The effect of the use of a TNF-alpha inhibitor in hypothermic machine perfusion on kidney function after transplantation

Contemporary Clinical Trials 59 (2017) 44–50

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The effect of the use of a TNF-alpha inhibitor in hypothermic machine perfusion on kidney function after transplantation

MARK

Piotr Diuwea, Piotr Domagalaa,⁎, Magdalena Durlikb, Janusz Trzebickic, Andrzej Chmuraa, Artur Kwiatkowskia a b c

Department of General and Transplantation Surgery, The Medical University of Warsaw, Warsaw, Poland Department of Transplant Medicine, Nephrology and Internal Medicine, The Medical University of Warsaw, Warsaw, Poland Department of Anaesthesiology and Intensive Care, The Medical University of Warsaw, Warsaw, Poland

A R T I C L E I N F O

A B S T R A C T

Keywords: Brain death Ischemia-reperfusion injury Kidney transplantation Hypothermic machine perfusion Isolated organ treatment Regenerative medicine

One of the most important problems in transplantation medicine is the ischemia/reperfusion injury of the organs to be transplanted. The aim of the present study was to assess the effect of tumor necrosis factor-alpha (TNFalpha) inhibitor etanercept on the machine perfusion hypothermia of renal allograft kidney function and organ perfusion. No statistically significant differences were found in the impact of the applied intervention on kidney machine perfusion during which the average flow and vascular resistance were evaluated. There were no statistically significant differences in the occurrence of delayed graft function (DGF). Fewer events in patients who received a kidney from the etanercept treated Group A compared to the patients who received a kidney from the control Group B were observed when comparing the functional DGF and occurrence of acute rejection episodes, however, there was no statistically significant difference. In summary, no effect of treatment with etanercept an inhibitor of TNF-alpha in a hypothermic machine perfusion on renal allograft renal survival and its perfusion were detected in this study. However, treatment of the isolated organ may be important for the future of transplantation medicine.

1. Introduction One of the most important problems in transplantation medicine is the ischemia/reperfusion injury of the organs, developing at different stages: in the body of the donor undergoing brain death, in organ harvesting and storage with the potential for ischemia, in reperfusion of the organ, and in the early post-transplantation stage. This damage may affect both early and late results of organ transplantation [1–4]. The factors that have an effect on kidney injury before the transplantation include, but are not limited to: free oxygen radicals [5], proteolytic enzymes, reactive oxygen species [6,7], and apoptosis [8,9]. Endothelial cells, leukocytes, platelets [10], and many cytokines and modulators of inflammatory reactions are also activated, including the proinflammatory tumor necrosis factor alpha (TNF-alpha) cytokine [11–13]. Its expression increases in relation to brain death and after organ reperfusion [14–17]. Increased levels of circulating proinflammatory mediators that accompany brain death and reperfusion may

enhance the immunogenic potential of the transplanted organs and thus contribute to alloimmunization of the recipient and to increased probability of transplant rejection reaction [4,18,19]. Interventions to limit these changes may be directed at the time before organ harvesting, when the organ is in situ in the body of the donor, during organ storage, during transplantation, and early posttransplantation. The purpose of these interventions is to modify biochemical and physical processes related to ischemia and reperfusion, and to minimize the factors that may have a destructive effect on the transplanted organ. The use of mechanical perfusion (hypothermic machine perfusion; HMP) during hypothermic kidney storage reduced the risk of delayed graft function (DGF) and improved graft survival [20–23]. Using HMP for kidney storage is the standard in many centers worldwide. It also enables the assessment of ischemic organ injury through analysis of perfusion parameters, such as vascular resistance and flow, and the relevant chemistry tests of the perfusion fluid. A potential for organ

Abbreviations: AR, acute rejection; CI, confidence interval; CIT, cold ischemia time; CS, cold storage; DGF, delayed graft function; ECD, expanded criteria donors; fDGF, functional delayed graft function; HD, hemodialysis; HMP, hypothermic machine perfusion; HR, hazard ratio; PD, peritoneal dialysis; PNF, primary non-function; PRA, panel reactive antibody; PRU, peripheral resistance unit; RA, rheumatoid arthritis; SCD, standard criteria donors; TIT, total ischemia time; TNF, tumor necrosis factor; Tx, treatment; UW, university of Wisconsin; WIT 2, second warm ischemia time ⁎ Corresponding author at: Department of General and Transplantation Surgery, The Medical University of Warsaw, 59 Nowogrodzka Street, 02-006 Warsaw, Poland. E-mail address: [email protected] (P. Domagala). http://dx.doi.org/10.1016/j.cct.2017.05.013 Received 7 March 2017; Received in revised form 24 May 2017; Accepted 29 May 2017 Available online 29 May 2017 1551-7144/ © 2017 Elsevier Inc. All rights reserved.

