Delayed graft function (DGF) is a common early complication after kidney transplantation from donors after neurological or circulatory determination of death. A paired kidney analysis showed that DGF-associated risk of graft failure was greatest in the first year after transplantation, especially in recipients who subsequently develop acute rejection.1 Delayed graft function is primarily a consequence of ischemia/reperfusion (IR) injury, which results in postischemic acute tubular necrosis. The degree of posttransplant injury depends on a complex interplay of preexisting (pretransplant) donor kidney damage and pathogenic processes initiated upon reperfusion in the recipient.2
The mechanisms underlying posttransplant IR injury involve reactive oxygen species generation, which initiates innate immune activation, Toll-like receptor signaling and complement cascade activation.3,4 Emerging evidence from rodent and large animal models implicate a crucial role for the complement system, in particular as a proximal mediator of kidney IR injury. Kidney IR injury is significantly reduced in animals deficient in C3 or complement factor B, a component of the alternative pathway for complement activation. Likewise, deficiency in complement regulators decay accelerating factor (DAF, CD55) or factor H exacerbated IR injury.5,6 Complement activation after tissue injury results in local production of anaphylatoxins C3a and C5a. After binding to their specific receptors (C3aR and C5aR, respectively) which are expressed on both kidney cells and in bone marrow-derived immune cells, C3a and C5a function as vasodilators, chemo-attractants, and activators of endothelial cells, epithelial cells and infiltrating innate immune cells.7 Downstream complement activation causes membrane attack complex (MAC) formation which inserts pores into cell membranes, additionally activating endothelial cells via noncanonical nuclear factor-kB mediated signaling3 (Figure S1, SDC, http://links.lww.com/TP/B646).
In solid organ transplantation, ischemia and organ injury may commence in the deceased donor. Brain death has well-known effects on hemodynamic instability, hormone dysregulation, and donor organ immunogenicity.8,9 Current hypotheses include that this brain death-triggered local inflammation is partially responsible for inferior posttransplant outcomes (eg, higher incidence of DGF and acute rejection with poorer graft survival) of recipients of kidneys from donors who had neurological determinations of death compared with living donor kidneys.10 Experimental evidence indicates that brain death itself activates systemic complement, generating C5a, which upon binding to its receptor on kidney tubular cells contributes to kidney inflammation.11,12
These published studies on complement activation, brain death, and kidney injury raised the present hypothesis that C3a and C5a concentrations in donor urine before transplantation could help assess risk of early posttransplant IR injury and DGF. To test this hypothesis, we conducted a large, prospective, observational cohort study in which we measured C3a and C5a in urine samples of deceased donors at the time of kidney procurement and determined associations with early posttransplant outcomes in their recipients.
STUDY COHORT AND METHODS
The study cohort was selected from a prospective, multicenter, observational cohort study conducted via collaboration between 5 organ procurement organizations (OPOs) and 5 academic centers (Icahn School of Medicine at Mount Sinai, New York; Yale University School of Medicine, New Haven; Wayne State University, Detroit; Barnabas Health, Livingston; University of Pennsylvania, Philadelphia). Data collection methods are described in detail elsewhere.13 The subset of donors we used in this analysis resulted in kidney transplants at the 5 transplant centers, where detailed chart abstraction was performed for recipient outcomes.
Participating OPOs enrolled deceased kidney donors between May 2010 and December 2013. Yale University served as the sample and data-coordinating center. Organ procurement organization personnel followed institutional protocol for managing donors. Healthy volunteers included 5 men and 5 women with a median age of 32 (range, 24-61), recruited by oral invitation and general announcements at Mount Sinai under an IRB. The OPO scientific review committees as well as institutional review boards for the investigators approved the study, and waiver of consent was granted for recipient chart reviews.
This study also used data from the Organ Procurement and Transplantation Network (OPTN). The OPTN data system includes data on all donors, wait-listed candidates, and transplant recipients in the U.S., submitted by the members of the OPTN. The Health Resources and Services Administration, which is part of the US Department of Health and Human Services, oversees the activities of the OPTN.
