CORM-401 Reduces Ischemia Reperfusion Injury in an Ex Vivo Renal Porcine Model of the Donation After Circulatory Death

Abstract

Background

Carbon monoxide (CO) inhalation protects organ by reducing inflammation and cell death during transplantation processes in animal model. However, using CO in clinical transplantation is difficult due to its delivery in a controlled manner. A manganese-containing CO releasing molecules (CORM)-401 has recently been synthesized which can efficiently deliver 3 molar equivalents of CO. We report the ability of this anti-inflammatory CORM-401 to reduce ischemia reperfusion injury associated with prolonged cold storage of renal allografts obtained from donation after circulatory death in a porcine model of transplantation.

Methods

To stimulate donation after circulatory death condition, kidneys from large male Landrace pig were retrieved after 1 hour warm ischemia in situ by cross-clamping the renal pedicle. Procured kidneys, after a brief flushing with histidine-tryptophan-ketoglutarate solution were subjected to pulsatile perfusion at 4°C with University of Wisconsin solution for 4 hours and both kidneys were treated with either 200 μM CORM-401 or inactive CORM-401, respectively. Kidneys were then reperfused with normothermic isogeneic porcine blood through oxygenated pulsatile perfusion for 10 hours. Urine was collected, vascular flow was assessed during reperfusion and histopathology was assessed after 10 hours of reperfusion.

Results

We have found that CORM-401 administration reduced urinary protein excretion, attenuated kidney damage markers (kidney damage marker-1 and neutrophil gelatinase-associated lipocalin), and reduced ATN and dUTP nick end labeling staining in histopathologic sections. CORM-401 also prevented intrarenal hemorrhage and vascular clotting during reperfusion. Mechanistically, CORM-401 appeared to exert anti-inflammatory actions by suppressing Toll-like receptors 2, 4, and 6.

Conclusions

Carbon monoxide releasing molecules-401 provides renal protection after cold storage of kidneys and provides a novel clinically relevant ex vivo organ preservation strategy.

Kidney transplantation remains the best option for patients suffering from end-stage renal diseases. Shortage of donors necessitates the use of kidneys from donation after circulatory death (DCD) donor and older, less healthy individuals (expanded criteria donors). Compared with neurologic brain death donor kidneys, older DCD kidneys have a markedly higher delayed graft function (DGF) rate.1 Evidence indicates that this is due to ongoing damage incurred by ischemia-reperfusion injury (IRI) associated with warm ischemic time incurred during the DCD process, as well as cold storage damage and the reperfusion injury that occurs as a result of the return of warm perfusion to the organ.2,3 Prolonged ischemia is a known factor for DGF and duration of ischemia time had the single most impact on graft survival in recipients.4 This early injury may result in an inflammatory cascade that increases the release of damage-associated molecular patterns (DAMPs), cytokine and chemokine expression, MHC upregulation, with innate immunity induction of adaptive immunologic responses, with subsequent increased rejection and permanent damage ultimately characterized as interstitial fibrosis and tubular atrophy.5,6 However, although current methods of organ storage in a static cold solution or cold pulsatile perfusion can increase storage times, they still fail to optimally protect kidneys from IRI. It has been shown that transplantation of DCD kidneys preperfused in a normothermic condition for 8 hours had better graft function and lowered graft injury compared with cold static storage.7

Inflammation associated with IRI activates many inflammatory pathways including receptors called Toll-like receptors (TLRs). Toll like receptors are type 1 transmembrane proteins expressed primarily by macrophages, dendritic cells and neutrophils.8 Toll like receptors are also constitutively expressed within solid organs, such as the kidney, liver, and heart.9 The typical ligands for TLRs are microbial products (eg, bacterial CpG DNA, and lipopolysaccharides). Interestingly, molecules, such as high-mobility group box 1 (HMGB1), heat shock proteins, and necrotic nucleosome-DNA (collectively called DAMPs) released from the necrotic cells or organs undergoing prolonged ischemia can also act as endogenous TLR ligands to promote inflammation.10,11 Expression of TLRs by the kidney is functionally important, as parenchymal cell deficiency of TLR4 dramatically limits kidney injury after IRI and diminishes intrarenal inflammation.12 Therefore, strategies that limit TLR signaling or their expression are likely to have a benefit in controlling IRI-related processes.