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therapy has emerged, because specific drugs may be administered during perfusion. These treatments include attempts to inhibit secretion of proinflammatory molecules and their precursors by inhibiting their expression at the genetic level, by blocking their target sites, or by using monoclonal antibodies against specific antigens. Such interventions may contribute to expansion of therapeutic opportunities for contemporary transplantation medicine, to improve of transplantation outcomes and to increase of the pool of organs used for transplantation.

Table 1 Kidney donor characteristics (mean ± SD). Donors n = 47 SCD/ECD Age ( ± SD) (years) Female/male BMI ( ± SD) (kg/m2) Cause of death CNS trauma Cerebrovascular accidents Other Hypertension (%) Hypotension (%) ICU stay (days) (IQR) Vasopressors (%) Urine output (last day) (mL) Terminal creatinine level (mg/dL)

2. Patients and methods The study included 100 kidneys harvested from 50 organ donors deceased between April 2011 and February 2014 and 94 organ recipients who were transplanted with these organs. After harvest in the facility of the donor, the kidneys were placed in a preservation fluid in simple hypothermia (simple cold storage; CS) and transported in thermostable containers filled with crushed ice (to maintain the temperature of about 4 °C) to the transplantation clinic. The kidneys were surgically prepared in operating theater conditions. After identification of anatomic structures, a vascular cannula was placed on the renal artery and kidneys (each one separately), and the kidneys were placed in the cassette hypothermic organ perfusion LifePort Kidney Transporter (Organ Recovery System, IL) cassette filled with the KPS-1 fluid. Kidneys from each pair were randomly, alternately (left/right) assigned to one of two study groups: Group A (etanercept +), where a p75 Fc receptor protein (TNF-alpha inhibitor) known as etanercept was added to the perfusion fluid after the first hour of perfusion, or the control Group B (etanercept −) without any intervention. Six of 100 kidneys included in the study were disqualified from transplantation. Four kidneys were disqualified due to elevated values of vascular resistance during machine perfusion and two kidneys were disqualified due to the diagnosis of prostate cancer in the donor found upon autopsy. In all, 94 kidney transplantation procedures were performed and each group included 47 kidneys. The kidneys that were not transplanted were excluded from the study.

42/5 (89.4%/10.6%) 45.3 ± 12.2 14/33 (29.8%/70.2%) 25.8 ± 3.6 16 (34%) 26 (55.3%) 5 (10.6%) 17.0 59.6 5 (4. 7) 87.2 4392 ± 1643 1.24 ± 0.6

Abbreviations: BMI, body mass index; CNS, central nervous system; ECD, expanded criteria donors; ICU, intensive care unit; SCD, standard criteria donors. Table 2 Recipient characteristics of treated (etanercept +) and untreated (etanercept −) patient groups.

Age (years) Female/male BMI (kg/m2) HD treatment prior to transplant PD treatment prior to transplant Duration of HD before transplantation (months) (IQR) Duration of PD before transplantation (months) (IQR) Preemptive transplant Hypertension (%) Diabetes (%) Ischemic heart disease (%) Urine output before transplantation (per day) (mL)

2.1. Organ donors Kidneys were harvested from deceased people after the relevant commission had confirmed brain death. The organs were obtained from 47 donors, 70.2% of whom were males. The mean age of donors was 45.3 years. The most prevalent cause of death of the donors was the cerebrovascular disease (55.3%). The donors that died of craniocerebral injuries represented 34% of the study population. Donors' mean serum creatinine levels preceding death was 1.2 mg/dL and the mean urea level was 45.1 mg/dL. The kidneys were collected from standard and expanded criteria donors. United Network for Organ Sharing criteria were adopted for the expanded criteria donors (ECD). Of 47 donors, five (10.6%) met the ECD criteria (Table 1).