Definitions of Variables and Outcomes
Expanded criteria donors (ECD) were defined as age of 60 years or older, or age 50 to 59 years with 2 of the following: history of high blood pressure, serum creatinine level greater than or equal to 1.5 mg/dL, or death resulting from cerebral vascular accident (CVA). We calculated the Kidney Donor Profile Index (KDPI) as described, accounting for the following donor-related risk factors: age, height, weight, ethnicity, history of hypertension, diabetes, CVA as cause of death, terminal serum creatinine concentration, hepatitis C virus status, and donation after circulatory death (DCD) status.14 The Kidney Donor Risk Index (KDRI) was then mapped to the corresponding KDPI, representing a cumulative percentage scale relative to all US deceased donors in 2010.15
Donor acute kidney injury (AKI) was defined according to AKI Network criteria based on admission to terminal serum creatinine (irrespective of time between measurements and of urine output cutoffs) as follows: stage 1, increase in serum creatinine by ≥0.3 mg/dL or 1.5 to less than twofold increase; stage 2, 2 to less than threefold increase; and stage 3, threefold or greater increase, or terminal serum creatinine 4.0 mg/dL or greater after a rise of at least 0.5 mg/dL (no donors were dialyzed). Recipient outcomes were obtained from the OPTN/United Network for Organ Sharing (UNOS) database and were compared between study donor groups. Delayed graft function was defined conventionally as any need for dialysis in the first week after transplantation. Graft failure was defined as return to chronic dialysis or a repeat transplant and was censored for patient death. Estimated glomerular filtration rate (eGFR) was calculated from 12-month serum creatinine value reported in UNOS follow-up forms using the Chronic Kidney Disease Epidemiology Collaboration equation.16 For recipients with graft failure (n = 40), we imputed 12-month eGFR as 10 mL/min per 1.73 m2 and carried forward the last available serum creatinine value for recipients that died (n = 27).
Sample Collection and Complement Measurement
Ten milliliters of fresh donor urine was collected from the Foley catheter tube in the procurement operating room, immediately placed on wet ice and transferred to the OPOs for temporary storage at −80°C before batched shipments. Samples were processed after a single controlled thaw, separated into 1 ml barcoded aliquots, and then stored at −80°C without the addition of protease inhibitors until biomarker measurement. Laboratory personnel at Mount Sinai performed all measurements for complement proteins using commercially available enzyme-linked immunosorbent assay kits (OptEIA, BD Biosciences). Standards and samples were added to 96-well plates, washed, combined with a mixture of biotinylated antihuman C3a or C5a antibody and streptavidin horseradish peroxidase, and then read at 450 nm for quantitation.
Descriptive statistics were reported as mean (standard deviation) or median [interquartile range] for continuous variables and as frequency (percentage) for categorical variables. Comparisons were made using Wilcoxon rank sum tests for continuous variables and Pearson χ2 test for categorical variables. To directly estimate the relative risk (RR) between the urinary biomarkers and DGF, we fit multivariable modified Poisson regression models.17 When Poisson models are used on a binary outcome, the error of the RR is overestimated. To avoid this issue we are using Zou's approach to modify the Poisson regression models with a sandwich estimation procedure for a robust error variance.17 These models were adjusted for KDRI, cold ischemia time, HLA mismatches, recipient race, recipient body mass index (BMI), and preemptive transplantation. To determine if donor AKI status modified the association between urinary complement and recipient DGF, an interaction term between urinary biomarker and donor AKI was included in each model. The interaction term for urine C5a was significant (P = 0.05) and thus results were stratified by donor AKI status. Biomarkers were log-base 2 transformed. To determine if donor AKI status modified the association between biomarkers and DGF, interaction terms were included in the models. All regression models were clustered at the level of the kidney donor.
To estimate the discriminatory ability of the biomarkers, we calculated area under the receiver-operating characteristic curve statistics and calculated positive and negative predicted values. SAS 9.3 statistical software for Windows (SAS Institute, Cary, NC) was used for statistical testing, and all statistical tests and confidence intervals (CI) were 2-sided with a cutoff significance level of 0.05.
Deceased Donor Characteristics According to AKI Status
Of 469 donors, 114 (24%) were classified as having AKI stage 1 or higher. Factors that were significantly associated with donor AKI included black race, ECD status, CVA as cause of death, and high KDPI (Table 1). Eighty-four percent of the donors received a vasoactive agent (dopamine, epinephrine, neosynephrine, levophed, pitressin) within 24 hours before cross clamp.
Deceased Donor and Recipient Characteristics According to DGF Status
We analyzed outcomes in 902 recipients of kidneys from 469 deceased donors. Most donors had both of their kidneys transplanted. In 36 donors, only 1 kidney was transplanted; the discarded organ was included in the donor analysis. The incidence of DGF in the cohort was 32% (n = 291). Recipient factors associated with DGF included black race, high BMI, machine perfusion, longer dialysis vintage and cold ischemia time. Comparison of clinical donor characteristics for kidneys with and without subsequent DGF showed that KDPI, DCD status, older donor age, CVA as cause of death, AKI, and donor terminal serum creatinine were all associated with DGF. There were no differences in the proportion of ECD kidneys between the DGF and non-DGF groups (Table 2).
