our outpatient clinic for 1-year follow-up, withdrew their consent, changed the transplant center, died (n = 7) or lost their graft (n = 2) in the first year after KTx. Mean follow-up time was 4.35 ± 1.02 years, median follow-up time 4.35 years (IQR 3.80-5.35 years), respectively. suPAR was measured in two different blood samples. The first sample was collected within 24 h before KTx, and the second was collected during a routine 1-year patient visit to our outpatient clinic. The initial immunosuppressive regimen consisted of basiliximab, tacrolimus (target trough 6-12 ng/mL), mycophenolate mofetil, and prednisolone. Anti-thymocyte globulin was administered to re-transplanted or highly immunized patients (PRA > 85%). AB0-incompatible patients received rituximab four weeks before KTx. Three patients with atypical hemolytic uremic syndrome (aHUS) as an underlying disease were given eculizumab in addition to basiliximab. Oral CMV-prophylaxis with valganciclovir was administered for 100 days in R + and 200 days in the D + /R−constellation.
ELISA. Plasma concentrations of suPAR were measured using the Quantikine Human uPAR ELISA assay (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The assay range is 62.5-4000 pg/ml, with a sensitivity of 33 pg/ml to suPAR. The samples above the concentration limit of the test were re-measured after tenfold dilution in Calibrator Diluent RD 6-10 reagent according to the manufacturer's directions. Blood samples were collected after gaining informed consent at hospital admission within 24 h prior to KTx. The baseline and one-year samples were collected from each patient and immediately sent to the research core laboratory. The serum was obtained by centrifugation for 10 min at 2000 g using a refrigerated centrifuge, transferred into clean polypropylene tubes, and stored at − 80 °C until time of assay.
Patient characteristics were taken from the hospital's electronic patient records. The data of all the participating patients was made anonymous prior to conducting an analysis. Moreover, written informed consent was obtained from all the participants. All experiments were performed in accordance with the current transplantation guidelines and the declarations of Istanbul and Helsinki. This study was approved by the local ethics committee, Ethik Kommission der Ärztekammer Westfalen-Lippe und der Medizinischen Fakultät der Westfälischen Wilhelms-Universität (No. 2013-364-f-S and No. 2019-109-f-S).
Outcome measures. The main outcome measures involved renal function (eGFR calculated using the CKD-EPI equation 20 and urine-protein/creatinine ratio (UPCR)) at years one to four after KTx. Other outcome parameters were patient and overall graft survival. Patient survival was defined as the time from KTx to death (due to any cause). When patients were lost to follow-up, the time from KTx to a patient's last contact was recorded. They were indicated as "alive". Overall graft survival was defined as the time from KTx to death (from any cause), graft failure, or the last contact, whichever occurred first. Re-initiation of dialysis or re-transplantation was considered as graft failure. Recipients after LD and DD were considered separately 21 .
Patients were subjected to kidney biopsy in case of increased creatinine levels (≥ 0.3 mg/dL) and/or a significant increase in proteinuria. The kidney biopsies were evaluated by two independent pathologists. The whole blood was analyzed for creatinine (enzymatic assay; Creatinine-Pap, Roche Diagnostics, Mannheim, Germany). Organ rejections were diagnosed as per the BANFF classification 22 .
Statistical analysis. Data was analyzed using IBM SPSS Statistics 26 (IBM Corp., Armonk, New York, USA). Normally distributed continuous variables have been presented as mean ± standard deviation (SD), and non-normally distributed continuous variables were presented as median and 1st and 3rd quartiles (interquartile range, IQR). Absolute and relative frequencies have been provided for categorical variables 21 .
Pairs of independent groups were compared using the Student's t-test for normally distributed data, the Mann-Whitney U test for non-normal data, and the Fisher's exact test or Chi-Quadrat-Test for categorical variables. To compare the paired data, we used the Wilcoxon test for continuous variables and the McNemar test for categorical variables.
The cumulative probability of developing eGFR loss > 30% in our KTx cohort was calculated using the Kaplan-Meier analysis, and the curves were compared using the log-rank test. A cut-off for suPAR after one year for patients at higher risk for eGFR-loss was identified by calculating Youden-indices based on a ROC-analysis.
To evaluate independent risk factors for the onset of eGFR loss > 30%, we performed multivariable Coxregression analyses. Patients' outcome data have been presented in Table 2.