Group A (etanercept +)

Group B (etanercept −)

P value

47.1 ± 14 14/33 (29.8%/ 70.2%) 24.1 ± 4.2 39 (83%)

51.9 ± 13.8 15/32 (31.9%/ 68.1%) 24.5 ± 3.6 38 (80.9%)

0.102 0.823

9 (19.1%)

12 (25.5%)

0.620

32 (18. 48)

32 (17. 60)

0.763

12 (8. 39)

28 (15. 41)

0.319

0 (0%) 93.6 21.3 14.9 514 ± 593

1 (2.1%) 97.9 8.5 19.1 824 ± 764

1.0 0.617 0.082 0.583 0.037

1.0

Abbreviations: BMI, body mass index; PD, peritoneal dialysis; HD, hemodialysis. Table 3 Immunological status of treated (etanercept +) and untreated (etanercept−) patient groups.

2.2. Organ recipients No statistically significant differences were found when comparing characteristics of Group A and Group B kidney recipients (Table 2). No significant differences were found in the immunological match between the recipients and the donors or in the pre-transplantation recipient immunization, measured by the level of PRA (the maximum and the last measured values) (Table 3). The most common basic immunosuppression regimen was a three-drug regimen combining a corticosteroid, tacrolimus, and mycophenolic acid ester or sodium salt. No differences were shown between groups with respect to immunosuppression regimens used.

HLA mismatch (average)

Group A (etanercept +)

Group B(etanercept −)

P value

loc A loc B loc DR Totality ( ± SD) Most recent PRA (%) Highest PRA (%)

0.98 1.07 0.56 2.56 ± 1.2 2.59 4.21

1.13 1.2 0.74 3.07 ± 1.4 3.22 5.88

NS NS NS 0.071 0.295 0.868

Abbreviations: PRA, panel reactive antibody.

2.3. Etanercept Etanercept (Enbrel) is a receptor protein, p75 Fc (manufactured by use of genetic DNA recombination) that binds the human tumor necrosis factor (TNF). Etanercept is a dimer of a chimeric protein. It is a combination of the human TNF receptor 2 domain (TNFR2/p75), 45

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ing tests were used for group comparison, as appropriate: the Student's t-test, the Mann–Whitney test, the chi–square test, the Fisher's exact test, and the Cochran–Mantel–Haenszel test. Overall survival and the time to the composite endpoint (death or graft loss) were estimated using Kaplan–Meier curves and the log-rank test and were analyzed with use of the Cox proportional hazard model. The effect of the study intervention on criteria was reported using hazard ratios, 95% confidence intervals, and statistical significance test results. Creatinine levels during patient follow-up after kidney transplantation were analyzed using the linear model for repeated measurements. Statistical significance was defined as P = 0.05.

which binds extracellular ligands, and the human IgG1 Fc domain. Etanercept mechanism of action involves competitive inhibition of TNF binding to its cell surface receptor TNFR, which prevents TNF-mediated cell response and makes TNF biologically inactive. Etanercept may also modulate biological responses controlled by other molecules (e.g., cytokines, adhesion molecules, or proteinases) that are induced or regulated by TNF. Etanercept is used in the treatment of rheumatoid arthritis (RA), multiarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, and vulgar psoriasis (large plaque psoriasis). Etanercept (1.5 mg; 3 mL) was added to the perfusion fluid. 2.4. Organ storage

2.7. Ethics After transport in simple hypothermia from the site of the donor, the kidneys were stored with use using hypothermic machine perfusion (LifePort Kidney Transporter). Each kidney was stored in a separate perfusion device, in a cassette filled with 1 L of perfusion fluid KPS-1 that was constantly cooled, and its temperature was maintained at about 4 °C. Constant flow of the perfusion fluid through the kidney was maintained and systolic pressure during perfusion was maintained at 30 mm Hg. After the first hour of perfusion, 3 mL (1.5 mg) of etanercept solution was added to the perfusion fluid of one kidney from the perfused pair (alternately, the right or the left kidney). The volume of the perfusion fluid amounted 1000 mL per one kidney therefore the concentration of etanercept was 1.5 mg/L. Mean pressure, vascular resistance and flow were measured during perfusion after the first hour (before drug administration) and in the fourth hour of perfusion. All kidneys were perfused up to the start of transplantation therefore there were differences in the period of perfusion time of each kidney in both groups. Hence the time of treatment with etanercept differed in kidneys from Group A.

