Donor noradrenaline use is associated with better allograft survival in recipients of pancreas transplantation

Introduction Outcomes following pancreas transplantation are suboptimal and better donor selection is required to improve this. Vasoactive drugs (VaD) are commonly used to correct the abnormal haemodynamics of organ donors in intensive care units. VaDs can differentially affect insulin secretion positively (dobutamine) or negatively (noradrenaline). The hypothesis was that some VaDs might induce beta-cell stress or rest and therefore impact pancreas transplant outcomes. The aim of the study was to assess relationships between VaD use and pancreas transplant graft survival. Methods Data from the UK Transplant Registry on all pancreas transplants performed between 2004 and 2016 with complete follow-up data were included. Univariable- and multivariable-adjusted Cox regression analyses determined risks of graft failure associated with VaD use. Results In 2,183 pancreas transplants, VaDs were used in the following numbers of donors: dobutamine 76 (3.5%), dopamine 84 (3.8%), adrenaline 161 (7.4%), noradrenaline 1,589 (72.8%) and vasopressin 1,219 (55.8%). In multivariable models, adjusted for covariates and the co-administration of other VaDs, noradrenaline use (vs non-use) was a strong predictor of better graft survival (hazard ratio [95% confidence interval] 0.77 [0.64–0.94], p = 0.01). Conclusions Noradrenaline use was associated with better graft survival in models adjusted for donor and recipient variables – this may be related to inhibition of pancreatic insulin secretion initiating pancreatic beta-cell ‘rest’. Further research is required to replicate these findings and establish whether relationships are causal. Identification of alternative methods of inducing beta-cell rest could be valuable in improving graft outcomes.


Introduction
Pancreas transplantation is a highly effective life-saving therapy for patients with insulin-dependent diabetes and end-organ damage from renal failure or unavoidable episodes of severe hypoglycaemia.Owing to a welldocumented shortage of organs for transplantation, there is a need to optimise methods for the assessment and selection of high-quality pancreases.Outcomes are improving, but historically remain far from ideal: six of ten pancreas grafts have failed at 5 years when transplanted alone, and three of ten have failed at 5 years when the pancreas is transplanted together with a kidney. 1 Technical considerations provide one explanation for these poorer outcomes and these have evolved and improved considerably over time. 2 Immunological factors including sensitisation from prior kidney transplants is a probable factor behind the discrepancy in outcomes between synchronous pancreases and kidney transplants compared with pancreases transplanted after kidney transplantation. 3n organ donors, the process of brainstem death results in high levels of systemic catecholamines affecting all organs, including the pancreas. 4,5Catecholamine-based vasoactive drugs (VaDs) are commonly used to correct the hypotension and reduced cardiac output experienced by organ donors in intensive care units.However, this may inadvertently affect organ quality.The donor care bundle produced by NHS Blood and Transplant recommends aiming to achieve mean arterial pressure values between 60 and 80mmHg by correction of hypovolaemia, with the administration of fluid boluses in the first instance and then vasopressors. 6Vasopressin is recommended as the vasopressor of choice, and it is advised to wean or stop catecholamines.If inotropes are to be used then dopamine is recommended as the agent of choice, although dobutamine is often used.
Pancreatic beta cells receive sympathetic autonomic innervation and express α2-adrenergic receptors that inhibit insulin secretion and β2-adrenergic receptors that *Martin K Rutter and David van Dellen contributed equally to this work.stimulate insulin secretion. 7,8Adrenergic receptors are G protein-coupled receptors (GPCRs).VaDs vary in their affinity for certain GPCRs and as such may have variable impacts on insulin secretion (Table 1).
The relationship between VaDs and outcomes after pancreas transplantation is unknown.Because these drugs have effects on insulin secretion, we hypothesised that some VaDs might impact pancreas transplant outcomes.This study aimed to assess relationships of VaD use to pancreas transplant graft survival.