In parallel, it was found that kidney function improved one year after KTx to a mean eGFR of 57.3 ± 20.1 ml/ min/1.73 m 2 (Table 1). Although LD recipients showed a tendency toward a higher eGFR compared to DD recipients (60.0 ± 20.3 ml/min/1.73 m 2 vs. 53.9 ± 19.5 ml/min/1.73 m 2 , p = 0.075), the difference was not statistically significant. Dialysis vintage tended to be associated with suPAR levels prior to KTx (n = 100, p = 0.067,). Upon a closer analysis, it was seen that preemptive recipients who never underwent dialysis had significantly lower suPAR levels (suPAR 5249 (2302, 7806) pg/ml, n = 8) compared to patients on any mode of dialysis before transplantation (suPAR 8392 (6011, 11,503) pg/ml, n = 92) (p = 0.006) (see Fig. 3A). The suPAR levels of patients on hemodialysis (suPAR 8322 pg/ml (5956, 11,475), n = 71) and patients treated with peritoneal dialysis (8075 (6936, 12,492) pg/ ml, n = 12) were comparable (p = 0.928) (see Fig. 3A). One year after KTx, the suPAR levels of patients on any mode of dialysis prior to transplant compared with preemptively transplanted patients became equal (Fig. 3B). www.nature.com/scientificreports/ The suPAR levels in patients one year after KTx were not correlated to the eGFR at the same time (p = 0.24, r = − 0.119). However, it was associated with the development of the eGFR between the second and fourth year after KTx. Higher suPAR levels one year after KTx could be associated with a higher eGFR-loss in the following three years (Fig. 4, n = 82 (18 patients lost of follow-up), p = 0.021, r = − 0.255).
In our study, a correlation between the suPAR levels and the incidence or the number of biopsy-proven allograft rejections could not be detected.
We found that only four patients experienced terminal graft failures by the time of the follow-up. Moreover, two patients died during the course of the study, and two patients died without losing their graft before. Due to these small number of events, we did not perform survival analyses with these endpoints.
Instead, we took a loss of renal function in terms of more than 30% of eGFR loss from year one as an endpoint 23,24 . eGFR-loss > 30% was stated, when it was constant for at least one month and did not increase subsequently. Our patient collective was divided into two groups based on a cut-off for suPAR below and above 6212 pg/ml by calculating the Youden-indices for a receiver operating characteristic analysis according to suPAR measured after one year. (n = 82 vs. n = 18). Patients with allograft loss were defined as eGFR-loss > 30%, patients who died with unimpaired allograft function were handled as negative for eGFR-loss > 30%. The Kaplan-Meier analysis and Log-rank test showed a significantly reduced time to eGFR-loss > 30% for patients with suPAR levels above 6212 pg/ml (33.5 vs. 61.9 months, Fig. 5).
The suPAR levels after one year as an independent risk factor for eGFR-loss > 30% subsequently. To evaluate whether suPAR after one year is independently associated with accelerated eGFRloss survival, apart from other known risk factors, we performed a multivariable Cox-regression analysis that included several known risk factors causing inferior allograft survival (Table 3).
Besides the well-known risk factors for kidney allograft failure such as age at the time of KTx (p = 0.017, HR 1.134) and previous KTx (p = 0.02, HR 39.639), the Cox-regression analysis confirmed suPAR one year after transplantation as an independent risk factor for eGFR-loss > 30% (p < 0.001, HR 1.001).

Discussion
suPAR levels are high in patients with chronic proteinuric and non-proteinuric kidney diseases and may predict disease courses 7,10 . A recent study by Hayek et al. provided evidence that suPAR may be directly involved in the pathogenesis of acute kidney injury. Higher preprocedural suPAR levels were shown to be associated with subsequent acute kidney injury after different medical procedures, e.g. coronary angiography. These findings were experimentally confirmed in a mouse model for acute kidney injury. Mechanistically, Hayek et al. described a sensitization of kidney proximal tubules by suPAR to injury through modulation of cellular bioenergetics and increased oxidative stress in a cell culture model, which was not found for podocytes 25 . However, Wei et al. described a mechanism for suPAR leading to podocyte foot process effacement through the activation of αvβ3 integrin in podocytes 5 , thereby acting as a driver of kidney injury. Moreover, Hahm et al. reported that BM-derived immature myeloid cells are responsible for the elevated pathological levels of suPAR in the case of LPS-treated mice with proteinuric kidney disease and thus, they described suPAR as a key contributor to glomerular dysfunction 1 .