The study was approved by The Ethics Committee of The Medical University of Warsaw. 3. Results 3.1. Kidney storage and perfusion parameters There were no differences between groups with respect to the mean vascular flow and the mean resistance measured in the first and in the fourth hours of perfusion (Table 4). The mean cold ischemia time (CIT) for the study group was 29 h and 58 min: Group A, 26 h and 22 min; Group B, 28 h and 24 min (P = 0.334) (Table 5). Kidneys from Group A were exposed for etanercept with different time and the mean time was 21 h and 7 min. 3.2. Kidney transplantation results Patient survival at 12 months after transplantation was the same in both groups (97.8%; P = 1.0). Patient survival with functioning graft at 12 months after transplantation (the primary endpoint) was 91.5% in Group A and 95.7% in Group B (P = 0.435). In a selected group of 65 patients (69.1%) with a longer follow-up (those who were enrolled into the study earlier than other patients), patient survival at 24 months of follow-up was 94.2% vs. 97.8% (Fig. 1), and survival of patients with a functioning graft was 86.2% vs. 93.5%, in Group A vs. Group B, respectively (Fig. 2). Death of six kidney transplant recipients was noted before the end of the observation period. There were four deaths in Group A and two deaths in Group B, and function of the graft was preserved in all these patients. Seven recipients from the entire group (five from Group A and two from Group B) lost graft function and were placed on treatment with hemodialysis. Two episodes of primary non-function (PNF) of the

2.5. Assessment of the recipients after kidney transplantation The study analyzed 94 enrolled kidney transplantations performed between April 2011 and February 2014. The follow-up was 12 to 39 months. The observation period was at least 24 months in 64 patients and at least 12 months in 30 patients. Immediate function (IF), delayed function (DGF), and primary non-function (PNF) of the transplanted kidney were analyzed. DGF was defined as the necessity to perform at least one hemodialysis procedure within the first seven days after transplantation. Functional delayed graft function (fDGF) was diagnosed when the serum creatinine level increased, remained unchanged, or decreased by < 10% per day for three consecutive days in the first week after transplantation. Incidence of acute rejection (AR) was diagnosed based on the results of kidney biopsy histopathology per the Banff 2009 criteria. Kidney survival and recipient survival 6, 12, and 24 months after transplantation and transplanted kidney function expressed as the level of creatinine on day 7 and at months 1, 3, 6, 12, and 24 after transplantation were assessed. The primary analysis in this study was testing the difference in patient survival with functioning graft between groups. Secondary endpoints were DGF, fDGF, AR and kidney function expressed as the level of creatinine at 12 months after transplantation. According to approved protocol, analyses of all secondary endpoints were declared as exploratory therefore there is no need for multiplicity adjustment.

Table 4 Perfusion parameters of treated (etanercept +) and untreated (etanercept −) patient groups.

Kidney left/right (n) HMP Time of CS prior to start HMP (min) Mean vascular flow, 1st h (mL/min) Mean vascular flow, 4th h (m/min) Mean renal resistance, 1st h (PRU*) Mean renal resistance, 4th h (PRU)

2.6. Statistical analysis Characteristics of the study groups were analyzed using standard descriptive statistics. For quantitative variables, arithmetic mean along with standard deviation was given, and if a variable showed a distribution different from normal statistical measures of position were used with median values and interquartile range. For qualitative variables, absolute and relative frequencies were reported. The follow-

Group A (etanercept +)

Group B (etanercept −)

P value

23/24 100% 254.3 ± 119.4

24/23 100% 257.4 ± 125.3

0.837 1.0 0.903

123.8 ± 39.8

124.7 ± 39.7

0.909

130.7 ± 35.5

130.4 ± 35.1

0.963

0.22 ± 0.11

0.2 ± 0.09

0.345

0.18 ± 0.07

0.18 ± 0.07

0.818

Abbreviations: CS, cold storage; HMP, hypothermic machine perfusion; PRU, peripheral resistance unit, (mmHg x min/mL).

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Table 5 Characteristics of transplants of treated (etanercept +) and untreated (etanercept -) patient groups.

WIT 2 ( ± SD) (min) CIT ( ± SD) (hr) TIT ( ± SD) (hr)

Group A (etanercept +)

Group B (etanercept −)

P value

36.6 ± 8.2 26 h 22 min ± 8 h 47 min 26 h 58 min ± 8 h 49 min

35.8 ± 10.1 28 h 24 min ± 10 h 58 min 29 h 9 min ± 11 h 5 min

0.667 0.334 0.308

Abbreviations: WIT 2, second warm ischemia time; CIT, cold ischemia time; TIT, total ischemia time.

