A novel redox-active metalloporphyrin reduces reactive oxygen species and inflammatory markers but does not improve marginal mass engraftment in a murine donation after circulatory death islet transplantation model

ABSTRACT

Islet transplantation is a highly effective treatment for stabilizing glycemic control for select patients with type-1 diabetes. Despite improvements to clinical transplantation, single-donor transplant success has been hard to achieve routinely, necessitating increasing demands on viable organ availability. Donation after circulatory death (DCD) may be an alternative option to increase organ availability however, these organs tend to be more compromised. The use of metalloporphyrin anti-inflammatory and antioxidant (MnP) compounds previously demonstrated improved in vivo islet function in preclinical islet transplantation. However, the administration of MnP (BMX-001) in a DCD islet isolation and transplantation model has yet to be established. In this study, murine donors were subjected to a 15-min warm ischemic (WI) period prior to isolation and culture with or without MnP. Subsequent to one-hour culture, islets were assessed for in vitro viability and in vivo function. A 15-minute WI period significantly reduced islet yield, regardless of MnP-treatment relative to yields from standard isolation. MnP-treated islets did not improve islet viability compared to DCD islets alone. MnP-treatment did significantly reduce the presence of extracellular reactive oxygen species (ROS) (p < 0 .05). Marginal, syngeneic islets (200 islets) transplanted under the renal capsule exhibited similar in vivo outcomes regardless of WI or MnP-treatment. DCD islet grafts harvested 7 d post-transplant exhibited sustained TNF-α and IL-10, while MnP-treated islet-bearing grafts demonstrated reduced IL-10 levels. Taken together, 15-minute WI in murine islet isolation significantly impairs islet yield. DCD islets do indeed demonstrate in vivo function, though MnP therapy was unable to improve viability and engraftment outcomes.

Introduction

Islet transplantation has become a well-established treatment therapy for a subset of patients with type 1 diabetes mellitus.¹ The establishment of the “Edmonton Protocol” by Shapiro and colleagues demonstrated a high rate of insulin independence up to 1 y post-transplant.² Follow-up of these patients revealed a decline in graft function, with some patients returning to modest amounts of exogenous insulin, though still maintaining the benefit of the absence of hypoglycemic unawareness.³ To date, considerable improvements in clinical islet transplantation outcomes have been observed, revealing insulin independence in at least 50% of recipients at 5 y post-transplant, matching outcomes achieved by whole organ pancreas transplantation.⁴

Despite numerous advances in clinical islet transplantation, most recipients require more than one intraportal islet infusion to establish and maintain periods of insulin independence.⁵ Cadaveric donor pancreata are currently the sole, scarce source of islets. As such, to meet clinical demand, transplant centers routinely process extended criteria donor organs to expand the donor pool. Recently, donation after circulatory death (DCD) donors have been identified as a potential source of extended donors.⁵ In whole pancreas transplantation, DCD and neurological determination of death (NDD) organ recipients have demonstrated similar rates of patient survival and graft function.⁶ Alternatively, DCD donation is associated with poor graft function in liver transplantation.^7,8

Prior to transplantation, islet loss and impaired β-cell metabolic function are the result of cellular insults during organ procurement and islet isolation.⁹ The isolation procedure itself contributes to islet injury as a result of mechanical, ischemic and oxidative stress.^10,11 With reduced endogenous antioxidant capacity, islets are highly susceptible to oxidative stress and subsequent over-production of reactive oxygen species (ROS).^9,12 The generation of ROS during islet isolation has been linked to the up-regulation of the transcription factor NF-κB, generation of pro-inflammatory cytokines and subsequent cell death.⁹ Evidence suggests that redox modulation of islets through treatment with metalloporphyrin anti-inflammatory and antioxidant (MnP) compounds in culture can abrogate the deleterious consequences of oxidative stress, thus preserving islet mass in vitro.⁹ Moreover, administration of MnP to islets prior to transplantation demonstrated improved graft function in a murine, marginal syngeneic model, as well as delayed allograft rejection in an MHC-mismatched islet transplant model.¹² Within the context of human islets, MnP administration has demonstrated beneficial viability and engraftment outcomes in rodent transplant models.^9,10,12

To expand on the growing utility of DCD organs in islet transplantation, herein, we sought to establish a murine DCD islet isolation model and assess whether the reduction of ROS through acute redox modulation via a novel MnP-agent, BMX-001, could improve in vitro and in vivo islet function in a syngeneic, marginal transplantation model.

