By contrast, donor DDAVP was associated with improved 2-year graft survival (85.4% vs. 73.6%, log-rank P=0.003) (Fig. 2A). The survival benefit persisted after censoring for death (91.1% vs. 82.0%, log-rank P=0.01) (Fig. 2B), which was confirmed in multiple Cox regression analyses of both model 1 and model 2 (Table 3). Because there were some differences in donor management between the two procurement regions, we performed separate analyses in strata of Bavarian and non-Bavarian kidneys. The magnitude of the 2-year survival advantage was quite similar in BW (89.2% vs. 73.4%) and Bavaria (83.9% vs. 73.9%). However, the survival benefit was not statistically significant in Bavarian organs due to the limited number of donors unexposed to DDAVP. Differences in concomitant donor treatment regarding the administration of prednisolone (HR 0.90, 95% CI 0.53–1.52; P=0.69), insulin (HR 1.43, 95% CI 0.89–2.29; P=0.14), noradrenaline (HR 1.27, 95% CI 0.66–2.45; P=0.47), and the usage of UW (HR 1.28, 95% CI 0.77–2.11; P=0.34) were no significant explanatory variables of graft survival.
Stratifying the analyses by the median of cold ischemic time indicated that survival curves diverged further, if the kidney was transplanted after a cold storage period under 14 hr (DDAVP 91.3% vs. no DDAVP 77.8%, log-rank P=0.008) (Fig. 3A), whereas the beneficial effect of DDAVP was abrogated in the subgroup of recipients who were transplanted with a prolonged cold ischemic time exceeding 14 hr (DDAVP 91.2% vs. no DDAVP 86.5%, log-rank P=0.39) (Fig. 3B). This survival analysis yielded analogous findings, when death was classified as failure. Because it was recently demonstrated that donor pretreatment with dopamine improves early graft function (3), we reanalyzed death-censored graft survival by DDAVP exposure in the respective subgroups of dopamine pretreatment. DDAVP was associated with profoundly improved 2-year graft survival, if the donor was pretreated with dopamine (DDAVP 92.7% vs. 78.6%, log-rank P=0.006), which was not recognized in donors from the nondopamine group (DDAVP 89.7% vs. no DDAVP 84.8%, log-rank P=0.37). Redoing the analysis without censoring yielded analogous findings.
This study indicates that DDAVP administration in the DBD is associated with improved graft survival after kidney transplantation. Censoring for death confirms that the advantageous effect is actually related to graft survival itself, rather than to all-cause mortality. Numerous efforts have been made to reduce the deleterious effects of prolonged cold ischemia (12). Hence, it is particularly interesting that the survival advantage attributable to DDAVP occurred exclusively in patients who were transplanted after a short cold ischemic time below 14 hr, while the benefit was abrogated when cold storage exceeded 14 hr.
Apart from its antidiuretic effect, DDAVP binds to endothelial V2-vasopressin receptors, which results in cyclic adenosine monophosphate-mediated exocytosis of WPB (13). These secretory storage granules harbor preformed pro-thrombotic and pro-inflammatory constituents, whose rapid release enables the activated endothelium to react to stressors within minutes without necessitating previous protein biosynthesis (14). In organ transplantation, exposure to hypoxia during cold preservation and shear stress during reperfusion induce the exocytosis of WPBs (9, 15), releasing von Willebrand factor, interleukin-8, the adhesion molecule P-selectin, and the Tie2-antagonist angiopoietin-2. These WPB constituents account, at least in part, for the deleterious effects of reperfusion injury, because von Willebrand factor facilitates platelet adhesion (13), while interleukin-8 attracts leukocytes (16) which are tethered to the endothelium by P-selectin (17). Angiopoietin-2 plays a critical role in subsequent leukocyte transmigration by destabilizing the endothelial cells and in sensitizing the endothelium to the effects of tumor necrosis factor alpha-induced gene transcription (9, 18). This positive feedback loop after reperfusion may ultimately translate in accelerated transplant vasculopathy (19), a major drawback for long-term graft survival.
The disadvantage of fully viable WPB constituents does not necessarily challenge the benefit of a shorter cold ischemic time (20). Notably, in our cohort 80% of transplants were performed within the 18-hr time window where cold ischemia was not found to be detrimental for the outcome of the renal graft (20). Pretreatment of the DBD with DDAVP will disarm the graft's endothelium from injurious WPB constituents before transplantation, thereby ameliorating reperfusion injury. During cold storage, the endothelium undergoes depletion of the WPBs in a time-dependent manner (15), which may explain why DDAVP was ineffective when cold ischemic time exceeded 14 hr. Cold preservation hampers the constitutive turnover and repletion of WPB before reperfusion.