In the past, we and others have used carbon monoxide (CO) to prevent IRI and improve renal function at reperfusion.13,14 Carbon monoxide is a gaseous endogenous molecule which has been shown to be vasoregulatory and prevents apoptotic cell death and inflammation associated injury such as IRI.15 Our group has administered CO in a stable chemical form bound to transition carbonyls called CO-releasing molecule (CORM). We found that when infused directly into the murine donor kidney during cold storage, CORM-3 reduces cell death, and inflammation associated with improvement in graft survival and function.16 In this study, we used the fourth generation of this family of molecules named CORM-401. It is based on a clinically utilizable manganese-based agent which is not only more potent than previously synthesized CORMs but also highly controlled in CO release.17 We assessed the ability of CO to be released at 37°C and 4°C. We also assessed the ability of CORM-401 to protect the kidney against IRI at different temperatures in a preclinical porcine transplant model, using simulated DCD kidneys stored with a clinical pulsatile perfusion pump. Reperfusion was achieved using warm, oxygenated porcine blood in a physiologic pulsatile perfusion circuit. We thus hypothesized and tested whether CORM-401 could prevent IRI-related damage. We present data that the benefit we observed with CORM-401 is highly significant and clinically feasible, and is associated mechanistically with reduction of TLRs and a direct effect on limiting intrarenal inflammation and cell death pathways.

MATERIALS AND METHODS

Animals

Under the approval by the University of Western Ontario research ethics board, all experiments were conducted on large (40-50 kg) white male Landrace pig. To simulate DCD condition, kidneys were retrieved after 1 hour warm ischemia by cross-clamping the renal pedicle in situ. During the kidney harvesting procedure, the incision was extended towards the chest to expose the heart. Once the kidneys are ready to be procured, direct cardiac puncture was used to drain the blood and collect it in a special container (approximately 1.5 L) with 25 000 units of heparin. After a brief flushing with 100 mL of histidine-tryptophan-ketoglutarate (HTK) cold solution, kidneys were subjected to pulsatile perfusion at 4°C with University of Wisconsin (UW) for 4 hours in LifePort pump (Organ Recovery Systems, USA) at an initial pressure set at 30 mm Hg.

Perfusion and Reperfusion of Kidneys

Along with static cold preservation, one of the current clinical standards to reduce storage injury is the hypothermia (4°C) LifePort pump (Figure 1A). This hypothermic storage device perfuses the kidneys with cold pulsatile solution that places cells in a lower metabolic state. However, compared with static cold storage, it has very little impact upon rejection, graft function and survival despite some improvement in DGF rates.18 In contrast, another study on expanded criteria donors has found that hypothermic machine perfusion (HMP) was associated with a reduced incidence of DGF with increased 1-year survival.19 In all experiments, kidneys are stored for 4 hours on the LifePort pump prior to reperfusion.

FIGURE 1.

A, Clinical cold organ storage system used to perfuse DCD kidneys at 4°C in all experiments. B, A commercially available renal preservation pump which provides pulsatile flow and measures resistance (RM3 Medical system) was modified for normothermic blood perfusion and reperfusion in the same unit. Components including oxygenator, water heater and flow probe are highlighted. Kidneys were placed in cassette reservoir on a perfusion stage as pictured with the artery, vein and ureter cannulated as designed in the schematic (C). D, Study design for ex vivo CORM-401 experiments at 37°C.

To simulate organ reperfusion, we provide pulsatile normothermic (37°C) isogeneic oxygenated blood (FiO₂ = 40%) using physiologic blood pressures (mean BP of 60). The perfusion circuit utilizes a modified version of the clinically relevant RM3 (Water Instruments Inc., MN) pulsatile perfusion system originally developed for hypothermic nonoxygenated pulsatile preservation of kidneys. Figure 1B shows the composition of the circuit including peristaltic pump, disposable cassette, a membrane oxygenator, venous blood reservoir and water heating unit. A simplified design is shown in schematic (Figure 1C). We have modified this system to allow normothermic perfusion using blood via vascular access by cutting in a double-luer connector to provide both inflow and outflow. In this way, deoxygenated blood leaving the reservoir was rerouted through the oxygenator so that a regulated amount of oxygen can be maintained. Views of the DCD kidneys placed in the chamber at perfusion stage were photographed (Figure 1B).