4. Discussion

graft occurred in Group A, with no statistical significance between the analyzed groups (P = 0.495). Delayed graft function (DGF) occurred with the same frequency in both groups and was 23.4% (11/47). Immediate function (IF) after transplantation was observed in 72.3% (34/47) in Group A and in 76.6% (36/47) in Group B (P = NS). In fDGF analysis, fewer events were observed in the treated Group A (10/47; 21.3%) than in Group B (15/47; 31.9%) (P = 0.234). Similarly, the number of AR episodes was lower in Group A (5/47; 10.6%) than in Group B (9/47; 19.1%) (P = 0.386). These differences were not statistically significant (Table 6). There were no statistically significant differences between groups in serum creatinine levels on day 7 or at months 1, 3, 6, 12, and 24 after kidney transplantation (Fig. 3). An additional evaluation of serum creatinine level and creatinine clearance for day 7 was performed after exclusion of patients with DGF and with DGF/fDGF combined from the analysis. No statistically significant differences were found. The percentage of patients with creatinine concentration < 2 mg/dL after 12 and 24 months were comparable in both groups: 91.7% vs. 87.8% (P = 0.717) and 92% vs. 93.5% (P = 1.0) for Group A vs. Group B, respectively. There was no impact of the treatment time on kidney function, measured in the first period after transplantation as well as in the end of observation (data not presented). An analysis using the proportional hazard Cox model was performed that evaluated the effect of the TNF-α-inhibitor used during hypothermic machine kidney perfusion on the risk of recipient's death and the loss of kidney graft. The comparison used the hazard ratio (HR) with 95% confidence interval (CI) analysis. The intervention (etanercept) caused a 2.3-fold increase in the risk of the recipient's death (CI: 0.57 to 9.65, P = 0.241), and a 2.6-fold increase in the risk of graft loss (CI: 0.48 to 14.11, P = 0.264). The risk of the occurrence of the composite endpoint (occurrence of any event) was 2.5-fold higher in the treated group (CI: 0.88 to 7.05, P = 0.087).

To our knowledge, this study is the first in Poland, and one of the first in the world, to examine prospective clinical human trials evaluating the treatment of an isolated organ stored using hypothermic machine perfusion. Most studies to treat an isolated organ in vitro were conducted in animal models and applied to kidneys, lungs, and heart. Organ damage associated with ischemia and reperfusion is a key factor that influences the early and late results of organ transplantation. The problem for modern transplantation is to identify factors that affect transplanted organs and attempt to counteract of these processes. Machine perfusion helps to limit ischemic injury of a kidney [24]. Application during the storage, could reduce ischemic renal injury, and may be appropriate. This could improve the results of transplantation but also increase the number of transplants using organs that were disqualified previously because of the severity of ischemic injury. In animal studies, after 6 h of induced brain death, the increase in mRNA expression in kidney tubules and interstitium of cytokines interleukin 1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein (MCP-1), and the adhesion molecule ICAM-1 was observed [14]. In another animal study 6 h after induction of brain death, the increase in concentration of proinflammatory cytokines IL-1β, IL-6, and TNF-α in serum was demonstrated [16]. Higher concentrations of cytokines IL-6, IL-8, TNF-α, and IL-2R were found in the serum of brain dead kidney donors than in living kidney donors [17]. In the study of Kim et al., mRNA expression of the proinflammatory cytokines TNF-α, IFN-γ, and IL-10 in kidney biopsies taken from brain dead donors was higher than that found in living kidney donors. We concluded that increased expression of genes for inflammatory cytokines in the tissues of potential brain dead organ donors and negative impact on their immunogenicity may contribute to worse outcomes for transplant recipients [25]. The aim of the present study was to assess the effect of usage of TNFalpha inhibitor etanercept in the machine perfusion hypothermia on renal allograft kidney function and organ perfusion. There were no

Fig. 1. Overall survival - graft loss censored (N = 94). Group A (etanercept +), red line; Group B (etanercept −), black line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Survival with working graft - ‘composite endpoint’ (N = 94). Group A (etanercept +), red line; Group B (etanercept−), black line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Table 6 Results of kidney transplantation in groups.