A recent human study indicates that the inflammatory response after reperfusion does not necessarily determine intermediate graft prognosis (21). This is in line with our observation that DDAVP increases long-term graft survival, while early clinical events such as DGF and BPAR remained unaffected. Our suggestion of a novel pharmacologic approach to protect the graft through WPB depletion parallels previous experimental studies. Qian et al. (22) reported that inhibition of WPB release by inducible nitric oxide synthase resulted in superior graft survival after murine cardiac transplantation. In experimental kidney transplantation, blocking P-selectin prevented ischemia/reperfusion-induced graft failure (17, 23).
Our findings extend an earlier RCT from Paris showing that donor treatment with DDAVP does not compromise initial graft function after kidney transplantation. Neither was any effect on long-term function detectable, because the trial was not designed to investigate graft survival. More importantly, the reported mean cold ischemia of 28 hr may have prevented the detection of DDAVP's protective potential (11). Because mean cold ischemic time has decreased to 14 hr in the Eurotransplant area at present (12), the generalizability of the French trial to current transplant conditions seems to be limited. Our results are supported by an incidental finding of Nijboer et al. (24) who observed superior renal function 1 and 3 years after transplantation, when the donor was treated with DDAVP. DDAVP seems to exert protection particularly in recipients of dopamine pretreated grafts in our study. Both dopamine and a short cold ischemic time preserve endothelial cell integrity (25–28), which is apparently a prerequisite for the WPBs' critical role in the endothelial stress response. Given that DDAVP's action on WPB exocytosis in donors before organ retrieval is causative for improving transplant outcome, it is conceivable that the survival advantage only became apparent in kidney grafts that were either pretreated with dopamine or subjected to short cold ischemia.
The effects of donor DDAVP on renal allograft survival are robust and of considerable clinical relevance. Nonetheless, several study limitations have to be acknowledged: First, the post hoc character indicates that our results remain nonconfirmatory. Second, as in all observational studies, our data are susceptible to potential confounding. To address the latter issue, we reanalyzed all outcome measures in two separate models of multivariate adjustment. For bias reduction, the technique of propensity scoring was applied through regression adjustment (29). In addition, the data were reanalyzed in strata of the procurement regions. None of the principal study findings was affected hereby. Nevertheless, it cannot be fully excluded that an unknown extraneous factor may have distorted results leading to a nonrandom error, because DDAVP exposure was driven by the clinical assessment of the donor centers. Third, we used the administration of DDAVP as a dichotomous variable for data analysis, because we lack detailed information on cumulative doses, timing, and duration of DDAVP treatment from the standardized donor information forms. Contrasting the drug's antidiuretic effect (8), DDAVP depletes the endothelial WPBs in a dose-independent manner. In fact, endothelial cells become progressively unresponsive to repeated administration of DDAVP over short intervals (<24 hr) (13, 30). Therefore, we consider this limitation to be of minor relevance.
A major strength of our study is that it is nested in the database of a large multicenter RCT (3). This implicates that the data were prospectively assessed and all study endpoints were prespecified, which enhances the internal validity of the study. The outcome measure of long-term graft survival is definitely the hardest endpoint available in kidney transplantation. Our study was carried out under real-life conditions in a multicenter setting, where transplant recipients were not exposed to protocol-mandated interventions. This enhances the generalizability of the presented results. Given DDAVP's pharmacologic mechanism of action, we can also provide a biologic plausibility to explain our findings. Nonetheless, the proposed interaction between DDAVP and WPBs lacks biopsy or other measured evidence in our study to move beyond the theoretical at present.
In conclusion, this study indicates that optimized care of the DBD before organ retrieval has the potential to substantially improve the outcome after kidney transplantation. In so doing, both preserving the graft's endothelial viability during cold ischemia and disarming endothelial cells from their injurious WPB constituents before reperfusion seems to offer a complementary pharmacologic approach. Standard use of combined donor pretreatment with low-dose dopamine and DDAVP should receive further evaluation by a RCT in kidney transplantation.
MATERIALS AND METHODS
Study Design and Patients
Rationale, design, and execution of the dopamine trial have been delineated elsewhere (3). The randomized dopamine trial (ClinicalTrials.gov Identifier: NCT00115115) was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice, and the International Conference on Harmonisation guidelines. The protocol was reviewed and approved by the Institutional Review Board (Independent Ethics Committee of the Medical Faculty of Mannheim, Germany) and the kidney advisory committee of the Eurotransplant International Foundation, Leiden, The Netherlands. In brief, between March 2004 and August 2007, 264 DBDs from two procurement regions in Germany—Bavaria and BW—were included in the trial. After confirmed brain death, and consent for donation, in accordance with national guidelines and transplantation legislation, eligible DBDs needed to be hemodynamically stable without catecholamine support, except norepinephrine at a dose not exceeding 0.4 μg/kg/min. All donors had to have a serum creatinine less than 1.3 mg/dL on admission and less than 2 mg/dL before randomization. All eligible donors were closely monitored to maintain hemodynamic stability within predefined thresholds for intervention.