Study Design

After perfusion in cold for 4 hours, artery, vein, and ureter were cannulated and flushed with plasmaLyte solution to get rid of HTK solution. This step represents an immediate initial storage of organ in cold after procurement to minimize injury. At this stage, left and right kidneys (n = 5) were treated with 200 μM inactive CORM-401 (iCORM-401) and CORM-401, respectively. Inactive CORM-401 (iCORM-401) was prepared by incubating CORM-401 stock solution (5 mM; dissolved in plasmaLyte solution) for 36 hours in a cell culture incubator at 37°C to liberate CO. The iCORM-401 stock solution was frozen (−80°C) and used in all consecutive experiments.

Both CORMs were diluted (×200) in plasmaLyte solution and delivered through pulsatile action at 37°C over the period of 20 minutes. Kidneys were then reperfused with oxygenated (40%) autologous blood in a moist chamber with externally added creatinine (10 mg/L) at 37°C for 10 hours. Perfusion chamber and perfusate were maintained at this temperature by an open bath thermostat.

Detection of CO Release

CO release from CORM-401 was carried out as suggested.20 Human hemoglobin (Hb) (Sigma) was dissolved at 20 mg/mL in dH₂O, reduced by 1.5 mg/mL sodium dithionite and passed through a 5 mL Zeba desalt spin column (Thermo) to remove excess dithionite. It was then diluted to 50 μM in the indicated air-sparged buffers prior to spectrophotometry. Hemoglobin solutions were incubated with either 25 μM CORM-401 or no drug for 20 minutes at 37°C or 4 hours at 4°C. CO release was determined using the difference in absorbance at 568 nm, with absorbances at 560 and 578 nm in the untreated Hb sample used to correct for any possible deoxyhemoglobin in the solution. Millimolar absorption values were obtained from Zijlstra and Buursma.21

Pump Data Analysis

Renal blood flow rate was recorded every hour. Total urine output indicative of overall performance of ex vivo kidney during reperfusion was measured. Biochemical analysis of urine samples (total protein, protein/creatinine ratio), CO toxicity (formation of carboxyhemoglobin [COHb]) in postperfusion blood was carried in hospital core facility and clinical medical laboratory.

Histological Studies to Determine Renal Tissue Injury

Kidney tissue section (5 μm thickness) after ex vivo reperfusion was fixed in 10% formalin in PBS solution, embedded in paraffin and stained with H&E for necrosis, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for apoptosis, and Martius Scarlet Blue (MSB) for blood (yellow), fibrin (red), and collagen (blue). All the slides were examined and scored by a pathologist in a blinded manner. The degree of acute tubular injury was determined by a qualified renal pathologist by using a semiquantitative graded scale: 0, no change; 1, less than 10%; 2, 11% to 25%; 3, 26% to 45%; 4, 46% to 75% and 5, 76% to 100%. The injury was assessed as percentage of tissue involved with histological changes in the border area between cortex and medulla. Scores were averaged for at least 3 independent tissue sections from the similar treatment with 3 nonoverlapping fields for each kidney.

Caspase-3 Activity Assay

Caspase-3 activity was determined as per the manufacturer’s protocol (GeneTex, USA). Briefly, total protein was isolated from 50 mg of fresh kidney tissues taken from the area surrounding cortex and medulla. Thoroughly homogenized tissues in lysis solution were centrifuged at 16 000g for 10 minutes. Reaction buffer and caspase substrate were added in tissue lysate followed by incubation at 37°C for an hour. Detection was based on colorimetric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA. Absorbance was quantified using a microtiter plate reader at 400 or 405 nm. Caspase-3 activity in the experimental groups was normalized to the control group.

Enzyme-Linked Immunosorbent Assay

The amount of porcine urinary kidney injury markers neutrophil gelatinase-associated lipocalin (NGAL) (Enzo Life Sciences) and kidney injury molecule 1 (KIM1; MyBioSource) was determined by enzyme-linked immunosorbent assay (ELISA) as per manufacturer’s protocol. Briefly, total urine was collected aseptically, centrifuged to remove particulate matter and diluted to 1:10 in the buffer supplied with the kits and assessed immediately. Standards and samples (100 μL) were added to the micro ELISA plate wells and combined with NGAL and KIM1 specific antibodies. Biotinylated detection antibodies (100 μL) and Avidin-HRP conjugate were added and incubated in room temperature until a suitable intensity of blue color appeared. The reaction was terminated by the addition of sulphuric acid solution and measured spectrophotometrically at a wavelength of 450 ± 2 nm. Concentration of NGAL and KIM1 were determined from the standard curve.