Results

DCD mouse islets cultured in the presence of MnP exhibit reduced extracellular ROS

Supernatants from DCD islets cultured in media alone exhibited a greater fold-increase in extracellular ROS production, which was ameliorated in the presence of MnP (DCD: 2.68 ± 0.11vs. DCD+MnP-treated: 1.65 ± 0.15) (n = 3 isolations, P < 0 .001) (Fig. 1A).

Figure 1.

In vitro assessment of control, DCD and DCA+MnP-treated islets. (A) Fold-change in extracellular ROS assessed from cell-free supernatants was significantly reduced in DCD+MnP-treated (blue) islets than DCD islets (black) (p < 0 .001, t-test). (B) Control islet yield per pancreas (red) was significantly greater than DCD and DCD+MnP-treated islets (blue) (p < 0 .05, ANOVA). In contrast, supplementation with MnP did not improve islet isolation yields in DCD donors. (C) DCD and DCD+MnP-treated islets exhibited similar islet viability as assessed by dual-fluorescence staining (p > 0 .05, t-test). (D) Islet function, as assessed by static glucose stimulated insulin secretion, demonstrated no significant difference between DCD and DCD+MnP-treated islets (p > 0 .05, t-test).

Fifteen minute warm ischemia significantly impairs islet yield in a murine DCD model

Subsequent to islet isolation and culture, islets were handpicked and quantified to determine islet yield. For standard control conditions, an islet yield of 192.6 ± 24.2 islets/pancreas was achieved. In contrast, islets harvested from DCD and DCD+MnP-treated donors exhibited a significantly reduced islet yield (77 ± 12.9 islets/pancreas vs. 79.1 ± 17.3 islets/pancreas, respectively, p < 0 .05). Notably, MnP-treatment during organ procurement and 1 h culture did not improve islet yield outcomes in DCD donors (p > 0 .05) (Fig. 1B).

MnP administration does not improve in vitro islet viability and function

Dual-fluorescence staining assessing islet viability in DCD and DCD+MnP-treated islets revealed no discernable difference between groups when assessed 1 h post-culture (DCD: 77.23 ± 4.9 vs. DCD+MnP-treated: 75.32 ±5 .3) (Fig. 1C). Similarly, glucose static challenge revealed that murine DCD islets secreted insulin in response to glucose (Stimulation index: 1.38 ± 0.33) to a similar degree to that of DCD+MnP-treated islets (Stimulation index: 1.29 ± 0.24, p > 0 .05) (Fig. 1D).

Efficacy of DCD islets pre-treated with MnP

Islet engraftment efficacy of islets isolated from DCD donors pre-treated with or without MnP was evaluated in a marginal islet transplant mass model (200 islets per recipient, n = 14 per group). As a means to compare engraftment efficiency, an additional group of diabetic recipients were transplanted with a marginal dose under the kidney capsule from standard control donors (Control: n = 7). Recipients of control islet transplants became euglycemic, 5 of 7 (71%), on average 14.5 ± 5.7 d post-syngeneic transplant (red). DCD islet recipient mice became euglycemic, 11 of 14 (78.6%) by 24.5 ± 6.4 d post-transplant, while 10 of 14 (71.4%) DCD+MnP-treated islet recipients became euglycemic in 12.10 ± 3.7 d (data non-significant) (Fig. 2A). Daily non-fasting blood glucose monitoring of euglycemic transplant recipients revealed no difference between control, DCD or DCD+MnP-treated islet recipients (Fig. 2B).

Figure 2.

Efficacy of syngeneic, marginal islet transplants under the renal capsule of BALB/c recipients. (A) Percent euglycemia of syngeneic, marginal islet transplant recipients was indistinguishable between control (red, n = 5 of 7), DCD (black, 11 of 14) and DCD+MnP-treated (blue, n = 11 of 14) recipients (p > 0 .05, Mantel-Cox). (B) Non-fasting blood glucose measurements of euglycemic recipients post-transplant. Recipients of control, DCD or DCD+MnP-treated marginal islets exhibited robust glycemic control until graft retrieval (arrow).