Methods
Data on all pancreas transplants performed between 1 January 2004 and 1 November 2016 from the UK Transplant Registry, held by NHS Blood and Transplant, were reviewed.All data were collected prospectively with written informed consent from donor families and transplant recipients.
All-cause pancreas graft failure was defined as the time from transplant to loss of insulin independence.Patients dying with a functioning graft were censored.
The distributions of all donor and recipient variables were assessed for form and completeness.Cases with missing outcome data (n = 88) were excluded.Data for cold ischaemic time were missing in 9% of cases, donor creatinine in 12% of cases and recipient body mass index (BMI) in 27% of casesthis was addressed by using pooled results from multiple imputation using the fully conditional specification (Markov Chain Monte Carlo) algorithm.Missing data for all other variables were infrequent (< 1%) and cases with missing data relating to independent variables were excluded from individual analyses at that level.

Statistical analyses
We assessed relationships between exposures (VaDs) and donor or recipient clinical variables to identify potential confounders of relationships between exposures (VaDs) and outcome (graft failure) using Fisher's exact test, Student's t-test or the Mann-Whitney U-test as appropriate.Univariable Cox regression determined hazard ratios (HR [95% confidence interval {CI}]) for graft failure associated with clinical variables identified as being related to exposures to increase understanding of their potential role as confounders (p < 0.1).The association between VaD use and graft failure was determined using a Cox regression model, which was adjusted for co-administration of VaDs and also clinical variables related to graft failure in a Cox regression model (p < 0.05).
Statistical analyses were performed using IBM SPSS statistics version 22 (IBM, Armonk, NY).
Use of noradrenaline in donors was associated with both donor and recipient non-White ethnicity, previous history of hypertension, previous history of cardiac disease, peri-retrieval hypotension, donation after brain death (DBD), trauma and hypoxic brain injury as cause of  2 and 3).
In a univariable Cox regression model, use of noradrenaline was related to a lower risk of pancreas graft failure (HR [95% CI] 0.74 [0.61-0.89],p = 0.001) (Table 4).In the same analysis the following were related (p < 0.1) to all-cause pancreas graft failure: increasing donor age; increasing donor BMI; donor history of previous hypertension; cardiac arrest (but not its duration) during the donation phase; donor cause of death involving trauma, intracranial haemorrhage and hypoxic brain injury; increasing recipient age; type of transplant; and increasing cold ischaemic time ( After adjusting for co-administration and non-administration of donor drugs, only noradrenaline use was associated with better graft survival (HR [95% CI] 0.70 (0.53-0.92), p = 0.01) although dobutamine use was associated with a non-significant trend for adverse graft survival (1.50 [0.99-2.27],p = 0.055) (Table 5).After including all potential confounders in a multivariableadjusted Cox regression model, donor noradrenaline use was significantly related to a 26% lower risk of pancreas graft loss (HR [95% CI] 0.77 [0.64-0.94],p = 0.01) (Table 6; Figure 1).

Discussion
This study demonstrates that donor use of noradrenaline was associated with better pancreas graft survival after pancreas transplantation.Noradrenaline use appeared beneficial irrespective of the type of pancreas transplant (PTA or PAK procedures rather than SPK).If validated, these data have implications for donor selection and the optimal management of pancreas transplant donors.

Prior studies
Only one previous study investigated the relationship between VaDs and outcome after pancreas transplantation.In this small case series of 59 pancreas transplant recipients, there was no difference in outcomes in relation to the use of catecholamine-based VaDs or desmopressin. 9This small study may have been underpowered to detect differences in outcomes in relation to the exposure.
Poorer graft survival in patients receiving PAK remains a topic of on-going interest and debate regarding the factors accounting for this difference.Immunological factors are likely to be key contributors, and our data report that more than 60% of patients undergoing pancreas transplantation were in mismatch group 4 ([1 DR and 2 B] or [2 DR]).The effects of high mismatch in the PAK cohort are likely to compound the effects of prior sensitisation from initial kidney transplantation.These data are corroborated by an analysis of UK Transplant Registry data comparing live donor kidney transplantation with SPK, where only 30% of live donor kidney transplants were in group 4. 10 In patients with a pancreas graft functioning for more than 90 days, survival was superior to that experienced by recipients of live donor kidney transplantation.These data illustrate the important beneficial effects of good glycaemic control on patient survival.Interestingly, in a single-centre analysis from Spain, pancreas graft survival was worse with live donor kidney transplantation compared with deceased donor transplantation. 3