Patient survival 6th month 12th month 24th month (n = 65) Patient survival with functioning graft 6th month 12th month 24th month (n = 65) PNF DGF fDGF AR Urine output 7th day after Tx ( ± SD) (mL) Serum creatinine ( ± SD) (mL/dL) 7th day 7th daya (n = 72) 7th dab (n = 65) 1st month 3rd month 6th month 12th month 24th month Serum creatinine level < 2 mg/dL after 12 months 24 months

Group A (etanercept +) n = 47

Group B (etanercept −) n = 47

P value

100% 97.7% 94.2% (n = 32)

100% 97.8% 97.8% (n = 33)

1.0 1.0

93.6% 91.5% 86.2% (n = 32) 2 (4.3%) 11 (23.4%) 10 (21.3%) 5 (10.6%) 2878 ± 1089

97.9% 95.7% 93.5% (n = 33) 0% 11 (23.4%) 15 (31.9%) 9 (19.1%) 2967 ± 1200

0.617 0.435 0.495 1.0 0.243 0.386 0.708

3.29 2.38 2.19 2.32 1.89 1.69 1.62 1.32

3.51 ± 2.37 2.58 ± 1.59 2.08 ± 0.67 1.8 ± 1.0 1.8 ± 1.07 1.47 ± 0.51 1.51 ± 0.82 1.36 ± 0.46

0.685 0.594 0.689 0.213 0.780 0.223 0.626 0.750

87.8% 93.5% (n = 33)

0.717 1.0

± ± ± ± ± ± ± ±

2.85 1.61 1.42 2.61 1.41 0.79 1.1 0.48

91.7% 92% (n = 32)

Abbreviations: PNF, primary non-function; DGF, delayed graft function; fDGF, functional delayed graft function; AR, acute rejection; Tx, treatment. a Patients without DGF. b Patients without DGF and fDGF.

performed on small groups of animals, involved different organs, and different drugs and different mechanisms of action. Few reports on the use of a TNF-α inhibitor during perfusion and organ storage have been published. The use of antibodies against TNF-α during lung perfusion with UW (University of Wisconsin) solution in an animal model caused a reduction in ischemia-reperfusion injury and showed that the addition of anti-TNF-α enhanced the potential to limit injury by UW fluid. This procedure improved blood flow through the lung by reducing microvascular permeability after reperfusion and vascular resistance during perfusion. This intervention also reduced biopsy evaluated inflammatory infiltration [26]. A similar intervention using TNF-α suppressor during perfusion was presented by Oshima et al. [27]. This improved hemodynamics after reperfusion and resulted in a statistically significant reduction of TNF-α concentration measured in the serum of the heart recipient in the third

statistically significant differences between groups in the impact of the applied interventions on kidney machine perfusion during which the average flow and vascular resistance were evaluated. There were no significant differences in the occurrence of DGF. The lower number of events in patients who received a kidney from the treated Group A compared to the patients who have received a kidney from the control Group B were observed comparing the functional DGF and occurrence of AR episodes. These values did not differ statistically significantly. The concentration of serum creatinine after 12 months of transplantation was 1.61 vs. 1.51 mg/dL, and after 24 months 1.43 vs. 1.34 mg/dL in Groups A and B, respectively. This was not statistically significant. Survival of a recipient and the survival of a recipient with functioning graft 12 and 24 months' post-transplantation did not differ significantly between the groups compared. Comparison of these results to other published data is difficult. Most previous studies investigating a treatment during organ storage were 48

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Fig. 3. Mean serum creatinine levels. Group A (etanercept +), red line; Group B, (etanercept −), black line. Abbreviations: D7, day seven; M1, month 1; M3, month 3; M6, months 6; M12, months 12; M24, months 24. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Enrichment of fluid used for flushing and storage of organs in hypothermia of different substances is likely to increase their protective effects. Genescà et al. [29] supplemented the UW fluid with fructose1,6-bisphosphonate, and Xu et al. [30] added diazoxide to the Celsior fluid. Thus, it was possible to reduce ischemia-reperfusion injury of organs in animal models. Southard et al. reported the addition of glutathione and adenosine to UW fluid was important for ATP recovery after reperfusion [31]. Chiang'a et al. [32] confirmed the theory that modification of the composition of the UW fluid could increase its protective capacity by reducing the levels of TNF-α in preservation fluid. The addition of prostaglandin E1 (PGE1) to a perfusion fluid during HMP reduced DGF and improved kidney perfusion parameters per Polyak'a et al. [33]. These results show that different drugs used in hypothermic conditions can influence organ injury. TNF-α is one of the cytokines involved in the inflammatory response cascade and is released during apoptosis and brain death. Its effect on ischemia-reperfusion organ injury, however, is so significant that Pecha et al. [34,35] proposed to supplement standard postoperative immunosuppressive therapy with TNF inhibitors. A possible beneficial effect of perioperative use of etanercept was reported by Hering et al. in the pancreatic islet transplantation human model [36] and by Choi et al. in the renal ischemia-reperfusion injury mouse model [37]. Blocking TNFα during hypothermic organ storage may not be sufficient to inhibit injury processes or reduce damage and improve organ function posttransplantation. Blocking gene expression may be a good alternative. Studies on the use of artificially synthesized double stranded short interfering RNA molecules (siRNA) that selectively affect the selected target gene and block the expression of its mRNA have been conducted. The use of siRNA directed to the genes encoding TNF-α during hypothermic storage of mouse heart was presented by Zheng et al. [38]. Blocking TNF-α expression is one of the therapeutic options, however, the complexity of the processes at the cellular, tissue, and organ levels may make this impossible. It may be possible to treat isolated organs in vitro during the storage, which fits with the assumptions of regenerative medicine, however, uncertainties about the most appropriate methods and materials remain.