Quantitative Real-time PCR

Total RNA was purified with PerfectPure RNA and Tissue kits (Applied Biosystems) from 50 mg of fresh kidney tissues. Thoroughly homogenized tissues in lysis solution were centrifuged at 16 000g for 1 minute. Four hundred microliters of tissue lysate was then passed through preclear filter to remove debris. Next, the lysates were applied to the purification column to bind the RNA and washed with 400-μL wash 1 and wash 2 solutions to remove proteins and DNA. Finally, the purified RNA was eluted with DEPC-treated water and measured by nanodrop. cDNA was synthesized with the First-strand synthesis System according to the manufacturer’s protocol (Invitrogen). Primers were designed using primer designing software from Applied Biosystems. The gene sequences were obtained from the NCBI database. Real-time quantitative PCR reaction was performed in the presence of Brilliant SYBR Green QPCR Master Mix kit (Invitrogen). Amplification of β-actin mRNA was used as an internal control. The normalized delta threshold cycle value and relative expression levels (2-ΔΔCt) were calculated according to the manufacturer’s protocol.

Statistical Analysis

Creatinine clearance was calculated by using the standard equation (C_x = Urine_x × volume total urine output in mL)/plasma_x where x denotes creatinine. Data were compared using Student t test for paired values. A P value of 0.05 or less was considered a significant difference. Data are represented as means ± SD. All statistical analyses were performed with GraphPad Prism version 6.

RESULTS

Optimizing CO Release from CORM-401 in PlasmaLyte and UW Solution at 37°C and 4°C

In the first set of experiments we assessed release of CO from CORM-401 under cold (eg, 4°C) and normothermic (eg, 37°C) temperatures into 2 different solutions used clinically as well as in our experimental settings, for example, UW preservation solution and PlasmaLyte cardiac perfusion solution (Baxter, Canada). As shown in Figure 2A, the levels of CO released from CORM-401 were similar in both solutions when assessed either at 37°C or 4°C, respectively. Important to note, however, that CORM-401 releases approximately 15 times more CO within the same period of time in both solutions at 37°C, as compared with the levels of CO released at 4°C. These findings provide the first evidence that temperature of the solution used for delivery of CORM-401–derived CO is a critical factor and should be taken into account when treating DCD kidneys for transplantation.

FIGURE 2.

Detection of CO released in perfusion solutions from CORM-401 at 37°C and 4°C. A, Human Hb was dissolved in dH₂O (20 mg/mL) and diluted to 50 μM in both PlasmaLyte and UW solutions. CORM-401 was incubated with Hb in the presence of either 25 μM CORM-401 or no drug for 20 minutes at 37°C or 4 hours at 4°C. In both solutions, the amount of CO released was determined using the difference in absorbance at 568 nm by measuring the conversion of hemoglobin to COHb. The rate of CO release was calculated (mean ± SD.) from the fitted curve as explained in the materials and methods. B, Determination of nontoxic dose of CORM-401. In pig DCD kidneys, intra-arterial delivery of 200 μM of CORM-401 with plasmaLyte solution resulted in completely nontoxic level of COHb (0.3-3.5%) over the period of 10 hours of reperfusion at 37°C as measured in clinical core medical laboratory at London Health Sciences Centre.

Development of COHb after CORM-401 Administration

In our experiments we used CORM-401/iCORM-401 at total 200 μM concentration given in 4 different intervals (50 μM) each over the period of 20 minutes. This concentration was chosen based on our recent work demonstrating noncytotoxic and protective effects of CORM-401 (10-40 μM) in an in vitro model of anoxia/reoxygenation (data not shown). Importantly, CORM-401 (200 μM) administered through our circuit ex vivo over the period of 8 hours had no major impact on COHb formation in reperfused blood (Figure 2B).