The 1-year acute rejection rate in the cohort was 9% and was not different in DGF versus non-DGF (9% vs 8%; P = 0.8). Median 1-year eGFR was 40.7 (33.1-54.5) mL/min per 1.73 m2 in recipients with rejection and 58.5 (46.3-74.7) mL/min per 1.73 m2 in recipients without rejection (P < 0.0001).
Anaphylatoxins Are Detectable in Deceased Donor Urine
Both C3a and C5a were detected in deceased-donor urine samples. Compared with donors with brain death urine samples from DCD donors contained more C3a (0.32 [0.21-0.47] ng/mL vs 0.38 [0.25-0.63] ng/mL; P = 0.04) and more C5a (0.28 [0.13-0.78] ng/mL vs 1.0 [0.24-2.17] ng/mL; P < 0.001).
C3a concentrations were slightly higher in donors with KDPI less than 60% compared with those with KDPI above 60% (P = 0.04). Of note, urinary C5a was detected in only low concentrations in healthy controls (0.11 ± 0.05 ng/mL; n = 10) (Figure 1).
Urinary C5a Correlates With Donor AKI
We observed a significant association between donor AKI and urinary C5a, with threefold higher median C5a concentration in donors with AKI stage 2/3 versus those without AKI (0.92 [0.32-1.62] ng/mL vs 0.27 [0.14-0.83] ng/mL, P < 0.001; Figure 1, Table S1, SDC, http://links.lww.com/TP/B646). These findings persisted after indexing urine C5a to urine creatinine concentration (Table S1, SDC, http://links.lww.com/TP/B646). Urinary C3a values were not associated with AKI status (Table S1, SDC, http://links.lww.com/TP/B646).
Thirteen (18%) of the 71 DCD donors met criteria for AKI. In this DCD subset, we observed a nonsignificant trend toward higher C5a concentration in the setting of donor AKI compared with no AKI (1.57 [0.47, 2.17] ng/mL vs 0.72 [0.24, 2.18] ng/mL).
To test whether C5a correlates with other donor injury biomarkers we analyzed our data on urinary KIM-1, neutrophil gelatinase–associated lipocalin (NGAL), IL-18. We observed a strong direct correlation between C5a and these injury markers, supporting a mechanistic relationship (Table S2, SDC, http://links.lww.com/TP/B646).
Donor Urine C5a Correlates With Risk of Developing Posttransplant DGF
Of the 902 transplanted kidneys, 32% experienced DGF. We observed significantly higher donor urine C5a values for recipients who developed DGF compared with those who did not (0.42 [0.15-1.28] ng/mL vs 0.28 [0.13-0.87] ng/mL; P = 0.002). In contrast, donor urinary C3a was not different by DGF status (Table 3). In multivariable analyses, donor urine C5a remained associated with DGF independent of clinical variables known to associate with DGF, including KDRI, cold ischemia time, HLA mismatch, and the following recipient characteristics: race, BMI, and pretransplant dialysis (Table 4).
We previously showed that donor AKI stage is associated with recipient DGF.18 Donor AKI showed significant effect modification with C5a (P = 0.05) but not C3a (P = 0.9) on the outcome of DGF. Because we saw the effect modification with C5a, we presented C3a with the same approach. Because we observed that donor AKI stage was strongly associated with increased donor urinary C5a concentrations in the current analysis (P < 0.001; Figure 1, Table S1, SDC, http://links.lww.com/TP/B646), we tested whether donor C5a was associated with recipient DGF independent of donor AKI. We first analyzed the relationship between urinary C5a and DGF stratified by donor AKI. Of the 216 recipients of donor kidneys with AKI stage ≥1 (24% of the total cohort, n = 902), 95 (44%) developed DGF. In the 680 recipients of kidneys from donors without AKI (72% of the total cohort), 192 (28%) developed DGF. In this DGF cohort, we observed a nonsignificant trend toward higher donor urine C5a levels in the donor AKI group compared with non-AKI group (0.56 [0.18, 1.54] ng/mL vs 0.37 [0.15, 1.27] ng/mL; P = 0.35; Table 3). Figure S2 (SDC, http://links.lww.com/TP/B646) summarizes the occurrence of donor AKI and DGF according to donor type. Analyses adjusted for donor and recipient characteristics showed donor urinary C5a independently associated with recipient DGF only in the non-AKI donor subset, thereby identifying an additional high-risk group (Table 4).