Glucose tolerance testing

Intraperitoneal glucose tolerance tests (IPGTTs) were performed on all euglycemic recipients 60 d post-transplant. Mice in all transplant groups exhibited a physiological response to glucose bolus, with a prompt restoration of normoglycemia up to 120 min post-dextrose infusion (Fig. 3A). Furthermore, there was no significant difference in mean area under the curve (AUC) ± s.e.m. (AUC Control: 1773 ± 93.4 mmol/L/120min vs. DCD: 2090 ± 177.6 mmol/L/120min vs. DCD+MnP-treated: 1753 ± 105.9 mmol/L/120min, p > 0 .05, ANOVA, Fig. 3B).

Figure 3.

Intraperitoneal glucose tolerance test (IPGTT) of syngeneic, marginal islet mass recipients transplanted with control, DCD or DCD+MnP-treated islets 60 d post-transplant. (A) Blood glucose prolife post-dextrose bolus of control (red, n = 5), DCD (black, n = 11) and DCD+MnP-treated islets (blue, n = 10) (B) Blood glucose area under the curve (AUC) analysis did not differ between control, DCD and DCD+MnP-treated islet recipients (p > 0 .05, ANOVA). Mice were administered 3 mg/kg 25% dextrose i.p. Blood glucose measurements were monitored at t = 0, 15, 30, 60, 90 and 120 minutes.

Pro-Inflammatory cytokine profile

Islet-bearing kidney grafts from control, DCD and DCD+MnP-treated islet recipients were assessed for the pro-inflammatory cytokines TNF-α, KC-GRO, IFN-γ, IL-1β, IL-10, IL-6 and IL-12p70, 24 hours and 7 d post-transplant. Control islet-bearing grafts harvested 7 d post-transplant exhibited a significant reduction in IL-10, IL-1β and TNF-α cytokine levels compared to grafts harvested 24 h post-transplant (p < 0 .05, p < 0 .05, and p < 0 .001, respectively) (Fig. 4A–C). In contrast, DCD islet-bearing grafts exhibited indistinguishable IL-10 and TNF-α levels at 24 h and 7 d post-transplant, but reduced IL-1β levels at 7 d post-transplant (p < 0.05) (Fig. 4D–F). With the exception of TNF-α, DCD+MnP-treated islets exhibited significantly reduced IL-10 and IL-1β cytokine levels for grafts harvested at 7 d post-transplant in comparison to grafts harvested at 24 h post-transplant (p < 0 .05 and p < 0 .01, respectively) (Fig. 4G–I). Inflammatory levels for cytokines KC-GRO, IFN-γ, IL-6 and IL-12p70 were non-detectable in grafts harvested at the aforementioned time points (data not shown).

Figure 4.

Pro-Inflammatory profile of islet-bearing kidney grafts harvested 24 h and 7 d post-transplant. (A–C) Control islet-bearing kidney grafts exhibited significantly reduced pro-inflammatory profiles for IL-10, IL-1β and TNF-α at 7 d post-transplant (gray, n = 3) relative to 24 h post-transplant (black, n = 3). (D–F) DCD islet-bearing kidney grafts exhibited significantly reduced IL-1 β (p < 0.05), but persistent IL-10 and TNF-α levels at 7 d post-transplant (gray, n = 3) compared to 24 h post-transplant (black, n = 3). ((G–I) DCD+MnP-treated islet-bearing kidney grafts exhibited significantly reduced pro-inflammatory profiles for IL-10 and IL-1β (p < 0 .05), but sustained TNF-α cytokine levels at 7 d post-transplant (gray, n = 3) relative to 24 h post-transplant (black, n = 3) (p < 0 .05).