hour after reperfusion. The small number of patients in groups A and B may explain the lack of statistically significant differences in this study. Forty seven patients in each group may not have been sufficient to reveal the effect of the applied intervention. In each group, only a few episodes of death of the recipient, graft loss, or return to hemodialysis before the end of the observation period were reported. Therefore, it was not possible to conduct multivariate data analysis with an assessment of these impacts. The analyzed intervention resulted in a 2.3-fold increased risk of death of the recipient, 2.6-fold increased risk of graft loss, and 2.5-fold increased risk of a complex endpoint calculated using the Cox proportional hazards model. The observed trend could be a coincidence due to the small number of globally reported events during the observation period and large 95% CIs. The result is surprising because it contradicts predicted and expected baseline assumptions. Prolonged observation and increased the numbers of patients in groups could allow for more accurate assessment and analysis. Because patients were included in the study gradually, the shortest period of follow-up was 12 months and the longest was 46 months. In 29 recipients, follow-up was at least 12 months (15 in Group A and 14 in Group B), and in 65 patients it was at least 24 months (32 in Group A and 33 in Group B). Extending the observation period in recipients and unifying this time in all patients, could increase the number of observed events and increase the power of the statistical analysis. The hypothermic environment with the use of the TNF-α inhibitor is another factor that may have influenced the results. Cellular metabolism is slowed and affects the activity of enzymes and proteins responsible for the distribution and metabolism of drugs, which may restrict or enhance their activity and bioavailability. According to data provided by the manufacturer of the drug, the chemical and physical stability of the prepared solution of Enbrel (etanercept) is maintained for 48 h at a temperature of 2 °C to 8 °C. Therefore, the drug should have been active in this study. However, no data describes whether hypothermia changes the mechanism of action of etanercept, which acts by competitive inhibition of TNF with its cellular surface receptor TNFR. Such a change could reduce the potential for a therapeutic effect of preventing TNF-dependent cellular response. There are single publications of animal studies that demonstrated the ability to use the etanercept therapy during hypothermia. Use of anti-TNF-α during lung perfusion was presented by Chiang et al. [26], and Oshima et al. [27] used a drug to suppress TNF-α during heart perfusion. In the study of Bunegina et al. [28] carried out on animals, UW fluid was enriched with the addition of oxygen during the hypothermic mechanical perfusion of the kidney.

5. Conclusions In summary, no effect of treatment of kidneys with an inhibitor of TNF-alpha in a hypothermic machine perfusion on renal allograft renal perfusion and survival were detected in this study. It is difficult to 49

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determine which stage of the therapeutic intervention to target due to the complexity of the processes involved with organ ischemia and brain death. However, treatment of the isolated organ may be the future of transplantation medicine.

[15]

[16]

Funding sources [17]

The analysis was funded by the Polish Ministry of Science and Higher Education: Limitation of ischemic damage of a kidney stored in machine perfusion in hypothermia – evaluation of the impact on allogeneic kidney transplant function” (project nr N N403 589338; agreement nr 9014/B/P01/2010/38).

[18] [19]

[20]

Disclosure [21]

The analysis was not supported by any companies, including pharmaceutical ones. There is no conflict of interest related to this study.

[22]

Acknowledgments

[23]

We thank our colleagues from The Transplantation Institute of The Medical University of Warsaw who provided continuous care and treatment of patients post-transplantation.

[24]

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