Mechanistic insights
It is unclear how noradrenaline use is associated with better graft outcomes and what underlying mechanisms may contribute to this finding.However, we can speculate that the inhibition of pancreatic insulin secretion leads to a period of beta-cell 'rest'.Beta-cell rest is an important concept in diabetes medicine, and can lead to improvements in glycaemic control and a reduction in beta-cell death. 11This hypothesis is supported by our data: donors who received noradrenaline were significantly more likely to have also required insulin to treat hyperglycaemia.
Noradrenaline has a high affinity for α2 receptors and little effect on β2 receptors.Noradrenaline exerts its potent inhibitory effect on insulin secretion by activating GPCRs in several ways.The predominant mechanism is through inhibition of the exocytosis of secretory granules, also termed the 'distal' effect, by its action on G βγ protein. 12,13Heterotrimeric Gi proteins (subsets 1 and 2) also act by retarding the process of refilling empty insulin secretory granules, thus disrupting the state of the readily releasable pool of insulin-containing granules. 14o proteins reduce the number of docked granules (i.e.those granules at an immediate state of secretory readiness). 14Activation of K ATP channels by Gi/Go proteins hyperpolarises the beta cell and prevents gating of voltage-dependent Ca 2+ channels, which increase intracellular Ca 2+ concentration and trigger insulin release. 157][18] Finally, Gz proteins regulate endocytosis of insulin secretory vesicles. 19he data in this study were suggestive of a potentially adverse effect of donor dobutamine use on graft survival, although the results were not statistically significant.Dobutamine is a synthetic catecholamine with a complex mixture of affinity to adrenergic receptors.The (-)-isomer of dobutamine is a potent partial alpha agonist, whereas the (+)-isomer is a potent beta agonist and alpha antagonist. 20When dobutamine is administered clinically (in its racemic form) it exerts a partial alpha agonist effect and antagonises the alpha effect in physiological circumstances of high sympathetic nervous activity, such as brain death. 21It exerts a strong beta-1 activity, but only partial agonism of the beta-2 receptor and competitively inhibits the effect of adrenaline at the beta-2 receptor. 20,22acemic dobutamine is reported to have a higher affinity for the β1-adrenergic receptor than for the β2-adrenergic receptor in a 3:1 ratio. 23As such, dobutamine has been shown to stimulate insulin secretion in healthy normal men. 24drenaline, which is synthesised through the methylation of the primary amine of noradrenaline, has a lower affinity for α2-adrenoceptors (AR) but a greater affinity for β2-receptors. 20Its mechanism of action upon the α2-AR in the pancreatic beta cell is the same as for norepinephrine.However, the underlying mechanism of action and mediators of β2-AR-induced insulin secretion are less well understood.Nonetheless, there is a clear causal 21 , and dose-dependent relationship between β2 agonists and insulin secretion from islet cells. 22,23][26] Dopamine, the most basic catechol structure, is the upstream molecule from which both norepinephrine and epinephrine are derived.Dopamine receptors can be classified into two groups: D1-like, which include D1 and D5 receptors; and D2-like, which include D2, D3 and D4.The presence of D2-like receptors on pancreatic beta cells has been confirmed, and agonism of these receptors inhibits glucose-stimulated insulin secretion via modulation of Gα i receptors. 27,28These findings were confirmed by Simpson et al who also demonstrated an autocrine role for dopamine in the regulation of insulin secretion. 29asopressin, meanwhile, via Gq proteins, stimulates both insulin and glucagon secretion. 30,31Vasopressin receptors have been identified on both pancreatic alpha and beta cells, and the co-stimulation of both insulin and glucagon secretion may account for the absence of any effect on graft outcomes in this study.
Co-activation of α2 and β2 receptors has been a poorly understood aspect of adrenergic receptor research.There is some evidence to support an acceleration of agonist-stimulated α2-receptor endocytosis under β2 co-activating conditions. 32Other research, however, suggests that co-activation of α2 and β2 receptors leads to desensitisation and downregulation of α2 receptors and results in a 67-fold reduction in the threshold concentration of adrenaline required for α2 downregulation. 33Given that noradrenaline was co-administered with dobutamine in 88% of cases, this hypothesis may provide an explanation for the lost attenuation of α2 receptor activity in the dobutamine subgroup.
Beta-cell rest may also be induced by the administration of exogenous insulin, and use of supplementary insulin therapy in both organ donors and pancreas and islet transplant recipients has been investigated. 34We have previously shown that use of insulin therapy to treat hyperglycaemia (glucose > 10mmol/L) in organ donors is associated with worse glycaemic control at 3 months after islet transplantation and also higher rates of isolated islet failure at 3 months following pancreas transplantation. 35,36It is conceivable that outcomes may have been poorer still in the cohort of donors experiencing hyperglycaemia had they not Sex (male) 338 ( 57   Adverse outcomes in the DCD subgroup are more commonly associated with warm ischaemia arising between the withdrawal of life-sustaining therapies and the perfusion of cold preservation solutions.