Discussion

Islet transplantation is limited, in part, by availability of cadaveric donor pancreata. Expansion of the donor pool to include extended criteria and DCD donors would considerably enhance availability of clinical islet transplants. To date, the success of DCD islets in the context of clinical islet transplantation has been evaluated at few single centers worldwide, with limited long-term successful outcomes.^13-15 The necessity to establish preclinical models evaluating the utility of DCD islets could provide insights into their clinical application. Herein, we sought to establish a murine DCD model and evaluate its efficacy in a marginal, syngeneic islet transplant model, as well as determine if supplementation with a novel MnP during organ procurement, islet isolation and brief culture could improve DCD islet transplant function.

In the present study, we determined that a 15 min warm ischemic (WI) model in mice significantly impaired islet yield per pancreas relative to standard islet donors without WI. A study by Giraud and colleagues also established that mice exposed to WI exhibited a significantly reduced islet yield relative to islets not exposed to periods of WI.¹⁶ The authors reported that 30 min of WI in humans is equivalent to 3.5 min of WI in mice based on metabolic differences between the 2 species, like oxygen consumption and resting heart rate.¹⁶ Our 15-minute WI model greatly exceeds this equivalence rate, and though MnP administration could reduce extracellular ROS, it could not improve islet yield or in vitro viability outcomes.

Despite our in vitro observations, DCD islets exhibited engraftment outcomes similar to standard procured islets. Similar to our findings, in a porcine islet isolation model, pancreata subjected to 30 min of warm ischemia exhibited a significantly reduced islet yield in comparison to non-warm ischemic pancreata. In contrast, ischemic porcine islets exhibited reduced graft function compared to their non-warm ischemic counterparts in a diabetic nude mouse transplant model.¹⁷ Within the clinical setting, a recent report by Andres and colleagues revealed that clinical islet isolations from 15 human DCD pancreata experiencing a maximal warm ischemia limit of <30 min exhibited no discernable difference in islet yield in comparison to standard neurological determination of death (NDD) pancreata. Moreover, examination of insulin requirement one month post-transplant in recipients of islets from NDD or DCD pancreata revealed no significant difference between groups.⁵ WI significantly impaired islet yield in our murine model, but DCD islets exhibited similar in vivo islet function to standard control islet recipients, as evidenced by the restoration of euglycemia and IPGTT responses at 60 d post-transplant. Though MnP administration did not enhance islet function, the findings in the present study demonstrate that MnP supplementation is safe and non-toxic to islets.

Control islet recipients exhibited significantly reduced pro-inflammatory cytokine levels at 7 d post-transplant. To the contrary, DCD islets exhibited a sustained pro-inflammatory cytokines IL-10 and TNF-α up to 7 d post-transplant relative to standard control islet recipients. Short-term culture of DCD+MnP islets did not significantly improve TNF-α profiles, though a significant reduction was observed in IL-1β and IL-10. Though heightened pro-inflammatory cytokines in DCD islet recipients did not impact the long-term engraftment outcomes in this syngeneic model, the consequences in allotransplantation may prove detrimental. The interplay between the innate immune system, pro-inflammatory cytokines and the adaptive immune system is critical in transplantation.¹⁸ Within the context of clinical islet transplantation, Bellin and colleagues observed significantly improved long-term insulin independence rates in recipients administered a TNF-α inhibitor in the peri-transplant period along with T-cell depleting antibody.¹⁹ Tse et al. demonstrated that redox modulation through MnP administration greatly diminished ROS production and subsequently ameliorated the synergism between the innate and adaptive immune response, and subsequent inflammatory cytokine production.²⁰ In parallel to measuring pro-inflammatory cytokine markers, it may have been of added benefit to examine the levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) in the acute transplant period to determine whether MnP administration impacted these levels. Previous bodies of work demonstrated that increased MCP-1 levels negatively impacted islet engraftment outcomes.^21,22 These events may account for the observation that 25% of DCD-islet recipients were euglycemic 10 d post-transplant, as compared to 50% of DCD+MnP-treated islet recipients. Furthermore, our observation that MnP treatment reduced some pro-inflammatory cytokines, exploring the affect of MnP administration paired with an effective immunosuppressive regimen in a preclinical allograft model could improve islet engraftment outcomes.