Treatment with CORM-401 After Cold Perfusion Reduces Injury and Cell Death

To stimulate DCD conditions in porcine model, both kidneys were subjected to 1 hour in situ warm ischemia by clamping the hilum of both kidneys. We have previously found that when these kidneys are preserved only in HTK solution at 4°C for longer periods of time (eg, 8-24 hours), the degree of organ injury (histological analysis), and level of kidney injury related markers (eg, KIM1 and NGAL) were much higher than unclamped controls (data not shown). In the current study, DCD kidneys stored for 4 hours in the LifePort system under cold conditions (4°C) (Figure 1A) were perfused with warm 37°C plasmaLyte solution containing 200 μM CORM-401 or iCORM-401. As shown in Figure 3, CORM-401 (but not iCORM-401) dramatically reduced the degree of IRI-related damage with respect to: (1) acute tubular necrosis [ATN; confirmed by H&E staining and semiquantitative analysis (Figures 3A and D, respectively); ATN score of 3 = 26% to 45% death versus 5 = 76% death; P = 0.0016]. iCORM-401-treated kidneys had more severe necrosis and intrarenal hemorrhage vs CORM-401 treated organs (Figure 3C; left panel), 2) apoptosis [TUNEL staining (Figure 3b) and caspase 3 activity (Figure 3E; twofold changes), respectively], 3) kidney injury molecule 1 (KIM1) and NGAL levels in the urine produced during 10 hours of blood reperfusion (Figure 3F and G, respectively). Consistent with ATN score, both injury markers appeared significantly lower (P = 0.0003 and P = 0.0017, respectively) in urine obtained from CORM-401–treated kidneys.

FIGURE 3.

Ex vivo perfusion with CORM-401 reduces IRI in porcine DCD kidneys. A, Both porcine kidneys subjected to 1 hour warm ischemia in situ were perfused by UW solution in 4°C for 4 hours followed by intrarenal delivery of 200 μM of inactive CORM-401 or iCORM-401 (left) and CORM-401 (right) in warm plamaLyte solution for 20 min. After 10 hours of blood reperfusion at 37°C, kidney sections were stained with H&E (A), TUNEL (B) and MSB (C) to morphologically assess IRI, quantify apoptosis and to highlight fibrin thrombi and/or intrarenal hemorrhage respectively. D, Percent of acute tubular necrosis from H&E sections (40×) was determined using representative images from 5 independent experiments by a qualified Pathologist. CORM-401 treatment showed significant reduction in ATN (score 2; 11-25% death; P = 0.0016) associated with (E) reduced induction of proapoptotic caspase3 (colorimetric assay) as well as 2 urinary kidney injury markers (F) KIM1 (P = 0.003) and (G) NGAL (P = 0.0017). Blue and yellow circles highlight areas of tubular necrosis and interstitial blood hemorrhage respectively. Extensive hemorrhage and blood clot observed during warm reperfusion was also prevented (C, right) by CORM-401 treatment.

CORM-401 Therapy is Associated with Improved Renal Function at Reperfusion

CORM-401 (200 μM) treatment of DCD kidneys was associated with increased renal blood flow rate versus iCORM-401 kidneys (Figure 4A). Important to note at the beginning of reperfusion, both CORM-401 and iCORM-401 treated kidneys exhibited poor flow (>50 mL/min), but by 4 hours, the blood flow was doubled (>100 mL/min) in CORM-401–treated kidneys, whereas blood flow in iCORM-401 (200 μM) treated kidneys remained below 50 mL/min. In addition, CORM-401 treated kidneys produced a higher total urine volume (P = 0.0444), and reduced urinary protein (P = 0.0166) and protein/creatinine ratio (P = 0.0247) (Figures 4B, C, and D, respectively). Renal function was significantly improved in the CORM-401 treated kidneys. It produced more urine and had higher level of creatinine clearance compared to the iCORM-401(P = 0.0195) (Figure 4E).

FIGURE 4.

Ex vivo perfusion with CORM-401 improves kidney function ex vivo (A) DCD kidneys treated with iCORM-401 and CORM-401 were monitored for perfusion flow rate (mL/min) during blood reperfusion over the period of 4 hours. An increase in perfusion rate was observed by kidneys treated with CORM-401. B, CORM-401 treatment produced higher amount of urine during 10 hours postreperfusion (P = 0.0444) (C) reduced proteinuria (P = 0.0166) and (D) significantly lowered protein-creatinine ratio (P = 0.0247). E, Creatinine clearance was improved in CORM-401 treated kidneys (P = 0.0195). All are indicative of superior kidney function ex vivo. Each line or bar represents the mean±SD of n = 5 experiments for each group.