We also tested the predictive value of C5a for development of DGF, considering values within the 1st tertile of C5a level as negative and values within the second or third tertiles as positive results. In donors with AKI, C5a had positive and negative predictive values of 44% and 56%, respectively. This suggests that donor urine C5a does not add to the predictive value of donor AKI for the development of DGF. In non-AKI kidneys, C5a had positive and negative predictive values of 32% and 78%, respectively, suggesting that low urinary C5a concentrations and the absence of donor AKI represent low risk for DGF (Table S3, SDC, http://links.lww.com/TP/B646). The area under the curve for predicting DGF with C3a and C5a were 0.51 (95% CI, 0.47-0.55) and 0.56 (95% CI, 0.52-0.60), respectively (Figure S3, SDC, http://links.lww.com/TP/B646).
Donor Urinary C3a/C5a And Posttransplant Kidney Function
To test whether donor urine C3a or C5a concentrations were associated with 12-month posttransplant graft function, we compared urinary C5a levels within the 1st tertile with the second or third tertiles. We observed a nonsignificant trend toward higher eGFR in the lowest C5a tertile for the total cohort (T1: 59.4 mL/min per 1.73 m2 vs T2&3: 56.5 mL/min per 1.73 m2; P = 0.09). However, stratifying by donor AKI revealed no clear association for urinary C3a (data not shown) or C5a with 12-month eGFR (Table S4, SDC, http://links.lww.com/TP/B646). To determine if 12-month eGFR differed by C5a tertile when stratified by both the donor AKI profile and corresponding recipient DGF status, we tested a 3 way interaction term in an ANOVA model. The interaction term of urinary C5a tertile X donor AKI X recipient DGF was not significant (P = 0.23).
This a prospective, observational, multicenter cohort study to test the potential utility of C3a and C5a within deceased donor urine samples as biomarkers for posttransplant outcomes. Our findings indicate that C5a detected in donor urine obtained at the time of organ procurement can be used to assess the risk of developing DGF in the recipient. Elevated donor urinary C5a was independently associated with DGF and provides clinically useful information beyond donor terminal serum creatinine values or AKI stages. For patients who received kidneys from donors without AKI, elevated urinary C5a was associated with an elevated risk of DGF and low C5a had a high negative predictive for DGF. For recipients of donor organs with AKI, urinary C5a provided no additional predictive utility for DGF.
Deceased-donor AKI is associated with organ discard and DGF, but data on longer-term allograft survival are limited.2 Nevertheless, to advance DGF prevention and treatment, it is important to understand the effects of IR-induced AKI and dissect and evaluate the influence of factors related to the donor and to the recipient. Our current inability to accurately predict or quickly diagnose DGF leads to misclassification of patients in clinical trials aimed at preventing or treating DGF.19 For example, ameliorating IR injury after transplant would likely only reduce DGF when IR injury is the primary cause of DGF. The same treatments would be ineffective when DGF is due to recipient comorbidities, such as diastolic heart disease.
Taken together with clinical information, biomarkers measured in the donor could guide perioperative recipient management. We have previously shown that the level of kidney injury molecule-1 (KIM-1) renal tissue expression before reperfusion correlated inversely with renal function at the time of procurement, but not with the occurrence of DGF.20 In addition, our group found that donor urinary concentrations of NGAL, KIM-1, IL-18, and liver-type fatty acid binding protein (L-FABP) each strongly associated with donor AKI but were of limited value in predicting recipient DGF.21
In contrast to previous donor biomarkers, we found that doubling of donor urinary C5a concentration was independently associated with 23% increased risk of DGF. Our findings also support the emerging concepts that complement activation is pathogenically linked to renal damage and IR-initiated transplant kidney injury in humans.22,23 Brain death is a trigger for complement activation and associates with increased circulating C3a, C5a, and C5b-9 levels; whereas systemic C5a is believed to mediate renal inflammation via tubular C5a-C5aR interaction.11,24 Systemic and renal complement activation is thought to exacerbate IR injury and the inhibition of complement-improved graft function after transplantation in preclinical models.12,25 Anti-C5a antibody treatment of pretransplant donor rat kidneys during cold ischemia protected renal function and prolonged graft survival.26
Our data show that complement activation is similarly ongoing in humans because we find more C5a in urine samples from deceased donors than from normal volunteers (analogous to living donors). We focused on urinary and not blood complement levels because urinary C3a and C5a reflect filtered as well as locally (especially by tubular cells) produced and activated complement.27 An important goal of future studies is to clarify the triggers and mechanisms of complement activation during brain death and other events before organ retrieval.