The results from the current study demonstrated that short-term administration of a novel MnP, BMX-001, was capable of significantly reducing extracellular ROS production in a DCD islet isolation model. Work in our laboratory has demonstrated that human islets cultured with 30 μM BMX-001 improves islet recovery up to 7 d in culture, and exhibited cytoprotection in the presence of tacrolimus, relative to islets cultured without MnP (data not published). These results provided the rationale for utilizing this dose in our DCD model. Numerous studies have utilized and observed cytoprotection and improved engraftment outcomes when human or murine islets were cultured in the presence of early-generation MnPs for 24 h or more.^9,10,12 It may be possible that short-term culture with MnP in our model cannot feasibly confer protection and that longer islet culture may be required to observe a significant benefit of MnP treatment. Due to an initial low islet yield, a prolonged islet culture period was not incorporated into the study to avoid further islet loss in culture. We are in the process of utilizing this novel MnP treatment in a standard murine transplant model to determine if MnP administration can confer improved engraftment outcomes, which may translate to improved clinical islet transplantation success. Despite these observations, our data reveal that DCD mouse islets are capable of restoring euglycemia comparable to control islets which is an encouraging finding, strengthening their utility in the islet transplant setting. It is clear that the DCD WI model is especially challenging for islet isolation in mice, and likely does not parallel a similar process in human islet isolation. Therefore, we cannot extrapolate from the current studies how protective MnP therapies will be in human islet isolation and transplantation. However, the impact upon inflammatory markers and ROS is strongly positive. Further studies in large animals and human islet isolation are now required to fully understand the potential benefit of this approach.

Materials and methods

Murine donation after circulatory death model

Mouse care was in accordance with the guidelines approved by the Canadian Council on Animal Care. Animals were housed under conventional conditions having access to food and water ad libitum. 8 to 12 week old male BALB/c mice (Jackson Laboratories, Canada) were placed under anesthetic with 5% isoflurane and euthanized via cervical dislocation. Animals were confirmed deceased by cardiac palpation and were maintained under a heat lamp to maintain an internal body temperature of 37°C, as measured by a rectal thermometer, for a total warm ischemic (WI) period of 15 min. DCD BALB/c mice were randomly assigned as non-treated (DCD) or MnP donors (DCD+MnP-treated) and subsequently were administered 1 ml of cold histadine-tryptophan-ketoglutarate (HTK) with 1U/μl heparin (Sandoz Canada Inc., Boucherville, QC, CA) supplemented with or without 30 μmol/L MnP through the abdominal aorta in DCD donors.

Mouse pancreatectomy and islet isolation

Pancreatic islets were isolated from standard (Control) or DCD BALB/c mice (Jackson Laboratories, Canada). Prior to pancreatectomy, the common bile duct was cannulated with a 27-gauge needle and the pancreas was distended with 0.125 mg/mL cold Liberase TL Research Grade enzyme (Roche Diagnostics, Laval, QC, CA) in Hank's Balanced Salt Solution (HBSS, Sigma-Aldrich Canada Co., Oakville, ON, CA). Islets were isolated by digesting the pancreases in a 50 ml Falcon tube placed in a 37°C water bath for 14 min with light agitation. Following the pancreatic digestion phase, islets were purified using histopaque-density gradient centrifugation (1.108, 1.083 and 1.069 g/mL, Sigma-Aldrich Canada Co., Oakville, ON, Canada).

Administration of MnP

BMX-001 (MnTnBuOE-2-PhP⁵⁺ MN(III) meso-tetrakis(N-b-butoxyethylpyridinium-2-yl)porphyrin) was provided by BioMimetix JV LLC. BMX-001 is one of the most potent metalloporphyrins with regard to anti-inflammatory and catalytic antioxidant function.²³ BMX-001 was administered during organ procurement, islet isolation, as well as during brief islet culture. DCD BALB/c mice were randomly assigned as non-treated (DCD) or MnP donors (DCD+MnP-treated). During mouse pancreatectomy and islet isolation, MnP (concentration 30 μmol/L) was delivered to pancreatic tissue with Liberase, as described above. In the non-treated group, Liberase with vehicle was delivered. DCD+MnP-treated and DCD pancreata were maintained in cold HBSS supplemented with or without MnP, respectively, until islet isolation (as described above).