Strengths and weaknesses
This study has several strengths: it is the largest to assess relationships between VaDs and graft failure after pancreas transplantation; the entire UK experience of pancreas transplantation between 2004 and 2016 is reported; and several variables were accounted for that could confound relationships between exposures and outcome.
We acknowledged some limitations.First, the majority of donors received at least two VaDs, which means that there may be uncertainty around whether the data indicate that noradrenaline is 'beneficial' or that comparator drugs are 'harmful'.However, for the mechanistic reasons outlined above, the former explanation may be more likely.We addressed the issue by also reporting data from the cohort of patients that received no other drugs, and by adjusting for all permutations of drug use and co-linearity in the multivariable model.Second, the study used routinely collected registry data with its inherent potential limitations: retrospective design, potential for variation in data quality and missing data.However, we were fortunate that the data quality in the study was relatively high with limited missing data.Third, even though the study was large, there were limited numbers of events in some subgroups.For example, important relationships between dobutamine use and graft failure may not have been adequately captured because of limited statistical power.Finally, the focus on graft failure limits the ability to identify relationships that could lead to the early identification of deteriorating graft function.The Igls criteria on defining outcomes in beta-cell replacement therapy addresses this to some extent, but because of the small numbers of transplants performed annually, accrual of a comprehensive dataset may take some time. 40

Conclusion
Noradrenaline use was associated with better graft survival after adjusting for potentially confounding donor and recipient variablesthese benefits may be related to inhibition of pancreatic insulin secretion initiating beta-cell 'rest'.Further research is required to validate these findings and to establish whether relationships are causal.Identification of alternative methods of initiating beta-cell rest could be valuable in improving graft outcomes.

Figure 1
Figure 1 Kaplan-Meier curves showing the survival probability after pancreas transplantation stratified by vasoactive drug use: (a) noradrenaline, (b) dobutamine

Table 2
Donor and recipient characteristics associated with noradrenaline use in pancreas transplant donors CIT = cold ischaemic time; DBD = donation after brain death; HLA = human leucocyte antigen; n.a.= not applicable; SPK = simultaneous pancreas-kidney.Alcohol excess = ≥ 7units/day; cardiac arrest = cessation of circulation during the acute event that led to organ donation; cardiac disease = either ischaemic heart disease of valvular disease; donor drug use = presented as use vs non-use; methylprednisolone use = 15mg/kg to a maximum of 1g as outlined in the donor care bundle (data on total prescribed dosage for other drugs were not available); smoking = either past or present *Age (years), BMI (kg/m 2 ) and creatinine μmol/L) are continuous data presented as mean ± SD.Missing data were handled by multiple imputation (creatinine, 12%; CIT, 9%; recipient BMI, 27% of cases).†Amylase is continuous data presented as median (interquartile range).All binary data are presented as n (%).All available variables were included in analysis.

Table 3
HLA mismatch groups

Table 4
Donor and recipient factors predicting risk of graft failure after pancreas transplantation