TLRs Are Highly Activated in DCD Kidneys and CORM-401 Suppresses Its Expression

As proinflammatory immune activation in DCD organs contributes to IRI,22 we assessed the ability of CORM-401 to modulate the levels of TLRs, one of the predominant inflammatory systems involved in IRI.23,24 To this end, we assessed expression of TLRs, MyD88, NF-κB, and HMGB1 in DCD kidneys treated with CORM-401 or iCORM-401 (200 μM). We found that TLRs 2, 6, and 7 were highly expressed in surgically induced IRI in pig kidneys (data not shown). Treatment of DCD pig kidney with CORM-401 resulted in significant inhibition of expression of multiple TLRs, including TLR2 (P = 0.0216), 4 (P = 0.0027) and 6 (P = 0.0192) (Figure 5A), MyD88 (P = 0.0060) (Figure 5B), and NF-κB(0.0340) (Figure 5C) and HMGB1 (P = 0.0058) (Figure 5D) as assessed by quantitative RT-PCR. However, no significant changes were found in other TLRs (TLR3, TLR5, TLR7, and TLR9) and MAPK (P = 0.7567); Jun amino-terminal kinases (Figure 5E).

FIGURE 5.

Toll-like receptors are highly expressed in IRI and CORM-401 inhibits its expression. QRT-PCR- mRNA expressions of (A) selective TLRs (TLR2, P = 0.0216; TLR4, P = 0.0027; TLR6, P = 0.0192) (B) their common adaptor protein MyD88 (P = 0.0060) (C) a key transcription factor of TLR signaling pathway NF-κB (P = 0.0340) (D) a damage associated molecule HMGB1(P = 0.0058) released after necrotic tissue injury were evaluated from the RNA isolated from the pig kidney tissues perfused with iCORM-401 or CORM-401. Relative changes of the genes are shown after normalizing it with a housekeeping gene. Treatment of DCD pig kidney with CORM-401 during normothermic blood perfusion resulted in significant inhibition of expression of multiple TLRs (eg, TLR2, 4, and 6,), MyD88, NF-κB and HMGB1. No significant changes were found in TLR5, 7, 8, and 9 (data not shown) and MAPK expression (P = 0.7567) (E).

DISCUSSION

Few changes have been made in protective strategies against IRI in organ preservation techniques over the past 3 decades.25,26 Previously, we have shown that addition of CORM-3 to the preservation solution reduced cell death and improved kidney function in a murine model of renal transplantation.16 We wanted to show that CORM, with its potential to reduce inflammation, prevent cell death and reduce vascular spasm in small animal models, could be used with clinically relevant pulsatile perfusion devices to improve renal viability and function in a porcine model of transplantation. In these preclinical experiments, CORM-401, in which CO is bound to a physiologically relevant manganese molecule, was used instead of CORM-2 or 3, which are bound to a heavy metal core. To further address the issue of safety and potential development of toxic levels COHb during reperfusion in organ transplant recipients, we measured levels of COHb saturation in the blood during reperfusion. We have found that delivery of 200 μM of CORM-401 in our circuit over the period of 20 minutes produces COHb ranges between 0.3 to 3.5% in reperfused blood over the period of 10 hours. By comparison, smoking 1 to 2 packs of cigarette per day generates COHb levels of 1.5 to 5.0% in an individual’s blood, whereas 66% COHb levels in blood is considered as lethal.27 This provides a rationale to administer CORM-401 in a normothermic temperature for superior CO liberation during organ storage, which we have previously shown to be optimal in the protection of organs.16 In this study, a porcine DCD model, which is associated with a high incidence of DGF, was utilized in order to assess the effects of CORM-401 on IR-injury. We used the clinical LifePort hypothermic pulsatile perfusion machine to perfuse organs before reperfusion with normothermic oxygenated blood to simulate transplantation conditions.