Islet culture

Subsequent to islet isolation, control, DCD and DCD+MnP-treated islets were cultured in Connaught Medical Research Laboratories (CMRL-1066) medium supplemented with 10% fetal bovine serum, L-glutamine (2mM), penicillin (50 000 units), streptomycin (50 mg), HEPES (5mM), nicotinamide (10mM) and sodium pyruvate (5mM) at 37°C/5%CO₂ for 1 h. DCD+MnP-treated islets were cultured in CMRL with 30 μM MnP.

Reactive oxygen species analysis

Subsequent to one hour culture, cell-free supernatant samples from the study groups were assayed for ROS released into the culture media by Acridan Lumigen PS-3 assay (Amersham ECL Plus Kit; Fisher Scientific Inc., Ottawa, ON, Canada).²⁴ Acridan Lumigen PS-3 is excited by ROS and reactive nitrogen species in the presence of hydrogen peroxide, producing chemiluminescense at 430 nm. Media samples were stored at −20°C until time of analysis. CMRL and CMRL+MnP culture medium served as controls for each group, and results were expressed as fold-change increase compared to each respective control.

Assessment of islet yield and viability

One hour post-culture, islets were handpicked and counted to determine yield, and represented as islets per pancreas. Islet viability was determined by simultaneous staining of live and dead cells using a 2-color fluorescence assay (SytoGreen 13 and ethidium bromide, Invitrogen, Oregon, USA). The percentage of viable and dead cells was determined for DCD and DCD+MnP-treated islets.

Static glucose-stimulated insulin secretion (s-GSIS)

Handpicked islets from DCD and DCD+MnP-treated groups were subjected to s-GSIS. Islets were incubated in RPMI-1640 containing low (2.8 mmol/l) glucose for one hour, followed by high (16.7 mmol/l) glucose for an additional hour. Subsequent to glucose challenge, cell-free supernatants were harvested and insulin levels were measured by enzyme-linked immunosorbent assay (ELISA) (Mercodia, Uppsala, Sweden).

Diabetes induction and marginal islet mass transplantation

One week prior to transplantation, recipient BALB/c mice were rendered diabetic by chemical induction with intraperitoneal streptozotocin (STZ) (Sigma-Aldrich Canada Co., Oakville, ON, Canada), at 185 mg/kg in acetate phosphate buffer, pH 4.5. Diabetes was confirmed when non-fasting blood glucose levels exceeded 15 mmol/L for 2 consecutive daily readings. One hour post-islet culture, marginal mass islets from Control, DCD or DCD+MnP-treated islets (200 islets ± 10% per diabetic recipient) with purity of 90 ± 5%, were aspirated into polyethylene (PE-50) tubing using a micro-syringe, and centrifuged into a pellet suitable for transplantation. A left lateral paralumbar incision was made and the left kidney delivered. The renal capsule was incised and the islets were infused.

Evaluation of islet graft function

Transplant efficacy was assessed 3 times per week in recipients through non-fasting blood glucose measurements (mmol/L), using a portable glucometer (OneTouch Ultra 2, LifeScan, Canada) in all groups tested. Graft function and reversal of diabetes was defined as 2 consecutive readings ≤ 11.1 mmol/L and maintained until study completion. To assess metabolic capacity of the islet graft, intraperitoneal glucose tolerance tests (IPGTTs) were conducted on euglycemic mice 60 d post-transplant. Mice were fasted overnight prior to receiving an intraperitoneal glucose bolus (3g/kg). Blood glucose levels were evaluated at baseline (time 0), 15, 30, 60, 90 and 120 min post-injection. Blood glucose area under the curve (AUC-blood glucose) was calculated and analyzed between transplant groups.

Islet graft retrieval

In order to corroborate graft-dependent euglycemia, islet transplants were retrieved by nephrectomy. Islet transplant recipients were placed under anesthesia, and their graft-bearing kidney was exposed. Using a LT200 Ligaclip (Johnson & Johnson, Inc., Ville St-Laurent, QC, CA), the renal vessels and ureter were ligated and the islet graft-bearing kidney was removed. Non-fasting blood glucose measurements were monitored up to 7 d post-graft removal to confirm hyperglycemia and thus post-transplant graft function.