In summary, whether suPAR acts as an originator of kidney injury, is produced and/or released as a consequence of (kidney) injury, or both can occur concurrently has not been completely elucidated yet 26 . Since data on suPAR and outcome after KTx apart from FSGS patients is still absent, we performed this prospective study. www.nature.com/scientificreports/ Our data shows that after KTx suPAR levels improved significantly (Fig. 1), in parallel with an increase in renal function (Table 1). Notably, there were distinct differences between LD and DD recipients. The suPAR levels in DD recipients were significantly higher compared to those receiving LD. This could perhaps be related to the fact that eight (16%) of the LD were preemptive with significantly lower suPAR values compared to those on dialysis prior to transplantation (Fig. 3A). Moreover, their dialysis vintage was shorter, and these patients were younger than DD recipients (Table 1). Interestingly, dialysis vintage was significantly correlated to suPAR levels prior to transplantation (p = 0.013, r = 0.258). In congruence, among other factors, dialysis vintage as well as the age of the patient have recently been linked to the suPAR levels in dialysis patients 11,12 . Additionally, since suPAR is excreted by the kidneys, end-stage renal disease may lead to an accumulation in the serum 27 . After KTx, the suPAR levels of both LD and DD recipients decreased to a comparable level at the 1-year mark (Fig. 2).
Similarly, morbidity and mortality of DD recipients are usually higher which is, among others, related to longer dialysis vintage 28,29 . Notably, higher suPAR levels have been shown to translate into all-cause and both CVD and non-CVD mortality in hemodialysis patients 11,12 . Thus, one may only speculate that lower suPAR levels in preemptive LD might contribute to preferable outcomes of LD KTx 28 . However, our study sample was too small to allow us to draw such conclusions.
Serum suPAR levels can indicate CKD prior to measurable function loss 10 . As the suPAR levels decline after KTx and as the native recipient kidneys usually remain in-situ after transplantation, the pathologically altered kidneys seem to be ineligible as a relevant suPAR source (due to progressive loss of function); otherwise, suPAR could be excreted by the working graft after transplantation, hence, kidneys seemed to have cleared suPAR from www.nature.com/scientificreports/ the circulation, at least in healthy volunteers 30 . However, our data confirmed the prognostical value of suPAR to predict the decline in functionality of the allograft (Figs. 4 and 5). We observed that the suPAR level one year after KTx is predictive of future graft function (eGFR) to some extent, and suPAR levels above a cut-off of 6212 pg/ml serve as a risk factor for a significantly accelerated decrease of eGFR. Notably, even after adjustment for several other known risk factors for accelerated loss of allograft function, suPAR levels remained significantly predictive for eGFR loss one year after KTx (see Table 3). This is in line with the observations made in CKD patients 9,10 . One should keep in mind that different suPAR tests from different manufacturers may result in different suPAR cut-off values despite high sensitivity and specificity, as recently reported by Winnicki et al. 17 .
Interestingly, there are interferences in the αvβ3 integrin pathway, which is induced by suPAR and calcineurin-mediated processes. These clacineurin-mediated processes are suppressed in KTx patients through the administration of calcineurin inhibitors (CNI) such as tacrolimus or cyclosporine. CNI exhibit antiproteinuric effects by stabilizing the actin cytoskeleton and stress fibers of podocytes through synaptopodin re-storage 31,32 . Furthermore, in lupus nephritis, a combination therapy of tacrolimus and mycophenolate mofetil (a standard of care after KTx) 33 , had an additive protective effect for the podocyte actin cytoskeleton by tacrolimus-induced synaptopodin-mediated activation of RhoA and mycophenolate mofetil-mediated VAV1 inhibition of Rac1 32 .  www.nature.com/scientificreports/ However, our study has limitations, as it is a single-center study and observational in nature. Further, for our study, we analyzed data of a relatively small cohort, which did not include data on non-renal causes for eGFR decline or proteinuria such as infections, CNI toxicity, or surgical problems. Another study limitation is that GFR was estimated using serum creatinine-based CKD-EPI formula instead of cystatin C-based calculation, which has some advantages, such as higher sensitivity and specificity, over creatinine measurement 34 .
In conclusion, on the one hand, our observations implicate that elevated suPAR is a consequence of kidney disease and chronical inflammation, as KTx drops suPAR levels significantly after resolving the state of end-stage renal disease. On the other hand, suPAR seems capable as an early marker for allograft dysfunction after KTx.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Variable
Hazard Ratio 95% CI P-value