The TLR signaling pathway has been shown to initiate powerful inflammatory processes during IRI. In fact, organ damage in IRI is primarily caused by activated TLRs expressed in dendritic cells, graft infiltrating cells and kidney tubular epithelial cells.28 Several groups showed an activated TLR2 and TLR4 during IRI can ultimately affect transplant outcomes.29,30 The best-described ligand in IRI, HMGB1 induces proinflammatory signaling via TLRs in a similar fashion in microbial pattern recognition by TLR. Most of the TLRs signaling pathways involved a common adaptor protein MyD88 and transcription factor nuclear factor κB (NF-κB).31 We and others have previously shown that low concentrations of CO have strong anti-inflammatory properties and can protect tissues against IRI.13 However, the exact mechanism(s) of its anti-inflammatory action were not well understood. For the first time, we have shown that several TLRs are overexpressed in renal tissues obtained from reperfusion of our surgically stimulated DCD kidneys. It also appears that intrarenal delivery of CORM-401 through pulsatile perfusion can suppress the expression of multiple TLRs, including TLR2, TLR4, and TLR6. We noted a similar effect in other molecules associated with TLR2/TLR6 signaling such as NF-κB and MyD88. NF-κB is the key transcription factor in inflammation central to TLR-mediated production of inflammatory cytokines and chemokines. Finally, the expression of HMGB1, which is one of the prominent DAMPs in TLR2/TLR6 signaling, was significantly reduced upon CORM-401 treatment.

It is possible that overall protective effects of CO at the organ level are achieved through multiple pathways. Primarily, CO may protect organs from IRI through reduction of DAMPs (eg, reduced HMGB1) that initiate TLRs responses. Second, a reduced expression of TLRs may ameliorate inflammatory response resulting in reduced IRI as reflected in our experiments. We observed improved protection of the kidney as verified by reduced apoptosis, acute tubular necrosis, and renal hemorrhage, as well as decreased expression of apoptotic marker caspase3 in CORM-401 treated renal tissues. Consistent with this finding, decreased level of kidney damage markers (KIM1 and NGAL) were detected in the urine generated during reperfusion post CORM-401 treatment.

Cold preservation induces vasoconstriction, which creates reduced renal blood flow during reperfusion.32 This reduced renal blood flow may contribute to renal injury, which further compounds reduced renal blood flow. In this study, CORM-401–treated kidneys had a significantly improved blood flow and reduced vascular resistance as early as 1 hour of reperfusion while iCORM-401–treated kidneys remained stagnant. We believe that in addition of its anti-inflammatory role, its vasodilatory properties contribute to increased urine production seen in the CORM-401–treated kidneys. We believe that the efficient release of CO plays an important role in vasodilation and reduction of vascular resistance during this ex vivo process. The production of higher volumes of urine was accompanied with reduced urinary protein and albumin/creatinine ratios and increased creatinine clearance indicating less damage to the reperfused organs. Our findings show that the benefit we observed with oxygenation and CORM401 is highly significant and clinically feasible, and is associated with a reduction of TLRs and a direct effect of this compound on intra-renal inflammation and cell death pathways. Prolonged cold storage damages the organ but to increase storage time immediately after procurement, cold preservation is still a clinical reality. Our experimental design is based on the fact that we can minimize cold storage time and replace it by warm oxygenated perfusion. However, we cannot completely eliminate the cold preservation method used in the clinic because there are no viable pumping options in warm. Although normothermic perfusion and preservation with blood is the most physiologically relevant method to protect organs and their function ex vivo, use of autologous oxygenated blood for ex vivo preservation requires establishing a complex network of supply chain management and logistics to get this blood. Moreover, perfusing kidneys with blood is not a feasible option since a large amount of matching blood is needed to perfuse them. Most importantly, one of the limitations we encountered with blood-based perfusion for prolonged periods of time was the development of extensive hemorrhage and blood clots. Although CORM-401 could mitigate these effects due to its vasodilatory properties, a comparison of different temperature conditions and blood perfusate alternatives to carry oxygen needs to be undertaken. In fact, we have already assessed these conditions in our studies and our findings suggest that 22°C is an optimal temperature condition to perfuse DCD kidneys ex vivo than hypothermia and normothermia (under review-Transplantation). Furthermore, we have perfused these kidneys at room temperature (21-22°C) with a hemoglobin-based cell-free oxygen carrier solution. We found that hemoglobin-based cell-free oxygen carrier can deliver oxygen as competently as blood at 22°C and confers the best available way to preserve kidneys for the prolonged period of time required for pretransplant preparation (pending submission).

In summary, we have shown for the first time that perfusate infused CORM-401 protects the kidney postreperfusion through reduction of vascular resistance, apoptosis and necrosis. This is associated with reduced HMGB1 expression and TLR 2/6 inflammatory pathways.