Pro-inflammatory cytokine assessment

Pro-inflammatory cytokines were analyzed from islet-bearing kidney grafts harvested 24 h and 7 d post-transplant. Three mice per group underwent recovery nephrectomy at the aforementioned time points, and grafts were assessed for mouse tumor necrosis factor (TNF)-α, KC-GRO, interferon (IFN)-γ, interleukin (IL)-1β, IL-10, IL-6 and IL-12p70. Pro-inflammatory levels were measured using a Mouse ProInflammatory 7-Plex Tissue Culture Kit according to manufacturer instructions (Meso Scale Diagnostics, K15012B-1). The plate was loaded into an MSD-SECTOR® instrument for analysis where a voltage was applied and the bound label emitted a quantitative (0 – 1.0 × 10⁶ pg/mL) measure of light. Values were normalized to weight of tissue homogenized.

Statistical analysis

All data are represented as the mean ± standard error of mean (s.e.m.). In vitro islet viability data comparisons between DCD and DCD+MnP-treated islets were conducted through unpaired Student's t-test. Blood glucose AUC analysis for glucose tolerance test data was conducted through parametric one-way ANOVA using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Tukey's post-hoc tests were used following the analysis of variances for multiple comparisons between study groups. Kaplan-Meyer survival function curves were compared using the log-rank statistical method (Mantel-Cox). P < 0 .05 was considered significant.

Abbreviations

AUC

area under curve

BMX-001

MnTnBuOE-2-PhP⁵⁺ MN(III) meso-tetrakis(N-b-butoxyethylpyridinium-2-yl)porphyrin

DCD

donation after circulatory death

ELISA

enzyme-linked immunosorbent assay

HBSS

Hank's Buffered Saline Solution

HTK

histadine-tryptophan-ketoglutarate

IFN-γ

interferon-gamma

IL

interleukin

IPGTT

intraperitoneal glucose tolerance test

KC

kidney capsule

MCP-1

monocyte chemoattractant protein-1

MnP

metalloporphyrin anti-inflammatory and catalytic antioxidant functions

NDD

neurological determination of death

PBS

phosphate buffered saline

ROS

reactive oxygen species

SEM

standard error of mean

TBS

tris-buffered saline

TNF-α

tumor necrosis factor- α

WI

warm ischemia

Disclosure of Potential Conflicts of Interest

The authors declare no conflict of interest. James Crapo is manager of BioMimetix JV, LLC, and holds equity. Jon Piganelli has equity in BioMimetix JV, LLC.

Funding

This work was supported in part by the Diabetes Research Institute Foundation of Canada (DRIFCan) and the Canadian National Transplant Research Program (CNTRP). Dr. A.M James Shapiro is supported through a Senior Clinical Scholarship from Alberta Innovates Healthcare Solutions, and holds a Canada Research Chair in Transplantation Surgery and Regenerative Medicine funded through the Government of Canada. A.M.J.S is also supported by AIHS CRIO Team Award #201201154. Antonio Bruni is supported through scholarships from the University of Alberta, the Alberta Diabetes Institute and the Alberta Diabetes Foundation. Dr. Andrew R. Pepper is supported through an AIHS Postdoctoral Fellow- 550 ship and a Fellowship from the Juvenile Diabetes Research Foundation Canadian Clinical Trials Network. Dr. Boris GalaLopez is supported through the AIHS Clinical Fellowship. Dr. James D. Crapo is supported by BioMimetix JV, LLC, by NCI HHSN 261201500002C and NIH 1R4CA195749 to BioMime- 555 tix JV, LLC, and by NIH R01 HL089897 to National Jewish Health. Dr. Jon D. Piganelli is supported by the American Diabetes Association (CDA 7.07 CD-16 and 1-12-BS-161), and the Juvenile Diabetes Research Foundation (5-2013-91, 47- 2013-517, and 2-SRA-2014-296-Q-R).

Contributions

AB participated in the research design, performance of the research, data analysis and writing of the manuscript. ARP, BGL, RP, and NA participated in the performance of the research and reviewing of the manuscript. JC and JDP. participated in research design and writing of the manuscript. AMJS participated in research design and writing of the manuscript.