We found that there was significantly higher C3a and C5a generation in DCD donors compared with brain dead donors. DCD donors result in prolonged warm-ischemia during kidney procurement, and the increased anaphylatoxin levels seen with DCD are likely due to complement activation through the coagulation activation pathway.28 Thrombin can cleave C5 to C5a and C5b to activate terminal complement pathway.29 Our studies build upon previous reports in mice supporting a predominant role for C5a and a minor role for C3a in the pathogenesis of AKI. Although both C3a and C5a were upregulated in postischemic mouse kidneys in our study, the pathogenic role of C5a dominated.7 C5a is downstream of C3a and there is further amplification (Figure S1, SDC, http://links.lww.com/TP/B646). It is possible that this amplification cascade is one of the reasons why we observe higher amount of and more pronounced effects with C5a and not C3a.30 C5a is a potent chemoattractant, but the role of C3a in chemotaxis is less clear. In addition, C5a has multiple downstream effects including direct proinflammatory effects on tubular and endothelial cells.7,31 These cells express C5aR and C5a/C5aR binding has been shown to induce inflammasome activation that results in IL-18 release.32 C5a is 1 of 2 cleavage products of C5, the other being C5b. C5b initiates the MAC formation and MAC insertion on endothelial cells induces noncanonical NFkB activation, which again results in release of cytokines and, potentially other markers of injury.3 The strong direct correlations we observed between C5a and the injury marker NGAL, IL-18, and KIM1 support the mechanistic relationship. Hypoxia promotes reactive oxygen species production, causing cell injury and up-regulation of Toll-like receptor and other molecules like HMGB1, leading to further complement production and immune cell recruitment.4,33
Our newly documented association between donor urine C5a levels and recipient DGF has important clinical implications. The finding that anaphylatoxins are released in donors before organ removal provides a strong rationale to initially target complement in the donor and then extend this treatment into the recipient. For example, eculizumab blocks C5 convertase activity, thereby preventing C5a production and MAC formation. Eculizumab has been tested to evaluate its potential to reduce the rate of peritransplant ischemic acute injury in humans. Preliminary results of the Prevention of Delayed Graft Function Using Eculizumab Therapy (PROTECT) study (ClinicalTrials ID NCT02145182) found a nonsignificant difference in the incidence of DGF, graft loss, or loss to follow up (35.9% with eculizumab vs 41.7% in placebo-treated controls).34 However, several more anticomplement drugs are in development,2,35 and it may be possible to improve outcomes by providing anti-DGF therapy only in those recipients at highest risk of developing DGF. Our study suggests that an approach to such enrichment could be restricting this therapy to recipients of kidneys with elevated donor urine C5a levels. Similar to KIM-1, for example, it may be possible to develop urine C5a assays that utilize rapid microsphere-based dipstick technology and require only small sample volumes.
The strengths of our study include standardized, prospective and a large sample size permitting adjusted analyses of donor, transport and recipient variables. However, there are important limitations to the study. First, we could not analyze specific donor events that could have influenced complement production such as duration of brain death or degree of hemodynamic instability. Second, despite adjustment for critical donor and recipient factors, unmeasured confounding variables is possible given the observational study design. Third, we only assessed at a single timepoint and could not extend C3a/C5a measurement into the peritransplant period, which can also influence graft outcome. In addition, other markers of complement activation like properidin, a key factor in alternative pathway complement activation, and the formation of C5b-9, are other potential makers of complement activation in the context of transplantation.
The finding that urinary C5a independently associates with the severity of renal injury in the donor and the recipient highlights the important role of C5a in renal damage in humans. Further innovative studies are needed to test the utility of combining C5a measurements with complement-targeted therapies.
The authors thank Isabel Butrymowicz and Rowena Kemp for their assistance with data and sample coordination for this multicenter study. We also thank the OPOs Gift of Life Philadelphia (Sharon West, Vicky Reilly), the New York Organ Donor Network (Harvey Lerner, Anthony Guidice, Allison Hoffman), the Michigan Organ and Tissue Donation Program (Burton Mattice, Susan Shay), and the New Jersey Sharing Network (William Reitsma, Cindy Godfrey, Alene Steward, Joel Padilla Benitez).
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