Donor characteristics were obtained from standardized donor information forms, including dichotomous data on the coadministration of DDAVP. DDAVP was administered using intravenous bolus doses of 1 to 2 μg, followed by repeated maintenance doses of 0.5 to 1 μg every 2 to 12 hr, to limit urine output less than 200 mL/hr. All kidneys were preserved using static cold storage before transplantation.
Eligible recipients were aged 18 years or older and had to fulfill the usual criteria of a renal transplant candidate. Before wait-listing, they gave their consent that depersonalized medical data will be transmitted to Eurotransplant for scientific analyses. Transplant and recipient baseline characteristics were obtained from the Eurotransplant database. Recipient follow-up data were assessed using a standardized case report form. Only routine parameters were requested, including class and doses of immunosuppressive therapy during the 24 hr before and after transplantation, dialysis requirement, occurrence, and severity of BPAR and date and cause of allograft failure, and recipient death when this occurred. Anonymity of donors and recipients was ensured by the use of Eurotransplant code numbers for data collection.
We analyzed the effects of donor DDAVP by using the predefined study endpoints of the randomized dopamine trial (3). Grouping was based on donor DDAVP administration during intensive care before organ recovery. Efficacy endpoints were the incidence of DGF, as assessed by the requirement of single or multiple dialyzes sessions within the first week after transplantation, the incidence and severity of BPAR within 30 days posttransplant, and patient/allograft survival during 2 years after transplantation.
The study database provides a statistical power of 84% to detect a 10% difference in graft survival at a significance level of 0.05 given a cumulative event rate of 18% in the entire study population and taking into account that 362 kidney grafts were exposed to DDAVP whereas 125 remained unexposed. Quantitative data were evaluated using the two-sample Student t test. For qualitative data, the χ2- or the 2-sided Fisher exact test was applied, as appropriate. Cumulative and death-censored graft survival was calculated according to the Kaplan-Meier method, and group differences were assessed by the log-rank test. Multiple logistic regression analyses of the dichotomous study endpoints—DGF and BPAR—were carried out to control for putative confounding factors. In analogy, a multiple Cox proportional hazards model was applied for the analysis of graft survival until 2 years after transplantation, with and without censoring for death with a functioning graft. All outcome measures were evaluated by two separate regression models. In the first model, donor and recipient age and gender, cause of brain death, concomitant donor treatment (insulin and/or prednisolone), preservation solution, cold ischemic time, number of HLA mismatches, panel reactivity more than 5%, waiting time, and history of a previous transplant were included as possible explanatory variables. For bias reduction, the technique of propensity scoring was used through covariance adjustment in the second regression model (29). The propensity score defined as the conditional probability of assignment to a particular treatment was estimated by stepwise logistic regression of DDAVP administration on donor-specific covariates (31, 32). Propensity scoring considered the following variables: age, gender, cause of brain death, duration of brain death, occurrence of hypotensive episodes or cardiac arrest, serum creatinine, hypernatremia (defined as serum sodium concentration >145 mmol/L), systolic and diastolic blood pressure, urine output during the 24 hr before organ recovery, concomitant treatment with dopamine, noradrenaline, glucocorticoids, insulin, plasma expanders, and the region of organ procurement (Bavaria/BW). Stepwise backward selection was applied by defining a P more than or equal to 0.10 as an exit criterion for removal of the covariate from the full model. Significant variables in the propensity score were subsequently incorporated as additional covariates in the second regression models.
All results are presented as OR or HR, respectively, with a 95% CI for a one-unit change in the variable. Significance was defined according to a two-sided P less than 0.05 in all analyses. Statistical analyses were carried out with Stata Statistical Software for MS Windows (Stata Corp., College Station, TX).
This study is embedded in the database of an investigator-driven clinical trial conducted by the University Medical Centre Mannheim, Germany. The authors thank John H. Clorius (German Cancer Research Center, Heidelberg) for proofreading the manuscript and all collaborating transplantation centers who provided data to the trial. None of the collaborators were compensated for their contributions.
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
Brain-dead donor; Renal transplantation; Graft survival; Endothelium; Reperfusion injury